The Pennsylvania State University

The Graduate School

Department of Molecular Medicine

ROLE OF SPHINGOLIPID SIGNALING IN PATHOGENESIS OF LARGE

GRANULAR LYMPHOCYTE LEUKEMIA

A Dissertation in

Molecular Medicine

by

Mithun Vinod Shah

© 2009 Mithun Vinod Shah

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

May 2009

ii The dissertation of Mithun Vinod Shah was reviewed and approved* by the following:

Thomas P. Loughran, Jr. Professor of Medicine Dissertation Advisor Co-Chair of Committee

Rosalyn B. Irby Assistant Professor of Medicine Co-chair of Committee

Gary Clawson Professor of Pathology, and Biochemistry and Molecular Biology

Edward J. Gunther Assistant Professor of Medicine

Charles H. Lang Director, Molecular Medicine Graduate Program

*Signatures are on file in the Graduate School

iii ABSTRACT

Large granular lymphocyte (LGL) leukemia is a disorder of mature cytotoxic cells. LGL leukemia is characterized by accumulation of cytotoxic cells in blood and infiltration in bone marrow and other tissues. Leukemic LGL could arise from expansion of either CD3+ CD8+ T-cells (T-cell LGL leukemia or T-LGL leukemia) or those arising

from CD3- natural killer (NK)-cells (NK-cell LGL leukemia or NK-LGL leukemia).

LGL leukemia is a rare disorder consisting of less than 5% of non-B cell leukemia.

Clinically, LGL leukemia can manifest along a spectrum of disorders ranging from

slowly progressing indolent disorder to an aggressive leukemia that could be fatal within

months. About fifty percent of LGL leukemia patients also present with variety of

autoimmune conditions, rheumatoid arthritis being the most common one.

Normally, following antigen clearance, cytotoxic T-lymphocytes (CTL) become

sensitive to Fas-mediated apoptosis resulting in activation-induced cell death (AICD). In

contrast, the leukemic LGLs are resistance to Fas-mediated apoptosis. It is believed that constitutively active survival signals keep leukemic LGLs alive in the face of functional

Fas-apoptotic machinery. Previously, constitutive activation of Jak-Stat signaling

pathway, PI3k-Akt pathway, and Ras-Mek-Erk signaling pathways have been described in leukemic LGL.

In this study, we identified unique gene signature from PBMC of thirty LGL leukemia patients. Pathway-based analysis of gene signature identified that sphingolipid metabolism and signaling are differentially regulated in leukemic LGL. We also showed that unlike CD8+ cells from healthy donors, sphingosine-1-phosphate (S1P) receptor

iv

S1P5 (EDG8) is the predominant S1P-receptor (S1PR) leukemic LGL. Disruption of balance between pro- and anti-apoptotic sphingolipids (also known as sphingolipid rheostat) leads to selective induction of apoptosis in leukemic LGL. We further showed that sphingosine-1-phosphate (S1P)-mediated signaling induces resistance to Fas- mediated apoptosis in normal activated PBMC. On the other hand, inhibition of S1P- mediated signaling by FTY720 restored Fas-sensitivity in leukemic LGL. Collectively these results suggest that S1P-S1P5 mediated signaling may contribute to abnormal

survival of leukemic LGL that is mediated by Gα12 signaling.

We further investigated how FTY720 induced cell death in leukemic LGL.

FTY720 induced cell death in leukemic LGL that is insensitive to pertussis toxin (PTX) suggesting a role of Gα12/13-mediated signaling in survival of leukemic LGL. FTY720-

mediated cell death is independent of sodium orthovanadate or okadaic acid (OA)

suggesting that FTY720 mediates cell death independent of phosphatases. Our results

indicate that FTY720-mediated cell death in leukemic LGL was independent of action of extrinsic- or intrinsic caspase cascades suggesting a possibility that FTY720 mediated apoptosis-like programmed cell death rather than classical apoptosis. FTY720 damaged mitochondrial membrane prior to induction of cell death and downregulated MCL1 independent of STAT3 activity in one patient sample but not in others.

FTY720 is a novel immunomodulatory agent currently being evaluated for post renal transplant patients and in patients with autoimmune conditions such as multiple sclerosis. Recent work has identified its role as a novel antineoplastic drug as well. Due to significant association of LGL leukemia with autoimmune conditions, it is possible that FTY720 may be of therapeutic benefit to these patients.

v TABLE OF CONTENTS

LIST OF FIGURES ...... ix

LIST OF TABLES...... xii

ACKNOWLEDGEMENTS...... xiii

Chapter 1 Clinical features of Large Granular Lymphocytes...... 1

Clinical features of LGL leukemia ...... 2

Signs and Symptoms...... 2 Immunophenotype and Diagnosis ...... 3

Treatment ...... 4

Survival Signaling in LGL Leukemia...... 5

Role of Ras-Mek-Erk Signaling Pathway...... 5

Dysregulation of Ras-Mek-Erk signaling in pathogenesis of LGL leukemia ...... 6 Therapeutic implications ...... 7

Role of PI3k-Akt Signaling Pathway...... 8

Dysregulation of PI3k-Akt pathway in LGL leukemia ...... 9 Therapeutic Implications...... 10

Role of Nuclear factor kappa-B (NF-κB) Signaling Pathway ...... 10

Dysregulated NF-κB signaling in survival of leukemic LGL ...... 11

Role of Jak-Stat Signaling Pathway...... 12

Dysregulated Jak-Stat pathway in pathogenesis of LGL leukemia ...... 13 Therapeutic implications ...... 14

References...... 16

Chapter 2 Activation-induced Cell Death and its Dysregulation in Leukemic LGL..20

Activation-induced cell death in T-cell homeostasis...... 20

Uncoupling of AICD in LGL leukemia...... 22 Biology of Fas-mediated apoptosis ...... 22

vi Fas-resistance in LGL leukemia ...... 23

Dysregulation of Fas-mediated apoptosis in leukemic LGL ...... 23 Role of soluble Fas and FasL in LGL leukemia...... 26 Abnormal formation of DISC in leukemic LGL ...... 27

Materials and Methods...... 28

Patient consent...... 28 Cell culture and CD8+ T-cell enrichment ...... 28 RNA isolation and global gene expression analysis...... 30 EASE analysis ...... 32

Results ...... 32

Comparison of the gene expression signature of naïve normal cells with activated normal cells...... 32 Comparison of the gene expression signature of naïve normal cells with leukemic LGL...... 34 Comparison of the gene expression signature of activated normal cells with leukemic LGL...... 35 Dysregulated expression of apoptosis related genes in leukemic LGL...... 37 Apoptosis related genetic signature unique to leukemic LGL...... 40 GenMAPP analysis of apoptosis related genes in leukemic LGL...... 41

Validation of microarray analysis...... 43

NKp46 ...... 43 Serine Proteinase Inhibitor 9 (SERPINB9) ...... 45 Other genes...... 47

Discussion...... 48

References...... 50

Chapter 3 Role of Sphingolipid Rheostat in LGL Leukemia ...... 53

Introduction...... 53

Role of sphingolipids in cancer ...... 54 Role of sphingolipids in inflammatory and auto-immune conditions ...... 55

Materials and methods ...... 56

Patient consent...... 56 Chemicals and reagents ...... 56 Antibodies...... 57

vii Pathway-based analysis...... 57 Real-time PCR analysis...... 58 Western blot assay...... 60 Apoptosis assay ...... 60

Results ...... 61

Pathway-based (GSEA) microarray analysis of leukemic LGL...... 61

Alpha-subunit of acid ceramidase is downregulated in leukemic LGL: ...... 63 S1P-receptors in CD8+ T-cells: ...... 64 S1PR profiling in leukemic LGL: ...... 65 Differential regulation of sphingolipid metabolism and signaling play an important role in survival of leukemic LGL...... 66 Inhibition of acid ceramidase leads to selective induction of apoptosis in leukemic LGL ...... 68 Inhibition of S1P-mediated signaling leads to selective induction of apoptosis in LGL leukemia PBMC ...... 70 Differential regulation of S1P-mediated signaling contributes to abnormal survival of leukemic LGL ...... 71

Discussion...... 75

Model of sphingolipid rheostat in LGL leukemia: ...... 79

Therapeutic opportunities ...... 80

References...... 82

Chapter 4 Mechanism of FTY720 mediated cell death in LGL leukemia...... 86

Introduction...... 86

Mechanism of FTY720-induced cell death ...... 88

Materials and Methods...... 89

Patient consent...... 89 Chemicals and reagents ...... 89 Antibodies...... 90 Real-time PCR analysis...... 90 Western blot assay...... 91 Apoptosis assay ...... 91

Results ...... 92

FTY720 mediated cell death is independent of Gαi signaling...... 92

viii FTY720 mediated cell death is independent of actions of caspases...... 96 FTY720 damages mitochondrial membrane prior to inducing cell death in leukemic LGL...... 102 Real-time PCR analysis of putative S1P5 targets in leukemic LGL...... 107 Mechanism of FTY720 mediated cell death in leukemic LGL is independent of phosphatases...... 108

Discussion...... 111

References...... 115

Chapter 5 Conclusions and Future Directions ...... 118

Future Directions ...... 120

Appendix A Gene Signature activated normal cells vs. resting normal cells...... 122

Appendix B Gene signature leukemic LGL vs. resting normal cells ...... 141

Appendix C Gene Signature: Leukemic LGL vs activated normal cells...... 149

Appendix D Differentially expressed apoptosis related (Gene ontology category # 006915) in leukemic LGL vs. activated enriched CD8+ cells...... 188

ix LIST OF FIGURES

Figure 1: Cross-talk among various signaling pathways in leukemic LGL...... 15

Figure 2: Activation-induced cell death (AICD) in T-cells and role of Fas ...... 21

Figure 3: Model of apoptosis-resistance in leukemic LGL ...... 25

Figure 4: Preparation of samples and experimental procedure...... 29

Figure 5: Preparation of samples and experimental procedure...... 30

Figure 6: Microarray profiling of naïve normal PBMC and activated normal PBMC using gene- and theme-based approach...... 33

Figure 7: Microarray profiling of leukemic LGL compared to naive normal PBMC using gene- and theme-based approach...... 35

Figure 8: Constitutive gene expression signature of leukemic LGL compared to activated normal cells ...... 37

Figure 9: Differentially regulated apoptosis related genes in leukemic LGL compared to activated enriched normal CD8+ cells...... 41

Figure 10: GenMAPP analysis of differentially regulated apoptosis related genes in leukemic LGL...... 42

Figure 11: NKp46 is expressed in T-LGL leukemia cells ...... 44

Figure 12: SERPINB9 is overexpressed in leukemic LGL compared to normal phenotypes ...... 46

Figure 13: Sphingolipid metabolism and signaling pathway is differentially regulated in leukemic LGL...... 62

Figure 14: Gene-based analysis of microarray experiments for ASAH1 mRNA expression ...... 63

Figure 15: Differential expression of ASAH1 in LGL leukemia PBMC ...... 64

Figure 16: Relative abundance of S1P-receptors in naïve enriched normal CD8+ cells...... 65

Figure 17: Relative expressions of S1PR in leukemic LGL compared to normal cells...... 66

x Figure 18: Inhibition of enzymes outside sphingolipid rheostat does not induce selective apoptosis in leukemic LGL...... 68

Figure 19: Inhibition of acid ceramidase induced differential apoptosis in leukemic LGL...... 69

Figure 20: Inhibition of S1P-mediated signaling by FTY720 induced differential apoptosis in leukemic LGL...... 71

Figure 21: FTY720 selectively induces apoptosis in leukemic LGL but not in LGL from healthy donors ...... 72

Figure 22: Treatment with FTY720 sensitizes LGL leukemia PBMC to Fas- mediated apoptosis...... 73

Figure 23: Exogenous S1P produces Fas-resistance phenotype in activated cells from healthy donors...... 74

Figure 24: Sphingolipid rheostat and its role in survival of leukemic LGL ...... 80

Figure 25: FTY720, an inhibitor of S1P-mediated signaling ...... 93

Figure 26: FTY720 mediated apoptosis is insensitive to PTX ...... 95

Figure 27: CH11-mediated apoptosis in Jurkat T-cells is dependent of action of caspases. z-VAD-FMK is a cell permeable pan-caspase inhibitor...... 97

Figure 28: CH11-mediated apoptosis in Jurkat T-cells is dependent of action of caspases...... 98

Figure 29: FTY720 mediated cell death in leuekmemic LGL is independent of the action of caspases...... 99

Figure 30: FTY720 mediated cell death in leukemic LGL is independent of caspase-3...... 100

Figure 31: FTY720 induced cell death in leukemic LGL is independent of action of caspases -8 and -9...... 102

Figure 32: Induction of cell death by FTY720 correlates with breach in mitochondrial integrity ...... 104

Figure 33: FTY720 mediated induction of cell death is correlated with downregulation of myeloid cell leukemia-1 but not with cleavage of Bcl-2 associated X-protein (BAX) ...... 106

xi

Figure 34: Quantitative real-time PCR analysis of putative S1P5 targets in leukemic LGL...... 108

Figure 35: FTY720 mediated cell death is independent of Sodium Orthovanadate (VA) pre-treatment in leukemic LGL...... 109

Figure 36: Okadaic acid (OA) does not rescue FTY720-treated leukemic LGL from undergoing cell death...... 111

xii LIST OF TABLES

Table 1: List of differentially expressed genes in leukemic LGL compared to activated enriched CD8+ cells that belong to GO category ‘apoptosis’ (GO:0006915). Fold change referes to fold-expression observed in leukemic LGL compared to activated enriched CD8+ cells...... 39

Table 2: List of Primers Used for Real-Time PCR...... 59

xiii ACKNOWLEDGEMENTS

I would like to acknowledge my gratitude towards my parents. They have supported me in all the endeavors I undertook. They have made innumerable sacrifices to get me where I am today. I would like to thank my family for their support and encouragement when it mattered the most.

I would like to thank my mentor, Dr. Thomas P. Loughran, Jr., for his continued support throughout my graduate career. At times, when I questioned my abilities, he placed insurmountable amount of confidence in me and guided me in calm manner. I would like to thank Dr. Rosalyn Irby for all her support. I would like to thank my committee members Dr. Gary Clawson and Dr. Edward Gunther for their continued support and guidance in keeping me on track to finish this adventure in time. I would also like to thank Dr. Gary Clawson, Dr. Edward Gunther and Dr. Rosalyn Irby for their immense help during research rotations I did. Their patience is really appreciated.

Finally, I would like to thank my colleagues and members of Loughran lab for supporting me and their ever-readiness to help me out with my research. My special thanks to Ranran Zhang for helping me with experiments and carefully scrutinizing manuscripts as well as this thesis.

Chapter 1

Clinical features of Large Granular Lymphocytes

Large granular lymphocyte (LGL) leukemia is a rare disorder of cytotoxic

lymphocytes. It was first described as a clonal proliferation of LGL involving blood, marrow, and spleen (Loughran, Kadin et al. 1985). There are two varieties of LGL leukemia depending upon the cell of origin – leukemia of cytotoxic T-lymphocytes (T- cell LGL leukemia or T-LGL leukemia) and that of natural killer (NK) cells (NK-cell

LGL leukemia or NK-LGL leukemia) (Loughran 1993). T-LGL leukemia is characterized by expansion of clonal CD3+ CD8+ T-cells (cytotoxic T-lymphocytes,

CTL), while NK-LGL is characterized by expansion of CD3- LGL cells.

Despite important differences in the origin and functions, CTL share several characteristics with NK-cells (Smyth, Kelly et al. 2001). Together, they are commonly referred to as cytotoxic cells. In peripheral smear examination, LGL are larger than most lymphocytes (almost double the size of a red blood cell) and contain azurophilic granules in cytoplasm.

LGL are a crucial effector arm of the immune system. They actively survey for virus-infected or transformed cells. Upon recognition, LGL make brief contact with target cells and induce apoptosis in the latter (Smyth, Kelly et al. 2001; Van Lier, Ten

Berge et al. 2003). LGL use various strategies to induce apoptosis in the target cells.

One way to induce apoptosis in target cells is by delivering an extracellular signal through death receptors such as Fas (CD95). Interaction of Fas with its ligand known as

2 Fas ligand (CD95L, FasL) induces apoptosis in Fas-bearing cells. While expression of

Fas is widely distributed, expression of FasL is relatively restricted to activated LGL

(Krammer 2000). This distribution pattern allows for surveying of a variety of cells by

LGL and mounting immune response if needed.

Another mechanism of apoptosis induction is through actions of cytotoxins. LGL contain various cytotoxins such as perforin (pore forming protein, pfp) and granzyme B

(GrB) in azurophilic granules that can be localized to the synapse formed between effector and target cells. Perforin punches pores in target cells and facilitates delivery of other cytotoxins into the target cells. Among known cytotoxins, GrB is a powerful inducer of apoptosis. Once in target cells, GrB cleaves and activates various proteins such as caspases, filamin, nuclear poly(ADP-ribose) polymerase (PARP) and Bid leading to cell death in both caspase dependent and independent manners (Krammer 2000;

Smyth, Kelly et al. 2001).

Clinical features of LGL leukemia

Signs and Symptoms

Clinically, LGL leukemia is a disorder of middle-aged individuals with median age being around 50 years. Both T- and NK-LGL leukemia may manifest as an indolent disorder or an aggressive leukemia. Aggressive NK-LGL leukemia is one of the most aggressive tumors known, with median survival being a few months following diagnosis.

3 Fortunately, more than 85% of LGL leukemia manifest as indolent T-cell disease with median survival of more than 10 years (Loughran 1993; Sokol and Loughran 2006).

About a third of patients with T-LGL are asymptomatic and diagnosed coincidentally. The rest present with an array of symptoms depending upon underlying pathology. Recurrent infections due to coexistent neutropenia is a common feature of

LGL leukemia Other hematologic conditions may be associated with LGL leukemia, including hemolytic anemia, pure red cell aplasia, cyclic neutropenia and aplastic anemia.

Some patients present with B-symptoms such as fever, unexplained weight loss and night sweats. While rheumatoid arthritis is the most common autoimmune condition seen in

LGL leukemia, LGL leukemia may be associated with a wide spectrum of autoimmune conditions (Lamy and Loughran 1998; Sokol and Loughran 2006).

Immunophenotype and Diagnosis

T-cell LGL leukemia is characterized by expansion of CD3+ CD8+ T-cell receptor (TCR)-αβ T-cells though rarely CD3+ CD4+ or CD4- CD8- TCRγδ T-cells are involved (Loughran 1993). It was recently shown that leukemic T-LGL have a CD3+

CD8+ CD45RA+ CD62L- phenotype consistent with effector-memory RA T-cells

(TEMRA) (Loughran 1993; Yang, Epling-Burnette et al. 2008). Leukemic T-LGL can be

considered as a malignant expansion of TEMRA. Leukemic T-LGL often express CD57+

which is a marker for mature T-cells. NK-LGL leukemia is characterized by CD3-

CD56+ and/or CD16+ cells (Loughran 1993).

4 LGL leukemia is diagnosed using hematological investigations including the examination of peripheral smear, flow cytometry, and polymerase chain reaction (PCR) for TCR (TCR rearrangement assay). Persistent elevated LGL count, as demonstrated by a typical LGL morphology on peripheral smear, points towards LGL leukemia. Flow cytometry and molecular clonality studies (such as TCR rearrangement assays) can be useful in suggesting or establishing clonal origin of cells in T-LGL leukemia. Absence of

TCR-restriction can make diagnosis difficult for NK-LGL, but the study of NK-receptor repertoire may suggest clonality in NK-LGL leukemia patients (Loughran 1993; Lamy and Loughran 2003; Sokol and Loughran 2006).

Treatment

The mainstay of treatment in LGL leukemia is immunosuppressive therapy rather than chemotherapy. LGL leukemia patients with indolent course usually do not require any treatment. In such cases, regular follow-up is important to establish the course of the disease and monitor associated symptoms (Lamy and Loughran 2003; Sokol and

Loughran 2006). Indications for therapy include symptomatic anemia/neutropenia or severe anemia (transfusion dependent) or neutropenia (absolute neutrophil count

<500/µl). While there is no established standard of care, choices of immunosuppressive therapy include methotrexate, cyclophosphamide, and cyclosporine A. Corticosteroids such as prednisone may be used to hasten the clinical response. Treatment options such as fludarabine, anti-CD52 monoclonal antibody (alemtuzumab), antithymocyte globulin

5 (ATG) or splenectomy are considered second-line options in case of failure with the first- line treatment (Sokol and Loughran 2006).

Survival Signaling in LGL Leukemia

Role of Ras-Mek-Erk Signaling Pathway

Ras has a well-established role in tumor biology. Mutations of Ras occur in about

30% of all human cancers (Schubbert, Shannon et al. 2007). Ras family of proteins belongs to guanidine triphosphatase (GTPase). In its active (GTP-bound) form, Ras engages various downstream effector pathways that play an essential role in cellular responses such as survival, proliferation and differentiation (Schubbert, Shannon et al.

2007).

Ras cascade is the major pro-survival regulator following T-cell activation (Mor and Philips 2006). Ras is activated following activation signal delivered to receptor tyrosine kinases (RTK). For cytokines and growth factors, this means binding of ligand to its receptor. For T-cells, immunoreceptor tyrosine-based activation motifs (ITAM) of

TCR translate the signal of antigen engagement to Ras. Ras signaling can be activated by

Src-family kinases (SFK), platelet-derived growth factor (PDGF), or sphingosine-1- phosphate (S1P) through Gα12 (Heldin and Westermark 1999; Pyne and Pyne 2002;

Samelson 2002; Veillette, Latour et al. 2002). This allows Ras to bind to GTP and

become activated.

6 Ras activity is also regulated by post-transcriptional regulations. Prenyl transferases are a class of enzymes that include farnesyl transferases (FT) and geranylgeranyl transferases (GGT). Prenyl-transferases modify Ras activity by adding either one or two hydrophobic moieties on C-terminus of RAS. This modification is essential to anchor Ras on cytosolic leaflet of cellular membranes which is a pre-requisite for activation (Mor and Philips 2006; Schubbert, Shannon et al. 2007).

Once activated, Ras phosphorylates and activates Raf-1. Raf-1 phosphorylates

MAPK-extracellular regulated kinase (ERK) kinase (MEK) resulting in its activation.

Activated MEK in turn phosphorylates ERK resulting in its translocation to the nucleus.

In the nucleus, ERK activates various transcription factors, including Fos and Jun of Ets family, which contribute to resulting in proliferation, differentiation or survival of cells

(Mor and Philips 2006; Schubbert, Shannon et al. 2007; Steelman, Abrams et al. 2008).

Ras signaling also promotes survival by directly promoting the transcription of FLIP and

MCL1 (Budd, Yeh et al. 2006; Huntington, Puthalakath et al. 2007). In Jurkat T-cells

MAPK/ERK activity is inversely proportional to Fas-sensitivity and anti-apoptotic activity of MAPK/ERK signaling overrides Fas-mediated apoptotic signals (Holmström,

Schmitz et al. 2000).

Dysregulation of Ras-Mek-Erk signaling in pathogenesis of LGL leukemia

LGL leukemia patients harbor constitutively active form of Ras (H-Ras-GTP) in their PBMC (Epling-Burnette, Bai et al. 2004). Ras-Mek-Erk signaling was found to be constitutively activated in leukemic LGL.

7 Inhibition of RAS, MEK or ERK induced apoptosis as well as restored Fas- sensitivity in leukemic LGL (Epling-Burnette, Bai et al. 2004). Inhibition of Ras either using chemical inhibitor FTI2153 or by overexpressing dominant negative form of Ras, induced apoptosis in leukemic LGL by inhibiting ERK activity. Similar results were obtained using MAPK inhibitors (PD98059 or U0216) that induced apoptosis in leukemic

LGL and restored Fas-sensitivity in Erk-dependent manner. These results suggest that overactive RAS and MEK lies upstream to ERK in mediating survival signals in leukemic LGL (Epling-Burnette, Bai et al. 2004).

Therapeutic implications

R1150777 (Zarnestra, Tipifarnib) is a farnesyl transferase inhibitor (FTI) designed to inhibit Ras pathway. Zarnestra is being investigated as a potential treatment in various tumors including leukemia (Armand, Burnett et al. 2007). Since Ras isoforms such as H-

Ras requires farnesylation for malignant transformation activity,(Zhu, Gerbino et al.

2005) it was hypothesized that by inhibiting farnesylation of Ras, Zarnestra would inhibit

Ras-mediated signaling in LGL leukemia. A clinical trial was conducted on eight LGL leukemia patients using this drug. While none of the patients achieved clinical response, interesting biological responses were observed in most of these patients. One patient with NK-LGL leukemia had improvement in symptoms and signs of pulmonary hypertension while receiving Zarnestra (Epling-Burnette, Sokol et al. 2008).

8

Role of PI3k-Akt Signaling Pathway

Among downstream effector pathways of Ras cascade, phosphoinositide-3-kinase

(PI3k) -v-akt murine thymoma viral oncogene homolog (Akt) mediated signaling has well established role in metabolism, survival and proliferation. With such cell stimulating profile, it is hardly any surprise that dysregulation of PI3k-Akt signaling is seen in various human tumors. This dysregulation is commonly caused by the deletion or mutation of a negative regulator of PI3k-Akt signaling, phosphatase and tensin homolog (PTEN) (Vivanco and Sawyers 2002).

In T-cells, PI3k-Akt signaling plays role in T-cell receptor mediated activation and proliferation. PI3K is activated upon membrane relocation. This relocation is mediated by the interaction of PI3K with RAS or SFK, or through the direct interaction of PI3K with cytokine receptors at the SH2-domain binding site (Vivanco and Sawyers

2002; Schade, Powers et al. 2006; Steelman, Abrams et al. 2008). The most studied component downstream of PI3K is a serine/threonine kinase - AKT (also known as protein kinase B, PKB). Activation of AKT accounts for many of the biological functions of PI3K. To promote proliferation, AKT acts through inducing cyclin D1, and mammalian target of rapamycin (mTOR) pathway, as well as downregulating forkhead box class O (FOXO) transcription factors and cyclin-dependent kinase inhibitors (CKI) such as p21WAF and p27KIP1.

One of the main targets downstream to Akt in pro-survival signaling is NF-κB

signaling (see below). Further, AKT phosphorylates Bcl-2 antagonist of cell death

9

(BAD), preventing its interaction with anti-apoptotic factor Bcl-xL. This leaves Bcl-xL to exert its anti-apoptotic functions. AKT phosphorylates and inhibits caspase-9, thus inhibiting apoptosis. Another anti-apoptotic phosphorylation target of AKT is MDM2 – a negative regulator of tumor suppressor p53. Phosphorylated MDM2 binds to p53, expediting its degradation and interfering with tumor suppression effects of the latter

(Mayo and Donner 2001; Wymann and Schneiter 2008). Following activation, PI3k-Akt signaling can enhance survival of T-cells through inhibition of Fas clustering and DISC formation. This mechanism may contribute to abnormal DISC formation and Fas- resistance phenotype observed in leukemic LGL (Lamy, Liu et al. 1998; Yang, Epling-

Burnette et al. 2008).

Dysregulation of PI3k-Akt pathway in LGL leukemia

In LGL leukemia PBMC, SFK maintain PI3K in its constitutively activated form as assessed by phosphorylation of AKT and glycogen synthase kinase-3 (GSK3).

Inhibition of SFK or PI3K induced apoptosis in leukemic LGL is accompanied by inhibition of ERK1/2 activity. It was proposed that SFK-mediated activation of PI3k-

Akt pathway results in constitutive ERK activity in leukemic LGL. This placed Akt upstream to Erk in LGL survival signaling. Inhibition of this pathway at any level – namely inhibition of SFK, AKT or ERK –induced apoptosis in leukemic LGL (Epling-

Burnette, Bai et al. 2004; Schade, Powers et al. 2006).

10 Therapeutic Implications

Due to their role in promoting proliferation and survival, PI3k and Akt are good candidate drug targets for anti-tumor therapy. Inhibitors of PI3K such as PI-103 and

ZSTK474 have shown promise as anti-tumor agents (Wymann and Schneiter 2008).

Role of Nuclear factor kappa-B (NF-κB) Signaling Pathway

NF-κB was first identified as an enhancer of immunoglobulin κ-light chain in activated B-cells (Sen and Baltimore 1986). Subsequently, it was realized that NF-κB plays an essential role in hematopoiesis, inflammation, as well as survival and proliferation of adaptive immune system cells. Thus, NF-κB is now recognized as a critical player in almost all the aspects of immune responses (Sen and Baltimore 1986;

Hayden, West et al. 2006). Normally, NF-κB is found in the cytoplasm as a complex with the inhibitor of NF-κB (IκB). This complex keeps NF-κB from both entering the nucleus and binding to DNA, thus depriving its transcriptional functions. This inhibition is void once IκB is phosphorylated by (IκB)-kinase complex (IKK). Phsophorylation of

IκB, leads to its ubiquitination and proteosomal degradation. IKK is a known AKT substrate, rendering NF-κB downstream to PI3K-AKT pathway. In summary, phosphorylation of IKK by AKT leads to activation of the IKK, which in turn leads to phosphorylation and degradation of IκB, leading to NF-κB activation (Wymann and

Schneiter 2008).

Activation of NF-κB is one of the best characterized pathways in antigen-receptor signaling in both B- and T-cells. Activation of NF-κB downstream of TCR ligation

11 facilitates antigen specific proliferation and maturation of lymphocytes into effector cells.

NF-κB orchestrates T-cell activation by providing a milieu to proliferate (such as inducing IL2 production) and acquire effector functions (such as promoting transcription of RANTES, Fas and FasL) (Hoffmann and Baltimore 2006).

The most important of NF-κB functions is protection of T-cells against AICD

(Rivera-Walsh, Cvijic et al. 2000). This function is executed primarily through promoting the expression of pro-survival Bcl-2 family members and inhibitors of apoptosis (IAPs) (Hoffmann and Baltimore 2006). Deficiency or inhibition of NF-κB activity results in failure of activation, either due to lack of proliferation or premature onset of apoptosis suggesting crucial role of NF-κB signaling in mounting effective T- cell response (Kontgen, Grumont et al. 1995; Jeremias, Kupatt et al. 1998; Wan and

DeGregori 2003; Hayden, West et al. 2006).

Dysregulated NF-κB signaling in survival of leukemic LGL

The pivotal role that NF-κB plays in CD8+ T-cell activation and survival along with an established role in various aspects of tumorigenesis as well as inflammation makes NF-κB an interesting candidate to study in LGL leukemia. Gene expression signature of leukemic LGL showed that c-Rel was overexpressed in leukemic LGL

(Shah, Zhang et al. 2008). We recently found that leukemic LGL show constitutive activity of NF-κB compared to normal PBMC. We further found that the inhibition of

NF-κB resulted in apoptosis of leukemic LGL. Inhibition of Akt led to inhibition of NF-

κB activity whereas, inhibition of NF-κB activity did not affect Akt phosphorylation.

12 This suggests that NF-κB acts downstream of PI3k-Akt pathway in leukemic LGL. We also found that NF-κB maintains expression of Mcl-1 independent of STAT3 activity

(Zhang, Shah et al. 2008).

Role of Jak-Stat Signaling Pathway

Janus kinase-signal transducers and activators of transcription (Jak-Stat) signaling cascade plays role in conferring survival in various tumors. It is also known to be activated following T-cell activation. JAK proteins are kinases that phosphorylate STAT proteins. STAT proteins are latent transcription factors that, upon activation, transcribe various known anti-apoptotic genes. Jak-Stat pathway plays an essential role following cytokine signaling. Four members of Jak family and seven members of Stat family are known in humans. Stat family members may form homo- or heterodimers resulting in various combinations that may have overlapping but distinct transcription profiles (Levy and Lee 2002; Murray 2007).

Upon activation of cytokine or growth factor receptor, aggregation of receptors leads to transphosphorylation of JAK leading to its activation. Activated JAK can now bind to STAT. STAT proteins contain an N-terminus dimerization domain, a central

DNA-binding domain and a C-terminus transactivation domain. Phosphorylation of tyrosine and threonine (believed to be mediated by ERK) of the dimerization domain allows STAT proteins to form homo- or heterodimers. Dimerized STAT proteins then translocate to the nucleus where they exert their transcriptional activities by binding to enhancer regions of the target genes including Bcl-xL, myeloid cell leukemia sequence 1

13 (Mcl-1), IAP-family of protein survivin (BIRC5), cell-cycle regulator cyclin D1, c-Myc and vascular endothelial growth factor (VEGF) (Levy and Inghirami 2006). While physiological activation of STATs last for a few minutes to a few hours, constitutively activated STATs are frequently found in a wide variety of human tumors (Steelman,

Abrams et al. 2008). Transformations mediated by oncogenes such as v-src, v-abl, v-fps, v-fes, v-eyk and Gα12 have been shown to be mediated by STAT especially by STAT3

(Epling-Burnette 2001; Buettner, Mora et al. 2002; Kumar, Shore et al. 2005).

Dysregulated Jak-Stat pathway in pathogenesis of LGL leukemia

Leukemic LGL harbor constitutively activated STAT1 and/or STAT3 but not

STAT5. STAT3 and/or STAT1 dimers from LGL leukemia patients’ PBMC showed

DNA binding activity equivalent to that of in vitro activated normal PBMC, suggesting

that leukemic LGL are activated in vivo. Inhibition of JAK2/3 using small molecular

tyrosine kinase inhibitor AG490 induced apoptosis in leukemic LGL and restored Fas-

sensitivity. In addition, specific inhibition of STAT3 using antisense to STAT3 induced

significant apoptosis and restored Fas-sensitivity in leukemic LGL. The promoter region

of human MCL1 - a BCL2 family member important for maintaining mitochondria

integrity - contains a STAT3 binding site. In leukemic LGL, STAT3 binds to this site

and induces expression of MCL1. The induction of apoptosis through STAT3 inhibition

correlates with decreased MCL1 expression, indicating a role of anti-apoptotic protein

MCL1 in survival of leukemic LGL (Epling-Burnette 2001).

14 Therapeutic implications

Recently, there has been a great interest in finding specific methods to inhibit

Stat3 signaling. Small molecules inhibitors (JSI124 and platinum (IV) compounds such as CPA-7 or peptide-based inhibitors specific to STAT3,(Turkson, Zhang et al. 2004;

Iwamaru, Szymanski et al. 2006; Schust, Sperl et al. 2006; Tan, Lan et al. 2006) have shown promising results in specifically inhibiting Stat3-mediated signaling. Given the role of Jak-Stat pathway in survival of leukemic LGL, it will be interesting to see if these novel agents can be used as therapeutic strategies in LGL leukemia patients.

Signaling cascades serve triple purposes – they carry message from the cell surface into the nucleus, amplify the signal, and regulate the cell in order to adapt to the environment. In doing so, survival pathways do not work in isolation. Instead, they are involved in dynamic and complex interaction. Figure 1 shows potential interactions

between various survival signaling pathways described above.

15

Figure 1: Cross-talk among various signaling pathways in leukemic LGL Various survival signaling pathways interact and cross-regulate each other at various levels. It is this dynamic interaction that leads to survival in leukemic LGL. Figure shows known survival signaling pathways and their interactions. Legends used: red - upregulation and/or constitutive activation, green - deregulation, blue – downregulation/or constitutive inhibition; white - no direct information available in LGL leukemia; golden – state of a cell or its component such as apoptosis and mitochondrial integrity loss, rectangle – intracellular component, circle - extracellular component; blue edges – activation/enhancement, red edges – inhibition or downregulation (Shah et al, Cancer, in press).

16

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Chapter 2

Activation-induced Cell Death and its Dysregulation in Leukemic LGL

Activation-induced cell death in T-cell homeostasis

In the periphery, an antigen encounter by an antigen-specific naïve T-cell can lead to proliferation of that T-cell. To become activated, CD8+ T-cells require three stimuli: i) antigen-receptor signaling that is mediated through TCR-CD3 complex, ii) co- stimulation provided by CD28 with its ligands CD80 or CD86, and iii) a ‘third signal’ which is provided by cytokines such as IL-12, type I interferons (IFN) -α and -β, or type

II interferon such as IFN-γ (Hayden, West et al. 2006; Mescher, Popescu et al. 2007).

Within days, vigorous proliferation leads to an increase in antigen-specific T-cells by about 50,000-fold (Williams and Bevan 2007). This is accompanied by acquisition of effector functions (Thome and Tschopp 2001). Since unchecked proliferation and cytotoxic potential of CTL is not desirable due to risk of autoimmunity and malignancy, most of these cells are selectively eliminated following antigen clearance (Thome and

Tschopp 2001) (Figure 2).

21

Figure 2: Activation-induced cell death (AICD) in T-cells and role of Fas Activation of T-cells may be divided in three phases for better understanding. The expansion phase is characterized by exponential expansion of antigen-specific T-cells due to proliferation. During this phase, T-cells are only minimally sensitive to Fas-mediated apoptosis. Fas-sensitivity increases and by around 10-days, T-cells are sensitive to Fas- mediated apoptosis leading to Fas-dependent deletion of activated CTL (Thome and Tschopp 2001).

The process of elimination of cytotoxic T-cells serves as a defense against autoimmunity. This process is called activation-induced cell death (AICD) in that apoptosis of CTL ensues and is induced by T-cell receptor mediated activation. AICD is important in maintaining T-cell homeostasis as well as tolerance to self-antigen in periphery (Zhang, Xu et al. 2004). One mechanism of AICD is through interaction of

22 death receptor Fas with FasL (Krammer 2000; Zhang, Xu et al. 2004). Activation of

CTLs upregulate the surface expression of both Fas and FasL on its surface. This elevation ensures that activated CTL can be effectively eliminated via FasL-mediated apoptosis either through autocrine or paracrine fashion after clearing the infection.

Uncoupling of AICD in LGL leukemia

Gene expression profiling carried out using microarray technology showed a unique gene expression signature in LGL leukemia PBMC (Shah, Zhang et al. 2008).

Expression Analysis Systematic Explorer (EASE) (Hosack, Dennis et al. 2003) suggested that while leukemic LGL shows an expression pattern in agreement with acquisition of effector functions, they show severe dysregulation in apoptotic machinery. Various genes known to have pro-apoptotic function were downregulated while those with known anti-apoptotic functions were upregulated. While genes related with ‘activation’ were expressed in agreement with normal activated cells, the apoptosis related genes were dysregulated. It was further suggested that uncoupling of activation and apoptotic signals in LGL leukemia may lead to inhibition of AICD (Shah, Zhang et al. 2008).

Biology of Fas-mediated apoptosis

Fas is a member of tumor necrosis factor receptor (TNFR) family of proteins that plays role in CTL-mediated apoptosis of target cells including other activated CTL

(Matiba, Mariani et al. 1997; Thome and Tschopp 2001). Interaction of FasL with its

23 receptor leads to trimerization of Fas. The cytosolic portion of the trimerized receptor complex binds to an adaptor protein called Fas-associated death domain (FADD).

Collectively, this complex is called death-inducing signaling complex (DISC) (Matiba,

Mariani et al. 1997). Formation of DISC allows for binding and activation of a zymogen known as procaspase-8. Binding of procaspase-8 leads to its cleavage and formation of heterotetramers of two p10 and p18 subunits each (Lavrik, Krueger et al.). Caspase-8 serves as an initiator caspase that activates other caspases (known as effector caspases) eventually culminating in cell death. DISC formation and activation of caspase-8 are, in part, regulated by cellular FADD-like IL1-converting enzyme (FLICE)-inhibitory protein

(c-FLIP) (Thome and Tschopp 2001).

Fas-resistance in LGL leukemia

Dysregulation of Fas-mediated apoptosis in leukemic LGL

Like activated normal CTL, leukemic LGL express abundant Fas and FasL on their surfaces. However, while normal activated CTL readily undergo Fas-FasL mediated apoptosis, leukemic LGL are resistant to FasL-mediated apoptosis (Lamy, Liu et al. 1998). This raises two possibilities: i) Fas-FasL apoptotic machinery is deficient in leukemic LGL or ii) some constitutively present survival signals keep leukemic LGL alive even in the face of Fas-mediated death signals. Both these possibilities have been investigated thoroughly.

24 Mouse models harboring defective Fas-FasL apoptotic machinery show severe defects in immune system homeostasis. While gld mice have mutated FasL that has defective functions, lpr mice carry a loss-of-function mutation in Fas. Both mice show a similar phenotype with accumulation of CD4+ CD8+ double positive lymphocytes, lymphadenopathy, splenomegaly and autoimmune diseases. Similar mutations in humans results in autoimmune lymphoproliferative syndrome or ALPS (Lamy, Liu et al. 1998;

Fleisher 2005).

LGL leukemia patients show persistent lymphocytosis, hypergammaglobulinemia, splenomegaly and significant association with autoimmune diseases (40-50% in leukemic

LGL patients compared to 1-3% in general population). However, they do not have lymphadenopathy and lymphocytosis is typically that of CD3+ CD4- CD8+ cells.

Moreover, in contrast with lpr and gld mice as well as ALPS patients, LGL leukemia patients do not carry any known mutation in Fas or FasL (Loughran 1993; Liu, Wei et al.

2002). In vitro treatment with interleukin-2 (IL2), phytohemagglutinin (PHA) and IL-2, or ceramide sensitizes leukemic LGL to Fas-mediated apoptosis (Epling-Burnette 2001;

Yang, Epling-Burnette et al. 2008). Also, inhibitors of various survival signaling pathways (such as AG490 and FTY720, see below) restore Fas-sensitivity in leukemic

LGL,(Epling-Burnette 2001; Shah, Zhang et al. 2008) suggesting intact Fas-FasL apoptotic machinery. Together, these data suggest that although leukemic LGL are capable of undergoing Fas-mediated apoptosis, various survival signals keep them from undergoing AICD, leading to the accumulation of leukemic LGL in the periphery

(Figure 3).

25

Figure 3: Model of apoptosis-resistance in leukemic LGL Cytotoxic cells kill the target cells by one of two mechanisms. Activated cytotoxic cells upregulate FasL on their surfaces, while target cells express Fas. This facilitates Fas- FasL mediated apoptosis in the target cells. Another mechanism is through exocytosis of cytolytic granules in the target cells. Similar mechanism operates to maintain T-cell homeostasis. Activation of CTL upregulates both Fas and FasL on their surfaces. CTLs use Fas-FasL mediated apoptosis to induce apoptosis either in self or another antigen- specific T-cells (middle panel). While leukemic LGL express abundant Fas and FasL, they are resistant to Fas-mediated apoptosis. It is believed that survival signaling overcomes Fas-mediated apoptotic signals in leukemic LGL leading to their accumulation.

26 Role of soluble Fas and FasL in LGL leukemia

While known mutation in Fas or FasL genes were not found in LGL leukemia, various isoforms of both Fas and FasL were found in serum of LGL leukemia patients.

Sera from LGL leukemia patients contain elevated levels of soluble form of Fas receptor

(sFas). sFas blocked Fas-mediated apoptosis in both activated normal PBMC and IL2- treated leukemic LGL. It was proposed that sFas may work as a decoy for FasL, resulting in Fas-resistance phenotype of leukemic LGL (Liu, Wei et al. 2002). Indeed, the source of these sFas variants in serum was traced to LGL leukemia PBMC. LGL leukemia PBMC contain alternatively spliced Fas variants not seen in normal naïve or activated PBMC. When overexpressed, these variants were secreted in supernatant and supernatant containing these sFas variants blocked Fas-mediated apoptosis of leukemic

LGL (Liu, Wei et al. 2002).

Interestingly, the presence of the soluble form of FasL is also known in LGL leukemia. sFasL can be produced by alternative splicing or by proteolytic cleavage of membrane bound FasL by matrix metalloproteinase (MMP) family of enzymes

(Kayagaki, Kawasaki et al. 1995; Ayroldi, D'Adamio et al. 1999). LGL leukemia patients have high amounts of sFasL in serum while normal serum does not contain any detectable levels of sFasL. Since normal T- and NK- cells express FasL only following activation, it was suggested that neutralizing antibody to sFasL or MMP inhibitors may be used in treating LGL leukemia with autoimmune diseases (Tanaka, Suda et al. 1996;

Liu, Wei et al. 2002).

27 Abnormal formation of DISC in leukemic LGL

As described above, DISC formation is the immediate downstream event of Fas-

FasL ligation in Fas-mediated apoptosis. Both short and long forms of cellular FLICE like inhibitory protein, c-FLIP (known as c-FLIPS and c-FLIPL respectively) contain

caspase-homologous regions, enabling them to be recruited to DISC. However, FLIP

lack proteolytic capabilities of caspase-8. Thus, the recruitment of FLIP into DISC

inhibits the execution of the Fas-induced apoptosis signal by caspases and promotes cell

survival. FLIP not only inhibit Fas-mediated apoptosis, but are also known to induce NF-

κB- and Erk-mediated proliferation in T-cells (Thome and Tschopp 2001).

Downregulation of FLIP is seen towards the end of CTL response and correlates with

increased sensitivity of CTL to Fas-mediated apoptosis. In contrast, leukemic LGL

express higher basal levels of both isoforms of FLIP that may contribute to Fas-resistant

phenotype of leukemic LGL (Yang, Epling-Burnette et al. 2008).

While the role of many pathways is described in abnormal survival of leukemic

LGL, a global gene expression profile of leukemic LGL has not been carried out. We

used the global gene expression profiling to identify the unique ‘molecular signature’ of

leukemic LGL. We were particularly interested in identifying potential survival

pathways that might render leukemic LGL resistant to AICD. We analyzed microarray

data using ‘theme-based’ bioinformatics approaches such as EASE (Hosack, Dennis et al.

2003).

28 Materials and Methods

Patient consent

All patients met the clinical criteria of T-cell (CD3+) LGL leukemia with increased LGL counts and clonal T-cell receptor (TCR) gene rearrangement and had not received treatment for LGL leukemia. Informed consents were signed by all patients and age- and gender-matched normal individuals to allow the use of their cells for these experiments. Buffy coats were obtained from Hershey Medical Center Blood Bank according to protocols observed by Milton S. Hershey Medical Center, Hershey, PA.

Cell culture and CD8+ T-cell enrichment

Buffy coats of normal donors were either used for RNA isolation or enriched for

CD8+ cells using Human CD8 Immunocolumn (Cedarlane Laboratories, Burlington,

NC). These samples are referred to as ‘naïve normal PBMC’ and ‘naïve normal enriched

CD8+’ respectively. 5X107 PBMC were cultured in RPMI-1640 with Glutamax

supplemented with 10% fetal bovine serum in presence of PHA (1µg/ml) for three days followed by IL-2 (500 IU/ml) for seven days. These cells are referred to as ‘activated

normal PBMC’. High dose of IL-2 resulted in preferential proliferation of CD3+ CD8+

cells. At the end of ten days, a fraction of cells was subjected to CD8+ enrichment using

Human CD8 Immunocolumn. Enriched CD8+ cells are referred to as ‘activated normal

enriched CD8+’ (Figure 4, 5). We chose this method for activation as it is consistent with induction of activation induced cell death in vitro culture. LGL leukemia patient

29 PBMC were isolated from patient blood samples and studied without culturing, sorting or activation in vitro.

Figure 4: Preparation of samples and experimental procedure Freshly isolated PBMC from normal individuals (naïve normal PBMC) were enriched for CD8+ T-cells using negative isolation (naïve normal enriched CD8+ cells) as described in materials and methods. 5X107 cells were activated using 1µg/ml of PHA for 3 days followed by 500 IU/ml of IL2 for 7 days (activated normal PBMC). Following activation, 108 cells were enriched for CD8+ cells (activated normal enriched CD8+ cells). Percentage of CD3+ CD8+ cells is shown in parenthesis.

30

Figure 5: Preparation of samples and experimental procedure The samples were used for RNA isolation and subjected to microarray analysis. LGL leukemia PBMC were obtained fresh from the patients and were not cultured, sorted or activated. The samples with similar phenotype were either pooled (pooled sample analysis) or analyzed without pooling (unpooled sample analysis). Genes differentially expressed in both analyses were considered to be differentially expressed.

RNA isolation and global gene expression analysis

Total RNA was isolated with TRIzol and quantified and verified for integrity using the Bioanalyzer 2100 (Agilent). The Human Genome U133A GeneChip

(Affymetrix, Santa Clara, CA) was used for gene expression profiling. cRNA synthesis, hybridization, and posthybridization staining were performed as previously described

Irby (Irby, Malek et al. 2005) and Malek (Malek, Irby et al. 2002) using standard protocols as recommended by the manufacturer (Affymetrix). Stained chips were scanned on a GeneChip Scanner 3000 7G (Affymetrix), and data files were processed using the

Affymetrix GeneChip Operating Software (GCOS) to provide probe set presence/absence detection calls (settings τ = 0.015, α1 = 0.05, α2 = 0.065), calculate signal intensities, determine quality of hybridizations, scale hybridization experiments, and calculate gene

31 expression change. All hybridizations were scaled (normalized) to an average intensity of 500. The mean scaling factor was 5.7 with no two values differing by more than 2- fold. Additional Affymetrix quality metrics for each hybridization included % presence calls > 42%, and Sig (3’/5’) ratios less than 2 for house keeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. A total of 44 hybridizations were performed for this study: naïve PBMC (3 hybridizations from individual donors, 1 hybridization from a pool of 10 donors), naïve CD8+ (3 hybridizations from individual donors, 1 hybridization from a pool of 10 donors), activated PBMC (3 hybridizations from individual donors), activated CD8+ (3 hybridizations from individual donors), leukemic LGL (30 hybridizations from individual patients).

Differences in gene expression were analyzed by an ANOVA (per gene) with implementation of false discovery rate (FDR) as defined by Benjamini and Hochberg to control for type I error resulting from multiple comparisons (Benjamini and Hochberg

1995; Reiner, Yekutieli et al. 2003). ANOVA with FDR is available in the ArraySTAT software package (Amersham Biosciences) as well as in FDRAME in the Bioconductor project (www.bioconductor.org). Genes identified as significantly differentially expressed were subjected to hierarchical cluster analysis by using TIGR Multi

Experiment Viewer (TMEV; available at www.tigr.org/softlab). Three criteria were used

in defining differentially expressed genes that constitute ‘gene signature’: i) expression

of a given gene should be changed by at least 2-fold; ii) FDR of 1% as the cut-off for

statistical significance; iii) gene signatures defining leukemic LGL vs. naïve normal cells

32 must include genes that are differentially expressed in both leukemic LGL vs. naïve normal PBMC as well as leukemic LGL vs. naïve enriched CD8+ cell comparisons.

EASE analysis

Biological themes associated with genes found to be differentially expressed were identified using the Expression Analysis Systematic Explorer (EASE) application

(Hosack, Dennis et al. 2003). EASE analysis identifies gene ontology (GO) categories

(‘biological themes’) containing an over-representation of differentially expressed genes, thereby denoting presumed biological relevance. For all the following EASE analyses,

Fisher’s Exact score<0.05 was considered as a cut-off for significance. We focused attention on GO categories underneath the parent categories Biological Process (GO:

00081500) and Molecular Function (GO: 0003674).

Results

Comparison of the gene expression signature of naïve normal cells with activated normal cells

First, we asked the question: what defines the gene-expression pattern of PBMC following activation in vitro. The genes that were differentially expressed in both naïve

PBMC vs. activated PBMC as well as naïve enriched CD8+ vs. activated enriched CD8+ cells were considered to be differentially expressed and used for further analysis. A total

33 of 775 genes showed at least 2-fold change in expression in naïve vs. activated samples at the false detection rate (FDR) of 1% (Figure 6 and Appendix 1).

Figure 6: Microarray profiling of naïve normal PBMC and activated normal PBMC using gene- and theme-based approach Differential expression of genes in normal PBMC following activation. A total of 775 genes are expressed differentially (at least 2 fold-change and 1% FDR) following activation. EASE analysis of two phenotypes show statistically significant upregulation of genes belonging to GO categories ‘Regulation of programmed cell death’ and ‘Positive regulation of apoptosis’. Each row in the cluster image represents an individual gene and each column represents an individual sample from LGL leukemia patient or healthy normal donor. The relative transcript abundance of each gene is color coded. A red color indicates high expression; black indicates intermediate expression and green indicates low expression.

To characterize the expression patterns and to identify biological themes, we

compared the expression profiles of naïve and activated enriched CD8+ T-cells using

EASE analysis. EASE analysis of naïve vs. activated cells shows that differentially

expressed genes are over-represented in 305 GO categories with Fisher’s Exact

34 Score<0.05. We focused on GO categories Biological process (GO: 00081500) and

Molecular Function (GO: 0003674). An overwhelming number of genes that exhibited differential expression between activated and naïve cells belonged to categories related to positive regulation of apoptosis and enhanced immune response.

Comparison of the gene expression signature of naïve normal cells with leukemic LGL

When comparing the constitutive gene expression signature of leukemic LGL to naïve normal PBMC and naïve normal enriched CD8+ cells, we noted 174 differentially expressed genes (Figure 7 and Appendix 2). The genes that were differentially expressed

in both naïve PBMC vs. leukemic LGL as well as naïve enriched CD8+ vs. leukemic

LGL were considered to be differentially expressed and used for further analysis.

EASE analysis shows a total of 201 GO categories that had significant over- representation of differentially expressed genes in leukemic LGL (Fisher’s exact score

<0.05) belonging to either ‘Biological Processes’ or ‘Molecular Function’ categories.

GO categories related to leukocyte activation and immune responses, inhibition of apoptosis, and response to virus were particularly prominent.

35

Figure 7: Microarray profiling of leukemic LGL compared to naive normal PBMC using gene- and theme-based approach Constitutive gene expression signature of leukemic LGL compared to naïve normal cells. A total of 174 genes were differentially expressed in leukemic LGL compared to naïve normal cells (at least 2 fold-change and 1% FDR). EASE analysis of leukemic LGL compared to naïve normal cells shows significant upregulation of genes belonging to GO categories such as Immune system process, immune response, viral life cycle, viral infectious cycle, and viral genome replication. Each row in the cluster image represents an individual gene and each column represents an individual sample from LGL leukemia patient or healthy normal donor. The relative transcript abundance of each gene is color coded. A red color indicates high expression; black indicates intermediate expression and green indicates low expression.

Comparison of the gene expression signature of activated normal cells with leukemic LGL

Further, comparing the gene expression signature of leukemic LGL to activated normal PBMC and activated normal enriched CD8+ cells showed 1492 genes to be

36 differentially expressed when compared to activated normal PBMC and activated normal enriched CD8+ cells (Figure 8 and Appendix 3). The genes that were differentially

expressed in both naïve PBMC vs. leukemic LGL as well as naïve enriched CD8+ vs.

leukemic LGL were considered to be differentially expressed and used for further

analysis. Identifying these genes would allow us to create a unique gene expression

signature of leukemic LGL compared to that of normal naïve and activated cells. EASE

analysis revealed that these differentially expressed genes were over-represented in 482

GO categories (Fisher’s Exact Score<0.05). Noteworthy were the categories related to

negative regulation of apoptosis, positive regulation of TCR signaling and enhanced

immune response related categories.

37

Figure 8: Constitutive gene expression signature of leukemic LGL compared to activated normal cells A total of 1492 genes were differentially expressed in leukemic LGL compared to naïve normal cells (at least 2 fold-change and 1% FDR). EASE analysis of leukemic LGL compared to activated normal cells shows upregulation of immune response related signaling and cytotoxicity related GO categories to be significantly upregulated in leukemic LGL compared to activated normal cells. Each row in the cluster image represents an individual gene and each column represents an individual sample from LGL leukemia patient or healthy normal donor. The relative transcript abundance of each gene is color coded. A red color indicates high expression; black indicates intermediate expression and green indicates low expression.

Dysregulated expression of apoptosis related genes in leukemic LGL

Previous work in our laboratory has suggested that leukemic LGL accumulate in vivo because of inhibited apoptotic pathways. Microarray analysis using various approaches confirms this hypothesis. In addition, global gene expression and EASE

38 analyses revealed a preponderance of differentially expressed genes related to apoptosis in leukemic LGL. Hence, we further focused on the genes belonging to GO category:

0006915 (‘apoptosis related genes’) that are differentially expressed in AICD prone – activated enriched CD8+ cells - and AICD resistant leukemic LGL. Table 1 lists some

of 128 genes belonging to GO: 0006915 that are constitutively differentially expressed in

leukemic LGL compared to activated enriched CD8+ cells (at least 2 fold-change and 1%

FDR; for full list, please see Appendix 4).

39

Table 1: List of differentially expressed genes in leukemic LGL compared to activated enriched CD8+ cells that belong to GO category ‘apoptosis’ (GO:0006915). Fold change referes to fold-expression observed in leukemic LGL compared to activated enriched CD8+ cells. Gene Fold-change Gene description symbol TNFAIP3 6.73 TNF-alpha-induced protein 3 GZMH 6.5 Granzyme H BCL2A1 4.47 BCL2-related protein A1 LITAF 3.78 Lipopolysaccharide-induced TNF factor TNFSF13 3.73 TNF superfamily, member 13 CTSB 3.46 Cathepsin B FAIM3 3.43 Fas apoptotic inhibitory molecule 3 CASP8 3.41 Caspase 8 SERPINB9 3.36 Serpin Peptidase Inhibitor, clade B GADD45B 3.36 Growth arrest and DNA-damage-inducible, beta RHOT2 2.71 Ras homolog gene family, member T2 RAF1 2.28 v-raf-1 leukemia viral oncogene homolog 1 Nuclear factor of kappa light polypeptide gene enhancer in B- NFKBIA 2.27 cells inhibitor, alpha MCL1 2.25 Myeloid cell leukemia sequence 1 (BCL2-related) PDCD4 2.19 Programmed cell death 4 TP53BP2 2.19 Tumor protein p53 binding protein, 2 PRF1 2.16 Perforin 1 RELA 2.13 v-rel,p65 TP53 0.5 Tumor protein p53 TNFAIP8 0.47 TNF alpha-induced protein 8 CASP3 0.46 Caspase 3 STAT1 0.45 Signal transducer and activator of transcription 1 IL2RB 0.44 Interleukin 2 receptor, beta LCK 0.41 Lymphocyte-specific protein tyrosine kinase FAS 0.37 Fas (TNFSF6) SIVA 0.33 CD27-binding (Siva) protein BAG2 0.23 BCL2-associated athanogene 2 BAX 0.22 BCL2-associated X protein BCL2 0.21 B-cell CLL GZMA 0.17 Granzyme A PRDX2 0.16 Peroxiredoxin 2 CASP6 0.15 Caspase 6 TNFSF10 0.15 TNF superfamily, member 10 TUBB 0.1 Tubulin, beta

40 Apoptosis related genetic signature unique to leukemic LGL

The genes that were differentially regulated between leukemic LGL and activated enriched normal CD8+ cells were identified (at least 2 fold-change and 1% FDR). A total of 128 genes belonging to GO category ‘apoptosis’ were differentially expressed in leukemic LGL compared to activated enriched normal CD8+ cells (AcCD8+).

The analysis shows that many genes typically considered to be pro-apoptotic are downregulated while those considered anti-apoptotic are upregulated in leukemic LGL.

An obvious short coming of the microarray technology is that it creates a signature at transcriptional level. Changes that occur at translational and post-translational levels

(e.g., post-translational modification – PTM) are not identified. Gene-based analysis of apoptosis related genes is shown in Figure 9.

41

Figure 9: Differentially regulated apoptosis related genes in leukemic LGL compared to activated enriched normal CD8+ cells Apoptosis related genetic signature unique to leukemic LGL. The genes that were differentially regulated between leukemic LGL and activated enriched normal CD8+ cells were identified (at least 2 fold-change and 1% FDR). A total of 128 genes belonging to GO category ‘apoptosis’ were differentially expressed in leukemic LGL compared to activated enriched normal CD8+ cells (AcCD8+). Figure shows that leukemic LGL have upregulated anti-apoptotic genes such as SERPINB9 while pro-apoptotic genes such as BAX are downregulated. Each row in the cluster image represents an individual gene and each column represents an individual sample from LGL leukemia patient or healthy normal donor. The relative transcript abundance of each gene is color coded. A red color indicates high expression; black indicates intermediate expression and green indicates low expression.

GenMAPP analysis of apoptosis related genes in leukemic LGL

Gene Map Annotator and Pathway Profiler (GenMAPP) is a software used to visualize and analyze genomic data in the context of pathways (Dahlquist, Salomonis et

42 al. 2002). It is used to integrate gene-based data to gain better insight in biology by integrating pathways or group of genes. The genes identified as significantly different between leukemic LGL and activated enriched CD8+ cells were imported in GenMAPP.

MAPP file ‘Hs_apoptosis’ was modified to include apoptosis related network of genes in

LGL leukemia. GenMAPP analysis further confirms the idea that leukemic LGL are characterized by dysregulation of apoptotic genes (Figure 10). GenMAPP analysis also

helps in understanding relationships between various apoptosis related genes.

Figure 10: GenMAPP analysis of differentially regulated apoptosis related genes in leukemic LGL Differentially regulated genes between leukemic LGL and activated enriched CD8+ cells were imported in GenMAPP for visualization. Known apoptosis related genes and their relation is shown. The genes constitutively upregulated in leukemic LGL compared to activated normal enriched CD8+ cells are shown in red, genes constitutively downregulated in leukemic LGL are shown in blue, while those in white show no change in expression between two phenotypes. The number accompanying each gene indicates fold-change in expression.

43 Validation of microarray analysis

NKp46

Natural killer cells are usually defined by absence of cell specific markers

(‘negative definition’). For example, NK cells are characterized by absence of T-cell

(CD3) and B-cell markers. NKp46 was initially identified as a marker specifically present in human NK-cells. NKp46, a member of the highly conserved natural cytotoxicity receptor (NCR) family of NK-activating receptors, best defines NK-cells across species (Pessino, Sivori et al. 1998; Walzer, Jaeger et al. 2007).

Recently, it was suggested that a few T-cells also express NKp46 in humans

(Caligiuri 2008). Characteristics, origin and function of these cells are unknown.

Interestingly, microarray analysis (Figure 11A) suggested that NKp46 was upregulated in

T-LGL (CD3+ cells). We validated this finding using three-color flow cytometry analysis using CD3, CD8, and NKp46 antibodies. Figure 11B shows that leukemic

CD3+ cells (T-cells), but not normal CD3+ (T-cells) cells express NKp46 on their

surfaces, further validating microarray analysis.

44

(A)

Figure 11: NKp46 is expressed in T-LGL leukemia cells (A) Microarray analysis shows that leukemic T-LGL shows overexpression of NKp46 which was previously considered to be specific marker for NK-cells (p-value T-LGL vs N PBMC 5.72E-07, T-LGL vs N CD8 1.05E-05, T-LGL vs. Ac PBMC 2.95E-07, T-LGL vs. Ac CD8+ 9.83 E-07) (B) Flow cytometry data confirms microarray predictions. Data shown is from one healthy donor and two T-LGL patients. PBMC are stained with CD3 and NKp46 antibodies. While very few CD3+ T-cells express NKp46 in healthy donor, CD3+ T-cells from T-LGL patients show abundant expression of NKp46.

45 Serine Proteinase Inhibitor 9 (SERPINB9)

During the down-phase of T-cell activation, activated CTL induce apoptosis in other activated CTL through granule exocytosis and death receptor pathways. Leukemic

LGL contain perforin and GrB as well as other proteases in their granules. Leukemic

LGL also express abundant Fas and FasL on their surface and possess intact Fas- machinery. Given these findings, it is puzzling that leukemic LGL are resistant to apoptosis mediated by either of these pathways. It is believed that there should be survival signals operating in leukemic LGL that would inhibit both granule exocytosis and Fas-FasL mediated apoptosis. One such candidate is a homologue of cowpox virus protein cytokine response modifier A (CrmA) known as SERPINB9 (PI6). SERPINB9 binds to and inactivates serine proteinases including GrB and caspase-8 (Sprecher,

Morgenstern et al. 1995; Sun, Bird et al. 1996; Smyth, Kelly et al. 2001).

We hypothesized that that upregulation of SERPINB9 may protect leukemic LGL from killing each other thus helping to escape AICD. Upregulation of SERPINB9 is sufficient to inhibit both GrB and Fas-FasL mediated apoptosis in various in vivo and in vitro models (Stout-Delgado, Getachew et al. 2007). Microarray analysis suggested that while activation led to downregulation of SERPINB9 in normal CD8+ cells, it was significantly upregulated in leukemic LGL. We confirmed these findings using quantitative real-time PCR (qRT-PCR). qRT-PCR analyses show that leukemic LGL express greater than 30-fold higher SERPINB9 compared to activated CD8+ cells from healthy donors (Figure 12).

46 (A) 1600 Expresson of SERPINB9

1400

1200

1000

800

600 Relative Flu Units Flu Relative

400

200

0 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 120 121 122 123 124 127 128 129 130 131 132 133 136 137 1233 1352 1375 JCD8 CCD8 KCD8 JPMBC CPMBC KPMBC JAcCD8

CAcCD8 KAcCD8 Sample 140 - CD8 140 JAcPMBC CAcPMBC KAcPMBC 139 - PBMC 139 (B)

Figure 12: SERPINB9 is overexpressed in leukemic LGL compared to normal phenotypes (A) Microarray analysis of normal PBMC and enriched CD8+ cells compared to leukemic LGL show that SERPINB9 is overexpressed in leukemic LGL (Black – naïve normal PBMC, green – naïve normal enriched CD8+ cells, blue –activated normal PBMC, and yellow – activated normal CD8+ cells, red – leukemic LGL). (B) Quantitative real-time PCR analysis of SERPINB9. qRT-PCR analysis was carried out using primers as described in Table-1. Statistical analysis using REST-MCS shows that SERPINB9 is upregulated in leukemic LGL by ~30-fold compared to activated enriched CD8+ cells thus validating microarray results (Shah et al, Clinical Leukemia, in press).

47 Other genes

Microarray analysis predicted that T-box expressed in T-cells (TBX21, T-bet), and c-Rel are upregulated in leukemic LGL compared to normal PBMC. It was recently shown that both T-bet and NF-κB are overexpressed and overactive in leukemic LGL compared to normal PBMC (Zhang, Shah et al. 2008).

We also found constitutive upregulation of numerous genes in leukemic LGL that are involved in CTL function, such as serine proteinases (granzyme B and granzyme H), cysteine proteinases (cathepsin C and cathepsin S), calpain (a cytosolic non-lysosomal cysteine protease), and perforin. Since these genes are expressed only after activation, these results confirm that leukemic LGL had been activated in vivo. We had previously found these genes to be upregulated in leukemic LGL utilizing Northern blot and RNAse protection assays, thus providing further validation of the current microarray studies

(Kothapalli, Ratna et al. 2003).

48

Discussion

In this study we showed that the unique molecular signature of LGL leukemia is characterized by dysregulation in expression of many apoptotic genes. Theme-based approach using EASE analysis showed that while the principal difference between naïve normal PBMC and leukemic LGL was in immune response related pathways, leukemic cells significantly differed from activated normal PBMC in terms of apoptosis related pathways. Activated normal PBMC show simultaneous upregulation of both pro-survival pathways (e.g. ‘Jak-Stat cascade related genes’) and pro-apoptotic (e.g. ‘induction of apoptosis’) pathways related genes, reinforcing the idea that activation and proliferation of T-lymphocytes is tightly coupled with pro-apoptotic signaling (Thome and Tschopp

2001). In contrast, leukemic LGL were characterized by over-representation of the genes belonging to GO categories such as ‘leukocyte activation’ along with categories such as

‘negative regulation of apoptosis’. This statistically significant difference (using Fisher’s exact score <0.05) suggests that the activation and apoptosis that are tightly coupled in normal activated cells are uncoupled in leukemic LGL resulting in the failure of AICD.

One possible mechanism of such dysregulation is chronic viral infection suggested by upregulation of genes belonging to virus infection-related GO categories in leukemic

LGL.

Many of the differentially regulated genes found in leukemic LGL compared to activated normal cells belong to GO category ‘apoptosis’ (Table 2; Figure 8). Most of the genes constitutively upregulated in leukemic LGL are anti-apoptotic while those

49 downregulated are known to have pro-apoptotic functions (Figure 9). TNFAIP3 (A20) was the apoptotic pathway gene that showed the highest upregulation in leukemic LGL in microarray analysis. TNFAIP3 is an NF-κB inducible gene that protects T-lymphocytes from undergoing TNF-induced apoptosis. In normal T-lymphocytes TNFAIP3 is downregulated after activation (Tewari, Wolf et al. 1995; He and Ting 2002).

Upregulated expression of TNFAIP3 in leukemic LGL was confirmed by Northern blot analysis, thus validating these microarray results (Shah, Zhang et al. 2008).

Differentially regulated apoptosis genes in LGL leukemia featured many of those belonging to the Bcl-2 family. The Bcl-2 family proteins are central to life-or-death decisions made by cells due to presence of both pro- and anti-apoptotic actions of its family members (Thome and Tschopp 2001). The expression of the Bcl-2 related X gene

(BAX) was down-regulated in leukemic LGL by about 4.5-fold. In contrast, the expression of myeloid cell factor-1 (MCL1) was upregulated in leukemic LGL by 2.25- fold. We have previously shown that inhibition or downregulation of STAT3 is associated with decreased MCL1 and increased apoptosis in leukemic LGL (Epling

Burnette PK 2001).

Collectively, these observations support our hypothesis that LGL leukemia is characterized by profound dysregulation of apoptosis in these chronically activated T- lymphocytes and that inhibition of apoptosis could play an important role in its pathogenesis.

50 References

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Chapter 3

Role of Sphingolipid Rheostat in LGL Leukemia

Introduction

The objective of this study was to use global gene expression profiling to identify the

survival mechanisms in LGL leukemia. Pathway-based microarray analysis using gene set

enrichment analysis (GSEA) identified genes related to sphingolipid metabolism and G-protein

signaling pathways (Gα12-related pathway) to be differentially expressed in leukemic LGL.

These pathways were of particular interest since our laboratory discovered the human

sphingosine 1-phosphate (S1P) receptor-5 (S1P5). S1P5 was cloned from an LGL leukemia library following initial identification as an expressed sequence tag (EST) overexpressed in LGL

leukemia (Kothapalli, Kusmartseva et al. 2002). Whether sphingolipid signaling plays any role

in apoptosis of CTL in vivo, and consequently in long-term survival of CTL, is not known.

Sphingolipids are biologically active lysophospholipids that act as second messengers or

directly regulate diverse biological functions, such as survival, proliferation, calcium

homeostasis and migration. We hypothesized that altered sphingolipid-mediated signaling is

important for survival of leukemic LGL.

Sphingolipid biosynthetic pathway is an intricate pathway with many rapidly inter-

changeable intermediate metabolites. A pro-apoptotic sphingolipid, ceramide, can be

synthesized in vivo by condensation of serine and palmytoyl CoA (de novo synthesis) or from

sphingomyelin (catabolic pathway). Ceramide is a pro-apoptotic molecule that is synthesized in

cells following wide variety of stress or death signals, including Fas-FasL interaction. Ceramide

54 can be deacytylated into sphingosine by ceramidases such as ASAH1. Sphingosine, then, can be phosphorylated by one of the two SphK into S1P. Though structurally closely related to ceramide, S1P is a pro-survival molecule in various cell types. Given that pro-apoptotic (such as ceramide and sphingosine) and anti-apoptotic (such as S1P, and ceramide-1-phosphate- C1P) sphingolipids exist in a fast exchanging equilibration, it is their relative amount (termed sphingolipid rheostat), rather than their absolute quantities, that determines cell fate (Spiegel and

Milstien 2002; Rosen and Goetzl 2005; Wymann and Schneiter 2008).

Role of sphingolipids - especially that of S1P - in oncogenesis, metastasis and angiogenesis is well established (Visentin, Vekich et al. 2006). S1P, presumably through S1PR, plays a protective role in T-cell survival by protecting cells against ceramide and Fas-FasL mediated apoptosis (Cuvillier, Rosenthal et al. 1998; Goetzl, Kong et al. 1999; Wymann and

Schneiter 2008). While any component that can lead to excessive S1P production or constitutive

S1P-mediated signaling may act as an oncogene, the role of three components of sphingolipid rheostat, ASAH1, SphK and Gα12, are most studied.

Role of sphingolipids in cancer

Acid ceramidase functions upstream to SphK in sphingolipid metabolism. Upregulation

of ceramidase is a survival mechanism used by variety of human tumors to combat ceramide

production that normally follows various apoptosis-inducing insults (Spiegel, Cuvillier et al.

1998; Kolesnick 2002; Park and Schuchman 2006). A chemical inhibitor of acid ceramidase,

NOE, selectively induced apoptosis in various tumor cells (Morales, Paris et al. 2006). This renders acid ceramidase as a potential target for anti-tumor treatment.

55

SphK is over expressed in variety of tumors and is considered an oncogene (Spiegel,

Cuvillier et al. 1998; Milstien and Spiegel 2006). Many growth factors implicated in tumorigenesis, such as PDGF, are known to activate SphK. Activated SphK promotes cell survival by increasing cellular concentration of S1P while reducing concentrations of ceramide and sphingosine. This puts SphK in very crucial position in lipid-mediated survival signaling and rendering it a candidate for anti-tumor therapy.

Gα12 or GNA12 is a G-protein that is coupled with various GPCR, the most notable being

S1P5 in leukemic LGL. Constitutive active form of Gα12 induces transformation that is partially

mediated by activating STAT3 via multiple pathways, including JAK3, PDGFα, and PI3K;

components known to be deregulated in LGL leukemia (Kumar, Shore et al. 2005).

Role of sphingolipids in inflammatory and auto-immune conditions

Many inflammatory cytokines such as IL-1, TNFα and VEGF, activate SphK to increase

intracellular levels of S1P (Chalfant & Spiegel, 2005). High S1P leads to decreased turn-over of

effector T-cells (Goetzl et al., 1999). As discussed in the previous chapter, inhibition of S1P-

mediated signaling leads to selective induction of apoptosis in leukemic LGL. Another

mechanism by which S1P contributes to inflammatory conditions is by the way of controlling

lymphocyte trafficking. S1P-mediated signaling is crucial in generating T-cell mediated

response. S1P receptor, S1P1, is downregulated in T-cells following activation. This renders

lymphocytes insensitive to S1P gradient while lymphocyte entry is not affected. This leads to

accumulation of lymphocytes in lymph nodes allowing more time for T-cells to become primed

by antigen-presenting cells.

56

Materials and methods

Patient consent:

All patients met the clinical criteria of T-cell (CD3+) LGL leukemia with increased LGL counts and clonal T-cell receptor (TCR) gene rearrangement and had not received treatment for

LGL leukemia. Informed consents were signed by all patients and age- and gender-matched normal individuals to allow the use of their cells for these experiments. Buffy coats were obtained from Hershey Medical Center Blood Bank according to protocols observed by Milton

S. Hershey Medical Center, Hershey, PA.

Chemicals and reagents:

Reagents and chemicals including phytohemagglutinin (PHA), TRIzol, RPMI-1640 with

Glutamax, and fetal bovine serum were obtained from Invitrogen Corporation (Carsbald, CA,

USA) and used according to manufacturer’s instructions. Human CD8 cell Immunocolumn was obtained from Cedarlane lab (Burlington, NC). N-oleoylethanolamine (NOE) was purchased from Calbiochem (San Diego, CA). FTY720 was purchased from Cayman Chemical Company

(Ann Arbor, MI) while D-erythro-sphingosine-1-phosphate (S1P) was purchased from Avanti

Polar Lipids (Alabaster, AL). The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr.

Maurice Gately, Hoffmann - La Roche Inc (Lahm and Stein 1985).

57

Antibodies:

Anti-Fas antibody (activating, human CH11 clone) was obtained from Upstate Cell

Signaling Solutions (Lake Placid, NY); Annexin V: PE Apoptosis Detection Kit-I, CD3-APC,

CD8-FITC, CD57-FITC with respective isotype controls, and monoclonal antibody to acid ceramidase were obtained from BD Biosciences (San Jose, CA).

Pathway-based analysis

The expression data matrix created using GCOS as described above was imported into

DNA Chip Analyzer (dChip) software(Li and Wong 2000) and normalized using default settings.

Gene expression profiles of thirty LGL leukemia patients vs. activated enriched CD8+ cells from three controls were used to generate a molecular signature and theme-based analysis. These expression matrices were exported to GenMAPP or GSEA softwares (Dahlquist, Salomonis et al.

2002; Mootha, Lindgren et al. 2003; Subramanian, Tamayo et al. 2005). List of genes identified as significantly different were visualized using GenMAPP. MAPP file ‘Hs_apoptosis’ was modified to include apoptosis related network of genes in LGL leukemia. GSEA approach was similar to methods reported previously(Mootha, Lindgren et al. 2003) with slight modifications as described below. All the default settings except ‘gene set permutation’ with 1000 iterations were used for the analysis. The gene set file (c2.mvs.symbols, supplementary data) was a modification of c2 (http://www.broad.mit.edu/gsea/msigdb/msigdb_index.html) to include customized gene sets.

58

Real-time PCR analysis

Total RNA was isolated using TRIzol as described. RNA was treated with amplification grade DNAse I (Invitrogen) according to manufacturer’s instructions. Real-time PCR analysis was done using SYBR® GreenER™ Two-Step qRT-PCR Kits Universal (Invitrogen, CA) according to manufacturer’s instructions. PCR Primers used are listed in Table 2. PCR was performed with Cepheid SmartCycler (Sunnyvale, CA) using following conditions: 2m at 50oC,

10m at 95oC, followed by 40 cycles of 1m at 95oC, 1m at 60oC and 2m at 72oC. Melt curve

analysis was performed following each run using default settings. Real-time PCR analysis for

comparison of multiple transcripts in a single sample, expression was normalized to GAPDH and

adjusted for primer efficiency. For relative abundance between phenotypes, primer efficiency was determined and analyzed using REST MCS® software (Pfaffl, Horgan et al. 2002). All the

samples were tested in duplicate and the transcripts were normalized to GAPDH expression.

59

Table 2: List of Primers Used for Real-Time PCR Gene Primers

Forward - 5’-GAGTCAACGGATTTGGTCGT-3’ GAPDH Reverse – 5’-TTGATTTTGGAGGGATCTCG-3’

Forward – 5’-CAGCAAATCGGACAATTCCT-3’ S1P1 Reverse – 5’-GCCAGCGACCAAGTAAAGAG-3’

Forward – 5’-ACCATGGGCAGCTTGTACTC-3’ S1P2 Reverse - 5’-GCAACAGAGGATGACGATGA-3’

Forward - 5’-AACCCGGTCATCTACACGCTGG-3’ S1P3 Reverse - 5’-GCAGGTCTTCCTTGACCTTCG-3’

Forward - 5’-CGGCTCATTGTTCTGCACTA-3’ S1P4 Reverse - 5’-AAGTTCTCCAGCACCACCAG-3’

Forward - 5’-CTGTCCTGGGAAGACCAAAA-3’ S1P5 Reverse - 5’-CAGCATCGCTGCATTTCTTA-3’

Forward - 5’-CAAGGGAAGCTTTTCCTCTC-3’ MCL1 Reverse - 5’-CATGGAAACCAAGCCAAAGT-3’

Forward - 5’-GTAACAACCGGGATTCTTCAG-3’ PAK3 Reverse - 5’-CTGGGAAGATAGAGCGAAGC-3’

Forward - 5’-CCCTATCCTATCCGCAAACA-3’ RAC1 Reverse - 5’-CGCACCTCAGGATACCACTT-3’

Forward - 5’-TGCTGATCGTGTTCAGTAAG -3’ RHOB Reverse - 5’-AGCACATGAGAATGA CGTCG-3’

Forward - 5’-GAAGAAAGAGAAGCTCGAGAAGAAGG-3’ ROCK1 Reverse - 5’-ATCTTGTAGCTCCCGCATCTGT-3’

60

Western blot assay

Cells were lysed in a buffer composed of 50 mM Tris-Cl (pH 7.6); 5 mM EDTA; 150 mM NaCl; 0.5% NP-40; 0.1% SDS containing 1:100 dilution of protease inhibitory cocktail; 1 mM sodium orthovanadate; and 0.5 mM PMSF (all from Sigma-Aldrich). Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, CA). 30 μg of total protein was boiled in Laemmli SDS-PAGE sample loading buffer containing 4% beta-mercaptoethanol and

80mM DTT; subjected to 15% SDS-PAGE. The proteins were then transferred to a membrane and Western blot assay was performed as described previously (Epling Burnette PK 2001).

Apoptosis assay

Freshly isolated PBMC of normal individuals or LGL patients were used for apoptosis assay. Briefly, 5X105 cells were seeded in 0.5 ml of RPMI-1640 medium supplemented with

10% FBS. The cells were untreated or treated with vehicle or compound for indicated duration.

Following treatment, cells were assessed by flow cytometry for apoptosis using Annexin-V conjugated with phytoerythrocin (PE) and 7-amino-actinomycin-D (7-AAD) staining (BD

Biosciences) according to manufacturer’s instructions. Briefly, the cells were washed once with

PBS and resuspended in 20 μg/ml Annexin-V-PE and 20 μg/ml 7-AAD in 500 μl of 1X Binding

Buffer. The cells were incubated for 10 minutes in the dark at room temperature and then immediately analyzed using BD FACSCalibur machine. All the live cells were gated and ten- thousand events were analyzed for Annexin-V-PE and 7-AAD. Annexin-V negative, 7-AAD positive cells were considered non-viable and excluded from further analysis. Apoptosis was calculated using the following formula:

61

Percentage specific apoptosis = (Annexin-V positive cells in treatment - Annexin-V positive cells in control) X 100 / (100 - Annexin-V positive cells in control)

Results

Pathway-based (GSEA) microarray analysis of leukemic LGL

We used GSEA to identify potential survival mechanisms in leukemic LGL compared to activated normal PBMC. These two phenotypes were chosen in order to identify ‘themes’ or

‘pathways’ that are differentially regulated in cells that upon activation can undergo AICD

(normal activated PBMC) and those that cannot (LGL leukemia samples).

GSEA of LGL leukemia PBMC compared to activated normal PBMC revealed a total of

13 gene sets to be constitutively enriched in LGL leukemia phenotype compared to activated normal PBMC phenotype at the FDR<15%. Two of the gene sets enriched in leukemic LGL were ‘EDG_Pathway’ (P=0.006) and ‘ST_GA12_Pathway’ (P=0.001) representing the genes that belong to S1PR- and Gα12- mediated signaling, respectively (Figure 13). In GSEA, the

enrichment score is driven by the group of genes within a gene set that shows the highest

correlation with the given phenotype – the core enriched genes (Table 2). The core-enrichment

analysis of leukemic LGL compared to activated normal PBMC showed that many of the genes

central to sphingolipid metabolism and signaling including acid ceramidase (ASAH1), S1P

receptor-5 (EDG8), sphingosine kinase 1 (SPHK1), sphingomyelin diesterase 1 and 2 (SMPD1

and SMPD2), and phosphatidyl inositol-3 kinase (PI3KR1) were core-enriched in the LGL

leukemia phenotype.

62

(A) (B)

Figure 13: Sphingolipid metabolism and signaling pathway is differentially regulated in leukemic LGL The expression profile of LGL leukemia PBMC (n=30) was compared to that of activated normal PBMC (n=3) using Gene Set Enrichment Analysis (GSEA). Two of the thirteen pathways enriched (FDR≤15%) in leukemic LGL are shown. The expression profile of the components of (A) ‘EDG_Pathway’ (P=0.006) and (B) ‘ST_GA_12_pathway’ (P=0.001) gene sets in leukemic LGL compared to activated normal PBMC. Each column represents individual sample from a LGL leukemia patient (gray) or normal healthy donor (yellow). Each row represents a gene. Red shows high expression, white denotes intermediate expression while blue denotes low expression.

We were interested in validating overexpression of these core components in leukemic

LGL. Microarray analysis had suggested that the expression of ASAH1 was downregulated following activation of normal CD8+ T-cells. In contrast, leukemic LGL constitutively express higher levels of ASAH1 mRNA (Figure 14).

63

Figure 14: Gene-based analysis of microarray experiments for ASAH1 mRNA expression The expression of ASAH1 mRNA in naïve normal PBMC (N PBMC, n=4), and activated normal PBMC (AC PBMC, n=3) compared to LGL leukemia PBMC (TLGL, n=30). Each bar represents mean relative fluorescence units while error bars represent standard error of mean (SEM).

Alpha-subunit of acid ceramidase is downregulated in leukemic LGL:

We further investigated if acid ceramidase is downregulated at protein level also.

Western blot assay using cell lysate from naïve normal PBMC, activated normal PBMC or leukemic LGL revealed that α-subunit of ASAH1 is downregulated in activated normal PBMC compared to leukemic LGL cells (Figure 15). These results are in agreement with microarray predictions further validating our microarray methodology.

64

Figure 15: Differential expression of ASAH1 in LGL leukemia PBMC Expression of α-subunit of acid ceramidase in naïve (N PBMC, n=3) and activated (AC PBMC, n=4) normal PBMC compared to LGL leukemia PBMC (TLGL, n=6). Samples were subjected to SDS-PAGE followed by membrane transfer. The blot was probed with antibody to α-subunit of acid ceramidase or β–actin and developed using chemiluminescence. Western blot analysis suggests that acid ceramidase is expressed in naïve PBMC constitutively (N PBMC, lane 1-3). Following activation of lymphocytes, α-subunit of acid ceramidase is downregulated to almost undetectable levels (AC PBMC, lane 4-7) while it is significantly upregulated in all LGL leukemia PBMC samples (TLGL, lane 8-13).

S1P-receptors in CD8+ T-cells:

In the immune system, S1P acts either in anautocrine or a paracrine manner, either

intracellularly or through one of the S1P-receptors (S1PR) on the cell surface (Spiegel and

Milstien 2002). There are at least five highly-specific S1PR known in humans – S1P1 through

S1P5. S1PR are G-protein coupled receptor (GPCR). S1P1 is the most predominant S1PR in

human naïve CD8+ T-cells, while other receptors are expressed at very low levels (Shah, Zhang

et al. 2008). In T-cells, S1PR-mediated signaling plays an important role in egress from

lymphoid organs following activation (Schwab and Cyster 2007).

We assessed relative abundance of S1P-receptors in PBMC samples from patients with

LGL leukemia using real-time PCR. S1P5 was the most abundant of these receptors in LGL

leukemia, while S1P2 and S1P3 were not expressed (Figure 16).

65

Figure 16: Relative abundance of S1P-receptors in naïve enriched normal CD8+ cells Enriched CD8+ cells from healthy donors (white dots, n=4) and leukemic LGL (black dots, n=5) were analyzed for S1PR expression using real-time PCR. The figure shows that S1P5 is the predominant S1P-receptor for leukemic LGL.

S1PR profiling in leukemic LGL:

Next, we compared expression of S1PR in leukemic LGL to naïve and activated normal phenotypes. We found that S1P1 mRNA was significantly down-regulated in leukemic LGL

compared to naïve phenotypes, but not in comparison to activated phenotypes. Comparing three

phenotypes with approximately similar percentage of CD3+ CD8+ cells, namely naïve enriched

CD8+ cells, activated enriched CD8+ cells and leukemic LGL, S1P5 mRNA was overexpressed

in leukemic LGL by 4.5-fold compared to naïve enriched CD8+ cells (P=0.008) and by 235-fold

compared to activated enriched normal CD8+ cells (P=0.001; Figure 17). S1P4 was not significantly altered in any of the phenotypes tested.

66

Figure 17: Relative expressions of S1PR in leukemic LGL compared to normal cells

Relative expression of S1P1 (white bars), S1P4 (gray bars) and S1P5 (black bars) in leukemic LGL (n=5) compared to normal phenotypes (n=3-5). A positive value indicates upregulation, while a negative value indicates downregulation of a gene in leukemic LGL. Error bars represent standard deviation of expression. S1P5 is upregulated in LGL leukemia PBMC compared to all normal phenotypes. S1P1 is upregulated in naïve phenotypes compared to both activated phenotypes and leukemic LGL. (*P<0.05, **P<0.001)

Differential regulation of sphingolipid metabolism and signaling play an important role in survival of leukemic LGL

Given the contrasting role of ceramide and S1P in cellular homeostasis, we hypothesized

that the collective differential expression of genes associated with sphingolipid metabolism and

related signaling could result in an altered rheostat of sphingolipid metabolites. This, in

association with collective upregulation of genes in the Gα12-mediated signaling pathway in

67 leukemic LGL, may confer abnormal survival on leukemic LGL resulting in the accumulation of leukemic cells.

To test our hypothesis functionally, we treated PBMC from healthy individuals or LGL leukemia patients with a series of inhibitors of sphingolipid metabolism pathway. Since these inhibitors act upon sphingolipid pathway ‘prior’ to sphingolipid rheostat, we hypothesized that treatment with these inhibitors should not induce apoptosis in leukemic LGL. Normal or LGL leukemia patient PBMC were treated with vehicle myriocin (inhibitor of SPCT), fumonisin

(inhibitor of ceramide synthase), desipramine (inhibitor of acid sphingomyelinase) or GW4869

(inhibitor of neutral sphingomyelinase) for up to 24h. The doses used are 5-10 times higher than doses known to induce apoptosis in many cell types. Induction of apoptosis was tested using flow cytometry. Figure 18 shows normalized apoptosis in normal or LGL leukemia PBMC. We

concluded that none of these inhibitors tested induced significant apoptosis in either normal or

LGL leukemia PBMC.

68

Figure 18: Inhibition of enzymes outside sphingolipid rheostat does not induce selective apoptosis in leukemic LGL Naïve normal PBMC (N PBMC, one representative sample of two independent experiments showed) or leukemic LGL (TLGL, n=3) were either left untreated or treated with vehicle or indicated concentrations of myriocin (left upper panel), fumonisin (right upper panel), desipramine (left lower panel), and GW4869 (right lower panel) for 24h. Induction of apoptosis was assessed using flow cytometry. There was no differential apoptosis of leukemic LGL compared to normal naïve PBMC using each of these inhibitors. (Each dot (o) represents mean percentage of apoptosis ± SEM of three separate experiments in an individual sample; marker (-) represents the mean of all samples in a given treatment; Y-axis represents percentage of apoptosis normalized to no treatment control)

Inhibition of acid ceramidase leads to selective induction of apoptosis in leukemic LGL

We hypothesized that disruption of sphingolipid rheostat by inhibition of acid ceramidase should lead to induction of apoptosis in leukemic LGL but not in normal PBMC. To

69 test this hypothesis, we used a selective small molecule inhibitor of ASAH1 known as N- oleoylethanolamine (NOE). Freshly isolated PBMC from healthy donors (N PBMC) or LGL leukemia patients (TLGL) were treated with vehicle or 100μM NOE for 6h. As an additional control, PBMC from a healthy donor were activated using PHA and IL2 for ten days. Following

10-days activation, activated normal PBMC (AC PBMC) was treated similarly. Cells undergoing apoptosis were identified using flow cytometry as described in materials and methods section.

NOE did not induce any significant apoptosis in naïve or activated normal PBMC. In contrast, treatment with NOE, but not vehicle, induced apoptosis in leukemic LGL. NOE induced apoptosis by greater than 30-fold compared to naive normal PBMC (P=0.00001,

Figure 19).

Figure 19: Inhibition of acid ceramidase induced differential apoptosis in leukemic LGL Naïve normal PBMC (N PBMC, white dots, n=5), activated normal PBMC (AC PBMC, gray dots, n=6), or leukemic LGL (TLGL, black dots, n=6) were either left untreated or treated with vehicle (methanol) or 100µM N OE for 6h. Induction of apoptosis was assessed using flow cytometry. NOE induced approximately 30-fold higher apoptosis in LGL leukemia PBMC compared to normal PBMC (*P=0.00001) (Each dot (o) represents mean percentage of apoptosis ± SEM of three separate experiments in an individual sample; marker (-) represents the mean of all samples in a given treatment; Y-axis represents percentage of apoptosis normalized to no treatment control)

70

Inhibition of S1P-mediated signaling leads to selective induction of apoptosis in LGL leukemia PBMC

We hypothesized that S1P-mediated signaling is crucial for survival of leukemic LGL in vivo. To test this hypothesis, we used a novel immunomodulator known as FTY720. FTY720 is an functional antagonist of inhibitor of S1P-mediated signaling. Freshly isolated PBMC from healthy donors (N PBMC) or LGL leukemia patients (TLGL) were treated with vehicle or 5μM

FTY720 for 6h. As an additional control, PBMC from a healthy donor were activated using

PHA and IL2 for ten days. Following 10-days activation, activated normal PBMC (AC PBMC) was treated similarly. Cells undergoing apoptosis were identified using flow cytometry as described in materials and methods section.

Treatment with FTY720, but not vehicle, induced apoptosis by greater than 13-fold compared to naive normal PBMC (P=0.0009, Figure 20). None of the inhibitors shown in

figures 13, 14 or 15 induced any significant apoptosis in normal PBMC.

71

Figure 20: Inhibition of S1P-mediated signaling by FTY720 induced differential apoptosis in leukemic LGL Naïve normal PBMC (N PBMC, white dots, n=5), activated normal PBMC (AC PBMC, gray dots, n=4), or leukemic LGL (TLGL, black dots, n=5) were either left untreated or treated with vehicle (DMSO) or 5µM FTY720 for 6h. Leukemic LGL showed approximately 13-fold higher apoptosis compared to naïve normal PBMC (*P=0.0009). (Each dot (o) represents mean percentage of apoptosis ± SEM of three separate experiments in an individual sample; marker (-) represents the mean of all samples in a given treatment; Y-axis represents percentage of apoptosis normalized to no treatment control)

Differential regulation of S1P-mediated signaling contributes to abnormal survival of leukemic LGL

Leukemic LGL are characterized by abnormal expansion of CD3+ CD57+ cells. We then asked if normal and leukemic CD3+ CD57+ cells (LGL) differed in their sensitivity towards inhibition of S1P-mediated signaling. Thus, in contrast to previous experiment, this experiment looks at induction of apoptosis in either normal or malignant LGL only. Freshly isolated PBMC from healthy donors or LGL leukemia patients were treated with vehicle or 5μM FTY720 for 6h.

Induction of apoptosis was assessed in CD3+ CD57+ double positive cells using flow cytometry.

72

As an additional control, PBMC from a healthy donor were activated using PHA and IL2 for ten days. Following 10-days activation, activated normal PBMC were treated with vehicle or 5μM

FTY720 for 6h. We found that FTY720 induced 20-fold higher apoptosis in leukemic LGL compared to naïve normal LGL and approximately 4-fold higher apoptosis compared to activated normal LGL (Figure 21 ).

Figure 21: FTY720 selectively induces apoptosis in leukemic LGL but not in LGL from healthy donors PBMC from healthy donors (N PBMC 1,2), normal activated PBMC (AC PBMC) or LGL leukemia patients (TLGL 1,2) were treated with DMSO (hollow bars) or 5μM FTY720 (filled bars) as described. Flow cytometric analysis for induction of apoptosis was done. Cells were gated for CD3+ CD57+ double-positive cells (LGL cells) and further analyzed for apoptosis. Leukemic LGL showed 20-fold higher apoptosis compared to naïve normal LGL. Y-axis represents percentage of apoptosis normalized to no treatment control.

To evaluate effects of S1P inhibition on Fas-mediated apoptosis in leukemic LGL, we

assessed effect of FTY720 treatment on Fas-mediated apoptosis in PBMC from LGL leukemia

patients. In agreement with our previous work, there was no significant induction of apoptosis

73 following treatment with vehicle and anti-Fas mAb (1μg/ml CH11). We showed that treatment with FTY720 induced apoptosis in leukemic LGL and also sensitized leukemic LGL to Fas- mediated apoptosis (P=0.001) (Figure 22)

Figure 22: Treatment with FTY720 sensitizes LGL leukemia PBMC to Fas-mediated apoptosis PBMC from LGL leukemia patients were incubated with vehicle or 5μM FTY720 for 1 h. Each treatment group was further divided into two and were left untreated or treated with 1μg/ml CH11 and further incubated with 6h. In addition to inducing spontaneous apoptosis, treatment with 5μM FTY720 further sensitizes leukemic LGL to Fas-mediated apoptosis (*P=0.001). (Results shown are representative of three independent experiments; Y-axis represents percentage of apoptosis normalized to no treatment control).

In order to assess if addition of S1P leads to inhibition of Fas-mediated apoptosis in

normal PBMC, PBMC from healthy donors were activated with PHA+IL2 as described in

methods section. After ten days, the cells were washed and resuspended in RPMI-1640

supplemented with 1% FBS for 18h. The cells were then treated with indicated amount of S1P

or vehicle for one hour and then either left untreated or treated with 1μg/ml CH11 for further

74

3.5h. Induction of apoptosis was assessed by flow cytometry. Addition of CH11 led to induction of apoptosis in activated normal cells as expected. Addition of S1P, but not vehicle, led to inhibition of Fas-mediated apoptosis in activated normal PBMC in a dose-dependent manner

(Figure 23). Addition of 0.5μM S1P to the culture medium inhibited Fas-mediated apoptosis by

more than 55% compared to addition of CH11 only (P<0.03).

Figure 23: Exogenous S1P produces Fas-resistance phenotype in activated cells from healthy donors Activated PBMC from normal healthy donors were cultured in RPMI-1640 supplemented with 1% FBS for 18 h. The cells were treated with either vehicle or indicated concentrations of S1P for 1 hr. 1μg/ml of CH11 was added to the wells and cells were further incubated for an additional 3.5h. The graph shows that increasing amount of S1P in culture protects cells from Fas-mediated apoptosis in a dose-dependent manner. At 0.5μM and 0.05μM concentrations, S1P inhibits Fas-mediated apoptosis by more than 55% and 30% (*P<0.03) respectively. (The results shown are a representative of one of the three individual experiments performed; Y-axis represents percentage of apoptosis normalized to no treatment control)

Discussion

GSEA of leukemic LGL compared to activated normal PBMC suggested that sphingolipid metabolism and signaling related gene set is enriched in leukemic LGL.

GSEA is a pathway-based approach to microarray analysis that determines whether a pre-

defined set of genes shows statistically significant difference in expression between two

phenotypes. It considers the behavior of a set of genes rather than individual genes

(Mootha, Lindgren et al. 2003). An approach such as GSEA is particularly important in

samples with subtle variations between two phenotypes or in those with high variances.

Such an approach can be valuable in suggesting potential involvement of a pathway; however this assertion needs to be validated functionally.

We hypothesized that inhibitors that alter ceramide-S1P rheostat would induce apoptosis in leukemic LGL, but inhibitors of sphingolipid metabolism not altering this rheostat would have no effect on survival of leukemic LGL. In the latter category, we tested myriocin, fumonisin, desipramine and GW4869. None of these agents induced apoptosis in leukemic LGL. We conclude that inhibition of pathways outside sphingolipid rheostat had no effect on survival of leukemic LGL.

In contrast, inhibition of acid ceramidase - an enzyme central to sphingolipid rheostat - led to apoptosis of leukemic LGL. Ceramidase is the enzyme regulating the rate-limiting step in conversion of ceramide into sphingosine and a free fatty acid.

Upregulation of acid ceramidase is seen in many human cancers. High acid ceramidase activity results in lower levels of ceramide in the cells even following ceramide-inducing

76 stimuli (Pyne and Pyne 2000; Park and Schuchman 2006). Gene-based analysis identified ASAH1 as one of the most significantly upregulated apoptosis related genes in leukemic LGL compared to activated enriched CD8+ cells. This observation was confirmed at the protein level by Western blot analysis. Acid ceramidase was also identified as a core enriched gene using GSEA – thus rendering it as an important potential target to perturb sphingolipid signaling. The activity of acid ceramidase can be inhibited by its specific chemical inhibitor NOE. NOE is known to induce apoptosis in human alveolar macrophages (Monick, Mallampalli et al. 2004); however, the effect of

NOE treatment in lymphocytes is not known. We found that inhibition of the activity of acid ceramidase led to significantly higher induction of apoptosis in leukemic LGL. This result suggests that high ceramidase activity, presumably due to higher expression of acid ceramidase, is one of the mechanisms of survival in leukemic LGL. Further studies using the overexpression of ASAH1 will be needed to confirm the importance of upregulation of acid ceramidase in the long-term survival of CTL.

Our results strongly suggest that S1P-mediated signaling is important in keeping

CD8+ cells alive following activation. S1P provides protection against Fas-mediated apoptosis in various T-lymphocyte cell lines (Cuvillier, Rosenthal et al. 1998; Goetzl,

Kong et al. 1999; Olivera, Kohama et al. 1999; Goetzl and Graler 2004). Our results showed that S1P can protect activated normal PBMC from Fas-mediated apoptosis, similar to the Fas-resistant phenotype observed in leukemic LGL. S1P-mediated signaling can be inhibited by FTY720 (Fingolimod). In peripheral T-lymphocytes,

FTY720 preferentially augments apoptosis of activated cells following TCR-mediated activation (Shimizu, Li et al. 1998). We found that 6h treatment with FTY720 induces

77 apoptosis in PBMC from LGL leukemia patients but not in naive or activated normal

PBMC. Importantly, we demonstrated that FTY720 selectively induced apoptosis in leukemic LGL but not in naïve or activated normal LGL. FTY720 is an immunomodulator currently being evaluated in clinical trials for use in autoimmune conditions such as multiple sclerosis (Massberg and von Andrian 2006; Brinkmann

2007). Immunosuppressive therapy has been the cornerstone of treatment in LGL leukemia. However, only about 50% of patients respond to an initial regimen and there is no known curative treatment (Sokol and Loughran 2006). Hence, FTY720 may be a potential therapeutic agent in LGL leukemia.

FTY720 (2-amino-2-(2-(4-octylphenyl) ethyl)-1, 3-propanediol hydrochloride)

was derived from extensive chemical derivatization based on the Chinese medicine Isaria

sinclairii. It is a chemical derivative of ISP-1 (myriocin – an inhibitor of SPCT enzyme

described above). However, it was soon apparent that the mechanism of action of

FTY720 was different from that of myriocin. While it is well-known that FTY720 is an immunomodulator and produces brief lymphopenia, the exact mechanism of

lymphopenia and immunesuppression are still unknown. FTY720 binds to all the S1P

receptors except S1P2 (Brinkmann 2007). Because of selective activity of FTY720 on

leukemic LGL, we were interested in further characterizing S1P-receptor expression in

leukemic LGL. We found that S1P1 is the predominant receptor in all normal

phenotypes. S1P1 is known to be downregulated in T-lymphocytes following in vitro or

in vivo activation (Graeler and Goetzl 2002; Brinkmann 2007). We confirmed these

results in activated normal CD8+ cells and also showed downregulation of S1P1 in leukemic LGL. These results further suggest that leukemic LGL are activated cells in

78 vivo. Previously we showed upregulation of S1P5 in leukemic LGL (Kothapalli,

Kusmartseva et al. 2002). Our results show that S1P5 is the predominant S1P-receptor in

leukemic LGL and that S1P5 is selectively upregulated in leukemic LGL compared to naïve and activated normal PBMC and enriched CD8+ cells. Recently, it was also shown

that S1P5 is the predominant receptor in mature NK cell subset. (Walzer, Chiossone et al.

2007)

GSEA of leukemic LGL compared to activated PBMC suggested enrichment of

Gα12 signaling pathway. S1P5 acts as GPCR upstream to both Gαi and Gα12/13.

Constitutive Gα12 signaling can mediate oncogenic transformation through Src and PI3k-

Akt mediated activation of STAT3 (Kumar, Shore et al. 2005). We and others have shown that Src-family kinases, PI3K-AKT, and STAT3 activation contribute to apoptotic resistance of leukemic LGL (Epling Burnette PK 2001; Schade, Powers et al. 2006).

Taken together, these facts suggest that altered sphingolipid mediated signaling through

S1P5-Gα12 may play an important role in survival of leukemic LGL.

Maintenance of long-lived functional CTL is necessary for generating vaccines

directed at cancer and chronic viral infections as well as in adoptive T-cell cancer

therapy. As recently reviewed, the biggest obstacle following adoptive T-cell therapy for

cancers is poor survival and maintenance of effector functions of desired T-cells (Curiel

2007; June 2007). Passive transfer of ‘primed’ CTL is not sufficient for generating long-

lasting effector response in vivo. In addition, many tumor cells express FasL on their

surfaces. This can be an effective mechanism of immune evasion by tumors since

activated T-lymphocytes express high levels of Fas on their surface, making the latter

susceptible to Fas/FasL mediated apoptosis (Leen, Rooney et al. 2007). LGL leukemia

79 patients show clonal expansion of functional cytotoxic CD8+ cells that are inherently resistant to Fas/FasL mediated apoptosis. Therefore, LGL leukemia might serve as a human model to investigate key pathways promoting CTL survival. Our findings suggest importance of sphingolipid-mediated signaling in long-term survival of CTL in vivo.

Model of sphingolipid rheostat in LGL leukemia:

The sphingolipid rheostat described above, is dysregulated in LGL leukemia

(Figure 24). The first clue about involvement of sphingolipid signaling in survival of

leukemic LGL came when human S1P5 was originally identified as an overexpressed

expressed sequence tag (EST) using DNA library from LGL leukemia PBMC

(Kothapalli, Kusmartseva et al. 2002). Later, analysis of gene signature established that

sphingolipid metabolism as well as Gα12-mediated signaling was enriched in LGL

leukemia PBMC.

Our results suggest that the balance of sphingolipid metabolites ‘favors’ survival

over apoptosis in leukemic LGL. Inhibition of enzymes ‘outside’ the rheostat, do not

revert this balance back to normal and thus, do not induce apoptosis in leukemic LGL.

However, inhibition of enzymes integral to sphingolipid rheostat selectively induces

apoptosis in leukemic LGL (Fig. 18-20).

80

Figure 24: Sphingolipid rheostat and its role in survival of leukemic LGL Sphingolipid signaling is determined by balance between pro- and anti-apoptotic sphingolipid molecules. It is proposed that deregulation in sphingolipid signaling shifts the balance towards survival in leukemic LGL. NOE is an inhibitor of acid ceramidase (ASAH1), while SKI-I and II selectively inhibit sphingosine kinase (SPHK). FTY720 is an immunomodulator that acts as an functional antagonist of sphingosine-1-phosphate (S1P)-mediated signaling. Treatment with any of these molecules selectively induced apoptosis in leukemic LGL.

Therapeutic opportunities

FTY720 or Fingolimod is a novel immunomodulatory compound in clinical trials for post-renal transplant and in autoimmune conditions such as multiple sclerosis (MS).

FTY720 is an analogue of S1P that binds to four out of five S1PR (except S1P2) and acts as a functional antagonist (Brinkmann 2007). Given that FTY720 selectively induces apoptosis in leukemic LGL and that LGL leukemia patients have significantly high co- existing autoimmune diseases, it is possible that FTY720 may have a therapeutic role in these patients.

81 Sphingosine kinase inhibitors (SKI-I and II) are known to have anti-tumor activity and have proven their anti-tumor activity in mice models (French, Upson et al. 2006).

Development of newer, more selective inhibitors offer exciting opportunities (Paugh,

Paugh et al. 2008).

Neutralizing antibody to S1P has been proposed as an interesting candidate in blocking various signaling cascades in solid tumors. It works as a ‘sponge’ in neutralizing S1P. When injected, these antibodies were successful neutralizing S1P thus providing protection against various solid tumors in mouse models. The mechanism was through inhibition of angiogenesis, inhibition of S1P-mediated proliferation, and reversed

S1P-mediated protection against apoptosis (Milstien and Spiegel 2006; Visentin, Vekich et al. 2006). While there have been no reports of efficacy of neutralizing antibody to S1P as a potential therapeutic in leukemia. It remains to be seen if it offers any advantage in treatment of LGL leukemia.

82

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Chapter 4

Mechanism of FTY720 mediated cell death in LGL leukemia

Introduction

Our previous work has identified that sphingolipid rheostat is dysregulated in

leukemic LGL. Disruption of this dysregulated balance readily induced apoptosis in leukemic LGL. One such way to disrupt sphingolipid rheostat is by treating leukemic

LGL with FTY720 (Fingolimod), an inhibitor of S1P-mediated signaling. FTY720 has been successfully tested in various models of autoimmunity as a potential therapeutic. It has also shown to be useful in extending graft survival. FTY720 is being tested in clinical trials for post-renal transplant patients and in patients with multiple sclerosis

(MS). It was recently proposed as a novel antineoplastic agent based on findings that it selectively induces apoptosis in various cancer cell models (Liu, Zhao et al. 2008).

Given that FTY720 selectively induces in leukemic LGL and that LGL leukemia patients have significantly high co-existing autoimmune diseases, it is possible that FTY720 may have a therapeutic role in T-LGL leukemia patients.

FTY720, an analogue of S1P, binds to four out of five S1PR (except S1P2) and acts as a functional antagonist (Brinkmann 2007). FTY720, due to its functional antagonist behavior, blocks S1P- S1P1 mediated egress from lymph nodes trapping the cells in lymph nodes producing lymphopenia. Interestingly, it is proposed that trapping

of lymphocytes in lymph nodes following FTY720 treatment, is not equal for all the T-

87 cell subsets. FTY720 preferentially traps naïve and central memory cells while sparing effector-memory cells, thereby enriching effector-memory T-cells in blood. This unique mechanism, it is hoped, will help patients by down-modulating immune response to new antigens, without significantly affecting response to antigens previously known.

FTY720 produces comparable lymphopenia in aly/aly mice that lack lymph nodes and Peyer’s patches suggesting that there exists an additional mechanism of lymphopenia induced by FTY720. A possible explanation for FTY720 mediated lymphopenia is that

FTY720 preferentially induces apoptosis in activated T-cells (Suzuki, Li et al. 1997;

Goetzl, Kong et al. 1999). It is still unclear how much this mechanism contributes towards FTY720 mediated immunomodulation in vivo. We previously showed that

FTY720 indeed selectively induces apoptosis in leukemic LGL while sparing LGL from healthy donors. However, the mechanism of FTY720 mediated cell death induction is not known.

In this report we show that FTY720 mediated cell death is independent of Gαi signaling raising a possibility that it is mediated by S1P5-Gα12/13 pathway. We also show

that unlike previous reports, FTY720-mediated cell death is independent of action of

phosphoserine/phosphothreonine and phosphotyrosine phosphatases (Sonoda, Kasahara et al. 1997). Our results also show that FTY720 mediated cell death in leukemic LGL is independent of the action of caspases. In all the patient samples tested, the treatment with FTY720 damages mitochondrial membrane. In one of the patients, induction of

apoptosis coincided with downregulation of MCL1, a protein known to be upregulated in

leukemic LGL. However, FTY720 induced apoptosis was not associated with

downregulation of MCL1 in other patient samples tested. We propose that FTY720

88 mediates apoptosis-like PCD by inducing mitochondrial damage independent of the action of caspases.

Mechanism of FTY720-induced cell death

FTY720 is structurally similar to sphingosine and is phosphorylated to form (S)- configured FTY720-phosphate by SphK in vivo. Due to similarity of structures, FTY720 competes with S1P for phosphorylation. Both the isoforms of SPHK (SPHK1 and

SPHK2) phosphorylate FTY720. However, SPHK2 is about 30-fold more efficient at phosphorylating FTY720 (Billich, Bornancin et al. 2003). It was also proposed that

SPHK2 was essential for lymphopenia produced by FTY720. Following phosphorylation, FTY720-phosphate but not FTY720 binds to all S1PR with high- affinity except to S1P2 (Figure 25).

FTY720 induces apoptosis in various kinds of leukemic cell lines in vitro (Jurkat,

HL60 and MM1S). It has been shown to induce apoptosis in chronic lymphocytic leukemia (Neviani, Santhanam et al. 2007; Liu, Zhao et al. 2008). It has been previously shown that FTY720 preferentially induces apoptosis in activated cells (Suzuki, Li et al.

1997; Goetzl, Kong et al. 1999). Various mechanisms of apoptosis induction have been described to explain activity of FTY720.

89 Materials and Methods

Patient consent

All patients met the clinical criteria of T-cell (CD3+) LGL leukemia with increased LGL counts and clonal T-cell receptor (TCR) gene rearrangement and had not received treatment for LGL leukemia. Informed consents were signed by all patients and age- and gender-matched normal individuals to allow the use of their cells for these experiments. Buffy coats were obtained from Hershey Medical Center Blood Bank according to protocols observed by Milton S. Hershey Medical Center, Hershey, PA.

Chemicals and reagents

Reagents and chemicals including phytohemagglutinin (PHA), TRIzol, RPMI-

1640 with Glutamax, and fetal bovine serum were obtained from Invitrogen Corporation

(Carsbald, CA, USA) and used according to manufacturer’s instructions. FTY720 was purchased from Cayman Chemical Company (Ann Arbor, MI). Pertussis toxin was obtained from Sigma Aldrich (St. Louis, MO). z-VAD-FMK and Ac-DEVD-FMK were obtained from Fisher Scientific (Pittsburgh, PA). Sodium orthovanadate and Okadaic acid were obtained from Calbiochem (San Diego, CA).

90 Antibodies

Anti-Fas antibody (activating, human CH11 clone) was obtained from Upstate

Cell Signaling Solutions (Lake Placid, NY); Annexin V: PE Apoptosis Detection Kit-I,

CD3-APC, CD8-FITC with respective isotype controls were all obtained from BD

Biosciences (San Jose, CA). Monoclonal antibodies to caspase-3, caspase-8, and polyclonal antibody to caspase-9 were obtained from Alexis Biosciences (San Diego,

CA). Monoclonal antibodies to MCL1 and phosphorylated STAT3 (pSTAT3) were obtained from Santa Cruz Biotech (Santa Cruz, CA). Monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Chemicon

International (Temecula, CA). Monoclonal antibody to Bax was obtained from (Cell

Signaling Technology, Boston, MA).

Real-time PCR analysis

Total RNA was isolated using TRIzol as described. RNA was treated with amplification grade DNAse I (Invitrogen) according to manufacturer’s instructions.

Real-time PCR analysis was done using SYBR® GreenER™ Two-Step qRT-PCR Kits

Universal (Invitrogen, CA) according to manufacturer’s instructions. PCR Primers used are listed in the Table 1 (Chapter 4). PCR was performed with Cepheid SmartCycler

(Sunnyvale, CA) using following conditions: 2m at 50oC, 10m at 95oC, followed by 40

cycles of 1m at 95oC, 1m at 60oC and 2m at 72oC. Melt curve analysis was performed following each run using default settings. Real-time PCR analysis for comparison of multiple transcripts in a single sample, expression was normalized to GAPDH and

91 adjusted for primer efficiency. For relative abundance between phenotypes, primer efficiency was determined and analyzed using REST MCS® software.(Pfaffl, Horgan et al. 2002) All the samples were tested in duplicate and the transcripts were normalized to

GAPDH expression.

Western blot assay

Cells were lysed in a buffer composed of 50 mM Tris-Cl (pH 7.6); 5 mM EDTA;

150 mM NaCl; 0.5% NP-40; 0.1% SDS containing 1:100 dilution of protease inhibitory cocktail; 1 mM sodium orthovanadate; and 0.5 mM PMSF (all from Sigma-Aldrich).

Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories,

CA). 30 μg of total protein was boiled in Laemmli SDS-PAGE sample loading buffer containing 4% beta-mercaptoethanol and 80mM DTT; subjected to 15% SDS-PAGE.

The proteins were then transferred to a membrane and Western blot assay was performed as described previously.(Epling Burnette PK 2001)

Apoptosis assay

Freshly isolated PBMC of normal individuals or LGL patients were used for cell death assay. Briefly, 5X105 cells were seeded in 0.5 ml of RPMI-1640 medium

supplemented with 10% FBS. The cells were untreated or treated with vehicle or

compound for indicated duration. Following treatment, cells were assessed by flow

cytometry for cell death using Annexin-V conjugated with phytoerythrocin (PE) and 7-

92 amino-actinomycin-D (7-AAD) staining (BD Biosciences) according to manufacturer’s instructions. Briefly, the cells were washed once with PBS and resuspended in 20 μg/ml

Annexin-V-PE and 20 μg/ml 7-AAD in 500 μl of 1X Binding Buffer. The cells were incubated for 10 minutes in the dark at room temperature and then immediately analyzed using BD FACSCalibur machine. All the live cells were gated and ten-thousand events were analyzed for Annexin-V-PE and 7-AAD. Annexin-V negative, 7-AAD positive cells were considered non-viable and excluded from further analysis. Percentage specific cell death was calculated using the following formula:

Percentage specific cell death = (Annexin-V positive cells in treatment -

Annexin-V positive cells in control) X 100 / (100 - Annexin-V positive cells in control)

Results

FTY720 mediated cell death is independent of Gαi signaling

Guanine nucleotide binding proteins (G-proteins) are important heterotrimeric

proteins that act as second-messengers in various signaling cascades. G-proteins receive

signals through their cognate GPCR. GPCR are promiscuous in coupling with their

downstream G-proteins, each GPCR pairing with one or more G-proteins. G-proteins, in

turn, engage various pathways regulating survival, proliferation, vascular tone,

endothelial barrier function, migration etc.

93 (A)

(B)

Figure 25: FTY720, an inhibitor of S1P-mediated signaling

(A) FTY720 is a structural analogue of S1PR. S1PRs are GPCR that couple with Gαi, Gαs, Gαq, or Gα12/13. FTY720 binds to four out of five S1PR (except S1P2). S1P1 predominantly signals through Gαi signaling cascade while S1P5 is known to signal through both Gαi and Gα12/13. Adapted from (http://www.postech.ac.kr/chem/skc- lab/research/lipidomics.htm; Sanchez and Hla 2004).

The homing response in T-lymphocyte is dependent upon Gαi protein

(Brinkmann, JBC 2002). A phenotype is considered to be mediated by Gαi signaling if

the phenotype is rescued following pre-treatment with pertussis toxin (PTX). PTX is an

exotoxin produced by the microorganism Bordetella pertussis. PTX causes ADP-

ribosylation of the α-subunits of the heterotrimeric G-proteins Gαi, and Gαo. This prevents the G-protein from interacting with its cognate GPCR on the cell membrane, thus interfering with intracellular communication.

94 Shinomya et al showed that apoptosis induced by FTY720 is insensitive to PTX suggesting that induction of apoptosis by FTY720 does not depend upon Gαi signaling

(Shinomiya, Li et al. 1997 ). Given the fact that S1P2 and S1P3 are not expressed in

either normal or leukemic CD8+ cells and that S1P4 is not differentially expressed

between the two phenotypes (Shah, Zhang et al. 2008), it can be hypothesized that in

leukemic LGL, either S1P1- or S1P5- mediated signaling plays role in FTY720 mediated

cell death Figure 26A).

S1P1 mediates signaling predominantly through Gαi signaling, which is sensitive

to PTX. On the other hand, S1P5 is known to mediate signaling either through Gαi or

Gα12/13. While Gαi is sensitive to PTX, Gα12/13 is not. Hence, if a phenotype is

insensitive to PTX, it can be suggested to work through Gα12/13 signaling pathway.

We hypothesized if survival signals in leukemic LGL are mediated through Gαi, pre-treatment with PTX should rescue the phenotype following FTY720 treatment. On the other hand, if induction of cell death is not inhibited following PTX pre-treatment, it can be considered to be mediated through Gα12/13, which may be mediated by S1P5.

As shown in Figure 2B, leukemic T-LGL were pre-treated with 100 ng/ml PTX for 2h or 18h to inhibit Gαi signaling. Following the pre-treatment, cells were treated

either with vehicle or 5 µM FTY720 for 6h. Induction of cell death was assessed using

Annexin-V and 7-AAD binding using flow cytometry as described. As described in

Figure 26B, PTX does not rescue FTY720 mediated cell death in leukemic LGL

suggesting that FTY720 interferes with signals independent of Gαi. This strongly

suggests that if survival signals in leukemic LGL are mediated by S1PR-dependent

pathways, they are mediated through S1P5 Æ Gα12/13.

95

(A)

(B)

Figure 26: FTY720 mediated apoptosis is insensitive to PTX (A) Model of mechanism of FTY720 mediated apoptosis in leukemic LGL. Leukemic LGL show downregulation of S1P1 while they overexpress S1P5. S1P4 is not expressed differentially between normal and leukemic LGL phenotypes. Both S1P2 and S1P3 are not expressed in either phenotype. This leads to a hypothesis that FTY720 might either work through S1P1 or S1P5 using Gαi or Gα12/13 as G-proteins. Gαi mediated signaling is sensitive to PTX while Gα12/13 signaling is insensitive to PTX treatment. (B) Leukemic LGL were pre-treated with 100- or 200 ng/ml of PTX for 2h (data not shown) or 18h and then treated with either vehicle or 5µM FTY720 for 6h. Induction of apoptosis was assessed using Annexin-V and 7-AAD binding using flow cytometry as described. The graph shows that PTCX does not rescue apoptosis phenomenon in FTY720 treated cells suggesting that FTY720 mediated induction of apoptosis is independent of Gαi signaling. (Data shown is representative graph of experiments conducted with three individual T- LGL patient samples. Each bar shows percentage of apoptosis induced normalized to no treatment control. Bar shows mean calculated from three biological replicates while error bars indicate standard error of mean)

96 FTY720 mediated cell death is independent of actions of caspases

One approach to study action of caspases in a given phenotype is to pre-treat the cells with a caspase-inhibitor to see if the phenotype is rescued. It has been suggested that in human myelogenous leukemia cell line HL-60, caspase-3 was cleaved in FTY720 treated cells as early as 3h. However, FTY720 mediated apoptosis was completely inhibited by pre-treatment with pan-caspase inhibitor z-VAD-FMK (carbobenzoxy-valyl- alanyl-aspartyl-[O-methyl]- fluoromethylketone). z-VAD-FMK is a cell permeable pan- caspase inhibitor. Acetyl-Asp-Glu-Val-Asp- fluoromethylketone (Ac-DEVD-FMK) is an irreversible inhibitor of caspase-3. At higher concentrations, Ac-DEVD-FMK also inhibits caspases-6, -7, -8, and -10. Fluoromethylketone (FMK) group in these inhibitors ensures irreversible inhibition of target caspases (Nagahara, Ikekita et al. 2000). These results suggested that FTY720 induces apoptosis in caspase-3 dependent manner.

In contrast, in B-cell malignancies and primary B cells from patients with chronic lymphocytic leukemia (CLL), FTY720 induced apoptosis that was independent of effects of caspases. Pan-caspase inhibitor failed to rescue cells following FTY720 treatment

(Liu, Zhao et al. 2008)

We asked if FTY720 mediated apoptosis in caspase-dependent or –independent manner. As a positive control, we used an established model of caspase-mediated apoptosis. Jurkat T-cells are known to undergo apoptosis following treatment with anti-

Fas antibody (CH11) that is dependent on action of caspases. In agreement with previously published results, pre-treatment with z-VAD-FMK rescued almost all of the

Jurkat T-cells from undergoing Fas-mediated apoptosis validating our experimental

97 methods (Ko, Johnson et al. 2000). Similar to z-VAD-FMK, pre-treatment with Ac-

DEVD-FMK for 2h rescued most of Jurkat T-cells from undergoing CH11 mediated apoptosis validating our experimental methods (Figure 27, 28).

CH11 (100 ng/ml) - - - + + + z-VAD-FMK - 50 100 - 50 100 (µM)

Figure 27: CH11-mediated apoptosis in Jurkat T-cells is dependent of action of caspases. z-VAD-FMK is a cell permeable pan-caspase inhibitor Jurkat T-cells were pre-treated with 50µM or 100µM of z-VAD-FMK or vehicle for 2h and then treated with either vehicle or 100 ng/ml CH11 for 6h more. Induction of

98 apoptosis was assessed using Annexin-V and 7-AAD binding using flow cytometry as described. In agreement with previously published results, pre-treatment with z-VAD- FMK rescued almost all Jurkat T-cells from undergoing apoptosis. It can be concluded that CH11-mediated apoptosis is dependent on actions of caspases. (Each bar shows percentage of apoptosis induced normalized to no treatment control. Bar shows mean of average calculated from three biological replicates while error bars indicate standard error of mean)

Figure 28: CH11-mediated apoptosis in Jurkat T-cells is dependent of action of caspases Ac-DEVD-FMK is a cell permeable inhibitor of caspases-3, -6, -7, -8. Jurkat T-cells were pre- treated with 50µM or 100µM of Ac-DEVD-FMK or vehicle for 2h and then treated with either vehicle or 100ng/ml CH11 for 6h more. Induction of apoptosis was assessed using Annexin-V and 7-AAD binding using flow cytometry as described. In agreement with previously published results, pre-treatment with Ac-DEVD-FMK rescued almost all Jurkat T-cells from undergoing apoptosis. It can be concluded that CH11-mediated apoptosis is dependent on actions of caspases. (Each bar shows percentage of apoptosis induced normalized to no treatment control. Bar shows mean of average calculated from three biological replicates while error bars indicate standard error of mean)

99 We asked if pre-treatment with either of these inhibitors can similarly rescue

FTY720 mediated cell death in leukemic LGL. Both z-VAD-FMK and Ac-DEVD-FMK were not toxic to the cells at concentrations tested. While FTY720 induced cell death as expected, pre-treatment of leukemic LGL with 50 µM or 100 µM of z-VAD-FMK or Ac-

DEVD-FMK for 2h failed to rescue FTY720 mediated cell death in leukemic LGL

(Figure 29, 30 shows representative data from one of the four T-LGL patient samples

tested). These results suggest that FTY720 mediated cell death is independent of the

action of caspases in leukemic LGL

FTY720 5µM - - + + +

z-VAD-FMK 50 100 - 50 100 (µM)

Figure 29: FTY720 mediated cell death in leuekmemic LGL is independent of the action of caspases z-VAD-FMK is a cell permeable pan-caspase inhibitor. Leukemic LGL were pre-treated with 50µM or 100µM of z-VAD-FMK for 2h and then treated with either vehicle or 5µM FTY720 for 6h. Induction of apoptosis was assessed using Annexin-V and 7-AAD

100 binding using flow cytometry as described. The graph shows that there was no statistically significant difference when the cells were pre-treated with z-VAD-FMK or not. Since induction of apoptosis was not rescued by z-VAD-FMK at any concentrations tested, it can be concluded that FTY720 mediated cell death is independent of actions of the caspases. (Data shown is a representative graph of experiments conducted with three individual T-LGL patient samples. Each bar shows percentage of apoptosis induced normalized to no treatment control. Bar shows mean of average calculated from three biological replicates while error bars indicate standard error of mean)

DEVD- FMK 50 100 - 50 100 (µM) FTY720 - - 5 5 5 5µM

Figure 30: FTY720 mediated cell death in leukemic LGL is independent of caspase-3 Ac-DEVD-FMK is an inhibitor of caspases-3, 6, 7 and 10. Leukemic LGL were pre- treated with 50µM or 100µM of DEVD-FMK for 2h and then treated with either vehicle or 5µM FTY720 for 6h. Induction of cell death was assessed using Annexin-V and 7- AAD binding using flow cytometry as described. The graph shows that there was no statistically significant difference when the cells were pre-treated with Ac-DEVD-FMK or not. Since induction of cell death was not rescued by Ac-DEVD-FMK at any concentrations tested, it can be concluded that FTY720 mediated cell death is independent of actions of caspases -3, 6, 7 or 10. (Data shown is a representative graph of experiments conducted with three individual T-LGL patient samples. Each bar shows percentage of cell death induced normalized to no treatment control. Bar shows mean of average calculated from three biological replicates while error bars indicate standard error of mean)

101

To confirm the finding that FTY720 induces cell death in caspase-independent manner, we treated leukemic LGL with 5 µM of FTY720 and harvested cells at 30m, 3h and 6h. Cell lysates were prepared and western blot analysis was performed as described in Materials and Methods section. Western blot analysis with caspases-8 and -9 antibodies revealed that there was no differential cleavage of caspases-8, or -9 in cells treated with vehicle or FTY720 for any time points tested. Western blot with caspase-3 antibody suggests that caspase-3 is cleaved (and thus activated) in time dependent manner at 3h and 6h time points in FTY720 treated cells but not in vehicle treated cells.

These results confirmed that FTY720 induces caspase-independent cell death in leukemic

LGL (Figure 31).

102

Figure 31: FTY720 induced cell death in leukemic LGL is independent of action of caspases -8 and -9 Leukemic LGL were treated with 5µM FTY720 for 30 minutes, 3h, or 6h. Cell lysates were prepared as described in materials and methods section. Western blot analysis was performed as described. Western blot analysis shows no differential cleavage of caspase- 9 or -9 at any time point tested. However, caspase-3 is cleaved in FTY720 treated samples at 3h and 6h time points. Membrane was stripped and re-blotted with anti- GAPDH antibody as loading control.

FTY720 damages mitochondrial membrane prior to inducing cell death in leukemic LGL

It was recently shown that in B-cell malignancies and primary B-cells from patients with CLL, FTY720 induced apoptosis by damaging mitochondrial integrity and by downregulating MCL1 in STAT3 dependent manner (Liu, Zhao et al. 2008). In

103 MM1S cells, FTY720 damaged integrity of mitochondrial membrane in these cells

(Yasui, Hideshima et al. 2005). One of the approaches to study integrity of mitochondrial membrane is by measuring mitochondrial membrane potential (∆ψm).

3,3'-Dihexyloxacarbocyanine iodide (DiOC6(3)) is a fluorescent cationic dye that is

selectively taken up by mitochondira when used at low concentrations (<50nM). Uptake

of DiOC6(3) correlates with ∆ψm. One of the hallmarks of damage to mitochondrial

membrane damage is reduction in ∆ψm leading to lesser accumulation of DiOC6(3) in

cells, which can be measured using flow cytometry. On flow cytometry analysis,

high DiOC6(3) cells were considered to have intact mitochondria, while those with

low DiOC6(3) were considered to be those with damaged mitochondrial membrane

(Zamzami, Susin et al. 1996).

We asked if this mechanism of action was responsible for cell death observed in

leukemic LGL following FTY720 treatment. For this experiment, leukemic LGL were

treated with vehicle or DMSO for 3h. During the last 30m of incubation, DiOC6(3) was added to the culture at 40nM final concentration. Cells were then incubated in the dark.

At the end of incubation, cells were washed once and resuspended in annexin-V binding buffer and stained with annexin-V PE as described above. Two-color flow cytometric analysis was performed to determine percentage of cells that showed reduction in ∆ψm.

As shown in Figure 32A, B, following FTY720 treatment, leukemic LGL showed reduction in ∆ψm as early as 3h. Interestingly, most of these cells did not take up annexin-V staining suggesting that mitochondrial membrane damage preceded induction of cell death in these cells. Figure 8B shows percentage of DiOC6(3)low annexin negative (lower left corner) cells in each of the three T-LGL patients tested.

104 (A)

(B)

Figure 32: Induction of cell death by FTY720 correlates with breach in mitochondrial integrity (A) Leukemic T-LGL were either left untreated or treated with 5 µM of FTY720 for 3h. This time point was selected since it coincides with induction of cell death as measured by annexin-V and 7-AAD staining. Figure shows representative flow cytometry experiment

105 results. Flow cytometry results show an increasing number of cells with decreased mitochondrial transmembrane potential as measured by decreased uptake of DiOC6(3) in cells. (B) Quantification of mitochondrial membrane damage in FTY720-treated cells. Flow cytometry data was collected as described in (A). DiOC6(3)low Annexin negative cells were quantified. Percentage of cell death was normalized to vehicle treated cells. Figure shows that there are a large number of cells show decreased mitochondrial transmembrane potential and are annexin negative at 3h suggesting that breach in mitochondrial membrane precedes induction of cell death in these cells. (Data shown is a representative graph of experiments conducted with three individual T-LGL patient samples. (Each bar shows percentage of cell death induced normalized to vehicle treated control)

In Jurkat T-cells and MM.1S (a multiple myeloma cell line) cells, FTY720

mediated apoptosis was associated with cleavage of BAX protein (Goetzl, Kong et al.

1999; Yasui, Hideshima et al. 2005). BAX is an pro-apoptotic member of Bcl-2 family

member proteins. Upon activation, Bax is cleaved to and cleaved BAX is more efficient

at inducing damage to mitochondrial membrane (Jürgensmeier, Xie et al. 1998). We

asked if FTY720 mediated cell death was associated with cleavage of BAX in leukemic

LGL. PBMC from T-LGL patients were treated with vehicle or 5 µM FTY720 as

described and cell lysates were prepared. Western blot analysis with anti-BAX antibody

shows that BAX is not differentially cleaved when leukemic LGL were treated with

FTY720 suggesting that FTY720 does not induce cell death in leukemic LGL by BAX

cleavage (Figure 33).

In MM.1S cells, FTY720 mediated apoptosis was associated with decreased

STAT3 activity (as assessed by phosphorylated STAT3 expression) and downregulation

of MCL1 (Yasui, Hideshima et al. 2005). While in CLL cells, FTY720 downregulated

MCL1 independent of STAT3 activity (Liu, Zhao et al. 2008).

Western blot analysis performed with anti-MCL1 and anti-phospho-

STAT3 antibodies revealed that FTY720 causes downregulation of MCL1 within 3h – a

106 time point that coincides with induction of cell death following FTY720 treatment.

Interestingly, phospho-STAT3 levels remain unchanged throughout the experiment

(Figure 33).

(A)

(B)

Figure 33: FTY720 mediated induction of cell death is correlated with downregulation of myeloid cell leukemia-1 but not with cleavage of Bcl-2 associated X-protein (BAX) A potential mechanism of cell death-induction by FTY720 is through cleavage of BAX protein. BAX is a pro-apoptotic protein that is known to cleave in response to FTY720 treatment. PBMC from T-LGL leukemia patient was treated with vehicle or FTY720 for 30m, 3h or 6h and cell lysates were prepared. Western blot analysis was performed with BAX, MCL1 and phospho-STAT3 antibodies. Western blot analysis shows no cleavage of BAX at any time point tested, suggesting a cell death mechanism that is independent

107 of BAX. MCL1 is downregulated 3h and 6h following FTY720 treatment while it is unchanged in vehicle treated sample. In leukemic LGL, downregulation of MCL1 and induction of cell death may be associated with or independent of STAT3 activity (Epling- Burnette 2001; Zhang, Shah et al. 2008). Our results show that FTY720 mediates downregulation of MCL1 independent of STAT3 activity as assessed by phospho-STAT3 level. (B) In other two T-LGL patient samples, FTY720 induced cell death was associated with mitochondrial membrane damage (Fig 8 TLGL1 and 2), but is not associated with downregulation of MCL1, suggesting more than one mechanisms of induction of cell death.

Real-time PCR analysis of putative S1P5 targets in leukemic LGL

Our lab has identified targets that are specifically downregulated following 24h or

48h treatment with siRNA against S1P5 (Liu et al, unpublished results). We asked if these targets changed following FTY720 treatment in leukemic LGL. Leukemic LGL were treated with vehicle or FTY720 for 3h. RNA was isolated and quantitative real-time PCR was performed as described in materials and methods section in Chapter 2. Analysis of real-time PCR shows that none of the targets showed statistically significant change following FTY720 treatment. These may raise two possibilities: i) FTY720 acts independent of S1P5 signaling or ii) since the targets were identified at 24h and 48h, an

experiment performed at longer duration is needed. Figure 34 shows real-time PCR

analysis with TLGL1 patient sample there is no statistically significant change in the

targets studied. T-LGL patient sample used for this experiment is the same that does not show any difference in MCL1 expression at the protein level further validating western blot analysis.

108

Figure 34: Quantitative real-time PCR analysis of putative S1P5 targets in leukemic LGL Leukemic LGL from a T-LGL leukemia patient were treated with vehicle or FTY720 for 3h. Total RNA was isolated and real-time PCR analysis was performed as described. The results were analyzed using REST MCS software. The analysis shows no difference between targets studied at the time point indicated.

Mechanism of FTY720 mediated cell death in leukemic LGL is independent of phosphatases

In T98G human glioblastoma cell lines, FTY720 readily induced apoptosis. Co- treatment with as little as 0.2nM of sodium orthovanadate (VA) rescued almost all the cells from FTY720-mediated apoptosis suggesting that FTY720 induced apoptosis in phosphotyrosine phosphatase dependent manner (Sonoda, Yamamoto et al. 2001).

In leukemic LGL, treatment with 0.4 nM VA alone did not induce significant cell death in 6h. While treatment with FTY720 induced cell death of about half of the cells as expected, co-treatment with VA did not rescue cells from FTY720 mediated cell death

109 (Figure 35) suggesting that FTY720 mediated death signals are not dependent upon

phosphotyrosine phosphatases (e.g. dephosphorylation of focal adhesive kinases (FAK)).

Figure 35: FTY720 mediated cell death is independent of Sodium Orthovanadate (VA) pre-treatment in leukemic LGL VA is a non-selective inhibitor of phosphotyrosine phosphatases (Sonoda, Kasahara et al. 1997; Sonoda, Yamamoto et al. 2001). Leukemic T-LGL were treated with FTY720 alone or with VA (0.4 nM) for 6h. Induction of cell death was assessed using Annexin-V and 7-AAD binding using flow cytometry as described. Figure shows that FTY720 mediated induction of cell death in leukemic LGL is independent of treatment with VA suggesting that induction of cell death phenotype does not depend upon signaling through phosphotyrosine phosphatases. (Data shown is a representative graph of experiments conducted with three individual T-LGL patient samples. Each bar shows percentage of cell death induced normalized to no treatment control. Bar shows mean of average calculated from three biological replicates while error bars indicate standard error of mean)

Recently, it was shown that in CLL cells, FTY720 induced apoptosis was readily

rescued by pre-treatment with okadaic acid (OA). OA was initially isolated from marine

sponge Halichondria okadai. OA is a major toxic component in diarrhetic shellfish

poisoning and a potent tumour promoter. It is known to be a potent inhibitor of protein

phosphatases (PP) 1, 2A and 2B. When used at concentrations 0.25 nM or less, it is

110 known to be a selective inhibitor of PP2A (Svensson 2003; Neviani, Santhanam et al.

2007). In CLL as well as chronic myelogenous leukemia (CML) cells, FTY720 was shown to induce apoptosis in PP2A dependent manner. Inhibition of PP2A with OA rescued partially rescued cells from undergoing apoptosis (Neviani, Santhanam et al.

2007; Liu, Zhao et al. 2008).

In leukemic LGL, treatment with up to 0.25 nM OA did not induce significant cell death in 6h. While treatment with FTY720 induced cell death of about half of the cells as expected, co-treatment with OA did not rescue cells from FTY720 mediated cell death

(Figure 36) suggesting that FTY720 mediated death signals are not dependent upon

phosphoserine/phosphothreonine phosphatases such as PP2A.

111

Figure 36: Okadaic acid (OA) does not rescue FTY720-treated leukemic LGL from undergoing cell death OA is an inhibitor of protein phosphatase 2A (PP2A) when used at 0.25nM concentration or lesser (Neviani, Santhanam et al. 2007; Liu, Zhao et al. 2008). Leukemic T-LGL were treated with FTY720 alone or with OA (0.025nM and 0.25nM) for 6h. Induction of cell death was assessed using Annexin-V and 7-AAD binding using flow cytometry as described. FTY720 mediated induction of cell death in leukemic LGL is independent of treatment with OA suggesting that induction of cell death phenotype does not depend upon signaling through PP2A. (Data shown is a representative graph of experiments conducted with three individual T-LGL patient samples. Each bar shows percentage of cell death induced normalized to no treatment control. Bar shows mean of average calculated from three biological replicates while error bars indicate standard error of mean)

Discussion

Here we investigated the mechanism of FTY720 mediated cell death in T-LGL leukemia. FTY720 is a novel immunomodulator currently in clinical trials for in

112 autoimmune diseases such as multiple sclerosis (MS). Oral administration of FTY720 prolongs allograft survival in experimental organ transplantation without producing any noticeable side effects (Yamasaki, Inoue et al. 1998; Nagahara, Ikekita et al. 2000;

Brinkmann, Davis et al. 2002). FTY720 is being tested in post-transplant renal patients to enhance graft survival (Tedesco-Silva, Pescovitz et al. 2006). Since LGL leukemia has a strong clinical correlation with a wide variety of autoimmune diseases, FTY720 might be of therapeutic importance in patients with T-LGL leukemia (Sokol and Loughran

2006; Shah, Zhang et al. 2008).

We previously showed that FTY720 selectively induces cell death in leukemic

LGL. Our results show that inhibition of Gαi signaling or inhibition of phosphatases did

not rescue leukemic LGL from undergoing cell death. In this chapter, we show that

FTY720 induces cell death in caspase-independent and mitochondria dependent manner.

Previously it was shown that STAT3 is overactive in leukemic LGL and that inhibition of

STAT3 activity leads to MCL1 downregulation coinciding with induction of cell death in

leukemic LGL (Epling-Burnette 2001). It was recently shown that induction of apoptosis

and downregulation of MCL1 can occur independent of STAT3 activity as well (Zhang,

Shah et al. 2008). In this study, following FTY720 treatment, downregulation of MCL1

in leukemic LGL was found to be independent of phosphorylation status (and hence

activity) of STAT3.

Induction of cell death usually follows one of the two signals – i) extrinsic or

receptor-mediated signals or ii) intrinsic or mitochondrial signals. The extrinsic pathway

is initiated by ligation of a death receptors (such as Fas or TNF receptor) with their

cognate ligands (such as FasL or TNF) to activate initiator caspases (caspase-8 and –10),

113 which in turn cleave and activate effector caspases such as caspase-3 and –7. The intrinsic pathway follows damage of the mitochondrial membrane and the release of mitochondrial proteins into the cytosol. For example, following mitochondrial damage, a mitochondrial protein cytochrome c is released into the cytosol and forms a protein complex known as apoptosome by combining with two cytosolic proteins apoptoic protease activating factor-1 (Apaf-1) and procaspase-9. This cleaves and activates caspase-9 thereby initiating apoptotic caspase cascade (Riedl and Salvesen 2007).

We found that FTY720 damaged mitochondrial membrane and reduced ∆ψm in all the patient samples tested. The breach in mitochondrial membrane integrity temporally preceded other markers of apoptosis induction, suggesting that mitochondrial membrane damage is a key event in cell death following FTY720 treatment. One of the patient samples treated with FTY720 showed no cleavage of Bax but did downregulation of MCL1, an anti-apoptotic protein known to be upregulated in LGL leukemia.

However, other two patient samples did not show any change in the expression of MCL1.

Our results show that while more than 50% leukemic LGL show apoptotic phenotype within 6h, neither caspase-8 nor caspase-9 are activated, thus excluding both intrinsic and extrinsic caspase cascades. However, caspase-3 is cleaved in FTY720 treated cells in time-dependent manner. Inhibition of all the caspases, or specific inhibition of caspase-3, did not rescue cells from FTY720 mediated apoptosis suggesting that activation of caspases was not an essential event in induction of cell death. These results, in presence of annexin positive phenotype of leukemic LGL, raises a possibility that leukemic LGL are indeed undergoing caspase-independent apoptosis like PCD rather than classical apoptosis (Jaattela and Tschopp 2003).

114 Our central hypothesis for this work is that abnormal sphingolipid rheostat leads to abnormal survival in leukemic LGL. Thus it is the balance of pro- and anti-apoptotic sphingolipids that determine cell fate. Ceramide, a pro-apoptotic sphingolipid is known to induce cell death in variety of cells. While exact mechanism of ceramide induced cell death is not known, it has been shown that ceramide can induce caspase-independent

PCD. Hence, adding ceramide would be analogous to inhibiting S1P-mediated signaling in our model. FTY720 is a functional antagonist of S1P-mediated signaling. Therefore, it can be argued that FTY720 may induce cell death in leukemic LGL independent of the action of caspases. Our results confirm these findings and reveal a novel mechanism of

FTY720 mediated cell death in mature lymphocytes.

115

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Chapter 5

Conclusions and Future Directions

Large granular lymphocyte leukemia is a chronic lymphoproliferative disorder of

CTL or NK cells. Clinically, LGL leukemia is a rare disease chiefly seen in middle aged

men and women with roughly equal prevalence. Increasing number of LGL in the

peripheral blood is associated with B symptoms and a strong association with

autoimmune disorders. Previous studies have shown that inhibition of apoptosis in

leukemic LGL as the chief mechanism of pathogenesis in LGL leukemia. Focus of this

study was T-cell type of LGL leukemia. Leukemic T-LGL are activated CTL, but unlike activated CTL from healthy subjects leukemic T-LGL are resistant to Fas-FasL mediated apoptosis resulting in accumulation of leukemic cells in the periphery.

While various survival pathways in LGL leukemia have been described, a global gene expression analysis has not been carried out. We used microarray technology to create unique gene signature of leukemic LGL. We then used gene- and pathway-based analyses to identify the basic mechanism of clonal proliferation and survival of these cells. Our results show that following stimulation, CD8+ cells from healthy controls showed upregulation of genes related to activation and pro-apoptosis suggesting coupling of activation and apoptosis. In contrast, while leukemic LGL showed upregulation of activation and cytotoxicity related genes, they also had upregulation of anti-apoptotic genes and downregulation of pro-apoptotic genes. This suggests uncoupling of activation and apoptosis in these cells. This uncoupling of activation and apoptosis may be the

119 fundamental defect leading to clonal proliferation and resistance to apoptosis in leukemic

LGL.

Pathway-based approach to microarray analysis also identified a novel pathway of survival in leukemic LGL. Our results show that sphingolipid rheostat (a delicate balance between pro- and anti-apoptotic sphingolipid molecules) is dysregulated in leukemic

LGL. We also showed that disruption of this rheostat selectively induces apoptosis in leukemic LGL. Among the chemical inhibitors used to disrupt sphingolipid rheostat,

FTY720 is of special interest. FTY720 selectively induced apoptosis in leukemic LGL.

FTY720 is being studied in various clinical trials for treatment of autoimmune diseases and in post-transplant patients for immunosupression. However, the exact mechanism of its actions is not known. Our results suggest that FTY720 may be acting through selective induction of apoptosis in CD3+ CD57+ T-cells. This may also explain therapeutic efficacy of FTY720 in various autoimmune diseases since clinically, LGL leukemia, especially T-LGL leukemia, is significantly associated with variety of autoimmune diseases.

While our initial results suggest that FTY720 induces apoptosis in leukemic LGL by disrupting sphingolipid rheostat, the exact mechanism of action is not yet known. We have shown that FTY720 induced apoptosis in leukemic LGL is not associated with cleavage of caspases-3, -8 or -9. Our initial results suggest that FTY720 disrupts mitochondrial membrane potential prior to induction of cell death and may represent the chief mechanism of its actions. FTY720 was shown to induce apoptosis in some other leukemia by inhibiting tyrosine and protein phosphatases. In contrast, our results suggest that FTY720 mediated cell death is independent of actions of such phosphatases.

120 Future Directions

This work is the first step towards identifying global gene expression profiles of cytotoxic lymphocytes. We have performed microarray analysis using gene-based and theme-based approaches to identify genes that are differentially expressed between

PBMC from LGL leukemia patients compared to healthy controls. This approach has identified target genes that may play role in long-term survival of CTL. For example, we have identified ASAH1 and SERPINB9 to be upregulated in leukemic LGL compared to normal naïve and activated CD8+ T-cells. It remains to be seen if selective upregulation of these genes can lead to increased in vivo survival of CTL.

We propose that lentivirus-based approach should be used to stably transduce

CD8+ T-cells from healthy control. Following in vitro culture, effects of upregulation of the target genes can be measured by testing for survival and resistance to Fas-mediated apoptosis. The hypothesis for the future work will be that upregulation of ASAH1 or

SERPINB9 should lead to enhancement of survival and resistance to Fas-mediated apoptosis (and hence, LGL leukemia-like phenotype).

The work describing upregulation of NKp46 in T-cell type of LGL leukemia is novel. While there are many ‘negative markers’ for identification of NK cells there is lack of consensus about markers that can identify human NK cells unequivocally.

Initially, NKp46 was described as a marker specific to human NK cells. Our work shows that in fact, NKp46 is also expressed in a small subset of CD3+ CD8+ T-cells in LGL leukemia patients, but not in normal controls. Whether this upregulation of NKp46 is

121 related to cytotoxicity, malignancy or as a result of the common origin of CTL and NK cells during development, is not known.

Overall, this work aims to identify a novel mechanism of long-term survival in

CTL. Abnormal sphingolipid rheostat can lead to abnormal long-term survival in leukemic LGL leading to its accumulation in periphery. One of the major features of this work is identification of FTY720 as a potential therapeutic agent for LGL leukemia.

FTY720 shows rapid and selective induction of apoptosis in leukemic CD3+ CD57+ cells but not in the cells with the same phenotype of healthy controls. While our results suggest that FTY720 might be working through disruption of sphingolipid rheostat in leukemic LGL, the exact mechanism of action and target of FTY720 in these cells still remain unknown. One of the targets proposed is sphingosine-1-phosphate receptor S1P5.

These findings can be of importance in two ways – i) strategies can be targeted to

disrupt sphingolipid rheostat to selectively kill leukemic LGL but not normal LGL cells

in the body and ii) when selective survival of a clone in the body is desired, it can be

achieved through manipulation of sphingolipid signaling in order to facilitate survival.

The later approach may be of help in designing CTL based vaccines where in vivo

survival and activity of the desired clone of CTL has proved to be Achilles’ heel.

Appendix A

Gene Signature activated normal cells vs. resting normal cells

Log Ratio (Ac Log Ratio (Ac Gene Symbol Gene Title PBMC/Resting CD8/ Resting PBMC) CD8) TYMS thymidylate synthetase 4.90 5.00 KIAA0101 KIAA0101 4.81 5.13 NUSAP1 nucleolar and spindle associated protein 1 4.16 4.35 PCNA proliferating cell nuclear antigen 4.12 3.21 RACGAP1 Rac GTPase activating protein 1 4.09 3.69 CKS2 CDC28 protein kinase regulatory subunit 2 4.03 3.01 ZWINT ZW10 interactor 3.88 4.05 MCM2 minichromosome maintenance deficient 2, mitotin (S. MCM2 3.81 3.61 cerevisiae) MLF1IP MLF1 interacting protein 3.69 3.62 CKS1B CDC28 protein kinase regulatory subunit 1B 3.51 3.52 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) MTHFD2 3.37 3.30 2, methenyltetrahydrofolate cyclohydrolase TARS threonyl-tRNA synthetase 3.16 3.03 phosphoribosylaminoimidazole carboxylase, PAICS 3.15 3.43 phosphoribosylaminoimidazole succinocarboxamide synthetase GPR171 G protein-coupled receptor 171 3.14 2.44 GMNN geminin, DNA replication inhibitor 3.13 2.78 MCM6 minichromosome maintenance deficient 6 (MIS5 MCM6 3.11 2.35 homolog, S. pombe) (S. cerevisiae) PTTG1 pituitary tumor-transforming 1 3.00 2.73 RFC4 replication factor C (activator 1) 4, 37kDa 2.99 2.52 MT1E metallothionein 1E (functional) 2.88 2.17 CETN3 centrin, EF-hand protein, 3 (CDC31 homolog, yeast) 2.87 2.21 TUBB tubulin, beta 2.87 3.03 IARS isoleucine-tRNA synthetase 2.85 2.34 SMC4 structural maintenance of chromosomes 4 2.85 2.72 solute carrier family 1 (glutamate/neutral amino acid SLC1A4 2.84 3.16 transporter), member 4 RFC3 replication factor C (activator 1) 3, 38kDa 2.83 2.77 NASP nuclear autoantigenic sperm protein (histone-binding) 2.82 2.15 MCM3 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) 2.81 2.69 RDX radixin 2.78 2.59 RPA3 replication protein A3, 14kDa 2.77 2.22 ubiquitin-conjugating enzyme E2S /// similar to Ubiquitin- conjugating enzyme E2S (Ubiquitin-conjugating enzyme E2-24 UBE2S 2.77 1.92 kDa) (Ubiquitin-protein ligase) (Ubiquitin carrier protein) (E2- EPF5) SLAMF1 signaling lymphocytic activation molecule family member 1 2.76 2.49

123 tumor necrosis factor (ligand) superfamily, member 10 /// tumor TNFSF10 2.75 4.29 necrosis factor (ligand) superfamily, member 10 XCL2 chemokine (C motif) ligand 2 2.75 1.71 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) MTHFD1 1, methenyltetrahydrofolate cyclohydrolase, 2.74 2.35 formyltetrahydrofolate synthetase SHMT2 serine hydroxymethyltransferase 2 (mitochondrial) 2.73 2.91 TUBB tubulin, beta /// tubulin, beta 2.73 3.01 ASF1A ASF1 anti-silencing function 1 homolog A (S. cerevisiae) 2.72 2.19 ENO1 enolase 1, (alpha) 2.65 2.58 NUP43 nucleoporin 43kDa 2.64 3.12 SMC4 structural maintenance of chromosomes 4 2.63 2.51 GIMAP6 GTPase, IMAP family member 6 2.62 2.00 NUP37 nucleoporin 37kDa 2.62 2.62 CDK4 cyclin-dependent kinase 4 2.62 2.56 HIST1H4C histone 1, H4c 2.60 1.75 LAPTM4B lysosomal associated protein transmembrane 4 beta 2.57 2.25 mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N- MGAT2 2.56 1.66 acetylglucosaminyltransferase cytidine monophosphate-N-acetylneuraminic acid hydroxylase CMAH 2.55 2.24 (CMP-N-acetylneuraminate monooxygenase) MINA MYC induced nuclear antigen 2.54 2.18 CCL4 chemokine (C-C motif) ligand 4 2.54 1.83 SOS1 son of sevenless homolog 1 (Drosophila) 2.53 2.40 GPI glucose phosphate isomerase 2.53 2.09 PSMD14 proteasome (prosome, macropain) 26S subunit, non-ATPase, 14 2.52 2.34 chemokine (C-C motif) ligand 3 /// chemokine (C-C motif) CCL3 /// ligand 3-like 1 /// chemokine (C-C motif) ligand 3-like 3 /// CCL3L1 /// similar to Small inducible cytokine A3-like 1 precursor 2.49 1.93 CCL3L3 /// (Tonsillar lymphocyte LD78 beta protein) (LD78-beta(1-70)) LOC643930 (G0/G1 switch regulat PFKP phosphofructokinase, platelet 2.45 1.87 solute carrier family 7 (cationic amino acid transporter, y+ SLC7A1 2.44 1.92 system), member 1 RRM1 ribonucleotide reductase M1 polypeptide 2.41 1.67 NME1 non-metastatic cells 1, protein (NM23A) expressed in 2.40 2.13 fatty acid binding protein 5 (psoriasis-associated) /// similar to FABP5 /// Fatty acid-binding protein, epidermal (E-FABP) (Psoriasis- 2.40 2.50 LOC653327 associated fatty acid-binding protein homolog) (PA-FABP) IL32 interleukin 32 /// interleukin 32 2.38 2.45 granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated GZMA serine esterase 3) /// granzyme A (granzyme 1, cytotoxic T- 2.38 1.37 lymphocyte-associated serine esterase 3) SAP30 Sin3A-associated protein, 30kDa 2.38 2.11 ASNS asparagine synthetase 2.37 1.99 LIMA1 LIM domain and actin binding 1 2.35 2.35 IRF4 interferon regulatory factor 4 2.34 2.28 HSPA1A heat shock 70kDa protein 1A 2.34 2.06 PEX3 peroxisomal biogenesis factor 3 2.33 1.24 HSPD1 heat shock 60kDa protein 1 (chaperonin) 2.33 1.64 G3BP Ras-GTPase-activating protein SH3-domain-binding protein 2.32 1.77

124 SMC1A structural maintenance of chromosomes 1A 2.32 2.52 EXOSC8 exosome component 8 2.32 1.50 solute carrier family 1 (glutamate/neutral amino acid SLC1A4 2.32 2.68 transporter), member 4 RRAS2 related RAS viral (r-ras) oncogene homolog 2 2.31 1.81 MCM7 MCM7 minichromosome maintenance deficient 7 (S. cerevisiae) 2.31 2.03 CASP6 caspase 6, apoptosis-related cysteine peptidase 2.30 1.83 THYN1 thymocyte nuclear protein 1 2.30 2.28 RTCD1 RNA terminal phosphate cyclase domain 1 2.29 1.82 DDB2 damage-specific DNA binding protein 2, 48kDa 2.28 1.78 phosphoribosylglycinamide formyltransferase, GART phosphoribosylglycinamide synthetase, 2.27 2.52 phosphoribosylaminoimidazole synthetase RANBP1 RAN binding protein 1 2.25 1.72 BNIP3 BCL2/adenovirus E1B 19kDa interacting protein 3 2.25 1.20 DARS aspartyl-tRNA synthetase 2.23 2.16 RNASEH2A ribonuclease H2, subunit A 2.22 3.02 HN1 hematological and neurological expressed 1 2.22 2.91 MGC2463 hypothetical protein LOC79037 2.22 1.43 ORC6L origin recognition complex, subunit 6 like (yeast) 2.21 2.37 TRIB3 tribbles homolog 3 (Drosophila) 2.21 2.96 USP1 ubiquitin specific peptidase 1 2.20 2.43 ACADM acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain 2.18 2.05 STAT1 signal transducer and activator of transcription 1, 91kDa 2.18 2.07 HSPD1 heat shock 60kDa protein 1 (chaperonin) 2.17 1.48 SLC39A8 solute carrier family 39 (zinc transporter), member 8 2.16 1.55 TAF9 RNA polymerase II, TATA box binding protein (TBP)- TAF9 2.16 1.92 associated factor, 32kDa dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing DPP4 2.15 2.73 protein 2) IDH2 isocitrate dehydrogenase 2 (NADP+), mitochondrial 2.15 2.19 CDK2 /// cyclin-dependent kinase 2 /// beta-carotene dioxygenase 2 2.15 1.58 BCDO2 SCP2 Sterol carrier protein 2 2.14 1.75 KIF22 kinesin family member 22 2.14 1.79 ENO1 enolase 1, (alpha) 2.13 2.15 PSMA5 proteasome (prosome, macropain) subunit, alpha type, 5 2.13 1.67 CLDND1 claudin domain containing 1 2.12 1.85 SNRPD1 small nuclear ribonucleoprotein D1 polypeptide 16kDa 2.12 1.69 tumor necrosis factor (ligand) superfamily, member 10 /// tumor TNFSF10 2.12 3.59 necrosis factor (ligand) superfamily, member 10 5-aminoimidazole-4-carboxamide ribonucleotide ATIC 2.11 1.63 formyltransferase/IMP cyclohydrolase SOS1 son of sevenless homolog 1 (Drosophila) 2.11 2.14 PPID peptidylprolyl isomerase D (cyclophilin D) 2.10 1.57 DSCR2 Down syndrome critical region gene 2 2.08 2.70 INSIG2 insulin induced gene 2 2.08 1.48 GLRX2 glutaredoxin 2 2.08 2.65 CPOX coproporphyrinogen oxidase 2.06 1.67 DUT dUTP pyrophosphatase 2.05 1.89

125 CCT7 chaperonin containing TCP1, subunit 7 (eta) 2.05 1.43 DARS aspartyl-tRNA synthetase 2.05 1.93 PCMT1 protein-L-isoaspartate (D-aspartate) O-methyltransferase 2.05 1.87 TPI1 triosephosphate isomerase 1 2.04 2.72 C11orf73 chromosome 11 open reading frame 73 2.04 1.84 C13orf34 chromosome 13 open reading frame 34 2.03 1.87 IL17RB interleukin 17 receptor B 2.03 1.51 macrophage migration inhibitory factor (glycosylation-inhibiting MIF 2.02 1.34 factor) LSM2 homolog, U6 small nuclear RNA associated (S. LSM2 2.02 2.21 cerevisiae) NUDT21 nudix (nucleoside diphosphate linked moiety X)-type motif 21 2.02 1.47 MCM5 minichromosome maintenance deficient 5, cell division MCM5 2.02 2.49 cycle 46 (S. cerevisiae) HNRPF heterogeneous nuclear ribonucleoprotein F 2.01 1.76 POLR3K polymerase (RNA) III (DNA directed) polypeptide K, 12.3 kDa 2.01 2.35 ENOSF1 enolase superfamily member 1 2.01 1.33 C3orf28 chromsome 3 open reading frame 28 2.01 1.36 IMPDH2 IMP (inosine monophosphate) dehydrogenase 2 2.01 1.41 FAS Fas (TNF receptor superfamily, member 6) 2.01 1.84 FH fumarate hydratase 2.00 2.38 AK2 adenylate kinase 2 1.99 2.38 FAS Fas (TNF receptor superfamily, member 6) 1.99 2.10 SLC39A8 solute carrier family 39 (zinc transporter), member 8 1.99 1.38 guanylate binding protein 1, interferon-inducible, 67kDa /// GBP1 1.98 1.75 guanylate binding protein 1, interferon-inducible, 67kDa PPID peptidylprolyl isomerase D (cyclophilin D) 1.96 1.77 KIF2 kinesin heavy chain member 2 1.96 1.52 RRM1 ribonucleotide reductase M1 polypeptide 1.95 1.54 STIL SCL/TAL1 interrupting locus 1.95 2.61 CCDC53 coiled-coil domain containing 53 1.95 1.70 acidic (leucine-rich) nuclear phosphoprotein 32 family, member ANP32E E /// acidic (leucine-rich) nuclear phosphoprotein 32 family, 1.95 1.87 member E SWI/SNF related, matrix associated, actin dependent regulator SMARCA3 1.95 1.40 of chromatin, subfamily a, member 3 C7orf24 chromosome 7 open reading frame 24 1.94 2.10 proteasome (prosome, macropain) subunit, beta type, 8 (large PSMB8 1.94 1.72 multifunctional peptidase 7) PRDX3 peroxiredoxin 3 1.94 3.18 MRPL39 mitochondrial ribosomal protein L39 1.93 1.27 CCT5 chaperonin containing TCP1, subunit 5 (epsilon) 1.93 2.27 SAE1 SUMO-1 activating enzyme subunit 1 1.93 2.05 DYNLL1 dynein, light chain, LC8-type 1 1.93 2.34 NCBP1 nuclear cap binding protein subunit 1, 80kDa 1.92 1.49 GIMAP4 GTPase, IMAP family member 4 1.92 1.90 IFNG interferon, gamma 1.92 1.20 C3orf60 chromosome 3 open reading frame 60 1.90 1.79 EED embryonic ectoderm development 1.90 1.25 RNF34 ring finger protein 34 1.90 1.33

126 USP1 ubiquitin specific peptidase 1 1.90 1.16 TUBB tubulin, beta 1.90 2.26 CHMP5 chromatin modifying protein 5 1.89 1.58 KRCC1 lysine-rich coiled-coil 1 1.89 1.54 EIF2S1 eukaryotic translation initiation factor 2, subunit 1 alpha, 35kDa 1.89 1.39 HAX1 HCLS1 associated protein X-1 1.89 1.81 ICOS inducible T-cell co-stimulator 1.89 1.36 HNRPAB heterogeneous nuclear ribonucleoprotein A/B 1.88 1.89 H2AFZ H2A histone family, member Z 1.88 1.70 XPOT exportin, tRNA (nuclear export receptor for tRNAs) 1.88 1.50 TPI1 triosephosphate isomerase 1 1.88 2.41 AHA1, activator of heat shock 90kDa protein ATPase homolog AHSA1 1.87 1.28 1 (yeast) MRPS33 mitochondrial ribosomal protein S33 1.87 1.42 VDAC1 voltage-dependent anion channel 1 1.87 1.72 glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate GOT2 1.87 1.40 aminotransferase 2) solute carrier family 16, member 1 (monocarboxylic acid SLC16A1 1.86 1.47 transporter 1) CARS cysteinyl-tRNA synthetase 1.85 1.36 guanylate binding protein 1, interferon-inducible, 67kDa /// GBP1 1.84 1.87 guanylate binding protein 1, interferon-inducible, 67kDa EIF2S2 eukaryotic translation initiation factor 2, subunit 2 beta, 38kDa 1.83 2.04 BAX BCL2-associated X protein 1.83 1.26 IFI16 interferon, gamma-inducible protein 16 1.82 1.66 MT1X metallothionein 1X 1.82 1.31 ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit CLPP 1.82 1.31 homolog (E. coli) GMPS guanine monphosphate synthetase 1.82 1.50 KPNB1 karyopherin (importin) beta 1 1.82 1.61 C12orf11 chromosome 12 open reading frame 11 1.81 1.89 BAG2 BCL2-associated athanogene 2 1.80 1.22 acetyl-Coenzyme A acetyltransferase 1 (acetoacetyl Coenzyme ACAT1 1.80 1.94 A thiolase) ORC5L origin recognition complex, subunit 5-like (yeast) 1.79 1.45 ARHGAP19 Rho GTPase activating protein 19 1.79 1.50 MCTS1 malignant T cell amplified sequence 1 1.79 1.64 PIGF phosphatidylinositol glycan anchor biosynthesis, class F 1.79 2.25 GARS glycyl-tRNA synthetase 1.78 1.81 EBP emopamil binding protein (sterol isomerase) 1.78 1.58 MRPS31 mitochondrial ribosomal protein S31 1.78 2.06 DONSON downstream neighbor of SON 1.77 2.08 GCLM glutamate-cysteine ligase, modifier subunit 1.76 1.32 ITGB3BP integrin beta 3 binding protein (beta3-endonexin) 1.76 1.53 HMGB3 high-mobility group box 3 1.75 1.64 proteasome (prosome, macropain) subunit, beta type, 2 /// PSMB2 1.75 1.97 proteasome (prosome, macropain) subunit, beta type, 2 KIAA0286 KIAA0286 protein 1.75 2.18 IMMT inner membrane protein, mitochondrial (mitofilin) 1.75 1.35 AUH AU RNA binding protein/enoyl-Coenzyme A hydratase 1.75 2.05

127 LSM4 homolog, U6 small nuclear RNA associated (S. LSM4 1.74 1.84 cerevisiae) GLO1 glyoxalase I 1.73 1.81 METTL5 methyltransferase like 5 1.73 1.49 MTERFD1 MTERF domain containing 1 1.73 1.65 PRDX2 peroxiredoxin 2 1.72 1.17 SSRP1 structure specific recognition protein 1 1.72 1.87 ETNK1 ethanolamine kinase 1 1.72 1.31 RECQL RecQ protein-like (DNA helicase Q1-like) 1.72 2.04 MDH1 malate dehydrogenase 1, NAD (soluble) 1.72 1.39 CEP57 centrosomal protein 57kDa 1.71 1.25 HSPA4 heat shock 70kDa protein 4 1.71 1.22 RPS27L ribosomal protein S27-like 1.71 1.59 PTGER2 prostaglandin E receptor 2 (subtype EP2), 53kDa 1.71 1.97 STOML2 stomatin (EPB72)-like 2 1.71 1.43 AK2 adenylate kinase 2 1.71 2.25 MT2A metallothionein 2A 1.71 1.14 SYNCRIP synaptotagmin binding, cytoplasmic RNA interacting protein 1.71 2.20 C1orf41 /// chromosome 1 open reading frame 41 /// interleukin 17 receptor 1.71 2.13 IL17RB B ALAS1 aminolevulinate, delta-, synthase 1 1.71 2.49 TAP1 transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) 1.71 1.19 CASP8AP2 CASP8 associated protein 2 1.70 1.35 BANF1 barrier to autointegration factor 1 1.70 2.03 CSE1L CSE1 chromosome segregation 1-like (yeast) 1.70 1.12 BAX BCL2-associated X protein 1.69 1.60 dihydrolipoamide S-succinyltransferase (E2 component of 2- DLST /// PA2G4 1.69 2.14 oxo-glutarate complex) /// proliferation-associated 2G4, 38kDa SNRPG small nuclear ribonucleoprotein polypeptide G 1.69 1.27 TPM4 tropomyosin 4 1.68 2.32 CENTB1 centaurin, beta 1 1.68 1.19 MRPL15 mitochondrial ribosomal protein L15 1.68 2.41 MSH6 mutS homolog 6 (E. coli) 1.68 1.31 HTRA2 HtrA serine peptidase 2 1.67 1.27 MT1M metallothionein 1M 1.67 1.27 COMMD3 COMM domain containing 3 1.67 1.52 C13orf7 chromosome 13 open reading frame 7 1.66 1.35 H2AFZ H2A histone family, member Z 1.66 1.31 hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan HPRT1 1.66 1.63 syndrome) asparagine-linked glycosylation 8 homolog (S. cerevisiae, alpha- ALG8 1.66 1.32 1,3-glucosyltransferase) ENO2 enolase 2 (gamma, neuronal) 1.65 1.42 CBX1 chromobox homolog 1 (HP1 beta homolog Drosophila ) 1.65 1.59 DUT dUTP pyrophosphatase 1.65 1.41 MRPS14 mitochondrial ribosomal protein S14 1.65 1.72 BAZ1B bromodomain adjacent to zinc finger domain, 1B 1.64 2.09 RALA v-ral simian leukemia viral oncogene homolog A (ras related) 1.64 1.27 MRPL13 mitochondrial ribosomal protein L13 1.64 2.14 VRK1 vaccinia related kinase 1 1.64 1.35

128 C1GALT1C1 C1GALT1-specific chaperone 1 1.64 1.60 YARS tyrosyl-tRNA synthetase 1.64 1.17 ARMC1 armadillo repeat containing 1 1.63 1.40 MFAP1 microfibrillar-associated protein 1 1.63 1.28 TMEM48 transmembrane protein 48 1.63 1.83 EIF2B2 eukaryotic translation initiation factor 2B, subunit 2 beta, 39kDa 1.63 1.50 FAS Fas (TNF receptor superfamily, member 6) 1.62 1.54 TM6SF1 transmembrane 6 superfamily member 1 1.62 1.82 SERBP1 SERPINE1 mRNA binding protein 1 1.62 1.25 NFE2L1 nuclear factor (erythroid-derived 2)-like 1 1.62 1.14 CACYBP calcyclin binding protein 1.61 1.34 MYC v-myc myelocytomatosis viral oncogene homolog (avian) 1.61 1.20 FH fumarate hydratase 1.61 1.62 CCT4 chaperonin containing TCP1, subunit 4 (delta) 1.61 1.37 ETFB electron-transfer-flavoprotein, beta polypeptide 1.60 1.45 POLR2H polymerase (RNA) II (DNA directed) polypeptide H 1.60 1.46 TSR1 TSR1, 20S rRNA accumulation, homolog (S. cerevisiae) 1.60 1.48 TFDP1 transcription factor Dp-1 1.60 1.92 PHLDA1 pleckstrin homology-like domain, family A, member 1 1.59 2.00 VDAC1 voltage-dependent anion channel 1 1.59 1.65 COPB2 coatomer protein complex, subunit beta 2 (beta prime) 1.58 1.54 ITPR1 inositol 1,4,5-triphosphate receptor, type 1 1.58 1.83 LOC653784 similar to hypothetical protein FLJ14346 1.58 1.18 LMNB1 lamin B1 1.58 2.39 MTX2 metaxin 2 1.58 1.89 LSM4 homolog, U6 small nuclear RNA associated (S. LSM4 1.57 1.84 cerevisiae) EPRS glutamyl-prolyl-tRNA synthetase 1.57 1.62 EEF1E1 eukaryotic translation elongation factor 1 epsilon 1 1.56 1.18 MARS methionine-tRNA synthetase 1.56 1.41 DNAJC9 DnaJ (Hsp40) homolog, subfamily C, member 9 1.56 1.33 CCND2 cyclin D2 1.56 1.28 NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa NDUFS1 1.56 1.13 (NADH-coenzyme Q reductase) CBFB core-binding factor, beta subunit 1.56 1.28 SSRP1 structure specific recognition protein 1 1.56 1.57 STAT1 signal transducer and activator of transcription 1, 91kDa 1.56 1.36 RPS6KA5 ribosomal protein S6 kinase, 90kDa, polypeptide 5 1.55 1.23 PTPN7 protein tyrosine phosphatase, non-receptor type 7 1.55 1.13 EIF3S1 eukaryotic translation initiation factor 3, subunit 1 alpha, 35kDa 1.55 1.22 C4orf27 chromosome 4 open reading frame 27 1.55 1.35 UBE2V1 /// ubiquitin-conjugating enzyme E2 variant 1 /// ubiquitin- 1.54 1.40 Kua-UEV conjugating enzyme E2 variant 1 ORMDL2 ORM1-like 2 (S. cerevisiae) 1.54 1.49 thioredoxin domain containing 9 /// thioredoxin domain TXNDC9 1.54 1.13 containing 9 MRPL18 mitochondrial ribosomal protein L18 1.54 1.85 gamma-glutamyl hydrolase (conjugase, GGH 1.54 2.35 folylpolygammaglutamyl hydrolase) C6orf130 chromosome 6 open reading frame 130 1.53 1.17

129 CACYBP calcyclin binding protein 1.53 1.22 EBNA1BP2 EBNA1 binding protein 2 1.53 1.13 dynein, cytoplasmic 1, intermediate chain 2 /// dynein, DYNC1I2 1.52 1.91 cytoplasmic 1, intermediate chain 2 WDR12 WD repeat domain 12 1.52 1.25 PECI peroxisomal D3,D2-enoyl-CoA isomerase 1.52 1.47 HMGB2 high-mobility group box 2 1.52 1.52 SUCLG1 succinate-CoA ligase, GDP-forming, alpha subunit 1.52 1.34 RAN RAN, member RAS oncogene family 1.51 1.15 CCDC56 coiled-coil domain containing 56 1.51 1.68 NARG2 NMDA receptor regulated 2 1.51 1.24 TXN thioredoxin 1.50 1.69 acetyl-Coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme ACAT2 1.50 1.22 A thiolase) SMC3 structural maintenance of chromosomes 3 1.50 1.29 C1QBP complement component 1, q subcomponent binding protein 1.49 1.21 DHFR /// dihydrofolate reductase /// similar to Dihydrofolate reductase 1.49 2.13 LOC643509 C11orf48 chromosome 11 open reading frame 48 1.49 1.24 MEA1 male-enhanced antigen 1 1.49 1.62 PRMT1 protein arginine methyltransferase 1 1.48 1.22 CSE1L CSE1 chromosome segregation 1-like (yeast) 1.48 1.12 GTF2E1 general transcription factor IIE, polypeptide 1, alpha 56kDa 1.48 1.44 IFI16 interferon, gamma-inducible protein 16 1.48 1.52 CTSC cathepsin C 1.48 2.19 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 ELAVL1 1.47 1.79 (Hu antigen R) CARS cysteinyl-tRNA synthetase 1.47 1.44 CLTA clathrin, light polypeptide (Lca) 1.47 1.93 MRPS18B mitochondrial ribosomal protein S18B 1.47 1.42 C12orf24 chromosome 12 open reading frame 24 1.47 1.80 STAMBP STAM binding protein 1.46 1.38 PSMD8 proteasome (prosome, macropain) 26S subunit, non-ATPase, 8 1.46 1.38 NAT1 N-acetyltransferase 1 (arylamine N-acetyltransferase) 1.45 1.23 MRPL3 mitochondrial ribosomal protein L3 1.45 1.29 K-ALPHA-1 alpha tubulin 1.45 1.81 MT1F metallothionein 1F (functional) 1.45 1.16 PSMB5 proteasome (prosome, macropain) subunit, beta type, 5 1.45 1.56 ACTR3 ARP3 actin-related protein 3 homolog (yeast) 1.44 1.68 SRM spermidine synthase 1.44 1.46 TPM4 tropomyosin 4 1.44 3.20 ACTR3 ARP3 actin-related protein 3 homolog (yeast) 1.44 1.31 ADAM19 ADAM metallopeptidase domain 19 (meltrin beta) 1.44 2.53 K-ALPHA-1 alpha tubulin 1.44 1.61 RAP1GDS1 RAP1, GTP-GDP dissociation stimulator 1 1.43 1.64 LAPTM4B lysosomal associated protein transmembrane 4 beta 1.43 1.95 DCK deoxycytidine kinase 1.43 1.25 SERBP1 SERPINE1 mRNA binding protein 1 1.42 1.25 DUT dUTP pyrophosphatase 1.42 1.38

130 TMPO thymopoietin 1.40 1.99 C19orf7 chromosome 19 open reading frame 7 1.40 1.25 NARS2 asparaginyl-tRNA synthetase 2 (mitochondrial)(putative) 1.39 1.28 TERF2 telomeric repeat binding factor 2 1.39 1.58 PANK2 pantothenate kinase 2 (Hallervorden-Spatz syndrome) 1.39 1.23 FAM121B /// family with sequence similarity 121B /// NODAL modulator 3 1.39 1.31 NOMO3 NFYC nuclear transcription factor Y, gamma 1.39 1.28 SRPK1 SFRS protein kinase 1 1.38 1.16 LYRM1 LYR motif containing 1 1.38 1.58 FAM111A family with sequence similarity 111, member A 1.38 1.24 C14orf130 chromosome 14 open reading frame 130 1.38 1.53 MLH1 mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) 1.37 1.34 PCMT1 protein-L-isoaspartate (D-aspartate) O-methyltransferase 1.37 2.07 SARS seryl-tRNA synthetase 1.37 1.15 HTATSF1 HIV-1 Tat specific factor 1 1.37 1.23 solute carrier family 25 (mitochondrial carrier; oxoglutarate SLC25A11 1.36 1.49 carrier), member 11 ITPR1 inositol 1,4,5-triphosphate receptor, type 1 1.36 1.24 splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate SFRS1 1.36 1.35 splicing factor) HIP2 huntingtin interacting protein 2 1.36 1.19 CRYZ crystallin, zeta (quinone reductase) 1.36 1.18 PRDX1 peroxiredoxin 1 1.36 1.15 proteasome (prosome, macropain) 26S subunit, ATPase, 4 /// PSMC4 /// similar to 26S protease regulatory subunit 6B (MIP224) (MB67- 1.36 1.46 LOC652826 interacting protein) (TAT-binding protein 7) (TBP-7) JTV1 JTV1 gene 1.36 1.39 TBL2 transducin (beta)-like 2 1.34 1.70 ras-related C3 botulinum toxin substrate 2 (rho family, small RAC2 1.34 1.77 GTP binding protein Rac2) PROSC proline synthetase co-transcribed homolog (bacterial) 1.34 1.68 DAXX death-associated protein 6 1.33 1.21 BCL2 B-cell CLL/lymphoma 2 1.33 1.29 MOBK1B MOB1, Mps One Binder kinase activator-like 1B (yeast) 1.32 1.16 PKM2 pyruvate kinase, muscle 1.32 2.69 STK39 serine threonine kinase 39 (STE20/SPS1 homolog, yeast) 1.31 1.40 FLJ21908 hypothetical protein FLJ21908 1.31 1.51 C18orf10 chromosome 18 open reading frame 10 1.31 1.81 ALOX5AP arachidonate 5-lipoxygenase-activating protein 1.31 1.70 PRKDC protein kinase, DNA-activated, catalytic polypeptide 1.31 1.32 dolichyl-phosphate (UDP-N-acetylglucosamine) N- DPAGT1 acetylglucosaminephosphotransferase 1 (GlcNAc-1-P 1.31 1.31 transferase) PDCD6 programmed cell death 6 1.31 1.14 VBP1 von Hippel-Lindau binding protein 1 1.30 1.16 C14orf143 chromosome 14 open reading frame 143 1.30 1.82 CD7 CD7 molecule 1.29 1.42 CCT3 chaperonin containing TCP1, subunit 3 (gamma) 1.29 1.28 SEC23B Sec23 homolog B (S. cerevisiae) 1.29 1.14

131 MAL mal, T-cell differentiation protein 1.29 1.43 6-Sep septin 6 1.29 1.48 interleukin 2 receptor, gamma (severe combined IL2RG 1.29 1.17 immunodeficiency) SRI sorcin 1.29 1.43 TMPO thymopoietin 1.28 1.55 SLC43A3 solute carrier family 43, member 3 1.28 1.86 COP9 constitutive photomorphogenic homolog subunit 3 COPS3 1.28 1.19 (Arabidopsis) HEMK1 HemK methyltransferase family member 1 1.28 1.42 ubiquinol-cytochrome c reductase, complex III subunit VII, UQCRQ 1.28 1.19 9.5kDa CSNK2B casein kinase 2, beta polypeptide 1.28 1.40 electron-transfer-flavoprotein, alpha polypeptide (glutaric ETFA 1.27 1.16 aciduria II) CALM3 calmodulin 3 (phosphorylase kinase, delta) 1.27 1.75 LMO4 LIM domain only 4 1.27 1.45 FANCL Fanconi anemia, complementation group L 1.26 1.33 dihydrolipoamide S-succinyltransferase (E2 component of 2- DLST 1.26 1.49 oxo-glutarate complex) HAT1 histone acetyltransferase 1 1.25 1.41 AKAP2 /// A kinase (PRKA) anchor protein 2 /// PALM2-AKAP2 protein 1.25 1.44 PALM2-AKAP2 CEP57 centrosomal protein 57kDa 1.25 1.17 SUCLA2 succinate-CoA ligase, ADP-forming, beta subunit 1.25 1.85 SOCS2 suppressor of cytokine signaling 2 1.24 1.98 C10orf61 chromosome 10 open reading frame 61 1.24 1.58 C10orf22 chromosome 10 open reading frame 22 1.24 1.18 VKORC1 vitamin K epoxide reductase complex, subunit 1 1.24 1.37 PMVK phosphomevalonate kinase 1.24 1.54 UBE2V2 ubiquitin-conjugating enzyme E2 variant 2 1.23 1.47 C6orf79 chromosome 6 open reading frame 79 1.23 1.78 KIF22 kinesin family member 22 1.23 1.30 ATP synthase, H+ transporting, mitochondrial F0 complex, ATP5G3 1.22 1.31 subunit C3 (subunit 9) nucleolar protein family A, member 3 (H/ACA small nucleolar NOLA3 1.22 1.56 RNPs) IPO7 Importin 7 1.21 1.39 NMI N-myc (and STAT) interactor 1.21 1.52 OAS3 2'-5'-oligoadenylate synthetase 3, 100kDa 1.21 2.21 IVNS1ABP influenza virus NS1A binding protein 1.21 1.28 proteasome (prosome, macropain) subunit, beta type, 9 (large PSMB9 1.20 1.30 multifunctional peptidase 2) TMEM4 transmembrane protein 4 1.20 1.20 K-ALPHA-1 alpha tubulin /// alpha tubulin 1.20 1.61 CHMP5 chromatin modifying protein 5 1.19 1.25 NEK7 NIMA (never in mitosis gene a)-related kinase 7 1.19 1.13 HADHSC L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain 1.19 1.19 DHFR /// dihydrofolate reductase /// similar to Dihydrofolate reductase 1.19 2.03 LOC643509 AK2 adenylate kinase 2 1.19 1.21

132 FAS Fas (TNF receptor superfamily, member 6) 1.18 1.28 C16orf33 chromosome 16 open reading frame 33 1.18 1.39 ASH2L ash2 (absent, small, or homeotic)-like (Drosophila) 1.17 1.40 RPA2 replication protein A2, 32kDa 1.17 1.13 TUBB2C tubulin, beta 2C 1.17 1.27 APIP APAF1 interacting protein 1.17 1.71 apolipoprotein B mRNA editing enzyme, catalytic polypeptide- APOBEC3C 1.17 1.69 like 3C SYNCRIP synaptotagmin binding, cytoplasmic RNA interacting protein 1.17 1.20 SQRDL sulfide quinone reductase-like (yeast) 1.17 1.68 ------1.17 1.25 SNRPF small nuclear ribonucleoprotein polypeptide F 1.17 1.47 ZNF410 zinc finger protein 410 1.16 1.46 FLOT1 flotillin 1 1.16 1.69 COX5A cytochrome c oxidase subunit Va 1.16 1.41 MRPL40 mitochondrial ribosomal protein L40 1.15 1.32 TXNRD1 thioredoxin reductase 1 1.13 1.25 IFI35 interferon-induced protein 35 1.13 1.21 GIMAP5 GTPase, IMAP family member 5 1.13 1.23 SSBP1 single-stranded DNA binding protein 1 1.12 1.28 RER1 retention in endoplasmic reticulum 1 homolog (S. RER1 1.12 1.36 cerevisiae) TUBB2C tubulin, beta 2C 1.12 1.25 GPIAP1 GPI-anchored membrane protein 1 1.12 1.41 PGK1 phosphoglycerate kinase 1 1.12 1.43 MAP2K4 mitogen-activated protein kinase kinase 4 1.12 1.28 NUCB2 nucleobindin 2 1.12 1.31 ETHE1 ethylmalonic encephalopathy 1 1.11 1.13 ARL3 ADP-ribosylation factor-like 3 1.09 1.56 CLTA clathrin, light polypeptide (Lca) 1.09 1.54 TMEM165 transmembrane protein 165 1.09 1.23 AHCY S-adenosylhomocysteine hydrolase 1.09 1.27 SMC6 structural maintenance of chromosomes 6 1.09 1.25 PRDX4 peroxiredoxin 4 1.09 1.79 Platelet-activating factor acetylhydrolase, isoform Ib, alpha PAFAH1B1 1.08 1.33 subunit 45kDa CORO1A coronin, actin binding protein, 1A 1.08 1.58 Mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl- MGAT5 -1.08 -1.30 glucosaminyltransferase RPL22 ribosomal protein L22 -1.09 -1.74 DNAJB1 DnaJ (Hsp40) homolog, subfamily B, member 1 -1.09 -2.19 RABGAP1L RAB GTPase activating protein 1-like -1.09 -1.81 C6orf111 chromosome 6 open reading frame 111 -1.09 -1.66 RORA RAR-related orphan receptor A -1.09 -2.31 KLF12 Kruppel-like factor 12 -1.09 -1.86 RBM39 RNA binding motif protein 39 -1.09 -1.50 KIAA0256 KIAA0256 gene product -1.09 -1.83 SMURF2 SMAD specific E3 ubiquitin protein ligase 2 -1.10 -1.35 TTC3 tetratricopeptide repeat domain 3 -1.10 -1.81

133 UTX ubiquitously transcribed tetratricopeptide repeat, X chromosome -1.10 -1.68 DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked -1.11 -1.35 RIOK3 RIO kinase 3 (yeast) /// RIO kinase 3 (yeast) -1.11 -1.17 TMBIM1 transmembrane BAX inhibitor motif containing 1 -1.12 -1.26 RB1CC1 RB1-inducible coiled-coil 1 -1.12 -1.79 BIN2 bridging integrator 2 -1.12 -1.41 PASK PAS domain containing serine/threonine kinase -1.12 -2.32 LEPROTL1 leptin receptor overlapping transcript-like 1 -1.12 -1.85 HLA-F major histocompatibility complex, class I, F -1.13 -1.24 CLK4 CDC-like kinase 4 -1.13 -1.92 VIL2 villin 2 (ezrin) -1.13 -1.67 CD84 CD84 molecule -1.13 -1.44 PTBP2 polypyrimidine tract binding protein 2 -1.13 -1.84 OAT ornithine aminotransferase (gyrate atrophy) -1.14 -1.14 PTDSR phosphatidylserine receptor -1.14 -2.16 TRIM44 tripartite motif-containing 44 -1.14 -1.37 TXNIP thioredoxin interacting protein -1.14 -1.72 NIFUN NifU-like N-terminal domain containing -1.15 -1.16 EP300 E1A binding protein p300 -1.15 -1.16 TXNIP thioredoxin interacting protein -1.15 -2.11 GADD45B growth arrest and DNA-damage-inducible, beta -1.15 -1.62 CD44 /// CD44 molecule (Indian blood group) /// mitogen-activated -1.16 -1.19 MAPK10 protein kinase 10 KIAA1718 KIAA1718 protein -1.16 -1.84 GADD45B growth arrest and DNA-damage-inducible, beta -1.16 -1.41 RPL23 ribosomal protein L23 -1.16 -1.83 REL v-rel reticuloendotheliosis viral oncogene homolog (avian) -1.16 -1.47 ATP6V1G1 ATPase, H+ transporting, lysosomal 13kDa, V1 subunit G1 -1.17 -1.81 6-Mar membrane-associated ring finger (C3HC4) 6 -1.17 -1.35 PFAAP5 Phosphonoformate immuno-associated protein 5 -1.17 -1.73 heterogeneous nuclear ribonucleoprotein K /// heterogeneous HNRPK -1.17 -1.12 nuclear ribonucleoprotein K TRA2A transformer-2 alpha -1.17 -1.80 FBXO21 F-box protein 21 -1.17 -1.81 pleckstrin homology, Sec7 and coiled-coil domains 1(cytohesin PSCD1 -1.17 -1.97 1) VPS13B vacuolar protein sorting 13B (yeast) -1.18 -1.44 USP24 ubiquitin specific peptidase 24 -1.18 -1.17 ATXN7 ataxin 7 -1.18 -1.54 PASK PAS domain containing serine/threonine kinase -1.18 -2.24 RABGGTB Rab geranylgeranyltransferase, beta subunit -1.18 -1.39 LONPL Peroxisomal LON protease like -1.18 -1.35 ABLIM1 actin binding LIM protein 1 -1.18 -1.78 RASSF3 Ras association (RalGDS/AF-6) domain family 3 -1.19 -1.25 ZBTB43 zinc finger and BTB domain containing 43 -1.19 -1.40 EIF1 eukaryotic translation initiation factor 1 -1.19 -1.28 TXNDC13 thioredoxin domain containing 13 -1.19 -1.71 NBPF14 /// neuroblastoma breakpoint family, member 14 /// KIAA1245 /// KIAA1245 /// neuroblastoma breakpoint family, member 11 /// neuroblastoma -1.20 -1.28 NBPF11 /// breakpoint family, member 15 /// neuroblastoma breakpoint

134 NBPF15 /// family, member 9 /// neuroblastoma breakpoint family, member NBPF9 /// 16 NBPF16 LDLRAP1 low density lipoprotein receptor adaptor protein 1 -1.20 -1.70 DAZAP2 DAZ associated protein 2 -1.20 -1.14 DNAJB6 DnaJ (Hsp40) homolog, subfamily B, member 6 -1.20 -2.19 HBP1 HMG-box transcription factor 1 -1.21 -1.82 NKTR natural killer-tumor recognition sequence -1.21 -1.50 RB1CC1 RB1-inducible coiled-coil 1 -1.22 -1.94 HNRPH3 heterogeneous nuclear ribonucleoprotein H3 (2H9) -1.23 -1.61 C1orf63 chromosome 1 open reading frame 63 -1.23 -1.65 USPL1 ubiquitin specific peptidase like 1 -1.23 -1.40 C14orf138 chromosome 14 open reading frame 138 -1.23 -1.93 UBE2E1 Ubiquitin-conjugating enzyme E2E 1 (UBC4/5 homolog, yeast) -1.23 -1.20 CD69 CD69 molecule -1.23 -1.71 RPL22 ribosomal protein L22 -1.23 -1.97 CASP8 caspase 8, apoptosis-related cysteine peptidase -1.24 -1.91 EIF1B eukaryotic translation initiation factor 1B -1.24 -1.63 MAN2A1 mannosidase, alpha, class 2A, member 1 -1.24 -1.67 SH3YL1 SH3 domain containing, Ysc84-like 1 (S. cerevisiae) -1.25 -2.47 FAM21B /// family with sequence similarity 21, member B /// family with FAM21C /// sequence similarity 21, member C /// similar to KIAA0592 -1.25 -1.24 RP11-56A21.1 protein /// similar to similar to KIAA0592 protein /// LOC653450 SETD1B SET domain containing 1B -1.25 -2.17 RPL31 /// ribosomal protein L31 /// similar to ribosomal protein L31 -1.25 -1.56 LOC653773 CSNK1A1 casein kinase 1, alpha 1 -1.26 -1.30 CRY1 cryptochrome 1 (photolyase-like) -1.26 -1.18 JMJD3 jumonji domain containing 3 -1.26 -1.37 phosphatidylinositol glycan anchor biosynthesis, class A (paroxysmal nocturnal hemoglobinuria) /// phosphatidylinositol PIGA -1.26 -1.42 glycan anchor biosynthesis, class A (paroxysmal nocturnal hemoglobinuria) LOC644617 hypothetical LOC644617 /// hypothetical LOC644617 -1.27 -1.40 DNA segment on chromosome X and Y (unique) 155 expressed RP13-297E16.1 -1.27 -1.85 sequence, isoform 1 RYK RYK receptor-like tyrosine kinase -1.27 -1.73 FBXO11 F-box protein 11 -1.28 -1.36 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 /// DEAD (Asp- DDX5 -1.28 -1.28 Glu-Ala-Asp) box polypeptide 5 HECA headcase homolog (Drosophila) -1.29 -1.83 SMURF1 SMAD specific E3 ubiquitin protein ligase 1 -1.29 -1.94 WWP1 WW domain containing E3 ubiquitin protein ligase 1 -1.29 -1.24 TMEM66 transmembrane protein 66 -1.30 -2.23 SH3GLB1 SH3-domain GRB2-like endophilin B1 -1.30 -1.45 THRAP2 thyroid hormone receptor associated protein 2 -1.30 -1.34 ------1.30 -2.18 HNRPH3 heterogeneous nuclear ribonucleoprotein H3 (2H9) -1.30 -1.65 PCMTD2 protein-L-isoaspartate (D-aspartate) O-methyltransferase -1.30 -1.77

135 domain containing 2 RNMT RNA (guanine-7-) methyltransferase -1.30 -2.14 PRPF4B PRP4 pre-mRNA processing factor 4 homolog B (yeast) -1.30 -1.52 SFRS5 splicing factor, arginine/serine-rich 5 -1.31 -2.00 RIOK3 RIO kinase 3 (yeast) /// RIO kinase 3 (yeast) -1.31 -1.16 IFRD1 interferon-related developmental regulator 1 -1.31 -2.35 protein phosphatase 1A (formerly 2C), magnesium-dependent, PPM1A alpha isoform /// protein phosphatase 1A (formerly 2C), -1.31 -1.54 magnesium-dependent, alpha isoform JUND jun D proto-oncogene -1.31 -1.34 TTC3 tetratricopeptide repeat domain 3 -1.32 -1.67 Fas apoptotic inhibitory molecule 3 /// Fas apoptotic inhibitory FAIM3 -1.32 -1.61 molecule 3 ARHGEF18 rho/rac guanine nucleotide exchange factor (GEF) 18 -1.32 -1.35 VCP valosin-containing protein -1.32 -1.60 VPS13C vacuolar protein sorting 13 homolog C (S. cerevisiae) -1.32 -1.21 DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked -1.33 -1.17 NCOA1 nuclear receptor coactivator 1 -1.34 -1.57 CPD carboxypeptidase D -1.34 -1.13 EIF1 eukaryotic translation initiation factor 1 -1.34 -1.35 EIF5 eukaryotic translation initiation factor 5 -1.35 -1.47 RNF38 ring finger protein 38 -1.35 -1.29 EIF1 eukaryotic translation initiation factor 1 -1.36 -1.38 CASP8 caspase 8, apoptosis-related cysteine peptidase -1.36 -2.27 RAPGEF2 Rap guanine nucleotide exchange factor (GEF) 2 -1.36 -1.78 RGC32 response gene to complement 32 -1.36 -2.46 BEXL1 brain expressed X-linked-like 1 -1.36 -1.65 THUMPD1 THUMP domain containing 1 -1.36 -1.25 SNX3 sorting nexin 3 -1.37 -1.21 PIK3C2A phosphoinositide-3-kinase, class 2, alpha polypeptide -1.37 -1.50 ZNF91 zinc finger protein 91 -1.37 -1.97 DAZAP2 DAZ associated protein 2 -1.38 -1.46 LAPTM5 lysosomal associated multispanning membrane protein 5 -1.39 -1.15 pleckstrin homology domain containing, family F (with FYVE PLEKHF2 -1.39 -1.67 domain) member 2 H3F3B H3 histone, family 3B (H3.3B) -1.39 -1.68 NXF1 nuclear RNA export factor 1 -1.40 -1.38 integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen ITGB1 -1.40 -1.59 CD29 includes MDF2, MSK12) ZFAND5 zinc finger, AN1-type domain 5 -1.40 -1.34 FNDC3B fibronectin type III domain containing 3B -1.40 -1.72 GALNACT-2 /// chondroitin sulfate GalNAcT-2 /// similar to chondroitin beta1,4 -1.40 -1.15 LOC644504 N-acetylgalactosaminyltransferase 2 protein phosphatase 1B (formerly 2C), magnesium-dependent, PPM1B -1.40 -1.13 beta isoform PTP4A1 protein tyrosine phosphatase type IVA, member 1 -1.40 -1.45 VAV3 vav 3 oncogene -1.41 -1.29 C4orf15 chromosome 4 open reading frame 15 -1.41 -2.48 BTG2 BTG family, member 2 -1.42 -1.99 PIK3R1 phosphoinositide-3-kinase, regulatory subunit 1 (p85 alpha) -1.42 -2.03

136 NGFRAP1 nerve growth factor receptor (TNFRSF16) associated protein 1 -1.42 -1.30 AHNAK AHNAK nucleoprotein (desmoyokin) -1.43 -1.51 PABPN1 poly(A) binding protein, nuclear 1 -1.43 -1.24 phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 PDE4B -1.44 -1.67 dunce homolog, Drosophila) EFHC2 EF-hand domain (C-terminal) containing 2 -1.44 -1.37 6-Mar membrane-associated ring finger (C3HC4) 6 -1.44 -2.18 C6orf32 chromosome 6 open reading frame 32 -1.44 -1.98 vacuolar protein sorting 37 homolog B (S. cerevisiae) /// VPS37B -1.44 -1.33 vacuolar protein sorting 37 homolog B (S. cerevisiae) PRG1 proteoglycan 1, secretory granule -1.44 -1.35 GADD45B growth arrest and DNA-damage-inducible, beta -1.45 -2.33 FOXP1 Forkhead box P1 -1.46 -1.79 C6orf111 chromosome 6 open reading frame 111 -1.46 -1.95 RAPGEF2 Rap guanine nucleotide exchange factor (GEF) 2 -1.46 -1.81 CXCR4 chemokine (C-X-C motif) receptor 4 -1.46 -2.17 BTG1 B-cell translocation gene 1, anti-proliferative -1.48 -1.76 8-Mar membrane-associated ring finger (C3HC4) 8 -1.48 -1.46 PFDN5 prefoldin subunit 5 -1.48 -1.78 NELL2 NEL-like 2 (chicken) /// NEL-like 2 (chicken) -1.48 -2.71 CD44 CD44 molecule (Indian blood group) -1.49 -1.15 Protein phosphatase 2, regulatory subunit B (B56), gamma PPP2R5C -1.50 -2.21 isoform chemokine (C-X-C motif) receptor 4 /// chemokine (C-X-C CXCR4 -1.50 -2.51 motif) receptor 4 NECAP1 NECAP endocytosis associated 1 -1.50 -1.54 LOC92482 hypothetical protein LOC92482 -1.51 -2.39 S100 calcium binding protein A4 (calcium protein, calvasculin, S100A4 -1.51 -1.18 metastasin, murine placental homolog) transforming growth factor, beta receptor III (betaglycan, TGFBR3 -1.51 -2.96 300kDa) CTSO cathepsin O -1.51 -1.62 DNAJB1 DnaJ (Hsp40) homolog, subfamily B, member 1 -1.51 -2.03 IL6ST Interleukin 6 signal transducer (gp130, oncostatin M receptor) -1.51 -1.59 LITAF lipopolysaccharide-induced TNF factor -1.52 -2.02 KIAA0404 hypothetical protein LOC23130 -1.52 -2.30 CXCR4 chemokine (C-X-C motif) receptor 4 -1.53 -2.34 PCNX pecanex homolog (Drosophila) -1.53 -1.58 KIAA1109 KIAA1109 -1.55 -1.65 FUBP1 far upstream element (FUSE) binding protein 1 -1.55 -1.80 TMEM1 transmembrane protein 1 -1.57 -2.30 SGSH N-sulfoglucosamine sulfohydrolase (sulfamidase) -1.57 -1.64 DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked -1.58 -2.15 TIPARP TCDD-inducible poly(ADP-ribose) polymerase -1.59 -2.25 --- CDNA FLJ40810 fis, clone TRACH2009743 -1.59 -1.79 PFDN5 prefoldin subunit 5 -1.59 -1.79 KLF2 Kruppel-like factor 2 (lung) -1.59 -2.69 LITAF lipopolysaccharide-induced TNF factor -1.60 -2.17 FUBP1 Far upstream element (FUSE) binding protein 1 -1.60 -1.42 RNF139 ring finger protein 139 -1.60 -1.59

137 TSPYL1 TSPY-like 1 -1.60 -1.82 SFRS15 splicing factor, arginine/serine-rich 15 -1.60 -1.37 --- Homo sapiens, clone IMAGE:4214654, mRNA -1.61 -2.89 TOB1 transducer of ERBB2, 1 -1.61 -2.22 C6orf111 chromosome 6 open reading frame 111 -1.62 -1.45 INSIG1 insulin induced gene 1 -1.62 -1.36 SKP1A S-phase kinase-associated protein 1A (p19A) -1.63 -1.79 TTC3 tetratricopeptide repeat domain 3 -1.64 -1.77 ------1.64 -1.58 DAZAP2 DAZ associated protein 2 -1.64 -1.74 USP3 ubiquitin specific peptidase 3 -1.65 -1.42 ELF2 E74-like factor 2 (ets domain transcription factor) -1.66 -1.96 SENP6 SUMO1/sentrin specific peptidase 6 -1.67 -1.80 A kinase (PRKA) anchor protein 13 /// A kinase (PRKA) anchor AKAP13 -1.67 -1.35 protein 13 PI4KII phosphatidylinositol 4-kinase type II -1.67 -1.43 BSDC1 BSD domain containing 1 -1.67 -1.40 RYK RYK receptor-like tyrosine kinase -1.67 -2.01 PPP1R2 protein phosphatase 1, regulatory (inhibitor) subunit 2 -1.67 -1.95 TRAF3IP3 TRAF3 interacting protein 3 -1.69 -1.87 ENTPD4 ectonucleoside triphosphate diphosphohydrolase 4 -1.69 -2.13 RPL10 /// LOC284393 /// ribosomal protein L10 /// similar to ribosomal protein L10 /// LOC285176 /// similar to ribosomal protein L10 /// similar to ribosomal protein LOC389342 /// L10 /// similar to ribosomal protein L10 /// similar to 60S -1.69 -2.24 LOC390364 /// ribosomal protein L10 (QM protein) (Tumor suppressor QM) LOC644039 /// (Laminin LOC647074 HNRPL heterogeneous nuclear ribonucleoprotein L -1.70 -1.44 SFRS5 splicing factor, arginine/serine-rich 5 -1.70 -2.20 FAM8A1 family with sequence similarity 8, member A1 -1.71 -1.89 PDCD4 programmed cell death 4 (neoplastic transformation inhibitor) -1.71 -2.09 SORL1 sortilin-related receptor, L(DLR class) A repeats-containing -1.71 -2.74 RBM38 RNA binding motif protein 38 /// RNA binding motif protein 38 -1.71 -2.01 ZFP36L2 zinc finger protein 36, C3H type-like 2 -1.72 -2.68 LAPTM4A lysosomal-associated protein transmembrane 4 alpha -1.73 -1.58 PIAS1 Protein inhibitor of activated STAT, 1 -1.74 -2.21 PIK3R1 phosphoinositide-3-kinase, regulatory subunit 1 (p85 alpha) -1.74 -2.66 SETD2 SET domain containing 2 -1.75 -2.92 nuclear factor of kappa light polypeptide gene enhancer in B- NFKBIA -1.75 -1.43 cells inhibitor, alpha C8orf60 chromosome 8 open reading frame 60 -1.75 -1.46 ANKRD12 Ankyrin repeat domain 12 -1.76 -1.85 PHF1 /// DLEC1 PHD finger protein 1 /// deleted in lung and esophageal cancer 1 -1.76 -1.71 IDS iduronate 2-sulfatase (Hunter syndrome) -1.76 -2.07 NR4A3 nuclear receptor subfamily 4, group A, member 3 -1.76 -2.29 UBL3 ubiquitin-like 3 -1.76 -2.89 phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 PDE4B -1.77 -1.95 dunce homolog, Drosophila) VAMP3 vesicle-associated membrane protein 3 (cellubrevin) -1.78 -1.45

138 Splicing factor proline/glutamine-rich (polypyrimidine tract SFPQ -1.79 -1.63 binding protein associated) DKFZp586I1420 hypothetical protein DKFZp586I1420 -1.79 -2.15 HLA-E major histocompatibility complex, class I, E -1.80 -1.71 IFNGR1 interferon gamma receptor 1 /// interferon gamma receptor 1 -1.83 -1.89 CTSK cathepsin K (pycnodysostosis) -1.83 -1.25 HGSNAT /// Heparan-alpha-glucosaminide N-acetyltransferase /// similar to -1.84 -1.13 LOC643642 transmembrane protein 76 ZFP36L2 zinc finger protein 36, C3H type-like 2 -1.85 -1.61 splicing factor proline/glutamine-rich (polypyrimidine tract SFPQ -1.85 -1.83 binding protein associated) ZFP36L2 zinc finger protein 36, C3H type-like 2 -1.86 -2.12 KIAA0831 KIAA0831 -1.86 -2.03 Heterogeneous nuclear ribonucleoprotein D (AU-rich element HNRPD -1.87 -1.79 RNA binding protein 1, 37kDa) SH2B3 SH2B adaptor protein 3 -1.88 -1.35 integrin, alpha M (complement component 3 receptor 3 subunit) ITGAM /// integrin, alpha M (complement component 3 receptor 3 -1.88 -1.40 subunit) USP36 ubiquitin specific peptidase 36 -1.90 -1.58 BTG1 B-cell translocation gene 1, anti-proliferative -1.90 -2.32 ferritin, heavy polypeptide pseudogene 1 /// ferritin, heavy FTHP1 -1.90 -1.64 polypeptide pseudogene 1 CLK1 CDC-like kinase 1 -1.90 -1.98 v-maf musculoaponeurotic fibrosarcoma oncogene homolog F MAFF -1.92 -3.04 (avian) SMAD7 SMAD, mothers against DPP homolog 7 (Drosophila) -1.92 -2.44 MOAP1 modulator of apoptosis 1 -1.92 -1.93 CIRBP cold inducible RNA binding protein -1.92 -2.40 TGIF TGFB-induced factor (TALE family homeobox) -1.92 -2.02 GOLGA4 golgi autoantigen, golgin subfamily a, 4 -1.93 -2.52 NACHT, leucine rich repeat and PYD (pyrin domain) NALP1 -1.93 -1.74 containing 1 RNF11 ring finger protein 11 -1.94 -1.93 TUBA3 tubulin, alpha 3 -1.95 -1.15 EIF4A1 Eukaryotic translation initiation factor 4A, isoform 1 -1.95 -1.69 FTH1 ferritin, heavy polypeptide 1 -1.97 -1.33 vesicle-associated membrane protein 3 (cellubrevin) /// vesicle- VAMP3 -1.97 -1.40 associated membrane protein 3 (cellubrevin) C11orf32 chromosome 11 open reading frame 32 -1.97 -2.29 CHD1 chromodomain helicase DNA binding protein 1 -1.98 -2.16 NRIP1 nuclear receptor interacting protein 1 -1.98 -2.03 PTPN12 protein tyrosine phosphatase, non-receptor type 12 -1.99 -1.70 C20orf111 chromosome 20 open reading frame 111 -1.99 -2.37 RIOK3 RIO kinase 3 (yeast) /// RIO kinase 3 (yeast) -1.99 -1.12 ITM2B integral membrane protein 2B -2.00 -1.34 MCL1 myeloid cell leukemia sequence 1 (BCL2-related) -2.01 -1.39 TSC22D1 TSC22 domain family, member 1 -2.02 -2.10 RNASET2 ribonuclease T2 -2.02 -1.26 ZDHHC7 zinc finger, DHHC-type containing 7 -2.02 -1.67

139 RNASET2 ribonuclease T2 -2.05 -1.17 CHMP1B chromatin modifying protein 1B -2.05 -1.70 ------2.05 -1.89 CCNL1 cyclin L1 -2.09 -2.10 RASGRP2 RAS guanyl releasing protein 2 (calcium and DAG-regulated) -2.13 -2.40 HNRPA1 /// heterogeneous nuclear ribonucleoprotein A1 /// hypothetical -2.13 -1.36 LOC644245 protein LOC644245 RNF10 ring finger protein 10 /// ring finger protein 10 -2.14 -1.94 USP3 Ubiquitin specific peptidase 3 -2.17 -3.34 JUN V-jun sarcoma virus 17 oncogene homolog (avian) -2.18 -1.93 Epstein-Barr virus induced gene 2 (lymphocyte-specific G EBI2 -2.22 -1.21 protein-coupled receptor) IFNGR1 interferon gamma receptor 1 -2.22 -1.94 CD44 CD44 molecule (Indian blood group) -2.23 -2.89 LOC54103 hypothetical protein LOC54103 -2.25 -2.44 CUTL1 cut-like 1, CCAAT displacement protein (Drosophila) -2.28 -1.95 BTB and CNC homology 1, basic leucine zipper transcription BACH1 -2.29 -1.47 factor 1 PELI1 pellino homolog 1 (Drosophila) -2.31 -2.07 JUNB jun B proto-oncogene -2.32 -2.91 ZNF394 zinc finger protein 394 -2.34 -2.80 SNN stannin -2.35 -1.57 FAM46C family with sequence similarity 46, member C -2.36 -2.80 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 -2.36 -3.86 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 -2.37 -3.60 GABARAPL1 GABA(A) receptor-associated protein like 1 -2.41 -3.01 SON SON DNA binding protein -2.43 -2.35 ZNF238 zinc finger protein 238 -2.46 -2.33 HIST2H2BE histone 2, H2be -2.46 -2.46 DNAJB6 DnaJ (Hsp40) homolog, subfamily B, member 6 -2.49 -2.40 JMJD1C jumonji domain containing 1C -2.50 -2.60 PTPRE protein tyrosine phosphatase, receptor type, E -2.54 -1.64 ATP2B1 ATPase, Ca++ transporting, plasma membrane 1 -2.55 -2.57 GLIPR1 GLI pathogenesis-related 1 (glioma) -2.56 -1.57 killer cell lectin-like receptor subfamily B, member 1 /// killer KLRB1 -2.57 -3.38 cell lectin-like receptor subfamily B, member 1 MGC17330 HGFL gene /// HGFL gene -2.59 -3.29 AMY1A /// amylase, alpha 1A; salivary /// single-stranded DNA binding -2.62 -2.26 SSBP1 protein 1 B-cell CLL/lymphoma 6 (zinc finger protein 51) /// B-cell BCL6 -2.63 -2.49 CLL/lymphoma 6 (zinc finger protein 51) ASAH1 N-acylsphingosine amidohydrolase (acid ceramidase) 1 -2.70 -1.29 KLF10 Kruppel-like factor 10 -2.70 -1.96 HIST2H2AA3 /// LOC653610 histone 2, H2aa3 /// similar to Histone H2A.o (H2A/o) (H2A.2) -2.72 -3.29 /// (H2a-615) /// histone 2, H2aa4 HIST2H2AA4 IRS2 insulin receptor substrate 2 -2.74 -2.59 EIF1 eukaryotic translation initiation factor 1 -2.77 -2.54 SKP1A S-phase kinase-associated protein 1A (p19A) -2.78 -2.89

140 LOC54103 hypothetical protein LOC54103 -2.78 -2.88 C1orf38 chromosome 1 open reading frame 38 -2.85 -1.19 MYLIP myosin regulatory light chain interacting protein -2.85 -3.18 SAT1 spermidine/spermine N1-acetyltransferase 1 -2.88 -1.55 CD83 CD83 molecule -2.94 -1.52 GABARAPL1 GABA(A) receptor-associated protein like 1 -2.94 -2.21 CKAP4 cytoskeleton-associated protein 4 -3.01 -1.78 GABARAPL1 GABA(A) receptor-associated protein like 1 /// GABA(A) /// -3.05 -3.18 receptors associated protein like 3 GABARAPL3 MGC17330 HGFL gene /// HGFL gene -3.07 -3.92 YPEL5 yippee-like 5 (Drosophila) -3.14 -3.70 ZNF331 zinc finger protein 331 -3.20 -3.96 SGK serum/glucocorticoid regulated kinase -3.22 -1.45 H3F3B H3 histone, family 3B (H3.3B) -3.30 -2.82 LYN v-yes-1 Yamaguchi sarcoma viral related oncogene homolog -3.36 -1.52 SAT1 spermidine/spermine N1-acetyltransferase 1 -3.49 -1.91 APLP2 amyloid beta (A4) precursor-like protein 2 -3.55 -1.29 PSAP /// prosaposin (variant Gaucher disease and variant metachromatic -3.63 -1.31 PLSCR3 leukodystrophy) /// phospholipid scramblase 3 FTH1 ferritin, heavy polypeptide 1 -3.64 -2.22 v-yes-1 Yamaguchi sarcoma viral related oncogene homolog /// LYN -3.69 -1.58 v-yes-1 Yamaguchi sarcoma viral related oncogene homolog CTSS cathepsin S -3.87 -1.21 SAT1 spermidine/spermine N1-acetyltransferase 1 -4.00 -1.33 PSAP /// prosaposin (variant Gaucher disease and variant metachromatic -4.00 -1.73 PLSCR3 leukodystrophy) /// phospholipid scramblase 3 NR4A2 nuclear receptor subfamily 4, group A, member 2 -4.06 -5.60 RGS2 regulator of G-protein signalling 2, 24kDa -4.45 -3.52 TYROBP TYRO protein tyrosine kinase binding protein -4.80 -2.40 DUSP1 dual specificity phosphatase 1 -5.89 -4.98

Appendix B

Gene signature leukemic LGL vs. resting normal cells

Log Ratio Log ratio (Leukemic (Leukemic Gene Symbol Gene Title LGL/Resting LGL/Resting PBMC) CD8) HBB hemoglobin, beta /// hemoglobin, beta 3.75 3.85 HBB hemoglobin, beta 3.27 3.71 HBA2 hemoglobin, alpha 2 /// hemoglobin, alpha 2 3.02 4.18 GPR56 G protein-coupled receptor 56 2.71 1.46 chemokine (C-C motif) ligand 3 /// chemokine (C-C motif) CCL3 /// ligand 3-like 1 /// chemokine (C-C motif) ligand 3-like 3 /// CCL3L1 /// similar to Small inducible cytokine A3-like 1 precursor 2.59 1.57 CCL3L3 /// (Tonsillar lymphocyte LD78 beta protein) (LD78-beta(1-70)) LOC643930 (G0/G1 switch regulat CCL4 chemokine (C-C motif) ligand 4 2.39 1.36 TOX thymus high mobility group box protein TOX 2.39 1.16 IFNG interferon, gamma 2.37 0.84 ZFHX1B zinc finger homeobox 1b 2.21 2.59 solute carrier family 1 (glutamate/neutral amino acid SLC1A4 2.12 2.08 transporter), member 4 killer cell immunoglobulin-like receptor, three domains, long KIR3DL2 2.06 0.96 cytoplasmic tail, 2 TBX21 T-box 21 1.94 1.29 guanylate binding protein 1, interferon-inducible, 67kDa /// GBP1 1.75 1.88 guanylate binding protein 1, interferon-inducible, 67kDa ADRB2 adrenergic, beta-2-, receptor, surface 1.75 0.69 killer cell immunoglobulin-like receptor, three domains, long KIR3DL2 1.71 0.73 cytoplasmic tail, 2 SERPINB9 serpin peptidase inhibitor, clade B (ovalbumin), member 9 1.70 1.18 fatty acid binding protein 5 (psoriasis-associated) /// similar to FABP5 /// Fatty acid-binding protein, epidermal (E-FABP) (Psoriasis- 1.67 1.55 LOC653327 associated fatty acid-binding protein homolog) (PA-FABP) perforin 1 (pore forming protein) /// perforin 1 (pore forming PRF1 1.59 0.73 protein) ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative ENPP4 1.58 0.60 function) FLJ20035 hypothetical protein FLJ20035 1.57 0.69 solute carrier family 1 (glutamate/neutral amino acid SLC1A4 1.57 1.67 transporter), member 4 GIMAP6 GTPase, IMAP family member 6 1.56 0.85 LPIN2 lipin 2 1.52 0.94 GLS glutaminase 1.50 1.29 CX3CR1 chemokine (C-X3-C motif) receptor 1 1.49 1.26 CD81 CD81 molecule 1.46 1.04

142 OPTN optineurin 1.44 0.76 STOM stomatin 1.43 1.11 TANK TRAF family member-associated NFKB activator 1.39 1.99 ZHX2 zinc fingers and homeoboxes 2 1.38 1.06 TBK1 TANK-binding kinase 1 1.37 1.37 FCGR3B Fc fragment of IgG, low affinity IIIb, receptor (CD16b) 1.36 1.91 HSPA1A heat shock 70kDa protein 1A 1.36 1.36 mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N- MGAT2 1.31 0.65 acetylglucosaminyltransferase OASL 2'-5'-oligoadenylate synthetase-like 1.31 1.22 guanylate binding protein 1, interferon-inducible, 67kDa /// GBP1 1.31 1.31 guanylate binding protein 1, interferon-inducible, 67kDa AYTL2 acyltransferase like 2 1.30 1.10 protein phosphatase 2 (formerly 2A), regulatory subunit B (PR PPP2R2B 1.27 0.86 52), beta isoform RAP2A /// RAP2A, member of RAS oncogene family /// RAP2B, 1.27 0.89 RAP2B member of RAS oncogene family STK39 serine threonine kinase 39 (STE20/SPS1 homolog, yeast) 1.26 0.89 integrin, alpha L (antigen CD11A (p180), lymphocyte ITGAL 1.25 0.84 function-associated antigen 1; alpha polypeptide) ARHGAP25 Rho GTPase activating protein 25 1.24 1.05 RYBP RING1 and YY1 binding protein 1.24 0.87 UTP14, U3 small nucleolar ribonucleoprotein, homolog C UTP14C 1.24 0.98 (yeast) SON SON DNA binding protein 1.23 0.81 GOLPH3 golgi phosphoprotein 3 (coat-protein) 1.21 1.38 GABPB2 GA binding protein transcription factor, beta subunit 2 1.20 1.23 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, B4GALT5 1.20 1.70 polypeptide 5 MAN1A1 Mannosidase, alpha, class 1A, member 1 1.19 0.91 SAP30 Sin3A-associated protein, 30kDa 1.18 0.81 TETRAN tetracycline transporter-like protein 1.18 0.87 MAT2B methionine adenosyltransferase II, beta 1.17 0.77 inhibitor of DNA binding 2, dominant negative helix-loop- ID2 /// ID2B helix protein /// inhibitor of DNA binding 2B, dominant 1.16 1.00 negative helix-loop-helix protein RAP2A RAP2A, member of RAS oncogene family 1.16 0.58 matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, MMP9 1.16 1.45 92kDa type IV collagenase) STOM stomatin 1.16 0.72 CTBP2 /// LOC645291 C-terminal binding protein 2 /// similar to C-terminal binding /// protein 2 /// similar to C-terminal binding protein 2 /// similar 1.14 1.58 LOC645508 to C-terminal binding protein 2 /// LOC650999 KIAA1466 KIAA1466 gene 1.14 1.16 SON SON DNA binding protein 1.14 0.73 GRK5 G protein-coupled receptor kinase 5 1.13 0.93 NPC1 Niemann-Pick disease, type C1 1.13 0.69 BAZ1A bromodomain adjacent to zinc finger domain, 1A 1.13 0.77

143 thioredoxin domain containing 9 /// thioredoxin domain TXNDC9 1.13 0.74 containing 9 RECQL RecQ protein-like (DNA helicase Q1-like) 1.11 1.08 STXBP3 syntaxin binding protein 3 1.10 0.81 IRF4 interferon regulatory factor 4 1.10 1.57 ACTR3 ARP3 actin-related protein 3 homolog (yeast) 1.09 0.97 KIAA0240 KIAA0240 1.08 0.68 STAT1 signal transducer and activator of transcription 1, 91kDa 1.08 1.15 IFI16 interferon, gamma-inducible protein 16 1.08 0.89 ARHGEF3 Rho guanine nucleotide exchange factor (GEF) 3 1.07 0.84 LPIN2 lipin 2 1.07 1.06 SP2 Sp2 transcription factor 1.06 0.92 KIAA0196 KIAA0196 1.05 0.85 CRI1 CREBBP/EP300 inhibitor 1 /// CREBBP/EP300 inhibitor 1 1.04 0.84 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, B4GALT5 1.04 1.66 polypeptide 5 UNC50 unc-50 homolog (C. elegans) 1.04 1.03 EPS15 epidermal growth factor receptor pathway substrate 15 1.03 1.06 solute carrier family 25 (mitochondrial carrier; phosphate SLC25A24 1.03 1.58 carrier), member 24 VEZF1 vascular endothelial zinc finger 1 1.03 0.87 restin (Reed-Steinberg cell-expressed intermediate filament- RSN 1.02 1.02 associated protein) CDYL chromodomain protein, Y-like 1.02 1.02 FAS Fas (TNF receptor superfamily, member 6) 1.01 0.73 HMGN4 high mobility group nucleosomal binding domain 4 1.00 1.03 ACADM acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain 1.00 0.74 dullard homolog (Xenopus laevis) /// dullard homolog DULLARD 0.99 1.07 (Xenopus laevis) FLJ22028 hypothetical protein FLJ22028 0.99 0.91 2,3-bisphosphoglycerate mutase /// 2,3-bisphosphoglycerate BPGM 0.98 0.82 mutase TBL2 transducin (beta)-like 2 0.98 0.98 ARID5B AT rich interactive domain 5B (MRF1-like) 0.98 1.36 ARHGAP25 Rho GTPase activating protein 25 0.96 0.78 ABCD3 ATP-binding cassette, sub-family D (ALD), member 3 0.95 1.15 aminoadipate-semialdehyde dehydrogenase- AASDHPPT 0.94 1.33 phosphopantetheinyl transferase HCRP1 hepatocellular carcinoma-related HCRP1 0.92 0.88 acetyl-Coenzyme A acetyltransferase 1 (acetoacetyl ACAT1 0.92 1.07 Coenzyme A thiolase) mediator of RNA polymerase II transcription, subunit 12 MED12 0.92 1.00 homolog (S. cerevisiae) platelet-activating factor acetylhydrolase, isoform Ib, alpha PAFAH1B1 0.91 0.85 subunit 45kDa ZNF294 zinc finger protein 294 0.91 0.82 PDLIM1 PDZ and LIM domain 1 (elfin) 0.89 1.36 IAPP Islet amyloid polypeptide 0.89 1.02 C5orf22 chromosome 5 open reading frame 22 0.88 0.95 SPTAN1 Spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) 0.87 0.90 C1orf156 chromosome 1 open reading frame 156 0.86 0.79

144 UBE2A ubiquitin-conjugating enzyme E2A (RAD6 homolog) 0.86 0.88 ZCCHC6 zinc finger, CCHC domain containing 6 0.86 0.93 CTSC cathepsin C 0.86 1.40 IGF2R insulin-like growth factor 2 receptor 0.85 0.79 C3AR1 complement component 3a receptor 1 0.85 1.25 KRAS v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog 0.84 0.80 SOLH small optic lobes homolog (Drosophila) 0.84 0.84 TRAFD1 TRAF-type zinc finger domain containing 1 0.83 1.02 CHMP5 chromatin modifying protein 5 0.82 0.71 SEC22B SEC22 vesicle trafficking protein homolog B (S. cerevisiae) 0.82 0.80 PCMT1 protein-L-isoaspartate (D-aspartate) O-methyltransferase 0.82 0.81 CSTF3 cleavage stimulation factor, 3' pre-RNA, subunit 3, 77kDa 0.81 0.91 PCF11, cleavage and polyadenylation factor subunit, homolog PCF11 0.81 0.79 (S. cerevisiae) EFHD2 EF-hand domain family, member D2 0.80 0.82 GTF2E1 general transcription factor IIE, polypeptide 1, alpha 56kDa 0.79 0.99 WSB2 WD repeat and SOCS box-containing 2 0.79 0.75 FANCL Fanconi anemia, complementation group L 0.79 0.88 cell division cycle 2-like 5 (cholinesterase-related cell division CDC2L5 0.79 0.64 controller) NAT1 N-acetyltransferase 1 (arylamine N-acetyltransferase) 0.78 0.58 FAS Fas (TNF receptor superfamily, member 6) 0.78 0.68 RAB7L1 RAB7, member RAS oncogene family-like 1 0.78 0.68 UCHL5 ubiquitin carboxyl-terminal hydrolase L5 0.76 0.63 CHMP5 chromatin modifying protein 5 0.74 0.63 solute carrier family 16, member 6 (monocarboxylic acid SLC16A6 0.74 0.96 transporter 7) STAT1 signal transducer and activator of transcription 1, 91kDa 0.72 0.91 FAS Fas (TNF receptor superfamily, member 6) 0.71 0.85 CTBP2 C-terminal binding protein 2 0.71 1.19 SAMD9 sterile alpha motif domain containing 9 0.69 0.79 TM2D1 TM2 domain containing 1 0.69 0.98 FAS Fas (TNF receptor superfamily, member 6) 0.69 0.79 NMI N-myc (and STAT) interactor 0.65 0.73 PCMT1 protein-L-isoaspartate (D-aspartate) O-methyltransferase 0.64 1.19 methylenetetrahydrofolate dehydrogenase (NADP+ MTHFD2 0.63 0.71 dependent) 2, methenyltetrahydrofolate cyclohydrolase NBR1 /// neighbor of BRCA1 gene 1 /// similar to neighbor of BRCA1 0.59 0.63 LOC653347 gene 1 6-Sep septin 6 -0.59 -0.70 solute carrier family 7 (cationic amino acid transporter, y+ SLC7A6 -0.63 -0.65 system), member 6 FAM102A family with sequence similarity 102, member A -0.65 -0.85 SPTBN1 spectrin, beta, non-erythrocytic 1 -0.66 -0.87 HIST1H1C histone 1, H1c -0.68 -1.13 LEPROTL1 leptin receptor overlapping transcript-like 1 -0.69 -1.44 GOLGA8B golgi autoantigen, golgin subfamily a, 8B -0.72 -1.31 HINT1 histidine triad nucleotide binding protein 1 -0.73 -1.17 IFRD1 interferon-related developmental regulator 1 -0.73 -2.26 GABARAPL1 GABA(A) receptor-associated protein like 1 /// GABA(A) -0.74 -1.06

145 /// receptors associated protein like 3 GABARAPL3 JMJD1C jumonji domain containing 1C -0.75 -0.76 C11orf32 chromosome 11 open reading frame 32 -0.75 -1.10 chemokine (C-C motif) receptor 7 /// chemokine (C-C motif) CCR7 -0.76 -1.12 receptor 7 PRKCA protein kinase C, alpha -0.76 -0.72 CD7 CD7 molecule -0.76 -1.32 CREM cAMP responsive element modulator -0.77 -2.07 heat shock protein 90kDa alpha (cytosolic), class B member 1 HSP90AB1 /// heat shock protein 90kDa alpha (cytosolic), class B member -0.77 -0.92 1 FHL1 four and a half LIM domains 1 -0.80 -0.71 EEF1G /// eukaryotic translation elongation factor 1 gamma /// similar to -0.83 -1.04 LOC654007 Elongation factor 1-gamma (EF-1-gamma) (eEF-1B gamma) JUN v-jun sarcoma virus 17 oncogene homolog (avian) -0.83 -2.25 SNF1LK SNF1-like kinase /// SNF1-like kinase -0.84 -1.11 PTP4A1 protein tyrosine phosphatase type IVA, member 1 -0.84 -1.06 BTB and CNC homology 1, basic leucine zipper transcription BACH2 factor 2 /// BTB and CNC homology 1, basic leucine zipper -0.85 -0.99 transcription factor 2 C6orf48 chromosome 6 open reading frame 48 -0.85 -1.10 solute carrier family 7 (cationic amino acid transporter, y+ SLC7A6 -0.86 -1.31 system), member 6 NELL2 NEL-like 2 (chicken) /// NEL-like 2 (chicken) -0.86 -2.35 ABLIM1 actin binding LIM protein 1 -0.87 -1.31 EEF1G /// eukaryotic translation elongation factor 1 gamma /// similar to -0.88 -1.04 LOC654007 Elongation factor 1-gamma (EF-1-gamma) (eEF-1B gamma) MAL mal, T-cell differentiation protein -0.88 -0.77 solute carrier family 2 (facilitated glucose transporter), SLC2A3 -0.88 -0.92 member 3 TRAPPC6A trafficking protein particle complex 6A -0.88 -0.93 DGKA diacylglycerol kinase, alpha 80kDa -0.89 -1.32 RHOH Ras homolog gene family, member H -0.90 -1.26 CD44 /// CD44 molecule (Indian blood group) /// mitogen-activated -0.90 -1.01 MAPK10 protein kinase 10 TMPO thymopoietin -0.90 -1.09 sparc/osteonectin, cwcv and kazal-like domains proteoglycan SPOCK2 -0.92 -1.66 (testican) 2 JUN v-jun sarcoma virus 17 oncogene homolog (avian) -0.92 -2.16 LDHB lactate dehydrogenase B -0.93 -1.35 NR4A3 nuclear receptor subfamily 4, group A, member 3 -0.93 -1.15 JUN V-jun sarcoma virus 17 oncogene homolog (avian) -0.94 -1.58 EEF1G /// eukaryotic translation elongation factor 1 gamma /// similar to -0.95 -1.15 LOC654007 Elongation factor 1-gamma (EF-1-gamma) (eEF-1B gamma) ZFP36L2 zinc finger protein 36, C3H type-like 2 -0.95 -1.63 FLT3LG fms-related tyrosine kinase 3 ligand -0.98 -1.18 GABARAPL1 GABA(A) receptor-associated protein like 1 -0.98 -1.07 special AT-rich sequence binding protein 1 (binds to nuclear SATB1 -0.99 -1.55 matrix/scaffold-associating DNA's) PLXDC1 plexin domain containing 1 -0.99 -1.48

146 IGFBP7 insulin-like growth factor binding protein 7 -0.99 -1.08 MGC17330 HGFL gene /// HGFL gene -0.99 -1.46 ------0.99 -0.87 MGC17330 HGFL gene /// HGFL gene -0.99 -1.72 RPL29 ribosomal protein L29 -1.00 -1.29 C20orf67 chromosome 20 open reading frame 67 -1.00 -1.33 HSP90AB1 heat shock protein 90kDa alpha (cytosolic), class B member 1 -1.01 -1.33 RPL10A ribosomal protein L10a /// ribosomal protein L10a -1.01 -1.44 RSL1D1 ribosomal L1 domain containing 1 -1.02 -1.02 GABARAPL1 GABA(A) receptor-associated protein like 1 -1.04 -0.90 PASK PAS domain containing serine/threonine kinase -1.05 -1.85 YPEL5 yippee-like 5 (Drosophila) -1.05 -2.01 nudE nuclear distribution gene E homolog like 1 (A. nidulans) NDEL1 /// nudE nuclear distribution gene E homolog like 1 (A. -1.06 -0.71 nidulans) RPL18 ribosomal protein L18 /// ribosomal protein L18 -1.08 -1.38 JUNB jun B proto-oncogene -1.08 -1.60 RPL4 ribosomal protein L4 -1.09 -1.44 RPL13 ribosomal protein L13 -1.09 -1.37 RGS10 regulator of G-protein signalling 10 -1.10 -1.17 BTG1 B-cell translocation gene 1, anti-proliferative -1.10 -1.51 RPL4 ribosomal protein L4 /// ribosomal protein L4 -1.11 -1.46 DGKA /// diacylglycerol kinase, alpha 80kDa /// beta-carotene -1.12 -1.07 BCDO2 dioxygenase 2 SORL1 sortilin-related receptor, L(DLR class) A repeats-containing -1.12 -1.61 solute carrier family 2 (facilitated glucose transporter), SLC2A3 -1.12 -1.31 member 3 RPL31 /// ribosomal protein L31 /// similar to ribosomal protein L31 -1.12 -1.01 LOC653773 TCF7 transcription factor 7 (T-cell specific, HMG-box) -1.12 -1.75 RGC32 response gene to complement 32 -1.13 -1.93 RPL15 ribosomal protein L15 -1.14 -1.67 LOC151162 hypothetical protein LOC151162 -1.14 -1.08 solute carrier family 2 (facilitated glucose transporter), SLC2A3 -1.14 -1.01 member 3 RPL24 /// ribosomal protein L24 /// acyl-CoA synthetase medium-chain ACSM3 /// family member 3 /// solute carrier family 36 (proton/amino -1.16 -1.55 SLC36A2 acid symporter), member 2 ------1.17 -1.48 NOSIP nitric oxide synthase interacting protein -1.17 -1.84 LASS6 LAG1 longevity assurance homolog 6 (S. cerevisiae) -1.18 -1.04 JAM3 junctional adhesion molecule 3 -1.19 -1.21 IL18RAP interleukin 18 receptor accessory protein -1.19 -2.27 TBC1D4 TBC1 domain family, member 4 -1.19 -1.21 SERINC5 Serine incorporator 5 -1.19 -1.38 RPS26 /// LOC644166 ribosomal protein S26 /// similar to 40S ribosomal protein S26 -1.20 -1.71 /// /// similar to 40S ribosomal protein S26 LOC644191 DNAJB1 DnaJ (Hsp40) homolog, subfamily B, member 1 -1.20 -2.35

147 MMD monocyte to macrophage differentiation-associated -1.22 -0.79 prion protein (p27-30) (Creutzfeldt-Jakob disease, Gerstmann- PRNP -1.23 -0.83 Strausler-Scheinker syndrome, fatal familial insomnia) RPLP0 /// ribosomal protein, large, P0 /// similar to ribosomal protein P0 -1.25 -1.58 RPLP0-like RPS21 ribosomal protein S21 -1.26 -1.69 RPL29 ribosomal protein L29 -1.26 -1.57 GPRASP1 G protein-coupled receptor associated sorting protein 1 -1.28 -1.27 IMPDH2 IMP (inosine monophosphate) dehydrogenase 2 -1.28 -1.59 INPP4B inositol polyphosphate-4-phosphatase, type II, 105kDa -1.29 -1.80 LSM7 homolog, U6 small nuclear RNA associated (S. LSM7 -1.33 -1.19 cerevisiae) ITGA6 integrin, alpha 6 -1.33 -1.23 BEXL1 brain expressed X-linked-like 1 -1.33 -1.54 HIST2H2AA3 /// histone 2, H2aa3 /// similar to Histone H2A.o (H2A/o) LOC653610 -1.33 -1.80 (H2A.2) (H2a-615) /// histone 2, H2aa4 /// HIST2H2AA4 LEF1 lymphoid enhancer-binding factor 1 -1.36 -1.62 TXK TXK tyrosine kinase -1.37 -2.12 GADD45A growth arrest and DNA-damage-inducible, alpha -1.37 -2.76 phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 PDE4B -1.38 -1.21 dunce homolog, Drosophila) CD28 CD28 molecule -1.38 -1.66 FOS v-fos FBJ murine osteosarcoma viral oncogene homolog -1.42 -2.57 solute carrier family 2 (facilitated glucose transporter), SLC2A3 -1.42 -1.02 member 3 FYB FYN binding protein (FYB-120/130) -1.47 -1.53 NXT1 NTF2-like export factor 1 -1.51 -1.83 ATG16L1 ATG16 autophagy related 16-like 1 (S. cerevisiae) -1.51 -1.47 ------1.52 -1.55 PIK3CA Phosphoinositide-3-kinase, catalytic, alpha polypeptide -1.52 -1.49 tumor necrosis factor receptor superfamily, member 7 /// TNFRSF7 -1.54 -1.98 tumor necrosis factor receptor superfamily, member 7 ZNF331 zinc finger protein 331 -1.56 -2.52 IL7R interleukin 7 receptor /// interleukin 7 receptor -1.61 -2.28 SNRK SNF related kinase -1.64 -1.35 GOLGA4 golgi autoantigen, golgin subfamily a, 4 -1.65 -2.48 IRS2 insulin receptor substrate 2 -1.65 -1.43 STK17B serine/threonine kinase 17b (apoptosis-inducing) -1.67 -1.70 NGFRAP1 nerve growth factor receptor (TNFRSF16) associated protein 1 -1.69 -1.18 LOC202134 hypothetical protein LOC202134 -1.70 -1.47 ------1.74 -2.74 AQP3 aquaporin 3 (Gill blood group) -1.78 -2.35 SKIL SKI-like -1.83 -2.51 IRS2 insulin receptor substrate 2 -1.85 -1.99 granzyme K (granzyme 3; tryptase II) /// granzyme K GZMK -1.90 -3.95 (granzyme 3; tryptase II) phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 PDE4B -1.91 -1.99 dunce homolog, Drosophila)

148 Phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 PDE4B -2.03 -2.50 dunce homolog, Drosophila) phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 PDE4B -2.09 -2.33 dunce homolog, Drosophila) LTB lymphotoxin beta (TNF superfamily, member 3) -2.11 -2.36 inhibitor of DNA binding 1, dominant negative helix-loop- ID1 -2.27 -2.76 helix protein

Appendix C

Gene Signature: Leukemic LGL vs activated normal cells

Log ratio Log ratio (leukemic (leukemic Gene Symbol Gene Title LGL/Ac LGL/Ac PBMC) CD8) DUSP1 dual specificity phosphatase 1 4.64 3.40 v-yes-1 Yamaguchi sarcoma viral related oncogene homolog /// v-yes-1 LYN 3.69 3.61 Yamaguchi sarcoma viral related oncogene homolog TYROBP TYRO protein tyrosine kinase binding protein 3.60 3.34 PSAP /// prosaposin (variant Gaucher disease and variant metachromatic leukodystrophy) 3.35 3.42 PLSCR3 /// phospholipid scramblase 3 v-yes-1 Yamaguchi sarcoma viral related oncogene homolog /// v-yes-1 LYN 3.21 3.09 Yamaguchi sarcoma viral related oncogene homolog TGFBR3 transforming growth factor, beta receptor III (betaglycan, 300kDa) 3.03 2.83 NR4A2 nuclear receptor subfamily 4, group A, member 2 3.02 3.27 RGS2 regulator of G-protein signalling 2, 24kDa 2.96 2.66 SOD2 superoxide dismutase 2, mitochondrial 2.89 2.59 PTPRE protein tyrosine phosphatase, receptor type, E 2.89 3.03 granzyme H (cathepsin G-like 2, protein h-CCPX) /// granzyme H (cathepsin G- GZMH 2.83 2.70 like 2, protein h-CCPX) ZFP36L1 zinc finger protein 36, C3H type-like 1 2.81 1.99 SAT1 spermidine/spermine N1-acetyltransferase 1 2.81 2.65 LYN v-yes-1 Yamaguchi sarcoma viral related oncogene homolog 2.80 2.96 RIOK3 RIO kinase 3 (yeast) /// RIO kinase 3 (yeast) 2.76 2.13 H3F3B H3 histone, family 3B (H3.3B) 2.76 2.47 SAT1 spermidine/spermine N1-acetyltransferase 1 2.75 2.18 APLP2 amyloid beta (A4) precursor-like protein 2 2.74 2.84 SAT1 spermidine/spermine N1-acetyltransferase 1 2.71 2.78 IFI30 interferon, gamma-inducible protein 30 2.70 3.39 FTH1 ferritin, heavy polypeptide 1 2.66 2.88 mitogen-activated protein kinase kinase 3 /// mitogen-activated protein kinase MAP2K3 /// kinase 3 /// similar to mitogen-activated protein kinase kinase 3 isoform A /// 2.64 2.80 LOC651423 similar to mitogen-activated protein kinase kinase 3 isoform A DNAJB6 DnaJ (Hsp40) homolog, subfamily B, member 6 2.58 1.91 FCER1G Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide 2.56 2.35 PSAP /// prosaposin (variant Gaucher disease and variant metachromatic leukodystrophy) 2.56 2.73 PLSCR3 /// phospholipid scramblase 3 HLA-DPA1 major histocompatibility complex, class II, DP alpha 1 2.54 2.59 CTSS cathepsin S 2.52 2.78 FUBP1 Far upstream element (FUSE) binding protein 1 2.51 2.15 SGK serum/glucocorticoid regulated kinase 2.49 2.42 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 2.49 2.75 ASAH1 N-acylsphingosine amidohydrolase (acid ceramidase) 1 2.46 3.04

150 MYLIP myosin regulatory light chain interacting protein 2.46 1.84 LOC54103 hypothetical protein LOC54103 2.46 2.74 CKAP4 cytoskeleton-associated protein 4 2.45 2.37 CTBP2 C-terminal binding protein 2 2.43 2.01 SKP1A S-phase kinase-associated protein 1A (p19A) 2.42 1.79 ------2.41 2.39 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 2.39 2.48 C1orf38 chromosome 1 open reading frame 38 2.38 2.59 GLUL glutamate-ammonia ligase (glutamine synthetase) 2.35 2.14 BCL2A1 BCL2-related protein A1 2.34 2.16 AMY1A /// amylase, alpha 1A; salivary /// single-stranded DNA binding protein 1 2.33 2.07 SSBP1 KLF2 Kruppel-like factor 2 (lung) 2.33 2.83 MXRA7 matrix-remodelling associated 7 2.33 2.04 LOC54103 hypothetical protein LOC54103 2.31 2.66 GABARAPL1 GABA(A) receptor-associated protein like 1 /// GABA(A) receptors associated /// 2.31 2.12 protein like 3 GABARAPL3 SETD2 SET domain containing 2 2.30 2.54 KIAA1109 KIAA1109 2.29 2.02 DKFZp586I1420 hypothetical protein DKFZp586I1420 2.29 2.29 PNRC1 proline-rich nuclear receptor coactivator 1 2.27 1.51 B4GALT5 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 5 2.26 1.84 RNF10 ring finger protein 10 /// ring finger protein 10 2.26 1.90 TGIF TGFB-induced factor (TALE family homeobox) 2.24 2.31 UBL3 ubiquitin-like 3 2.23 2.78 PHF1 /// DLEC1 PHD finger protein 1 /// deleted in lung and esophageal cancer 1 2.22 2.04 ASAH1 N-acylsphingosine amidohydrolase (acid ceramidase) 1 2.22 2.17 CTSK cathepsin K (pycnodysostosis) 2.21 1.85 CRTAM cytotoxic and regulatory T cell molecule 2.20 1.77 TSC22D1 TSC22 domain family, member 1 2.20 2.13 CTSS cathepsin S 2.20 2.46 EIF1 eukaryotic translation initiation factor 1 2.16 1.80 PTPN12 protein tyrosine phosphatase, non-receptor type 12 2.14 2.17 RYK RYK receptor-like tyrosine kinase 2.14 1.80 NPC2 Niemann-Pick disease, type C2 2.13 2.54 PRPF4B PRP4 pre-mRNA processing factor 4 homolog B (yeast) 2.10 2.09 8-Mar membrane-associated ring finger (C3HC4) 8 2.10 1.87 ------2.09 1.97 YPEL5 yippee-like 5 (Drosophila) 2.09 1.69 LITAF lipopolysaccharide-induced TNF factor 2.08 1.92 MGC17330 HGFL gene /// HGFL gene 2.08 2.19 ALOX5 arachidonate 5-lipoxygenase 2.07 2.92 BACH1 BTB and CNC homology 1, basic leucine zipper transcription factor 1 2.07 2.15 LITAF lipopolysaccharide-induced TNF factor 2.07 2.12 HNRPL heterogeneous nuclear ribonucleoprotein L 2.06 1.67 VAV3 vav 3 oncogene 2.06 1.68 LOC284988 hypothetical LOC284988 2.06 2.23 C1orf38 chromosome 1 open reading frame 38 2.06 2.19

151 FAM8A1 family with sequence similarity 8, member A1 2.06 1.92 B-cell CLL/lymphoma 6 (zinc finger protein 51) /// B-cell CLL/lymphoma 6 BCL6 2.06 2.56 (zinc finger protein 51) PPP2R5C Protein phosphatase 2, regulatory subunit B (B56), gamma isoform 2.05 1.92 TOX thymus high mobility group box protein TOX 2.05 2.02 SFRS15 splicing factor, arginine/serine-rich 15 2.05 1.71 INSIG1 insulin induced gene 1 2.05 2.01 TBX21 T-box 21 2.04 2.08 MKRN1 makorin, ring finger protein, 1 2.03 1.51 AIF1 allograft inflammatory factor 1 2.03 2.02 C6orf111 chromosome 6 open reading frame 111 2.02 1.72 PLEK pleckstrin 2.01 2.03 ITM2B integral membrane protein 2B 2.00 1.35 RNF11 ring finger protein 11 1.99 1.98 SON SON DNA binding protein 1.99 1.63 ENPP4 ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) 1.98 2.01 CD83 CD83 molecule 1.98 2.60 ANKRD12 Ankyrin repeat domain 12 1.98 1.74 SMURF2 SMAD specific E3 ubiquitin protein ligase 2 1.97 1.48 USP24 ubiquitin specific peptidase 24 1.96 1.62 CIRBP cold inducible RNA binding protein 1.94 1.89 RHOQ ras homolog gene family, member Q 1.94 1.30 VPS13C vacuolar protein sorting 13 homolog C (S. cerevisiae) 1.93 1.88 TRIM8 tripartite motif-containing 8 /// tripartite motif-containing 8 1.92 1.68 IL15 interleukin 15 1.92 1.72 GTF3C1 general transcription factor IIIC, polypeptide 1, alpha 220kDa 1.91 1.85 RUNX3 runt-related transcription factor 3 1.91 1.54 SERPINB9 serpin peptidase inhibitor, clade B (ovalbumin), member 9 1.90 1.75 FCGR3A /// Fc fragment of IgG, low affinity IIIa, receptor (CD16a) /// Fc fragment of IgG, 1.90 1.34 FCGR3B low affinity IIIb, receptor (CD16b) GABARAPL1 GABA(A) receptor-associated protein like 1 1.90 1.32 NCOA1 nuclear receptor coactivator 1 1.88 1.79 APLP2 amyloid beta (A4) precursor-like protein 2 1.88 1.88 ATP2B1 ATPase, Ca++ transporting, plasma membrane 1 1.86 2.18 NRIP1 nuclear receptor interacting protein 1 1.86 2.14 KIAA0831 KIAA0831 1.86 1.73 PI4KII phosphatidylinositol 4-kinase type II 1.85 1.73 CEBPB CCAAT/enhancer binding protein (C/EBP), beta 1.85 1.54 FBXL5 F-box and leucine-rich repeat protein 5 1.85 1.52 PIAS1 protein inhibitor of activated STAT, 1 1.84 1.53 TAF13 RNA polymerase II, TATA box binding protein (TBP)-associated TAF13 /// factor, 18kDa /// PRP39 pre-mRNA processing factor 39 homolog (S. 1.82 1.49 PRPF39 cerevisiae) SMAD7 SMAD, mothers against DPP homolog 7 (Drosophila) 1.82 1.32 KLF10 Kruppel-like factor 10 1.81 2.11 CSNK1D casein kinase 1, delta 1.81 1.50 RBM38 RNA binding motif protein 38 /// RNA binding motif protein 38 1.81 1.35 IGHMBP2 immunoglobulin mu binding protein 2 1.81 1.80 TMEM1 transmembrane protein 1 1.81 1.73

152 ZNF238 zinc finger protein 238 1.80 2.03 KIR3DL2 killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 2 1.80 1.07 IDS iduronate 2-sulfatase (Hunter syndrome) 1.80 1.66 RRN3 RRN3 RNA polymerase I transcription factor homolog (S. cerevisiae) 1.79 1.08 ATP6V0A1 ATPase, H+ transporting, lysosomal V0 subunit a1 1.79 1.62 ACSL1 acyl-CoA synthetase long-chain family member 1 1.78 2.25 TNFSF13 /// tumor necrosis factor (ligand) superfamily, member 13 /// tumor necrosis factor TNFSF12- 1.78 1.90 (ligand) superfamily, member 12-member 13 TNFSF13 EFHD2 EF-hand domain family, member D2 1.78 1.79 PTBP2 polypyrimidine tract binding protein 2 1.77 1.94 MAPKAPK2 mitogen-activated protein kinase-activated protein kinase 2 1.77 1.60 CLK1 CDC-like kinase 1 1.77 1.07 PPM1B protein phosphatase 1B (formerly 2C), magnesium-dependent, beta isoform 1.77 1.28 RAPGEF6 Rap guanine nucleotide exchange factor (GEF) 6 1.77 1.85 MARCKSL1 MARCKS-like 1 1.77 1.70 PECAM1 platelet/endothelial cell adhesion molecule (CD31 antigen) 1.76 0.90 HLA-F major histocompatibility complex, class I, F 1.75 1.52 JMJD1C jumonji domain containing 1C 1.75 1.84 CROP cisplatin resistance-associated overexpressed protein 1.75 1.67 NKG7 natural killer cell group 7 sequence 1.73 1.15 C2orf17 chromosome 2 open reading frame 17 1.73 1.42 MTMR3 myotubularin related protein 3 1.73 1.54 VCL vinculin 1.73 1.83 KIAA0404 hypothetical protein LOC23130 1.72 1.75 C20orf3 chromosome 20 open reading frame 3 1.72 1.26 RUNX3 runt-related transcription factor 3 1.71 1.35 EIF5 eukaryotic translation initiation factor 5 1.70 1.30 RBM25 RNA binding motif protein 25 1.70 1.74 TSPYL1 TSPY-like 1 1.69 1.34 RABGAP1L RAB GTPase activating protein 1-like 1.69 1.91 VAMP2 vesicle-associated membrane protein 2 (synaptobrevin 2) 1.69 1.73 GLS glutaminase 1.67 1.19 RPS10 /// ribosomal protein S10 /// hypothetical LOC376693 1.67 2.15 LOC376693 NIPBL Nipped-B homolog (Drosophila) 1.67 1.73 BSDC1 BSD domain containing 1 1.67 1.31 RYK RYK receptor-like tyrosine kinase 1.66 1.59 IFNGR1 interferon gamma receptor 1 1.66 1.87 IDS iduronate 2-sulfatase (Hunter syndrome) 1.66 1.51 PBEF1 /// RP11- pre-B-cell colony enhancing factor 1 /// pre-B cell enhancing factor 1 1.66 1.54 92J19.4 pseudogene FUBP1 far upstream element (FUSE) binding protein 1 1.65 1.71 splicing factor proline/glutamine-rich (polypyrimidine tract binding protein SFPQ 1.64 1.15 associated) hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and HERC1 1.64 1.89 RCC1 (CHC1)-like domain (RLD) 1 ILF3 interleukin enhancer binding factor 3, 90kDa 1.64 1.47 VAMP3 vesicle-associated membrane protein 3 (cellubrevin) 1.64 1.60 EP300 E1A binding protein p300 1.64 1.89

153 ZNF331 zinc finger protein 331 1.64 1.43 MKRN1 makorin, ring finger protein, 1 /// makorin, ring finger protein, 1 1.63 1.36 B4GALT1 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 1.63 1.19 FAM46C family with sequence similarity 46, member C 1.63 1.27 B4GALT5 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 5 1.62 1.44 DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked 1.62 1.39 OTUB1 OTU domain, ubiquitin aldehyde binding 1 1.62 1.76 SAFB2 scaffold attachment factor B2 1.62 1.61 protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform /// PPM1A 1.62 1.41 protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, PRKRIR 1.62 1.52 repressor of (P58 repressor) CASP8 caspase 8, apoptosis-related cysteine peptidase 1.61 1.77 C6orf62 chromosome 6 open reading frame 62 1.61 1.71 HOM-TES-103 hypothetical protein LOC25900, isoform 3 1.61 1.52 HIST2H2BE histone 2, H2be 1.61 1.61 TXNDC13 thioredoxin domain containing 13 1.60 1.56 ZNF710 Zinc finger protein 710 1.60 1.10 SFN stratifin 1.60 1.86 CD84 CD84 molecule 1.60 1.68 NECAP1 NECAP endocytosis associated 1 1.60 1.65 PLEK pleckstrin 1.60 1.37 MGC17330 HGFL gene /// HGFL gene 1.60 1.84 CTBP2 C-terminal binding protein 2 1.59 1.63 CBLL1 Cas-Br-M (murine) ecotropic retroviral transforming sequence-like 1 1.59 0.89 OTUD4 OTU domain containing 4 1.59 1.29 HLA-E major histocompatibility complex, class I, E 1.59 1.29 PELI1 pellino homolog 1 (Drosophila) 1.59 1.94 CCL5 chemokine (C-C motif) ligand 5 /// chemokine (C-C motif) ligand 5 1.58 1.04 UBR2 ubiquitin protein ligase E3 component n-recognin 2 1.58 1.80 RB1CC1 RB1-inducible coiled-coil 1 1.58 1.66 GLIPR1 GLI pathogenesis-related 1 (glioma) 1.57 1.33 AKAP13 A kinase (PRKA) anchor protein 13 1.57 1.43 UBP1 upstream binding protein 1 (LBP-1a) 1.57 1.21 BRD1 bromodomain containing 1 1.56 1.37 ELF2 E74-like factor 2 (ets domain transcription factor) 1.56 1.59 MTMR9 myotubularin related protein 9 1.56 1.39 RBM26 RNA binding motif protein 26 1.56 1.39 ITPKB inositol 1,4,5-trisphosphate 3-kinase B 1.56 1.52 nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, NFKBIA 1.55 1.18 alpha HNRPH3 heterogeneous nuclear ribonucleoprotein H3 (2H9) 1.55 1.63 PUM1 pumilio homolog 1 (Drosophila) 1.54 0.93 C14orf138 chromosome 14 open reading frame 138 1.54 1.58 6-Mar membrane-associated ring finger (C3HC4) 6 1.54 1.47 SH3GLB1 SH3-domain GRB2-like endophilin B1 1.54 1.61 SLC16A6 solute carrier family 16, member 6 (monocarboxylic acid transporter 7) 1.54 1.75 FAIM3 Fas apoptotic inhibitory molecule 3 /// Fas apoptotic inhibitory molecule 3 1.54 1.78 FBXO38 F-box protein 38 /// F-box protein 38 1.54 1.30

154 WWP2 WW domain containing E3 ubiquitin protein ligase 2 1.54 1.07 FAM21B /// family with sequence similarity 21, member B /// family with sequence FAM21C /// similarity 21, member C /// similar to KIAA0592 protein /// similar to similar to 1.53 1.56 RP11-56A21.1 KIAA0592 protein /// LOC653450 SGSH N-sulfoglucosamine sulfohydrolase (sulfamidase) 1.53 2.21 NARG1L NMDA receptor regulated 1-like 1.53 1.42 UAP1 UDP-N-acteylglucosamine pyrophosphorylase 1 1.53 1.50 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- GALNT10 1.53 1.14 acetylgalactosaminyltransferase 10 (GalNAc-T10) --- Homo sapiens, clone IMAGE:4214654, mRNA 1.53 2.19 --- CDNA FLJ40810 fis, clone TRACH2009743 1.52 1.65 KIR3DL2 killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 2 1.52 0.98 PIK3R1 phosphoinositide-3-kinase, regulatory subunit 1 (p85 alpha) 1.52 1.14 EP300 E1A binding protein p300 1.52 1.08 RAPGEF2 Rap guanine nucleotide exchange factor (GEF) 2 1.52 1.76 TCIRG1 T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3 1.52 1.39 PCGF3 polycomb group ring finger 3 1.52 1.40 PCF11 PCF11, cleavage and polyadenylation factor subunit, homolog (S. cerevisiae) 1.52 1.41 pleckstrin homology domain containing, family F (with FYVE domain) member PLEKHF2 1.52 1.46 2 CLK4 CDC-like kinase 4 1.52 1.60 PLA2G4B phospholipase A2, group IVB (cytosolic) 1.52 1.31 inhibitor of DNA binding 2, dominant negative helix-loop-helix protein /// ID2 /// ID2B 1.52 0.89 inhibitor of DNA binding 2B, dominant negative helix-loop-helix protein ANKRA2 ankyrin repeat, family A (RFXANK-like), 2 1.52 1.08 MORC3 MORC family CW-type zinc finger 3 1.51 1.04 CHD1 chromodomain helicase DNA binding protein 1 1.51 1.41 killer cell lectin-like receptor subfamily B, member 1 /// killer cell lectin-like KLRB1 1.51 1.43 receptor subfamily B, member 1 DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked 1.51 1.61 WWP1 WW domain containing E3 ubiquitin protein ligase 1 1.51 1.32 GALNACT-2 chondroitin sulfate GalNAcT-2 1.51 1.02 RAP2B RAP2B, member of RAS oncogene family 1.51 1.46 KLRD1 killer cell lectin-like receptor subfamily D, member 1 1.51 1.31 PIAS1 Protein inhibitor of activated STAT, 1 1.50 1.45 SETD1B SET domain containing 1B 1.50 1.75 FMR1 fragile X mental retardation 1 1.50 1.35 RFPL3 ret finger protein-like 3 1.50 1.02 ADIPOR1 adiponectin receptor 1 /// adiponectin receptor 1 1.49 1.21 CSNK1A1 casein kinase 1, alpha 1 1.49 1.44 GALNACT-2 /// chondroitin sulfate GalNAcT-2 /// similar to chondroitin beta1,4 N- 1.49 1.35 LOC644504 acetylgalactosaminyltransferase 2 DAZAP2 DAZ associated protein 2 1.49 1.50 ITM2A integral membrane protein 2A 1.48 1.45 PIK3C2A phosphoinositide-3-kinase, class 2, alpha polypeptide 1.48 1.50 TSPYL4 TSPY-like 4 1.48 1.04 ------1.48 1.10 FBXO11 F-box protein 11 1.47 1.50 RABGGTB Rab geranylgeranyltransferase, beta subunit 1.47 1.15

155 RASSF3 Ras association (RalGDS/AF-6) domain family 3 1.47 1.22 SENP6 SUMO1/sentrin specific peptidase 6 1.47 1.17 HGSNAT /// Heparan-alpha-glucosaminide N-acetyltransferase /// similar to transmembrane 1.47 1.10 LOC643642 protein 76 CUTL1 cut-like 1, CCAAT displacement protein (Drosophila) 1.46 1.30 PKN2 protein kinase N2 1.46 1.37 protein phosphatase 3 (formerly 2B), catalytic subunit, gamma isoform PPP3CC 1.46 1.14 (calcineurin A gamma) KIR3DL2 killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 2 1.46 1.03 PABPN1 poly(A) binding protein, nuclear 1 1.46 1.74 STOM stomatin 1.46 1.26 PRF1 perforin 1 (pore forming protein) /// perforin 1 (pore forming protein) 1.46 1.11 CSNK1G2 casein kinase 1, gamma 2 1.46 1.48 KIAA1718 KIAA1718 protein 1.46 1.80 ------1.46 1.26 PSTPIP2 proline-serine-threonine phosphatase interacting protein 2 1.45 1.10 CD44 CD44 molecule (Indian blood group) 1.45 1.82 UNC84B unc-84 homolog B (C. elegans) 1.45 0.88 PDCD4 programmed cell death 4 (neoplastic transformation inhibitor) 1.44 1.13 C22orf9 chromosome 22 open reading frame 9 1.44 1.72 KLRD1 killer cell lectin-like receptor subfamily D, member 1 1.44 1.38 N-PAC cytokine-like nuclear factor n-pac 1.44 0.98 TBK1 TANK-binding kinase 1 1.44 1.15 YTHDC2 YTH domain containing 2 1.43 1.09 GABARAPL1 GABA(A) receptor-associated protein like 1 1.43 1.94 PAX8 paired box gene 8 1.43 1.42 vacuolar protein sorting 37 homolog B (S. cerevisiae) /// vacuolar protein VPS37B 1.43 1.01 sorting 37 homolog B (S. cerevisiae) LIPA lipase A, lysosomal acid, cholesterol esterase (Wolman disease) 1.43 1.68 IGF2R insulin-like growth factor 2 receptor 1.43 1.35 EPS15 epidermal growth factor receptor pathway substrate 15 1.42 1.35 THUMPD1 THUMP domain containing 1 1.42 1.42 GNS glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) 1.42 1.67 SKP1A S-phase kinase-associated protein 1A (p19A) 1.42 1.16 AKAP13 A kinase (PRKA) anchor protein 13 1.41 1.25 CCNL1 cyclin L1 1.41 0.91 SNN stannin 1.41 1.14 C20orf111 chromosome 20 open reading frame 111 1.41 1.38 HNRPDL Heterogeneous nuclear ribonucleoprotein D-like 1.40 1.35 TANK TRAF family member-associated NFKB activator 1.40 1.39 CYLD cylindromatosis (turban tumor syndrome) 1.40 1.09 NCOA1 nuclear receptor coactivator 1 1.40 1.37 JMJD1B jumonji domain containing 1B 1.40 1.51 NIFUN NifU-like N-terminal domain containing 1.39 0.96 SFRS5 splicing factor, arginine/serine-rich 5 1.39 1.47 KIAA1128 KIAA1128 1.39 1.07 GADD45B growth arrest and DNA-damage-inducible, beta 1.39 1.75 CTSO cathepsin O 1.39 1.76 LAPTM4A lysosomal-associated protein transmembrane 4 alpha 1.39 1.59

156 PC2 (positive cofactor 2, multiprotein complex) glutamine/Q-rich-associated PCQAP 1.39 0.92 protein --- Transcribed locus 1.39 1.26 HIST2H2AA3 /// LOC653610 histone 2, H2aa3 /// similar to Histone H2A.o (H2A/o) (H2A.2) (H2a-615) /// 1.39 1.49 /// histone 2, H2aa4 HIST2H2AA4 SMCHD1 structural maintenance of chromosomes flexible hinge domain containing 1 1.39 1.43 LOC644617 hypothetical LOC644617 /// hypothetical LOC644617 1.39 1.41 RP3-377H14.5 hypothetical protein FLJ35429 1.39 1.21 PGRMC2 progesterone receptor membrane component 2 1.38 1.28 ATP9B ATPase, Class II, type 9B 1.38 1.83 DRAM damage-regulated autophagy modulator 1.38 1.40 ZFAND6 zinc finger, AN1-type domain 6 1.38 1.10 TTC3 tetratricopeptide repeat domain 3 1.38 1.02 ZFP36L2 zinc finger protein 36, C3H type-like 2 1.38 1.53 KIAA0226 KIAA0226 1.38 1.21 GDI1 GDP dissociation inhibitor 1 1.38 1.21 CENTB2 centaurin, beta 2 1.38 0.99 KLHL20 kelch-like 20 (Drosophila) 1.38 2.13 NALP1 NACHT, leucine rich repeat and PYD (pyrin domain) containing 1 1.37 1.35 GAK cyclin G associated kinase 1.37 1.44 UTX ubiquitously transcribed tetratricopeptide repeat, X chromosome 1.37 1.31 FBXO9 F-box protein 9 1.37 1.17 SMURF1 SMAD specific E3 ubiquitin protein ligase 1 1.36 1.90 ZNF394 zinc finger protein 394 1.36 1.25 TSN translin 1.36 1.21 PCTK2 PCTAIRE protein kinase 2 1.36 1.23 TNKS tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 1.36 0.88 FLJ11286 hypothetical protein FLJ11286 1.36 1.11 RBM39 RNA binding motif protein 39 1.36 1.27 GRK5 G protein-coupled receptor kinase 5 1.36 1.18 SCO2 SCO cytochrome oxidase deficient homolog 2 (yeast) 1.35 1.62 IFNGR1 interferon gamma receptor 1 /// interferon gamma receptor 1 1.35 1.85 C6orf111 chromosome 6 open reading frame 111 1.35 1.44 CPD carboxypeptidase D 1.35 1.29 ABCA2 ATP-binding cassette, sub-family A (ABC1), member 2 1.35 1.44 RB1CC1 RB1-inducible coiled-coil 1 1.35 1.45 RPL38 Ribosomal protein L38 1.34 1.18 AGTPBP1 ATP/GTP binding protein 1 1.34 1.21 CDC42EP3 CDC42 effector protein (Rho GTPase binding) 3 1.34 0.95 RNF38 ring finger protein 38 1.34 1.29 RAB5B RAB5B, member RAS oncogene family 1.34 1.30 RNF139 ring finger protein 139 1.34 1.15 PPP1R2 protein phosphatase 1, regulatory (inhibitor) subunit 2 1.33 1.41 ALMS1 Alstrom syndrome 1 1.33 1.42 RAB2 RAB2, member RAS oncogene family 1.33 1.66 ENTPD4 ectonucleoside triphosphate diphosphohydrolase 4 1.33 1.43 SERINC1 serine incorporator 1 1.33 1.19

157 AT rich interactive domain 4B (RBP1- like) /// AT rich interactive domain 4B ARID4B 1.33 1.37 (RBP1- like) VCP valosin-containing protein 1.33 1.50 AMIGO2 adhesion molecule with Ig-like domain 2 1.32 1.32 CENTA1 centaurin, alpha 1 1.32 1.40 similar to mitogen-activated protein kinase kinase 3 isoform A /// similar to LOC651423 1.32 1.64 mitogen-activated protein kinase kinase 3 isoform A RMND5A required for meiotic nuclear division 5 homolog A (S. cerevisiae) 1.32 1.04 C6orf61 chromosome 6 open reading frame 61 1.32 1.04 SH3BGRL SH3 domain binding glutamic acid-rich protein like 1.32 1.47 DERL1 Der1-like domain family, member 1 1.31 0.97 ATP6AP2 ATPase, H+ transporting, lysosomal accessory protein 2 1.31 1.24 ferritin, heavy polypeptide pseudogene 1 /// ferritin, heavy polypeptide FTHP1 1.31 1.93 pseudogene 1 SDCCAG1 serologically defined colon cancer antigen 1 1.31 1.19 MYL4 myosin, light polypeptide 4, alkali; atrial, embryonic 1.31 1.22 SLC2A3P1 Solute carrier family 2 (facilitated glucose transporter), member 3 pseudogene 1 1.30 1.31 MAN2A1 mannosidase, alpha, class 2A, member 1 1.30 1.54 PHF15 PHD finger protein 15 1.30 1.38 ADCY7 adenylate cyclase 7 1.30 1.07 UBE2D1 ubiquitin-conjugating enzyme E2D 1 (UBC4/5 homolog, yeast) 1.30 1.05 ZZEF1 zinc finger, ZZ-type with EF-hand domain 1 1.30 0.91 KIAA0310 KIAA0310 1.30 1.05 PCMTD2 protein-L-isoaspartate (D-aspartate) O-methyltransferase domain containing 2 1.30 1.31 PPP1CB protein phosphatase 1, catalytic subunit, beta isoform 1.29 1.07 VPS4B vacuolar protein sorting 4 homolog B (S. cerevisiae) 1.29 1.61 PRKD3 protein kinase D3 1.29 1.29 FYN FYN oncogene related to SRC, FGR, YES 1.29 1.10 F2R coagulation factor II (thrombin) receptor 1.29 1.10 BRD1 bromodomain containing 1 1.29 1.50 RPL38 ribosomal protein L38 1.28 1.07 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 /// DEAD (Asp-Glu-Ala-Asp) DDX5 1.28 0.99 box polypeptide 5 COQ10B coenzyme Q10 homolog B (S. cerevisiae) 1.28 1.39 KIAA0232 KIAA0232 gene product 1.28 1.44 Splicing factor proline/glutamine-rich (polypyrimidine tract binding protein SFPQ 1.28 0.87 associated) TMEM30A transmembrane protein 30A 1.28 1.36 EIF1B eukaryotic translation initiation factor 1B 1.28 1.54 CREBBP CREB binding protein (Rubinstein-Taybi syndrome) 1.28 1.33 JUND jun D proto-oncogene 1.28 1.06 TMEM2 transmembrane protein 2 1.28 1.32 MX2 myxovirus (influenza virus) resistance 2 (mouse) 1.27 1.50 ASCIZ ATM/ATR-Substrate Chk2-Interacting Zn2+-finger protein 1.27 1.06 DNAJB9 DnaJ (Hsp40) homolog, subfamily B, member 9 1.27 1.24 FLNA filamin A, alpha (actin binding protein 280) 1.27 1.02 LOC220594 TL132 protein 1.27 1.00 ZDHHC7 zinc finger, DHHC-type containing 7 1.27 1.02 ZF HCF-binding transcription factor Zhangfei 1.27 1.53 CTSB cathepsin B 1.27 1.28

158 KLF12 Kruppel-like factor 12 1.27 1.25 TSPAN14 tetraspanin 14 /// tetraspanin 14 1.27 1.22 INSIG1 insulin induced gene 1 1.27 1.11 GIT1 G protein-coupled receptor kinase interactor 1 1.26 1.20 RASGRP2 RAS guanyl releasing protein 2 (calcium and DAG-regulated) 1.26 1.33 LOC222070 hypothetical protein LOC222070 1.26 1.48 PRKCB1 Protein kinase C, beta 1 1.26 1.07 NDUFA5 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5, 13kDa 1.26 1.71 TMEM87A transmembrane protein 87A 1.26 1.18 CENTD1 centaurin, delta 1 1.26 1.06 LDLR low density lipoprotein receptor (familial hypercholesterolemia) 1.26 0.96 integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes ITGB1 1.26 1.56 MDF2, MSK12) CD81 CD81 molecule 1.26 1.10 PSCD1 pleckstrin homology, Sec7 and coiled-coil domains 1(cytohesin 1) 1.26 1.67 ARNTL aryl hydrocarbon receptor nuclear translocator-like 1.26 1.64 CASZ1 castor homolog 1, zinc finger (Drosophila) 1.25 1.21 VAMP1 vesicle-associated membrane protein 1 (synaptobrevin 1) 1.25 1.33 ELF1 E74-like factor 1 (ets domain transcription factor) 1.25 1.28 LONPL Peroxisomal LON protease like 1.25 1.15 FRAT1 frequently rearranged in advanced T-cell lymphomas 1.25 1.27 LAMP2 lysosomal-associated membrane protein 2 1.25 1.14 vesicle-associated membrane protein 3 (cellubrevin) /// vesicle-associated VAMP3 1.25 1.57 membrane protein 3 (cellubrevin) PXN paxillin 1.25 0.96 ATP6V0D1 ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d1 1.25 1.52 CCNT2 cyclin T2 1.25 1.41 SIGIRR single immunoglobulin and toll-interleukin 1 receptor (TIR) domain 1.24 0.94 PARP6 poly (ADP-ribose) polymerase family, member 6 1.24 1.15 SGPL1 sphingosine-1-phosphate lyase 1 1.24 0.87 CALM1 calmodulin 1 (phosphorylase kinase, delta) 1.24 1.31 MEGF9 multiple EGF-like-domains 9 1.24 1.44 matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV MMP9 1.24 2.13 collagenase) ZFAND5 zinc finger, AN1-type domain 5 1.24 1.38 JUNB jun B proto-oncogene 1.23 1.31 TTC17 tetratricopeptide repeat domain 17 1.23 1.28 PCNX pecanex homolog (Drosophila) 1.23 1.37 LAMP1 lysosomal-associated membrane protein 1 1.23 1.31 JMJD3 jumonji domain containing 3 1.23 1.25 Heterogeneous nuclear ribonucleoprotein D (AU-rich element RNA binding HNRPD 1.23 0.90 protein 1, 37kDa) CHKB /// choline kinase beta /// carnitine palmitoyltransferase 1B (muscle) /// Rho CPT1B /// 1.22 0.87 GTPase activating protein 29 ARHGAP29 VEZF1 vascular endothelial zinc finger 1 1.22 1.14 KIAA0143 KIAA0143 protein 1.22 1.27 C11orf32 chromosome 11 open reading frame 32 1.22 1.19 WDR45 WD repeat domain 45 1.22 1.03 ------1.22 1.42

159 ATP6V0C ATPase, H+ transporting, lysosomal 16kDa, V0 subunit c 1.22 1.02 PICALM phosphatidylinositol binding clathrin assembly protein 1.22 1.26 THRAP2 thyroid hormone receptor associated protein 2 1.22 1.37 SLTM SAFB-like, transcription modulator 1.22 1.07 SH2B3 SH2B adaptor protein 3 1.22 1.58 RNF103 ring finger protein 103 1.22 1.90 ATP6AP2 ATPase, H+ transporting, lysosomal accessory protein 2 1.22 1.07 phosphatidylinositol glycan anchor biosynthesis, class A (paroxysmal nocturnal PIGA hemoglobinuria) /// phosphatidylinositol glycan anchor biosynthesis, class A 1.21 1.11 (paroxysmal nocturnal hemoglobinuria) HNRPH1 heterogeneous nuclear ribonucleoprotein H1 (H) 1.21 0.96 CCR1 chemokine (C-C motif) receptor 1 1.21 1.02 RFP2 ret finger protein 2 1.21 1.39 EIF2AK3 eukaryotic translation initiation factor 2-alpha kinase 3 1.21 1.03 BTG1 B-cell translocation gene 1, anti-proliferative 1.21 1.08 ZNF91 zinc finger protein 91 1.21 1.32 VPS13B vacuolar protein sorting 13B (yeast) 1.21 1.15 PAIP1 poly(A) binding protein interacting protein 1 1.21 0.94 ATXN7 ataxin 7 1.20 0.95 DNAJA1 DnaJ (Hsp40) homolog, subfamily A, member 1 1.20 1.28 WDR37 WD repeat domain 37 1.20 1.26 RORA RAR-related orphan receptor A 1.20 1.29 ITSN2 intersectin 2 1.20 1.16 TMEM1 transmembrane protein 1 1.20 1.16 SFRS12 splicing factor, arginine/serine-rich 12 1.20 1.12 CHKB /// choline kinase beta /// carnitine palmitoyltransferase 1B (muscle) /// Rho CPT1B /// 1.19 0.94 GTPase activating protein 29 ARHGAP29 RBM16 RNA binding motif protein 16 1.19 0.92 C6orf111 chromosome 6 open reading frame 111 1.19 1.75 OASL 2'-5'-oligoadenylate synthetase-like 1.19 1.21 MYD88 myeloid differentiation primary response gene (88) 1.19 1.37 ANGEL2 angel homolog 2 (Drosophila) 1.19 0.93 LOC222070 hypothetical protein LOC222070 1.19 1.36 FAM21B /// family with sequence similarity 21, member B /// family with sequence FAM21C /// similarity 21, member C /// similar to KIAA0592 protein /// similar to similar to 1.19 1.23 RP11-56A21.1 KIAA0592 protein /// LOC653450 ATP13A3 ATPase type 13A3 1.19 1.05 PIK3R5 phosphoinositide-3-kinase, regulatory subunit 5, p101 1.18 1.12 TLE4 transducin-like enhancer of split 4 (E(sp1) homolog, Drosophila) 1.18 1.15 heterogeneous nuclear ribonucleoprotein K /// heterogeneous nuclear HNRPK 1.18 0.99 ribonucleoprotein K 6-Mar membrane-associated ring finger (C3HC4) 6 1.18 0.98 FYN FYN oncogene related to SRC, FGR, YES 1.18 1.19 THRAP1 thyroid hormone receptor associated protein 1 1.18 1.16 ZCCHC2 zinc finger, CCHC domain containing 2 1.18 1.07 AIF1 allograft inflammatory factor 1 1.18 0.95 LOC92482 hypothetical protein LOC92482 1.18 0.98 HIST1H2BK histone 1, H2bk 1.18 1.23

160 DGKD diacylglycerol kinase, delta 130kDa 1.18 0.90 KLRG1 killer cell lectin-like receptor subfamily G, member 1 1.17 0.88 ERBB2IP erbb2 interacting protein 1.17 1.07 FBXO21 F-box protein 21 1.17 1.41 RAPGEF2 Rap guanine nucleotide exchange factor (GEF) 2 1.17 1.44 CBX6 chromobox homolog 6 1.17 0.88 TIPARP TCDD-inducible poly(ADP-ribose) polymerase 1.17 0.87 TSC1 tuberous sclerosis 1 1.17 0.89 PFAAP5 phosphonoformate immuno-associated protein 5 1.17 1.40 LOC643287 similar to prothymosin, alpha (gene sequence 28) 1.17 1.46 TAF11 RNA polymerase II, TATA box binding protein (TBP)-associated TAF11 1.17 0.98 factor, 28kDa DEAD (Asp-Glu-Ala-Asp) box polypeptide 21 /// DEAD (Asp-Glu-Ala-Asp) DDX21 1.17 0.96 box polypeptide 21 RPS17 /// ribosomal protein S17 /// similar to 40S ribosomal protein S17 1.16 1.39 LOC402057 PTK9 PTK9 protein tyrosine kinase 9 1.16 1.35 SFRS15 splicing factor, arginine/serine-rich 15 1.16 0.93 hypothetical protein DKFZp566J091 /// hypothetical protein DKFZp566J091 /// LBH /// similar to hypothetical protein DKFZp566J091 /// similar to hypothetical 1.16 1.03 LOC653743 protein DKFZp566J091 NBPF10 neuroblastoma breakpoint family, member 10 1.16 1.43 TMUB2 transmembrane and ubiquitin-like domain containing 2 1.16 1.31 SMCHD1 structural maintenance of chromosomes flexible hinge domain containing 1 1.16 1.07 MAP3K8 mitogen-activated protein kinase kinase kinase 8 1.16 1.09 DVL3 dishevelled, dsh homolog 3 (Drosophila) 1.15 1.04 ATR ataxia telangiectasia and Rad3 related 1.15 1.25 RAB2 RAB2, member RAS oncogene family 1.15 1.05 RNMT RNA (guanine-7-) methyltransferase 1.15 1.31 v-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa RELA 1.15 1.09 light polypeptide gene enhancer in B-cells 3, p65 (avian) ZDHHC17 zinc finger, DHHC-type containing 17 1.15 1.08 H3F3A /// H3 histone, family 3A /// H3 histone, family 3A /// H3 histone, family 3A LOC440926 /// pseudogene /// H3 histone, family 3A pseudogene /// similar to H3 histone, 1.15 1.14 LOC644914 family 3B /// similar to H3 histone, family 3B PFAAP5 Phosphonoformate immuno-associated protein 5 1.15 1.43 NSFL1C NSFL1 (p97) cofactor (p47) 1.15 1.33 EZH1 enhancer of zeste homolog 1 (Drosophila) 1.15 1.38 MMP14 matrix metallopeptidase 14 (membrane-inserted) 1.15 1.20 SETD2 SET domain containing 2 1.14 1.17 NISCH nischarin 1.14 1.26 TRIM33 tripartite motif-containing 33 1.14 1.00 HRB /// HIV-1 Rev binding protein /// region containing hypothetical protein 1.14 1.26 LOC649094 LOC285086; HIV-1 Rev binding protein SIRT7 sirtuin (silent mating type information regulation 2 homolog) 7 (S. cerevisiae) 1.14 0.89 RUTBC1 RUN and TBC1 domain containing 1 1.14 1.23 ------1.14 1.56 protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta PPP2R2B 1.14 1.31 isoform PSCD4 pleckstrin homology, Sec7 and coiled-coil domains 4 1.14 0.97

161 MTSS1 metastasis suppressor 1 1.14 1.00 KIF13B kinesin family member 13B 1.13 1.29 C1QDC1 C1q domain containing 1 1.13 1.01 SNX3 sorting nexin 3 1.13 1.37 PAIP1 poly(A) binding protein interacting protein 1 1.13 1.02 STOM stomatin 1.13 1.00 JARID2 Jumonji, AT rich interactive domain 2 1.13 0.94 CRSP2 cofactor required for Sp1 transcriptional activation, subunit 2, 150kDa 1.13 1.34 USP16 ubiquitin specific peptidase 16 1.13 1.16 HBLD2 HESB like domain containing 2 1.13 0.91 OSTM1 osteopetrosis associated transmembrane protein 1 1.13 1.26 SKIP skeletal muscle and kidney enriched inositol phosphatase 1.13 1.16 MCL1 myeloid cell leukemia sequence 1 (BCL2-related) 1.12 1.17 CNOT2 CCR4-NOT transcription complex, subunit 2 1.12 1.09 MAN2B2 mannosidase, alpha, class 2B, member 2 1.12 1.07 STK19 serine/threonine kinase 19 1.12 1.01 ZNF226 zinc finger protein 226 1.11 0.87 YTHDC1 YTH domain containing 1 1.11 1.30 PFDN5 prefoldin subunit 5 1.11 1.38 ETS2 v-ets erythroblastosis virus E26 oncogene homolog 2 (avian) 1.11 1.16 RBBP6 retinoblastoma binding protein 6 1.11 1.07 ADAM8 ADAM metallopeptidase domain 8 /// ADAM metallopeptidase domain 8 1.11 1.54 BTAF1 RNA polymerase II, B-TFIID transcription factor-associated, 170kDa BTAF1 1.11 1.07 (Mot1 homolog, S. cerevisiae) EFHC2 EF-hand domain (C-terminal) containing 2 1.11 0.93 MYO1F myosin IF 1.11 0.88 Epstein-Barr virus induced gene 2 (lymphocyte-specific G protein-coupled EBI2 1.11 0.88 receptor) FAM21B /// family with sequence similarity 21, member B /// similar to KIAA0592 protein 1.11 0.98 RP11-56A21.1 CREG1 cellular repressor of E1A-stimulated genes 1 1.11 1.23 ZNF318 zinc finger protein 318 1.10 1.39 USP34 ubiquitin specific peptidase 34 1.10 1.36 TRAF3IP3 TRAF3 interacting protein 3 1.10 1.16 HNRPH3 heterogeneous nuclear ribonucleoprotein H3 (2H9) 1.10 1.01 EHD1 EH-domain containing 1 1.10 0.93 ATRX /// alpha thalassemia/mental retardation syndrome X-linked (RAD54 homolog, S. 1.10 1.08 LOC642995 cerevisiae) /// similar to transcriptional regulator ATRX isoform 1 GARNL1 GTPase activating Rap/RanGAP domain-like 1 1.10 1.06 TMEM9B TMEM9 domain family, member B 1.10 1.27 SF3A1 splicing factor 3a, subunit 1, 120kDa 1.10 1.01 IL10RA interleukin 10 receptor, alpha 1.10 0.94 USP3 Ubiquitin specific peptidase 3 1.09 1.49 TP53BP2 tumor protein p53 binding protein, 2 1.09 1.13 SMC5L1 SMC5 structural maintenance of chromosomes 5-like 1 (yeast) 1.09 0.99 LST1 leukocyte specific transcript 1 1.09 1.28 RORA RAR-related orphan receptor A 1.09 0.97 SAR1A SAR1 gene homolog A (S. cerevisiae) 1.09 1.13 BTN2A1 butyrophilin, subfamily 2, member A1 1.09 1.05

162 KIAA0256 /// KIAA0256 gene product /// trafficking protein particle complex 5 1.09 1.40 TRAPPC5 DNAJB6 DnaJ (Hsp40) homolog, subfamily B, member 6 1.08 1.31 IRS2 insulin receptor substrate 2 1.08 1.16 SRRM1 serine/arginine repetitive matrix 1 1.08 0.92 TTC3 tetratricopeptide repeat domain 3 1.08 1.12 PQLC1 PQ loop repeat containing 1 1.08 1.10 RABEP2 /// rabaptin, RAB GTPase binding effector protein 2 /// similar to Rab GTPase 1.08 0.99 LOC652743 binding effector protein 2 (Rabaptin-5beta) RAF1 v-raf-1 murine leukemia viral oncogene homolog 1 1.08 1.19 GALC galactosylceramidase 1.08 0.92 NPC1 Niemann-Pick disease, type C1 1.08 1.44 C12orf41 chromosome 12 open reading frame 41 1.08 1.09 mediator of RNA polymerase II transcription, subunit 31 homolog (S. MED31 1.08 1.07 cerevisiae) RSAD1 radical S-adenosyl methionine domain containing 1 1.07 0.91 protein phosphatase 3 (formerly 2B), catalytic subunit, gamma isoform PPP3CC 1.07 1.21 (calcineurin A gamma) MGLL monoglyceride lipase /// monoglyceride lipase 1.07 2.02 HNRPH3 heterogeneous nuclear ribonucleoprotein H3 (2H9) 1.07 1.13 TPP1 tripeptidyl peptidase I 1.07 0.89 CAPZA2 capping protein (actin filament) muscle Z-line, alpha 2 1.07 0.99 RAB1A RAB1A, member RAS oncogene family 1.07 1.10 PTGER4 prostaglandin E receptor 4 (subtype EP4) 1.06 0.94 ------1.06 1.29 UBE2J1 ubiquitin-conjugating enzyme E2, J1 (UBC6 homolog, yeast) 1.06 1.09 family with sequence similarity 21, member C /// family with sequence FAM21C /// similarity 21, member C /// similar to similar to KIAA0592 protein /// similar to 1.06 1.00 LOC653450 similar to KIAA0592 protein KIAA0226 KIAA0226 1.06 0.95 TMEM87A transmembrane protein 87A 1.06 1.30 RANBP2 RAN binding protein 2 1.06 1.09 integrin, alpha M (complement component 3 receptor 3 subunit) /// integrin, ITGAM 1.06 1.23 alpha M (complement component 3 receptor 3 subunit) RBM39 RNA binding motif protein 39 1.06 1.17 GABARAP GABA(A) receptor-associated protein 1.06 0.92 ATP8B1 ATPase, Class I, type 8B, member 1 1.05 0.93 ELMO2 engulfment and cell motility 2 1.05 0.94 DYRK2 dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 1.05 0.96 ZBTB7A zinc finger and BTB domain containing 7A 1.05 0.96 PARP8 poly (ADP-ribose) polymerase family, member 8 1.05 1.17 DDX24 DEAD (Asp-Glu-Ala-Asp) box polypeptide 24 1.05 0.97 BCLAF1 BCL2-associated transcription factor 1 1.05 0.91 AKAP13 A kinase (PRKA) anchor protein 13 /// A kinase (PRKA) anchor protein 13 1.04 1.15 MINK1 misshapen-like kinase 1 (zebrafish) 1.04 1.23 Full-length cDNA clone CS0DK002YF13 of HeLa cells Cot 25-normalized of --- 1.04 1.10 Homo sapiens (human) ST3GAL5 ST3 beta-galactoside alpha-2,3-sialyltransferase 5 1.04 1.40 UNC84A unc-84 homolog A (C. elegans) 1.04 1.12 KIAA0430 KIAA0430 1.04 1.08

163 KIAA1219 KIAA1219 /// KIAA1219 1.04 1.25 HNRPH2 heterogeneous nuclear ribonucleoprotein H2 (H') 1.04 1.25 SOD2 superoxide dismutase 2, mitochondrial 1.04 1.09 MADD MAP-kinase activating death domain 1.03 1.19 C1orf9 chromosome 1 open reading frame 9 1.03 1.28 C4orf30 chromosome 4 open reading frame 30 1.03 1.08 CHMP1B chromatin modifying protein 1B 1.03 1.01 ARF6 ADP-ribosylation factor 6 1.03 1.18 VPS11 vacuolar protein sorting 11 (yeast) 1.03 0.89 CTSB cathepsin B 1.03 1.50 ATP6AP1 ATPase, H+ transporting, lysosomal accessory protein 1 1.03 1.38 RCN2 reticulocalbin 2, EF-hand calcium binding domain 1.03 0.90 TIA1 TIA1 cytotoxic granule-associated RNA binding protein 1.02 1.06 ZNF500 zinc finger protein 500 1.02 0.98 PRDM2 PR domain containing 2, with ZNF domain 1.02 1.48 GCC2 GRIP and coiled-coil domain containing 2 1.02 1.03 INPP5D inositol polyphosphate-5-phosphatase, 145kDa 1.02 0.91 GSTA1 Glutathione S-transferase A1 1.02 1.50 7-Mar membrane-associated ring finger (C3HC4) 7 1.01 1.03 MTF1 metal-regulatory transcription factor 1 1.01 0.92 USP24 ubiquitin specific peptidase 24 1.01 0.89 EIF1AX eukaryotic translation initiation factor 1A, X-linked 1.01 0.93 XPO6 exportin 6 1.01 1.00 MFSD1 major facilitator superfamily domain containing 1 1.01 1.26 RIOK3 RIO kinase 3 (yeast) /// RIO kinase 3 (yeast) 1.01 1.02 CRSP3 cofactor required for Sp1 transcriptional activation, subunit 3, 130kDa 1.00 1.13 ARS2 ARS2 protein 1.00 0.97 DNAJB14 DnaJ (Hsp40) homolog, subfamily B, member 14 1.00 1.14 TBCC tubulin-specific chaperone c 1.00 0.88 KCTD2 potassium channel tetramerisation domain containing 2 1.00 0.90 --- Clone 23728 mRNA sequence 1.00 0.91 DRD5 dopamine receptor D5 1.00 0.88 EMP3 epithelial membrane protein 3 0.99 1.00 ZF HCF-binding transcription factor Zhangfei 0.99 1.09 CDC2L1 /// cell division cycle 2-like 1 (PITSLRE proteins) /// cell division cycle 2-like 2 0.99 1.02 CDC2L2 (PITSLRE proteins) TXNDC13 thioredoxin domain containing 13 0.99 1.12 RIOK3 RIO kinase 3 (yeast) /// RIO kinase 3 (yeast) 0.99 1.02 IDS iduronate 2-sulfatase (Hunter syndrome) 0.99 0.95 MTMR9 myotubularin related protein 9 0.99 0.87 KIAA0240 KIAA0240 0.99 1.02 AUTS2 autism susceptibility candidate 2 0.99 1.00 EIF3S10 eukaryotic translation initiation factor 3, subunit 10 theta, 150/170kDa 0.99 0.94 DPP8 dipeptidyl-peptidase 8 0.99 1.10 LMBRD1 LMBR1 domain containing 1 0.98 1.29 NOTCH1 Notch homolog 1, translocation-associated (Drosophila) 0.98 0.96 VEZF1 vascular endothelial zinc finger 1 0.98 0.99 PHKB phosphorylase kinase, beta 0.98 1.14 GALNT1 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- 0.98 0.87

164 acetylgalactosaminyltransferase 1 (GalNAc-T1) pleckstrin homology domain containing, family M (with RUN domain) member PLEKHM1 0.98 0.92 1 BTN2A1 butyrophilin, subfamily 2, member A1 0.98 1.02 PLAGL1 pleiomorphic adenoma gene-like 1 0.98 0.99 FNBP4 formin binding protein 4 0.98 1.08 SMAD3 SMAD, mothers against DPP homolog 3 (Drosophila) 0.97 1.10 CHPT1 choline phosphotransferase 1 0.97 0.92 C8orf60 chromosome 8 open reading frame 60 0.97 1.02 UGCG UDP-glucose ceramide glucosyltransferase 0.97 1.02 C14orf11 chromosome 14 open reading frame 11 0.97 0.99 KLF12 Kruppel-like factor 12 0.97 1.13 RBMS1 /// RNA binding motif, single stranded interacting protein 1 /// chromosome 2 open C2orf12 /// reading frame 12 /// region containing chromosome 2 open reading frame 12; 0.97 1.01 LOC648293 RNA binding motif, single stranded interacting protein 1 GMEB2 glucocorticoid modulatory element binding protein 2 0.97 0.94 H2AFV H2A histone family, member V 0.97 0.94 CUL5 cullin 5 0.96 1.10 TXNIP thioredoxin interacting protein 0.96 1.19 WWOX WW domain containing oxidoreductase 0.96 1.09 WIPI2 WD repeat domain, phosphoinositide interacting 2 0.96 0.93 CSDE1 cold shock domain containing E1, RNA-binding 0.96 0.90 GOLGA7 golgi autoantigen, golgin subfamily a, 7 0.96 1.09 BTG2 BTG family, member 2 0.96 1.36 TOB1 transducer of ERBB2, 1 0.96 1.12 PIK3R1 phosphoinositide-3-kinase, regulatory subunit 1 (p85 alpha) 0.95 0.97 TRIM52 tripartite motif-containing 52 0.95 0.99 N4BP1 /// Nedd4 binding protein 1 /// similar to Nedd4 binding protein 1 0.95 1.04 LOC653213 AHNAK AHNAK nucleoprotein (desmoyokin) 0.95 1.42 TRIM22 tripartite motif-containing 22 0.95 0.94 SPEN spen homolog, transcriptional regulator (Drosophila) 0.95 0.98 H3F3B H3 histone, family 3B (H3.3B) 0.95 1.08 NKTR natural killer-tumor recognition sequence 0.95 0.93 FNDC3B fibronectin type III domain containing 3B 0.94 0.91 MAN1A1 Mannosidase, alpha, class 1A, member 1 0.94 1.02 GGA1 golgi associated, gamma adaptin ear containing, ARF binding protein 1 0.94 0.87 GADD45B growth arrest and DNA-damage-inducible, beta 0.94 1.03 FLJ14154 hypothetical protein FLJ14154 0.94 0.94 SEC63 SEC63-like (S. cerevisiae) 0.94 1.05 RNASET2 ribonuclease T2 0.94 1.38 SMCR7L Smith-Magenis syndrome chromosome region, candidate 7-like 0.93 0.91 KIAA0143 KIAA0143 protein 0.93 1.08 family with sequence similarity 108, member A1 /// family with sequence FAM108A1 0.93 1.01 similarity 108, member A1 CPD carboxypeptidase D 0.93 0.96 CTRL chymotrypsin-like 0.93 0.98 MLPH melanophilin 0.93 0.93 ANXA11 annexin A11 0.93 0.89 CHFR checkpoint with forkhead and ring finger domains 0.93 1.15

165 UBE4A ubiquitination factor E4A (UFD2 homolog, yeast) 0.92 0.99 ACSL3 acyl-CoA synthetase long-chain family member 3 0.92 0.94 MSL2L1 male-specific lethal 2-like 1 (Drosophila) 0.92 1.19 PITPNA phosphatidylinositol transfer protein, alpha 0.92 0.96 C6orf32 chromosome 6 open reading frame 32 0.92 1.06 PPP4R1 protein phosphatase 4, regulatory subunit 1 0.92 0.99 HECA headcase homolog (Drosophila) 0.92 0.99 CXCR4 chemokine (C-X-C motif) receptor 4 0.92 1.07 SPAG9 sperm associated antigen 9 0.92 0.93 CDKN1B cyclin-dependent kinase inhibitor 1B (p27, Kip1) 0.91 1.16 PBX2 pre-B-cell leukemia transcription factor 2 0.91 0.93 TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor, TAF7 0.91 0.90 55kDa PSME4 proteasome (prosome, macropain) activator subunit 4 0.91 0.89 LOC162427 hypothetical protein LOC162427 0.91 1.20 IL6ST Interleukin 6 signal transducer (gp130, oncostatin M receptor) 0.91 1.22 ZFAND5 zinc finger, AN1-type domain 5 0.91 1.12 PIAS1 protein inhibitor of activated STAT, 1 0.91 0.89 C19orf22 chromosome 19 open reading frame 22 0.91 0.96 WIRE WIRE protein 0.90 1.07 ANKRD49 ankyrin repeat domain 49 0.90 0.94 PHF8 PHD finger protein 8 0.90 1.12 IRF8 interferon regulatory factor 8 /// interferon regulatory factor 8 0.90 1.23 CD46 CD46 molecule, complement regulatory protein 0.90 1.25 DRD2 dopamine receptor D2 0.90 1.16 PFDN5 prefoldin subunit 5 0.90 1.12 MBD4 methyl-CpG binding domain protein 4 0.90 1.06 VPS4A vacuolar protein sorting 4 homolog A (S. cerevisiae) 0.90 0.94 SAPS3 SAPS domain family, member 3 0.90 0.90 R3HDM2 R3H domain containing 2 0.89 1.15 --- ELISC-1 0.89 0.94 CDC2L5 cell division cycle 2-like 5 (cholinesterase-related cell division controller) 0.89 1.07 FLJ10404 hypothetical protein FLJ10404 0.89 1.12 FBXO7 F-box protein 7 0.89 0.92 --- MRNA; cDNA DKFZp667B0924 (from clone DKFZp667B0924) 0.89 1.01 SIRT3 sirtuin (silent mating type information regulation 2 homolog) 3 (S. cerevisiae) 0.89 0.97 EGFR-coamplified and overexpressed protein /// EGFR-coamplified and ECOP 0.88 0.89 overexpressed protein PARP12 poly (ADP-ribose) polymerase family, member 12 0.88 0.87 ABI1 abl-interactor 1 0.88 1.03 POFUT2 protein O-fucosyltransferase 2 0.88 1.03 AMPD2 adenosine monophosphate deaminase 2 (isoform L) 0.88 0.88 SDCBP syndecan binding protein (syntenin) 0.88 1.19 NXF1 nuclear RNA export factor 1 0.88 0.89 C18orf8 chromosome 18 open reading frame 8 0.88 1.03 SR140 U2-associated SR140 protein 0.87 1.01 LST1 leukocyte specific transcript 1 0.87 0.95 PDLIM1 PDZ and LIM domain 1 (elfin) 0.87 0.95 ZMYM2 zinc finger, MYM-type 2 0.87 0.98

166 RNASET2 ribonuclease T2 0.87 1.21 STX12 syntaxin 12 0.87 1.04 TMBIM1 transmembrane BAX inhibitor motif containing 1 0.87 1.11 ARMC8 armadillo repeat containing 8 0.87 0.97 MZF1 myeloid zinc finger 1 0.86 0.94 DNA segment on chromosome X and Y (unique) 155 expressed sequence, RP13-297E16.1 0.86 0.89 isoform 1 VPS16 vacuolar protein sorting 16 (yeast) 0.86 0.91 TXNIP thioredoxin interacting protein 0.86 1.25 ARHGAP25 Rho GTPase activating protein 25 0.85 1.18 CASP8 caspase 8, apoptosis-related cysteine peptidase 0.85 0.87 EPM2AIP1 EPM2A (laforin) interacting protein 1 0.85 1.57 TIMP1 TIMP metallopeptidase inhibitor 1 0.84 0.94 RABAC1 Rab acceptor 1 (prenylated) 0.84 0.96 DKFZP566N034 Hypothetical protein DKFZp566N034 0.84 1.15 CEP170 centrosomal protein 170kDa 0.84 0.96 USP34 ubiquitin specific peptidase 34 0.84 0.90 KIAA0317 KIAA0317 0.84 1.03 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, YWHAZ 0.83 0.89 zeta polypeptide CXCR4 chemokine (C-X-C motif) receptor 4 0.83 1.07 SYNE2 spectrin repeat containing, nuclear envelope 2 0.83 0.91 YES1 v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 0.83 1.31 ARID4A AT rich interactive domain 4A (RBP1-like) 0.83 1.00 TUFT1 tuftelin 1 0.82 1.07 MOBK1B MOB1, Mps One Binder kinase activator-like 1B (yeast) 0.82 0.97 HLA-DRB1 major histocompatibility complex, class II, DR beta 1 0.82 0.93 WDR48 WD repeat domain 48 0.82 0.94 CNIH4 cornichon homolog 4 (Drosophila) -0.83 -1.00 PREB prolactin regulatory element binding -0.83 -1.20 SUCLA2 succinate-CoA ligase, ADP-forming, beta subunit -0.83 -1.17 DLEU1 /// deleted in lymphocytic leukemia, 1 /// SPANX family, member C -0.83 -0.91 SPANXC TRFP Trf (TATA binding protein-related factor)-proximal homolog (Drosophila) -0.84 -1.02 IL16 interleukin 16 (lymphocyte chemoattractant factor) -0.84 -0.90 EBP emopamil binding protein (sterol isomerase) -0.85 -1.25 FLJ20125 hypothetical protein FLJ20125 -0.85 -0.94 SIP1 survival of motor neuron protein interacting protein 1 -0.85 -0.96 karyopherin alpha 2 (RAG cohort 1, importin alpha 1) /// similar to Importin KPNA2 /// alpha-2 subunit (Karyopherin alpha-2 subunit) (SRP1-alpha) (RAG cohort -0.85 -1.15 LOC643995 protein 1) ME2 /// malic enzyme 2, NAD(+)-dependent, mitochondrial /// protein kinase, cAMP- -0.85 -1.04 PRKAR2B dependent, regulatory, type II, beta PHYH phytanoyl-CoA 2-hydroxylase -0.86 -0.98 RALA v-ral simian leukemia viral oncogene homolog A (ras related) -0.86 -1.15 cyclin-dependent kinase 7 (MO15 homolog, Xenopus laevis, cdk-activating CDK7 -0.86 -1.02 kinase) STCH stress 70 protein chaperone, microsome-associated, 60kDa -0.86 -0.88 FKBP1A FK506 binding protein 1A, 12kDa -0.86 -1.43 ESD esterase D/formylglutathione hydrolase -0.86 -0.93

167 ANP32E acidic (leucine-rich) nuclear phosphoprotein 32 family, member E -0.87 -0.96 GTF2H5 general transcription factor IIH, polypeptide 5 -0.87 -1.10 HTATSF1 HIV-1 Tat specific factor 1 -0.87 -0.96 DNAJC15 DnaJ (Hsp40) homolog, subfamily C, member 15 -0.87 -1.21 lymphocyte cytosolic protein 2 (SH2 domain containing leukocyte protein of LCP2 -0.87 -1.17 76kDa) STYK1 serine/threonine/tyrosine kinase 1 /// serine/threonine/tyrosine kinase 1 -0.87 -1.23 UQCRH ubiquinol-cytochrome c reductase hinge protein -0.87 -0.91 KIAA0746 KIAA0746 protein -0.87 -0.90 SRP72 signal recognition particle 72kDa -0.88 -0.90 CHCHD3 coiled-coil-helix-coiled-coil-helix domain containing 3 -0.88 -1.25 SEPHS1 selenophosphate synthetase 1 -0.88 -0.97 solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), SLC25A5 -0.88 -1.08 member 5 ATP5J ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 -0.88 -0.98 NUCB1 nucleobindin 1 -0.88 -1.15 PRKDC protein kinase, DNA-activated, catalytic polypeptide -0.88 -0.93 DRG1 developmentally regulated GTP binding protein 1 -0.88 -0.88 ACAT1 acetyl-Coenzyme A acetyltransferase 1 (acetoacetyl Coenzyme A thiolase) -0.88 -0.87 HSPC111 hypothetical protein HSPC111 -0.89 -1.46 LRPPRC leucine-rich PPR-motif containing -0.89 -0.96 COQ2 coenzyme Q2 homolog, prenyltransferase (yeast) -0.89 -1.14 GCLM glutamate-cysteine ligase, modifier subunit -0.89 -0.96 DEK DEK oncogene (DNA binding) -0.89 -1.29 RP11-82K18.3 kynurenine aminotransferase III -0.89 -0.97 TES testis derived transcript (3 LIM domains) -0.90 -0.93 AIP aryl hydrocarbon receptor interacting protein -0.90 -1.01 PRPSAP2 phosphoribosyl pyrophosphate synthetase-associated protein 2 -0.90 -0.89 ZNF544 zinc finger protein 544 -0.90 -0.91 DEGS1 degenerative spermatocyte homolog 1, lipid desaturase (Drosophila) -0.90 -1.36 NDUFB11 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 11, 17.3kDa -0.90 -0.94 MT1X metallothionein 1X -0.90 -1.16 ENY2 enhancer of yellow 2 homolog (Drosophila) -0.91 -0.87 C14orf166 chromosome 14 open reading frame 166 -0.91 -0.93 DKC1 dyskeratosis congenita 1, dyskerin -0.91 -0.99 TNFSF5IP1 tumor necrosis factor superfamily, member 5-induced protein 1 -0.91 -1.00 PPM1G protein phosphatase 1G (formerly 2C), magnesium-dependent, gamma isoform -0.91 -1.32 PHLDA1 pleckstrin homology-like domain, family A, member 1 -0.91 -1.30 interleukin enhancer binding factor 2, 45kDa /// interleukin enhancer binding ILF2 -0.91 -1.05 factor 2, 45kDa UBE2V2 ubiquitin-conjugating enzyme E2 variant 2 -0.91 -1.14 NSUN5 NOL1/NOP2/Sun domain family, member 5 -0.91 -0.96 COPS5 COP9 constitutive photomorphogenic homolog subunit 5 (Arabidopsis) -0.91 -1.07 PDHB pyruvate dehydrogenase (lipoamide) beta -0.91 -1.17 CMAS cytidine monophosphate N-acetylneuraminic acid synthetase -0.91 -1.01 CEBPG CCAAT/enhancer binding protein (C/EBP), gamma -0.92 -1.37 GTF2F1 general transcription factor IIF, polypeptide 1, 74kDa -0.92 -1.01 VDAC3 voltage-dependent anion channel 3 -0.92 -0.92 BET1 BET1 homolog (S. cerevisiae) -0.93 -1.10

168 AHCYL1 S-adenosylhomocysteine hydrolase-like 1 -0.93 -1.10 C14orf130 chromosome 14 open reading frame 130 -0.93 -1.13 C21orf59 chromosome 21 open reading frame 59 -0.93 -0.93 MOCS2 molybdenum cofactor synthesis 2 -0.93 -1.04 TRAPPC3 trafficking protein particle complex 3 -0.93 -0.97 SNX1 sorting nexin 1 -0.94 -0.89 SYNCRIP synaptotagmin binding, cytoplasmic RNA interacting protein -0.94 -1.12 HDHD1A haloacid dehalogenase-like hydrolase domain containing 1A -0.94 -0.94 TPRKB TP53RK binding protein -0.94 -1.16 COX5B cytochrome c oxidase subunit Vb /// cytochrome c oxidase subunit Vb -0.94 -1.11 vesicle-associated membrane protein 4 /// vesicle-associated membrane protein VAMP4 -0.94 -0.93 4 LRPPRC leucine-rich PPR-motif containing /// leucine-rich PPR-motif containing -0.94 -0.90 C19orf7 chromosome 19 open reading frame 7 -0.95 -0.98 PARP2 poly (ADP-ribose) polymerase family, member 2 -0.95 -0.91 LMAN2 lectin, mannose-binding 2 -0.95 -1.09 HDGFRP3 hepatoma-derived growth factor, related protein 3 -0.95 -1.36 YIF1A Yip1 interacting factor homolog A (S. cerevisiae) -0.95 -0.90 YME1L1 YME1-like 1 (S. cerevisiae) -0.95 -1.10 PRIM2A primase, polypeptide 2A, 58kDa -0.95 -0.91 RAB5A RAB5A, member RAS oncogene family -0.95 -0.97 DNAPTP6 DNA polymerase-transactivated protein 6 -0.96 -0.98 TXNL4A thioredoxin-like 4A -0.96 -0.96 solute carrier family 3 (activators of dibasic and neutral amino acid transport), SLC3A2 -0.96 -1.24 member 2 PRKDC protein kinase, DNA-activated, catalytic polypeptide -0.96 -1.23 DDOST dolichyl-diphosphooligosaccharide-protein glycosyltransferase -0.96 -0.97 RAD23B RAD23 homolog B (S. cerevisiae) -0.96 -0.88 PDIA6 protein disulfide isomerase family A, member 6 -0.96 -0.99 D15Wsu75e DNA segment, Chr 15, Wayne State University 75, expressed -0.96 -1.26 CCDC85B coiled-coil domain containing 85B -0.97 -0.93 CEP57 centrosomal protein 57kDa -0.97 -1.22 POT1 POT1 protection of telomeres 1 homolog (S. pombe) -0.97 -1.04 UGP2 UDP-glucose pyrophosphorylase 2 -0.97 -0.98 WDR68 WD repeat domain 68 -0.97 -1.03 NFYB nuclear transcription factor Y, beta -0.97 -1.01 NARG2 NMDA receptor regulated 2 -0.97 -1.11 HSPA4 heat shock 70kDa protein 4 -0.97 -0.88 STAMBP STAM binding protein -0.97 -1.08 ASMTL acetylserotonin O-methyltransferase-like -0.98 -0.91 PHB2 prohibitin 2 -0.98 -1.00 PSMC5 proteasome (prosome, macropain) 26S subunit, ATPase, 5 -0.98 -0.99 TCEA1 transcription elongation factor A (SII), 1 -0.98 -0.93 TMOD3 tropomodulin 3 (ubiquitous) -0.98 -1.40 PRDX4 peroxiredoxin 4 -0.98 -1.04 GYPC glycophorin C (Gerbich blood group) -0.98 -1.10 CLTA clathrin, light polypeptide (Lca) -0.99 -1.20 VIM vimentin -0.99 -1.16 TIPRL TIP41, TOR signalling pathway regulator-like (S. cerevisiae) -0.99 -1.07

169 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit ATP5G3 -0.99 -1.08 9) CTPS CTP synthase -0.99 -1.13 NDUFB1 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7kDa -0.99 -1.02 DCK deoxycytidine kinase -0.99 -1.19 TIMM8B translocase of inner mitochondrial membrane 8 homolog B (yeast) -0.99 -0.96 eukaryotic translation initiation factor 4A, isoform 1 /// eukaryotic translation EIF4A1 -0.99 -1.01 initiation factor 4A, isoform 1 GNL3 guanine nucleotide binding protein-like 3 (nucleolar) -0.99 -0.92 LDHB lactate dehydrogenase B -0.99 -1.09 IDH3A isocitrate dehydrogenase 3 (NAD+) alpha -0.99 -1.08 KIF22 kinesin family member 22 -0.99 -1.16 DNM1L dynamin 1-like -1.00 -0.97 GRHPR glyoxylate reductase/hydroxypyruvate reductase -1.00 -0.97 X-ray repair complementing defective repair in Chinese hamster cells 5 XRCC5 -1.00 -0.97 (double-strand-break rejoining; Ku autoantigen, 80kDa) ------1.00 -1.17 FAS Fas (TNF receptor superfamily, member 6) -1.00 -1.10 UEVLD UEV and lactate/malate dehyrogenase domains -1.00 -1.04 RRAS2 related RAS viral (r-ras) oncogene homolog 2 -1.00 -0.95 KIAA0020 KIAA0020 -1.00 -0.89 LRP8 low density lipoprotein receptor-related protein 8, apolipoprotein e receptor -1.00 -0.96 YME1L1 YME1-like 1 (S. cerevisiae) -1.00 -1.00 GOLGA8G /// GOLGA8D /// golgi autoantigen, golgin subfamily a, 8G /// golgi autoantigen, golgin LOC388189 /// subfamily a, 8D /// similar to golgi autoantigen, golgin family member /// golgi -1.00 -1.17 GOLGA8E /// autoantigen, golgin subfamily a, 8E /// golgi autoantigen, golgin subfamily a, GOLGA8C /// 8C /// golgi autoantige GOLGA8F OAS3 2'-5'-oligoadenylate synthetase 3, 100kDa -1.00 -1.36 MDH2 malate dehydrogenase 2, NAD (mitochondrial) -1.01 -1.08 ANP32B acidic (leucine-rich) nuclear phosphoprotein 32 family, member B -1.01 -1.02 HSPA8 heat shock 70kDa protein 8 -1.01 -0.95 CAND1 cullin-associated and neddylation-dissociated 1 -1.01 -1.19 PTBP1 polypyrimidine tract binding protein 1 -1.01 -1.24 XPNPEP1 X-prolyl aminopeptidase (aminopeptidase P) 1, soluble -1.01 -1.18 TRAPPC6A trafficking protein particle complex 6A -1.02 -0.96 TCEB1 transcription elongation factor B (SIII), polypeptide 1 (15kDa, elongin C) -1.02 -1.08 TNFAIP8 tumor necrosis factor, alpha-induced protein 8 -1.02 -1.10 CD2 CD2 molecule /// CD2 molecule -1.02 -1.04 TTF1 transcription termination factor, RNA polymerase I -1.02 -1.00 WDR77 WD repeat domain 77 -1.02 -1.24 CREB1 cAMP responsive element binding protein 1 -1.02 -1.25 CCNG1 cyclin G1 -1.02 -0.93 PSMA2 proteasome (prosome, macropain) subunit, alpha type, 2 -1.03 -0.92 CREM cAMP responsive element modulator -1.03 -0.90 RFK riboflavin kinase -1.03 -1.01 NUCB2 nucleobindin 2 -1.03 -1.71 C6orf130 chromosome 6 open reading frame 130 -1.03 -0.94 MSH2 mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) -1.03 -1.35

170 CRYBB2 crystallin, beta B2 -1.03 -1.24 proteasome (prosome, macropain) 26S subunit, ATPase, 4 /// similar to 26S PSMC4 /// protease regulatory subunit 6B (MIP224) (MB67-interacting protein) (TAT- -1.04 -1.27 LOC652826 binding protein 7) (TBP-7) PARP1 poly (ADP-ribose) polymerase family, member 1 -1.04 -0.88 MRPS31 mitochondrial ribosomal protein S31 -1.04 -1.50 RPS26 /// ribosomal protein S26 /// similar to 40S ribosomal protein S26 /// similar to 40S LOC644166 /// -1.04 -1.02 ribosomal protein S26 LOC644191 PTPN7 protein tyrosine phosphatase, non-receptor type 7 -1.04 -1.26 PTGER2 prostaglandin E receptor 2 (subtype EP2), 53kDa -1.04 -1.30 NCF4 neutrophil cytosolic factor 4, 40kDa -1.04 -1.12 CTNNAL1 catenin (cadherin-associated protein), alpha-like 1 -1.05 -0.98 VBP1 von Hippel-Lindau binding protein 1 -1.05 -1.17 MRPS18B mitochondrial ribosomal protein S18B -1.05 -0.90 TRAPPC2 /// trafficking protein particle complex 2 /// spondyloepiphyseal dysplasia, late, -1.05 -1.15 SEDLP pseudogene ACAT2 acetyl-Coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme A thiolase) -1.05 -1.02 CEP57 centrosomal protein 57kDa -1.05 -0.99 ------1.05 -0.94 DEF6 differentially expressed in FDCP 6 homolog (mouse) -1.05 -1.19 PPAP2A phosphatidic acid phosphatase type 2A -1.05 -1.66 MED6 mediator of RNA polymerase II transcription, subunit 6 homolog (S. cerevisiae) -1.06 -1.03 SUZ12 suppressor of zeste 12 homolog (Drosophila) -1.06 -1.11 CREB1 cAMP responsive element binding protein 1 -1.06 -1.11 C18orf1 chromosome 18 open reading frame 1 -1.06 -1.08 LGALS8 lectin, galactoside-binding, soluble, 8 (galectin 8) -1.06 -1.13 PSMA1 proteasome (prosome, macropain) subunit, alpha type, 1 -1.06 -0.89 ADH5 alcohol dehydrogenase 5 (class III), chi polypeptide -1.06 -1.23 NUTF2 nuclear transport factor 2 -1.07 -1.01 RAB27A RAB27A, member RAS oncogene family -1.07 -1.25 TXNRD1 thioredoxin reductase 1 -1.07 -1.12 CCT6A chaperonin containing TCP1, subunit 6A (zeta 1) -1.07 -0.95 uridine monophosphate synthetase (orotate phosphoribosyl transferase and UMPS -1.07 -1.19 orotidine-5'-decarboxylase) MT1F metallothionein 1F (functional) -1.07 -1.17 IL2RB interleukin 2 receptor, beta /// interleukin 2 receptor, beta -1.07 -1.20 GIMAP6 GTPase, IMAP family member 6 -1.07 -1.14 GOLGA8B golgi autoantigen, golgin subfamily a, 8B -1.07 -1.19 CEP63 centrosomal protein 63kDa -1.07 -1.04 ROD1 ROD1 regulator of differentiation 1 (S. pombe) -1.07 -1.13 SLBP stem-loop (histone) binding protein -1.07 -1.03 ACY1 aminoacylase 1 -1.07 -1.13 DMC1 dosage suppressor of mck1 homolog, meiosis-specific homologous DMC1 -1.07 -1.39 recombination (yeast) NUP85 nucleoporin 85kDa -1.07 -0.99 AURKAIP1 /// aurora kinase A interacting protein 1 /// similar to Aurora kinase A-interacting -1.07 -0.98 LOC643556 protein (AURKA-interacting protein) CCR7 chemokine (C-C motif) receptor 7 /// chemokine (C-C motif) receptor 7 -1.08 -0.99 FDFT1 farnesyl-diphosphate farnesyltransferase 1 -1.08 -1.00

171 SMC3 structural maintenance of chromosomes 3 -1.08 -1.18 EMG1 EMG1 nucleolar protein homolog (S. cerevisiae) -1.08 -1.14 FURIN furin (paired basic amino acid cleaving enzyme) -1.08 -1.40 PGM1 phosphoglucomutase 1 -1.08 -0.93 PSIP1 PC4 and SFRS1 interacting protein 1 -1.09 -1.10 SIVA CD27-binding (Siva) protein -1.09 -1.15 SS18 synovial sarcoma translocation, chromosome 18 -1.09 -1.17 PRSS15 protease, serine, 15 -1.09 -0.90 HNRPC heterogeneous nuclear ribonucleoprotein C (C1/C2) -1.09 -0.89 C1orf25 chromosome 1 open reading frame 25 /// chromosome 1 open reading frame 25 -1.09 -0.97 IL18RAP interleukin 18 receptor accessory protein -1.09 -1.42 ETFDH electron-transferring-flavoprotein dehydrogenase -1.09 -0.98 IPO7 importin 7 -1.09 -1.01 C11orf48 chromosome 11 open reading frame 48 -1.09 -1.29 DARS aspartyl-tRNA synthetase -1.09 -1.41 C6orf96 chromosome 6 open reading frame 96 -1.09 -1.36 PRPF4 PRP4 pre-mRNA processing factor 4 homolog (yeast) -1.09 -1.09 GPR18 G protein-coupled receptor 18 -1.09 -1.17 LARS2 leucyl-tRNA synthetase 2, mitochondrial -1.09 -0.95 C5orf13 chromosome 5 open reading frame 13 -1.09 -1.07 ARL3 ADP-ribosylation factor-like 3 -1.10 -1.11 TMEM39B transmembrane protein 39B -1.10 -1.21 dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate DLST -1.10 -1.33 complex) CCDC51 coiled-coil domain containing 51 -1.10 -0.94 GANAB glucosidase, alpha; neutral AB -1.10 -1.24 MT1M metallothionein 1M -1.10 -0.98 TAGLN2 transgelin 2 -1.11 -0.93 APIP APAF1 interacting protein -1.11 -1.43 DNAJC9 DnaJ (Hsp40) homolog, subfamily C, member 9 -1.11 -1.48 HSPA8 heat shock 70kDa protein 8 -1.11 -1.08 MRPS17 mitochondrial ribosomal protein S17 -1.11 -1.11 COX8A cytochrome c oxidase subunit 8A (ubiquitous) -1.11 -0.94 integrin, alpha E (antigen CD103, human mucosal lymphocyte antigen 1; alpha ITGAE -1.11 -1.20 polypeptide) HNRPA3P1 /// heterogeneous nuclear ribonucleoprotein A3 pseudogene 1 /// heterogeneous HNRPA3 /// nuclear ribonucleoprotein A3 /// heterogeneous nuclear ribonucleoprotein A3 -1.11 -1.12 LOC643689 /// pseudogene /// similar to heterogeneous nuclear ribonucleoprotein A3 LOC647474 uridine monophosphate synthetase (orotate phosphoribosyl transferase and UMPS -1.11 -1.17 orotidine-5'-decarboxylase) PDCD6 programmed cell death 6 -1.11 -1.05 FAM96B family with sequence similarity 96, member B -1.11 -1.03 SRI sorcin -1.11 -1.20 RAB11FIP1 RAB11 family interacting protein 1 (class I) -1.11 -1.06 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1 /// ATP ATP5F1 -1.11 -1.18 synthase, H+ transporting, mitochondrial F0 complex, subunit B1 MTF2 metal response element binding transcription factor 2 -1.11 -1.20 KARS lysyl-tRNA synthetase /// lysyl-tRNA synthetase -1.11 -0.93 NDUFV1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa -1.12 -1.13

172 ING2 inhibitor of growth family, member 2 -1.12 -1.00 PSMA1 proteasome (prosome, macropain) subunit, alpha type, 1 -1.12 -0.88 IL2RG interleukin 2 receptor, gamma (severe combined immunodeficiency) -1.12 -1.25 ORC5L origin recognition complex, subunit 5-like (yeast) -1.12 -1.05 PSIP1 PC4 and SFRS1 interacting protein 1 -1.12 -1.13 C10orf61 chromosome 10 open reading frame 61 -1.12 -1.22 --- CDNA clone IMAGE:4842353 -1.12 -1.39 C17orf75 chromosome 17 open reading frame 75 -1.12 -1.17 MRPS31 mitochondrial ribosomal protein S31 -1.13 -1.45 CEP57 centrosomal protein 57kDa -1.13 -1.21 LCK lymphocyte-specific protein tyrosine kinase -1.13 -1.04 CCT2 chaperonin containing TCP1, subunit 2 (beta) -1.13 -1.23 APEX1 APEX nuclease (multifunctional DNA repair enzyme) 1 -1.13 -0.95 PPIE peptidylprolyl isomerase E (cyclophilin E) -1.13 -1.24 HNRPA2B1 heterogeneous nuclear ribonucleoprotein A2/B1 -1.13 -1.15 C6orf211 chromosome 6 open reading frame 211 -1.13 -1.29 histidine triad nucleotide binding protein 1 /// histidine triad nucleotide binding HINT1 -1.13 -0.99 protein 1 C4orf27 chromosome 4 open reading frame 27 -1.13 -1.35 MRPS28 mitochondrial ribosomal protein S28 -1.13 -1.05 phosphoglycerate mutase 1 (brain) /// similar to Phosphoglycerate mutase 1 PGAM1 /// (Phosphoglycerate mutase isozyme B) (PGAM-B) (BPG-dependent PGAM 1) LOC642969 /// -1.13 -0.94 /// similar to Phosphoglycerate mutase 1 (Phosphoglycerate mutase isozyme B) LOC643576 (PGAM-B) (BPG-dependent PGAM 1) MLH1 mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) -1.13 -1.11 SFXN1 sideroflexin 1 -1.14 -1.40 GPKOW G patch domain and KOW motifs -1.14 -1.07 TXN thioredoxin -1.14 -1.03 CLTA clathrin, light polypeptide (Lca) -1.14 -1.27 GTF3A general transcription factor IIIA -1.14 -0.97 RER1 RER1 retention in endoplasmic reticulum 1 homolog (S. cerevisiae) -1.14 -0.89 CSE1L CSE1 chromosome segregation 1-like (yeast) -1.14 -1.13 PGAP1 GPI deacylase -1.14 -1.50 TMEM97 transmembrane protein 97 -1.15 -1.46 PGK1 phosphoglycerate kinase 1 -1.15 -1.08 DGKA /// diacylglycerol kinase, alpha 80kDa /// beta-carotene dioxygenase 2 -1.15 -1.08 BCDO2 SNAP29 synaptosomal-associated protein, 29kDa -1.15 -0.97 RPA1 replication protein A1, 70kDa -1.15 -1.15 PECI peroxisomal D3,D2-enoyl-CoA isomerase -1.15 -1.27 MT1F metallothionein 1F (functional) -1.15 -1.32 EIF3S8 /// eukaryotic translation initiation factor 3, subunit 8, 110kDa /// similar to -1.15 -0.97 LOC653352 eukaryotic translation initiation factor 3, subunit 8 ALDOA aldolase A, fructose-bisphosphate -1.15 -1.03 UTP6 UTP6, small subunit (SSU) processome component, homolog (yeast) -1.16 -0.89 HADH2 hydroxyacyl-Coenzyme A dehydrogenase, type II -1.16 -0.91 CYC1 cytochrome c-1 -1.16 -1.09 HMGB2 high-mobility group box 2 -1.16 -1.39 CHMP5 chromatin modifying protein 5 -1.16 -0.95 C19orf10 chromosome 19 open reading frame 10 -1.16 -0.93

173 PGK1 phosphoglycerate kinase 1 -1.16 -1.13 ATPIF1 ATPase inhibitory factor 1 -1.16 -1.15 TMEM106B transmembrane protein 106B -1.16 -1.32 CDC27 cell division cycle 27 -1.16 -1.09 PRPS1 phosphoribosyl pyrophosphate synthetase 1 -1.17 -1.21 GSTO1 glutathione S-transferase omega 1 -1.17 -1.05 CDC37 CDC37 cell division cycle 37 homolog (S. cerevisiae) -1.17 -1.11 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide ATP5C1 -1.17 -1.05 1 KARS lysyl-tRNA synthetase -1.17 -1.04 SKP2 S-phase kinase-associated protein 2 (p45) -1.17 -1.43 NDUFB6 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6, 17kDa -1.17 -1.28 HSPA4 heat shock 70kDa protein 4 -1.17 -1.05 METAP2 methionyl aminopeptidase 2 -1.18 -1.15 CASP3 caspase 3, apoptosis-related cysteine peptidase -1.18 -1.12 ACTR3 ARP3 actin-related protein 3 homolog (yeast) -1.18 -1.28 PDHA1 pyruvate dehydrogenase (lipoamide) alpha 1 -1.18 -1.19 NDUFV2 NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa -1.18 -1.17 ACADM acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain -1.18 -1.30 DDX48 DEAD (Asp-Glu-Ala-Asp) box polypeptide 48 -1.19 -1.42 CACYBP calcyclin binding protein /// calcyclin binding protein -1.19 -0.95 PMVK phosphomevalonate kinase -1.19 -1.38 TXNL2 thioredoxin-like 2 -1.19 -1.18 PRR11 proline rich 11 -1.19 -1.07 NT5C 5', 3'-nucleotidase, cytosolic -1.19 -1.31 IFI16 interferon, gamma-inducible protein 16 -1.19 -1.13 NUP107 nucleoporin 107kDa -1.19 -0.99 ESD esterase D/formylglutathione hydrolase -1.19 -1.37 ORMDL2 ORM1-like 2 (S. cerevisiae) -1.19 -1.04 CCT6A chaperonin containing TCP1, subunit 6A (zeta 1) -1.19 -0.88 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, SMARCA3 -1.19 -1.31 subfamily a, member 3 CYB5B cytochrome b5 type B (outer mitochondrial membrane) -1.19 -1.21 SAP30 Sin3A-associated protein, 30kDa -1.19 -1.30 HSD17B12 hydroxysteroid (17-beta) dehydrogenase 12 -1.19 -1.07 INPP4A inositol polyphosphate-4-phosphatase, type I, 107kDa -1.20 -1.28 ACYP1 acylphosphatase 1, erythrocyte (common) type -1.20 -1.05 ARMC1 armadillo repeat containing 1 -1.20 -1.09 NEDD9 neural precursor cell expressed, developmentally down-regulated 9 -1.20 -1.22 RPLP0 ribosomal protein, large, P0 -1.20 -0.93 MRPL18 mitochondrial ribosomal protein L18 -1.20 -1.39 GADD45A growth arrest and DNA-damage-inducible, alpha -1.20 -1.41 PFKL phosphofructokinase, liver -1.20 -1.06 HDGFRP3 hepatoma-derived growth factor, related protein 3 -1.20 -1.55 GPIAP1 GPI-anchored membrane protein 1 -1.20 -1.19 HAT1 histone acetyltransferase 1 -1.21 -1.51 SNRPD2 small nuclear ribonucleoprotein D2 polypeptide 16.5kDa -1.21 -1.06 ALDOA aldolase A, fructose-bisphosphate -1.21 -1.04 PDE3B phosphodiesterase 3B, cGMP-inhibited -1.21 -1.04

174 YEATS4 YEATS domain containing 4 -1.21 -1.62 5,10-methenyltetrahydrofolate synthetase (5-formyltetrahydrofolate cyclo- MTHFS -1.21 -1.05 ligase) SNRPD3 small nuclear ribonucleoprotein D3 polypeptide 18kDa -1.21 -1.01 PSMA3 proteasome (prosome, macropain) subunit, alpha type, 3 -1.21 -1.15 TRADD TNFRSF1A-associated via death domain -1.21 -1.09 FAS Fas (TNF receptor superfamily, member 6) -1.21 -1.42 TEX264 testis expressed sequence 264 -1.21 -1.25 VLDLR very low density lipoprotein receptor -1.21 -1.57 IVNS1ABP influenza virus NS1A binding protein -1.21 -1.33 EIF2B2 eukaryotic translation initiation factor 2B, subunit 2 beta, 39kDa -1.21 -1.10 NARS2 asparaginyl-tRNA synthetase 2 (mitochondrial)(putative) -1.21 -1.29 CCT3 chaperonin containing TCP1, subunit 3 (gamma) -1.22 -1.42 TM6SF1 transmembrane 6 superfamily member 1 -1.22 -1.11 MRPS14 mitochondrial ribosomal protein S14 -1.22 -1.18 C16orf61 chromosome 16 open reading frame 61 -1.22 -1.32 COPB2 coatomer protein complex, subunit beta 2 (beta prime) -1.22 -1.01 APOBEC3G apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G -1.22 -1.17 HSPC111 hypothetical protein HSPC111 -1.22 -1.01 C16orf80 chromosome 16 open reading frame 80 -1.22 -1.02 asparagine-linked glycosylation 8 homolog (S. cerevisiae, alpha-1,3- ALG8 -1.22 -1.04 glucosyltransferase) PDXK pyridoxal (pyridoxine, vitamin B6) kinase -1.22 -1.18 NOLC1 nucleolar and coiled-body phosphoprotein 1 -1.23 -1.49 CAPZB capping protein (actin filament) muscle Z-line, beta -1.23 -1.30 IPO7 Importin 7 -1.23 -1.32 LCK lymphocyte-specific protein tyrosine kinase -1.23 -1.28 FXR1 fragile X mental retardation, autosomal homolog 1 -1.23 -1.03 CAB39 calcium binding protein 39 -1.23 -0.88 DDT D-dopachrome tautomerase -1.23 -1.37 NOL5A nucleolar protein 5A (56kDa with KKE/D repeat) -1.23 -1.66 CHD1L chromodomain helicase DNA binding protein 1-like -1.23 -1.29 RPP30 ribonuclease P/MRP 30kDa subunit -1.23 -1.25 SNUPN snurportin 1 -1.23 -1.20 FAM82B family with sequence similarity 82, member B -1.23 -1.53 BUB3 BUB3 budding uninhibited by benzimidazoles 3 homolog (yeast) -1.24 -1.33 QRSL1 glutaminyl-tRNA synthase (glutamine-hydrolyzing)-like 1 -1.24 -1.02 NP nucleoside phosphorylase -1.24 -1.15 CSNK2A1 casein kinase 2, alpha 1 polypeptide -1.24 -1.03 IVNS1ABP influenza virus NS1A binding protein -1.24 -1.23 PSMA7 proteasome (prosome, macropain) subunit, alpha type, 7 -1.24 -1.02 DARS aspartyl-tRNA synthetase -1.24 -1.44 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide ATP5C1 -1.24 -1.14 1 NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23kDa (NADH-coenzyme NDUFS8 -1.24 -1.29 Q reductase) SFRS1 splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing factor) -1.25 -1.11 ROD1 ROD1 regulator of differentiation 1 (S. pombe) -1.25 -1.27 RNF34 ring finger protein 34 -1.25 -1.03

175 ARMET arginine-rich, mutated in early stage tumors -1.25 -0.99 SSBP1 single-stranded DNA binding protein 1 -1.25 -1.44 CCNB1IP1 cyclin B1 interacting protein 1 -1.25 -1.29 HMGB3 high-mobility group box 3 -1.25 -1.31 OBFC1 oligonucleotide/oligosaccharide-binding fold containing 1 -1.25 -1.25 MGAT2 mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase -1.25 -1.01 ICMT isoprenylcysteine carboxyl methyltransferase -1.25 -1.24 ITPR1 inositol 1,4,5-triphosphate receptor, type 1 -1.25 -1.38 MRPL39 mitochondrial ribosomal protein L39 -1.25 -1.31 CALM3 calmodulin 3 (phosphorylase kinase, delta) -1.25 -1.55 BAZ1B bromodomain adjacent to zinc finger domain, 1B -1.25 -1.31 ETFA electron-transfer-flavoprotein, alpha polypeptide (glutaric aciduria II) -1.25 -1.28 MOBK1B MOB1, Mps One Binder kinase activator-like 1B (yeast) -1.25 -1.07 NONO non-POU domain containing, octamer-binding -1.26 -1.06 CSNK2A2 casein kinase 2, alpha prime polypeptide -1.26 -1.12 RAD1 RAD1 homolog (S. pombe) -1.26 -1.35 RPL26L1 ribosomal protein L26-like 1 -1.26 -0.97 ETHE1 ethylmalonic encephalopathy 1 -1.27 -1.03 ILF3 interleukin enhancer binding factor 3, 90kDa -1.27 -1.47 BOLA2 /// bolA-like 2 (E. coli) /// bolA-like 2B (E. coli) -1.27 -1.16 BOLA2B RAD51C RAD51 homolog C (S. cerevisiae) -1.27 -1.30 HMOX2 heme oxygenase (decycling) 2 -1.27 -1.38 PDHB pyruvate dehydrogenase (lipoamide) beta -1.27 -1.13 MSH6 mutS homolog 6 (E. coli) -1.28 -1.39 CTA-126B4.3 CGI-96 protein -1.28 -1.17 ETNK1 ethanolamine kinase 1 -1.28 -1.28 DUT dUTP pyrophosphatase -1.28 -1.11 CKLF chemokine-like factor -1.28 -1.50 POLR2E polymerase (RNA) II (DNA directed) polypeptide E, 25kDa -1.28 -1.27 VDAC1 voltage-dependent anion channel 1 -1.28 -1.41 ELAVL1 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 (Hu antigen R) -1.28 -1.49 VKORC1 vitamin K epoxide reductase complex, subunit 1 -1.28 -1.03 SIVA CD27-binding (Siva) protein -1.28 -1.61 TRAF3IP3 TRAF3 interacting protein 3 -1.28 -1.32 IVNS1ABP influenza virus NS1A binding protein -1.28 -1.51 HSPE1 heat shock 10kDa protein 1 (chaperonin 10) -1.29 -1.35 C1orf41 /// chromosome 1 open reading frame 41 /// interleukin 17 receptor B -1.29 -1.83 IL17RB ECH1 enoyl Coenzyme A hydratase 1, peroxisomal -1.29 -1.04 EIF5B eukaryotic translation initiation factor 5B -1.29 -1.26 WIPI1 WD repeat domain, phosphoinositide interacting 1 -1.29 -1.50 ACLY ATP citrate lyase -1.29 -1.31 WDR1 WD repeat domain 1 -1.29 -1.71 TAF9 RNA polymerase II, TATA box binding protein (TBP)-associated factor, TAF9 -1.29 -1.74 32kDa PPP1CA protein phosphatase 1, catalytic subunit, alpha isoform -1.29 -1.14 SARS seryl-tRNA synthetase -1.29 -1.22 FAM69A family with sequence similarity 69, member A -1.29 -0.89

176 ANP32B acidic (leucine-rich) nuclear phosphoprotein 32 family, member B -1.29 -1.27 TMEM4 transmembrane protein 4 -1.29 -1.33 TMEM48 transmembrane protein 48 -1.29 -1.61 TPD52 tumor protein D52 -1.29 -1.09 SP140 SP140 nuclear body protein -1.30 -1.00 RBBP4 retinoblastoma binding protein 4 -1.30 -1.08 ELOVL family member 6, elongation of long chain fatty acids (FEN1/Elo2, ELOVL6 -1.30 -1.48 SUR4/Elo3-like, yeast) MRPL40 mitochondrial ribosomal protein L40 -1.30 -1.40 TSTA3 tissue specific transplantation antigen P35B -1.30 -0.99 COX5A cytochrome c oxidase subunit Va -1.30 -1.36 EIF3S2 eukaryotic translation initiation factor 3, subunit 2 beta, 36kDa -1.30 -0.98 PSMA7 proteasome (prosome, macropain) subunit, alpha type, 7 -1.31 -1.17 SUCLG1 succinate-CoA ligase, GDP-forming, alpha subunit -1.31 -1.38 TFRC transferrin receptor (p90, CD71) -1.31 -1.10 NME2 /// non-metastatic cells 2, protein (NM23B) expressed in /// NM23-LV -1.31 -1.23 NME1-NME2 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide ATP5C1 -1.31 -1.22 1 SNRPF small nuclear ribonucleoprotein polypeptide F -1.31 -1.57 SEC23B Sec23 homolog B (S. cerevisiae) -1.31 -1.22 RANGNRF RAN guanine nucleotide release factor -1.32 -1.31 HNRPC heterogeneous nuclear ribonucleoprotein C (C1/C2) -1.32 -1.64 MyoD family inhibitor domain containing /// MyoD family inhibitor domain MDFIC -1.32 -1.39 containing MEA1 male-enhanced antigen 1 -1.32 -1.56 SNRPB small nuclear ribonucleoprotein polypeptides B and B1 -1.32 -1.35 BRCC3 BRCA1/BRCA2-containing complex, subunit 3 -1.32 -1.31 DHPS deoxyhypusine synthase -1.33 -1.23 SERBP1 SERPINE1 mRNA binding protein 1 -1.33 -1.35 YARS tyrosyl-tRNA synthetase -1.33 -1.37 dolichyl-phosphate (UDP-N-acetylglucosamine) N- DPAGT1 -1.33 -0.92 acetylglucosaminephosphotransferase 1 (GlcNAc-1-P transferase) LEF1 lymphoid enhancer-binding factor 1 -1.33 -1.16 DEGS1 degenerative spermatocyte homolog 1, lipid desaturase (Drosophila) -1.33 -1.42 LGTN ligatin -1.34 -0.88 SLC5A3 solute carrier family 5 (inositol transporters), member 3 -1.34 -1.11 ALAS1 aminolevulinate, delta-, synthase 1 -1.34 -1.28 NONO non-POU domain containing, octamer-binding -1.34 -1.19 APOBEC3C apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3C -1.34 -1.86 SEPHS1 selenophosphate synthetase 1 -1.34 -1.59 CDC5L CDC5 cell division cycle 5-like (S. pombe) -1.34 -1.44 K-ALPHA-1 alpha tubulin /// alpha tubulin -1.34 -1.36 CDK2 /// cyclin-dependent kinase 2 /// beta-carotene dioxygenase 2 -1.34 -1.26 BCDO2 DYNLL1 dynein, light chain, LC8-type 1 -1.34 -1.41 PPIH peptidylprolyl isomerase H (cyclophilin H) -1.35 -1.45 AK2 adenylate kinase 2 -1.35 -1.43 RPA1 replication protein A1, 70kDa -1.35 -1.24 SNAPC1 small nuclear RNA activating complex, polypeptide 1, 43kDa -1.35 -1.25

177 ------1.35 -1.29 POLR2H polymerase (RNA) II (DNA directed) polypeptide H -1.35 -1.24 LSM7 LSM7 homolog, U6 small nuclear RNA associated (S. cerevisiae) -1.35 -1.22 ELAVL1 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 (Hu antigen R) -1.35 -1.33 KRCC1 lysine-rich coiled-coil 1 -1.35 -1.12 C13orf34 chromosome 13 open reading frame 34 -1.35 -1.46 EIF3S1 eukaryotic translation initiation factor 3, subunit 1 alpha, 35kDa -1.35 -1.37 C20orf172 chromosome 20 open reading frame 172 -1.36 -2.06 RAD21 RAD21 homolog (S. pombe) -1.36 -1.42 NCF4 neutrophil cytosolic factor 4, 40kDa /// neutrophil cytosolic factor 4, 40kDa -1.36 -1.67 TPD52 tumor protein D52 -1.36 -1.61 ATP5H ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d -1.36 -1.44 MRPL34 mitochondrial ribosomal protein L34 /// mitochondrial ribosomal protein L34 -1.36 -1.40 TUBA6 tubulin, alpha 6 /// tubulin, alpha 6 -1.36 -1.51 MFNG manic fringe homolog (Drosophila) -1.37 -1.47 CSNK2B casein kinase 2, beta polypeptide -1.37 -1.33 splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing factor) SFRS1 /// splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing -1.37 -1.01 factor) RPS27L ribosomal protein S27-like -1.37 -1.39 CREM cAMP responsive element modulator -1.37 -1.49 PSMB4 proteasome (prosome, macropain) subunit, beta type, 4 -1.38 -1.07 CBFB core-binding factor, beta subunit -1.38 -1.47 SNRPA small nuclear ribonucleoprotein polypeptide A -1.38 -1.36 PELO pelota homolog (Drosophila) -1.38 -1.42 --- MRNA from HIV associated non-Hodgkin's lymphoma (clone hl1-98) -1.38 -1.23 UQCRC1 ubiquinol-cytochrome c reductase core protein I -1.38 -1.27 KIAA0746 KIAA0746 protein -1.38 -1.17 CPOX coproporphyrinogen oxidase -1.38 -1.12 MMD monocyte to macrophage differentiation-associated -1.38 -1.40 NANS N-acetylneuraminic acid synthase (sialic acid synthase) -1.38 -1.15 CCNE2 cyclin E2 -1.39 -1.54 NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23kDa (NADH-coenzyme NDUFS8 -1.39 -1.68 Q reductase) MRPS12 mitochondrial ribosomal protein S12 -1.39 -1.38 CCND2 cyclin D2 -1.39 -1.52 MPG N-methylpurine-DNA glycosylase -1.39 -1.25 UQCRQ ubiquinol-cytochrome c reductase, complex III subunit VII, 9.5kDa -1.39 -1.20 GIMAP4 GTPase, IMAP family member 4 -1.40 -1.26 NUDT21 nudix (nucleoside diphosphate linked moiety X)-type motif 21 -1.40 -1.27 TMPO thymopoietin -1.40 -1.24 ARHGAP19 Rho GTPase activating protein 19 -1.40 -1.55 COX4NB COX4 neighbor -1.40 -1.36 LGALS8 lectin, galactoside-binding, soluble, 8 (galectin 8) -1.40 -1.16 NFE2L3 nuclear factor (erythroid-derived 2)-like 3 -1.40 -1.41 NFE2L1 nuclear factor (erythroid-derived 2)-like 1 -1.40 -0.92 farnesyl diphosphate synthase (farnesyl pyrophosphate synthetase, FDPS -1.40 -1.35 dimethylallyltranstransferase, geranyltranstransferase) CEP76 centrosomal protein 76kDa -1.40 -1.31

178 HADHSC L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain -1.41 -1.27 FBL fibrillarin /// fibrillarin -1.41 -1.31 GPIAP1 GPI-anchored membrane protein 1 -1.41 -1.44 CSE1L CSE1 chromosome segregation 1-like (yeast) -1.41 -1.45 WHSC1 Wolf-Hirschhorn syndrome candidate 1 -1.41 -1.15 PARP2 poly (ADP-ribose) polymerase family, member 2 -1.41 -1.67 ITPR2 /// inositol 1,4,5-triphosphate receptor, type 2 /// voltage-dependent anion channel -1.41 -1.23 VDAC3 3 PCCB propionyl Coenzyme A carboxylase, beta polypeptide -1.42 -1.27 HMGB1 high-mobility group box 1 -1.42 -1.49 MTX2 metaxin 2 -1.42 -1.54 USP1 ubiquitin specific peptidase 1 -1.43 -1.33 H2AFZ H2A histone family, member Z -1.43 -1.22 ORC5L origin recognition complex, subunit 5-like (yeast) -1.43 -1.48 PKM2 pyruvate kinase, muscle -1.43 -1.71 LGALS1 lectin, galactoside-binding, soluble, 1 (galectin 1) -1.43 -1.32 EEF1E1 eukaryotic translation elongation factor 1 epsilon 1 -1.43 -1.35 MTERFD1 MTERF domain containing 1 -1.44 -1.41 HPRT1 hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan syndrome) -1.44 -1.56 TMEM156 transmembrane protein 156 -1.44 -1.50 PSMD4 proteasome (prosome, macropain) 26S subunit, non-ATPase, 4 -1.44 -1.21 MRP63 mitochondrial ribosomal protein 63 -1.44 -1.27 BTB and CNC homology 1, basic leucine zipper transcription factor 2 /// BTB BACH2 -1.44 -1.52 and CNC homology 1, basic leucine zipper transcription factor 2 FAM111A family with sequence similarity 111, member A -1.44 -1.48 TUBA6 tubulin, alpha 6 -1.44 -1.52 K-ALPHA-1 alpha tubulin -1.44 -1.58 NOLA2 nucleolar protein family A, member 2 (H/ACA small nucleolar RNPs) -1.44 -1.37 RANBP5 RAN binding protein 5 -1.44 -1.36 ENO2 enolase 2 (gamma, neuronal) -1.44 -1.62 K-ALPHA-1 alpha tubulin /// alpha tubulin -1.45 -1.55 NOP17 NOP17 -1.45 -1.25 RPS21 ribosomal protein S21 -1.45 -1.38 FTS fused toes homolog (mouse) -1.45 -1.60 DDB2 damage-specific DNA binding protein 2, 48kDa -1.45 -1.37 NOC3L nucleolar complex associated 3 homolog (S. cerevisiae) -1.46 -1.31 NOLA3 nucleolar protein family A, member 3 (H/ACA small nucleolar RNPs) -1.46 -1.23 STAT1 signal transducer and activator of transcription 1, 91kDa -1.46 -1.16 TRA@ /// TRDV2 /// T cell receptor alpha locus /// T cell receptor delta variable 2 /// T cell receptor -1.46 -1.40 TRAV20 /// alpha variable 20 /// T cell receptor alpha constant TRAC DNA2L DNA2 DNA replication helicase 2-like (yeast) -1.46 -1.34 MT1E metallothionein 1E (functional) -1.46 -1.51 CEP57 centrosomal protein 57kDa -1.47 -1.45 AKR7A2 aldo-keto reductase family 7, member A2 (aflatoxin aldehyde reductase) -1.47 -1.55 TALDO1 transaldolase 1 -1.47 -1.26 DGKA diacylglycerol kinase, alpha 80kDa -1.47 -1.54 IL17RB interleukin 17 receptor B -1.47 -1.49

179 UNG uracil-DNA glycosylase -1.47 -1.85 GAPDH glyceraldehyde-3-phosphate dehydrogenase -1.47 -1.31 LDHB lactate dehydrogenase B -1.47 -1.44 PKP4 plakophilin 4 -1.48 -1.16 AK2 adenylate kinase 2 -1.48 -1.50 GNA15 guanine nucleotide binding protein (G protein), alpha 15 (Gq class) -1.48 -1.26 EIF2S1 eukaryotic translation initiation factor 2, subunit 1 alpha, 35kDa -1.48 -1.39 POLR2J polymerase (RNA) II (DNA directed) polypeptide J, 13.3kDa -1.48 -1.20 RSL1D1 ribosomal L1 domain containing 1 -1.49 -1.22 HIGD1A HIG1 domain family, member 1A -1.49 -1.17 NEK7 NIMA (never in mitosis gene a)-related kinase 7 -1.49 -1.62 LYRM1 LYR motif containing 1 -1.49 -1.56 JOSD3 Josephin domain containing 3 -1.49 -1.49 GDI2 GDP dissociation inhibitor 2 /// GDP dissociation inhibitor 2 -1.49 -1.20 GDI2 GDP dissociation inhibitor 2 /// GDP dissociation inhibitor 2 -1.49 -1.33 ANAPC5 anaphase promoting complex subunit 5 -1.49 -1.59 NDUFB5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa -1.50 -1.33 ALDH18A1 aldehyde dehydrogenase 18 family, member A1 -1.50 -1.20 CARS cysteinyl-tRNA synthetase -1.50 -1.58 HINT1 histidine triad nucleotide binding protein 1 -1.50 -1.13 FLT3LG fms-related tyrosine kinase 3 ligand -1.50 -1.60 MICAL2 microtubule associated monoxygenase, calponin and LIM domain containing 2 -1.50 -1.62 SLC16A1 solute carrier family 16, member 1 (monocarboxylic acid transporter 1) -1.51 -1.40 MTIF2 mitochondrial translational initiation factor 2 -1.51 -1.43 EBP emopamil binding protein (sterol isomerase) -1.51 -1.54 SLC16A3 solute carrier family 16, member 3 (monocarboxylic acid transporter 4) -1.51 -1.68 PPID peptidylprolyl isomerase D (cyclophilin D) -1.51 -1.49 heat shock protein 90kDa alpha (cytosolic), class B member 1 /// heat shock HSP90AB1 -1.51 -1.44 protein 90kDa alpha (cytosolic), class B member 1 INSIG2 insulin induced gene 2 -1.51 -1.18 MARS methionine-tRNA synthetase -1.51 -1.38 ITPR1 inositol 1,4,5-triphosphate receptor, type 1 -1.51 -1.47 G3BP Ras-GTPase-activating protein SH3-domain-binding protein -1.52 -1.45 glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase GOT2 -1.52 -1.22 2) PLP2 proteolipid protein 2 (colonic epithelium-enriched) -1.52 -1.35 EPRS glutamyl-prolyl-tRNA synthetase -1.52 -1.61 ACTL6A actin-like 6A -1.53 -1.82 SSRP1 structure specific recognition protein 1 -1.53 -1.71 CARS cysteinyl-tRNA synthetase -1.53 -1.59 SNRPG small nuclear ribonucleoprotein polypeptide G -1.53 -1.64 MSH3 mutS homolog 3 (E. coli) -1.54 -1.19 GAPDH glyceraldehyde-3-phosphate dehydrogenase -1.54 -1.29 dynein, cytoplasmic 1, intermediate chain 2 /// dynein, cytoplasmic 1, DYNC1I2 -1.54 -1.30 intermediate chain 2 LIMA1 LIM domain and actin binding 1 -1.54 -1.49 NIT2 nitrilase family, member 2 -1.55 -1.27 RAB33A RAB33A, member RAS oncogene family -1.55 -1.64 MARS methionine-tRNA synthetase -1.55 -1.60

180 RANBP5 RAN binding protein 5 -1.55 -1.40 TUBB2C tubulin, beta 2C -1.55 -1.64 FAM121B /// family with sequence similarity 121B /// NODAL modulator 3 -1.55 -1.55 NOMO3 CKAP2 cytoskeleton associated protein 2 -1.56 -1.42 ------1.56 -1.36 H2AFZ H2A histone family, member Z -1.56 -2.03 C18orf10 chromosome 18 open reading frame 10 -1.56 -1.88 TSR1 TSR1, 20S rRNA accumulation, homolog (S. cerevisiae) -1.56 -1.79 PSMA6 proteasome (prosome, macropain) subunit, alpha type, 6 -1.56 -1.31 MRPL15 mitochondrial ribosomal protein L15 -1.56 -1.71 METTL5 methyltransferase like 5 -1.56 -1.66 EED embryonic ectoderm development -1.56 -1.22 erythrocyte membrane protein band 4.1 (elliptocytosis 1, RH-linked) /// EPB41 /// erythrocyte membrane protein band 4.1 (elliptocytosis 1, RH-linked) /// -1.56 -1.34 MRPS15 mitochondrial ribosomal protein S15 /// mitochondrial ribosomal protein S15 MRPS35 mitochondrial ribosomal protein S35 -1.57 -1.23 MRPL13 mitochondrial ribosomal protein L13 -1.57 -1.63 SQRDL sulfide quinone reductase-like (yeast) -1.57 -1.32 PPA2 /// RNF36 pyrophosphatase (inorganic) 2 /// ring finger protein 36 -1.57 -1.45 CLNS1A chloride channel, nucleotide-sensitive, 1A -1.57 -1.33 PEX3 peroxisomal biogenesis factor 3 -1.58 -1.41 GCHFR GTP cyclohydrolase I feedback regulator -1.58 -1.96 UCHL3 ubiquitin carboxyl-terminal esterase L3 (ubiquitin thiolesterase) -1.59 -1.49 CD7 CD7 molecule -1.59 -2.37 POLD3 polymerase (DNA-directed), delta 3, accessory subunit -1.59 -1.44 CLEC2D C-type lectin domain family 2, member D -1.59 -1.67 MGC2463 hypothetical protein LOC79037 -1.59 -1.84 RGS10 regulator of G-protein signalling 10 -1.59 -1.46 HNRPF heterogeneous nuclear ribonucleoprotein F -1.59 -1.49 ARHGEF6 Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 -1.59 -1.45 REXO2 REX2, RNA exonuclease 2 homolog (S. cerevisiae) -1.60 -1.45 LAPTM4B lysosomal associated protein transmembrane 4 beta -1.60 -2.02 LSM2 LSM2 homolog, U6 small nuclear RNA associated (S. cerevisiae) -1.60 -1.87 FLJ14346 hypothetical protein FLJ14346 -1.60 -1.58 COPS3 COP9 constitutive photomorphogenic homolog subunit 3 (Arabidopsis) -1.60 -1.54 T cell receptor alpha locus /// T cell receptor alpha locus /// T cell receptor alpha TRA@ /// TRAC -1.60 -1.47 constant /// T cell receptor alpha constant C13orf27 chromosome 13 open reading frame 27 -1.60 -1.57 NPM1 nucleophosmin (nucleolar phosphoprotein B23, numatrin) -1.60 -1.47 CCR2 chemokine (C-C motif) receptor 2 /// chemokine (C-C motif) receptor 2 -1.61 -1.10 NUCB1 nucleobindin 1 -1.61 -1.76 FH fumarate hydratase -1.61 -1.58 PGK1 phosphoglycerate kinase 1 -1.61 -1.77 RAD23A RAD23 homolog A (S. cerevisiae) -1.61 -1.50 AGA aspartylglucosaminidase -1.61 -1.38 LDHA lactate dehydrogenase A -1.61 -1.59 PSMB5 proteasome (prosome, macropain) subunit, beta type, 5 -1.61 -1.49 APOBEC3G /// apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G /// -1.62 -1.61 APOBEC3F apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F

181 MRPL23 mitochondrial ribosomal protein L23 -1.62 -1.59 EBNA1BP2 EBNA1 binding protein 2 -1.62 -1.41 DONSON downstream neighbor of SON -1.62 -1.98 MCCC1 methylcrotonoyl-Coenzyme A carboxylase 1 (alpha) -1.62 -1.45 TPI1 triosephosphate isomerase 1 -1.63 -1.66 TUBB tubulin, beta -1.63 -1.75 IMMT inner membrane protein, mitochondrial (mitofilin) -1.63 -1.52 TFDP1 transcription factor Dp-1 -1.63 -1.57 ENOSF1 enolase superfamily member 1 -1.63 -1.86 SERBP1 SERPINE1 mRNA binding protein 1 -1.63 -1.67 NDUFB8 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19kDa -1.63 -1.52 ACOT7 acyl-CoA thioesterase 7 -1.63 -1.81 TUBB2C tubulin, beta 2C -1.64 -1.77 LMNB1 lamin B1 -1.64 -2.01 HEMK1 HemK methyltransferase family member 1 -1.64 -1.62 SLC39A8 solute carrier family 39 (zinc transporter), member 8 -1.64 -1.75 SLC7A1 solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 -1.64 -1.61 C14orf143 chromosome 14 open reading frame 143 -1.65 -1.84 ADSL adenylosuccinate lyase -1.65 -1.29 BZW2 basic leucine zipper and W2 domains 2 -1.65 -1.54 EIF4EBP1 eukaryotic translation initiation factor 4E binding protein 1 -1.65 -1.67 proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional PSMB8 -1.66 -1.51 peptidase 7) K-ALPHA-1 alpha tubulin -1.66 -1.71 TMPO thymopoietin -1.66 -1.73 NFYC nuclear transcription factor Y, gamma -1.66 -1.31 PIGF phosphatidylinositol glycan anchor biosynthesis, class F -1.67 -1.70 C14orf139 chromosome 14 open reading frame 139 -1.67 -1.82 dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate DLST /// PA2G4 -1.67 -2.04 complex) /// proliferation-associated 2G4, 38kDa C14orf92 chromosome 14 open reading frame 92 -1.67 -1.48 CBFB core-binding factor, beta subunit -1.67 -1.62 EPRS glutamyl-prolyl-tRNA synthetase -1.68 -1.43 MDH1 malate dehydrogenase 1, NAD (soluble) -1.68 -1.63 AHCY S-adenosylhomocysteine hydrolase -1.69 -1.66 TPM4 tropomyosin 4 -1.69 -2.40 RAP1GDS1 RAP1, GTP-GDP dissociation stimulator 1 -1.69 -1.67 special AT-rich sequence binding protein 1 (binds to nuclear matrix/scaffold- SATB1 -1.69 -1.75 associating DNA's) DHFR /// dihydrofolate reductase /// similar to Dihydrofolate reductase -1.69 -1.75 LOC643509 HAX1 HCLS1 associated protein X-1 -1.70 -1.72 UBE2V1 /// ubiquitin-conjugating enzyme E2 variant 1 /// ubiquitin-conjugating enzyme E2 -1.70 -1.70 Kua-UEV variant 1 SET translocation (myeloid leukemia-associated) /// similar to SET protein SET /// (Phosphatase 2A inhibitor I2PP2A) (I-2PP2A) (Template-activating factor I) -1.70 -1.77 LOC642869 (TAF-I) (Liver regeneration-related protein LRRGR00002) COMMD3 COMM domain containing 3 -1.70 -1.35 TPD52 tumor protein D52 -1.71 -1.64 GZMK granzyme K (granzyme 3; tryptase II) /// granzyme K (granzyme 3; tryptase II) -1.71 -1.98

182 GLRX2 glutaredoxin 2 -1.71 -1.84 HSPA4 heat shock 70kDa protein 4 -1.71 -1.48 karyopherin alpha 2 (RAG cohort 1, importin alpha 1) /// karyopherin alpha 2 KPNA2 /// (RAG cohort 1, importin alpha 1) /// similar to Importin alpha-2 subunit -1.71 -1.97 LOC643995 (Karyopherin alpha-2 subunit) (SRP1-alpha) (RAG cohort protein 1) /// similar to Importin alpha-2 subunit TFB1M transcription factor B1, mitochondrial -1.71 -1.30 RRAS2 related RAS viral (r-ras) oncogene homolog 2 -1.71 -1.79 FBXO5 F-box protein 5 -1.72 -1.87 CENTB1 centaurin, beta 1 -1.72 -2.09 C1QBP complement component 1, q subcomponent binding protein -1.72 -1.66 TXK TXK tyrosine kinase -1.72 -1.62 nucleophosmin (nucleolar phosphoprotein B23, numatrin) /// nucleophosmin NPM1 -1.72 -1.45 (nucleolar phosphoprotein B23, numatrin) CBX1 chromobox homolog 1 (HP1 beta homolog Drosophila ) -1.72 -1.77 C12orf24 chromosome 12 open reading frame 24 -1.72 -1.98 CCT8 chaperonin containing TCP1, subunit 8 (theta) -1.73 -1.39 ARHGAP15 Rho GTPase activating protein 15 -1.73 -1.55 GARS glycyl-tRNA synthetase -1.74 -1.89 DHFR /// dihydrofolate reductase /// similar to Dihydrofolate reductase -1.74 -2.25 LOC643509 proteasome (prosome, macropain) 26S subunit, non-ATPase, 4 /// proteasome PSMD4 -1.74 -1.51 (prosome, macropain) 26S subunit, non-ATPase, 4 HIP2 huntingtin interacting protein 2 -1.75 -1.44 C1GALT1C1 C1GALT1-specific chaperone 1 -1.75 -1.49 H2AFV H2A histone family, member V -1.75 -1.54 AUH AU RNA binding protein/enoyl-Coenzyme A hydratase -1.75 -1.59 RPLP0 /// ribosomal protein, large, P0 /// similar to ribosomal protein P0 -1.75 -1.64 RPLP0-like BLM Bloom syndrome -1.75 -1.96 MRPL3 mitochondrial ribosomal protein L3 -1.75 -1.82 PRMT1 protein arginine methyltransferase 1 -1.75 -1.64 SOS1 son of sevenless homolog 1 (Drosophila) -1.75 -1.72 TRIB3 tribbles homolog 3 (Drosophila) -1.76 -1.74 TMEM4 transmembrane protein 4 -1.76 -1.63 C1QBP complement component 1, q subcomponent binding protein -1.76 -1.67 PAFAH1B1 Platelet-activating factor acetylhydrolase, isoform Ib, alpha subunit 45kDa -1.76 -1.80 SFRS3 splicing factor, arginine/serine-rich 3 -1.76 -1.56 WDR12 WD repeat domain 12 -1.76 -1.58 VRK1 vaccinia related kinase 1 -1.77 -1.65 CAPG capping protein (actin filament), gelsolin-like -1.77 -1.89 PPA1 pyrophosphatase (inorganic) 1 -1.78 -1.58 TIMM13 translocase of inner mitochondrial membrane 13 homolog (yeast) -1.78 -1.70 CCDC53 coiled-coil domain containing 53 -1.79 -1.92 CCT2 chaperonin containing TCP1, subunit 2 (beta) -1.79 -1.50 CCNA2 cyclin A2 -1.79 -1.85 C6orf79 chromosome 6 open reading frame 79 -1.80 -2.02 GGH gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) -1.80 -1.96 TPI1 triosephosphate isomerase 1 -1.80 -1.95 C11orf73 chromosome 11 open reading frame 73 -1.81 -1.88

183 CACYBP calcyclin binding protein -1.81 -1.75 GAPDH glyceraldehyde-3-phosphate dehydrogenase -1.81 -1.52 MAPKAPK3 mitogen-activated protein kinase-activated protein kinase 3 -1.82 -1.81 MRPL12 mitochondrial ribosomal protein L12 -1.82 -1.72 KIF22 kinesin family member 22 -1.83 -1.94 C7orf24 chromosome 7 open reading frame 24 -1.83 -1.99 RAN RAN, member RAS oncogene family -1.83 -1.85 RFC5 replication factor C (activator 1) 5, 36.5kDa -1.83 -1.77 6-Sep septin 6 -1.84 -2.41 BANF1 barrier to autointegration factor 1 -1.84 -1.81 IDH2 isocitrate dehydrogenase 2 (NADP+), mitochondrial -1.84 -2.07 CLTA clathrin, light polypeptide (Lca) -1.84 -1.85 HSPH1 heat shock 105kDa/110kDa protein 1 -1.84 -1.60 WEE1 WEE1 homolog (S. pombe) -1.85 -1.87 XPOT exportin, tRNA (nuclear export receptor for tRNAs) -1.85 -1.72 PPID peptidylprolyl isomerase D (cyclophilin D) -1.85 -1.80 USP1 ubiquitin specific peptidase 1 -1.85 -2.35 K-ALPHA-1 alpha tubulin -1.85 -1.71 NCBP1 nuclear cap binding protein subunit 1, 80kDa -1.85 -1.71 SERBP1 SERPINE1 mRNA binding protein 1 -1.86 -1.72 SAR1B SAR1 gene homolog B (S. cerevisiae) -1.86 -1.90 MYC v-myc myelocytomatosis viral oncogene homolog (avian) -1.86 -1.55 KIF2 kinesin heavy chain member 2 -1.86 -1.87 SCP2 Sterol carrier protein 2 -1.86 -1.90 hCAP-D3 KIAA0056 protein -1.86 -1.96 C12orf11 chromosome 12 open reading frame 11 -1.87 -1.92 GLO1 glyoxalase I -1.87 -2.01 FH fumarate hydratase -1.87 -1.92 MCM5 minichromosome maintenance deficient 5, cell division cycle 46 (S. MCM5 -1.88 -1.85 cerevisiae) proteasome (prosome, macropain) subunit, beta type, 2 /// proteasome PSMB2 -1.88 -1.85 (prosome, macropain) subunit, beta type, 2 HSPA1A /// heat shock 70kDa protein 1A /// heat shock 70kDa protein 1B -1.88 -1.51 HSPA1B CCT4 chaperonin containing TCP1, subunit 4 (delta) -1.89 -1.75 SERBP1 SERPINE1 mRNA binding protein 1 -1.89 -1.56 HINT1 histidine triad nucleotide binding protein 1 -1.89 -1.48 P2RY14 purinergic receptor P2Y, G-protein coupled, 14 -1.89 -2.15 SNRPD1 small nuclear ribonucleoprotein D1 polypeptide 16kDa -1.91 -1.81 CETN3 centrin, EF-hand protein, 3 (CDC31 homolog, yeast) -1.91 -1.66 MRPL42 mitochondrial ribosomal protein L42 -1.92 -1.86 CCT5 chaperonin containing TCP1, subunit 5 (epsilon) -1.92 -1.84 CORO1A coronin, actin binding protein, 1A -1.92 -1.92 PSMC3 proteasome (prosome, macropain) 26S subunit, ATPase, 3 -1.93 -1.64 BHLHB2 basic helix-loop-helix domain containing, class B, 2 -1.93 -1.72 DNAJC9 DnaJ (Hsp40) homolog, subfamily C, member 9 -1.93 -2.11 SLC39A8 solute carrier family 39 (zinc transporter), member 8 -1.94 -1.88 ANP32A acidic (leucine-rich) nuclear phosphoprotein 32 family, member A -1.94 -1.86 BAG2 BCL2-associated athanogene 2 -1.94 -2.13

184 PFKP phosphofructokinase, platelet -1.94 -1.76 CSE1L CSE1 chromosome segregation 1-like (yeast) -1.94 -1.96 DUT dUTP pyrophosphatase -1.94 -1.92 FLJ20152 hypothetical protein FLJ20152 -1.96 -1.75 CD96 CD96 molecule -1.96 -1.92 SRM spermidine synthase -1.97 -2.09 POLR2E polymerase (RNA) II (DNA directed) polypeptide E, 25kDa -1.97 -1.79 THYN1 thymocyte nuclear protein 1 -1.97 -2.09 acidic (leucine-rich) nuclear phosphoprotein 32 family, member E /// acidic ANP32E -1.97 -2.14 (leucine-rich) nuclear phosphoprotein 32 family, member E ALOX5AP arachidonate 5-lipoxygenase-activating protein -1.98 -2.11 HSP90AB1 heat shock protein 90kDa alpha (cytosolic), class B member 1 -1.98 -1.95 MCM7 MCM7 minichromosome maintenance deficient 7 (S. cerevisiae) -1.98 -2.20 SMC1A structural maintenance of chromosomes 1A -1.99 -2.02 CACYBP calcyclin binding protein -1.99 -1.83 TMEM97 transmembrane protein 97 -2.00 -1.88 MCTS1 malignant T cell amplified sequence 1 -2.00 -1.95 LSM4 LSM4 homolog, U6 small nuclear RNA associated (S. cerevisiae) -2.00 -1.82 ITGB3BP integrin beta 3 binding protein (beta3-endonexin) -2.00 -1.76 phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide GART -2.01 -2.04 synthetase, phosphoribosylaminoimidazole synthetase RCC1 regulator of chromosome condensation 1 -2.01 -1.93 SYNCRIP synaptotagmin binding, cytoplasmic RNA interacting protein -2.03 -1.99 GMPS guanine monphosphate synthetase -2.03 -1.95 PSMD8 proteasome (prosome, macropain) 26S subunit, non-ATPase, 8 -2.04 -1.88 C3orf60 chromosome 3 open reading frame 60 -2.04 -1.94 BAX BCL2-associated X protein -2.04 -2.20 tumor necrosis factor receptor superfamily, member 7 /// tumor necrosis factor TNFRSF7 -2.04 -1.99 receptor superfamily, member 7 HNRPAB heterogeneous nuclear ribonucleoprotein A/B -2.04 -2.09 TPM4 tropomyosin 4 -2.04 -2.22 PSMA5 proteasome (prosome, macropain) subunit, alpha type, 5 -2.05 -1.92 SMC4 structural maintenance of chromosomes 4 -2.05 -2.20 NEIL3 nei endonuclease VIII-like 3 (E. coli) -2.05 -1.65 CD7 CD7 molecule -2.06 -2.74 ETFB electron-transfer-flavoprotein, beta polypeptide -2.07 -1.85 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP ATIC -2.07 -1.87 cyclohydrolase SAE1 SUMO-1 activating enzyme subunit 1 -2.07 -2.24 PRDX3 peroxiredoxin 3 -2.08 -1.95 ENTPD1 ectonucleoside triphosphate diphosphohydrolase 1 -2.08 -2.31 SMC4 structural maintenance of chromosomes 4 -2.10 -2.26 EXOSC8 exosome component 8 -2.10 -2.05 ENOSF1 enolase superfamily member 1 -2.10 -1.88 POLR3K polymerase (RNA) III (DNA directed) polypeptide K, 12.3 kDa -2.10 -2.14 RRM1 ribonucleotide reductase M1 polypeptide -2.10 -2.24 NUDT21 nudix (nucleoside diphosphate linked moiety X)-type motif 21 -2.11 -2.04 cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-N- CMAH -2.12 -1.91 acetylneuraminate monooxygenase) GPR171 G protein-coupled receptor 171 -2.12 -2.31

185 PHB prohibitin -2.12 -1.69 DSCR2 Down syndrome critical region gene 2 -2.13 -2.25 AK2 adenylate kinase 2 -2.16 -2.21 CDK4 cyclin-dependent kinase 4 -2.17 -2.12 MAL mal, T-cell differentiation protein -2.17 -2.20 tumor necrosis factor (ligand) superfamily, member 10 /// tumor necrosis factor TNFSF10 -2.17 -2.45 (ligand) superfamily, member 10 HN1 hematological and neurological expressed 1 -2.18 -2.24 RFC4 replication factor C (activator 1) 4, 37kDa -2.18 -2.15 VDAC1 voltage-dependent anion channel 1 -2.19 -2.13 STIL SCL/TAL1 interrupting locus -2.21 -2.56 ORC6L origin recognition complex, subunit 6 like (yeast) -2.22 -2.34 MCM3 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) -2.22 -2.28 IARS isoleucine-tRNA synthetase -2.22 -2.16 ADAM19 ADAM metallopeptidase domain 19 (meltrin beta) -2.23 -2.37 BNIP3 BCL2/adenovirus E1B 19kDa interacting protein 3 -2.23 -1.77 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, MTHFD1 -2.23 -2.33 methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase LOC653784 similar to hypothetical protein FLJ14346 -2.23 -2.17 NUP37 nucleoporin 37kDa -2.26 -2.31 ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding RAC2 -2.26 -2.42 protein Rac2) NME1 non-metastatic cells 1, protein (NM23A) expressed in -2.26 -2.17 EZH2 enhancer of zeste homolog 2 (Drosophila) -2.26 -2.27 EGFL6 EGF-like-domain, multiple 6 -2.27 -2.69 TMPO thymopoietin -2.28 -2.14 RBBP8 retinoblastoma binding protein 8 -2.28 -2.12 PSMD14 proteasome (prosome, macropain) 26S subunit, non-ATPase, 14 -2.30 -2.24 AK2 adenylate kinase 2 -2.31 -2.25 tumor necrosis factor (ligand) superfamily, member 10 /// tumor necrosis factor TNFSF10 -2.32 -2.60 (ligand) superfamily, member 10 RTCD1 RNA terminal phosphate cyclase domain 1 -2.32 -2.40 MIF macrophage migration inhibitory factor (glycosylation-inhibiting factor) -2.33 -2.16 RFC2 replication factor C (activator 1) 2, 40kDa -2.34 -2.21 RDX radixin -2.34 -1.88 STOML2 stomatin (EPB72)-like 2 -2.35 -2.05 CCT7 chaperonin containing TCP1, subunit 7 (eta) -2.37 -2.22 CDC6 CDC6 cell division cycle 6 homolog (S. cerevisiae) -2.37 -2.78 SSRP1 structure specific recognition protein 1 -2.38 -2.68 RFC3 replication factor C (activator 1) 3, 38kDa -2.38 -2.21 RPA3 replication protein A3, 14kDa -2.38 -2.30 granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) GZMA /// granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase -2.38 -2.59 3) ASF1A ASF1 anti-silencing function 1 homolog A (S. cerevisiae) -2.39 -2.38 PTTG1 pituitary tumor-transforming 1 -2.39 -2.20 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, YWHAE -2.39 -2.90 epsilon polypeptide TRA@ T cell receptor alpha locus -2.40 -1.99 EIF5A eukaryotic translation initiation factor 5A -2.40 -2.28

186 MCM6 minichromosome maintenance deficient 6 (MIS5 homolog, S. pombe) MCM6 -2.40 -2.53 (S. cerevisiae) FEN1 flap structure-specific endonuclease 1 -2.40 -2.66 CEP57 centrosomal protein 57kDa -2.40 -2.00 BCL2 B-cell CLL/lymphoma 2 -2.41 -2.27 ATAD2 ATPase family, AAA domain containing 2 -2.42 -2.10 SLAMF1 signaling lymphocytic activation molecule family member 1 -2.42 -2.39 HSPD1 heat shock 60kDa protein 1 (chaperonin) -2.42 -2.28 NUP43 nucleoporin 43kDa -2.43 -2.48 STIP1 stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizing protein) -2.45 -2.11 tumor necrosis factor (ligand) superfamily, member 10 /// tumor necrosis factor TNFSF10 -2.45 -2.73 (ligand) superfamily, member 10 RNASEH2A ribonuclease H2, subunit A -2.46 -2.67 C3orf28 chromsome 3 open reading frame 28 -2.49 -2.34 SOCS1 suppressor of cytokine signaling 1 -2.49 -1.75 MAD2L1 MAD2 mitotic arrest deficient-like 1 (yeast) -2.49 -2.71 SOCS2 suppressor of cytokine signaling 2 -2.50 -2.17 FRMD4B FERM domain containing 4B -2.51 -2.21 XCL2 chemokine (C motif) ligand 2 -2.51 -3.07 INPP4B inositol polyphosphate-4-phosphatase, type II, 105kDa -2.55 -2.67 XCL1 /// XCL2 chemokine (C motif) ligand 1 /// chemokine (C motif) ligand 2 -2.56 -3.18 GPI glucose phosphate isomerase -2.58 -2.39 GMNN geminin, DNA replication inhibitor -2.58 -2.88 ENO1 enolase 1, (alpha) -2.58 -2.35 ASNS asparagine synthetase -2.59 -2.49 CLDND1 claudin domain containing 1 -2.60 -2.56 TARS threonyl-tRNA synthetase -2.61 -2.60 LSM4 LSM4 homolog, U6 small nuclear RNA associated (S. cerevisiae) -2.61 -2.68 MINA MYC induced nuclear antigen -2.62 -2.49 SOS1 son of sevenless homolog 1 (Drosophila) -2.64 -2.52 MCM4 MCM4 minichromosome maintenance deficient 4 (S. cerevisiae) -2.64 -3.21 EIF2S2 eukaryotic translation initiation factor 2, subunit 2 beta, 38kDa -2.68 -2.66 H2AFX H2A histone family, member X -2.69 -2.87 PRDX2 peroxiredoxin 2 -2.70 -2.61 RRM1 ribonucleotide reductase M1 polypeptide -2.72 -2.63 RANBP1 RAN binding protein 1 -2.72 -2.49 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, MTHFD2 -2.74 -2.59 methenyltetrahydrofolate cyclohydrolase DUT dUTP pyrophosphatase -2.75 -2.69 KIF2C kinesin family member 2C -2.78 -2.77 HSPD1 heat shock 60kDa protein 1 (chaperonin) -2.84 -2.66 NASP nuclear autoantigenic sperm protein (histone-binding) -2.89 -3.09 CASP6 caspase 6, apoptosis-related cysteine peptidase -2.92 -2.69 SLC35E1 /// solute carrier family 35, member E1 /// hypothetical protein LOC146909 -2.93 -2.94 LOC146909 IL32 interleukin 32 /// interleukin 32 -2.94 -3.02 SHMT2 serine hydroxymethyltransferase 2 (mitochondrial) -3.06 -3.00 PCNA proliferating cell nuclear antigen -3.14 -3.45 UBE2S /// ubiquitin-conjugating enzyme E2S /// similar to Ubiquitin-conjugating enzyme -3.15 -3.09 LOC651816 E2S (Ubiquitin-conjugating enzyme E2-24 kDa) (Ubiquitin-protein ligase)

187 (Ubiquitin carrier protein) (E2-EPF5) MCM2 MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae) -3.16 -3.10 CKS2 CDC28 protein kinase regulatory subunit 2 -3.19 -3.11 CHEK1 CHK1 checkpoint homolog (S. pombe) -3.21 -3.32 TUBB tubulin, beta /// tubulin, beta -3.22 -3.27 LTB lymphotoxin beta (TNF superfamily, member 3) -3.24 -2.71 IMPDH2 IMP (inosine monophosphate) dehydrogenase 2 -3.29 -3.00 TUBB tubulin, beta -3.34 -3.32 CKS1B CDC28 protein kinase regulatory subunit 1B -3.43 -3.37 phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole PAICS -3.47 -3.50 succinocarboxamide synthetase ENO1 enolase 1, (alpha) -3.47 -3.13 RACGAP1 Rac GTPase activating protein 1 -3.47 -3.56 HIST1H4C histone 1, H4c -3.51 -3.41 MLF1IP MLF1 interacting protein -3.63 -3.77 KIF14 kinesin family member 14 -3.73 -4.06 ZWINT ZW10 interactor -3.75 -4.02 NUSAP1 nucleolar and spindle associated protein 1 -3.76 -3.78 HCAP-G chromosome condensation protein G -4.13 -3.76 KIAA0101 KIAA0101 -4.84 -5.07 TYMS thymidylate synthetase -4.92 -5.21

Appendix D

Differentially expressed apoptosis related (Gene ontology category # 006915) in leukemic LGL vs. activated enriched CD8+ cells

log2 Gene Symbol Gene Title (LGL/AcCD8) TNFAIP3 tumor necrosis factor, alpha-induced protein 3 2.75 granzyme H (cathepsin G-like 2, protein h-CCPX) /// granzyme H (cathepsin G-like GZMH 2.70 2, protein h-CCPX) TNFAIP3 tumor necrosis factor, alpha-induced protein 3 2.48 SGK serum/glucocorticoid regulated kinase 2.42 BCL2A1 BCL2-related protein A1 2.16 LITAF lipopolysaccharide-induced TNF factor 2.12 LITAF lipopolysaccharide-induced TNF factor 1.92 TNFSF13 /// tumor necrosis factor (ligand) superfamily, member 13 /// tumor necrosis factor TNFSF12- 1.90 (ligand) superfamily, member 12-member 13 TNFSF13 EP300 E1A binding protein p300 1.89 CTSB cathepsin B 1.79 FAIM3 Fas apoptotic inhibitory molecule 3 /// Fas apoptotic inhibitory molecule 3 1.78 CRTAM cytotoxic and regulatory T cell molecule 1.77 CASP8 caspase 8, apoptosis-related cysteine peptidase 1.77 SERPINB9 serpin peptidase inhibitor, clade B (ovalbumin), member 9 1.75 GADD45B growth arrest and DNA-damage-inducible, beta 1.75 CROP cisplatin resistance-associated overexpressed protein 1.67 SON SON DNA binding protein 1.63 SH3GLB1 SH3-domain GRB2-like endophilin B1 1.61 RUNX3 runt-related transcription factor 3 1.54 CTSB cathepsin B 1.50 VCP valosin-containing protein 1.50 RHOT2 ras homolog gene family, member T2 1.44 IFI6 /// IGH@ interferon, alpha-inducible protein 6 /// immunoglobulin heavy locus /// /// IGHG1 /// immunoglobulin heavy constant gamma 1 (G1m marker) /// immunoglobulin heavy IGHG2 /// 1.44 constant gamma 2 (G2m marker) /// immunoglobulin heavy constant gamma 3 (G3m IGHG3 /// marker) /// immunoglobulin he IGHM ABCA2 ATP-binding cassette, sub-family A (ABC1), member 2 1.44 SNCA synuclein, alpha (non A4 component of amyloid precursor) 1.37 NALP1 NACHT, leucine rich repeat and PYD (pyrin domain) containing 1 1.35 RUNX3 runt-related transcription factor 3 1.35 CTSB cathepsin B 1.34 SIAH1 seven in absentia homolog 1 (Drosophila) 1.30 CTSB cathepsin B 1.28 RAF1 v-raf-1 murine leukemia viral oncogene homolog 1 1.19 NFKBIA nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha 1.18

189 TPT1 tumor protein, translationally-controlled 1 1.18 MCL1 myeloid cell leukemia sequence 1 (BCL2-related) 1.17 HSPA9B heat shock 70kDa protein 9B (mortalin-2) 1.16 IER3 immediate early response 3 1.14 PDCD4 programmed cell death 4 (neoplastic transformation inhibitor) 1.13 TP53BP2 tumor protein p53 binding protein, 2 1.13 PRF1 perforin 1 (pore forming protein) /// perforin 1 (pore forming protein) 1.11 CUL5 cullin 5 1.10 F2R coagulation factor II (thrombin) receptor 1.10 v-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light RELA 1.09 polypeptide gene enhancer in B-cells 3, p65 (avian) EP300 E1A binding protein p300 1.08 BTG1 B-cell translocation gene 1, anti-proliferative 1.08 FASTKD5 FAST kinase domains 5 1.07 TIA1 TIA1 cytotoxic granule-associated RNA binding protein 1.06 CD40 CD40 molecule, TNF receptor superfamily member 5 1.05 GADD45B growth arrest and DNA-damage-inducible, beta 1.03 CDC2L1 /// cell division cycle 2-like 1 (PITSLRE proteins) /// cell division cycle 2-like 2 1.02 CDC2L2 (PITSLRE proteins) ANXA5 annexin A5 1.02 OPA1 optic atrophy 1 (autosomal dominant) 1.01 TNFRSF14 tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator) 1.00 PLAGL1 pleiomorphic adenoma gene-like 1 0.99 ELMO2 engulfment and cell motility 2 0.94 BCLAF1 BCL2-associated transcription factor 1 0.91 CASP8 caspase 8, apoptosis-related cysteine peptidase 0.87 SGPL1 sphingosine-1-phosphate lyase 1 0.87 PREI3 preimplantation protein 3 0.86 DIDO1 death inducer-obliterator 1 0.86 FEM1B fem-1 homolog b (C. elegans) 0.83 RRAGC Ras-related GTP binding C 0.82 FOXO3A forkhead box O3A 0.82 BECN1 beclin 1 (coiled-coil, myosin-like BCL2 interacting protein) 0.81 BTG1 B-cell translocation gene 1, anti-proliferative 0.81 BAG5 BCL2-associated athanogene 5 0.81 TNFRSF10B tumor necrosis factor receptor superfamily, member 10b 0.80 MAP3K5 mitogen-activated protein kinase kinase kinase 5 0.80 PTPRC protein tyrosine phosphatase, receptor type, C 0.77 SNRK SNF related kinase 0.77 UBE4B ubiquitination factor E4B (UFD2 homolog, yeast) 0.75 MOAP1 modulator of apoptosis 1 0.74 PDCD4 programmed cell death 4 (neoplastic transformation inhibitor) 0.74 RIPK1 receptor (TNFRSF)-interacting serine-threonine kinase 1 0.71 BID BH3 interacting domain death agonist /// BH3 interacting domain death agonist 0.71 CASP1 caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase) 0.70 MAP3K5 mitogen-activated protein kinase kinase kinase 5 0.69 CUL3 cullin 3 0.66 ITGB2 integrin, beta 2 (complement component 3 receptor 3 and 4 subunit) 0.63 BAG5 BCL2-associated athanogene 5 0.63

190 PTPRC protein tyrosine phosphatase, receptor type, C 0.62 RHOT1 ras homolog gene family, member T1 0.60 CUL2 cullin 2 0.59 CFLAR CASP8 and FADD-like apoptosis regulator 0.58 NUP62 nucleoporin 62kDa -0.64 TNFAIP8 tumor necrosis factor, alpha-induced protein 8 -0.64 HSPA9B heat shock 70kDa protein 9B (mortalin-2) -0.65 HSPA1A heat shock 70kDa protein 1A -0.70 PDCD11 programmed cell death 11 -0.71 HTRA2 HtrA serine peptidase 2 -0.72 MALT1 mucosa associated lymphoid tissue lymphoma translocation gene 1 -0.76 CASP8AP2 CASP8 associated protein 2 -0.76 FAF1 Fas (TNFRSF6) associated factor 1 -0.78 PRKCA protein kinase C, alpha -0.79 MALT1 mucosa associated lymphoid tissue lymphoma translocation gene 1 -0.79 CD3E CD3e molecule, epsilon (CD3-TCR complex) -0.80 ATG5 ATG5 autophagy related 5 homolog (S. cerevisiae) -0.82 PPP2R1B protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), beta isoform -0.82 NUP62 nucleoporin 62kDa -0.83 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta YWHAH -0.85 polypeptide CDKN2A cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) -0.87 E2F1 E2F transcription factor 1 -0.90 TRAF5 TNF receptor-associated factor 5 -0.93 ESD esterase D/formylglutathione hydrolase -0.93 CTNNAL1 catenin (cadherin-associated protein), alpha-like 1 -0.98 TP53 tumor protein p53 (Li-Fraumeni syndrome) -1.00 FXR1 fragile X mental retardation, autosomal homolog 1 -1.03 RNF34 ring finger protein 34 -1.03 LCK lymphocyte-specific protein tyrosine kinase -1.04 CD2 CD2 molecule /// CD2 molecule -1.04 PDCD6 programmed cell death 6 -1.05 PERP PERP, TP53 apoptosis effector -1.09 LDHB lactate dehydrogenase B -1.09 TRADD TNFRSF1A-associated via death domain -1.09 TNFAIP8 tumor necrosis factor, alpha-induced protein 8 -1.10 FAS Fas (TNF receptor superfamily, member 6) -1.10 CASP3 caspase 3, apoptosis-related cysteine peptidase -1.12 IFI16 interferon, gamma-inducible protein 16 -1.13 CSE1L CSE1 chromosome segregation 1-like (yeast) -1.13 SIVA CD27-binding (Siva) protein -1.15 STAT1 signal transducer and activator of transcription 1, 91kDa -1.16 PHLDA1 pleckstrin homology-like domain, family A, member 1 -1.16 IL2RB interleukin 2 receptor, beta /// interleukin 2 receptor, beta -1.20 GSTP1 glutathione S-transferase pi -1.22 EIF2AK2 eukaryotic translation initiation factor 2-alpha kinase 2 -1.28 LCK lymphocyte-specific protein tyrosine kinase -1.28 PHLDA1 pleckstrin homology-like domain, family A, member 1 -1.30 LGALS1 lectin, galactoside-binding, soluble, 1 (galectin 1) -1.32

191 HSPE1 heat shock 10kDa protein 1 (chaperonin 10) -1.35 EEF1E1 eukaryotic translation elongation factor 1 epsilon 1 -1.35 CEBPG CCAAT/enhancer binding protein (C/EBP), gamma -1.37 YARS tyrosyl-tRNA synthetase -1.37 ESD esterase D/formylglutathione hydrolase -1.37 CALR calreticulin -1.38 VDAC1 voltage-dependent anion channel 1 -1.41 GADD45A growth arrest and DNA-damage-inducible, alpha -1.41 CKAP2 cytoskeleton associated protein 2 -1.42 FAS Fas (TNF receptor superfamily, member 6) -1.42 RAD21 RAD21 homolog (S. pombe) -1.42 APIP APAF1 interacting protein -1.43 LDHB lactate dehydrogenase B -1.44 CSE1L CSE1 chromosome segregation 1-like (yeast) -1.45 nucleophosmin (nucleolar phosphoprotein B23, numatrin) /// nucleophosmin NPM1 -1.45 (nucleolar phosphoprotein B23, numatrin) ARHGEF6 Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 -1.45 NPM1 nucleophosmin (nucleolar phosphoprotein B23, numatrin) -1.47 HMGB1 high-mobility group box 1 -1.49 HSPA1A /// heat shock 70kDa protein 1A /// heat shock 70kDa protein 1B -1.51 HSPA1B FTS fused toes homolog (mouse) -1.60 SIVA CD27-binding (Siva) protein -1.61 TUBB2C tubulin, beta 2C -1.64 PHB prohibitin -1.69 TRIB3 tribbles homolog 3 (Drosophila) -1.74 TUBB tubulin, beta -1.75 ITGB3BP integrin beta 3 binding protein (beta3-endonexin) -1.76 TUBB2C tubulin, beta 2C -1.77 BNIP3 BCL2/adenovirus E1B 19kDa interacting protein 3 -1.77 GLRX2 glutaredoxin 2 -1.84 ARHGDIA Rho GDP dissociation inhibitor (GDI) alpha -1.91 CSE1L CSE1 chromosome segregation 1-like (yeast) -1.96 tumor necrosis factor receptor superfamily, member 7 /// tumor necrosis factor TNFRSF7 -1.99 receptor superfamily, member 7 GLO1 glyoxalase I -2.01 VDAC1 voltage-dependent anion channel 1 -2.13 BAG2 BCL2-associated athanogene 2 -2.13 MIF macrophage migration inhibitory factor (glycosylation-inhibiting factor) -2.16 NME1 non-metastatic cells 1, protein (NM23A) expressed in -2.17 SOCS2 suppressor of cytokine signaling 2 -2.17 BAX BCL2-associated X protein -2.20 MAL mal, T-cell differentiation protein -2.20 BCL2 B-cell CLL/lymphoma 2 -2.27 HSPD1 heat shock 60kDa protein 1 (chaperonin) -2.28 tumor necrosis factor (ligand) superfamily, member 10 /// tumor necrosis factor TNFSF10 -2.45 (ligand) superfamily, member 10 granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) /// GZMA -2.59 granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 /// tumor necrosis factor -2.60

192 (ligand) superfamily, member 10 PRDX2 peroxiredoxin 2 -2.61 HSPD1 heat shock 60kDa protein 1 (chaperonin) -2.66 CASP6 caspase 6, apoptosis-related cysteine peptidase -2.69 tumor necrosis factor (ligand) superfamily, member 10 /// tumor necrosis factor TNFSF10 -2.73 (ligand) superfamily, member 10 TUBB tubulin, beta /// tubulin, beta -3.27 TUBB tubulin, beta -3.32

Curriculum Vitae Mithun Vinod Shah

Education: PhD in Molecular Medicine: The Pennsylvania State University, Hershey, PA (Aug 2003-May 2009) Bachelor of Medicine & Surgery (MBBS), B.J. Medical College, Gujarat University, India (1996-2002) Experience: Graduate Fellow in Molecular Medicine 2003 - 2009 Dr. Thomas P. Loughran, Jr., The Pennsylvania State University, Hershey, PA Summer Research Intern May 2005 – Aug 2005 Dr. Paul August, Head, Pathway Mapping and Elucidation Cambridge Genomic Centre, Sanofi-Aventis Inc., Cambridge, MA Resident Clinician, Microbiology and Immunology 2002-2003 Dr. Parijath Goswami, Head, Department of Microbiology and Immunology Gujarat Cancer and Research Institute Volunteer, World Health Organization – Gujarat 2001-2002 Dr. Nilesh Buddha, Team leader, WHO – Gujarat Rotating Houseman Clinician, Medicine and Surgery, B.J. Medical College 2001-2002 Dean, B.J. Medical College, Ahmedabad Publications: Shah MV, Zhang R et al. Molecular profiling of large granular lymphocyte leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes. Blood, 2008; 112(3):770-81 Zhang R, Shah MV et al. Modeling for a cure: a dynamic model of the survival signals in T-cell large granular lymphocyte leukemia. Proc Natl Acad Sci USA, 2008; 105(42):16308-13 Shah MV, Zhang R, Loughran TP, Jr. Mechanism of FTY720 induced apoptosis in T-LGL leukemia (in preparation) Shah MV, Loughran TP, Jr. Antibody profiling of LGL leukemia (in preparation) Reviews and Book chapters: Bayliss T, Shah MV, Zhang R, Loughran TP, Jr., Large granular lymphocyte leukemia: a review. Textbook of advances in T-cell malignancies. (invited review, submitted) Shah MV, Zhang R, Loughran TP, Jr., Never say die: Survival signaling in LGL leukemia. Clinical Leukemia (invited review, submitted) Zhang R, Shah MV, Loughran TP, Jr. Associated disorders of LGL leukemia. Hematol Oncol (invited review, in preparation) Shah MV. ‘Instructive Immunology’: Interplay between the innate and the adaptive immune system. Ind J Allergy Asthma Immunol, 2004; 18(2):87-92 Poster presentations: Zhang R, Shah MV, et al. Modeling for cure: A survival signaling network for T-large granular lymphocyte leukemia; Joint CSHL/Wellcome Trust Conference in Network Biology. Aug 2008, Cambridge, UK Shah MV, Zhang R, et al. Role of sphingolipid signaling in survival of cytotoxic lymphocytes. Poster Presentation at FASEB Summer Research Conference, Jun 2007, Tucson, AZ Shah MV and Goswami P. Changing Pattern of Fungal Infections in Post-Operative Immunocompromised Patients at GCRI. Best Poster Presentation Award Recipient at 2nd Gujarat State Chapter of Indian Association of Medical Microbiologists, March 2003, Ahmedabad, India.