SETD2 Loss Research Articles

A Novel Epigenetic Marker Predicts Treatment Response in Glioblastoma and Provides a Target for Overcoming Treatment Resistance

<h3>Purpose/Objective(s)</h3> Glioblastoma (GBM) is a highly aggressive primary brain tumor. A major challenge in GBM treatment is tumor resistance to radiation and chemotherapy. An important hallmark of GBM is the frequent mutation of epigenetic modifiers resulting in alteration of epigenetic signaling pathways in tumor cells. However, the effect of epigenetic signaling on chemotherapy response in GBM remains to be elucidated. SETD2 is a unique histone methyl transferase that facilitates H3K36 tri-methylation. Here, we unveil the role of SETD2 mutations that frequently occur in GBM in tumor resistance to temozolomide chemotherapy. <h3>Materials/Methods</h3> We have performed next-generation sequencing of the SETD2 gene and immunohistochemical staining if its product H3K36me3 in 129 GBM patient samples that was supplemented with TCGA database analysis of additional 279 GBM samples. We used primary patient-derived GBM lines to investigate the mechanism of chemotherapy resistance in SETD2-mutant GBM. <h3>Results</h3> We demonstrate that inactivating SETD2 mutations occur in up to 13% of GBM patients. Our investigation reveals that SETD2 mutations are associated with reduced progression-free (Median PFS 0.93 vs. 0.44 years, p=0.0022) and overall survival (Median survival 1.69 vs. 1.30 years, p=0.0044) in patients with methylated MGMT (methyl-guanine methyl transferase) promotor who received temozolomide suggesting that mutation of SETD2 is a predictive marker for treatment outcomes in GBM. Consequently, we demonstrate that loss of SETD2 results in reduced H3K36me3 levels in patient samples and a profound resistance to temozolomide in primary patient-derived GBM lines. MGMT-deficient tumors can acquire chemoresistance due to disrupted mismatch repair (MMR), a DNA repair pathway that plays a critical role in converting primary temozolomide-induced DNA lesions into toxic DNA double-strand breaks. Strikingly, we found that SETD2 loss abrogates the expression of the MMR factor MSH6 indicating that chemoresistance in SETD2-deficient cells us due to disrupted MMR. Mechanistically, we show that SETD2 regulates MMR by promoting transcription of the <i>MSH6</i> gene in GBM. Epigenetic modifiers have specific antagonists capable of reversing chromatin alterations induced by these modifiers. This provides a unique opportunity to restore chemotherapy response in SETD2-mutant GBM by targeting the antagonists of SETD2. We demonstrate that combined targeting of H3K36me3-specific histone de-methylases KDM4A and NO66 restores H3K36me3 levels along with MSH6 expression and sensitivity to temozolomide in SETD2-deficient GBM cells. <h3>Conclusion</h3> Our findings establish SETD2 mutation as a novel molecular marker predictive of treatment response in GBM and provide a framework for a novel approach to overcome chemotherapy resistance in this malignant brain tumor by targeting an epigenetic signaling pathway.

Abstract 3725: SETD2 loss in renal carcinoma cells induces the unfolded protein response

Abstract Introduction: SETD2 encodes a histone H3-K36 methyltransferase which is frequently inactivated in clear cell renal carcinomas (ccRCCs) and papillary RCCs via 3p deletion/LOH and deleterious mutations. Histone H3-K36 trimethylation is facilitated by SETD2, which is necessary for proper pre-mRNA intron splicing. Improperly spliced mature mRNA may lead to aberrant translation of retained introns (ATaRI), which represents potential therapeutic vulnerabilities. We explored this hypothesis using real world data and RCC models. Methods and Results: Gene set enrichment analysis comparing SETD2-mutant to WT tumors using samples from the TCGA KIRC data set revealed that the unfolded protein response (UPR) was strongly enriched, as well as several immunotherapy-relevant pathways. This suggested that peptides arising from ATaRI may be present, since they would not be expected to fold properly and thus need to be addressed by the UPR pathway to maintain homeostasis. To investigate this further, we generated Setd2-isogenic RENCA cells using CRISPR. Knockout was confirmed by sequencing and immunoblot. H3K36 trimethylation was decreased or eliminated in monoclonal knockout cell lines, confirming a functional effect. Markers of UPR activation, including Atf4 and cleaved Atf6, were found to be upregulated in Setd2-mutant RENCA cells compared to controls as measured by immunoblot. Cleaved ATF6 translocates to the nucleus to induce the UPR transcriptional program. Consistent with this, Atf6 was found to localize to the nucleus in Setd2-knockout cells using immunofluorescence (IF). Analysis of tissue microarrays of human SETD2-mutant vs -WT ccRCC revealed increased ATF6 signal in areas with low H3K36 tri-methylation, indicating UPR activation in vivo. Interestingly, CHOP, another downstream effector of the UPR pathway which predominantly regulates cell death, did not become upregulated in Setd2-knockout cells, suggesting activation of a compensatory cell survival pathway. Geldanamycin was shown to destabilize the Perk and Ire1a arms of the UPR, which resulted in increased cell death in Setd2 mutant cells using Cell Titre glo. Additionally, H3K36 trimethylation has been implicated in directing homologous repair of DNA, and blunted responses to agents that induce DNA double-strand breaks of p-Atm, p53, and Rad51 were observed by immunoblot and IF. The inability to detect DNA damage in Setd2 mutant cells however did not confer sensitivity to PARP inhibitors. Conclusions: We identify activation of the UPR upon Setd2 loss and suggest that activation of part of the UPR pathway may represent a new therapeutic vulnerability for exploitation as a rationale for personalized medicine. We further characterize the altered DNA damage response in the setting of Setd2 loss. We continue to evaluate the generation of peptides arising from ATaRI in Setd2-mutant contexts. Citation Format: Alexander Metz, Marya Kozinova, Robert Uzzo, Jessica Peskin, Michael Slifker, Janusz Franco-Barraza, Edna Cukierman, Philip Abbosh. SETD2 loss in renal carcinoma cells induces the unfolded protein response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3725.

SETD2 loss perturbs the kidney cancer epigenetic landscape to promote metastasis and engenders actionable dependencies on histone chaperone complexes.

SETD2 is a H3K36 trimethyltransferase that is mutated with high prevalence (13%) in clear cell renal cell carcinoma (ccRCC). Genomic profiling of primary ccRCC tumors reveals a positive correlation between SETD2 mutations and metastasis. However, whether and how SETD2 loss promotes metastasis remains unclear. In this study, we used SETD2-mutant metastatic ccRCC patient-derived cell line and xenograft models and showed that H3K36me3 restoration greatly reduced distant metastases of ccRCC in mice in an MMP1-dependent manner. An integrated multi-omics analysis using ATAC-seq, ChIP-seq, and RNA-seq established a tumor suppressor model in which loss of SETD2-mediated H3K36me3 activates enhancers to drive oncogenic transcriptional output through regulation of chromatin accessibility. Furthermore, we uncovered mechanism-based therapeutic strategies for SETD2-deficient cancer through the targeting of specific histone chaperone complexes including ASF1A/B and SPT16. Overall, SETD2 loss creates a permissive epigenetic landscape for cooperating oncogenic drivers to amplify transcriptional output, providing unique therapeutic opportunities.

DDRE-32. SETD2 HISTONE METHYLTRANSFERASE MUTATION STATUS PREDICTS TREATMENT RESPONSE IN GLIOBLASTOMA: STRATEGIES TO OVERCOME CHEMORESISTANCE

Abstract A major challenge in GBM treatment is tumor resistance to radiation and chemotherapy. A hallmark of GBM is the frequent mutation of epigenetic modifiers resulting in alteration of epigenetic signaling pathways. However, the effect of epigenetic signaling on chemotherapy response in GBM remains unknown. SETD2 is a histone methyl transferase that facilitates H3K36 tri-methylation. Here, we unveil the role of SETD2 mutations that frequently occur in GBM in tumor resistance to temozolomide chemotherapy. Targeted sequencing of the SETD2 gene in GBM tumor samples revealed that SETD2 mutations are associated with reduced overall survival in patients with methylated MGMT (methyl-guanine methyl transferase) promotor who received temozolomide. Consequently, we demonstrate that loss of SETD2 results in reduced H3K36me3 levels and a profound temozolomide resistance in GBM cells. MGMT-deficient tumors can acquire chemoresistance due to disrupted mismatch repair (MMR), a DNA repair pathway that converts primary temozolomide-induced DNA lesions into toxic DNA double-strand breaks. Strikingly, we found that SETD2 loss abrogates the expression of the MMR factor MSH6 indicating that chemoresistance in SETD2-deficient cells us due to disrupted MMR. Mechanistically, we show that SETD2 regulates MMR by promoting transcription of the MSH6 gene in GBM. Epigenetic modifiers have specific antagonists capable of reversing chromatin alterations induced by these modifiers. This provides a unique opportunity to restore chemotherapy response in SETD2-mutant GBM by targeting the antagonists of SETD2. We demonstrate that combined targeting of H3K36me3-specific histone de-methylases KDM4A and NO66 restores H3K36me3 levels along with MSH6 expression and sensitivity to temozolomide in SETD2-deficient GBM cells. Thus, our findings establish SETD2 mutation as a novel molecular marker predictive of chemotherapy response in GBM and provide a framework for a novel approach to overcome chemotherapy resistance in this malignant brain tumor by targeting an epigenetic pathway.

Pharmacologic Inhibition of the Histone Methyltransferase SETD2 with EZM0414 As a Novel Therapeutic Strategy in Relapsed or Refractory Multiple Myeloma and Diffuse Large B-Cell Lymphoma

Pharmacologic Inhibition of the Histone Methyltransferase SETD2 with EZM0414 As a Novel Therapeutic Strategy in Relapsed or Refractory Multiple Myeloma and Diffuse Large B-Cell Lymphoma

CML-182: SETD2 Loss of Function Induces Genomic Instability in CML and May Contribute to Disease Progression to Blast Crisis

Context: Genetic/genomic instability is a hallmark of CML. SETD2, a histone methyltransferase that trimethylates histone H3 on K36 (H3K36me3), has recently demonstrated a crucial role in preserving genomic integrity by modulating DNA Mismatch Repair (MMR) and Homologous Recombination (HR) repair. By Western blotting (WB), we previously observed SETD2 and H3K36me3 loss (resulting from aberrant SETD2 turnover) in 85% of blast crisis (BC) CML patients but not in newly diagnosed chronic phase (CP) patients who will achieve optimal responses. Objectives: 1. To investigate whether SETD2 is involved in the maintenance of genetic/genomic stability in CML. 2. To assess whether SETD2 loss precedes or follows progression from CP to blast crisis BC. Materials: SETD2-proficient (LAMA84) and -deficient (KCL22) CML cell lines and primary patient samples (n=86) were studied. Results: To investigate whether SETD2/H3K36me3 loss impinges on the activation and proficiency of HR, we used UV rays to induce DNA damage in SETD2 siRNA-depleted LAMA 84 (SETD2-proficient) cells. Compared to control cells, cells silenced for SETD2 displayed a marked increase in γH2AX (a marker of DNA damage) and fewer RAD51 foci (markers of ongoing HR). Assessment of MMR proficiency is ongoing. To further confirm the role of SETD2 as a tumor suppressor implicated in maintaining genomic stability in CML, we transfected KCL22 (SETD2-deficient) cells with an ectopic SETD2 plasmid. Preliminary results showed that SETD2 re-expression induced a reduction in cell doubling time, an accumulation of cells at G1/S checkpoint and a significant reduction in clonogenic potential. Studies are ongoing to assess the effects on DNA damage repair pathways. We next wondered when during disease history SETD2/H3K36me3 loss occurs. WB analysis ofpaired (diagnosis/progression) samples from four CML patients suggested that patients who will progress still have intact and functional SETD2 at diagnosis. Additional analyses are ongoing to assess whether SETD2/H3K36me3 loss precedes, hence allows to predict, progression to BC. Conclusion: Loss of SETD2/H3K36me3 is a novel, BCR-ABL1-independent mechanism of genetic instability in CML. Further investigations are needed to establish to what extent SETD2/H3K36me3 loss contributes to (and may be predictive of) disease progression. Supported by AIRC project 23001.

Poster: CML-182: SETD2 Loss of Function Induces Genomic Instability in CML and May Contribute to Disease Progression to Blast Crisis

Poster: CML-182: SETD2 Loss of Function Induces Genomic Instability in CML and May Contribute to Disease Progression to Blast Crisis

Abstract 2027: SETD2 loss in renal carcinoma cells induces the unfolded protein response

Abstract Introduction: SETD2 encodes a histone H3-K36 methyltransferase which is frequently inactivated in clear cell renal carcinomas (ccRCCs) and papillary RCCs via 3p deletion/LOH and deleterious mutations. Histone H3-K36 trimethylation is facilitated by SETD2, which is necessary for proper pre-mRNA intron splicing. Improperly spliced mature-mRNA may lead to aberrant translation of retained introns (ATaRI), which represents potential therapeutic vulnerabilities. We explored this hypothesis using real world data and RCC models. Methods and Results: Gene set enrichment analysis comparing SETD2-mutant to WT tumors using samples from the TCGA KIRC data set revealed that the untranslated protein response (UPR) was strongly enriched, as well as several immunotherapy-relevant pathways. This suggested that peptides arising from ATaRI may be present, since they would not be expected to fold properly and thus need to be addressed by the UPR pathway to maintain homeostasis. To investigate this further, we generated Setd2-isogenic RENCA cells using CRISPR. Knockout was confirmed by sequencing and immunoblot. H3K36 trimethylation was decreased or eliminated in monoclonal knockout cell lines, confirming a functional effect. Markers of UPR activation, including cleaved Atf6 and Atf4, were found to be upregulated in Setd2-mutant RENCA cells compared to controls as measured by immunoblot. Cleaved ATF6 is known to translocate to the nucleus to take part in a UPR transcriptional program. Consistent with this, ATF6 was found to localize to the nucleus in Setd2-knockout cells using immunofluorescence. Interestingly, CHOP, another downstream effector of the UPR pathway which predominantly regulates cell death, did not become upregulated in Setd2-knockout cells, suggesting activation of a compensatory cell survival pathway. Conclusions: We identify activation of the UPR upon Setd2 loss and suggest that activation of part of the UPR pathway may represent a new therapeutic vulnerability for exploitation as a rationale for personalized medicine. We continue to evaluate the generation of peptides arising from ATaRI in Setd2-mutant contexts. Citation Format: Alexander Metz, Robert Uzzo, Philip Abbosh. SETD2 loss in renal carcinoma cells induces the unfolded protein response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2027.

SETD2 Loss of Function Is a Recurrent Event in Advanced-Phase Chronic Myeloid Leukemia

Abstract In recent years, deregulation of epigenetic modifiers has turned out to be a recurrent event in cancer evolution. The SETD2 tumor suppressor gene encodes a histone methyltransferase that catalyzes the trimethylation of lysine 36 on histone H3 (H3K36Me3), the major chromatin mark associated with active transcription and alternative splicing. Recent studies have also implicated SETD2 in homologous recombination repair, mismatch repair, mitosis and cytokinesis. SETD2 loss of function was initially uncovered in clear cell renal cell carcinomas and was subsequently found in many other solid tumors. More recently, it has also been reported in a variety of hematologic myeloid and lymphoid malignancies, including acute lymphoblastic leukemia,acute myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphoma, systemic mastocytosis and myeloproliferative neoplasms. We thus wondered whether advanced-phase chronic myeloid leukemia (CML), that shareS similarities with acute leukemias including a high level of genetic instability, may also exhibit SETD2 loss of function. To this purpose, we studied a relatively large cohort of 69 advanced phase CML patients (pts), including pts with accelerated phase (AP; n=15), myeloid blast crisis (my-BC; n=30), and lymphoid blast crisis (ly-BC; n=15) and pts with tyrosine kinase inhibitor (TKI)-resistant chronic phase harboring 2 or more BCR-ABL kinase domain mutations (mut-CP; n=9). Samples collected at diagnosis from CP pts (n=8) who achieved optimal response to therapy were also studied for comparison. A recent study in advanced systemic mastocytosis has demonstrated that SETD2 loss of function may result not only from genomic disruption but also from post-translational events, suggesting that the frequency of SETD2 involvement in solid tumors or hematologic malignancies might be higher than sequencing and copy number data alone would suggest. We thus decided to use a Western Blotting (WB) approach to assess H3K36Me3 levels as a surrogate marker of SETD2 loss of function and to screen for the expression of SETD2 full-length vs truncated protein isoforms, that are the most frequent result of inactivating nonsense or frameshift mutations. Reduced or null H3K36Me3 (as compared to a pool of healthy donors) was detected in the great majority of pts with advanced-phase CML (58/69, 84.1%; 12/15 AP, 24/30 my-BC, 12/15 ly-BC, 8/9 mut-CP). In contrast, CP pts at diagnosis showed H3K36Me3 levels superimposable to those of healthy donors. In some of them, however, the CD34+ cell fraction was available and reduced H3K36Me3 levels were detected in all cases. No evidence of abnormal SETD2 protein isoforms was detected, but pts with no H3K36Me3 also had no detectable SETD2 protein and pts with reduced H3K36Me3 had similarly reduced levels of full-length SETD2 protein as assessed by densitometric analysis of WB (Spearman R=0.95, p To rule out the possibility that SETD2 loss of function might be another age-associated alteration in an epigenetic regulator, we collected a series of 80 healthy individuals with an age ranging from 65 to 95 years. In this cohort, we checked for SETD2 mutations by sequencing and for SETD2 and H3K36 trimethylation levels by WB. No mutations were detected and protein levels were not significantly lower in older adults than in a pool of younger adults. Advanced-phase CML is characterized by a profound genetic heterogeneity. We here show the first evidence for a novel, nearly unifying alteration. However, SETD2 downmodulation and, consequently, H3K36Me3 deficiency seem to be most frequently accomplished at the post-translational level rather than being the results of genetic/genomic hits or transcriptional repression. Further studies aimed to assess whether H3K36Me3 loss might contribute to the genetic instability that is the hallmark of advanced-phase CML are warranted. Restoring physiological H3K36Me3 levels may help improve the outcome of this critical subset of pts. Supported by AIL and AIRC (project code 16996) Disclosures Castagnetti: Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Gugliotta: Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rosti: Incyte: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Iurlo: Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Abruzzese: Novartis: Consultancy; Pfizer: Consultancy; Incyte: Consultancy; BMS: Consultancy. Crugnola: BMS: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Bonifacio: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Tiribelli: Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Galimberti: Bristol-Myers Squibb: Speakers Bureau; Incyte: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Speakers Bureau. Baccarani: Incyte ARIAD: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Martinelli: Celgene: Consultancy; Amgen: Consultancy; JohnsonJ Pfizer: Consultancy; Ariad/Incyte: Consultancy; Roche: Consultancy. Soverini: Bristol-Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.

SETD2 Non-Genomic Loss of Function and p53 Functional Inactivation in Advanced Systemic Mastocytosis (SM): Pathogenetic and Therapeutic Implications

KIT constitutive activation is thought to be necessary but not sufficient for the development of aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). The identification and dissection of cooperating pathogenetic pathways may set the basis for the development of novel therapeutic strategies.We have recently found that the HMC-1.1 and -1.2 MCL cell lines and ASM/MCL patients (pts) 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, homologous recombination (HR), mismatch repair (MMR). Inhibition of proteasome-mediated degradation by bortezomib restored SETD2 protein expression and H3K36Me3. Reversing the detrimental effects of SETD2 and H36K36Me3 deficiency may be a promising therapeutic strategy.In this study, we aimed to investigate the effects of SETD2 loss of function in ASM and MCL and to assess the in vitro efficacy of second-generation proteasome inhibitors as therapeutic agents. To investigate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting and immunofluorescence to assess phosphorylated histone 2A.X (γH2AX), which marks ongoing DNA damage signaling and DNA double strand breaks and Rad51, which is a surrogate for active HR. Compared to cells from healthy controls, cells from SETD2- and H3K36Me3-deficient MCL pts had significantly higher levels of γH2AX and Rad51. To assess whether the increased DNA damage may translate into a greater number of submicroscopic genomic lesions, and whether the degree of SETD2 down-modulation may be inversely correlated with the degree of genomic complexity, SNP-array data from 13 pts were examined. Structural complexity score (SCS) and genome instability index (GII) were not significantly different (SCS, 387 vs 376; GII, 0.293 vs 0.337) between pts with > or <50% residual SETD2 and H3K36Me3 levels, suggesting that the activity of HR repair may counteract DNA damage events. Moreover, SETD2 down-modulation was not associated with reduced MMR proficiency, as assessed by lack of microsatellite instability. On the other hand, 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.In renal cell carcinoma cell lines, SETD2 has been shown to bind and activate p53. After proteasome inhibition, SETD2 was indeed found to immunoprecipitate 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 SP-141, a new class of MDM2 inhibitors that promote MDM2 auto-ubiquitination and degradation, also 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 for 24-48-72-96 hours induced cytostatic but not cytotoxic effects as shown by cell growth curves. Cytostatic effects resulted from a G2/M block of cell cycle induced by Gadd45a overexpression. Clonogenic assays supported the cytostatic effects of SP141 in HMC-1.1 and -1.2 cells (LD50=0.133µM and 0.050µM respectively).Finally, treatment with the second-generation proteasome inhibitors carfilzomib and ixazomib potently induced apoptosis and reduced colony growth in HMC-1.1 (LD50=0.10nM and 0.66nM respectively) and -1.2 (LD50=0.13nM and 0.72nM respectively) cells.In conclusion: 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. Approaches aimed to inhibit MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies worth to be further explored.Supported by AIL and AIRC (16996) DisclosuresZanotti:Deciphera: Consultancy. Ciceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Valent:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; Ariad: Honoraria, Research Funding; Incyte: Honoraria; Deciphera: Honoraria, Research Funding; Pfizer: Honoraria; Blueprint: Research Funding; Teva: Honoraria. Martinelli:Amgen: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Roche: Consultancy; Johnson&Johnson: Consultancy; Ariad/Incyte: Consultancy. Soverini:Incyte Biosciences: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy.

SETD2 Histone Methyltransferase Mutation Status Predicts Treatment Response In Glioblastoma: Strategies To Overcome Chemoresistance

Glioblastoma (GBM) is a highly aggressive primary brain tumor. A major challenge in GBM treatment is tumor resistance to radiation and chemotherapy. An important hallmark of GBM is the frequent mutation of epigenetic modifiers resulting in alteration of epigenetic signaling pathways in tumor cells. However, the effect of epigenetic signaling on chemotherapy response in GBM remains to be elucidated. SETD2 is a unique histone methyl transferase that facilitates H3K36 tri-methylation. Here, we unveil the role of SETD2 mutations that frequently occur in GBM in tumor resistance to temozolomide chemotherapy. We have performed a TCGA database analysis to assess the role of SETD2 mutations in treatment response in GBM patients. We have utilized an in vitro model of GBM and multiple molecular and biochemical approaches to investigate the role of SETD2 in tumor response to temozolomide and establish a strategy to overcome chemoresistance in SETD2-mutant GBM. Our TCGA database analysis reveals that SETD2 mutations are associated with reduced overall survival in patients with methylated MGMT (methyl-guanine methyl transferase) promotor who received temozolomide suggesting that mutation of SETD2 is a predictive marker for chemotherapy resistance in GBM. Consequently, we demonstrate that loss of SETD2 results in reduced H3K36me3 levels in GBM patient samples and a profound resistance to temozolomide in GBM cells. MGMT-deficient tumors can acquire chemoresistance due to disrupted mismatch repair (MMR), a DNA repair pathway that plays a critical role in converting primary temozolomide-induced DNA lesions into toxic DNA double-strand breaks. Strikingly, we found that SETD2 loss abrogates the expression of the MMR factor MSH6 indicating that chemoresistance in SETD2-deficient cells is due to disrupted MMR. Mechanistically, we show that SETD2 regulates MMR by promoting transcription of the MSH6 gene in GBM. Epigenetic modifiers have specific antagonists capable of reversing chromatin alterations induced by these modifiers. This provides a unique opportunity to restore chemotherapy response in SETD2-mutant GBM by targeting the antagonists of SETD2. We demonstrate that combined targeting of H3K36me3-specific histone de-methylases KDM4A and NO66 restores H3K36me3 levels along with MSH6 expression and sensitivity to temozolomide in SETD2-deficient GBM cells. Our findings establish SETD2 mutation as a novel molecular marker predictive of chemotherapy response in GBM and provide a framework for a novel approach to overcome chemotherapy resistance in this malignant brain tumor by targeting a specific epigenetic signaling pathway.

CML-206: ReSETting SETD2/H3K36Me3 Deficiency as a New Therapeutic Strategy in Blast Crisis Chronic Myeloid Leukemia Patients

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.

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.

Systematic Analysis of Aberrant Biochemical Networks and Potential Drug Vulnerabilities Induced by Tumor Suppressor Loss in Malignant Pleural Mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is driven by the inactivation of tumor suppressor genes (TSGs). An unmet need in the field is the translation of the genomic landscape into effective TSG-specific therapies. Methods: We correlated genomes against transcriptomes of patients’ MPM tumors, by weighted gene co-expression network analysis (WGCNA). The identified aberrant biochemical networks and potential drug targets induced by tumor suppressor loss were validated by integrative data analysis and functional interrogation. Results: CDKN2A/2B loss activates G2/M checkpoint and PI3K/AKT, prioritizing a co-targeting strategy for CDKN2A/2B-null MPM. CDKN2A deficiency significantly co-occurs with deletions of anti-viral type I interferon (IFN-I) genes and BAP1 mutations, that enriches the IFN-I signature, stratifying a unique subset, with deficient IFN-I, but proficient BAP1 for oncolytic viral immunotherapies. Aberrant p53 attenuates differentiation and SETD2 loss acquires the dependency on EGFRs, highlighting the potential of differentiation therapy and pan-EGFR inhibitors for these subpopulations, respectively. LATS2 deficiency is linked with dysregulated immunoregulation, suggesting a rationale for immune checkpoint blockade. Finally, multiple lines of evidence support Dasatinib as a promising therapeutic for LATS2-mutant MPM. Conclusions: Systematic identification of abnormal cellular processes and potential drug vulnerabilities specified by TSG alterations provide a framework for precision oncology in MPM.

Abstract 1249: SetD2 histone methyltransferase mutation status predicts treatment response in glioblastoma: Strategies to overcome chemoresistance

Abstract Purpose: Glioblastoma (GBM) is a highly aggressive primary brain tumor. A major challenge in GBM treatment is tumor resistance to radiation and chemotherapy. An important hallmark of GBM is the frequent mutation of epigenetic modifiers resulting in alteration of epigenetic signaling pathways. However, the effect of epigenetic signaling on chemotherapy response in GBM remains to be elucidated. SETD2 is a unique histone methyl transferase that facilitates H3K36 tri-methylation. Here, we unveil the role of SETD2 mutations that frequently occur in GBM in tumor resistance to temozolomide chemotherapy. Method: We have generated CRISPR/Cas9-mediated SETD2-KO GBM cell lines to study the role of SETD2 in temozolomide response. DNA methylome and whole exome sequencing data from the TCGA databank were analyzed to determine the MGMT (methyl-guanine methyl transferase) promotor methylation status and SETD2 mutations, respectively. H3K36me3 and MSH6 levels were determined in GBM patient samples by immunohistochemical staining. SETD2 mutation status was determined in GBM patient samples by targeted sequencing of the SETD2 gene using the Agilent SureSelect Target technique. Results: Our TCGA database analysis reveals that SETD2 mutations are associated with reduced overall survival in patients with methylated MGMT promotor who received temozolomide suggesting that mutation of SETD2 is a predictive marker for chemotherapy resistance in GBM. Consequently, we demonstrate that loss of SETD2 results in reduced H3K36me3 levels and a profound resistance to temozolomide in GBM cells. MGMT-deficient tumors can acquire chemoresistance due to disrupted mismatch repair (MMR), a DNA repair pathway that plays a critical role in converting primary temozolomide-induced DNA lesions into toxic DNA double-strand breaks. Strikingly, we found that SETD2 loss abrogates the expression of the MMR factor MSH6 indicating that chemoresistance in SETD2-deficient cells is due to disrupted MMR. Accordingly, low H3K36me3 levels in GBM tumor samples correlate with loss of MSH6 expression. Mechanistically, we show that SETD2 regulates MMR by promoting transcription of the MSH6 gene in GBM. Finally, we establish a strategy to re-sensitize SETD2-deficient GBM cells to temozolomide. Epigenetic modifiers have specific antagonists capable of reversing chromatin alterations induced by these modifiers. This provides a unique opportunity to restore chemotherapy response in SETD2-mutant GBM by targeting the antagonists of SETD2. We demonstrate that combined targeting of H3K36me3-specific histone de-methylases KDM4A and NO66 restores H3K36me3 levels along with MSH6 expression and sensitivity to temozolomide in SETD2-deficient GBM cells. Conclusions: Our findings establish SETD2 mutation as a novel molecular marker predictive of chemotherapy response in GBM and provide a framework for a novel approach to overcome chemotherapy resistance in this malignant brain tumor by targeting a specific epigenetic signaling pathway. Fig.1: Immunohistochemical staining of H3K36me3 and MSH6 in GBM tumor sections demonstrating that tumor areas with low H3K36me3 expression lack MSH6 expression. Citation Format: Nishanth Gabriel, Sonika Dahiya, Xiaowei Wang, Michael Goldstein. SetD2 histone methyltransferase mutation status predicts treatment response in glioblastoma: Strategies to overcome chemoresistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1249.

Aurora Kinase a/MDM2-Mediated SETD2 Loss of Function in Chronic Myeloid Leukemia Patients in Blast Crisis Can be Therapeutically Targeted Inducing Apoptotic Cell Death in a Caspase-Dependent Way

Aurora Kinase a/MDM2-Mediated SETD2 Loss of Function in Chronic Myeloid Leukemia Patients in Blast Crisis Can be Therapeutically Targeted Inducing Apoptotic Cell Death in a Caspase-Dependent Way

Loss of Setd2 promotes Kras-induced acinar-to-ductal metaplasia and epithelia–mesenchymal transition during pancreatic carcinogenesis

ObjectiveSETD2, the sole histone H3K36 trimethyltransferase, is frequently mutated or deleted in human cancer, including pancreatic ductal adenocarcinoma (PDAC). However, whether SETD2/H3K36me3 alteration results in PDAC remains largely unknown.DesignTCGA(PAAD) public...

PS1437 SETD2 NON‐GENOMIC LOSS OF FUNCTION IN ADVANCED SYSTEMIC MASTOCYTOSIS (SM): PATHOGENETIC AND THERAPEUTIC IMPLICATIONS

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.

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

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.

Roles of SETD2 in Leukemia-Transcription, DNA-Damage, and Beyond.

The non-redundant histone methyltransferase SETD2 (SET domain containing 2; KMT3A) is responsible for tri-methylation of lysine 36 on histone H3 (H3K36me3). Presence of the H3K36me3 histone mark across the genome has been correlated with transcriptional activation and elongation, but also with the regulation of DNA mismatch repair, homologous recombination and alternative splicing. The role of SETD2 and the H3K36me3 histone mark in cancer is controversial. SETD2 is lost or mutated in various cancers, supporting a tumor suppressive role of the protein. Alterations in the SETD2 gene are also present in leukemia patients, where they are associated with aggressive disease and relapse. In line, heterozygous SETD2 loss caused chemotherapy resistance in leukemia cell lines and mouse models. In contrast, other studies indicate that SETD2 is critically required for the proliferation of leukemia cells. Thus, although studies of SETD2-dependent processes in cancer have contributed to a better understanding of the SETD2–H3K36me3 axis, many open questions remain regarding its specific role in leukemia. Here, we review the current literature about critical functions of SETD2 in the context of hematopoietic malignancies.