Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that, despite being treatable with well- defined chemotherapy regimens, is ultimately fatal in over half of all cases categorized as high-risk AML. Mutations in MLL, FLT3, DNMT3A and P53 are associated with high-risk AML. Even targeted FLT3 anti kinase therapy, which constitutes 30 % of AML, failed to engender durable response in this group of AML. Co-operative oncogenic signaling” was attributed to poor therapeutic outcome, but lacks mechanistic understanding. Based on our recent publication and new preliminary data, we found that co-operative oncogenic signaling converges on c-FOS and DUSP1, which results in an increased apoptotic threshold in cancer cells and confers drug resistance. Thus, genetic or pharmacologic inhibition of c-FOS and DUSP1 sensitizes cancer cells to chemotherapy (Kesarwani, et. al. Nature Medicine 2017). We show greater expression of c-FOS and DUSP1 in high-risk AML patients, but not in low risk-AML patients. Both genetic and chemical inhibition of c-FOS and DUSP1 results in increased drug sensitivity to both TKI and conventional chemotherapeutic drugs in a model of high-risk AML (FLT3ITD+MLLAF9). Thus, we hypothesize that co-operative oncogenic signaling in AML induces the expression of c-FOS and DUSP1 resulting to drug resistance and disease relapse due to elevated apoptotic threshold. In Aim 1, we will determine whether c-FOS and DUSP1 are necessary and sufficient for transformation in a most frequent, aggressive, and fatal AML driven by FLT3ITD+DNMT3Amut+NPM1C and FLT3ITD+P53mut mutations. We will examine the cellular basis of c-FOS and DUSP1 dependency in the high- risk AML mouse models and primary patient samples by genetic deletion and pharmacological inhibition of c- FOS and DUSP1. Next, we propose experiments to understand the mechanistic basis for how co-operative oncogenic signaling via c-FOS and DUSP1 contributes to transformation and treatment failure in AML, with the goal for novel treatment strategies. Based on our preliminary data, we hypothesize that c-FOS and DUSP1 signaling converges upon oncogenically-activated enhancers mediated by specific AP-1 transcriptional complexes. In the presence of c-FOS and DUSP1, AP-1 complexes consist of c-FOS-JUN, which mediate oncogenically-active enhancers, while in the absence of c-FOS and DUSP1, Jun family homodimers (JUN- JUNB, JUNB-JUND, JUN-JUND) predominate which are unable to support the leukemic cell state. In Aim 2, we will molecularly link c-FOS-JUN AP-1 and DUSP1 activity to global enhancer chromatin dynamics. Moreover, we will exploit chromatin-embedded target-gene-reporter alleles to provide a detailed analysis of functionally-relevant downstream genes at a single-cell level in high-risk AML. The proposed work is expected to delineate the necessity of c-FOS and DUSP1 signaling in high-risk AML, as well as to provide deep molecular insight into the mechanisms underlying leukemic transformati...