The role of HECTD1 and MCL-1 in the regulation of normal and malignant haematopoiesis

Abstract

Cellular processes important for haematopoiesis are frequently perturbed in malignant cells. Accordingly, healthy immature progenitor cells and malignant cancer cells often share certain properties, e.g. rapid proliferation and self-renewal. Deciphering these developmental pathways can provide information about the critical drivers involved in neoplastic transformation and sustained cancer cell growth. Results in this thesis addresses the role of two proteins, HECTD1 and MCL-1, in haematopoiesis and haematological malignancies.

HECTD1 is an E3 ubiquitin ligase required for mouse embryonic development, as homozygous loss of Hectd1 leads to embryonic lethality. HECTD1 is widely expressed in diverse tissues, including haematopoietic cells. However, its role in adult tissues in vivo has not been described. Therefore, we generated mice in which HECTD1 deletion was restricted to the haematopoietic system of adult mice. Analysis of these mice at steady state revealed small perturbations in certain T cell subsets. However, competitive reconstitution experiments revealed that HECTD1 deletion affects the haematopoietic stem and progenitor cell (HSPC) populations. Serial transplantation assays showed that loss of HECTD1 results in a defect in the self-renewal properties of mouse HSPCs. Interestingly, RNA sequencing of Hectd1-/- HSPCs revealed that HECTD1-deficiency led to increased expression of interferon regulated genes, suggesting that HECTD1 plays a critical role in the maintenance of HSPC populations by negatively regulating the interferon signalling pathway. Additionally, I employed the MLL AF9 mouse model of acute myeloid leukaemia and showed that HECTD1-deficiency significantly delayed the latency of tumour development in vivo compared to control mice.

MCL-1 is a pro-survival regulator of the intrinsic apoptosis pathway. MCL-1 expression is integral to the survival of many different blood cell types, and to the development and sustained growth of many haematological malignancies. Recently a highly specific MCL-1 inhibitor, S63845, showing 6-fold higher affinity to human MCL-1 compared to mouse MCL-1 was described. To accurately test the efficacy and tolerability of S63845 in preclinical models of disease, we developed a humanised Mcl 1 (huMcl-1) mouse strain in which the genomic region of the murine Mcl-1 locus was replaced with the coding regions for human MCL-1. These mice are phenotypically indistinguishable from wild-type mice, and the intrinsic apoptotic pathway remains intact in their cells. However, as anticipated, huMcl-1 mice were more sensitive to S63845 than wild-type mice. To test whether malignant cells from the humanised MCL-1 mice also show higher sensitivity to S63845, we generated Eµ-Myc lymphomas on a huMcl-1 background. Lymphoma cell lines derived from huMcl-1;Eµ-Myc mice were ~6 times more sensitive to S63845 in vitro compared to Eµ-Myc lymphoma cells expressing mouse MCL-1. Transplantation of huMcl-1;Eµ-Myc lymphoma cells into huMcl-1 mice and treatment with S63845 resulted in tumour-free survival in >60% of mice. Furthermore, combining low doses of S63845 with sub-optimal doses of cyclophosphamide led to almost complete tumour regression. These results show that our huMcl-1 mouse model represents a valuable preclinical tool to test MCL-1 inhibitors, either alone or in combination with other anti-cancer agents, for a broad range of cancers, allowing accurate prediction of efficacy against tumour cells and on target toxicity to normal tissues.