Mechanism of Hyperactive PLCγ1 Signaling in T-Cell Leukemia/Lymphoma

The phospholipase PLCγ1 is essential for T cell activation. It is frequently mutated in Adult T‐cell leukemia/lymphoma (ATLL), Peripheral T-cell lymphomas (PTCL), and Cutaneous T-cell lymphoma (CTCL). However, the molecular mechanisms of PLCγ1 mutation in pathogenesis of these T cell malignancies remains unclear. Liquid-liquid phase separation (LLPS) is an emerging principle in organizing cellular signaling. The dysregulation of LLPS derived by aberrant protein aggregation is progressively implicated as pathological mechanism in tumorigenesis. We previously reported that PLCγ1 structurally promotes LLPS of linker for activation of T cells (LAT) to form condensates in physiological TCR signaling. These lead us to hypothesize that leukemia associated PLCγ1 mutations enhanced TCR signaling and T cell growth by promoting abnormal LLPS.

Three frequent and persistent PLCγ1 mutations (R48W, S345F, and D1165H) were chosen in this study. The mutated PLCγ1 recombinant proteins significantly boosted membrane associated LAT condensation compared with wild type (WT) at physiological concentration in a membrane-based biochemical reconstitution system. Increased condensation of LAT was confirmed in live T cells through total internal reflection fluorescence (TIRF) microcopy. Consequently, the downstream signaling including calcium influx and ERK phosphorylation were enhanced in T cell cancer lines harboring PLCγ1 mutations. PLCγ1 mutations also significantly induced the secretion of cytokines and chemokines such as IL-2 and CXCL10. Ectopically expression of PLCγ1 mutants in human primary T cells promotes T cell proliferation, CD69 expression, and effector memory T cell development. Together, these results suggested that leukemia-associated PLCγ1 mutations drive abnormal LLPS to boost TCR signaling and promote T cell proliferation.

We also determined if PLCγ1 mutations render any drug resistance by testing a few T cell lymphoma drugs in clinical use. The T cell lymphoma cell line Hut78 expressing PLCγ1 mutations showed resistance to histone deacetylase (HDAC) inhibitors. Moreover, PLCγ1 mutations reduced apoptosis under HDAC inhibition, with a company of enhanced Bcl-2 expression. The Human protein kinase phosphorylation array assay was performed to reveal the mechanism underlying drug resistance. We found that inhibiting MAPK could reverse the HDAC inhibitor resistance at non-cytotoxicity concentrations. These results demonstrated that PLCγ1-MAPK signaling axis confers the HDAC inhibitor resistance.

In summary, leukemia-associated PLCγ1 mutations facilitate abnormal LLPS formation to enhance the T cell receptor signaling. Gain-of-function of PLCγ1 mutations cause drug resistance to HDAC inhibitors, which can be rehabilitated by inhibiting the MAPK pathway. Our work reveals molecular mechanisms underlying T cell lymphomagenesis and provides solutions to patients suffering from drug resistance.