Zhou, Xin

Abstract BACKGROUND Medulloblastoma (MB) is a highly malignant childhood cerebellar tumor comprising a collection of molecularly and clinically distinct entities. Group 3 and Group 4-MB subgroups originate from overlapping progenitor pools of the developing rhombic lip when neuronal differentiation hierarchies are subverted through somatic alterations. Mutations suspected to deregulate the activity of chromatin-modifying genes are pervasive in Group 3 and Group 4-MB. However, the molecular consequences of these alterations remain largely undefined. METHODS We characterized the chromatin landscape of fifty-two primary MBs using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for six histone modifications and performed multi-modal integration with sample-matched somatic lesions, genome-wide DNA methylation, and transcriptomic profiles. RESULTS Analysis of differential chromatin states across MB subgroups using a Hidden Markov Model revealed a significant enrichment of the bivalent enhancer state (EnhBiv; marked by coincident H3K4me1 and H3K27me3) in Group 4-MB which localized to promoters of neurodevelopmental genes and transcription factors. To functionally dissect the pathogenetic significance of the enriched EnhBiv state in Group 4-MB, we sought to identify key regulators that confer this state. Integrative bioinformatics coupled with CUT&RUN analysis revealed significant enrichment of KDM2B, a histone lysine demethylase overexpressed in Group 4-MB, in gene promoters marked by the EnhBiv state. CRISPR gene targeting or targeted protein degradation of KDM2B selectively suppressed growth of Group 3 and Group 4-MB models in vitro and in vivo. Mechanistically, inhibition of KDM2B disrupted chromatin bivalency by blocking PRC2 complex recruitment and reducing H3K27me3 deposition, profoundly derepressing PRC2 targets involved in neuronal differentiation. CONCLUSIONS Our comprehensive characterization of the MB chromatin landscape in a large cohort of primary tumors provides unprecedented insights into the epigenetic basis of MB subgroups, implicating novel mechanisms of tumorigenesis, and nominating KDM2B as a selective dependency in Group 3/4-MB that should be prioritized as an attractive therapeutic target.

Abstract CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. CIViC contains peer-reviewed, published literature curated and expertly-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViC’s functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new Evidence Types (Predisposing, Oncogenic and Functional). The growing CIViC knowledgebase has over 300 contributors and distributes clinically-relevant cancer variant data currently representing >3200 variants in >470 genes from >3100 publications.

Abstract Introduction B progenitor acute lymphoblastic leukemia (B-ALL) is a leading cause of childhood cancer death. Many chimeric genes have been identified and led to a refined classification of B-ALL and tailored therapies. Still, up to 30% of B-ALL cannot be classified into established subtypes, and the outcome for many is poor. Methods To identify novel subtypes of B-ALL, we performed integrative genomic analysis including transcriptome sequencing (RNA-seq) of 1,988 cases from St. Jude, Children's Oncology Group and adult cooperative group studies and analyzed chromosomal rearrangements, gene-expression profiles (GEP), somatic mutations and chromosome-level copy-number alterations. Cases lacking known or putative subtype-defining alterations underwent whole genome sequencing. Effects on proliferation and transformation of novel lesions were assessed by retroviral expression in cell lines and point-mutation knock-in mice using CRISPR/Cas9 genome editing. Results Using integrated genetic alterations and gene expression profiling, we classified 23 B-ALL subtypes (Table and Figure). Three groups included cases with similar GEP as canonical subtypes (ETV6-RUNX1, KMT2A-rearranged, and ZNF384-rearranged), but lacking the expected drivers (e.g., ETV6-RUNX1-like, n=42). Eighteen cases (0.9%) had rearrangements of BCL2, MYC and/or BCL6 showing a distinct GEP; they were mostly adults (n=16) with very poor outcome. These alterations, rarely seen in ALL, are identical to those observed in "double/triple hit" lymphoma, and are of pre-B immunophenotype. Eight cases with tightly clustered GEP comprised a novel subtype defined by IKZF1 N159Y missense mutation. N159Y is in the DNA-binding domain of IKZF1, and is known to perturb IKZF1 function, with distinct nuclear mislocalization and induction of aberrant intercellular adhesion. We identified two subtypes with distinct GEP characterized by PAX5 alterations. One, herein termed PAX5 altered (PAX5alt), comprised 148 (7.4%) cases, was characterized by diverse PAX5 alterations including rearrangements (n=57), sequence mutations (n=46) and/or focal intragenic amplifications (n=8). These PAX5 alterations were found in 73.6% of PAX5alt cases and different alteration types were mutually exclusive. Other PAX5 alterations, including deletions and large-scale amplifications were also assessed using SNP array, but were not enriched in the PAX5alt group. Clinically, PAX5alt pediatric and adult patients had favorable (96.8±3.2%) and intermediate (42.1±10.2%) 5-year overall survival (OS), respectively. The other GEP distinct subtype comprised 44 cases, all with PAX5 P80R missense mutations. In 30 of these cases, PAX5 P80R was homozygous due to deletion of the wild-type (WT) PAX5 allele or copy-neutral loss of heterozygosity. Of the other 14 cases with heterozygous PAX5 P80R mutations, 13 had a second frameshift (n=7), nonsense (n=2) or deleterious missense (n=4) PAX5 mutation. Four of the remaining 1,944 cases also had the PAX5 P80R mutation, but all were heterozygous with preservation of a WT PAX5 allele, consistent with the notion that homozygous or compound heterozygous PAX5 P80R mutation is the hallmark of this subtype. Adult PAX5 P80R cases (n=14) showed better 5-year OS (61.9±13.4%) than those in PAX5alt subtype (42.1±10.2%). To examine the effects of PAX5 P80R on B-cell maturation, WT PAX5, PAX5 P80R, V26G and P34Q were expressed in Pax5-/- lineage-depleted bone marrow cells. Expression of WT PAX5, PAX5 V26G and P34Q resulted in near complete rescue of B cell differentiation; however, expression of PAX5 P80R blocked the differentiation at the pre-pro-B stage of B-cell maturation. Further, Pax5 P80R heterozygous or homozygous mice developed pre-B-ALL with a median latency of 166 and 87 days, respectively, with heterozygous mice acquiring alterations on the second allele. In contrast, Pax5+/- mice, and those harboring G183S mutation observed in familial leukemia, do not spontaneously develop B-ALL. Conclusions These results show the utility of transcriptome sequencing in defining subtypes and founding genetic alterations in B-ALL, provide a revised taxonomy of the disease across the age spectrum, and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis. Disclosures McKay: ImmunoGen Inc.: Employment. Tallman:Orsenix: Other: Advisory board; AROG: Research Funding; BioSight: Other: Advisory board; Cellerant: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Konopleva:Stemline Therapeutics: Research Funding. Relling:Shire Pharmaceuticals: Research Funding. Mullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.