ALBERT

Description: Survival through periods of resource scarcity depends on the balance between metabolic demands and energy storage. The opposing effects of predation and starvation mortality are predicted to result in trade-offs between traits that optimize fitness during periods of resource plenty (e.g., during the growing season) and those that optimize fitness during periods of resource scarcity (e.g., during the winter). We conducted a common environment experiment with two genetically distinct strains of rainbow trout to investigate trade-offs due to (1) the balance of growth and predation risk related to foraging rate during the growing season and (2) the allocation of energy to body size prior to the winter. Fry (age 0) from both strains were stocked into replicate natural lakes at low and high elevation that differed in winter duration (i.e., ice cover) by 59 days. Overwinter survival was lowest in the high-elevation lakes for both strains. Activity rate and growth rate were highest at high elevation, but growing season survival did not differ between strains or between environments. Hence, we did not observe a trade-off between growth and predation risk related to foraging rate. Growth rate also differed significantly between the strains across both environments, which suggests that growth rate is involved in local adaptation. There was not, however, a difference between strains or between environments in energy storage. Hence, we did not observe a trade-off between growth and storage. Our findings suggest that intrinsic metabolic rate, which affects a trade-off between growth rate and overwinter survival, may influence local adaptation in organisms that experience particularly harsh winter conditions (e.g., extended periods trapped beneath the ice in high-elevation lakes) in some parts of their range. Survival through periods of resource scarcity depends on the balance between metabolic demands and energy storage. In rainbow trout, we did not observe a trade-off between growth and predation risk related to foraging rate, nor did we observe a trade-off between growth and storage. Our findings suggest that there may be a trade-off between growth rate and starvation resistance mediated by metabolic rate.

Description: NPM1 mutations are one of the most common alterations observed in acute myeloid leukemia (AML). When coupled with wild type FLT3 status in cytogenetically normal (CN) patients, NPM1 mutations confer favorable prognoses compared with other alterations. However, a subset of CN NPM1Mut :FLT3Wt patients with AML have dismal outcomes, suggesting that uncharacterized alterations influence the outcomes in these patients. To address this, we performed reverse phase protein array (RPPA) analysis on CD34+ bone marrow cells isolated from 43 de novo CN NPM1Mut :FLT3Wt AML patient as well as healthy donor controls. Through these analyses, we observed that overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) associated with extremely poor outcomes within this a priori favorable prognostic group, as almost 90% of patients with increased hnRNP K expression died within 12 months of diagnosis while nearly 40% of individuals with normal hnRNP K expression survived seven years (Figure 1A). hnRNP K is a multifunctional RNA and DNA binding protein whose expression is often altered in cancers. To directly examine the functional relationship between hnRNP K overexpression and mutant NPM1 in hematologic malignancies, we generated tissue-specific transgenic mouse models with the ability to overexpress hnRNP K (hnRNP KTg) in the presence or absence of mutant Npm1 (Npm1Tg). By crossing these mice with Vav-Cre expressing mice, we specifically activated hnRNP K overexpression and mutant NPM1 expression in the hematological compartment. Using Lin-CD117+ hematopoietic stem cells (HSCs) from hnRNP KTg, Npm1Tg, and hnRNP KTg;Npm1Tg mice, we observed significant changes in differentiation and proliferation potential in colony formation assays. Overexpression of hnRNP K alone significantly increased the number of colonies compared to wild type and Npm1Tg HSCs while expression of mutant Npm1Tg resulted in increased numbers of cells compared to wild type and hnRNP KTg HSCs. Importantly, the combination of hnRNP K overexpression and mutant Npm1 resulted in a cumulative increase in both the number of colonies and number of cells, indicating that hnRNP K and mutant NPM1 cooperate to dictate differentiation and proliferation potential in HSCs (Figure 1B). Next, we examined the in vivo impact of hnRNP K overexpression and mutant Npm1 expression by analyzing the bone marrows of Npm Tg, hnRNP KTg, and Npm1Tg;hnRNP KTg mice. Within the first six months of life, these mice rapidly developed significant myeloid hyperplasias as determined by flow cytometry and pathologic analyses (Figure 1C). Together, our findings reveal that mutant Npm1 and hnRNP K overexpression result in similar myeloid phenotypes. However, these genetic alterations are also cooperative, suggesting both increased hnRNP K expression and mutant NPM1 synergize to impact hematopoietic phenotypes and drive AML progression through similar pathways but potentially via unique molecular processes. Currently, we are investigating the direct interaction and global relationship between hnRNP K and mutant Npm1 in regulating tumor suppressor and oncogenic programs (e.g.; p53- and c-Myc pathways). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Deletion of the 9q21.32 locus is observed in some patients with AML, suggesting an undescribed tumor suppressor resides in this region. HNRNPK is an attractive candidate, as it is one of six genes mapped to this region and is thought to drive AML progression when mutated. Mechanistically, hnRNP K functions as a DNA and RNA binding protein that transcriptionally and translationally governs gene expression. Together, these findings suggest hnRNP K may serve as a currently uncharacterized tumor suppressor and that its haploinsufficiency plays a pivotal role in AML progression. To interrogate a potential relationship between HNRNPK haploinsufficiency and AML, we performed fluorescence in situ hybridization (FISH) using bone marrow aspirates from newly diagnosed AML patients. These analyses revealed the HNRNPK gene is specifically lost in a subset of AML patients and results in a significant decrease in HNRNP K expression in CD34+ cells from patients harboring this deletion (Fig. 1A). To directly examine the potential tumor-suppressive activities of hnRNP K in vivo, we generated a haploinsufficient hnRNP K (hnRNP K+/-) mouse model. hnRNP K haploinsufficiency resulted in reduced survival and hematologic neoplasms with marked genomic instability (Fig. 1B). Critically, hnRNP K+/- hematopoietic stem progenitor cells (HSPCs) were transplantable and had the capacity to develop hematopoietic neoplasms in recipient mice. To examine the mechanism driving these tumor phenotypes, we analyzed the cytokine profile of hnRNP K+/- mice. This examination revealed that several critical pro-proliferation and myeloid differentiation cytokines were significantly overexpressed (e.g.; IL-3, IL-6, GM-CSF, and G-CSF), resulting in activation of the JAK-STAT pathway (Fig. 1C). Next, we analyzed the proliferation potential of hnRNP K+/- HSPCs and mouse embryo fibroblast (MEFs) using cell based studies. Here, we observed a significant increase in the number of colony forming units in HSPCs and proliferation potential in both HSPC and MEFs between cells from hnRNP K+/- and wild type mice. Critical to the proliferative advantaged observed in hnRNP K+/- cells, activation of the p53/p21 pathway was significantly reduced in hnRNP K+/- MEFs following exposure to ionizing radiation. To more fully explore the molecular mechanisms responsible for these phenotypes, we performed whole-transcriptome analyses. Pathway analysis revealed that hnRNP K haploinsufficiency resulted in the attenuation of critical p53- and C/EBP pathways (Fig. 1D). We next validated expression changes in critical genes known to govern tumorigenesis, myeloid differentiation, and proliferation. Here, we observed a significantly down regulation in C/EBPa, C/EBPβ, and p21 at both the transcript and protein level (Fig. 1E-F). To assess the relationship between hnRNP K expression and regulation of these genes, we performed Chromatin Immunoprecipitation (ChIP) and RNA immunoprecipitation (RIP) analyses. ChIP analysis revealed the transcriptional activities of hnRNP K through its direct interaction with the promoter of these genes. Critically, reduced hnRNP K expression significantly diminished these interactions, resulting in their reduced expression. Interestingly, RIP analyses revealed that hnRNP K may also specifically regulate the expression of the tumor suppressive p42 isoform of C/EBPα through its translation regulation of the C/EBPα transcript. Together, these results suggest hnRNP K is a bona fide tumor suppressor and that its loss contributes to the development of hematologic malignancies. Given its critical regulation of the p53 pathway, drug therapies targeting the p53 pathway may serve as efficacious treatment options for patients with 9q deletions. To address this, we are currently examining the efficacy of Mdm2-p53 antagonist (e.g.; Nutlin-3 and AMG-232) using primary patient samples and primary cells from hnRNP K+/- mice with hematologic malignancies. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Background The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML), particularly in cases with high-risk cytogenetics. It has been shown that p53 stabilization, which frequently occurs when the protein is mutated, can compromise its function. We have shown that p53 stabilization, regardless of the presence of mutations, suggesting alterations of other components in the p53 pathway. Methodology p53 expression was determined using high-throughput reverse phase protein array (RPPA) technology in 719 samples from 511 pts. Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status of p53 alleles was assessed by Sanger sequencing of exons 5 through 9. Expression of components of the p53 pathway was determined using standard immunohistochemical techniques. Nutlin-3a was used in in vitro culture experiments. Results Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). p53 expression correlated directly with CD34 (p=0.001) and inversely correlated with WBC (p=0.007), PB and BM blast burden (p=0.0001), and survival (p=0.01). High p53 (p53high) expression was more associated with unfavorable cytogenetics, particularly -5 (p=0.00001). p53high resulted in lower complete remission (CR) rates (51% vs 56%; p=??), higher relapsed rates (82% vs 62%; p=??), and shorter median overall survival (OS; 29.8 vs. 51 wks, p=0.009) compared to p53low pts. Most cases with p53high had unfavorable cytogenetics. We next correlated p53 stabilization with the presence of p53 mutations in 68 pts. p53 mutations were detected in 20/54 (37%) p53high pts and in 0/14 (0%) pts with p53low. p53high, either in the presence (29 wks) or in the absence (24 wks) of p53 mutations (p=1.0), was associated with significantly shorter OS compared with p53low pts (56 wks; p=0.05). Multivariate analysis revealed p53 expression to be an independent risk factor for survival in AML (p=0.02). p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), BAD (p=0.0001), cleaved PARP (p=0.002), and cleaved PARP (p=0.01), and negatively with p21 (p=0.01), and MDM2 (p=0.001).Given the similar OS in p53high pts carrying mutant or wild-type p53, we scored the immunohistochemical expression of MDM2, MDM4, and p21 in 30 p53high pts (9 p53 mutated, 21 wild-type p53). Overexpression of MDM2 was observed in 44% vs 48% pts with mutant vs wild-type p53, respectively, whereas rates were 67% vs 62% for MDM4, and 0% vs 19% for p21, for each respective genotype. Overall, of the 21 p53high pts carrying wild-type p53, 15 (71%) had overexpression of MDM2 and/or MDM4, whereas 81% had no p21 expression, indicating deficient activation of the p53 pathway similar to those cases carrying mutant p53. We are currently assessing response to nutlin-3a therapy in 24 primary AML samples (4 mutant p53, 20 wild-type p53). Results showing the impact of p53 mutation and/or stabilization, and expression levels of MDM2, MDM4, and p21 on nutlin-3a therapy will be presented. Conclusions p53 stabilization (p53high) is a powerful predictive and prognostic factor in AML, which is independent of the presence of mutant p53 alleles. Poor outcomes in pts with p53high lacking p53 mutations are very frequently associated with overexpression of negative regulators of p53 such as MDM2 and/or MDM4 and p21 downregulation, indicating a functionally altered p53 pathway. These findings may have implications for therapies targeting the MDM2/p53 axis in AML. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Internal tandem duplication (ITD) mutation in Fms-like kinase 3 (FLT3) are observed in approximately 25% of newly diagnosed acute myeloid leukemia (AML) patients. FLT3 ITD is generally associated with poor survival outcome. A recent study has demonstrated that the TAM (Tyro3/MER/AXL) tyrosine kinase AXL is required for FLT3 inhibitor resistance in FLT3 ITD AML cells (Park et al Leukemia 2014). AXL expression is prognostic for poor outcome in AML (Ben-Batalla et al Blood 2013). These findings suggest that AXL is an excellent candidate for AML therapy particularly in patients with FLT3 ITD. ONO-9330547 (Ono Pharmaceutical Co, Osaka, Japan) is a highly specific AXL/MER inhibitor being developed for AML therapy. Here we report on the in vitro and in vivo effects of ONO-9330547 in AML models. Methods: FLT3 WT (HL60, OCI-AML3) and FLT3 ITD (Molm13, MV4;11) cells were treated with varying doses of ONO-9330547 and effect on cell viability and induction of apoptosis was assessed by flow cytometry using DAPI, Annexin V, and counting beads. Cell cycle was assessed by flow cytometry using EdU incorporation and FX Cycle Violet dye. Protein was isolated and reverse phase protein array (RPPA) analysis was performed on ONO-9330547 treated cells. RPPA results were validated by immunoblot analysis. RNA was collected and qRT-PCR performed. The drug was also tested on FLT3 ITD cells in an in vitro model of the AML microenvironment using co-culture with mesenchymal stromal cells (MSC). Finally, efficacy of ONO-9330547 in an in vivo AML xenograft model was tested using Molm13 cells expressing luciferase/GFP in NSG mice. Drug was given in feed at 0.013% (estimated ~ 20 mg/kg/day) and 0.004% (estimated ~ 6 mg/kg/day). Leukemia burden was assessed by IVIS imaging. Results: FLT3 ITD cell lines were highly sensitive to ONO-9330547. A dose of 10 nM drug induces apoptosis after 48 hours and 50 nM eliminates 〉 95% after 72 hours. MSC protect leukemia cells from ONO-9330547, but killing was still effective with higher doses. ONO-9330547 was effective arresting cell growth of Molm13 and MV4;11 cells. After 24 hours, 5 nM ONO-9330547 resulted in accumulation of cells in G1/G0. RPPA analysis of cells treated with the AXL inhibitor revealed suppression of known AXL targets (e.g. p-S6RP likely via AKT). RPPA also revealed novel targets including CDK1, p-RB, Cyclin B1, and PLK1. Immunoblot analysis also demonstrated that the drug suppressed expression of MCL-1 (predicted due to block of ERK and AKT signaling by the drug). Analysis of PLK1 gene expression by qRT-PCR revealed potent suppression of PLK1 mRNA by ONO-9330547. These findings suggest that inhibition of AXL results in block of CDK1 activity with concomitant loss of RB phosphorylation leading to suppression of PLK1 expression (presumably via RB association with E2F). This model is consistent with the observed effect of ONO-9330547 blocking cell entry into S phase. Finally, both low dose (~ 6 mg/kg) and high dose (~ 20 mg/kg) ONO-9330547 was effective reducing leukemia burden and significantly enhancing survival of mice bearing Molm13 leukemia cells. Interestingly, while mice receiving control feed displayed leukemia infiltrate in the liver, ONO-9330547 greatly reduced or prevented Molm13 cell infiltration of the liver. Conclusions: These results suggest that ONO-9330547 is effective killing AML cells with FLT3 ITD. The identification of p-RB and PLK1 as novel targets of the drug suggests for the first time that AXL regulates cell cycle in AML cells via CDK/RB/PLK1 axis. Finally, ONO-9330547 proved effective in in vivo AML xenograft models and the drug prevented AML infiltration of the liver. These results suggest that ONO-9330547 is a promising candidate for AML therapy particularly in AML patients with FLT3 ITD. Disclosures Yasuhiro: 3Ono Pharmaceutical Co. Ltd: Employment. Yoshizawa:3Ono Pharmaceutical Co. Ltd: Employment. Cortes:Ariad: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; Arog: Research Funding; Astellas: Research Funding; Ambit: Research Funding.

Description: hnRNP K is a transcriptional and translational regulatory protein critical for maintaining hematopoietic homeostasis. We have recently shown that alterations in hnRNP K expression are sufficient to drive hematological malignancies, including lymphomas. Through reverse phase protein array (RPPA) analyses, we observed that hnRNP K is overexpressed in patients with hematological malignancies and that this overexpression is directly correlated with increased c-Myc levels and activation of putative c-Myc targets. Given the critical role of c-Myc during lymphomagenesis, these findings point to a novel mechanism by which c-Myc is overexpressed in lymphoma patients that do not carry c-Myc amplifications or translocations. In order to examine the oncogenic potential of hnRNP K overexpression in hematological cancers and evaluate its role in driving c-Myc dependent lymphomas, we generated multiple transgenic hnRNP K mice that specifically overexpress hnRNP K in the B-cell compartment (Eµ-hnRNP K). Characterization of these Eµ-hnRNP K mice revealed that hnRNP K is significantly overexpressed in multiple transgenic lines (Fig. 1A) and resulted in a significant reduction in survival (Fig. 1B) with marked enlargement of hematopoietic tissues (spleens, lymph nodes, and thymi) and highly penetrant lymphomas capable of invasion into distant organs (Fig. 1C). Flow cytometry and CBC analyses of malignantperipheral blood and bone marrows revealed a significant increase in immature lymphoblasts consistent with an aggressive disease state. To examine the malignant potential of these hnRNP K overexpression dependent malignancies, we performed transplantation assays. Malignant cells from the Eµ-hnRNP K micerapidly engrafted in recipient NSG mice and the facilitated the development of aggressive lymphomas of immature origin (Fig. 1D). Together, these results demonstrate that when overexpressed, hnRNP K behaves as a bona-fide oncogene. To address the molecular mechanisms driving the oncogenic potential of hnRNP K overexpression, we evaluated c-Myc expression in tissues from these transgenic mice. Here, we observed a direct correlation between hnRNP K overexpression and an increase in c-Myc expression in multiple transgenic lines. To further investigate the relationship between hnRNP K overexpression and c-Myc expression, we transiently transfected HEK-293 cells with hnRNP K and observed a significant increase in c-Myc expression. Next, we performed RNA immunoprecipitation (RIP) assays using murine tissues and cell lines to determine whether hnRNP K translationally regulates c-Myc expression. These experiments revealed that hnRNP K directly interacted with the Myc transcript to facilitate its translation (Fig. 1E). Given that c-Myc overexpression is often observed in hematologic malignancies without amplification or translocation, these finding strongly suggest that hnRNP K overexpression may represent a novel mechanism to drive c-Myc expression. Thus, a more robust understanding of this cooperative mechanism may lead to more effective and personalized treatment strategies. To this end, we are now directly evaluating our hypothesis that malignant cells that overexpress hnRNP K will be uniquely sensitive to compounds that inhibit c-Myc expression. Until recently, c-Myc was an undruggable target; however, the discovery of small molecules that inhibit the function of Bromodomain and extra terminal (BET) family members (e.g.; JQ1) have shown tremendous efficacy in disrupting c-Myc activities. Using both JQ1 and a proprietary molecule from Arvinas® (BRD4-Protac), that uses Proteolysis Targeting Chimeras (Protac), we are currently examining the therapeutic efficacy of pharmacological targeting c-Myc in hnRNP K-mediated malignancies. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: The heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA/DNA-binding proteins that consist of several family members: A1, A2, B1, C2 and K. hnRNPs have been implicated in numerous cellular processes and when dysregulated, have been suggested to impact tumorigenesis. hnRNP A2/B1 is overexpressed in some lung cancers and has been suggested to be a useful early detection marker for lung carcinoma. hnRNP K has been reported to associate with colon cancer and directly interacts with DNA, RNA, and proteins to regulate gene expression in numerous cellular processes involved in mitogenic responses and tumorigenesis. Loss of hnRNP K expression results in defects in differentiation and apoptosis, and increased hnRNP K expression has been associated with loss of apoptosis and an increase in cellular proliferation. To explore the possibility of altered hnRNP K expression or mutations in primary myelofibrosis, we isolated mRNA from primary blood or bone marrow mononuclear cells from patients with myelofibrosis (n=62) and healthy controls (n=19). We examined hnRNP K levels and determined that hnRNP K is significantly overexpressed in myelofibrosis (p=

Description: hnRNP K is an RNA binding protein that controls a multitude of cellular processes and is aberrantly expressed in cancers. We have previously shown that hnRNP K functions as a haploinsufficient tumor suppressor in AML patients with 9q deletions. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon. We have recently discovered that hnRNP K overexpression in patients with diffuse large B-cell lymphoma correlates with dismal outcomes and directly resulted in the development of lymphomas in transgenic mice. To understand the mechanistic basis for the oncogenicity of hnRNP K overexpression and to identify therapeutic vulnerabilities, we performed RNA-sequencing, RNA immunoprecipitation following by sequencing (RIP-Seq), mass spectrometry, and polysome assays. We observed that hnRNP K regulates both global transcription and translational processes within the cell via modulation of 7SK and translation initiation proteins (such as the eIFs and PABP), respectively. Consequently, we hypothesized that aberrant hnRNP K expression primarily perturbs oncogenes with short half-lives. Mechanistically, we identified that hnRNP K binds to the RNA and regulates the expression of a plethora of critical oncogenes and tumor suppressors involved in hematologic malignancies such as c-Myc, RUNX1, and Cyclin D1. As proof of concept for clinical applications, we have demonstrated that hnRNP K-driven c-Myc overexpression renders tumors susceptible to bromodomain inhibition. Given that hnRNP K directs global transcription and translation, it is likely that hnRNP K overexpressing tumors will also be sensitive to transcriptional and translational inhibitors such as CDK9 inhibitors and omacetaxine mepesuccinate, respectively. However, since hnRNP K also regulates a plethora of additional cellular processes that extend far beyond mRNA and protein synthesis, there is a need to develop hnRNP K-specific inhibitors that will only target these activities. Thus, we have recently begun to identify small molecule compounds that can directly inhibit hnRNP K-RNA binding function on specific targets using an in vitro fluorescent binding assay. Using this assay, we are currently screening a library of 70,000 small molecule compounds to identify agents that can prevent hnRNP K-RNA interactions. In summary, we have established that hnRNP K is a bona fide oncogene that drives lymphomagenesis. Global analyses have revealed therapeutic vulnerabilities of hnRNP K overexpressing tumors. Furthermore, using our in vitro RNA binding assays, we anticipate identification of novel hnRNP K-specific inhibitors. Disclosures No relevant conflicts of interest to declare.

Description: Background: Internal tandem duplication (ITD) mutation in Fems-like kinase 3 (FLT3) occur in ~ 25% of newly diagnosed acute myeloid leukemia (AML) patients and are associated with poor survival outcome. We recently demonstrated that highly specific AXL/MER inhibitor ONO-7475 (Ono Pharmaceutical Co, Osaka, Japan) was effective as a single agent against FLT3 ITD AML cells studied with in vitro co-culture and murine xenograft models (Ruvolo et al Haematologica, 2017). Based on expression of TAM kinases in the AML cells tested, mechanism for ONO-7475 killing in the FLT3 ITD AML cells was due to AXL inhibition. Here we report on the in vitro and in vivo effects of ONO-7475 when combined with FLT3 inhibitor Sorafenib in FLT3 ITD AML models. Methods: FLT3 ITD AML cell lines Molm13 and MV4;11 as well as Sorafenib resistant Molm13 cells were treated with varying doses of ONO-7475 in the presence and absence of FLT3 inhibitor Sorafenib and effect on cell viability and induction of apoptosis was assessed by flow cytometry using DAPI, Annexin V, and counting beads. RNA was isolated and gene expression profiling (GEP) analysis using microarray was performed on ONO-7475 treated cells. GEP results for a number of genes including CDK1, PLK1, and other cell cycle regulators were validated by qRT-PCR. The efficacy of ONO-7475 combination with Sorafenib in an in vivo AML xenograft model was tested using Molm13 cells expressing luciferase/GFP in NSG mice. Both drugs were given by oral gavage (ONO-7475 at 10 mg/kg and Sorafenib at 5 mg/kg). Drugs were given 5 days a week. Leukemia burden was assessed by IVIS imaging. Results: Molm13 and MV4;11 cells were highly sensitive to ONO-7475 combination with Sorafenib. A dose of 50 nM ONO-7475 with 25 nM Sorafenib potently induces apoptosis and eliminates 〉 90% of cells after 72 hours. Sorafenib resistant Molm13 cells were more resistant to either drug compared to parental cells. However, combination of 50 nM ONO-7475 with 100 nM Sorafenib potently induces apoptosis in the Sorafenib resistant cells with nearly all leukemic cells eliminated after 72 hour treatment. GEP analysis of cells treated with the AXL inhibitor revealed suppression of many genes involved in cell cycle control including various CDKs (e.g. CDK1, CDK4), Cyclins (e.g Cyclin B1), and other cell cycle regulators such as PLK1. ONO-7475 inhibition of gene expression of these genes was verified by qRT-PCR. GEP also revealed induction of genes associated with more mature myeloid cells including HLA-DR, CD114, and CD115. Finally, single agent use of ONO-7475 (10 mg/kg) or Sorafenib (5 mg/kg) had limited effect in the FLT3 ITD AML xenograft model; however, the combination of both drugs at single agent dose was effective reducing leukemia burden and significantly enhancing survival of mice bearing the Molm13 leukemia cells. Conclusions: These results suggest that ONO-7475 combination with Sorafenib is effective killing AML cells with FLT3 ITD including those that are resistant to the FLT3 inhibitor. The identification of gene expression of cell cycle regulators as novel targets of the drug suggests for the first time that AXL regulates cell cycle via a complex transcriptional mechanism. The ability of the drug to induce expression of genes associated with mature myeloid cells suggest the drug may have the potential to promote differentiation of FLT3 AML cells. Finally, combination of ONO-7475 with Sorafenib proved effective in an in vivo AML xenograft model suggesting that combination of ONO-7475 with a FLT3 inhibitor could be efficacious for therapy of AML patients with FLT3 ITD. Disclosures Yasuhiro: Ono Pharmaceutical: Employment. Tanaka:Ono Pharmaceutical: Employment. Yoshizawa:Ono Pharmaceutical: Employment. Cortes:Pfizer: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Arog: Research Funding. Andreeff:AstraZeneca: Research Funding.