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Supplementary Figure S1. Response of Tet2-/- Flt3ITD leukemia model to 5-Aza therapy Tet2-/-Flt3ITD leukemia model treated with vehicle or 5-Aza (n=5). Supplementary Figure S2. Idh2R140QFlt3ITD leukemia model phenotype. Supplementary Figure S3. Idh2R140QFlt3ITD leukemia model treatment response to AG-221 Idh2R140QFlt3ITD leukemia model treated with vehicle or AG-221 (n=6). Supplementary Figure S4. Genomic methylation analysis of Idh2R140Q Flt3ITD leukemia model response to AG-221 therapy. Supplementary Figure S5. GSEA analysis of 5-Aza and AG-221 response. Supplementary Figure S6. Response of Tet2-/-Flt3ITD leukemia model to 5-Aza and AC220 therapy. Supplementary Figure S7. Idh2R140QFlt3ITD and Tet2-/-Flt3ITD leukemia model response to combination therapy. Supplementary Figure S8. Methylation and expression response of Tet2-/-Flt3ITD and Idh2R140QFlt3ITD leukemia model to combination therapy. Supplementary Figure S9. Response of Tet2-/- Flt3ITD and Idh2R140QFlt3ITD leukemia model to combination therapy.

Supplementary Methods, Figure Legends

Supplementary Table S1. List of Synergy Genes. Supplementary Table S2. A, Statistical testing of DMCs identified in combination therapy compared to single treatment. B, Differentially methylated CpGs in response to combination therapy.

As health systems are adapting to increased accountability for quality outcomes, population health, and collaborative care, medical schools are adapting curricula to better prepare physicians to function in health systems. Two components of this educational transformation are (1) increasing physician competence in Health Systems Science, including quality, population health, social determinants of health, and interprofessional collaboration, and (2) providing roles for students to act as change agents while adding value to the health system. The authors, three medical students who served as patient navigators during their first year of medical school, provide perspectives regarding their clinical systems learning roles, which spanned the levels of individual patients, clinic operations, and the health system. Specifically, authors describe working with a struggling patient, developing an intake assessment tool to aid clinical operations, and creating a directory of community-based resources. Authors discuss educational benefits, including understanding social determinants of health, barriers to care, and inefficiencies within the healthcare system. Several challenges are explored, including the importance of student initiative and concerns about traditional curricular outcomes. Through early experiences, students describe developing a professional identity as a change agent, while also learning key competencies required for clinical practice.

Objectives: The objective of this study is to understand the effect that operative patellar stabilization (without concomitant distalization) has on patellar height. Specifically, we studied MPFL reconstruction (MPFLr), non-distalizing tibial tubercle osteotomy (TTO), and trochleoplasty. There are several established risk factors for patellar instability, including patella alta and trochlear dysplasia. Traditionally, in patients with patellar instability with associated significant patella alta, the elevated patellar height can be corrected with distalizing TTO or patellar tendon shortening. Shortening the patellar tendon or moving the tubercle inferiorly corrects the alta and, in turn, causes the patella to engage the trochlea earlier in flexion providing increased bony restraint. Patients with trochlear dysplasia can be treated with trochleoplasty, where the abnormally contoured trochlea is corrected. MPFL reconstruction (MPFLr) is often performed in the absence of predisposing anatomic abnormality or in addition to either of the aforementioned procedures. The reconstruction restores the medial restraint to the patella. Recent literature suggests that MPFLr without distalizing TTO decreases patellar height. Luceri et al showed a decrease in patellar height at a mean follow up of 18.4 ± 12.0 months in a population with an average age of 25.0 using a technique with two patellar tunnels and tensioning the knee in 30 degrees of flexion. Lykissas showed similar results in an adolescent population (mean age 14.2) with a mean follow up of 6 months using a technique with a single patellar limb and tensioning their grafts in 45 degrees of knee flexion. Fabricant also found a decrease in patellar height in adolescents (mean age 14.9) at 3 months of follow up. Their technique featured 2 tunnels in the patella with the graft tensioned in 20 degrees of knee flexion. Roessler, however, did not observe a change in patellar height in a series of 32 patients with an average age of 29.7. We studied the effect of patellar instability surgery on patellar height to better elucidate why there are conflicts in the literature. While the current literature focuses on MPFLr, we also studied non-distalizing TTO and trochleoplasty. We hypothesized that patellar stabilization procedures that were not performed to address patella alta decrease patellar height. Methods: A multi-center, retrospective chart review was conducted at Johns Hopkins, University of Virginia, Cleveland Clinic, and Massachusetts General Hospital. All patients who underwent MPFLr, non-distalizing TTO, trochleoplasty, or a combination of the three operations from 2016-2021 by one of the participating attending surgeons were included. Radiographs obtained pre-operatively, 2 weeks post-operatively, and 3 months post-operatively were analyzed. Caton-Deschamps Index (CDI) and a linear measure from the distal pole of the patella to the proximal, anterior tibial plateau were collected for each patient. Data was deidentified and pooled across the four centers. Patients were excluded if they did not have pre-operative imaging stored in the medical record or if their lateral knee x-ray did not meet study criteria (15-65 degrees of flexion and less than 8 millimeters of overhang of the femoral condyles). 1x2 repeated measures ANOVA for two time points (pre-operative and 2 weeks post-operative) and 1x3 repeated measures ANOVA for three time points (pre-operative, 2 weeks post-operative, and 3 months post-operative) were conducted. p-values less than 0.05 were considered statistically significant. Results: A total of 262 knees met inclusion criteria (Table 1). 117 underwent isolated MPFLr, 106 MPFLr and TTO, and 39 MPFLr and trochleoplasty. CDI was 1.13, 1.11, and 1.09 preoperatively in each group, respectively. CDI decreased by a statistically significant amount 2 weeks post-operatively in all groups. The decrease was -0.08 for isolated MPFLr, -0.10 for MPFLr and TTO, and -0.15 for MPFLr and trochleoplasty (p=0.001 for each). A decrease of over a millimeter was also seen in all groups at 2 weeks when looking at the linear measure from the distal poll of the patella to the proximal, anterior tibial plateau. Exact changes were MPFLr = -1.20 mm (p=0.028), MPFLr and TTO = -1.28 mm (p=0.001), and MPFLr and trochleoplasty = -1.85 mm (p=0.006). At three months, CDI remained decreased in the MPFLr and TTO and MPRLr and trochleoplasty groups with exact decreases of -0.10 and -0.14, respectively (p = 0.001 for both). The linear measure also remained decreased at three months, with changes of -2.59 mm for MPFLr and TTO and -2.29 mm for MPFLr and trochleoplasty (p = 0.001 for both). The isolated MPFLr group failed to reach statistical significance for either CDI or the linear measure at three months. Conclusions: Our results demonstrate that patellar instability surgery affects patellar height, even when a distalizing tibial tubercle osteotomy or patellar tendon shortening are not performed. A decrease was seen in patellar height 2 weeks post operatively, whether the patient underwent MPFLr, MPFLr and TTO, or MPFLr and trochleoplasty. This was also observed at 3 months in the linear measure of MPFLr and TTO group and the CDI of the MPFLr and trochleoplasty group. All other groups did not reach statistical significance at 3 months. These results have important clinical implications. They suggest that these patellar instability surgeries decrease patellar height. Surgeons can expect a decrease in CDI of approximately 0.1 and a shortening of 1-2 mm in the distance between the distal patella and the proximal plateau when performing these procedures. This means patients with borderline patellar alta may have their alta satisfactorily treated without a distalizing TTO. However, if a CDI change of more than 0.1 is needed, distalizing osteotomy or patellar tendon shortening may be indicated.

Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617Fand mutant IDH1R132Hor Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617FIdh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mutand IDH2mutmutations. Taken together, these data suggest that combined JAK and IDH inhibition May offer a therapeutic advantage in this high-risk MPN subtype., Damon Runyon Cancer Research Foundation (DRG-2241-15), Howard Hughes Medical Institute (Faculty Scholars Award), Stand Up To Cancer, National Cancer Institute (U.S.) (P50CA165962), National Cancer Institute (U.S.) (P30CA14051), Koch Institute for Integrative Cancer Research ( Dana-Farber Harvard Cancer Center Bridge Project), Leukemia & Lymphoma Society of America. Specialized Center of Research (SCOR) Program, National Institutes of Health (U.S.) (grant U54OD020355-01), National Institutes of Health (U.S.) (grant NCI R01CA172636), National Institutes of Health (U.S.) (grant R35CA197594), National Cancer Institute (U.S.) (Cancer Center Support Grant (P30 CA008747).

The objective of this study was to evaluate the hemodynamic performance and gaseous microemboli (GME) handling ability of a simulated neonatal extracorporeal life support (ECLS) circuit with an in-line continuous renal replacement therapy (CRRT) device. The circuit consisted of a Maquet RotaFlow centrifugal pump or HL20 roller pump, Quadrox-iD Pediatric diffusion membrane oxygenator, 8-Fr arterial cannula, 10-Fr venous cannula, and Better-Bladder (BB) with “Y” connector. A second Quadrox-I Adult oxygenator was added postarterial cannula for GME experiments. The circuit and pseudo-patient were primed with lactated Ringer's solution and packed human red blood cells (hematocrit 40%). All hemodynamic trials were conducted at ECLS flow rates ranging from 200 to 600 mL/min and CRRT flow rate of 75 mL/min at 36°C. Real-time pressure and flow data were recorded with a data acquisition system and GME were detected and characterized using the Emboli Detection and Classification Quantifier System. CRRT was added at distinct locations such that blood entered CRRT between the pump and oxygenator (A), recirculated through the pump (B), or bypassed the pump (C). With the centrifugal pump, all CRRT positions had similar flow rates, mean arterial pressure (MAP), and total hemodynamic energy (THE) loss. With the roller pump, C demonstrated increased flow rates (293.2–686.4 mL/min) and increased MAP (59.4–75.5 mm Hg) (P

As it is common for patients treated with extracorporeal life support (ECLS) to subsequently require continuous renal replacement therapy (CRRT), and neonatal patients encounter limitations due to lack of access points, inclusion of CRRT in the ECLS circuit could provide advanced treatment for this population. The objective of this study was to evaluate an alternative neonatal ECLS circuit containing either a Maquet RotaFlow centrifugal pump or Maquet HL20 roller pump with one of seven configurations of CRRT using the Prismaflex 2000 System. All ECLS circuit setups included a Quadrox-iD Pediatric diffusion membrane oxygenator, a Better Bladder, an 8-Fr arterial cannula, a 10-Fr venous cannula, and 6 feet of ¼-inch diameter arterial and venous tubing. The circuit was primed with lactated Ringer's solution and packed human red blood cells resulting in a total priming volume of 700 mL for both the circuit and the 3-kg pseudopatient. Hemodynamic data were recorded for ECLS flow rates of 200, 400, and 600 mL/min and a CRRT flow rate of 50 mL/min. When a centrifugal pump is used, the hemodynamic performance of any combined ECLS and CRRT circuit was not significantly different than that of the circuit without CRRT, thus any configuration could potentially be used. However, introduction of CRRT to a circuit containing a roller pump does affect performance properties for some CRRT positions. The circuits with CRRT positions B and G demonstrated decreased total hemodynamic energy (THE) levels at the post-arterial cannula site, while positions D and E demonstrated increased post-arterial cannula THE levels compared to the circuit without CRRT. CRRT positions A, C, and F did not have significant changes with respect to pre-arterial cannula flow and THE levels, compared to the circuit without CRRT. Considering hemodynamic performance, for neonatal combined extracorporeal membrane oxygenation (ECMO) and CRRT circuits with both blood pumps, we recommend the use of CRRT position A due to its hemodynamic similarities to the ECMO circuit without CRRT.

Genomic studies in acute myeloid leukemias (AML) have identified mutations that drive altered DNA methylation, including TET2 and IDH2. Here, we show that models of AML resulting from TET2 or IDH2 mutations combined with FLT3ITD mutations are sensitive to 5-azacytidine or to the IDH2 inhibitor AG-221, respectively. 5-azacytidine and AG-221 treatment induced an attenuation of aberrant DNA methylation and transcriptional output and resulted in a reduction in leukemic blasts consistent with antileukemic activity. These therapeutic benefits were associated with restoration of leukemic cell differentiation, and the normalization of hematopoiesis was derived from mutant cells. By contrast, combining AG-221 or 5-azacytidine with FLT3 inhibition resulted in a reduction in mutant allele burden, progressive recovery of normal hematopoiesis from non-mutant stem-progenitor cells, and reversal of dysregulated DNA methylation and transcriptional output. Together, our studies suggest combined targeting of signaling and epigenetic pathways can increase therapeutic response in AML. Significance: AMLs with mutations in TET2 or IDH2 are sensitive to epigenetic therapy through inhibition of DNA methyltransferase activity by 5-azacytidine or inhibition of mutant IDH2 through AG-221. These inhibitors induce a differentiation response and can be used to inform mechanism-based combination therapy. Cancer Discov; 7(5); 494–505. ©2017 AACR. See related commentary by Thomas and Majeti, p. 459. See related article by Yen et al., p. 478. This article is highlighted in the In This Issue feature, p. 443

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3AR882), have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3AR882 AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy, suggesting that DNMT3AR882 cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3ITD) and the nucleophosmin gene (Npm1c) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3AR882 mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3AR882 cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3AR882 mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.

Supplemental Digital Content is available in the text, Despite recent advances, the myeloproliferative neoplasms (MPNs) are attended by considerable morbidity and mortality. Janus kinase (Jak) inhibitors such as ruxolitinib manage symptoms but do not substantially change the natural history of the disease. In this report, we show the effects of IMG-7289, an irreversible inhibitor of the epigenetically active lysine-specific demethylase 1 (LSD1) in mouse models of MPN. Once-daily treatment with IMG-7289 normalized or improved blood cell counts, reduced spleen volumes, restored normal splenic architecture, and reduced bone marrow fibrosis. Most importantly, LSD1 inhibition lowered mutant allele burden and improved survival. IMG-7289 selectively inhibited proliferation and induced apoptosis of JAK2 V617F cells by concomitantly increasing expression and methylation of p53, and, independently, the pro-apoptotic factor PUMA and by decreasing the levels of its antiapoptotic antagonist BCLXL. These data provide a molecular understanding of the disease-modifying activity of the LSD1 inhibitor IMG-7289 that is currently undergoing clinical evaluation in patients with high-risk myelofibrosis. Moreover, low doses of IMG-7289 and ruxolitinib synergize in normalizing the MPN phenotype in mice, offering a rationale for investigating combination therapy.

Summary Mutations in epigenetic modifiers and signaling factors often co-occur in myeloid malignancies, including TET2 and NRAS mutations. Concurrent Tet2 loss and Nras G12D expression in hematopoietic cells induced myeloid transformation, with a fully penetrant, lethal chronic myelomonocytic leukemia (CMML), which was serially transplantable. Tet2 loss and Nras mutation cooperatively led to decrease in negative regulators of mitogen-activated protein kinase (MAPK) activation, including Spry2, thereby causing synergistic activation of MAPK signaling by epigenetic silencing. Tet2/Nras double-mutant leukemia showed preferential sensitivity to MAPK kinase (MEK) inhibition in both mouse model and patient samples. These data provide insights into how epigenetic and signaling mutations cooperate in myeloid transformation and provide a rationale for mechanism-based therapy in CMML patients with these high-risk genetic lesions.

Viny et al. show that depletion of the cohesin subunit Smc3 affects HSC function through disruption of chromatin structure and that cohesin haploinsufficiency cooperates with Flt3-ITD to induce transformation of stem/progenitor cells and leukemia development., Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation; however, cohesin mutant leukemias do not show genomic instability. We hypothesized that reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Biallelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation and revealed an absolute requirement for cohesin in hematopoietic stem cell (HSC) function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose dependency for cohesin in regulating chromatin structure and HSC function.

SummaryAlthough clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasm (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated whether CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2 and MPL mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients.

Specific combinations of acute myeloid leukemia (AML) disease alleles, including FLT3 and TET2 mutations, confer distinct biologic features and adverse outcome. We generated mice with mutations in Tet2 and Flt3, which resulted in fully penetrant, lethal AML. Multipotent Tet2(-/-);Flt3(ITD) progenitors (LSK CD48(+)CD150(-)) propagate disease in secondary recipients and were refractory to standard AML chemotherapy and FLT3-targeted therapy. Flt3(ITD) mutations and Tet2 loss cooperatively remodeled DNA methylation and gene expression to an extent not seen with either mutant allele alone, including at the Gata2 locus. Re-expression of Gata2 induced differentiation in AML stem cells and attenuated leukemogenesis. TET2 and FLT3 mutations cooperatively induce AML, with a defined leukemia stem cell population characterized by site-specific changes in DNA methylation and gene expression.

Recent studies have reported that activation-induced cytidine deaminase (AID) and ten-eleven-translocation (TET) family members regulate active DNA demethylation. Genetic alterations of TET2 occur in myeloid malignancies, and hematopoietic-specific loss of Tet2 induces aberrant hematopoietic stem cell (HSC) self-renewal/differentiation, implicating TET2 as a master regulator of normal and malignant hematopoiesis. Despite the functional link between AID and TET in epigenetic gene regulation, the role of AID loss in hematopoiesis and myeloid transformation remains to be investigated. Here, we show that Aid loss in mice leads to expansion of myeloid cells and reduced erythroid progenitors resulting in anemia, with dysregulated expression of Cebpa and Gata1, myeloid/erythroid lineage-specific transcription factors. Consistent with data in the murine context, silencing of AID in human bone marrow cells skews differentiation toward myelomonocytic lineage. However, in contrast to Tet2 loss, Aid loss does not contribute to enhanced HSC self-renewal or cooperate with Flt3-ITD to induce myeloid transformation. Genome-wide transcription and differential methylation analysis uncover the critical role of Aid as a key epigenetic regulator. These results indicate that AID and TET2 share common effects on myeloid and erythroid lineage differentiation, however, their role is nonredundant in regulating HSC self-renewal and in myeloid transformation.

As it is common for patients treated with extracorporeal life support (ECLS) to subsequently require continuous renal replacement therapy (CRRT), and neonatal patients encounter limitations due to lack of access points, inclusion of CRRT in the ECLS circuit could provide advanced treatment for this population. The objective of this study was to evaluate an alternative neonatal ECLS circuit containing either a Maquet RotaFlow centrifugal pump or Maquet HL20 roller pump with one of seven configurations of CRRT using the Prismaflex 2000 System. All ECLS circuit setups included a Quadrox-iD Pediatric diffusion membrane oxygenator, a Better Bladder, an 8-Fr arterial cannula, a 10-Fr venous cannula, and 6 feet of ¼-inch diameter arterial and venous tubing. The circuit was primed with lactated Ringer's solution and packed human red blood cells resulting in a total priming volume of 700 mL for both the circuit and the 3-kg pseudopatient. Hemodynamic data were recorded for ECLS flow rates of 200, 400, and 600 mL/min and a CRRT flow rate of 50 mL/min. When a centrifugal pump is used, the hemodynamic performance of any combined ECLS and CRRT circuit was not significantly different than that of the circuit without CRRT, thus any configuration could potentially be used. However, introduction of CRRT to a circuit containing a roller pump does affect performance properties for some CRRT positions. The circuits with CRRT positions B and G demonstrated decreased total hemodynamic energy (THE) levels at the post-arterial cannula site, while positions D and E demonstrated increased post-arterial cannula THE levels compared to the circuit without CRRT. CRRT positions A, C, and F did not have significant changes with respect to pre-arterial cannula flow and THE levels, compared to the circuit without CRRT. Considering hemodynamic performance, for neonatal combined extracorporeal membrane oxygenation (ECMO) and CRRT circuits with both blood pumps, we recommend the use of CRRT position A due to its hemodynamic similarities to the ECMO circuit without CRRT.

DNA methyltransferase 3A (DNMT3A) mutations are observed in myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Transplantation studies have elucidated an important role for Dnmt3a in stem cell self-renewal and in myeloid differentiation. Here, we investigated the impact of conditional hematopoietic Dnmt3a loss on disease phenotype in primary mice. Mx1-Cre-mediated Dnmt3a ablation led to the development of a lethal, fully penetrant MPN with myelodysplasia (MDS/MPN) characterized by peripheral cytopenias and by marked, progressive hepatomegaly. We detected expanded stem/progenitor populations in the liver of Dnmt3a-ablated mice. The MDS/MPN induced by Dnmt3a ablation was transplantable, including the marked hepatomegaly. Homing studies showed that Dnmt3a-deleted bone marrow cells preferentially migrated to the liver. Gene expression and DNA methylation analyses of progenitor cell populations identified differential regulation of hematopoietic regulatory pathways, including fetal liver hematopoiesis transcriptional programs. These data demonstrate that Dnmt3a ablation in the hematopoietic system leads to myeloid transformation in vivo, with cell-autonomous aberrant tissue tropism and marked extramedullary hematopoiesis (EMH) with liver involvement. Hence, in addition to the established role of Dnmt3a in regulating self-renewal, Dnmt3a regulates tissue tropism and limits myeloid progenitor expansion in vivo.

The discovery of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to clinical development of Janus kinase (JAK) inhibitors for treatment of MPN. These inhibitors improve constitutional symptoms and splenomegaly but do not significantly reduce mutant allele burden in patients. We recently showed that chronic exposure to JAK inhibitors results in inhibitor persistence via JAK2 transactivation and persistent JAK-signal transducer and activator of transcription signaling. We performed genetic and pharmacologic studies to determine whether improved JAK2 inhibition would show increased efficacy in MPN models and primary samples. Jak2 deletion in vivo led to profound reduction in disease burden not seen with JAK inhibitors, and deletion of Jak2 following chronic ruxolitinib therapy markedly reduced mutant allele burden. This demonstrates that JAK2 remains an essential target in MPN cells that survive in the setting of chronic JAK inhibition. Combination therapy with the heat shock protein 90 (HSP90) inhibitor PU-H71 and ruxolitinib reduced total and phospho-JAK2 and achieved more potent inhibition of downstream signaling than ruxolitinib monotherapy. Combination treatment improved blood counts, spleen weights, and reduced bone marrow fibrosis compared with ruxolitinib alone. These data suggest alternate approaches that increase JAK2 targeting, including combination JAK/HSP90 inhibitor therapy, are warranted in the clinical setting.

Epigenetic modifiers and signaling factors are frequently mutated and often co-occur in various myeloid malignancies. However, precisely how these mutations cooperate to cause myeloid leukemia is not fully understood. Here, we show that cells with concurrent Ten-eleven-translocation 2 (Tet2) loss and Nras mutation can cause lethal chronic myelomonocytic leukemia (CMML) like disease in vivo and synergistically activate Ras signaling through epigenetic silencing of Sprouty2 (Spry2), thereby making cells with both disease alleles dependent on MAPK signaling and highly sensitive to MEK inhibition. To assess if Tet2 loss and Nras mutation cooperate in myeloid transformation, we crossed Tet2 conditional knockout mice (Mx1-Cre+Tet2f/f) and Nras mutant mice (Mx1-Cre+Nras+/G12D) to generate Mx1-Cre+Tet2f/fNras+/G12D mice (Tet2Δ/ΔNras+/G12D). These mice, compared to single mutant mice with either allele alone, had more significant monocytosis, expansion of Lineage- Sca-1+ c-Kit+ (LSK) and myeloid progenitors in both bone marrow (BM) and spleen and development of lethal CMML-like disease (median survival 264 days). Moreover, serial transplantation of splenic cells derived from leukemic Tet2Δ/ΔNras+/G12D mice caused similar CMML-like disease in recipients, which emanates from LSK-positive stem/progenitor cells as the disease propagating population. To delineate how Tet2 loss and Nras mutation synergize in leukemic transformation, we next performed western blot and phospho-flow analysis of MAPK and PI3K signaling in primary hematopoietic cells. Interestingly, pErk, pAkt and pS6 expression were significantly higher in Tet2Δ/ΔNras+/G12D cells compared to WT or single mutant cells, indicating that Tet2 loss and Nras mutation cooperates to further activate Ras signaling (Figure 1). Consistent with our murine model, TET2 silencing in NRAS mutant human leukemia cells increased MAPK output, consistent with augmentation of signaling by concurrent TET2/NRAS alterations in human leukemia cells. To unravel the molecular mechanism of Ras signaling activation, we assessed mRNA / protein expression and performed bisulfite sequencing of known regulators of MAPK signaling, including Sprouty family members. We observed significant decrease of Spry2 expression, stepwise and specific hyper-methylation of CpG islands in the Spry2 promoter region in Tet2Δ/ΔNras+/G12D cells compared to WT or single mutant cells, consistent with progressive epigenetic remodeling in these leukemia cells in vivo (Figure 2). Genome wide methylation profiling of WT, single mutant and Tet2Δ/ΔNras+/G12D LSK cells using enhanced reduced representation bisulfite sequencing demonstrated clear separation of leukemic Tet2Δ/ΔNras+/G12D LSKs from WT or single mutant LSKs. Most importantly, restoration of Spry2 expression in Tet2Δ/ΔNras+/G12D cells led to decrease in pErk / pAkt level and significantly reduced colony formation, which functionally validates Spry2 as a key epigenetic target in Tet2/Nras mutant leukemia cells. We next assessed whether the increased MAPK signaling seen in Tet2Δ/ΔNras+/G12D cells leads to differential sensitivity to MEK inhibition by performing studies with the clinical MEK inhibitor binimetinib (ARRY162). Tet2Δ/ΔNras+/G12D cells showed significantly higher sensitivity to binimetinib compared to Nras+/G12D cells in vitro (IC50, 6.948nM vs. 690.4nM). Moreover, in vivo treatment of Tet2Δ/ΔNras+/G12D leukemic recipients with binimetinib restored splenomegaly, significantly reduced disease burden in BM and spleen and improved overall survival compared to vehicle treatment (median survival 24.5 days vs. 44.5 days, p=0.0018, Figure 3). Of note, knockdown of human TET2 in NRAS mutant human leukemia cells sensitized to MEK inhibition in a similar manner demonstrating this approach may have value in leukemia patients with concurrent TET2 and NRAS mutations. These data clearly indicate that Tet2 loss and Nras mutation synergize in myeloid transformation and cooperatively remodel DNA methylation, which leads to epigenetic silencing of Spry2 and synergistic activation of MAPK signaling, which can be leveraged through therapeutic MEK inhibition. Our studies provide novel insights into how signaling and epigenetic mutations cooperate in leukemic transformation and provide a rationale for mechanism based therapy in CMML patients with these high risk genetic lesions. Disclosures Melnick: Janssen: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Among BCR-ABL-negative myeloproliferative neoplasms (MPN), primary myelofibrosis (PMF) and post PV/ET myelofibrosis (MF) are associated with the highest degree of morbidity and mortality, including progressive bone marrow (BM) fibrosis and resultant BM failure. Although the JAK inhibitor ruxolitinib is now approved for the treatment of MF-associated splenomegaly and systemic symptoms, JAK inhibitor therapy does not reduce the proportion of JAK2-mutant cells in MPN patients. The limited ability of JAK inhibition to induce molecular or clinicopathologic responses in the majority of MPN patients underscores the need for the development of more effective therapies for JAK kinase-dependent malignancies. Recent studies have shown that the lysine-specific histone demethylase, LSD1 (KDM1A), participates in the balance between proliferation and differentiation in vivo by influencing state-specific gene expression patterns. In physiologic hematopoiesis, LSD1 is essential for normal myeloid differentiation affecting the erythroid, megakaryocytic and granulocytic lineages. Small molecule inhibitors of LSD1 have shown promising results in preclinical models of acute myeloid leukemia (AML) and solid cancers and have recently entered clinical trials in AML. However, the role and requirement for LSD1 in the pathogenesis of MPNs and the therapeutic targeting of LSD1 in MPN has not been investigated. In this study, we first tested the effects of IMG-98, a potent, selective LSD1 inhibitor, in the MPLW515L-driven ET/MF mouse model. After disease was established, mice were treated with IMG-98 or vehicle for 28 days. LSD1 inhibition in mice markedly suppressed myeloproliferation reducing granulocyte counts and spleen weights compared to mice treated with vehicle thus establishing therapeutic efficacy (Fig. 1a). Pathologic analysis of BM and spleen confirmed a marked reduction in myeloproliferation as well as a reversal of extramedullary hematopoiesis (EMH). Most notably, we observed a marked reduction in reticulin fibrosis with IMG-98 treatment (Fig. 1b). We next investigated the impact of IMG-98 therapy on inflammatory cytokine signaling; in contrast to the broad anti-cytokine effects of JAK1/2 inhibition, we observed a more specific anti-cytokine effect of IMG-98, a significant reduction in the secretion of the inflammatory cytokine Cxcl5 (Fig. 1c), a key participant in pathologic inflammatory states. We then investigated the in vivo impact of IMG-98 therapy on mutant disease burden. IMG-98 therapy reduced mutant allele burden to a degree not seen with JAK1/2 inhibitor therapy: whereas 74.6% of circulating cells in mice treated with vehicle were GFP-positive cells, only 43.2% of circulating cells were GFP-positive in IMG-98-treated mice (Fig. 1d). Flow cytometry analysis of spleen and BM revealed reduced numbers of CD11b/Gr1-positive myeloid cells and CD41-positive megakaryocytes. The numbers of mutant GFP-positive myeloid cells and megakaryocytes in these tissues were also significantly reduced by IMG-98 treatment. Studies of the impact of LSD1 inhibition on MPN stem cell function and on epigenetic regulation in MPN cells will be presented in detail. In summary, the LSD1 inhibitor IMG-98 had a highly significant therapeutic effect in an established preclinical model of ET/MF. LSD1 inhibition in diseased mice reduced JAK-STAT-driven myeloproliferation, markedly reversed EMH and BM fibrosis, and reduced the mutant clone burden. These data suggest LSD1 is a valid target in MPN and that clinical studies of LSD1 inhibitor IMG-98 alone and in combination with JAK inhibitors are warranted. Figure 1. a, b) LSD1 inhibition results in reduced white blood cell counts (WBC) and platelet counts (PLT). (a), and in near-complete elimination of BM fibrosis (b). c) Profound reduction of Cxcl5 serum levels in IMG-98 treated mice compared to vehicle treated mice. d) Significantly lower mutant allele burden in the peripheral blood of IMG-98 treated mice. * P Disclosures Riehnhoff: Imago: Employment, Equity Ownership. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation, however cohesin-mutant leukemias do not show genomic instability suggesting an alternate role in malignant transformation. We hypothesized reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic stem/progenitor cells. We therefore investigated the impact of both complete loss and haploinsufficiency of Smc3, an obligate member of the cohesin complex, in normal hematopoiesis and in myeloid transformation by developing a conditional Smc3 knockout allele. Somatic loss of Smc3 in hematopoietic cells induced lethal bone marrow aplasia (median survival 11 days; p To further explore the mechanism by which Smc3 loss cooperates with Flt3ITD to induce leukemia, we investigated chromatin cis-regulatory architecture with transposase hypersensitivity assays (ATAC-seq). We hypothesized that increased accessibility at cis-regulatory elements and the alterations in gene expression seen in cells with combined Smc3 haploinsufficiency and Flt3ITD may be in a large part driven by potentiated Stat signaling at chromatin. We analyzed 146 transcription factor recognition motifs within the THS differentially observed in Smc3Δ/+Flt3ITD and wild-type cells. Chromatin accessibility gained in Smc3Δ/+Flt3ITD cells are enriched in Stat family transcription factor binding sites, including Stat5. We also observed enrichment of the Stat5 gene expression signature in the Smc3Δ/+Flt3ITD cells compared to Smc3Δ/+, Flt3ITD and wild-type cells, suggesting the divergent mutations cooperate to potentiate oncogenic Stat5 signaling in HSPCs. Our results demonstrate a key dose-dependent role for the cohesin complex in hematopoiesis, and show that reduced cohesin functions to alter enhancer-mediated transcription and contribute to aberrant self-renewal and myeloid transformation. Figure 1. Figure 1. Disclosures Levine: Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are observed in patients with acute myeloid leukemia (AML). Leukemia-associated IDH1/2 mutations result in aberrant accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). The observation that IDH1/2 mutations are mutually exclusive with TET2 mutations led to the finding that IDH1/2-mutant production of 2-HG inhibits TET2 function and induces changes in DNA methylation. These data suggested that small molecule inhibition of mutant IDH enzymes might reverse the aberrant epigenetic remodeling of IDH-mutant leukemia cells and restore normal hematopoietic differentiation. We therefore investigated the in vivo efficacy of AG-221, a potent and selective mutant IDH2 inhibitor in early-phase clinical trials, in murine models of IDH2-mutant leukemia. We first assessed the impact of AG-221 on 2-HG production in hematopoietic cells expressing mutant IDH2-R140Q. AG-221 treatment (10mg/kg or 100mg/kg bid) led to a reduction in 2-HG in vivo (96.7% below pre-treatment levels). Moreover, AG-221 treatment restored megakaryocyte-erythroid progenitor (MEP) differentiation that is suppressed by mutant IDH2 expression in vivo (mean MEP% mean, 39% Veh vs 50% AG-221). We next investigated the impact of mutant IDH2 inhibition with AG-221 on DNA methylation in vivo. We used eRRBS, a bisulfite-based next-generation sequencing platform, to assess the effect of AG-221 therapy on DNA methylation. AG-221 or vehicle therapy treated LSK stem cells (lin- Sca+ c-Kit+) were sorted from mice expressing IDH2-R140Q and evaluated by eRRBS. AG-221 therapy reversed the effects of mutant IDH2; we observed a significant reduction in DNA methylation, including 180 genes that had 20 or more hypomethylated differentially methylated cytosines (DMCs) following treatment. 84 of these genes had reduced methylation at 10 or more DMCs in the gene promoter with AG-221 therapy compared to vehicle. Mutant IDH2 inhibition with AG-221 reversed aberrant methylation at many genes with a known role in hematopoietic proliferation and differentiation, including the master transcriptional factor RUNX1. We next assessed in vivo effects of the small-molecule IDH2-R140Q inhibitor in a mouse model of IDH2-mutant leukemia. We generated mice that simultaneously expressed a constitutive Flt3ITD knock-in allele and a conditional mutant IDH2R140Q knock-in allele. As reported recently using retroviral/transgenic models, Mx1-Cre IDH2R140QFlt3ITD developed fully penetrant, transplantable AML with expansion of c-Kit+ positive blasts in the peripheral blood, and widespread leukemic infiltration. AG-221 inhibited the serial replating capacity of IDH2R140QFlt3ITD expressing cells in vitro. We competitively transplanted IDH2R140QFlt3ITD AML cells and normal bone marrow cells into secondary recipients, and then assessed the effect of AG-221 therapy on leukemia in vivo and on disease burden. AG-221 (100mg/kg bid) treatment of mice engrafted with Mx1-Cre IDH2R140QFlt3ITD AML cells markedly reduced 2HG levels consistent with on target inhibition in vivo. AG-221 therapy induced differentiation of leukemic cells, with an increase in the CD11b+ population and a decrease in the c-Kit+ population in the peripheral blood at 2wks. We next assessed the impact of treatment with both AG-221 therapy with AC220, a potent, specific Flt3 inhibitor in late phase clinical trials. Combined IDH2R140Q and Flt3ITD inhibition resulted in a marked decrease in leukemic burden to vehicle-treated mice, with a significant reduction in leukemic cell chimerism in vivo in the setting of combined inhibition at 2 wks, (mean 45.2 fraction 88% veh, 73% AG-221, p These data demonstrate that AG-221 inhibits mutant IDH2-mediated 2-HG production in vivo and reverses the effects of mutant IDH2 on DNA methylation in mutant stem/progenitor cells. AG-221 induces differentiation and impairs self-renewal of IDH2-mutant leukemia cells, effects that are further enhanced by simultaneous inhibition of Flt3ITD. Clinical trials combining IDH2 inhibitors with other targeted AML therapies are warranted in order to increase therapeutic efficacy. Disclosures Intlekofer: Foundation Medicine, Inc: Consultancy. Thompson:Agios: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Straley:Agios: Employment, Equity Ownership. Gliser:Agios Pharmaceuticals: Employment, Stockholder Other. Yen:Agios: Employment, Equity Ownership. Levine:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are frequent in normal karyotype de novo acute myeloid leukemia (AML) (20-35%), chronic myelomonocytic leukemia (CMML) (10-20%) and myelodysplastic syndrome (MDS) (8%). Hematopoietic-specific loss of Dnmt3a in a mouse model leads to acquisition of aberrant self-renewal by the HSCs and expansion of the stem/progenitor compartment in bone marrow transplantation studies. Despite these important insights, the impact of hematopoietic deletion of Dnmt3a on disease phenotype in primary, non-transplanted mice has not been described. Mx1-Cre-mediated Dnmt3a ablation in the hematopoietic system in primary mice led to the development of a myeloproliferative neoplasm (MPN) with a 100% penetrance (n=14) and a median age of onset at 47.7 weeks (survival difference between Dnmt3a KO and control animals p Myeloproliferation induced by Dnmt3a loss was characterized by marked, progressive hepatomegaly (liver weights 7.25±1.195 g in Dnmt3a-deleted animals vs 1.61±0.266 g in wild-type controls, p Notably, the myeloid disease phenotype induced by Dnmt3a loss was fully transplantable, including the marked hepatomegaly; these data demonstrate that the liver-specific expansion reflects a cell-autonomous mechanism. To assess relative tropism for different target organs, we next performed homing studies where Dnmt3a-deleted bone marrow cells were competed against wild-type counterparts in lethally irradiated hosts. 48 hours after transplantation, we observed increased tropism of the Dnmt3aΔ/Δ BM cells to the liver and spleen, whereas control cells preferentially localized to the bone marrow (difference between homing to bone marrow and spleen/liver p In conclusion, our studies show that ablation of Dnmt3a in the hematopoietic system leads to myeloid transformation in vivo, with cell autonomous liver tropism and marked extramedullary hematopoiesis. These data demonstrate, in addition to its established role in controlling self-renewal, Dnmt3a serves as an important regulator of the myeloid compartment that limits expansion of myeloid progenitors in vivo. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The identification of JAK-STAT pathway mutations in the majority of patients with the myeloproliferative neoplasms (MPN) polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF) led the to clinical development of JAK inhibitors, and the resultant approval of ruxolitinib for the treatment of PMF. However, despite this important therapeutic advance, there are significant limitations to JAK inhibitor therapy both with respect to efficacy and toxicity. First, although JAK inhibitors reduce splenomegaly, ameliorate symptoms, and improve long-term outcome, they do not achieve molecular or pathologic remission at currently utilized dosing strategies. Second, JAK2 has a role in hematopoiesis and other biological processes, and JAK inhibition leads to significant hematologic toxicities including anemia and thrombocytopenia. We recently used genetic and pharmacologic studies to demonstrate that JAK inhibitor persistent cells which survive JAK inhibitor therapy in vitro and in vivo remain JAK2 dependent, consistent with incomplete target inhibition. As such, we hypothesized that alternate dosing regimens which allow for intermittent, maximal target inhibition might increase efficacy and reduce toxicity. We therefore used experimental and modeling approaches to investigate the potential efficacy of alternate dosing regimens. We first explored the effects of chronic vs intermittent dosing in vitro by altering the treatment regimen in cell lines. To this end, we treated the JAK2 V617F mutant cell line, SET-2, and JAK2-wild-type (control) cell lines with ruxolitinib (1µM vs 0.5 µM) on a chronic or intermittent (alternating 1 week on and 1 week off the drug) basis. We then performed cell viability assays using flow cytometry to estimate the effect of the drug on the cell division and death rates of each cell population. Using this data, we developed a mathematical model to predict responses to varying dose therapy. Cell proliferation was described using an exponential growth model (pt2 = pt1 e(birth rate-death rate)Dt, p=population size). Birth and death rates as a function of the drug concentration was fitted using a simple iterative least squares estimation from the in vitro collected data, where death(c) = 0.0046log(1.5014 + 30.4910c) and birth(c) = 0.0098 + 0.0051e-1.2946c. Treatment cycles were modeled by ton + toff for pulsitile versus chronic (toff = 0) regimens for time on and off drug. We also added a toxicity constraint based on preclinical testing and the mathematical model T(c) = (α/c) –β, where α = 539 and β=5.2, which will inform our in vivo studies. Inputting these rates into a mathematical model to predict optimal treatment schedule, our in silico analysis suggest that high dose pulse treatment of INCB18424 has the same efficacy as chronic dosing and is associated with reduced toxicity. We are currently testing our dosing and administration schedules using in vivo models of MPN, and we will present these data at the meeting. Preliminary studies suggest intermittent JAK inhibition shows similar efficacy as chronic JAK inhibition, with reduced toxicity, suggesting our in silico models inform the development of more optimal dosing regimens. We are now testing higher doses of JAK inhibitors in an intermittent administration regimen in order to maximize efficacy and mitigate hematologic and non-hematologic toxicity. In conclusion, our proof-of-principle studies show that intermittent treatment with JAK kinase inhibitors demonstrates equivalent efficacy in vitro and our in silico data suggests that we will see reduced toxicity with intermittent dosing in the mouse models. Our in vivo data will inform further clinical optimization of treatment regiments for patients with myeloproliferative neoplasms Disclosures Koppikar: Amgen: Employment. Levine:Novartis: Consultancy, Grant support Other.

Despite significant advances in cancer research and treatment, therapeutic resistance remains a major obstacle for achieving stable remission in cancer patients. Acute myeloid leukemia (AML) is no exception, and most AML patients develop resistance to chemotherapy/targeted therapies, which results in disease relapse and progression. Recurrent mutations in the DNA methyltransferase 3A (DNMT3A) gene have been identified in 20-30% of AML cases and are predictive of unfavorable prognosis in patients treated with standard anti-leukemic regimens. In addition, DNMT3A-mutant AMLs appear to be relatively refractory to anthracycline family chemotherapeutics, such as daunorubicin. Half of all DNMT3A mutations affect amino acid residue R882, and recent work has shown that these mutants display decreased enzymatic activity and aberrant binding properties. In addition, previous studies have shown that wild-type DNMT3A functions as a pro-apoptotic switch in response to genotoxic stress induced by another anthracycline doxorubicin. We propose that mutant DNMT3A protects cells from apoptosis in response to DNA damage by altering molecular machinery involved in DNA-damage sensing, response and/or repair, through DNA methylation-dependent or independent mechanisms. Specifically, our data show that mutant DNMT3A affects recruitment of DNA repair proteins to chromatin, including aberrant distribution of homologous recombination marker RAD51. We are currently investigating molecular changes in DNA damage response in DNMT3A-mutant cells in vitro and ex vivo, and leukemogenic potential of the mutant Dnmt3a allele in vivo, whether alone or in combination with other cooperating oncogenes. Citation Format: Olga A. Guryanova, Kaitlyn Shank, Luisa Luciani, Evangelia Loizou, Matthew D. Keller, Abby R. Weinstein, Omar Abdel-Wahab, Siddhartha Mukherjee, Stephen S. Nimer, Ross L. Levine. Leukemia-associated DNMT3A R882 mutations and their role in anthracycline-induced DNA damage response and therapeutic resistance. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 44. doi:10.1158/1538-7445.CANSUSC14-44

The JAK1/2 dual kinase inhibitor INC424 (ruxolitinibª, Jakafi¨) has been recently approved for the treatment of intermediate or high-risk myelofibrosis (MF) and is in late-stage clinical trials for polycythemia vera (PV). Although JAK inhibitors have been shown to improve patient constitutional symptoms and reduce splenomegaly, JAK inhibitor monotherapy does not significantly reduce mutant allele burden in the majority of MPN patients. Furthermore, the therapeutic window of JAK inhibitors is limited due to the essential role of the JAK-STAT signaling pathway in normal hematopoiesis, which has been observed in the clinic where these inhibitor have been associated with dose limiting toxicities including anemia and thrombocytopenia. Therefore, there is a need to identify additional pathways that might be involved in the development and maintenance of MPN mutant clones, which could then be targeted in combination with JAK2 for improved therapeutic benefit. Recent studies have shown that the hedgehog (Hh) signaling pathway has important roles in normal hematopoiesis as well as in the pathogenesis of myeloid malignancies. We therefore investigated whether the Hh pathway was activated in MPN patients and whether it represented a therapeutic target in MPN preclinical models. We observed 20-100 fold increase in expression of Hh target genes including Gli1 and Ptch1 by quantitative PCR in granulocytes isolated from MPN patients as compared to normal controls. We also observed activation of the pathway in a murine bone marrow transplant (BMT) model of PMF using a Gli-luciferase reporter system. These data demonstrate the Hh pathway is active in primary MPN patient samples and in preclinical, models of PMF. Small molecule inhibitors of Hh signaling have shown promising results in Phase I clinical trials in patients with myeloid malignancies. In this study, we tested the effects of LDE225 (Sonidegib), a selective Smo inhibitor in combination with INC424 in a murine BMT model of ET/MF. Mice were treated with vehicle, 90mg/kg INC424 or a combination of INC424 and 80mg/kg LDE225 for 28 days. We observed a significant reduction in WBC (32.74K/ul vs. 9.85K/ul, p Reduction in blood counts and spleen weights observed in combination of INC424 and LDE225 as compared to INC424 monotherapy. Disclosures: Rose: Novartis Pharmaceutical: Employment. Amakye:Novartis Pharmaceutical: Employment.

Specific combinations of Acute Myeloid Leukemia (AML) somatic mutations are associated with distinct clinical and biologic features. However, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Concurrent mutations in FLT3 and TET2 are associated with adverse outcome. We hypothesized that activating mutations in FLT3 would cooperate with inactivating mutations in TET2to induce AML in vivo, and that we could investigate AML pathogenesis and therapeutic response using a genetic model of this poor-risk AML genotype. To understand how these genes cooperate to induce AML, we generated Vav+Tet2fl/flFlt3-ITD mice, which resulted in fully penetrant, lethal disease in all recipient mice. Flow cytometric analysis revealed expansion of mac1+ cells in the peripheral blood, with progressive expansion of a c-Kit+, blast population which was apparent in the blood and bone marrow at 28 days, leading to lethal AML in all Vav+Tet2fl/flFlt3-ITD mice with a median survival of 12 months. Consistent with genetic data demonstrating most AML patients have monoallelic TET2 mutations, Vav+Tet2fl/+Flt3-ITD mice also develop AML, suggesting haploinsufficiency for Tet2 is sufficient to cooperate with the Flt3-ITD mutation to induce AML. All mice developed leukocytosis (median 85K/uL), splenomegaly (median 554mg) and hepatomegaly (median 2900mg) with evidence of extramedullary disease cell infiltration by leukemic blasts. Flow cytometric analysis of stem/progenitor populations revealed expansion of the granulocyte-macrophage progenitor (GMP) population and the lin- sca+ kit+ (LSK) stem cell population. Detailed analysis of the LSK population revealed a decrease in the LT-HSC population (LSK CD150+ CD48-) that was replaced by a monomorphic CD48+ CD150- multipotent progenitor population. Given previous studies have shown that LSK and GMP cells can contain leukemia stem cells (LSC) in other models of AML, we performed secondary transplant studies with LSK and GMP populations. LSK (CD48+ CD150-) cells, but not GMP cells, were able to induce disease in secondary and tertiary recipients in vivo. In order to assess the sensitivity of Tet2/Flt3-mutant AML and specifically the LSCs, to targeted therapies, we treated primary and transplanted mice with chronic administration of AC220, a FLT3 inhibitor in late-stage clinical trials. AC220 treatment inhibited FLT3 signaling in vivo, and reduced peripheral blood counts/splenomegaly. However, FLT3 inhibition did not reduce the proportion of AML cells in the bone marrow and peripheral blood. AC220 therapy in vivo reduced the proportion of GMP cells, but not LSK cells, demonstrating LSCs in this model are resistant to FLT3-targeted anti-leukemic therapy. We hypothesized that Tet2/Flt3-mutant LSCs possess a distinct epigenetic/transcriptional signature that contributes to leukemic cell self-renewal and therapeutic resistance. We performed RNA-seq using the Lifetech Proton sequencer to profile the expression landscape of Vav+Tet2fl/flFlt3-ITD mutant LSKs compared to normal stem cells. We were able to obtain an average of 62 million reads per sample. We identified over 400 genes differentially expressed in LSCs relative to normal hematopoietic stem cells (FC>2.5, padj Our data demonstrate that TET and FLT3 mutations can cooperate to induce AML in vivo, with a defined LSC population that is resistant to targeted therapies and characterized by site-specific changes in DNA methylation and gene expression. Current studies are aimed to assess the functional role of specific gene targets in LSC survival, and at defining therapeutic liabilities that can be translated to the clinical context. Disclosures: No relevant conflicts of interest to declare.

Abstract 3394 Hematopoietic stem cells (HSC) replenish the cellular components of the blood throughout life by a homeostatic process in which the majority of HSCs remain quiescent while a small percentage enter the cell cycle to either self-review or differentiate. During inflammatory responses to infections, Interferons (IFNa, IFNg) perturb HSC homeostasis, presumably in response to the demand for increased numbers of inflammatory cells. Previous studies have highlighted an apparent paradox, i.e. IFNs suppress the proliferation of normally cycling murine hematopoietic progenitor cells (HPCs), yet increase the fraction of normally quiescent Sca+ HSCs that proliferate. To investigate the mechanisms underlying this paradox, we dissected the dynamics of cell surface phenotypes, cell cycle kinetics, pro- and anti-apoptotic pathways within the HSC and HPC compartments in response to pIpC and IFNs both in vivo and in vitro. Forty-eight hours after pIpC injection, bone marrow (BM) cellularity declined by 60%, the proportion of Sca- kit+ HPCs fell from 0.45% to 0.05%, while the proportion of BM cells with the Sca+ kit+ HSC phenotype increased from 0.17 to 0.26%. To determine whether the increase in Sca+kit+ cells was due to proliferation of HSCs or upregulation of Sca-1 on HPCs, we cultured purified CD150+ Sca-Kit+ HPCs and CD150+Sca+kit+ HSCs in vitro with IFNa, IFNg, or PBS. Sca expression was induced on previously Sca- HPCs, and the level of Sca expression on HSCs was also increased. This induction was detectable as early as 6 hours after treatment and accompanied by an increase in Sca mRNA. BrdU incorporation into both HPC and HSC populations decreased from pre-treatment baselines, further indicating that the increase in cells with the HSC phenotype was not due to HSC proliferation, but rather the appearance of cycling HPCs within the HSC staining gate following IFN-induced upregulation of Sca. Staining with FITC-DEVD-FMK identified active cleaved capase-3 in pIpC- or IFN-treated cells, suggesting that the reduced cellularity following IFN reflected a cellular stress that killed Lin+ precursors cells and some HPCs, but spared HSCs. In contrast to lin+kit- precursors, all kit + HPCs and HSCs expressed bcl-2, suggesting that expression of anti-apoptotic proteins may prevent IFN-induced stress from resulting in HSC/HPC apoptosis despite the initial triggering of caspase-3 cleavage. In summary, acute treatment with IFNs has anti-proliferative effects on all hematopoietic cells, including precursors, HPCs and HSCs, with the apparent increase in HSC proliferation the result of HPCs masquerading as Sca+HSCs after exposure to IFN. Unlike precursors, HSCs and some HPCs survive treatment to IFNs despite activation of cleaved caspase-3, possibly due to their expression of bcl-2, and likely related anti-apoptotic regulators. The previously observed increase in HSC proliferation days and weeks following IFN treatment is most likely due to the homeostatic response of HSCs to the depopulation of the precursor and HPCs caused by acute IFN exposure. Disclosures: No relevant conflicts of interest to declare.