STAG2-Mutant MDS Development Is Associated with Accumulation of R-Loops

STAG2-Mutant MDS Development Is Associated with Accumulation of R-Loops

Lin Han^1,2, Benjamin Braun^1,2, Caroline Conway^1,3, Amy Wang^1,2, Qingqing Yan⁴, Phillip Wulfridge⁴, Johann-Christoph Jann^1,2, William Doyle^1,2, Caroline Pitton^1,2, Kavitha Sarma⁴, Zuzana Tothova^1,2

¹Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA

²Cancer Program, Broad Institute, Cambridge, MA

³Department of Molecular and Cellular Biology, Harvard University, Boston, MA

⁴Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, PA

Myelodysplastic syndromes (MDS) are clonal bone marrow disorders characterized by ineffective hematopoiesis, abnormal cell morphology, and cytopenias in one or more lineages. Recurrent somatic mutations in genes encoding the cohesin complex, including STAG2, are observed in MDS and secondary acute myeloid leukemia (sAML) and associated with poor overall survival. However, how STAG2 mutations mediate the transformation to MDS remains elusive. Our previous studies identified increased DNA damage across multiple models of STAG2-mutant MDS and AML. Our preliminary data in STAG2-mutant AML cell lines show that overexpression of the DNA/RNA (R-loop)-specific nuclease RNaseH resulted in almost a complete rescue of the DNA damage phenotype. Here, we describe a study aimed to understand the mechanisms driving the R-loop mediated accumulation of DNA damage in a new model of Tet2/Stag2-mutant MDS.

Loss of function mutations of STAG2 strongly co-occur with mutations in TET2, one of the most frequently mutated genes in clonal hematopoiesis of indeterminate potential (CHIP). We generated a new mouse model characterized by conditional knockout of a single allele of Tet2 and both alleles of Stag2 in the hematopoietic compartment and investigated their impact on hematopoiesis and disease development. Tet2/Stag2 double mutant mice developed monocytosis, thrombocytopenia and lymphopenia as compared to wild type (WT) or Tet2 heterozygous littermate controls as early as 4 weeks post poly(I:C) administration. Bone marrow analysis demonstrated an increase in the number of hematopoietic stem cells (HSCs) and multi potent progenitor (MPP)1, MPP2 and MPP3 cells, as well as myeloid skewing with expansion of the common myeloid progenitor (CMP) and granulocyte monocyte progenitor (GMP) compartments, with an accompanying loss of common lymphoid progenitors (CLPs) and megakaryocyte/erythroid progenitors (MEPs). RNA-Seq analysis on sorted Lin-negative c-Kit+ cells from the bone marrow of Tet2/Stag2 double mutant mice demonstrated a strong enrichment of HSC programs and loss of DNA damage repair signature. Bone marrow morphologic analysis showed evidence of erythroid and megakaryocyte dysplasia. Altogether, these data support that our new model faithfully recapitulates features of STAG2-mutant MDS.

Next, we sought to examine the effect of Stag2 loss on DNA damage in vivo. Our new mouse model of Tet2/Stag2-mutant MDS model demonstrated increased DNA damage as evidenced by high levels of H2Ax in the bone marrow of Tet2/Stag2 double mutant but not WT or single mutant Tet2 or Stag2 mice. Furthermore, we investigate the impact of Stag2 loss on transcription-coupled stress in the form of R-loops, which have been previously shown to associate with mutations in other epigenetic regulators in myeloid malignancies, such as splicing factors. We assessed the level of R-loops in sorted Lin-negative c-Kit+ bone marrow cells using a recombinant catalytically inactive GFP-RNaseH1 protein fusion with preserved ability to bind but not cleave R-loops and observed a significant increase of R-loop formation in Tet2/Stag2-mutant mice. Interestingly, single Stag2-KO cells also showed an increase in R-loop formation, although to a much lesser extent, suggesting that Stag2 is important in R-loop regulation. We mapped the genome-wide distribution of R-loops in c-Kit+ bone marrow cells isolated from WT, single and double mutant mice using MapR and detected a significant increase in R-loop formation in the Tet2/Stag2-mutant MDS. Integrative analysis of R-loops, gene expression, open chromatin and DNA damage in Lin-negative c-Kit+ cells showed association of new R-loop formation with transcriptional dysregulation and DNA damage accumulation.

In sum, we present a new model of Tet2/Stag2-mutant MDS and demonstrate the impact of Stag2 loss on R loop accumulation during CHIP to MDS disease progression, resulting in accumulation of DNA damage. Our studies imply R loop dysregulation as an important mechanism during cohesin-mutant MDS development and suggest new opportunities for therapeutic targeting.

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