Li, Hu

Abstract BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. Significance: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.

Abstract Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of B-cell Non-Hodgkin Lymphoma (NHL), representing a third of all new cases. DLBCL is further sub-divided into various molecular sub-types based on gene expression and co-occurring genetic alterations. Gene expression based- subtypes include the germinal center B-cell (GCB) and the activated B-cell type (ABC) subtypes, with the ABC sub-type having a poorer prognosis than the GCB sub-type. Interestingly, 30-40% of all DLBCL patients harbor mutations in key epigenetic regulators, EZH2, KMT2D, CREBBP, EP300, and mutations in histone proteins themselves. Through an unbiased cell-based phenotypic screen, we discovered that inhibition of lysine demethylases, specifically KDM4C and KDM4A, represents a vulnerability across 15 different DLBCL cell lines including germinal center B-cell (GCB) and activated B-cell (ABC) type lines, with a GI50 value between 75nM to ~200nM, while sparing leukemia lines. Consistently, treatment of xenograft-based animal models of DLBCL with a low dose of KDM4A/KDM4C inhibitor delivered intra-peritoneally three times a week, results in a drastic reduction of tumor burden. Both KDM4A and KDM4C catalyze the removal of histone H3 K9 di- and tri-methylation (H3K9me2/3) and H3K36 di- and tri-methylation (H3K36me2/3). H3K9me2/3 is associated with promoter and enhancer repression, while H3K36me2/3 is present in gene bodies during transcription but also functions as a chromatin repressor Consistently, we have identified key enhancers, including those associated with IKZF1, that are "decommissioned" after inhibition of KDM4A/KDM4C. Repressed enhancer activity, through loss of H3K27 acetylation and H3K4me1, and gain of H3K9me2/3, results in a rapid transcriptional downregulation of IKZF1 and its partners including IKZF3. Given the role of IKZF1 in the transcriptional regulation of key B Cell Receptor (BCR) signaling components, we show that KDM4A/KDM4C inhibition leads to a downregulation of SYK, a proximal BCR-signaling component, which likely precedes DLBCL cell apoptosis. In addition, we observed an activation of extra-lineage transcription factors such as CEBPA and CEBPB, which are normally repressed by IKZF1 in the lymphoid lineage. A concomitant downregulation of the B-cell gene expression program and an upregulation of the myeloid (CD14+ monocytic) gene expression program is also observed, implying a "trans-differentiation" of DLBCL cells into the monocyte lineage. This lineage-switch correlates with an increased population of CD14+ expressing cells. Finally, using DLBCL patient data sets, we can show that over-expression of either KDM4A or KDM4C is associated with poor prognosis in DLBCL patients. In summary, we have discovered that KDM4A/KDM4C inhibition results in an increase of repressive histone modifications at several intra-genic "enhancers" of genes that are responsible for the survival and proliferation of DLBCL cells. The elucidation of this unique epigenetic mechanism provides a strong rationale for the development of novel targeted therapies against both multiple subtypes of DLBCL. Figure. Figure. Disclosures Shipp: Bayer: Research Funding; AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Neuroblastoma is an embryonal tumor arising in the peripheral sympathetic nervous system (PSNS), and often presents with widespread metastasis at diagnosis. We recently demonstrated that overexpression of LMO1 can synergize with MYCN to promote tumor metastasis in our zebrafish model system. Here, we identify a “metastasis suppressor” gene – growth arrest specific 7 (GAS7) – whose loss of function or reduced expression can promote dissemination and migration of neuroblastoma cells with MYCN overexpression, both in vitro and in vivo. GAS7 resides on chromosome 17p13.1, a region that is deleted in high-risk neuroblastomas with higher frequency in MYCN-amplified cases. Expression levels of GAS7 are also significantly downregulated in MYCN-amplified or otherwise MYCN-overexpressing human neuroblastoma cells, as well as MYCN-overexpressing zebrafish PSNS cells, suggesting an inverse relationship between the oncogenic activities of MYCN and GAS7downregulation. To determine whether loss of function or reduced expression of GAS7 could contribute to neuroblastoma pathogenesis in the context of MYCN overexpression, we developed a gas7 knockout zebrafish line using a transcription activator-like effector nucleases (TALEN) method. Loss of function of gas7 promoted hematogenous metastasis of MYCN-overexpressing tumor cells to tissues and organs that paralleled the common sites of metastasis in neuroblastoma patients. Further RNA-sequencing and qRT-PCR analyses showed that representative signature genes encoding proteins involved in tumor cell-cell or cell-extracellular matrix interactions were significantly downregulated in fish tumors with loss of function of gas7, consistent with our electron microscopic finding in the fish model showing a loss of contact among tumor cells in fish lacking functional gas7, suggesting that GAS7 may physically act as a metastasis suppressor. Thus, in high-risk neuroblastoma with MYCNamplification or overexpression, we propose that deletion or reduced expression of GAS7 cooperates with MYCN to promote neuroblastoma cell dissemination. Although the precise mechanism of this interaction remains unclear, we suggest that it relies, in part, on the downregulation of pivotal genes involved in tumor cell-cell or cell-extracellular matrix contacts. Citation Format: Zhiwei Dong, Gonzalo Lepez Garcia, Cheng Zhang, Erin N. Dankert, Jo Lynne Harenza, Xiaonan Hou, Hu Li, John M. Maris, Shizhen (Jane) Zhu. Loss of function of GAS7 promotes neuroblastoma metastasis in the context of MYCN overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-127.

e14237 Background: Targeting myeloid-derived suppressor cells (MDSCs), key mediators of intrinsic and adaptive immune resistance, remains a significant challenge in kidney cancer immunotherapy. One potential driver of MDSC mediated tumor immunosuppression is the cytokine interleukin-1 beta (IL-1β). Although large cancer prevention studies show that targeting IL1 decreases cancer risk, the exact mechanism of anti-IL-1 mediated anti-tumor efficacy is not yet defined. To understand the immunogenic effects of IL-1β blockade and effects on MDSCs, we used the RENCA model of RCC. Methods: We inoculated BALB/c mice with 1x106 RENCA cells. On Day +10 we treated mice (n = 8/group) with vehicle, αPD1, αIL1β, cabozantinib, combination αPD1/αIL1β, or cabozantinib/αIL1β. We harvested day +18 tumors to quantify tumor size and immune cells by flow cytometry. We then isolated immune cells for differential gene expression profiling by RNAseq. Results: Treatment with αIL1β significantly decreased tumor size compared to vehicle or αPD1 monotherapy. Combining αPD1/αIL1β or cabozantinib/αIL1β potentiated the anti-tumor effects of αPD1 or cabozantinib monotherapy respectively. Anti-IL1β therapy decreased polymorphonuclear (PMN)-MDSC infiltration (αIL1β: 0.76% +/- 0.21 vs. vehicle: 1.89% +/- 0.37, P = 0.014). Relative to PD1 monotherapy, combining αPD1 and αIL1β decreased PMN-MDSCs (αPD1: 2.14% +/- 0.56 vs αPD1/αIL1β: 0.91% +/- 0.15, P = 0.033) and increased M1-like tumor associated macrophages (TAM) (αPD1: 0.798% +/- 0.21 vs. αPD1/αIL1β: 12.04% +/- 2.9, P = 0.0008). Gene expression profiling from sorted immune cells from αIL1β treated tumors revealed multiple differentially regulated cytokines including interleukin-6, IFN-γ, and IL-10. Conclusions: Blockade of IL-1β depletes intratumoral PMN-MDSCs and increases M1-like TAM with minimal effects observed on the T cell compartment. Treatment with cabozantinib or αPD1 in combination with αIL1β led to sustained control of tumor growth. IL-1β represents a promising target for kidney cancer immunotherapy. Based on the preclinical data, a clinical trial of neoadjuvant αPD1 and αIL1β therapy is planned for patients with high risk RCC.

Deleterious variants in dihydropyrimidine dehydrogenase (DPD, DPYD gene) can be highly predictive of clinical toxicity to the widely prescribed chemotherapeutic 5‐fluorouracil (5‐FU). However, there are very limited data pertaining to the functional consequences of the >450 reported no‐synonymous DPYD variants. We developed a DPYD‐specific variant classifier (DPYD‐Varifier) using machine learning and in vitro functional data for 156 missense DPYD variants. The developed model showed 85% accuracy and outperformed other in silico prediction tools. An examination of feature importance within the model provided additional insight into functional aspects of the DPD protein relevant to 5‐FU toxicity. In the absence of clinical data for unstudied variants, prediction tools like DPYD‐Varifier have great potential to individualize medicine and improve the clinical decision‐making process.

Background— Genetic studies of cardiomyopathy and heart failure have limited throughput in mammalian models. Adult zebrafish have been recently pursued as a vertebrate model with higher throughput, but genetic conservation must be tested. Methods and Results— We conducted transcriptome analysis of zebrafish heart and searched for fish homologues of 51 known human dilated cardiomyopathy–associated genes. We also identified genes with high cardiac expression and genes with differential expression between embryonic and adult stages. Among tested genes, 30 had a single zebrafish orthologue, 14 had 2 homologues, and 5 had ≥3 homologues. By analyzing the expression data on the basis of cardiac abundance and enrichment hypotheses, we identified a single zebrafish gene for 14 of 19 multiple-homologue genes and 2 zebrafish homologues of high priority for ACTC1 . Of note, our data suggested vmhc and vmhcl as functional zebrafish orthologues for human genes MYH6 and MYH7 , respectively, which are established molecular markers for cardiac remodeling. Conclusions— Most known genes for human dilated cardiomyopathy have a corresponding zebrafish orthologue, which supports the use of zebrafish as a conserved vertebrate model. Definition of the cardiac transcriptome and fetal gene program will facilitate systems biology studies of dilated cardiomyopathy in zebrafish.

Whole exome sequencing (WES) provides an unprecedented opportunity to identify the potential aetiological role of rare functional variants in human complex diseases. Large-scale collaborations have generated germline WES data on patients with a number of diseases, especially cancer, but less often on healthy controls under the same sequencing procedures. These data can be a valuable resource for identifying new disease susceptibility loci if study designs are appropriately applied. This review describes suggested strategies and technical considerations when focusing on case-only study designs that use WES data in complex disease scenarios. These include variant filtering based on frequency and functionality, gene prioritisation, interrogation of different data types and targeted sequencing validation. We propose that if case-only WES designs were applied in an appropriate manner, new susceptibility genes containing rare variants for human complex diseases can be detected.