Matthew S. Koletsky

Sequencing of pediatric gliomas has identified missense mutations Lys27Met (K27M) and Gly34Arg/Val (G34R/V) in genes encoding histone H3.3 (H3F3A) and H3.1 (HIST3H1B). We report that human diffuse intrinsic pontine gliomas (DIPGs) containing the K27M mutation display significantly lower overall amounts of H3 with trimethylated lysine 27 (H3K27me3) and that histone H3K27M transgenes are sufficient to reduce the amounts of H3K27me3 in vitro and in vivo. We find that H3K27M inhibits the enzymatic activity of the Polycomb repressive complex 2 through interaction with the EZH2 subunit. In addition, transgenes containing lysine-to-methionine substitutions at other known methylated lysines (H3K9 and H3K36) are sufficient to cause specific reduction in methylation through inhibition of SET-domain enzymes. We propose that K-to-M substitutions may represent a mechanism to alter epigenetic states in a variety of pathologies.

The commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX, DAXX, and MEN1. We genotyped 64 PanNETs and found 58% carry ATRX, DAXX, and MEN1 mutations (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis to reveal two distinct subgroups with one consisting entirely of A-D-M mutant PanNETs. Two genes differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha-cells (FDR q-value < 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells.

Abstract The most commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX , DAXX , and MEN1 . Little is known about the cells-of-origin for non-functional neuroendocrine tumors. Here, we genotyped 64 PanNETs for mutations in ATRX , DAXX , and MEN1 and found 37 tumors (58%) carry mutations in these three genes (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis on 33 randomly selected cases to reveal two distinct subgroups with one group consisting entirely of A-D-M mutant PanNETs. Two biomarkers differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX gene expression and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha cells (pval &lt; 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells.

Abstract Pediatric gliomas such as glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG) are aggressive brain tumors for which there is no effective therapy. The reason for the poor therapeutic outcome for children diagnosed with glioblastomas might be due to the therapeutic approach, which is not significantly different from the treatment of adult gliomas. Recent studies have shown that the gene expression profile of pediatric gliomas is distinct from histologically similar tumors in adults. These findings strongly suggest that the underlying mechanisms of tumor progression are different in these gliomas. Whole-genome sequencing of these pediatric tumors has revealed mutations in genes of H3F3A and HIST1H3B encoding histone H3.3 or H3.1, respectively. The mutations resulted in a substitution of lysine (K) to methionine (M) at position 27 of the protein (H3K27M). The lysine 27 residue of histone H3 is a posttranslational modification site, methylated by the polycomb repressive complex 2 (PRC2). A mutation like H3K27M might play a significant role in tumorigenesis and tumor progression since histones and their posttranslational modifications are key epigenetic regulators. In collaboration with the laboratory of David Allis, we raised antibodies against the K27M mutation of histone H3. Using this antibody, Lewis et al. corroborated the presence of H3 K27M protein in DIPG samples containing the mutant allele. Furthermore, they provided evidence that this mutation can alter the molecular processes in the nucleus through a gain-of-function mechanism in glioblastoma. Here we show that this Anti-Histone H3, K27M mutant antibody (Cat. No.: ABE419), together with other modified histone antibodies, are useful tools for the characterization and classification of different type of brain tumors and can be used in several applications including immunoblotting and chromatin immunoprecipitation (ChIP). Citation Format: Anita Lozsa, Robin T. Clark, Peter W. Lewis, Matthew S. Koletsky, Oren J. Becher, John Rosenfeld, Sturges Michael, David C. Allis, Trinette Chuang, Wayne Speckmann, Alejandra Solache. A specific antibody for Histone H3 Lys27Met mutation for the characterization of pediatric glioblastomas. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A32.

Abstract Pancreatic neuroendocrine tumors (PanNETs) or islet cell tumors are a rare neuroendocrine malignancy with an annual incidence of less than 1 per 100,000 per year. Current classification scheme for PanNETs include grade and stage. While well-differentiated PanNETs can be successfully treated with surgery, there are few treatments for metastatic PanNETs. A greater understanding of the cells of origin of PanNETs, tumor progression and pathway pathogenesis may guide the development of novel therapeutic options. The most commonly mutated genes in PanNETs are ATRX, DAXX, and MEN1. Little is known about the cells-of-origin for non-functional neuroendocrine tumors. Here, we genotyped 64 PanNETs for mutations in ATRX, DAXX, and MEN1 and found 37 tumors (58%) carry mutations in these three genes (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing (n=33) and Illumina 450K DNA methylation (n=32) analysis on randomly selected PanNETs to reveal two distinct subgroups with one group consisting exclusively of A-D-M mutant PanNETs. Pair-wise correlation of gene expression between all PanNETs, showed A-D-M mutant PanNETs are more homogeneous as a group than A-D-M WT PanNETs. Among A-D-M mutant PanNETs, mutants with the same genotype (mutations in ATRX/DAXX/MEN1) did not show greater gene expression correlation. Two biomarkers differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX gene expression and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature similar to that of alpha cells (pval &amp;lt; 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. We validated our subtype classification and A-D-M mutant panNET alpha signature using two independent PanNETs expression dataset. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells. Citation Format: Saurabh V. Laddha, Chang S. Chan, Peter Lewis, Matthew Koletsky, Kenneth Robzyk, Edaise Da Silva, Paula J. Torres, Brian Untch, Promita Bose, Timothy Chan, David S. Klimstra, David C. Allis, Laura H. Tang. ATRX, DAXX or MEN1 mutant pancreatic neuroendocrine tumors are a distinct “alpha-cell signature” subgroup [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1799.