The data presented in this study support the repurposing of mebendazole as a combination treatment with radiation therapy in TNBC patients.

No abstract

Despite aggressive treatments, pancreatic ductal adenocarcinoma (PDAC) remains an intractable disease, largely because it is refractory to therapeutic interventions. To overcome its nutrient-poor microenvironment, PDAC heavily relies on autophagy for metabolic needs to promote tumor growth and survival. Here, we explore autophagy inhibition as a method to enhance the effects of radiotherapy on PDAC tumors. Hydroxychloroquine is an autophagy inhibitor at the focus of many PDAC clinical trials, including in combination with radiotherapy. However, its acid-labile properties likely reduce its intratumoral efficacy. Here, we demonstrate that EAD1, a synthesized analogue of HCQ, is a more effective therapeutic for sensitizing PDAC tumors of various KRAS mutations to radiotherapy. Specifically, in vitro models show that EAD1 is an effective inhibitor of autophagic flux in PDAC cells, accompanied by a potent inhibition of proliferation. When combined with radiotherapy, EAD1 is consistently superior to HCQ not only as a single agent, but also in radiosensitizing PDAC cells, and perhaps most importantly, in decreasing the self-renewal capacity of PDAC cancer stem cells (PCSC). The more pronounced sensitizing effects of autophagy inhibitors on pancreatic stem over differentiated cells points to a new understanding that PCSCs may be more dependent on autophagy to counter the effects of radiation toxicity, a potential mechanism explaining the resistance of PCSCs to radiotherapy. Finally, in vivo subcutaneous tumor models demonstrate that combination of radiotherapy and EAD1 is the most successful at controlling tumor growth. The models also confirmed a similar toxicity profile between EAD1 and Hydroxychloroquine.

The current 5-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is ~8%. The dismal prognosis in PDAC reflects in part an exceptional level of resistance to available therapies. Approximately half of PDAC patients present with localized tumors amenable to local therapies, though only a minority are candidates for potentially curative surgery due to unresectable disease, which leaves radiation therapy (RT) as the only other option. The clinical reality is that two thirds of all PDAC patients succumb to local disease burden regardless of clinical stage at diagnosis. Therefore, improving the effectiveness of PDAC RT has the potential to transform overall outcomes for this lethal disease. More than a century after its discovery, RT remains a powerful therapeutic agent against many cancers, but has largely failed to offer much benefit to PDAC patients according to randomized, controlled clinical trials. The biologic reasons for such exceptional radiation resistance remain obscure. However, studies show that activating KRAS mutations in PDAC drive a NRF2-controlled antioxidant program that endows tumor cells with a reduced intracellular environment and lower levels of reactive oxygen species (ROS). Such persistent NRF2 pathway activation would poise PDAC cells to resist oxidative stress induced by radiation and in all likelihood limits the effectiveness of RT in the clinic. Supporting this hypothesis, our study shows that radiation activates the NRF2 pathway, as well as autophagy, and inhibition of either pathway sensitizes PDAC cells to radiation. Moreover, the radiosensitizing effect is enhanced when both the NRF2 and autophagy pathways are inhibited and, perhaps most importantly, NRF2 or autophagy inhibition radiosensitizes the notoriously therapy-resistant PDAC cancer stem cells. Supporting a role for NRF2 in protecting PDAC cells from radiation-induced oxidative stress, irradiated PDAC cells with depleted NRF2 have elevated ROS levels compared to NRF2-wt controls. Interestingly, irradiated PDAC cells reprogram their glucose and glutamine metabolism in an NRF2-dependent manner and seem to reroute glucose through the antioxidant pentose phosphate pathway (PPP), likely for the generation of reducing equivalents in the form of NADPH. This suggests that NRF2 drives metabolic rewiring in irradiated PDAC cells in favor of cellular antioxidant responses. Taken together, our data strongly suggest that NRF2 plays a pivotal role in promoting radiation resistance of KRAS-mutated PDAC tumors by driving powerful antioxidant responses through metabolic reprogramming and cytoprotective autophagy. If this is correct, the NRF2-autophagy-metabolism axis may be targeted therapeutically to reverse the chemo/radioresistant phenotype of PDAC. Citation Format: Justine Bailleul, Taha Yazal, David Sung, Daisy Palomera, Anahita Sehgal, Amy Dao, Erina Vlashi. NRF2 drives metabolic reprogramming in irradiated pancreatic cancer cells and promotes radioresistance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C59.

CD31 immunofluoresence staining was performed on tumor sections to measure endothelial cell area. Results: Among miRs targeting SMPD1, miR-15a exhibited the greatest differential change in HUVECs at 6 hours post-IR between low and high dose radiation. Lower dose was associated with higher miR-15a and vice versa. Exogenous miR-15a mimic significantly decreased SMPD1 mRNA and protein expression. miR-15a levels were reciprocal in magnitude to SMPD1 mRNA levels. miR-15a inhibition decreased cell proliferation in both HUVECs and CT26 colorectal cells and increased apoptosis when combined with radiation. Associated with increased apoptosis, miR-15a inhibition significantly increased Fas expression in HUVECs compared to radiation alone. Further, miR-15a inhibition increased inflammasomeassociated caspase 1, GSDMD expression, and inflammatory CXCL10 production. Both systemic and vascular targeted delivery of miR-15a inhibitor significantly diminished tumor growth and decreased endothelial cell density in vivo in our CRC flank tumor model. Conclusion: Acting upon the vasculature, our data demonstrates miR-15a inhibition increases SMPD1 in ECs, decreases proliferation in vitro and in vivo, and stimulates pyroptosis to abrogate tumor growth.