A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells

Development of adult T-cell leukemia/lymphoma (ATL) involves human T-cell leukemia virus type 1 (HTLV-1) infection and accumulation of somatic mutations. The most frequently mutated gene in ATL (36 % of cases) is phospholipase C gamma1 (PLCG1). PLCG1 is also frequently mutated in other T-cell lymphomas. However, the functional consequences of the PLCG1 mutations in cancer cells have not been characterized. We compared the activity of the wild-type PLCγ1 with that of a mutant carrying a hot-spot mutation of PLCγ1 (S345F) observed in ATL, both in cells and in cell-free assays. To analyse the impact of the mutation on cellular properties, we quantified cellular proliferation, aggregation, chemotaxis and apoptosis by live cell-imaging in an S345F+ ATL-derived cell line (KK1) and a KK1 cell line in which we reverted the mutation to the wild-type sequence using CRISPR/Cas9 and homology-directed repair. The PLCγ1 S345F mutation results in an increase of basal PLC activity in vitro and in different cell types. This higher basal activity is further enhanced by upstream signalling. Reversion of the S345F mutation in the KK1 cell line resulted in reduction of the PLC activity, lower rates of proliferation and aggregation, and a marked reduction in chemotaxis towards CCL22. The PLCγ1-pathway inhibitors ibrutinib and ritonavir reduced both the PLC activity and the tested functions of KK1 cells. Consistent with observations from clinical studies, our data provide direct evidence that activated variants of the PLCγ1 enzyme contribute to the properties of the malignant T-cell clone in ATL. MRC (UK) Project Grant (P028160).