Cohesin Complex Mutations Impair Differentiation of Human Hematopoietic Stem and Progenitor Cells
Authors: Mazumdar Claire , Li Rui , Buenrostro Jason , Chang Howard Y. , Majeti Ravi 
Published: October 2019

The cohesin complex is a multiprotein complex involved in a number of cellular processes including sister chromatid cohesion in mitosis, replication fork organization, and regulation of chromatin accessibility for gene expression. Mutations in genes encoding the members of the cohesin complex (SMC1A, SMC3, STAG2, and RAD21) occur in about 10-15% of de novo acute myeloid leukemia (AML) patients. Apart from AML, cohesin mutations have been found in many human cancers indicating a central role for this complex in oncogenesis. In AML, our prior studies have demonstrated that cohesin mutations occur in pre-leukemic hematopoietic stem and progenitor cells (HSPC) that retain normal differentiation potential. Thus, these mutations are likely key initiating events in leukemia pathogenesis. Due to their importance in AML evolution, we sought to determine the effect of these mutations on human hematopoiesis.

Cohesin mutations typically occur as heterozygous mutations throughout the genes suggesting either a haploinsufficiency or dominant negative effect. Co-immunoprecipitation experiments in primary human AML samples showed marked decrease in binding between RAD21 and SMC1A in RAD21/SMC1A-mutant AML. These results suggest a dominant negative effect of cohesin mutants on complex formation.

In an effort to characterize the phenotype of cohesin complex mutations in AML, we generated human AML cell lines engineered to express wildtype (WT) or mutant cohesin components under the control of a doxycycline-inducible promoter. We chose the TF-1 erythroleukemia cell line due to its ability to differentiate down the erythroid lineage in response to erythropoietin (EPO). We found that cohesin mutant cell lines showed a significant decrease in erythroid differentiation upon exposure to EPO as determined by surface expression of glycophorin A (GPA) and RNA expression of fetal hemoglobin and KLF-1, a key erythroid transcription factor, suggesting that cohesin mutations act in a dominant negative manner to impair differentiation.

We next investigated the impact of cohesin complex mutations on normal HSPCs from primary human cord blood. We transduced CD34+ cord blood cells with lentivirus encoding constitutive expression of either WT or mutant cohesin components. Transduced cells were isolated and cultured under several conditions. First, cells were cultured with cytokines designed to promote retention of HSPCs, and cord blood cells expressing mutant cohesin showed significant retention of CD34+ expression as compared to WT or control cells. Second, cells were cultured under conditions designed to promote granulocytic/monocytic differentiation, and cohesin mutant-expressing cells showed a significant decrease in CD14+ expression compared to controls. Third, cells were cultured under conditions designed to promote erythroid differentiation, and cohesin mutant cells showed a significant decrease in CD71 and GPA-double positive erythroid cells. Together, this data suggests that cohesin complex mutations impart a differentiation block on primary human HSPCs. Finally, we investigated whether cohesin mutations affected the serial colony replating ability of human HSPCs in vitro. Primary human cord blood HSPCs were transduced with cohesin mutant-encoding lentivirus, sorted, and cultured in methylcellulose for 14 days. No differences were observed in the colony number or type in the primary plating. However, cohesin-mutant cells exhibited increased serial replating potential beyond the 3rd replating, with essentially no control or WT colonies after the 2ndreplating.

In summary, our results indicate that cohesin complex mutations impair HSPC differentiation and increase in vitro replating of primary human cells. The mechanisms by which this occurs are currently being investigated, but preliminary data suggests that mutations in cohesin affect global chromatin accessibility. These results are consistent with a model of mutational acquisition in AML that we have proposed, in which pre-leukemic mutations occur in genes involved in global regulation of gene expression through epigenetic mechanisms that impair differentiation and/or affect self-renewal (such as IDH1/2, TET2, DNMT3A, and cohesin), whereas late mutations occur in genes that generally lead to an increase in activated signaling and proliferation (such as FLT3 and RAS).

Disclosures

No relevant conflicts of interest to declare.