FTY720-P) Nor SET Dimerization Or Ceramide Induction but Depends on Interaction with SET K209

Antagonistic activities of the immunomodulator and protein phosphatase 2A (PP2A)-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven myeloproliferative neoplasms.

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the degree

Doctor of Philosophy

in the Graduate School of The Ohio State University

Joshua John Oaks, B.S.

Integrated Biomedical Sciences Graduate Program

The Ohio State University

2013

Dissertation Committee

Danilo Perrotti MD, PhD

Roger Briesewitz, PhD

Denis Guttridge, PhD

Harold Fisk, PhD

Joshua John Oaks

ABSTRACT

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant

in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-

activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of

cancer. We show here that PP2A is inactive in Polycythemia Vera (PV) and other myeloproliferative neoplasms (MPNs) characterized by the expression of the transforming Jak2V617F oncogene. PP2A inactivation occurs in a Jak2V617F dose/kinase- dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET and by modulation of nitric oxide synthase (NOS2)-induced nitrosylation of

PP2A. Genetic and/or PAD-mediated PP2A reactivation induces Jak2V617F

inactivation/downregulation and impairs clonogenic potential of Jak2V617F cell lines and

PV but not normal CD34+ progenitors. Likewise, FTY720 decreases leukemic allelic

burden, reduces splenomegaly and significantly increases survival of Jak2V617F leukemic mice without adverse effects. Mechanistically, we show that in Jak2V617F cells, FTY720 anti-leukemic activity neither requires FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we showed that Jak2V617F also utilizes an alternative sphingosine kinase-1 (SPHK1)- mediated pathway to inhibit PP2A, and that FTY720-P, acting as a sphingosine-1- phosphate-receptor-1 (S1PR1) agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-

SET-mediated PP2A inhibition. Thus, PADs (e.g. FTY720) represent suitable therapeutic

alternatives for Jak2V617F MPNs.

ACKNOWLEDGEMENTS

This work would not be possible without the help and guidance of many people to whom I am indebted.

Firstly, I would like to thank my advisor, Dr. Danilo Perrotti. He has given me the inspiration, direction and opportunity to pursue the research I love. He is the spark that drives all of us who work for him and without his guidance and experience none of this work would be possible. Likewise, all of the current and former members of the Perrotti

laboratory I have had the pleasure to work with have been essential making this work

possible.

I would also like to thank: Dr. J. Van Brocklyn who has served as my

“sphingolipid expert” throughout this project. He was always willing to assist me with

protocols, discussions, or experiments and was never reluctant to do so despite his busy

schedule; Dr. Briesewitz for his technical support but more for his willingness to be

involved with my professional development; and, finally, Drs. Landesman and Kalid, and

Ms. Shechter, and the crew at Karyopharm for their enthusiasm for our research on

pharmacologic development of SET inhibitors and their generosity allowing the

continuation of this project.

iii

Vita et Studiorum

Born March 18, 1977 . . . . ………………………………………………………. Dayton, Ohio

2003 ………………………………………………B.S. Dietetics, University of Cincinnati

Field of Study

Major Field: Integrated Biomedical Sciences

Area of Emphasis: Pharmacology

Table of Contents

Abstract…………………………………………………………………..……………..……ii

Acknowledgements…………………………………………………………………..…….iii

Vita et Studiorum…..………………………………………………………….……..……..iv

List of Tables……………………………………………………………………………….vi

List of Figures………………………………………………………………………….…..vii

Chapters

1. Introduction……...………………………………………………….………………..1

2. Regulation of Jak2- and Jak2V617F-driven erythropoiesis…………...…...... …..16

3. Methods…………………..…………………………………………………….…...92

4. Results………………………………………………………..………..……….….109

5. Discussion……………………………………………………………….………...139

6. Future Directions……………………………………………………………….....146

References………………………………………………………………………..…..……158

List of Tables

1 List of major kinases regulated by PP2A…………………………….……..…42

2 S1P receptor G-protein coupling…………………………………………...….58

3 S1P1 downstream inhibitors……………………………………………...….…93

List of Figures

1 Jak2 and common Jak2 mutations ………………...……………………..…………4

2 Myeloid hematopoiesis …..………………………..………………..……...……..……13

3 Wild type and V617F Jak2 signaling ...…….………………..…………………...…14

4 Protein Phosphatase 2a (PP2A) ……………………….………..………………...... 17

5 Nitric oxide synthase (NOS) activates PP2A …………………..……………..……….53

6 Sphingolipids ………………………………………………..……………...... …….57

7 FTY720 and FTY720-(S)-Phosphate …….……………………...………………..……84

8 FTY720 Immunosuppressive activity …...……………...…………….…………….85

9 FTY720 anti-cancer activity ………….....……………...…………….…………….89

10 Jak2V617F suppresses PP2A activity in a SET-dependent manner A and B……….110

10 Jak2V617F suppresses PP2A activity in a SET-dependent manner C and D………112

11 Jak2V617F/PI3Kγ/PKC signals inhibit PP2A through SET phosphorylation A.….…114

11 Jak2V617F/PI3Kγ/PKC signals inhibit PP2A through SET phosphorylation B.….…115

11 Jak2V617F/PI3Kγ/PKC signals inhibit PP2A through SET phosphorylation C and

D…………………………………………………………………………………………...115

12 In vivo FTY720 anti-leukemic effects and lack of toxicity A and B

……………………………………………………………………………………….…….120

12 In vivo FTY720 anti-leukemic effects and lack of toxicity C…………………………121

12 In vivo FTY720 anti-leukemic effects and lack of toxicity D…………………………123

12 In vivo FTY720 anti-leukemic effects and lack of toxicity E…………………………124

13 FTY720 phosphorylation is dispensible for its anti-leukemic activity A….………125

13 FTY720 phosphorylation is dispensible for its anti-leukemic activity B….………126

vii

13 FTY720 phosphorylation is dispensible for its anti-leukemic activity C….………128

14 FTY720-P promotes Jak2 and suppresses PP2A activity A and B…..………....…131

14 FTY720-P promotes Jak2 and suppresses PP2A activity C…..………………....…132

14 FTY720-P promotes Jak2 and suppresses PP2A activity D and E…..………....…134

15 FTY720-dependent PP2A activation in myeloid cells depends on the SET K209-

FTY720 interaction but not on SET dimerization or ceramide levels A and B...….136

15 FTY720-dependent PP2A activation in myeloid cells depends on the SET K209-

FTY720 interaction but not on SET dimerization or ceramide levels C and D...….138

16 Antagonizing effect of FTY720 and FTY720-P on leukemogenesis……………….145

17 Novel FTY720 analogs …..…….………...... …150

18 SET inhibitor development strategy……… ……………………..…….………..….…152

19 Molecular docking of FTY720 and SET………..………..……..………….……….…153

20 Novel SET inhibitors ……………………………….………………...... …………..…155

viii

CHAPTER 1

INTRODUCTION

Polycythemia Vera (PV) is a rare hematopoietic malignancy that involves the erythroid

(red blood cell) system. It is currently defined as a myeloproliferative neoplasm (MPN)

and affects fewer than 2 people in one million. This disorder was first described by Louis

Vaquez in 1892 (1) and by William Osler in 1903 (2) and was originally called “Osler-

Vaquez disease”. Initially, the disorder was associated to altitude sickness “the torpor, mental and physical; the sensation of fullness in the head, with headache, vertigo and in some cases nausea and vomiting, remind us of the symptoms to which mountain climbers and aeronauts are subject” (2).

By modern definitions, PV typically manifests as uncontrolled erythropoiesis (3-5) with a hematocrit of >52% in Caucasian men and >47% in men of African origin or in women.

Splenomegaly and pruritus are also common sympthoms used for diagnosis of PV (6).

The high erythroid cell content in the peripheral blood (PB) of PV patients leads to hyperviscosity, which can result in thrombosis (7-12) and, therefore, in high risk for heart attack or stroke.

While red blood cell count is still part of the PV diagnostic panel, a polymerase chain reaction (PCR)-based test is now used to assess presence of a mutated Janus Kinase 2

(Jak2) oncogene that in the majority of PV cases represents the hallmark of this hematologic disorder (see below).

1.1 The Jak2 receptor tyrosine kinase

Janus Kinase 2 (Jak2) is a receptor-bound cytosolic tyrosine kinase consisting of 7

JH (Jak homology) domains: a Src homology 2 domain (SH2), a tyrosine kinase domain

(JH1), two c-terminal domains, and a pseudokinase domain (JH2). The JH3-7 region forms a FERM (4.1 protein, ezrin, radixin, moesin-homology) domain, which is required

for binding to the erythropoietin (EPO) receptor. Jak2 translates a conformational change

caused by ligand [e.g. EPO] binding to cytokine receptors (e.g. EpoR) into an

intracellular signaling cascade (13). This is accomplished by the phosphorylation of specific tyrosine residues on the receptor N-terminus (intracellular) that provides a

docking site for signal transducer and activator of transcription (STAT) proteins (14, 15),

which upon Jak2-dependent phosphorylation dimerize and relocate into the nucleus

where they act as modulators of transcription (16-18).

Chromosomal alterations and gene mutations have been reported for the Jak2

gene which results in a mutated Jak2 protein (Figure 1). The best described chromosomal abnormality is the gene translocation leading to the formation of chimeric tyrosine kinase protein TEL-Jak although a PCM1-Jak2 fusion gene was also found in a few leukemia patients (19). TEL-Jak (or TEL-Jak2) is a fusion protein combining the 5’ region of the

TEL (also called ETC6) transcription factor with the kinase (JH1) domain of Jak2. The chromosomal alteration leads to the loss of inhibitory regulatory domains resulting in constitutively active Jak2 signaling that has been associated with the development of acute lymphoblastic leukemia (ALL) (20-22). Likewise, the PCM1-Jak2 fusion protein 2

has been found only in patients with atypical chronic myelogenous leukemia (CML).

Unlike TEL-Jak and other Jak2-related chromosomal aberrations, Jak2 point mutations

(e.g. V617F and exon 12 mutations) were found in PV patients. Interestingly, these mutations were located within or adjacent to the Jak2 pseudokinase domain (23-29).

Specifically, exon 12 mutations are present in a few cases of PV while the V617F mutation is the most common Jak2 mutation found in nearly 90% of PV patients(30).

Figure 1. Jak2 and Common Jak2 Mutations. Jak2 contains 7 “Jak-Homology” (JH) domains and can be dividing into functional sections. All known mutations found in leukemia/MPDs retain the JH1 “kinase” domain. Common mutations are also found in or near the JH2 “pseudokinase” domain, which produces a loss of inhibitory control.

Jak2V617F is caused by a G to T single nucleotide mutation at nucleotide 1849 on chromosome 9 resulting in a valine to phenylalanine substitution in exon 14 of the inhibitory JH2 “pseudokinase” domain of Jak2. This mutation leads to constitutive Jak2 3

activition through the loss of inhibition normally provided by the JH2 domain. This

results in cytokine hypersensitivity and uncontrolled proliferation of erythroid

progenitors.

Wild-type Jak2 activity is controlled at the post-translational level by a variety of cellular

regulators that control proliferation and maturation of erythroid progenitors (31-34).

Among these, there are the suppressor of cytokine signaling (SOCS) proteins, SOCS1,

SOCS3, protein phosphatase 2A (PP2A), and Src homology region 2-domain

phosphatase-1 SHP-1(35). Notably, the SOCSs proteins lose their inhibitory function in

cells expressing Jak2V617F and, paradoxically, SOCS3 can even enhance proliferation in

erythroid cells expressing the Jak2V617F mutated protein (28), resulting in greater

expansion of the erythroid population and, therefore, increased hematocrit (ispissatio

sanguinis). Thus, it is through escape of these regulatory inhibitory mechanisms that

Jak2V617F exerts its oncogenic activity by inducing and sustaining hyperproliferative

aberrant signals, the inhibition of which still represents a therapeutic challenge.

While the measurement of total Jak2 expression levels is straightforward, the assessment of Jak2 activation presents some complications. The most common laboratory-based method for measuring Jak2 activation is to employ immunoblotting to assess Jak2 phosphorylation on tyrosine residues 1007/1008 even if several other tyrosine residues have been shown to correlate with Jak2 activity. These include the tyrosines 221,

813, 868, 966, and 972, which are associated with Jak2 activation, and the tyrosines 119

and 570 that, instead, correlated with reduced kinase activity (36-38). Real-time PCR is 4

now used in clinic quantification of Jak2 oncogenic allele burden and assessment of the

Jak2 mutation status.

1.2 Polycythemia Vera: biology, clinical features and therapy.

As stated above, PV is associated with an aberrant and constitutive Jak2 tyrosine

kinase activity that depends on a valine to phenylalanine (V617F) substitution in the Jak2 inhibitory JH2 “pseudokinase” domain. The mutated Jak2V617F oncogene, discovered by

Vainchenker et. al. in 2005 (34), is found in over 90% of PV cases (39) and ~40% of

these patients are homozygous for the mutation (4, 34). Thus, the uncontrolled Jak2V617F

activity is essential for the pathogenesis of PV although other additional oncogenic events may contribute to the maintenance of the disease (see below).

PV affects patients with a median age of 50 and clinically presents itself with an asymptomatic developmental phase that it may last for as many as five years (40). This phase is followed by an erythrocytosis phase of five to twenty years. During this symptomatic and pathognomonic phase, characterized by the excessive production of red blood cells, patients may also show signs of bone marrow myelofibrosis. If the disease is not diagnosed and treated at this stage with phlebotomy or medication (see below) the patient has a high likelihood of dying of cardiac events. Over time, PV patients can develop a post-polycythemic myeloid metaplasia (PPMM) or “spent phase”, which is characterized by a hypocellular bone marrow and is indicative of the inability of hematopoietic progenitors to develop into mature erythrocytes. At this stage, which

occasionally responds to interferon treatment or bone marrow transplantation (see

below), the patient experiences anemia and shows pronounced splenomegaly.

PV patients also present non-specific symptoms such as headache, dizziness, and excessive sweating (41). Pruritus (itchy, irritated skin) is also commonly found in patients

with PV, possibly due to histamine release or IL-7 secretion (42). Gastric distress is likely

related to histamine levels and is also found with PV (43). Patients with particularly

thrombocythemia (high platelet counts; >400,000/µL), including those affected by the

PV-related myeloproliferative disorder Essential Thrombocythemia (ET), experience redness with burning hands and feet (44, 45). The most serious concern for PV patients is an increased risk of thrombosis which can result in a potentially fatal heart attack or stroke, as no specific symptomatic signs announce the advent of thrombosis (46).

Despite of the discovery of Jak2 mutations, PV is still diagnosed primarily through measurement of increased red blood cell mass (>36 mL/kg in men and >32 mL/kg in women). This method was tweaked by taking into account other factors such as body surface area (47), elevated hematocrit, low EPO levels. However, the presence of the mutated Jak2 serves now as a primary diagnostic criterion.

PV has traditionally been treated primarily with phlebotomy to reduce the hematocrit; nearly 100% of patients with PV receive this treatment alone or in combination with drugs that reduce the risk of thrombosis (48). Other therapies have included traditional myelosuppressive agents such as hydroxyurea (HU) or anagrelide. HU can reduce the risk of thrombosis by reducing platelet counts; however it has also been associated with 6

an increased risk of leukemic transformation (48). Anagrelide is typically used to reduce

the thrombosis risk associated with PV but, unlike HU, has not been shown to increase

the risk of leukemic transformation. However, anagrelide therapy presents other adverse

effects such as heart palpitation (49). Interferon alpha is also used to treat PV (50), and

the new PEG-lyated formulation seems to achieve good clinical outcomes (51) albeit rare

serious complications like induction of diabetes have also been reported (52).

The newest treatments for PV patients are the so-called tyrosine kinase inhibitors

(TKI) which target Jak2 activity. After the success of imatinib mesylate (Gleevec) in the

therapy of CML, there were great expectations for these small molecules inhibiting Jak2

activity. However, although they have been rapidly introduced into the clinic, Jak2

inhibitors show some success at reducing splenomegaly but they were ineffective in

reducing both the leukemic cell burden and the bone marrow fibrosis (27, 53-65). In fact,

levels of the mutated Jak2V617F allele remains unchanged in PV patients treated with Jak2

TKIs when used alone as first line therapy (53, 54, 57-59, 62-64).

Because PV patients, if left untreated, have a dramatically shortened lifespan (66) and current treatments can improve outcome but not cure the disease; the overall mortality is still 1.5 times higher than that of a normal population (12, 67). Thus, it is clear that a better understanding of the biology of erythro-myelopoiesis in normal individuals and PV patients is necessary for unraveling the molecular mechanisms responsible for the emergence and controlling the development of this disease and for the development of more successful therapies. 7

1.3 Erythropoiesis 101: understanding the normal to rationally treat the pathologic process.

At the core of PV is an inability to control erythropoiesis. Erythropoiesis is the process of producing red blood cells (RBCs), also known as “erythrocytes”, the main function of which is to transport oxygen from the lungs to each cell of the organism. In adults, erythropoiesis occurs in the bone marrow of the pelvis, vertebrae and sternum, and produces 1010 erythrocytes per hour. In young adults, the marrow of tibia and femur is involved as well. Erythropoiesis is strictly monitored by the kidneys that secrete EPO in response low oxygen conditions; in fact, EPO stimulates the proliferation of erythroid progenitors leading to the formation of reticulocytes (nucleated red cells) which are released into the peripheral blood while continuing their differentiation process that, within 24 hours, will result in loss of nuclear content to form mature RBCs. Notably, reticulocytes are usually detectable as a minute population of circulating blood cells and their increase is indicative of red blood disorders. A mature RBC will continue to circulate and transport oxygen for three to four months after which the RBC will have accumulated enough damage due to forced passage through capillaries and small blood vessels to make it unstable at which point it is phagocytized and replaced by new erythrocytes.

Erythropoiesis is part of the myeloid differentiation arm of hematopoiesis (Figure 2), and is initiated in a limited number of pluripotent hematopoietic stem cells, which differentiate into myeloid progenitors known as CFU-GEMM (granulocyte, erythrocyte, 8

monocyte and megakaryocyte colony-forming unit). From here, cells differentiate into

BFU-E (burst-forming unit, erythroid) and CFU-E (colony-forming unit, erythroid) that

will mature into reticulocytes. The lymphoid lineage also originates at the stem cell level

but will not be discussed here.

Figure 2. Myeloid hematopoiesis. Hematopoietic pluripotent stem cells produce mature erythrocytes through several differentiation steps. Jak2 V617F produces too many erythrocytes in PV but can influence a parallel pathway and result in excessive platelets producing the related MPD known as essential thrombocytopenia (ET).

Although erythrocyte production is stimulated primarily by erythropoietin (EPO), other

cytokines such as interleukin-3 (IL-3, produced by basophils and T-cells) and

granulocyte-macrophage colony stimulating factor (GM-CSF, secreted by endothelial and

hematopoietic cells) also participate to erythropoiesis specifically at the BFU-E and

CFU-E stages. In this scenario, activation of Jak2 serves to transmit stimulator signals

originated by the ligand-mediated triggering of the erythropoietin receptor (EpoR), GM- 9

CSF receptor (GM-CSFR) and IL-3 receptor (IL-3R), which are all Jak2 kinase-linked

(68-70) although it seems that only the alteration in the EpoR signaling plays a pivotal role in the etiopathogenesis of PV.

Figure 3. Wild type and V617F Jak2 Signaling. (left) In normal erythroblasts, Jak2 is activated upon ligand-mediated triggering of the Epo receptor. This produces a phosphorylation cascade that leads to Jak2-mediated recruitment and activation of STAT proteins. Jak2 kinase activity is thereafter turned off by dephosphorylation mediated by either Suppressor Of Cytokine Signaling (SOCS) proteins or SHP-1 (Src homology region 2 domain-containing phosphatase-1) or by disengagement of Epo from its receptor; (right) In Jak2V617F erythroid cells, Jak2 is constitutively active and continues signaling through STATs in the absence of cytokines and despite SOCS activity. Notably, we found that PP2A is inactive in these cells.

The EpoR is a type I cytokine receptor encoded by the EPOR gene. The binding of EPO induces EpoR dimerization that, in turn, triggers the auto- or trans-phosphorylation of the

Jak2 bound to the cytoplasmic tail of the EpoR itself. The phosphorylated/activated Jak2 kinases phosphorylate the receptor leading to STAT5 recruitment, phosphorylation and translocation into the nucleus where it acts as a transcription factor (Figure 3).

CHAPTER 2

Regulation of Jak2- and Jak2V617F-driven Erythropoiesis

2.1 The tumor Suppressor Protein Phosphatase 2A (PP2A)

Although Jak2V617F is resistant to regulation by SOCS, erythroid progenitors can use PP2A to deactivate the tyrosine kinase-driven proliferation triggered by the EPO-

EpoR interaction.

PP2A is a trimeric holoenzyme (Figure 4) consisting of a “C” catalytic subunit, an “A” scaffolding subunit that as an A-C dimer (termed PP2AD) (71) associates to a specific

“B” regulatory subunit which confers substrate specificity. PP2A is a widely distributed regulator of cell proliferation, survival, differentiation and cell cycle progression. PP2A is a serine/threonine phosphatase that regulates cell signaling by directly and/or indirectly targeting serine/threonine kinases, tyrosine kinases, phosphatases and other proteins regulating transcription (e.g. c-Myc, beta-catenin), apoptosis (e.g. BAD, 14-3-3), and many other regulators of different cellular processes (72). PP2A regulates the cell cycle through interaction with Aurora kinases A and B, Src, FOXO1A, and many others (73-

77) and has been shown to restrain the effects of oncogenic kinases such as BCR-ABL1

and mutated c-Kit as well as that of Jak2, Src-family kinases and others (73-76, 78-85).

Loss of PP2A can be tumorigenic (86, 87), while increased expression is oncosuppressive

(88) and induces apoptosis (89, 90). 12

2.1.1 PP2A structure

Regulation of such a wide variety of cellular substrates is achieved primarily

through either regulation of the phosphatase activity produced by the catalytic subunit or

by alterations in the expression of specific regulatory B subunits (91-93). There are two

Figure 4. Protein Phosphatase 2A (PP2A). PP2A functions as a trimeric holoenzyme: an “A” scaffolding subunit, a “B” regulatory subunit, which regulates substrate specificity, and a catalytic “C” subunit which is itself regulated by post- translational modifications such as phosphorylation, nitration, and methylation. The catalytic subunit can also be regulated by endogenous proteins such as the inhibitors SET, I1PP2A, and CIP2A.

described isoforms of the C subunit (α and β) which are nearly identical in sequence (94,

95) with the α subunit being the much more abundant isoform (96) due to a more active

promoter (97). There are also two isoforms (α and β) of the scaffolding A subunit which

also have high sequence similarity (98), with the α subunit predominating in expression

(99). The different associations of these subunits, their post-translational modifications and interactions with other regulatory proteins all influence the activity of PP2A toward a specific substrate. Notably, the combination of different A, C and B subunits leads to the possible formation of more than one hundred different PP2A holoenzymes.

PP2A has been shown to bind to, and be inhibited by Src-kinases (100) and Jak2 (83, 84,

101). Conversely, PP2A appears activated upon interaction with the tyrosine phosphatase

SHP-1 (78). Indeed, PP2A forms a complex with both Jak2 and SHP-1 (101), in which the latter is proposed as a controller of the inhibitory effects of Jak2 on PP2A phosphatase activity. It is because of this interplay and of the essential role played by

PP2A in cell survival and proliferation that pharmacologic targeting (re-activating) of

PP2A by the different PP2A Activating Drugs (PADs; e.g. FTY720, OSU-2S, 1,9- dideocy forskolin etc.) represents a promising avenue by which the activity of the oncogenic Jak2V617F can be neutralized.

2.1.2 PP2A regulation

PP2A can be regulated through several independent mechanisms. The phosphatase activity of PP2A can be altered, the subcellular localization of PP2A can be influenced and, finally, the substrate specificity of PP2A can also be controlled.

The catalytic activity of PP2A can be controlled by post-translational modifications. The best described of these is tyrosine phosphorylation, particularly on tyrosine 307 of the catalytic subunit. Phosphorylation at this residue as been primarily associated with inactivity of PP2A (84, 102-104). Moreover, phosphorylation of tyrosine

307 has been demonstrated to reduce binding of the regulatory subunits PR55B and

PR61B αβɛ to the A/C dimer which can alter the activity of PP2A to specific substrates

(104). Interestingly, tyrosine phosphorylation at this site as also been shown to influence

leucine methylation (discussed later) (105). An additional phosphorylation on threonine

304 has also been associated with regulation of the binding to the B subunit known as

PR55 (105).

Another post-translational modification, nitrosylation, may control PP2A activity through

inhibition of tyrosine phosphorylation. Nitration of PP2A occurs on tyrosine residues,

again likely 307, as a result of nitric oxide synthase (NOS2) activity (106, 107). Nitration

is not believed to have any function on it’s own but is used to prevent PP2A

phosphorylation. Loss of inhibitory phosphorylation yields active PP2A following

nitration. Finally, methylation of leucine 309 has been reported (108). Methylation of

PP2A is controlled by two known enzymes: leucine carboxyl methyltransferase 1

(LCMT1) and protein methylesterase 1 (PME-1), which add and remove a methyl group,

respectively (109, 110). The role of methylation of PP2A has not been fully verified; in

fact, PP2A methylation has been associated to increase, decrease or no change in PP2A

activity depending on cell type evaluated (108, 109, 111). Another potential but still disputed role for methylation is its involvement in the regulation of the PP2AAC dimer

binding to regulatory B subunits (112-114). Nonetheless, PME-1 may also regulate PP2A

activity independently of methylation (115).

PP2A methylation also controls binding of PP2A phosphatase activator (PTPA). PTPA is

an ATP-dependent isomerase that has been shown to activate PP2A when bound to the

A-C dimer (116). Recent findings suggest that the binding of PTPA to the A scaffolding subunit occurs in a PME-1-dependent manner and is important for activation of PP2A 15

phosphatase activity toward phosphoserine/phosphothreonine resisdues (115).

Conversely, the loss of PTPA increases the phosphotyrosine phosphatase activity of

PP2A likely through a conformational change in the catalytic subunit and, has been

associated with apoptosis of HeLa cells (117).

Like the catalytic C subunit, the scaffolding A subunit is also required for PP2A

activity and its binding to the other subunits is also post-translationally regulated. The A

subunit consists of 15 highly similar HEAT (Huntingtin, Elongation, A subunit, TOR motifs where TOR is PI3K Target of Rapamycin). Modification of any of these motifs can result in loss of PP2A phosphatase activity. When measured in a cell-free system, increasing concentrations of PP2A A added to the C subunit produced a reduction in

PP2A activity primarily when measured by dephosphorylation of a phosphoprotein while

PP2A activity towards a smaller phosphopeptide was only marginally changed (118).

While the best described sources of PP2A regulation involve activity modulation through the C subunit which can be post-translationally modified and can accept binding partners such as SET or through substrate selectivity via the regulatory B subunits (described below) PP2A can also be regulated through interactions with the scaffolding A subunit.

The most basic function of the A subunit is to allow the recruitment of regulatory

B subunits to the catalytic C subunit; because of this the A subunit is required for PP2A activity (86). The A subunit through its HEAT domains interacts with both the C and B subunits. Mutations of any of these HEAT regions reduce the affinity for these subunits and, therefore, induce a loss of PP2A activity (119, 120). Moreover, the A subunit 16

participates in the regulation of the PP2A substrate binding specificity. Specifically, the

PP2A A subunit is necessary for the binding of human telomerase reverse transcriptase

(hTERT) to PP2A in both yeast (121) and breast cancer cells (122). Indeed, while PP2A has been shown to bind and inhibit the activity of hTERT the PKC activator phorbol myristate acetate (PMA) activates hTERT. Notably, we also found PKC to participate in the signaling leading to PP2A inhibition in leukemia (discussed below).

The A subunit also is essential for binding to heat shock factor 2 (HSF2) (123) and to the

tumor suppressor HRSL3 (124). PP2A complexed with HRSL3 results in PP2A inhibition. Other binding partners to the A subunit include the cystic fibrosis transmembrane conductance regulator protein (CFTR) (125), AMP-activated protein kinase (AMPK) (126), importin 9 (127), and SMG-5 (128). Finally, it has been reported that the A subunit plays an important role in the stabilization of the C subunit as unbound subunits are subjected to rapid proteasomal degradation (129, 130).

The catalytic activity of PP2A has also been shown to be influenced by the presence of cations. In one study Galadari et al. examined the role of cations on ceramide-induced activation of PP2A (131). It seems that chromium, manganese, iron, nickel, copper and zinc all inhibited PP2A regardless of whether the PP2A had been stimulated with ceramide or not. Conversely, calcium stimulated basal PP2A activity but was inhibitory towards those PP2A complexes activated by ceramide. In another study,

Turowski et al looked at the influence of the polycation protamine sulfate on PP2A activation in the presence of different concentrations of the PR65 (A subunit) using as 17

substrates both the full regulatory subunit of PKA and a peptide

(DLDVPIPGRFDRRVSVAAE) of this phosphoprotein (118). They found that the catalytic activity of the C subunit is reduced when PR65 is added; however, the addition of protamine reverted the effect of PR65 from inhibitory to stimulatory (118). When instead the shorter phosphopeptide was used as substrate, the inhibitory activity of PR65 in the absence protamine was suppressed (118), suggesting that the presence of cations can modulate the activity of the whole PP2A holoenzyme toward a specific substrate.

The regulatory B subunits are the major players in determining PP2A substrate specificity. These subunits have little in common and each is coded by a separate gene that frequently exhibit tissue specificity (93, 132). The B subunits can be divided into four families designated as B, B’, B’’, and B’’’ (93, 132). Within each family are specific isoforms further designed by Greek letters (α, β, etc). Moreover, several species have been found to have multiple splice variants, further expanding the variation (93, 132). As the C and A subunits, the B subunits are also subject to post-translational modifications.

For example, B56γ is phosphorylated on serine 337 by ERK (extracellular signal regulated kinase) signals, thus enabling the PP2A trimer to act in an auto-regulatory loop and consequently dephosphorylaties ERK (133). Another example of B-subunit activation by phosphorylation is with B56γ3, which is phosphorylated by the ataxia- telangiectasia mutated (ATM) protein. This serine phosphorylation increases binding between the B subunit at the PP2AAC complex. In this context, the B56γ3 directs PP2A phosphatase activity towards the p53 tumor suppressor (134). 18

2.1.3 Additional PP2A regulators.

In addition to the post-translational regulation of individual PP2A subunits, there

are PP2A-associated proteins that have a direct regulatory role in altering PP2A phosphatase activity. The most relevant in the regulation of PP2A activity are I1PP2A,

SET (also know as I2PP2A) and CIP2A. While I1PP2A and SET (see section 2.1.4) are

two closely related proteins, the recently discovered CIP2A is structurally different

although they all interact with PP2AC. Indeed, it is possible that their inhibitory function

relies on the ability to act as scaffold proteins capable of recruiting kinase(s) (e.g. Jak2

and/or src kinases) capable of blocking PP2A activity through phosphorylation.

I1PP2A is also known as acidic leucine-rich nuclear phosphoprotein 32 member

A (ANP32A), putative HLA associated protein-I (PHAP1) and pp32 (135). I1PP2A has been shown to regulate PP2A activity and responds through loss of inhibition to sphingosine or dimethylsphingosine (DMS) (136). Like SET, I1PP2A is also part of the histone acetyltransferase regulatory INHAT complex (discussed below) (137).

Reportedly, I1PP2A is important for the regulation of tau phosphorylation which plays a role in the development of Alzheimer’s disease (138). In addition, I1PP2A seems to have a role in cancer prevention. In fact, forced I1PP2A expression in drug-resistant non- small-cell lung cancer (NSCLC) cells was able to restore caspase-dependent apoptosis

(139). Similarly, the tumor-suppressing activity of I1PP2A in HeLa cells was attributed to the ability of I1PP2A to induce apoptosis in stressed cells through activation of caspase

3; in fact, deletion of the C-terminal and M-regions of I1PP2A significantly reduced the 19

induction and acyivation of caspase 3. Interestingly, pp32R1 is a protein almost identical

to I1PP2A; however, due to a minute difference within the M-region, it lose the ability to

induce apoptosis and acquires oncogenic properties (140).

Another PP2A inhibitor is Anp32e, an I1PP2A closely related protein that was originally

called cerebellar developmental-regulated protein 1 (CPD1) because it was expected to

play a role in neuronal development (141). Anp32e is found as three isoforms of different

molecular weights (34, 66 and 76kDa), different subcellular localizations and different distributions within the mouse brain where it was initially characterized (142). Among the three isoforms, the 76kDa protein bound most robustly to PP2A and potently inhibited PP2A activity. The 34kDa isoform is expressed within the nucleus only transiently during development of the mouse brain; it is undetectable in the nucleus from cells from an adult animal. Interestingly, a proteomics-based study with tumorigenic and non tumorigenic cells suggests that Anp32e is likely a tumor suppressor and in all probability functions similar to Anp32a (143).

CIP2A (Cancerous Inhibitor of PP2A) is an endogenous PP2A inhibitor found overexpressed in a variety of cancers (144). CIP2A binds directly to PP2A causing inhibition and results in stabilization of c-Myc, a well characterized PP2A target (144). In leukemia, CIP2A was found as a MLL partener in an AML patient (145), and subsequently it was described to be a potent inducer of leukemia cell proliferation (146) and a predictor of CML blastic transformation (147). Accordingly, CIP2A impairs the

ability of PP2A to inhibit the activity and induce degradation of the c-Myc oncogene, a transcription factor that is itself found to be dysregulated in a variety of cancers (144).

Another protein that has been demonstrated to bind directly to PP2Ac is α4; however, α4 inhibits PP2A activity by displacing the PP2Aa subunit (148). Much like with PP2Ac methylation, the effects of α4 are ambiguous; studies report both an increase and a decrease in phosphatase activity following binding of α4 to PP2Ac (149-152).

Binding to α4 is modulated by both PP2Ac tyrosine phosphorylation on amino acid residue 307 and leucine methylation at 309. Using a Y307F (non-phosphorylatable mutant) and L309Q (non-methylatable mutant) together, Chung et al were able to greatly increase binding of α4 to PP2Ac, indicating that these two sites are responsible for regulating binding (149). Importantly, a single mutation alone was insufficient to induce binding of α4. Another PP2A inhibitory factor, also known as the target of rapamycin signaling pathway regulator-like (TIPRL), binds to PP2Ac directly even in the presence

of α4 indicating the two proteins do not share a binding region but may cooperate to

suppress PP2A activity (153). Indeed, TIPRL has been described as a necessary

component of ATM-mediated PP2A inhibition (154).

2.1.4 The PP2A inhibitor SET oncoprotein.

SET (Suvar 3-9, enhancer of zeste, trithorax,; TAF-1β or I2PP2A ) is an

endogenous physiologic inhibitor of PP2A (155-159). SET is a ubiquitously expressed,

39 kDa protein that is phosphorylated mostly on serine residues and is primarily, but not

only, localized in the nucleus (160, 161).

SET has been shown to play a role in a variety of cancers (155, 162). SET was

originally discovered as a part of the chimeric SET-CAN protein composed of the entire

SET cDNA fused in frame with the carboxy-terminal end of the nuclear pore complex

CAN protein in a acute non-lymphocytic leukemia patient sample (163, 164). Likewise,

Carlson et al. found increased SET expression in Wilms’ tumors (155). The role of SET in cancer is not always clear but likely involves one or both of its known functions: to regulate chromatin assembly or gene transcription, and inhibit PP2A activity. An

example of a potential scenario where the former is involved in carcinogenesis was found

by White el al when studying environmental alkylphenolic compounds. Several of the

chemicals studied not only induced proliferation of breast cancer cells but also increased

protein expression of SET (termed TAF-1 in this study) suggesting a possible role for

SET in chemically induced, estrogen sensitive breast cancer (165). Conversely, the

PP2A-inhibitory function of SET plays an important role in hematopoietic malignancies.

Indeed, we were the first to show that increased SET expression plays a fundamental role in leukemia progression; in fact, we have shown that SET overexpression is necessary for

BCR-ABL1-mediated inhibition of PP2A in Ph+ B-ALL leukemia (78) and CML

progenitors from patients in chronic phase and undergoing blastic transformation (78).

Because SET undergos phosphorylation and is a Jak2 target (166), it is highly plausible

that it might exert its PP2A inhibitory function not only in BCR-ABL1 leukemias but also in other MPDs (e.g. PV, ET, PMF) characterized by constitutive Jak2 activation.

In normal cells, SET serves not only as regulator of PP2A (see below) but also as a regulator of gene expression. In fact, SET is part of the INHAT (inhibitor of acetyltransferase) complex where it prevents the acetylation of histones thereby reducing gene transcription (167, 168) by altering chromatin structure (169-171). In this regard, it was reported that the chromatin remodelling function of SET plays a role in the pathogenesis of primary microencephaly (172). In fact, genetic loss of SET or loss of

SET/MCPH1 (microcephalin) binding obstructs mitosis by allowing premature chromatin condensation (172). Interestingly, SET can also increase gene expression levels (173) in both a histone acetylation dependent and independent manner (174). Nevertheless acetylation potentiates the SET-mediated increase in gene expression. Although it is still unknown which factors determine the negative or positive effect of SET on gene expression/histone acetylation, it has been demonstrated that SET must dimerize to increase gene expression (175) providing a potential mechanism for post-translation modification to control SET activity.

Regarding SET function as PP2A inhibitor, initial studies showed that SET specifically inhibits PP2A at nanomolar concentrations without having an effect on the related phosphatases PP1 or PP2B (158). To act as a PP2A inhibitor, SET requires its phosphorylation on serines 9 and/or 24, two potential target sites for protein kinase C

(PKC) (160). Interestingly, SET phosphorylation on serine 9 also prevents its 23

dimerization and causes its relocation into the cytoplasm (176). Using a S9A mutant to mimic non-phosphorylated SET and an S9E mutant as a phosphomimetic, it was confirmed that phosphorylation on serine 9 controls SET subcellular localization as the phosphomimetic mutant showed enhanced export into the cytoplasm (177). Vasudevan, et al (178) added serine 93 to the list of SET phosphorylation sites and identified PI3Kγ as the kinase responsible for this phosphorylation. Using both non-phosphorylatable

(serine to alanine) and phosphomimetic (serine to aspartic acid) SET mutants, they showed that SET binding to PP2A required SET serine 93 phosphorylation (178). Finally, a recent study also demonstrated a role for SET serine 171 phosphorylation in the inhibition of PP2A activity (179). Specifically, PKD (protein kinase D) was identified as being responsible for phosphorylating SET serine 171. Notably, the three SET-targeting kinases (PKC, PI3K and PKD) all function in series to support cell proliferation and survival; PI3K is known to activate PKC through PIP3 (see below) and PKC activates

PKD through phosphorylation (180, 181). Surprisingly, when serine 171 was mutated to either alanine or glutamic acid PP2A activity was not inhibited, suggesting that phosphorylation of SET serine 171 per se is not sufficient for modulating PP2A activity and that cooperating phosphorylation events are necessary for conferring a full PP2A inhibitory activity to SET.

The SET inhibitory effect on PP2A has also been shown to be regulated by the sphingolipid signaling molecule ceramide (182), a well known PP2A activator (183).

Ceramide activates PP2A through binding directly to SET on helix 7. This binding likely 24

produces a conformational change in the SET protein and/or disrupts the binding face

causing SET to be displaced from PP2A (182).

Another detailed study by Saito et al also examined the various domains within

the SET protein (termed TAF-1β in this study) to determine their role in both chromatin

remodeling and PP2A inhibition (184). Here they demonstrated that the C-terminal region is required for chromatin remodeling while the N-terminal region is necessary for

PP2A inhibition (184) therefore suggesting the two functions of SET might be

independent. Specifically, SET is a 277 amino acid-long protein that retains full PP2A inhibitory function when truncated at amino acid 225 or 119 but loses it when deleted from amino acid 37 to amino acid 123 (184). Additionally, a dimerization-deficient SET protein (175) retained the ability to inhibit PP2A when assayed in a cell-free system.

Because SET dimerization robustly increases DNA replication and transcription (175), it was concluded that PP2A inhibitory activity and chromatin remodeling SET functions are totally independent although it cannot be excluded that they may act at the same time for regulating gene expression.

Despite of its role in gene regulation, SET is currently the target of anti-cancer studies because it can be pharmacologically targeted to reactivate PP2A activity (185) .

In one study, Fujiwara et al treated canine lymphoma cells with the peptide OP449 which targets the C-terminal of the SET protein (186). Cells expressing high levels of SET protein were found to be most sensitive to OP449-induced apoptosis. Additionally, cells treated with OP449 had a 1.5 fold increase in PP2A activity suggesting that activated 25

PP2A may play a role in inducing apoptosis in these cells. Moreover, peptide-induced

SET inhibition not only reactiveted PP2A but also influenced other SET-binding partners

(187). In fact, Switzer et al showed that the peptide COG112 not only induced PP2A reactivation but, upon binding to SET, it also disrupted the SET interaction with the nm23-H1 tumor suppressor that, in turn, re-acquired its anti-metastatic activity, a function primarily described in neuroblastoma (188).

Another SET binding partner that is liberated through SET inhibition is the Rac GTPase; indeed, Rac1 binding to phosphorylated SET allows Rac1-dependent cell migration as demonstrated by ability of a membrane-localized and myristoylated SET to enhance

HEK293 cell migration (176) . Although a role for SET in the regulation of tumor metastasis is still unclear, it is possible that such an effect is mediated by the ability of

SET to reactivate tumor suppressors as nm23-H1 and PP2A.

2.1.5 SETBP1

SET Binding Protein 1 (SETBP1, SEB) is a 170 kDa primarily nuclear protein (189) originally found fused with NUP98 (Nuclear core complex protein) in a leukemia patient

(190). Although the function of the SETBP1-NUP98 fusion is not known, its presence suggests a role for SETBP1 in cancer. Indeed, it seems that SETBP1 stabilizes SET protein and inhibit PP2A, thus promoting the proliferation of leukemic cells (191).

Accordingly, SETBP1 was found overexpressed in over half of cancer patients sampled

(n=192) and patients with SETBP1 overexpression had a worse prognosis compared to

those without. Finally, SETBP1 gene mutations were found in a set of atypical CML samples (192). The locations of the mutations within the protein were found to be in

within the Ski (Sloan-Kettering Institute protooncoprotein) binding region and no

mutations were found in the SET-binding domain. In addition, these mutations increase

SETBP1 binding affinity to SET suggesting an important role for SET-SETBP1 interplay

in leukemogenesis. Indeed functional studies indicated that SETBP1 expression

enhances hematopoietic progenitor self-renewal (a process critical for leukemia) through induction of the transcription factors Hoxa 9/10 (193).

2.1.6 Effect of the microenvironment on PP2A tumor suppressor activity.

Recently, attention has been focused on the role of the microenvironment as it

relates to cancer initiation, maintenance and progression. Hypoxia, a low oxygen

condition common in the tumor microenvironment, has been shown to influence PP2A

activity in glioblastoma multiforme (GBM) cells (194). Indeed, the protein level of the

PP2A A, B55 and C subunits were found to be reduced whereas levels of the hypoxia- inducible factor 1α (HIF-1α), a transcription factor induced by low oxygen and important for cancer stem cell self-renewal were found variable among both normal and tumor samples (194). Interestingly, PP2A activity directly correlated with HIF-1α expression in

GBM and normal tissue (194). In a similar study, it was reported that astrocytes deprived

of both glucose and oxygen had a decrease in several kinase-dependant signaling

molecules including phosphorylated ERK1/2 and Akt (195); inhibition of PP2A by

okadaic acid restored phosphorylation of these proteins. Because low glucose/low oxygen

conditions induce apoptosis in astrocytes, it is highly possible that PP2A is active during

hypoxia and that PP2A is at least in part responsible for the death of astrocytes deprived

of oxygen. Altogether, these data strongly suggest the importance of the hypoxic

microenvironment in the regulation of PP2A activity and the relationship between PP2A,

microenvironment and cancer cell survival.

IL-11, a cytokine produced by bone marrow (BM) stromal cells has been shown to induce the colocalization of Jak2 and PP2A in 3T3-L1 fibroblast-like cells (101).

Cytokine stimulation also resulted in the association of Yes (a proto-oncogenic tyrosine kinase) and PI3K with Jak2. The specific relationship between PP2A and these kinases was not evaluated in this study, however two of these kinases (Jak2 and PI3K) are able to inhibit PP2A activity (see below). As IL-11 is a BM-localized cytokine, it is possible that

PP2A may be inhibited in cancer cells residing in the BM microenvironment. Similarly,

IL-2 has also been demonstrated to decrease PP2A activity and plays a role in CD4+ T-

cell migration (196). Colony stimulating factor-1 (CSF-1), a proliferation-inducing

cytokine produced by osteoblasts and therefore believed to be abundant in the BM

microenvironment also has a relationship with PP2A (197). Macrophages collected from

murine BM stimulated with CSF-1 produced a robust increase in PP2Ac protein level and

PP2A activity was increased following CSF-1 stimulation, suggesting an active role for

PP2A in the regulation of macrophage proliferation (197).

One additional factor found to regulate PP2A is the anti-proliferative Transforming

Growth Factor β (TGF-β) (198). In EpH4 mammary epithelial cells TGF-β induced cell cycle arrest through PP2A-mediated dephosphorylation of p70s6k serine/threonine kinase,

the loss of which prevents cell proliferation by inducing G1arrest.

2.2 Role of PP2A in Cancer

The notion that PP2A functions as a tumor suppressor originates from the finding that

okadaic acid (a known carcinogen) specifically inhibits the phosphatase activity of PP2A

when used at low nanomolar concentrations (90, 199, 200). The earliest research

describing the role of PP2A as a tumor suppressor focused on the tumor-promoting

effects of okadaic acid when administered in combination with the carcinogen DMBA

(7,12-Dimethylbenz(a)anthracene) (200). In accordance with the “two-hit”

carcinogenesis theory (201), tumors were rarely observed in mice exposed to either

okadaic acid or DMBA, but nearly 100% of mice developed cancer when compounds

were used in combination (200). Accordingly, the tumor-inducer polyomavirus SV40

(simian vacuolating virus 40) small-T antigen achieves oncogenic properties through direct inhibition of PP2A (202). Indeed, PP2A negatively regulates cell cycle progression by counteracting SV40 replication and its ability to promote a G1 to S phase transition (203, 204). A copious number of factors, including several kinases (Table 1), that control cell proliferation, survival, differentiation and resistance to apoptosis were

also found to be stimulated by okadaic acid and/or targets of PP2A phosphatase activity

(205).

Table 1: List of major kinases inactivated by PP2A

31-kDa S6 kinase (206) Jak2 (84) Akt (207) MAPKAP kinase 2 (218) AMP-activated kinase (208) Mck1 (219) Aurora Kinase (80) MEK (220) CaM kinase I (209) p38/RK 66 (221) CaM kinase II (210) p70 S6 kinase (222) CaM kinase IV (211) p90 RSK1, p90 RSK3 (223) cAMP-dependent kinase (212) PKB (215) CDC2 (CDK1) (213) PKC (224) CDK2 (214) PKD (225) cGMP-dependent kinase A Polo-like kinase (Plk) (214) (215) RAF-1 (226) ERK (216) Ste7 (227) IkB kinase (IKK) (217)

Additionally, mutations in the PP2AA subunit, like those found in the MCF-7 breast cancer cell line, impair binding of the A subunit to either B or C subunits (228), thereby leading to increased beta1-integrin serine phosphorylation which depends on PP2A loss of function (229). Mutations in the genes encoding different PP2AB subunits leading to

PP2A loss of function have also been described in several solid tumors (e.g. renal and breast carcinoma and melanoma)(230, 231). Interestingly, genes encoding for these different PP2A subunits are all located in fragile sites or in chromosomal region frequently involved in loss-of-heterozygosity (LOH) thus justifying the susceptibility of

PP2A to dysregulation and its role as tumor suppressor (232).

2.3 PI3K and its Role in the Regulation of PP2A activity.

Phosphatidylinositol 3-kinases (PI-3Ks) are a family of enzymes involved in a variety of cellular functions particularly proliferation and apoptosis. There are three defined classes of PI3K, class I, class II and class III each distinguished by both structure and function. Class I PI3Ks includes two subsets (IA and IB) and generate

phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol (3,4)-bisphosphate

[PI(3,4)P2] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3].

These PI3Ks are activated by G protein-coupled receptors and tyrosine kinase receptors. This class consists of heterodimeric enzymes containing a regulatory and a catalytic subunit. Class IA PI3K is composed of a p110 catalytic and a p85 regulatory subunit (233). There are five variants of the p85 regulatory subunit (p85α, p55α, p50α, p85β, and p55γ). Likewise, four variants of the p110 catalytic subunit (p110α, β, δ and γ) have also been described (234-236). The class II category of PI3K is comprehensive of

three catalytic isoforms (C2α, C2β, and C2γ). Interestingly, the C-terminal domain of

these kinases lacks the ability to bind calcium thereby indicating that calcium influx is

not required for their kinase activity. Finally, class III is similar to class I in structure as

also these PI3Ks exist as heterodimers consisting of a ~97kD catalytic subunit and

regulatory subunit (236).

Because our data identified the class I PI3Kγ as a key regulator of the Jak2/PP2A

interplay, we will discuss below the relationship between PI3K and each of the factors

individually. 31

2.3.1 Jak2 and PI3K interplay.

Although research on Jak2 signaling has been primarily focused on the Jak-STAT

pathway, there is evidence of Jak2 signaling through PI3K as well. For example, Garcia-

Ramirez et al found that stimulation of the human retinal pigment epithelial ARPE-19 cell line with erythropoietin protected the cells from high sugar conditions (237). This effect was lost upon Jak2 inhibition by AG490 as well as after treatment with the pan-

PI3K inhibitor LY294002 indicating that both Jak2 and PI3K are likely participating in the same pathway. A similar Jak2-PI3K interplay was described in EpoR+ Ba/F3 cells expressing the Jak2V617F oncoprotein (238). In these cells, PI3K-mediated Akt activation was independent from cytokine stimulation and was inhibited by inhibition of PI-3K activity by LY294002 treatment (238). By contrast, PI3K-mediated Akt activation required cytokine stimulation in cells transduced with a wild type Jak2 kinase (238).

Jak2V617F, as described above, retains kinase activity in the absence of cytokines while

wild type Jak2 must be stimulated to function. LY294002-sensitive Akt phosphorylation,

which also depends on Jak2 stimulation (either through cytokines or gain-of-function

mutations), seems also to directly involve Jak2 and PI3K activities (238). Importantly,

tyrosine 479 phosphorylation of the erythropoietin receptor was found to be required to

induce Jak2-mediated phosphorylation of Akt (238), suggesting that PI3K binds to the

EpoR in response to phosphorylation by Jak2. In a separate study conducted in non- hematopoietic cell lines, Akt phosphorylation was also detected upon EpoR activation

(239). Importantly, Akt activation by EpoR/Jak2 was suppressed in this system by using 32

the PI3K inhibitor PI-103, which selectively impairs the activity of class I PI3Ks thus indicating that Jak2 and PI3K are components of a common molecular network (239).

2.3.2 The PI3K and PP2A interplay.

While the relationship between PI3K and PP2A is well described in terms of the

inhibitory effects of PP2A on PI3K (240), much less is known on the effect of PI3K on

PP2A activity. It was reported that the SET oncoprotein may become active as PP2A

inhibitor upon serine phosphorylation by PI3Kγ (178), however a direct and robust

evidence of a direct effect of PI3K on SET is still lacking. By contrast, there is

convincing evidence suggesting Protein Kinase C (PKC, discussed below), which is

downstream of PI3K, as the kinase phosphorylating SET.

Finally, another evidence of the PI3K-PP2A interplay was found in HER2/neu-

expressing breast cancer cells in which PP2A phosphorylation on the inhibitory tyrosine

307 site was reduced upon treatment with the PI3K inhibitor LY294002, indicating that

PP2A is inactivated through PI3K-mediated signals in HER2/neu+ breast cancer cells

(241).

2.3.2 Role of PI3K in Polycethemia Vera.

The role of PI3K in PV has not been well studied to date. An increase in phosphorylated Akt (a downstream target of PI3K) was found in PV patient samples stimulated with EPO but not with normal EPO-stimulated controls indicating a dysregulated response to cytokine stimulation in PV that includes PI3K in addition to the 33

Jak2-stimulated STAT signaling pathway (242). Likewise, PI3K activity is essential for

the differentiation of myeloid progenitors from PV patients (243). In support of this observation, Barnache et al found that PI3K activity was increased and essential for the development of the EPO-independent proerythroblastic cell in a Friend virus

erythroleukemia model (244).

2.4 The Protein Kinase C (PKC) family.

Protein kinase C (PKC) is a family of lipid-regulated serine/threonine kinases which serve as signaling proteins and consists of a family of fifteen isoforms divided into three families (245). The families are categorized based on cofactor requirements: The first is termed “conventional” and requires DAG (diacylglycerol) and calcium for

activation; the second group called “novel” requires only DAG; and, the third PKC group named “atypical” do not require either cofactors for activation.

PKC is regulated through the binding of cofactors or surrogate cofactors that can act as a

substitute for DAG like phorbol 12-myristate 13-acetate (PMA) or fumonisin B1

(FB1)(246). PKC specificity for cofactors is determined by the “C” domains (C1 and C2)

at the N-terminus of the kinase. The “C1” domain recognizes DAG while the “C2” domain recognizes calcium. Novel PKC isoforms contain both C1 and C2 domains but

the C2 domain is not functional. Seemingly, atypical PKCs do not bind ligands at the C1

domain and lack a C2 domain entirely, hence the absence of requirement for these two

cofactors (247).

To act as a kinase, PKC must be first primed through post-translational modification

before undergoing full activation by DAG or calcium. The best described priming post

translational modification is mediated by its serine/threonine phosphorylation (248, 249).

The most prominent kinase to provide this phosphorylation is pyruvate dehydrogenase

lipoamide kinase isozyme 1 (PDK-1). Interestingly, PDK-1 serves as the intermediate between PI3K and PKC wherein PI3K activity induces PDK-1 activity through phosphorylation of the PDK-1 regulatory domain (250).

Subcellular localization also controls the function of specific PKC isoforms. For example, when stimulated by FB1 or PMA both PKCα and PKCγ translocate to the cytoplasmic membrane (251, 252). Importantly both of these compounds are known carcinogens (253-255). Likewise, the tumor promoter/PMA analog 12-deoxyphorbol 13- tetradecanoate induced PKC translocation from the cytoplasm to the plasma and then to the nuclear membrane whereas the chemically-related tumor inhibitor 12-deoxyphorbol

13-phenylacetate produced primarily nuclear membrane localization (256).

In contrast to activation by calcium or DAG, PKC activity can be reduced by nitric oxide, at least in part, through post-translational modification of the C1 region

(257). In a study using pulmonary artery endothelial cells, Kahlos et al found that 8 ppm of nitric oxide reduced the activity of three isoforms of PKC (α, ε, and ζ) each one belonging to one of the different PKC classes. While all three enzymes were inhibited by

NO the novel PKC, PKCε experienced the greatest percentage decline in activity following NO exposure. While the specific mechanism of inhibition was not explained in 35

detail it is likely that a combination of factors may have played a role in NO-induced loss of PKC activity. Although NO decreases PKC protein levels (258), it can also produce a reduction in PKC activity as was found following the administration of NO or of NO- donating reagents (259). Interestingly, a high concentration of NO prevented the binding of phorbol ester to PKC (259). Phorbol ester is used as a mimic for DAG that binds and activates conventional and novel forms of PKC proteins through binding the “C1” region,

which is also affected by NO. Notably, PKC has also been shown to associate with PP2A

and, in particular, it associates with the B56δ subunit in N2a cells (260). In vitro PKCδ binds, phosphorylates and directly activates purified PP2A (261). Likewise PP2A activity was reduced in PKCδ dominant-negative cells when compared to control cells N27 cells.

Interestingly, the PP2A activator FTY720 (discussed elsewhere) increased threonine and serine phosphorylation (activation) of PKCδ in hepatocellular carcinoma cells (262)

although we provide strong evidence that such effect is SET mediated which becomes

active as PP2A inhibitor upon PI-3K-induced PKCδ-dependent phosphorylation.

2.5 Regulation of PP2A by nitrosylation and role of Jak2 in the induction of

Nitric Oxide (NO).

Nitric oxide (NO) acts as a second messenger in a variety of human tissues. NO is primarily known for its role in vasodilation (263); in fact, it is also known as

“endothelium-derived relaxation factor” or “EDRF” (264). NO is produced through the activity of the nitric oxide synthase (NOS) which uses L-arginine, oxygen and NADPH

as substrates and co-factors (265). There are three known NOS enzymes in mammalian

systems, each one is encoded by a separate gene, termed neuronal NOS (nNOS or

NOS1), inducible NOS (iNOS or NOS2) and endothelial NOS (eNOS or NOS3), which

show 50-60% sequence homology (266-272).

Nitric oxide has been shown to have an inhibitory effect on leukemia cell growth.

For example, treatment of CML and normal cells with a variety of NO-releasing compounds including SIN-1 resulted in NO formation that induced apoptosis in leukemia but not in normal cells (273). The apoptosis-inducing effects of NO were counteracted by co-administration of iron-containing compounds that, upon sequestering radical oxygen, prevent the formation of peroxynitrite (PN) which, in turn, induces cell death (273). In fact, metalloporphyrins containing either iron or manganese have been demonstrated to reduce peroxynitrite concentration through scavenging (274). Thus, SIN-1 may kill CML cells through the induction of NO and formation of peroxynitrite.

Interestingly, PN has also been shown to activate PP2A through nitration of

PP2AC on tyrosines 284 or 307 (106, 107, 275). In fact, Wilson and Wu demonstrated

that PN-mediated PP2A activation correlated with decreased PP2A phosphorylation

(107), suggesting that PP2AC nitrosylation antagonizes the inhibitory effect of tyrosine

phosphorylation on PP2A activity thus leading to PP2A activation (Figure 5).

Supporting the important role played by nitrosylation in the regulation of PP2A activity stands the evidence that the apoptotic effects of the NO-inducer hydroxytyrosol is antagonized by the administration of the PP2A inhibitor okadaic acid (276). 37

Figure 5. Nitric oxide synthase activates PP2A. Nitric oxide synthase (NOS) generates nitric oxide (NO) which will react with radical oxygen within the cell to produce peroxynitrite (ONOO-). Peroxynitrite, in turn, produces nitrotyrosine on regulatory tyrosine residues on PP2A thereby resulting in PP2A activation.

Additionally, the PP2A activator ceramide was shown by Li et al to increase the expression of eNOS (NOS3) in endothelial cells (277). Notably, the concentration of bioactive NO was not increased despite this upregulation of NOS that, as reported, was expected to produce additional bioactive NO. In this study, the ceramide-induced increase in radical oxygen species (ROS) might account for the lack of NO induction; in fact, the ceramide-induced NOS may favor the generation of ROS at the expense of NO

(277); however, we cannot exclude that ceramide increases the concentration of both NO and ROS which may contribute to PN production. Because both PN and ceramide activate PP2A, it is possible that also NO can potentiate the effect of the sphingolipid ceramide in the induction of PP2A.

A relationship between nitric oxide synthase and Jak2 has been described, at least in terms of Jak2 inducing NOS expression. A study using the ADTC5 chondrocyte cell line found that stimulation with interleukin-1 (IL-1) alone and in combination with the hormone leptin produced an increase in NOS2 expression that was prevented either by 38

treatment with the Jak inhibitor AG490 or by the PI3K inhibitor LY294002 (278, 279),

suggesting that nitric oxide synthase expression is regulated, at least in part, by the

activity of both Jak2 and PI3K. Similar results were obtained by using the macrophage cell line RAW 264.7; in these cells, the lipopolysaccharide (LPS)-induced NOS2 expression which could be prevented by co-treatment with the Jak2 inhibitor AG490

(280). Thus, Jak2 appears to be required for NO production in different cell types and may require PI3K activity as well. Interestingly, NO shares a role with SET as well. NO has been shown primarily to cause an increase in acetylation through two different mechanisms: an increase in HAT (histone acetyltransferase) activity or a decrease in

HDAC (histone deacetylase) activity/expression. An increase in NO, often found during inflammatory processes, has been demonstrated to decrease total levels of HDAC2 protein (281). This process, described using human airway epithelial cells, involves the generation of PN through the reaction of NO with radical oxygen; the same process involved in the nitrosylation of PP2A. If this NO-induced NOS2-mediated mechanism, which leads to activation of PP2A and is somehow regulated by Jak2, is a mechanism active to balance Jak2 kinase activity in a PP2A-mediated manner remains to be

demonstrated.

2.6 Role of Sphingolipid Signaling in Cancer

Sphingolipids are aliphatic lipids that were discovered in brain tissue in the 1870s

and subsequently identified as key molecules playing a variety of roles in cellular

structure and function. Sphingolipids are divided into classes and classified based on the

number of carbons on the aliphatic chain. The primary base of all sphingolipids is sphingosine (Figure 6).

Figure 6. Sphingolipids. Sphingolipids retain a base carbon chain (sphingosine) which can have a fatty acid chain added to create ceramide or a phosphate group added to create phosphate derivatives.

The addition of a fatty acid chain to the amino group of sphingosine produces the

next group represented by the ceramide. Other groups include glycosphingolipids, which

contain a ceramide molecule with a sugar at the 1-hydroxyl position, and sphingomyelins

that have a phosphocholine or phosphoethanolamine at the 1-hydroxyl position. Each of

these classes can be further modified. D-erythro-sphingosine, sphingosine-1-phosphate

(S1P) and ceramide (N-Caproyl-sphingosine) are three sphingolipids with a defined role as signal transducers in normal hematopoiesis as well as in malignant cells (282-284).

Sphingosine serves as a substrate for sphingosine kinase 1 (SPHK1) (285, 286) and sphingosine kinase 2 (SPHK2) (287) to produce the signaling lipid S1P. 40

Most of the properties attributed to SPHK1 depend on the effect of the S1P interaction

with a specific S1P receptor. S1P is thereafter processed and degraded primarily by

activity regulated by S1P lyase (288). Once inside the cells, S1P has been known since

1990 to act as a second messenger and induce changes within target cells (289, 290); however, it was not until the discovery of the seven-transmembrane S1P receptor proteins

[previously termed endothelial differentiation G-protein-coupled (EDG) receptor] in 1998 that it became clear that S1P could also function as an extracellular cytokine (291). There are currently five known S1P receptors (S1P1-5) each coupled with a G-protein (Table 2)

and specifically expressed in different tissue types (292).

Table 2. G-protein coupled S1P receptors (S1PR). Each of the S1P receptors has been shown to be G-protein coupled, however the specific G-protein species and resultant downstream targets vary among receptor type.

2.6.1 SPHINGOSINE

Sphingosine is the most basic of the sphingolipids and serves as a scaffold for all

other sphingolipids including sphingosine-1-phosphate (S1P) and ceramide. Sphingosine is generated from palmitoyl CoA and serine through a condensation reaction that 41

produces dehydrosphingosine. This compound is further modified first to dihydrosphingosine (DHS) and then to sphingosine by reaction with NADPH and FAD, respectively. Most of the functions attributed to sphingosine are actually induced by sphingosine products such as S1P and ceramide. Notably, sphingosine itself is primarily regarded as a pro-apoptotic signaling lipid.

The first described molecular target of sphingosine is Protein Kinase C (PKC)

(293, 294). Sphingosine was found to inhibit PKC in human HL60 leukemia cells and

A431 epidermoid carcinoma cells where it simultaneously stimulates the Epidermal

Growth Factor Receptor (EGFR) and increases its expression. In both of these studies, sphingosine administration also induced apoptosis of cancer cells (293, 294).

Sphingosine activates specific kinases, termed Sphingosine Dependent Kinases

(SDKs), which are not responsive to most lipids including dimethylsphingosine (DMS).

The identities and targets of some of these kinases remains still unknown; however, defined targets include 14-3-3 proteins (295), HSP90 and calreticulin (296). Interestingly, one of these kinases termed SDK1 mediates the proapoptotic activity of sphingosine in

MKN-28 gastric cancer cells (297) and is a caspase-cleaved fragment of the kinase domain of PKCδ (298) that, as stated above, is also a lipid-dependant kinase. Other cancer cell types found to be subject to sphingosine/SDK1-mediated apoptosis include mesothelioma and human monocytic leukemia U937cells (299). Note that most of the sphingosine generated within cancer cells often causes a decrease in apoptosis due to its conversion to enzymatic conversion to S1P. 42

2.6.2 Sphingosine-1-phosphate (S1P)

The bioactive lipid S1P controls a variety of biological functions including cell

migration, cellular signaling and apoptosis. The type of response produced by S1P

depends upon cell type, presence or absence of malignancy, surface receptor expression

and possibly on the aberrant activation of signaling molecules controlling sphingolipid

pathway signaling. S1P has functions other than those related to carcinogenesis (such as

cardiac regulation); only those functions related to cancer will be discussed here. The

functions of S1P are divided based on the generating enzyme (SPHK1 or SPHK2) or on

the receptor(s) involved; a similar system is used below to describe the role of S1P.

2.6.3 Sphingosine kinase 1 (SPHK1).

Although both the SPHK enzymes share a kinase domain, use the same substrate

and produce the same product, they serve different functions (283). SPHK1 is primarily a

cytoplasmic kinase with anti-apoptotic properties (282). There are three SPHK1 isoforms

(a, b, and c) generated by alternative splicing. They differ at the amino acids residues

characterizing their N-terminus (300) and for their subcellular localization. SPHK1a is secreted into the extracellular space and diffuses into blood plasma (301), while the b and c isoforms are retained inside the cytosolic compartment.

Most of the published data regarding the SPHK1-mediated S1P signaling in cancer describe an oncogenic/metastatic-enhancing function of the S1P lipid with the only exception made for signals generated upon interaction with S1PR2. A recent study

examined the role of the systemic S1P vs the S1P produced by an allograft tumor

engrafted into mice (302). To differentiate the effects of systemic S1P produced by the

recipient animal from that produced by the tumor itself, wild-type and SPHK1-/- mice

were used as donors or recipients for the tumors. The tumors themselves expressed either

scrambled siRNA or siRNA directed at SPHK1. SPKH1-/- mice infused with TRAMP

prostate cancer cells lived longer and had a reduced tumor score compared to normal

mice infused with these same cells. Similarly, SPHK1-/- mice infused with MB49 murine

urothelial carcinoma cells had tumors with a significantly lower volume compared to

normal mice infused with these cells. Likewise, the tumor volume of normal mice infused

with SPHK1-knock down MB49 cells was significantly reduced; the tumor volume in

these mice was similar to that of SPHK-/- mice given scrambled siRNA. There was not a

significant potentiation in tumor reduction with SPHK-/- mice infused with SPHK1

knock-down cells when compared to either knock-down alone (302). Moreover, normal mice given an S1P-neutralizing antibody (Sphingomab) had reduced tumor volume similar to those found in the SPHK1-/- mice; this effect was partly lost following shRNA-

mediated knock-down of the tumor suppressor Brms1 indicating that systemic S1P likely

exacerbates tumor development through inhibition of Brms1 (302). These data indicate

the importance of S1P in tumor development and highlight the potential interactions between S1P and (in this case) a tumor suppressor.

SPHK1 is the better-studied of the two known SPHK isoforms. This isoform is

also generally regarded as driving proliferation and reducing apoptosis thus making it an

interesting candidate as an oncogene-regulated factor.

One simple study evaluating the effects of SPHK1 activity in colon cancer found that

increased SPHK1 expression (as driven by a transduced expression vector) increased the

viability and reduced baseline apoptosis levels of LOVO colon cancer cells (303).

Moreover, increased SPHK1 activity also increased cellular motility as measured by a

cell invasiveness assay (303). A reduction in SPHK1 activity, either through

administration of a chemical inhibitor (DMS) or through shRNA-mediated knock down produced the opposite effect; these cells presented with greatly increased apoptosis/decreased viability in combination with a reduction in cellular motility (303).

Mechanistically, the authors found an increase in ERK1/2 activity in cells overexpressing

SPHK1 and a significant reduction in ERK1/2 expression in cells treated with DMS or

expressing SPHK1 shRNA (303). Conversely, both p38 (a stress-responsive kinase) and its target MAPKAPK2 were upregulated in the cells with reduced SPHK1 activity (303),

suggesting these cells were undergoing cellular stress as a consequence of reduced

SPHK1 expression/activity. Finally, SPHK1 was found to be overexpressed in

tumorigenic proerythroblasts (304). The enhanced activity of SPHK1 in these cells made the cells more resistant to serum starvation also through activation of the mitogenic and survival kinases ERK1/2 and PI3K; however, increased SPHK1 activity did not confer

EPO-independence (304). 45

2.6.4 Sphingosine kinase 2 (SPHK2).

In contrast to SPHK1, there are only two known SPHK2 isoforms, one of which is located primarily in the cytoplasm (a) and the other one in the nucleus (b) due to the presence of a nuclear localization sequence (301, 305). Different studies attempted to identify a SPHK2 specific function; however, the real role of this kinase remains elusive although it seems that SPHK2 transduces pro-apoptotic signals (306). Notably, a well described function of SPHK2 is its ability to phosphorylate the sphingosine analogue

FTY720 and positively regulate its immunosuppressive activity (307, 308). As a nuclear kinase, SPHK2 seems to antagonize and inhibit DNA synthesis (305). Of the two forms of SPHK2, the longer SPHK2a is usually the most expressed isoform (309).

Serum-starved NIH3T3 cells presented increased apoptosis when SPHK2 was overexpressed (contrasted to an increased survival when SPHK1 was overexpressed)

(310). Mechanistically, the SPHK2 interaction with and sequestration of Bcl-xL through the SPHK2 BH3 domainis is required for SPHK2-induced apoptosis (310). Likewise, reduced SPHK2 activity was observed in HEK293 cultured in serum-free conditions, and ectopic expression of both short and long SPHK2 isoforms further reduces proliferation with the short form (b) producing the greatest inhibition. However, overexpression of

SPHK2a has no effect on proliferation while SPHK2b reduces it when cells are grown in the presence of serum (309). Despite most of the reports indicate a pro-apoptotic function for SPHK2, Gao et al, found that SPHK2 also possesses an anti-apoptotic function in

A498 cells (311). The SPHK2 functions as a pro- or anti-apoptotic protein may depend 46

on the cell microenvironment; indeed, MCF7 cells expressing SPHK2 shRNA grew faster

in vitro than untransfected controls (312). However, the SPHK2 shRNA-expressing

MCF7 cells produced a more aggressive phenotype with reduced macrophage infiltration

when subcutaneously implanted into nude mice (312).

2.6.5 Regulation of SPHK expression and activity

The sphingosine kinase isoforms can be regulated through several mechanisms

including allosteric inhibition, substrate concentration, expression level and post-

translational modification.

• Substrate concentration. Both isoforms of SPHK use sphingosine, which is produced from ceramide, as the source for the lipid backbone of S1P. Ceramide itself can be generated through the degradation of sphingomyelin by sphingomyelinase or through a multiple step de novo synthesis pathway originating from palmityl CoA and serine. The loss of ceramide from either of these sources will, obviously, limit the functionality of the

enzyme as will loss of ATP.

• Expression/distribution. Stimulation of C2C12 myoblasts with either insulin-like

growth factor 1 (IGF-1) or platelet-derived growth factor (PDGF) induces SPHK1 activity and expression (313, 314). Interestingly, PDGF stimulation increased cell

proliferation which was refractory to S1PR1 knock-down or treatment with the S1PR1

antagonist W146 (313, 314), suggesting that PDGF induces cell proliferation, at least in

part, through SPHK1. By contrast, IGF-1-stimulated cells relied upon the S1PR2 for

induction of differentiation, suggesting that SPHK1 is an intermediate signaling molecule

which depending on the stimulus and the type of receptor triggered is capable of

transducing different signals likely through its involvement in different multiprotein

complexes.

SPHK1 activity also increases in response to hypoxia as a result of an increase of ROS in

PC-3 prostate cancer cells (315). Another regulator of SPHK1 is the tumor suppressor

p53 (316); SPHK1 protein levels were reduced through a p53-induced caspace 2-

dependent proteolysis in the wild-type cells following irradiation (which activates p53),

and modestly increased in irradiated p53-/- MEF cells (316).

Interestingly, also some drugs can induce an increase in expression of SPHKs. For example, the thiazolidinediones rosiglitazone and troglitazone increased SPHK1 activity and expression in mouse mesangial cells by inducing an increase in SPHK1 mRNA levels (317). By contrast, the polysaccharide MDG-1 increases S1P production through an increase in SPHK1 activity (318), an effect also observed in lipopolysaccharide

(LPS)-stimulated rat astrocytes (319). SPHK1 levels were also found elevated in imatinib-resistant K562 CML blast crisis (CML-BC) cells (320). When compared to imatinib-sensitive cells, the resistant K562 cells had an upregulation of SPHK1 protein and a corresponding increase in SPHK1 activity (320). Through the use of chemical inhibitors and siRNA-mediated knock-down, the authors defined that SPHK1 was upregulated through the BCR-ABL1-regulated PI3K/mTOR pathway (320).

• SPHK Post-translational modification. SPHK1 is phosphorylated on

serine 225, likely by ERK1/2 or PKC (321, 322). Phosphorylation of this site increases

SPHK1 activity without changing its affinity for ceramide or ATP, induces its relocation to the plasma membrane, and is removed by PP2A (321). Moreover, SPHK1 expression has been reported to be regulated by acetylation in virtue of the presence of two conserved “GK” sequences (323). Mutation analysis revealed that SPHK1 lysine acetylation serves to increase protein stability through inhibition of ubiquitination and block of proteasome-dependent degradation (323).

SPHK2 is phosphorylated on five different serines, 351, 363, 368, 378 and 448)(324-326) although threonine 578 is also suspected to be a true phosphorylation site (327).

Currently, all described phosphorylation sites are believed to induce kinase activity when phosphorylated.

• Chemicals regulating SPHK activity. A variety of natural and synthetic chemical

inhibitors are currently available for SPHK inhibition. The best described is the SPHK

inhibitor dimethylsphingosine (DMS). DMS is a sphingosine metabolite that is found

naturally in some tumors and is a competitive inhibitor of SPHK1 and a non-competitive

inhibitor of SPHK2 (287, 328). DMS is not used clinically due to poor pharmacokinetic

properties and to the severe off-target effects (329, 330). More recently, two SPHK inhibitors have been isolated from microbes. These two compounds, termed F-12509A and B-5354c were found to elicit SPHK inhibition of both isoforms in a competitive and

non-competitive manner, respectively (331). Although still not in clinical use, both compounds have the potential to be used in cancer therapy as adjuvants (332, 333).

Despite the promise of natural product SPHK inhibitors, the bulk of the compounds currently studied are synthetic small molecules. The synthetic SPHK inhibitors can be further divided into two groups, the lipid-like and the non-lipid like.

One of the first lipid-like synthetic SPHK inhibitors discovered is D,L-threo- dihydrosphingosine (DHS) (334). DHS shares many properties of DMS in that it lacks clinical usefulness due to off-target effects and is subject to quick metabolism within the cell. Unlike DMS, DHS is an inhibitor of only SPHK1. This compound is also rarely used experimentally as more robust products are available. Most of the lipid-like molecules are based on the semi-synthetic immunosuppressant FTY720 (fingolimod). Although

FTY720 is a known SPHK1 inhibitor modification of base molecule has been performed to alter the properties of the compound (335). An example is (R)-FTY720-OMe. This compound functions as a competitive SPHK2 inhibitor (contrasted to FTY720 which is a competitive inhibitor of SPHK1) (336). This compound has been demonstrated to induce apoptosis of MCF-7 breast cancer cells partly due to degradation of the SPHK2 protein.

The non-lipid small molecule inhibitors represent the fastest growing category of

SPHK inhibitors. An early non-lipid small molecule inhibitor is SK1-II. This compound inhibits both isoforms of SPHK and, unlike DMS, lacks activity towards PI3K or PKC

(337). Moreover, this compound was found to be an effective anti-cancer agent even towards tumor cell lines which are resistant to chemotherapeutic agents (e.g. MCF-7VP 50

cells). This compound has been further refined into more specific analogs and continues

to be developed (338).

2.6.6 The intracellular S1P targets and the S1P receptors (S1PR)

• Intracellular S1P targets. Although the majority of the cellular effects of S1P

have been attributed to the S1P receptors found on the cell surface (see below) there have

been verified intracellular receptors for S1P. The first of these proteins to be discovered

were the nuclear histone deacetylase inhibitors HDAC1 and HDAC2 (339). SPHK2 was

found to bind histone H3, H2B and H4, and siRNA-induced loss of SPHK2 activity

resulted in a loss of histone acetylation without affecting binding (339). Moreover, LC-

MS/MS analysis found S1P to be bound to immunoprecipitated HDAC1 (339). The

second intracellular protein identified as a S1P target is the E3 ubiquitin ligase and TNF

receptor-associated factor 2 (TRAF2) (340). TRAF2 functions to ubiquitinate another member for the TNF complex, IKK which is bound to NF-κB. IKK is degraded following ubiquitination thus liberating NF-κB which then migrates to the nucleus where it functions as a transcription factor. However, the importance of the TRAF2 association with SPHK1 had not yet been described although it was reported that SPHK1 expression was necessary to elicit a molecular response to TNF stimulation in MEF cells (341).

Furthermore, addition of exogenous S1P (which does not readily increase intracellular

S1P concentration) did not activate a TNF reporter suggesting that intracellular S1P (the product of SPHK1) but not extracellular S1P is required for TNF signaling (341). Again

using LC- MS/MS, S1P was found to be the only sphingolipid found bound to

immunoprecipitated TRAF2 (341).

• The Sphingosine-1-Phosphate Receptors (S1PR)

S1PR1: The sphingosine-1-phosphate receptor 1 (S1PR1, formerly EDG-1 receptor)

is the first described and best studied of the 5 known S1P receptors. S1PR1 is primarily known as a Gi-coupled receptor but its ability to transduce Jak/STAT signaling properties

has been recently described (342). Because the primary function of this receptor is to

transduce mitogenic, survival and cellular migration signals, it is not surprising that it

plays a prominent role in the maintenance and development of several types of cancer

cells (343) . In is diffuse large B-cell lymphoma (DLBCL), S1PR1 has a pro-proliferative

function upon signaling through a non-canonical Jak/STAT3 pathway (344). In these lymphoma cells, STAT3 colocalizes with the S1PR1 and becomes deactivated

(dephosphorylated) upon shRNA-mediated S1PR1 knock-down thereby leading to apoptosis (344). Interestingly, STAT3 also serves as a transcription factor to induce expression of the S1PR1 thus providing the opportunity for a positive-feedback loop

(342). Finally, the authors use the S1PR1-targeting drug FTY720 as a S1PR1 antagonist;

FTY720 administration to Ly3 and Ly10 cells mimics the effects of S1PR1 knock-down and triggers apoptosis.

Activation the S1PR1 by S1P also induces angiogenesis (345), an crucial event for solid tumor development and metastasis. An experimental S1PR1 inhibitor, TASP0277308, decreased vascular endothelial growth factor (VEGF) –driven angiogenesis in cell culture 52

and reduced in vivo angiogenesis induced by fibrosarcoma HT1080 cells transplanted into mice (345). Similarly, expression of the S1PR1 was found to be necessary for precancerous endothelial remodeling in response to arsenic exposure (346); the

remodeling process requires increased vascularization of the tissue bed, a known S1PR1

function (347).

Adherent ovarian cancer cells displayed increased proliferation when cultured with S1P

(348). Notably, increased S1P levels were also found in the ascitic liquid from ovaraian cancer patients (348) consistent with the reported role of S1PR1 on cell migration and

cancer metastisis. Conversely, S1PR1 expression was found to be downregulated in

glioblastoma cells when compared to normal astrocytes (349). Furthermore, forced

S1PR1 expression inhibited proliferation while siRNA-mediated S1PR1 knock-down in

glioblastoma cells increased cellular expansion. Finally, low S1PR1 expression correlated

with a worse prognosis in glioblastoma patients. This particular work highlights the

importance of cell type when examining the effects of S1P because opposite S1PR1

signals can be elicited according on the cell type albeit it is generally accepted that

S1PR1 is primarily involved in transducing proliferative/anti-apoptotic signals.

S1PR2: S1PR2 (formerly Edg-5) is generally pro-proliferative although, like the

S1PR1, the functions of this receptor are dependent upon cell type. S1PR2 also negatively regulates hypoxia-induced pathological neovascularization in the mouse eye

(350). Indeed, S1PR2 knock-out mice show decreased tumor vascularization as well as

lowered tumor volume when used in Lewis lung carcinoma mouse xenograph model

(351). Furthermore, aged S1PR2-/- mice developed diffuse large B-cell lymphoma

(DLBCL) while S1PR2+/+ mice did not, again suggesting that the S1PR2 receptor

antagonizes tumor formation (352). Accordingly, high frequency (26%) of S1PR2 mutations was reported in DLBCL patients (352). S1PR2 expression is also increased in

Wilm’s tumor, in which S1PR2 is the primary driver of COX-2 (353), an event associated with increased tumor aggressiveness. Interestingly, S1PR2 triggering by S1P induces activation of Jun N-terminal kinase (JNK) and stress-activated protein kinase

(SAPK)(354).

In contrast with S1PR1, S1PR2 activation inhibits Rac and Rho GTPases-mediated cellular migration (355). It was reported that activation of S1PR2 inhibits Rac activity and reduces cell motility without interfering with Rho activity (355). However, in hepatocytes, stimulation of S1PR2 also prevented cellular proliferation primarily through modulation of Rho activity (356).

S1PR3: The S1PR3 (EDG-3) is a Gi/q/12/13-coupled S1P receptor found expressed

in most of human and mouse tissues although it is highly expressed in liver, kidney and heart (357). Treatment of MCF7 breast cancer cells with S1P caused internalization

(activation) of the epidermal growth factor receptor (EGFR, a receptor that stimulates cell growth when active) and rapid recycling of the S1PR3 (358). S1PR3 is highly expressed

in lung (359), breast (360) and prostate (361) cancer cells where it seem to positively

regulate their metastatic potential (362). Indeed, it seems that S1P-triggering of S1PR3 may induce chemotherapy resistance in prostate cancer cells and, furthermore, resistant cells may be primed for an augmented response to S1P due to increased expression of

S1P receptors (361). Based on these studies, the S1PR3 is described as a potential target

for cancer therapeutics. In fact, a recently developed monoclonal antibody targeting the

S1PR3 (7H9) effectively reduces the growth of a breast cancer xenograph (363).

S1PR4: The S1PR4 (formerly Edg-6) is a lymphocyte specific S1P receptor originally identified as an orphan receptor with high affinity for S1P and moderate

affinity for sphingosylphosphorylcholine (364). Stimulation of CHO cells expressing the

2+ S1PR4 induced [Ca ]I (364). Pretreatment of the S1PR4-expressing CHO cells with the

PLC inhibitor U73122 prevented the increase in calcium concentration suggesting that

S1PR4 signals though PLC and that calcium release is downstream of S1PR4-induced

PLC activation. Likewise, stimulation of the S1PR4 with S1P also induced ERK1/2 phosphorylation (365).

S1PR4 was later found to be Gαi and Gα12/13-coupled (291, 366) and, like the S1PR1,

S1PR4 induces cell motility upon stimulation with S1P (367). Mice lacking S1P lyase,

which have elevated blood neutrophils due to an inability to break down S1P, have partial

normalization of leukocyte counts when the S1PR4 is deleted (368).

S1PR5: The S1PR5 (formerly Edg-8), which is down regulated in esophageal cancer cells by S1P, inhibits proliferation and migration (369). Indeed, it was reported

that the S1PR5 inhibits ERK1/2 activation/phosphorylation following simulation with serum (354). S1PR5 like the other S1PRs is also a G protein-coupled receptor; it reduces cellular proliferation in a Gi- and Gα12-dependent manner as evidenced by sensitivity to pertussis toxin that, as reported (354)targets the Gi protein (370).

A unique feature of the S1PR5 is its ability to control centrosome function (371).

In fact, Gilles et al found colocalization of SPHK1 and SPHK2 as well as S1PR5 within the centrosome of HEK 293 cells (371), suggesting that S1PR5 may play a role in cell division.

• The Ceramide-mediated Pathways in Cancer Cells

Ceramide is a sphingolipid with characteristics distinct from those of S1P; in fact, while

S1P has chemotactic (372) and anti-apoptotic (373, 374) activities, ceramide is regarded as a pro-apoptotic factor (375, 376) in part because of its ability to activate PP2A (183).

For example, treatment of TKI-resistant CML cells with ceramide restored sensitivity to

TKI-induced apoptosis (377). Interestingly, the dual Src and ABL TKI dasatinib seems to exert its apoptotic activity partly by promoting downregulation of SPHK1(378), which is overexpressed in several leukemic cell types, including CML (320). This is not surprising as overexpression of SPHK1 reduces the sensitivity of acute myeloid leukemia (AML)

HL60 cells to chemotherapeutic agents (332) most likely by increasing the intracellular or extracellular levels of S1P that, in turn, prevent apoptosis by upregulating the expression of the anti-apoptotic factor MCL-1 (379). Consistent with the ability to induce and amplify anti-apoptotic signals, we also recently reported that activation of the 56

SPHK1/S1PR1 enhances stability of the BCR-ABL1 protein thereby providing survival and proliferation signal to CML cells (380).

Importantly, ceramide also reduces erythroid colony formation, a consequence that is reversed by exposure to S1P (381). These lipids are exceptionally interesting as ceramide is a well described activator of PP2A (183) whereas S1P is a robust inhibitor of

PP2A (see Results Section).

• Ceramide-1-Phosphate (C1P).

Ceramide-1-Phosphate (C1P), is a phosphorylated ceramide largely analogous to

S1P; the biological effects of C1P are larger similar to S1P as it has been shown to induce

proliferation/inhibit apoptosis as well as mediate migration (382). C1P was first discovered in the human leukemia HL60 cells in which it was found to be produced from the action of sphingomyelinase on sphingomyelin (383) by the activity of ceramide kinase (384). C1P blocks apoptosis in BM-derived macrophages cultured without macrophage-colony-stimulating factor (M-CSF), which is normally necessary to maintain macrophage viability through inhibition of ceramide synthesis (385). Likewise, C1P (0.5-

1.0µM) increases proliferation and reduced apoptosis of A549 lung adenocarcinoma cells and NIH3T3 fibroblasts (386).

Like S1P, C1P also mediates migration of RAW264.7 macrophages through a Gi-coupled

surface receptor, although the receptor itself has yet to be identified (387). Induction of

intracellular C1P production via stimulation of these cells with either IL-1β or the ion carrier A23187 failed to induce cell migration whereas exposure to exogenous C1P 57

caused a robust increase in ERK1/2 and JNK phosphorylation (387), suggesting a role for

C1P similar to that of S1P.

2.7 Anti-cancer activity of the clinically-relevant sphingosine analog FTY720

FTY720 is a synthetic sphingolipid currently used clinically for patients with relapsing multiple sclerosis (MS) because of its immunosuppressive activity (388). The compound itself is considered a pro-drug lacking activity until converted by sphingosine kinase 2 (SPHK2) into the phosphorylated FTY720-S-phosphate (FTY720-P) (Figure 7), which is secreted into the extracellular compartment and re-internalized upon interaction with S1P receptors (307, 308). The FTY720 phosphorylation is reverted in vivo primarily by the activity of the lipid phosphate phosphatases 1a and 3 (LPP1a, LPP3) (389, 390).

Conversely, S1P lyase, originally believed to be the primary dephosphorylating enzyme, is unable to dephosphorylate FTY720-P (391).

Figure 7. FTY720 and FTY720-(S)- Phosphate. Parental FTY720 (top) is reversibly phosphorylated by sphingosine kinase 2 (SPHK2) to become the immunosuppressive FTY720-(S)-Phosphate. (FTY720-P; bottom).

FTY720-P interacts and modulates activity of four out of five S1P receptors (FTY720-P has no activity on S1PR2) (392). The primary mechanism of FTY720 immunosuppressive

activity rests on internalization of the S1PR1. When the natural ligand of S1PR1 (S1P)

occupies the receptor, the complex is internalized and the receptor is recycled to the cell

surface. By contrast, FTY720-P binding to S1PR1 triggers internalization of the complex

but the receptor is sequestered in the cytoplasm and is thereafter degraded (393).

FTYT720-P-induced loss of surface S1PR1 expression impairs the response of T and B cells to chemotactic/migration signals normally provided by endogenous S1P in response to inflammatory cytokines thereby resulting in block of the egress of lymphocytes to the peripheral blood and their accumulation in the lymph nodes (392, 394) (Figure 8).

Figure 8. FTY720 Immunosuppression. FTY720 is phosphorylated in vivo by sphingosine kinase 2 (SPHK2). Following phosphorylation within the cytoplasm, FTY720-P is exported into the extracellular space making it available to the S1PR1 (sphingosine-1- phosphate receptor 1). Agonism/internalization of this receptor leads to sequestration of lymphocytes which produces immunosuppression.

FTY720 was first synthesized through modification of myriocin (ISP-1), a natural product produced by the fungus Isaria sinclairii. Although the parental compound is active as an immunosuppressive agent as it inhibits the activity of serine palmitoyltransferase, FTY720 is a much stronger and less toxic immunosuppressant that, as we stated above, works through modulation of the S1PR1 signaling (395-400).

FTY720 was first successfully tested in preclinical renal transplant setting; however, its

use in post-transplant patients was discontinued as better outcomes were achieved with

cyclosporine A derivatives (401, 402). Despite this lackluster performance, research on

FTY720 continued with a particular focus on the autoimmune disease multiple sclerosis

(MS). In treating MS, FTY720 was found to be effective at reducing disease relapse and preventing disease progression as defined by a decrease in the number of lesions

(scleroses) detected using MRI measurements (403). FTY720 has a strong safety profile with limited and transient side effects similar to those found in earlier transplant studies

(transient bradycardia, respiratory infection, and basal-cell carcinoma) but much reduced in magnitude due to the lower doses required to achieve a therapeutic response in relapsing MS (0.5mg- 1.25mg/day)(404-407). Importantly, these adverse effects can be all ascribed to the signals generated by the phosphorylated FTY720-P (408) . In 2010

FTY720 (also knows ad Fingolimod), now named Gilenya, was approved by the FDA for its use in MS patients.

Studies performed early after its development indicated that FTY720 also has

very strong anti-cancer properties. One of the earliest studies showed FTY720 induced 60

caspase-dependent apoptosis in prostate cancer cells, albeit at a dose of 40µM (409).

Later studies used more physiologically relevant concentrations and found effectiveness

of FTY720 at killing glioma (410) and breast cancer (411) cells. Importantly, it was reported that exposure of leukemic Jurkat human T cells to FTY720 led to serine/threonine dephosphorylation of Akt and of p70S6K (412). Interestingly, FTY720-

induced Akt-dephosphorylation was prevented with the co-administration of the

phosphatase inhibitor okadaic acid used at a dose that specifically inhibits the activity of

PP2A (0.1-1nM) but not that of other cellular phosphatases, suggesting FTY720 was

activating PP2A. The PP2A-activating function of FTY720 was confirmed by measuring

PP2A activity in living cells treated with FTY720 and in phosphatase assays with

purified PP2A ABC complexes (412).

We recently reported that FTY720 but not FTY720-P has strong anti-cancer activity in

imatinib- and dasatinib-sensitive and –resistant chronic and blastic phase CML, Ph+ ALL

and AML with a mutated c-kit (79, 413). Note that these leukemias are characterized by activation of Jak2 and SET-dependent inhibition of PP2A (78, 166) Furthermore, we showed that the anti-cancer activity of FTY720 does not require SPHK2 phosphorylation or S1PR1 interaction (79, 414) but depends, at least in Ph+ leukemias, on its ability to

restore PP2A function that, in turn, promotes BCR-ABL1 inactivation/proteasome-

degradation and inhibition of key proliferation/survival factors such as Jak2, Akt and

ERK1/2 (38, 78, 79). In fact, FTY720 markedly suppresses cell proliferation and induces

apoptosis of CD34+ progenitors from tyrosine kinase inhibitor (TKI)-sensitive and – 61

resistant Ph+ leukemia patients but not of BM progenitors from healthy individuals, which

already present highly active PP2A(78, 79).

Figure 9. FTY720 Anti-cancer Activity. (A) BCR-ABL1 inhibitory effect on PP2A are mediated by overexpression of SET. (B) PP2A activating drugs like Forskolin (Neviani et al., Cancer Cell 2005) and FTY720 (Neviani et a., J. Clin. Invest 2007) impair leukemogenesis by inducing inactivation of the BCR-ABL1 oncogene and that of its downstream effectors. FTY720 does not require phosphorylation to work as a PP2A activating drug and inhibit leukemia development and progression.

Accordingly, long-term (27 weeks) FTY720 treatment (10 mg/Kg/day) of leukemic animals extensively prolongs survival (80-90% survival) and restores normal myelopoiesis without exerting any toxic effects in hematopoietic and non-hematopoietic organs (79), although it reversibly reduces B and T lymphocytes (185, 415). The history

of FTY720 so far has described it as a safe compound with uses as both an MS treatment

as well as a cancer therapeutic drug both in vitro and in animal studies.

In this study we show that FTY720 (Fingolimod, Gilenya) is a potent protein phosphatase

2A (PP2A)-activating drug (PAD). As we previously discussed, PP2A is a tumor

suppressor found inactivated in different types of cancer. We found that PP2A is inactive

in Polycythemia Vera (PV) and other myeloproliferative neoplasms (MPNs)

characterized by the expression of the transforming Jak2V617F oncogene. PP2A

inactivation occurs in a Jak2V617F dose/kinase-dependent manner through the PI-3Kγ-

PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic and/or PAD-mediated

PP2A reactivation induces Jak2V617F inactivation/downregulation and impairs clonogenic

potential of Jak2V617F cell lines and PV but not normal CD34+ progenitors. Likewise,

FTY720 decreases leukemic allelic burden, reduces splenomegaly and significantly increases survival of Jak2V617F leukemic mice without adverse effects. Mechanistically,

we show that in Jak2V617F cells, FTY720 anti-leukemic activity neither requires FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we showed that Jak2V617F also utilizes an

alternative sphingosine kinase-1 (SPHK1)-mediated pathway to inhibit PP2A, and that

FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 (S1PR1) agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs

(e.g. FTY720) represent suitable therapeutic alternatives for Jak2V617F MPNs.

CHAPTER 3

Material And Methods

3.1 Primary cells and cell lines

Non-identifiable Jak2V617F MPN (BM) and peripheral blood (PB) patient samples

were obtained from The Ohio State University (OSU) Comprehensive Cancer Center

(Columbus, OH), MD Anderson Cancer Center (Houston, TX), Hammersmith Hospital

(London, UK) and Memorial Sloan-Kettering Cancer Center (New York, NY) leukemia

tissue banks. Frozen samples of healthy donor CD34+ BM cells (NBM) were purchased

from Cincinnati Children’s Hospital (Cincinnati, OH).

Frozen samples from Jak2V617F PV, PMF and/or ET patients were Ficoll separated and/or

selected using MACS CD34 MultiSort kit (Miltneyi Biotec). All human primary cells

were maintained in IMDM, 30% FBS, 1% penicillin/streptomycin, 1% L-glutamine

(Gibco) plus rhIL-3 (20 ng/ml), rhIL-6 (20 ng/ml), rhFlt-3 ligand (100 ng/ml) and rhKL

(100 ng/ml) (Stem Cell Technologies). The murine pro-B Ba/F3, the human erythroleukemia TF-1 and HEL cell lines, and their derivatives were cultured in IMDM,

10% FBS, 1% penicillin/streptomycin, 1% L-glutamine (Gibco). Parental Ba/F3 cells were grown in medium supplemented with 10% WEHI-conditioned medium as a source of crude IL-3.

Retroviral and lentiviral infections were carried out as follows; briefly, HEK 293T cells

(for lentivirus) or phoenix cells (for retrovirus) were transiently transfected overnight with construct to be expressed using, VSVG, and psPAX (lentivirus only) using calcium 64

phosphate (Promega Profection kit). Following transfection, medium on cells was

changed and left for 48 hours to collect virus. Virus containing medium was then

collected, filtered through a 0.45µm filter and incubated with cells to be infected for 24

hours.

Retro/lentivirally-transduced cells were sorted for GFP+ (green fluorescent protein) or

underwent antibiotic-mediated selection. All studies with human specimens were

performed with The Ohio State University Institutional Review Board approval.

3.2 Methylcellulose-based clonogenic assay

Colony Forming Cell (CFC) methylcellulose assays were carried out by plating

103 cells from Jak2V617F cell lines or 104 CD34+ PV or NBM cells in 0.9% MethoCult

M3234 or H4435 (Stem Cell Technologies Inc.), respectively. Colonies (> 125 µm) were

scored after one (cell lines) and two (primary cells) weeks.

3.3 Chemical and biological Reagents.

• Chemicals

Cells were treated with the following kinase, phosphatase and/or sphingolipid

pathway inhibitors and/or activators used at concentrations, times and schedules indicated

in Results. Jak inhibitors: Jak inhibitor I (Calbiochem), AG490 (Cayman Chemical) and

TG101348 (SAR302503, TargeGen/Sanofi-Aventis); PP2A activity inhibitors: okadaic

acid (Calbiochem); PP2A-activators: FTY720 (Cayman Chemical); its non- immunosuppressive derivatives S-FTY720-Regioisomer (CUNY Univ., NY) and OSU- 65

2S (OSUCC Pharmacology Core Lab), and its phenoxy-conjugated FTY720-phenoxy- biotin and FTY720-phenoxy-NBD (Cayman Chemical); Sphingolipid pathway

modulators: the immunosuppressive S1PR1,3-5 agonist phosphorylated FTY720-P

(Cayman Chemicals), the S1PR1-selective agonist SEW2871 (Cayman Chemical), the

pan-sphingosine-1-phosphate-receptor agonist S1P (Cayman Chemicals), the S1PR1/3

antagonist/inverse agonist VPC23019 (Avanti Polar Lipids), and the sphingosine kinase

inhibitor DMS (dimethylsphingosine, Sigma Aldrich); Kinase inhibitors: pan-PI-3K

inhibitor LY294002, PI-3Kγ inhibitor AS-604850, PKC inhibitor PKC-412, and MEK1

inhibitor PD98059 (Sigma Aldrich).

• Plasmids

MSCV-puro-Jak2 V617F and MigR1-Jak2 wild type and Jak2 V617F. Jak2 wild-

type cDNA was purchased from Openbiosystems in a pSPORT6 vector. Wild-type Jak2 was directly subcloned into MigR1. Jak2 V617F was produced via site-directed mutagenesis and also and ligated into MigR1 and MSCV-puro using Bgl II and EcoR1 sites of MSCV-puro and the bicistronic GFP-containing MigRI vector.

MigRI-HA-PP2Ac. The HA-tagged PP2Ac cDNA was PCR amplified from

pHM6-HA-PP2Ac and subcloned into the HpaI/EcoRI sites of MigR1.

pLL3.7-shSET. The shRNA SET construct was obtained by subcloning the

double-stranded 60 mer oligonucleotide containing the SET target sequence (5’-

TGAAATAGACAGACTTAAT-3’) into the pLL3.7 vector (Luk Van Parijs (416)).

pCDH-Flag-SET. The human SET cDNA was obtained from K562 mRNA by

RT-PCR using an upstream primer containing an HpaI site, the FLAG epitope, and the

first 16 nucleotides of SET cDNA, and a downstream primer containing the last 21

nucleotides of SET linked to an EcoRI restriction site. The XbaI/EcoRI digested PCR

product was subcloned into the pCDH-CMV-MCS-EF1-copGFP vector.

pCDH-SETK209D. K209D mutated SET cDNA was generated using site-directed

mutagenesis and subcloned into pCDH using the same sites as wild-type FLAG-SET.

S9A, S9E, S24A and S24E mutated SETs were created using site-directed mutagenesis of

with-type FLAG-SET in PCDH.

pCDH-SET-ND. Non-dimerizable SET (termed TAF-1βPME in original publication (175). Was cut from pCHA at XbaI and BglII and ligated into pCDH at Xba1

and BamH1.

pGipz Jak2 shRNA was purchased from Openbiosystems (#V2LHS_61653). This

vector contains the sequence 5’-GTACAGATTTCGCAGATTT-3’

3.4 In vivo Studies with FTY720 in Jak2V617F PV mouse model

Four- to six-week-old immunocompromised ICR-SCID mice (n=50) were

intravenously (i.v.)-injected with 105 Ba/F3-Jak2V617F cells (n=30) or used as controls

(n=20). After engraftment (one week), 15 mice were intraperitoneal (i.p.) FTY720-treated

daily, and 15 mice were vehicle (saline solution)-treated. As controls, 10 age-matched

SCID mice received neither cells nor treatment and the other 10 received FTY720 at the

same regimen. At 6 and 8-week post-transplant, 3 mice/group were sacrificed and organs

(BM, spleen, liver, kidney and heart) were subjected to macroscopic and microscopic

(H&E staining) evaluation of leukemic-cell infiltration. Simultaneously, PB was collected by retro-orbital survival bleeding for SNaPshot analysis of Jak2 alleles (see below). Following 10 weeks of treatment, untreated (age-matched) and FTY720-treated

(10 weeks; 10 mg/kg/d) control mice (n=3/group) were used to assess the effect of long- term FTY720 treatment on cardiac function (see below). The remaining mice were used for Kaplan-Meier survival analysis. All the animal protocols/procedures were approved by OSU IACUC.

• SNaPshot analysis of Jak2 V617F mutants: RNA was trizol-extracted from PB of

untreated and FTY720-treated leukemic mice and reverse transcribed using the

Superscript III First-Strand cDNA Synthesis Kit (Life Technologies

Corporation/Invitrogen) Polymerase chain reaction (PCR) amplification of JAK2

covering the V617F mutation was carried out using primers ′ V617(F) 5 -

tggaatttttgttcttcttcagg-3′ and V617(R) 5′-tcaactgcacaaaacggaag-3′ under the following conditions: 10’ at 96 oC, 40 cycles (94 oC for 15”, 56 oC for 15”, 72 oC for 1’ ) followed

by 5’ at 72 oC. PCR products were purified by using 2l ExoSAP-IT (Affymetrix) for 5l

PCR product. The SNaPshot reaction was performed as previously described(417) using

the SNaPshot Multiplex Kit (Life Technologies Corporation/Applied Biosystems) and the

primer V617F(SS) (5′ -ggttttgaattatggtgtctgt-3′) under the conditions: 5’ at 95 oC, 26

cycles (95 oC for 10”, 50 oC for 5”, 60 oC for 30”) followed by Shrimp Alkaline 68

Phosphatase treatment (Affymetrix). SNaPshots were analyzed as previously

described(417). The peak areas representing allele-specific extended primers were

determined using GeneMapper Software (Life Technologies Corporation/Applied

Biosystems). Changes in the leukemic allelic burden (mutant versus wild-type allele)

were determined by creation of a ratio using the absolute peak heights of both peaks.

Serial dilutions of homozygous mutant and wild type samples were performed to create a

standard curve as a reference. A reduction in the ratio was considered as an absolute

reduction of the respective allele expression.

• Cardiologic Effects of long-term FTY720 treatment: Cardiomyocyte isolation and

cardiac measurements: Ventricular myocytes were isolated from untreated (age-matched) and FTY720-treated (10 weeks; 10 mg/kg/d) control mice (n=3 per group) as previously described(418). Briefly, the heart was cannulated and hung on a Langendorff apparatus.

It was then perfused with Ca2+ free tyrode solution for 4 min. The solution was then

switched to Tyrode solution (in mmol/L): 140 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 10

glucose, 5 HEPES, pH 7.4 adjusted with NaOH or HCl also containing Liberase

Blendzyme II (0.077 mg/ml) (Roche Applied Science, Indianapolis, IN). After 3-5 min,

the heart was taken down, the ventricles minced, and myocytes were dissociated by

trituration. Subsequently the myocytes were filtered, centrifuged, and resuspended in

Tyrode solution containing 200 μmol/L Ca2+. Myocytes were used within 4 hours of

isolation. Myocyte contraction was evaluated by simultaneous measurement of cell

shortening, via edge detection, and calcium transients, via epifluorescence (Fluo4-AM) in 69

the absence or presence of freshly prepared (daily) isoproterenol (ISO, 1 μM, a non-

selective β-AR agonist). The Ca2+ transient measurements were also performed as described(418). Briefly, myocytes were loaded at room temperature with Fluo-4 AM (10

μmol/L, Molecular Probes, Eugene, OR) for 30 min, and then another 30 min were

allowed for intracellular de-esterification. The solution for de-esterification was Tyrode

solution containing 200 μmol/L Ca2+. The instrumentation used for cell fluorescence

measurements was a Cairn Research Limited (Faversham, UK) epifluorescence system.

2+ [Ca ]i was measured by Fluo-4 epifluorescence with excitation at 480±20 nm and emission at 535±25 nm. The illumination field was restricted to collect the emission of a single cell. Data were expressed as ΔF/F0, where F is the fluorescence intensity and F0 is

the intensity at rest. Myocytes were stimulated at 1 Hz via platinum electrodes connected

to a Grass Telefactor S48 stimulator (West Warwick, RI).

• Heart rate: Electrocardiograms were recorded in conscious untreated (age-

matched) and FTY720-treated (10 weeks) control mice (n=3 per group) using the

ECGenie, a non-invasive instrument for cardiac function by placing the mouse onto a

recording platform. Following a sufficient amount of time for the animals to acclimate,

heart rates were taken from the triggered recordings when the animal’s paws were in

contact with the electrodes.

3.5 Immunoblots and Protein-based Assays.

Cells (106 for cell lines, 104 for primary patient samples) were lysed in RIPA buffer (150

mM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris [pH 8.0]) supplemented with protease

and/or phosphatase inhibitor cocktails (Complete and PhosStop mix; Roche) and 10 mM

β-glycerol-phosphate. After incubation on ice (20 min), lysates were clarified (13,000×g,

20 min, 4°C), denatured and subjected to SDS-PAGE and immunoblotting. The primary antibodies used were: anti-SET/I2PP2A (Santa Cruz), -Jak2 (Cell Signaling), - pJak2Y1007/1008 (Cell Signaling), -pJak2Y972 (Millipore), -PP2Ac (Upstate), - pPP2AcY307

(Epitomics) and -Grb2 (BD TransLab Inc.).

For immunoprecipitation (IP) and to detect the SET-FTY720 interaction, cell lysates

(1mg) in RIPA buffer were incubated for 1 hour at 4ºC with either the primary antibody or FTY720-phenoxy-biotin (Cayman Chemical) followed by the anti-NBD antibody (see below). For SET dimerization assays, lysates of Ba/F3-expressing FLAG-SET and GFP-

SET cells were co-incubated and used in anti-Flag IPs and anti-GFP immunoblotting (see

below). PP2A, Sphingosine Kinase activities, DAG kinase-mediated ceramide

quantitation and mass spectrometry of ceramide subspecies are described below in this

chapter.

For classical immunoprecipitation (IP) and to detect the SET-FTY720 interaction, cell

lysates (1mg) were prepared in RIPA buffer and incubated for 1 hour at 4℃ with either

the primary antibody or with 10µM FTY720-phenoxy-biotin (Cayman Chemical)

followed by an overnight incubation with 10ng anti-NBD antibody or 10ng of rabbit IgG

control antibody. 71

For FTY720-biotin pull-down assay, Ba/F3 cell lysates (1mg) were diluted in

10mL of binding buffer (50 mM ammonium carbonate, 0.5 M NaCl, pH 10.0) and

incubated with 10µM of either FTY720 or FTY720-phenoxy-biotin with gentle agitation for 4 hours. Lysates were subjected to two-rounds of monomeric avidin chromatography column (Pierce) and the FTY720-containing complex was eluted with 20 mL 2mM biotin in PBS, concentrated using 10 kDa cut-off Centricon columns (Millipore), mixed with 2X denaturing loading buffer, fractionated onto SDS-PAGE and analyzed by western blotting.

For SET dimerization assays, Ba/F3 cells expressing FLAG-SET or GFP-

SET were lysed in NP40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.2, 1% NP-

40) supplemented with protease and phosphatase inhibitor cocktails (Roche). FLAG-SET

lysates (2 mg) were incubated with 20 µl Anti-FLAG beads (Sigma) for 3 hours at 4 oC

with gentle agitation. Beads were washed 2x with ice-cold TBS. 2mg GFP-SET cell lysate was added along with either FTY720 (10 µM) or DMSO control and incubated for

2 hours at 4 oC with gentle agitation. Beads were then washed 2x with ice-cold TBS and proteins eluted with SDS buffer and subject to SDS-PAGE and WB for the GFP-SET.

3.6 Kinase and Phosphatase Assays

• PP2A activity by immuno-colorimetric phosphatase assay: PP2A phosphatase

assays from whole cell lysates were carried out using the PP2Ac immunoprecipitation phosphatase assay kit (Millipore, Billerica, MA). Briefly, 50 g of protein lysate in 100

µl of 20mM Hepes [pH 7.0]/100mM NaCl, 5µg of PP2Ac antibody (Millipore) and 25 µl of Protein A-agarose were added to 400 µl of 50mM Tris [pH 7.0], 100mM CaCl2, and

immunoprecipitation were carried out at +4oC for 2h. Immunoprecipitates were washed

and used in the phosphatase reaction according to the manufacturer’s protocol.

Specificity of the reported protocol for PP2A has been reported (79).

• Sphingosine Kinase Assay: Untreated and FTY720 (2.5µM 6 hours)-treated

Ba/F3-Jak2V617F cells were lysed in 200 mM Tris pH 7.4, 20% glycerol, 1mM β-ME, 1

mM EDTA, 1mM NaVO3, and protease inhibitor cocktail. 100µg of cell lysates were

incubated with 32P-γATP and 1mM sphingosine (1 hour; 37 ºC) and subjected to acidic

lipid extraction. Radioactive S1P was TLC separated and visualized by autoradiography.

To assess PP2A dependence cells were treated with FTY720 (2.5µM 6 hours) and

okadaic acid (0.25 nM, 1.5 hours).

• DAG kinase-mediated Ceramide quantitation: DAG kinase-mediated ceramide

quantitation was performed as described (419). Briefly, sphingolipids were extracted

from 106 untreated or FTY720-treated (2.5µM, 6 hours) Ba/F3-Jak2V617F cells by using 3

mL of CHCL3:MeOH:H2O (1:1:1). Extracted lipids (200µL) were then supplemented

with 40 µl of 7.5% octyl-β-D-glucopyranoside (Calbiochem), 5 mM cardiolipin in 10

mM imidazole. This suspension was subjected to 5 freeze-thaw cycles followed by 10

minutes of sonication. Thereafter, the following reagents were added to the suspension:

100 µl of reaction buffer (100 mM imidazole-HCl (pH 6.6), 100 mM NaCl, 25 mM

MgCl2, and 2 mM EGTA), 30 µl fresh 13mM DTT solution, 10 µl DAG kinase, 20 µl of 73

ATP mixture (ATP consists of 1 µl radioactive ATP to 19 µl cold 10 mM ATP). Samples

were incubated at room temperature for 1 hour, and the reaction was terminated by

adding 0.5 ml of CHCl3:MeOH:HCl (100:100:1). Samples were vortexed thoroughly,

and after adding 100 µl of 1 M NaCl, samples were centrifuged for 5 minutes at 2,000

RPM and 50 µl of each sample were spotted onto TLC plates. DAG activity was

developed in chlorform:acetone:methanol:acetic acid:water (10:4:3:2:1) and the

radioactive S1P produced by the reaction visualized by autoradiography.

3.7 Mass spectrometry analysis of ceramide subspecies.

Endogenous ceramide species from treated (FTY720, 2.5µM for 24 hours) and

untreated Ba/F3-Jak2V617F cells were analyzed by mass spectrometry using normal phase high performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization. Separations were performed using a ThermoFinnigan (Foster City,

CA) LCQ ion trap mass spectrometer.

3.8 Real-time Polymerase Chain Reaction (PCR)

Real-time PCR was performed as described previously (301). Briefly, total RNA (1 μg),

isolated from TF-1 cells retrovirally-transfected with MigR1 or MigR1-Jak2V617F, was

reverse transcribed and used for quantitative Real-time PCR (SYBR Green technology).

Individual SPHK isoforms were quantified by using sequence-specific primers: hSphk-

1a: forward (F) 5’-gtcgaggttatggatccagcg-3’, reverse (R) 5’-ttccgccgctcagtgagcat-3’;

Sphk-1b: (F) 5’-atggatccagtggtcggttgcg-3’, (R) 5’-ttccgccgctcagtgagcat-3’; Sphk-1c (F)

5’-atgtccgctcaagttctgggat-3’, (R) 5’-tcctgccttcagctccttat-3’; hSphk-2a (F) 5’- agatgaatggacaccttgaagc-3’, (R) 5’-ctgggtaggagccaaactcg-3’; Sphk-2b (F) 5’- gccaccatggccccgcccccaccgccact-3’, (R) 5’-cagggtgcagcagcctgagac-5’.

Triplicates were normalized using GAPDH levels.

3.9 Statistical analysis.

For in vitro studies, data were statistically compared with the 2-tailed Student’s t- test. For animal studies, the estimated probabilities for survival were calculated by the

Kaplan-Meier method, and the log-rank test was used to evaluate the differences among survival distributions. A p-value of less than 0.01 (mouse model p<0.05) was considered statistically significant.

CHAPTER 4

Results

4.1 PP2A tumor suppressor activity is inhibited in polycythemia vera in a

Jak2V617F/SET-dependent manner.

To evaluate the role of PP2A in Jak2 V617F-expressing cells, the IL-3-dependent pro-B lymphoid cell line Ba/F3 was transduced with a retroviral vector encoding for wild type (WT) or V617F Jak2. Cells were then sorted for high- and low-GFP expression to produce two separate populations with graded Jak2 expression levels (Figure 10A, inset). PP2A phosphatase activity assays showed that overexpression of Jak2 induced a significant decrease in PP2A activity with the highest Jak2 expression levels corresponding to the greatest decrease of phosphatase activity (~55% vs ~99% inhibition)

(Figure. 10A). Notably, while the cells expressing wild-type Jak2 produced an inhibition of PP2A activity similar to that of the Jak2V617F cells, only the Ba/F3-Jak2V617F (high)

cells acquired cytokine-independence. In these cells, restoration of PP2A activity by

ectopic HA-PP2Ac expression resulted in decreased Jak2V617F activity and expression

(Figure 10A) and, consequently, marked reduction of cytokine-independent survival as

indicated by the ~90% reduction of their clonogenic potential (Figure 10D, blue).

Figure 10A and 10B. Jak2V617F suppresses PP2A activity in a SET-dependent manner. (A): Graph: PP2A activity in Ba/F3 cells transduced with the MigR1 vector (white), Ba/F3 cells expressing low (pink) and high (red) levels of wild type (wt) and V617F Jak2 kinase, and in HA-PP2A-expressing (blue) Ba/F3-Jak2V617F cells. Inset: Jak2 levels in Ba/F3 cells transduced with wt or V617F Jak2, and sorted for high and low GFP expression. Right: immunoblots show Jak2 expression and activity, and PP2Ac expression in vector- and HA- PP2A-transduced Ba/F3-Jak2V617F cells. (B): PP2A activity in untreated and/or Jak inhibitor [25µM AG490; 1µM Jak Inhibitor I; 1µM TG101348] -treated primary CD34+ normal bone marrow (NBM), polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocytosis (ET) patient samples, Ba/F3-Jak2V617F and HEL cells.

PP2A assays in BM and/or PB cells from healthy individuals (n=3) and PV, MF and ET patients (n=9) revealed that marked suppression (~ 80% inhibition) of PP2A activity is a common feature of myeloproliferative neoplasms (MPN) carrying the oncogenic

Jak2V617F allele (Figure 10B). In fact, inhibition of Jak2 activity by treatment of the

Ba/F3-Jak2V617F and the Jak2V617F homozygous human leukemic HEL cells, and primary

CD34+ progenitors from PV patients (n=3) with the Jak inhibitors AG490 (25µM), Jak

Inhibitor I (1µM) and/or TG101348 (1µM) restored PP2A activity to levels similar to those observed in primary CD34+ BM progenitors from healthy individuals and in non-

transformed Ba/F3 cells (Figure 10A and 10B). Similarly, downmodulation of Jak2V617F

expression by short-hairpin RNA interference (Jak2-shRNA) in both HEL cells and

CD34+ PV progenitors rescued PP2A activity as indicated by PP2A assay and by the

decreased levels of Y307-phosphorylated PP2Ac (inactive)(104) in Jak2-shRNA-

expressing HEL and primary PV progenitors (Figure 10C). To further understand the

mechanism whereby Jak2V617F suppresses PP2A activity, we used shRNAs in primary

CD34+ PV progenitors, Ba/F3-Jak2V617F and HEL cells to negatively modulate expression of SET, a direct PP2A inhibitor that, reportedly, blocks PP2A activity in CML progenitors through a BCR-ABL1 kinase-dependent Jak2-mediated manner (78, 160,

166).

Figure 10C and 10D. Jak2V617F suppresses PP2A activity in a SET-dependent manner. (C): PP2A activity in Jak2 shRNA-expressing HEL cells and SET shRNA- expressing Ba/F3-Jak2V617F, HEL and primary CD34+ PV cells. Inset: levels of inactive PP2Ac (pY307) in Jak2 shRNA-expressing PV CD34+ progenitors. Top right: SET downmodulation in SET shRNA-expressing Ba/F3-Jak2V617F and HEL cells. (D): Percentage of colony forming cells (CFC) in HEL and Ba/F3-Jak2V617F cells transduced with empty vector (red) or with the V617F SET shRNA (white), and in HA-PP2Ac-transduced Ba/F3-Jak2 cells (blue). 78

Lentiviral (pLL3.7)-mediated SET knock-down (Figure 10C, inset) fully restored PP2A

activity in all cell types (Figure 10C). Consistent with the pro-apoptotic role of active

PP2A, cells expressing SET shRNA had a reduction in colony forming cells (CFC) of

91% and 80% for Ba/F3-Jak2V617F+ and HEL cells, respectively (Figure 10D). Thus,

Jak2V617F uses SET to inactivate PP2A in a dose- and kinase-dependent manner and to maintain survival of leukemic cells.

4.2 Jak2V617F-dependent and PI-3Kγ/PKC-mediated SET phosphorylation leads

to inhibition of PP2A.

In CML, suppression of PP2A activity depends on the BCR-ABL1-induced and Jak2-

mediated overexpression of SET (78, 166); however, expression of Jak2V617F oncogene

did not increase SET levels (Figure 11B, top), suggesting that post-translational

modification(s) control(s) the ability of SET to act as a PP2A inhibitor in Jak2V617F- driven hematologic neoplasms. Reportedly, SET undergoes PI-3Kγ-mediated serine 9 and 24 phosphorylation by PKC that controls SET nuclear export and, therefore, its ability to act as a physiologic inhibitor of cytoplasmic PP2A (160, 176, 178). Thus, it is possible that Jak2V617F-generated signals suppresses PP2A activity through the PI-3Kγ-

PKC-mediated SET phosphorylation on these two serine residues (Figure 11A).

Figure 11A. Jak2V617F-driven PI- 3Kγ/PKC-mediated signals induce inhibition of PP2A through SET phosphorylation. Molecular pathway model of Jak2/Jak2V617F-mediated PP2A inhibition. Chemical inhibitors are indicated in black

Ectopic expression of wild type (WT) and phosphomimetic mutation of SET serine 9 into

glutamic acid (S9E) resulted in marked suppression of PP2A activity (~80% inhibition)

in parental Ba/F3 cells (Figure 11B, bottom left). Conversely, only a ~40% decrease in

PP2A activity was observed upon ectopic expression of the non-phosphorlatable SET

S9A mutant (Figure 11B, bottom left) that, as expected, also markedly accumulated into the nucleus and (Figure 11B, bottom right). Because PP2A activity was not differentially and significantly affected upon expression of SET serine 24 mutants (not shown), it is highly plausible that serine 9 phosphorylation accounts for most of the SET inhibitory function on PP2A.

Figure 11B. Jak2V617F- driven PI-3Kγ/PKC- mediated signals induce inhibition of PP2A through SET phosphorylation. (B): Top: SET and Grb2 protein levels in Ba/F3 and Ba/F3- Jak2V617F cells. Bottom: PP2A activity in Ba/F3 cells transduced with empty vector (pCDH), wild type (WT), non-phosphorylatable S9A and phosphomimetic S9E mutant SET Flag- tagged constructs. Inset: Ectopic wild type and mutant SET proteins (nuclear fraction).

Accordingly, treatment of Ba/F3-Jak2V617F cells with the pan-PI-3K (LY294002; 25µM,

6 hrs.), the selective PI-3Kγ (AS-604850; 1µM, 6 hrs.) and an inhibitor of conventional

PKCs (PKC-412(420); 2µM, 6hrs.) markedly reduced SET serine phosphorylation

(Figure 11C) and restored PP2A activity to levels similar to those in non-transformed

cells (Figure 11D). Likewise, reduced SET serine phosphorylation and rescued PP2A activity was achieved upon exposure of Ba/F3-Jak2V617F cells to the Jak2 inhibitors

TG101348 (1µM; 6hrs.), AG490 (25µM; 6 hrs.) and Jak Inhibitor I (1µM; 6 hrs) (Figure

11C and 11D). Further supporting the existence of a Jak2V617F-PI-3Kγ/PKC-SET axis, inhibition of Jak2 activity also resulted in decreased levels of conventional PKCs (Figure

11C, bottom). Moreover, consistent with the negative effect of PP2A on PKCs, treatment

of Ba/F3-Jak2V617F cells with FTY720 reduced both SET phosphorylation (Figure 11C, top) and conventional PKCs expression (Figure 11C, bottom). As expected, inhibition of a non-related pathway (e.g. MAPK) by the MEK1 inhibitor PD98059 (10µM; 6 hrs) did not alter either SET phosphorylation or PP2A activity (Figure 11C and 11D).

Reportedly, insulin as well as engagement/activation of the sphingosine-1-phosphate receptor 1 (S1PR1) enhances Jak2 activity, which can recruit PI-3K into a PP2A- containing complex thereby resulting in PP2A inhibition (342, 420, 421).

Figure 11C and 11D. Jak2V617F-driven PI-3Kγ/PKC-mediated signals induce inhibition of PP2A through SET phosphorylation. (C): Levels of serine phosphorylated SET (pSET) in SET immunoprecipitates (SET) from lysates of untreated, kinase (Jak2, PI-3K, PI-3Kg, PKC and MEK1) inhibitors- and FTY720-treated Ba/F3- Jak2V617F cells. (D): PP2A activity in untreated and drug-treated Ba/F3-Jak2V617F cells, and in Ba/F3-Jak2V617F cells transduced with empty vector or expressing a sphingosine-1- phosphate receptor 1 (S1PR1) shRNA and treated with 2.5µM FTY720. Inset: S1PR1 V617F levels in parental and S1PR1 shRNA-expressing Ba/F3-Jak2 cells.

Although Jak2V617F expression did not increase S1PR1 expression (Figure 11D, inset), to

rule out the possibility that suppression of PP2A in Jak2V617F-transformed cells depends

on Jak2V617F-dependent or –independent S1PR1 activation that, in turn, may suppress

PP2A activity through enhancement of wild type Jak2 signaling, S1PR1 shRNAs were

used to downregulate S1PR1 expression in Ba/F3-Jak2V617F cells (Figure 11D, inset).

Inhibition of S1PR1 expression did neither rescue PP2A activity nor halted FTY720

PP2A-activating function (Figure 11D), suggesting that Jak2V617F does not require

S1PR1-mediated signals to suppress PP2A activity and, likewise, the FTY720 PP2A-

activating function is independent on the expression of S1PR1 in Jak2V617F-transformed hematopoietic cells.

4.3 FTY720 long-term treatment increases survival and decreases leukemia allelic burden without any cardiologic toxicity.

Reportedly, PP2A-activating drugs (e.g. FTY720) have strong anti-leukemic activity and safety profile in animal models of different types of acute and chronic leukemias (78, 79,

422). To evaluate the potential of using PADs and, specifically, FTY720 in Jak2V617F

MPN patients, we used an aggressive mouse model of Jak2V617F leukemia in which each

transplanted cytokine-independent Ba/F3-Jak2V617F cell represents a potential leukemia-

initiating cell (423). SCID mice (n=30) were i.v. injected with Ba/F3-Jak2V617F cells (5 ×

105 cells/mouse). After 7 days all mice were engrafted (presence in PB of GFP+ Jak2V617F

cells) and FTY720 was i.p. administered (10 mg/kg/d; LD50 [50% lethal dose] = 300

mg/kg) to 15 mice. As controls, 10 age-matched mice only received daily treatment with

FTY720, whereas 15 cell-injected mice were left untreated. After 4 weeks of FTY720 treatment (5 weeks post-transplant), PB was collected by retro-orbital survival bleeding from untreated and FTY720-treated leukemic mice (n=3 per group) and used for assessing Jak2V617F allelic burden by SNaPshot assay. Reduction of the leukemic allelic

burden in FTY720-treated animals (Figure 12A, red box) was clearly evidenced by the significant reduction of their V617F burden (red curves) or by the almost sole expression of wild-type Jak2 allele (blue curves).

Figure 12A and 12B. In vivo anti-leukemic effects of FTY720 and lack of toxicity in long-term treated animals. (A): Jak2V617F leukemic burden at 6 weeks after Ba/F3-Jak2V617F cell-injection measured by SnapShot assay in untreated (grey) and FTY720 (red)-treated (5 weeks; 10mg/kg/d) mice. Age-matched mice (white) were used as a control. Levels of wild type and V617F Jak2 alleles are showed by the blue and red curves, respectively. (B): Average spleen weights of untreated or FTY720-treated age-matched and leukemic mice.

As expected, the Jak2 allelic status was nearly 100% mutated in cells from PB of

untreated leukemic animals (Figure 12A, grey box), and totally wild type in PB isolated

from an age-matched healthy control (Figure 12A, white box). Mice were thereafter

sacrificed and organs were evaluated by visual inspection and light microscopy. Mice

injected with Ba/F3-Jak2V617F cells (grey) showed massive splenomegaly (n=3/group;

P<0.05), whereas the morphology of spleens from FTY720-treated Ba/F3-Jak2V617F cell–

injected mice (red) resembled that of control age-matched (white) or FTY720-only

treated (green) mice (Figure 12B).

H&E-stained sections of spleen, bone marrow, liver and kidney of Ba/F3-Jak2V617F mice

treated with vehicle (saline solution) showed extensive infiltration of blast cells with a

low degree of maturation typical of an overt leukemia–like process (Figure 12C). In

contrast, spleen, bone marrow, liver and kidney from the FTY720-treated leukemic mice

were similar to that of the age-matched and FTY720-injected control groups (Figure

12C). Notably, cardiac tissue did not appear infiltrated by Ba/F3-Jak2V617F cells (Figure

12C).

Almost at the end of 7th week of treatment (8 post-transplant), 4 untreated animals were moribund and were sacrificed together with 4 FTY720-treated mice from the group and

organ subjected to histopathology analysis. Marked splenomegaly was observed in the

untreated animals while spleens of FTY720-treated Jak2V617F animals were similar to those of age-matched controls (not shown).

Figure 12C. In vivo anti-leukemic effects of FTY720 and lack of toxicity in long- term treated animals. H&E-stained sections of spleen, bone marrow, liver, heart and kidney of untreated (white) and FTY720-treated (green) age-matched control mice and, untreated (grey) and FTY720-treated (red) leukemic mice.

Consistent with these findings, survival of FTY720-treated mice injected with Ba/F3-

Jak2V617F cells was significantly increased with a median survival time of 10.4 weeks

(P<0.0001) and all were alive at 8 weeks post-transplant when untreated mice were all

succumbed (Figure 12D). By contrast, the median survival of mice injected with Ba/F3-

Jak2V617F cells only was 6.7 weeks (Figure 12D). As expected, 100% of control mice were still alive after 22 weeks of daily i.p. treatment with FTY720 (10 mg/kg)

Furthermore, no significant changes in body weight (not shown) and no signs of toxicity were observed in control or Ba/F3-Jak2V617F SCID mice that received 10 mg/kg FTY720 for 22 weeks.

Figure 12D. In vivo anti- leukemic effects of FTY720 and lack of toxicity in long- term treated animals. (D): Kaplan–Meier curve show survival of untreated leukemic (grey), FTY720-treated leukemic (red) and age-matched control (green) mice.

Because the FDA-approved FTY720 may in some cases mildly affect cardiac performance (424), we conducted an in-depth assessment of cardiac function in non- diseased animals long-term treated (10 weeks) with FTY720 (10mg/kg/d) compared to untreated control mice. Electrocardiography analysis showed that the shortening amplitude, a measurement of myocyte contraction, is not decreased following FTY720 treatment (Figure 12E, top). In addition, stimulation with isoproterenol (1µM) produced 87

the expected increase in amplitude, an affect that was unchanged following FTY720

treatment (Figure 12E, top). Likewise, relengthening time was no different in FTY720-

treatment regardless of isoproterenol stimulation (Figure 12E, top right). Calcium

transient, another measure of contractile function, was also unchanged in FTY720-treated

mice (Fig. 12E, middle). Finally, the heart rate of treated mice remained identical to that of untreated mice and bradycardia was completely absent (Figure 12E bottom).

Figure 12E. In vivo anti-leukemic effects of FTY720 and lack of toxicity in long-term treated animals. (E): Electrocardiographic measurements of untreated or FTY720-treated (10 weeks) control mice.

4.4 Conversion into FTY720-P is dispensable for FTY720 anti-leukemic activity

in Jak2V617F cells.

Bradycardia is one of the mild side effects due to the FTY720 converted by sphingosine kinase 2 (SPHK2) from its prodrug (unphosphorylated) into the phosphorylated immunosuppressant FTY720-P (425) (Figure 13A); however, FTY720 efficiently restores PP2A tumor suppressor activity in hematopoietic progenitors (Figure 13A)(79,

422).

Figure 13A. FTY720 phosphorylation is dispensable for its anti-leukemic activity on Jak2V617F cells. (A): Schematic representation of FTY720 conversion into FTY720-P and FTY720/PP2A interplay; inhibitors are indicated in black.

In fact, treatment of CD34+ PV progenitors (n=4) and Ba/F3-Jak2V617F cells with FTY720

(2.5µM, single dose) reduced the clonogenic potential of Ba/F3 Jak2V617F+ by 84% and

CD34+ PV patient samples by 62% (Figure 13B) without impairing the colony formation

of parental Ba/F3 cells (Figure 13B) and that of CD34+ NBM(79).

Figure 13B. FTY720 phosphorylation is dispensable for its anti-leukemic activity on Jak2V617F cells. (B): Left: Effect of FTY720 and its non-immunosuppressive derivatives (OSU-2S and FTY720-S-Regioisomer) on the clonogenic potential, expressed as percentage of colony forming cells (CFC), of Ba/F3, Ba/F3-Jak2V617F and/or primary CD34+ PV cells. Right top: Jak2 expression in vehicle- and FTY720-treated CD34+ progenitors derived from the bone marrow (BM) or peripheral blood (PB) of PV patients. Right bottom: Jak2 expression in Ba/F3-Jak2V617F cells treated with the PP2A activator FTY720 alone or in combination with okadaic acid used at concentration (0.25nM) that inhibits PP2A activity only.

Consistent with the effect of expression of ectopic HA-PP2Ac, SET shRNA and Jak2 shRNA (Figure 10), FTY720-induced decreased survival of Jak2V617F primary cells and cell lines corresponded to restoration of PP2A activity to the physiological levels detected in CD34+ NBM progenitors and parental Ba/F3 cells (Figure 13C), and to downregulation of Jak2V617F expression in both primary PV CD34+ BM and PB progenitors and Ba/F3-Jak2V617F cells (Figure 13B, right). Notably, dependence of

Jak2V617F downregulation on PP2A reactivation was counteracted by co-treatment with

okadaic acid (0.25nM) used at a concentration that specifically inhibits PP2A

phosphatase activity only (90) (Figure 13B). To assess whether PP2A activation by

FTY720 results from intracellular reconversion of phosphorylated (immunosuppressive)

FTY720 into its non-phosphorylated native form, primary CD34+ PV progenitors (n=3),

HEL and Ba/F3-Jak2V617F cells were treated for 6 hours with a synthetically

phosphorylated FTY720 (FTY720-P). FTY720-P neither restored PP2A activity (Figure

13C, yellow bars) nor triggered Jak2V617F downregulation (Figure 13C, right – lane 4).

Accordingly, co-treatment with the sphingosine kinase inhibitor DMS (2.5µM), which prevents the de novo conversion of FTY720 to FTY720-P, did not antagonized the ability of FTY720 to restore PP2A activity in Ba/F3-Jak2V617F and HEL cells (Figure

13C, green bars). Indeed, DMS potentiated the loss of Jak2V617F protein induced by

FTY720 treatment (Figure 13C, right panel – lane 3) and greatly enhanced the FTY720

(500 nM)-induced killing of Ba/F3-Jak2V617F cells (Figure 13C, bottom right – compare

red and green lines) likely by preventing the conversion (inactivation) of FTY720 to

FTY720-P and effectively increasing the amount of active compound. As expected(79),

parental Ba/F3 cells continued to proliferate when exposed to 500 nM FTY720 (Figure

13C, bottom right – blue line).

Figure 13C. FTY720 phosphorylation is dispensable for its anti-leukemic activity on Jak2V617F cells. (C): Left: PP2A activity in Ba/F3-Jak2V617F, HEL, primary CD34+ NBM and PV cells untreated and/or treated with FTY720 (2.5µM) alone or in combination with the sphingosine kinase inhibitor DMS (2.5µM), the immunosuppressive and S1PR1 agonist FTY720-P (2.5µM), or with the non- immunosuppressive FTY720 derivatives (FTY720-S-regioisomer and OSU-2S). Right top: Jak2 protein levels in HEL cells untreated and treated with FTY720 (2.5µM) alone or in combination with DMS (2.5µM), or treated with FTY720-P (2.5µM). Left bottom: Proliferation of Ba/F3-Jak2V617F cells (or Ba/F3-MigR1 used as a control) untreated and/or treated with FTY720 (500nM) alone or in combination with DMS (500nM).

To further demonstrate that conversion to FTY720-P is unnecessary for the FTY720 anti- tumor activity, Ba/F3-Jak2V617F cells were treated with two FTY720 derivatives (S-

FTY720-regioisomer(426) and OSU-2S(427)) that neither interact with S1PR1 nor

undergo SPHK2-dependent phosphorylation in myeloid progenitors, and=induce neither

B- nor T-cell lymphopenia (Neviani et. al. manuscript submitted 2013)(428, 429). Both

non-phosphorylatable FTY720 derivatives restored PP2A activity, decreased Jak2V617F

levels and impaired clonogenic potential of Ba/F3-Jak2V617F but not that of parental

Ba/F3 cells (Figure 13B and 13C).

4.5 FTY720-P (immunosuppressive) augments Jak2 and suppresses PP2A activities.

Although FTY720-P does not rescue PP2A and downregulation of S1PR1, a receptor mediating FTY720-P immunosuppressive activity on mature B- and T-cells (392, 430), does not prevent PP2A activation by FTY720 in Jak2V617F-transformed hematopoietic

progenitors (Figure 11), it is unclear whether the interaction between extracellular

FTY720-P and S1PR1 elicits signals that synergize or antagonize with the PP2A- dependent FTY720 anti-tumor activity. By using non-transformed hematopoietic Ba/F3

progenitors and their derivative Ba/F3-Jak2V617F cells, expressing both wild type and mutated Jak2 proteins, we found that FTY720-P suppresses PP2A through a mechanism that closely parallels that of oncogenic Jak2V617F (Figure 14A).

In fact, treatment of Ba/F3 cells with FTY720-P (2.5 µM; 6 hrs.) strongly increased levels of Y972 phosphorylated (active) Jak2 (Figure 14B, lane 4) and suppressed PP2A

activity (~80% reduction) (Figure 14C, lane 5).

Figure 14A and 14B. FTY720-P promotes Jak2 and suppresses PP2A activities. (A): Schematic representation of the FTY720-P-induced and SIPR1/Jak2 (wild type and V617F)- mediated PP2A inhibition. (B): Western blots show levels of active Jak2 (pY972) and Grb2 in parental Ba/F3 and Ba/F3-Jak2V617F cells untreated and treated with the S1PR1 agonists FTY720-P (2.5 µM), SEW 2871 (10µM) and S1P (1µM), and with the PP2A activators FTY720 (2.5µM), OSU-2S (2.5µM) and (S)-FTY720-regioisomer (2.5µM), respectively.

Likewise, Jak2 was strongly activated whereas PP2A activity was strongly inhibited upon exposure of Ba/F3 cells to the natural S1PR1-5 ligand S1P (1µM; 6 hrs.) and to the

S1PR1-specific agonist SEW 2871 (10µM, 6 hrs.). Furthermore, shRNA-mediated

S1PR1 downregulation blunted the effect of FTY720-P on PP2A (Figure 14C, lane 6), whereas treatment with the FTY720 derivatives OSU-2S and (S)-FTY720-regioisomer

(2.5 µM; 6 hrs.), which do not undergo phosphorylation and do not interact with S1PR1, did not enhance but, like FTY720, marked inhibited levels of active Jak2 in Ba/F3-

Jak2V617F cells (Figure 14B, lanes 5-8).

Interestingly, rescued PP2A activity to levels similar to those found in parental Ba/F3 cells was observed upon co-treatment of Ba/F3 cells with FTY720-P and, the Jak

inhibitors AG490 (not shown) and TG101348 (1µM, Figure 14C lane 7), PI-3K and PI-

3Kγ inhibitors LY294002 (not shown) and AS-604850 (1µM, Figure 14C lane 8), respectively, or the conventional PKC inhibitor PKC-412 (2µM; Figure 14C lane 9), suggesting that S1PR1 agonists like FTY720-P induce signals that activate the Jak2 (wild type)/PI-3Kγ/PKC pathways that, in turn, will lead to inhibition of PP2A (tumor suppressor) activity.

Figure 14C. FTY720-P promotes Jak2 and suppresses PP2A activities (C): PP2A activity in Ba/F3 cells untreated and treated with FTY720 (2.5µM), SEW 2871 (10µM), S1P (1µM), FTY720-P (2.5µM) and FTY720-P in combination with S1PR1 shRNA, TG101348 (1µM), AS-604850 (1µM) or PKC-412 (2µM).

Consistent with the existence of a S1PR1-dependent and Jak2 wild type-mediated PP2A inhibitory pathway in addition to that initiated by Jak2V617F oncogenic kinase activity, expression of Jak2V617F in cytokine-dependent TF-1 erythroleukemia cells markedly enhanced mRNA and protein levels of all SPHK1 isoforms (1A, 1B and 1C) (Figure

14D, left and middle panels). Conversely, levels of the FTY720 conversion kinase,

SPHK2, remained unchanged upon ectopic Jak2V617F expression (Figure 14D, left).

Figure 14D and 14E. FTY720-P promotes Jak2 and suppresses PP2A activities. (D): Left: Effect of Jak2V617F expression on the mRNA levels (qRT-PCR) of specific SPHK1/2 isoforms in TF-1 cells. Fold change are relative to levels of SPHK1 and SHPK2 isoforms in parental TF-1 cells. Middle top: Effect of Jak2V617F expression on sphingosine kinase 1 (SPHK1) expression in TF-1 cells. Middle bottom: SPHK1 kinase activity measured as fold change in the amount of sphingosine converted to sphingosine-1-phosphate (S1P) in Ba/F3-Jak2V617F cells untreated and treated with FTY720 (2.5µM) alone or in combination with okadaic acid (0.25 nM); Right: PP2A activity in Ba/F3 cells and in parental and S1PR1 shRNA-expressing Ba/F3-Jak2V617F cells left untreated or treated with the S1PR1 antagonist VPC 23019.

Interestingly TLC-based SPHK1 assay showed that FTY720 (2.5µM) treatment of Ba/F3-

Jak2V617F cells impaired SPHK1 activity (decreased sphingosine to S1P conversion) in a

PP2A-dependent manner as co-treatment with 0.25nM okadaic acid antagonized the effect of FTY720 (Figure 14D, middle lower panel). Accordingly, S1PR1 inhibition, achieved by exposure of Ba/F3-Jak2V617F cells to the inverse S1PR1/3 agonist VPC

23019 (10µM), efficiently rescued PP2A activity (Figure 14D, right). Thus, restoration

of PP2A activity by FTY720 also prevents S1P production thereby decreasing the

negative effect of S1PR1 signaling on PP2A.

4.6 FTY720 sequesters SET and activates PP2A in the absence of SET

dimerization or induction of ceramide.

In solid tumors, the pro-apoptotic sphingolipid ceramide has recently been shown to

activate PP2A (183) through binding directly to SET lysine 209 (K209) on helix 7 (182,

431); thus, we evaluated whether FTY720 treatment of hematopoietic cells induces

activation of PP2A by directly interacting with SET. Ba/F3 cells were transfected with

wild type and the K209D SET mutant and assayed for PP2A activity after treatment with

either 2.5 µM FTY720 or vehicle. Cells expressing the mutated SET had PP2A activity

suppressed to a similar degree as cells transfected with wild-type SET (Figure 15A). As

expected, overexpression of SET suppressed PP2A activity in Ba/F3 cells (Figure 15A);

however, phosphatase activity was efficiently restored by FTY720 treatment (Figure

15A). By contrast, FTY720 was unable to rescue PP2A activity in cells expressing the 97

SETK209D mutant, suggesting that FTY720, like ceramide, requires interaction with SET lysine 209 for activating PP2A.

To further confirm that FTY720-SET interaction is occurring in hematopoietic cells, anti-

NBD (a fluorochrome used as an affinity tag here) immunoprecipitations and monomeric avidin-based chromatography were performed upon treatment of Ba/F3-Jak2V617F cells with FTY720-phenoxy-NBD and FTY720-phenoxy-biotin, respectively (Figure 15B, left). Note that, both FTY720 conjugates were still capable of inducing PP2A activity in of Ba/F3-Jak2V617F cells (Figure 15B, right).

Figure 15A and 15B. FTY720-dependent PP2A activation in myeloid cells depends on the SET K209-FTY720 interaction but not on SET dimerization or increased ceramide levels. (A): PP2A activity in untreated and FTY720-treated Ba/F3 cells expressing WT or K209D SET proteins. (B): Left: pull-down assays demonstrate FTY720-SET interaction in NBD immunoprecipitates of Ba/F3-Jak2V617F cell lysates treated with NBD-conjugated FTY720 (top), and in avidin-mediated affinity chromatography with lysates of Ba/F3-Jak2V617F cells treated with biotin-tagged FTY720. Right: PP2A activity in Ba/F3-Jak2V617F cells treated with FTY720 (2.5µM), FTY720-NBD (2.5µM) and FTY720-biotin (2.5µM).

Anti-SET western blots clearly showed association of SET with either of the two

FTY720 molecules (Figure 15B, lanes 3). As expected, SET was detected neither in anti-

IgG immunoprecipitates nor in chromatography with lysates of cells treated with non- conjugated FTY720 (Figure 15B, lane 2). Because FTY720 increases ceramide levels in specific culture conditions (432), and can displace SET from binding to ceramide (not shown), we investigated whether FTY720 altered gross ceramide levels and, specifically, the C18 ceramide (Figure 15C, yellow bar) that, reportedly, is responsible for PP2A induction. DAG kinase/TLC assay showed that exposure to FTY720 did not alter gross ceramide levels in Ba/F3-Jak2V617F cells (Figure 15C, top).

Figure 15C and 15D. FTY720-dependent PP2A activation depends on the SET K209- FTY720 interaction but not on SET dimerization or increased ceramide levels. (C): Left: PP2A activity in parental and, WT or non-dimerizing (TAF-1β PME mutant) SET-expressing Ba/F3 cells. Right: GFP-SET levels in Flag-immunoprecipitates from lysates of Ba/F3-Flag- SET co-incubated with equal amount of Ba/F3-GFP-SET cell lysates in the presence of DMSO or FTY720. (D): Top: DAG kinase assay shows gross ceramide levels in untreated and FTY720-treated Ba/F3-Jak2V617F cells. Bottom: LC-MS/MS measurement of specific ceramide levels in FTY720-treated Ba/F3-Jak2V617F cells expressed as percentage of those in untreated cells. The ceramide C18 able to induce PP2A activation is depicted in yellow. 99

Likewise, highly sensitive LC/MS indicated that FTY720 did not induce changes in

levels of specific ceramide species (Figure 15C, bottom), further suggesting that ceramide is not responsible for induction of PP2A in FTY720-treated Ba/F3-Jak2V617F

cells. Finally, anti-GFP immunoblot on anti-Flag immunoprecipitates from vehicle

(DMSO)- and FTY720-treated Ba/F3-Flag-SET cell lysate co-incubated with Ba/F3-

GFP-SET lysates (1:1) showed that FTY720 decreased SET dimerization (Figure 15D, right), suggesting that SET binds FTY720 as a single molecule. Moreover, a dimerization-deficient SET (TAF-1β PME) (175) inhibited PP2A as well as wild type

SET (Figure 15D, left), indicating that its dimerization is not required to suppress PP2A.

100

CHAPTER 5

Discussion

PP2A tumor suppressor activity plays a pivotal role in cancer emergence, development

and progression (422). In strong similarity with BCR-ABL1+ leukemias (78) and in agreement with the notion that higher but not lower levels of Jak2V617F circumvents the requirement of Epo-receptor for transformation of myeloid progenitors (433), we showed here that PP2A activity is impaired in CD34+ MPN progenitors expressing the Jak2V617F

oncogene, and that inhibition of PP2A phosphatase activity through tyrosine 307

phosphorylation occurs in a oncogene dose- and kinase-dependent manner and is mediated by the PP2A inhibitor SET.

The possibility that Jak2V617F can directly phosphorylate PP2A is supported by the fact

that IL-3-stimulation induces Jak2-PP2A association and tyrosine phosphorylation of

PP2A in 32Dcl3 myeloid cells (83), and by our data indicating that the Jak2V617F-driven

PI-3Kγ/PKC activation is responsible for PKC-dependent SET activation. This is not unexpected if we consider that in other cell types SET was already suggested to be a PKC substrate (160), and that Jak2 associates and activates PI-3K that, in turn, recruits PP2A and induces canonical PKC activation via PIP3 production (421, 434). In this scenario,

SET might work as scaffold protein recruiting the Jak2V617F-containing complex to PP2A.

Seemingly, we previously reported that Jak2 is recruited by BCR-ABL1 to mediate SET-

dependent PP2A inactivation (166). To ensure that SET is the primary inhibitor of PP2A

in Jak2V617Fcells, we also targeted the related PP2A inhibitor I1PP2A (inhibitor 1 of 101

PP2A) by administering DMS and sphingosine that, as reported (136), target this inhibitor and restore PP2A activity when it is repressed by I1PP2A. Treatment of Ba/F3-

Jak2V617F+ cells with these compounds failed to induce PP2A activity (not shown),

indicating that, I1PP2A is unlikely to play a role in Jak2V617F-induced inhibition of PP2A.

Inhibition of PP2A in MPNs appears to be essential for leukemogenesis; in fact, we show

that genetic (ectopic PP2Ac or SET-shRNA expression) and/or pharmacologic (FTY720,

S-FTY720-regioisomer, and OSU-2S) reactivation of PP2A results in

inactivation/downregulation of Jak2V617F , reduced clonogenic potential of PV progenitors

and Jak2V617F cell lines, and impaired in vivo leukemogenesis (e.g. decreased Jak2V617F

allelic burden and normal splenic morphology) and significantly increased animal

survival. Interestingly, the importance of PP2A in the regulation of Jak2V617F oncogenic

network is further supported by the evidence that FTY720 and/or its non-

immunosuppressive derivatives also selectively suppresses, in a PP2A-dependent, BCR-

ABL1 kinase-independent and Jak2-mediated manner, the survival and self-renewal of

TKI-resistant CML but not normal quiescent HSCs both in vitro and in serial BM transplantation assays (Neviani et al., manuscript submitted 2013)(426-429). Moreover, as previously observed (79, 422), administration of pharmacologic doses of FTY720 had neither unexpected adverse effects on normal hematopoiesis nor toxicity in non- hematopoietic organs. In this regard, FTY720-treated animal had normal cardiac cell function, consistent with the notion that a clinically manageable bradycardia and atrioventricular conduction block are only observed in MS patients at the time of FTY720 102

therapy initiation (435), and depend on the interaction between the immunosuppressive

FTY720-P and S1PR1 (436, 437).

Independent from phosphorylation is instead the in vitro and in vivo FTY720 anti-

leukemic activity against Jak2V617F MPN cells; in fact, in analogy with the effects of

SPHK2 modulation and FTY720-P treatment in CML and KitD816V AML cells (79, 438),

the S1PR1 agonist FTY720-P did neither induce PP2A activity nor antagonize Jak2V617F

or Jak2V617F-driven cell proliferation. Conversely, we showed that FTY720 non- immunosuppressive derivatives (OSU-2S and S-FTY720-regioisomer), which do not interact with S1PR1 (427, 429), efficiently downregulated Jak2V617F kinase activity. This

is of high importance as exposure of hematopoietic precursors to S1PR1 agonists,

including the endogenous S1P and the immunomodulator FTY720-P, led to inhibition of

PP2A tumor suppressor activity. In addition, Jak2 phosphorylation on Y972 and

activation was increased in Ba/F3 treated with S1PR1 agonists, including FTY720-P,

despite the notion that S1P receptors are G-protein coupled receptors (GPCR) that

typically do not signal through receptor-bound kinases such as Jak2 (439). This is not

surprising as Jak2 Y972 was also found phosphorylated in response to Jak2 activation by

another GPCR (440). In this scenario, it is not surprising that an inverse modulation of

SHPK1 was observed in response to FTY720 treatment and Jak2V617F expression. In fact, in accordance with previous research (441), not only SPHK1 activity was decreased following PP2A activation by FTY720 and increased by Jak2V617F, but the anti-apoptotic

S1P (442), product of SPHK1 activity, also inhibits PP2A upon triggering the S1PR1- 103

Jak2-Pi-3Kγ/PKC-SET pathway in non-transformed and Jak2V617F hematopoietic

precursors. This seems not to be the case for the FTY720 conversion enzyme, SPHK2,

the levels of which remained unaltered by Jak2V617F expression. However,

LC/ESI/MS/MS analysis indicated that most of the intracellular FTY720 given to

myeloid precursors remains or returns into a non-phosphorylated state (Neviani et al., manuscript submitted 2013), suggesting that the predominant effect of FTY720 is to reactivate PP2A in malignant hematopoietic progenitors. Conversely, FTY720 in its phosphorylated form seems to act primarily as a S1PR1 agonist only in mature lymphocytes to induce immunosuppression (430). In spite of this, our data indicate that

S1PR1 agonists suppress PP2A also in hematopoietic progenitors; thus, the limiting factor for a wide use of FTY720 as an anti-leukemia agent depends on the levels and activity of SPHK2 that, if aberrantly elevated, can antagonize FTY720 anti-cancer activity.

Mechanistically, we showed that the activity of FTY720 as a PAD in Jak2V617F MPNs depends on the sequestration of SET through interaction with SET K209 but not on SET dimerization or induction of ceramide. SET dimerization has been shown to be critical for

SET-enhanced DNA replication (175) but not for PP2A inhibition in a cell-free system

(184); thus it is likely that the PP2A inhibitory effects of SET are independent of its nuclear role, and that the importance of SET serine phosphorylation is limited to determining its cytoplasmic localization and ability to interact and inhibit cytoplasmic

PP2A that, otherwise, would suppress Jak2V617F leukemogenic activity. Because PP2A 104

has a pleiotropic anti-cancer activity (422), our data suggest that the use of FTY720 and, better, other PADs lacking S1PR1 agonist activity (Figure 16) might be used together with TKIs in the treatment of Jak2V617F+ MPNs and other malignancies (e.g. Ph+ leukemias, Kit-mutated AML, etc (422)) characterized by inactivation of the PP2A tumor suppressor.

Figure 16. Antagonizing effect of FTY720 and FTY720-P on leukemogenesis. Schematic representation on the effect of PP2A-activating drugs (PADs, e.g. FTY720) and Sphingosine-1- phosphate-receptor-1 agonists (FTY720-P) on leukemogenesis through the opposite effect on the interplay between oncogenic kinase (e.g. Jak2V617F and BCR- ABL1) signaling and tumor suppressor (i.e. PP2A) activity.

105

CHAPTER 6

Future Directions

Our continuing projects all build on our successes achieved so far with the

targeting of the SET/PP2A interplay through the use of PADs (79) (e.g. FTY720) as antileukemic agents. We are embarking on a two-tiered project designed to produce new

PADs and, specifically, non-immunosuppressive FTY720 analogs and novel SET inhibitors. We expect that the successful completion of these projects will facilitate the introduction of PADs in therapeutic protocols for Ph+ and Jak2V617F+ MPNs, CML-BC,

Ph+ B-ALL, and all other cancers characterized by functional inactivation of the PP2A

tumor suppressor through a SET-dependent mechanism.

We have learned that FTY720 works in a manner independent of the mechanism by which FTY720-P operates. While the immunosuppressive FTY720-P goes through a two-step process involving the activity SPHK2 and S1PR1, the antileukemic FTY720

interacts directly with SET with no known involvement of SPHK2 or S1PR1. In fact, the

limitations of using a FTY720-P are not restricted to its immunosuppressive action but

also include the PP2A inhibitory effects which also rely on the triggering of the

SPHK2/S1PR1 axis. This knowledge provides us with an opportunity to tease apart these

two functions and focus on only the antileukemic properties of FTY720. With this in

mind we are embarking on finding new and more potent FTY720 derivatives that retain

the PP2A activating effects of FTY720 but lack receptor activity and, therefore, are

unable to induce S1PR1-mediated adverse effects (e.g. bradychardia). 106

For this project we are taking a two-tiered approach. The first segment of this

program, which is already underway, is centered on FTY720 as a lead compound. The

structure of FTY720 not only provides the compound with affinity to SET but also gives

the chemical “drug like” properties that make it therapeutically relevant (e.g. low

molecular weight, LogP, etc.). By maintaining the core structure of FTY720 we hope to

retain the favorable properties of the compound while eliminating receptor-mediated

immunosuppressive and adverse effects. A side benefit of this project is the possibility of

developing a compound that can be manufactured more efficiently than the parental

FTY720 thus reducing costs and increasing patient access. To begin this project, we have

been testing analogs of FTY720 to evaluate them for the ability to activate PP2A,

selective toxicity towards leukemia cells, and immunosuppression.

Thus far, we have been able to demonstrate the potential for some of these compounds.

We currently have three analogs that have been verified to be anti-leukemogenic without producing immunosuppression. As shown in Figure 17A and 17D, each of these compounds activates PP2A similarly to the parental FTY720. We used two methods for determining the immunosuppressive ability of these new compounds. Because FTY720-P induces immunosuppression through internalization of the S1PR1, we used a GFP-tagged

S1PR1 receptor and confocal microscopy to follow the localization of the receptor following exposure to the FTY720 derivatives. The receptor is normally found on the cell surface in untreated cells (Figure 17B, far left) but becomes internalized upon binding of

FTY720-P (Figure 17B, 2nd panel). 107

Figure 17. Novel non-immunosuppressive FTY720 derivatives. (A) PP2A activity in cells treated with three FTY720 analogs demonstrating similar ability to activate PP2A in CML cells. (B) Confocal microscopy showing internalization of a GFP-tagged S1PR1 upon treatment with immunosuppressive FTY720-P but not with three FTY720 analogs. (C) Flow cytometric data demonstrating the effect of FTY720 and its derivatives on circulating B220+/CD19+ B-cells (D) PP2A activation (left) and colony forming assay (right) in Ba/F3 Jak2V617F cells treated with FTY20 and its non-immunosuppressive derivatives.

108

This internalization is expected to happen upon binding with any ligands for this receptor therefore we used the GFP-S1PR1 as an indirect indicator of the immunosuppressive potential of our test compounds. As shown (Figure 17B, panels 2-4), none of the

FTY720 analogs induce receptor internalization suggesting that they are not immunosuppressive. To confirm the loss of immunosuppressive activity, , FVB/N mice were treated with a single dose (10mg/kg; i.p.) of FTY720 and of its dervivatives (OSU-

2S, S-FTY720-Me and S-FTY720-regioisomer). FTY720 demonstrated a significant reduction in circulating B220+/CD19+ lymphocytes whereas mice given any of the

FTY720 analogs at the same dose showed no change in circulating B220+/CD19+ B-cells

(Figure 17C). Similar effects were noted on circulating T-cells (not shown).

Thus, to find other SET interacting PADs, we will screen additional compounds in a similar manner: checking for PP2A activation, evaluating receptor activity, and determining their effect on circulating B- and T-cells in vivo. Compounds that activate

PP2A but lack receptor/immunesuppressive function will undergo studied in more detail.

These experiments will be aimed at determining binding of the selected compounds to

SET through the use of affinity chromatography-based binding assays, and by PP2A assays performed on lysate of cells expressing a mutated forms of SET that are unable to bind FTY720.. It is important to focus on compounds that target the ability of SET to interact and inhibit PP2A. Importantly, these compounds might also have strong selectivity toward cancer cells as SET has been described as overexpressed only in tumor 109

tissues . We will then determine essential functional properties of the compounds including selective killing, toxicity, and half-life. The most favorable compounds, as determined by all of the work to this point, will then be moved into more rigorous preclinical testing on primary patient samples and in in vivo mouse models. We expect that by the end of this project to have one or more FTY720-like compounds with robust PP2A activating potential and lacking the immunosuppressive effects of FTY720. This project, by expanding on a known compound, has the potential to produce novel, effective drugs that may undergo further pharmacokinetic and pharmacodynamics studies within a relatively short time frame. A more comprehensive program is developed in the second tier of this project (Figure 18).

Figure 18. SET inhibitors development strategy. An initial screening will be performed using a database of approximately 1 million compounds. Molecules were selected for drug-like properties and docked within helix 7 of SET. Positive “hits” will be screened for inducing cancer cell death and PP2A activation. Compounds that not meet these criteria will be excluded from further study. Remaining compounds will be assayed for direct binding to SET.

110

The second tier of this project builds upon out data regarding FTY720/SET

binding. Here, we will begin with a ground-up approach starting with an entirely new,

FTY720 unrelated leads rather than modifying a known SET-interacting drugs (e.g.

FTY720).. Following the flow chart outlined in Figure 18, an in silico screen was

performed first.

Two libraries of commercially available compounds from Maybridge and Bionet were

docked onto SET. Because SET forms a dimer both in solution and in vivo to form a

“headphone” shape (170), only one of the two components of this dimer has an “open”

conformation that allows for the binding of small molecules; thus, only this conformation

was used for docking.

Figure 19. Molecular docking of FTY720 and SET. A computational model was used to bind FTY720 (green) to the open confirmation of SET. Several residues were found to be important for FTY720 to bind including several glutamic acid (E) residues. Phenylalanine (F) 68 and arginine (R) 64 produce a hydrophobic pocket.

111

SET amino acid residues found to be important for the binding of FTY720 were E57,

E114, E116 and K209 (Figure 19). F68 and R64, while not important for the FTY720

pharmacophore binding, produced a hydrophobic pocket into which the lipid tail of

FTY720 would be positioned (Figure 19).

The initial drug screening started with approximately 1 million compounds. Compounds

that were found to have high affinity when docked within the helix 7 of SET, and those that possess drug-like properties have been selected for further testing.

We initially expected 100-200 compounds to be selected by the in silico screen; however,

only approximately 80 compounds met the criteria for both binding and drug-like

properties. Each of the ~80 compounds was tested for ability to activate PP2A in BCR-

ABL1+ cell lines (Figure 20).

Compounds that activated PP2A at a concentration of 2.5µM (concentration used for

+ FTY720) and that displayed an EC50 ≤10µM in the BCR-ABL1 cell line K562 (data not

shown) were then assayed for SET binding.

SET binding assays consisted of incubating cell lysates with the test compound and FTY720-biotin. Following incubation the mixture is passed through a monomeric avidin column. A reduction in the amount of SET found in the lysate upon SET immunoblotting, indicates direct binding between the test compound and SET. This will also ensure that the drug-induced PP2A activation is not produced through other pathways. Furthermore, during the molecular docking process we identified, in addition to the SET K209, other residues within the helix 7 that may play a role in ligand binding. 112

We have produced viral expression vectors containing a mutated version (glutamic acid to alanine) of several of the residues (e.g. E57, E114, and E116).

Figure 20. Screening for Novel SET Inhibitors. (A) Representative PP2A activity assay demonstrating a mixture of positive and negative “hits” from 80 test compounds selected by in silico screen. (B) Selected compounds that possess PP2A activating function were assayed for SET binding by competing with FTY720-Biotin.

113

Cells expressing these mutant SETs will allow us to determine which residues are

critical for ligand binding. This will enable us to refine our array of lead compounds and, likely, to increase affinity. For this purpose, we will use non-transformed Ba/F3

lymphoblasts cells expressing a specific SET mutant. These cells have reduced PP2A

activity through an increase in total SET levels alone, and they do not express a mutated

tyrosine kinase or other factor that could inhibit PP2A independently of SET. We will

treat these cells with the compound of interest and measure PP2A activity. Each tested

SET mutant cell line will be compared to cells overexpressing wild-type SET. The

importance of a residue is established when the PP2A response is reduced upon

expression of a specific SET mutant.

We are confident that we will succeed in finding new SET-interacting drugs that possess

the ability to reactivate the PP2A tumor suppressor as it is clearly emerging from several

studies that there is an obvious advantage of using PADs in clinic because of their

potentially high therapeutic index, as they selectively activate PP2A in leukemic cells

without adverse effects on normal hematopoiesis.

114

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