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BackgroundBoth modified FOLFIRINOX (mFFX) and gemcitabine/nab-paclitaxel chemotherapy regimens have been shown to improve clinical outcomes in patients with pancreatic cancer, and are often used interchangeably as the standard of care. Preclinical studies often do not use these regimens, since administering these multiagent approaches can be difficult. In this study, we assessed the feasibility of administering these two chemotherapy regimens in spontaneous pancreatic tumors using KPC mice with the ultimate goal of advancing preclinical studies.MethodsKPC mice were created by breeding KrasLSL-G12D/+ to Trp53fl/fl;Ptf1αCre/+, resulting in KrasLSL-G12D/+;p53fl/+;Ptf1αCre/+ mice. At 14 weeks of age, mice were palpated for spontaneous tumor growth that was verified using ultrasounds. Mice with tumors under 15 mm in diameter were used. The mice were assigned to one of seven treatment regimens: 1 cycle of mFOLFIRINOX (FFX X1), 2 cycles of mFOLFIRINOX (FFX X2), 1 cycle of mFOLFIRINOX with 40Gy SBRT (FFX SBRT), 1 cycle of gemcitabine/nab-paclitaxel (GEM/AB X1), 2 cycles of gemcitabine/nab-paclitaxel (GEM/AB X2), 2 cycles of gemcitabine/nab-paclitaxel with 40Gy SBRT (GEM/AB SBRT), or no chemotherapy treatment (control).ResultsIn total, 92 mice were included. The median OS in the FFX X2 group was slightly longer that the median OS in the FFX X1 group (15 days vs 11 days, P=0.003). Mice in the GEM/AB X2 group had longer OS when compared to mice in the GEM/AB X1 group (33.5 vs 13 days, P=0.001). Mice treated with chemotherapy survived longer than untreated control animals (median OS: 6.5 days, P P ConclusionsWe demonstrate the utility and feasibility of clinicall relevant mFOLFIRINOX and gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer.

Purpose Chemotherapy combined with radiation therapy is the most commonly used approach for treating locally advanced pancreatic cancer. The use of curative doses of radiation in this disease setting is constrained because of the close proximity of the head of the pancreas to the duodenum. The purpose of this study was to determine whether fasting protects the duodenum from high-dose radiation, thereby enabling dose escalation for efficient killing of pancreatic tumor cells. Methods and Materials C57BL/6J mice were either fed or fasted for 24 hours and then exposed to total abdominal radiation at 11.5 Gy. Food intake, body weight, overall health, and survival were monitored. Small intestines were harvested at various time points after radiation, and villi length, crypt depth, and number of crypts per millimeter of intestine were determined. Immunohistochemistry was performed to assess apoptosis and double-strand DNA breaks, and microcolony assays were performed to determine intestinal stem cell regeneration capacity. A syngeneic KPC model of pancreatic cancer was used to determine the effects of fasting on the radiation responses of both pancreatic cancer and host intestinal tissues. Results We demonstrated that a 24-hour fast in mice improved intestinal stem cell regeneration, as revealed by microcolony assay, and improved host survival of lethal doses of total abdominal irradiation compared with fed controls. Fasting also improved survival of mice with orthotopic pancreatic tumors subjected to lethal abdominal radiation compared with controls with free access to food. Furthermore, fasting did not affect tumor cell killing by radiation therapy and enhanced γ-H2AX staining after radiation therapy, suggesting an additional mild radiosensitizing effect. Conclusions These results establish proof of concept for fasting as a dose-escalation strategy, enabling ablative radiation in the treatment of unresectable pancreatic cancer.

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.

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.

Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5 Gy × 5 fractions (total of 62.5 Gy, EQD2 = 140.6 Gy). This radioprotection enables ablative radiation therapy in a mouse model of pancreatic cancer and nearly triples the median survival compared to controls. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

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.

Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5Gy x 5 fractions (total of 62.5Gy, D2EQ=140.6Gy), which would likely ablate pancreatic cancer. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

Locally advanced pancreatic cancer (LAPC) almost always fatal since it is unresectable and chemotherapy is only modestly effective. The efficacy of radiation therapy (RT) for LAPC is limited by the potentially fatal toxicity to nearby intestines. There are no FDA-approved medications that can prevent this radiotoxicity, but we find that FG-4592, a small molecule inhibitor of EGLN proteins, significantly reduces radiation damage to the intestines without radioprotecting tumors. KPC (KrasLSL/+; Trp53FL/+; Ptf1aCre/+) animals received dose-escalated radiation treatments with and without FG-4592 for radioprotection. High-dose RT reduced death from local progression, improved survival, and shifted the patterns of failure to a late metastatic death compared to controls. The addition of FG-4592 to RT further improved survival compared to vehicle controls by eliminating radiation-induced gastrointestinal toxicity. Thus, selective protection of the intestinal tract by EGLN inhibition may enable higher, and potentially definitive doses of cytotoxic therapy to be delivered to LAPC.One Sentence SummaryThe EGLN inhibitor FG-4592 allows higher, and potentially definitive, doses of radiation to be delivered to pancreatic cancer by reducing normal tissue toxicity without protecting tumors.

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 cancer is the fourth leading cause of cancer-related deaths in the US. Surgical resection is the only potentially curative treatment; however, only 15%-20% of patients present with tumors that can be resected. There is no consensus regarding standard of care in unresectable cases, however many academic centers use stereotactic body radiotherapy (SBRT) to give tumor-directed radiotherapy (RT). Unfortunately, even this conformal technique can still cause severe gastrointestinal (GI) toxic effects caused by the proximity of the pancreatic head to the duodenum. Protecting the intestine from the toxic effects of radiation may enable dose escalation that could achieve more effective local control of disease. We and others have previously shown that a prolonged fast of 24 hours protects mice from lethal doses of etoposide. In this study, we extend and build on our previous finding to demonstrate that a similar 24 hour fast also protects from lethal doses of total abdominal radiation. Histologic analyses, using the Withers-Elkind microcolony assay, show that fasting protected small intestinal (SI) stem cells from radiation damage and promoted early regeneration. To show a proof-of-principle for the use of this radioporotective maneuver in cancer therapy, we developed an orthotopic model of pancreatic cancer using KPC tumor cells syngeneic to C57BL/6. Here, we show that fasting-mediated intestinal protection enabled dose escalated SBRT for treatment of these orthotopic tumors. RT with fasting radioprotection delayed tumor growth and improved survival compared to controls. Given this robust phenotype, we developed a 3D culture ex vivo assay using intestinal stem cell enriched epithelial spheroid cultures. We modified these intestinal spheroids with a bioluminescent reporter and used these cells to develop a modified clonogenic assay for 3D culture that can be used to identify novel radioprotectors, such as a fasting mimetic. Taken together, these results suggest that fasting protects small intestinal stem cells sufficiently to allow animals to receive potentially curative doses of abdominal radiation that would other wise be lethal. Future work will aim to identifying the mechanism by which fasting confers intestinal protection and drug candidates that can be used to mimic this fasting-mediated protection. Citation Format: Marimar de la Cruz Bonilla, Kristina M. Stemler, Tara N. Fujimoto, Sabrina Jeter-Jones, Jessica M. Molkentine, Gabriela M. Asencio Torres, Cullen M. Taniguchi, Helen Piwnica-Worms. Fasting protects mice from lethal radiation by promoting small intestinal stem cell survival [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 4164.

273 Background: There are no useful preclinical models that resemble relevant fractionated radiation, such as SBRT, with relevant chemotherapy. Most preclinical studies use a single fraction of radiation to large fields, causing significant toxicity and/or insufficient dose which leads to accelerated repopulation and worsened outcomes. Here, we employ mice with spontaneous pancreatic tumors, and treat them in a preclinical trial utilizing gemcitabine/nab-paclitaxel +/-SBRT. Methods: Mice with an activated KRas (G12D) allele and heterozygous for p53 expression only in the pancreas (KPC) were created and spontaneous tumors were diagnosed via weekly palpation and ultrasound. Mice with single tumors that were between 3-7mm at diagnosis were sequentially randomized to received either no treatment, gemcitabine/nab-paclitaxel x 2 weeks, or gemcitabine/nab-paclitaxel x 2 weeks followed by 8Gy x 5 daily fractions of SBRT to the pancreatic tumor. SBRT was delivered using X-RAD 225Cx (Precision X-Ray, Inc) small animal irradiator under image guidance by cone beam micro CT using AP/PA technique with a 10mm collimator. Every mouse was subjected to necropsy and a cause of death assigned. Tumor growth was monitored by small animal ultrasound. Kaplan-Meier survival analysis was performed to assess efficacy of treatments. Results: A total of 40 mice were enrolled with 12 receiving chemotherapy alone, 13 receiving chemotherapy +SBRT and 15 mice had no further treatment. The addition of induction chemotherapy improves lifespan compared to untreated mice. The addition of SBRT further improved survival. Causes of death in untreated animals of the gemcitabine/nab-paclitaxel group were largely from local progression, whereas the addition of SBRT provided local control causing most of the mice in this cohort to die from metastatic progression. Conclusions: SBRT improves survival when added to gem/nab-paclitaxel in a preclinical model, which may reflect favorable biology for this approach. Our novel system employs similar SBRT fields and doses, and may thus be useful for the evaluation of other chemotherapeutic regimens with SBRT.