Novel targeted therapies demonstrate improved survival in specific subgroups (defined by genetic variants) of acute myeloid leukemia (AML) patients, validating the paradigm of molecularly targeted therapy. However, identifying correlations between AML molecular attributes and effective therapies is challenging. Recent advances in high-throughput in vitro drug sensitivity screening applied to primary AML blasts were used to uncover such correlations; however, these methods cannot predict the response of leukemic stem cells (LSCs). Our study aimed to predict in vitro response to targeted therapies, based on molecular markers, with subsequent validation in LSCs. We performed ex vivo sensitivity screening to 46 drugs on 29 primary AML samples at diagnosis or relapse. Using unsupervised hierarchical clustering analysis, we identified group with sensitivity to several tyrosine kinase inhibitors (TKIs), including the multi-TKI, dasatinib, and searched for correlations between dasatinib response, exome sequencing and gene expression from our dataset and from the Beat AML dataset. Unsupervised hierarchical clustering analysis of gene expression resulted in clustering of dasatinib responders and non-responders. In vitro response to dasatinib could be predicted based on gene expression (AUC=0.78). Furthermore, mutations in FLT3/ITD and PTPN11 were enriched in the dasatinib sensitive samples as opposed to mutations in TP53 which were enriched in resistant samples. Based on these results, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. Our results demonstrate that in a subgroup of FLT3/ITD AML (4 out of 9) dasatinib significantly inhibits LSC engraftment. In summary we show that dasatinib has an anti-leukemic effect both on bulk blasts and, more importantly, LSCs from a subset of AML patients that can be identified based on mutational and expression profiles. Our data provide a rational basis for clinical trials of dasatinib in a molecularly selected subset of AML patients.

With aging, humans accumulate preleukemic mutations (pLMs) in hematopoietic stem and progenitor cells (HSPCs) which was termed age-related clonal hematopoiesis (ARCH). To gain a better insight changes in the HSPCs-environment crosstalk upon aging that might contribute to ARCH, it is critical to develop a multilayer perspective that integrates information on mutations, epigenetics the cellular context and the bone marrow (BM) microenvironment, since all these layers are changing during ageing. Accordingly, a key question in the field is how the ageing BM microenvironment influences clonal expansion of HSPCs. Fatty bone marrow (FBM) is one of the environmental factors that may influence clonal hematopoiesis (CH) with age. As we age, our bone marrow shifts from red to adipocyte-enriched yellow BM. We hypothesize that age related BMF accumulation may provide a selective advantage to specific pre-leukemic stem and progenitor cells (preL-HSPCs) carrying pLM. To support this hypothesis, we established a FBM model in NSG mice to enable the study of both human and rodent preL-HSPCs. Transplantation of primary human preL-HSPCs from AML patients (DNMT3A, NPM1 mutations ) into FBM resulted in enhanced engraftment compared to control mice without FBM. We further demonstrate that DNMT3A-R882H+/- mice derived BM HSPCs, engrafted significantly higher in NSG mice with FBM compared to controls. Interestingly, when DNMT3A-R882H+/- derived BM cells from middle-aged mice (12-month old) were injected into FBM mice, engraftment increased tenfold. Secondary engraftment of aged DNMT3A-R882H +/-BM derived cells resulted in an increase in engraftment upon transplantation into to FBM, suggesting enhanced in vivo self-renewal capacity of HSPCs in FBM. To study the underlying molecular mechanisms provided by the FBM to preL-HSPCs carrying DNMT3A-R882H +/-, we used a multiplex cytokine assay. In this approach we analyzed 17 common cytokines in BM following transplantation of young, two-month old, or middle-aged, 12-month old, DNMT3A-R882H +/-or control - BM derived cells into FBM. Our results show that transplanting two months old, middle-aged DNMT3A-R882H +/-or control BM derived cells to FBM resulted in a significant increase in BM mIL-6 secretion when compared to transplants into control, non-FBM mice. mIL-6 was secreted by adipocytes following irradiation regardless of which cells are transplanted. We then transplanted middle-aged DNMT3A-R882H+/- BM derived cells to FBM mice that had been treated intraperitoneally with a neutralizing IL-6 Ab. The administration of neutralizing IL-6 Ab resulted in a significant decrease in engraftment of DNMT3A-R882H+/- BM derived cells, confirming that IL6 contributes to the expansion of the DNMT3a-R882H+/- cells in FBM. In summary, these results demonstrate for the first time that the FBM provides a selective advantage to pre-leukemic cells carrying DNMT3A-R882H. Importantly, we show that IL-6 is a one of the major players in the molecular mechanism that confers the FBM advantage specifically to preL-HSPCs carrying R882H both in vitro and in vivo. Disclosures Mueller-Tidow: Janssen Cilag: Consultancy, Research Funding; Bioline: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding.

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Background: Recent advances in acute myeloid leukemia(AML) targeted therapy improve overall survival. While these targeted therapies can achieve prolonged remissions, most patients will eventually relapseunder therapy. Our recent studies suggest that relapse most often originates from several sub-clones of leukemic stem cells (LSCs), present before therapy initiation, and selected due to several resistance mechanisms. Eradication of these LSCs during treatment induction /remission could thus potentially prevent relapse. The overall goal of the current study was to identify drugs which can be safely administrated to patients at diagnosis and that will target LSCs. Since simultaneously testing multiple drugs in vivo is not feasible, we used an in vitrohigh throughput drug sensitivity assay to identify new targets in primary AML samples. Methods: Drug sensitivity and resistance testing (DSRT) was assessed in vitro (N=46 compounds) on primary AML samples from patients in complete remission (N=29). We performed whole exome sequencing and RNAseq on samples to identify correlations between molecular attributes and in vitro DSRT. Results:Unsupervised hierarchical clustering analysis of in vitro DSRT, measured by IC50, identified a subgroup of primary AML samples sensitive to various tyrosine kinase inhibitors (TKIs). In this subgroup, 52% (9/17) of AML samples displayed sensitivity to dasatinib (defined as a 10-fold decrease in IC50 compared to resistant samples). Dasatinib has broad TKI activity, and is safely administered in the treatment of leukemia. We therefore focused our analysis on predicting AML response to dasatinib, validating our results on the Beat AML cohort. Enrichment analysis of mutational variants in dasatinib-sensitive and resistant primary AML samples identified enrichment of FLT3/ITD (p=0.05) and PTPN11(p=0.05) mutations among dasatinib responders. Samples resistant to dasatinib were enriched with TP53 mutations (p=0.01). No global gene expression changes were observed between dasatinib-sensitive and resistant samples in our cohort, nor in the Beat AML cohort. Following this, we tested the differential expression of specific dasatinib-targeted genes between dasatinib-responding and resistant samples. No significant differences were identified. However, unsupervised hierarchical clustering of dasatinib targeted genes expression in our study and in the Beat AML cohort identified a subgroup of AML samples (enriched in dasatinib responders) that demonstrated overexpression of three SRC family tyrosine kinases:FGR, HCK and LYN as well as PTK6, CSK, GAK and EPHB2. Analysis of the PTPN11 mutant samples revealed that the IC50 for dasatinib in 23 carriers of the mutant PTPN11 was significantly lower compared to the IC50 of PTPN11 wild type samples (p=0.005). LYN was also upregulated (p<0.001) in the mutant samples. We therefore hypothesized that gene expression of dasatinib-targeted genes could be used as a predictive biomarker of dasatinib response among FLT3/ITD carriers. We found that among FLT3/ITD AML carriers in the Beat AML cohort LYN, HCK, CSK and EPHB2 were significantly over-expressed in the dasatinib responding samples (N=27) as compared to the dasatinib resistant samples (N=35). To predict response to dasatinib among FLT3/ITD carriers we used a decision tree classifier based on the expression levels of these four genes. Our prediction model yielded a sensitivity of 74% and specificity of 83% for differentiating dasatinib responders from non-responders with an AUC of 0.84. Based on our findings, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. We found that in a subset of these samples, dasatinib significantly inhibited LSCs engraftment. This subset of FLT3/ITD AML samples expressed higher levels of LYN, HCK,FGR and SRC as compared to the FLT3/ITD samples that were not sensitive to dasatinib therapy in vivo. In summary, we identified a subgroup of AML patients sensitive to dasatinib, based on mutational and expression profiles. Dasatinib has anti-leukemic effects on both blasts and LSCs. Further clinical studies are needed to demonstrate whether selection of tyrosine kinase inhibitors, based on specific biomarkers, could indeed prevent relapse. Disclosures Tavor: Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS companies: Membership on an entity's Board of Directors or advisory committees.

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