Clonal evolution and therapeutic vulnerabilities in SETBP1-mutated leukemia: Insights from physiological mouse model and integrative functional screens Free

Somatic mutations in SET binding protein 1 (SETBP1), a putative chromatin regulator, occur in ~30% of high-risk myeloid malignancies, including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and secondary acute myeloid leukemia (AML), and strongly correlate with adverse clinical outcomes. Despite this significance, the precise roles of SETBP1 mutations in blood cancer remain enigmatic, primarily due to the lack of physiologically relevant cell lines or animal models that recapitulate the clonal evolution. Indeed, our 23 attempts to establish SETBP1-mutated PDX models failed, as the clones did not show advantageous effects. While conventional wisdom suggests that SETBP1 mutations escape proteasomal degradation to drive oncogenesis, our Setbp1 knockout mice revealed that SETBP1 is dispensable for normal and malignant hematopoiesis (Tanaka et al. Leukemia 2023), challenging prevailing paradigms.

To resolve these contradictions, we developed a blood-specific Setbp1 mutant locus knock-in (KI) mouse model using Vav1-iCre, corresponding to the most prevalent D868N mutation. Unlike retroviral overexpression that immortalizes murine hematopoietic stem and progenitor cells (HSPCs), our KI model did not perturb HSPC function or transcriptome in vivo, indicating that SETBP1 acts as a cooperative rather than an initiating driver. As ASXL1 mutations are the most frequent founder mutations in SETBP1-mutated MDS/AML, we created double KI mice with Asxl1/Setbp1 physiologic mutations for transplant studies. These mice exhibited striking monocytosis (~40% of peripheral white blood cells) and HSPC expansion, rescuing stemness defects imposed by ASXL1 mutations. scRNA-seq/ATAC-seq revealed unique transcriptional clusters enriched for Hoxa9/10, Eya1, Myb, and Myc pathway alongside increased chromatin accessibility, suggesting that both mutants cooperate to remodel chromatin and amplify oncogenic transcriptional programs. Over time, the transplant model with double KI developed a leukemia phenotype, including serially transplantable AML and CMML-like diseases, all spontaneously acquired RAS pathway-activating mutations in Nras, Flt3, and Ptpn11. This reflects findings from over 7,000 patient samples, showing a significant correlation between SETBP1, ASXL1, and RAS pathway mutations. Indeed, the presence of RAS mutations predicts poor outcomes in SETBP1-mutated cases. Functional validation confirmed that all three alterations are required for complete transformation toward AML, establishing a three-step model: ASXL1mutations initiate clonal evolution, SETBP1 mutations enhance oncogenic programs, and RAS pathway activation completes the transformation.

Leveraging these unprecedented in vivo models and cell lines with physiologically relevant SETBP1 mutations, we conducted comprehensive therapeutic screens using whole-genome CRISPR/sgRNA and pan-cancer drug libraries. The integrated results revealed a critical dependency on the druggable Exportin 1 (XPO1), shown by a 20.8-fold reduction (p = 0.00004) in the 7-day CRISPR screen and a 0.0003-fold growth under Leptomycin B treatment compared to DMSO at day 4. This unique dependency was robustly validated through genetic and pharmacological approaches using shXpo1, Selinexor, and Leptomycin B, resulting in monocytic differentiation and apoptosis in Setbp1-mutated AML cells. Notably, Selinexor monotherapy achieved sustained remissions exceeding 110 days in vivo, profoundly extending survival compared to controls, which succumbed to leukemia with a median survival of 59.5 days (p = 0.0004), demonstrating its potential to eradicate SETBP1-mutated leukemia. Mechanistically, XPO1 physically interacts with SETBP1 through the nuclear export signal groove and acts as a chromatin regulator beyond its canonical nuclear-cytoplasmic transport role. XPO1 inhibition suppressed the transcription of leukemogenic genes, including Hoxacluster genes, Myb, Eya1, and, critically, Myc pathway, with altered XPO1-mediated chromatin accessibility, as shown by integrated ChIP/RNA/ATAC-seq. These results uncover a critical epigenetic vulnerability that represents a promising target for therapeutic intervention in SETBP1-mutated leukemia.

In summary, our study elucidates the cooperative leukemogenic roles of SETBP1, ASXL1, and RAS pathway mutations and identifies XPO1 as a targetable epigenetic vulnerability. These findings support precision treatment strategies for aggressive myeloid malignancies.