Stag2-Cohesin Loss Pertubes Npm1c and Flt3ITD Mutant Hematopoietic Stem and Progenitor Differentiation

Stag2-Cohesin Loss Pertubes Npm1c and Flt3ITD Mutant Hematopoietic Stem and Progenitor Differentiation

Jane J. Xu^{1, 2}, Besmira Alija^{1, 2}, John Pantazi^{1, 2}, Varun Sudunagunta^{1, 2, 3}, Stephanine Braustein^{1, 2}, Robert L. Bowman⁴, Aaron D. Viny^{1, 2, 3}

¹ Columbia Stem Cell Initiative, Columbia Irving Medical Centre, New York City, NY

² Department of Medicine, Hematology & Oncology, Columbia Irving Medical Centre, New York, NY

³ Vagelos College of Physicians and Surgeons, Columbia University, New York, NY

⁴ Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA

Acute myeloid leukemia (AML) is an aggressive myeloid malignancy with high relapse (40-60%) and low median survival rate (5-7 months post treatment in relapse setting), particularly for patients over 65 years old. Genomic investigations of AML have identified recurrent mutations, such as FMS-like tyrosine kinase 3 (FLT3, 20-25%) and Nucleophosmin (NPM1, 30%). When mutated, FLT3 acquires an internal tandem duplication (FLT3-ITD) and causes aberrant STAT5/AKT signaling. For NPM1, mutation frequently occurs at nuclear export signal, which results in cytoplasmic mislocalization (NPM1c). In AML, these driver mutations do co-occur with other genetic lesions, particularly in epigenetic regulators. In the clinical setting, the FLT3-ITD patients have a very poor prognosis. While the pathophysiology is not completely understood, direct inhibition of FLT3 by tyrosine kinase inhibitors have, to-date, been underwhelming. In contrast, the NPM1c mutation in AML patients is favorable and chemotherapy sensitive when NPM1c is the sole driver mutation. Yet, co-mutation of NPM1c with other known AML mutations, such as Flt3-ITD, negates this favorable prognosis.

STAG2 (Stromal Antigen 2) is a member of cohesin complex that is recurrently mutated in >10 cancers and is essential in maintaining the integrity of the 3-dimensional genome partitioning structure known as topologically structural domains (TADs). Previously, our 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 impairment of sub-TADs and effects the capability of key hematopoietic transcription factors, such as PU.1, to access and engage their target genes. In the context of AML, disruption of TADs can lead to altered transcriptional output and cellular behavior. Mutation of STAG2 frequently arises in AML (16%), particularly in AML arising from antecedent myelodysplastic syndrome, and co-occurs with NPM1 and FLT3 oncogene.

To examine the consequences of co-occurring Stag2/Npm1c or Stag2/Flt3-ITD mutations, we generated two compound mutant murine models crossed to conditional alleles of Stag2, Npm1c and constitutively active Flt3^ITD in the Ubc-CreER background. At 6-8 weeks of age, tamoxifen was administered to mice harboring Stag2/Npm1c or Stag2/Flt3-ITD mutations to delete Stag2 or activate Npm1c alleles respectively, while the Flt3^ITD mutation is active since birth. Immunophenotyping analysis was performed at 4 weeks post mutation activation in the bone marrow and peripheral blood. Comparing to WT, there is an expansion of hematopoietic stem and progenitor LSK population (HSPC, lin- cKit+ Sca1+). Within the HSPC, Stag2Npm1^c/+ mice had an elevated myeloid-biased multipotent progenitor population (MPP3, LSK flk2- CD150- CD48+). However, there was no changes in myeloid progenitor (MP) and granulocyte-monocyte progenitors (GMP). Single cell RNA-seq analysis of Stag2Npm1^c/+ LSK cells confirmed a delayed in progression in the MPP3 population via RNA velocity and pseudotime analysis. Bulk ATAC-seq of Stag2Npm1^c/+ MPP3 population revealed an increased chromatin accessibility comparing to WT. 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^+/-

Npm1^c/+ mice, suggesting Stag2-cohesin loss obstructs differentiation in Npm1^c/+ HSPC.

Comparing to Npm1c mutation, loss of Stag2-cohesin resulted in no changes in Stag2Flt3^ITD MPP3 cells compared to monogenic Flt3-ITD mutation. However, we did observe a normalization of MP and GMPs, suggesting that Stag2-cohesin is crucial for abnormal myeloid differentiation in Flt3^ITD background. In addition to the constitutively active Flt3^ITD model, we additionally generated a stepwise Stag2/Flt3-ITD mutational model by deleting Stag2 first (via Mx1-Cre) followed by tamoxifen-inducible Frt activated Flt3^ITD mutation (Flt3 frt-ITD/+). The novel model recapitulates the genetic evolution of secondary AML, where STAG2 mutation is highly enriched. Interestingly, there was no difference in the normalization effect between constitutive and conditional activation of Flt3^ITD mutation, suggesting the loss of Stag2-cohesin is not required prior to Flt3^ITD acquisition. Surprisingly, Stag2^1stITD^2nd mice has preserved hematopoietic stem cell (HSC, LSK flk2- CD150+ CD48-) identified via immunophenotyping and scRNAseq analysis. Comparing to ITD mutant, Stag2^1stITD^2nd LSK cells have decreased expression of Socs2 and Cish, the canonical downstream targets of ITD aberrant signaling.

In summary, the chromatin structure facilitated by Stag2-cohesin has unique roles in leukemia mutant cells via Stag2/Npm1c and Stag2/Flt3^ITD. Loss of Stag2-cohesin leads to expansion of MPP3 population in the Npm1^c/+ cells and LSK to MP differentiation block in Flt3^ITD cells, regardless of mutational order. In addition, we identified a persistent HSC population in the Stag2Flt3^ITD mice. Our data highlight an important regulatory role of Stag2-cohesin in Npm1c and Flt3^ITD mediated leukemogenesis with potential therapeutic implications.

This research is funded by NIH K08CA215317 to ADV, ASH scholar award to ADV.

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