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Radiation therapy for abdominal tumors is challenging because the small intestine is exquisitely radiosensitive. Unfortunately, there are no FDA-approved therapies to prevent or mitigate GI radiotoxicity. The EGLN protein family are oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs). Our group has previously shown that stabilization of HIF2 through genetic deletion or pharmacologic inhibition of the EGLNs mitigates and protects against GI radiotoxicity in mice by improving intestinal crypt stem cell survival. Here we aimed to elucidate the molecular mechanisms by which HIF2 confers GI radioprotection. We developed duodenal organoids from mice, transiently overexpressed non-degradable HIF2, and performed bulk RNA sequencing. Interestingly, HIF2 upregulated known radiation modulators and genes involved in GI homeostasis, including Wnt5a. Non-canonical Wnt5a signaling has been shown by other groups to improve intestinal crypt regeneration in response to injury. Here we show that HIF2 drives Wnt5a expression in multiple duodenal organoid models. Luciferase reporter assays performed in human cells showed that HIF2 directly activates the WNT5A promoter via a hypoxia response element. We then evaluated crypt regeneration using spheroid formation assays. Duodenal organoids that were pre-treated with recombinant Wnt5a had a higher cryptogenic capacity after irradiation, compared to vehicle-treated organoids. Conversely, we found that Wnt5a knockout decreased the cryptogenic potential of intestinal stem cells following irradiation. Treatment with recombinant Wnt5a prior to irradiation rescued the cryptogenic capacity of Wnt5a knockout organoids, indicating that Wnt5a is necessary and sufficient for duodenal radioprotection. Taken together, our results suggest that HIF2 radioprotects the GI tract by inducing Wnt5a expression.

Purpose Patients with pancreatic cancer often receive radiation therapy before undergoing surgical resection. We compared the clinical outcomes differences between stereotactic body radiation therapy (SBRT) and 3-dimensional (3D)/intensity-modulated radiation therapy (IMRT). Methods and Materials We retrospectively collected data from the University of Texas MD Anderson Cancer Center. Patients with borderline resectable/potentially resectable or locally advanced pancreatic cancer receiving neoadjuvant SBRT (median, 36.0 Gy/5fx), 3D conformal radiation (median, 50.4 Gy/28 fx) or IMRT (median, 50.4 Gy/28 fx) were included. Overall survival (OS) and progression-free survival were analyzed using Cox regression. Results In total, 104 patients were included in our study. Fifty-seven patients (54.8%) were treated with SBRT, and 47 patients (45.2%) were treated with 3D/IMRT. Patients in the SBRT group were slightly older (median age: 70.3 vs 62.7 in the 3D/IMRT group). Both groups had similar proportions of patients with locally advanced pancreatic cancer (SBRT: 30, 52.6%; 3D/IMRT: 24, 51.1%). All patients were treated with chemotherapy. Patients in the SBRT group underwent more surgical resection compared with the 3D/IMRT group (38.6% vs 23.4%, respectively). At a median follow-up of 22 months, a total of 60 patients (57.7%) died: 25 (25/57, 43.9%) in the SBRT group, and 35 (35/47, 74.5%) in the 3D/IMRT group. Median OS was slightly higher in the SBRT group (29.6 months vs 24.1 months in the 3D/IMRT group). On multivariable Cox regression, the choice of radiation therapy technique was not associated with differences in OS (adjusted hazard ratios [aHR] = 0.5; 95% confidence interval [CI], 0.2%-1.3%, P = .18). Moreover, patients that underwent surgical resection had better OS (aHR = 0.3, 95% CI, 0.1%-0.8%, P = .01). Furthermore, progression-free survival was also similar between patients treated with SBRT and those treated with 3D/IMRT (aHR = 0.9, 95% CI, 0.5%-1.8%, P = .81) Conclusions SBRT was associated with similar clinical outcomes compared with conventional radiation techniques, despite being delivered over a shorter period of time which would spare patients prolonged treatment burden. Future prospective data are still needed to better assess the role of SBRT in patients with pancreatic cancer.

IMPORTANCEPatients with localized squamous cell carcinoma of the anus (SCCA) who experience treatment toxicity or recurrences have few therapeutic options.Investigation into the microbiome’s influence on treatment toxicity or its potential use as a predictive biomarker in this rare disease could improve these patients’ outcomes.OBJECTIVETo longitudinally characterize the SCCA tumor microbiome and assess its association with treatment-related toxicities.DESIGNProspective cohort study.SETTINGSingle tertiary cancer center.PARTICIPANTSTwenty-two patients with biopsy-confirmed non-metastatic SCCA receiving standard-of-care chemoradiotherapy as part of an Institutional Review Board-approved study from April 2017 to July 2019.MAIN OUTCOMES AND MEASURESDiversity and taxonomic characterization of the SCCA microbiome throughout chemoradiotherapy using swab-based anorectal microbial specimen collection and 16S rRNA gene sequencing.RESULTSTwenty-two SCCA patients were included in this study with a median (range) age of 58.5 (39-77), and 18 (82%) were women. Alpha diversity remained relatively stable throughout chemoradiotherapy, except for decreases in the Chao1 (P=0.03) and Observed Features (P=0.03) indices at week 5 relative to baseline. Tumor microbial compositions measured using weighted UniFrac changed significantly by the end of treatment (P=0.03). Linear discriminant analysis effect sizes revealed differential enrichment of bacteria at specific time points, including the enrichment of Clostridia at both baseline and follow-up and the enrichment of Corynebacterium at week 5. Patients experiencing high toxicity at week 5 had higher relative abundances of Clostridia, Actinobacteria, and Clostridiales at baseline (P=0.03 for all).CONCLUSIONS AND RELEVANCEOur study presents the first longitudinal characterization of the SCCA microbiome throughout chemoradiation. The tumor microbiome undergoes significant changes during and after chemoradiotherapy, and patient-reported toxicity levels are associated with patients’ microbial profiles. Further studies into these microbial characterizations and associations are needed to elucidate the tumor microbiome’s role in predicting treatment-related outcomes for SCCA patients.Key PointsQUESTIONHow does the squamous cell anal tumor microbiome change during chemoradiotherapy, and how do these changes influence treatment-related toxicity?FINDINGSProspective longitudinal characterization of anal cancer patients revealed significant modulation of the local tumor microbiome in response to chemoradiotherapy including shifts in overall composition and differential enrichment of key taxa.Additionally, enrichment of specific taxa at baseline was associated with increased levels of treatment-related toxicities by the end of treatment.MEANINGThe anal tumor microbiome is significantly altered by chemoradiotherapy and could potentially serve as a biomarker for treatment-related toxicities.

Mitochondria are dynamic organelles that constantly alter their shape through the recruitment of specialized proteins, like mitofusin-2 (Mfn2) and dynamin-related protein 1 (Drp1). Mfn2 induces the fusion of nearby mitochondria, while Drp1 mediates mitochondrial fission. We previously found that the genetic or pharmacological activation of mitochondrial fusion was tumor suppressive against pancreatic ductal adenocarcinoma (PDAC) in several model systems [1]. The mechanisms of how these different inducers of mitochondrial fusion reduce pancreatic cancer growth are still unknown. Here, we characterized and compared the metabolic reprogramming of these three independent methods of inducing mitochondrial fusion in KPC cells: overexpression of Mfn2, genetic editing of Drp1, or treatment with leflunomide. We identified significantly altered metabolites via robust, orthogonal statistical analyses and found that mitochondrial fusion consistently produces alterations in the metabolism of amino acids. Our unbiased methodology revealed that metabolic perturbations were similar across all these methods of inducing mitochondrial fusion, proposing a common pathway for metabolic targeting with other drugs.

Background & AimsPancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma, which contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown.MethodsWe used a dual recombinase mouse model to delete Hif1α or Hif2α in α-smooth muscle actin (αSMA)-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF-Hif2α expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, a HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade.ResultsCAF-specific deletion of HIF2, but not HIF1, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = 0.0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models.ConclusionsTogether, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.

Purpose Localized pancreatic cancer is commonly treated with stereotactic body radiation therapy (SBRT), which often requires the placement of fiducial markers. We compared the clinical outcomes of patients with and without fiducial markers. Methods and Materials We retrospectively collected data on patients with pancreatic cancer treated with neoadjuvant SBRT at a single institution. Patients were divided into 2 groups based on the placement of a fiducial marker. Local recurrence was the primary outcome. Time to event endpoints were analyzed using COX regression. Results We included 96 patients with unresectable pancreatic cancer: 46 patients (47.9%) did not have a fiducial marker, and 50 patients (52.1%) had a fiducial placed. Patients in the fiducial group were older and had more locally advanced pancreatic cancer compared with those who did not have a fiducial placed. Most patients in both groups (92.7%) received chemotherapy before SBRT treatment. SBRT was delivered to a median of 36 Gy over 5 fractions in the no-fiducial group, and 38 Gy over 5 fractions in the fiducial group. At a median follow-up of 20 months, local recurrence was similar irrespective of fiducial placement (adjusted hazard ratio [aHR] 0.6, 95% CI 0.3-1.3, P = .59). Furthermore, no difference in overall survival was noted between the 2 groups (aHR 0.8, 95% CI 0.3-1.9, P = .65). In patients who eventually underwent surgery post-SBRT, no difference in surgical margins (P = .40) or lymphovascular invasion (P = .76) was noted between the 2 groups. No patient developed acute pancreatitis after fiducial placement. Conclusions Our data suggest that the use of fiducial markers does not negatively affect clinical outcomes in patients with localized pancreatic cancer. Prospective confirmation of our results is still needed.

Although bariatric surgery is proven to sustain weight loss in morbidly obese patients, long-term adverse effects have yet to be fully characterized. This study compared the long-term consequences of two common forms of bariatric surgery: one-anastomosis gastric bypass (OAGB) and Roux-en-Y Gastric Bypass (RYGB) in a preclinical rat model. We evaluated the influence of biliopancreatic limb (BPL) length, malabsorption, and bile acid (BA) reflux on esogastric mucosa. After 30 weeks of follow-up, Wistar rats operated on RYGB, OAGB with a short BPL (15 cm, OAGB-15), or a long BPL (35 cm, OAGB-35), and unoperated rats exhibit no cases of esogastric cancer, metaplasia, dysplasia, or Barrett’s esophagus. Compared to RYGB, OAGB-35 rats presented higher rate of esophagitis, fundic gastritis and perianastomotic foveolar hyperplasia. OAGB-35 rats also revealed the greatest weight loss and malabsorption. On the contrary, BA concentrations were the highest in the residual gastric pouch of OAGB-15 rats. Yet, no association could be established between the esogastric lesions and malabsorption, weight loss, or gastric bile acid concentrations. In conclusion, RYGB results in a better long-term outcome than OAGB, as chronic signs of biliary reflux or reactional gastritis were reported post-OAGB even after reducing the BPL length in a preclinical rat model.

Despite ongoing efforts, patients with locally advanced pancreatic cancer (LAPC) continue to have a dismal prognosis. Such tumors are unresectable, and optimal treatment with chemotherapy and/or radiation therapy is still not established. While chemotherapy is conventionally aimed at preventing metastatic spread of disease, radiation therapy acts locally, improving local control which can potentially improve overall survival and most importantly quality of life. Here, we aim to review the primary literature assessing the role of diverse radiation therapy strategies for patients with LAPC. Many radiation regimens can be considered, and no standard treatment has demonstrated a clear improvement in clinical outcomes. We advise that the modality of choice be dependent on the availability of equipment, the dose and fractionation of treatment, as well as the dose received by normal tissue. Moreover, a candid discussion with the patient concerning treatment goals is equally as essential. Three notable strategies for LAPC are intensity-modulated radiation therapy, volumetric modulated arc therapy, and proton. These radiation modalities tend to have improved dose distribution to the target volumes, while minimizing the radiation dose to surrounding normal tissues. Stereotactic body radiation therapy can also be considered in LAPC patients in cases where the tumor does not invade the duodenum or other neighboring structures. Because of the high doses delivered by stereotactic body radiation therapy, proper respiratory and tumor motion management should be implemented to reduce collateral radiation dosing. Despite improved clinical outcomes with modern radiation modalities, evolving techniques, and more accurate planning, future studies remain essential to elucidate the optimal role for radiation therapy among patients with LAPC.

PURPOSE/OBJECTIVE(S) Patients with pancreatic cancer often receive radiation therapy before undergoing surgical resection. We compared the clinical outcomes differences between SBRT and 3D/IMRT after receiving current, modern standard-of-care chemotherapy. MATERIALS/METHODS We retrospectively identified patients with borderline resectable or locally advanced pancreatic cancer treated with neoadjuvant SBRT (median: 36.0Gy/5fx), 3D-CRT (median: 50.4Gy/28fx) or IMRT (median: 50.4Gy/28fx) at one institution 2015-2020. Overall survival (OS) and progression-free survival (PFS) were analyzed using COX regression. RESULTS In total, 111 patients were included. Sixty patients (54.1%) were treated with SBRT, and 51 patients (45.9%) were treated with 3D/IMRT. Patients in the SBRT group were slightly older (median age: 70.0 vs 62.7 in the 3D/IMRT group). Both groups had similar proportions of patients with locally advanced pancreatic cancer (SBRT: 32, 53.3%; 3D/IMRT: 27, 52.9%). All patients were treated with chemotherapy. Patients in the SBRT group underwent slightly more surgical resection when compared to the 3D/IMRT group (36.7% vs 21.6%, respectively, P = 0.08). At a median follow-up of 22 months, a total of 65 patients (58.6%) died: 28 (28/60, 46.7%) in the SBRT group, and 37 (37/51, 72.5%) in the 3D/IMRT group. Median OS was slightly higher in the SBRT group (28.7 months vs 24.2 months in the 3D/IMRT group). On multivariable COX regression, the choice of radiation therapy technique was not associated with differences in OS (aHR = 0.6, 95% CI [0.3-1.1], P = 0.10). Moreover, patients that underwent surgical resection had better OS (aHR = 0.3, P = 0.003). Furthermore, PFS was also similar between patients treated with SBRT and those treated with 3D/IMRT (aHR = 0.8, P = 0.31). CONCLUSION SBRT was associated with similar clinical outcomes when compared to conventional radiation techniques, despite being delivered over a shorter period of time which would spare patients prolonged treatment burden. Future prospective data are still needed to better assess the role of SBRT in patients with pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) requires mitochondrial oxidative phosphorylation (OXPHOS) to fuel its growth; however, broadly inhibiting this pathway might also disrupt essential mitochondrial functions in normal tissues. PDAC cells exhibit abnormally fragmented mitochondria that are essential to the oncogenicity of PDAC, but it was unclear if this mitochondrial feature was a valid therapeutic target. Here, we present evidence that normalizing the fragmented mitochondria of pancreatic cancer via the process of mitochondrial fusion reduces OXPHOS, which correlates with suppressed tumor growth and improved survival in preclinical models. Mitochondrial fusion was achieved by genetic or pharmacologic inhibition of dynamin-related protein-1 (Drp1) or through overexpression of mitofusin-2 (Mfn2). Notably, we found that oral leflunomide, an FDA-approved arthritis drug, promoted a 2-fold increase in Mfn2 expression in tumors and was repurposed as a chemotherapeutic agent, improving the median survival of mice with spontaneous tumors by 50% compared with vehicle. We found that the chief tumor-suppressive mechanism of mitochondrial fusion was enhanced mitophagy, which proportionally reduced mitochondrial mass and ATP production. These data suggest that mitochondrial fusion is a specific and druggable regulator of pancreatic cancer growth that could be rapidly translated to the clinic.

Simple Summary Cancer therapies should improve patient survival or at least improve the quality of their life as they receive treatment for their disease. This is particularly important in pancreatic cancer, where current treatments often have to balance between limiting tumor growth and minimizing patient toxicity. There has been an increasing appreciation among physicians to capture the patient’s voice using tools called patient-reported outcome measures (PROM). In this article, we describe the available PROMs and their relative strengths and weaknesses to help oncologists make sense of this rapidly growing field. Finally, we present a decision-making tool that can help researchers and clinicians select the ideal PROM that fits their needs. Abstract Pancreatic cancer and its treatment often dramatically impact patients’ quality of life (QoL). Given this, as well as increased focus on QoL measures in clinical oncology, there has been a rise in the number of instruments that measure patient-reported outcomes (PROs). In this review, we describe the landscape of different PRO instruments pertaining to pancreatic cancer, with specific emphasis on PRO findings related to pancreatic cancer patients receiving radiotherapy (RT). Twenty-five of the most commonly utilized PROs are compared in detail. Notably, most of the PRO tools discussed are not specific to pancreatic cancer but are generic and have been used in various malignancies. Published findings concerning PROs in pancreatic cancer involving RT are also extracted and summarized. Among the measures used, the European Organization for Research and Treatment Cancer QLQ-C30 was the most commonly utilized. We recommend a careful selection of PRO measures in clinical pancreatic cancer research and care and encourage the use of a combination of symptom-specific and global QoL tools to more fully capture patients’ perspectives.

Background: Pancreatic ductal adenocarcinoma (PDAC) is driven by activating Kras mutations which cause a fragmented mitochondrial phenotype. We recently demonstrated that leflunomide, an FDA-approved drug for rheumatoid arthritis, could be repurposed for pancreatic cancer therapy. The mode of action is through the activation of mitochondrial fusion, which inhibits PDAC growth in a KRAS-dependent fashion. Leflunomide also has the off-target effect of inhibiting dihydroorotate dehydrogenase (DHODH), which is critical for pyrimidine biosynthesis. Gemcitabine is part of the standard of care for pancreatic cancer and also inhibits nucleoside biosynthesis. Accordingly, we hypothesized that leflunomide might synergize with gemcitabine chemotherapy to further improve outcomes. In this pre-clinical study, we attempt to provide proof-of-concept for combining leflunomide with gemcitabine/nab-paclitaxel, the current gold standard for PDAC, in order to characterize a synergistic effect and to provide evidence for potentially repurposing the drug for clinical use. Methods: We heterotopically implanted patient-derived pancreatic cancer cell lines with an activating KRAS mutations (MDA-PATC148) or WT KRAS (MDA-PATC153) into the flanks of immunocompromised (NOD-SCID) mice. The tumors were grown to 5mm in size then assigned to one of four treatment groups: (1) vehicle control, (2) gemcitabine/nab-paclitaxel only, and (3) leflunomide + gemcitabine/nab-paclitaxel. Leflunomide was dosed at 20 mg/kg and given concurrently with gemcitabine and nab-paclitaxel. Gemcitabine was administered IP 2x/week at 100 mg/kg and nab-paclitaxel given once per week by tail vein at 10 mg/kg. Results: The combination of leflunomide with gemcitabine/nab-paclitaxel synergistically impeded tumor proliferation and reduced the median tumor volume by more than 50% when compared to vehicle (102.6 vs. 294.8 mm3, ****P-value < 0.0001 by Mann-Whitney U test) or gemcitabine/nab-paclitaxel only group (102.6 mm3 vs. 211.7 mm3, **P-value < 0.01 by Mann-Whitney U test) in KRAS mutant PATC148 mice. Interestingly, the addition of leflunomide to gemcitabine/nab-paclitaxel did not further improve tumor responses in KRAS wild-type PATC153 tumors, suggesting a dependence on mutant KRAS for therapeutic efficacy of the repurposed mitochondrial fusion actiator, leflunomide. Conclusions: Leflunomide exhibits therapeutic synergy with standard-of-care gemcitabine/nab-paclitaxel in patient-derived PDAC lines that harbor KRAS mutations, but are ineffective in KRAS WT lines, suggesting that this combination might be effective not only in PDAC, but in other cancer with mutant KRAS, such as cancers of colon, lung, or bile ducts. Citation Format: Nicholas D. Nguyen, Meifang Yu, Tara N. Fujimoto, Abagail Delahoussaye, Yanqing Huang, Manuel A. Estrada, Cullen M. Taniguchi. Mitochondrial fusion exerts KRAS-dependent therapeutic synergy with gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 555.