Absence of Mcm6 mimics haemopoietic ageing and has implications for leukaemic transformation

Abstract

Maintenance of genomic stability is critical for tumour suppression. Deregulation of the DNA replication machinery leads to accumulation of replication stress and increased mutational events often leading to oncogenesis. Replication stress occurs during normal cell ageing; aged cells acquire more background mutations and show inefficient DNA damage repair response (DDRR). Here, we aimed to investigate whether background mutations affect haemopoietic stem and progenitor cell (HSPC) functionality and leukaemic transformation. We generated an ageing-like haemopoietic model using CRISPR lentiviral vectors to knockout (KO) Mcm6, a key factor of the DNA replication machinery whose expression decreases with age. Murine HSPCs were transduced with CRISPR empty vector (EV) or carrying guide RNAs (gRNAs) targeting Mcm6. HSPC functionality was assessed in colony forming cell assays. KO cells formed fewer, but morphologically bigger and more compact colonies indicating a defect in self-renewal/stemness. Flow cytometry analysis revealed that KO cells expressed significantly lower levels of ter119 and c-Kit markers and higher levels of CD71 suggesting a block in erythropoiesis in vitro. To elucidate Mcm6 KO effects in vivo, transduced HSPCs were injected into lethally irradiated mice. Mice that received KO cells (KO chimeras) had lower levels of CD19 B cell marker and higher CD11b and Gr-1 myeloid markers in the peripheral blood. Bone marrow cells isolated from the KO chimeric mice accumulated replication stress evident by an increase in γH2AX, and showed poor DDRR supported by the lower expression of key DDR genes. Finally, using EV and KO transduced 32D cells, we assessed leukaemic transformation following MLL-AF9 (MA9) oncogene expression. 32D-KO-MA9+ cells exhibited a proliferative advantage and expressed lower levels of the c-kit progenitor marker compared to controls indicating that DNA replication stress as a result of Mcm6 deficiency changes the leukaemic phenotype. Collectively, our data indicates that replication stress alters both haemopoietic differentiation and leukaemic transformation, reminiscent of HSPC aged cells and the increased incidence of AML in the aged.