Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer.

Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance.

Tumor-selective effects of active RAS inhibition in pancreatic ductal adenocarcinoma.

Broad-spectrum RAS inhibition holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. However, the impact of inhibiting RAS functions in normal tissues is not known. RMC-7977 is a highly selective inhibitor of the active (GTP-bound) forms of KRAS, HRAS, and NRAS, with affinity for both mutant and wild type (WT) variants. As >90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models, including human and murine cell lines, human patient-derived organoids, human PDAC explants, subcutaneous and orthotopic cell-line or patient derived xenografts, syngeneic allografts, and genetically engineered mouse models. We observed broad and pronounced anti-tumor activity across these models following direct RAS inhibition at doses and concentrations that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS inhibition in the setting of PDAC.

Phase Ib and Expansion Study of Gemcitabine, Nab-Paclitaxel, and Ficlatuzumab in Patients With Metastatic Pancreatic Cancer.

BACKGROUND: In preclinical pancreatic ductal adenocarcinoma (PDAC) models, inhibition of hepatocyte growth factor (HGF) signaling using ficlatuzumab, a recombinant humanized anti-HGF antibody, and gemcitabine reduced tumor burden. METHODS: Patients with previously untreated metastatic PDAC enrolled in a phase Ib dose escalation study with 3 + 3 design of 2 dose cohorts of ficlatuzumab 10 and 20 mg/kg administered intravenously every other week with gemcitabine 1000 mg/m2 and albumin-bound paclitaxel 125 mg/m2 given 3 weeks on and 1 week off. This was followed by an expansion phase at the maximally tolerated dose of the combination. RESULTS: Twenty-six patients (sex, 12 male:14 female; median age, 68 years [range, 49-83 years]) were enrolled, 22 patients were evaluable. No dose-limiting toxicities were identified (N = 7 pts) and ficlatuzumab at 20 mg/kg was chosen as the maximum tolerated dose. Among the 21 patients treated at the MTD, best response by RECISTv1.1: 6 (29%) partial response, 12 (57%) stable disease, 1 (5%) progressive disease, and 2 (9%) not evaluable. Median progression-free survival and overall survival times were 11.0 months (95% CI, 7.6-11.4 months) and 16.2 months (95% CI, 9.1 months to not reached), respectively. Toxicities attributed to ficlatuzumab included hypoalbuminemia (grade 3, 16%; any grade, 52%) and edema (grade 3, 8%; any grade, 48%). Immunohistochemistry for c-Met pathway activation demonstrated higher tumor cell p-Met levels in patients who experienced response to therapy. CONCLUSION: In this phase Ib trial, ficlatuzumab, gemcitabine, and albumin-bound paclitaxel were associated with durable treatment responses and increased rates of hypoalbuminemia and edema.

PTHrP Drives Pancreatic Cancer Growth and Metastasis and Reveals a New Therapeutic Vulnerability.

Pancreatic cancer metastasis is a leading cause of cancer-related deaths, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Here, we report that the parathyroid hormone-related protein (PTHrP encoded by PTHLH) is frequently amplified as part of the KRAS amplicon in patients with pancreatic cancer. PTHrP upregulation drives the growth of both primary and metastatic tumors in mice and is highly enriched in pancreatic ductal adenocarcinoma metastases. Loss of PTHrP-either genetically or pharmacologically-dramatically reduces tumor burden, eliminates metastasis, and enhances overall survival. These effects are mediated in part through a reduction in epithelial-to-mesenchymal transition, which reduces the ability of tumor cells to initiate metastatic cascade. Spp1, which encodes osteopontin, is revealed to be a downstream effector of PTHrP. Our results establish a new paradigm in pancreatic cancer whereby PTHrP is a driver of disease progression and emerges as a novel therapeutic vulnerability. SIGNIFICANCE: Pancreatic cancer often presents with metastases, yet no strategies exist to pharmacologically inhibit this process. Herein, we establish the oncogenic and prometastatic roles of PTHLH, a novel amplified gene in pancreatic ductal adenocarcinoma. We demonstrate that blocking PTHrP activity reduces primary tumor growth, prevents metastasis, and prolongs survival in mice.This article is highlighted in the In This Issue feature, p. 1601.

EMT, MET, Plasticity, and Tumor Metastasis.

Cancer cell identity and plasticity are required in transition states, such as epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), in primary tumor initiation, progression, and metastasis. The functional roles of EMT, MET, and the partial state (referred to as pEMT) may vary based on the type of tumor, the state of dissemination, and the degree of metastatic colonization. Herein, we review EMT, MET, pEMT, and plasticity in the context of tumor metastasis.

Regulation of Epithelial Plasticity Determines Metastatic Organotropism in Pancreatic Cancer.

The regulation of metastatic organotropism in pancreatic ductal a denocarcinoma (PDAC) remains poorly understood. We demonstrate, using multiple mouse models, that liver and lung metastatic organotropism is dependent upon p120catenin (p120ctn)-mediated epithelial identity. Mono-allelic p120ctn loss accelerates KrasG12D-driven pancreatic cancer formation and liver metastasis. Importantly, one p120ctn allele is sufficient for E-CADHERIN-mediated cell adhesion. By contrast, cells with bi-allelic p120ctn loss demonstrate marked lung organotropism; however, rescue with p120ctn isoform 1A restores liver metastasis. In a p120ctn-independent PDAC model, mosaic loss of E-CADHERIN expression reveals selective pressure for E-CADHERIN-positive liver metastasis and E-CADHERIN-negative lung metastasis. Furthermore, human PDAC and liver metastases support the premise that liver metastases exhibit predominantly epithelial characteristics. RNA-seq demonstrates differential induction of pathways associated with metastasis and epithelial-to-mesenchymal transition in p120ctn-deficient versus p120ctn-wild-type cells. Taken together, P120CTN and E-CADHERIN mediated epithelial plasticity is an addition to the conceptual framework underlying metastatic organotropism in pancreatic cancer.

Pancreas 3D Organoids: Current and Future Aspects as a Research Platform for Personalized Medicine in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is one of the most aggressive forms of cancer, and the third leading cause of cancer-related mortality in the United States. Although important advances have been made in the last decade, the mortality rate of pancreatic ductal adenocarcinoma has not changed appreciably. This review summarizes a rapidly emerging model of pancreatic cancer research, focusing on 3-dimensional organoids as a powerful tool for several applications, but above all, representing a step toward personalized medicine.

ETV5 regulates ductal morphogenesis with Sox9 and is critical for regeneration from pancreatitis.

BACKGROUND: The plasticity of pancreatic acinar cells to undergo acinar to ductal metaplasia (ADM) has been demonstrated to contribute to the regeneration of the pancreas in response to injury. Sox9 is critical for ductal cell fate and important in the formation of ADM, most likely in concert with a complex hierarchy of, as yet, not fully elucidated transcription factors. RESULTS: By using a mouse model of acute pancreatitis and three dimensional organoid culture of primary pancreatic ductal cells, we herein characterize the Ets-transcription factor Etv5 as a pivotal regulator of ductal cell identity and ADM that acts upstream of Sox9 and is essential for Sox9 expression in ADM. Loss of Etv5 is associated with increased severity of acute pancreatitis and impaired ADM formation leading to delayed tissue regeneration and recovery in response to injury. CONCLUSIONS: Our data provide new insights in the regulation of ADM with implications in our understanding of pancreatic homeostasis, pancreatitis and epithelial plasticity. Developmental Dynamics 247:854-866, 2018. © 2018 Wiley Periodicals, Inc.

ETS-Transcription Factor ETV1 Regulates Stromal Expansion and Metastasis in Pancreatic Cancer.

BACKGROUND & AIMS: The ETS-transcription factor ETV1 is involved in epithelial-mesenchymal transition during pancreatic development and is induced in mouse pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC). We investigated the function of ETV1 in stromal expansion of PDAC and metastasis, as well as its effects on a novel downstream target Sparc, which encodes a matricellular protein found in PDAC stroma that has been associated with invasiveness, metastasis and poor patient outcomes. METHODS: Pancreatic ductal cells were isolated from Pdx1Cre;Kras(G12D/+) mice (PanIN), Pdx1Cre;Kras(G12D/+);p53(fl/+) and Pdx1Cre;Kras(G12D/+);p53(fl/+);Rosa26(YFP) mice (PDAC), and Pdx1Cre;Kras(G12D/+);p53(fl/+);Sparc(-/-) mice. Cells were grown in 3-dimensional organoid culture to analyze morphology, proliferation, and invasion. Human PanIN and PDAC tissues were evaluated for ETV1 expression. Orthotopic pancreatic transplants of ETV1-overexpressing PDAC and respective control cells were performed. RESULTS: ETV1 expression was significantly increased in human PanINs and, even more so, in primary and metastatic PDAC. Analyses of mouse orthotopic xenografts revealed that ETV1 induced significantly larger primary tumors than controls, with significantly increased stromal expansion, ascites and metastases. In 3-dimensional organoids, ETV1 disrupted cyst architecture, induced EMT, and increased invasive capacity. Furthermore, we identified Sparc as a novel functional gene target of Etv1 by luciferase assays, and SPARC and ETV1 proteins co-localized in vivo. Disruption of Sparc abrogates the phenotype of stromal expansion and metastasis found with ETV1 overexpression in vivo. We identified hyaluronan synthase 2 (Has2) as another novel downstream factor of Etv1; that may mediate ETV1's significant expansion of hyaluronic acid in PDAC stroma. Conversely, disruption of Etv1 in PDAC mice (Pdx1Cre;Kras(G12D/+);p53(fl/+);Rosa26(YFP);Cre;Etv1(fl/fl)) reduced levels of SPARC and hyaluronic acid in the stroma. CONCLUSIONS: ETV1 is critical in the desmoplastic stromal expansion and metastatic progression of pancreatic cancer in mice, mediated functionally in part through Sparc and Has2.

Prrx1 isoform switching regulates pancreatic cancer invasion and metastatic colonization.

The two major isoforms of the paired-related homeodomain transcription factor 1 (Prrx1), Prrx1a and Prrx1b, are involved in pancreatic development, pancreatitis, and carcinogenesis, although the biological role that these isoforms serve in the systemic dissemination of pancreatic ductal adenocarcinoma (PDAC) has not been investigated. An epithelial-mesenchymal transition (EMT) is believed to be important for primary tumor progression and dissemination, whereas a mesenchymal-epithelial transition (MET) appears crucial for metastatic colonization. Here, we describe novel roles for both isoforms in the metastatic cascade using complementary in vitro and in vivo models. Prrx1b promotes invasion, tumor dedifferentiation, and EMT. In contrast, Prrx1a stimulates metastatic outgrowth in the liver, tumor differentiation, and MET. We further demonstrate that the switch from Prrx1b to Prrx1a governs EMT plasticity in both mouse models of PDAC and human PDAC. Last, we identify hepatocyte growth factor ( HGF) as a novel transcriptional target of Prrx1b. Targeted therapy of HGF in combination with gemcitabine in a preclinical model of PDAC reduces primary tumor volume and eliminates metastatic disease. Overall, we provide new insights into the isoform-specific roles of Prrx1a and Prrx1b in primary PDAC formation, dissemination, and metastatic colonization, allowing for novel therapeutic strategies targeting EMT plasticity.

Spontaneous Pancreatitis Caused by Tissue-Specific Gene Ablation of Hhex in Mice.

BACKGROUND & AIMS: Perturbations in pancreatic ductal bicarbonate secretion cause chronic pancreatitis. The physiologic mechanism of ductal secretion is known, but its transcriptional control is not. We determine the role of the transcription factor hematopoietically expressed homeobox protein (Hhex) in ductal secretion and pancreatitis. METHODS: We derived mice with pancreas-specific, Cremediated Hhex gene ablation to determine the requirement of Hhex in the pancreatic duct in early life and in adult stages. Histologic and immunostaining analyses were used to detect the presence of pathology. Pancreatic primary ductal cells were isolated to discover differentially expressed transcripts upon acute Hhex ablation on a cell autonomous level. RESULTS: Hhex protein was detected throughout the embryonic and adult ductal trees. Ablation of Hhex in pancreatic progenitors resulted in postnatal ductal ectasia associated with acinar-to-ductal metaplasia, a progressive phenotype that ultimately resulted in chronic pancreatitis. Hhex ablation in adult mice, however, did not cause any detectable pathology. Ductal ectasia in young mice did not result from perturbation of expression of Hnf6, Hnf1ß, or the primary cilia genes. RNA-seq analysis of Hhex-ablated pancreatic primary ductal cells showed mRNA levels of the G-protein coupled receptor natriuretic peptide receptor 3 (Npr3), implicated in paracrine signaling, up-regulated by 4.70-fold. CONCLUSIONS: Although Hhex is dispensable for ductal cell function in the adult, ablation of Hhex in pancreatic progenitors results in pancreatitis. Our data highlight the critical role of Hhex in maintaining ductal homeostasis in early life and support ductal hypersecretion as a novel etiology of pediatric chronic pancreatitis.

Isolation, culture and genetic manipulation of mouse pancreatic ductal cells.

The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles duct cells morphologically and, to some extent, at a molecular level. Recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and ductlike cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. This approach is fast (~4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas.