Talazoparib Treatment Preferentially Depletes Cohesin-Mutant Clones in New In Vivo Models of Cohesin-Mutant Myeloid Diseases

Cohesin is a multisubunit protein complex that is involved in sister chromatid cohesion, post-replicative DNA repair and transcriptional regulation, and is frequently mutated in cancer. STAG2 is the most recurrently mutated cohesin gene in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML) but no STAG2-mutant disease models currently exist to study the natural evolution of cohesin-mutant myeloid disease, or to test therapeutic vulnerabilities. Our previous workimplicates PARP1 inhibition as a therapeutic vulnerability in STAG2-mutant AML cell lines, which display impaired replication fork progression and accumulation of double-strand DNA breaks.We developed a syngeneic mouse model of cohesin-mutant myeloid disease mimicking the phenotype seen in human MDS, in which STAG2 mutations arise as clonal secondary lesions in the background of clonal hematopoiesis. We used this model to test the effect of sequential acquisition of Tet2 and Stag2 mutations on mouse hematopoiesis, and in vivo therapeutic vulnerabilities including sensitivity to PARP1 inhibition. In parallel, we developed patient-derived xenograft (PDX) models of cohesin-mutant AML to extend our findings.

Hematopoietic stem and progenitor cells (Lineage-, Sca1+, c-Kit+ cells) harvested from Mx1-Cre;Cas9 heterozygous C57BL.6 mice were transduced with sgRNA targeting Tet2 or non-targeting sgRNA and transplanted into lethally irradiated SJL recipient mice. Engraftment and clonal expansion of Tet2-mutant cells was confirmed with fluorescent protein reporter expression and next-generation sequencing (NGS), and was not associated with any overt phenotype, similar to human clonal hematopoiesis of indeterminate potential (CHIP). c-Kit enriched bone marrow cells from mice with Tet2-mutant CHIP were subsequently transduced with sgRNA targeting Stag2 or non-targeting sgRNAs and re-transplanted into recipient mice. Mice were serially followed for up to 9 months and showed increased clonal expansion of Tet2/Stag2-mutant cells over Tet2-only mutant cells. In contrast to Tet2-only mutant mice, Tet2/Stag2-mutant mice developed leukocytosis, absolute monocytosis, anemia, and thrombocytopenia. Bone marrow evaluation of Tet2/Stag2 mice demonstrated increased hemophagocytosis and fewer megakaryocytes in comparison to Tet2-only mice and NGS confirmed predicted loss of function frameshift mutations in Tet2 and Stag2. We therefore generated a new model of cohesin-mutant myeloid disease recapitulating sequential acquisition of Stag2mutations in the context of an initiating epigenetic lesion as seen in patients with cohesin-mutant MDS.

Tet2/Stag2 andTet2-mutant clones and the associated hematologic phenotypes were serially transplantable and allowed for genotype-specific in vivo drug testing of the PARP1 inhibitor talazoparib. Forty recipient mice transplanted with Tet2 or Tet2/Stag2 mutant bone marrow cells were stratified into treatment groups with talazoparib or vehicle. Expression of congenic markers and fluorescent reporters linked to Tet2 and Stag2 sgRNA expression were used to monitor mice during 4 weeks of treatment. We observed normalization of leukocytosis, monocytosis and thrombocytopenia in Tet2/Stag2 mutant mice treated with talazoparib. In addition, Tet2/Stag2-mutant mice but not Tet2-only mice treated with talazoparib demonstrated a significant loss of mutant clones as determined by flow cytometry and NGS. To further extend these findings in primary human cells, we developed serially transplantable PDX models of cohesin-mutant AML and performed in vivo drug testing with talazoparib. We noted increased survival of cohesin-mutant PDX models treated with talazoparib as compared to vehicle.

We demonstrate the development of a syngeneic mouse model of Stag2-mutated myeloid disease through sequential introduction of Tet2 or Stag2 mutations in primary mouse hematopoietic stem and progenitor cells. Mice with loss of Tet2 and Stag2 recapitulate hematologic parameters mimicking myelodysplastic/myeloproliferative neoplasms in humans. In vivo treatment with talazoparib demonstrates selective depletion of cohesin mutations in syngeneic and PDX models. The effect of talazoparib monotherapy in cohesin-mutated AML or MDS with excess blasts is under investigation in a pilot proof-of-concept study (ClinicalTrials.gov Identifier NCT03974217).