Mutations In KDM6A Research Articles

A pilot study of tazemetostat and pembrolizumab in advanced urothelial carcinoma (ETCTN 10183).

4574 Background: Mutations of COMPASS-related proteins KMT2D, KMT2C, and KDM6A are observed in 66% of advanced urothelial carcinomas (UC). In vivo, the administration of tazemetostat, an enzymatic EZH2 inhibitor (EZH2i), reduced tumor burden and enhanced the immune response in animal models of UC. We hypothesized that tazemetostat might improve response to immunotherapy in advanced UC. Methods: ETCTN 10183 (NCT03854474) is a multicenter pilot study evaluating the safety and efficacy of tazemetostat 800 mg BID+ pembrolizumab 200 mg given every three weeks for up to two years in patients (pts) with platinum-refractory (Arm A) or cisplatin/chemo-ineligible advanced UC (Arm B). The primary objective was to identify the recommended phase two dose (RP2D) of tazemetostat in combination with pembrolizumab. Secondary objectives included safety, response rate, and progression-free survival. Translational objectives included determining tumor mutational burden in COMPASS-related genes, tumor subtyping, TCR clonality, T cell infiltration, and PDL-1 expression. Results: There were no dose-limiting toxicities in the safety lead-in phase (n=6 pts), and the RP2D for tazemetostat was established at 800 mg BID. Baseline characteristics for Arm A N=12, Arm B N=13: 72% males; 96% white, 4% Black; 88% ECOG PS 0-1; 88% had bladder primary, 20% Stage III, 72% stage IV; 80% had visceral or bone metastases. The median number of cycles was 5 in Arm A (range 1-35) and 4 in Arm B (range 2-12). Treatment related G3/4 adverse events (AEs) were observed in 8 patients, most common were anemia and lymphopenia (n=2 each). Treatment related G1/2 AEs included nausea (n=6); skin and subcutaneous tissue disorders (n=3); anemia, lymphopenia, and thrombocytopenia (n=2 each). Among evaluable patients (n=10 in each arm), partial response rate was: Arm A 30%, Arm B 20%; stable disease: Arm A 30%, Arm B 30%; and progressive disease: Arm A 30%, Arm B 20%. 8 pts were on study treatment for ≥ 9 cycles (5 in Arm A, 3 in Arm B). All pts are off protocol treatment. Median progression-free survival was 3.06 (95% CI: 1.38-7.75) months for Arm A and 3.02 (95%CI: 2.4-5.65) for Arm B. Conclusions: Tazemetostat 800 mg BID and pembrolizumab combination is feasible and well-tolerated in patients with advanced UC. In this pilot study, improvement in efficacy relative to historical controls of pembrolizumab alone appears to be modest. Response in tumors with COMPASS-related mutations and EZH2 activity will be evaluated. Clinical trial information: NCT03854474 .

Mutation patterns and prognostic implications in acute myeloid leukemia with chromosomal 7 deletions.

6547 Background: Chromosomal 7 deletions (del7/7q) are the most common adverse cytogenetic event in acute myeloid leukemia and are associated with high rate of relapse. We analyzed gene mutation profiles in del7/7q AML patients who relapsed after initial remission to identify molecular patterns associated with clinical outcomes. Methods: Out of 351 newly diagnosed AML patients with del7/7q, 115 (32%) achieved complete remission after first-line treatment between 2012-2023. Of the 115 patients, 77 (67%) patients relapsed during the study follow-up. Kaplan-Meier and Cox regression were used to analyze survival outcomes. Next-generation sequencing was performed on a targeted panel of 81 genes for 59 patients who relapsed on bone marrow samples at diagnosis and relapse for paired analysis. Results: The median age of the 115 patients at diagnosis was 74 (41-93). Thirty-four patients (30%) received intensive treatment. With 30.8-month median follow-up, the median overall survival (OS) was 10.4 months, with improved survival in patients without TP53 mutation (13.04 vs. 8.6 months, p=0.005) or complex karyotype (12.4 vs. 8.6 months, p=0.03). The median duration of response for the 77 patients who relapsed was 5 months (2-7). Allo-HSCT significantly improved median OS (not reached vs. 8.5 months, P=6e-06), and was identified as the key variable impacting survival in multivariate analysis. TP53 was the most commonly mutated gene at diagnosis (67%) and relapse (71%). Mutations in CBLC and KDM6A were cleared after induction therapy, while BCOR and BCORL1 were commonly acquired at relapse (Table). At diagnosis, a lower co-mutation burden was observed in TP53 mutant patients as compared with wild type (average 1.84 vs. 3.65 respectively, p=0.0001). Patients with mutated TP53 and coexisting mutations in GATA2, NF1, BCORL1, or RUNX1 at diagnosis had shorter relapse free survival (RFS, 2 vs. 5 months, p=0.01) and OS (7.2 vs. 10.4 months, p=0.01) compared to patients with solely mutated TP53 (HR 2.2; 95% CI 1.2-4; P = 0.004). Allo-HSCT significantly improved the OS in patients with mutated TP53 (13 vs. 8 months, p=0.01). Conclusions: Mutated TP53 clones persist from diagnosis to relapse in patients with del7/7q AML. In patients with mutated TP53, co-occurring mutations in GATA2, NF1, BCORL1or RUNX1 at diagnosis were linked to a shorter RFS and indicate a particularly high-risk subgroup that require proceeding to allo-HSCT in CR1 without delay. [Table: see text]

Abstract B018: KDM6A and MTAP expression loss enable identification of a large fraction of low grade non-invasive urothelial neoplasia of the urinary bladder

Abstract The identification of low-grade urothelial neoplasia in cytologic samples or in biopsies with dysplastic lesions poses a particular problem for pathologists in routine diagnostics. Improved diagnosis of low-grade urothelial neoplasia could help to reduce the number of cystoscopic follow-up examinations. KDM6A, also known as UTX (ubiquitously transcribed X chromosome tetratricopeptide repeat protein) is an epigenetic regulator which is frequently mutated in urothelial carcinoma. Because KDM6A is localized on chromosome Xp11.3, and because of the male predominance in bladder cancer, many KDM6A mutations should result in a complete loss of KDM6A protein expression and become detectable by immunohistochemistry (IHC). Similarly, S-methyl-5′-thioadenosine phosphorylase (MTAP), a critical enzyme for DNA and RNA synthesis, is co-deleted with the CDKN2A tumor suppressor located on chromosome 9p21 in about 25% of bladder cancers, resulting in reduced or lost protein expression. To study the prevalence and diagnostic potential of immunohistochemical loss of KDM6A and MTAP expression, 928 tumors were analyzed by IHC in a tissue microarray format. The cohort included 628 non-invasive papillary tumors (pTa) and 300 tumors from patients who underwent radical cystectomy for muscle-invasive disease (pT2-4). A complete KDM6A expression loss occurred in 35% of 342 pTa G2 low-grade, 23% of 150 pTa G2 high-grade, 17.8% of 90 pTa G3 tumors, and 20.1% of 243 interpretable muscle invasive (pT2-4) cancers (p<0.0001). Within pTa tumors, there was a strong correlation between KDM6A loss and low tumor grade (p=0.0003), whereas no significant difference was found between pT3 G3 and muscle invasive cancers (p=0.8828). Complete absence of MTAP expression occurred in 11% of 297 pTa G2 low-grade, 29.6% of 125 pTa G2 high-grade, 18.4% of 76 pTa G3 tumors, and 26.3% of 217 muscle invasive (pT2-4) cancers (p<0.0001). A combined analysis of 702 cancers with interpretable data for KDM6A and MTAP showed that 6.6% of tumors had expression loss of both KDM6A and MTAP, 20% had KDM6A loss only, and 13% had MTAP loss only. The rate of expression loss of KDM6A and/or MTAP was markedly higher in pTa G2 low (41.2%) and in pTa G2 high (40.3%) than in pTa G3 tumors (29%). In summary, our data demonstrate that about 40% of low-grade non-invasive urothelial neoplasias – which are usually not detected by urine cytology – had a completed expression loss of either KDM6A or MTAP. In patients with these alterations, follow-up could be potentially facilitated by immunohistochemistry. Citation Format: Florian Viehweger, Neele Heckmann, Natalia Gorbokon, Henning Plage, Sebastian Hofbauer, Sarah Weinberger, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Maximilian Lennartz, Tim Mandelkow, Elena Bady, Margit Fisch, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Tobias Klatte, Thorsten Schlomm, David Horst, Henrik Zecha, Martina Kluth, Sarah Minner. KDM6A and MTAP expression loss enable identification of a large fraction of low grade non-invasive urothelial neoplasia of the urinary bladder [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B018.

Abstract 1049: Prevalence of KDM6A expression loss in human cancer: A tissue microarray study on 6,560 cancers from 114 different tumor types

Abstract KDM6A, also known as UTX (ubiquitously transcribed X chromosome tetratricopeptide repeat protein) is an epigenetic regulator constituting a critical part of the COMPASS-like complex. KDM6A mutations occur commonly in cancer. These are of interest because KDM6A loss results in a dependency on EZH2, a potential therapeutic target. Most data on KDM6A have been obtained by next generation sequencing (NGS). However, since most KDM6A mutations are truncating and KDM6A is located on the X chromosome, many mutations result in a complete expression loss which can be detected by immunohistochemistry (IHC). To learn more on the prevalence of KDM6A expression loss and its role in cancer, a tissue microarray containing 6,560 samples from 114 different tumor entities and 608 samples of 76 different normal tissue types was analyzed. In normal tissues, KDM6A staining was rather ubiquitously seen in nuclei. In cancer, KDM6A staining was considered weak (1+) in 581 (11.2%), moderate in 1,927 (37%), and strong in 2,362 (45.4%) of 5,206 interpretable tumors. A complete loss of KDM6A staining occurred in 336 (6.5%) of tumors. At least one case with a complete KDM6A expression loss was observed in 51 of 114 tumor categories. KDM6A staining was most commonly lost in different categories of urothelial carcinomas (19.5-39.2%), squamous cell carcinomas of the esophagus (27.1%), hepatocellular carcinoma (18.4%), gastric carcinoma (3.2-12.5%), papillary renal cell carcinoma (9.5%), adenocarcinoma of the prostate (5.9-9.4%), chromophobe renal cell carcinoma (8.9%), Ewing sarcoma (8.3%), pheochromocytoma (8.3%), and metastatic malignant melanoma (8%). A KDM6A expression loss in less than 8% of cases was observed in 32 further tumor entities. KDM6A expression loss was more than twice as frequent in tumors from male (8.4% of 2,526) than in tumors from female patients (3.9% of 2,024; p<0.0001). It is concluded that KDM6A IHC is a suitable method for the detection of truncating KDM6A mutations, especially in males. These make up for about 2/3 of all KDM6A mutations and may exert specific biological effects than differ from the various KDM6A missense mutations known to also occur. Citation Format: Florian Viehweger, Natalia Gorbokon, Maximilian Lennartz, Viktor Reiswich, Florian Lutz, Till Krech, Andreas M. Luebke, Claudia Hube-Magg, Guido Sauter, Ronald Simon, Stefan Steurer, Andreas H. Marx, Christian Bernreuther, Martina Kluth, Sarah Minner. Prevalence of KDM6A expression loss in human cancer: A tissue microarray study on 6,560 cancers from 114 different tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1049.

Abstract 6449: KDM6A expression loss is a common feature in low grade non-invasive urothelial carcinomas of the urinary bladder

Abstract KDM6A, also known as UTX (ubiquitously transcribed X chromosome tetratricopeptide repeat protein) is an epigenetic regulator which is frequently mutated in urothelial carcinoma. Because KDM6A loss causes a dependency on EZH2, a potential therapeutic target, KDM6A analysis may have therapeutic importance. Most data on KDM6A mutations are derived from next generation sequencing (NGS). However, because of the combination of a high rate of truncating KDM6A mutations, the localization of KDM6A on chromosome Xp11.3, and the male predominance in bladder cancer, many KDM6A mutations should result in a complete loss of KDM6A protein expression and become detectable by immunohistochemistry (IHC). To study the prevalence and the potential diagnostic and clinical role of KDM6A expression loss, more than 2,100 tumors were analyzed by IHC in a tissue microarray format. The cohort included 1,128 patients who underwent radical cystectomy for muscle-invasive disease (pT2-4). A complete KDM6A expression loss occurred in 36% of 350 pTa G2 low-grade, 23% of 152 pTa G2 high-grade, and 18.5% of 92 pTa G3 tumors (p=0.0002). As compared to pTa G3 tumors, the frequency of KDM6A expression loss did not differ significantly in pT2 (17.2%), pT3 (21.9%), and pT4 (18.2%) cancers. Within pT2-4 carcinomas, KDM6A staining was unrelated to pT, pN, grade, L-status, V-status, overall survival, recurrence-free survival, and cancer-specific survival (p≥0.18 each). A KDM6A loss was more common in male (22.2%) than in female patients (15.4%; p<0,0001), and within male patients, a KDM6A loss was more frequent in tumors with Y-chromosome loss (36.1%) than in cancers without a Y-chromosome loss (16.3%; p<0.0001). KDM6A expression loss was significantly associated with increased p63 expression (p<0.0001) and high uroplakin 1b staining (p=0.0013) but unrelated to CK20, GATA3, UpK1b, and p53 staining. In summary, our data demonstrate that truncating KDM6A mutations occur in a significant subset of urothelial carcinomas which is linked to low-grade non-invasive cancer, male gender and loss of the Y chromosome. The predominance of KDM6A loss in low grade tumors makes KDM6A IHC a promising new tool for identification of low grade dysplasia in biopsies and early bladder cancer detection in cytology. Citation Format: Florian Viehweger, Natalia Gorbokon, Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberger, Bernhard Ralla, Antonia Franz, Annika Fendler, Michela de Martino, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Maximilian Lennartz, Tim Mandelkow, Elena Bady, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Joachim Weischenfeldt, Tobias Klatte, Thorsten Schlomm, David Horst, Henrik Zecha, Martina Kluth, Sarah Minner. KDM6A expression loss is a common feature in low grade non-invasive urothelial carcinomas of the urinary bladder [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6449.

Abstract 4889: Integrative multi-omic analysis reveals novel prognostic biological entities in pediatric medulloblastoma

Abstract Medulloblastoma is a cerebellar tumor for which relapses are associated with poor survival rates of less than 5%. Molecular heterogeneity and dose-limiting toxicities that occur with standard of care approaches, which include surgical resection, craniospinal irradiation, and chemotherapy, complicate the treatment of both primary and recurrent medulloblastoma due to adverse long-term sequela. While the integration of molecular analyses into the histopathology of pediatric medulloblastomas has changed the way these diseases are diagnosed, classified, and treated, there remains an unmet need to further understand the underpinnings of the disease to improve clinical outcomes and minimize neuronal, cognitive, and hormonal toxicities that occur with standard-of-care therapies. We therefore aimed to investigate novel subgroups of pediatric medulloblastoma as a basis for further defining biological properties and targets for the disease through multi-omic characterization. We derived single-nucleotide variant, copy number variant, expression, alternative splicing, and methylation array data from pediatric medulloblastomas profiled as part of the Children’s Brain Tumor Network (CBTN) and the Open Pediatric Cancer (OpenPedCan) projects. Using non-negative matrix factorization across the five datasets derived from genomic, transcriptomic, and epigenomic profiling, we identified 14 clusters found to be prognostically significant by Kaplan-Meier analysis of overall survival (p < 0.0001). Our model further subdivided sonic hedgehog (SHH) and Group 3/4 tumors, but not Wnt-driven tumors, suggesting that the latter forms a homogeneous molecular subgroup. Among our subgroups, we recapitulated the four classically defined medulloblastoma subtypes known to be associated with mutations in KDM6A, KMT2C, CTNNB1, PTCH1, TP53, KBTBD4, MYC, and MYCN. Cascading biological relationships across the copy number, methylation, and expression domains were observed among novel subgroups, potentially driving migratory, immuno-regulatory, GPCR-related, growth factor-related, and histone methylation biological programs. A comparison of subgroup expression profiles to transcriptional signatures from the Library of Integrated Network-Based Cellular Signatures (LINCS) program identified canonical oncogenic pathways unique to each subgroup as potentially targetable, including, but not limited to, MAPK, VEGFA, inflammatory, fatty acid metabolic, and PI3K/AKT signaling. Our work uncovers novel biological entities in pediatric medulloblastoma potentially driven by higher order mechanisms across the genome, transcriptome, and epigenome, with the possibility of informing novel precision medicine approaches for the disease. Citation Format: Komal Rathi, Varun Kesherwani, Ammar S. Naqvi, Yuankun Zhu, Alex Sickler, Xiaoyan Huang, Bo Zhang, Brian Rood, Adam C. Resnick, Adam A. Kraya. Integrative multi-omic analysis reveals novel prognostic biological entities in pediatric medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4889.

Abstract 883: Genetic characteristics between males and females with urothelial bladder cancer

Abstract Urothelial bladder cancer (UBC) affects the epithelial cells of the urinary tract and is the most common type of bladder cancer worldwide. Statistically, males are four times more likely to develop UBC than females. It is known that decreased expression of the KDM6A gene, coded on the X chromosome, corresponds with a higher incidence of UBC. KDM6A mutations are known to be more prevalent in females with non-muscle-invasive UBC. These mutations are targetable by inhibiting EZH2, an enzyme coded by its namesake gene. On the environmental side, males are at a higher risk of developing carcinogenic complications due to smoking and environmental toxins. However, this discrepancy fails to exist when comparing males and females who smoke similar levels. Though environmental factors may play a role in influencing behavior and altering levels of risk to develop UBC, research suggests that they do not single-handedly explain sex-based disparities in UBC. In this study, we characterize the molecular mechanisms associated with altered overall survival by uncovering previously unreported data on the expression of KDM6A and FGFR3, a commonly altered oncogene, in UBC patients. Our in silico approach consisted of broad survival data analysis through cBioPortal (n=1486), sequencing-based gene expression data through ICGC Portal (n=295), and single nucleotide polymorphism identification with the PLCO Atlas (n=69,429). The 5-year overall survival (OS) curves of 13 therapeutic and/or significantly altered genes between males and females were analyzed. BRCA2 was the only gene associated with worse survival for females with alterations. Interestingly, males with alterations to the following genes had significantly better OS outcomes: ERCC2, KDM6A, BRCA2, FGFR3. However, there was no gene whose alterations were associated with significantly better OS outcomes for females. KDM6A and FGFR3, two genes whose alterations are typically associated with worse OS outcomes in males, exhibit the opposite effect. Additionally, males exhibited significantly worse OS outcomes when presenting with CDKN2A and CDKN2B alterations, while females exhibited significantly worse OS outcomes when presenting with CDKN2A and NTRK1. A majority of European males had SNPs on the following genes: CDKN2B, ERBB2, CREBBP, while a majority of European females had SNPs on BRCA2. Differential expression also revealed 714 genes that were significantly expressed in one sex when compared to the other. Therefore, we suggest that there are unique molecular differences between sex that conflict with previous analyses; further research should be conducted to explore the effect of sex via the tumor-suppressive characteristics of KDM6A as well as oncogenic properties of FGFR3. Citation Format: Rishi Nair, Roy Khalife, Anthony Magliocco. Genetic characteristics between males and females with urothelial bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 883.

Abstract 4248: Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development

Abstract Introduction: Approximately 30% of advanced bladder cancer (BCa) tumors exhibit MTAP gene deletion, which has been associated with luminal subtype BCa, poor response to chemotherapy, and shorter progression to metastatic disease (De Souza 2023). MTAP deletion has been proposed as a potential biomarker for response to pemetrexed and an emerging class of PRMT5 and MAT2A inhibitors in development for MTAP-deleted tumors. Here, we describe the first patient-derived 3D model of BCa with MTAP deletion and its translational potential as a drug sensitivity platform. Methods: A patient (pt) with metastatic BCa harboring MTAP deletion underwent a pelvic lymph node biopsy. A fresh tumor sample was processed under collagenase/dispase solution for 30 minutes and filtered through a 70-um cell strainer. Cells were cultures in low attachment plates with MammoCult™ Basal Medium (Stem Cell Technologies). After 5 passages and 3D spheroid formation, immunostaining for cytokeratin (CK) 5 and 6, GATA3, CK20, and Ki67 staining were performed to confirm the urothelial origin. The MTAP-del was confirmed by a western blot of the organoid specimen and compared to other BLCa cell lines (UMUC, RT4, J82, and 5637). The viability assays were performed using the CellTiter-Glo® 3D Cell kit. Next-generation sequencing (NGS) of primary BCa tumor revealed deletion of MTAP, CDKN2A, CDKN2B, and mutations in p53, TERT, and KDM6A. Results: The patient organoid sample was positive for the luminal CK5/6 markers and negative for GATA3. Ki67 and CK20 were primarily expressed in the nucleus of the same sample. The organoid retained morphologic and immunohistochemical features of the patient’s primary bladder tumor, showing the same squamous differentiation and similar Ki-67 proliferative index. The organoid displayed no evidence of the MTAP protein by western blot (similar findings with the MTAP-del cell lines UMUC and RT4). The organoid showed sensitivity to pemetrexed [pemetrexed IC50=0.12 uM (other MTAP-del BLCa IC50: 0.1-0.23 uM)] and resistance to gemcitabine and cisplatin [Gem IC50=55 nM (other BLCa IC50: 1-29 nM), Cis IC50=90.5 uM (other BLCa IC50: 2-17 uM). Conclusions: We describe the first patient-derived MTAP-deleted BCa organoid recapitulating the features of the primary tumor. The organoid displayed sensitivity to pemetrexed as described with MTAP-deleted BCa. The organoid represents a unique platform for testing novel therapeutic strategies for MTAP-deleted BCa. Citation Format: Maximilian Schwermann, Benedito Carneiro, Shaolei Lu, Andre De Souza, Matthew Hadfield, Anthony Mega, Galina Lagos, Sari Khaleel, Dragan Golijanin, Sheldon Holder, Praveen Srinivasan, William MacDonald, Andrew George, Lanlan Zhou, Leiqing Zhang, Wafik El-Deiry. Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4248.

Abstract 1123: Genomic characterization of radiotherapy-associated bladder cancer

Abstract Background: Bladder cancer (BCa) is the 4th most common cancer diagnosed in men, with 82,290 new cases predicted in 2023. As BCa frequently occurs at an advanced patient age, its diagnosis may follow radiotherapy (RT) for the treatment of prostate cancer. Currently, data is limited regarding the influence of RT on the clinical course and genomic landscape of subsequent BCa, mainly due to a paucity of samples. From prior studies, pelvic radiation confers an increased risk of BCa, with resulting tumors that are largely genomically similar to non-RT tumors with the exception of an increase rate of genomic insertions. Our current study looks to expand on these findings by characterizing the genomes of the largest cohort of RT-associated bladder cancers to date. Methods: We identified BCa cases from patients with a prior history of pelvic radiation for prostate cancer, and tumor only targeted sequencing was performed using Foundation Medicine (FMI) on FFPE tissue (n=41). For control cases (CTRL), patients with no prior record of pelvic radiation and who had previously underwent standard of care FMI sequencing were included (n=41). Sequencing data were re-aligned to hg38 and mutation calling was performed on the newly aligned sequences. Results: CTRL and RT cohorts had similar distributions of patients based on race and smoking status. While cT stage was similar between the two cohorts, the CTRL cohort was biased toward higher cN and cM stages with no difference in overall survival (median OS: CTRL = 20m, RT = 23m), though the advanced stage of the CTRL cohort could mask a worse prognosis in the RT cohort. Latency specific analysis revealed short latency tumors (<10 years from RT) had a significantly shorter overall survival than long latency tumors (p<0.01, HR=5.59, CI=1.81-17.3), and a numerically, but not significantly, shorter survival than CTRL patients (p=0.17, HR=1.55, CI=0.82-2.94). Long latency tumor patients had a significantly longer overall survival than CTRL patients (p<0.01, HR = 0.22, CI=0.07-0.63). In line with prior studies, RT samples had increased numbers of insertions (p<0.01). In addition, we also detected an increase in SNVs in the RT samples (p<0.01). Of note, KDM6A alterations were enriched within the RT cohort (CTRL = 10/41, RT = 30/41, p<0.001), with series of splice site mutations exclusive to RT samples (p<0.001). Conclusion: Patients diagnosed with BCa within a short interval following pelvic radiation have a worse prognosis. Genomic analysis showed RT-associated tumors having an increased number of insertions, SNVs, and KDM6A splice site mutations compared to non-RT tumors. Overall, the spectrum of recurrent alterations within the cohorts were consistent with previous studies, with the exception of the KDM6A mutations. To our knowledge, this is the first report of a RT-associated alteration in a specific gene in bladder cancer and additional studies will be needed to validate and understand the consequences of this finding. Citation Format: N. Ari Wijetunga, Kathryn H. Gessner, Krishna Kanchi, Sara E. Wobker, David Corcoran, Matthew D. Galsky, Matthew I. Milowsky, William Y. Kim, Tracy L. Rose, Jeffrey S. Damrauer. Genomic characterization of radiotherapy-associated bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1123.

Gender disparities in clinical outcomes of urothelial carcinoma linked to X chromosome gene KDM6A mutation

ObjectiveKDM6A, a representative tumour suppressor gene with sex bias, is frequently altered in urothelial carcinoma (UC). The specific impacts of KDM6A mutations on gender-based clinical outcomes in UC remain poorly...

Kdm6a Modulates Hematopoiesis and Leukemogenesis Via Demethylase-Dependent Epigenetic Programming

Hematopoietic stem and progenitor cells (HSPCs) maintain their differentiation by precisely regulating gene expression patterns at the transcriptional and epigenetic levels. KDM6A encodes an epigenetic regulator of the repressive chromatin mark histone 3 lysine 27 tri-methylation (H3K27me3). Growing evidence suggested the involvement of KDM6A in hematological malignancies. KDM6A mutations in leukemia frequently occur in males, reflecting that KDM6A escapes X chromosome inactivation. Therefore, female patients with single-allele loss-of-function KDM6A mutations will partially retain KDM6A expression, which is called haploinsufficiency. Furthermore, KDM6A haploinsufficiency was reported to be associated with refractory and/or relapsed leukemia, while the role of KDM6A haploinsufficiency in hematopoiesis remains unexplored. We established kdm6a-null zebrafish using CRISPR-Cas9 gene editing. We found that kdm6a loss impaired HSPCs formation. kdm6a-null embryos showed reduced expression of the hemogenic endothelial and HSPC markers runx1 at 28 hours post-fertilization (hpf). Using Tg(kdrl:mCherry;cmyb:en-GFP) embryos, we also found that the number of hemogenic endothelium and emerging HSPCs in kdm6a-null was significantly decreased. To study the importance of Kdm6a-mediated demethylase activity, we ectopically expressed kdm6a constructs (wild-type kdm6a, or catalytically-dead kdm6a) in kdm6a-null embryos. The result showed that, unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for decreased HSPC numbers. Then, we aimed to understand the potential role of kdm6a haploinsufficiency in hematopoiesis and leukemogenic mechanisms. We found that zebrafish mutants that are haploinsufficient in kdm6a ( kdm6a +/-) exhibited uncontrolled myeloid differentiation. The expression of myeloid-specific markers substantially increased in kdm6a +/- mutants, compared with WT siblings at 72 hpf. Confocal analysis showed that the ratio of myeloid-primed HSPCs was markedly increased in kdm6a +/- embryos compared to WT siblings. Consistently, a significant upregulation in transcripts encoding key myeloerythorid TFs including cebps, irf8 and gata1a was observed in kdm6a +/- embryonic HSPCs. Furthermore, kdm6a +/- adults exhibited the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or exhibited the phenotype of increased progenitors, which mimics acute myeloid leukemia. We next sorted HSPCs from WT or kdm6a +/- at 72 hpf for RNA-sequencing and ATAC-sequencing. Enrichment analysis showed transcriptional dysregulation of genes involving “Purine metabolism”, “Cysteine and methionine metabolism”, “TGF-beta signaling pathway”, and “Cell cycle”. GSEA further uncovered positive enrichment of gene sets in kdm6a +/- embryonic HSPCs, including “Oxidative phosphorylation”, “mTORC1 signaling” and “DNA repair”. These dysregulated pathways in kdm6a +/- HSPCs are highly involved in cellular homeostasis and hallmarks of aged hematopoietic system. ATAC analysis also revealed robust changes in chromatin accessibility, especially including genes related to aged hematopoietic system. Integrated RNA-seq and ATAC-seq analyses showed that the chromatin accessibility at promoters and transcription of 63 genes were decreased in kdm6a +/- HSPCs. And we found that the negative regulator of Jak-Stat signaling- socs3a was on the list. Ectopic expression of socs3a in kdm6a +/- could partially rescue the uncontrolled myeloid differentiation of HSPCs. Consistently, abnormal myeloid differentiation from kdm6a +/- mutations was rescued by Jak or Stat3 inhibitors. We also tested the importance of Kdm6a-mediated demethylase activity. Unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for dysregulated expression of socs3a and key myeloerythorid genes including cebps. In conclusion, our data demonstrate the multiple roles of kdm6a in regulating HSPC homeostasis and provide new insight into the mechanism of kdm6a haploinsufficiency in uncontrolled myeloid differentiation through demethylase-dependent epigenetic programming.

Mutations in Histone Lysine Methyltransferase Genes Are Associated with Autoimmune Cytopenias

Introduction Epigenetic mechanisms are important in regulation of gene expression by modifying chromatin structure to facilitate DNA accessibility (Lau et al Nat Immunol. 2018). DNA methylation and histone modifications are critical for the pathogenesis of hematologic malignancies and autoimmune diseases (Surace et al Front Immunol. 2019) Alterations in enzymes involved in histone modification are associated with immunodeficiencies, including Kabuki syndrome (KS), a rare genetic disorder associated with autoimmune cytopenias, such as immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) (Margot et al J Am Coll Med Genet 2020) caused by loss-of-function KMT2D and KDM6A mutations. Somatic mutations in the KMT2D gene have also been observed in patients with Cold Agglutinin Disease. (Małecka et al Br J Haematol. 2018). Aims: The aims of this study are: 1) to identify the frequency of mutations in histone methyltransferase genes (MT) KMT2D, KMT2A, KMT2C and KDM6A in patients with autoimmune cytopenias, and 2) to compare clinical, laboratory and immunological characteristics, and response to therapy in patients with or without mutation in MT and autoimmune cytopenias (AIC). Methods Data mining software from the electronic medical record was used to identify patients ≥ 18 years with a diagnosis of AIC, including ITP, warm and cold AIHA, autoimmune neutropenia, and Evans Syndrome (ES), who had Next Generation Sequencing (NGS) performed. Cytopenias due to malignancy, nutritional deficiency, medications, pregnancy, or infection were excluded. Patients were categorized into two groups based on the presence or absence of mutation in the MT genes (MT+, MT-) with variant allelic frequency (VAF) > 2%. Demographic, laboratory, and clinical characteristics, treatment outcomes, and NGS sequencing data were collected and compared between groups. Next, KMT2 mutations seen in patients with AIC were cross-referenced against the 1000 Genome Project (1000GP) population database to evaluate the frequency of polymorphisms in the general population (Auton et al Nature 2015). CADD in silico prediction algorithm was used to score the potential deleterious effect of MT mutations (Kirchner Nat Genet. 2014). Chi-Square test was used for categorical variables. Statistical analysis was completed in R software. Results Among 495 patients who underwent NGS, 79 (16%) had a mutation in MT genes. These mutations were present in 36% of patients with AIC as compared to 13% in those without AIC (p=0.0005). Among 55 patients with AIC, mutations in the MT genes were present in 20 patients (MT+), 35 patients did not have these mutations (MT-). KMT2C mutations were seen in 10 (50%) patients, KMT2D mutations in 9 patients (45%, one patient had two KMT2D mutations), 1 patient (5%) had both KMT2C & KMT2D variants (Figure 1). The median VAF was 39.5% (IQR, 15.45 - 49.80). The MT+ group had a significantly higher proportion of Hispanic (60% vs 31%) and non-Hispanic White patients (25% vs 14%), p=0.038, Table 1. The median age at diagnosis for the MT+ cohort was younger compared to MT- (45.5 vs 58 years old, respectively), though not statistically significant. AIC diagnoses in both groups were ITP (44%) wAIHA (22%) cAIHA (9%) Autoimmune Neutropenia (2%), and ES (24%). Compared to the MT- group, the MT+ group had lower IgG levels (p=0.0041) and absolute lymphocyte count (p=0.041). MT+ patients with AIHA were more likely to have positive Direct Coombs and complement involvement. No significant differences were found in median nadir hemoglobin and platelet counts, need for therapy, or median number of lines of therapy. Best overall response to therapy was similar in the two groups (93%), with more complete responses in MT- group (55% vs 33%) though not statistically significant. Next, we assessed KMT2 polymorphisms in the general population. Most of the polymorphisms were not present in the 1000GP database and none had a frequency > 0.001. The average Phred score was 19.8; 12/20 patients had Phred score > 20, suggesting that most mutations are in the top 1% of the deleterious variants. Conclusion We report for the first time that patients with autoimmune cytopenias have a high frequency of variants in MT genes. Median allelic frequency close to 50% suggests potential germline predisposition to immune dysregulation and autoimmunity however further studies are needed to better understand the impact of these observations.

Impact of PTPN11 Mutations in De Novo, Secondary and Relapsed/Refractory Acute Myeloid Leukemia

Background Somatic mutations in the protein phosphatase non-receptor type 11 ( PTPN11) gene occur frequently in children with juvenile myelomonocytic leukemia and less commonly in adults with AML, being present in less than 5% of reported cases. Uncertainty exists regarding the prognostic impact of PTPN11 mutations in AML, largely owing to their observed rarity. Some retrospective studies have identified PTPN11 mutations as deleterious prognostic markers in AML, though others have noted that their negative impact is mitigated by concurrent NPM1 mutations, which frequently co-occur. We analyzed the outcomes of patients at our institution with AML and PTPN11 mutations in order to add to the literature regarding the relevance of this mutation. Methods We conducted a retrospective case series of patients at a single-center, tertiary-care institution who were diagnosed with AML between January 1 st, 2020 and June 1 st, 2023. Inclusion criteria included: 18 years of age or older, primary follow-up at the study institution, documented diagnosis of AML and identification of a PTPN11 mutation by NGS. Outcomes of interest included cytogenetic and molecular profiles, response to induction or salvage therapy, progression-free survival (PFS) and overall survival (OS). Results Twenty-two (12 men and 10 women) patients with PTPN11 mutant AML were identified. The median age at diagnosis was 60 years (range, 31-89 years). Nineteen patients had a PTPN11 mutation identified at diagnosis and 3 had a PTPN11 mutation appear at the time of relapse. Nine patients with de novo AML had PTPN11 mutations identified at diagnosis. Six were female and the average age at diagnosis was 60 years (range, 44-89 years). The most common co-occurring mutations were NPM1 (8 patients), DNMT3A (3 patients), TET2 (2 patients) and FLT3-TKD (2 patients). Cytogenetics were normal or non-adverse in 8 patients, and complex/adverse-risk in 1 patient. Seven patients received treatment - 4 with intensive chemotherapy and 3 with a hypomethylating agent (HMA) + venetoclax. Treatment response was CR/CRi in 6 patients (85.7%). At a median follow-up of 195 days, median PFS and OS were not reached. Two patients relapsed at 76 and 139 days after response. No patients received an allogeneic transplant in first remission. Ten patients with therapy-related or secondary-AML (t/s-AML) were identified, all with PTPN11 mutations noted at the time of AML diagnosis. Two patients had preceding MDS and 3 had preceding MDS/MPN. Median age was 63 years (range, 31-70 years). The most common co-occurring mutations were RUNX1 (5 patients), FLT3-ITD (2 patients) and NRAS/KRAS (3 patients). Chromosome 7 abnormalities were present in 4 of 5 patients with RUNX1 mutations. Cytogenetics were complex or adverse-risk in 9 patients. Eight patients received treatment - 6 with intensive chemotherapy and 2 with HMA + venetoclax. Treatment response was CR/CRi in 3 patients (37.5%). Median PFS was 183 days and median OS in treated patients was 307 days. The only patient with an ongoing response at the time of data cut-off received an allogeneic transplantation in CR1. Three patients with AML had emergence of a PTPN11 mutation at the time of relapse, at 245, 343 and 2262 days after initial diagnosis. PTPN11 was the only new mutation identified in 2 patients, and emerged in conjunction with an NRAS and KDM6A mutation in 1 patient. One patient received treatment, attaining a CRi after HMA + venetoclax, but relapsed 112 days later. The other 2 patients passed at 10 and 21 days after relapse. Conclusions PTPN11 mutations were relatively rare, occurring in only 22 patients with AML during a 3.5-year time span. Stark differences were noted in patients with de novo AML versus t/s-AML and PTPN11 mutations. De novo AML patients frequently demonstrated NPM1 mutations and a normal karyotype, while t/s-AML patients frequently had RUNX1 and FLT3 mutations, as well as adverse cytogenetics. Although the impact of PTPN11 mutations in AML may be influenced by concurrent NPM1 mutations or cytogenetic characteristics, the context of AML may ultimately have the largest impact.

Haematopoietic loss of KDM6A impairs cardiac recovery post myocardial infarction via pro-inflammatory myeloid cells

Abstract Background Ischaemic heart failure (IHF) remains a leading cause of death worldwide. Clonal haematopoiesis has emerged as a major risk factor for IHF and patients with CH have worse outcomes after acute myocardial infarction (AMI). CH is characterized by clonal expansion of a haematopoietic stem cell that carries a leukaemogenic mutation. Recently, acquired loss-of-function mutations in the histone demethylase KDM6A were identified as CH-drivers in IHF patients. KDM6A is frequently mutated in human cancer and acts as a tumour suppressor in acute myeloid leukaemia. However, the role of KDM6A in the regulation of cardiac damage after MI is not understood. Purpose Investigate the effect of leukocyte specific loss of KDM6A in the development of IHF after AMI in mice and humans. Methods/Results A mouse model of haematopoietic specific loss of Kdm6a (VaviCre-Kdm6aflox) combined with a model of myocardial injury was employed to investigate the role of Kdm6a in leukocytes in post-MI cardiac recovery. Firstly, following 28 days (D) post-MI, mice lacking Kdm6a in haematopoietic cells (KDM6A KO) showed significantly worse cardiac ejection fraction compared to controls (controls 43.98 ± 2.55 % vs KDM6A KO 31.86 ± 3.23 % recovery from baseline, P=0.009). This was accompanied by an increase in cardiac scar size at D28 post-MI (controls 22.46±5.25% vs KDM6A KO 56.83±11.1 % area, P<0.0001). Moreover, KDM6A KO mice showed peripheral blood monocytosis (controls 33793±5644 vs KDM6A KO 76227±14035 monocytes/ml blood, P =0.008) at D3 post-MI, which was accompanied by an increased influx of CD11b+ myeloid cells in the injured heart (7804±1023 vs 11444±1436 CD11b+ cells/mg tissue, P=0.05), as well as by an increased expression of pro-inflammatory marker TNF-α (controls 1.01±0.14 vs KDM6A KO 2.16±0.45 fold change, P=0.04) in the ischaemic myocardium. These data were corroborated by single-cell RNA sequencing (scRNA-seq) profiling of CD45+ cardiac leukocytes 3D post-MI. Here, transcriptomically distinct subsets of macrophages/monocytes showed upregulation of pro-inflammatory markers such as Il1-β, Nlrp3, Cxcl2, and Nfkb1 (P < 0.05, Bonferroni correction). Finally, scRNA-seq of circulating blood cells from IHF patients carrying KDM6A CH-driver mutations showed elevated levels of pro-inflammatory genes such as IL32, IL7R and CCL5 in CD14+ classical monocytes (P < 0.05, Bonferroni correction) compared to non-carriers. Conclusions In summary, this study shows that loss of KDM6A in myeloid cells amplifies acute cardiac inflammation and impairs recovery of left ventricular function after AMI accompanied by increased pro-inflammatory myeloid cells in the heart. Further interrogation of genes identified in the aforementioned murine and human sequencing data and subsequent in vitro mechanistic studies will be essential to identify the mechanism by which loss of KDM6A results in altered leukocyte function and worse HF prognosis post-MI.

Ipsilateral synchronous papillary renal neoplasm with reverse polarity and urothelial carcinoma in a renal transplant recipient: a rare case report with molecular analysis and literature review

BackgroundRenal transplant recipients (RTRs) have a 3- to 5-fold higher risk of developing malignant tumors than the general population, with new malignant tumors after transplantation considered to be the leading cause of death in RTRs. In pathological practice, it is rare for neoplasms with different histology to be located in the same organ. We report the first case of a synchronous papillary renal neoplasm with reverse polarity (PRNRP) and urothelial carcinoma (UC) in the ipsilateral kidney in an RTR. Molecular detection was conducted by next-generation sequencing.Case presentationA 68-year-old female suffered from uremia 19 years ago and underwent renal transplantation (RT) after receiving dialysis for 6 months. Hematuria occurred one month ago and an enhanced CT showed that there were two abnormal density foci in the middle and lower parts of the autologous left kidney. A laparoscopic left nephrectomy and ureterectomy were performed. Gross examination revealed a mass (I) in the left renal parenchyma, 2*1.8*1.5 cm in size, that protruded from the renal capsule, and a cauliflower-like mass (II), 5*2.5*2 cm in size, adjacent to the mass (I). Microscopic findings revealed these lesions were PRNRP and UC, respectively. PCR analysis revealed a KRAS gene mutation (G12D in exon 2) in the PRNRP, while NGS analysis revealed FGFR3 (S249C in exon 7) and KDM6A (Q271Ter in exon 10 and A782Lfs in exon 17) mutations in the UC.ConclusionsWe report here for the first time an extraordinarily rare case of synchronous renal tumors of a PRNRP and UC in the ipsilateral kidney of an RTR. We identified simultaneous KRAS, FGFR3, and KDM6A mutations in two different renal masses in the ipsilateral kidney. Pathologic assessment with comparative molecular analysis of mutational profiles facilitates tumor studies after RT and may be of great value in clinical management strategies.

Clinical and genomic characterisation of adenoid cystic carcinoma of the head and neck, lung, and breast.

6094 Background: Adenoid cystic carcinoma (ACC) arises most commonly within the major salivary glands and other exocrine glands. ACC is typically a slow growing disease with high rates of recurrence many years after diagnosis. To inform post-operative surveillance and to provide comparator data for non-randomised studies, we sought to perform clinical and genomic characterisation of a cohort of 450 ACC patients, comparing outcomes between salivary ACC and other primary sites. Methods: 450 patients with ACC underwent clinical review at 4 UK NHS institutions. Electronic records were reviewed to extract clinical and demographic data. In 225 patients, DNA-based next-generation sequencing was performed to identify alterations in the most frequently altered genes in ACC (NOTCH1, ARID1A, KDM6A, SETD2, or TP53). Kaplan-Meier survival analyses calculated the time to first confirmed recurrence (TTFR) and overall survival from diagnosis (OS-diag) and from first confirmed recurrence (OS-rec). Results: Of 450 ACC patients, the primary site was most frequently the major salivary gland (44.7%; 201/450) comprising parotid (86), submandibular (96), sublingual (9) and salivary NOS (12). Other primary sites were sinonasal (99/450; 22.0%), upper aerodigestive (69/450; 15.3%), tracheobronchial (46/450; 10.2%), breast (16/450; 3.6%), skin (10/450; 2.2%), lacrimal (7/450; 1.6%), and other (2/450; 0.4%). Complete survival data were available in 440 patients. Recurrent/metastatic disease was confirmed in 69.8% (314/450). For these patients, the median TTFR was 4.5 yrs and for those with recurrence, the median OS from recurrence (OS-rec) was 4.8 yrs. The median TTFR in major salivary ACC was 5.8 yrs (95% CI 4.3 – 7.0). In comparison, the shortest median TTFR was seen in tracheobronchial (2.9 yrs, 95% CI 1.6 – 6.8; p = 0.019) and sinonasal ACC (3.8 yrs, 95% CI 2.9 – 5.0; p = 0.004). Breast ACC showed a differing survival pattern compared with major salivary ACC. Although the OS-diag in these two groups was not significantly different (9.75 yrs and 11.3 yrs respectively, p = ns), breast ACC showed a significantly increased TTFR (9.8 versus 4.4 yrs, p = 0.067), and a significantly reduced OS-rec (0.75 versus 4.8 yrs, p = < 0.001). KDM6A mutation was seen more frequently in primary sites outside the major salivary glands (p = 0.005). However, there was no relationship between primary tumour site and mutations in NOTCH1, ARID1A, SETD2 or TP53. Conclusions: In this real-world multi-centre UK study, tracheobronchial and sinonasal ACC have significantly shorter TTFR than major salivary ACC. This study suggests that breast ACC has a similar OS from diagnosis to major salivary ACC, but has an increased TTFR and shorter OS following recurrence. This may have implications for follow-up protocols after primary treatment of breast ACC, and initiation of systemic therapy following recurrence.

Next-Generation Sequencing–Based Genomic Profiling of Children with Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML BFM protocols. Targeted next-generation sequencing (NGS) of 54 genes revealed 17 genes that were recurrently mutated in more than 5% of patients. Considerable differences were observed in the mutational profiles compared to previous studies as BCORL1, CUX1, KDM6A, PHF6 and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, WT1) mutations were found to be associated with induction failure and shorter event-free survival suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% vs. 44.4%) and transcription factors (35.1 vs. 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identified previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.

Abstract 4733: Effect of KDM6A mutation in bladder cancer

Abstract As of 2020, bladder cancer is the tenth most common cancer in the world, with a heavier toll exerted on males. Its higher tumor recurrence and progression rates cause major treatment challenges. One of the most frequent mutations in bladder cancer is in lysine-specific demethylase 6A (KDM6A). This gene encodes for ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), which catalyzes the demethylation of di- and tri- methylated histones H3 lysine 27 and works as part of “COMPASS-LIKE” protein complexes to coordinate many transcriptional processes. The molecular basis for how KDM6A mutations promote carcinogenesis is still under investigation. In this study, we use single-cell RNA sequencing data from bladder cancers with and without KDM6A mutations in both human and mice to examine the effects of KDM6A mutations on the cellular composition and gene expression of tumor and normal bladders. In mice without tumors, KDM6A mutations seem to correlate with enrichment of macrophages in the bladder tissue microenvironment. The opposite is true, however, in the presence of tumors, as there were not only more macrophages but also more dendritic cells in wildtype mice. This pattern was also observed in human, suggesting differences in immune infiltrates in the tumor microenvironment are part of the phenotype of KDM6A mutation. Lastly, KDM6A mutations may also impact urothelial cell states at baseline, as KDM6A KO mouse bladders contain higher proportions of intermediate cell populations compared with their wildtype counterparts. Together, these observations contribute to a better understanding of the role KDM6A plays in bladder cancer. Citation Format: Ninh B. Le, Hong Qiu, Emily E. Fink, Surbhi Sona, Angela H. Ting, Byron H. Lee. Effect of KDM6A mutation in bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4733.

Opposing effects of KDM6A and JDP2 on glucocorticoid sensitivity in T-ALL.

Glucocorticoids (GCs) are the cornerstone of acute lymphoblastic leukemia (ALL) therapy. Although mutations in NR3C1, which encodes the GC receptor (GR), and other genes involved in GC signaling occur at relapse, additional mechanisms of adaptive GC resistance are uncertain. We transplanted and treated 10 primary mouse T-lineage acute lymphoblastic leukemias (T-ALLs) initiated by retroviral insertional mutagenesis with GC dexamethasone (DEX). Multiple distinct relapsed clones from 1 such leukemia (T-ALL 8633) exhibited discrete retroviral integrations that upregulated Jdp2 expression. This leukemia harbored a Kdm6a mutation. In the human T-ALL cell line CCRF-CEM, enforced JDP2 overexpression conferred GC resistance, whereas KDM6A inactivation unexpectedly enhanced GC sensitivity. In the context of KDM6A knockout, JDP2 overexpression induced profound GC resistance, counteracting the sensitization conferred by KDM6A loss. These resistant "double mutant" cells with combined KDM6A loss and JDP2 overexpression exhibited decreased NR3C1 mRNA and GR protein upregulation upon DEX exposure. Analysis of paired samples from 2 patients with KDM6A-mutant T-ALL in a relapsed pediatric ALL cohort revealed a somatic NR3C1 mutation at relapse in 1 patient and a markedly elevated JDP2 expression in the other. Together, these data implicate JDP2 overexpression as a mechanism of adaptive GC resistance in T-ALL, which functionally interacts with KDM6A inactivation.

KDM6A Loss Triggers an Epigenetic Switch That Disrupts Urothelial Differentiation and Drives Cell Proliferation in Bladder Cancer.

A gain-of-function epigenetic switch that disrupts differentiation is triggered by inactivating KDM6A mutations in bladder cancer and can serve as a potential target for novel therapies.