PROJECT SUMMARY/ABSTRACT: Project 2 Outcomes for acute myeloid leukemia (AML) patients have improved little over four decades. This is largely due to the development of acquired drug resistance and refractory disease. Thus, there is an urgent need for new strategies to target residual AML cells or refractory clones before they trigger disease relapse. The tumor microenvironment is a key factor in the development of malignant progression and therapy resistance. The bi- directional communication between cancer cells and microenvironmental cells is much more complex than initially perceived. Thus, one strategy to reduce relapse is to target the signaling mediated by growth factors secreted by stromal or immune cells within the bone marrow microenvironment that play a critical role in promoting leukemia cell survival, development of drug resistance, and immune evasion. Therefore, the long- term goal of this project is to deconvolute the comprehensive network of events and cellular heterogeneity that contribute to acquired resistance in a context dependent manner to identify new therapeutic approaches. As part of our prior U54 DRSN funding, omics-based analysis of 350 primary AML patient samples found that multiple secreted cytokines and immune factors may contribute to drug resistance. Therefore, we hypothesize that reprogramming of the bone marrow niche modulates drug response and drives acquired resistance in AML. Comprehensive understanding of these mechanisms will lead to the identification of new combination therapies. We will focus on 5 essential therapeutic targets in AML: BCL2, FLT3, JAK2, MEK, and epigenetic pathways and will address this hypothesis following three well integrated aims. In Aim 1, we will map microenvironmental signatures and tumor cross-talk mechanisms against drug response features of AML by performing single cell gene expression and epigenetic analyses in the context of bone marrow niche. Tumor microenvironment cross- talk will be further explored in Aim 2 focusing on monocytes and stromal cells and in the context of NK and T cells in Aim 3 by performing CRISPR/Cas screening with co-culture platforms. Identified targets/pathways will be validated to fully define microenvironmental mechanisms of acquired drug resistance. These results will build predictive models of signaling crosstalk in response to therapeutic stress and identify combination therapies to overcome acquired resistance, particularly in the context of the microenvironment. We will test the effect of these inhibitors on cell viability, cellular heterogeneity, differentiation, and target inhibition using multi-parametric flow cytometry and immunofluorescence analysis. We will integrate these findings with Project 1 and prioritized targets will be tested for their translational impact in Project 3. Overall, elucidating the influence of microenvironment-driven signaling on drug response and survival of AML cells will help identify novel tractable targets for combination therapies to overcome acquired drug resistance in AML.