Thesis Written by Shorog Al Omair B.S., University of Dmmam, 2010 M.S

Thesis written by

Shorog Al Omair

B.S., University of Dmmam, 2010

M.S., Kent State University, 2015

Approved by

Gail Fraizer, Associate Professor, Ph.D., Masters Advisor, School of Biomedical Sciences

Ernest J. Freeman, Ph.D., Director, School of Biomedical Sciences

James Blank, Ph.D., Dean, College of Arts and Sciences

Regulators of VEGF-a major isoforms in leukemia

A thesis submitted

To Kent State University in partial

Fulfillment of the requirements for the

Degree of Master of Science

Shorog Al Omair

July 2015

Except for previously published materials

TABLE OF CONTENTS

TABLE OF CONTENTS………………………………………………………………………………….… iii

LIST OF FIGURES……………………………………………………………………………...………….… iv

LIST OF TABLES……………………………………………………………………………………………… v

LIST OF ABBREVIATIONS…………………………………………………………………………….… vi

ACKNOWLEDGMENTS………………….………………………………………………...………...….. viii

I. INTRODUCTION…………………………………………………………………….………1

II. METHODOLOGY………………………………………………………….…………… 24

III. RESULTS…………………..………………………………………………………………29

IV. DISCUSSION……………...………………………………………………....….………..45

V. FUTURE DIRECTIONS……………...... ………………...………………..………….52

VI. REFERENCES……………...……………………………...………………..….…….….55

iii LIST OF FIGURES

Figure 1. Alternative splicing regulatory sequences and splicing factors………...... ….7

Figure 2. The SR proteins shuttle between the cytoplasm and the nucleus……………12

Figure 3. WT1 structure.…………….….….……………………………………………………….....….14

Figure 4. Major VEGF-a isoforms.…………….….….…..……….………………....……………..….18

Figure 5. Characterization of VEGF-a major isoforms in K562 cells...... 32

Figure 6. WT1 inhibited VEGF121 without reducing VEGF165 in K562 cells……...…33

Figure 7. SRPK expression levels were not altered by WT1 transfection…………....34

Figure 8. SRPIN340 reduced VEGF-a isoforms in leukemia cells…………………..……….37

Figure 9. SRPIN340 significantly reduced growth of U937 leukemia cells ...... 38

Figure 10. EGF up-regulated VEGF-a in K562 leukemia cells…………………………...……40

Figure 11. SRPIN340 attenuated EGF-mediated induction of VEGF-a isoforms…..42

Figure 12. SRPIN340 reduces the nuclear intensity of SR proteins in K562………..44

Figure 13. Proposed model of EGF/SRPK1/SR pathway regulating VEGF isoform expression ………………………………………………………………………………………….54

iv LIST OF TABLES

Table 1. WHO classification of hematopoietic tumors of non-lymphoid origin…….…..3

Table 2. WHO classification of tumors of lymphoid origin.…………..……………….…….…..4

Table 3. The nomenclature of the 12 SR splicing factors.……………………..……...... …….8

v LIST OF ABBREVIATIONS

ALL Acute Lymphocytic Leukemia

AML Acute Myeloid Leukemia

AS Alternative Splicing

CML Chronic Myeloid Leukemia

Clk/sty CDC-like kinase

CLL Chronic Lymphocytic leukemia

ECM Extra Cellular Matrix

EGF Epidermal Growth Factor

EGFR Epidermal Growth Factor Receptor

ESE Exonic splicing Enhancer

ESS Exonic splicing suppressor hnRNPs Heterogeneous Nuclear Ribonucleoparticles

ISE Intronic Splicing Enhancer

ISS Intronic Splicing Suppressor

vi MDS Myelodysplastic Syndrome

NRPs Neuropilins snRNPs Small Nuclear Ribonucleoproteins

SR Serine-Arginine-Rich

SRPK Serine-Arginine Protein Kinases

SRSF Serine-Arginine-Rich Splicing Factor

RRMs RNA Recognition Motif

VEGF-a Vascular Endothelial Growth Factor-a

VEGFR Vascular Endothelial Growth Factor Receptor

WHO World Health Organization

vii ACKNOWLEGEMENT

The completion of this thesis would not have been possible without the support of many peoples. First, I would like to express my deep appreciation and gratitude to my advisor, Dr. Gail Fraizer, for her continuous guidance, motivation, and support. I value everything I learned from her about all aspects of scientific research. Her advice, and editorial comments guided me through during the process of writing the thesis.

I would like to thank my committee members, Dr. Gary Koski, and Dr. Steven

Kuerbitz for their time, advice, and suggestions that helped improve this work.

I would also like to thank all members of our lab for their cooperation and teamwork. I would like to thank, Sony Pandey, for her mentoring, and contribution with experiments.

The support of my family was essential for the successful completion of this work. I would like to thank my parents for believing in me, and raising me to think beyond social stereotypes. Finally, I would like to express my endless gratitude to my lovely husband, Abdulaziz, for his endless support, and encouragement during this challenging journey.

viii

CHAPTER 1

Introduction

Overview of hematologic malignancies

Blood cells are formed in the bone marrow in a physiological, developmental process called hematopoiesis. Irregularities in hematopoiesis lead to uncontrolled growth of a subset of blood cells resulting in leukemia (Kumar, Abbas, & Aster,

2014). Leukemia refers to a heterogeneous group of hematologic malignancies.

Leukemia is classified into chronic and acute leukemia and further classified based on cell lineage into myeloid and lymphoid. There are four major four types of leukemia: chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL).(Jones, 2010)

The cytogenetic translocations, or alterations, that are linked to the etiology of a subset of leukemia, are commonly used to categorize that subset. One example is chronic myeloid leukemia that is caused by the Philadelphia chromosome translocation (Bcr-abl t(9;22))(Jones, 2010). Lymphoma is another neoplastic disorder of lymphatic cells that originates in the lymphoid system. However, there is a fine line that separates lymphoma from leukemia because it can disseminate into the bone marrow and present a leukemia-like picture (Kumar et al., 2014). More detailed classifications of myeloid and lymphoid leukemia are described in the 2008

1 World Health Organization (WHO) classification (Table.1 and 2)(Jones, 2010).

Discoveries of molecular mechanisms contributing to leukemia have helped in the advances in leukemia therapeutic approaches and the increase in survival rate.

(Kimura, Ando, & Kojima, 2014) However, more work needs to be done to understand the underlying molecular mechanisms contributing to leukemia.

2 Table.1

2008 WHO classification of hematopoietic tumors of nonlymphoid origin. adapted from (Jones, 2010)

3 Table.2

2008 WHO classification of tumors of lymphoid origin, adapted from (Jones, 2010).

4 Pre-mRNA splicing:

The numbers of genes encoded in the human genome does not reflect the complexity and diversity of proteins expressed. One explanation for such diversity is the tightly regulated process of Alternative Splicing (AS)(Graveley, 2001). There are two types of pre-mRNA splicing, the regulated alternative pre-mRNA splicing and the constitutive pre-mRNA splicing (Black, 2003; Kornblihtt et al., 2013). The constitutive pre-mRNA splicing refers to the process where the transcript is always spliced the same way, like in the case of the splicing of introns, and the constitutive removal of certain exon(s). Alternative splicing is the process where exons can be alternatively spliced (either excluded or included) under different conditions allowing the differential expression of varying splice isoforms from the same gene.

The mechanism of RNA splicing involves a biochemical reaction accomplished by large complex machinery called the spliceosome. The spliceosome is composed of small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5; and non-small nuclear ribonucleoproteins (splicing factors) (Black, 2003; Krämer, 1996). Splicing factors recognize and bind specific sequences of the pre-mRNA that can either be enhancers of the spliceosome recognition of the splice site (Splicing Enhancer) or negative suppressor sequence (splicing Silencer) blocking the spliceosome recognition of the splice site (Kornblihtt et al., 2013). These enhancer and suppressor sequences can be either intronic or exonic. Thereby splicing factors can bind intronic splicing enhancer (ISE), exonic splicing Enhancer (ESE), intronic splicing suppressor (ISS), or exonic splicing suppressor (ESS) (Figure1).

5 There are two major families of splicing factors, the serine- and arginine-rich protein family (SR splicing factors) and the hnRNPs proteins (heterogeneous nuclear ribonucleoparticles) (Figure2) (S. Lin & Fu, 2007; Martinez-Contreras et al.,

2007; Zahler, Lane, Stolk, & Roth, 1992). The serine- and arginine-rich proteins (SR splicing factors) are RNA-binding proteins that have an amino terminus RNA recognition domain, and SR rich carboxyl terminus (Graveley, 2000; Krämer,

1996; Shepard & Hertel, 2009; Twyffels, Gueydan, & Kruys, 2011). SR proteins shuttle between the cytoplasm and the nucleus to function in constitutive and alternative splicing of pre-mRNA. Other functions of SR proteins have also been described, such as involvement in transcriptional elongation, mRNA export, mRNA stability and translational efficiency (Twyffels et al., 2011). The number of defined

SR proteins is currently 12 (Table.3) and they have now been re-named for consistency (Manley & Krainer, 2010).

Figure 1. Alternative splicing regulatory sequences and splicing factors. Splicing factors (SR or hnRNP) bind intronic splicing enhancer (ISE), exonic splicing Enhancer (ESE), intronic splicing suppressor (ISS), or exonic splicing suppressor (ESS). When splicing factor binds an enhancer, this promotes the spliceosome recognition of the nearby splice site(s). Splicing factor binding to a suppressor sequence blocks the recognition of the nearby splice site and inhibits splicing. Adapted from (Kornblihtt et al., 2013)

7 Table.3

Table.3 The nomenclature of the 12 SR splicing factors, as described by Manley & Krainer,2010. Table adapted from (Manley & Krainer, 2010)

8 Alternative splicing in leukemia

In cancer, aberrant regulation of the process of alternative splicing can lead to favoring certain isoforms and changing the tightly regulated normal ratio of isoforms. Unbalanced alternative RNA splicing has been linked to promoting cancer cell proliferation, avoidance of apoptosis and survival (Ladomery, 2013; Venables,

2006). In leukemia there are several studies that demonstrated evidence for the significant role of aberrant alternative splicing and showed that Bcr-abl fusion protein is a major player (Venables, 2006). Bcr-abl has been linked to the aberrant alternative splicing of several genes including the pre–B cell linker protein SLP6,

Bruton’s tyrosine kinase (BTK), tyrosine kinase Pyk2, and the lymphocyte specific transcription factor Ikaros. In CML the p210BCR/ABL fusion protein up-regulated expression of many genes involved in alternative splicing, such as splicing factor kinases, helicase II, and splicing factors (Salesse, Dylla, & Verfaillie, 2004).

Although the mechanism(s) causing inappropriate alternative splicing are not fully understood, it`s likely they would involve irregularities in splicing factor expression or function. SRSF1, a member of the SR splicing factors family (Table 3), is considered an oncoprotein and is overexpressed in several types of cancers (Karni et al., 2007). SRSF1 is also a critical target of myc transcriptional regulation (S. Das,

Anczuków, Akerman, & Krainer, 2012). Recently SRSF1 overexpression was reported in ALL patients’ samples and SRSF1 expression was necessary for avoidance of apoptosis in ALL(Zou et al., 2012). Additionally, function-altering mutations of SR splicing factors are commonly found in myeloid dysplastic

9 syndromes (MDS) and considered a novel pathway of leukemogenesis (Makishima et al., 2012; Yoshida et al., 2011). For example, hematopoiesis is altered in-vivo by a mutation changing the binding preference of the snRNP U2AF1. U2AF1 is commonly mutated in myelodysplastic syndrome (MDS)(Shirai et al., 2015). Other common mutations in MDS are SRSF2 mutations that alter it`s function in regulating alternative splicing (Kim et al., 2015). Another mechanism that affects alternative splicing is the post-translational regulation of SR splicing factors, i.e. phosphorylation (Ngo et al., 2005a). One common mechanism of SR proteins phosphorylation and activation is discussed below.

Serine Arginine rich proteins kinases (SRPKs):

Most SR splicing factors have been shown to be substrates for the Serine-arginine protein kinases (SRPK), and in theory all SR splicing factors can be targets for SRPK mediated phosphorylation. The phosphorylation of SR protein by SRPK is necessary for the activation and transport of SR splicing factors to the nucleus (Figure2) (Ngo et al., 2005b; Twyffels et al., 2011; Yeakley et al., 1999). However, aberrant SRPK expression has been described in several types of solid tumors such as breast, pancreas and colon cancers (Giannakouros, Nikolakaki, Mylonis, & Georgatsou,

2011). In leukemia SRPK1 was found overexpressed in acute T-cell leukemia induced by human T-cell leukemia virus-1 (Hishizawa et al., 2005), and in myeloid leukemia up-regulation of SRPK1 was dependent on the kinase function of p210BCR/ABL (Salesse et al., 2004). Investigating SRPKs as pharmaceutical targets has led to the development of a small molecular weight inhibitor, SRPIN340 that inhibits

10 the kinase function of SRPKs without inhibiting other splicing kinases (Fukuhara et al., 2006)

Regulation of SRPK

The control of SRPK localization or cellular partitioning is considered a major way of regulating it`s activity. SRPK is found mainly in the cytoplasm and less in the nucleus of the interphase cell, and then it is translocated to the nucleus prior to G2/M phase

(Ding et al., 2006). How SRPK translocation is regulated in response to extracellular signals is not fully understood although mechanisms of post-translational modifications of SRPK or its chaperone complex have been described (Giannakouros et al., 2011). One example of a signal that induces post-translational modification of

SRPKs is epidermal growth factor (EGF). A systematic analysis of factors that mediate the effect of EGF on pre-mRNA splicing revealed that EGF mainly activated the AKT-SRPKs-SR splicing factor pathway (Zhou et al., 2012). Another method of regulating the SRPK pathway is by transcriptional regulators of SRPK. The zinc finger transcription factor, WT1, was shown to transcriptionally suppress SRPK1 in conditionally immortalized podocytes (Amin et al., 2011). Since WT1 is overexpressed in some types of leukemia (Barragan et al., 2004; Menssen et al.,

1995; Miwa, Beran, & Saunders, 1992), this suggests a potential reduction of SRPK levels in these leukemia types. However, it is unknown if WT1 regulates SRPK expression in leukemia.

SRSF1

Figure 2. The SR proteins shuttle between the cytoplasm and the nucleus. SRSF1 (ASF/SF2) structure is shown at the top, with its RNA recognition motif (RRMs) shown in orange, docking motif (tan), and the two arginine –serine rich domains (yellow). SRPK1 (shown in blue) phosphorylates SRSF1 which triggers it`s transport into the nucleus by transportin-SR (purple). Once in the nucleus SRSF1 reside in the nuclear speckles. The nuclear kinase, CDC-like kinase (Clk/sty) further phosphorylates SRSF1allowing SRSF1 to leave the nuclear speckles and function in RNA splicing. Adapted from Ngo et al., 2005 (Ngo et al., 2005a)

12 The zinc finger transcription factor WT1

WT1 is a zinc finger transcription factor that can bind DNA and RNA (Figure3). WT1 gene encodes four zinc fingers and can be alternatively spliced creating different isoforms. In cancer WT1 has been described as both a tumor suppressor and as oncogene (Yang, Han, Saurez Saiz, & Minden, 2007) indicating that WT1 mediated effects in cancer might be specific depending on cellular conditions. WT1 is overexpressed in several types of acute leukemia (Barragan et al., 2004; Menssen et al., 1995; Miwa et al., 1992), and mutations are found in 15% of acute myeloid leukemia (King-Underwood, Renshaw, & Pritchard-Jones, 1996). WT1 mutations have been linked to poor clinical outcome in acute myeloid leukemia (Hollink et al.,

2009; Owen, Fitzgibbon, & Paschka, 2010; Virappane et al., 2008). Furthermore, in acute myeloid leukemia WT1 expression has been suggested as a marker for minimal residual disease after allogeneic stem cell transplantation (Candoni et al.,

2009). The mechanisms by which WT1 can contribute to leukemia are not fully understood. Recently WT1 was described to regulate murine hematopoiesis

(Cunningham, Palumbo, Grosso, Slater, & Miles, 2013). WT1 null hematopoietic progenitor cells had less proliferative potential to reconstitute, as a result of the increase in apoptosis when compared to control hematopoietic progenitors (that expressed normal levels of WT1)(Cunningham et al., 2013). Thus, these results suggest a potentially significant role of WT1 in the growth and avoidance of apoptosis in leukemia.

Figure3

Figure 3. WT1 structure. WT1 has an RNA and DNA binding domain composed of four zinc fingers on the carboxyl-terminus, and a dimerization domain on the amino terminus. Alternative splicing of WT1 generates four major WT1 isoforms depending on the exclusion or the inclusion of exon 5 and the KTS tripeptide (red). Adapted from (Morrison, Viney, & Ladomery, 2008).

14 Molecular mechanisms coordinately regulating transcription and RNA splicing

RNA splicing, including alternative splicing, can be post-transcriptional or co-transcriptional. The process of transcription and alternative splicing are tightly and coordinately regulated (Kornblihtt et al., 2013; Kornblihtt, de la Mata, Fededa,

Munoz, & Nogues, 2004). Alternative splicing has been shown to be regulated by cellular signaling, transcription factors, and chromatin status (Kornblihtt et al.,

2013; Kornblihtt et al., 2004). For example, a study showed that changing the promoter of RNA polymerase II (PolII) resulted in alterations of its isoforms ratios, providing evidence that transcription can affect alternative splicing (Cramer et al.,

1999; Cramer, Pesce, Baralle, & Kornblihtt, 1997; Kornblihtt et al., 2013; Kornblihtt et al., 2004). Secondly, it has been shown that different transcription factors have different effects on alternative splicing in mammalian cells (Nogues, Kadener,

Cramer, Bentley, & Kornblihtt, 2002). Co-activators recruited by steroid receptors also had a differential effect on alternative splicing, and different steroid hormone/receptor interactions had different effects on alternative splicing

(Auboeuf et al., 2004; Dowhan et al., 2005). Another way to link transcription to

RNA splicing is the recruitment of splicing factors by general transcriptional machinery. For example, Pol II is a key player in recruiting SR splicing factors to the nascent transcript (R. Das et al., 2007).

15 Vascular Endothelial Growth Factor-a and splice isoforms:

Vascular Endothelial Growth Factor-a (VEGF-a) is a growth factor that binds two types of receptor tyrosine kinase VEGF receptors (VEGFR1 and 2) and Neuropilins

(NRPs) to activate cell signaling (Ferrara, Gerber, & LeCouter, 2003). VEGF-a is key factor in regulating several physiological developmental processes and also pathological processes like cancer. In solid tumors, VEGF-a is well known as a growth factor and angiogenic factor permitting the growth of large tumors by increasing blood flow to the tumor cells (Ferrara et al., 2003). VEGF-a has been shown to contribute to leukemia in a couple of ways; by binding to receptors found on leukemia cells (Autocrine growth stimulating pathway), and by inducing proliferation and growth of endothelial cells in the bone marrow microenvironment, i.e. augmenting bone marrow angiogenesis (Podar & Anderson, 2005). Several studies provided evidence for VEGF-a over-expression in leukemia patients’ samples and showed it is correlated with worse clinical outcome (Aguayo, Alvaro Estey,

Elihu Kantarjian, Hagop Mansouri, Taghi Gidel, Cristi Keating, Michael Giles, Francis

Estrov, Zeev Barlogie,Bart Albitar, Maher, 1999; de Bont et al., 2002; Kampen, ter

Elst, & de Bont, 2013; Koomagi, Zintl, Sauerbrey, & Volm, 2001; Mourah et al., 2009;

Padro et al., 2002; Verstovsek et al., 2002; Wang et al., 2007).

However, VEGF-a can be alternatively spliced generating several isoforms with varying biological characteristics and different binding affinities to receptors and

ECM, depending on the alternative splicing of exon 6 and exon 7, which encode heparin-binding domains (Ferrara et al., 2003). The three most prevalent VEGF isoforms include VEGF121, VEGF165, and VEGF189 (Figure 4). Each are named

16 after the number of their amino acids. VEGF189 has the highest affinity to ECM and is the least diffusible isoform because it has both exon 6 and 7. VEGF165 retains exon 6 but lacks exon 7, so it has some binding affinity to the ECM but can also diffuse away from the cell of origin. VEGF121 is the shortest isoform lacking exons 6 and 7, and the most freely diffusible of these three major. The second known difference in the biological characteristics of VEGF-a isoforms is their binding to

VEGFRs and NRPs. VEGF165 and VEGF189 can bind VEGFR1/2 and NRPs while

VEGF121 can only bind VEGFR1/2 (Ferrara et al., 2003). Although variable in their biological characteristics, all three isoforms of VEGF-a are needed, and they all contribute to the complexity of VEGF-a signaling (Arcondeguy, Lacazette, Millevoi,

Prats, & Touriol, 2013; Grunstein, Masbad, Hickey, Giordano, & Johnson, 2000; Guo et al., 2001; Kanthou et al., 2014). In-vivo, mice expressing single isoforms suffered developmental abnormalities, except for VEGF165 mice, suggesting that all VEGF-a isoforms are required for normal development and that VEGF165, which is somewhat diffusible and can bind VEGFRs and NRPs, have the ability to compensate for the absence of other isoforms (Ng, Rohan, Sunday, Demello, & D'Amore, 2001).

VEGF189

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8

VEGF165

Exon 1 Exon 2, Exon 3 Exon 4 Exon 5 Exon 7 Exon 8

VEGF121

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 8

Figure 4: Major VEGF-a isoforms: Major isoforms of VEGF are 121 and 165 amino acids in length. Shown in yellow are the locations of Taqman probes used to specifically quantify VEGF121 and VEGF165. 1) VEGF189 transcript with eight exons. 2) VEGF165 transcript, which lacks exon6. 3) VEGF121 transcript lacking both exon6 and exon7.

Regulation of VEGF-a alternative splicing:

Despite growing knowledge of the significance of VEGF-a isoforms in cancer

(Bates et al., 2012; Catena et al., 2007; Fenton et al., 2004; Grunstein et al., 2000;

Jayson et al., 2011; H. Lin et al., 2008; Mourah et al., 2009; Yu, Rak, Klement, &

Kerbel, 2002; Zhang et al., 2000), little is known about the molecular mechanisms regulating the alternative splicing of VEGF-a (Arcondeguy et al., 2013). In

Endometrial Carcinoma cells hypoxia/low pH caused a shift in VEGF-a isoforms favoring VEGF121, which correlated with activation of SR proteins (SRSF1, SRSF3 or

SRSF4), and indeed knockdown of any of these SR proteins abolished that shift

(Elias & Dias, 2008). Conversely a reduction of VEGF121 isoforms was reported following WT1 over-expression in cells; and WT1 null murine hematoprogenitors had higher levels of VEGF121 compared to control cells normally expressing WT1

(Cunningham et al., 2013). This suggests a potentially significant contribution of

WT1 to leukemia by controlling VEGF isoforms. Consistently, VEGF-a has been described as a target of WT1 mediated regulation in prostate cancer cells, Ewing carcinoma, and cervical cancer (Hanson, Gorman, Reese, & Fraizer, 2007; Katuri et al., 2014; McCarty, Awad, & Loeb, 2011; Yamanouchi et al., 2014). One potential hypothesis for the mechanism of WT1 mediated regulation of VEGF-a isoforms is the inhibition of SRPK1 as described in podocytes(Amin et al., 2011), but it`s is not known if WT1 inhibits SRPK1 expression in leukemia too.

Specific inhibition of SRPK using SRPIN340 and VEGF-a

Investigating SRPKs as pharmaceutical targets has led to the development of a small molecular weight inhibitor, SRPIN340 that specifically inhibits the kinase function of SRPKs. SRPIN340 was found effective in inhibiting the phosphorylation of SR splicing factors. Consequently the nuclear localization and functions of SR-splicing factors in alternative splicing were inhibited. SRPIN340 was found to specifically inhibit SRPK1, and it also inhibits SRPK2, but much higher concentrations are required (Fukuhara et al., 2006). SRPIN340 has been used to inhibit angiogenesis in cellular and mouse models of solid tumors. It successfully inhibited angiogenesis in conditionally immortalized podocytes, colon carcinoma, prostate cancer and melanoma by inhibiting VEGF-a (Oltean et al., 2012). Knockdown studies showed that the reduction in several SR splicing factors appears to affect VEGF121 expression without reducing VEGF165 (Elias & Dias, 2008). However, the effect of

SRPK inhibition, using SRPIN340, on specific VEGF-a isoforms has not been characterized and the question of whether it affects VEGF121and VEGF165 differently has not been addressed. Inhibition of SRPK using SRPIN340 inhibits

VEGF-a expression even following the induction of VEGF by growth factors (Zhou et al., 2012). One growth factor that is known to activate SRPK and induce VEGF-a expression is discussed below.

20 The link between EGF and VEGF-a includes expression of EGF receptors in leukemia

Studies investigating the effect of EGF signaling showed that EGF up-regulates the expression of VEGF-a in glioblastoma cells (Maity, Pore, Lee,

Solomon, & O'Rourke, 2000) and prostate cancer cells (Zhong et al., 2000).

Conversely, inhibition of EGF receptor (EGFR) inhibited VEGF expression in several types of carcinoma cells and tumors (Pore et al., 2006). Interestingly, in leukemia, it was previously thought that EGFRs are not expressed on leukemia cells, however recent studies have shown that EGFRs are expressed in some myeloid leukemia cells derived from patients and some leukemia cell lines. Specifically, K562, MEG-01, CEM and SKO-007 were positive for EGFR, based on RT-PCR results. Importantly, EGFR expression in AML samples correlated with poor prognosis (Sun et al., 2012). This provides initial support for a potentially significant contribution of EGF to leukemia.

The effect of EGF over-expression on leukemia is not fully understood.

21 Hypothesis and specific aims:

Our studies included two major hypotheses. First, we hypothesized that in leukemia cells WT1 overexpression would differentially control alternative splicing of VEGF-a

(inhibiting VEGF121 compared to VEGF165 levels) by acting as an inhibitor of

SRPK1 transcription. Secondly, we hypothesized that other inhibitors of SRPK`s activity (such as SRPIN340) would reduce VEGF121 compared to VEGF165 levels, despite EGF induction of VEGF-a.

Specific Aims:

Aim1: Study the effect of overexpressing WT1 on VEGF165 and VEGF121 isoform expression in leukemia cells.

A. Does WT1 overexpression favor VEGF165 and inhibit VEGF121?

B. Does WT1 overexpression alter VEGF splicing by inhibiting splicing

kinase SRPK1 transcription?

Aim2: Study the effect of altered SRPK1 activity on VEGF165 and VEGF121 isoform expression in leukemia cells.

Aim2: Study the role of splicing kinase SRPK1 in VEGF-a alternative splicing

A. Would a specific inhibitor for the kinase function of SRPKs differentially

inhibit VEGF isoforms?

B. Does EGF signaling differentially induce splice isoforms of VEGF-a in

leukemia?

C. Does SRPIN340 inhibit EGF induction splice isoforms of VEGF-a?

Hypothesized model:

WT1è êSRPK1 expression è ê phosph.SR splicing factors è ê VEGF121 : VEGF165

SRPIN340 è êSRPK1 activityè ê phosph.SR splicing factors è êVEGF121: VEGF165.

EGFè é SRPK activity è éphosph. SR splicing factors èé VEGF121 and VEGF165.

EGF+ SRPIN340èê SRPK activity è êphosph. SR splicing factors èê VEGF121 and VEGF165.

CHAPTER 2

Methodology

Cell culture and transfections:

Leukemia cell lines K562, U937, and Nalm6 were maintained in RPMI containing

10% fetal bovine serum (FBS) and antibiotics (100IU/ml penicillin and

o streptomycin) in a 5% CO2 humidified incubator at 37 C. Cells were seeded at cell density of (2.5-5 X105 per ml) in 6 or 12-well plate and transfected with 250-500 nanogram of plasmid DNA. The cytomegalovirus promoter driven pcB6+WT1 (A) construct encoding the human WT1 gene (lacking both KST insertion and exon 5) and the pcB6+ empty vector control were previously described (Hanson et al, 2012).

The VEGF luciferase reporter construct, VEGF411, containing the proximal 400 bp promoter region was previously described (Hanson et al, 2012). Transfection was done in triplicate using the Lipofectamine LTX and Plus Reagent (Life Technologies,

Carlsbad, CA) for K562 cells, following manufacturer’s recommendations. U937 and

Nalm6 were transfected using TransIT®-2020 Transfection Reagent from Mirus

(Mirus Bio Corp, WI).

24 EGF and SRPIN340 treatments:

K562 and U937 cells were serum starved for at least 16 hours, then seeded at 2.5

X105 per well in a 12 well plate, treated with or without 100 ng/ml recombinant hEGF (Lonza Group, Switzerland), and incubated for 48 hours. For SRPK inhibitor treatments, 5 X105 per well of K562 or U937 cells were seeded in 6 well plate and treated for 48 hours with 10uM SRPK specific inhibitor, SRPIN340 (Sigma, MO) or control diluent (0.5-1% DMSO). For combination treatments, 2.5 X105 K562 cells were treated as described above except cells were pre-treated with 10uM SRPIN340

1 hr prior to combined EGF and SRPIN treatment.

RNA, and cDNA preparation and Quantitative PCR Analysis:

Total RNA was harvested using GenElute™ Mammalian Total RNA Miniprep Kit

(Sigma, St.Louis, MO) as per manufacturer’s recommendations and RNA concentrations were determined using a Nanodrop ND-1000 spectrophotometer

(Nanodrop Technologies, Inc, Wilmington, DE). RNA (1 microgram) was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Life

Technologies, Carlsbad CA). For quantitative real-time PCR (qPCR) cDNA samples were diluted 10-fold, and amplification of cDNA (10 ng ) samples was done in triplicate using Brilliant III Ultra-Fast QPCR master mix (Agilent Technologies, La

Jolla, CA) and specific Taqman Gene Expression assays (Applied Biosystems, Foster

City, CA). Taqman primers used were: 1) Hs00900057_m1 for VEGF165, 2)

Hs03929005_m1 for VEGF121 and 3) Hs99999901_s1 for 18S (housekeeping gene).

QPCR amplification was done in a Stratagene 3000MxPro thermocycler (Agilent

25 Technologies, La Jolla CA) using conditions recommended by manufacturer. Briefly initial denaturation was at 95o C for 2 min, followed by 40 cycles of 95o C/ 60o C

(for 5 sec/20 sec). To analyse gene expression, the comparative CT method was used. First average CT values were normalized to the18s rRNA gene, an endogenous

“housekeeping” control (CTmeasured-CT18S). These dCT values were transformed

(2-dCT), and the average 2 –dct of the treated samples were plotted relative to the average 2 –dct of controls to represent the average fold change in mRNA expression following treatments.

MTT assay:

For the cell viability assays, 2.5 x 104 U937 cells were seeded in a 96 well plate and treated with either control diluent (0.5% DMSO), or 10 micro molar SRPIN340 and incubated for 48 hours. Following manufacturer’s recommendations, the MTT cell proliferation assay (Trevigen, Gaithersburg MD) was used to measure the effect of inhibition of SRPK activity. Briefly, 10 μl of MTT reagent was added to each well and incubated for 3 hr (in the dark) to permit time for the formation of formazan crystals, indicating metabolically active cells. Then 100 μl of detergent was added, and cells incubated an additional 2 hr (in the dark) at 37 °C. Using a Biotek microplate reader, the OD was measured at 570 nm and 630nm. Substrate absorbance values of 630 nm were subtracted from 570 nm values to cancel the background effect. The average values for the normalized triplicate wells were determined and the percentage of metabolically active cells (representing those

26 surviving SRPIN 340 treatment) was determined by dividing the absorbance of the treated by the DMSO control wells.

Statistical Analysis:

Student`s T-test was used to determine statistical significance of the change between three samples of control (untreated) and three test (treated) samples. One asterisk (*) is used to represents P-value <0.05, ** for P-value <0.005, *** for P-value of <0.0005. Standard Error bars shows the standard error of mean.

Cytoplasmic and nuclear fractionation and western blot

2 ml of 1*10^7/ml per well of K562 were seeded in a 6 well plate (2 wells/treatment), treated with either 10 micro molar of SRPIN340, or control diluent (DMSO), and incubated for 48 hours. Pellets from 2 wells were pooled, and cells were lysed on ice for 10 minutes using 400 ul of cytoplasmic buffer (10 mMHEPES pH7.9, 10 mM KCL, 3 mM MgCL2, 0.5% NP-40, and 5% glycerol). Halt

(Pierce, Rockford, IL) protease inhibitors (1 ul/100ul lysate) and EDTA (1 ul/100ul lysate) were added to the lysate. Lysates were centrifuged at 6000rpm for 10 minutes at 4°C. The supernatant was saved as cytoplasmic lysate and stored in -20°C freezer. The pellet was suspended in 200 ul of 1X RIPA lysis with 10% SDS and rocked on ice for two to three hours with frequent mixing by pipetting up and down with 200ul pipette. Halt protease inhibitors (1 ul/100ul lysate) and EDTA (1 ul/100ul lysate) were added to the lysate. Homogeneous lysates were centrifuged at

14000 rpm for 10 minute at 4°C. Supernatant was saved as nuclear lysate and

27 stored in -20 °C freezer. Nuclear and cytoplasmic lysates were sonicated and their protein concentration was measured using the Bicinchoninic acid (BCA) Protein assay (Pierce, Rockford, IL) and calculated based on the BSA standard curve. 30ug of protein was denatured using 6X loading buffer containing β-Mercaptoethanol and

DTT. Protein was then boiled for 5 minutes. Protein preparations were loaded into

10% SDS-PAGE gels and run for 90 minutes at 150 volts. Proteins were transferred onto PVDF membrane for 1 hour at 100 Volts, followed by washing 3 times in PBS with 0.1% Tween 20 (PBST). Membranes were blocked in PBST with 5% non-fat dry milk (PBST-blocking buffer) for 1 hour and then incubated overnight with primary antibodies in PBST-blocking buffer, either 1:200 anti-pan SR proteins (Santa Cruz,

Dallas, Texas), 1:1000 anti-B-actin (GenScript, Piscataway, NJ), or 1:500 anti-acetyl histone 3 (Millipore, Billerica, MA). The membrane was washed with PBST and incubated for one hour at rom temperature with the appropriate secondary antibody, and washed with PBST twice for five minutes each. Membranes were developed by incubating the membrane in a luminol ECL solution followed by chemiluminescent detection using a Fuji LAS 3000 detection system (R&D Systems,

Minneapolis, MN). Digital images were saved and bands intensities were quantified using ImageJ software.

CHAPTER 3

Results

WT1 mediated inhibition of VEGF-121 was not a result of SRPK inhibition

To assess the relative levels of VEGF-a isoforms (VEGF121 and VEGF165) we quantified the endogenous mRNA levels of VEGF165 and VEGF121 using isoform specific quantitative Taqman PCR assays of triplicate samples of K562, U937, and

Nalm-6 cells maintained as described in the methods. The VEGF121 isoform was significantly higher than VEGF165 in all three cell lines (Figure 5). The statistical significance was determined using T-test with P-value <0.05. The efficiency of

VEGF121 and VEGF165 Taqman assays was verified, and the two primers had very similar amplification efficiencies (data not shown). These results indicate that VEGF

165 mRNA is not the predominant VEGF isoform in leukemia cells, similar to observations in carcinomas (Catena et al., 2007; Fenton et al., 2004; Zhang et al.,

2000). Although many factors may regulate VEGF isoform ratios in leukemia cells, we have examined the transcription factor WT1, reported to be elevated in acute leukemia (Candoni et al., 2009; Miwa et al., 1992; Owen et al., 2010; Yang et al.,

2007).

To determine whether WT1 transfection alters the ratio of major isoforms of

VEGF-a in leukemia cells, we transfected leukemia cells (K562, U937 and Nalm-6) with WT1 cDNA expression plasmid construct. K562 cells transfected with WT1 expression construct showed a consistent 2-fold repression of VEGF121 mRNA compared to control cells transfected with the empty vector cB6+ (T-test, P<0.05).

On the other hand, VEGF165 mRNA expression was not significantly changed in

K562 cells transfected with WT1 compared to empty vector (Figure 6A) indicating that WT1 favors VEGF121 expression relative to VEGF165. Conversely, there was no significant change in the mRNA expression of VEGF121 or VEGF165 in U937

(Figure 6B), and Nalm6 cells (Figure 6C), transfected with WT1 expression construct compared to empty vector control. This suggests that WT1 effect on

VEGF-a isoform expression depends on cellular context. Unlike the K562 cells, the pre-B leukemia cell line, Nalm-6, and the myelo-monocytic lymphoma derived cell line, U937, did not respond to WT1 over-expression, which implies lineage differences might account for differential WT1 effect. Possibly the chronic myeloid leukemia derived K56 cells contain co-factors necessary for WT1 mediated regulation of VEGF-a isoforms.

To identify a potential mechanism for the repression of VEGF121 by WT1 in

K562 cells, we examined its transcriptional target, SRPK, an SR splicing factor kinase. We measured SRPK1 mRNA expression in WT1 transfected leukemia cells to see if the reduction in VEGF121 was a result of WT1 mediated transcriptional

30 inhibition of SRPK1, as described in other systems (Amin et al., 2011). Despite the

2-fold inhibition of VEGF121 mediated by WT1, shown in Figure 6A, there was no significant change in SRPK1 mRNA expression in leukemia cells (K562) transfected with WT1 compared to empty vector controls (Figure 7) suggesting that

WT1-mediated inhibition of VEGF121 isoforms was not due to SRPK1 inhibition.

A K562 5 * 4 3 2

Fold change 1 0 VEGF165 VEGF121

B C Nalm-6 U937 12 12 *** 10 10 ** 8 8 6 6 4 4 Fold change 2 2

0 0 VEGF165 VEGF121 VEGF165 VEGF121

Figure 5. Characterization of VEGF-a major isoforms in K562 RNA was isolated from K562, Nalm-6, and U937 cells, reverse transcribed and assayed for VEGF121 and VEGF165 transcript levels as described in the text using isoform specific VEGF-a assays. Values were normalized to the endogenous control 18S. Shown are ratios of each isoform relative to average VEGF165 levels. A student T-test was performed and significance was determined (* for P-value <0.05, ** for P-value <0.005, and *** for P-value<0.0005). Standard error bars represent standard error of mean. Experiments were done in triplicate, each sample was measured in triplicate and results were reproduced.

A 2 K562

1.5 control 1 WT1A

0.5 Fold Change

0 VEGF121 VEGF165

B C U937 2 Nalm-6 2

1.5 1.5

Control 1 Control 1

Fold change WT1 WT1 0.5 0.5 Fold Change

0 0 VEGF121 VEGF165 VEGF121 VEGF165

Figure 6. WT1 inhibited VEGF121 without reducing VEGF165 in K562 cells. RNA was isolated from WT1 transfected K562 (A), Nalm-6 (B), and U937 (C), and reverse transcribed and assayed for VEGF121. Values were normalized to the endogenous control 18S. Bars show average fold change of mRNA expression in cells transfected with WT1 compared to the average expression of the empty vector controls. A student T-test was performed and significance was determined (* for P-value <0.05). Standard error bars represent standard error of mean. The experiment was done in triplicate, and results were reproduced.

K562

1.5

SRPK1

Fold change 0.5

0 cont WT1

Figure 7. SRPK expression levels were not altered by WT1 transfection RNA was isolated from WT1 transfected K562, reverse transcribed, and assayed for of SRPK1 in K562 cells transfected with WT1. Values were normalized to the endogenous control 18S. Bars show average fold change of relative mRNA expression in cells transfected with WT1 compared to the average expression of the empty vector controls. Standard error bars represent standard error of mean and statistical significance was determined using T-test. The experiment was done in triplicate.

Specific inhibition of SRPK activity significantly inhibited expression of

VEGF-a isoforms.

Although our results ruled out WT1 mediated regulation of SRPK expression, they did not test SRPK involvement in VEGF-a isoform expression. To investigate the role of the kinase activity of SRPK in the regulation of VEGF-a isoform expression

K562 and U937 cells were treated with SRPIN340, an inhibitor of SRPK kinase activity. 10 micro molar of SRPIN340 was used to assess the requirement for

SRPK-mediated phosphorylation of SR splicing factors in the RNA alternative splicing of VEGF-a. In K562 cells SRPIN340 treatment resulted in a significant 2-fold inhibition of both VEGF121 and VEGF165 compared to their levels in the diluent controls (Figure 8A). (T-test, P-value <0.05). These results indicate that SRPK mediated phosphorylation of SR protein is necessary for the efficient processing of the two splice isoforms of VEGF-a. The experiment was done in triplicate, and results were reproduced. Since SERPIN340 affected both isoforms in K562 cells, we asked whether it had a general effect on U937 cells, as well.

In U937 a greater reduction in VEGF121 and VEGF165 was observed in

SRPIN340 treated samples compared to three DMSO control samples (~10 fold suppression, P-value of <0.05) (Figure 8B). However treatment of U937 cells with

SRPIN34 also appeared to alter cell appearance somewhat, raising concerns about cell viability, so an assay for metabolic activity (MTT) was performed. Cellular

35 metabolic activity was measured as an indicator of leukemia cell growth and viability (Figure 9), We compared activity of U937 cells treated with 10 micro molar SRPIN340 to that of cells treated with control diluent (DMSO). On average,

SRPIN340 significantly reduced metabolic activity of treated U937 cells to less than

40% of control cell activity. This reduction was statistically significant (T-test,

P<0.005) and represents reduction in cell viability, potentially due to enhanced cell death or growth arrest (Figure 9). Overall, these results suggest that SRPIN340`s action on SR proteins has broad sweeping effects on cell metabolism and viability in

U937 cells that are not limited to VEGF-a expression.

! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ! ! A" ! 1.5! K562! !

! 1! Control! *! ! *! SRPIN340!

! Fold!change! 0.5!

! 0! VEGF121! VEGF165! ! B B" " U937% ! 1.5" !

1" ! DMSO" ! 0.5"Fold%%change% SRPIN340" *! * ! 0" VEGF121" VEGF165"

Figure 8. SRPIN340 reduced VEGF-a isoforms in leukemia cells RNA was isolated from K562 (A) and U937 (B) treated with 10 micro molar of SRPK`s specific inhibitor (SRPIN340), reverse transcribed, and assayed for VEGF121 and VEGF165. Values were normalized to the endogenous control 18S. Bars show average fold change of VEGF-a mRNA expression in cells treated with SRPIN340 compared to the average expression of controls. Statistical significance was determined using T-test. Experiments were repeated and similar results were observed.

K562

120

100

80 ** 60 40

Metabolic activity 20 0 Control SIRPIN340

Figure 9. SRPIN340 significantly reduced growth of U937 leukemia cells. MTT assay used to measure cellular metabolic activity of K562 cells. We compared K562 cells treated with 10 micro molar SRPIN340 to control diluent (DMSO) cells. Bars shows average percentage of SRPIN340 treated cells compared to activity of cells treated with control diluent (DMSO). A student T-test was performed and significance was determined. (T-test, P<0.005). The experiment was done triplicate. Results were reproduced.

The effect of epidermal growth factor (EGF) on expression of VEGF-a

isoforms in leukemia cells

Since suppression of SRPK activity by SRPIN340 reduced both VEGF121 and

VEGF165 isoform expression in K562, we asked whether the converse would be true for activators of SRPK, such as EGF. Both K562 and U937 cells were serum starved overnight then treated with 100 ng/ml of human epidermal growth factor

(hEGF) for 48 hours. Quantitative PCR of VEGF-a major isoforms (Figure 10A) showed significant up-regulation (~ 2 fold) of both VEGF121 and VEGF165 mRNA expression in EGF treated K562 cells compared to untreated controls (T-test,

P-value<0.05). The experiment was repeated three times with consistent results.

Conversely, in U937 cells, which do not express EGF receptor (EGFR) (Sun et al.,

2012), EGF did not significantly affect the mRNA levels of VEGF-a isoforms (Figure

10B) in treated cells compared to untreated controls. Thus, EGF appears to require

EGFR activation to enhance VEGF-a isoform expression in leukemia cells.

K562 A 3 *

2.5 * B

2 * 1.5 control

Fold change 1 EGF

0.5

0 VEGF121 VEGF165

B U937 3

2.5

2 1.5 control

EGF 1

0.5 0 VEGF165 VEGF121

Figure 10. EGF up-regulated VEGF-a in leukemia cells (K562) RNA was isolated from K562 (A) and the EGFR negative cells, U937 (B) cells treated for 48 hours with 100 ng of human EGF, as described in the text, reverse transcribed, and assayed for VEGF121 and VEGF165. Bars show average fold change of VEGF-a isoforms mRNA expression in EGF treated cells compared to the average expression of untreated controls. A student T-test was performed and significance was determined (P<0.05). The experiments were done in triplicate and results were reproduced.

SRPIN340 attenuated EGF induction of VEGF-a isoform expression

While these results showed that EGF enhanced and SRPIN340 suppressed

VEGF 121 and 165 expression in K562 cells, it was unclear whether EGF-mediated induction of VEGF-a isoforms could be inhibited by SRPK inhibitors. We measured

VEGF-a isoform mRNA expression levels in K562 leukemia cells pretreated with

SRPK`s inhibitor (SRPIN340) prior to the EGF induction. Quantitative PCR was used to measure expression of VEGF-a major isoforms, VEGF121 and VEGF165, in serum starved K562 cells treated with either control diluent (DMSO), or 10micro molar

SRPIN340; or 100ng/ml human recombinant EGF, or both SRPIN340 and EGF (as described in methods). Consistent with our previous results, SRPIN340 alone significantly reduced both VEGF121 and VEGF165 isoforms (~2 fold, P-value <0.05) compared to DMSO control treated cells (Figure 11). And K562 cells treated with

EGF alone had a significant ( >2 fold) increase in VEGF 121 and 165 isoform expression, compared to cells treated with both the SRPK`s inhibitor (SRPIN340) and EGF . (T-test, P<0.05). This experiment was performed in triplicate and was reproduced with consistent results. Since our results showed that EGF did not have an effect on VEGF-a isoform expression in the EGFR negative U937 cells, there was no rationale for testing the EGF and SRPIN340 combination treatment.

K562

3 VEGF165 Fold change 2 VEGF121 1

Figure 11. SRPIN340 attenuated EGF-mediated induction of VEGF-a isoforms. RNA was isolated from K562 cells treated with control diluent, SRPIN340, human EGF or pretreated with SRPIN340 and then induced with EGF. Values were normalized to the endogenous control 18S. Average. Bars show average fold change of VEGF121 and VEGF165 mRNA expression in K562 cells treated relative to the expression in cells treated with control diluent. Experiments were done in triplicate. Significance was determined, as described in the text, by t-test (*p<0.05). The experiment was repeated and similar results were observed.

SRPIN340 reduces the nuclear intensity of SR proteins in K562

Western blot analysis of nuclear and cytoplasmic proteins from K562 lysates showed reduced levels of SR proteins in nuclear extracts of SRPIN340 treated K562 compared to cells treated with control diluent. Normalized intensities of SR protein bands were reduced by 20-60% reflecting an inhibition of SR protein nuclear localization in SRPIN340 treated cells. The strongest reductions were in nuclear proteins representing SRSF1/2 and SRSF5, which were reduced to only 40% of that seen in DMSO treated control cells. Of note, the intensity of the 30 kDa band

(SRSF1/2) in cytoplasmic lysates showed a 40% increase in SRPIN340 treated lysates compared to DMSO treated controls..

A 42kDa! B6Ac9n# K562#

!!!!!!!!!!Nuclear!!!!!!!!!!!!!!Cytoplasmic! SRPIN340!!!!!!!DMSO#######SRPIN340!!!!!!DMSO# # SR#proteins# SRSF4! 72kDa! 55kDa! SRSF6!

SRSF5! 43kDa! SRSF1/2!

18kDa! Acetyl#Histone#3#

C B

140 140 120 120 100 100 Nuclear 80 Cytoplasmic 80 DMSO DmSO 60 60 Nuclear Cytoplasmic 40 SRPIN340 40 SRPIN340

Intensity of SR proteins Intensity of SR proteins 20 20

0 0

Figure 12 SRPIN340 reduced levels of nuclear SR proteins in K562 cells. (A) K562 were treated with either 10 micromolar SRPIN340, or control diluent DMSO, then nuclear and cytoplasmic protein lysates were extracted. 30ug of protein was separated by SDS-PAGE and transferred to PVDF membrane for incubation with anti-pan SR proteins (Santa Cruz), anti-B-actin (GenScript), or anti-acetyl histone 3 (Millipore), followed by the appropriate secondary antibody. Signal was detected with luminol reagent using a FujiFilm LAS-3000 detection system. Shown are captured images with size markers. SR bands are labeled based on size estimates provided by manufacturer: SRSF4 (75kDa), SRSF6 (55kDa), SRSF (40kDa), and

44 SRSF1/2(30kDa). Quantitation of SR protein bands in nuclear (B) and cytoplasmic (C) extracts was determined by Image J analysis and normalized to acetyl histone 3 and B-actin loading controls, respectively.

CHAPTER 4

Discussion

Our group and others identified VEGF-a as a target of WT1 mediated transcriptional regulation in carcinoma cells (Hanson et al., 2007; Katuri et al., 2014;

McCarty et al., 2011; Yamanouchi et al., 2014). However, the effect of WT1 on

VEGF-a expression in leukemia and on specific isoform expression of VEGF-a had not been studied before. Here we report that WT1 alters the ratio of VEGF121 relative to VEGF165 in K562 chronic myelogenous leukemia cells independent of

SRPK inhibition. Our results in K562 cells transfected with WT1 showed that WT1 down-regulated the shorter isoform of VEGF-a (VEGF121), which is consistent with a recent report suggesting that WT1 regulates hematopoiesis by decreasing the ratio of VEGF121 relative to VEGF165 in murine hematoprogenitors (Cunningham et al., 2013).

The vascular endothelial growth factor-a (VEGF-a) has been shown to contribute to leukemia in a couple of ways; by binding to receptors found on leukemia cells (and activating an autocrine growth stimulatory pathway) and by inducing endothelial cells proliferation and growth in the bone marrow microenvironment, i.e. augmenting bone marrow angiogenesis (Podar &

45 Anderson, 2005). Several studies showed that when VEGF-a is overexpressed it correlates with poor prognosis in leukemia patients (Aguayo, et al., 1999; de Bont et al., 2002; Kampen et al., 2013; Koomagi et al., 2001; Mourah et al., 2009; Padro et al.,

2002; Verstovsek et al., 2002; Wang et al., 2007). Specifically VEGF-a splice isoform

VEGF121 was significantly overexpressed in AML patients’ samples, and importantly, elevated VEGF121 was correlated with poor prognosis (Mourah et al.,

2009). A poor prognosis (an increase risk of relapse) has also been associated with both WT1 loss of function mutations and elevated expression (Candoni et al., 2009;

Hollink et al., 2009; King-Underwood et al., 1996; Miwa et al., 1992; Owen et al.,

2010; Virappane et al., 2008; Yang et al., 2007). Our work suggests that WT1 expression is involved in the molecular mechanism(s) regulating the alternative splicing of VEGF-a isoforms, specifically reducing VEGF 121 isoform levels in K562 cells, although we have not yet examined the effect of WT1 mutations or silencing in these cells. In contrast, WT1 over-expression in U937 and Nalm-6 did not change

VEGF-a isoform expression suggesting that WT1-mediated regulation of VEGF-a isoform expression depends on cell type, stage or condition. K562 is a CML cell line with the Philadelphia chromosome translocation (Bcr-abl fusion protein), which had been linked to several irregularities in alternative splicing (Venables, 2006). How

WT1 might relate to the effect of Bcr-abl on alternative splicing is not yet understood.

WT1 transcriptionally suppresses the Serine/Arginine splicing factors` kinase (SRPK1) in podocytes (Amin et al., 2011). However, unlike what has been reported in immortalized podocytes, our results in K562 cells indicate that the WT1

46 mediated down-regulation of the ratio of VEGF121 relative to VEGF165 was not a result of the down-regulation of SRPK1 mRNA expression. The mechanism by which WT1 controls the expression of the alternative splice isoforms of VEGF-a in leukemia still needs to be clarified. One possible hypothesis is that WT1, as a bound transcription factor, could recruit certain splicing factors as a way to coordinately regulate transcription and alternative splicing. It`s known that the process of transcription and alternative splicing are tightly and coordinately regulated (Smith

& Valcárcel, 2000). Work is in progress to examine WT1 association with SR splicing factors, a potential mechanism for directly regulating the isoform ratio of VEGF-a by recruiting specific SR splicing factors that favor the exclusion of both exon6 and exon7. SR splicing factors have been described as links between the regulation of transcription and RNA splicing as they are recruited to the nascent transcript by transcription complexes (R. Das et al., 2007).

Here, we demonstrated that even though transcription of the SRPK1 gene was not affected by WT1, the kinase activity of SRPK was necessary for the RNA splicing of VEGF-a isoforms. The use of an SRPK specific inhibitor

(SRPIN340)(Fukuhara et al., 2006) down-regulated both isoforms similarly suggesting that SRPK is required for the proper splicing of both VEGF165 and

VEGF121, and it does not differentially control the ratio of VEGF165 relative to

VEGF121 in leukemia cells (K562 and U937). Possibly both isoforms were affected because SRPK inhibition can affect multiple SR splicing factors, since most SR proteins require phosphorylation by SRPK`s for their activation and the nuclear translocation (Giannakouros et al., 2011). In addition to regulation alternative

47 splicing, SR proteins functions in regulating transcriptional elongation and mRNA stability (R. Das et al., 2007; Shepard & Hertel, 2009; Twyffels et al., 2011).

Future work must be done to identify specific SR splicing factors that favor

VEGF121, and others that favor VEGF165 isoform. Initial clues have been provided by knockdown experiments in endometrial carcinoma cells showing that knocking-down specific SR splicing factors SRSF1, SRSF3 or SRSF5 abolished the hypoxia mediated induction of VEGF121 but did not seem to affect the longer isoforms (Elias & Dias, 2008). This suggests that SRSF1, SRSF3 or SRSF5 favor

VEGF121 while other splicing factors might favor VEGF165. Also, studying the consequences of the inhibition of SR proteins (using SRPIN340) on VEGF-a isoforms mRNA stability, and transcriptional elongation can help clarify the broad functions of SR proteins in regulating VEGF-a isoform expression that might not be limited to alternative splicing.

Previous studies of the effect of SRPK`s specific inhibitor (SRPIN340) on

VEGF-a were done in several solid tumors and showed that SRPIN340 inhibits

VEGF-a expression without affecting viability or proliferation of cell lines and significantly reduces tumor angiogenesis in mouse models (Oltean et al., 2012).

However, the effect of SRPIN340 on VEGF121 and VEGF165 isoform expression and relative ratios was not examined.

One previously described mechanism of SRPIN340 mediated inhibition of

VEGF-a isoforms is the suppression of SR proteins nuclear translocation due to the reduction in SRPK activity (Amin et al., 2011). Our preliminary protein studies, showing a reduction in the nuclear intensity of SR proteins in K562 cells treated

48 with SRPIN340, supports our hypothesis that SRPIN340 reduces VEGF-a isoforms expression by inhibiting the nuclear localization of SR proteins (Figure 13).

Our results showed that SRPIN340 significantly inhibited U937 growth and metabolic activity suggesting that the impact of SRPIN340 on splicing factor activation and alternative splicing in hematopoietic malignancies may be significant.

Taken together, we showed that in K562 and U937, SRPIN340 significantly inhibited

VEGF-a expression (K562), a key element in leukemia autocrine stimulatory growth

(Podar & Anderson, 2005), and inhibited leukemia cells growth and viability (U937).

The growth inhibition can be a result of VEGF-a inhibition in addition to other broader changes in alternative splicing

Here, we have demonstrated that in the EGFR positive leukemia cells (K562) epidermal growth factor, EGF, can up-regulate VEGF-a expression. In contrast, in

U937 cells lacking EGFR, EGF treatment did not up-regulate VEGF-a, consistent with the notion that EGF mediated induction of VEGF-a expression appears to be mediated through EGFR. Our results are also in agreement with studies linking EGF to the up-regulation of VEGF-a expression in several solid tumor types, including glioblastoma (Maity et al., 2000) and prostate cancer cell lines (Zhong et al., 2000)

Consistent with this, EGFR inhibition reduced VEGF expression in several types of carcinoma cells and tumors (Pore et al., 2006). Our results link EGF and VEGF-a expression and provide much-needed supporting evidence of a role for EGF in leukemia. Consistent with our observations, EGFR expression correlates with poor prognosis in acute myeloid leukemia patients (Sun et al., 2012) and therapies targeting EGFR induced apoptosis (Moon et al., 2007) and differentiation (Stegmaier

49 et al., 2005) of myeloid leukemia cells, although mechanisms are not clear. Despite the lack of EGFR in U937 cells, inhibitors of EGFR (gefitinib) induced apoptosis

(Moon et al., 2007). Also, Erlotinib, another inhibitor of EGFR prompted growth arrest, differentiation, and apoptosis in EGFR negative myeloid cell lines (P39, KG-1, and HL 60) and myeloid blasts derived from patients (Boehrer et al., 2008). How

EGFR inhibitors are affecting EGFR negative cells is not clear, but the authors speculate that EGFR inhibitors caused an off-target effect (Boehrer et al., 2008). This is consistent with another study showing that gefitinib induces differentiation of myeloid cell lines (HL-60, Kasumi-1, and U937); and in primary patient-derived

AML blasts (Stegmaier et al., 2005). However, clinical studies investigating the use of gefitinib as a single agent treatment of AML were not successful (Deangelo et al.,

2014).

Overall, our results showed that treatment of K562 cells with the SRPK`s inhibitor prior to EGF attenuated up-regulation of VEGF-a isoforms, and emphasizes the necessity of SRPK activity in the RNA splicing of both VEGF-a isoforms

(VEGF121 and VEGF165). The SRPKs-SR splicing factor pathway has been found to be a major transducer of EGF effect on mRNA splicing in other systems (Zhou et al.,

2012). Therefore, we expected SRPK inhibition to affect the activation of SR factors by EGF and thereby, dampen EGF induction of VEGF-a isoforms. Preliminary studies showed that SRPIN340 treatment reduced nuclear localization of SR splicing factors in K562 cells. These results support the proposed model of SRPIN340 inhibiting the

SRPK/SR splicing factor pathway and thereby inhibiting VEGF121 and VEGF165 mRNA expression. Finally, by identifying EGF and SRPK as regulators of VEGF-a

50 expression in leukemia, our work provides support for current and future therapies targeting EGF signaling and SRPK activity in leukemia.

CHAPTER 5

Future directions

Use of WT1 knockdown and mutant WT1 expression constructs in leukemia cells can help clarify the role of WT1 in regulating VEGF-a expression in leukemia and determine whether subsequent altered isoform ratios correlate with changes in leukemia cell proliferation and/or apoptosis (using flow-cytometry). Secondly, to understand the mechanism of WT1 mediated regulation of VEGF-a isoforms, independent of SRPK1, it will be important to determine whether splicing factors are activated or recruited by WT1 expression. Co-immunoprecipitation assays of

WT1 with SR-splicing factors can be done to determine whether WT1 associates with splicing factors, and whether it directly regulates the isoform ratios of VEGF-a by recruiting specific splicing factors to the transcript. It will be important to identify SR factors involved, but initially we can screen using pan-SR Abs.

Further protein studies measuring the nuclear intensity of SR proteins can confirm that SRPIN340 affects VEGF-a isoforms by inhibiting the nuclear translocation of SR proteins. Other mechanisms to regulate isoform expression, other than alternative splicing, such as isoform mRNA stability and translational efficiency can also be investigated to help identify the broad functions of SR proteins

52 in regulating VEGF-a isoform expression that might not be limited to alternative splicing.

Finally, since the SRPIN340 mediated suppression of VEGF-a expression suggests a potential inhibition of leukemia cell growth and viability in some types of leukemia cells, the effect of SRPIN340 on cell proliferation and/or apoptosis can be examined in future studies.

Summary'model!

EGF$ Un#phosphorylated/ /SR/protein/ +$

SRPK1/ −$ SRPIN340$ Cytoplasm$

Ac7ve/phosphorylated/ /SR/protein/ Nucleus$

5$$$$$$8$

Figure 13. Proposed model of EGF/SRPK1/SR pathway regulating VEGF isoform expression. EGF activates SRPK1 resulting in phosphorylation of cytoplasmic SR splicing factors, and subsequent nuclear translocation. Conversely, SRPIN340 blocks this translocation by inhibiting SRPK1 activity. Expression of VEGF 121 and 165 isoforms is enhanced by activation of this pathway by EGF, and suppressed by the SRPK inhibitor, SRPIN340.

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