Project Summary. Acute leukemias are aggressive hematological tumors and the most common malignancy in children and adolescents. Though cure rates have steadily improved over the last 50 years with the implementation of intensive chemotherapy regimens, today 20% of pediatric acute lymphoblastic leukemia (ALL) patients will relapse or develop refractory disease. Moreover, therapeutic outcomes in acute myeloid leukemia (AML) are significantly worse with cure rates in the range of 65-70%. Despite much progress in the characterization of the genetic and molecular basis of leukemia, the specific mechanisms mediating resistance, disease progression and relapse remain largely unknown. Thus, genomic profiling analyses have shown marked genetic heterogeneity at relapse which together with the complex combination chemotherapy protocols used in treatment, make it difficult to directly assign precise roles in resistance for most of relapse-associated mutations. My central hypothesis is that genetic mutations, signaling and epigenetic mechanisms controlling self-renewal, response to stress and resistance converge on a restricted number of master regulators that drive the acquisition of a drug-tolerant persistent phenotype at minimal residual disease and ultimately relapse. The objective of this proposal is to establish the specific role of remission- and relapse-associated transcriptional and epigenetic regulators as drivers of drug-tolerant persistent phenotypes and chemotherapy resistance across pediatric acute leukemias. These studies will be impactful to our understanding of the mechanisms conducive to therapeutic failure and will facilitate the rationale design of therapies directed to the eradication of high-risk minimal residual disease and the prevention of leukemia relapse. Towards this goal I propose the following specific aims: Aim 1 (mentored phase): To interrogate by single cell mutational profiling the impact of minimal residual disease composition and clonal dynamics in ALL relapse; Aim 2 (mentored/independent phase): To identify and target master regulators of single cell transcriptional and epigenetic states driving ALL persistence, disease progression and relapse; and Aim 3 (independent phase): To map and target genetic, transcriptional and epigenetic drivers of minimal residual disease and leukemia relapse in pediatric AML. To address these questions, I will use diagnostic-remission-relapse matched leukemia primary samples and leukemia xenografts, combined with single-cell genomics, transcriptomics and epigenomics to functionally characterize convergent mechanisms driving minimal residual disease, resistance and relapse. These analyses will inform functional testing of master regulators and direct single-cell reverse genetic CRISPR screens to define the role of specific genes as drivers of chemoresistance and relapse. These results will ultimately facilitate the rational design of therapies to improve the treatment of this devastating child...