Crispino, John D.

Progression of myeloproliferative neoplasms to blast phase (BPMPN) is associated with lack of response to conventional therapies and dire clinical outcomes. Consequently, there is a major unmet need to develop new therapies for BPMPN. Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a key contributor to somatic variation in cancer, but this phenomenon has not yet been described in BPMPN. More broadly, whether chromothripsis might result in actionable molecular events that are amenable to targeting remains an open question. To characterise the contribution of structural variants to BPMPN, we first performed integrated copy number and mutation profiling in 64 BPMPN patients by SNP karyotyping and targeted next generation sequencing. We observed a recurrent pattern of chromothripsis that involved chromosome 21, which together with other structural variants led to amplification of a common region of chromosome 21 (‘chr21amp‘) in ~25% of patients (GISTIC q-val<0.01, Fig 1A). Chr21amp was associated with TP53 mutations and a higher number of copy number alterations. Patients with chr21amp had a particularly aggressive and treatment-resistant phenotype, with 0% surviving 12 months compared to 46% in the non-chr21amp pts ( p=0.0007), retaining significance in multivariate analysis including after correction for TP53 mutation status. Whole genome sequencing confirmed that the chromosomal rearrangements resulting in chr21amp occurred by different mechanisms, ranging from simple amplification to highly complex chromothriptic events involving multiple chromosomes. There were no recurrent translocation partners or mutations. The minimally amplified region (MAR) spanned 2.7Mb and contained 24 genes, with a median copy number of 3.5 (range 2.7-8.3) Single-cell transcriptomics combined with allelic resolution genotyping revealed that chr21amp was present in the dominant subclone and occurred subsequent to JAK2V617F and mut TP53 acquisition. Chr21amp was detectable in phenotypic HSCs and throughout early stages of hematopoiesis, but not in mature erythroid cells, consistent with a differentiation block. Of the 24 genes in the minimally amplified region, only one gene, DYRK1A, a serine threonine kinase linked to cell proliferation and survival, was both differentially expressed (single-cell and bulk RNAseq) and differentially accessible (ATACseq). To explore the functional role of DYRK1A in BPMPN we performed shRNA and CRISPR-mediated DYRK1A-knockdown and knockout (KO) in BPMPN cell lines (HEL and SET-2), which led to impaired cell proliferation. The DYRK1A inhibitors EHT1610 and GNF2133 also led to dose-dependent growth inhibition. DYRK1A-KO BPMPN HEL or SET2 cell clones showed a reduced ability to propagate leukemia in vivo with a significant survival advantage vs. wild type control mice. BPMPN chr21amp+ primary patient CD34+ cells were highly sensitive toDYRK1A inhibitors, while healthy control CD34+ cells were unaffected Prior studies have shown that DYRK1A activates the DREAM complex, a transcriptional repressor of DNA-repair pathways. In chr21amp patient cells, the DREAM DNA repair gene signature was significantly downregulated (NES -1.74, q-val <0.001), while conversely in CRISPR DYRK1A KO SET2 cells the transcriptional DNA repair signature was upregulated (NES 1.76, q-val <0.001). In functional assays, CRISPR DYRK1A KO was protective against DNA damage, with a reduction in γ-H2AX foci after etoposide treatment or irradiation ( p<0.01 for both). A second mechanism of leukemogenesis emerged from geneset enrichment analyses, which suggested enhanced JAK-STAT signaling in chr21amp BPMPN cells and downregulation in the CRISPR DYRK1A KO context. We validated this by showing that DYRK1A overexpression activates and potentiates STAT5B transcriptional activity in a luciferase reporter assay. Finally, we noted that the STAT target BCL2 was selectively upregulated in chr21amp cells. BCL2 inhibition showed strong synergy with DYRK1A inhibitors for induction of BPMPN cell apoptosis (Bliss synergy score 15). Collectively, these findings define the chr21amp event as a novel prognostic biomarker in BPMPN. We pinpoint DYRK1A amplification as a central driver of genomic instability and exacerbated JAK-STAT signalling, for the first time linking chromothripsis to a specific druggable target ( Fig 1B).

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell–specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

Abstract Primary myelofibrosis (PMF) is one of three myeloproliferative neoplasms (MPN) that are morphologically and molecularly inter-related, the other two being polycythemia vera (PV) and essential thrombocythemia (ET). MPNs are characterized by JAK-STAT–activating JAK2, CALR, or MPL mutations that give rise to stem cell–derived clonal myeloproliferation, which is prone to leukemic and, in case of PV and ET, fibrotic transformation. Abnormal megakaryocyte proliferation is accompanied by bone marrow fibrosis and characterizes PMF, while the clinical phenotype is pathogenetically linked to ineffective hematopoiesis and aberrant cytokine expression. Among MPN-associated driver mutations, type 1–like CALR mutation has been associated with favorable prognosis in PMF, while ASXL1, SRSF2, U2AF1-Q157, EZH2, CBL, and K/NRAS mutations have been shown to be prognostically detrimental. Such information has enabled development of exclusively genetic (GIPSS) and clinically integrated (MIPSSv2) prognostic models that facilitate individualized treatment decisions. Allogeneic stem cell transplantation remains the only treatment modality in MF with the potential to prolong survival, whereas drug therapy, including JAK2 inhibitors, is directed mostly at the inflammatory component of the disease and is therefore palliative in nature. Similarly, disease-modifying activity remains elusive for currently available investigational drugs, while their additional value in symptom management awaits controlled confirmation. There is a need for genetic characterization of clinical observations followed by in vitro and in vivo preclinical studies that will hopefully identify therapies that target the malignant clone in MF to improve patient outcomes.

Abstract The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. Significance: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression. This article is highlighted in the In This Issue feature, p. 1307

Nearly 20% of patients with myelofibrosis progress to blast phase disease; an aggressive form of acute myeloid leukemia. Although previous studies have implicated loss of TP53 or JARID2 in progression, by and large the genetic events that lead to conversion to blast phase remain unknown. To identify genes whose loss drives progression, we performed a focused CRISPR/Cas9 screen in which murine Jak2V617F bone marrow cells expressing Cas9 were transduced with two separate sgRNA libraries of known tumor suppressor genes and subjected to colony replating assays. Transduction of one of the two libraries led to serial replating and enhanced self-renewal of the Jak2V617F cells. Subsequent DNA sequencing revealed enrichment of all four guides targeting STK11, the gene that encodes LKB1 which regulates a number of key cellular pathways including energy utilization by activation of AMPK. To confirm that loss of Stk11 is the event that leads to increased clonogenicity, we collected cells from Jak2V617F/Vav-Cre+ and control Vav-Cre+ mice and induced Stk11 knockout by electroporating Cas9-Stk11 sgRNA ribonucleoprotein complexes. Consistent with the screening results, only Jak2V617F Vav-Cre+ cells with Cas9-Stk11 sgRNA showed serial replating. To determine whether Stk11 is required for growth of cells with a different driver of enhanced JAK/STAT signaling, we doubly transduced Stk11 homozygous floxed bone marrow cells with MPLW515L-mCherry and Cre-GFP to delete Stk11. As expected, cells with both MPLW515L and Cre recombinase showed enhanced self-renewal, while singly infected control cells failed to replate. These results demonstrate that activation of JAK/STAT signaling can overcome the requirement for Stk11 in normal hematopoiesis and suggest that STK11 loss may be a strong driver of malignant transformation in combination with enhanced JAK-STAT signaling. We next investigated the mechanism by which loss of STK11 cooperates with enhanced JAK/STAT signaling to promote leukemia. RNA-sequencing of wild-type, Stk11+/+/ MPLW515L, and Stk11-/-/MPLW515L hematopoietic cells revealed enrichment of a number of pathways related to hypoxia, oxidative phosphorylation and mitochondrial translation in cells lacking LKB1. Western blot assays confirmed activation of mTOR signaling as well as HIF1a stabilization and pathway activation, both of which have been reported to lie downstream of LKB1 loss. We also performed a number of studies to determine the relevance of reduced LKB1 expression to leukemic progression. First, we induced deletion of Stk11 in mice that were transplanted with HSPCs expressing MPLW515L after development of the MPN phenotype. Loss of Stk11 caused a rapid lethality that was associated with enhanced bone marrow fibrosis and osteosclerosis. We also observed accumulation of leukemic blasts in small clusters consistent with AML transformation arising in the spent phase MPN. Additionally, we deleted STK11 by CRISPR/Cas9 in primary MPN patient samples and monitored their engraftment in immunocompromised mice. We observed enhanced engraftment and increased reticulin fibrosis and osteosclerosis in mice that received the STK11 edited cells compared to those with non-targeted sgRNA. Third, we compared the expression of STK11 in paired blast and chronic phase myelofibrosis patient samples by RT-PCR. Consistent with the hypothesis that loss of STK11 facilitates leukemia, we found that its expression was decreased by more than 50% in five of seven paired post-MPN AML patient samples, with two having STK11 levels below 20%. We further validated downregulation of LKB1 by immunohistochemistry on paired chronic and blast phase MPN specimens and observed little staining in the blast phase specimens. Finally, to further show that the mechanism of in vitro enhanced self-renewal is related to leukemia progression, we stained the paired marrows for HIF1a and saw a dramatic increase in staining at the AML phase. We also analyzed RNA-seq data of paired chronic versus blast phase MPNs specimens and observed that there is a strong congruence of enriched pathways that are associated with the in vitro mouse HSPC phenotype and the human blast phase progression, such as oxidative phosphorylation and hypoxia. Together, our study demonstrates that loss of LKB1/STK11 promotes transformation of cells with activated JAK/STAT signaling and that STK11 is a prominent candidate tumor suppressor gene in post-MPN AML. Disclosures Gurbuxani: UpToDate: Honoraria. Hoffman:Dompe: Research Funding; Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees. Levine:Astellas: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Rampal:Galecto: Consultancy; Incyte: Consultancy, Research Funding; Constellation: Research Funding; Stemline: Consultancy, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; CTI Biopharma: Consultancy; Abbvie: Consultancy; Pharmaessentia: Consultancy; Promedior: Consultancy; Blueprint: Consultancy.

Abstract Background: The selective AURKA inhibitor alisertib (MLN8237) exhibits disease modifying activity in murine models of myelofibrosis by eradicating atypical megakaryocytes resulting in reduction of marrow fibrosis (Nat Med 2015). Here, we present long term follow-up results from the investigator initiated pilot study of alisertib in patients with myelofibrosis (clinical trials.gov Identifier NCT 02530619). Methods: 24 patients with DIPSS intermediate 1, intermediate-2, or high risk myelofibrosis who were in need of therapy, refractory/intolerant or unlikely to respond to JAK inhibitors with neutrophil count ≥ 1 x109/L, and platelet count ≥ 50 x109/L, received alisertib (provided by Millennium Pharmaceuticals Inc) at a dose of 50 mg twice daily for one week every 21 days. Toxicity assessment was performed by the standard common terminology criteria (Version 4.0). Response was assessed by the international working group for myelofibrosis research and treatment (IWGMRT) criteria. Correlative studies included assessments of JAK2V617F, CALR, and MPL mutant allele burden, degree of fibrosis and GATA1 expression in bone marrow samples obtained pre and post therapy. Results: We enrolled 17 patients with primary myelofibrosis, 4 with post essential thrombocythemia myelofibrosis and 3 with post polycythemia vera myelofibrosis. Median age was 72 years with 66% males. 79% of patients were DIPSS intermediate risk, and the remainder were high risk with 15 patients (62.5%) having received prior JAK inhibitor therapy. Driver mutational status was as follows; 58% JAK2V617F, 29% CALR, and 13% MPL mutated. At study entry, 54% of patients demonstrated palpable splenomegaly ≥ 5 cm below the left costal margin, 54% were transfusion dependent with all patients experiencing constitutional symptoms. At the time of data cut-off, patients received a median of 7.5 cycles (range; 1-29 cycles) of therapy. The 7 patients presently on study have received a median of 23 cycles (range; 8-29 cycles). Reasons for treatment discontinuation included progressive disease/lack of response in 11 (65%) patients, toxicity in 4 (24%) patients and refusal of further therapy in 2 (11%) patients.Safety and Efficacy assessments The most common treatment-emergent grade 3/4 adverse events included neutropenia (42%), thrombocytopenia (29%) and anemia (21%), with 4% each experiencing neutropenic fever, diarrhea, vertigo, elevated creatinine and elevated alanine aminotransferase. 22 patients were considered for response evaluation with 4 of 14 patients (29%) with palpable splenomegaly ≥ 5 cm achieving a spleen response, 1 of 13 patients (8%) becoming transfusion independent, and 5 of 22 patients (23%) experiencing symptom response with ≥ 50% reduction in the MPN-SAF total symptom score. However, when response assessment was restricted to 13 patients who had received a minimum of 5 cycles of therapy, spleen responses were observed in 4 of 7 (57%) patients, 1 of 5 (20%) achieved transfusion independence and 5 of 13 (38%) achieved symptom response. All patients presenting with leukocytosis (n=4) and thrombocytosis (n=2) had resolution with therapy. Of the 7 patients presently on study, four patients continue to demonstrate symptom response, two patients with both spleen and symptom response, and another patient with sustained anemia response. Correlative assessments We compared the intensity of staining of GATA1, a factor that is required for maturation, in sequential bone marrow biopsies from six patients at baseline and after a minimum of five cycles and observed a striking increase in the numbers of GATA1-positive megakaryocytes in five of six cases (Figure 1a). In addition, we observed a one grade reduction in marrow fibrosis in 4 of 6 paired samples (Figure 1b). This reduction in fibrosis was accompanied by sustained responses to the drug. Finally, we compared JAK2, MPL or CALR mutant allele burden in eight paired baseline and cycle 5 or 6 samples and observed decreases in 4 of 8 patients (Figure 1c). Conclusions: Alisertib is safe and well tolerated in patients with myelofibrosis with prolonged administration up to 1.7 years. In addition to providing clinical benefit, alisertib restored normal morphology and GATA1 expression in atypical megakaryocytes and reduced marrow fibrosis and mutant allele burdens. These findings demonstrate that AURKA inhibition should be further explored as a therapeutic option in myelofibrosis. Figure 1. Figure 1. Disclosures Swords: AbbVie: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding. Frankfurt:Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Altman:Cyclacel: Other: payment to the institution to conduct clinical trial work; Epizyme: Other: payment to the institution to conduct clinical trial work; Ariad: Other: payment to the institution to conduct clinical trial work; Bayer: Other: payment to the institution to conduct clinical trial work; Celator: Other: payment to the institution to conduct clinical trial work; FujiFilm: Other: payment to the institution to conduct clinical trial work; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: payment to the institution to conduct clinical trial work; Agios: Other: Payment to the institution to conduct the trial ; Astellas Pharma: Other; Genetech: Other: Payment to the institution to conduct clinical trial work; Syros: Membership on an entity's Board of Directors or advisory committees; Incyte: Other: payment to the institution to conduct clinical trial work; GSK: Other: payment to the institution to conduct clinical trial work; Immune Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Boeringer Ingelheim: Other: payment to the institution to conduct clinical trial work; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: payment to the institution to conduct clinical trial work. Rampal:Celgene: Honoraria; Stemline: Research Funding; Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership. Crispino:Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

Abstract Abstract 64 Megakaryocytes are one of the few cell types that undergo a modified form of the cell cycle termed endomitosis, in which cells skip the late stages of mitosis to become polyploid. Murine and human megakaryocytes commonly reach modal ploidy states of 32N and 16N, respectively, and can sometimes achieve DNA contents as high as 64N. Polyploidization is associated with upregulation of megakaryocyte lineage specific genes, proplatelet formation and expression of genes related to apoptosis. RNA expression array studies have shown that high ploidy states are strongly correlated with megakaryocyte differentiation and maturation. Importantly, the choice of a megakaryocyte to undergo polyploidization and differentiation is inextricably linked to exit from the proliferative cell cycle. Given that megakaryocytes in patients with essential thrombocythemia are hyperproliferative and that those in primary myelofibrosis fail to undergo normal differentiation or polyploidization, we hypothesized that small molecule inducers of polyploidization would drive these cells to exit the proliferative cell cycle and undergo terminal differentiation or death. In collaboration with the Broad Institute, we performed a high throughput screen and identified small molecules that induce polyploidization and proliferative arrest of malignant megakaryocytes, including those that express MPLW515L and JAK2 V617F. We have shown that these compounds, including the Rho kinase inhibitor dimethylfasudil (diMF), selectively increase polyploidization, expression of megakaryocyte cell surface markers, and apoptosis of murine and human megakaryocytic cell lines and primary cells. Furthermore, diMF blocked the growth of primary human AMKL blasts both in vitro and in vivo. With respect to MPNs, diMF showed potent activity against megakaryocytic cell lines and primary cells expressing either JAK2 V617F or MPL W515L alleles commonly associated with these disorders. diMF inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis of megakaryocytes transduced with JAK2 V617F or MPLW515L. diMF also significantly reduced megakaryocyte colony forming units (CFU-MK), and induced polyploidization and differentiation of bone marrow and fetal liver megakaryocytes from Gata1 mutant mice, which develop a PMF-like disease. Given that diMF induces growth arrest, polyploidization and apoptosis of cells that express activated mutants of JAK2 and MPL, we predicted that it, as well as other small molecule inducers of polyploidy, would be efficient at restraining aberrant megakaryocyte proliferation in both PMF and ET. To assay the effectiveness of diMF in these disorders, we treated peripheral blood mononuclear cells from patients with either PMF or ET with diMF and monitored growth and maturation of megakaryocytes. We discovered that diMF induced polyploidization and subsequent apoptosis of both types of MPN primary samples. diMF also reduced CFU-MK of these MPN patient samples. Next, we assessed the activity of diMF in a model of MPN in which congenic recipients of MPLW515L transduced Balb/C bone marrow cells develop a rapid MPN characterized by leukocytosis, thrombocytosis, bone marrow fibrosis, and death. diMF led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen of a mouse model of PMF. diMF also led to a significant reduction in the platelet count and a trend towards decreased white cell count, with no effect on hematocrit. Overall, diMF results were comparable to intermediate doses of INCB16562. These encouraging results strongly suggest that diMF induces a decline in megakaryocyte lineage, which leads to reduction in platelet count, and support pre-clinical development of diMF for megakaryocytic subtypes of MPNs. Of note, diMF did not inhibit the phosphorylation of Stat5 or Stat3, suggesting that it acts through a mechanism distinct from JAK2 inhibitors. Interestingly, combination of diMF with a selective JAK2 inhibitor greatly enhanced the efficacy of diMF to inhibit proliferation and induce apoptosis in MPLW515L transduced megakaryocytic cell line. These data support combining JAK inhibition and induction of megakaryocyte polyploidy as a new therapeutic strategy for MPNs. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3862 Somatic mutations in JAK2 and MPL are associated with the BCR-ABL negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). While oral JAK2 inhibitors improve peripheral blood counts and splenomegaly, these drugs show varying effects on JAK2 mutant allele burden and do not eliminate the malignant clone in humans or in animal models of MPN. Although much of the research to date has focused on JAK/STAT signaling, other pathways downstream of the class I cytokine receptors, including PI3K/AKT and ERK are also activated in MPNs. Our hypothesis is that persistent activation of these signaling pathways contributes to the progression of myeloproliferative neoplasms (MPNs). Multiple studies have shown that overexpression of activated JAK2 and MPL mutants (e.g. JAK2 V617F or MPL W515L) in primary murine bone marrow cells causes robust AKT, STAT3, and STAT5, JNK, ERK phosphorylation. To determine the extent to which these signaling pathways are involved in the disease, we cultured JAK2 V617F and MPL W515L expressing clones of the G1ME and 6133 megakaryocytic cell lines with a panel of small molecule kinase inhibitors. We discovered that inhibition of the PI3K/AKT and MAPK/JNK signaling pathways with triciribine and SP600125 respectively, potently suppressed growth of these cells by inducing G1 arrest and apoptosis. In contrast, inhibitors of Ras, ERK, Src family tyrosine kinase, and PKC failed to significantly inhibit proliferation of the JAK2 or MPL mutant expressing cells. Murine bone marrow cells transduced with MPL W515L show a dramatic expansion of megakaryocytes (CFU-MKs) and this expansion was abrogated in the presence of PI3K/AKT inhibitors suggesting a requirement for this pathway in aberrant megakaryocyte expansion. Since AKT inhibition showed the strongest effect, we assayed the activity of MK-2206, a potent and selective allosteric AKT inhibitor, on multiple models of MPNs. We discovered that MK-2206 induced proliferative arrest and apoptosis accompanied by suppression of PI3K/AKT signaling in G1ME and 6133 cells expressing MPLW515L. MK-2206 also potently inhibited liquid culture growth and colony formation of PMF patient CD34+ cells in vitro. Finally, in preliminary murine transplants experiments with MPLW515L expressing bone marrow progenitors, treatment with MK-2206 led to significant reductions in peripheral blood leukocytosis and extramedullary hematopoiesis. Together, these findings demonstrate that the PI3K/AKT axis represents a rational target for therapy in human myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

Abstract We recently reported that the induction of polyploidization of malignant megakaryocytes shows great promise as a new therapy for acute leukemia. Polyploidization inducers such as dimethylfasudil (diMF) and MLN8237, both of which target Aurora A kinase (AURKA), induce proliferation arrest, polyploidization, expression of megakaryocyte differentiation markers and apoptosis of leukemic megakaryocytes in vitro and in vivo. Since megakaryocytes in primary myelofibrosis (PMF) show impaired polyploidization and maturation, and likely directly contribute to the disease, we predicted that polyploidization inducers would provide a new therapeutic strategy. To determine the effect of these compounds on the growth of MPN cells, we first treated the JAK2 V617F mutant megakaryocytic SET2 cell line with varying doses of MLN8237 and diMF. Both compounds effectively and dose dependently inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis. Furthermore, MLN8237 synergized with ruxolitinib to induce apoptosis of the SET2 cells and also potently induced growth arrest of JAK2 inhibitor persistent SET2 cells. We observed a similar polyploidization and differentiating activity of MLN8237 and diMF on megakaryocytes derived from primary human PMF progenitors. The ability of these agents to induce polyploidization was specific, as the non-megakaryocyte fractions of the cultures were not affected. Next, we assayed the activity of polyploidization inducers on progression of MPNs in two mouse models: JAK2V617F conditional knockin mice and mice engrafted with MPLW515L expressing bone marrow progenitors. Of note, spleens from both mouse models displayed a robust increase in both total and phosphorylated forms of AURKA relative to control animals, further suggesting that AURKA is a rational target in this disease. We first assayed the activities of MLN8237 and diMF in the MPLW515L bone marrow transplantation model. Recipient mice develop a rapid MPN characterized by leukocytosis, thrombocytosis and bone marrow fibrosis. Both MLN8237 and diMF reduced the disease burden, as evidenced by significant reductions in the liver and spleen weights, white cell counts and platelet counts. Both compounds also led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen. Moreover, plasma levels of TGF-β a known myelofibrogenic cytokine, were decreased by more than 3-fold by the drug treatment. Both diMF and MLN8237 led to selective polyploidization of megakaryocytes in the spleen as well as marked reductions in the levels of p-AURKA. Of note, neither agent affected the extent of phosphorylation of STAT3 or STAT5. Therefore, we tested whether the combined use of a JAK inhibitor and a polyploidy inducer would show enhanced activity in vivo. Indeed, the combination of MLN8237 and ruxolitinib led to greater reductions in tumor burden in the MPLW515L mouse model than either agent alone. Similar results were obtained using the JAK2V617F knock-in model. To further validate our conclusion that AURKA is a target in PMF, we infected Aurkafl/fl floxed bone marrow progenitors with MPLW515L and transplanted the cells to irradiated recipients. Excision of both alleles of Aurka by Cre mediated recombination completely resolved the disease, while heterozygous deletion of Aurka significantly reduced the disease burden, in a manner similar to treatment with MLN8237. Given that heterozygous deletion of Aurka does not alter normal hematopoiesis in mice, the fact that a 50% reduction in kinase expression was associated with a significant decrease in disease burden suggests that there is an effective therapeutic window in which AURKA inhibitors will be effective against MPN while sparing normal hematopoiesis. Although JAK inhibitors provide symptomatic relief, it is becoming clear that they are not curative. Thus, there is an urgent need to develop new agents to use in combination with JAK inhibitors. Our data reveal that inducing polyploidization and differentiation of dysplastic megakaryocytes in PMF ameliorates features of the disease both in vitro and in vivo. These results support the initiation of clinical studies that combine a JAK inhibitor with an AURKA inhibitor. Disclosures: Crispino: Sanofi: Research Funding.

Abstract Abstract 2867 Primary myelofibrosis (PMF) is a clonal hematologic malignancy, which results from the transformation of a pluripotent hematopoietic progenitor cell. A major consequence of this transformation is increased hematopoiesis and an overproduction of abnormal blood cells. PMF is associated with bone marrow fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their bone marrow and spleen. PMF patients can present well known mutations including JAK2V617F (65%), MPL (10%), TET2 (17%), CBL (6%), IDH (4%,), which are not specific to the disease and are also present in polycythemia vera, essential thrombocythemia and AML. We hypothesize that the genetic events associated with PMF, including MPL and JAK2 mutations, contribute to defects in Mk maturation, but that additional changes are needed to explain the striking abnormalities seen in PMF relative to the other myeloproliferative diseases. Although there have been studies to examine the aberrant gene expression program of CD34+ cells of PMF patients, we chose to examine the changes that occur in gene expression specifically in Mks as a way to better understand their abnormal differentiation and to determine their contribution to the disease. Primary CD34+ cells from PMF patients and healthy donors were cultivated in serum free media supplemented with recombinant TPO, BSA, liposomes, insulin and transferrin to support the growth of Mks. After 10 days of differentiation, we evaluated the cultures for proliferation, apoptosis and differentiation by flow cytometry. We found that PMF specimens gave rise to a lower percentage of mature (CD41+CD42+) cells as compared to healthy donors, but showed, a lower ploidy level, a greater proliferation and increased survival. These observations are consistent with the clinical observations that PMF bone marrow is characterized by an increased number of immature, dysplastic Mks. We used flow cytometry to collect two populations of cells for analysis: immature CD41+CD42− Mks, and CD41+CD42+ mature MKs. After sorting, we extracted RNA and performed whole genome microarray analysis with Illumina Human HT12-v4 arrays on cohorts of PMF and control specimens. Gene expression data were analyzed by GeneSpring and Gene Set Enrichment Analysis (GSEA). We found that the CD41+CD42− MKs derived from PMF progenitors showed reduced expression of GATA1 as compared to control cells, as expected based on previous study by Dr. Alessandro Vannuchi. GeneSpring analysis revealed that myeloid transcription factors, including CEBPa, GFI1, and SPI1 (PU.1), which are not expressed in normal MKs, are strikingly and significantly overexpressed in PMF samples. Moreover, c-myb, which regulates the erythroid/Mk cell fate decision, FOG-1 and AML1, are also overexpressed in PMF Mks. This aberrant myeloid gene expression program in PMF Mks is reminiscent of a similar defect we observed in Mks with reduced expression of GATA-1 and GATA-2. We predict that reduced levels of GATA-1 protein in PMF Mks, as reported by Dr. Alessandro Vannucchi and colleagues, is in part responsible for the aberrant growth and differentiation of the PMF Mks. Our data support the model that PMF Mks are defective in their ability to properly regulate expression of hematopoietic regulators. Further analysis by GSEA revealed that hematopoietic and cytokine pathways are among those that are highly enriched in PMF Mks. We recently reported that the molecules dimethylfasudil (diMF) and MLN9237 are able to selectively increase ploidy, Mk surface marker expression, and apoptosis of malignant Mks. We treated Mks derived from PMF progenitor cells with diMF and observed a high increase in polyploidization accompanied with a reduction of Mks proliferation. Thus, diMF is able to partially restore Mk differentiation of PMF cells, supporting the testing of polyploidy inducers in myelofibrosis patients. Disclosures: Pardanani: Sanofi-Aventis: Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other.