Dose-Dependent Role of the Cohesin Complex in Normal and Malignant Hematopoiesis

Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation, however cohesin-mutant leukemias do not show genomic instability suggesting an alternate role in malignant transformation. We hypothesized reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic stem/progenitor cells. We therefore investigated the impact of both complete loss and haploinsufficiency of Smc3, an obligate member of the cohesin complex, in normal hematopoiesis and in myeloid transformation by developing a conditional Smc3 knockout allele. Somatic loss of Smc3 in hematopoietic cells induced lethal bone marrow aplasia (median survival 11 days; p<0.001), with premature sister chromatid separation and abnormal nucleolar organization. Competitive transplant assays showed that Smc3 loss completely abrogated stem cell self-renewal in vivo. These data are consistent with an absolute requirement for the cohesin complex in hematopoietic stem/progenitor cells. By contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, with increased serial replating, expanded hematopoietic stem/progenitor cells, and a self-renewal/engraftment advantage in competitive transplantation assays in vivo (Figure a). Smc3 haploinsufficiency altered coordinated transcriptional output, including reduced expression of master regulatory transcription factors governing lineage commitment. Consistent with these data, Smc3 loss resulted in expanded Cd150+ Cd48+ ST-HSC (p=0.008), reduction in Cd150+ Cd48- LT-HSC (p=0.001), and altered chromatin architecture with dysregulated expression of genes with specific chromatin architecture footprints. Smc3 haploinsufficiency cooperated with Flt3^ITD to induce acute leukemia in vivo (Figure b), with dysregulated expression of hematopoietic master regulators and altered nucleolar topology similar to that observed in germline cohesinopathy syndromes and in AML patients with cohesin mutations (Figure c).

To further explore the mechanism by which Smc3 loss cooperates with Flt3^ITD to induce leukemia, we investigated chromatin cis-regulatory architecture with transposase hypersensitivity assays (ATAC-seq). We hypothesized that increased accessibility at cis-regulatory elements and the alterations in gene expression seen in cells with combined Smc3 haploinsufficiency and Flt3^ITD may be in a large part driven by potentiated Stat signaling at chromatin. We analyzed 146 transcription factor recognition motifs within the THS differentially observed in Smc3^Δ/+Flt3^ITD and wild-type cells. Chromatin accessibility gained in Smc3^Δ/+Flt3^ITD cells are enriched in Stat family transcription factor binding sites, including Stat5. We also observed enrichment of the Stat5 gene expression signature in the Smc3^Δ/+Flt3^ITD cells compared to Smc3^Δ/+, Flt3^ITD and wild-type cells, suggesting the divergent mutations cooperate to potentiate oncogenic Stat5 signaling in HSPCs. Our results demonstrate a key dose-dependent role for the cohesin complex in hematopoiesis, and show that reduced cohesin functions to alter enhancer-mediated transcription and contribute to aberrant self-renewal and myeloid transformation.

Figure 1.

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Figure 1.

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