Faculty Bibliography

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options¹. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families². We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases³. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

Ncoa4 mediates autophagic degradation of ferritin, the cytosolic iron storage complex, to maintain intracellular iron homeostasis. Recent evidence also supports a role for Ncoa4 in systemic iron homeostasis and erythropoiesis. However, the specific contribution and temporal importance of Ncoa4-mediated ferritinophagy in regulating systemic iron homeostasis and erythropoiesis is unclear. Here, we show that Ncoa4 has a critical role in basal systemic iron homeostasis and both cell autonomous and non-autonomous roles in murine erythropoiesis. Using an inducible murine model of Ncoa4 knockout, acute systemic disruption of Ncoa4 impaired systemic iron homeostasis leading to tissue ferritin and iron accumulation, a decrease in serum iron, and anemia. Mice acutely depleted of Ncoa4 engaged the Hif2α-erythropoietin system to compensate for anemia. Mice with targeted deletion of Ncoa4 specifically in the erythroid compartment developed a pronounced anemia in the immediate postnatal stage, a mild hypochromic microcytic anemia at adult stages, and were more sensitive to hemolysis with higher requirements for the Hif2α-Epo axis and extra-medullary erythropoiesis during recovery. These studies demonstrate the importance of Ncoa4-mediated ferritinophagy as a regulator of systemic iron homeostasis and define the relative cell autonomous and non-autonomous contributions of Ncoa4 in supporting erythropoiesis in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent tumorigenic growth, but the role of KRAS in supporting autophagy has not been established. We show that, to our surprise, suppression of KRAS increased autophagic flux, as did pharmacological inhibition of its effector ERK MAPK. Furthermore, we demonstrate that either KRAS suppression or ERK inhibition decreased both glycolytic and mitochondrial functions. We speculated that ERK inhibition might thus enhance PDAC dependence on autophagy, in part by impairing other KRAS- or ERK-driven metabolic processes. Accordingly, we found that the autophagy inhibitor chloroquine and genetic or pharmacologic inhibition of specific autophagy regulators synergistically enhanced the ability of ERK inhibitors to mediate antitumor activity in KRAS-driven PDAC. We conclude that combinations of pharmacologic inhibitors that concurrently block both ERK MAPK and autophagic processes that are upregulated in response to ERK inhibition may be effective treatments for PDAC.

Pancreatic cancer cells are characterized by deregulated metabolic programs that facilitate growth and resistance to oxidative stress. Among these programs, pancreatic cancers preferentially utilize a metabolic pathway through the enzyme aspartate aminotransferase 1 (also known as glutamate oxaloacetate transaminase 1, GOT1) to support cellular redox homeostasis. As such, small molecule inhibitors that target GOT1 could serve as starting points for the development of new therapies for pancreatic cancer. We ran a high-throughput screen for inhibitors of GOT1 and identified a small molecule, iGOT1-01, with in vitro GOT1 inhibitor activity. Application in pancreatic cancer cells revealed metabolic and growth inhibitory activity reflecting a promiscuous inhibitory profile. We then performed an in silico docking analysis to study inhibitor-GOT1 interactions with iGOT1-01 analogs that possess improved solubility and potency properties. These results suggested that the GOT1 inhibitor competed for binding to the pyridoxal 5-phosphate (PLP) cofactor site of GOT1. To analyze how the GOT1 inhibitor bound to GOT1, a series of GOT1 mutant enzymes that abolished PLP binding were generated. Application of the mutants in X-ray crystallography and thermal shift assays again suggested but were unable to formally conclude that the GOT1 inhibitor bound to the PLP site. Mutational studies revealed the relationship between PLP binding and thermal stability of GOT1, while highlighting the essential nature of several residues for GOT1 catalytic activity. Insight into the mode of action of GOT1 inhibitors may provide leads to the development of drugs that target redox balance in pancreatic cancer.

Oncogenic KRAS is the key driver of pancreatic ductal adenocarcinoma (PDAC). We previously described a role for KRAS in PDAC tumor maintenance through rewiring of cellular metabolism to support proliferation. Understanding the details of this metabolic reprogramming in human PDAC may provide novel therapeutic opportunities. Here we show that the dependence on oncogenic KRAS correlates with specific metabolic profiles that involve maintenance of nucleotide pools as key mediators of KRAS-dependence. KRAS promotes these effects by activating a MAPK-dependent signaling pathway leading to MYC upregulation and transcription of the non-oxidative pentose phosphate pathway (PPP) gene RPIA, which results in nucleotide biosynthesis. The use of MEK inhibitors recapitulates the KRAS-dependence pattern and the expected metabolic changes. Antagonizing the PPP or pyrimidine biosynthesis inhibits the growth of KRAS-resistant cells. Together, these data reveal differential metabolic rewiring between KRAS-resistant and sensitive cells, and demonstrate that targeting nucleotide metabolism can overcome resistance to KRAS/MEK inhibition.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is extremely refractory to the therapeutic approaches that have been evaluated to date. Recently, it has been demonstrated that PDAC tumors are dependent upon a metabolic pathway involving aspartate aminotransferase 1, also known as glutamate-oxaloacetate transaminase 1 (GOT1), for the maintenance of redox homeostasis and sustained proliferation. As such, small molecule inhibitors targeting this metabolic pathway may provide a novel therapeutic approach for the treatment of this devastating disease. To this end, from a high throughput screen of ∼800,000 molecules, 4-(1H-indol-4-yl)-N-phenylpiperazine-1-carboxamide was identified as an inhibitor of GOT1. Mouse pharmacokinetic studies revealed that potency, rather than inherent metabolic instability, would limit immediate cell- and rodent xenograft-based experiments aimed at validating this potential cancer metabolism-related target. Medicinal chemistry-based optimization resulted in the identification of multiple derivatives with >10-fold improvements in potency, as well as the identification of a tryptamine-based series of GOT1 inhibitors.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive cancer that is highly refractory to the current standards of care. The difficulty in treating this disease is due to a number of different factors, including altered metabolism. In PDA, the metabolic rewiring favors anabolic reactions which supply the cancer cell with necessary cellular building blocks for unconstrained growth. Furthermore, PDA cells display high levels of basal autophagy and macropinocytosis. KRAS is the driving oncogene in PDA and many of the metabolic changes are downstream of its activation. Together, these unique pathways for nutrient utilization and acquisition result in metabolic plasticity enabling cells to rapidly adapt to nutrient and oxygen fluctuations. This remarkable adaptability has been implicated as a cause of the intense therapeutic resistance. In this review, we discuss metabolic pathways in PDA tumors and highlight and how they contribute to the pathogenesis and treatment of the disease.

While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P₂) in mammalian cells is generated by the conversion of phosphatidylinositol-4-phosphate (PI-4-P) to PI-4, 5-P₂, a small fraction can be made by phosphorylating phosphatidylinositol-5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2a^-/-Pip4k2b^-/- mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P₂ synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.