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ObjectiveHere, we evaluate the contribution of AT-rich interaction domain-containing protein 1A (ARID1A), the most frequently mutated member of the SWItch/sucrose non-fermentable (SWI/SNF) complex, in pancreatic homeostasis and pancreatic ductal adenocarcinoma (PDAC) pathogenesis using mouse models.DesignMice with a targeted deletion of Arid1a in the pancreas by itself and in the context of two common genetic alterations in PDAC, Kras and p53, were followed longitudinally. Pancreases were examined and analysed for proliferation, response to injury and tumourigenesis. Cancer cell lines derived from these models were analysed for clonogenic, migratory, invasive and transcriptomic changes.Results Arid1a deletion in the pancreas results in progressive acinar-to-ductal metaplasia (ADM), loss of acinar mass, diminished acinar regeneration in response to injury and ductal cell expansion. Mutant Kras cooperates with homozygous deletion of Arid1a, leading to intraductal papillary mucinous neoplasm (IPMN). Arid1a loss in the context of mutant Kras and p53 leads to shorter tumour latency, with the resulting tumours being poorly differentiated. Cancer cell lines derived from Arid1a-mutant tumours are more mesenchymal, migratory, invasive and capable of anchorage-independent growth; gene expression analysis showed activation of epithelial-mesenchymal transition (EMT) and stem cell identity pathways that are partially dependent on Arid1a loss for dysregulation.ConclusionsARID1A plays a key role in pancreatic acinar homeostasis and response to injury. Furthermore, ARID1A restrains oncogenic KRAS-driven formation of premalignant proliferative IPMN. Arid1a-deficient PDACs are poorly differentiated and have mesenchymal features conferring migratory/invasive and stem-like properties.

Cholangiocarcinoma (CCA) is a deadly and heterogeneous type of cancer characterized by a spectrum of epidemiologic associations as well as genetic and epigenetic alterations. We seek to understand how these features inter-relate in the earliest phase of cancer development and through the course of disease progression. For this, we studied murine models of liver injury integrating the most commonly occurring gene mutations of CCA – including Kras, Tp53, Arid1a and Smad4 – as well as murine hepatobiliary cancer models and derived primary cell lines based on these mutations. Among commonly mutated genes in CCA, we found that Smad4 functions uniquely to restrict reactive cholangiocyte expansion to liver injury through restraint of the proliferative response. Inactivation of Smad4 accelerates carcinogenesis, provoking pre-neoplastic biliary lesions and CCA development in an injury setting. Expression analyses of Smad4-perturbed reactive cholangiocytes and CCA lines demonstrated shared enriched pathways, including cell-cycle regulation, MYC signaling and oxidative phosphorylation, suggesting that Smad4 may act via these mechanisms to regulate cholangiocyte proliferation and progression to CCA. Overall, we showed that TGFβ/SMAD4 signaling serves as a critical barrier restraining cholangiocyte expansion and malignant transformation in states of biliary injury.

Background The genetics of advanced biliary tract cancers (BTC), which encompass intra- and extra-hepatic cholangiocarcinomas as well as gallbladder carcinomas, are heterogeneous and remain to be fully defined. Methods To better characterize mutations in established known oncogenes and tumor suppressor genes we tested a mass spectrometric based platform to interrogate common cancer associated mutations across a panel of 77 formalin fixed paraffin embedded archived BTC cases. Results Mutations among three genes, KRAS, NRAS and PIK3CA were confirmed in this cohort. Activating mutations in PIK3CA were identified exclusively in GBC (4/32, 12.5%). KRAS mutations were identified in 3 (13%) intra-hepatic cholangiocarcinomas and 1 (33%) perihillar cholangiocarcinoma but were not identified in gallbladder carcinomas and extra-hepatic cholangiocarcinoma. Conclusions The presence of activating mutations in PIK3CA specifically in GBC has clinical implications in both the diagnosis of this cancer type, as well as the potential utility of targeted therapies such as PI3 kinase inhibitors.

Pancreas ductal adenocarcinoma (PDAC) is a highly lethal cancer that typically presents as advanced, unresectable disease. This invasive tendency, coupled with intrinsic resistance to standard therapies and genome instability, are major contributors to poor long-term survival. The genetic elements governing the invasive propensity of PDAC have not been well elucidated. Here, in the course of validating resident genes in highly recurrent and focal amplifications in PDAC, we have identified Rio Kinase 3 (RIOK3) as an amplified gene that alters cytoskeletal architecture as well as promotes pancreatic ductal cell migration and invasion. We determined that RIOK3 promotes its invasive activities through activation of the small G protein, Rac. This genomic and functional link to Rac signaling prompted a genome wide survey of other components of the Rho family network, revealing p21 Activated Kinase 4 (PAK4) as another amplified gene in PDAC tumors and cell lines. Like RIOK3, PAK4 promotes pancreas ductal cell motility and invasion. Together, the genomic and functional profiles establish the Rho family GTP-binding proteins as integral to the hallmark invasive nature of this lethal disease.

Gain-of-function mutations in isocitrate dehydrogenase ( IDH ) are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly cancer of the liver bile ducts; now mutant IDH is shown to block liver cell differentiation through the suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence, leading to expansion of liver progenitor cells primed for progression to IHCC. Mechanism of induction of a liver cancer Cancer-associated gain-of-function isocitrate dehydrogenase (IDH) mutations produce the 'oncometabolite' 2-hydroxyglutarate (2HG) that can inhibit a-ketoglutarate-dependent dioxygenase enzymes. Nabeel Bardeesy and colleagues show here that 2HG plays an active role in carcinogenesis: mutant IDH blocks liver progenitor cells from undergoing hepatocyte lineage progression through the production of 2HG and suppression of HNF4a, a master regulator of hepatocyte differentiation. Moreover, where mutant IDH coexists with activated Kras , it drives the expansion of liver progenitor cells, development of premalignant biliary lesions and progression to metastatic intrahepatic cholangiocarcinoma. The transgenic mouse model used here should facilitate further study of IDH function, particularly important in relation to cholangiocarcinoma, which is resistant to current treatments. Mutations in isocitrate dehydrogenase 1 ( IDH1 ) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer 1 , 2 , 3 , 4 , 5 . Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation 6 , 7 , 8 , 9 , 10 . However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs 4 , 5 , cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

ObjectiveIntrahepatic cholangiocarcinoma (iCCA) is rising in incidence, and at present, there are limited effective systemic therapies. iCCA tumours are infiltrated by stromal cells, with high prevalence of suppressive myeloid populations including tumour-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Here, we show that tumour-derived granulocyte–macrophage colony-stimulating factor (GM-CSF) and the host bone marrow is central for monopoiesis and potentiation of TAMs, and abrogation of this signalling axis facilitates antitumour immunity in a novel model of iCCA.MethodsBlood and tumours were analysed from iCCA patients and controls. Treatment and correlative studies were performed in mice with autochthonous and established orthotopic iCCA tumours treated with anti-GM-CSF monoclonal antibody.ResultsSystemic elevation in circulating myeloid cells correlates with poor prognosis in patients with iCCA, and patients who undergo resection have a worse overall survival if tumours are more infiltrated with CD68+ TAMs. Mice with spontaneous iCCA demonstrate significant elevation of monocytic myeloid cells in the tumour microenvironment and immune compartments, and tumours overexpress GM-CSF. Blockade of GM-CSF with a monoclonal antibody decreased tumour growth and spread. Mice bearing orthotopic tumours treated with anti-GM-CSF demonstrate repolarisation of immunosuppressive TAMs and MDSCs, facilitating T cell response and tumour regression. GM-CSF blockade dampened inflammatory gene networks in tumours and TAMs. Human tumours with decreased GM-CSF expression exhibit improved overall survival after resection.ConclusionsiCCA uses the GM-CSF-bone marrow axis to establish an immunosuppressive tumour microenvironment. Blockade of the GM-CSF axis promotes antitumour T cell immunity.

Assessing clinical tumor response following completion of total neoadjuvant therapy (TNT) in patients with locally advanced rectal cancer is paramount to select patients for watch-and-wait treatment. To assess organ preservation (OP) and oncologic outcomes according to clinical tumor response grade. This was secondary analysis of the Organ Preservation in Patients with Rectal Adenocarcinoma trial, a phase 2, nonblinded, multicenter, randomized clinical trial. Randomization occurred between April 2014 and March 2020. Eligible participants included patients with stage II or III rectal adenocarcinoma. Data analysis occurred from March 2022 to July 2023. Patients were randomized to induction chemotherapy followed by chemoradiation or chemoradiation followed by consolidation chemotherapy. Tumor response was assessed 8 (±4) weeks after TNT by digital rectal examination and endoscopy and categorized by clinical tumor response grade. A 3-tier grading schema that stratifies clinical tumor response into clinical complete response (CCR), near complete response (NCR), and incomplete clinical response (ICR) was devised to maximize patient eligibility for OP. OP and survival rates by clinical tumor response grade were analyzed using the Kaplan-Meier method and log-rank test. There were 304 eligible patients, including 125 patients with a CCR (median [IQR] age, 60.6 [50.4-68.0] years; 76 male [60.8%]), 114 with an NCR (median [IQR] age, 57.6 [49.1-67.9] years; 80 male [70.2%]), and 65 with an ICR (median [IQR] age, 55.5 [47.7-64.2] years; 41 male [63.1%]) based on endoscopic imaging. Age, sex, tumor distance from the anal verge, pathological tumor classification, and clinical nodal classification were similar among the clinical tumor response grades. Median (IQR) follow-up for patients with OP was 4.09 (2.99-4.93) years. The 3-year probability of OP was 77% (95% CI, 70%-85%) for patients with a CCR and 40% (95% CI, 32%-51%) for patients with an NCR (P < .001). Clinical tumor response grade was associated with disease-free survival, local recurrence-free survival, distant metastasis-free survival, and overall survival. In this secondary analysis of a randomized clinical trial, most patients with a CCR after TNT achieved OP, with few developing tumor regrowth. Although the probability of tumor regrowth was higher for patients with an NCR compared with patients with a CCR, a significant proportion of patients achieved OP. These findings suggest the 3-tier grading schema can be used to estimate recurrence and survival outcomes in patients with locally advanced rectal cancer who receive TNT. ClinicalTrials.gov Identifier: NCT02008656.

Background Diabetes mellitus (DM) is identified as a negative prognostic indicator in hepatocellular carcinoma (HCC), though the basis for this is unknown. Methods This is a retrospective analysis of a prospectively collected database of 191 HCC patients treated at the University of Rochester Medical Center (URMC) with orthotopic liver transplantation between 1998–2008. Clinical characteristics were compared between patients with and without DM prior to liver transplantation and logistic regression analyses were conducted to assess the effect of DM on clinical outcomes including vascular invasion. Results Eighty-four of 191 (44%) transplanted patients had DM at time of transplantation. An association of DM with invasive disease was found among transplanted HCC patients where histologically confirmed macrovascular invasion was found in 20.2% (17/84) of diabetics compared to 9.3% of non-diabetics (10/107) (p=0.032). This difference also remained significant when adjusting for tumor size, number of nodules, age, obesity and etiologic risk factors in multivariate logistic regression analysis (OR=3.2, p=0.025). Conclusions DM is associated with macrovascular invasion among a cohort of transplanted HCC patients.

Activating KRAS mutations and$p16^{Ink4a}$inactivation are near universal events in human pancreatic ductal adenocarcinoma (PDAC). In mouse models,$Kras^{G12D}$initiates formation of premalignant pancreatic ductal lesions, and loss of either$Ink4a/Arf$($p16^{Ink4a}/p19^{Arf}$) or p53 enables their malignant progression. As recent mouse modeling studies have suggested a less prominent role for$p16^{Ink4a}$in constraining malignant progression, we sought to assess the pathological and genomic impact of inactivation of$p16^{Ink4a}$,$p19^{Arf}$, and/or p53 in the$Kras^{G12D}$model. Rapidly progressive PDAC was observed in the setting of homozygous deletion of either p53 or$p16^{Ink4a}$, the latter with intact germ-line p53 and$p19^{Arf}$sequences. Additionally,$Kras^{G12D}$in the context of heterozygosity either for p53 plus$p16^{Ink4a}$or for$p16^{Ink4a}/p19^{Arf}$produced PDAC with longer latency and greater propensity for distant metastases relative to mice with homozygous deletion of p53 or$p16^{Ink4a}/p19^{Arf}$. Tumors from the double-heterozygous cohorts showed frequent$p16^{Ink4a}$inactivation and loss of either p53 or$p19^{Arf}$. Different genotypes were associated with specific histopathologic characteristics, most notably a trend toward less differentiated features in the homozygous$p16^{Ink4a}$/p19^{Arf}$mutant model. High-resolution genomic analysis revealed that the tumor suppressor genotype influenced the specific genomic patterns of these tumors and showed overlap in regional chromosomal alterations between murine and human PDAC. Collectively, our results establish that disruptions of$p16^{Ink4a}$and the$p19^{ARF}-p53$circuit play critical and cooperative roles in PDAC progression, with specific tumor suppressor genotypes provocatively influencing the tumor biological phenotypes and genomic profiles of the resultant tumors.

Germline mutations in LKB1 (also known as STK11 ) are associated with Peutz–Jeghers syndrome (PJS), a disorder with predisposition to gastrointestinal polyposis and cancer 1 . PJS polyps are unusual neoplasms characterized by marked epithelial and stromal overgrowth but have limited malignant potential 2 . Here we show that Lkb1 +/- mice develop intestinal polyps identical to those seen in individuals affected with PJS. Consistent with this in vivo tumour suppressor function, Lkb1 deficiency prevents culture-induced senescence without loss of Ink4a / Arf or p53 . Despite compromised mortality, Lkb1 -/- mouse embryonic fibroblasts show resistance to transformation by activated Ha- Ras either alone or with immortalizing oncogenes. This phenotype is in agreement with the paucity of mutations in Ras seen in PJS polyps 3 , 4 and suggests that loss of Lkb1 function as an early neoplastic event renders cells resistant to subsequent oncogene-induced transformation. In addition, the Lkb1 transcriptome shows modulation of factors linked to angiogenesis, extracellular matrix remodelling, cell adhesion and inhibition of Ras transformation. Together, our data rationalize several features of PJS polyposis—notably its peculiar histopathological presentation and limited malignant potential—and place Lkb1 in a distinct class of tumour suppressors.