PS1437 SETD2 NON‐GENOMIC LOSS OF FUNCTION IN ADVANCED SYSTEMIC MASTOCYTOSIS (SM): PATHOGENETIC AND THERAPEUTIC IMPLICATIONS
Authors: Mancini M. , Monaldi C. , De Santis S. , Papayannidis C. , Rondoni M. , Bavaro L. , Martelli M. , Ficarra E. , Paciello G. , Fontana M.C. , Zanotti R. , Pagano L. , Albano F. , Ciceri F. , Elena C. , Tosi P. , Delledonne M. , Valent P. , Martinelli G. , Cavo M. , Soverini S. 
Published: June 2019

Background:

We have recently found that advanced SM patients (pts) and the HMC‐1.1 and −1.2 MCL cell lines display histone H3 Lys36 trimethylation (H3K36Me3) deficiency as a result of non‐genomic loss of function of the SETD2 methyltransferase. SETD2 plays a key role in the control of transcription and splicing fidelity and homologous recombination (HR). Inhibition of proteasome‐mediated degradation by bortezomib restored SETD2 protein expression and H3K36Me3.

Aims:

In this study, we aimed to identify the different players involved in SETD2 loss of function in ASM and MCL and to verify if they may be considered as promising therapeutic targets.

Methods:

Western blotting (WB) and immunofluorescence (IF) were used to assess proteins expression. Co‐immunoprecipitation assays were performed to investigate proteins interactions. Apoptosis and clonogenic assays were performed to test drugs efficacy.

Results:

Compared to cells from healthy donors, SETD2‐ and H3K36Me3‐deficient cell lines and pts had significantly higher levels of γH2AX and lower levels of Rad51. RNA‐seq in SETD2‐deficient pts showed evidence of transcription and splicing defects like transcription‐induced chimeras and intron retention not observed in healthy donors. Moreover we found that SETD2 co‐immunoprecipitates (co‐IP) with p53 in HMC‐1.1 and −1.2 cells. Rescuing the interaction between SETD2 and p53 with bortezomib stabilized p53 and upregulated the expression levels of p53 targets including p21, p27, Bax and Gadd45a. This restored inhibition of cell proliferation, block of the transition towards cell cycle checkpoints and consequent activation of apoptosis. The ubiquitin E3 ligase MDM2 was also found to complex with SETD2 after proteasomal inhibition. Treatment with the MDM2 inhibitor SP‐141 rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP‐141 treatment of HMC‐1 cells at micromolar doses induced cytostatic but not cytotoxic effects. Clonogenic assays supported the cytostatic effects of SP141 in HMC‐1.1 and −1.2 cells. siRNA‐mediated knock‐down of MDM2 also rescued SETD2 expression and activity, supporting the hypothesis that SETD2 hyper‐ubiquitination by MDM2 plays a role in SETD2 reduced stability and proteasomal degradation. Co‐IP also showed that SETD2 interacts with Aurora Kinase A (AKA), that is overexpressed in advanced SM. We found that AKA phosphorylates SETD2 and this phosphorylation may be involved in SETD2 loss of function. In fact, both inhibition by Danusertib and siRNA‐mediated silencing rescued SETD2 expression and activity. Finally, we performed clonogenic assays to evaluate the therapeutic potential of bortezomib, carfilzomib and ixazomib in neoplastic mast cells from 3 patients with advanced SM and we observed in all cases that both first and second generation inhibitors induced a significant reduction of clonogenic activity at nanomolar doses.

Summary/Conclusion:

AKA triggered MDM2‐mediated ubiquitination contributes to SETD2 non‐genomic loss of function in ASM and MCL. Loss of SETD2 and H3K36Me3 is associated with increased DNA damage and transcription and splicing defects in ASM and MCL pts and is likely to afford an alternative mechanism for the inactivation of the p53‐mediated checkpoint without the need for TP53 gene mutations. Inhibiting AKA or MDM2 activity or proteasome‐mediated degradation are promising therapeutic strategies. Supported by AIRC (project 16996) and AIL.