Biology and targeting of mutant STAG2-mediated R loop accumulation

Dana-Farber Cancer Inst, Boston MA

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Abstract

Project Summary There is a fundamental gap in our understanding of how mutations in the cohesin complex, a multimeric protein complex essential for sister chromatid cohesion, chromatin organization and gene regulation, cause myeloid neoplasms (MDS) and acute myeloid leukemia (AML). Continued existence of this gap represents an important problem because despite the high frequency of cohesin mutations in MDS and AML and its association with high risk of disease and poor outcomes, there are currently no treatment strategies to target cohesin-mutant cells in MDS. STAG2 mutations most frequently co-occur with mutations in ASXL1, however models representing this combination are lacking. Furthermore, cohesin proteins have been shown to bind DNA-RNA hybrids (R loops) and when mutated, lead to R-loop associated DNA damage. New genetically engineered models of cohesin- mutant MDS offer a unique opportunity to test the contribution of disease alleles to R loop regulation, to dissect the role of R loops in MDS development, and to test their potential as therapeutic targets. The long-term goal is to improve outcomes in cohesin-mutant MDS/AML by deeper mechanistic understanding of disease biology. The overall objective is to determine whether R loops play a causal role in STAG2-mutant MDS development and could serve as therapeutic targets. The central hypothesis is that mutant STAG2 induced downregulation of R loop regulators leads to accumulation of R loops, which contribute to transcriptional and chromatin dysregulation of STAG2-mutant. Guided by strong preliminary data from the Tothova laboratory, the hypothesis will be tested by pursuing three specific aims: 1) Characterize the altered R loop regulation in STAG2-mutant cells; 2) Determine the role of R loop dysregulation in STAG2-mutant disease; and 3) Examine the clinical efficacy of targeting cGAS and STING. Under the first Aim, we will develop and characterize a novel genetic murine model of progression from ASXL1-mutant clonal hematopoiesis of indeterminate potential (CHIP) to ASXL1/Stag2- mutant MDS and perform genome-wide mapping of R loops in hematopoietic stem and progenitor cells. Under the second Aim, a combination of chromatin accessibility, gene expression profiling, H3K27Ac ChIP-Seq and RNAseH1 overexpression experiments will be used to functionally interrogate the role of R loops in STAG2- mutant cells on gene regulation, chromatin organization, and MDS development. Under the third aim, cGAS and STING modulators will be used to test the potential of therapeutic targeting of STAG2-mutant cells in vitro and in vivo. The approach is innovative through the application of novel murine models and innovative techniques of R loop detection. The downstream effects of R loop dysregulation in cohesin-mutant MDS have not been studied, and the mechanisms driving this accumulation are unknown. The proposed research is significant, because it is expected to define the biological role of cohesin mutations in a high-risk subset of MDS associated with poor outcomes. The expected output for the proposed research is that the knowledge gained will be immediately clinically significant for patients with cohesin-mutant MDS and inform new treatment strategies.