ALBERT

Description: Key Points NrasG12D/+ induces proliferation and increases self-renewal and myeloid differentiation bias in HSCs. ERK1/2 is constitutively hyperactivated in NrasG12D/+ HSCs and downregulation of the MEK/ERK signaling attenuates NrasG12D/+ HSC phenotypes.

Description: BCL2 is an antiapoptotic protein commonly expressed in hematologic malignancies. Overexpression of BCL-2 is a poor prognostic factor in acute myeloid leukemia (AML). Venetoclax (ABT-199) is a highly selective BCL2 inhibitor that can induce cell death in multiple leukemia cell lines. Recently, venetoclax received an FDA breakthrough therapy designation for use in combination with hypomethylating agents in treatment-naïve patients with AML who are unfit for intensive chemotherapy. However, venetoclax was only modestly effective as monotherapy in relapsed/refractory AML (19% CR/CRi). The aim of the current study is to integrate genomic and functional screen data to identify biomarkers to predict venetoclax sensitivity and resistance in AML, and to identify potential venetoclax combination treatment strategies. In this study, we investigated approximately 200 AML patient samples and correlated clinical parameters, whole exome sequence data, and RNAseq gene expression data with in vitro drug screening data (drug area under the curve (AUC)) to identify subsets of AML samples with sensitivity or resistance to venetoclax alone and in combinations with 10 small molecular inhibitors (Array-382, dasatinib, JQ-1, idelalisib, quizartinib, palbociclib, panobinostat, ruxolitinib, sorafenib, and trametinib). For gene expression, we observed that venetoclax correlated with 3 gene expression clusters (coefficient frequency: 0.94, 0.80 and 0.71 respectively) among 21 gene expression clusters in AML, associated with innate immune system, neutrophil degranulation, and interleukin-10 signaling. Among the BCL2 gene family, venetoclax AUC positively correlated with BCL2A1 (r=0.59, p

Description: Chronic myelomonocytic leukemia (CMML) primarily occurs in the elderly with the median age ranging from 65 to 75 years. As defined by WHO, CMML is characterized by persistent monocytosis in peripheral blood, hepatosplenomegaly, and the absence of BCR-ABL fusion gene. CMML is a devastating cancer for multiple reasons, one of which is that approximately 20% of CMML cases evolve into acute myeloid leukemia (AML) soon after their first diagnosis. However, little is known about the cellular and molecular mechanisms underlying this malignant transformation. Recently, our lab developed a CMML mouse model induced by oncogenic NrasG12D/+ expressed from its endogenous locus. Above 90% of recipient mice with NrasG12D/+ bone marrow cells developed CMML-like phenotypes with a median survival of ∼56 weeks. Interestingly, none of these mice spontaneously transform to AML. To identify the pathogenetic origins underlying CMML transformation to AML, we further deleted p53 expression in NrasG12D/+ bone marrow cells using p53fl/fl allele and Mx1-Cre because deletion of p53 is a common genetic event observed in oncogenic Ras-driven cancers. We found that ERK1/2 is significantly hyperactivated in NrasG12D/+; p53-/- hematopoietic stem/progenitor cells (enriched for myeloid progenitors) in the absence of cytokines or in the presence of low concentration of GM-CSF. Concomitantly, the mutant myeloid progenitors show significantly increased self-renewal in a serial replating assay in vitro. We transplanted NrasG12D/+, p53-/-, or NrasG12D/+; p53-/- bone marrow cells into lethally irradiated mice. Unlike recipients with p53-/- cells that died of a T-cell disease with 100% penetrance and a median survival of 24 weeks, ∼70% of recipients with NrasG12D/+; p53-/- cells died of AML or acute myeloid sarcoma with a median survival of 16 weeks. These malignant myeloid diseases are transplantable in secondary recipients. Interestingly, only mutant hematopoietic stem cells (HSCs) could initiate and maintain leukemia phenotypes in the NrasG12D/+ induced CMML model, whereas both NrasG12D/+; p53-/- HSCs and myeloid progenitors could initiate AML or acute myeloid sarcoma. Our results indicate that deletion of p53 cooperates with NrasG12D/+ mutation to transform CMML into an acute phase. This malignant transformation is initiated by mutant myeloid progenitors, which show increased self-renewal and potentially serve as leukemia initiating cells. Disclosures: No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML), the most common acute leukemia in adults, remains a challenging disease to treat due to its heterogeneity and high level of relapsed/refractory disease. Exploration of molecular pathways that drive AML have implicated broad activation of the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pro-survival pathways in the pathogenesis of the disease. Among the most frequent genetic perturbations, direct mutation of N- or K-RAS as well as mutation of nearby pathway regulators (PTPN11, NF1) are identified in ~20% of patients with AML. An additional ~35% of AML patients exhibit mutations in FLT3 or KIT, upstream receptor tyrosine kinases known to activate the RAS pathway. Moreover, relapsed/refractory disease in response to novel molecularly targeted FLT3 inhibitors often results in RAS pathway mutations underpinning the connection between these molecular pathways in AML. These findings resulted in several preclinical studies and ongoing clinical trials testing the efficacies of MEK1/2 inhibitors in AML either as a single agent or in combination with FLT3 inhibitors. To elucidate pathways leading to changes in trametinib sensitivity and resistance in a FLT3-ITD genomic landscape, we performed a genome-wide CRISPR screen for trametinib sensitivity in MOLM13 AML cells, which carry a FLT3-ITD mutation. Results from both the genome-wide screen and independently generated cell lines with decreased sensitivity to trametinib indicated involvement of a diversity of genes and pathways, including the tumor-suppressor, PTEN (a negative regulator of PI3K/AKT/mTOR), AMBRA1 (an autophagy regulator via the mTOR pathway), and DUSP7 (a phosphatase negatively regulating downstream ERK activity). Cells engineered to have loss-of-function for these genes as well as cells cultured for resistance to trametinib showed perturbed signaling in downstream PI3K/AKT/mTOR and MEK/ERK signaling cascades. Our work identified genes whose loss of function in the disease-implicated pathways confer trametinib resistance in AML and provide a rationale for selecting combinatorial trametinib/FLT3 inhibitors treatment based on unique patient mutational and gene expression landscapes. Disclosures Tyner: Incyte: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Takeda: Research Funding; Genentech: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Genentech: Research Funding; Syros: Research Funding; AstraZeneca: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Array: Research Funding; Agios: Research Funding; Agios: Research Funding; Aptose: Research Funding; Array: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Petra: Research Funding; Syros: Research Funding; Petra: Research Funding.

Description: To identify new therapeutic targets in acute myeloid leukemia (AML), we performed small-molecule and small-interfering RNA (siRNA) screens of primary AML patient samples. In 23% of samples, we found sensitivity to inhibition of colony-stimulating factor 1 (CSF1) receptor (CSF1R), a receptor tyrosine kinase responsible for survival, proliferation, and differentiation of myeloid-lineage cells. Sensitivity to CSF1R inhibitor GW-2580 was found preferentially in de novo and favorable-risk patients, and resistance to GW-2580 was associated with reduced overall survival. Using flow cytometry, we discovered that CSF1R is not expressed on the majority of leukemic blasts but instead on a subpopulation of supportive cells. Comparison of CSF1R-expressing cells in AML vs healthy donors by mass cytometry revealed expression of unique cell-surface markers. The quantity of CSF1R-expressing cells correlated with GW-2580 sensitivity. Exposure of primary AML patient samples to a panel of recombinant cytokines revealed that CSF1R inhibitor sensitivity correlated with a growth response to CSF1R ligand, CSF1, and other cytokines, including hepatocyte growth factor (HGF). The addition of CSF1 increased the secretion of HGF and other cytokines in conditioned media from AML patient samples, whereas adding GW-2580 reduced their secretion. In untreated cells, HGF levels correlated significantly with GW-2580 sensitivity. Finally, recombinant HGF and HS-5–conditioned media rescued cell viability after GW-2580 treatment in AML patient samples. Our results suggest that CSF1R-expressing cells support the bulk leukemia population through the secretion of HGF and other cytokines. This study identifies CSF1R as a novel therapeutic target of AML and provides a mechanism of paracrine cytokine/growth factor signaling in this disease.

Description: Abstract 4086 How oncogenes regulate adult stem cells to promote tumorigenesis is poorly understood. We and others previously reported that recipient mice transplanted with Nras G12D/+ or Nras G12D/G12D bone marrow cells develop distinct haemtopoietic malignancies. Mice with G12D/+ cells predominantly develop a myeloproliferative neoplasm (MPN) closely resembling chronic myelomonocytic leukemia (CMML), while animals with G12D/G12D cells develop acute T- or B-cell lymphoblastic leukemia (TALL or BALL) and/or MPN, with varying penetrance, which appear to be regulated by the activity of G12D/G12D haematopoietic stem cells (HSCs). Consistent with this notion, we found that G12D/+ HSCs are required to initiate and maintain CMML-like phenotypes in recipient mice and serve as MPN initiating cells. Therefore, we further investigated how endogenous oncogenic Nras signaling regulates the self-renewal and differentiation of HSCs to promote leukemia development in different lineages of cells. Here we show that G12D/+ signaling results in moderate hyperproliferation and increased self-renewal of HSCs, promoting expansion of myeloid progenitors and consequently myeloid malignancies. In contrast, G12D/G12D leads to excessive hyperproliferation, decreased self-renewal, and depletion of HSCs, which promote expansion of myeloid and lymphoid progenitors and subsequently malignancies in both compartments. Because leukemia development in Nras G12D/+ and G12D/G12D models is tightly associated with ERK1/2 hyperactivation in haematopoietic stem/progenitor cells (HSPCs), we studied the MEK/ERK signaling in HSCs and their downstream multipotent progenitors (MPPs) using a “HSC phosphor-flow” method we developed. Our data demonstrate that ERK1/2 is hyperactiavated in G12D/+ and G12D/G12D HSCs in a dose-dependent manner, while AKT is not affected in G12D/+ and G12D/G12D HSCs. In contrast, both ERK1/2 and AKT are not changed in G12D/+ and G12D/G12D MPPs. As expected, inhibition of MEK/ERK signaling by AZD6244 (a MEK1 inhibitor) rescues the HSC phenotypes and attenuates myeloproliferative neoplasm phenotypes in G12D/+ and G12D/G12D mice. Mechanistic analysis identifies that a cohort of MAPK pathaway genes regulating cell cycle and signaling are significantly differentially expressed in G12D/+ HSCs compared to control or G12D/G12D HSCs. Unlike the prevailing theory based on Ras overexpression studies, depletion of G12D/G12D HSCs is not associated with overexpression of cell senescence genes. Rather, the Wnt and Notch pathways are significantly downregulated in G12D/G12D but not G12D/+ HSCs. Therefore, we propose that endogenous Nras G12D signaling differentially regulates HSCs self-renewal and differentiation through a dose-dependent hyperactivation of ERK1/2. Disclosures: No relevant conflicts of interest to declare.

Description: Background Kras is a small GTPase essential for mouse embryonic development. Although Kras-/- fetal liver cells reconstitute recipient mice indistinguishably from wild-type cells, chimeric mice generated from injection of Kras-/- embryonic stem cells into wild-type blastocysts show little contribution of knockout cells to hematopoietic tissues even when these cells contribute to all the other tissues to a high degree. These results suggest that Kras plays an important role in adult hematopoiesis. However, early embryonic lethality of Kras-/- mice prevents further investigation of Kras functions in adulthood. To overcome this problem, we generated Kras conditional knockout mice (Krasfl/fl), which allow the deletion of Kras by the Cre recombinase in desired tissues and at desired developmental stages. Method We used two transgenic Cre lines, Mx1-Cre and Vav-Cre, to knockout Kras in adult hematopoietic system. The Mx1 promoter is induced by interferon signaling, which can be triggered by injections of polyinosinic-polycytidylic acid (pI-pC). The Vav promoter drives Cre expression specifically in fetal liver hematopoietic cells since E11.5 as well as in adult hematopoietic tissues. Both Cre lines efficiently deleted Kras expression in above 95% of hematopoietic cells as judged by single hematopoietic stem cell (HSC) genotyping. Results obtained from these two different Cre lines were essentially same. Results We found that the frequency and absolute number of Kras-/- HSCs, multipotent progenitors (MPPs), LSK (Lin- Sca1+ cKit+) cells, myeloid progenitors and common lymphoid progenitors are comparable to wild-type control cells. Consistent with this observation, cytokine signaling in Kras-/- hematopoietic stem/progenitor cells (HSPCs, Lin- cKit+) is indistinguishable from control HSPCs. In contrast, the percentage of CD19+ B-cells is moderately but significantly reduced in Kras-/- spleens and concomitantly cytokine-evoked ERK1/2 activation is greatly reduced in differentiated blood cells. To determine whether Kras plays an important role in regulating HSC functions, we performed a competitive bone marrow reconstitution assay using CD45.2+ control or Kras-/- bone marrow cells mixed together at ratios 1:1 and 3:1 with congeneic competitor cells (CD45.1+ bone marrow cells). Kras-/- bone marrow cells show significantly reduced long-term reconstitution in recipient mice compared to control cells (10% vs 45%). The reduced reconstitution is persistent in the secondary and tertiary recipients. However, detailed analysis in primary and secondary recipients revealed that the frequency of Kras-/- HSCs and MPPs is comparable to that of control cells and Kras-/- progenitor cells are also largely normal, indicating that Kras is dispensable for adult HSC functions but might play an important role in generating differentiated blood cells. The reduced generation of myeloid cells is further validated in an in vitro culture assay, in which we quantitatively measured the myeloid cell production from Lin- progenitor cells. Conclusions Our results indicate that loss-of-Kras could be compensated by other Ras isoforms in adult HSCs. However, in mature blood cells, Kras deficiency results in greatly reduced cytokine-evoked ERK1/2 activation. Under a stressed condition (e.g. competitive bone marrow transplantation), the generation of Kras-/- blood cells is defective. Taken together, our study reveals a novel and unique function of Kras in regulating adult hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) is a fast progressing blood malignancy with impaired differentiation and proliferation of myeloid precursors. It is one of the most common leukemias in adults and is known for its molecular and biological heterogeneity, with a variety of genetic lesions implicated in the disease. Among these variants, internal tandem duplication (ITD) or point mutations in the tyrosine kinase domain (TKD) of FLT3 tyrosine kinase are found in around 30% of AML patients. Sorafenib, a multi-kinase inhibitor that targets FLT3, RAF, VEGFR, FGFR, KIT and RET, is approved for use in hepatocarcinoma, renal cell carcinoma, and thyroid carcinoma treatments. Addition of different FLT3 inhibitors such as sorafenib to standard-of-care chemotherapy treatment prolongs AML patient survival with or without FLT3 mutations, although relapse caused by drug resistance remains a clinical challenge. Understanding the mechanisms of resistance to FLT3-targeted drugs, therefore, is necessary to improve treatment options and patient outcomes in AML. We aimed to elucidate resistance mechanisms to sorafenib by subjecting MOLM13 AML cells to genome-wide CRISPR screening to identify genes whose loss-of-function contributes to reduced drug sensitivity. Using Mageck along with an internally developed tiering system for screen hit prioritization, we identified negative regulators of MAPK as well as mTOR pathways as main players in sorafenib resistance. We validated prioritized hit genes using individual sgRNAs to generate single gene deficient cell models for LZTR1, NF1, TSC1 or TSC2. Drug sensitivity assays confirmed an increase in sorafenib resistance in these knockout cells. LZTR1-, TSC1- or TSC2-deficient cells also exhibited reduced sensitivity to a panel of additional FLT3 inhibitors. RNA sequencing results from 271 AML patient peripheral blood or bone marrow samples revealed a correlation between sorafenib sensitivity and lower expression of LZTR1, NF1, TSC1, and TSC2. MOLM13 cell lines resistant to crenolanib, quizartinib, and sorafenib were independently generated by incremental increase in concentration of each drug in cell culture media. Similarly, western blot analysis demonstrated up-regulation of MAPK and/or mTORC1 activity in these resistant cell lines. In addition, these cells were sensitive to MEK inhibitors, and the combination of FLT3 + MEK inhibitors showed synergistic efficacy over single agents in both resistant and parental cells. Taken together, our work identifies the contribution of the MAPK and PI3K/mTOR pathways to FLT3 inhibitor resistance in AML and suggests the combination of FLT3 + MEK inhibitors may be effective for AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors. Disclosures Tyner: Aptose: Research Funding; Array: Research Funding; Agios: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Syros: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Array: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Gilead: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Syros: Research Funding; Aptose: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Petra: Research Funding; Constellation: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding.

Description: Ras proteins control a complex intracellular signaling network. Gain-of-function mutations in RAS genes lead to RASopathy disorders in humans, including Noonan syndrome (NS). NS is the second most common syndromic cause of congenital heart disease. Although conditional expression of the NrasG12D/+ mutation in adult hematopoietic system is leukemogenic, its effects on embryonic development remain unclear. Here, we report that pan-embryonic expression of endogenous NrasG12D/+ by Mox2-Cre in mice caused embryonic lethality from embryonic day (E) 15.5 and developmental defects predominantly in the heart. At E13.5, NrasG12D/+; Mox2Cre/+ embryos displayed a moderate expansion of hematopoietic stem and progenitor cells without a significant impact on erythroid differentiation in the fetal liver. Importantly, the mutant embryos exhibited cardiac malformations resembling human congenital cardiac defects seen in NS patients, including ventricular septal defects, double outlet right ventricle, the hypertrabeculation/thin myocardium, and pulmonary valve stenosis. The mutant heart showed dysregulation of ERK, BMP, and Wnt pathways, crucial signaling pathways for cardiac development. Endothelial/endocardial-specific expression of NrasG12D/+ caused the cardiac morphological defects and embryonic lethality as observed in NrasG12D/+; Mox2Cre/+ mutants, but myocardial-specific expression of NrasG12D/+ did not. Thus, oncogenic NrasG12D mutation may not be compatible with embryonic survival.