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When pancreatic cancer cannot be removed surgically, patients frequently experience morbidity and death from progression of their primary tumor. Radiation therapy (RT) cannot yet substitute for an operation because radiation causes fatal bleeding and ulceration of the nearby stomach and intestines before achieving tumor control. There are no FDA-approved medications that prevent or reduce radiation-induced gastrointestinal injury. Here, we overcome this fundamental problem of anatomy and biology with the use of the oral EGLN inhibitor FG-4592, which selectively protects the intestinal tract from radiation toxicity without protecting tumors. A total of 70 KPC mice with autochthonous pancreatic tumors received oral FG-4592 or vehicle control ± ablative RT to a cumulative 75 Gy administered in 15 daily fractions to a limited tumor field. Although ablative RT reduced complications from local tumor progression, fatal gastrointestinal bleeding was observed in 56% of mice that received high-dose RT with vehicle control. However, radiation-induced bleeding was completely ameliorated in mice that received high-dose RT with FG-4592 (0% bleeding, P < 0.0001 compared with vehicle). Furthermore, FG-4592 reduced epithelial apoptosis by half (P = 0.002) and increased intestinal microvessel density by 80% compared with vehicle controls. EGLN inhibition did not stimulate cancer growth, as treatment with FG-4592 alone, or overexpression of HIF2 within KPC tumors independently improved survival. Thus, we provide a proof of concept for the selective protection of the intestinal tract by the EGLN inhibition to enable ablative doses of cytotoxic therapy in unresectable pancreatic cancer by reducing untoward morbidity and death from radiation-induced gastrointestinal bleeding. Significance: Selective protection of the intestinal tract by EGLN inhibition enables potentially definitive doses of radiation therapy. This might allow radiation to be a surgical surrogate for unresectable pancreatic cancer.

Introduction: 16S RNA sequencing and whole-genome sequencing (WGS) techniques have been previously shown to have a significant degree of correlation, particularly for higher-order-level taxa. However, the large majority of those studies utilize data derived from samples collected in similar but not identical contexts. This project provides a unique opportunity in that both 16S RNA and WGS sequencing datasets were derived from a single fecal sample originating from each patient while undergoing chemoradiation therapy. Using this high-quality information, we investigated the correlation of these two datasets in terms of microbial composition and taxa abundances. Methods: Forty-one rectal swab samples were collected at the time of pelvic examination before the initiation of chemoradiation treatment and sequenced via Illumina platforms. Alpha and beta diversity was evaluated using PCoA ordination derived from several distance metrics (Bray-Curtis, Jaccard, Weighted and Unweighted UniFrac, and Euclidean). Diversity measures from different data analysis tools were also compared to highlight differences in data processing. Taxa with the highest relative abundances within each set of sequencing data were used to evaluate consensus between the datasets. Results: Alpha diversity assessed by different measures from WGS analysis correlated (Spearman rho value) with the same measures derived from 16S rRNA sequencing. These measures include OTU abundances (rho = 0.763, p = 6.61e−09), Shannon diversity (rho = 0.809, p = 4.40e−09), and InvSimpson (rho = 0.797, p = 2.11e−08). Beta diversity analysis highlighted a greater level of similarity within WGS samples than 16sRNA samples and a high degree of dissimilarity between the two datasets. Among the top fourteen most abundant taxa identified in 16S rRNA sequencing, the four best correlated with WGS results were Faecalibacterium, rho = 0.475, p = 1.68e−03; Mollicutes, rho = 0.302, p = 5.48e−02; Clostridiales, rho = 0.021, p = 8.98e−01; and Bacteria, rho = 0.016, p = 9.23e−01. The remaining ten taxa were negatively correlated. Conclusions: The use of whole-genome sequencing approaches in microbiome analysis is expanding in order to provide greater depth of information about microbial function. However, comparisons of results from 16S rRNA sequencing and WGS will need to be evaluated with caution due to differences in the interpretation of relative abundances of specific taxa. Future efforts may help to better harmonize these analyses to facilitate comparisons across platforms. Citation Format: Greyson Biegert, Xiaogang Wu, Tatiana V. Karpinets, Melissa Mezzari, Jianhua Zhang, Lauren Colbert, Ann Klopp. Comparisons of microbial composition identified via 16s rRNA sequencing and whole-genome sequence-based analysis using gut samples for patients with cervical cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B11.

Rationale: Pancreatic cancer is almost always fatal since chemotherapy is only modestly effective and surgery is often not possible. Radiation therapy (RT) can be a definitive treatment in localized pancreatic cancer, but gastrointestinal toxicity to the nearby duodenum limits its efficacy. There are no known medications that can prevent or treat this gastrointestinal radiotoxicity, but we find that FG-4592, a small molecule inhibitor of EGLN proteins, reduces radiation damage to the intestines by selectively enhancing HIF expression within normal intestines but not within tumors. Methods: We developed a murine model of spontaneous pancreatic cancer (KrasLSL/+; Trp53FL/+; Ptf1aCre/+) that behaves similarly to locally advanced disease. KPC mice were screened for tumors by weekly ultrasounds and upon diagnosis were sequentially assigned to receive one of four possible treatments: Vehicle only (VEH), FG-4592 (FG), RT concurrent with vehicle (RT+VEH), or RT concurrent with FG-4592 (RT+FG). Mice were enrolled on a rolling basis and the total time from diagnosis to treatment end was approximately three weeks. Radiation treatments were planned to administer a total of 75Gy in 15 daily fractions. Results: A total of 70 KPC mice were evaluated. The administration of high-dose radiation therapy of >65Gy (high-dose) with or without radioprotection improved survival compared to all animals that received ≤65Gy (low-dose) of treatment or no RT (37 days [95% CI 33-41] vs. 15 days [10-21] vs. 15 days [10-20], respectively; p=0.005). KPC animals that received dose-escalated radiation lived significantly longer compared to unirradiated controls that received vehicle or FG-4592 alone. The median overall survival was compared amongst the treatment groups and was highest (p Citation Format: Cullen M. Taniguchi, Tara N. Fujimoto, Lauren E. Colbert, Jessica M. Moilkentine, Amit Deorukhkar, Laura J. Baseler, Ramesh Tailor, Gabriel O. Sawakuchi. EGLN inhibition reduces gastrointestinal radiation toxicity and improves survival in a murine model of locally advanced pancreatic cancer [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 979.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor outcomes with current therapies. Gemcitabine is the primary adjuvant drug used clinically, but its effectiveness is limited. In this study, our objective was to use a rationale-driven approach to identify novel biomarkers for outcome in patients with early-stage resected PDAC treated with adjuvant gemcitabine. Using a synthetic lethal screen in human PDAC cells, we identified 93 genes, including 55 genes linked to DNA damage responses (DDR), that demonstrated gemcitabine sensitization when silenced, including CHD7, which functions in chromatin remodeling. CHD7 depletion sensitized PDAC cells to gemcitabine and delayed their growth in tumor xenografts. Moreover, CHD7 silencing impaired ATR-dependent phosphorylation of CHK1 and increased DNA damage induced by gemcitabine. CHD7 was dysregulated, ranking above the 90th percentile in differential expression in a panel of PDAC clinical specimens, highlighting its potential as a biomarker. Immunohistochemical analysis of specimens from 59 patients with resected PDAC receiving adjuvant gemcitabine revealed that low CHD7 expression was associated with increased recurrence-free survival (RFS) and overall survival (OS), in univariate and multivariate analyses. Notably, CHD7 expression was not associated with RFS or OS for patients not receiving gemcitabine. Thus, low CHD7 expression was correlated selectively with gemcitabine sensitivity in this patient population. These results supported our rationale-driven strategy to exploit dysregulated DDR pathways in PDAC to identify genetic determinants of gemcitabine sensitivity, identifying CHD7 as a novel biomarker candidate to evaluate further for individualizing PDAC treatment. Cancer Res; 74(10); 2677–87. ©2014 AACR.

Background/ Objectives Chromodomain helicase DNA Binding protein 7 (CHD7) modulates chromatin remodeling during genome maintenance. Its role in pancreatic cancer (PAC) is unestablished, but preliminary data for Gemcitabine (GEM) sensitivity in pancreatic cell lines and pilot clinical data is promising. This study will evaluate CHD7 as a prognostic and predictive factor in resected PAC using 1) immunohistochemistry (IHC) of patient samples from RTOG 97-04, 2) prospective evaluation of CHD7 over-expression and treatment response in patients receiving FOLFIRINOX and GEM-based chemoradiotherapy (CRT) for locally advanced PAC at our institution and 3) a Phase II molecular-driven randomized trial of personalized therapy based on CHD7 expression. Methodology A loss of function siRNA screen identified genes, including CHD7, that conferred GEM sensitivity when silenced using GEM-treated Mia PaCa-2 cells. CHD7 expression was assessed by IHC scoring in 80 patients who underwent curative intent resection of PAC between 1/2000 and 10/2008. Kaplan-meier survival analysis was performed for recurrence-free survival (RFS) and overall survival (OS). Univariate (UV) and Multivariate (MV) Cox regression analyses using clinically relevant covariates were used to correlate CHD7 expression levels with RFS and OS in patients receiving GEM therapy (n=42). This analysis will be validated in 200 patient samples from RTOG 97-04. CHD7 expression will be correlated with RFS and OS in GEM and 5-Fluorouracil (5-FU) treatment arms. CHD7 expression will also be measured in pre-chemotherapy core biopsies from locally advanced PAC patients treated at our institution. Patients will receive either two cycles of FOLFIRINOX (FOL) followed by GEM-based CRT at a dose of 1000mg/m2 once weekly or two cycles of FOL followed by Capecitabine (CAP)-based CRT. CHD7 expression will again be correlated with survival. These results will be followed by an ECOG- sponsored multi-institutional Phase II randomized trial of FOL and GEM-based CRT or FOL and CAP-based CRT based on CHD7 gene expression. Preliminary Data/ Anticipated Results In patients receiving GEM (n=42), high CHD7 was associated with poor RFS (7 months vs 15 months; p=.025) and poor OS (10 months vs 18 months; p=.015). On MV analysis, high CHD7 expression remained a predictor of poor RFS (HR 8.2 [95% CI 2.4-28]; p=.001) and poor OS (HR 11.6 [95% CI 3.4-40); p We expect our RTOG 97-04 validation study to demonstrate stronger correlation of low CHD7 expression with improved RFS and OS in GEM-treated patients. Similarly, we expect our institutional trial to demonstrate improved RFS and OS with low CHD7 in the GEM-treated arm. Citation Format: Lauren E. Colbert, William A. Hall, Claire W. Hardy, Sarah B. Fisher, David S. Yu, Shishir K. Maithel, Bassel El-Rayes, Burcu Saka, N Volkan Adsay, Aleksandra Petrova, Brooke Pantazides, Jeanne Kowalski, Khanjan Gandhi, David Kooby, Charles Staley, Jerome C. Landry. A pilot clinical trial of chromodomain-helicase-DNA-binding protein 7 (CHD7) expression as a prognostic and predictive biomarker in patients with early-stage pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-168. doi:10.1158/1538-7445.AM2013-LB-168