S123 SETD2 LOSS OF FUNCTION IS A RECURRENT EVENT IN ADVANCED‐PHASE CHRONIC MYELOID LEUKEMIA INDUCED BY POST‐TRANSLATIONAL MECHANISMS THAT CAN BE THERAPEUTICALLY TARGETED
Authors: M. Mancini, S. De Santis, C. Monaldi, L. Bavaro, M. Martelli, F. Castagnetti, G. Gugliotta, G. Rosti, M.C. Fontana, E. Dan, B. Sinigallia, A. Iurlo, N. Orofino, E. Abbruzzese, M. Salvucci, P. Pregno, A. Gozzini, M. Crugnola, F. Albano, M. Bonifacio, E. Calistri, M. Tiribelli, G. Binotto, A. Imovilli, E. Trabacchi, S. Galimberti, C. Baratè, E. Tenti, M. Baccarani, G. Martinelli, M. Cavo, S. Soverini
Journal: HemaSphere (2019)

Background: Inactivating mutations in the SETD2 tumor suppressor occur in solid tumors and acute leukemias. SETD2 trimethylates histone H3 Lysine 36 (H3K36Me3) and plays a key role in transcription and splicing, homologous recombination (HR) and mismatch repair. In Systemic Mastocytosis, we have demonstrated that SETD2 loss of function may occur at the post‐translational level. Aims: In this study, we aimed to investigate post‐translational mechanisms involved in SETD2 loss of function and to asses if SETD2/H3K36me3 loss may contribute to genetic instability in CML. Methods: Western Blotting (WB) was used to assess proteins expression in a cohort of 90 advanced‐phase CML patients (pts). SETD2 mutations and transcript levels were investigated by NGS and real time PCR. Co‐immunoprecipitation (co‐IP) was used to study protein interactions. Immunofluorescence (IF) with an anti‐phospho‐histone2A.X (γH2AX) and an anti‐Rad51 antibody was used to evaluate DNA damage and HR repair. Apoptosis and clonogenic assays were performed to test sensitivity to proteasome, MDM2 and Aurora kinases inhibitors. siRNA‐mediated silencing of MDM2 and Aurora kinase A (AKA) was performed to assess MDM2 and AKA involvement in SETD2 reduced stability and proteasomal degradation. Results: Reduced or null SETD2 and H3K36Me3 were detected in 88% of pts as compared to a pool of healthy donors and to CP pts at diagnosis who achieved optimal responses to TKIs, but neither mutations/deletions nor mRNA down‐regulation were found. Proteasome inhibition in primary cells from pts with undetectable SETD2 restored H3K36Me3 and led to accumulation of hyper‐ubiquitinated SETD2. Moreover, it induced apoptosis and reduced clonogenic growth. In K562 cells (SETD2/H3K36Me3low), co‐IP performed before and after proteasome inhibition showed that SETD2 interacts with MDM2 and, as a result, it is hyper‐ubiquitinated. MDM2 inhibition by SP‐141 resulted in cytostatic effects and rescued SETD2 expression and activity. The latter was also achieved by siRNA‐mediated silencing of MDM2, suggesting that MDM2 is implicated in SETD2 reduced stability. Co‐IP also showed that SETD2 interacts with Aurora Kinase A (AKA) a Ser‐Thr kinase frequently overexpressed in CML. We found that AKA phosphorylates SETD2, and both pharmacological inhibition by Danusertib and siRNA‐mediated silencing rescued SETD2 expression and activity. Next, to investigate whether SETD2/H3K36Me3 loss may contribute to genetic instability, LAMA 84 (SETD2/H3K36Me3high) and K562 (SETD2/H3K36Me3low) cells were studied by WB and immunofluorescence (IF) to assess γH2AX and Rad51 foci in steady state conditions and after sub‐lethal DNA damage by UV exposure. The same studies were performed after SETD2 silencing for 14 weeks. Cells with low or silenced SETD2 had significantly higher levels of γH2AX and were unable to induce homologous recombination (HR) repair after DNA damage. Clonogenic assays performed in LAMA 84 cells before and after SETD2 silencing, in K562 (SETD2/H3K36Me3low) and in imatinib‐resistant (IM‐R) K562 cells which have lost SETD2 expression and activity, suggested that reduction of clonogenic growth after proteasomal or MDM2 inhibition is partly dependent on SETD2 expression and functional status. Summary/Conclusion: Phosphorylation by AKA and ubiquitination by MDM2 contribute to SETD2 non‐genomic loss of function in advanced‐phase CML. Loss of SETD2/H3K36Me3 result in increased DNA damage and impaired HR repair. Restoring physiological H3K36Me3 levels may help improve the outcome of this critical subset of pts. Supported by AIRC (project 16996) and AIL.