HMGA1 Chromatin Regulators Drive Progression in Myeloproliferative Neoplasms through Epigenetic Rewiring to Induce Networks Involved in GATA2 and Proliferation

Abstract Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemic transformation. The High Mobility Group A1 (HMGA1) gene encodes oncogenic chromatin regulators which are overexpressed in diverse tumors where they portend adverse outcomes (Resar Cancer Res 2010; Xian et al Nature Commun 2017). Hmga1 induces leukemic transformation in transgenic mice and HMGA1 is overexpressed in refractory myeloid malignancies (Resar et al Cancer Res 2018). Further, germline lesions within the HMGA1 loci increase the risk for developing MPN (Bao et al Nature 2020). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by rewiring transcriptional networks to foster leukemogenesis, and, 2) identify mechanisms underlying HMGA1 that could be targeted with therapy. Methods: To elucidate the function of HMGA1, we disrupted HMGA1 expression via CRISPR/Cas9 or short hairpin RNA (shRNA) targeting 2 different sequences per gene and assessed proliferation, colony formation, apoptosis, and leukemogenesis. We also generated JAK2 V617F transgenic mouse models of MF with Hmga1 deficiency. To dissect molecular mechanisms underlying HMGA1, we integrated RNAseq, ATACseq, and chromatin immunoprecipitation (ChIP) from MPN-AML cell lines (DAMI, SET-2). Next, we tested whether HMGA1 depletion synergizes with ruxolitinib in preventing leukemic engraftment in mice. To identify drugs to target HMGA1 networks, we applied the Broad Institute Connectivity Map (CMAP). Results: HMGA1 is overexpressed in CD34 + cells from patients with JAK2 V617F MPN with highest levels after transformation to MF or AML in 3 independent cohorts. CRISPR/Cas9 inactivation or shRNA-mediated HMGA1 silencing disrupts proliferation, decreases the frequency of cells in S phase, increases apoptosis, and impairs clonogenicity in human MPN-AML cell lines. HMGA1 depletion also prevents leukemic engraftment in mice. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2 V617Fmurine models of MPN, decreasing erythrocytosis, thrombocytosis, and preventing splenomegaly and fibrosis of the spleen and bone marrow. Further, Hmga1 deficiency preferentially prevents expansion in long-term HSC, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors in JAK2 V617F mice. RNAseq revealed genes induced by HMGA1 that govern cell cycle progression (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) and cell fate decisions (GATA2 networks), including the GATA2 master regulator gene. Silencing GATA2 recapitulates anti-leukemia phenotypes observed with HMGA1 deficiency whereas restoring GATA2 in MPN-AML cells with HMGA1 silencing partially rescues leukemia phenotypes, increasing clonogenicity and leukemic engraftment. Mechanistically, HMGA1 binds directly to AT-rich sequences near the GATA2 developmental enhancer (+9.5), enhances chromatin accessibility, and recruits active histone marks (H3K4me1/3) to induce GATA2 expression. HMGA1 depletion enhances responses to the JAK/STAT Inhibitor, ruxolitinib, delaying leukemic engraftment and prolonging survival in murine models of JAK2 V617F MPN-AML. Further, epigenetic drugs predicted to target HMGA1 transcriptional networks using CMAP synergize with JAK inhibitors to disrupt proliferation in human MPN-AML cells. HMGA1 and GATA2 are co-expressed and up-regulated with progression from MF to AML in matched patient samples. Moreover, HMGA1 transcriptional networks are activated in leukemic blasts, thus underscoring the role of HMGA1 in human MPN progression. Conclusions: We uncovered a previously unknown epigenetic program whereby HMGA1 enhances chromatin accessibility and recruits activating histone marks to induce transcriptional networks required for progression in MPN, including direct transactivation of GATA2. Further, HMGA1 networks can be targeted with epigenetic therapy and synergize with ruxolitinib. Together, our studies reveal a new paradigm whereby HMGA1 up-regulates GATA2 and proliferation networks to drive disease progression and illuminate HMGA1 as a novel therapeutic target in MPN. Figure 1 Figure 1. Disclosures Rampal: Jazz Pharmaceuticals: Consultancy; Incyte: Consultancy, Research Funding; Kartos: Consultancy; Constellation: Research Funding; Pharmaessentia: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; Stemline: Consultancy, Research Funding; BMS/Celgene: Consultancy; Novartis: Consultancy; Sierra Oncology: Consultancy; CTI: Consultancy; Abbvie: Consultancy; Memorial Sloan Kettering: Current Employment. Stubbs: Incyte Research Institute: Current Employment, Current holder of individual stocks in a privately-held company.

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Development of a Murine Model for Leukemic Transformation of Myeloproliferative Neoplasms for Preclinical Therapeutic Studies

Blood ◽

10.1182/blood.v120.21.808.808 ◽

2012 ◽

Vol 120 (21) ◽

pp. 808-808 ◽

Cited By ~ 1

Author(s):

Raajit K. Rampal ◽

Suveg Pandey ◽

Omar Abdel-Wahab ◽

Jennifer J Tsai ◽

Taghi Manshouri ◽

...

Keyword(s):

Murine Model ◽

Research Funding ◽

Somatic Mutations ◽

Myeloproliferative Neoplasms ◽

Survival Curve ◽

Philadelphia Chromosome ◽

In Vivo Studies ◽

Leukemic Transformation

Abstract Abstract 808 A subset of patients with Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) (Polycythemia Vera (PV), Essential Thrombocytosis (ET), and Primary Myelofibrosis (PMF)) subsequently transform to acute myeloid leukemia (AML). Leukemic transformation (LT) after MPN occurs in as many as 23% of PMF patients within 10 years of diagnosis, and in 4–8% of PV and ET patients in the first 18 years after diagnosis. The development of AML after an antecedent MPN is associated with a dismal clinical outcome, and is associated with a poor response to conventional anti-leukemic therapies. Although somatic mutations in the JAK-STAT signaling pathway, including in JAK2 and MPL, occur in the majority of MPN patients, the somatic mutations that drive LT from a pre-existing MPN have not been fully delineated. Recent candidate mutational studies have identified recurrent somatic mutations in a subset of known leukemogenic disease alleles at the time of transformation from MPN to AML, including mutations in TP53, IDH1/2, TET2 and SRSF2 as well as deletions in IKZF1. However, the functional contribution of these specific genetic events to LT has not been delineated, and genetically accurate models of transformation of Philadelphia-chromosome negative MPN to AML have not been reported to date. In order to develop a genetically accurate murine model of LT, we have modeled expression of JAK2V617F mutation in combination with TP53 loss in vivo to further our understanding of progression from MPN to AML and to use this preclinical model of LT to test novel therapies. Bone marrow (BM) cells from C57/Bl6 Tp53−/− and littermate control mice were infected with JAK2V617F-IRES-GFP retrovirus, followed by transplantation of transduced cells into lethally irradiated congenic recipients. Of note, transplantation of JAK2V617F/Tp53−/− cells, but not JAK2V617F positive cells was associated with impaired survival; 50% of mice injected with JAK2V617F/Tp53−/− cells died by day 100, whereas all mice injected with JAK2V617F positive cells survived 100 days or longer (p=0.011) (figure 1). Mice injected with JAK2V617F/Tp53−/− cells presented with significant leukocytosis, with a mean WBC of 38.4 in mice engrafted with JAK2V617F/Tp53−/− cells compared with 11.4 in JAK2V617F/Tp53 wildtype mice. At the time of sacrifice, all mice engrafted with JAK2V617F/Tp53−/− cells had increased numbers of blasts in the peripheral blood and bone marrow, as assessed by morphologic evaluation and flow cytometric analysis which noted CD117 expression on leukemic blasts. BM cells from mice engrafted with JAK2V617F/Tp53−/− cells were characterized by increased serial replating (>10 platings), which was not observed in plating studies with JAK2V617F positive cells. In addition, we noted that the disease from JAK2V617F/Tp53−/− cells, but not JAK2V617F positive cells, was transplantable into secondary recipients consistent with increased self-renewal in vivo. We have begun testing the efficacy of novel therapies in this murine model, using both in vitro assays and in vivo studies in secondary transplantation studies. Treatment with the JAK kinase inhibitors INCB18424 and CYT 387 resulted in dose-dependent inhibition of colony formation in vitro. The combination of INCB18424 and Decitabine (which has demonstrated clinical efficacy in post-MPN-AML) is associated with synergistic inhibitory effects in vitro. Based on these results, we are performing in vivo studies with INCB18424, Decitabine, and INCB18424 + Decitabine, and results from these preclinical therapeutic studies will be presented in detail. Taken together, our data demonstrate that expression of JAK2V617F plus Tp53 loss, a genoptype commonly seen in patients who transform to AML after MPN, efficiently models LT in vivo. This model can now be utilized to examine the mechanisms of leukemic transformation, including assessment of the leukemic cell of origin in transformed disease. In addition this model can be utilized to test novel therapeutic strategies in a preclinical setting, which can be used to inform clinical trials in this poor-risk hematologic malignancy. Figure 1: Survival curve of mice transplanted with JAK2V617F in presence and absence of Tp53 Figure 1:. Survival curve of mice transplanted with JAK2V617F in presence and absence of Tp53 Disclosures: Verstovsek: Incyte Corporation: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding; Celgene: Research Funding; SBIO: Research Funding; Lilly Oncology: Research Funding; Bristol-Myers: Research Funding; Geron Corp.: Research Funding; Gilead: Research Funding; YM Biosciences: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Infinity Pharmaceuticals: Research Funding.

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Preclinical Evaluation of the BET-Bromodomain (BET-BRD) Inhibitor OTX015 in Leukemia Cell Lines Harboring the JAK2 V617F Mutation

Blood ◽

10.1182/blood.v124.21.873.873 ◽

2014 ◽

Vol 124 (21) ◽

pp. 873-873

Author(s):

Maria Eugenia Riveiro ◽

Lucile Astorgues-Xerri ◽

Charlotte Canet-jourdan ◽

Mohamed Bekradda ◽

Esteban Cvitkovic ◽

...

Keyword(s):

Cell Cycle ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Leukemia Cell ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Mrna Levels ◽

Protein Levels ◽

V617f Mutation

Abstract Background: Exposure of cancer cells to BET-BRD protein inhibitors has been associated with a significant downregulation of C-MYC expression, leading to suppression of the transcriptional program linked to proliferation and survival. C-MYC mRNA expression, mediated by STAT5 activation, is induced by the JAK2 (V617F) mutation (JAK2mu) in transfected BA/F3 cells (Funakoshi-Tago, et al. 2013). We selected JAK2mu leukemia-derived cell lines for preclinical evaluation of OTX015 (Oncoethix, Switzerland), a selective orally-bioavailable inhibitor of BET-BRD proteins with promising early results in an ongoing phase I study in hematologic malignancies (Herait et al, AACR 2014, NCT01713582). Material and Methods: Antiproliferative effects of OTX015 and JQ1 were evaluated in three established JAK2mu human myeloid leukemia cell lines (SET2, MUTZ8, HEL 92.1.7). GI50 (OTX015 concentration inducing 50% growth inhibition) and Emax (% cell proliferation at 6 µM OTX015) values were determined by MTT assay after 72h exposure. Protein levels were analyzed by Western blot, and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. Induction of apoptosis was evaluated by Annexin-V. Simultaneous schedules of OTX015 combined with ruxolitinib, a JAK2 inhibitor, were evaluated. Combination index (CI) was determined using the Chou & Talalay method; CI<1 reflects synergy, CI=1 additivity and CI>1 antagonism. Results: After 72h exposure, SET2 was the most sensitive cell line (GI50=0.12 µM and Emax=15%), and HEL92.1.7 cells had a GI50=1.9 µM with an Emax=23%. MUTZ8 was the most resistant cell line with an Emax=61%. Similar GI50 and Emax values are observed with JQ1. A significant increase in the fraction of apoptotic cells was observed in SET2 cells after 72h 500 nM OTX015 exposure. Non-significant increases in Annexin-positive cells were seen in HEL92.1.7 and MUTZ8 cells. Cell cycle analysis revealed a significant increase in the percentage of SET2 cells in subG0/G1 after 24, 48, and 72h 500 nM OTX015, correlating with the increase in apoptosis. Conversely, an increase in the percent cells in the G1 phase was observed in HEL 92.1.7 cells. After 4h 500 nM OTX015, BRD2 mRNA levels were significantly increased in all three cell lines, whereas BRD3 levels were not modified. BRD4 mRNA levels increased significantly after 48h in SET2 cells. OTX015 treatment induced a transitory reduction of C-MYC mRNA levels after 4h with an increase at 24h in all cell lines. At the protein level, C-MYC decreased substantially in SET2 cells after 4h, with complete disappearance after 48h without recovery, while in the less sensitive MUTZ8 cell line, the decrease in C-MYC protein levels was transitory. Conversely, this proto-oncogene was not modified in HEL92.1.7 cells. In addition, p-STAT5 protein was downregulated by OTX015 in SET2 cells, but was increased in MUTZ8 cells after longer exposure time. Furthermore, BCL2 mRNA and protein levels decreased in SET2 cells, correlating with the apoptosis induction seen with OTX015 treatment. In HEL92.1.7 cells, P21 mRNA levels and cyclin D1 protein levels increased after 4h and 48h OTX015 treatment, respectively. Moreover, concomitant combination of OTX015 with ruxolitinib showed a highly antagonist effect (CI>7) in SET2 cells, the most sensitive cell line to both agents. On the other hand, very strong synergy was observed in HEL92.1.7 (CI=0.19) and MUTZ8 (CI=0.41), despite their low sensitivity to single agent OTX015. Conclusions. Our findings demonstrate that OTX015 exhibits potent activity against cultured leukemic cells expressing the JAK2 V617F mutation, inducing apoptosis or cell cycle arrest at submicromolar concentrations. This activity correlates with modulation of C-MYC, p-STAT5, BCL2, P21 and cyclin D1 mRNA and protein levels following OTX015 treatment. Our study highlights the novel and synergistic activity of the combination of a BRD antagonist and a JAK inhibitor in human leukemic cells harboring the JAK2 V617 F mutation, supporting the rationale for in vivo testing of OTX015 in combination with JAK inhibitors in leukemic JAK2mu models. Disclosures Riveiro: Oncoethix SA: Research Funding. Astorgues-Xerri:Oncoethix SA: Research Funding. Canet-jourdan:Oncoethix SA: Research Funding. Bekradda:Oncoethix SA: Research Funding. Cvitkovic:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Shareholder and CSO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Raymond:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Epidemiology of Patients with Classical Philadelphia-Chromosome Negative Myeloproliferative Neoplasms at a Single Academic Medical Center in Singapore

Blood ◽

10.1182/blood-2018-99-116728 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5478-5478

Author(s):

Priscella Shirley Chia ◽

Vanessa CL Chong ◽

Ting Yuan Tay ◽

Eng Soo Yap ◽

Wee Joo Chng ◽

...

Keyword(s):

Southeast Asia ◽

Ethnic Groups ◽

Research Funding ◽

Myeloproliferative Neoplasms ◽

Local Population ◽

Philadelphia Chromosome ◽

Leukemic Transformation ◽

Female Ratio ◽

Platelet Counts ◽

The Mean

Abstract Introduction: Myeloproliferative Neoplasms (MPNs) represents a disorder that involves abnormal proliferation of cells originating from the myeloid line. The proliferation of these cells can lead to complications that are at times fatal. Despite its potential to cause life threatening complications, there is little data on this disease in Southeast Asia. As Singapore is a multiracial country in Southeast Asia, there may be some disease characteristics exclusive to patients here due to its unique population composition. The data from this Southeast Asian cohort would be useful to determine disease homogeneity in Asian countries. Methods: A retrospective review of the MPN database from National University Hospital, Singapore (NUHS) revealed 320 patients who were clinically diagnosed with MPN from 2008 to 2017. This data included patients with Essential Thrombocythemia (ET), Polycythemia Rubra Vera (PRV), Primary Myelofibrosis (PMF), Myeloproliferative Neoplasm, unclassifiable (MPN-U) and Chronic Eosinophilic Leukemia (CEL) (Figure 1A) as per the 2017 WHO classification. For this analysis, we included only the classical Philadelphia chromosome negative MPN and focused on the epidemiology, transformation and overall survival rate. Results: There was a slight male predominance with a male to female ratio of 1.3:1. The ethnic groups within this cohort consisted of 65.8% ethnic Chinese, 20.7% Malay, 5.5% Indian and the rest were made up of other ethnic groups within the region such as Eurasians, Thai, Filipinos, Burmese, Indonesians, Bangladeshi, Vietnamese and Arabian patients. The mean age at diagnosis for this group was 60.5. The mean age was 59.2 years for ET, 61.2 for PRV and the mean age of PMF was the oldest at 63.8. The mean age of diagnosis for ET and PMF patients in our cohort was slightly older compared to the Korean cohort (55.4 and 59.5 years) (Byun, et al., 2016). The majority of this cohort was made up of ET patients (53.1%) followed by PRV (35.3%) and PMF (11.6%). 77.5% of these patients were JAK2 V617F mutation (JAK2) positive. The percentage of patients who were JAK2 positive for ET, PRV and PMF were 69.2%, 96.9% and 56.3% respectively. The percentage of JAK2 positive patients for the three subtypes were higher in our local population compared to the Chinese and Japanese cohorts. Only 120 patients were tested for Calreticulin Exon 9 (CALR) mutations as this molecular test was only available in our institution from 2015 onwards. ET patients make up 68.4% of CALR positive patients. It was noted that CALR positive patients had comparatively higher mean platelet counts of 925.2 than CALR negative patients with mean platelet counts of 691.7. This phenomenon is seen in both CALR positive ET and CALR positive MF patients. In the 10-year period, 25 patients were lost to follow up and 8 patients transferred their care to another institution. Overall, 27 patients were deceased, with a mean survival of 3.5 years. The death-to-case ratio was 11.5 per 100 cases. The death-to-case ratio for ET, PRV and PMF is 6.1 per 100, 8.2 per 100 and 31.3 per 100 respectively. During this period, only 6 patients had transformation. Three patients progressed to post-ET myelofibrosis and 3 had leukemic transformation. Those who had leukemic transformation were initially diagnosed with PRV (1 patient) and PMF (2 patients). All patients who had leukemic transformation were deceased and had a mean survival of 1.4 years from the transformation event. Conclusion: Whilst there were some observable differences between our data and existing Asian data, there is still insufficient information to determine disease homogeneity. This is partly due to the rapid growth of molecular knowledge in this field and the regular revision of the WHO diagnostic criteria of MPNs over the last decade or so. There needs to be coordinated efforts within the region to ensure that our patients have equal access to these diagnostic platforms and that they receive an accurate diagnosis. Disclosures Chng: Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Merck: Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses.

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Identification of the Epigenetic Reader BRD4 As a Novel Therapeutic Target in JAK2 V617F+ MPN Cells

Blood ◽

10.1182/blood.v126.23.2829.2829 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2829-2829

Author(s):

Alexandra Keller ◽

Barbara Peter ◽

Johannes Zuber ◽

Philipp Bernhard Staber ◽

Peter Bettelheim ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Research Funding ◽

Jak2 V617f ◽

G1 Arrest ◽

Mrna Levels ◽

Neoplastic Cells ◽

Hel Cells ◽

Ic50 Values ◽

Brd4 Inhibition

Abstract Myeloproliferative neoplasms (MPN) are characterized by clonal expansion and accumulation of erythrocytes, platelets, and myeloid cells in the bone marrow (BM) and other organs. Classical MPN are polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2 V617F mutation is frequently detected in neoplastic cells in patients with MPN. Although MPN are chronic and indolent diseases in most patients, fatal progression may occur. So far, the only curative approach for these patients is hematopoietic stem cell transplantation. Therefore, current research is evaluating new therapeutic targets and the effects of various targeted drugs. The epigenetic reader bromodomain-containing protein 4 (BRD4) has recently been identified as a promising target in acute myeloid leukemia. In the present study, we investigated the potential value of BRD4 as a molecular target in MPN. We employed two JAK2 V617F+ cell lines, SET-2 and HEL, as well as BM samples obtained from 18 MPN patients (ET: n=7; PV: n=7; PMF: n=4). Three BRD4 inhibitors were applied: JQ1, BI2536, and BI6727. As assessed by qPCR, primary MPN cells as well as SET-2 cells and HEL cells were found to express BRD4 mRNA. In 3 H-thymidine uptake experiments, all three BRD4 blockers were found to suppress the proliferation of the two MPN cell lines and of primary MPN cells in 8/8 patients tested. The effects of these drugs were dose-dependent, with the following IC50 values obtained in SET-2 cells: JQ1, 50-100 nM; BI2536, 20-40 nM; BI6727, 50-75 nM; and in HEL cells: JQ1, 100-500 nM; BI2536, 20-40 nM; BI6727, 30-50 nM. In primary MPN cells, all three agents tested produced IC50 values between 500 and 1000 nM. In normal BM cells, JQ1 did not produce a reasonable IC50 value (>5000 nM). In one patient sample (PMF), we analyzed the effect of JQ1 on the percentage of putative (neoplastic) stem cells (CD34+/CD38-). In this experiment, exposure to JQ1 was followed by a decrease in the percentage of CD34+/CD38- cells compared to control medium (control: 0.16% vs JQ1: 0.045%). To confirm the role of BRD4 as a potential target in MPN cells, we performed target-knockdown experiments in SET-2 cells and HEL cells using two different BRD4 shRNAs (#602 and #1817) and a random shRNA as control. In these experiments, the shRNA-induced knockdown of BRD4 was found to block proliferation in SET2 cells and HEL cells when compared to untransfected cells or random shRNA-transduced cells. In a next step, we examined the mechanism of drug-induced growth inhibition. In cell cycle experiments, BI2536 and BI6727 were found to induce a G2/M phase arrest in both cell lines. By contrast, JQ1 induced a G1 arrest in HEL cells, but did not show a significant effect on cell cycle progression in SET-2 cells. We also asked whether BRD4 inhibition is associated with induction of apoptosis in MPN cells. All three BRD4 blockers induced apoptosis in SET-2 cells and HEL cells at relatively high concentrations after 48 hours, with ED50 values of >5 µM for JQ1 and 0.5-5.0 µM for BI2536 and BI6727. Finally, we asked whether exposure to BRD4 inhibitors is associated with modulation of BRD4 mRNA or MYC mRNA expression. As assessed by qPCR, JQ1, BI2536, and BI6727 were found to downregulate BRD4 mRNA levels as well as MYC mRNA levels in SET-2 cells and HEL cells. In conclusion, our data show that BRD4 is expressed in JAK2 V617F+ MPN cells and that BRD4 inhibition is associated with decreased proliferation and survival of neoplastic cells. The clinical value of BRD4 as a novel target in MPN cells remains to be determined. Disclosures Zuber: Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Staber:Genactis: Research Funding; Morphosys: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda-Millenium: Research Funding; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Valent:Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

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Targeted Exome Sequencing Identifies Novel Mutations in Familial Myeloproliferative Neoplasms Patients in the State of Qatar

Blood ◽

10.1182/blood.v124.21.5570.5570 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5570-5570 ◽

Cited By ~ 1

Author(s):

Nader I Al-Dewik ◽

Bruno Cassinat ◽

Jean-Jacques Kiladjian ◽

Alexander Knuth ◽

Mohamed A. Yassin

Keyword(s):

Exome Sequencing ◽

Research Funding ◽

Germ Line ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Genetic Alterations ◽

Molecular Techniques ◽

Research Fund ◽

Healthy Individuals ◽

Targeted Exome Sequencing

Abstract Background: Myeloproliferative Neoplasms (MPNs) are clonal hematopoietic disorders characterized by excessive proliferation of one or more myeloid cell lineages. Philadelphia negative MPNs include Polycythemia Vera (PV), Essential Thrombocytosis (ET) & Primary Myelofibrosis (PMF). MPNs are associated with the presence JAK2 V617F mutation in 95% of PV & 50% of ET & PMF patients. Several molecular techniques such as RQ-PCR, HRM & Sequencing are currently used to detect common mutations. However, there are still significant numbers of MPNs that are negative to the most common genetic anomalies & many mutations are still unknown. The advent of Next Generation Sequencing (NGS) gives the opportunity to study relevant mutations in several genes. Aim: Utilizing NGS to identify potential genetic anomalies causing familial MPNs patients in Qatar. Methods: 6 MPNs patients from consanguineous families & 5 healthy individuals were consented into the study & peripheral blood samples were collected. gDNA was extracted & used for multiplex PCR amplification of amplicons targeting cancer associated mutations in 28 key genes (JAK2, MPL, THPO, CBL, LNK, SH2B3, NF1, SOCS1/2/3, TP53, NRAS/KRAS, NF1, IDH1/2, EZH2, ASXL1, TET2, ATM, KIT, RB, TP53, IKZF1, RUNX1, PDGFRB, TERT & CALR) using the Ion AmpliSeq Kit. NGS was performed via the Ion Torrent using the 318 chip & data was analyzed with the Torrent Suite Software. Mutation details were obtained from COSMIC database. A hg 19 sequence was used as reference. The confirmation of NGS data was performed using RQ-PCR or Sequencing. Results: 11 samples were successfully sequenced, with a mean depth of 1500 reads & the FASTQC plugin indicated good quality sequencing metrics. JAK2 V617F, JAK2 exon 12-15 & MPL (S505N, W515 L/K) negative samples tested before via RQ-PCR, HRM & sequencing were called negative by NGS. NGS identified novel deleterious mutations in MPNs patients. Out of 6 familial cases, 5 patients (P1- P5) were ET & 1 patient (P6) was PV. P1 had JAK2 V617F, ASXL1 T600P, CBFB G180S, THPO S184R &ITGA2R76Q, P2 had JAK2 V617F, MPL A554G & ATM F582L, the other three Patients (P3, P4 & P5) had CLAR K385fs*47 & one PV patient (P6) had TYK2 E1163G, ASXL1 P808H, PDGFRB P4L & TERT G300fs. Among the patients & healthy individuals, mutations/SNVs such as MPL P106L, K553N, SH2B3 L476F, ATM F1036F KIT N564S & TET2 T730R were also found Discussion & conclusion: Initial screening of known common genes (JAK2 V617F, JAK2 exon 12-15 & MPL W515 L/K) mutations did not reveal the causative mutations in 3% of 180 PV patients, 52% of 200 ET patients & 77% of 20 PMF patients. In this study, several deleterious somatic & germ-line mutations & SNVs were identified using Targeted Exome Sequencing approach. A complex combination of mutations in JAK2, THPO, ITGA2 & MPL genes occurred in ET patients & coexistence of several oncogenic events in TYK2, ASXL1, PDGFRB & TERT occurred in PV patient. This ﬁnding may also suggest that the MPNs phenotype may depend on presence of other mutations. It is worth mentioning that the presence of ATM variant in P2 is associated with increased risk of CLL. Somatic CALR type-2 mutation was identified in 3 ET (nonmutated JAK2 or MPL) patients. This mutation is 5-bp TTGTC insertion in exon 9 that generates a mutant protein with a novel C-terminal (p.K385fs*47). In patients & healthy individuals, a heterozygous germ-line mutation in exon 3 of the MPL gene (MPL P106L) has been observed. it has previously been described as a rare autosomal-dominant disorder. However, this mutation is considered to be frequent in Arabic populations, leading to severe thrombocytosis in homozygotes & occasionally to mild thrombocytosis in heterozygotes. In addition, several unreported variants of uncertain significance were identified. Our preliminary results suggested that MPNs patients in Qatar have several potential disease- associated variants & mutations. Evidences show that there exists a possibility of the disease arising out of the accumulation of genetic alterations & not as the consequence of a single genetic-hit event. This could possibly be due to the high rate of consanguineous marriages in Qatar i.e. the "Founder Effect". Our results recommended carrying out WES to explore & identify mutations which will be crucial to characterize many cases of MPNs with unknown molecular causes, gain a deep understanding of genotype-phenotype correlations & MPNs pathogenesis. Disclosures Al-Dewik: Qatar National Research Fund: Patents & Royalties, Research Funding. Yassin:Qatar National research fund: Patents & Royalties, Research Funding.

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RMC-4550, an Allosteric Inhibitor of SHP2, Displays Therapeutic Efficacy in Pre-Clinical Models of Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2019-128937 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4198-4198 ◽

Cited By ~ 1

Author(s):

Garima Pandey ◽

Nathan Horvat ◽

Narmin E. Amin ◽

Afua A. Akuffo ◽

Christelle Colin ◽

...

Keyword(s):

Research Funding ◽

Myeloproliferative Neoplasms ◽

Critical Role ◽

Philadelphia Chromosome ◽

Jak2 V617f ◽

Ras Signaling ◽

Jak2 Inhibitor ◽

Stat Signaling ◽

Drug Naïve

Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are JAK2-driven disorders resulting from mutations in JAK2, MPL, or CALR. Ruxolitinib, the only FDA-approved JAK2 inhibitor for MPNs, alleviates patient symptomology and improves quality of life, but has little effect on reducing mutant allele burden. This persistent survival of MPN cells in the face of ruxolitinib, as well as other JAK2 inhibitors that have been clinically tested, is a major clinical bottleneck to the development of an effective targeted therapy for MPN patients. Identifying new therapeutic targets which play critical roles in MPN cells and/or in JAK2 inhibitor persistence may lead to improved MPN therapies. SHP2 is an oncogenic tyrosine phosphatase that is an effector of growth factor and cytokine receptor signaling. SHP2 plays a critical role in the activation of the RAS-ERK pathway and regulates JAK-STAT signaling via numerous phosphatase-dependent mechanisms. Activating mutations of SHP2(PTPN11) have been identified in leukemia, including 8% of MPN patients whose disease progressed to acute myeloid leukemia (AML). In addition, SHP2 has been shown to mediate adaptive resistance to targeted therapies in several cancers. Given the role of SHP2 in cytokine and JAK-STAT signaling, we envisaged a potential role of SHP2 in MPN cell growth and/or survival and ruxolitinib persistence. Treatment of JAK2-V617F-driven MPN model cell lines (UKE1, SET2, and BaF3-JAK2-V617F) with ruxolitinib blocked constitutive tyrosine phosphorylation of SHP2, including phosphorylation of Y542, a marker for activated SHP2. This phosphorylation, however, was restored in ruxolitinib persistent cells. Combination treatment of the allosteric SHP2 inhibitor RMC-4550 (Revolution Medicines) with ruxolitinib prevented the development of ruxolitinib persistent cells and pre-established persistent cells remained sensitive to SHP2 inhibition. RMC-4550 treatment led to significantly reduced levels of pERK consistent with the role of SHP2 in RAS signaling. Interestingly, pERK levels in persistent cells were more sensitive to SHP2 inhibition compared to drug naïve cells suggesting pERK was more dependent on SHP2 in ruxolitinib persistent cells. SHP2 inhibitor treatment increased pSTAT5(Y694) in drug naïve cells but this increase was not observed in similarly treated persistent cells. Furthermore, while ruxolitinib inhibited pERK levels in UKE1 and SET2 cells, pERK levels recovered within 24 hrs of treatment. SHP2 inhibition prevented the recovery of pERK in the presence of ruxolitinib. Collectively, these data suggest that signaling pathways in MPN cells treated with ruxolitinib can become rewired, gaining greater dependence on SHP2, concomitant with sustained pERK and cell survival/growth. Interestingly, we identified a known activating SHP2 mutation (F71L) in UKE1 cells obtained from two independent sources - consistent with the presence of PTPN11 mutations in post-MPN AML. The persistent survival of UKE1 cells in ruxolitinib was antagonized by CRISPR-mediated reduction of SHP2 expression, providing further evidence that SHP2 contributes to ruxolitinib persistence. To assess the effects of a SHP2 inhibitor on MPN progression in vivo, we employed the MPLW515Lbone marrow transplant mouse model of MPN. Initial assessment of therapeutic treatment of mice with an established MPN phenotype indicated that once daily treatment of RMC-4550 (10 or 30 mg/kg) antagonized the MPN phenotype. Complete blood counts indicated a significant reduction in white blood cells, monocytes, and neutrophils compared to vehicle treated mice, while flow cytometry analysis indicated RMC-4550 diminished CD11b+ cell numbers to near that observed in mice transplanted with MPLWT-transduced bone marrow. RMC-4550 improved the overall health of diseased mice, as indicated by increased weight, and significantly reduced organomegaly of the spleen and liver compared to vehicle treated MPN mice. Finally, erythropoietin independent erythroid colony formation of JAK2V617F-positive MPN patient cells was suppressed following SHP2 inhibition, which synergized or enhanced the inhibition induced by ruxolitinib in this assay. In summary, our results suggest that SHP2 inhibition may represent a potential MPN therapy in both ruxolitinib naïve and resistant patients and is an attractive therapeutic target for future clinical investigation. Disclosures Epling-Burnette: Incyte Corporation: Research Funding; Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding. Reuther:Incyte Corporation: Research Funding.

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The HDAC INHIBITOR ITF2357 MODULATES KEY HEMATOPOIETIC GENES in JAK2V617F CELLS From MYELOPROLIFERATIVE Neoplasm PATIENTS

Blood ◽

10.1182/blood.v116.21.797.797 ◽

2010 ◽

Vol 116 (21) ◽

pp. 797-797

Author(s):

Ariel Amaru ◽

Katia Todoerti ◽

Anna Pellicioli ◽

Luca Donadoni ◽

Giacomo Tuana ◽

...

Keyword(s):

Cell Cycle ◽

Molecular Mechanism ◽

Cell Lines ◽

Hdac Inhibitor ◽

Research Funding ◽

Myeloproliferative Neoplasms ◽

Differentially Expressed ◽

Wild Type ◽

Western Blots ◽

Erythroid Progenitor

Abstract Abstract 797 We have previously shown that the pan-HDAC inhibitor ITF2357 has strong cytotoxic activity against cells from patients with myeloproliferative neoplasms (MPN) bearing JAK2 mutation at position 617. Indeed ITF2357 inhibited colony growth of JAK2V617F positive cells at doses 5–10 fold lower than those required to block JAK2 wild type cells. We have therefore investigated here the molecular mechanism of this effect. Three cell lines homozygotes (HEL, UKE1) or heterozygotes (SET2) for the JAK2V617F mutation were used along with cell lines bearing JAK2 wild type (K562 and KG1). We confirmed the higher sensitivity of mutated with respect to unmutated cell lines in colony formation assay (mean IC50 42 nM versus 179 nM) and alamar blue assay (mean IC50 84 nM vs 325 nM, respectively). In proliferation assays measuring number of live and dead cells at different time points, we observed that 100 nM ITF2357 blocked the proliferation of both JAK2 mutated and unmutated cell lines to a similar extent, with mean inhibition of 31–69% at 72 hours, but induced apoptosis more efficiently in JAK2 mutated (mean 34%) versus unmutated cells (mean 2%). By cell cycle analysis we could show a block in G1 phase of cell cycle in JAK2V617F cells treated with 100 nM drug. In order to unravel the mechanism of specific inhibition of JAK2 mutated cells by ITF2357, we first investigated expression of HDAC isoforms in the different cell lines. We could detect HDAC1, HDAC2 and HDAC3 proteins in Western blots but these were not differentially expressed in a panel of 3 JAK2 mutated and 3 wild type cell lines. We then set out to analyse the molecular mechanism of action of ITF2357 by global gene expression analysis. Using the Rank Product method with a false positive prediction (pfp) of 0.05 and a 2 fold change cut off parameters, we observed 716 and 863 genes modulated at 6 hours by 250 nM ITF2357 in HEL and UKE-1 cell lines, respectively; 293 of these, (179 up- and 114 down-regulated), were common between both cell lines and 10 were subsequently validated by Q-RT-PCR. Among differentially expressed genes, a number are known to play an important role in the control of proliferation and /or apoptosis, most notably APAF1, BCL2L11, CCNG2, NFKB2, MXD1 and TP53INP1, while additional 6 genes (C-MYB, A-MYB, TAL1, NFE2, MLF1, NOTCH2) are involved in the control of hematopoietic differentiation. Of particular interest is NFE2, which was down modulated 2.7 fold by ITF2357 at 6 hours at the RNA level and by about 2 fold at 24 hours at the protein level. NFE2 has been reported to be hyperexpressed in JAK2V617 MPN patients. We also showed that ITF2357 downmodulated NFE2 expression 2 fold also in CD34+ cells purified from these patients. Given the accepted role of NFE2 in the control of erythroid progenitor cell proliferation and differentiation, and its enhanced expression in MPN patients, our data suggest that NFE2 down-regulation by ITF2357 may at least partially explain the drug effect on growth of MPN progenitor cells. The regulation of NFE2 expression and that of other hematopoietic transcription factors and regulatory proteins in response to ITF2357 is under investigation in our laboratory and data will be presented. Disclosures: Fossati: Italfarmaco SpA: Employment. Rambaldi:Italfarmaco SpA: Research Funding. Golay:Italfarmaco SpA: Research Funding.

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PI3Kδ Inhibitor Idelalisib Inhibits AKT Signaling In Myelofibrosis Patients On Chronic JAK Inhibitor Therapy

Blood ◽

10.1182/blood.v122.21.4065.4065 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4065-4065

Author(s):

Sarah A Meadows ◽

Huong (Marie) Nguyen ◽

Christophe Queva ◽

Brian J. Lannutti ◽

Adam Kashishian ◽

...

Keyword(s):

Cell Lines ◽

Research Funding ◽

Jak2 V617f ◽

Akt Signaling ◽

Jak2 V617f Mutation ◽

Employment Equity ◽

Cell Lysates ◽

Cd34 Cells ◽

Equity Ownership ◽

V617f Mutation

Abstract Background Myelofibrosis (MF) is characterized by activation of the JAK-STAT pathway, with the JAK2 V617F mutation found in 50-60% of patients. Although JAK inhibitors, such as FDA-approved ruxolitinib, have been effective in reducing splenomegaly and mitigating symptoms, patients uniformly exhibit “disease persistence” which is equated with a lack of hematologic or molecular remissions, or with loss of clinical improvement over time. Prior studies using cell lines or primary patient samples have shown that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is dysregulated in MPNs and is a potential therapeutic target (Kamishimoto et al, Cell Signal 2011; Huang et al, ASH 2009 Abstract 1896; Vannucchi et al, ASH 2011 Abstract 3835; Khan et al, Leukemia 2013). In CLL and other B-cell malignancies, the PI3K pathway is constitutively upregulated and is dependent on PI3Kδ. Idelalisib is a δ-isoform-specific PI3K inhibitor that is efficacious in patients with CLL and indolent NHL. Herein, the specific aims of our study were: 1) to determine whether the PI3Kδ isoform is expressed in progenitor cells from MF patients, and 2) to evaluate the inhibitory effects of idelalisib on basal and thrombopoietin (TPO)-stimulated AKT/S6RP phosphorylation (p-AKT/p-S6RP) in cell lines and in primary samples from MF patients who were either on chronic ruxolitinib (RUX) therapy or were not exposed to ruxolitinib (RUX-naïve or off-therapy at the time of sample collection). Methods To evaluate isoform expression, CD34+ cells from the peripheral blood of MF patients were sorted by FACSAria and cell lysates were analyzed by Simple Western using Peggy (ProteinSimple) with recombinant protein as a positive control. For cell line studies, BaF3/MPL W515L and UT-7/TPO cells were stimulated with recombinant human TPO and incubated with idelalisib. Whole cell lysates were analyzed by Western blot to quantify the % of p-AKT and p-S6RP levels compared to idelalisib-untreated cells. For MF patient samples, PBMCs were isolated from the whole blood of MF patients who were either RUX-naïve or on chronic RUX therapy and treated for 2 hours with idelalisib. Antibodies specific to p-AKT Ser473 and pS6RP Ser235/236 were used to quantify the proportion of p-AKT and pS6RP in basal and TPO-stimulated CD34+/CD3-/CD14-/CD19-/CD66- gated cells. Results The PI3Kδ isoform was found to be the predominant isoform expressed in 3 of 3 RUX-naïve and 4 of 4 chronic RUX patients tested; PI3Kβ was expressed at lower levels and no PI3Kα or γ was detected (Figure 1). In BaF3/MPL cells, p-AKT levels decreased by 51%, 64% and 67%, with 0.1, 1.0, 2.0 µM idelalisib, respectively, when compared to idelalisib-untreated cells; p-S6RP levels decreased by 24%, 27%, and 41%, respectively. Similarly, for UT-7/TPO cells, p-AKT decreased by 11%, 44%, and 55%, and p-S6 decreased by 13%, 28% and 48%, respectively. In CD34+ cells from RUX-naïve patients (n=3), p-AKT and p-S6RP levels decreased with increasing concentrations of idelalisib (0.02, 0.2, 2 µM). All patients on chronic RUX treatment demonstrated decreased p-AKT (n=3) and p-S6RP (n=4 basal, n=3 TPO-induced; patient 4 was only tested for basal) levels with increasing concentrations of idelalisib in both basal (Figure 2A) and TPO-stimulated (Figure 2B) assays. All 4 chronic RUX and 2 of 3 RUX-naïve patients tested carried the JAK2 V617F mutation. Conclusions The PI3Kδ isoform was identified as the predominant isoform expressed in CD34+ cells from MF patients. In both cell lines and patient samples, idelalisib inhibits the PI3K/AKT pathway, with a dose-dependent decrease of p-AKT and p-S6RP. Inhibition was observed for both RUX-naïve and chronic RUX-treated patients. Studies are underway to evaluate the effects of idelalisib on progenitor colony formation and induction of cell cycle arrest and apoptosis. * Meadows and Nguyen are first co-authors Disclosures: Meadows: Gilead: Employment, Equity Ownership. Queva:Gilead: Employment, Equity Ownership. Lannutti:Gilead, Acetra, Effector: Consultancy, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Kashishian:Gilead: Employment, Equity Ownership. Jun:Gilead: Employment, Equity Ownership. Coutre:Gilead: Research Funding. Dansey:Gilead: Employment, Equity Ownership. Gotlib:Gilead: Consultancy, Research Funding.

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EXTH-37. TARGETING EPIGENETIC VULNERABILITIES IDENTIFIED FROM A CRISPR SCREEN IN H3.3K27M DIPG

Neuro-Oncology ◽

10.1093/neuonc/noaa215.391 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii95-ii95

Author(s):

Eshini Panditharatna ◽

Neekesh Dharia ◽

Deyao Li ◽

Alexander Beck ◽

McKenzie Shaw ◽

...

Keyword(s):

Cell Lines ◽

Single Gene ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

Epigenetic Therapy ◽

Epigenetic Landscape ◽

Chromatin Regulators ◽

Drug Concentrations ◽

Crispr Screen ◽

Novel Strategy

Abstract Children diagnosed with diffuse intrinsic pontine glioma (DIPG), a type of high grade glioma in the brainstem, currently have a dismal 5-year overall survival of only 2%. The majority of DIPG patients harbor a K27M mutation in histone 3.3 encoding genes (H3.3K27M). To understand if the aberrant epigenetic landscape induced by H3.3K27M provides an opportunity for novel targeted therapies, we conducted the first CRISPR/Cas9 screen using a focused library of 1,350 epigenetic regulatory and cancer related genes in six H3.3K27M DIPG patient-derived primary neurosphere cell lines. We identified gene dependencies in chromatin regulators, polycomb repressive complexes 1 and 2 (PRC1 and PRC2), histone demethylases, acetyltransferases and deacetylators as novel tumor cell dependencies in DIPG. We hypothesized that targeting dysregulated functions of chromatin regulators by genetically deleting and chemically targeting these epigenetically induced vulnerabilities, we could ameliorate, or even reverse the downstream oncogenic effects of the aberrant epigenetic landscape of DIPG. In our secondary CRISPR nanoscreen, we first used six single guide RNAs (sgRNA) to knockout each gene using CRISPR/Cas9 ribonucleoprotein nucleofections, followed by use of three best sgRNAs combined with homology directed repair templates. Compared to lentiviral delivery, nucleofection is a rapid method, with reduced off-target toxicity, suitable for single gene knockouts in DIPG neurospheres. Secondary CRISPR validations confirmed dependencies in BMI1, CBX4, KDM1A, EZH2, EED, SUZ12, HDAC2, and EP300. Next, we conducted a chemical screen using 20 inhibitors and degraders to target the aberrant activity of HDAC, KDM1A, P300/CBP, PRC1 and PRC2. We identified eight chemical compounds that were effective in H3.3K27M DIPG neurosphere cell lines at low drug concentrations. Among these, an inhibitor and degrader targeting P300/CBP activity indicates a novel strategy of epigenetic therapy in DIPG. Through our combinatorial testing, we will identify a synergistic combination of epigenetic therapy for treating children diagnosed with H3.3K27M DIPG.

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Modeling Calreticulin-Mutant Myeloproliferative Neoplasms with Isogenic Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2018-99-111512 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4319-4319 ◽

Cited By ~ 1

Author(s):

Wei Wang ◽

Tiansu Wang ◽

Andriana G. Kotini ◽

Camelia Iancu-Rubin ◽

Ronald Hoffman ◽

...

Keyword(s):

Research Funding ◽

Ex Vivo ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Mutant Type ◽

C Terminus ◽

Calr Mutations

Abstract Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.

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