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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.

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.

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. 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.