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954 Synergy of Stag2-Cohesin Loss Results in Expansion of Npm1c-Mutant Hematopoietic Stem and Progenitor Cells

Program: Oral and Poster Abstracts
Type: Oral
Session: 602. Myeloid Oncogenesis: Basic: Mechanisms and Models of Myeloid Malignancy
Hematology Disease Topics & Pathways:
Research, Fundamental Science
Monday, December 11, 2023: 5:45 PM

Jane J Xu, PhD1,2*, Yi Chen, MD, PhD3*, John Pantazi3*, Sebastian Fernando4*, Besmira Alija3*, Varun Sudunagunta, BA5, Govind Bhagat, MD6 and Aaron D. Viny, MD, MS7

1Columbia University Irving Medical Center, Department of Medicine, Division of Hematology / Oncology, New York, NY
2Columbia Stem Cell Initiative, New York, NY
3Columbia Stem Cell Initiative, Columbia Irving Medical Centre, New York, NY
4Columbia Stem Cell Initiative, Columbia Irving Medical Centre, New York
5Columbia University Vagelos College of Physicians and Surgeons, NEW YORK, NY
6Department of Pathology, Columbia University Irving Medical Center, New York, NY
7Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY

STAG2 is a member of cohesin complex that is recurrently mutated in >10 cancers and is essential in maintaining the integrity of the 3D genome partitioning structure known as topologically structural domains (TADs). Our previous work has demonstrated that depletion of various cohesin factors, including Stag2, leads to increased hematopoietic stem and progenitor population (HSPC) self-renewal and myeloid-biased differentiation. Loss of Stag2 leads to impaired sub-TADs and affects key hematopoietic transcription factors, such as PU.1, to access and engage their target genes. Yet, the extent and cooperative transformational role of Stag2-cohesin with leukemia driver mutations, such as Npm1c, remains unexplored.

To determine whether Stag2-cohesin regulates leukemic 3D chromatin organization, we generated dual Stag2Δ Npm1c/+ murine models with tamoxifen inducible UbcCreERT2. After 4 weeks, HSPCs (LSK cells; Lin-Sca1+Kit+) are increased in Stag2Δ Npm1c/+ double mutant mice (Figure 1A). Within LSK, Stag2Δ Npm1c/+ but not Npm1c/+ has marked expansion of the myeloid bias MPP2 (LSK+Flk2-Cd150+Cd48+) and MPP3 (LSK+Flk2-Cd150-Cd48+) compartment. Interestingly, Stag2Δ single mutant exhibit LSK and MPP3 expansion at 16 weeks, suggesting the early expansion of HSPC compartment is the result of Stag2ΔNpm1c/+ synergistic acceleration. No difference in serial replating of LSK and GMP (LSK-Cd34+FcyRII+) cells were observed between Npm1c/+ and Stag2Δ Npm1c/+. However, Stag2Δ Npm1c/+ LSK cells had impaired reconstitution capacity and myeloid biased output in primary and secondary transplantation. To determine if the blockage is specific to Stag2-cohesin loss, we generated the Smc3/Npm1c double mutant mice, where both Stag1-cohesin and Stag2-cohesin complexes were partially depleted upon heterozygous deletion of Smc3. Interestingly, there were no changes in LSK or MPP3 population in Smc3+/- Npm1c/+ mice, suggesting it is specific to Stag2-cohesin loss which obstructs differentiation in Npm1c/+ HSPC.

Mechanistically, we performed bulk ATAC-seq on sorted HSCLT (LSK+Flk2-Cd150+Cd48-) and MPP3 population, as poor long-term reconstitution suggests functional alteration in HSCLT. Comparing to WT, both Stag2Δ and Stag2Δ Npm1c/+ HSCLT have increased chromatin accessibility, while only Stag2Δ Npm1c/+ MPP3 have persistent opening of chromatin. Motif analysis of increased accessible peaks identified RUNX2, GATA3 and MEIS1 are among the increased accessible region in Stag2Δ Npm1c/+ MPP3, which reflects the increased myeloid output in the transplantation. Recently, various group have suggested that Npm1c directly binds to chromatin and drives the overexpression of genes, especially at HOX clusters and MEIS1. This suggests a possible synergistic mechanism which Stag2-cohesin regulates chromatin accessibility of key TF loci, which influences normal and leukemic stem and progenitor differentiation. To comprehensively profile the LSK population, we performed scRNAseq at 4 weeks post mutation activation, results showed a marked immune pathway activation in Stag2Δ Npm1c/+ LSK cells. To inter cell fate transition, we employed CellDancer, an RNA velocity based algorithm and found that Stag2Δ Npm1c/+ MPP cells have altered differentiation trajectory and delayed kinetics at MPP3 stages, suggesting a differentiation block (Figure 1B). In conjunction with our results, we postulate that Stag2-cohesin regulates TF interactions with regulatory elements during cell fate transitions. We are currently investigating the direct interaction of Stag2-cohesin with regulatory elements in Npm1c/+ cells and compare to WT and Stag2Δ Npm1c/+ cells. We will investigate the 3D genome organization of Npm1c/+ and Stag2Δ Npm1c/+ cells to determine whether Stag2 loss induces the phenotypic changes via Ctcf-dependent or independent manner.

Disclosures: Viny: Arima Genomics: Membership on an entity's Board of Directors or advisory committees.

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