Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of pre-existing subclones, remains unclear. In EGFR-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires AURKA activity. Non-genetic resistance through the activation of AURKA by its co-activator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in pre-clinical models. Treatment induced activation of AURKA was associated with resistance to EGFR inhibitors in-vitro, in-vivo and in individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA driven residual disease and acquired resistance.

In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types.

Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies. In the case of KRAS, we identify that published synthetic lethal screen hits significantly overlap at the pathway rather than gene level. Analysis of pathways encoded as protein networks could identify synthetic lethal candidates that are more reproducible than those previously reported. Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes are strongly modulated by changes in cellular conditions or genetic context, the latter determined using a pairwise genetic interaction map that identifies numerous interactions that suppress synthetic lethal effects. Accounting for pathway, cellular and genetic context nominates a DNA repair dependency in KRAS-mutant cells, mediated by a network containing BRCA1. We provide evidence for why most reported synthetic lethals are not reproducible which is addressable using a multi-faceted testing framework.

UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)–TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs.

Pancreatic ductal adenocarcinomas (PDACs) have enhanced nutrient uptake requirements and rapid metabolic processing. The enzyme UDP-glucose pyrophosphorylase 2 (UGP2) rests at the convergence of multiple metabolic pathways, however the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an essential role for UGP2 in the maintenance of PDAC growth in both in vitro and in vivo tumor models. Transcription of UGP2 is directly regulated by the YAP/TEAD complex. Loss of UGP2 leads to decreased intracellular glycogen and defects in N-glycosylation targets important for cell growth including epidermal growth factor receptor (EGFR). In murine xenograft models, knockdown of UGP2 halted tumor growth and repressed expression of EGFR. The critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer new avenues of therapy for otherwise intractable PDACs.

Radiotherapy plays an essential role in the treatment of more than half of all patients with cancer. In recent decades, advances in devices that deliver radiation and the development of treatment planning software have helped radiotherapy attain precise tumour targeting with minimal toxicity to surrounding tissues. Simultaneously, as more targeted drug therapies are being brought into the market, there has been significant interest in improving cure rates for cancer by adding drugs to radiotherapy to widen the therapeutic window, the difference between normal tissue toxicity and treatment efficacy. The development of new combination therapies will require judicious adaptation of preclinical models that are routinely used for traditional drug discovery. Here we highlight the strengths and weaknesses of each of these preclinical models and discuss how they can be used optimally to identify new and clinically beneficial drugeradiotherapy combinations.

Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies. Using KRAS as a model, we identified that published synthetic lethal screens significantly overlap at the pathway rather than gene level. Analysis of pathways encoded as protein networks identified synthetic lethal candidates that were more reproducible than those previously reported. Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes were strongly modulated by changes in cellular conditions or genetic context, the latter determined using a pairwise genetic interaction map that identified numerous interactions that suppress synthetic lethal effects. Accounting for pathway, cellular and genetic context nominates a DNA repair dependency in KRAS-mutant cells, mediated by a network containing BRCA1. We provide evidence for why most reported synthetic lethals are not reproducible which is addressable using a multi-faceted testing framework. STATEMENT OF SIGNIFICANCESynthetic lethal screens have the power to identify new targets in cancer, although have thus far come up short of expectation. We use computational and experimental approaches to delineate principles for identifying robust synthetic lethal targets that could aid in the development of effective new therapeutic strategies for genetically defined cancers.

UDP-glucose pyrophosphorylase 2 (UGP2) is a lynchpin metabolic enzyme that rests at the convergence of multiple metabolic pathways regulating both glycogen synthesis and glycosylation modifications. Here we elucidated the essential role of UGP2-mediated glycosylation in the maintenance of KRAS-driven cancer using both in vitro and in vivo models. A key effector of KRAS oncogenic function is the transcription factor YAP. We identified the YAP/TEAD transcription factor complex as a major regulator of UGP2 mRNA expression and enzymatic activity using genomic perturbations, correlative protein immunohistochemistry in PDAC samples, and ChIP-seq and targeted ChIP-qPCR at the UGP2 promoter. Loss of YAP or UGP2 leads to a decrease in glycogen and defects in key glycosylation targets such as EGFR. In murine xenograft models using pancreatic cancer lines, knockdown of UGP2 prevented tumor growth and decreased expression of the critical glycosylation target EGFR. This essential role for UGP2 in cancer maintenance may lead to new avenues of therapy in otherwise intractable KRAS-driven cancers. Citation Format: Andrew L. Wolfe, Jacqueline Galeas, Eneda Toska, Qing Zhou, Angel Ku, Richard P. Koche, Sourav Bandyopadhyay, Carlito B. Lebrilla, Maurizio Scaltriti, Frank McCormick, Sung Eun Kim. UGP2 is a critical regulator of protein glycosylation in 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 1470.