Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate “reprogramming” by opening new chromatin sites for expression that can attract transcription factors from the starting cell’s enhancers. Here we report that Sox4 is sufficient to initiate hepatobiliary metaplasia in the adult liver. In lineage-traced cells, we assessed the timing of Sox4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, Sox4 directly binds to and closes hepatocyte regulatory sequences via a motif it overlaps with Hnf4a, a hepatocyte master regulator. Subsequently, Sox4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

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Physiological reprogramming in vivo mediated by Sox4 pioneer factor activity

Pancreatic ductal adenocarcinoma (PDAC) is associated with mutations in Kras, a known oncogenic driver of PDAC; and the KRASG12D mutation is present in nearly half of PDAC patients. Recently, a non-covalent small molecule inhibitor (MRTX1133) was identified with specificity to the KrasG12D mutant protein. Here we explore the impact of KrasG12D inhibition by MRTX1133 on advanced PDAC and its influence on the tumor microenvironment. Employing different orthotopic xenograft and syngeneic tumor models, eight different PDXs, and two different autochthonous genetic models, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8+ effector T cells, decreases myeloid infiltration, and reprograms cancer associated fibroblasts. Autochthonous genetic mouse models treated with MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8+ T cells and immune checkpoint blockade therapy (iCBT) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of mutant Kras in advanced PDAC and human patient derived organoids (PDOs) induces Fas expression in cancer cells and facilitates CD8+T cell mediated death. These results demonstrate the efficacy of MRTX1133 in different mouse models of PDAC associated with reprogramming of stromal fibroblasts and a dependency on CD8+ T cell mediated tumor clearance. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with iCBT in clinical trials.

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Oncogenic KrasG12D specific non-covalent inhibitor reprograms tumor microenvironment to prevent and reverse early pre-neoplastic pancreatic lesions and in combination with immunotherapy regresses advanced PDAC in a CD8+ T cells dependent manner

Nutrient stress in the tumor microenvironment requires cancer cells to adopt adaptive metabolic programs to maintain survival and proliferation. Therefore, knowledge of microenvironmental nutrient levels and how cancer cells cope with such nutrition is critical to understand the metabolism underpinning cancer cell biology. Previously, we performed quantitative metabolomics of the interstitial fluid (the local perfusate) of murine pancreatic ductal adenocarcinoma (PDAC) tumors to comprehensively characterize nutrient availability in the microenvironment of these tumors (Sullivan et al., 2019a). Here, we develop Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains nutrient levels representative of the PDAC microenvironment, enabling study of PDAC metabolism under physiological nutrition. We show that PDAC cells cultured in TIFM, compared to standard laboratory models, adopt a cellular state more similar to PDAC cells in tumors. Further, using the TIFM model we identified arginine biosynthesis as a metabolic adaptation PDAC cells engage to cope with microenvironmental arginine starvation driven by myeloid cells in PDAC tumors. Altogether, these data show that nutrient availability in tumors is an important determinant of cancer cell metabolism and behavior, and cell culture models that incorporate physiological nutrient availability have improved fidelity and enable the discovery of novel cancer metabolic phenotypes.

/pdf/pancreatic-tumors-activate-arginine-biosynthesis-to-adapt-to-2bpevtc6.pdf

Pancreatic tumors activate arginine biosynthesis to adapt to myeloid-driven amino acid stress

KRASG12C inhibitors have revolutionized the clinical management of patients with KRASG12C-mutant lung adenocarcinoma. However, patient exposure to these inhibitors leads to the rapid onset of resistance. In this study, we have used genetically engineered mice to compare the therapeutic efficacy and the emergence of tumor resistance between genetic ablation of mutant Kras expression and pharmacological inhibition of oncogenic KRAS activity. Whereas Kras ablation induces massive tumor regression and prevents the appearance of resistant cells in vivo, treatment of KrasG12C/Trp53-driven lung adenocarcinomas with sotorasib, a selective KRASG12C inhibitor, caused a limited antitumor response similar to that observed in the clinic, including the rapid onset of resistance. Unlike in human tumors, we did not observe mutations in components of the RAS-signaling pathways. Instead, sotorasib-resistant tumors displayed amplification of the mutant Kras allele and activation of xenobiotic metabolism pathways, suggesting that reduction of the on-target activity of KRASG12C inhibitors is the main mechanism responsible for the onset of resistance. In sum, our results suggest that resistance to KRAS inhibitors could be prevented by achieving a more robust inhibition of KRAS signaling mimicking the results obtained upon Kras ablation. 

/pdf/kras-oncogene-ablation-prevents-resistance-in-advanced-lung-3b8biy2m.pdf

Kras oncogene ablation prevents resistance in advanced lung adenocarcinomas

Pancreatic enzymes assist metabolic digestion, and hormones like insulin and glucagon play a critical role in maintaining our blood sugar levels. A malignant pancreas is incapable of doing its regular functions, which results in a health catastrophe. To date, there is no effective biomarker to detect early-stage pancreatic cancer, which makes pancreatic cancer the cancer with the highest mortality rate of all cancer types. Primarily, mutations of the KRAS, CDKN2A, TP53, and SMAD4 genes are responsible for pancreatic cancer, of which mutations of the KRAS gene are present in more than 80% of pancreatic cancer cases. Accordingly, there is a desperate need to develop effective inhibitors of the proteins that are responsible for the proliferation, propagation, regulation, invasion, angiogenesis, and metastasis of pancreatic cancer. This article discusses the effectiveness and mode of action at the molecular level of a wide range of small molecule inhibitors that include pharmaceutically privileged molecules, compounds under clinical trials, and commercial drugs. Both natural and synthetic small molecule inhibitors have been counted. Anti-pancreatic cancer activity and related benefits of using single and combined therapy have been discussed separately. This article sheds light on the scenario, constraints, and future aspects of various small molecule inhibitors for treating pancreatic cancer—the most dreadful cancer so far.

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Molecular Research in Pancreatic Cancer: Small Molecule Inhibitors, Their Mechanistic Pathways and Beyond

Mutations in the KRAS oncogene are found in more than 90% of patients with pancreatic ductal adenocarcinoma (PDAC), with Gly-to-Asp mutations (KRASG12D) being the most common. Here, we tested the efficacy of a small-molecule KRASG12D inhibitor, MRTX1133, in implantable and autochthonous PDAC models with an intact immune system. In vitro studies validated the specificity and potency of MRTX1133. In vivo, MRTX1133 prompted deep tumor regressions in all models tested, including complete or near-complete remissions after 14 days. Concomitant with tumor cell apoptosis and proliferative arrest, drug treatment led to marked shifts in the tumor microenvironment (TME), including changes in fibroblasts, matrix, and macrophages. T cells were necessary for MRTX1133's full antitumor effect, and T-cell depletion accelerated tumor regrowth after therapy. These results validate the specificity, potency, and efficacy of MRTX1133 in immunocompetent KRASG12D-mutant PDAC models, providing a rationale for clinical testing and a platform for further investigation of combination therapies.Pharmacologic inhibition of KRASG12D in pancreatic cancer models with an intact immune system stimulates specific, potent, and durable tumor regressions. In the absence of overt toxicity, these results suggest that this and similar inhibitors should be tested as potential, high-impact novel therapies for patients with PDAC. See related commentary by Redding and Grabocka, p. 260. This article is highlighted in the In This Issue feature, p. 247. 

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Efficacy of a Small-Molecule Inhibitor of KrasG12D in Immunocompetent Models of Pancreatic Cancer

Solid tumors possess heterogeneous metabolic microenvironments where oxygen and nutrient availability are plentiful (fertile regions) or scarce (arid regions). While cancer cells residing in fertile regions proliferate rapidly, most cancer cells in vivo reside in arid regions and exhibit a slow-cycling state that renders them chemoresistant. Here, we developed an in vitro system enabling systematic comparison between these populations via transcriptome analysis, metabolomic profiling, and whole-genome CRISPR screening. Metabolic deprivation led to pronounced transcriptional and metabolic reprogramming, resulting in decreased anabolic activities and distinct vulnerabilities. Reductions in anabolic, energy-consuming activities, particularly cell proliferation, were not simply byproducts of the metabolic challenge, but rather essential adaptations. Mechanistically, Bcl-xL played a central role in the adaptation to nutrient and oxygen deprivation. In this setting, Bcl-xL protected quiescent cells from the lethal effects of cell-cycle entry in the absence of adequate nutrients. Moreover, inhibition of Bcl-xL combined with traditional chemotherapy had a synergistic antitumor effect that targeted cycling cells. Bcl-xL expression was strongly associated with poor patient survival despite being confined to the slow-cycling fraction of human pancreatic cancer cells. These findings provide a rationale for combining traditional cancer therapies that target rapidly cycling cells with those that target quiescent, chemoresistant cells associated with nutrient and oxygen deprivation.The majority of pancreatic cancer cells inhabit nutrient- and oxygen-poor tumor regions and require Bcl-xL for their survival, providing a compelling antitumor metabolic strategy. 

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Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer

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Robert G. Underwood

Papers

Efficacy of a Small-Molecule Inhibitor of KrasG12D in Immunocompetent Models of Pancreatic Cancer

Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer

Rapid in vivo multiplexed editing (RIME) of the adult mouse liver