Genome engineering of HMC-1.2 cells renders a human mast cell line with a single KIT mutation: an improved preclinical model for mastocytosis research

Abstract

Abstract The HMC-1.2 human mast cell (huMC) line is often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. HMC-1.2 cells express constitutively active KIT, an essential growth factor receptor for huMC survival and function, due to the presence of two oncogenic mutations (D816V and V560G). However, systemic mastocytosis is commonly associated with a single D816V-KIT mutation. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9 engineering to reverse the V560G mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single monoallelic D816V-KIT variant. Transcriptome analyses predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable. Phosphorylation levels of ERK1/2, AKT and STAT5 were also similar between HMC-1.2 and HMC-1.3 cells. However, HMC-1.3 cells were more susceptible to KIT, JAK2, and BCL2 inhibitors than HMC-1.2. Our study reveals that the additional V560G-KIT oncogenic variant modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases neoplasia, suggesting that huMCs with a single D816V-KIT (HMC-1.3) variant may represent an improved preclinical model for mastocytosis. This work was supported by the Division of Intramural Research of the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID).