The Experts below are selected from a list of 2976 Experts worldwide ranked by ideXlab platform
Jianru Xiao - One of the best experts on this subject based on the ideXlab platform.
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histone methyltransferase SETD2 regulates osteosarcoma cell growth and chemosensitivity by suppressing wnt β catenin signaling
Biochemical and Biophysical Research Communications, 2018Co-Authors: Chaoying Jiang, Jianru XiaoAbstract:SETD2 is a histone methyltransferase that catalyzes the trimethylation of lysine 36 on histone 3. SETD2 is frequently found to be mutated or deleted in a variety of human tumors, whereas the role of SETD2 in oncogenesis of osteosarcoma has never been defined. Here in our study, we uncovered that SETD2 regulates tumor growth and chemosensitivity of osteosarcoma. Overexpression of SETD2 significantly inhibited osteosarcoma cell growth in vitro and in vivo. Moreover, SETD2 significantly enhanced cisplatin-induced apoptosis in osteosarcoma cells and inhibited cancer stem cell properties in OS cells. SETD2 regulates Wnt/β-catenin signaling and its downstream gene c-myc, CD133 and cyclin D1. We further revealed that SETD2 upregulates H3K36me3 modification in GSK3B loci and promotes its transcription, which lead to β-catenin degradation. Together, our study delineates SETD2 function in osteosarcoma as an important regulator of Wnt/β-catenin signaling, and suggests SETD2 as a novel target in diagnosis and combined chemotherapy of osteosarcoma.
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Histone methyltransferase SETD2 regulates osteosarcoma cell growth and chemosensitivity by suppressing Wnt/β-catenin signaling.
Biochemical and biophysical research communications, 2018Co-Authors: Chaoying Jiang, Jianru XiaoAbstract:SETD2 is a histone methyltransferase that catalyzes the trimethylation of lysine 36 on histone 3. SETD2 is frequently found to be mutated or deleted in a variety of human tumors, whereas the role of SETD2 in oncogenesis of osteosarcoma has never been defined. Here in our study, we uncovered that SETD2 regulates tumor growth and chemosensitivity of osteosarcoma. Overexpression of SETD2 significantly inhibited osteosarcoma cell growth in vitro and in vivo. Moreover, SETD2 significantly enhanced cisplatin-induced apoptosis in osteosarcoma cells and inhibited cancer stem cell properties in OS cells. SETD2 regulates Wnt/β-catenin signaling and its downstream gene c-myc, CD133 and cyclin D1. We further revealed that SETD2 upregulates H3K36me3 modification in GSK3B loci and promotes its transcription, which lead to β-catenin degradation. Together, our study delineates SETD2 function in osteosarcoma as an important regulator of Wnt/β-catenin signaling, and suggests SETD2 as a novel target in diagnosis and combined chemotherapy of osteosarcoma.
Chaoying Jiang - One of the best experts on this subject based on the ideXlab platform.
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histone methyltransferase SETD2 regulates osteosarcoma cell growth and chemosensitivity by suppressing wnt β catenin signaling
Biochemical and Biophysical Research Communications, 2018Co-Authors: Chaoying Jiang, Jianru XiaoAbstract:SETD2 is a histone methyltransferase that catalyzes the trimethylation of lysine 36 on histone 3. SETD2 is frequently found to be mutated or deleted in a variety of human tumors, whereas the role of SETD2 in oncogenesis of osteosarcoma has never been defined. Here in our study, we uncovered that SETD2 regulates tumor growth and chemosensitivity of osteosarcoma. Overexpression of SETD2 significantly inhibited osteosarcoma cell growth in vitro and in vivo. Moreover, SETD2 significantly enhanced cisplatin-induced apoptosis in osteosarcoma cells and inhibited cancer stem cell properties in OS cells. SETD2 regulates Wnt/β-catenin signaling and its downstream gene c-myc, CD133 and cyclin D1. We further revealed that SETD2 upregulates H3K36me3 modification in GSK3B loci and promotes its transcription, which lead to β-catenin degradation. Together, our study delineates SETD2 function in osteosarcoma as an important regulator of Wnt/β-catenin signaling, and suggests SETD2 as a novel target in diagnosis and combined chemotherapy of osteosarcoma.
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Histone methyltransferase SETD2 regulates osteosarcoma cell growth and chemosensitivity by suppressing Wnt/β-catenin signaling.
Biochemical and biophysical research communications, 2018Co-Authors: Chaoying Jiang, Jianru XiaoAbstract:SETD2 is a histone methyltransferase that catalyzes the trimethylation of lysine 36 on histone 3. SETD2 is frequently found to be mutated or deleted in a variety of human tumors, whereas the role of SETD2 in oncogenesis of osteosarcoma has never been defined. Here in our study, we uncovered that SETD2 regulates tumor growth and chemosensitivity of osteosarcoma. Overexpression of SETD2 significantly inhibited osteosarcoma cell growth in vitro and in vivo. Moreover, SETD2 significantly enhanced cisplatin-induced apoptosis in osteosarcoma cells and inhibited cancer stem cell properties in OS cells. SETD2 regulates Wnt/β-catenin signaling and its downstream gene c-myc, CD133 and cyclin D1. We further revealed that SETD2 upregulates H3K36me3 modification in GSK3B loci and promotes its transcription, which lead to β-catenin degradation. Together, our study delineates SETD2 function in osteosarcoma as an important regulator of Wnt/β-catenin signaling, and suggests SETD2 as a novel target in diagnosis and combined chemotherapy of osteosarcoma.
Jerry L. Workman - One of the best experts on this subject based on the ideXlab platform.
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The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain.
Nature communications, 2021Co-Authors: Saikat Bhattacharya, Ning Zhang, Michaella J. Levy, Laurence Florens, Michael P. Washburn, Jerry L. WorkmanAbstract:Heterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery. The methylation of Histone 3 at Lysine 36 (H3K36) has been implicated in the regulation of transcription and coupled processes such as mRNA splicing. Here the authors show that the histone methyltransferase SETD2 interacts with hnRNP L to mediate the crosstalk between the transcription and splicing machineries.
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Regulation of SETD2 stability is important for the fidelity of H3K36me3 deposition
Epigenetics & chromatin, 2020Co-Authors: Saikat Bhattacharya, Jerry L. WorkmanAbstract:Background The histone H3K36me3 mark regulates transcription elongation, pre-mRNA splicing, DNA methylation, and DNA damage repair. However, knowledge of the regulation of the enzyme SETD2, which deposits this functionally important mark, is very limited. Results Here, we show that the poorly characterized N-terminal region of SETD2 plays a determining role in regulating the stability of SETD2. This stretch of 1-1403 amino acids contributes to the robust degradation of SETD2 by the proteasome. Besides, the SETD2 protein is aggregate prone and forms insoluble bodies in nuclei especially upon proteasome inhibition. Removal of the N-terminal segment results in the stabilization of SETD2 and leads to a marked increase in global H3K36me3 which, uncharacteristically, happens in a Pol II-independent manner. Conclusion The functionally uncharacterized N-terminal segment of SETD2 regulates its half-life to maintain the requisite cellular amount of the protein. The absence of SETD2 proteolysis results in a Pol II-independent H3K36me3 deposition and protein aggregation.
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The disordered regions of SETD2 govern H3K36me3 deposition by regulating its proteasome-mediated decay
2020Co-Authors: Saikat Bhattacharya, Ning Zhang, Jerry L. WorkmanAbstract:ABSTRACT SETD2 is the sole methyltransferase that tri-methylates histone H3 at lysine 36 in mammals. It has an extended N-terminal region which is absent in its yeast homolog Set2. The function of this poorly characterized region in regulating SETD2 stability has been reported. However, how this region regulates SETD2 half-life and the consequences of the cellular accumulation of SETD2 is unclear. Here we show that the SETD2 N-terminal region contains disordered regions and is targeted for degradation by the proteasome. The marked increase in global H3K36me3 that occurs on the removal of the N-terminal segment results in a non-canonical distribution including reduced enrichment over gene bodies and exons. An increased SETD2 abundance leads to widespread changes in transcription and alternative splicing. Thus, the regulation of SETD2 levels through intrinsically disordered region-facilitated proteolysis is important to maintain the fidelity of transcription and splicing related processes.
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The histone methyltransferase SETD2 couples transcription and splicing by engaging pre-mRNA processing factors through its SHI domain
2020Co-Authors: Saikat Bhattacharya, Ning Zhang, Michaella J. Levy, Laurence Florens, Michael P. Washburn, Jerry L. WorkmanAbstract:Heterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2- hnRNP L Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2 by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.
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regulation of SETD2 stability is important for the fidelity of h3k36me3 deposition
bioRxiv, 2020Co-Authors: Saikat Bhattacharya, Jerry L. WorkmanAbstract:ABSTRACT The histone H3K36me3 mark regulates transcription elongation, pre-mRNA splicing, DNA methylation, and DNA damage repair. However, knowledge of the regulation of the enzyme SETD2, which deposits this functionally important mark, is very limited. Here we show that the poorly characterized N-terminal region of SETD2 plays a determining role in regulating the stability of SETD2. This stretch of 1-1403 amino acids contributes to the robust degradation of SETD2 by the proteasome. Besides, the SETD2 protein is aggregate-prone and forms insoluble bodies in nuclei especially upon proteasome inhibition. Removal of the N-terminal segment results in the stabilization of SETD2 and leads to a marked increase in global H3K36me3 which, uncharacteristically, happens in a Pol II-independent manner. Thus, the regulation of SETD2 levels through proteasomal mediated decay is important to maintain the fidelity of H3K36me3 deposition.
Sara De Santis - One of the best experts on this subject based on the ideXlab platform.
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cml 206 resetting SETD2 h3k36me3 deficiency as a new therapeutic strategy in blast crisis chronic myeloid leukemia patients
Clinical Lymphoma Myeloma & Leukemia, 2020Co-Authors: Sara De Santis, Manuela Mancini, Cecilia Monaldi, Margherita Martelli, Fausto Castagnetti, Gabriele Gugliotta, Gianantonio Rosti, Elisa Dan, Barbara Sinigaglia, Alessandra IurloAbstract:Context SETD2 is a tumor suppressor encoding a histone methyltransferase that trimethylates histone H3 at lysine 36 (H3K36Me3) and is involved in various cellular processes, including transcriptional regulation and repair of DNA damage. SETD2 loss of function due to mutations or deletions has been observed in some solid tumors and acute leukemias. Objective We aimed to investigate the frequency and underlying mechanisms of SETD2 loss of function in advanced phase of Chronic Myeloid Leukemia (CML). Patients A large cohort of 90 CML patients with advanced phase CML was screened for SETD2 and H3K36Me3 status. Results Reduced or null SETD2 and H3K36Me3 were detected by western blotting in 88% of advanced-phase CML patients, as compared to a pool of healthy donors and to CP patients at diagnosis. No genetic abnormalities or transcript downregulation were observed and enhanced SETD2 proteasome-mediated degradation was rather found to occur. After proteasome inhibition, accumulation of hyper-ubiquitinated SETD2 bound to MDM2 was observed. Both MDM2 pharmacological inhibition by SP-141 and siRNA-mediated silencing suggested that MDM2 is implicated in SETD2 loss. Aurora Kinase A, that is overexpressed in CML, was also found to co-immunoprecipitate with SETD2. Both pharmacological inhibition and knock-down of Aurora A rescued SETD2 and H3K36Me3. SETD2/H3K36Me3 loss was found to impair the proficiency of homologous recombination (HR) repair as demonstrated by comparison of phosphorylated histone 2A.X and Rad51 foci in steady-state conditions and after sub-lethal DNA damage by UV exposure in SETD2-deficient CML cells as well as in SETD2-proficient CML cells subjected to SETD2 silencing. Reduction of clonogenic growth after treatment with proteasome, MDM2, and Aurora kinase A inhibitors at nanomolar or subnanomolar concentrations was observed both in cell lines and in primary cells from SETD2-deficient advanced-phase CML patients. Conclusions In advanced-phase CML, phosphorylation by Aurora kinase A and hyper-ubiquitination by MDM2 are responsible for SETD2 non-genomic loss of function. Loss of SETD2/H3K36Me3 was associated with increased DNA damage and impaired HR repair. Restoring physiological H3K36Me3 levels should further be explored as a therapeutic strategy to help improve the outcome of this critical subset of patients.
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CML-206: ReSETting SETD2/H3K36Me3 Deficiency as a New Therapeutic Strategy in Blast Crisis Chronic Myeloid Leukemia Patients
Clinical Lymphoma Myeloma and Leukemia, 2020Co-Authors: Sara De Santis, Manuela Mancini, Cecilia Monaldi, Margherita Martelli, Fausto Castagnetti, Gabriele Gugliotta, Gianantonio Rosti, Elisa Dan, Barbara Sinigaglia, Alessandra IurloAbstract:Context SETD2 is a tumor suppressor encoding a histone methyltransferase that trimethylates histone H3 at lysine 36 (H3K36Me3) and is involved in various cellular processes, including transcriptional regulation and repair of DNA damage. SETD2 loss of function due to mutations or deletions has been observed in some solid tumors and acute leukemias. Objective We aimed to investigate the frequency and underlying mechanisms of SETD2 loss of function in advanced phase of Chronic Myeloid Leukemia (CML). Patients A large cohort of 90 CML patients with advanced phase CML was screened for SETD2 and H3K36Me3 status. Results Reduced or null SETD2 and H3K36Me3 were detected by western blotting in 88% of advanced-phase CML patients, as compared to a pool of healthy donors and to CP patients at diagnosis. No genetic abnormalities or transcript downregulation were observed and enhanced SETD2 proteasome-mediated degradation was rather found to occur. After proteasome inhibition, accumulation of hyper-ubiquitinated SETD2 bound to MDM2 was observed. Both MDM2 pharmacological inhibition by SP-141 and siRNA-mediated silencing suggested that MDM2 is implicated in SETD2 loss. Aurora Kinase A, that is overexpressed in CML, was also found to co-immunoprecipitate with SETD2. Both pharmacological inhibition and knock-down of Aurora A rescued SETD2 and H3K36Me3. SETD2/H3K36Me3 loss was found to impair the proficiency of homologous recombination (HR) repair as demonstrated by comparison of phosphorylated histone 2A.X and Rad51 foci in steady-state conditions and after sub-lethal DNA damage by UV exposure in SETD2-deficient CML cells as well as in SETD2-proficient CML cells subjected to SETD2 silencing. Reduction of clonogenic growth after treatment with proteasome, MDM2, and Aurora kinase A inhibitors at nanomolar or subnanomolar concentrations was observed both in cell lines and in primary cells from SETD2-deficient advanced-phase CML patients. Conclusions In advanced-phase CML, phosphorylation by Aurora kinase A and hyper-ubiquitination by MDM2 are responsible for SETD2 non-genomic loss of function. Loss of SETD2/H3K36Me3 was associated with increased DNA damage and impaired HR repair. Restoring physiological H3K36Me3 levels should further be explored as a therapeutic strategy to help improve the outcome of this critical subset of patients.
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MPN-204: Midostaurin Synergizes with Nilotinib and Dasatinib Restoring SETD2 Expression and Activity in Advanced Systemic Mastocytosis
Clinical Lymphoma Myeloma and Leukemia, 2020Co-Authors: Cecilia Monaldi, Sara De Santis, Manuela Mancini, Margherita Martelli, Cristina Papayannidis, Michela Rondoni, Chiara Sartor, Roberta Zanotti, Massimiliano Bonifacio, Federica GrifoniAbstract: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.
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Aurora Kinase a/MDM2-Mediated SETD2 Loss of Function in Chronic Myeloid Leukemia Patients in Blast Crisis Induces Genetic Instability and Can be Therapeutically Targeted
Blood, 2018Co-Authors: Manuela Mancini, Sara De Santis, Cecilia Monaldi, Margherita Martelli, Fausto Castagnetti, Gabriele Gugliotta, Gianantonio Rosti, Luana Bavaro, Maria Chiara Fontana, Elisa DanAbstract:Abstract The SETD2 protein is a histone methyltransferase that specifically catalyzes the trimethylation of Lysine 36 on histone H3 (H3K36me3). SETD2/H3K36me3 are implicated in transcript elongation and splicing, DNA repair, chromosome segregation. SETD2 gene deletions and/or mutations (mostly frameshift or nonsense) have been reported in solid tumors (clear cell renal cell carcinoma, bladder cancer, lung cancer, melanoma, endometrial cancer) and in acute leukemias. Using a Western Blotting (WB) approach to screen for SETD2 protein expression and for H3K36me3 levels in a relatively large cohort of 80 advanced-phase chronic myeloid leukemia (CML) patients (pts), we could detect reduced or null SETD2 and H3K36me3 in 86% of pts as compared to a pool of healthy donors and to chronic phase (CP) pts at diagnosis who achieved optimal responses to TKI, but neither mutations/deletions nor transcriptional down-regulation were the underlying causes. Inhibition of proteasome-mediated degradation in primary cells from pts with undetectable SETD2 restored H3K36me3 and led to accumulation of hyper-ubiquitinated SETD2, suggesting that a functional protein is produced but rapidly degraded. Moreover, proteasome inhibition was found to induce apoptosis and to reduce clonogenic growth. In K562 cells (SETD2/H3K36me3low), co-immunoprecipitation (co-IP) performed before and after proteasome inhibition showed accumulation of the hyper-ubiquitinated form of SETD2 bound to MDM2. MDM2 inhibition by SP-141 resulted in cytostatic effects and restored SETD2 expression and activity. Superimposable results were 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 a Ser-Thr kinase frequently overexpressed in CML. We found that Aurora Kinase A 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 phosphorylated histone 2A.X (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 3 months. 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 strictly dependent on SETD2 expression and functional status (Figure 1A). First and second generation proteasome inhibitors (bortezomib, carfilzomib and ixazomib) inhibited the clonogenic potential of the mononuclear cell fraction from both CP (n=2) and blast crisis (BC) (n=4) CML pts at subnanomolar concentrations, with the extent of anti-tumor activity clearly anti-correlated with SETD2 expression and H3K36me3 levels: pts with lower SETD2 expression showed lower LD50 when compared with pts with higher SETD2 expression and H3K36me3 levels (Figure 1B). Similarly, clonogenic assays performed by administrating increasing doses of SP-141 (from 0.25 to 1.25 µM) suggested that MDM2 specific inhibition had more significant effects in BC-CML pts showing low SETD2 levels and activity as compared to BC-CML pts showing intermediate SETD2 levels and activity and to CP CML pts. In conclusion, phosphorylation by Aurora Kinase A and ubiquitination by MDM2 contribute to SETD2 non-genomic loss of function in advanced-phase CML. Loss of SETD2/H3K36me3 is associated with increased DNA damage and impaired HR repair. Restoring physiological H3K36me3 levels may help improve the outcome of this critical subset of pts. Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). Figure 1. Figure 1. Disclosures Castagnetti: Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Novartis: Honoraria; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Incyte: Honoraria. Abruzzese:Pfizer: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy. Bonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Martinelli:Ariad/incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Roche: Consultancy. Cavo:Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Bristol Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.
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MDM2 and Aurora Kinase a Contribute to SETD2 Loss of Function in Advanced Systemic Mastocytosis: Implications for Pathogenesis and Treatment
Blood, 2018Co-Authors: Manuela Mancini, Sara De Santis, Cecilia Monaldi, Margherita Martelli, Luana Bavaro, Cristina Papayannidis, Michela Rondoni, Abbenante Maria Chiara, Antonio Curti, Elisa FicarraAbstract:Abstract The SETD2 gene encodes the only methyltransferase responsible for histone H3 lysine 36 trimethylation (H3K36Me3) in humans. H3K36me3 play a key role in preserving the fidelity of transcription elongation and splicing. In addition, SETD2/H3K36me3 have more recently been implicated in the maintenance of genomic integrity by regulating homologous recombination (HR) repair, Mismatch Repair (MMR) mitotic spindle assembly and chromosome segregation. SETD2 deletions and/or inactivating mutations occur in many solid tumors and have recently been found also in acute leukemias. We have reported that the HMC-1.1 and -1.2 mast cell leukemia (MCL) cell lines and many advanced systemic mastocytosis (SM) patients (pts) display H3K36Me3 deficiency as a result of non-genomic loss of function of SETD2. Proteasome inhibition restored SETD2 protein expression and H3K36me3, suggesting that a functional protein is produced but rapidly degraded. In an attempt to uncover the mechanisms underlying this phenomenon, we used an in silico approach to identify candidate SETD2-interacting proteins, followed by experimental confirmation by co-immunoprecipitation (co-IP). We found that, after proteasomal inhibition, SETD2 co-immunoprecipitates with the ubiquitin E3 ligase MDM2. 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 as shown by cell growth curves. 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, further 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, as it was suggested in silico. We found that Aurora A is overexpressed in advanced SM and may target SETD2 for phosphorylation. Both pharmacological inhibition by Danusertib and siRNA-mediated silencing of Aurora A rescued SETD2 expression and activity, raising the hypothesis that phosphorylation by Aurora A might be the trigger for MDM-2 mediated degradation of SETD2. To evaluate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting (WB) and immunofluorescence (IF) to assess phosphorylated histone 2A.X (γH2AX) and Rad51 foci. Compared to cells from healthy controls, 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, intron retention and non-canonical splicing patterns not observed in healthy donors. Next, the ROSAD816V cell line, which displays SETD2 and H3K36me3 levels superimposable to healthy donors, was studied by WB and IF to assess γH2AX and Rad51 in steady state and after sub-lethal DNA damage by UV exposure. The same experiments were carried out after SETD2 silencing for 2 months. Cells with silenced SETD2 had significantly higher levels of γH2AX and were unable to activate the HR repair. Interestingly, clonogenic assays in ROSAD816V cells before and after SETD2 silencing showed that reduction of clonogenic potential after proteasomal or MDM2 inhibition is indeed SETD2-dependent (Figure 1A). 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 (Figure 1B). Taken together, our results suggest that AKA and MDM2-mediated post-translational modifications contribute to SETD2 non-genomic loss of function in advanced SM. Loss of SETD2 and H3K36me3 is associated with increased DNA damage and transcription and splicing defects in patients. Inhibiting AKA or MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies in patients with low SETD2 expression levels. Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). Figure 1. Figure 1. Disclosures Bonifacio: Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Pagano:Janssen: Speakers Bureau; Merck: Speakers Bureau; Gilead: Speakers Bureau; Basilea: Speakers Bureau; Pfizer: Speakers Bureau. Valent:Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Novartis: Consultancy; Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy.
Cecilia Monaldi - One of the best experts on this subject based on the ideXlab platform.
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cml 206 resetting SETD2 h3k36me3 deficiency as a new therapeutic strategy in blast crisis chronic myeloid leukemia patients
Clinical Lymphoma Myeloma & Leukemia, 2020Co-Authors: Sara De Santis, Manuela Mancini, Cecilia Monaldi, Margherita Martelli, Fausto Castagnetti, Gabriele Gugliotta, Gianantonio Rosti, Elisa Dan, Barbara Sinigaglia, Alessandra IurloAbstract:Context SETD2 is a tumor suppressor encoding a histone methyltransferase that trimethylates histone H3 at lysine 36 (H3K36Me3) and is involved in various cellular processes, including transcriptional regulation and repair of DNA damage. SETD2 loss of function due to mutations or deletions has been observed in some solid tumors and acute leukemias. Objective We aimed to investigate the frequency and underlying mechanisms of SETD2 loss of function in advanced phase of Chronic Myeloid Leukemia (CML). Patients A large cohort of 90 CML patients with advanced phase CML was screened for SETD2 and H3K36Me3 status. Results Reduced or null SETD2 and H3K36Me3 were detected by western blotting in 88% of advanced-phase CML patients, as compared to a pool of healthy donors and to CP patients at diagnosis. No genetic abnormalities or transcript downregulation were observed and enhanced SETD2 proteasome-mediated degradation was rather found to occur. After proteasome inhibition, accumulation of hyper-ubiquitinated SETD2 bound to MDM2 was observed. Both MDM2 pharmacological inhibition by SP-141 and siRNA-mediated silencing suggested that MDM2 is implicated in SETD2 loss. Aurora Kinase A, that is overexpressed in CML, was also found to co-immunoprecipitate with SETD2. Both pharmacological inhibition and knock-down of Aurora A rescued SETD2 and H3K36Me3. SETD2/H3K36Me3 loss was found to impair the proficiency of homologous recombination (HR) repair as demonstrated by comparison of phosphorylated histone 2A.X and Rad51 foci in steady-state conditions and after sub-lethal DNA damage by UV exposure in SETD2-deficient CML cells as well as in SETD2-proficient CML cells subjected to SETD2 silencing. Reduction of clonogenic growth after treatment with proteasome, MDM2, and Aurora kinase A inhibitors at nanomolar or subnanomolar concentrations was observed both in cell lines and in primary cells from SETD2-deficient advanced-phase CML patients. Conclusions In advanced-phase CML, phosphorylation by Aurora kinase A and hyper-ubiquitination by MDM2 are responsible for SETD2 non-genomic loss of function. Loss of SETD2/H3K36Me3 was associated with increased DNA damage and impaired HR repair. Restoring physiological H3K36Me3 levels should further be explored as a therapeutic strategy to help improve the outcome of this critical subset of patients.
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CML-206: ReSETting SETD2/H3K36Me3 Deficiency as a New Therapeutic Strategy in Blast Crisis Chronic Myeloid Leukemia Patients
Clinical Lymphoma Myeloma and Leukemia, 2020Co-Authors: Sara De Santis, Manuela Mancini, Cecilia Monaldi, Margherita Martelli, Fausto Castagnetti, Gabriele Gugliotta, Gianantonio Rosti, Elisa Dan, Barbara Sinigaglia, Alessandra IurloAbstract:Context SETD2 is a tumor suppressor encoding a histone methyltransferase that trimethylates histone H3 at lysine 36 (H3K36Me3) and is involved in various cellular processes, including transcriptional regulation and repair of DNA damage. SETD2 loss of function due to mutations or deletions has been observed in some solid tumors and acute leukemias. Objective We aimed to investigate the frequency and underlying mechanisms of SETD2 loss of function in advanced phase of Chronic Myeloid Leukemia (CML). Patients A large cohort of 90 CML patients with advanced phase CML was screened for SETD2 and H3K36Me3 status. Results Reduced or null SETD2 and H3K36Me3 were detected by western blotting in 88% of advanced-phase CML patients, as compared to a pool of healthy donors and to CP patients at diagnosis. No genetic abnormalities or transcript downregulation were observed and enhanced SETD2 proteasome-mediated degradation was rather found to occur. After proteasome inhibition, accumulation of hyper-ubiquitinated SETD2 bound to MDM2 was observed. Both MDM2 pharmacological inhibition by SP-141 and siRNA-mediated silencing suggested that MDM2 is implicated in SETD2 loss. Aurora Kinase A, that is overexpressed in CML, was also found to co-immunoprecipitate with SETD2. Both pharmacological inhibition and knock-down of Aurora A rescued SETD2 and H3K36Me3. SETD2/H3K36Me3 loss was found to impair the proficiency of homologous recombination (HR) repair as demonstrated by comparison of phosphorylated histone 2A.X and Rad51 foci in steady-state conditions and after sub-lethal DNA damage by UV exposure in SETD2-deficient CML cells as well as in SETD2-proficient CML cells subjected to SETD2 silencing. Reduction of clonogenic growth after treatment with proteasome, MDM2, and Aurora kinase A inhibitors at nanomolar or subnanomolar concentrations was observed both in cell lines and in primary cells from SETD2-deficient advanced-phase CML patients. Conclusions In advanced-phase CML, phosphorylation by Aurora kinase A and hyper-ubiquitination by MDM2 are responsible for SETD2 non-genomic loss of function. Loss of SETD2/H3K36Me3 was associated with increased DNA damage and impaired HR repair. Restoring physiological H3K36Me3 levels should further be explored as a therapeutic strategy to help improve the outcome of this critical subset of patients.
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MPN-204: Midostaurin Synergizes with Nilotinib and Dasatinib Restoring SETD2 Expression and Activity in Advanced Systemic Mastocytosis
Clinical Lymphoma Myeloma and Leukemia, 2020Co-Authors: Cecilia Monaldi, Sara De Santis, Manuela Mancini, Margherita Martelli, Cristina Papayannidis, Michela Rondoni, Chiara Sartor, Roberta Zanotti, Massimiliano Bonifacio, Federica GrifoniAbstract: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.
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Aurora Kinase a/MDM2-Mediated SETD2 Loss of Function in Chronic Myeloid Leukemia Patients in Blast Crisis Induces Genetic Instability and Can be Therapeutically Targeted
Blood, 2018Co-Authors: Manuela Mancini, Sara De Santis, Cecilia Monaldi, Margherita Martelli, Fausto Castagnetti, Gabriele Gugliotta, Gianantonio Rosti, Luana Bavaro, Maria Chiara Fontana, Elisa DanAbstract:Abstract The SETD2 protein is a histone methyltransferase that specifically catalyzes the trimethylation of Lysine 36 on histone H3 (H3K36me3). SETD2/H3K36me3 are implicated in transcript elongation and splicing, DNA repair, chromosome segregation. SETD2 gene deletions and/or mutations (mostly frameshift or nonsense) have been reported in solid tumors (clear cell renal cell carcinoma, bladder cancer, lung cancer, melanoma, endometrial cancer) and in acute leukemias. Using a Western Blotting (WB) approach to screen for SETD2 protein expression and for H3K36me3 levels in a relatively large cohort of 80 advanced-phase chronic myeloid leukemia (CML) patients (pts), we could detect reduced or null SETD2 and H3K36me3 in 86% of pts as compared to a pool of healthy donors and to chronic phase (CP) pts at diagnosis who achieved optimal responses to TKI, but neither mutations/deletions nor transcriptional down-regulation were the underlying causes. Inhibition of proteasome-mediated degradation in primary cells from pts with undetectable SETD2 restored H3K36me3 and led to accumulation of hyper-ubiquitinated SETD2, suggesting that a functional protein is produced but rapidly degraded. Moreover, proteasome inhibition was found to induce apoptosis and to reduce clonogenic growth. In K562 cells (SETD2/H3K36me3low), co-immunoprecipitation (co-IP) performed before and after proteasome inhibition showed accumulation of the hyper-ubiquitinated form of SETD2 bound to MDM2. MDM2 inhibition by SP-141 resulted in cytostatic effects and restored SETD2 expression and activity. Superimposable results were 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 a Ser-Thr kinase frequently overexpressed in CML. We found that Aurora Kinase A 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 phosphorylated histone 2A.X (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 3 months. 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 strictly dependent on SETD2 expression and functional status (Figure 1A). First and second generation proteasome inhibitors (bortezomib, carfilzomib and ixazomib) inhibited the clonogenic potential of the mononuclear cell fraction from both CP (n=2) and blast crisis (BC) (n=4) CML pts at subnanomolar concentrations, with the extent of anti-tumor activity clearly anti-correlated with SETD2 expression and H3K36me3 levels: pts with lower SETD2 expression showed lower LD50 when compared with pts with higher SETD2 expression and H3K36me3 levels (Figure 1B). Similarly, clonogenic assays performed by administrating increasing doses of SP-141 (from 0.25 to 1.25 µM) suggested that MDM2 specific inhibition had more significant effects in BC-CML pts showing low SETD2 levels and activity as compared to BC-CML pts showing intermediate SETD2 levels and activity and to CP CML pts. In conclusion, phosphorylation by Aurora Kinase A and ubiquitination by MDM2 contribute to SETD2 non-genomic loss of function in advanced-phase CML. Loss of SETD2/H3K36me3 is associated with increased DNA damage and impaired HR repair. Restoring physiological H3K36me3 levels may help improve the outcome of this critical subset of pts. Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). Figure 1. Figure 1. Disclosures Castagnetti: Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Novartis: Honoraria; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Incyte: Honoraria. Abruzzese:Pfizer: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy. Bonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Martinelli:Ariad/incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Roche: Consultancy. Cavo:Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Bristol Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.
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MDM2 and Aurora Kinase a Contribute to SETD2 Loss of Function in Advanced Systemic Mastocytosis: Implications for Pathogenesis and Treatment
Blood, 2018Co-Authors: Manuela Mancini, Sara De Santis, Cecilia Monaldi, Margherita Martelli, Luana Bavaro, Cristina Papayannidis, Michela Rondoni, Abbenante Maria Chiara, Antonio Curti, Elisa FicarraAbstract:Abstract The SETD2 gene encodes the only methyltransferase responsible for histone H3 lysine 36 trimethylation (H3K36Me3) in humans. H3K36me3 play a key role in preserving the fidelity of transcription elongation and splicing. In addition, SETD2/H3K36me3 have more recently been implicated in the maintenance of genomic integrity by regulating homologous recombination (HR) repair, Mismatch Repair (MMR) mitotic spindle assembly and chromosome segregation. SETD2 deletions and/or inactivating mutations occur in many solid tumors and have recently been found also in acute leukemias. We have reported that the HMC-1.1 and -1.2 mast cell leukemia (MCL) cell lines and many advanced systemic mastocytosis (SM) patients (pts) display H3K36Me3 deficiency as a result of non-genomic loss of function of SETD2. Proteasome inhibition restored SETD2 protein expression and H3K36me3, suggesting that a functional protein is produced but rapidly degraded. In an attempt to uncover the mechanisms underlying this phenomenon, we used an in silico approach to identify candidate SETD2-interacting proteins, followed by experimental confirmation by co-immunoprecipitation (co-IP). We found that, after proteasomal inhibition, SETD2 co-immunoprecipitates with the ubiquitin E3 ligase MDM2. 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 as shown by cell growth curves. 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, further 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, as it was suggested in silico. We found that Aurora A is overexpressed in advanced SM and may target SETD2 for phosphorylation. Both pharmacological inhibition by Danusertib and siRNA-mediated silencing of Aurora A rescued SETD2 expression and activity, raising the hypothesis that phosphorylation by Aurora A might be the trigger for MDM-2 mediated degradation of SETD2. To evaluate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting (WB) and immunofluorescence (IF) to assess phosphorylated histone 2A.X (γH2AX) and Rad51 foci. Compared to cells from healthy controls, 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, intron retention and non-canonical splicing patterns not observed in healthy donors. Next, the ROSAD816V cell line, which displays SETD2 and H3K36me3 levels superimposable to healthy donors, was studied by WB and IF to assess γH2AX and Rad51 in steady state and after sub-lethal DNA damage by UV exposure. The same experiments were carried out after SETD2 silencing for 2 months. Cells with silenced SETD2 had significantly higher levels of γH2AX and were unable to activate the HR repair. Interestingly, clonogenic assays in ROSAD816V cells before and after SETD2 silencing showed that reduction of clonogenic potential after proteasomal or MDM2 inhibition is indeed SETD2-dependent (Figure 1A). 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 (Figure 1B). Taken together, our results suggest that AKA and MDM2-mediated post-translational modifications contribute to SETD2 non-genomic loss of function in advanced SM. Loss of SETD2 and H3K36me3 is associated with increased DNA damage and transcription and splicing defects in patients. Inhibiting AKA or MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies in patients with low SETD2 expression levels. Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). Figure 1. Figure 1. Disclosures Bonifacio: Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Pagano:Janssen: Speakers Bureau; Merck: Speakers Bureau; Gilead: Speakers Bureau; Basilea: Speakers Bureau; Pfizer: Speakers Bureau. Valent:Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Novartis: Consultancy; Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy.