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Targeting of PIM1 Kinase in Myeloproliferative Neoplasms Induced by JAK2V617F

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Targeting of PIM1 Kinase in Myeloproliferative Neoplasms Induced by JAK2V617F

Abstract:

Myeloproliferative neoplasms (MPNs) are stem cell-derived blood disorders. The most common mutation found in MPN patients is the JAK2V617F mutation. JAK2 is a non-receptor tyrosine kinase involved in STAT signaling. The JAK2V617F mutation is a single amino acid substitution of a phenylalanine for valine, which causes JAK2 to be constitutively activated. This mutation can cause a hematopoietic transformation. Eventually this transformation can lead to the development of one of the three different Philadelphia-negative MPN diseases: Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF). The JAK2V617F mutation has been identified in 95% PV patients, and 50-60% of ET and PMF patients.  A JAK1/2 inhibitor (ruxolitinib) has been approved for MF and PV patients and, though it provides initial benefits, it is not effective enough to cause long-term remission in patients. This creates a critical need to identify new therapeutic targets for MPN patients.

We found that PIM1 levels were significantly increased in MPN patients, as well as our JAK2V617F mouse model of MPN. We observed that knockdown of PIM1 caused a significant decrease in proliferation of JAK2V617F expressing cells. We also found that PIM1 knockdown had no effect on the proliferation of hematopoietic cells not expressing JAK2V617F, leading us to believe PIM1 is only required in JAK2V617F mediated proliferation. Pharmacological inhibition of PIM kinases, using TP-3654, (kindly provided by Tolero pharmaceuticals) also led to a significant decrease in proliferation of JAK2V617F-expressing cells, but had no effect on cells lacking the mutation.

We also found that the PIM inhibitor, TP-3654, works synergistically with ruxolitinib to achieve an even greater decrease in proliferation. We found that using the combination of ruxolitinib and TP-3654, we could use both drugs at lower concentrations and achieve an even greater decrease in proliferation and an increase apoptosis. Furthermore, we found that inhibition of PIM kinases using TP-3654 can resensitize ruxolitinib-resistant cells to ruxolitinib treatment.

These important findings show that PIM1 plays an important in the proliferation of hematopoietic cells expressing the JAK2V617F mutation, but is dispensable for the maintenance of cells lacking the mutation. We also found that targeting PIM kinases with TP-3654, significantly decreased the proliferation, and increase apoptosis activation of JAK2V617F expressing cells. We also showed that TP-3654 and ruxolitinib can work synergistically. Lastly, we showed that inhibition of PIM kinases, using TP-3654, caused ruxolitinib-resistant cells to become resensitized to ruxolitinib. These findings helped us come to the conclusion PIM1 kinase, is an important therapeutic target in JAK2V617F-induced MPNs.

Table of Contents:

Acknowledgements………………………………………………………………………………………………………………….i

Abstract………………………………………………………………………………………………………………………………………ii

Table of Contents……………………………………………………………………………………………………………………iv

List of Figures & Tables………………………………………………………………………………………………………..vi

List of Abbreviations……………………………………………………………………………………………………………vii

Introduction………………………………………………………………………………………………………………………………1

Materials & Methods……………………………………………………………………………………………………………12

Patient Data…………………………………………………………………………………………………………………12

Cell Culture………………………………………………………………………………………………………………….12

Gene knockdown and Proliferation assay…………………………………………………………….13

Quantitative Real-Time PCR…………………………………………………………………………………….15

Lysing and Immunoblotting…………………………………………………………………………………….15

Apoptosis Assay…………………………………………………………………………………………………………16

Statistical Analysis…………………………………………………………………………………………………….16

Results………………………………………………………………………………………………………………………………………17

PIM1 expression is significantly increased in MPN patients…………………………….17

Knockdown of PIM1 kinase significantly reduces proliferation in JAK2V617F-               expressing cell lines…………………………………………………………………………………………………..20

Inhibition of PIM kinases significantly reduces proliferation of JAK2V617F-              expressing cell line…………………………………………………………………………………………………….22

PIM kinase inhibitor TP-3654 synergizes with ruxolitinib to inhibit cell               proliferation and induce apoptosis in JAK2V617F-expressing cells………………24

JAK2 inhibitor resistant BA/F3-EpoR-JAK2V617F cells are not inhibited by               ruxolitinib ……………………………………………………………………………………………………………………28

PIM1 expression is significantly increased in the BA/F3-EpoR-JAK2V617F               ruxolitinib-resistant cell line……………………………………………………………………………………30

BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cells are sensitive to PIM               kinase inhibition by TP-3654…………………………………………………………………………………..32

Pharmacological inhibition of PIM kinase can resensitize ruxolitinib-resistant               cells to ruxolitinib treatment…………………………………………………………………………………..33

Discussion………………………………………………………………………………………………………………………………..37

References……………………………………………………………………………………………………………………………….42

List of Figures/Tables:

Table 1. Frequency of different mutations in Myeloproliferative Neoplasms….3

Figure 1. Signaling pathways that are constitutively activated by JAK2V617F…4

Figure 2. PIM1 level is increased in the JAK2V617F expressing mouse model…8

Figure 3. Structure and selectivity of TP-3654…………………………………………………………..11

Table 2. Sequences of PIM1 shRNAs………………………………………………………………………………13

Table 3. Quantitative Real-Time PCR Primers……………………………………………………………15

Figure 4. PIM1 expression is significantly increased in Ph MPN patients……….18

Figure 5. PIM1 expression is significantly increased in JAK2V617F-positive MPN patients………………………………………………………………………………………………………………………….19

Figure 6. PIM1 knockdown causes a significant decrease in proliferation of JAK2V617F expressing cells……………………………………………………………………………………………..21

Figure 7. Pharmacological inhibition of PIM kinase causes a significant decrease in proliferation of JAK2V617F expressing cells………………………………………23

Figure 8. TP-3654 and Ruxolitinib synergize to cause a greater decrease in proliferation than either drug individually…………………………………………………………………25

Figure 9. TP-3654 and Ruxolitinib can synergize and significantly increase apoptosis of JAK2V617F cells……………………………………………………………………………………………27

Figure 10. Ruxolitinib did not effect the proliferation or signaling of BA/F3-EpoR-JAK2V617F resistant cells………………………………………………………………………………………29

Figure 11. PIM1 expression level is significantly increased in BA/F3-EpoR-V617F ruxolitinib-resistant cell line………………………………………………………………………………31

Figure 12. Pharmacological inhibition of PIM kinase significantly decreases proliferation of BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cells………………..32

Figure 13. PIM inhibition can resensitize BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line to Ruxolitinib treatment…………………………………………………………………34 

Figure 14. TP-3654 and Ruxolitinib synergize to inhibit the phosphorylation of downstream targets…………………………………………………………………………………………………………….36

List of Abbreviations:

Abbreviation         Full Word

BA/F3              Murine bone marrow-derived pro-B cell line

CLP              Common Lymphoid Progenitor

CMP                   Common Myeloid Progenitor

DHS           Donor Horse Serum

DMSO             Dimethyl Sulfoxide

EpoR               Erythropoietin Receptor

ET        Essential Thrombocythemia

FBS                Fetal Bovine Serum

HEL           Human Erythroleukemia cell line

HSC              Hematopoietic Stem Cell

IL-3                        Interleukin-3

IMDM                   Iscove’s Modified Dulbecco’s Media

JAK              Janus Family of nonreceptor tyrosine kinase

LT-HSCs                Long Term-Hematopoietic Stem Cell

MPN                  Myeloproliferative Neoplasm

MPP                 Multipotent Progenitor

PBS           Phosphate Buffered Saline

pI:pC          poly-Inosine:poly-Cytosine

PMF                   Primary Myelofibrosis

PV             Polycythemia Vera

RPMI                             Rosewell Park Memorial Institute medium

Ruxolitinib                JAK1/2 Inhibitor

SDS-Page          Sodium Dodecyl-Polyacrylamide Gel Electrophoresis

STAT     Signal Transducer and Activator of Transcription

ST-HSC               Short Term-Hematopoietic Stem Cell

TP-3654            Pan-PIM Kinase Inhibitor

Introduction:

Hematopoietic stem cells (HSCs) are the specialized stem cells, which produce a variety of blood cells. The process by which HSCs produce blood cells is hematopoiesis. Hematopoiesis can produce white blood cells (used in immunity) platelets (which are involved in coagulation) and erythrocytes (which transport oxygen throughout the body).1 The process of hematopoiesis occurs in the bone marrow. HSCs are classified into two subsets, long-term (LT-HSCs) and short term (ST-HSCs). LT-HSCs are capable of self-renew indefinitely, while ST-HSCs cannot. Typically, LT-HSCs can self-renew for the entire life of the host, while ST-HSCs can only self-renew for approximately 8 weeks.2 Both LT-HSCs and ST-HSCs exhibit multipotency, meaning they can develop into more than one type of cell. LT-HSCs give rise to ST-HSCs, which then go on to differentiate into multi-potential progenitors (MPPs).1,2 These MPPs cannot self-renew but are multipotent. MPPs can then go on to differentiate into one of two lineages, and either become common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs).1,3 CLPs give rise to T-lymphocytes, B-lymphocytes and natural killer cells (NKs), while CMPs are the common progenitors for megakaryocyte-erythrocyte progenitors (MEPs) and granulocyte-monocyte progenitors (GMPs).3 MEPs will eventually differentiate into either erythrocytes, or megakaryocytes, while GMPs will differentiate into granulocytes, or monocytes.4 In the bone marrow niches there are typically micro-environmental signals, which signal the ST-HSCs to undergo differentiation into a certain lineage depending on the body’s needs.4 A mutation in multipotent cells can lead to the development of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs), also known as myeloproliferative disorders (MPDs), were first proposed in 1951 by Dr. William Dameshek.5 MPNs are defined by a rare population of stem cells in the bone marrow that contain a certain initiating mutation, undergo clonal expansion, and self-renew indefinitely.6 The World Health Organization categorizes MPNs into two categories: 1) Philadelphia-chromosome positive (Ph+), also known as chronic myeloid leukemia (CML) and 2) Philadelphia-chromosome negative MPNs (Ph– MPNs). Philadelphia-chromosome positive means the BCR-ABL fusion gene is detected in the patient. 7 In classical Ph MPNs, there is clonal expansion of a mutated HSCs, which can give rise to all the myeloid cells, B cells, and NK cells. 8 The three classical, Ph MPNs include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). ET is most commonly characterized by a significant increase in platelet counts. PV is most commonly characterized by an increase in erythrocytes. PMF is characterized by the finding of fibrosis of the bone marrow with excess amount of collagen fibers. In all three MPNs a significant enlargement of spleen size (splenomegaly) is seen in the majority of patients.6 A group in 2012 reported the incidences of each of these MPNs as 22 to 27 per 100,000 for PV, 22 to 31 per 100,000 for ET, and approximately 2.4 per 100,000 for MF.9 They also reported that the mean age of the patients that presented with one of these MPNs was 53-61 years, but some patients presented at younger ages, which led them to suggest that MPNs can develop anytime in a person’s life. Interestingly, they also found that PV affects more males, while ET affects females more.9 They go on to conclude there must be some type of gender discrepancy in susceptibility to different MPNs.9

In 2005, four different groups discovered the same mutation in the pseudokinase domain of Janus kinase 2 (JAK2), which is an important kinase in the JAK-STAT pathway.  The mutation identified was the somatic mutation of guanine to thymine on exon 14 at nucleotide 1849 of JAK2.10–13 This mutation causes a valine to be switched for a phenylalanine at codon 617 in the JH2 psuedokinase domain of JAK2 (JAK2V617F).14 This JAK2V617F mutation was found to be prevalent in all three Ph MPNs. Researchers found that the JAK2V617F mutation is the most common mutation in all three Ph MPNs. As table 1 shows, recent data shows that JAK2V617F is much more prevalent than any other commonly found mutation in Ph– MPNs such as MPL, CBL, TET2 or EZH2. In approximately 95-97% of all PV patients and 50-60% of ET and MF patients, the JAK2V617F mutation has been reported.15  

JAK2 is typically regulated by the JH2 inhibitory domain and its interaction with the activation site JH1. JAK2 becomes activated when phosphorylated at the activation loop site of JH1, which can disrupt its interaction with JH2 and relieves the JH2 domain of inhibitory ability.7 When JAK2 undergoes the V617F mutation it becomes constitutively activated. In normal JAK2 signaling, a ligand  must binds to it’s receptor causing it dimerize. This dimerization can recruit JAK2, which then becomes phosphorylated and activated as previously described. When the JAK2 JH2 domain undergoes this V617F mutation it can no longer inhibit the phosphorylation of the JH1 site of JAK2 and it leads to activation of STAT, PI3K and MAPK, pathways even in the absence of ligand (Fig 1.). 12

Specifically when JAK2 is mutated to JAK2V617F it has been shown that there is phosphorylation and activation of STAT5 even in the absence of ligand.16 STAT5 is very important to JAK2V617F-derived MPNs and it has been shown that loss of STAT5 can prevent JAK2V617F-induced MPNs from developing.17 When STAT5 is phosphorylated it can form homodimers, which can be translocated into the nucleus. Inside the nucleus, dimers of STAT5 can recruit transcription elements and increase the transcription of specific genes.18 The genes regulated by STAT5 can play important roles in cell proliferation, apoptosis, cell differentiation, and inflammation.

In patients, the JAK2V617F mutation has been detected in LT-HSC (CD34+ CD38) and all HSC-derived lineages, demonstrating the mutation must be in a multipotent cell.19 Another study using a mouse model has shown that the disease-initiating cells must be LT-HSCs, and that it is a mutated LT-HSC that is responsible for initiating and maintaining the MPN disease.20 In MPNs, the initiating mutation gives a LT-HSC a selective advantage over normal LT-HSCs and causes clonal expansion of the mutated LT-HSC. In the case of JAK2V617F, this mutation causes the mutated LT-HSCs to have a selective advantage over normal HSCs and causes the mutated LT-HSC to preferentially clonally expand into CMPs rather than CLPs.20 This causes an increase in CMPs and eventually results in the phenotype seen in myeloproliferative patients.

The first JAK1/2 inhibitor to become FDA approved was Jakafi® (otherwise known as ruxolitinib, or INCB18424), which is approved for intermediate to high risk MF and for PV patients that do not respond to or are intolerant to hydroxyurea. The FDA approved ruxolitinib in November 2011 for these MF patients. Ruxolitinib oral tablets are available in doses of 5, 10, 15, 20, 25 mg that allows for individualized dosing regiments.  The COMFORT studies showed that treatment with ruxolitinib led to overall improvements in survival in MF patients when compared to placebo or best available therapy (BAT), which was typically hydroxyurea. Ruxolitinib treated patients showed a decrease in splenomegaly in 41.9% of patients versus 0.7% of the placebo treated patients during the COMFORT-I study.21,22 In the COMFORT-II study, ruxolitinib treated patients showed a decrease in splenomegaly in 28% compared to 0% in the BAT group.9,10 The most common side effect of the ruxolitinib compared to BAT was an increase in diarrhea (23 vs 12%) and physical weakness (18 vs 10%).22

One major problem in many cancer settings is the ability of cancer to become resistant to the drug the physician is treating it with. This leads to clinically short-termed remission, and many patients relapse with a much more aggressive cancer. Though ruxolitinib has shown positive effects for MPN patients, specifically MF patients, such as reduction in symptoms and spleen size, there is a need for secondary treatments. One reason there is a need for another treatment is some patients present with intrinsic resistance to ruxolitinib treatment. In one study, researchers measured the phosphorylation levels of STAT3 and STAT5, two known targets of JAK2, in 29 MPN patients and 6 normal donors.23 They found that in the MF patients there are some intrinsic properties that caused the cells to be resistant, and maintain high levels of p-STAT3 and p-STAT5, when exposed to a JAK2 inhibitor.23 Another study in 2013, studied 39 MPN patients who received ruxolitinib treatments between November 2009 and May 2013. Physicians defined 16 of the 39 (41%) patients as ruxolitinib-resistant.  Though 80% of non ruxolitinib-resistant patients showed spleen size reduction, only 60% of all the patients presented reductions in spleen size.24 This shows that though ruxolitinib has had positive results in many patients, other patients still do not respond to it well, and are in need of a secondary treatment for MPNs.

In vitro, there have also been studies done to show that ruxolitinib-sensitive cells can eventually become resistant. One of the way cells may become resistant to ruxolitinib treatment is by mutations in JAK1 and JAK2. In 2011, Hornakova and colleagues showed that mutations in Phe958 and Pro960 of JAK1 made the kinase constitutively active and resistant to JAK inhibitors.25 They also showed that a mutation in Tyr931 of JAK2 or JAK2V617F can cause JAK2 to be resistant to JAK inhibitors such as ruxolitinib.25 Another group in 2012 found that the mutations G935R, Y931C, and E864K, all made the JAK2 kinase resistant to JAK inhibitors.26 Though there have been no reported cases of these mutations happening in a patient and causing them to become resistant to ruxolitinib treatment, this is always a possibility. As mentioned earlier resistance is a serious problem in cancer treatment, and there is a need for a second line of defense against these MPNs.

In order to better study this JAK2V617F mutation and MPNs our lab previously generated a conditional JAK2V617F knock-in mouse. Using site-directed mutagenesis, the JAK2V617F floxed mouse was developed. This JAK2V617F- expressing mouse was then crossed with an MxCre mouse to generate an MxCre; JAK2V617F expressing mouse. Cre expression was then induced by intraperitoneal injections of polyinosine-polycytosine (pI:pC). After injection with pI:pC the JAK2V617F mutation is activated and the phenotype of the mouse becomes PV-like. This mouse model was then used to do gene analysis.  Using the MxCre; JAK2V617F and a control mouse, LT-HSCs were harvested and used for microarray gene analysis to identify differentially expressed genes. The heatmap (Fig. 2A) shows the differences in gene expression in the LT-HSCs of the JAK2V617F mouse when compared to the control mouse. Although many genes had differences in expression one specific gene of interest we saw an increase in was PIM1.  We also confirmed the increase in PIM1 expression, by running quantitative real-time PCR (Fig. 2B) on the LT-HSCs and by a Western blot of lysates from bone marrow of the WT mouse compared to the JAK2V617F expressing mouse (Fig. 2C).

PIM1 is serine/threonine kinase that has been shown to be important in many other cancers such as prostate cancer, breast cancer, and gastric cancer. 27 PIM1 is named after the proviral integration site of Moloney virus.28 It was originally found in mouse lymphoma samples due to the integration of the Moloney leukemia virus, which integrates into the 3′- region of PIM1 gene. This integration caused a premature stop codon, which led to the PIM1 transcript becoming more stable and long-lasting.27 This increased stability of the transcript led to increased translation of the transcript and more PIM1 being made at the ribosomes.27,28 PIM1 has been shown to regulate cell proliferation and inhibit apoptosis, which made researchers believe it could be an important kinase in cancer biology.

PIM1 has been shown to directly interact with the B-cell lymphoma-extra large (BCL-xl)/Bcl-2 associated death promoter (BAD). PIM1 can phosphorylate the protein BAD on Ser112. 27,29 This phosphorylation causes BAD to bind to 14-3-3 proteins and results in cytoplasmic sequestration of the p-BAD 14-3-3 complexes.27,30 Normally, when the cell is not undergoing apoptosis, Bcl-2 and Bcl-xl are in a heterodimer complex with BAX in the mitochondrial membrane. When BAD is dephosphorylated it enters the mitochondria and can displace BAX by binding Bcl-xl and Bcl-2.  Displaced BAX can then form homodimers and create pores in the mitochondria membrane that allow cytochrome c to be released from the mitochondria into the cytoplasm. 27,30 The release of cytochrome c can then induce apoptosis in the cell through the Caspase 9/3 pathway.

PIM1 can also cause an increase in cellular proliferation. The first known mechanism of how increased PIM1 expression caused an increase in cell proliferation is through CDC25A. CDC25A is a cell cycle phosphatase, which can be phosphorylated by PIM1.31 Phosphorylation of CDC25A by PIM1 can help promote the transition of the cells from the G1 phase to the S phase.27,31 PIM1 can also cause an increase in proliferation by phosphorylating CDC25C-associated kinase 1 (C-TAK1). When C-TAK1 is phosphorylated by PIM1, its activity is decreased. When C-TAK1 activity is decreased it reduces phosphorylation and inactivation of CDC25C, another phosphatase that promotes the transition of G2 to M phase in cell cycle. 27,32

Lastly, PIM1 has been shown to alter proliferation rates by phosphorylating and inactivating p21Cip1/WAF1 and p27Kip1. p21Cip1/WAF1 and p27Kip1 are potent cell cycle inhibitors.33,34 During times when the cell is not proliferating p21Cip1/WAF1 binds to proliferating cell nuclear antigen (PCNA). Phosphorylation of p21Cip1/WAF1 by PIM1 has been shown to increase the dissociation of PCNA and p21Cip1/WAF1, which can lead to an increase in proliferation. 27,33 PIM1 can also phosphorylate p27Kip1, which can promote its binding to the 14-3-3 protein. This complex can be exported to the cytoplasm, which then leads to its proteasomal degradation.27,34 By decreasing the effects of p21Cip1/WAF1 and p27Kip1, PIM1 can increase the proliferation in cells.

Other studies have revealed mechanisms by which PIM1 can increase protein synthesis. One study showed that PIM1 transfected cells are able to maintain high levels of phosphorylation of 4E-BP1.27,35 This increased phosphorylation can lead to the release of eIF-4E, which can go on to initiate translation of any available mRNAs. 27 These are some of the ways increased PIM1 expression has been shown to increase cell proliferation, decrease apoptosis, and initiate protein translation.

Recently, Tolero Pharmaceuticals designed a second-generation, selective PIM kinase inhibitor called TP-3654. TP-3654 can inhibit PIM1 and PIM3 at its lower concentrations, and inhibits PIM2 at higher concentrations.36 Like all kinase inhibitors TP-3654 does inhibit other kinases at higher concentrations, but as the in vitro selectivity profile below shows it is highly specific for PIM1 (Fig. 3B). TP-3654 is a promising pharmaceutical inhibitor because as shown in the selectivity profile (Fig. 3B), TP-3654 can inhibit all isoforms of PIM while only slightly inhibiting FLT3 and hERG.36 A first-generation PIM kinase inhibitor SGI-1776 went into phase I clinical trials. SGI-1776 was removed from clinical trials because it had cardiotoxicity through suppression of hERG cardiac potassium channel.37 TP-3654 needs to be further investigated, but as of now the results have been promising. TP-3654 can selectively inhibit PIM1 with only limited off-target effects.

The goal of this project was to investigate the effects of targeting PIM1 in these Ph JAK2V617F-positive myeloproliferative neoplasms. To do this, we used lenti-viral shRNA mediated knockdowns of PIM1 in JAK2V617F-positive cell lines to study the effects. We also used the PIM inhibitor TP-3654 to examine the effects of pharmacological PIM kinase inhibition on these cell lines. Lastly, we studied whether PIM kinase inhibition could resensitize ruxolitinib-resistant cell lines.

Materials/Methods:

Patient Data:

Publically available microarray data was used to check PIM1 expression in 94 MPN patients and 10 healthy controls. Using geodata set GSE54646 published by Rampal and collegues, in June of 2014, we checked the levels of PIM1 expression of PV, ET, and MF patients when compared to healthy individuals.38 Students t-tests were used to check for significant differences.

Cell Culture:

Murine Ba/F3-EpoR, Ba/F3-EpoR-JAK2V617F, human erythroleukemia (HEL), and UKE-1 cells were used.  Ba/F3 cells are a murine derived pro-B cell line derived from mouse bone marrow. Ba/F3-EpoR cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in the presence of 1 ng/mL murine interleukin-3 (IL-3) and antibiotics (penicillin/streptomycin). The Ba/F3-EpoR-JAK2V617F cell line was generated by transducing Ba/F3-EpoR cell line with MSCV-IRES-GFP retrovirus expressing JAK2V617F and sorting for GFP positive cells. Ba/F3-EpoR-JAK2V617F cells were cultured in RPMI-1640 medium supplemented with 10% FBS and antibiotics. HEL cells were developed from the bone marrow of a patient with erythroleukemia. HEL cells were used because they are a human cell line that expresses the JAK2V617F mutation. HEL cells were cultured in RPMI-1640 with 10% FBS and antibiotics. UKE-1 cells were developed from the bone marrow of a patient that had ET, which eventually transformed into acute leukemia. UKE-1 cells, like the HEL cells, were used because they are a human cell line that expresses the JAK2V617F mutation. UKE-1 cells were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10% FBS, 10%

horse serum, 1 µM hydrocortisone, and antibiotics.

Gene knockdown and Proliferation assays:

To assess cell proliferation, we used two lenti-viral shRNAs. Lenti-viral shRNA sequences are shown in table 2.  All lenti-virus sequences were cloned into pLKO.1 puro vectors. Lenti-viral shRNAs were produced by plating 5 x 106 human embryonic kidney 293T cells (HEK-293T) into  6 cm dishes with 4 mL of DMEM media supplemented with 10% FBS and antibiotics. 24 hours after plating the cells, the HEK-293T cells were transfected with 3 µG of the DNA of the lenti-virus, 1.5 µg REV, 1.5 µg RRE, 1.5 µg of VSVG, and 30 µL of PEI (1 mg/mL). REV, RRE, and VSVG are packaging components that help the virus, enter the cells, replicate, and be released.

24 hours post transfection, the media was changed and fresh DMEM with 10% FBS and antibiotics was added. 48 hours after the transfection the lenti-virus was harvested and filtered through a 0.45 µm filter. After harvesting the virus, fresh DMEM with 10% FBS and antibiotics was added to the plate. The next day, 72 hours post transfection, the virus was harvested for the second time and plates were discarded. Viruses were stored at -80° for long-term storage.

Viruses were then used to infect the different cell lines and knockdown PIM1. pLKO.1 puro scramble vectors were also used to infect the cells as a control. 5 x 105 cells were plated in 1 mL of media in wells of a 6 well plates. Then 2 milliliters of virus, 30 µL HEPES (1 M) and 4 µL of Polybrene (6 µg/µL), were added to infect the cells with the lenti-virus. Since the vectors we used for the infections had a puromycin resistant gene, 48 hours after infection, cells were treated for 48 hours with 2µM of puromycin to select out any cells that did not get infected. Western blot was used to confirm the knockdown for each infection. To assess cell proliferation 5 x 10cells were plated in duplicate into a 6 well plate. Using trypan blue exclusion, viable cell counts were performed at 24-hour intervals for 5 consecutive days.

All drug proliferation assays were done by plating 5×105 -1×10cells in wells of a 6 well plate. Drugs used were INCB018424 (ruxolitinib) and TP-3654 (PIM inhibitor provided by Tolero Pharmaceuticals), and were dissolved into dimethyl sulfoxide (DMSO) to the appropriate concentrations. Drugs were added to the wells at appropriate concentrations at 24-hour intervals. Cells were counted by using trypan blue exclusion every 24 hours for 5 consecutive days.

Quantitative Real-Time PCR

Total RNA was isolated from cells using RNeasy Mini Kit (Qiagen) and reverse transcribed using QuaniTect Reverse Transcription kit (Qiagen). Quantitative real-time PCR was done using SYBER Green PCR master mix (Qiagen) and was analyzed using the LightCycler 480 Real-Time PCR system (Roche Applied Science). GAPDH quantification was used to normalize the results. The primers that were used in these experiments are as follows in table 3.

Lysing and Immunoblotting

Cells were lysed 24 hours after puromycin selection or 8 hours after drug treatment using RIPA lysis buffer and a cocktail of proteasome inhibitors. RIPA Lysis buffer was prepared using 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 1% Sodium Deoxycholate, 0.1% SDS, and dH2O. Lysis buffer was prepared using RIPA lysis buffer with 5mM Na3VO4, 15 mM NaF, 30 mM β-Glycol Phosphate, 30 µg/mL, Leupeptin, 30 µg/mL Aprotonin, 6 µg/mL Pepstatin A, 6 µg/mL Antipain, and 300 µg/mL PMSF (Sigma-Aldrich). Samples were then lysed and 2X sample buffer was added. Samples were then boiled at 100° C for 10 minutes. Immunoblotting was performed after running an SDS page gel. Antibodies used are as follows: phospho-STAT5 (Tyr694), phospho-S6 ribosomal (Ser235/236), S6 ribosomal, phospho-4EBP1 (Thr37/46), phospho-p70S6K (Thr389), p70S6K, PIM1 (Cell Signaling); 4EBP1, STAT5, PIM2 (Santa Cruz); and β-actin (Sigma-Aldrich).

 

Apoptosis Assay:

Apoptosis assays were performed 48 or 72 hours after drug treatments. Cells were harvested and washed twice with cold phosphate-buffered saline (PBS). Cells were then counted and resuspended at 1 x 106/mL in 1X binding buffer (eBioscience).  1 x 105 cells was transferred to a FACS tube and stained with 3 µL of APC-Annexin V and 3 µL of Propidium iodide (PI) for 15 minutes. Then 400 µL of 1X binding buffer was added and cells were analyzed using BD LSR II Flow Cytometer.

Statistical Analysis:

The Student’s t-test was used to compare between the groups. Results are expressed as mean values ± standard error of mean (SEM). * stands for p-value <0.05 and ** stands for p-value <.005.

 

 

Results:

PIM1 expression is significantly increased in MPN patients:

Using publically available microarray data we examined PIM1 levels in MPN patients.38 This data set was comprised of 94 MPN patients and 10 healthy controls. Using this data set we analyzed PIM1 levels in these patients and found that PIM1 levels were significantly increased in MPN patients compared to the healthy controls. Figure 4A-C shows that in all three Ph MPNs PIM1 levels were significantly increased. This data set also showed whether the patient was JAK2V617F-positive or negative. Figure 5A-C shows that there was trend of increased PIM1 expression in JAK2V617F negative MPN patients relative to healthy controls, while patients that were JAK2V617F-positive had a significant increase in PIM1 expression.

 

 

 

 

 

 

 

 

 

 

 

 

Knockdown of PIM1 significantly reduces proliferation of JAK2V617F- expressing cell lines:

 

Using two different pLKO.1 puro lentiviruses that targeted PIM1, and pLKO.1 puro scramble vector, we infected 4 different cell lines to knock down PIM1 expression. We then selected out uninfected cells by using puromycin. After selection we then confirmed knockdown in each cell group by Western blot. Upon successful knockdown of PIM1 we plated the same number of cells per plate and monitored the 5-day proliferation of the cells. Live cells were counted every 24 hours using trypan blue exclusion. We found that when we knock down PIM1 in JAK2V617F-negative BA/F3 cells there was little to no effect on the proliferation (Fig. 6A). When we did the knockdown of PIM1 in cells expressing JAK2V617F, such as BA/F3-EpoR-V617F (Fig. 6B), HEL (Fig. 6C), and UKE-1 (Fig. 6D) we saw a significant decrease in cell proliferation. This led us to the conclusion that knocking down PIM1 in JAK2V617F expressing cells leads to a significant decrease in proliferation. This suggests that PIM1 plays an important role in the JAK2V617F mediated cell growth.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inhibition of PIM kinases significantly reduces proliferation of JAK2V617F-expressing cell lines.

 

We next used TP-3654, a pan PIM kinase inhibitor designed by Tolero pharmaceuticals, to evaluate the effect of pharmacological inhibition of PIM kinases on cell lines expressing the JAK2V617F mutation. We plated an equal number of cells per well in a 6 well plate, and added the drug at different concentrations. We counted the live cells in each well at 24-hour intervals using trypan blue exclusion and a hemocytometer. We found that pharmacological inhibition of PIM kinase had only a slight effect on proliferation of BA/F3 cells that aren’t expressing the JAK2V617F mutation (Fig. 7A). However, when we treated the JAK2V617F expressing cells, such as BA/F3-EpoR-JAK2V617F (Fig. 7B), HEL (Fig. 7C), and UKE-1 (Fig. 7D), with the PIM kinase inhibitor (TP-3654) we saw a significant dose dependent decrease in proliferation. Figure 7 below shows the day 5 percentage of proliferation when compared to the control of each cell type. This led us to the conclusion that pharmacological inhibition of PIM kinases can significantly decrease proliferation of JAK2V617F-expressing cells, while only exhibiting a slight effect on JAK2V617F-negative cells.

 

 

 

 

 

Figure 7.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PIM inhibitor synergizes with ruxolitinib to inhibit cell proliferation and induce apoptosis in JAK2V617F-expressing cells.

 

Using the PIM inhibitor TP-3654 and the FDA approved JAK1/2 inhibitor ruxolitinib, we wanted to check to see if these two drugs could work synergistically to inhibit proliferation and increase apoptosis. By plating the same number of cells per well, we again did a 5-day cell proliferation assay by counting the cells at 24-hour intervals using trypan blue exclusion. Using two different JAK2V617F expressing cell lines, BA/F3-EpoR-V617F (Fig. 8A) and UKE-1 (Fig. 8B), we show in figure 8 that treatment with the combination of TP-3654 and ruxolitinib leads to a greater decrease in proliferation at lower concentrations than achievable by either of the drugs alone at the same concentration. From this data we concluded that the combination of ruxolitinib and TP-3654 can inhibit proliferation more at lower concentrations when used against JAK2V617F expressing cells. This finding could someday be part of the rationale which allow physicians to prescribe the combination of each drug in lower concentrations for JAK2V617F-positive patients and achieve better results.

Not only did we show that the combination of TP-3654 and ruxolitinib leads to a greater decrease in proliferation, we also showed that the combination therapy can significantly increase apoptosis in JAK2V617F expressing cell lines. Using annexin V and propidium iodide staining and flow cytometry, we looked for increases in apoptotic cells in the drug treatments. TP-3654 and ruxolitinib had no significant effects alone, or in combination, on apoptosis in BA/F3 cells not expressing JAK2V617F (Fig. 9A). However in BA/F3-EpoR-JAK2V617F cells TP-3654 and ruxolitinib were able to significantly increase apoptosis alone and the combination therapy showed an even greater increase in apoptosis (Fig. 9B).  This demonstrates that TP-3654 and ruxolitinib can work synergistically to inhibit proliferation and increase apoptosis in JAK2V617F expressing cell lines. This is an important finding because, by using the combination, we can use lower concentrations of both drugs, which should help reduce side effects while achieving a greater effect on JAK2V617F-expressing cells.

JAK2 inhibitor resistant BA/F3-EpoR-JAK2V617F cells are not inhibited by ruxolitinib treatment.

 

We utilized a BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line that was created by culturing the cell in increasing concentrations of ruxolitinib. Before doing any experiments with these cells we wanted to make sure that this BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line was truly resistant to ruxolitinib treatment. To do this we treated the BA/F3-EpoR-JAK2V617F sensitive cell line with ruxolitinib and compared the results to those obtained by treating the ruxolitinib-resistant cell line with ruxolitinib. Normally when BA/F3-EpoR-JAK2V617F cells are treated with ruxolitinib it leads to a decrease in the phosphorylation of STAT5 (Fig. 10A). When we treated the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cells with ruxolitinib, we saw no decrease in the phosphorylation of STAT5 (Fig. 10B). We then wanted to confirm ruxolitinib treatment wouldn’t decrease proliferation in the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line. By plating the same number of cells and doing 24-hour interval cell counts, using trypan blue exclusion, we checked the cells proliferation. We saw that treatment with ruxolitinib did not decrease proliferation in the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line (Fig. 10C). This convinced us that this cell line was truly resistant to ruxolitinib treatment and that further experiments could be done using the cells.

 

PIM1 expression is significantly increased in the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line.

 

Since in other settings, such as prostate cancer, PIM1 expression has been shown to be increased in drug resistant cells, we wanted to asses the PIM1 expression in this BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line.39 By using quantitative real-time PCR, we first checked the PIM1 mRNA levels in BA/F3, BA/F3-EpoR-V617F, and BA/F3-EpoR-V617F ruxolitinib-resistant cell lines. We found that the cells containing the V617F mutation had a significant increase in PIM1 mRNA expression levels compared to the BA/F3 cells (Fig. 11A). We also found that the BA/F3-EpoR-V617F ruxolitinib-resistant cell line had a significant increase in PIM1 mRNA levels when compared to the BA/F3-EpoR-V617F cell line (Fig. 11A). We confirmed that the BA/F3-EpoR-V617F ruxolitinib-resistant cell line had increased PIM1 expression by checking the protein levels by Western blot (Fig. 11B). By using Western blot and real-time PCR, we concluded that the BA/F3-EpoR-V617F ruxolitinib-resistant cell line has a significant increase in PIM1 expression when compared to the BA/F3-EpoR-JAK2V617F sensitive cell line.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

BA/F3-EpoR-V617F ruxolitinib-resistant cells are sensitive to PIM kinase inhibition.

Since the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line had increased PIM1 expression, we hypothesized that the PIM inhibitor TP-3654 would be able to inhibit this cell line’s proliferation. To check whether the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cells were sensitive to PIM kinase inhibition we did a 5-day proliferation assay by counting the live cells at 24-hour intervals, by trypan blue exclusion.  Though this cell line was resistant to ruxolitinib, we found that it was still sensitive to the PIM kinase inhibitor TP-3654. We found that treatment of the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cells with TP-3654 could significantly reduce the cells proliferation (Fig. 12). This is an important finding because a PIM inhibitor could in the future help patients that don’t respond to ruxolitinib treatment in the future.

Pharmacological inhibition of PIM kinase can resensitize ruxolitinib-resistant cells to ruxolitinib treatment.

 

Since overexpressing PIM1 has been shown to lead to drug resistance in some settings, we hypothesized that inhibiting PIM kinases using TP-3654 would resensitize the ruxolitinib-resistant cells to ruxolitinib treatment.39 To see whether we could resentize the cells we did a 5-day proliferation by plating equal amounts of the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cells and added either ruxolitinib, TP-3654, or a combination of both drugs. By counting the cells at 24-hour intervals using trypan blue exclusion, we found that the combination of TP-3654 and ruxolitinib had an even greater decrease in proliferation than TP-3654 alone, and as expected ruxolitinib alone had no effect (Fig 13.). This led us to the conclusion that by adding the PIM kinase inhibitor TP-3654, we were effectively resensitizing ruxolitinib-resistant cells to ruxolitinib treatment.

To further investigate whether we were truly resensitizing the BA/F3-EpoR-JAK2V617F cells to ruxolitinib by using the PIM inhibitor, we did some Western blots to check known targets of JAK1/2 inhibition or PIM kinase inhibition. One verified target of JAK1/2 inhibition or ruxolitinib treatment is a decrease in STAT5 phosphorylation. As stated earlier, when we treated the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line with ruxolitinib we did not see any decrease in the phosphorylation levels of STAT5. However, when we treated the ruxolitinib-resistant cells with the combination of TP-3654 and ruxolitinib, we saw decreases in phospho-STAT5 levels which helped support the conclusion that PIM kinase inhibition, using TP-3654, was resensitizing the resistant cells to ruxolitinib treatment (Fig. 14). We then further looked into some of the downstream signaling effects of the combination of PIM and JAK inhibition and found that in the combination drug treatment there was the largest decrease in phosphorylation of p70S6K, S6 ribosomal kinase, and 4EBP1. S6 ribosomal and 4EBP1 are proteins that have been found to interact with 40S ribosomal unit, and decreases in phosphorylation of these kinases lead to decreases in translation. This further strengthened our hypothesis that these two drugs work synergistically to inhibit proliferation in these JAK2V617F cell lines (Fig. 14).

 

 

 

 

 

Discussion:

Currently many cancer research groups are researching targeted therapies to try to find a more specific way to kill cancerous cells, with limited side effects to noncancerous cells. In Ph MPNs the most common mutation found is the JAK2V617F mutation, which has been found in ~95% of PV patients and 50-60% of all PMF and ET patients.16 This JAK2V617F mutation causes constitutive activation of JAK2 and its downstream signaling such as STAT5. JAK2 and STAT5 have been shown to be required for the majority of the phenotypes found in JAK2V617- mediated MPNs.17 The first kinase inhibitor to be approved by the FDA for targeted therapy in these Ph JAK2V617F-positive MPNs was the JAK1/2 inhibitor ruxolitinib. Though ruxolitinib showed promising results in vitro, in the clinical setting it is not providing long-term remission, and some patients are nonresponsive to ruxolitinib treatment. Additionally, since clinical treatment with ruxolitinib treatment only began fairly recently, many patients may eventually become resistant to JAK1/2 inhibitors. This led to the need for novel targets in JAK2V617F-induced MPNs. We become interested in finding novel targets that can be inhibited in order to achieve response and remission in ruxolitinib unresponsive patients, or to have as a second line of defense for patients who may eventually become ruxolitinib-resistant.

PIM kinases, specifically PIM1, are quickly becoming important cancer targets. One paper recently showed that PIM1 is important in cell death, tumor growth, and chemotherapy resistance in triple negative breast cancer.40 A review paper also states that PIM1 plays an important in prostate cancer, mesothelioma, esophageal cancer, and T-cell acute lymphoblastic leukemia (T-ALL).41 PIM1 has been shown in other settings to prevent apoptosis by phosphorylating BAD on Ser112 and preventing its association with BCL-xl and BCL-2. This phosphorylation of BAD on Ser112 prevents BAX displacement from BCL-2 or BCL-xl, therefore cytochrome c not being released.41 PIM1 has been shown to play an important role in proliferation of cells by phosphorylating CDC25A, a phosphatase that can transition cells from G1 phase to S phase.  PIM1 has also been known to alter proliferation rates by phosphorylating p21 and p27 resulting in increased degradation of these proteins, which can cause an increase in the proliferation of the cells. 33,34

We became interested in PIM1 after designing an inducible JAK2V617F mouse model, and finding PIM1 expression was increased in the mouse expressing JAK2V617F compared to the WT mouse. First we checked the levels of PIM1 in Ph MPN patients by using publically available microarray data published by Rampal et al. and we found that PIM1 levels were significantly increased in MPN patients as a whole (Fig. 4).38 We further looked into patient data and found that there was a trend of increased PIM1 expression in JAK2V617F negative patients, and a significant increase in PIM1 expression in JAK2V617F-positive MPN patients (Fig. 5).

In our study we found, using two different approaches, PIM inhibition by shRNAs or PIM inhibitor TP-3654, results in decreased proliferation of JAK2V617F-positive cell lines.  We also showed that TP-3654 works synergistically with ruxolitinib to inhibit proliferation and induce apoptosis in cell lines containing the JAK2V617F mutation. These findings are significant because it suggests that the combination of ruxolitinib and TP-3654 would be a more beneficial treatment for JAK2V617F positive patients, than just ruxolitinib alone. Though further studies need to be done on the safety of taking ruxolitinib and TP-3654 together, but as of now, our in vitro results show that we can use lower concentrations of each drug to someday possibly treat JAK2V617F-induced MPNs.

In many cancer settings acquired resistance in patients or patients not responding to the targeted therapy is a big problem. Since there is literature that identifies increased PIM1 expression can lead to resistance, we generated a ruxolitinib-resistant cell line, and checked the expression of PIM1.39 Using the BA/F3-EpoR-JAK2V617F ruxolitinib-resistant cell line we found increased PIM1 expression, and that TP-3654 could significantly inhibit this cell lines proliferation. This suggests that in the future it would be beneficial for physicians to check PIM1 levels in patients who become resistant to ruxolitinib. The in vitro data we generated suggests that if the physician finds increased PIM1 levels in resistant patients it may be a better idea to treat with a PIM inhibitor, rather than ruxolitinib. This is a very important finding because it identifies a new therapeutic target in ruxolitinib-resistant JAK2V617F-induced MPNs, and suggests that physicians could use PIM inhibitors to resensitize these resistant patients.

Further studies are still being conducted to identify the role of PIM1 in these JAK2V617F-induced MPNs. Firstly, our lab is working on generating a PIM1 knock-out, MxCre; JAK2V617F knock-in mouse and analyzing how this mouse progresses compared to a MxCre;JAK2V617F knock-in mouse. Using this mouse model we can see how complete deletion of PIM1 in a mouse model affects the development of JAK2V617F induced MPNs. Also to further investigate the role of PIM1 in the progression of JAK2V617F-induced MPNs, we are currently treating JAK2V617F-expressing mice with the inhibitor TP-3654. We also are treating some mice with the combination of TP-3654 and ruxolitinib to further identify how these drugs may be working synergistically to inhibit proliferation of JAK2V617F-positive cells. Lastly, some initial data suggests that PIM2 levels are also increased in MPN patients, and our JAK2V617F mouse model. We need to further investigate what role PIM2, or even PIM3, play in JAK2V617F-induced MPNs.

In conclusion PIM1 plays an important role in JAK2V617F-positive cells proliferation, apoptosis, and resistance to drugs. Though in vivo data of PIM1 knockout, or PIM kinase inhibition is still being generated the in vitro data is very promising. An inhibitor such as TP-3654 that is very specific for PIM kinases could someday help physicians with targeted therapy in MPN patients. The overall hypothesis of this study was targeting PIM1 kinase we would be able to reduce proliferation of JAK2V617F expressing cells, which we examined two different ways using the knockdown and inhibitor. We also found that by targeting PIM kinases with inhibitor TP-3654 can synergize with ruxolitinib and decrease proliferations and increase apoptosis at lower concentrations. Lastly our studies show that by using TP-3654 we could resensitize ruxolitinib-resistant cells to ruxolitinib treatment. This all led us to our final conclusion that PIM1 plays a very important role in the JAK2V617F induced MPNs and by inhibiting PIM1 we could effectively decrease proliferation and increase apoptosis. This is an important finding because PIM1 is a novel target in JAK2V617F-induced MPNs and as stated earlier, there is a need for novel targets in these patients.

 

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