MPN-204: Midostaurin Synergizes with Nilotinib and Dasatinib Restoring SETD2 Expression and Activity in Advanced Systemic Mastocytosis

Context: Histone H3 lysine 36 trimethylation (H3K36me3) by SETD2 is implicated in transcriptional regulation, splicing fidelity, and DNA damage response signaling. SETD2 non-genomic loss of function, due to proteasome-mediated protein degradation, is a common event in advanced systemic mastocytosis (advSM). Objective: To investigate the mechanisms underlying SETD2 loss of function in advSM to highlight possible druggable targets. Patients or other participants: DNA and protein samples from a retrospective cohort of 88 patients with systemic mastocytosis, classified according to WHO criteria were used for sequencing, western blotting (WB), and immunoprecipitation studies. Neoplastic mast cells (MCs) from 10 pts with advSM were used for ex-vivo drug testing. Results: Aurora kinase A (AKA) was overexpressed and hyper-activated in advSM and this inversely correlated with SETD2 protein expression. siRNA-mediated silencing of AKA and pharmacological inhibition by danusertib rescued SETD2 expression and activity, suggesting that AKA is implicated in SETD2 degradation. The new gold standard of therapy in advSM is midostaurin that targets not only mutant KIT but also other kinases, including AKA. To investigate whether midostaurin treatment may result in efficient AKA inhibition and consequent SETD2/H3K36me3 rescue, the HMC-1 cell line was treated with 5 μM midostaurin for 24 h and phospho-AKA (T288), SETD2, and H3K36me3 expression were evaluated by WB. Midostaurin reduced AKA phosphorylation by 60%, partially restoring SETD2 expression and activity, but did not induce cytotoxic effects. We next tested the efficacy of combined treatment with midostaurin and nilotinib or dasatinib in inducing cytotoxic rather than cytostatic effects. Cytofluorimetric analysis of apoptosis and clonogenic assays in HMC-1 cells and in neoplastic MCs showed an important advantage in using the midostaurin + dasatinib/nilotinib combination compared to each single agent alone, as underlined by the significant reduction of drug doses necessary to obtain cytotoxic effects and cell growth arrest. Of note, we observed that midostaurin + nilotinib completely de-phosphorylated AKA and restored SETD2 expression and activity. Conclusions: AKA overexpression contributes to SETD2 non-genomic loss of function in advSM. KIT and AKA targeting by midostaurin in combination with second-generation tyrosine kinase inhibitors is a promising therapeutic alternative in patients with SETD2/H3K36me3 deficiency. Supported by AIRC project 23001.