Preclinical Development of a Non-Immunosuppressive FTY720 Derivative OSU-2S for Chronic Lymphocytic Leukemia and Other B-Cell Malignancies

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Preclinical development of a non-immunosuppressive FTY720 derivative OSU-2S for chronic lymphocytic leukemia and other B-cell malignancies

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Rajeswaran Mani, BVSc & AH

Graduate Program in Comparative and Veterinary Medicine

The Ohio State University

2014

Dissertation Committee:

Natarajan Muthusamy, Advisor

John C. Byrd

Ching-Shih Chen

Cheryl A. London

Copyrighted by

Rajeswaran Mani

2014

Abstract

Chemotherapeutics remains a choice of treatment for several malignant diseases.

However, selective cytotoxicity against cancer cells without compromising their normal counterparts pose a huge challenge for traditional drug design. Current therapies for chronic lymphocytic leukemia (CLL), the most prevalent adulthood leukemia in the western world are not curative rendering drug adverse effects and immunosuppression.

Here we developed a novel non-immunosuppressive FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells. OSU-2S induces activation of protein phosphatase 2A, phosphorylation and nuclear translocation of SHP1S591 and deregulation of multiple cellular processes in CLL. Moreover, with relevant to CLL disease TCL1A expression that was identified to be down regulated in response to OSU-2S in the gene expression profile was independently confirmed to be significantly down regulated both at the mRNA and protein levels. Exposure of OSU-2S to unintended cells is expected to adversely affect physiological functions of these ubiquitous phosphatases. To selectively deliver OSU-2S to leukemic cells, we developed tumor antigen targeted delivery of immunonanoparticle carrying a OSU-2S (2A2-OSU-2S-ILP). 2A2-OSU-2S-ILP immunonanoparticles mediated selective cytotoxicity of CLL but not normal B cells through targeting receptor tyrosine kinase ROR1 expressed in leukemic but not normal B cells. Developing a novel spontaneous CLL mouse model expressing human ROR1 in all

leukemic B cells, we demonstrate the therapeutic benefit of enhanced survival with 2A2-

OSU-2S-ILP in-vivo. The newly developed non-immunosuppressive OSU-2S, its delivery using human CLL specific surface antigen ROR1 directed immunonanoparticles and the novel transgenic mouse model of CLL that expresses human ROR1 exclusively in leukemic B cell surface are highly innovative and can be applied to CLL and other

ROR1+ malignancies including mantle cell lymphoma (MCL) and B-lineage acute lymphoblastic leukemia (ALL).

Further, treatment of MCL with OSU-2S induced PARP cleavage and increased the cell surface expression of CD74 a therapeutic target. OSU-2S in combination with anti-CD74 antibody milatuzumab had additive cytotoxicity in MCL cells. Moreover, ROR1 targeted

2A2-OSU-2S-ILP mediated selective cytotoxicity of MCL. Treatment with 2A2-OSU-

2S-ILP significantly reduced the tumor weight in MCL xenografted mice. Thus OSU-2S and its delivery using tumor antigen ROR1 directed immunonanoparticles could increase the width of existing armamentarium for MCL.

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Dedicated to my parents and my brother

Acknowledgments

I am grateful to my advisor and my advisory committee members for their professional mentorship and Veterinary Biosciences department for supporting my studentship. I wholeheartedly thank Drs. Muthusamy and Byrd lab members for their kind assistance and their continuous support while I carried out my research.

I thank Mr. Frank W. Frissora in particular for his support and in laboratory assistance all through the years since I joined the graduate program. I appreciate Dr.Yicheng Mao and

Mr. Chi-Ling Chiang who supported the research by synthesizing targeted liposomal nanoparticles and for help with the experiments. Special thanks to Drs.Ching-Shih Chen and Ribai Yan for synthesizing and providing us the OSU-2S drug for conducting preclinical studies. I am also thankful to the patients and healthy donors who supported the research by providing their blood without which the preclinical investigation of OSU-

2S could not have been possible.

I would like to extend my gratefulness to Dr. Byrd for providing translational insights to the experimental designs and Dr. London for her invaluable comments and providing me the opportunity for evaluation of OSU-2S in the spontaneous canine lymphoma samples.

Vita

April 2002 ...... Carmel Matriculation Higher Secondary

School, Erode, India

2007...... BVSc & AH (Veterinary Medicine)

Tamilnadu Veterinary and Animal Sciences

University (TANUVAS), Chennai, India

2009 to present ...... Graduate Research Associate, Department

of Veterinary Biosciences & Comprehensive

Cancer Center, The Ohio State University

Publications

1. Mani, R., Y. Mao, F.W. Frissora, C.L. Chiang, J. Wang, Y. Zhao, Y. Wu, B. Yu,

R. Yan, X. Mo, L. Yu, J. Flynn, J. Jones, L. Andritsos, S. Baskar, C. Rader, M.A. Phelps,

C.S. Chen, R.J. Lee, J.C. Byrd, L.J. Lee, and N. Muthusamy, Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B cell cytotoxicity in chronic lymphocytic leukemia. Leukemia, 2014. doi: 10.1038/leu.2014.199.

2. Lapalombella, R., Q. Sun, K. Williams, L. Tangeman, S. Jha, Y. Zhong, V.

Goettl, E. Mahoney, C. Berglund, S. Gupta, A. Farmer, R. Mani, A.J. Johnson, D. Lucas, vi

X. Mo, D. Daelemans, V. Sandanayaka, S. Shechter, D. McCauley, S. Shacham, M.

Kauffman, Y.M. Chook, and J.C. Byrd, Selective inhibitors of nuclear export show that

CRM1/XPO1 is a target in chronic lymphocytic leukemia. Blood, 2012. 120(23): p. 4621-

34.

3. Alinari, L., C.J. Prince, R.B. Edwards, W.H. Towns, R. Mani, A. Lehman, X.

Zhang, D. Jarjoura, L. Pan, A.D. Kinghorn, M.R. Grever, R.A. Baiocchi, and D.M.

Lucas, Dual Targeting of the Cyclin/Rb/E2F and Mitochondrial Pathways in Mantle Cell

Lymphoma with the Translation Inhibitor Silvestrol. Clin Cancer Res, 2012.

4. Lapalombella, R., Y.Y. Yeh, L. Wang, A. Ramanunni, S. Rafiq, S. Jha, J. Staubli,

D.M. Lucas, R. Mani, S.E. Herman, A.J. Johnson, A. Lozanski, L. Andritsos, J. Jones,

J.M. Flynn, B. Lannutti, P. Thompson, P. Algate, S. Stromatt, D. Jarjoura, X. Mo, D.

Wang, C.S. Chen, G. Lozanski, N.A. Heerema, S. Tridandapani, M.A. Freitas, N.

Muthusamy, and J.C. Byrd, Tetraspanin CD37 Directly Mediates Transduction of

Survival and Apoptotic Signals. Cancer Cell, 2012. 21(5): p. 694-708.

5. Alinari, L., E. Mahoney, J. Patton, X. Zhang, L. Huynh, C.T. Earl, R. Mani, Y.

Mao, B. Yu, C. Quinion, W.H. Towns, C.S. Chen, D.M. Goldenberg, K.A. Blum, J.C.

Byrd, N. Muthusamy, M. Praetorius-Ibba, and R.A. Baiocchi, FTY720 increases CD74 expression and sensitizes mantle cell lymphoma cells to milatuzumab-mediated cell death. Blood, 2011. 118(26): p. 6893-903.

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6. Bai, L.Y., Y. Ma, S.K. Kulp, S.H. Wang, C.F. Chiu, F. Frissora, R. Mani, X. Mo,

D. Jarjoura, J.C. Byrd, C.S. Chen, and N. Muthusamy, OSU-DY7, a novel D-tyrosinol derivative, mediates cytotoxicity in chronic lymphocytic leukaemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway. Br J Haematol, 2011. 153(5): p.

623-33.

7. Liu, Q., L. Alinari, C.S. Chen, F. Yan, J.T. Dalton, R. Lapalombella, X. Zhang,

R. Mani, T. Lin, J.C. Byrd, R.A. Baiocchi, and N. Muthusamy, FTY720 shows promising in vitro and in vivo preclinical activity by downmodulating Cyclin D1 and phospho-Akt in mantle cell lymphoma. Clin Cancer Res, 2010. 16(12): p. 3182-92.

Fields of Study

Major Field: Comparative and Veterinary Medicine

Experimental Therapeutics

Molecular Pharmacology

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Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vi

Table of Contents ...... ix

List of Tables ...... xii

List of Figures ...... xiii

List of Abbreviations ...... xviii

Introduction ...... 1

Chronic Lymphocytic Leukemia ...... 1

Role of Kinases in leukemia ...... 3

Role of Phosphatases in leukemia...... 7

FTY720 ...... 10

FTY720 in solid tumors ...... 10

FTY720 in hematologic malignancies ...... 11

OSU-2S a non-immunosuppressive FTY720 analogue ...... 13

Materials and Methods ...... 15 ix

Chapter 1: Preclinical activity of OSU-2S in CLL ...... 30

Chapter 2: Molecular pharmacology of OSU-2S in CLL ...... 46

OSU-2S activates PKC in CLL...... 54

OSU-2S induces ROS generation in CLL ...... 61

Gene expression analysis (GEA) of OSU-2S mediated cytotoxicity in CLL ...... 68

OSU-2S modulates TCL1 in CLL ...... 72

ROR1 targeted delivery of OSU-2S ...... 79

Chapter 3: In-vivo activity of OSU-2S in mouse models of CLL ...... 88

Maximum tolerated dose (MTD) studies ...... 90

Activity in Raji cell xenografted SCID mouse model ...... 92

Activity in E-TCL1 transgenic mouse model of CLL ...... 94

Activity in E-ROR1-TCL1 splenocytes engrafted mouse model of CLL ...... 96

E-ROR1-TCL1 double transgenic mouse development ...... 96

E-ROR1-TCL1 splenocytes engrafted mouse model development ..... 103

Chapter 4: OSU-2S in B-Cell Lymphomas ...... 109

OSU-2S and Mantle Cell Lymphoma ...... 109

Synopsis ...... 109

OSU-2S and Canine Diffuse Large B-Cell Lymphoma ...... 124

Canine spontaneous lymphoma ...... 124 x

Activity of OSU-2S in canine lymphoma ...... 125

Discussion ...... 129

Future Directions ...... 139

Target identification ...... 139

Role of OSU-2S-induced phospho SHP1S591 in cytotoxicity in CLL ..... 141

Mechanism of TCL1 down regulation by OSU-2S ...... 143

Effect of TCL1 down regulation on activator protein-1 function ...... 143

References ...... 145

Appendix A: IPA of OSU-2S treated CLL cells...... 166

Appendix B: Sensitivity of NCI-60 cancer cell lines to OSU-2S ...... 179

List of Tables

Table 1: List of micro-RNAs modulated by OSU-2S in CLL primary cells...... 75

Table 2: ROR1 expression by microarray (gene expression-A) and flow cytometry (cell surface-B) on CLL cells after OSU-2S treatment...... 87

Table 3: Differential gene expression detected by microarray in OSU-2S treated CLL cells...... 167

Table 4: Functional molecules in cancer affected by OSU-2S...... 178

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List of Figures

Figure 1: Activity of OSU-2S and FTY720 in lymphoid leukemic cell lines...... 33

Figure 2: Dose dependent cytotoxicity of OSU-2S in CLL primary cells...... 34

Figure 3: Time dependent cytotoxicity of OSU-2S in CLL primary cells...... 35

Figure 4: Influence of cell density on OSU-2S cytotoxicity in CLL primary cells...... 36

Figure 5: Activity of OSU-2S in CLL primary cells. (N=25) ...... 37

Figure 6: OSU-2S is active in 17p deleted CLL primary cells...... 38

Figure 7: OSU-2S is active in IGVH unmutated CLL primary cells...... 39

Figure 8: Activity of OSU-2S in rituximab resistant Raji clone 2R...... 40

Figure 9: Activity of OSU-2S in fludarabine resistant MEC2 cell line...... 41

Figure 10: OSU-2S promotes cytotoxicity in CLL primary cells that are sensitive and refractory to fludarabine...... 42

Figure 11: Caspase independent cytotoxicity of OSU-2S in CLL primary cells...... 43

Figure 12: Combinational activity of OSU-2S and anti-CD37 SMIP on CLL primary cells...... 45

Figure 13: Activation of PP2A by OSU-2S in CLL primary cells...... 48

Figure 14: Association of PP2A and SHP1(left) and SHP1 phosphorylation (right) in response to OSU-2S treatment in CLL primary cells...... 49

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Figure 15: Inverse correlation between OSU-2S-induced phospho SHP1S591 and viability in CLL primary cells...... 50

Figure 16: OSU-2S does not affect SHP1 enzyme activity in CLL...... 51

Figure 17: OSU-2S does not affect phospho Y416 of Src family kinases in CLL...... 52

Figure 18: Nuclear localization of phospho SHP1S591 in CLL...... 53

Figure 19: PKC inhibitor BIS reduces OSU-2S induced phospho SHP1S591...... 56

Figure 20: PKC inhibitor BIS partially rescues OSU-2S induced cytotoxicity in CLL. .. 57

Figure 21: OSU-2S activates PKC in CLL cells...... 58

Figure 22: OSU-2S activates purified PKC in in-vitro kinase assay...... 59

Figure 23: SFK inhibition does not affect OSU-2S induced phospho SHP1S591...... 60

Figure 24: ROS generation in OSU-2S treated CLL primary cells...... 63

Figure 25: ROS inhibition partially rescues OSU-2S cytotoxicity in CLL...... 64

Figure 26: ROS inhibition does not affect OSU-2S induced phospho SHP1S591...... 65

Figure 27: ROS generation in response to OSU-2S in CLL (time course)...... 66

Figure 28: Viability of CLL cells treated with OSU-2S in parallel to ROS time course. 67

Figure 29: Heat map of OSU-2S responsive genes in CLL primary cells...... 69

Figure 30: Top functions affected by OSU-2S in CLL...... 70

Figure 31: OSU-2S decreases BCR induced CD86 expression in CLL...... 71

Figure 32: OSU-2S down regulates TCL1A oncogene in CLL...... 73

Figure 33: TCL1 over expression does not protect MEC1 cell line from OSU-2S cytotoxicity...... 76

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Figure 34: OSU-2S decreases TCL1 protein expression in MEC1 cell lines over expressing TCL1...... 77

Figure 35: OSU-2S does not affect protein stability of TCL1 in MEC1 cell line over .... 78

Figure 36: Cell surface ROR1 expression on CLL and normal B cells...... 82

Figure 37: 2A2 mAb treated CLL have reduced surface ROR1 expression...... 83

Figure 38: ROR1 targeted delivery of OSU-2S induces cytotoxicity in CLL...... 84

Figure 39: 2A2-OSU-2S-ILP promoted comparable levels of cytotoxicity as CD20-OSU-

2S-ILP in CLL...... 85

Figure 40: Long term effect of OSU-2S-LP on normal B cells...... 86

Figure 41: OSU-2S does not alter B:T ratio in C57BL/6 wild type mouse...... 89

Figure 42: OSU-2S dose escalation study graphical representation...... 91

Figure 43: Leukemia reduction by OSU-2S in bone marrow of Raji cell xenografted

SCID mouse...... 93

Figure 44: Leukemia reduction by OSU-2S in peripheral blood of E-TCL1 transgenic mouse...... 95

Figure 45: E-ROR1-TCL1 double transgenic mouse development...... 98

Figure 46: ROR1 expression in Eµ-ROR1-TCL1 double transgenic mouse...... 99

Figure 47: ROR1 expression in ROR1 Tg mice peripheral blood by flow cytometry. .. 100

Figure 48: Effect of OSU-2S immunonanoparticles (ILPs) on Eµ-TCL1 mouse splenocytes (ex-vivo)...... 101

Figure 49: Effect of OSU-2S immunonanoparticles (ILPs) on Eµ-ROR1-TCL1 mouse splenocytes (ex-vivo)...... 102

Figure 50: Eµ-ROR1-TCL1 splenocytes engrafted mouse model of CLL...... 105

Figure 51: Reduced peripheral blood leukemic burden by 2A2-OSU-2S-ILP treatment.

...... 106

Figure 52: Survival curve of Eµ-ROR1-TCL1 splenocytes engrafted mouse model of

CLL treated with OSU-2S immunonanoparticles (ILP)...... 107

Figure 53: Survival curve of Eµ-ROR1-TCL1 splenocytes engrafted mouse model of

CLL treated with OSU-2S (plain drug)...... 108

Figure 54: OSU-2S is cytotoxic in MCL primary cells...... 114

Figure 55: Effect of OSU-2S on Cyclin D1 in JeKo and Mino cells...... 115

Figure 56: Cell surface CD74 induction by OSU-2S in MCL cell lines...... 116

Figure 57: OSU-2S and Milatuzumab combination in MCL cell lines...... 117

Figure 58: Cell surface CD74 induction by OSU-2S in MCL primary cells...... 118

Figure 59: OSU-2S and Milatuzumab combination effect in MCL primary cells ...... 119

Figure 60: ROR1 expression in MCL cell lines and primary cells...... 120

Figure 61: Cytotoxicity by ROR1 targeted OSU-2S nanoparticles in MCL cell lines. .. 121

Figure 62: Cytotoxicity by ROR1 targeted OSU-2S nanoparticles in MCL primary cells.

...... 122

Figure 63: In-vivo activity of ROR1 targeted OSU-2S in Xenograft model of MCL. ... 123

Figure 64: Activity of OSU-2S in canine DLBCL cell lines...... 126

Figure 65: OSU-2S induces apoptosis in canine spontaneous DLBCL...... 127

Figure 66: OSU-2S induces ROS mediated cytotoxicity in canine DLBCL...... 128

Figure 67: Activity of OSU-2S in NCI-60 cancerous cell lines...... 180

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Figure 68: Total growth inhibitory concentration of OSU-2S in NCI-60 cancer cell lines.

...... 181

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List of Abbreviations

2FaraA 9-beta-D-Arabinofuranosyl-2-fluoroadenine

AKT Serine/Threonine Protein Kinase Akt

ALL Acute Lymphoblastic Leukemia

AML Acute Myeloid Leukemia

ANOVA Analysis of Variance

BC Blast Crisis

BCA Bicinchoninic Acid

BCR B Cell Receptor

BCR-ABL Breakpoint Cluster Region - Abelson Murine Leukemia fusion gene

BIS Bisindolylmaleimide

BLNK B Cell Linker Protein

BTK Bruton's Tyrosine Kinase

CD Cluster of Differentiation cDNA Complementary Deoxyribonucleic Acid

CHX Cycloheximide

CLL Chronic Lymphocytic Leukemia

CML Chronic Myeloid Leukemia

CNS Central Nervous System

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cRNA Complementary Ribonucleic Acid

Ct Threshold Cycle Number

DAPI 4,6 diamidino-2-phenylindole

DHE Dihydroethidium

DLBCL Diffuse Large B-Cell Lymphoma

DMSO Dimethyl Sulfoxide

DSPE Distearoyl Phosphatidyl Ethanolamine

DUSP Dual Specificity Phosphatase

Eµ Enhancer of Immunoglobulin Heavy Chain µ

EDTA Ethylenediaminetetraacetic Acid

FcγRIIb Fc γ receptor II B

FDA Food and Drug Administration

FITC Fluorescein Isothiocyanate

GEA Gene Expression Analysis

GEP Gene Expression Profiling

H2O2 Hydrogen Peroxide

HCC Hepatocellular Carcinomas

HCP Hematopoietic Cell Phosphatase

HPLC High Performance Liquid Chromatography

HPβCD Hydroxypropyl-β-Cyclodextrin hROR1 Human ROR1

HRP Horseradish Peroxidase

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IACUC Institutional Animal Care and Use Committee

IC50 50% Inhibitory Concentration

IGVH Immunoglobulin Heavy chain Variable region

ILPs Liposomal Immunonanoparticles

IPA Ingenuity Pathway Analysis

JAK Janus kinase kDa Kilo-Dalton

LC-MS Liquid Chromatography–Mass Spectrometry mAb Monoclonal Antibody

MAPK Mitogen Activated Protein Kinase

MCL Mantle Cell Lymphoma me Moth Eaten

MFI Mean Fluorescence Intensity mRNA Messenger RNA

MS Multiple Sclerosis

MTD Maximum Tolerated Dose

mTOR Mechanistic Target of Rapamycin (serine/threonine kinase)

N-AC N-Acetyl Cysteine

NFκB Nuclear Factor kappa-light-chain-enhancer of activated B cells

NHL Non-Hodgkin's Lymphoma

NK Cell Natural Killer Cell

P32 Phosphorous 32

PARP Poly (ADP-ribose) Polymerase 1

PBS Phosphate Buffered Saline

PC Phosphatidylcholine

PCR Polymerase Chain Reaction

PEG Polyethylene Glycol

PI Propidium Iodide

PI3K P hosphatidylinositol-4,5-bisphosphate 3-kinase

PKC Protein Kinase C

PLCγ Phosholipase C γ

PMA Phorbol-12-myristate-13-acetate

PP2A Protein Phosphatase 2A

PTEN Phosphatase and Tensin Homolog

PTPN Protein Tyrosine Phosphatase, Non-receptor type

RBC Red Blood Cell

RMA Robust Multi-array Average

RNA Ribonucleic Acid

ROR1 Receptor tyrosine kinase like Orphan Receptor 1

ROS Reactive Oxygen Species

S1PR Sphingosine-1-Phosphate Receptor

SCID Severe Combined Immunodeficiency

SD Standard Deviation

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

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SFK Src Family Kinase

SH Src Homology

SHP1 Src Homology containing Phosphatase

SLP76 SH2-domain-containing leukocyte protein of 76 kDa

SMIP Small Modular Immunopharmaceutical

SNP Single Nucleotide Polymorphism

STAT Signal Transducer and Activator of Transcription

SYK Splenic Tyrosine Kinase

TBST Tris Buffered Saline with Tween

TCL1 T Cell Leukemia 1

TCR T Cell Receptor

Tg Transgene

TKI Tyrosine Kinase Inhibitor

Tris Tris(hydroxymethyl)aminomethane

WBC White Blood Cell

ZAP-70 Zeta-Chain (TCR) Associated Protein Kinase

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Introduction

Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia (CLL) is the most prevalent adult hood leukemia in the western world with estimated new cases of 15720 for 2014 in the United States(1) . The disease is characterized by accumulation of immune in-competent mature B cells expressing CD19, CD5, CD23 and low levels of membrane IgM, IgD in blood, bone marrow and other lymphoid organs (2, 3). The disease is heterogonous in nature that some patients need early therapy intervention while others do not need treatment until the symptoms worsen. The disease course can be staged according to the lymphocytosis, nodal involvement, organomegaly, anemia and thrombocytopenia. The clonal proliferation and accumulation of B cells is not only due to cell intrinsic defects but also nourished by the signals from the microenviromental cellular components including T cells, macrophages, stromal dendritic cells. These signals are mediated through cytokines, chemokines, growth factors, and cell surface ligands(4, 5). The Rai (0-IV) and Binet (A-

C) staging of CLL allows one to carefully select when the treatment is of appropriate value (6-8). However, based on the differential expression of markers like immunoglobulin heavy chain variable region (IgVH) mutation status, CD38 and ZAP70,

CLL can be classified into indolent and aggressive prognostic groups (9-11). The

indolent course of the disease is characterized by accumulation of IgVH gene mutated

CLL clones with low number of CD38 or ZAP-70 expressing B cells, In contrast, the aggressive course is distinguished by accumulation of IgVH-gene unmutated clones with high CD38 or ZAP-70 expressing B cells (10-14). Although these classifications are available, early intervention do not provide survival advantage. Identification of novel therapeutic or prognostic markers may change the paradigm to bring survival advantage with early interventions.

Current therapies for CLL

Current standard of care for CLL patients mostly include a combination of chemotherapeutics and biologicals. Alkylating agents like chlorambucil and bendamustine and purine analogues like pentostatin, cladarabine and fludarabine are cytotoxic agents that are currently available for treating CLL as first and second line therapy agents. Even though purine analogues, as single agents, yielded higher complete remission compared to other conventional treatment regimen like CHOP

(cyclophosphamide, doxorubicin, vincristine, prednisone) or CAP (cyclophosphamide,

Adriamycin /doxorubicin, prednisone) or chlorambucil, purine analogues had not increased the over-all survival of CLL patients(5, 15). The drug adverse effect associated with cytotoxic agents necessitated the evaluation of antibody therapeutics in CLL.

Rituximab, an anti-CD20 antibody was effective in combination with chemotherapy, although it was less active as low dose single agent(16). Several modifications to the

functional domains of the antibody led to emergence of new anti-CD20 antibodies such as Ofatumumab with increased CDC, Obinutuzumab (GA101) with potent direct cytotoxicity with different cytotoxic mechanisms, which are in clinical trials(17).

Alemtuzumab is a humanized monoclonal antibody against CD52 that is used as salvage therapy in high risk 17p deleted, 11q deleted or p53 mutated patients and in patients who have failed second line therapies(18, 19). Various combinations of chemo immunotherapy are utilized as frontline therapies and the selection of drug is based on the clinical stage; fitness of the patients; genetic abnormalities and the history of previous treatments and response(20). Additionally several novel agents are being developed and are in different phases of preclinical stages or clinical trials(5, 21). Although only curative against CLL, allogenic hematopoietic stem cell transplantation (HSCT) is being considered only for high risk younger patients because of transplant related mortality in elder patients mainly due to graft versus host disease(21). The recently approved orally bioavailable irreversible BTK inhibitor, ibrutinib for treating CLL patients that have undergone at least one previous therapy is notable(22, 23).

Role of Kinases in leukemia

The tumor signaling pathways are largely affected by protein phosphorylation, which is controlled by kinases and their functional antagonist phosphatases. While kinases with altered functions have known to induce certain tumors, it is logical to assume that phosphatases play a key role in tumorogenesis. Taking this cue, many studies have

identified phosphatases as dynamic and regulated enzymes affecting tumorogenic pathways(24, 25). In addition to their diversified roles in cell growth, development, differentiation and death, several kinases have been reported to play important role in tumor genesis and metastasis of various cancers at least in part by over expression at cancer amplicons (26-29). The primary pathogenic role of kinases in cancers is mediated through their deregulation or increased expression as a result of fusion gene. The kinases are classified based on the substrate specificity into tyrosine and serine-threonine kinases.

Based on their structure and function tyrosine kinases can be further classified into receptor and non-receptor kinases. Kinases can also be grouped based the sequence of the substrates they phosphorylate(27). Receptor tyrosine kinases have extracellular ligand binding domain and intracellular catalytic domain. The kinases can be targeted by inhibiting its activity by use of small molecules that block ATP binding site, interfering binding partner interaction or by use of RNA interference(30). In patients with CML, and in some patients with AML or ALL, the BCR-ABL fusion gene, the so called

Philadelphia-chromosome formed as result of chromosomal translocation 9:22 underlies the disease pathogenesis and targeting the BCR-ABL kinase with inhibitor significantly increased the survival outcome(31, 32). In hairy cell leukemia(HCL), BRAF V600E activating mutation seems to have etiological role and inhibition of BRAF is persuaded in on-going clinical trials (33, 34). The gain of function or activating point mutations in c-kit have been described in mast cell tumors, AML blast and other solid tumors contributing tumor cell survival and proliferation(35, 36). The cyclin dependent kinases

(CDK) include large family of serine-threonine protein kinases activated by

phosphorylation by other kinases and in association with cyclins (37) and control the cell cycle. Emerging CDK inhibitors are proved to induce apoptosis in a variety of leukemia including CLL(38-42). FMS-like tyrosine kinase-3(Flt-3) is expressed in CD34+ uncommitted cells in bone marrow and in blasts of AML. Domain duplication and point mutations activating FLT3 kinase are seen in AML and are therapeutically targeted with inhibitors(43). BTK is a member of TEC family of kinases that is over expressed at mRNA and protein levels in CLL. BTK kinase activity is important for the survival and expansion of CLL cells and treatment with ibrutinib inhibited the microenvironmental signal-induced proliferation of CLL cells(44, 45). Further, results from phase 2 clinical trials concluded that ibrutinib treatment resulted in durable remission in relapsed or refractory CLL patients including patients with high-risk genetic aberrations(23).

However, with the advent of high-tech gene sequencing new kinase mutants providing different constitutive signaling through downstream kinases or signal proteins are being identified(46, 47). A minority of CLL patients treated with ibrutinib developed resistance to the drug by acquiring C481S point mutation in BTK and therefore affecting ibrutinib binding or R665W/L845F gain-of-function mutations in PLCγ2 leading to autonomous

B cell receptor signaling(47).

The B cell receptor(BCR) signaling, that involves interaction of proteins that are often modified by phosphorylation through the interplay of kinases and phosphatases, determines the cell survival and death fate (48-50). The antigenic BCR stimulation after ligand binding results in phosphorylation of immunoreceptor tyrosine-based activation

motif (ITAM) of Igα/Igβ by SRC and creates a docking site for SYK, which in turn activated by phosphorylation by SRC(51). SYK transmits the BCR signal through activation of downstream kinases BTK, PI3K leading to increased activation of other kinases such as PKC, MAPK, AKT and mTOR some of which eventually promote the nuclear localization of transcription factors like NFκB and NFAT, resulting in cell survival and differentiation(52, 53). The BCR signaling plays an important role in pathophysiology of CLL especially from signals derived from microenvironment and targeting BCR is of vital importance in CLL (54-58). The microenvironment include extracellular matrix, lymphoma associated macrophages (LAM), nurse like cells(NLC), mesenchymal stromal cells, other blood cells including T cells and NK cells(56). Some of the mediators secreted or contributed by microenvironment include BAFF, APRIL,

CD31 by NLC; CXCL12, CXCL13, VCAM-1, fibronectin by stromal cells and CD154 by T-cells(4, 56, 57). The kinases associated with BCR signaling also contribute to

CXCL12/13 chemokine dependent migration by signaling through CXCR4/5 in CLL cells(4). It is also indicated that antigen independent tonic signaling of BCR occurs in

CLL cells and contributes to cell survival(59, 60). Currently several inhibitors targeting

BCR signaling kinases SYK, BTK, PI3K, mTOR are being tested in CLL in different clinical trials. (50, 55, 61-66). These kinase inhibitors also affect the tumor microenvironment resulting in redistribution of the leukemic cells from the nodes resulting in peripheral lymphocytosis(55). Use of inhibitors against kinases in BCR signaling may further increase the risk of infection in already immunocompromised leukemic patients (55). Despite extensive focus on the kinases, phosphatases as

therapeutic target in CLL has not been explored.

Role of Phosphatases in leukemia

The limited number of phosphatases and their broad substrate specificity make modulation of phosphatases in a specific cell type amenable and therapeutically beneficial(67). Multiple phosphatases have been demonstrated to be tumor suppressors

(24, 25, 68, 69). While targeting kinases using specific inhibitors is actively pursued by several groups, activation of phosphatases as therapeutic approach has not been explored due to lack of phosphatase activators. This project is aimed at modulating protein phosphatase SHP-1 and PP2A, two well known tumor suppressor protein phosphatases, using a small molecule OSU-2S. Phosphatase targeted therapy may provide alternative to kinase inhibitors in situations where kinase inhibitors are ineffective due to escape mutations/polymorphisms of drug targets yielding drug resistance(47). Further, many of the kinases involved in BCR signaling have overlapping function(70) and therefore inhibiting one specific kinase may not be advantageous. Given the broad substrate specificity of phosphatases and restricted expression and/or lowered activity in many leukemia, there is a high potential for modulating phosphatases for therapy. Down- regulation or inactivation of PTEN, SHP1 orPP2A have been reported in many leukemia(71-80). Postnatal knockdown of PTEN in hematopoietic cells in mice resulted in development of T-cell leukemia(81). More recently, RAG mediated micro deletions of

PTEN genes have been identified in T-cell acute lymphoblastic leukemia(T-ALL)(82).

Reduced enzyme activity of PTEN despite the high protein levels is seen associated with adult B-cell acute lymphoblastic leukemia with hyper activated PI3K-AKT signaling axis(75).

SHP1/PTPN6 is a tyrosine phosphatase primarily expressed in hematopoietic cells and functions as a negative regulator of many receptor tyrosine kinase signaling. It balances the kinase activity and cellular functions and is a well recognized tumor suppressor in leukemia/lymphomas (83, 84). At least four isoforms of SHP1 have been reported which arises by alternate splicing and exon skipping in N and C terminal region of the protein

(85, 86). SHP1 is predominantly expressed in hematopoietic cells but other cell types like epithelial, muscle cells also express SHP1. The function of SHP1 became unveiled after the identification of pathology of motheaten mice. Spontaneous point mutations in gene coding hematopoietic cell phosphatase (HCP) causes alternate splicing leading to non- functional protein products in motheaten mice characterized by their massive expansion of myeloid cells leading to systemic autoimmunity and patchy dermatitis (87-89). SHP1 is the human orthologue of mouse HCP and negatively regulates BCR signaling through binding to ITIM of FcγRIIb (90, 91). Though the complete spectrum of SHP1 substrates are not identified, some of its substrates in immune cell signaling include Src family kinases, SYK, ZAP70, BLNK/SLP76, STAT (92, 93). SHP1 also plays inhibitory roles in other hematopoietic cell types including T cells, NK cells, monocytes, macrophages and granulocytes affecting MAPK, JAK-STAT, PI3K-AKT and NFκB pathways (84, 93, 94).

The expression of SHP1 is drastically lowered in many leukemia and lymphomas due to

DNA promoter hyper methylation (68, 79, 95, 96) implying negative role of SHP-1 in development of leukemia/lymphoma (69, 97). Moreover, restoration of SHP1 in leukemia having low expression or devoid of SHP1 resulted in sensitization or direct death of tumor cells (25, 78, 98). Further, it was recently shown that SHP1 is directly involved in promoting death signals in CLL cells after ligation of tetraspanin CD37 (99).

Experimental data exists for SHP1 suppression mediated transformation of AML cells as one of the mechanisms of FLT3 internal tandem duplication mutations(100).

The reduced activity of PP2A due to activated oncogenic kinases plays a critical role in pathogenesis of CML(101). PP2A activating drugs have been shown efficacious in TKI resistant CML stem cells(102). It has also been demonstrated that PP2A activation by inhibition of SET, an endogenous PP2A inhibitor resulted in increased anti-leukemic activity of tyrosine kinase inhibitors and underscores the phosphatase activation as a parallel phenomenon that can be pursued as therapeutic approach(103). Moreover, PP2A mediated apoptotic effect of phenothaizines have been described in T-ALL(104). Not all phosphatases exhibit anti-leukemic role; PTPN2 have been associated with stemness in

CML with activation of the STAT5A and β-catenin and DUSP6 expression may promote

FLT3 ITD-mediated AML transformation (105, 106).

FTY720

FTY720 (Fingolimod) is a synthetic drug derived by modification of an immunosuppressive metabolite myriocin (ISP-1) from fungus Isaria sinclairii (107, 108).

FTY720 has been shown to affect the trafficking of lymphocytes and prolong the graft survival in the transplantation models (108, 109). FTY720 acts as a sphingosine analogue which gets phosphorylated by sphingosine kinases in-vivo and effluxes out of the cell where it binds to sphingosine-1-phosphate receptors (S1PR) on the cell surfaces in autocrine or paracrine fashion (110-113). In lymphocytes binding of phosphorylated

FTY720 to S1PR1 causes internalization and degradation of receptors leading to S1PR1 receptor down regulation. Thus FTY720 acts as a functional antagonist of S1PR1 and thwart the egress of lymphocytes from lymph nodes (114-116). As the lymphocytes are dependent on the S1PR1 signaling to egress out of lymph node, treatment with FTY720 causes reversible reduction in peripheral lymphocyte counts (117, 118). This immunosuppressive property of FTY720 has led to promising clinical trials in treating

Multiple Sclerosis (MS). FTY720 is approved by FDA in 2010 for treating relapsing MS where FTY720 reduces the peripheral blood lymphocytes and thereby decreasing the auto reactive T cells in CNS (119, 120).

FTY720 in solid tumors

Antitumor effects of FTY720 have been studied in different types of solid tumors and heme-malignancies (121-129). Bcl-2 associated, apoptosis with decreased ERK activity

was reported in renal cell carcinomas treated with FTY720 (123). FTY720 was also tested in hepatocellular carcinomas (HCC) and found to be active in in-vivo orthotropic and ectopic mouse models. The antitumor activity was attributed to the production of reactive oxygen species (ROS) resulting in PKCδ activation and caspase cleavage (126).

Though PKC function as survival kinase, apoptosis driving signal of PKCδ is dependent on the phosphorylation status of its tyrosine residues 64, 187 and 311. This preclinical study also emphasized the suitability of FTY720 in treating cancers with high cellular

ROS and less antioxidant. Early administration of FTY720 impaired the tumor development in urethane-induced lung tumor in mouse models but delayed administration lead to aggressive form presumably due to reduced peripheral leukocytes which are necessary elements in tumor surveillance (127). Treatment of gastric cancer cells with

FTY720 caused PTEN up regulation leading to impaired PI3K-Akt-MDM2 axis and increased p53, eventually promoting G1 arrest and apoptosis (130). In androgen independent prostrate tumor xenograft model, FTY720 administration caused potent antitumor effect linked to anti-angiogenic effect as evidenced by reduced angiogenic marker VEGF. (131). FTY720 treatment of PC-3 prostate cancer cells lowered the expression and transcription activity of Runx and promoted cadherin switching reversal and prevented type I collagen invasion (132).

FTY720 in hematologic malignancies

Preclinical studies of FTY720 in hematologic malignancies especially in myeloid and lymphoid leukemia have uncovered the potential action of FTY720 beyond its

immunosuppressive property. An early investigation suggested that anti tumor activity of

FTY720 in leukemic cell lines involved activation of protein phosphatase 2A (PP2A) or

PP2A like phosphatase culminating in dephosphorylation of phospho AKT (133). PP2A is one of the major serine-threonine phosphatases which regulates variety of cellular events ranging from cell cycle, transcription, translation and transduction of signals involving kinases (134). Loss of PP2A activity has been reported in blast crisis chronic myelogenous leukemia (CML-BC) and functional inactivation of PP2A by its physiologic inhibitor SET is necessary for BCR-ABL leukemogenesis in Philadelphia-chromosome positive leukemia like CML and Ph(+) B cell acute lymphoblastic leukemia(B-ALL)

(97). Activation of PP2A using FTY720 in CML-BC and Ph (+) ALL primary cells caused potent cytotoxicity selective to that of leukemic population. The study also highlighted that anti leukemic effects of FTY720 is independent of its phosphorylation and sphingosine-1-phosphate receptor signaling. More over pharmacological administration of FTY720 inhibited BCR-ABL leukemogenesis in xenograft mouse models without evidence of adverse effects (135). FTY720 also caused PP2A dependent,

Bcl-2–independent mechanism of cytotoxicity in CLL primary cells and lymphoblastic leukemia cell line models and xenograft animal models (125). Treatment of mantle cell lymphoma (MCL) cells with FTY720 resulted in down modulation of cyclin D1 leading to cell cycle arrest and reduced phospho AKT (128). In addition, FTY720 in MCL increased the cytosolic and surface expression of CD74 a target for milatuzumab through blockage of autophagy, thereby sensitizing MCL cells to milatuzumab cytotoxicity both in-vitro MCL cells and in in-vivo mouse models (129). In spite of its potent antitumor

effect and clinically approved for treating MS, the T-cell immunosuppressive property of

FTY720 hindered its further development as an anti-cancerous agent and warranted the development of successor analogues preserving the anti-tumor effect without immunosuppressive property.

OSU-2S a non-immunosuppressive FTY720 analogue

OSU-2S {(S)-2-amino-2-(4-{(6-methylheptyl)-oxy}phenethyl)pentan-1-ol} is a synthetic derivative of FTY720 by structure activity relationship with more potent anti tumor activity but lacks the S1PR mediated immunosuppressive effects (136). The non- immunosuppressiveness of OSU-2S has been attributed to three reasons: i) Unlike

FTY720, OSU-2S is not phosphorylated by sphingosine kinase-2 in in-vitro system. ii)

Synthetic phosphorylated OSU-2S failed to internalize S1PRs in cell lines expressing

S1PR but are internalized when treated with FTY720 or phosphorylated FTY720. iii)

Administration of OSU-2S to CD2F1 wild type mouse did not cause peripheral blood T cell count reduction but administration of FTY720 caused drastic reduction in T cell count in periphery(137). Both FTY720 and OSU-2S mediated cytotoxicity in HCC cells and involve activation of NADPH oxidase system and PKCδ, but the potency of OSU-2S is two-fold higher than FTY720 as phosphorylation of FTY720 is conceived to inactivate its anti tumor activity. The IC50 of OSU-2S in HCC cell lines Huh7, Hep3B, and PLC5 after 24 hours treatment were 2.4µM, 2.4µM and 3.5µM respectively. OSU-2S activated

NADPH oxidase in HCC cells through up regulation of gp91phox leading to ROS

generation which in turn activated PKCδ and subsequently the effector caspase 3.

However, OSU-2S cytotoxicity was independent of PP2A, PTEN, p53 and Mcl1 in HCC.

Moreover, daily administration of OSU-2S at 10 mg/kg for 42 days reduced the xenografted tumor volume by > 50% in nude mouse. The potency of OSU-2S as single agent established in in-vivo ectopic and orthotropic HCC mouse models further corroborate its translational significance. To support the proof-of-principle we have generated a custom mouse model expressing human ROR1 (hROR1) surface protein on all leukemic B cells which further closely resembled CLL. This dissertation illustrates the preclinical studies of OSU-2S in CLL and other B cells malignancies. The outlined studies include detailed description of methodology, mechanistic and preclinical evaluation of OSU-2S in B cell lines and CLL patient derived primary cells, molecular pharmacology of OSU-2S in CLL primary cells, in-vivo activity of OSU-2S in novel mouse models of CLL and mantle cell lymphoma using targeted delivery formulations, and activity of OSU-2S in canine B lymphoma. The implications of these findings, potential clinical application of OSU-2S for CLL and other B cell malignancies including

MCL and canine lymphoma are included in the final discussion.

Materials and Methods

Cells

Peripheral blood was obtained from CLL patients after informed consent in accordance with the Declaration of Helsinki and under protocol approved by The Ohio State

University hospital internal review board. All primary CLL cells used for the study are immunophenotypically defined as CLL as outlined by the modified 1996 National Cancer

Institute criteria(138). CLL B cells were isolated from freshly collected patient blood using “Rosette-Sep” kit (#15064, Stem Cell Technologies; Vancouver, BC, Canada) and ficoll density gradient centrifugation (#17-1440-03, Ficoll-Paque Plus; GE Healthcare,

Amershan Biosciences, Piscataway, NJ) according to the manufacturer’s instructions.

Normal B cells were purified from leukopaks purchased from American Red Cross,

Central Ohio (Columbus, OH). MCL primary cells were obtained from patients blood or lymph node biopsy under approved protocol of the institutional review board (IRB).

Mononuclear cells were isolated by ficoll density gradient centrifugation. Isolated mononuclear cells were cultured in RPMI 1640 media (#21870-084/-700, Gibco, Life

Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (#F4135-500ml, Sigma-Aldrich, St Louis, MO); 2 mM L-glutamine (#25030,

Gibco) and penicillin (100 U/mL); streptomycin (100 µg/mL) (#15140, Gibco) at 37°C with 5% CO2 using tissue culture flasks (BD Falcon). Human Burkitt lymphoma cell

lines Raji and Ramos, MCL cell lines JeKo and Mino were obtained from American

Type Culture Collection (ATCC, Manassas, VA) and MEC-1 and MEC-2 cells were obtained from DSMZ-German Collection of Microorganisms and Cell Cultures

(Braunschweig, Germany). Canine DLBCL cells were obtained by fine needle aspiration biopsy(FNAB) of lymphoma mass from canine patients diagnosed with DLBCL in OSU

Veterinary Medical Center under approved protocol and were cultured as described earlier.

Cell viability and apoptosis

Cell death was assessed by dual staining with annexin-V-FITC(#556419) and propidium iodide (PI) (#556463) (BD Bioscience, San Jose, CA) followed by FACS analysis using

Beckman-Coulter model EPICS XL cytometer. Briefly one million cells were suspended in 200µl of annexin binding buffer (# 556454, BD Bioscience, San Jose, CA) and stained with annexin-V-FITC and PI for 15 minutes in dark and read in cytometer. Metabolic activity of the cell lines was measured by CellTiter96® MTS {3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium} assay (#G111A,

Promega, Madison, WI) according to manufacturer’s instruction. Live dead stain

(Invitrogen) was used in combination with surface markers for flow cytometric experiments. Flow cytometric data were analyzed using Kaluza software (Beckman-

Coulter). Q-VD-OPH pan-caspase inhibitor (#030PH10901, MP Biochemicals, Solon,

OH) was used at 20µM for primary CLL cells.

Staining of cell surface markers

1x106 cells from culture were pipetted into a 5ml round bottomed tube (BD Falcon) and washed with 1ml of PBS and resuspended in 200 µl ice cold PBS containing required flow cytometric antibodies or staining reagents. The tubes were incubated on ice for 30 minutes in dark. For tubes that needed secondary antibody, it was added 20 minutes after the addition of primary antibody. The cells were then washed with 1ml of ice cold PBS and resuspended in 200-500µl ice cold PBS and read in the flow cytometer. Gating was done using the FMN (fluorescence minus one) staining technique and the flow cytometric data were analyzed used Kaluza software.

Staining for ROS

1x106 cultured CLL primary cells were taken in a 5ml round bottomed tube and stained with 10µM dihydroethidium (DHE) in the culture media for 30 minutes at room temperature along with live dead stain (0.5µl/tube). The cells were then washed with room temperature PBS and resuspended in 200µl of PBS before analyzed in flow cytometer. ROS is detected by fluorescence excitation⁄emission of 518⁄605nm on DHE stained cells (DHE positive). Hydrogen peroxide (H2O2) treated CLL cells (0.5% final concentration) for 15 minutes was used a positive control.

Immunoblotting and immunoprecipitation

Cells were washed with PBS and lysed using ice cold lysis buffer containing 10mM Tris

(tris(hydroxymethyl)aminomethane) pH 7.4, 150mM sodium chloride, 1% Triton X-100,

1% deoxycholic acid, 10% SDS and 5mM EDTA with protease inhibitor (#P8340,

Sigma), phosphatase inhibitor cocktails II and III (#P5726 and #P0044, Sigma) and 100 mM PMSF(# P7626,Sigma) added just before lysing the cells at 1:100 proposition. Total cell lysates were sonicated for complete nuclear lysis. Cytoplasmic and nuclear extracts were prepared with NE-PER extraction kit (#78833/78835, Pierce, Thermo scientific,

Rockford, IL). Proteins were quantified by bicinchoninic acid (BCA) method (#BCA

PASS R-A 23223 and R-B 23224, Pierce, Thermo Scientific) and separated by SDS-

PAGE gel, transferred to 0.2µM nitrocellulose membrane (#162-0112, Bio-Rad).

Precision plus protein standards (Kaledioscope, #161-0375, Bio-Rad Laboratories,

Hercules, CA) were used for protein size determination in kDa. The blots were probed with commercially obtained indicated antibodies, followed by horseradish peroxidase

(HRP) conjugated secondary antibodies (goat anti-rabbit #170-6515 and goat anti-mouse

#170-6516 Bio-Rad) and detected by addition of chemiluminescent substrate (Super signal West Pico #34080 or Femto #34095 Pierce, Thermo Scientific). Quantification was done with Chemi-Doc system with Quantity one software (Bio-Rad Laboratories,

Hercules, CA). Immunoprecipitation was carried out by incubating cell lysates with primary antibody and protein A agarose beads (#16-125, Millipore, Billerica, MA) in Tris buffered saline-tween20 (TBST) overnight at 4°C in a rocker, followed by washing the beads 6 times with ice cold TBST. The beads were boiled with 30µl of 6x Laemmeli buffer before separation by SDS-PAGE.

Phosphatase assay

SHP1 and PP2A enzyme activity were measured using DuoSet®IC (#DYC 2808, R&D

Systems, Minneapolis, MN) and PP2A immunoprecipitation phosphatase assay kit (#17-

313, Upstate/Millipore, Billerica, MA) respectively as per manufacturer’s instructions. In brief, phosphatase enzymes were immunoprecipitated from CLL cell extracts using anti-

SHP1/PP2A antibody conjugated/and agarose beads and incubation of enzyme bound beads with phospho peptide substrate to release free phosphate, followed by detection using malachite green solution and molybdic acid. The phosphatase activity is expressed as amount of free phosphate released from the reaction normalized to the levels of enzyme immunoprecipitated as quantified by immunoblotting to avoid variability in immunoprecipitation across the samples.

Chemicals and reagents

OSU-2S was synthesized at the Ohio State University Medicinal Chemistry Shared

Resource (MCSR) as described previously(136). Nuclear magnetic resonance and mass spectrometry were used to confirm the purity and identity of the compound. FTY720 was purchased from ChemieTek (# CT-FTY720, Indianapolis, IN) and used for in-vitro and in-vivo studies. Di-methyl sulfoxide (DMSO) ( #D128-500, Fisher Scientific) was used to dissolve FTY720/OSU-2S for in-vitro studies. 25% (2-Hydroxypropyl)-β-cyclodextrin

(HPβCD) (#H107, Sigma-Aldrich) was used to dissolve OSU-2S for in-vivo studies.

Dulbecco's Phosphate-Buffered Saline (PBS) (#21600-069, Gibco), Phorbol-12- myristate-13-acetate (PMA) (#524400, Calbiochem, Millipore, Billerica, MA);

Bisindolylmaleimide (#9841, Cell Signaling Technology, Danvers, MA) were obtained from the indicated vendors. Egg phosphatidylcholine (Egg PC, LIPOID E PC, Lipoid,

Newark, NJ), methoxy-polyethylene glycol (MW≈2,000Da)-distearoyl phosphatidyl- ethanolamine (PEG-DSPE, #880128P, Avanti Polar Lipids, Alabaster, AL), 2-

Iminothiolane (Traut's reagent, #26101, Thermo, Rockford, IL), cholesterol (Chol, #

C8667, Sigma, St. Louis, MO), DSPE-PEG-maleimide (DSPE-PEG-mal, # 880126P,

Avanti Polar Lipids) were used for immunoliposomal nanoparticle formulation. PKC activity was measured using PepTag assay kit (#V5330, Promega, Madison, WI). Steritop filter units with pore size 0.22µm (#SCGPT05RE, Millipore) were used for filter sterilizing the reagents.

Antibodies for indicated proteins were purchased from following vendors phospho S591

SHP1(#SP1531, ECM Biosciences); GAPDH(Clone 6C5, #MAB374), SHP1(Clone

HG213, #05738), PP2Ac (Clone C1D6, #05421) (Millipore); Actin(Clone I-19, #SC-

1616), Brg1(Clone G-7, #SC-17796), Cyclin D1(Clone DCS-6, #SC-20044) (Santa Cruz

Biotechnology, Dallas, TX); TCL1A (Clone 27D6/20, #K0028-3, MBL International),

Caspase 3(#9662), PARP(#9542), phospho-Src Family (Tyr416) (#2101) (Cell Signaling

Technology, Danvers, MA). Goat F(ab’)2 against human IgA+IgG+IgM (H+L) (#109-

006-064, Jackson ImmunoResearch Laboratories, West Grove, PA) was used at 6.5

µg/ml for cross linking BCR in cultured CLL cells. Goat anti-human IgG Fc fragment specific (#109-005-008, Jackson ImmunoResearch Laboratories) was used to cross link the humanized antibodies in cell cultures. Human CD19-FITC/PE (Clone HIB19), CD20-

PE (Clone L27), CD3-FITC/PE (Clone UCHT1), CD45-APC (Clone HI30), CD74-FITC

(Clone M-B741), CD86-FITC (Clone FUN-1) and mouse B220-FITC/PE/APC-Cy7

(Clone RA3-6B2), CD19-PE (Clone 1D3), CD3-FITC/PE, CD3-PE-Cy7 (Clone 17A2),

CD5-FITC/PE (Clone 53-7.3), CD45-APC (Clone 30-F11), Streptavidin-PE (#349023,

BD Bioscience); human ROR1 Biotinylated/ Alexa Fluor 488 (#FAB2000G, R&D

Systems) were used for flow cytometric analyses. 2A2-IgG against human ROR1 was kind gift from Dr.Christoph Rader, CCR, NCI.

Quantitative RT-PCR

Cells were suspended in TRizol®(Ambion, Life Technologies) reagent and kept at -70°C before RNA was extracted according to manufacturer’s instruction and cDNA was made using random primers (Invitrogen, Life Technologies). Real-time PCR was performed using TaqMan (Life Technologies) gene expression assay probe-primer sets for TCL1A

(ID:Hs00951350_m1) and 18S (ID:HS03003631_g1) and ViiA™ 7 Real-Time PCR

System (Applied Biosystems). The expression of target genes relative to internal control gene was calculated using the threshold cycle number (Ct). The relative target gene expression for each condition was normalized to vehicle control and fold change determined using the comparative method (2ΔΔCt).

Gene expression-microarray analysis

RNA extracted from primary CLL cells that responded to OSU-2S was used for gene expression profiling (GEP) performed in Microarray Shared Resource (MASR) in the

Ohio State University Comprehensive Cancer Center. RNA quality was analyzed using

RNA Nano bioanalyzer to assess the integrity of the samples. The labeled, fragmented cRNA samples were hybridized to GeneChip Human Genome U133 plus 2.0

(Affymetrix, GPL570)(139). The data were normalized by RMA method and two-sample t-tests were used to detect differentially expressed genes. Smoothing method was applied to improve variance estimates in the tests(140). The expected false positive rate was controlled at 0.0005 (five false positive out of 10000 tests)(141). To identify possible molecular and cellular functions affected by OSU-2S, genes changed by >2 fold

(p<0.0005) were selected for function and network analyses using Ingenuity pathway analysis (IPA) (http://www.ingenuity.com).

Microarray Heat Map

Construction of heat map was done using the robust multichip average(RMA) values of vehicle and drug treated CLL samples and setting vehicle treated conditions close to zero on log2 scale. The average RMA value of vehicle treated CLL samples was calculated for each gene and then subtracted from both vehicle and drug treated CLL samples for the same gene for normalization. The normalized values were then divided by the vehicle treated CLL average RMA value for each gene for compression in the heat map. The resulting values were inputted in the open source software MultiExperiment Viewer

(MeV) obtained from http://www.tm4.org/mev.html for building heat map.

miR analysis

RNA was isolated from CLL primary cells treated with (OSU-2S 8µM) for 16hr using the acid-phenol:chloroform reagent and mirVana™ miRNA isolation kit (#AM1561,

Ambion, Life Technologies). miR expression analysis was done at the Nucleic Acid

Shared Resource (NASR), OSU using nCounter® Human v1 miRNA expression assay probe (NanoString Technologies, Seattle, Washington) as recommended by the manufacturer. The positive spike-in controls were used for a direct assessment of sample preparation conditions. Negative controls were used to assess background hybridization and for filtering out low expression probes. A quantile normalization method was used to normalize samples. Linear mixed models for correlated data was then used to detect differentially expressed miRs. In order to improve the estimates of variability and statistical tests for differential expression, a variance smoothing method with fully moderated t-statistic was employed. The significance level was adjusted by the mean number of false positives.

Preparation of immunonanoparticle 2A2-ILPs

Cholestrol: Egg-PC: PEG-DSPE (molar ratio 33.5: 65: 1.5) were used to prepare liposomes by forcedly injecting the above mixture prepared in absolute ethanol into 10X volume phosphate buffered saline(PBS) pH 7.4. Traut’s reagent was used to reduce 2A2-

IgG at room temperature to form 2A2-IgG-SH which was further incubated with DSPE-

PEG-mal pH 6.8 (molar ratio1:10) to form 2A2-IgG micelles. The liposomes and 2A2-

IgG micelles were combined (lipid : 2A2-IgG molar ratio 2000:1) and incubated at 37oC

for one hour to form 2A2-ILPs which were filter sterilized before used for cell culture or in-vivo experiments.

OSU-2S was encapsulated in liposomes by ethanol injection method before tethering of

IgG on the liposomal surface. OSU-2S was dissolved in absolute ethanol at 10µM before addition of lipids (Cholestrol, Egg-PC and PEG-DSPE) and the fully mixed OSU-2S and lipids (1:20) were forcedly injected into 10X volume PBS pH 7.4 to form OSU-2S-LP.

The encapsulation of OSU-2S inside the liposome had very minimal effect on mean diameter by volume of nanoparticles, however altered the zeta potential of nanoparticles from - 4.10±0.34 to 5.41±0.12 mV. The drug entrapment efficiency measured by dialysis and LC-MS/MS was 90.09 ± 2.69% for the liposomal formulation and 88.25 ± 4.81% after immobilization of 2A2-IgG on the surface.

Confocal fluorescence microscopy

Experimental cells were pre stained with membrane labeling dye PKH26 (#MINI26,

Sigma-Aldrich, St Louis, MO) just before the start of the experiment. After the experiment, cells were adhered to microscopic slides by centrifugation in a Cytospin 3

(Shandon) centrifuge and followed by fixation in ice cold acetone. Boundary of cell population was marked with glass marker and cells were blocked with 2% bovine serum albumin in PBS and stained with indicated primary antibodies overnight at 4°C, followed by Alexa Fluor 488 (#A11008, Molecular Probes; Life Technologies, Grand Island, NY) florescent labeled secondary antibody for 1 hour. Nuclei were stained with DAPI (4,6

diamidino-2-phenylindole) (#H-1200, Vector laboratories) along with mounting medium.

Images were collected using Olympus Fluoview 1000 Laser Scanning confocal microscope. Z stacks of 20 to 30 slices through the cell (0.4 µm) were collected for each slide. Images were processed with Olympus Fluoview (Version 3.0) software and represent 1 slice per slide through middle of nucleus.

In-vivo experiments

All animal experiments were carried out under protocols approved by The Ohio State

University Institutional Animal Care and Use Committee (IACUC). C57BL/6 animals

(Taconic Farm, Germantown, NY) were used for immunosuppressive studies comparing

FTY720 and OSU-2S. Eµ-TCL1 mice(142) with white blood cell (WBC) count >15 x

103/µl were used for cytoreduction studies. Animals meeting the criterion de-novo were grouped into Vehicle or OSU-2S and received three daily doses (5mg/kg) of treatment and were bled to assess WBC count by staining peripheral blood smear. CD19 and CD3 percentages were analyzed by flow cytometer after RBC depletion by incubating with

RBC lysis buffer (Ammonium Chloride 150mM, Potassium Carbonate 10mM and Di-

Sodium EDTA 0.1mM) for 15 minutes on ice followed by washing with PBS and staining for markers.

Eµ-ROR1 transgenic mice generation

A 3.5kb Not1 human ROR1 cDNA fragment from pCMV6-XL6 plasmid (kindly provided by Christoph Rader, NCI) was cloned into the Bgl2 site of the pBH vector that

was shown to direct B cell specific expression of transgenes(143). Expression of hROR1 was confirmed in 70/z3 mouse pre-B cell lines transfected with the above construct containing hROR1 cDNA. Transgenic mice were generated by pronuclear injection of about 6.5kb fragment released with Not1/Kpn1 from the pBH transgenic construct in fertilized oocytes from C57BL/6 animal at Genetically Engineered Mouse Modeling facility at the Ohio State University Comprehensive Cancer Center (OSUCCC). The founders were identified by southern blot analysis of tail DNA for the presence of human

ROR1 transgene using P32 labeled human ROR1 cDNA probe (Fragment size and restriction enzyme used to generate the probe). Two independent founder animals identified were bred to C57BL/6 to establish founder lines that were genotyped by PCR with primers specific for hROR1 using genomic tail DNA. Eµ-ROR1 transgenic mouse was crossed with Eµ-TCL1 mouse(142) (kindly provided by Carlo Croce, The Ohio State

University) to generate Eµ-ROR1-TCL1 double transgenic animals, that were confirmed for the expression of both human ROR1 and TCL1 transgenes. Mouse spleen single cell suspensions were made by smashing spleen in petri dishes using PBS and filtering the suspension in 70µM nylon strainer (#08-771-2, Falcon cell strainer, Fisher Scientific).

Mouse spleenocytes were cultured similar to human mononuclear cells with the addition of 2-Mercaptoethanol (#O3446I-100, Fisher Scientific) at the final concentration of 100

µM in the culture.

Engraftment of leukemic spleenocytes

Eµ-ROR1-TCL1 mouse engraftment studies were done by injecting 10x106 splenocytes isolated from Eµ-ROR1-TCL1 double transgenic animal with known frank leukemia and splenomegaly into Eµ-ROR1 single transgenic mouse by tail vein injection for leukemia adaptation. Upon confirmation of leukemia as evidenced by circulating

CD5+CD19+/B200+ cells and splenomegaly, the splenic cells were engrafted into syngenic C57BL/6 animals. Splenocytes isolated by ficoll density centrifugation were stained for B220, CD5, hROR1 expression and viability and about 10 x 106 cells were injected into tail vein of C57BL/6 recipient and the disease progression was monitored bi-weekly by flow cytometric analysis of peripheral blood. Animals that have developed high WBC count (>10 x 103/µl) and 5% B220+CD5+ leukemic cells in peripheral blood were grouped into treatment groups and dosed 10 mg/kg everyday by intra-peritoneal injection of ILP formulations. All animals were monitored for signs of disease and other early removal criteria, including greater than 20% weight loss or cancer induced cachexia, inability to feed or move around and other infections.

Peripheral blood staining for flow cytometry

Blood samples were collected from the mice by mandibular region needle stick using

26G needle into EDTA tubes(#02-669-33, BD Microtainer, Fisher Scientific). About 30-

50µl of blood was pipetted into 5ml round bottomed flow cytometric tubes and the required flow cytometric antibodies mixed in ~20-50 µl of PBS was added and mixed well with the blood samples and incubated on ice for 30 minutes in dark. Before reading

in the flow cytometer 50µl of CountBright™ Absolute Counting Beads (#C36950,

Invitrogen) and 500µl of ice cold PBS were added to each tubes. Events were collected using the CD45+ discriminator to read only the WBC. Gating was done using the FMN

(fluorescence minus one) staining technique. The absolute cell numbers were calculated using the bead events and cell events collected and the volume of blood sample and beads added in the tubes as per manufacturer instruction.

MCL Xenograft model

Animal experiments were done under the protocol approved by the Institutional Animal

Care and Use Committee (IACUC) of The Ohio State University. NOD.Cg-Prkdcscid

Il2rgtm1Wjl/SzJ (NSG) mice were purchased from Jackson laboratories(Bar Harbor,

Maine). 6 week old female NSG mice were engrafted with 4 million Mino cells in the left flank region by subcutaneous injection. All animals were monitored for signs of disease, tumor size by digital calipers and other exclusion criteria including tumors weighing

>20% of body weight, extended anorexia or other diseases. Treatment began after palpable tumors were noted (2 weeks after engraftment). Mice were randomly grouped and treated with 2A2-OSU-2S-ILP; IgG-OSU-2S-ILP or 2A2-Empty-ILP at the dose of

10mg/kg of OSU-2S daily for 10days by intra-peritoneal injections. All animals were sacrificed 25days post engraftment and the tumors were collected and weighed by a person blinded to the study.

Statistical analysis of data

All statistical analyses were performed by statisticians in Center for Biostatistics at the

Ohio State University using SAS 9.3 software (SAS, Inc, Cary, NC). For data with repeated measures, mixed effect models were used considering observational dependencies across subjects(144). Matched samples were compared by paired t-tests.

Analysis of variance (ANOVA) was used to compare means of multiple independent groups. The association between phospho SHP1 and change in cell viability was assessed by Spearman correlation test. The log-rank test was applied for analysis of animal survival study. Holm’s method was employed to adjust multiplicity to control the family wise error rate at 0.05(145).

Chapter 1: Preclinical activity of OSU-2S in CLL

To assess the activity of OSU-2S, we used various lymphoid leukemic cell lines representing malignant disease conditions and treated them with OSU-2S and measured the cellular activity after defined period of time. Raji, Ramos (Burkitt lymphoma), Jeko,

Mino (Mantle cell lymphoma), Jurkat (acute T cell leukemia) and MEC1 (CLL) were used. Parallel comparison of FTY720 and OSU-2S on the above cell lines indicated that

OSU-2S is more potent in reducing cellular activity compared to FTY720 by MTS assay which is measure of mitochondrial function (Figure 1). Cells (1x106/ml) were treated with indicated concentrations of FTY720/OSU-2S for 24hrs and values were normalized to vehicle treated conditions. Figures represent mean with SD of three independent experiments. Then we tested the cytotoxicity of OSU-2S in primary B-CLL cells isolated by negative selection from peripheral blood of CLL patients. OSU-2S had potent cytotoxicity with dose, time and cell density dependent manner in CLL as evident form increase in annexin V and/or PI positivity. Dose titration studies revealed IC50=1.72µM

(95%CI 1.59-1.84µM) for OSU-2S on CLL cells (1x106/ml) (N=6) at 24hr (Figure 2).

Moreover, the minimal exposure of OSU-2S for few hours had profound effect on the viability of CLL cells at 24hours which is revealed in the time kinetic study (Figure 3) where CLL cells (10x106/ml) (N=3) were treated with 8μM OUS-2S for definite period of time, washed and re-plated in culture media before viability was assessed at 24hours.

Interestingly, the effect of OSU-2S changed with cell density plated and more activity was seen in less dense cells (Figure 4). CLL cells of different seed density (10x106/ml;

5x106/ml and 1x106/ml) (N=5) were treated with 2μM OSU-2S and viability was assessed 24 hours later. The cell density dependency could be attributed to the paracrine soluble and direct survival signals that CLL cells are receiving when closely packed in the media. However, the survival of CLL cells seeded at higher density can be overcome by increasing the concentration of OSU-2S to 8 µM(not shown). The summary of activity of 24hour treatment with 2µM OSU-2S in patients (n=25) derived primary CLL cells is shown in figure 5.

Cytogenetically chromosome 17p deletions (tumor suppressor p53 locus) and immunoglobulin variable heavy chain region (IGVH) gene unmutated CLL patients have minimal response to treatment and have very poor prognosis (12, 146). The promising preclinical activity of OSU-2S in prognostically poor CLL patient groups like chromosome 17p deletions (N=5) (Figure 6) and IGVH gene unmutated (N=8) (Figure 7) prompted us to evaluate this molecule for further studies. Rituximab an anti-CD20 antibody and fludarabine a purine analogue are the standard therapies used commonly used as first line therapy in CLL patients. So we decided to evaluate if OSU-2S can perform well in situations where the current therapies are not effective like drug resistant cell lines. To determine if OSU-2S mediated cytotoxicity against fludarabine and rituximab resistant cells lines we tested effect of OSU-2S on fludarabine resistant MEC2

(147) and rituximab resistant Raji cell lines(148). Cell lines were treated with different 31

concentrations of OSU-2S for 48hrs and the cellular metabolic activity was measured by

MTS assay. We found OSU-2S is active in rituximab resistant established Raji cell line clone 2R with dose dependency (linear trend significant: p < 0.0001) (Figure 8). Further,

OSU-2S is active in fludarabine resistant MEC2 cells (Figure 9). Consistent with its activity in fludarabine resistant MEC2 cell line, OSU-2S (8 µM) promoted cytotoxicity in primary CLL cells(10x106/ml) that are sensitive to resistant/refractory to fludarabine

(10 µM) (Figure 10). Moreover, the OSU-2S induced cytotoxicity is not dependent on caspase activation as concentrations of pan caspase inhibitor Q-VD-OPH that rescued fludarabine mediated cytotoxicity failed to rescue the OSU-2S induced cytotoxicity

(p<0.0001) which is illustrated in Figure 11. The top image is immunoblotting for caspase 3 and PARP, where both got cleaved by OSU-2S and fludarabine in the absence of Q-VD-OPH. But in the presence of Q-VD-OPH, unlike fludarabine treated cells, OSU-

2S treated cells still entered the apoptotic pathway without caspase 3 and PARP cleavage as illustrated in the bottom of the viability graph.

Figure 1: Activity of OSU-2S and FTY720 in lymphoid leukemic cell lines.

Figure 2: Dose dependent cytotoxicity of OSU-2S in CLL primary cells.

Figure 3: Time dependent cytotoxicity of OSU-2S in CLL primary cells.

Figure 4: Influence of cell density on OSU-2S cytotoxicity in CLL primary cells.

Figure 5: Activity of OSU-2S in CLL primary cells. (N=25)

Figure 6: OSU-2S is active in 17p deleted CLL primary cells.

Figure 7: OSU-2S is active in IGVH unmutated CLL primary cells.

Figure 8: Activity of OSU-2S in rituximab resistant Raji clone 2R.

Figure 9: Activity of OSU-2S in fludarabine resistant MEC2 cell line.

Figure 10: OSU-2S promotes cytotoxicity in CLL primary cells that are sensitive and refractory to fludarabine.

Figure 11: Caspase independent cytotoxicity of OSU-2S in CLL primary cells.

OSU-2S combinational therapy with phosphatase activating therapeutics

CD37 is a tetraspanin found in surface of CLL leukemic cells and its potential as target for monoclonal antibody has been described(149). Cross linking of CD37 on surface of

CLL cells by CD37 monoclonal antibodies or anti-CD37 SMIP(149) triggers survival signals through PI3K-AKT pathway and death signals through SHP1 activation(99). To determine if OSU-2S can be favorably combined with a phosphatase activating therapeutics, we used SHP1 activating CD37 targeting therapeutics that is currently in clinical trials for CLL. Patient derived CLL cells (N=6, 10x106/ml) are treated with 8µM

OSU-2S and anti-CD37 SMIP (5µg/ml) with cross linker and the viability was measured at 24hr by flow cytometry. Combination of OSU-2S and anti-CD37 SMIP enhanced the cytotoxicity significantly than the two treatments alone (Figure 12).

Figure 12: Combinational activity of OSU-2S and anti-CD37 SMIP on CLL primary

cells.

Chapter 2: Molecular pharmacology of OSU-2S in CLL

Tumor suppressor PP2A activation dependent cytotoxicity of FTY720 was well documented in CLL. To determine if OSU-2S can activate PP2A in CLL we performed

PP2A phosphatase activity assay using CLL primary cells treated with FTY720 or OSU-

2S. As expected OSU-2S a derivative of FTY720 still preserved the PP2A activation property as evident from comparable levels of activation with FTY720 (Figure 13). Since

PP2A activation resulted in SHP1 dependent BCR-ABL dephosphorylation, in myeloid leukemia treated with FTY720(135), we evaluated if OSU-2S affected the SHP1 by checking if PP2A and SHP1 co associate in response to OSU-2S treatment in CLL cells.

Interestingly, OSU-2S treatment resulted in increased phospho SHP1S591 that was co- immunoprecipitated with PP2A (Figure 14 left) as early as 4hrs post treatment. When analyzed for the phosphorylation status of SHP1 in total cell lysates abundant phospho

SHP1S591 was noted in OSU-2S treated conditions (Figure 14 right). Moreover, comparison of phospho SHP1S591 levels (5hr post treatment) and viability (24hr post treatment) in OSU-2S treated CLL patient cells showed an inverse correlation(N=20; rs =

-0.64) (Figure 15). While OSU-2S consistently induced increased phospho SHP1S591 levels, there was no change in the levels or enzymatic activity of SHP1 in CLL cells

(Figure 16). Consistent with this OSU-2S failed to alter the phosphorylated Src Family

Kinase Tyrosine416 (SFKY416), an auto phosphorylation site and a substrate of

SHP1(150) (Figure 17). The OSU-2S-induced phospho Serine 591 is located in C- terminal nuclear localization sequence of SHP1 protein (151, 152). Immunoblotting of phospho SHP1S591 and total SHP1 in cytosolic and nuclear extracts of CLL cells treated with OSU-2S revealed abundant phospho SHP1S591 in nuclear fraction which was further confirmed by high resolution confocal immunofluorescence microscopy (Figure 18).

Vehicle or OSU-2S treated CLL cells were fixed onto slides at 5 hour time point and were stained for cell membrane (red), phospho SHP1S591 (green) and nuclei (blue) and imaged using Olympus Fluoview 1000 laser scanning confocal microscope with Z stacks of 0.4μm per slice and images were chosen from the middle of nuclei. The image is representative of N=5 CLL patient samples. Our preliminary experiments indicate that cell lines expressing low levels of SHP1 are less sensitive to OSU-2S and when reconstituted with SHP1 became sensitive to OSU-2S and happened only when SHP1

S591 was phosphorylated, altogether confirming the importance of S591 phosphorylation in OSU-2S cytotoxicity.

Figure 13: Activation of PP2A by OSU-2S in CLL primary cells.

Figure 14: Association of PP2A and SHP1(left) and SHP1 phosphorylation (right) in response to OSU-2S treatment in CLL primary cells.

Figure 15: Inverse correlation between OSU-2S-induced phospho SHP1S591 and viability in CLL primary cells.

Figure 16: OSU-2S does not affect SHP1 enzyme activity in CLL.

Figure 17: OSU-2S does not affect phospho Y416 of Src family kinases in CLL.

Figure 18: Nuclear localization of phospho SHP1S591 in CLL.

OSU-2S activates PKC in CLL

Protein kinase C (PKC) has been shown to co-associate with SHP1 and phosphorylate

SHP1S591 residue(153). Consistent with a role for PKC in OSU-2S induced phosphorylation of SHP1S591 in CLL cells, bisindolylmaleimide (BIS), an inhibitor of

PKC that inhibited PKC activator PMA induced phosphorylation of SHP1S591, also partially prevented OSU-2S induced phosphorylation of SHP1S591 (Figure 19) and cytotoxicity (Figure 20). Viability was done at 24 hour time point by PI staining on CLL cells treated with Vehicle or 8µM OSU-2S in the presence or absence of PKC inhibitor

BIS(2µM) (N=8); bars represent SE. PMA is used as control for BIS. Then we thought if

OSU-2S can increase the PKC activity and as expected there was increase in PKC activity in CLL cells treated with 8µM OSU-2S for 4hr (N=6) (Figure 21) and this led us pose a further question if this was an direct or indirect effect. To see if OSU-2S have direct stimulatory effect on PKC we carried out in-vitro kinase assays using purified PKC treated with OSU-2S. As seen the activity of rat purified PKC is increased in OSU-2S treated condition (N=3), providing further evidence that OSU-2S can activated PKC directly (Figure 22). The SFK activating phosphorylation siteY416 was not affected by

OSU-2S treatment in CLL cells and therefore OSU-2S might not have an effect through

SFK. Dasatinib is a potent inhibitor of SFK and ABL kinases that has been approved for treating subsets of Ph+ CML patients(154, 155). Therefore we used dasatinib to directly inhibit SFK to confirm the independency of SFK in OSU-2S induced SHP1 phosphorylation. As expected, inhibition of SFK by dasatinib (100nM) in CLL primary

cells prior to treatment with OSU-2S, failed to inhibit the phosphorylation of

SHP1S591(Figure 23). These evidences indicate that activation of PKC might be downstream of Src kinases or could be due to direct drug effect on PKC.

Figure 19: PKC inhibitor BIS reduces OSU-2S induced phospho SHP1S591.

Figure 20: PKC inhibitor BIS partially rescues OSU-2S induced cytotoxicity in CLL.

Figure 21: OSU-2S activates PKC in CLL cells.

Figure 22: OSU-2S activates purified PKC in in-vitro kinase assay.

Figure 23: SFK inhibition does not affect OSU-2S induced phospho SHP1S591.

OSU-2S induces ROS generation in CLL

ROS generation have been noted in many cell types undergoing cell death(156-159).

Although, mainly involved in cell growth, signaling and immune cell functions, exacerbated ROS accumulation can lead to oxidative stress, resulting from oxidative damage of cell metabolites and cell structures(159). Nevertheless, the role of ROS in apoptosis mediated through intrinsic apoptosis pathway considered as para-phenomenon unrelated cause of death remains ambiguous. But accumulating reports suggest that ROS can be involved caspase and mitochondrial independent cell death(158). As OSU-2S caused caspase independent cell death, we thought ROS generation could be potential mediator of this pathway. Since ROS induced PKC stimulation by OSU-2S in HCC had been reported(136), we speculated if OSU-2S treatment in CLL caused ROS generation and therefore PKC activation. CLL primary cells treated with 8µM OSU-2S stained positive for dihydroethidium (DHE) stain at 24 hours proving ROS generation by OSU-

2S (Figure 24). Further, pre-treatment of CLL primary cells with 10mM N-acetyl cysteine (N-AC) a ROS scavenger, for 1hour before exposure to 8µM OSU-2S resulted in decreased cell death at 24hr (Figure 25) proving partial ROS dependency. To figure if

ROS is essential for OSU-2S mediated SHP1 phosphorylation, we collected the lysates from OSU-2S treated CLL cells previously pre-treated with or without N-AC for 1 hour and immunoblotting revealed that N-AC had no effect on OSU-2S induced SHP1 phosphorylation (Figure 26). However, it is possible that the ROS generation in our model might had been a late phenomenon and so we did a time course for ROS induction

and found that ROS is not induced by OSU-2S any time within 4 hours of treatment

(Figure 27) during which the entire PKC dependent SHP1 phosphorylation occurs in response to OSU-2S in CLL. Moreover, the dead cells increased consistently along with

ROS positivity at later time points after OSU-2S treatment indicating ROS generation is a late effect that could have culminated in cell death (Figure 28).

Figure 24: ROS generation in OSU-2S treated CLL primary cells.

Figure 25: ROS inhibition partially rescues OSU-2S cytotoxicity in CLL.

Figure 26: ROS inhibition does not affect OSU-2S induced phospho SHP1S591.

Figure 27: ROS generation in response to OSU-2S in CLL (time course).

Figure 28: Viability of CLL cells treated with OSU-2S in parallel to ROS time course.

Gene expression analysis (GEA) of OSU-2S mediated cytotoxicity in CLL

Gene expression studies by microarray analysis of RNA isolated from OSU-2S treated

CLL cells for 16 hours revealed at least 260 genes that were altered by >2 fold

(p<0.0005) (Figure 29) compared to vehicle treated controls. More detailed description of gene expression analysis is listed in methods section. Ingenuity pathway analysis (IPA)

(Appendix A) was carried out using the knowledge bank for classifying OSU-2S responsive genes in CLL. These genes represent modulators of cellular function and maintenance; development; growth and proliferation; cell death and survival; and cell cycle (Figure 30). IPA of top 40 genes included BCR signaling pathway components, such as PI3Kγ, PLCγ, and MAP2K6. Consistent with this, OSU-2S treatment reduced

BCR activation of CLL cells stimulated with goat F(ab’)2 against human IgA+IgG+IgM

(H+L), as identified with reduced activation marker CD86 (Figure 31).

Figure 29: Heat map of OSU-2S responsive genes in CLL primary cells.

Figure 30: Top functions affected by OSU-2S in CLL.

Figure 31: OSU-2S decreases BCR induced CD86 expression in CLL.

OSU-2S modulates TCL1 in CLL

TCL1 was identified as proto-oncogene in T-prolymphocytic leukemia where TCL1 and

TCL1b genes were activated by chromosomal aberrations placing them near the TCR loci(160). The normal expression of TCL1 is limited to early embryogenesis, some adult tissues and precursors of B and T cells. Deregulated expression of TCL1 lead to increased cell survival and transformation of hematopoietic T or B cells. Induced expression of human TCL1 gene in mouse B or T-cell specific promoter yielded development of various B or T-cell malignancies respectively(142, 161). It was also shown that TCL1 proteins can bind and augment the activity of the onco-protein AKT

(also known as protein kinase B) and the nuclear translocation of AKT. However, the role of TCL1 in CLL pathogenesis seems to be independent of AKT activation because

PTEN-/- mice do not develop B cell malignancies in spite of activated AKT pathway but rather due to NF-κB pathway activation (162, 163).

TCL1A expression, which is involved in the molecular pathogenesis of CLL(163) and identified to be down regulated in response to OSU-2S in the gene expression profile was independently confirmed to be significantly down regulated both at the mRNA by quantitative real time PCR (N=7) and protein levels by immunoblotting (Figure 32) with the corresponding up regulation in cFOS and FRA2 two known inhibitory targets of

TCL1A(163).

Figure 32: OSU-2S down regulates TCL1A oncogene in CLL.

Since regulation of TCL1 by miR-29 and miR-181 has been reported(164), we performed nanostring analysis for miR expression after OSU-2S treatment in CLL cells. However, the above miRs were not the part of significantly altered miR(Table 1). To find out if reduction of TCL1 oncoprotein in CLL cells would be the cause of OSU-2S cytotoxicity, we used a CLL cell line MEC1 over expressing TCL1 for further studies. Wild type

TCL1A was induced in MEC1 cell line transfected with tetracycline controlled expression system pRetroX-Tight-Pur (Clonetech, Mountain View, CA) cloned with

TCL1A cDNA by addition of 500µg/ml doxycycline (DOXY) for 24hrs and then cells were treated with different concentrations of OSU-2S before viability assessed by flow cytometry at 24 and 48 hours after OSU-2S treatment. As seen in figure 33 over expression of TCL1 in MEC1 cell lines did not protect the cells from OSU-2S cytotoxicity. Even though the TCL1 expression in these cells is under the control of exogenous modified Tet-responsive promoter, we thought if OSU-2S can affect the TCL1 protein in this system. So we immunoblotted for TCL1 in MEC1 cells transfected with

Vector (MEC1-Vector) or TCL1 (MEC1-Tcl1) using pRetroX-Tight-Pur construct with or without addition of doxycycline. Surprisingly, OSU-2S still decreased the TCL1 protein expression in doxycycline induced TCL1 in MEC1-Tcl1cell line(Figure 34). This led us to investigate if the protein stability of TCL1 is affected by OSU-2S treatment.

Therefore, we used cycloheximide(CHX), a protein synthesis inhibitor, to block the protein translation in MEC1-Tcl1 cell line after pretreatment with doxycycline for 48hr, followed by OSU-2S treatment. Immunoblotting for TCL1 on lysates collected from the above samples showed that in the presence of cycloheximide, OSU-2S did not altered the

pre-existing TCL1 protein proving that OSU-2S did not affect the TCL1 protein stability in TCL1 over expressing MEC1 cell line (Figure 35). Mcl1 protein was probed on the same blot as a control protein inhibited by cycloheximide treatment, which was also found to be reduced in response to OSU-2S treatment which could be attributed to its cytotoxicity.

Table 1: List of micro-RNAs modulated by OSU-2S in CLL primary cells.

Fold change to Name P value Vehicle hsa-miR-187-3p 2.98 7.21E-07 hsa-miR-210 0.38 7.83E-06 hsa-miR-542-3p 1.76 8.43E-03 hsa-miR-630 1.72 9.11E-03 hsa-miR-1915-3p 2.34 8.17E-05 hsa-miR-4516 1.91 2.16E-04

Figure 33: TCL1 over expression does not protect MEC1 cell line from OSU-2S cytotoxicity.

Figure 34: OSU-2S decreases TCL1 protein expression in MEC1 cell lines over expressing TCL1.

Figure 35: OSU-2S does not affect protein stability of TCL1 in MEC1 cell line over

expressing TCL1.

ROR1 targeted delivery of OSU-2S

Modulation of ubiquitous phosphatases such as PP2A and SHP1 in unintended target cells and normal B cells where these enzymes are necessary for maintaining cellular functions and homeostasis poses serious limitations in clinical settings. Novel therapy options that specifically target leukemic B cells sparing normal B cells are lacking and will be highly promising for CLL patients. ROR1 is a surface receptor tyrosine kinase expressed in over 95% of CLL but not normal B cells whose surface expression is unaltered with severity of CLL disease(165, 166). The cell surface expression of ROR1 on CLL cells was confirmed using a goat polyclonal anti human ROR1 antibody. The figure 36 represents the ROR1 MFI on CLL cells and normal B cells from healthy donor with gating on CD19+ live cells. Moreover ROR1 surface density in CLL is relatively low cell and internalization property makes it an attractive target for armed rather than naked monoclonal antibodies(mAbs). Liposomal immunonanoparticles(ILPs) have high affinities for target antigens on cell surface and are used to selectively enhance drug payload to target cells, favorably alter the drug kinetics, overcome drug off-target effects and potentially the drug efflux pumps(167). To target hROR1 surface protein in CLL cells, we utilized a non-cytotoxic anti-hROR1 mAb 2A2 (2A2-IgG)(168) for immunonanoparticle formulation. Restricted ROR1 expression limited to CLL B cells and the specificity of 2A2-IgG binding to ROR1 with high rate of internalization has been reported(165, 168). To confirm the internalization in our system we cultured the

CLL cells (1x106/ml) treated with 2A2 mAb or non specific mouse IgG (10µg/ml) at

37°C for 45 minutes and then washed stained for ROR1 expression using goat polyclonal antibody. It was seen that ROR1 expression decreased in 2A2 mAb treated samples but not in untreated or non specific IgG treated conditions in CLL (Figure 37). Although, steric hindrance due to epitope masking by 2A2 mAb can occur, the polyclonal nature of our flow cytometric antibody makes it less likely. Moreover, a similar experiment done in the cold incubation showed that the ROR1 was not internalized after 2A2 pretreatment and the polyclonal goat anti-ROR1 antibody detected the ROR1 on cells in hROR1 transgenic mice B cells (not shown). Given the restricted expression of ROR1 to leukemic cells, targeting ROR1 in CLL patients spares normal B cells, a robust advantage compared to other B cell targeting antibodies such as CD20 or CD19. In addition to specific targeting to leukemic cells, high rate of internalization of 2A2-IgG compared to anti-CD19 or anti-CD37 antibodies was observed in CLL cells(137). OSU-2S liposomal nanoparticles (OSU-2S-LP) and immunonanoparticles with 2A2-Ig (2A2-OSU-2S-ILP) were formulated. 2A2-Empty-ILP without OSU-2S drug served as control for OSU-2S in most experiments. Treatment of CLL primary cells (10x106/ml N=7 patients) with 2A2-

OSU-2S-ILP (10µg/ml) have very profound cytotoxicity compared to 2A2-Empty-ILP at

24hr (Figure 38). The above experiment with 2A2-Empty-ILP treatment also supports that 2A2-IgG binding alone or with cross linking effect of liposomes does not causes cytotoxicity in CLL. In order to demonstrate that OSU-2S treatment has no impact on the target of the 2A2-OSU-2S-ILP, we analyzed the expression of ROR1 in CLL cells from our GEP analysis and found the ROR1 was not modulated at mRNA level; further the cell surface ROR1 expression was not changed due to OSU-2S in CLL primary cells after

6 or 18 hrs supporting that OSU-2S does not alter the ROR1 expression (Table 2). This is important especially with chemo-immunotherapy because down modulation of the target would create drug resistant and over expression of ROR1 would provide survival advantage in CLL cells. We also compared the CD20 targeted delivery of OSU-2S with

ROR1 targeted delivery in primary CLL cells and found both had comparable levels of cytotoxicity. CLL cells (1×106/ml, N=4 patients) or Jurkat (1×106/ml, N=4) were incubated with different ILP formulation at 5μM of OSU-2S and 0.1μg/ml mAbs or free

OSU-2S (5μM) for 24hr before viability was analyzed by flow cytometry. ROR1 targeting (2A2-OSU-2S-ILP) and CD20 targeting (CD20-OSU-2S-ILP) OSU-2S formulations have comparable levels of cytotoxicity in CLL cells (no difference between

2A2-OSU-2S-ILP and CD20-OSU-2S-ILP), but no cytotoxicity in ROR1-ve CD20-ve

Jurkat cells (Figure 39). More importantly, to prove that liposomal packaging of OSU-2S is an efficient way to prevent the cytotoxicity to normal B cells we tested the long term effect of OSU-2S-LP on normal B cells. Normal B cells (1×106 /ml, N=4 donors) isolated from healthy donors were incubated with OSU-2S-LP formulation at 5μM of OSU-2S or free OSU-2S (5μM) for indicated duration before viability was analyzed by flow cytometry. Free OSU-2S was cytotoxic to normal B cells but there was no cytotoxicity by

OSU-2S-LP formulation (Figure 40).

Figure 36: Cell surface ROR1 expression on CLL and normal B cells.

Figure 37: 2A2 mAb treated CLL have reduced surface ROR1 expression.

Figure 38: ROR1 targeted delivery of OSU-2S induces cytotoxicity in CLL.

Figure 39: 2A2-OSU-2S-ILP promoted comparable levels of cytotoxicity as CD20-OSU- 2S-ILP in CLL.

Figure 40: Long term effect of OSU-2S-LP on normal B cells.

Table 2: ROR1 expression by microarray (gene expression-A) and flow cytometry (cell surface-B) on CLL cells after OSU-2S treatment.

Gene expression Probe set P value Fold change to vehicle

ROR1 Probe1 1.067288348 0.262766 ROR1 Probe2 0.933725248 0.275637 ROR1 Probe3 0.92999831 0.265262 B

6hr (ns) 18hr (ns) Cell surface ROR1 ΔMFI ΔMFI CLL1 -0.42 0.43 CLL2 -0.68 0.69 CLL3 -0.05 1.86 CLL4 -0.13 -0.89 ns - not significant

Chapter 3: In-vivo activity of OSU-2S in mouse models of CLL

OSU-2S does not traffic peripheral T cells

OSU-2S cannot be enzymatically phosphorylated by sphingosine kinase and therefore no interaction of OSU-2S with S1PR1 was seen underscores the non immunosuppressive property of OSU-2S. Further to support the previous claim we evaluated the in-vivo effect of OSU-2S in wild type C57BL/6 animals by measuring the lymphocyte count after administration. Administration of FTY720 (5mg/kg) but not OSU-2S(5mg/kg) decreased the peripheral blood T cell count in C57BL/6 mice. Lymphocyte counts were expressed

B-to-T-cell ratios to avoid misinterpretation of cytotoxic effects and cell trafficking effect. The B-to-T-cell ratio remained unchanged before and after treatment with saline or OSU-2S but increased in FTY720 treated animals due to a decrease in peripheral T cell numbers (Figure 41). Mice peripheral blood samples were collected and stained for

B220 and CD3 surface markers before and 6 hours after administration of the above reagents.

Figure 41: OSU-2S does not alter B:T ratio in C57BL/6 wild type mouse.

Maximum tolerated dose (MTD) studies

To find the maximum tolerated dose of OSU-2S for administering in the mouse model of diseases, a pilot MTD study was conducted in C57BL/6 mice. It was seen that administration of a single dose of 50 mg/kg by intra-peritoneal (i/p) route was lethal for these mice with in 24hr while all animals that received 10 mg/kg i/p route were alive. Our preliminary pharmacokinetic studies showed that by 48hr less than 12.5% drug was present in plasma based on T1/2 attained by i/p dosing(169). So a dose escalation group was used to attain the maximum tolerated dose. This group had 5 animal which were injected with OSU-2S every 48hr by i/p route starting with 10mg/kg on day 0. The subsequent doses were escalated by the percentage of previous dose used and escalation was tapered after the third injection(170). It is seen from the graphical representation

(Figure 42) that >50% of the animals tolerated 38.61mg/kg. Moreover, we used a constant dose group(N=5), which was continuously dosed with 10mg/kg OSU-2S every

48hr by i/p route and all the animals tolerated the dose very well for more than two weeks.

Figure 42: OSU-2S dose escalation study graphical representation.

Activity in Raji cell xenografted SCID mouse model

The B cell leukemic mouse model was established by engrafting aggressive Raji B cell line in severe combined immunodeficiency (SCID) mice as reported in (171). SCID mice have LOF mutations in protein kinase-DNA activated, catalytic polypeptide (Prkdc) gene and therefore lack mature lymphocytes due to defective V(D)J recombination of Ig and

TCR genes (172). SCID mice allows engraftment and growth of xenografted human cells or cell lines. 1x106 Raji parental cells were injected into SCID mouse by tail vein injection and waited for 3days for engraftment. The mice were then randomized and treated with 5mg/kg of OSU-2S daily for 14 days. The groups were monitored for survival and the tumor load was assessed after their death in the spleen and bone marrow by flow cytometric staining for human CD19+ and CD20+ Raji cells. The median survival time and tumor load between the groups were then compared. Though, there was a survival advantage in OSU-2S treated group by few days it was not statistically significant as the model was very aggressive and median survival of ~16days in vehicle treated groups. However, analysis of tumor burden in bone marrow where Raji cell typically engraft showed evidence of anti-leukemic activity of OSU-2S (Figure 43).

Figure 43: Leukemia reduction by OSU-2S in bone marrow of Raji cell xenografted SCID mouse.

Activity in E-TCL1 transgenic mouse model of CLL

Eμ-TCL1 mice (N=9) with WBC count >15x103/μl were treated with low doses of OSU-2S

(5mg/kg, intra-peritoneal injection) daily for three days. WBC count by blood smear evaluation and B-cell (CD19) and T-cell (CD3) percentiles by flow cytometry were determined before initial dosing and 24 hours after last dosing. Absolute cell numbers per micro liter of blood are shown in figure 44. As seen OSU-2S treatment had profound effect in reducing the peripheral blood CD19+ leukemic population in Eμ-TCL1 mouse model of

CLL.

Figure 44: Leukemia reduction by OSU-2S in peripheral blood of E-TCL1 transgenic mouse.

Activity in E-ROR1-TCL1 splenocytes engrafted mouse model of CLL

E-ROR1-TCL1 double transgenic mouse development

For in-vivo evaluation of hROR1 targeted OSU-2S, we generated Eµ-ROR1 transgenic mouse expressing hROR1 exclusively in B cells using a transgenic construct containing the IgH promoter/enhancer elements upstream of hROR1 cDNA. The transgenic line was identified by transgene specific PCR primers and confirmed by Southern blotting(137). B cell specific hROR1 transgenic protein expression in the splenocytes was confirmed by flow cytometry using hROR1 antibody in combination with B (anti-B220) and T (anti-

CD3) cell specific markers. 2A2-ILPs loaded with FAM ODN fluorescence molecule showed selective binding of 2A2-ILPs to hROR1+B220+ splenocytes from hROR1 transgenic mouse but not age matched non-transgenic mouse that are hROR1-

B220+(137). Our preliminary experiments showed administration of 2A2-OSU-2S-ILP in

Eµ-ROR1 single transgenic animals resulted in specific depletion of hROR1+ B cells in the peripheral blood. To recapitulate the human CLL disease with surface hROR1 antigen, the hROR1 transgenic mouse was crossed with a CLL mouse model expressing human TCL1 transgene(142) to generate Eµ-ROR1-TCL1 double transgenic mice

(Figure 45). The development of CLL disease burden in these mice characterized by accumulation surface hROR1+B220+CD5+ leukemic cells in peripheral blood, spleen and bone marrow was confirmed and the splenic hROR1+B220+CD5+ leukemic cells are shown in Figure 46. Moreover, flow cytometric analysis of ROR1 expression in the

peripheral blood samples from the transgenic mice was possible by whole blood staining

(Figure 47). Then we evaluated the specificity of OSU-2S immunonanoparticles on the spleenocytes derived from Eµ-TCL1 single transgenic and Eµ-ROR1-TCL1 double transgenic mice. The splenocytes were cultured ex-vivo with different ILPs(5µg/ml) and viability was assessed after 16hr by flow cytometry. There was no significant difference in cytotoxicity between 2A2-OSU-2S-ILP and non specific IgG-OSU-2S-ILP in splenocytes from Eµ-TCL1 (N=6) mice in ex-vivo cultures (Figure 48). However, splenocytes from hROR1+ Eµ-ROR1-TCL1 (N=8) mice were significantly killed by

2A2-OSU-2S-ILP in ex-vivo cultures compared to non specific IgG-OSU-2S-ILP or

OSU-2S-LP (Figure 49).

Figure 45: E-ROR1-TCL1 double transgenic mouse development.

Figure 46: ROR1 expression in Eµ-ROR1-TCL1 double transgenic mouse.

Figure 47: ROR1 expression in ROR1 Tg mice peripheral blood by flow cytometry.

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Figure 48: Effect of OSU-2S immunonanoparticles (ILPs) on Eµ-TCL1 mouse splenocytes (ex-vivo).

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Figure 49: Effect of OSU-2S immunonanoparticles (ILPs) on Eµ-ROR1-TCL1 mouse splenocytes (ex-vivo).

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E-ROR1-TCL1 splenocytes engrafted mouse model development

To target leukemic B cells and to avoid off target effects and adverse reaction if any, we planned to use hROR1 targeted 2A2-OSU-2S-ILP in Eµ-ROR1-TCL1 double transgenic mice for therapy evaluation. However, given the time frame to develop disease (~10 months) in Eµ-ROR1-TCL1 double transgenic, we decided to use serially engrafted splenocytes mouse model (Figure 50). This would allow us to perform experiments in a reliable, uniform, aggressive and less time consuming model. Engraftment of

B220+CD5+hROR1+ leukemic splenocytes originating from Eµ-ROR1-TCL1 double transgenic mice with frank leukemia into experimental C57BL/6 mice developed aggressive leukemia (WBC counts >10 x103/µl) with hROR1+B220+CD5+ cells in peripheral blood. These mice were randomly grouped into 2A2-OSU-2S-ILP, IgG-OSU-

2S-ILP, OSU-2S-LP or 2A2-Empty-ILP treatment cohorts and dosed 5 days every week for 4 weeks by intra-peritoneal injection of respective drug formulations. The leukemic burden assessed by staining peripheral blood for CD45+B220+CD5+ cells every other week revealed decreased leukemic burden (Figure 51) and prolonged median survival (82 days) in 2A2-OSU-2S-ILP treated animals compared to 2A2-Empty-ILP group (22 days)

(Figure 52) as seen in the survival curve of Eµ-ROR1-TCL1 splenocytes engrafted mice treated with ROR1 targeted OSU-2S formulation.

To evaluate if OUS-2S by itself have in-vivo activity in splenocytes engraftment model,

10x106 Eμ-ROR1-TCL1 double transgenic splenocytes were engrafted into syngenic

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C57BL/6 animals and disease progression was monitored bi-weekly by flow cytometric analysis of peripheral blood for WBC count. Animals were grouped two weeks after engraftment and dosed 5 days every week for 4 weeks by intra-peritoneal injection of vehicle (HPβCD) or OSU-2S as single agent(plain drug) (10mg/kg) (N=7/group).

Survival curve for Eμ-ROR1-TCL1 double transgenic splenocytes engraftment model of

CLL treated with plain OSU-2S or vehicle is shown in figure 53. Though OSU-2S as plain drug was beneficial in prolonging the survival of these leukemic mice, few notable adverse effects such as peritonitis and skin lesions at the site of injection were seen.

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Figure 50: Eµ-ROR1-TCL1 splenocytes engrafted mouse model of CLL.

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Figure 51: Reduced peripheral blood leukemic burden by 2A2-OSU-2S-ILP treatment.

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Figure 52: Survival curve of Eµ-ROR1-TCL1 splenocytes engrafted mouse model of CLL treated with OSU-2S immunonanoparticles (ILP).

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Figure 53: Survival curve of Eµ-ROR1-TCL1 splenocytes engrafted mouse model of CLL treated with OSU-2S (plain drug).

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Chapter 4: OSU-2S in B-Cell Lymphomas

OSU-2S and Mantle Cell Lymphoma

Synopsis

Treatment of mantle cell lymphoma (MCL) remains challenging given its complex pathophysiology. Though it is a rare form of non-Hodgkins lymphoma, patients with

MCL have limited treatment options. We have previously shown the preclinical effect of

FTY720 against MCL through down modulation of cyclin D1 and phospho-Akt. Despite this activity, given its potent T-cell immunosuppressive nature, use of FTY720 as therapeutic agent in cancer patient is admonishing. Hence, we have designed a novel non- immunosuppressive FTY720 derivative OSU-2S which exhibits potent cytotoxicity in

MCL cell lines and primary cells from MCL patients. OSU-2S induced PARP cleavage and increased the cell surface expression of CD74 a therapeutic target. OSU-2S in combination with anti-CD74 antibody milatuzumab had additive cytotoxicity in MCL cells. Moreover, we have developed a tumor antigen ROR1 targeted immunonanoparticle carrying OSU-2S (2A2-OSU-2S-ILP) that mediated selective cytotoxicity of MCL. The newly developed OSU-2S, its delivery using tumor antigen ROR1 directed immunonanoparticles provide targeted chemo-immunotherapy for MCL and could increase width of existing armamentarium for MCL.

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FTY720 increased the cell surface expression of CD74, a therapeutic target for milatuzumab monoclonal antibody therapy, without affecting the messenger levels through blockage of autophaghic pathway and accumulation of autophagolysosome cargo proteins with increase in LC3 II an autophagosome marker in MCL cell lines JeKo, Mino and Z-138(129). These findings supported the combination of FTY720 and CD74 targeting monoclonal antibody therapeutics. In the present report, we describe a non- immunosuppressive FTY720 derivative OSU-2S(136) that can mediate potent cytotoxicity in MCL cells, increase the cell surface CD74 expression in MCL cells and evaluate tumor directed lipid based OSU-2S formulation. The T-cell sequestering immunosuppressive nature of FTY720 has hindered our repurposing of FTY720 for treating heme-malignancies. Hence, OSU-2S was synthesized by FTY720 structure modification so that it does not react with sphingosine-1-phosphate-receptor the property that is essential for T cell sequestration and immunosuppression by FTY720. Using in- vivo models we have previously reported that OSU-2S does not cause drastic change in T cell counts unlike FTY720(137). Nevertheless, OSU-2S had comparable to higher activity in JeKo and Mino cell lines by MTS assays(Figure 1). We have also assessed the cytotoxic potential of OSU-2S(4µM) in MCL patient derived primary cells (N=8,

2x106/mL)(Figure 54). Treatment of JeKo and Mino cells with OSU-2S(4µM) caused cleavage of PARP protein as detected by immunoblotting. While Cyclin D1 protein disappeared in JeKo cells treated with OSU-2S, it remained unchanged in Mino cell line

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(Figure 55) suggesting that OSU-2S may have ploetrophic effect on these cells or effects may be due to heterogeneity of MCL cell lines as JeKo(173) was derived from blast variant and Mino(174) from the classic variant MCL patients. However, the effect of

OSU-2S on PP2A on these cells cannot be excluded, given the variable expression of

PP2A(data not shown) since activation of PP2A by OSU-2S and FTY720 in B cells have been reported(125). Since OSU-2S treatment resulted in increased LC3 II by immunoblotting in MCL cell lines that was reported previously by us, we checked the

CD74 cell surface expression. Treatment of the JeKo and Mino cells(1x106/mL) with

OSU-2S(5µM) resulted in increased CD74 as revealed by mean fluorescence intensities

(Figure 56). While OSU-2S induced comparable levels of CD74 expression in Mino cell line, it induced slightly lower expression of CD74 compared to FTY720 in JeKo cells.

This could be attributed to higher LC3 processing in Mino than JeKo by OSU-2S on these cells as reported previously. More importantly, OSU-2S(4µM) increased the cell surface CD74 expression in primary MCL cells (2x106/mL) but not the CD20 expression

(Figure 58). These findings still support the combination of this class of drug with CD74 targeting therapeutics. Therefore, we tested OSU-2S and anti-CD74 antibody combination therapy by using sub-optimal concentration of OSU-2S(4µM) and milatuzumab (anti-CD74 monoclonal antibody, 5µg/mL for JeKo, 1µg/mL for Mino and

5µg/mL for primary cells and with cross linker(αFc) fivefold of antibody concentration) in cell lines JeKo and Mino (Figure 57) and primary MCL cells (Figure 59). The combination therapy had greater cytotoxicity compared to the either of the drugs alone.

The tumor directed cytotoxic cargo delivery would improve the therapeutic index and

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preserve the healthy cells. So we designed a lipid based nanoparticles that can promote the drug payload specifically to tumors by utilizing the unique expression of receptor tyrosine kinase ROR1 in tumor cells of MCL, CLL and subset of pediatric ALL which have been reported(165, 175-177). We checked the expression of ROR1 on these cells and found high levels of ROR1 in JeKo, Mino and primary MCL cells (Figure 60). The lipid based OSU-2S nanoparticles(OSU-2S-LP) were then conjugated with anti-

ROR1mouse monoclonal antibody (2A2) to create immunonanoparticle 2A2-OSU-2S-

ILP which can specifically bind and deliver drug to the ROR1+ tumor cells. Binding of

2A2 clone antibody resulting in ROR1 internalization has been reported in JeKo and

Mino cells which would facilitate our drug delivery strategy in those cells(168).

Moreover, expression of ROR1 on tumor B cells but not normal B cells will facilitate selective delivery of the drug to lymphoma cells through ROR1 thus precluding normal B cells from cytotoxicity. Testing of immunonanoparticle formulation (2µg/mL) on JeKo,

Mino cell lines (1x106/mL, N=3) (Figure 61) and primary cells (2x106/mL, N=8) (Figure

62) showed selective cytotoxicity of 2A2-OSU-2S-ILP in MCL compared to other controls: untargeted formulation (OSU-2S-LP), non-specific antibody (IgG-OSU-2S-ILP) or empty targeted formulation (2A2-Empty-ILP). We then evaluated 2A2-OSU-2S-ILP in a mouse model of MCL by subcutaneous engraftment of Mino cell line(178). Mino cells were engrafted into the left flank region of NSG mice and were randomly grouped and treated with 2A2-OSU-2S-ILP(n=7), IgG-OSU-2S-ILP(n=6), or 2A2-Empty-

ILP(n=6). In-vivo treatment with 2A2-OSU-2S-ILP in Mino cell line xenografted mice revealed decreased tumor weight compared to the controls at the end of the study (Figure

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63). These findings describe the novel OSU-2S molecule as a successor of FTY720 which is active against MCL and could enhance CD74 expression. Importantly, the tumor antigen ROR1 directed delivery system for increasing the payload selectively to the cancer cells would prevent the exposure of chemo drug to other body cells influenced by the pharmacological property of liposomal packaging.

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Figure 54: OSU-2S is cytotoxic in MCL primary cells.

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Figure 55: Effect of OSU-2S on Cyclin D1 in JeKo and Mino cells.

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Figure 56: Cell surface CD74 induction by OSU-2S in MCL cell lines.

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Figure 57: OSU-2S and Milatuzumab combination in MCL cell lines.

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Figure 58: Cell surface CD74 induction by OSU-2S in MCL primary cells.

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Figure 59: OSU-2S and Milatuzumab combination effect in MCL primary cells

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Figure 60: ROR1 expression in MCL cell lines and primary cells.

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Figure 61: Cytotoxicity by ROR1 targeted OSU-2S nanoparticles in MCL cell lines.

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Figure 62: Cytotoxicity by ROR1 targeted OSU-2S nanoparticles in MCL primary cells.

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Figure 63: In-vivo activity of ROR1 targeted OSU-2S in Xenograft model of MCL.

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OSU-2S and Canine Diffuse Large B-Cell Lymphoma

Canine spontaneous lymphoma

Canine spontaneous diffuse large B-cell lymphoma(DLBCL) is the common NHL occurring in dogs with similar incidence rate to that of human(179). Canine NHL represent low-high grade human NHL with about 70-80% of them with B-cell origin.

With the natural biology of disease, similar cellular signaling components and chemotherapeutic drug sensitivity to that of human, canine DLBCL serve as a perfect model for evaluation of anti-cancerous compounds that are in clinical development(180,

181). Unlike the cage bred rodents, the out bred nature of the dogs closely represent the human population in addition to sharing the environment that humans are exposed.

Moreover, the tumor size and kinetics of canine lymphoma are comparable to that of human. The larger body size of the canine patients compared to the rodents will allow the multiplicity of the sampling procedures and extend the scope of analysis. Further, the cost and the time effectiveness associated with the clinical trials in veterinary patients along with fewer regulations would allow faster validation of investigational new drugs.

However, the lack of species specific reagents and research tools impedes the above approach.

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Activity of OSU-2S in canine lymphoma

We evaluated the activity of OSU-2S in canine DLBCL cell lines CLB1-1(182) and 17-

71(183) and found OSU-2S promoted cytotoxicity in these cells at concentration as low as 2µM after 24hour in culture (Figure 64). Next we tested if OSU-2S will be active in canine spontaneous DLBCL samples ex-vivo. The samples were collected from the canine patients in OSU VMC by FNAB of lymphoma mass under approved protocol. The samples were washed in PBS and the mononuclear cells were separated by ficoll density centrifugation and cultured in RPMI1640 supplemented with 10%FBS and antibiotics at

0.5x 106 cells/ml in 48 well cell culture plate. Later, the cultures were treated with 2µM

OSU-2S for 24hr and viability was assessed by flow cytometer after staining with annexin and propidium iodide. OSU-2S induced apoptosis in canine spontaneous DLBCL

(N=7 patients)(Figure 65). Since, OSU-2S cytotoxicity was associated with ROS production in CLL, we stained the canine DLBCL cells for ROS by DHE stain.

Interestingly, OSU-2S treatment resulted in ROS production in DLBCL cell lines and pretreatment with N-AC partially prevented the cytotoxicity in the cell lines (Figure 66).

Further characterization of OSU-2S cytotoxicity after target validation in canine spontaneous tumor and conduction of MTD studies in healthy dogs would form a basis for fast tracking of this compound through veterinary clinical trials(170).

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Figure 64: Activity of OSU-2S in canine DLBCL cell lines.

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Figure 65: OSU-2S induces apoptosis in canine spontaneous DLBCL.

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Figure 66: OSU-2S induces ROS mediated cytotoxicity in canine DLBCL.

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Discussion

To maintain the cell homeostasis, signaling pathways must be tightly controlled. Any deregulation if not compensated will ultimately disturb the system. Reversible protein phosphorylation plays an important role in cell proliferation, cell development, cell signaling and apoptosis. The net phosphorylation state of protein, which most of times determines the protein functions, is therefore controlled by a balance between kinases and phosphatases(184). Though it was earlier considered phosphatases are non-specific targets and have signal dampening functions inside the cell, the paradigm has changed with phosphatases playing myriad role in cancer cell signal transduction. With most research focus on kinases and their inhibitors for cancer therapeutics, this project was aimed at modulating and activation of protein phosphatases SHP-1 and PP2A which are well known tumor suppressor proteins. Moreover, phosphatase targeted therapy may become alternative to kinase inhibitors especially in situations where those inhibitors may not be effective like single nucleotide polymorphism (SNP) or escape mutations of drug targets. OSU-2S, a novel FTY720 analogue is synthesized and patented in the Ohio State

University (OSU) which is unique of its kind and the testing of this molecule in CLL is exclusively conducted in the OSU.

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Given more than 98% of protein phosphorylation occurs on serine threonine residues and is mediated by more than 400 protein serine threonine kinases, the lower number of serine threonine phosphatases (slightly more than 20) entails these enzymes must have a wide range of substrate specificity(185). Thus selective cytotoxicity against cancer cells through modulating protein phosphatases without compromising their normal counterparts pose a challenge for traditional drug design. Here we described a CLL tumor antigen ROR1 targeted delivery of immunonanoparticle carrying a novel non- immunosuppressive phosphatase activating FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells sparing normal B cells. These studies have focused on rational preclinical development of FTY720 derivative OSU-2S, its in-vitro and in- vivo mechanistic and therapeutic evaluations using novel targeted immunoliposomal delivery formulations and humanized mouse models of leukemia developed for this purpose.

Despite promising in-vitro and in-vivo activity of FTY720 against a variety of solid tumors and heme-malignancies like CLL, MCL, CML (125, 126, 128, 131, 135), our attempts to repurpose FTY720, a FDA approved drug for relapsing multiple sclerosis, for oncology indications were hindered by its immunosuppressive property involving T cell homing to lymph nodes. Hence, OSU-2S was rederived to distinguish it from the parent

FTY720 compound in 3 aspects such that OSU-2S is not phosphorylated by sphingosine kinase-2 in in-vitro assays; synthetic phosphorylated OSU-2S failed to co cluster with

S1PRs in cell lines expressing S1PR and administration of OSU-2S to wild type mice did

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not cause drastic peripheral blood T cell count reduction in contrast to FTY720 which engender lymph node homing of T cells.

Congruent with FTY720, OSU-2S activated PP2A in CLL B cells. Activation of PP2A resulting in SHP1 mediated dephosphorylation and degradation of BCR/ABL has been demonstrated in 32D-BCR/ABL cells. This was attributed to co-association of SHP1 with the PP2A-BCR/ABL complex(97). Interestingly, OSU-2S induced association of PP2A and phospho SHP1S591 in CLL cells. Since SHP1 dependent PP2A mediated tumor growth suppression and apoptosis by reduced BCR/ABL expression and activity was established in CML(97), we sought to see if OSU-2S affected the enzyme activity of

SHP1. While OSU-2S failed to modulate the SHP1 phosphatase activity, it induced phosphorylation of SHP1S591 at the putative PKC substrate motif (K/RXS*XK/R)(186) on SHP1. Consistent with our speculation on the role for PKC in OSU-2S induced phosphorylation, concentrations of PKC inhibitor BIS that reduced the PMA induced

PKC activity also inhibited OSU-2S induced phosphorylation of SHP1S591 in CLL B cells. This was further confirmed by OSU-2S-induced increase in PKC activity in CLL cells. More importantly OSU-2S had a direct stimulatory effect on purified PKC in our in-vitro kinase assays. These finding were consistent with literature evidence suggesting

PKC as a relevant phosphorylating kinase of SHP1S591 residue(85, 153). Experimental conditions reducing the phosphorylation of SHP1 also significantly prevented the cytotoxicity of OSU-2S implying the potential role of SHP1 in OSU-2S mediated apoptosis. This is also consistent with our earlier findings on PKCδ dependent reactive

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oxygen species (ROS) production and apoptosis in hepatocellular carcinoma treated with

OSU-2S(136). It is also possible that OSU-2S can mediate its effect through PKCδ dependent ROS production in CLL, as CLL cells have high PKCβII and PKCδ expression but we saw delayed ROS generation in CLL cells treated with OSU-2S, which could also be attributed to sequel of apoptosis.

The expression of SHP1 is drastically lowered in many leukemia and lymphomas due to

DNA promoter hyper methylation implying negative role of SHP-1 in development of leukemia/lymphoma (69). The phosphorylation of SHP1S591 may modulate the function of SHP1 directly by altering the enzyme catalytic activity or through different sub cellular localization and spatiotemporal regulation or association with its binding partners to signal cytotoxic effects. The enzyme activity of SHP1 can be affected by phosphorylation of its C-terminal amino acid residues Y536, Y564, and S591, which confer change in the structure conformation of the protein regulated by these post transcriptional modifications (99, 187, 188). Phosphorylation of Y536 of SHP1 generally increases the enzyme activity of SHP1 by many folds but Y564 has less influence on the activity. However, we did not find change in enzyme activity of phosphorylated SHP1S591 in CLL. This was supported by established cell type specific roles of S591 including increase or decrease in enzyme activity and nuclear localization signals. Phorbol ester had been shown to induce SHP1 activation with serine phosphorylation in HL-60 pro- myelocytic cells accompanied by growth arrest (189). However, in our study we saw no change in the enzyme activity, but nuclear localization of phospho SHP1S591 and cell

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death after treatment with OSU-2S. It is possible that nuclear trans located SHP1 might act on nuclear substrates or interact with other nuclear proteins regulating transcription to bring cell death in our model.

The phosphatase targeted therapeutics is not only a new field in cancer therapy, but also in controlling other human diseases. The emerging knowledge of phosphatase holoenzyme structure will help us to devise new drugs and fine tune them. Nevertheless, many of the signaling cascades by phosphatases remain unveiled and revealing this will definitely make them more valuable candidates in therapeutics. Although, OSU-2S has potent activity in CLL primary cells there is substantial difference between patients. The

SHP1 phosphorylation, a PKC dependent mechanism seemed partially linked to OSU-2S cytotoxicity and this phosphorylation induction seem to vary among CLL patients cells exposed to OSU-2S. The variation could be attributed to differences in the levels of PKC,

SHP1, as well as the activity of PKC and the sensitivity of PKC to OSU-2S or different survival signals that CLL cells are dependent on. However, OSU-2S killed prognostically poor CLL (IgVH unmutated and 17p deleted) samples. Intrinsic variability associated with CLL samples obtained from either naïve patients or patients previously treated with different chemotherapeutics could also contribute to the observed differences.

Nevertheless, OSU-2S is still active in previously treated CLL cells as seen in our experiments. Prolonged exposure of all primary CLL cells from patients we have tested with OSU-2S led to increased cytotoxicity and decreased viable cells (comparison of

Vehicle and OSU-2S treated CLL cells for 24hr (difference -51.28; p<0.0001) and 48hr

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(difference -56.91; p<0.0001)). Moreover, our time kinetic study showed OSU-2S cytotoxicity magnitude although differ among patients (CLL1 more sensitive than

CLL3), it is time dependent and eventually OSU-2S is cytotoxic to CLL cells after prolonged exposure. We did not see residual CLL cells after treating with OSU-2S when analyzed at later time points.

The novel strategy to enhance the efficiency and specificity of OSU-2S drug delivery in

CLL using hROR1 targeting 2A2-IgG conjugated ILPs has promising clinical implications. ROR1 identified as oncofetal protein is expressed during embryogenesis and in some malignancies including CLL cells but absent in mature normal B cells (165,

166, 190). Recent discovery of ROR1 expression in B cell precursor, hematogones may indicate toxic potential of 2A2-OSU-2S-ILP on these population(191). But it should be noted that mature normal B cells or CD34+ precursors lack ROR1 and hematogones have low ROR1 expression and occur in very low frequencies compared to CLL. Furthermore, recent reports using different ROR1 antibodies indicate that ROR1 expression and its phosphorylation are directly correlated with disease severity in CLL and can enhance leukemogenesis in CLL(192-194). Thus, the unique expression pattern of ROR1 in CLL cells makes it an attractive target for therapy. This has led to development of various

ROR1 targeted therapeutics including anti-ROR1 antibodies raised against different epitopes of ROR1 and have different cytotoxic potential(168, 194-197). Moreover, Fc domain engineering of anti-ROR1 antibodies are undertaken to enhance the effector functions like antibody dependent cell-mediated cytotoxicity (ADCC), complement

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dependent cytotoxicity (CDC) or properties of antibody-drug conjugate (ADC). The recently reported ROR1 directed chimeric antigenic receptor (ROR1-CAR) designed using 2A2-ScFv on T cell demonstrating potent anti-leukemic property against ROR1+ cells is noteworthy(198). Consistent with previous reports, our results demonstrated that anti-hROR1 mAb, 2A2-IgG selectively binds to CLL cells, is internalized efficiently, and does not induce cytotoxicity as a single reagent or in the presence of antibody or liposomal cross linker (data not shown)(168). Further, due to the limited cell surface density, hROR1 is considered preferably a target for armed rather than naked mAbs, though other anti-hROR1 mAbs inducing apoptosis in CLL have been reported(196). It is conceivable that incorporation of cytotoxic anti-hROR1 mAbs as well as the use of human or humanized anti-hROR1 mAbs in ILP formulations has potential clinical utility.

Different anti-ROR1 antibodies are extremely different in detection of ROR1 positive cells as demonstrated in the literature. So we have used two different antibodies in our studies to identify the ROR1 positive cells. 2A2-ILP-FAM ODN (2A2-flourescent oligonucleotide-liposome) was used to identify ROR1 in hROR1 transgenic mouse B cells and CLL (data not shown) in addition to goat polyclonal anti-human ROR1 antibody

(R&D Systems). Our objective of using 2A2 clone anti-ROR1 antibody for drug formulation is mainly due to its lack of direct cytotoxic property. Not only as pointed that different anti-ROR1 antibodies are different in their ability to detect ROR1 positive cells, but also in their ability to mediate direct cytotoxicity and internalization (168, 194, 196).

Hence for our study for delivering OSU-2S we have used 2A2 clone that has inherent

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non-cytotoxic and internalization character.

The liposomal packaging of OSU-2S reduced the risk of other cells being exposed to the drug, however the minimal effect of OSU-2S-LP on normal B cell may be due to prolonged exposure of these cells to lingering OSU-2S-LP particles which could have caused the particles to be fusing to normal B cells. Moreover, in targeted therapy 2A2-

OSU-2S-ILP particles would be consumed by malignant cells and induce cytotoxicity and therefore fewer particles would be available for normal cells to get exposed and additional dosing can be used to eliminate the malignant cells if needed. Additionally, the concentration of targeted particles could be lowered and optimized in a range that can be toxic only to malignant cells for which extensive pharmacokinetic/pharmacoanalytical studies are needed and to confirm the integrity of the liposomes. Interestingly, 2A2-OSU-

2S-ILP promoted comparable levels of cytotoxicity as CD20-OSU-2S-ILP in

ROR1+CD20+ CLL cells. Moreover, OSU-2S did not change the expression pattern of

ROR1 in CLL which would support that OSU-2S neither lowers the target for 2A2-OSU-

2S-ILP nor it enhances the ROR1 expression mediated survival in CLL cells (Table 2).

While cell surface CD19 did not changed after OSU-2S treatment in CLL, there was mild decrease in CD20 surface protein although statistically insignificant. These findings would support that OSU-2S does not alter the targets CD19 or CD20 which can be used for salvage therapy in CLL

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We have tested the activity OSU-2S as a single agent in different B lymphoid and

CLL mouse models and cancerous cells (Appendix B). In order to test the drug in a clinically relevant setting and to avoid drug adverse reaction, if any, we decided to deliver OSU-2S using an ILP formulation that could selectively target leukemic B cells in a mouse model of CLL. To accomplish this, we generated Eµ-ROR1 transgenic mice and crossed with the Eµ-TCL1 leukemia mouse model. These double transgenic mice exhibited CLL like disease later in life (>9 months) with additional expression of hROR1 in B220+ CD5+ leukemic cells. Since these mice express hROR1 and to conduct an in- vivo study in a uniform leukemic model, we adapted the leukemic cells suitable for allograft model by passaging the leukemic clones in wild type background mice. By confirming the tumor burden in peripheral blood after engrafting ROR1+ leukemic splenocytes in experimental animals, we then started treatment with OSU-2S encapsulated in ILP targeted against ROR1 which resulted in cytotoxicity specific for leukemic cells. We analyzed the peripheral blood from all animals biweekly and the splenocytes after death using multicolor flow cytometry. 2A2-OSU-2S-ILP group animals which eventually died of progressive disease still had

CD45+B220+CD5+hROR1+ cells. It is important to note that we have utilized an aggressive mouse model by serial transplantation of B220+CD5+hROR1+ leukemic cells in syngenic mice. This provided extremely high chance of engraftment and shorter survival (median survival for vehicle treated animals is 22days after detection of leukemia >5% B220+CD5+ and WBC > 10000cells/µl). Equally important to recognize is that these mice had only short term treatment for 5 days every week for 4 weeks after

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the leukemia detection. Since the treatment ended, the 2A2-OSU-2S-ILP group animals slowly progressed and died of leukemia. So we believe the cause of death in this group mouse to be from discontinued treatment rather than drug resistant. Even though we did not eradicate the disease, an extensive pharmacokinetic study leading to dosage and treatment duration calculation might be helpful for eradication of the disease in the mice studies.

It is expected that targeted therapy with 2A2-OSU-2S-ILP preserve the non- leukemic population. However, the percentage of non-leukemic cells in figure 51 does not necessary reflect that adequate number of cells are present in the blood with the evidence of no toxicity by targeted ILP as the concurrent leukemic cells can efface the normal population. So it is difficult to distinguish the reduction in percentage or number of non-leukemic cells numbers is due to toxic nature of the treatment or effacement by leukemic population. Despite the potent in-vivo activity as a single agent the ubiquitous expression of the phosphatases in diverse cell types, precludes use of OSU-2S as a free drug. The OSU-2S immunonanoparticle formulation that selectively targets hROR1+ leukemic CLL but not normal B cells in-vitro and in a humanized CLL mouse model rendering therapeutic benefit adds credence to the approach described here and can be extended to other therapeutic agents and ROR1+ malignancies including MCL and ALL.

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Future Directions

Target identification

Rationale: The target identification and validation of OSU-2S would be very essential for moving forward the pre-clinical studies involving OSU-2S. The identification of target would not only provide the critical mechanism behind the cytotoxic effect of the drug but also the perspective on the signaling components essential for the action of the drug.

Moreover, identification of direct target of OSU-2S will allow stratification of leukemia patients based on expression and/or function of the relevant chemo sensitizing targets, so that responsive groups are easily determined. Moreover, less affinity targets that are non- essential for cytotoxicity would act as the component of drug adverse effects. Several methods have been demonstrated to identify the drug targets and can be utilized for OSU-

2S.

a) Label free chromatographic co-elution method(199) can be employed if drug

labeling or immobilization are more likely to affect functional moieties and

therefore affect bioactivity and reactivity of the drug molecule. OSU-2S treated

CLL lysates are made at early time points and proteins are fractionized by ion

exchange HPLC along with drug alone controls, untreated lysates controls or

lysates spiked with drug controls followed by analyses in LC-MS/MS to plot free

and ligand/target bound OSU-2S. Target proteins present in the OSU-2S bound

139

drug fractions are then identified using LC-MS/MS. Validation of target will be

done using appropriate functional assay after identification of co-eluting protein. b) Using RNA interference (RNAi) technology, OSU-2S dependent synthetic lethal

screens can be performed in a selected leukemic cell line for identifying gene

targets whose knock down potentiates cytotoxicity to sub-lethal concentrations of

OSU-2S, and gene targets whose knock down rescues cytotoxicity to moderately

lethal concentrations of OSU-2S. c) By photo affinity labeling of the OSU-2S (like diazirine)(200-202), the interacting

ligand of OSU-2S can be linked to OSU-2S after irradiation. The stable complex

then can be subjected to downstream analysis. d) Using radiolabelled OSU-2S, the target proteins could be probed from the whole

cell protein lysates. e) By employing a library of phage-display on immobilized OSU-2S, the drug

binding site could be identified after comparison of peptide sequence; however

targets like glycoprotein, lipoprotein could not be identified by this technique.

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Role of OSU-2S-induced phospho SHP1S591 in cytotoxicity in CLL

To determine the function of phospho SHP1S591 in OSU-2S mediated cytotoxicity in CLL by assessing if phospho SHP1S591 has specific cellular and nuclear binding partners.

Rationale: The phosphorylation of SHP1 may modulate the function of SHP1 directly by altering the enzyme catalytic activity or through different sub cellular localization and/or spatiotemporal regulation or association with its binding partners to bring its cytotoxic effects. The enzyme activity of SHP1 can be affected by phosphorylation of its amino acid residues Y536, Y564, S591 in C-terminal which confer change in the structure conformation of the protein(85, 187, 203). Cell type specific roles of S591 have been reported including increase/decrease in enzyme activity, localization signals etc(188).

However, our in-vitro phosphatase assay revealed no change in SHP1 enzyme activity after treatment with OSU-2S in CLL cells; rather we saw predominant localization of phospho SHP1S591 in nuclear lysates after treatment which was also confirmed by high resolution confocal immunofluorescence microscopic imaging. We hypothesize that

OSU-2S induced phospho SHP1S591 has functional role in OSU-2S cytotoxicity through spatiotemporal regulation of its binding partners which might modulate TCL1 expression.

Phosphorylation of SHP1 may bring its phospho substrates or other mediators close together and may exists in association which could be revealed by co- immunoprecipitation (co-IP) of phospho SHP1S591. To identify the binding partners of

141

phospho SHP1 in response to OSU-2S, CLL lysates from vehicle or OSU-2S treatment will be subjected to co-immunoprecipitation using phospho SHP1S591 specific antibody followed by resolving in PAGE and staining with Coomassie Brilliant Blue dye to determine the molecular mass. Immunoblotting of co-IP with phospho tyrosine or phospho serine/threonine specific antibody will also be done to find the phospho status of associated proteins. To determine the identity of co-IPed proteins, IPed beads or PAGE resolved co-IPed proteins will be trypsin digested, eluted and the concentrated peptides are subjected to LC-MS/MS analysis as reported in (99) . The protein IDs obtained are then verified with manual validation to avoid false positives. The identified co-associated proteins are also verified by reciprocal immunoprecipitation, and their functional role in apoptotic signal is studied focusing on but not limited to regulation of TCL1 expression.

If the usage of CLL cells is not permissive to study SHP1 proteomics, inducible SHP1 in a cell line model will be employed for studying phospho SHP1 binding partners along with creation of null S591A, or phosphomimetics S591D/S591E mutants.

142

Mechanism of TCL1 down regulation by OSU-2S

Rationale: Since OSU-2S reduced TCL1 expression at mRNA level in CLL primary cells, it would be critical to understand how TCL1 is down regulated. While the reduced stability of TCL1 mRNA mediated through microRNA is possible, our miR expression analysis (Table 1) did not revealed the known TCL1 inhibitory microRNA, miR-29 and miR-181(164). Therefore, it is more likely that OSU-2S has effects on TCL1 gene transcriptional process. To determine if the TCL1 promoter activity is affected, a reporter gene cloned under the TCL1 promoter can be expressed in cell line and the reporter expression is determined with and without OSU-2S treatment.

Effect of TCL1 down regulation on activator protein-1 function

Rationale: OSU-2S down regulates TCL1 and up regulates FOS family members c-FOS and FRA2 which are components of activator protein-1 (AP-1). Further, direct interaction and inhibition of transcriptional activity of AP-1 members by TCL1, thus playing pathogenic role in CLL was previously reported(163). Moreover convincing evidences suggest that TCL1 role in pathogenesis of CLL is beyond its AKT co activation, as AKT activation in B cell specific PTEN deficient mice did not develop B cell malignancy(162). Therefore we hypothesize that OSU-2S mediated down regulation of

TCL1 enhances AP-1 activity and promotes cell death in CLL.

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AP-1 proteins are heterodimers made up of JUN family member (cJun, JunB or JunD) and FOS family members (cFos, Fra-1, Fra-2 or FosB)(204). The function of AP-1 proteins is context dependent like cell type, stimulus and relative levels of AP-1 components. Therefore we plan to test if AP-1 activity is increased after treatment with

OSU-2S along the context of TCL1 involvement. The effect of OSU-2S will be tested in cell line transfected with AP-1 luciferase reporter with or without TCL1 co-expression for determining AP-1 activity. The viability of the cells will also be evaluated and FOS and JUN family members will be measured at protein and messenger levels. Moreover, pro apoptotic genes FasL and TNF-α induced by AP-1 will measured and the above findings will be validated in CLL primary cells.

144

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Appendix A: IPA of OSU-2S treated CLL cells.

Reference set: Human Genome U133 Plus 2.0 Array

Relationship to include: Direct and Indirect

Includes Endogenous Chemicals p-value = 5.00E-04 and Fold Change ≥ 2.000

Samples used: CLL primary cells (N=3 patients)

Conditions: Vehicle and OSU-2S

166

Table 3: Differential gene expression detected by microarray in OSU-2S treated CLL cells.

Fold Gene Title Gene Symbol Difference to P value Vehicle SLAM family member 7 SLAMF7 26.45741122 9.75E-09 Ras-related GTP binding D RRAGD 13.35029763 8.25E-07 lectin, galactoside-binding, soluble, 3 LGALS3 10.18447706 1.88E-05 kallikrein-related peptidase 2 KLK2 9.74525543 1.26E-08 2'-5'-oligoadenylate synthetase 3, OAS3 8.575376417 4.53E-05 100kDa stearoyl-CoA desaturase (delta-9- SCD 8.350137764 3.65E-06 desaturase) UDP-N-acteylglucosamine UAP1L1 6.947047684 4.32E-09 pyrophosphorylase 1-like 1 FBJ murine osteosarcoma viral FOS 6.159016529 3.38E-06 oncogene homolog lymphocyte antigen 9 LY9 5.608053063 2.95E-05 bruno-like 6, RNA binding protein BRUNOL6 5.138687785 6.74E-06 (Drosophila) Chromosome 1 open reading frame 95 C1orf95 5.114524081 4.59E-08 myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 MX1 5.079195483 2.70E-05 (mouse) solute carrier family 26, member 11 SLC26A11 5.002675935 4.98E-06 kelch domain containing 8B KLHDC8B 4.70501721 1.31E-05 squalene epoxidase SQLE 4.694592713 3.10E-06 signal-regulatory protein alpha SIRPA 4.639598618 4.86E-07 DNA-damage-inducible transcript 3 /// nuclear receptor subfamily 1, group H, DDIT3 /// NR 4.541597964 1.16E-06 member 3 Continued

167

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle N-acylsphingosine amidohydrolase (acid ASAH1 4.303170489 4.59E-05 ceramidase) 1 2',5'-oligoadenylate synthetase 1, OAS1 4.220751816 4.31E-04 40/46kDa 3-hydroxy-3-methylglutaryl-Coenzyme A HMGCS1 4.193631223 1.83E-06 synthase 1 (soluble) estrogen receptor 2 (ER beta) ESR2 4.119587827 2.49E-07 apolipoprotein D APOD 4.048815774 6.53E-05 sterol-C4-methyl oxidase-like SC4MOL 4.041805786 9.84E-06 Spectrin repeat containing, nuclear SYNE2 4.028939104 1.56E-06 envelope 2 cytochrome P450, family 51, subfamily CYP51A1 3.940011935 1.28E-05 A, polypeptide 1 mitogen-activated protein kinase kinase MAP3K8 3.699678739 2.95E-04 kinase 8 interferon, gamma-inducible protein 30 IFI30 3.644447202 6.34E-05 wingless-type MMTV integration site WNT3 3.639650699 6.12E-05 family, member 3 chemokine (C-C motif) receptor 6 CCR6 3.588298727 4.33E-05 hypothetical protein LOC153577 LOC153577 3.557589372 7.44E-07 renin binding protein RENBP 3.495260541 3.98E-07 nuclear paraspeckle assembly transcript 1 NEAT1 3.443796753 1.15E-04 (non-protein coding) Niemann-Pick disease, type C2 NPC2 3.370820957 1.22E-06 folliculin FLCN 3.322570934 6.60E-07 malignant fibrous histiocytoma amplified MFHAS1 3.319578342 3.00E-07 sequence 1 activating transcription factor 3 ATF3 3.280691645 4.46E-04 chromosome 1 open reading frame 85 C1orf85 3.272288617 2.17E-07 insulin induced gene 1 INSIG1 3.240687707 5.24E-05 Cementum protein 1 CEMP1 3.21986455 3.65E-05 nudix (nucleoside diphosphate linked NUDT14 3.183910981 3.26E-07 moiety X)-type motif 14 chromosome 1 open reading frame 93 C1orf93 3.168060749 8.25E-06 Continued

168

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle kelch-like 6 (Drosophila) KLHL6 3.149231906 4.73E-06 3-hydroxy-3-methylglutaryl-Coenzyme A HMGCR 3.144215279 5.90E-06 reductase lanosterol synthase (2,3-oxidosqualene- LSS 3.131817185 5.58E-05 lanosterol cyclase) dual specificity phosphatase 10 DUSP10 3.110184144 2.93E-05 FOS-like antigen 2 FOSL2 3.086988268 1.05E-04 calcium binding protein 39-like CAB39L 3.075455284 2.77E-05 toll-like receptor 7 TLR7 3.020526408 1.54E-05 adenylate cyclase 3 ADCY3 3.001325935 9.68E-07 protein phosphatase 1, regulatory PPP1R15A 3.001117907 1.56E-04 (inhibitor) subunit 15A phosphatidylinositol 4-kinase type 2 alpha PI4K2A 2.997999204 4.66E-07 v-maf musculoaponeurotic fibrosarcoma MAFG 2.986384653 2.10E-06 oncogene homolog G (avian) 7-dehydrocholesterol reductase DHCR7 2.981420774 1.47E-05 chromosome 17 open reading frame 79 C17orf79 2.945474371 7.05E-06 Ras-related GTP binding C RRAGC 2.941393906 3.28E-05 Hexosaminidase A (alpha polypeptide) HEXA 2.937319093 5.78E-06 glucosamine-6-phosphate deaminase 1 GNPDA1 2.935690749 3.30E-06 KIAA1466 gene KIAA1466 2.891263305 8.23E-07 basic helix-loop-helix family, member BHLHE40 2.889059667 2.00E-04 e40 kelch-like 21 (Drosophila) KLHL21 2.841002301 1.00E-05 hydroxysteroid (17-beta) dehydrogenase HSD17B14 2.818446144 4.17E-04 14 brain protein I3 BRI3 2.795681481 8.83E-07 fatty acid synthase FASN 2.784078712 1.52E-06 amidohydrolase domain containing 2 AMDHD2 2.725082675 1.18E-05 mannosyl (alpha-1,3-)-glycoprotein beta- MGAT1 2.723571988 2.94E-05 1,2-N-acetylglucosaminyltransferase nuclear receptor subfamily 1, group D, member 1 /// thyroid hormone receptor, NR1D1 /// TH 2.718291172 3.59E-05 alpha (erythroblastic leukemia viral (v- erb-a optineurin OPTN 2.696522366 2.83E-06 Cdk5 and Abl enzyme substrate 1 CABLES1 2.672148157 1.10E-05 Continued 169

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle oxysterol binding protein-like 3 OSBPL3 2.671962944 1.10E-05 sialidase 1 (lysosomal sialidase) NEU1 2.653506348 8.29E-07 purinergic receptor P2X, ligand-gated ion P2RX7 2.64964669 7.07E-05 channel, 7 NIMA (never in mitosis gene a)-related NEK6 2.637735674 5.28E-05 kinase 6 vascular endothelial growth factor B VEGFB 2.607920697 1.34E-05 hypothetical protein LOC100129034 LOC100129034 2.607197728 5.49E-05 hypothetical protein LOC729678 LOC729678 2.605210597 1.42E-05 interferon regulatory factor 5 IRF5 2.601240877 1.83E-06 SET domain, bifurcated 2 SETDB2 2.580946077 6.21E-07 ankyrin repeat domain 9 ANKRD9 2.580051744 2.02E-04 growth factor independent 1 transcription GFI1 2.574335349 2.27E-04 repressor sterol regulatory element binding SREBF2 2.561874849 4.65E-06 transcription factor 2 hypothetical LOC645644 FLJ42627 2.560632121 2.20E-06 GM2 ganglioside activator GM2A 2.527130209 1.93E-06 isopentenyl-diphosphate delta isomerase 1 IDI1 2.499604773 1.99E-04 RAB20, member RAS oncogene family RAB20 2.497872779 1.68E-06 Similar to hCG2045213 LOC100131014 2.491992973 3.97E-04 solute carrier family 17 (anion/sugar SLC17A5 2.468098547 1.51E-05 transporter), member 5 ST3 beta-galactoside alpha-2,3- ST3GAL5 2.466388384 6.11E-05 sialyltransferase 5 StAR-related lipid transfer (START) STARD4 2.465875566 3.87E-06 domain containing 4 kelch-like 24 (Drosophila) KLHL24 2.462630196 8.94E-06 chromosome 19 open reading frame 48 C19orf48 2.435807293 3.44E-06 solute carrier family 20 (phosphate SLC20A1 2.432938749 1.15E-04 transporter), member 1 chloride channel 6 CLCN6 2.432938749 3.88E-05 solute carrier family 11 (proton-coupled SLC11A2 2.420659235 7.91E-05 divalent metal ion transporters), member 2 ATPase, H+ transporting, lysosomal ATP6V1A 2.41780853 4.53E-05 70kDa, V1 subunit A Continued

170

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle interleukin 9 receptor IL9R 2.396451928 1.52E-04 aldehyde dehydrogenase 5 family, ALDH5A1 2.377919704 4.18E-06 member A1 ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mental CLN8 2.373802651 2.89E-05 retardation) tumor necrosis factor receptor superfamily, member 14 (herpesvirus TNFRSF14 2.354139805 9.25E-05 entry mediator) TBC1 domain family, member 24 TBC1D24 2.353813474 1.49E-06 GINS complex subunit 2 (Psf2 homolog) GINS2 2.351204459 2.94E-05 chromosome 5 open reading frame 32 C5orf32 2.345669898 5.00E-05 neurolysin (metallopeptidase M3 family) NLN 2.340472802 3.73E-04 phosphatase and actin regulator 1 PHACTR1 2.327853069 1.51E-04 potassium voltage-gated channel, shaker- KCNAB2 2.312574752 4.83E-04 related subfamily, beta member 2 zinc finger E-box binding homeobox 2 ZEB2 2.307770884 2.40E-05 caspase 3, apoptosis-related cysteine CASP3 2.297237472 4.51E-06 peptidase transmembrane protein 97 TMEM97 2.294691166 2.43E-04 tripeptidyl peptidase I TPP1 2.283742514 1.98E-05 insulin receptor substrate 2 IRS2 2.280894946 2.91E-05 sequestosome 1 SQSTM1 2.280736852 1.58E-05 chromosome 9 open reading frame 21 C9orf21 2.270484204 5.69E-06 solute carrier family 2 (facilitated glucose SLC2A13 2.267024527 4.87E-05 transporter), member 13 MAX dimerization protein 1 MXD1 2.263570122 2.29E-05 solute carrier family 19 (thiamine SLC19A2 2.26231528 3.00E-05 transporter), member 2 v-maf musculoaponeurotic fibrosarcoma MAFF 2.259494429 8.85E-05 oncogene homolog F (avian) regulator of chromosome condensation (RCC1) and BTB (POZ) domain RCBTB1 2.256364275 2.75E-04 containing protein 1 cathepsin A CTSA 2.245287207 4.50E-05 folliculin interacting protein 1 FNIP1 2.240778428 1.70E-06 Continued

171

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle meteorin, glial cell differentiation METRNL 2.235193918 1.18E-04 regulator-like chromosome 5 open reading frame 41 C5orf41 2.229005227 2.07E-06 junction mediating and regulatory protein, JMY 2.219138944 2.99E-05 p53 cofactor potassium voltage-gated channel, Shaw- KCNC4 2.20045215 8.60E-05 related subfamily, member 4 chromosome 1 open reading frame 163 C1orf163 2.198013123 9.85E-06 cytoplasmic FMR1 interacting protein 2 CYFIP2 2.197251482 2.76E-04 centrosomal protein 68kDa CEP68 2.197251482 6.77E-06 chromosome 6 open reading frame 108 C6orf108 2.188587403 2.03E-04 ribonuclease P/MRP 25kDa subunit RPP25 2.175429102 3.22E-05 arrestin domain containing 2 ARRDC2 2.174072422 3.03E-04 StAR-related lipid transfer (START) STARD9 2.168804482 7.00E-06 domain containing 9 mannosidase, alpha, class 1A, member 1 MAN1A1 2.167151482 8.07E-05 TCDD-inducible poly(ADP-ribose) TIPARP 2.156811359 5.83E-06 polymerase WD repeat domain 81 WDR81 2.143249787 5.21E-06 small glutamine-rich tetratricopeptide SGTB 2.136575058 3.29E-06 repeat (TPR)-containing, beta dynein heavy chain domain 1 DNHD1 2.134206834 8.13E-06 N-methylpurine-DNA glycosylase MPG 2.127265346 3.35E-06 cystinosis, nephropathic CTNS 2.120640397 5.48E-06 insulin-like growth factor 2 receptor IGF2R 2.108182847 2.18E-04 synaptogyrin 3 SYNGR3 2.105700132 2.70E-04 perilipin 2 PLIN2 2.105116389 8.03E-06 lipoma HMGIC fusion partner-like 2 LHFPL2 2.100889088 6.82E-06 ankylosis, progressive homolog (mouse) ANKH 2.098705885 5.24E-06 N-acetylneuraminic acid synthase NANS 2.097542438 5.49E-06 solute carrier family 35, member B2 SLC35B2 2.094491457 7.25E-05 chromosome 17 open reading frame 91 C17orf91 2.086811082 3.84E-04 PHD finger protein 11 PHF11 2.082476189 7.46E-05 protein arginine methyltransferase 2 PRMT2 2.078582485 1.46E-04 ubiquitin associated protein 2 UBAP2 2.075127527 1.12E-05 glucosamine (N-acetyl)-6-sulfatase GNS 2.071104 1.12E-05 Continued 172

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle farnesyl-diphosphate farnesyltransferase 1 FDFT1 2.068521568 4.71E-05 Transforming growth factor, beta receptor TGFBR1 2.06665848 7.86E-06 1 Heterogeneous nuclear ribonucleoprotein D (AU-rich element RNA binding protein HNRNPD 2.063080335 3.90E-04 1, 37kDa) zinc finger and BTB domain containing ZBTB10 2.060079481 2.97E-04 10 Hermansky-Pudlak syndrome 1 HPS1 2.052240772 6.34E-05 family with sequence similarity 102, FAM102A 2.05081876 2.50E-05 member A phosphofructokinase, muscle PFKM 2.048119651 1.29E-05 N-terminal asparagine amidase NTAN1 2.045565877 1.21E-04 transketolase TKT 2.041316662 5.00E-05 insulin receptor substrate 1 IRS1 2.041033695 1.52E-04 ATPase, H+ transporting, lysosomal ATP6V1F 2.038912693 1.74E-04 14kDa, V1 subunit F chloride channel 5 CLCN5 2.035805876 4.24E-05 3-ketodihydrosphingosine reductase KDSR 2.033408397 1.52E-05 N-sulfoglucosamine sulfohydrolase SGSH 2.031858591 4.44E-05 transcription factor 7 (T-cell specific, TCF7 2.030450702 1.79E-04 HMG-box) mucolipin 1 MCOLN1 2.014050699 4.12E-05 PTPRF interacting protein, binding PPFIBP2 2.011678839 3.73E-04 protein 2 (liprin beta 2) galactosidase, alpha GLA 2.010006268 4.44E-06 pyruvate dehyrogenase phosphatase PDP2 2.009866949 9.57E-05 catalytic subunit 2 WD repeat domain, phosphoinositide WIPI1 2.005969958 2.06E-04 interacting 1 sorting nexin family member 30 SNX30 2.004718959 1.79E-04 glutathione peroxidase 4 (phospholipid GPX4 2.000138634 2.17E-05 hydroperoxidase) leucine rich repeat containing 16A LRRC16A 0.499896039 1.78E-05 family with sequence similarity 129, FAM129C 0.498788463 1.36E-05 member C Continued

173

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle potassium intermediate/small conductance calcium-activated channel, subfamily N, KCNN4 0.498581065 3.41E-05 member 4 ribosome binding protein 1 homolog RRBP1 0.498339211 1.65E-04 180kDa (dog) hypothetical LOC401068 LOC401068 0.497717838 4.01E-04 actin, beta ACTB 0.497476403 2.01E-04 ligase IV, DNA, ATP-dependent LIG4 0.494691152 3.87E-05 chromosome 11 open reading frame 21 C11orf21 0.494245591 1.36E-05 hematopoietic SH2 domain containing HSH2D 0.491103837 1.08E-04 phosphoinositide-3-kinase, catalytic, PIK3CG 0.490661507 1.11E-04 gamma polypeptide TBC1 domain family, member 9 (with TBC1D9 0.489744094 1.34E-05 GRAM domain) selectin P (granule membrane protein SELP 0.489472598 1.51E-04 140kDa, antigen CD62) ATP-binding cassette, sub-family G ABCG1 0.488489684 4.42E-04 (WHITE), member 1 RAB37, member RAS oncogene family RAB37 0.488455825 8.61E-06 EH-domain containing 1 EHD1 0.488455825 2.10E-05 zinc finger protein 36, C3H type-like 2 ZFP36L2 0.487880592 3.22E-05 tumor necrosis factor (ligand) TNFSF10 0.487812962 1.02E-05 superfamily, member 10 lymphocyte-specific protein 1 LSP1 0.485855767 7.07E-05 MACRO domain containing 2 MACROD2 0.485822092 2.41E-04 plexin D1 PLXND1 0.485115439 3.07E-04 solute carrier family 25, member 45 SLC25A45 0.484913727 2.90E-05 placenta-specific 8 PLAC8 0.483202559 1.36E-04 chromosome 1 open reading frame 228 C1orf228 0.48260006 3.45E-04 chromosome 16 open reading frame 54 C16orf54 0.481931498 6.75E-05 phosphodiesterase 7A PDE7A 0.481297222 1.26E-05 RAS guanyl releasing protein 2 (calcium RASGRP2 0.48053053 7.35E-05 and DAG-regulated) methyltransferase like 8 METTL8 0.479864835 2.77E-04 chromosome 14 open reading frame 139 C14orf139 0.479100426 4.30E-04 CXXC finger 5 CXXC5 0.478668907 2.46E-04 Continued

174

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle GTPase activating Rap/RanGAP domain- GARNL4 0.47813834 6.93E-05 like 4 signal transducing adaptor family member STAP1 0.477939529 9.19E-05 1 hypothetical protein MGC29506 MGC29506 0.477442864 2.88E-05 ectonucleotide ENPP2 0.476979775 3.94E-06 pyrophosphatase/phosphodiesterase 2 lymphocyte-specific protein tyrosine LCK 0.475066045 4.11E-04 kinase kelch domain containing 7B KLHDC7B 0.475000191 2.13E-04 zinc finger protein 831 ZNF831 0.474901428 1.98E-04 interferon-induced protein with IFIT2 0.469663701 8.95E-05 tetratricopeptide repeats 2 caspase recruitment domain family, CARD16 0.469468414 2.19E-04 member 16 heparan sulfate (glucosamine) 3-O- HS3ST1 0.46852567 3.05E-04 sulfotransferase 1 solute carrier family 1 (glutamate/neutral SLC1A4 0.467001795 1.76E-04 amino acid transporter), member 4 myosin VC MYO5C 0.465160339 4.23E-05 integrin, beta 7 ITGB7 0.464001058 6.19E-05 salt-inducible kinase 1 SIK1 0.463743832 2.91E-04 sialic acid binding Ig-like lectin 10 /// SIGLEC10 /// 0.461787171 2.01E-05 sialic acid binding Ig-like lectin 12 EH-domain containing 3 EHD3 0.461019602 5.08E-05 cytohesin 4 CYTH4 0.458533804 1.06E-04 anthrax toxin receptor 2 ANTXR2 0.456251118 1.45E-05 ribosomal protein S6 kinase, 90kDa, RPS6KA2 0.455398044 7.89E-05 polypeptide 2 protein tyrosine phosphatase, receptor PTPRO 0.454389058 2.68E-04 type, O contactin associated protein-like 2 CNTNAP2 0.453476596 4.55E-04 hypothetical protein LOC100287598 /// LOC100287598 0.452628709 1.75E-04 similar to predicted protein wingless-type MMTV integration site WNT10A 0.452440506 2.81E-04 family, member 10A Z-DNA binding protein 1 ZBP1 0.450937689 2.45E-05 Continued

175

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle ankyrin 1, erythrocytic ANK1 0.45040664 8.03E-06 glutaminyl-tRNA synthase (glutamine- QRSL1 0.450344204 5.67E-06 hydrolyzing)-like 1 thioredoxin interacting protein TXNIP 0.447853877 3.12E-04 transducin-like enhancer of split 1 (E(sp1) TLE1 0.446706762 2.36E-04 homolog, Drosophila) CD74 molecule, major histocompatibility CD74 0.446706762 4.59E-04 complex, class II invariant chain LFNG O-fucosylpeptide 3-beta-N- LFNG 0.44500702 5.24E-05 acetylglucosaminyltransferase zinc finger protein 92 ZNF92 0.441045683 1.20E-05 ectonucleoside triphosphate ENTPD1 0.440953979 3.08E-04 diphosphohydrolase 1 phosphatidylinositol transfer protein, PITPNM2 0.438333243 5.00E-06 membrane-associated 2 protein tyrosine phosphatase, receptor PTPRCAP 0.429015001 1.54E-04 type, C-associated protein CD24 molecule CD24 0.426642621 1.64E-04 GRB2-binding adaptor protein, GAPT 0.426228805 1.20E-04 transmembrane Rap guanine nucleotide exchange factor RAPGEF3 0.423284627 3.22E-05 (GEF) 3 phosphodiesterase 7B PDE7B 0.420186 4.54E-04 natural cytotoxicity triggering receptor 3 NCR3 0.419691166 1.91E-06 lymphocyte antigen 86 LY86 0.41838412 5.93E-05 solute carrier family 16, member 6 SLC16A6 0.411111034 3.22E-05 (monocarboxylic acid transporter 7) solute carrier family 14 (urea transporter), SLC14A1 0.407847015 6.92E-05 member 1 (Kidd blood group) Fc receptor-like 1 FCRL1 0.399176861 2.97E-05 Immunoglobulin lambda locus IGL@ 0.396117405 8.33E-06 paternally expressed 10 PEG10 0.395788061 8.25E-05 chromosome 7 open reading frame 50 C7orf50 0.395020656 3.86E-05 integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; ITGAL 0.393244911 3.79E-05 alpha polypeptide) Continued

176

Table 3 Continued

Fold Gene Title Gene Symbol Difference to P value Vehicle guanylate cyclase 2C (heat stable GUCY2C 0.38667677 3.15E-04 enterotoxin receptor) limb bud and heart development homolog LBH 0.382332567 1.20E-04 (mouse) arrestin domain containing 3 ARRDC3 0.379402213 5.32E-06 arachidonate 5-lipoxygenase ALOX5 0.379244457 1.86E-05 glutamic pyruvate transaminase (alanine GPT2 0.378194425 2.95E-04 aminotransferase) 2 uncoupling protein 2 (mitochondrial, UCP2 0.369744348 2.29E-05 proton carrier) sushi domain containing 3 SUSD3 0.358339752 1.21E-04 chronic lymphocytic leukemia up- CLLU1 0.357199013 4.98E-04 regulated 1 NDC80 homolog, kinetochore complex NDC80 0.356259404 1.30E-04 component (S. cerevisiae) T-cell leukemia/lymphoma 1A TCL1A 0.32005683 3.27E-04 ATP-binding cassette, sub-family A ABCA1 0.316548837 3.17E-04 (ABC1), member 1 Phospholipase C, gamma 2 PLCG2 0.304919432 3.41E-06 (phosphatidylinositol-specific) chemokine (C-X-C motif) receptor 3 CXCR3 0.284480171 7.61E-06 GLI pathogenesis-related 1 GLIPR1 0.278954166 8.83E-05 chromosome 6 open reading frame 105 C6orf105 0.276969688 2.90E-05 mitogen-activated protein kinase kinase 6 MAP2K6 0.272929454 1.25E-05 lymphotoxin beta (TNF superfamily, LTB 0.269209271 1.02E-04 member 3) pre-B lymphocyte 3 VPREB3 0.211833109 1.00E-04 gamma-aminobutyric acid (GABA) A GABRB2 0.207891114 2.70E-07 receptor, beta 2 CD1d molecule CD1D 0.133665588 8.58E-06

177

Table 4: Functional molecules in cancer affected by OSU-2S.

Function Category Function p-value Molecules Annotation chronic B-cell chronic B-cell Cancer 2.61E-02 CD74, MSMO1 leukemia leukemia cutaneous T-cell cutaneous T-cell Cancer 3.62E-02 CXCR3 lymphoma lymphoma development of B Cancer development 1.82E-02 LFNG cell lymphoma cells formation of burkitt- Cancer formation 1.82E-02 TCL1A like lymphoma CD274, CD74, Cancer hematologic cancer hematologic cancer 4.09E-02 MSMO1 CD274, CD74, hematological hematological CXCR3, GFI1, Cancer 3.37E-03 neoplasia neoplasia MSMO1, TCL1A CD274, CD74, lymphohematopoieti lymphohematopoieti CXCR3, GFI1, Cancer 3.37E-03 c cancer c cancer MSMO1, TCL1A CXCR3, GFI1, Cancer lymphomagenesis lymphomagenesis 3.84E-02 TCL1A Cancer ovarian cancer ovarian cancer 3.62E-02 CD274 tumorigenesis of Cancer tumorigenesis 6.55E-03 CD274, IL9R leukocyte cell lines

178

Appendix B: Sensitivity of NCI-60 cancer cell lines to OSU-2S

Activity of OSU-2S was tested in NCI-60 cancer cell lines representing various human tumors as a screen test by NCI(205). The methodology of NCI-60 DTP Human Tumor

Cell Line Screen can be found at http://dtp.nci.nih.gov/branches/btb/ivclsp.html.The growth inhibition of 50 % cells (GI50) and total growth inhibitory concentration of OSU-

2S are shown in figure 67 and figure 68 respectively.

179

Figure 67: Activity of OSU-2S in NCI-60 cancerous cell lines.

180

Figure 68: Total growth inhibitory concentration of OSU-2S in NCI-60 cancer cell lines.

181