Mutations In Pancreatic Ductal Adenocarcinoma Research Articles

Dissected peripancreatic tissue margin is a critical prognostic factor and is associated with a K-ras gene mutation in pancreatic ductal adenocarcinoma.

We previously reported that the dissected pancreatic tissue margin (DPM) and the preoperative serum level of carbohydrate antigen 19-9 (preCA19-9) were independent prognostic factors in pancreatic ductal adenocarcinoma (PDAC). In the current study, the prognostic relevance of these factors, including their molecular associations, were validated. A total of 161 patients with PDAC underwent a pancreatectomy between 1986 and 2013, and a multivariate Cox proportional hazards model and a propensity score-based model validated the prognostic importance of DPM. The prognostic factors were compared with the mutation profiles of the K-ras and TP53 genes. Univariate prognostic analysis of disease-specific survival (DSS) demonstrated that DPM (P<0.0001), preCA19-9 (P<0.0001) and Union for International Cancer Control (UICC) stage (P<0.0001), were all significantly associated with poor outcome in PDAC. A multivariate Cox proportional hazards model confirmed that preCA19-9 (P=0.0002) and DPM (P=0.0002) remained as prognostic factors independent of UICC stage (P=0.0015). The combination of preCA19-9 and DPM to predict prognosis could accurately identify the long-term survivors of PDAC (70% 5-year DSS), and a multivariate logistic regression model identified that DPM was the most effective predictor of mortality. The prognostic relevance of DPM was also confirmed (P=0.0008) through propensity score-based background adjustment of patient bias. K-ras gene mutation was significantly associated with DPM (P=0.0002), and DPM-positive patients demonstrated recurrence of distant metastasis in 67% of cases. Therefore, DPM is a critical prognostic indicator in PDAC. In combination with preCA19-9, DPM may be useful to identify long-term survivors of PDAC. Furthermore, to the best of our knowledge, the current study was the first to discover that DPM can represent a poor prognosis based putatively on its association with the K-ras gene mutation.

Critical role of KRAS mutation in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers worldwide. Little progress has been made in recent years concerning its diagnosis and treatment. Genetic alterations can be found in approximately 97% of PDAC cases. Mutations in the KRAS gene, which encodes the KRAS protein that regulates cell proliferation, differentiation, and apoptosis via activation of downstream signal transduction pathways, occur most frequently. KRAS plays a pivotal role in modulating the tumor microenvironment in PDAC patients. Additionally, KRAS can regulate metabolic changes in PDAC cells in a variety of ways. Although previous studies have shown that KRAS mutation detection can be used for early diagnosis and to predict the prognosis of PDAC patients, and many paths have been proposed to suppress the effects of KRAS , there is still no single pathway that leads to effective treatment of KRAS -mutant PDAC. This review summarizes the role of KRAS mutation in PDAC and examines the association between KRAS mutation and clinical applications.

1903P - Prevalence of KRAS mutation subtypes and MSI status in pancreatic cancer

Background: KRAS is the most prevalent driver mutation in pancreatic ductal adenocarcinoma (PDAC) which impacts cell differentiation, proliferation, migration and apoptosis. KRAS subtypes may have prognostic significance but this finding remains uncertain. Microsatellite Instability / mismatch repair deficiency (MMR) has also been reported in PDAC but at a low frequency (0.8%) and these patients are eligible for immunotherapy. This study investigates the effect of KRAS subtype on survival rate and the prevalence of mismatch repair deficiency in our patient cohort. Methods: 91 patients enrolled from 2005 to 2012 with biopsy proven PDAC of all stages. All clinical data were collected from the medical records of each patient. Next generation sequencing was performed on all 91 samples. We also evaluated MMR expression in all resection specimens who underwent adjuvant treatment. Statistical analysis: Data were summarized as descriptive analysis statistics and analyzed using unpaired t-test. Overall survival (OS) was measured from the date of diagnosis to last known follow up or date of dead from disease. Results: The most common type of mutation in all stages is KRAS (95%). KRAS mutation subtypes in the order of frequency were: G12D, G12V, G12R, Q61H, Q61L and G12C. Among codon 12 mutations, G12V showed the best OS at 20.12 months. Mutations in codon 61 carry better OS compared to codon 12 by 24 months (P = 0.0002, 95% CI 11.85 to 37.03). The staining for MMR status was performed on 30 of 91 specimens and all were mismatch repair protein proficient (analogous to MSI-stable [MSS]). Conclusions: The prevalence and distribution of KRAS mutations from our study is similar to previous reports. Patients with a KRAS codon 61 mutation had better OS than with a codon 12 mutation. Interestingly, Q61 variants are far less common than the G12 variants which may explain why most of the pancreatic patients have an aggressive disease course. Our results support the conclusion that MMR defects are uncommon in PDAC. Germline mutation analysis could be considered to find other targetable mutations found in hereditary cancer syndromes such as BRCA1/2 or PALB2. Legal entity responsible for the study: Lloyd Hutchinson. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Abstract 2443: Oxygen metabolism and hypoxia tolerance in organoid models of pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) has extremely heterogeneous hypoxic microenvironments across patients and high levels of hypoxia are correlated with increased tumor aggressiveness and resistance to therapy. However, the underlying genetic contributors to variations in hypoxia and its importance to the disease is currently unknown. We hypothesize that genetic mutations in PDAC associated with two principal factors - oxygen metabolism and hypoxia tolerance - influence the steady state levels of hypoxia in individual tumors. The demand for oxygen, which is influenced by genetic driven changes in cellular metabolism, define the levels and steepness of hypoxia gradients around perfused vessels. Tolerance to hypoxia determines the time tumor cells can survive in severe microenvironments depleted of oxygen and other nutrients. Both factors are affected by the activation of adaptive hypoxia stress response pathways including the HIF, UPR, and autophagy pathways. Method: We developed patient-derived-organoids from PDAC tumors for in vitro studies of oxygen metabolism and glycolytic rates using the Seahorse XF96. We also characterized hypoxia tolerance through monitoring of organoid growth and secondary growth under defined levels of oxygenation. In addition, we have developed an immunofluorescence image analysis pipeline to evaluate in vivo oxygen demand/consumption through the quantification of oxygen and proliferation gradients around perfused blood vessels. Results: We observed significant heterogeneities in oxygen metabolism and hypoxia tolerance across our patient derived organoid models. We also demonstrated the importance of PERK/UPR pathway in mediating both oxygen metabolism and hypoxia tolerance through regulation of ULK1, a kinase involved in the initiation of autophagy. Inhibition or knockdown of ULK1 decreased cell survival and correspondingly sensitized cells to hypoxia in organoid and tumor models. This is accompanied by accumulation of mitochondria and a corresponding increase in oxygen consumption, resulting in increased development of hypoxic cells. Conclusion: These experiments demonstrate the dual importance of oxygen metabolism and hypoxia tolerance and set the stage for the evaluation of these parameters and identification of the underlying genetic drivers of the hypoxic microenvironment. These genetic markers would be used for patient-selection and development of hypoxia-targeted therapies. Citation Format: Ji Zhang, Qingquan Liu, Dan Cojocari, Mark Zaidi, Trevor McKee, Nikolina Radulovich, Ming-Sound Tsao, David Hedley, Marianne Koritzinsky, Bradly G. Wouters. Oxygen metabolism and hypoxia tolerance in organoid models of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2443.

A Phase II Study of Pelareorep (REOLYSIN®) in Combination with Gemcitabine for Patients with Advanced Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with 1 and 5-year survival rates of ~18% and 7% respectively. FOLFIRINOX or gemcitabine in combination with nab-paclitaxel are standard treatment options for metastatic disease. However, both regimens are more toxic than gemcitabine alone. Pelareorep (REOLYSIN®), a proprietary isolate of reovirus Type 3 Dearing, has shown antitumor activity in clinical and preclinical models. In addition to direct cytotoxic effects, pelareorep can trigger antitumor immune responses. Due to the high frequency of RAS mutations in PDAC, we hypothesized that pelareorep would promote selective reovirus replication in pancreatic tumors and enhance the anticancer activity of gemcitabine. Chemotherapy-naïve patients with advanced PDAC were eligible for the study. The primary objective was Clinical Benefit Rate (complete response (CR) + partial response (PR) + stable disease (SD) ≥ 12 weeks) and secondary objectives include overall survival (OS), toxicity, and pharmacodynamics (PD) analysis. The study enrolled 34 patients; results included one partial response, 23 stable disease, and 5 progressive disease. The median OS was 10.2 months, with a 1- and 2-year survival rate of 45% and 24%, respectively. The treatment was well tolerated with manageable nonhematological toxicities. PD analysis revealed reovirus replication within pancreatic tumor and associated apoptosis. Upregulation of immune checkpoint marker PD-L1 suggests future consideration of combining oncolytic virus therapy with anti-PD-L1 inhibitors. We conclude that pelareorep complements single agent gemcitabine in PDAC.

Abstract A28: Understanding the role of the tumor suppressor p53 in pancreatic cancer development

Abstract In the next decade, pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer-related death. This high rate of mortality is attributed to late-stage diagnosis and lack of effective therapeutic strategies. The tumor-suppressor gene TP53 is mutated in ~75% of PDAC patients. The most frequently observed TP53 mutations in PDAC occur within the DNA binding domain at codons 175 and 273. TP53 mutations may induce loss of wild-type p53 function and gain of novel, oncogenic functions. We aim to use different mouse models of PDAC to identify the molecular pathways deregulated when wild-type p53 is lost and mutant p53 is expressed, to elucidate the molecular mechanisms driving PDAC development. While previous studies have suggested that point mutant p53 drives gain-of-function phenotypes, our studies of aging Pdx1-Cre;KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato mice with conditional p53 knockout (p53Flox/Flox or p53Flox/Null) or p53 mutation (p53LSL-R172H/Flox or p53LSL-R172H/Null) did not demonstrate a clear mutant p53 gain-of-function phenotype in tumor latency. We hypothesize that tumor latency is too rapid in these models to manifest a clear gain-of-function phenotype, requiring the establishment of different model systems. Indeed, our preliminary studies using tumor-derived p53R172H cell lines and isogenic p53 null cell lines suggest a role for mutant p53 gain of function in later-stage tumorigenesis, specifically in metastatic seeding. Furthermore, we are evaluating the role of the putative PDAC cells of origin, pancreatic acinar and ductal cells, in PDAC development. Using both tumor-derived mouse cell lines and pancreatic lineage-specific genetically engineered mouse models of PDAC, we can evaluate the consequences of loss of wild-type p53 expression and gain of mutant p53 expression in different pancreatic compartments. Ultimately, using these systems we aim to elucidate the target genes and molecular pathways controlled by wild-type p53 or by mutant p53 to regulate PDAC initiation. Understanding the molecular mechanisms driving PDAC initiation is critical for the development of new therapeutic strategies for early detection and effective intervention in PDAC patients. Citation Format: Brittany M. Flowers, Patty B. Garcia, Barbara M. Grüner, Monte M. Winslow, Laura D. Attardi. Understanding the role of the tumor suppressor p53 in pancreatic cancer development [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A28.

Overexpression of HER2 in the pancreas promotes development of intraductal papillary mucinous neoplasms in mice

Pancreatic ductal adenocarcinoma (PDA) has a 5-year survival rate of less than 5% and is the sixth leading cause of cancer death. Although KRAS mutations are one of the major driver mutations in PDA, KRAS mutation alone is not sufficient to induce invasive pancreatic cancer in mice model. HER2, also known as ERBB2, is a receptor tyrosine kinase, and overexpression of HER2 is associated with poor clinical outcomes in pancreatic cancer. However, no report has shown whether HER2 and its downstream signaling contributes to the pancreatic cancer development. By immunohistochemical analysis in human cases, HER2 protein expression was detected in 40% of PDAs and 29% of intraductal papillary mucinous carcinomas, another type of pancreatic cancer. In a mouse model, we showed overexpression of activated HER2 (HER2NT) in the pancreas, in which cystic neoplastic lesions resembling intraductal papillary mucinous neoplasm-like lesions in humans had developed. We also found that HER2NT cooperated with oncogenic Kras to accelerate the development of pancreatic intraepithelial neoplasms. In addition, using pancreatic organoids in 3D cultures, we found that organoids cultured from HER2NT/Kras double transgenic mice showed proliferative potential and tumorigenic ability cooperatively. HER2-signaling inhibition was suggested to be an new therapeutic target in some types of PDAs.

Comparison of Practice Guidelines, BRCAPRO, and Genetic Counselor Estimates to Identify Germline BRCA1 and BRCA2 Mutations in Pancreatic Cancer.

Germline BRCA1 and BRCA2 (BRCA) mutation carriers with pancreatic ductal adenocarcinoma (PDAC) may benefit from precision therapies and their relatives should undergo tailored cancer prevention. In this study, we compared strategies to identify BRCA carriers with PDAC. Incident cases of PDAC were prospectively recruited for BRCA sequencing. Probands were evaluated using the National Comprehensive Cancer Network (NCCN) and the Ontario Ministry of Health and Long-Term Care (MOHLTC) guidelines. The probability of each proband carrying a mutation was estimated by surveying genetic counselors and using BRCAPRO. BRCA mutations were detected in 22/484 (4.5%) probands. 152/484 (31.2%) and 16/484 (3.3%) probands met the NCCN and MOHLTC guidelines, respectively. The NCCN guidelines had higher sensitivity than the MOHLTC guidelines (0.864 versus 0.227, P < 0.001) but lower specificity (0.712 versus 0.976, P < 0.001). One hundred and nineteen genetic counselors completed the survey. Discrimination was similar between genetic counselors and BRCAPRO (area-under-the-curve: 0.755 and 0.775, respectively, P = 0.702). Genetic counselors generally overestimated (P = 0.008), whereas BRCAPRO severely underestimated (P < 0.001), the probability that each proband carried a mutation. Our results indicate that the NCCN guidelines and genetic counselors accurately identify BRCA mutations in PDAC, while the MOHLTC guidelines and BRCAPRO should be updated to account for the association between BRCA and PDAC.

Mutant KRAS Circulating Tumor DNA Is an Accurate Tool for Pancreatic Cancer Monitoring.

Many new pancreatic cancer treatment combinations have been discovered in recent years, yet the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains grim. The advent of new treatments highlights the need for better monitoring tools for treatment response, to allow a timely switch between different therapeutic regimens. Circulating tumor DNA (ctDNA) is a tool for cancer detection and characterization with growing clinical use. However, currently, ctDNA is not used for monitoring treatment response. The high prevalence of KRAS hotspot mutations in PDAC suggests that mutant KRAS can be an efficient ctDNA marker for PDAC monitoring. Seventeen metastatic PDAC patients were recruited and serial plasma samples were collected. CtDNA was extracted from the plasma, and KRAS mutation analysis was performed using next-generation sequencing and correlated with serum CA19-9 levels, imaging, and survival. Plasma KRAS mutations were detected in 5/17 (29.4%) patients. KRAS ctDNA detection was associated with shorter survival (8 vs. 37.5 months). Our results show that, in ctDNA positive patients, ctDNA is at least comparable to CA19-9 as a marker for monitoring treatment response. Furthermore, the rate of ctDNA change was inversely correlated with survival. Our results confirm that mutant KRAS ctDNA detection in metastatic PDAC patients is a poor prognostic marker. Additionally, we were able to show that mutant KRAS ctDNA analysis can be used to monitor treatment response in PDAC patients and that ctDNA dynamics is associated with survival. We suggest that ctDNA analysis in metastatic PDAC patients is a readily available tool for disease monitoring. Avoiding futile chemotherapy in metastatic pancreatic ductal adenocarcinoma (PDAC) patients by monitoring response to treatment is of utmost importance. A novel biomarker for monitoring treatment response in PDAC, using mutant KRAS circulating tumor DNA (ctDNA), is proposed. Results, although limited by small sample numbers, suggest that ctDNA can be an effective marker for disease monitoring and that ctDNA level over time is a better predictor of survival than the dynamics of the commonly used biomarker CA19-9. Therefore, ctDNA analysis can be a useful tool for monitoring PDAC treatment response. These results should be further validated in larger sample numbers.

DNA Repair Dysfunction in Pancreatic Cancer: A Clinically Relevant Subtype for Drug Development.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, with a 5-year survival rate of ≤7% across all stages. The limited success of conventional therapies for PDAC is at least partially attributable to its genetic heterogeneity. Precision targeting of known PDAC subtypes may positively affect the outcome of this disease. An important actionable subtype in this cancer is associated with DNA repair dysfunction, including cases with germline BRCA mutations. This subtype can be targeted by inhibitors of poly(ADP-ribose) polymerase (PARP). BRCA mutation-associated PDAC may be the first biomarker-driven subtype in this disease that can be successfully targeted. However, DNA repair defects can extend beyond the narrow spectrum of BRCA1/2 mutations in PDAC and are present in a large proportion of patients with familial PDAC. This review describes the subgroup of patients with PDAC with aberrant DNA repair and discusses diagnostic and therapeutic options.

Recurrent noncoding regulatory mutations in pancreatic ductal adenocarcinoma.

The contributions of coding mutations to tumorigenesis are relatively well known; however, little is known about somatic alterations in noncoding DNA. Here we describe GECCO (Genomic Enrichment Computational Clustering Operation) to analyze somatic noncoding alterations in 308 pancreatic ductal adenocarcinomas (PDAs) and identify commonly mutated regulatory regions. We find recurrent noncoding mutations are enriched in PDA pathways, including axon guidance and cell adhesion, and novel processes including transcription and homeobox genes. We identify mutations in protein binding sites correlating with differential expression of proximal genes and experimentally validate effects of mutations on expression. We developed an expression modulation score that quantifies the strength of gene regulation imposed by each class of regulatory elements, and find the strongest elements are most frequently mutated, suggesting a selective advantage. Our detailed single-cancer analysis of noncoding alterations identifies regulatory mutations as candidates for diagnostic and prognostic markers, and suggests novel mechanisms for tumor evolution.

Oncogenic KRas-induced Increase in Fluid-phase Endocytosis is Dependent on N-WASP and is Required for the Formation of Pancreatic Preneoplastic Lesions

Fluid-phase endocytosis is a homeostatic process with an unknown role in tumor initiation. The driver mutation in pancreatic ductal adenocarcinoma (PDAC) is constitutively active KRasG12D, which induces neoplastic transformation of acinar cells through acinar-to-ductal metaplasia (ADM). We have previously shown that KRasG12D-induced ADM is dependent on RAC1 and EGF receptor (EGFR) by a not fully clarified mechanism. Using three-dimensional mouse and human acinar tissue cultures and genetically engineered mouse models, we provide evidence that (i) KRasG12D leads to EGFR-dependent sustained fluid-phase endocytosis (FPE) during acinar metaplasia; (ii) variations in plasma membrane tension increase FPE and lead to ADM in vitro independently of EGFR; and (iii) that RAC1 regulates ADM formation partially through actin-dependent regulation of FPE. In addition, mice with a pancreas-specific deletion of the Neural-Wiskott–Aldrich syndrome protein (N-WASP), a regulator of F-actin, have reduced FPE and impaired ADM emphasizing the in vivo relevance of our findings. This work defines a new role of FPE as a tumor initiating mechanism.

Abstract A27: Recurrent noncoding regulatory mutations in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with a 5-year survival rate of 6%, due in part to therapy resistance and late stage at diagnosis. A detailed understanding of the molecular alterations underlying PDA is required to uncover mechanisms of tumorigenesis and enable development of more effective therapies. As such, large-scale exome sequencing efforts have revealed genes (KRAS, TP53, CDKN2A, SMAD4) and pathways (Wnt/Notch, TGFβ, axon guidance, cell adhesion) important for PDA progression. However, the exome comprises less than 2% of the human genome. Whole-genome sequencing analyses have uncovered a somatic mutation rate of 1 mutation per megabase in PDA. Therefore, PDA tumors carry thousands of somatic mutations per genome, mostly located in noncoding regions, the vast majority of which are completely uncharacterized. While the contributions of coding mutations to tumorigenesis are relatively well known, few studies have detailed alterations in noncoding DNA. Here we describe a new computational pipeline, GECCO (Genomic Enrichment Computational Clustering Operation) to analyze somatic noncoding alterations in 308 pancreatic ductal adenocarcinomas (PDA) and systematically identify commonly mutated putative regulatory regions. We find that recurrent somatic noncoding regulatory mutations are present in PDA but uncommon near canonical PDA genes. Instead, using GECCO we find that the regulatory mutations are enriched in known PDA pathways, including axon guidance and cell adhesion, and novel processes including transcriptional regulation and homeobox genes. We identify mutations in specific protein binding sites that correlate with significant differential expression of proximal genes. Furthermore, to explore the effects of these candidate mutations on gene expression, we developed an expression modulation score that quantifies the strength of gene regulation imposed by each class of regulatory elements. Surprisingly, we find that the strongest regulatory elements are also the most frequently mutated, suggesting a selective advantage for mutations in these regions. Our analysis provides a detailed single-cancer view of noncoding alterations in tumorigenesis, identifies recurrent regulatory mutations as new candidates for diagnostic and prognostic markers, and suggests novel mechanisms for tumor genome evolution. Citation Format: Michael E. Feigin, Tyler Garvin, Peter Bailey, Nicola Waddell, David K. Chang, Shimin Shuai, Steven Gallinger, John D. McPherson, Sean M. Grimmond, Ekta Khurana, Lincoln D. Stein, Andrew V. Biankin, Michael C. Schatz, David A. Tuveson.{Authors}. Recurrent noncoding regulatory mutations in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A27.

Heterogeneity of KRAS Mutations in Pancreatic Ductal Adenocarcinoma.

Activating Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are the most common and frequent changes observed in pancreatic cancer. This study aimed to determine the frequency and extent of intratumoral and metastatic lymph node KRAS mutation heterogeneity of resected pancreatic ductal adenocarcinoma. Tumor tissues macrodissected from tumor centers, invasion fronts (n = 97), and lymph nodes (n = 11) were subjected to DNA extraction and mutation analysis of KRAS codons 12 and 13 by pyrosequencing. Activating mutations in codon 12 of KRAS were detected in 90 (92.8%) tumor centers. No mutations were detected in KRAS codon 13 in any patient. After a comparison of tumor centers and invasion fronts, intratumoral heterogeneity of KRAS was observed only in 4 (4.1%) cases. Additional invasion front tumor analysis revealed the same mutation status consistent with each tumor center. No heterogeneity was observed between primary tumors and metastatic lymph nodes. Intratumoral heterogeneity of the KRAS mutational status is rare in pancreatic ductal adenocarcinoma. In addition, no KRAS heterogeneity between primary tumors and metastatic lymph nodes was detected in this study. This finding is consistent with the hypothesis that oncogenic activation of KRAS is the first driver mutation in pancreatic cancer.

Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries. Unfortunately, the overall 5-year survival rate is still less than 5%. The most frequent oncogenic mutations in PDAC are loss-of function mutations in p53 and gain-of-function mutations in KRAS. Here we show that clofazimine (Lamprene), a drug already used in the clinic for autoimmune diseases and leprosy, is able to efficiently kill in vitro five different PDAC cell lines harboring p53 mutations. We provide evidence that clofazimine induces apoptosis in PDAC cells with an EC50 in the μM range via its specific inhibitory action on the potassium channel Kv1.3. Intraperitoneal injection of clofazimine resulted in its accumulation in the pancreas of mice 8 hours after administration. Using an orthotopic PDAC xenotransplantation model in SCID beige mouse, we show that clofazimine significantly and strongly reduced the primary tumor weight. Thus, our work identifies clofazimine as a promising therapeutic agent against PDAC and further highlights ion channels as possible oncological targets.

Reduced Expression of Histone Methyltransferases KMT2C and KMT2D Correlates with Improved Outcome in Pancreatic Ductal Adenocarcinoma.

Genes encoding the histone H3 lysine 4 methyltransferases KMT2C and KMT2D are subject to deletion and mutation in pancreatic ductal adenocarcinoma (PDAC), where these lesions identify a group of patients with a more favorable prognosis. In this study, we demonstrate that low KMT2C and KMT2D expression in biopsies also defines better outcome groups, with median survivals of 15.9 versus 9.2 months (P = 0.029) and 19.9 versus 11.8 months (P = 0.001), respectively. Experiments with eight human pancreatic cell lines showed attenuated cell proliferation when these methyltransferases were depleted, suggesting that this improved outcome may reflect a cell-cycle block with diminished progression from G0-G1 RNA-seq analysis of PDAC cell lines following KMT2C or KMT2D knockdown identified 31 and 124 differentially expressed genes, respectively, with 19 genes in common. Gene-set enrichment analysis revealed significant downregulation of genes related to cell-cycle and growth. These data were corroborated independently by examining KMT2C/D signatures extracted from the International Cancer Genome Consortium and The Cancer Genome Atlas datasets. Furthermore, these experiments highlighted a potential role for NCAPD3, a condensin II complex subunit, as an outcome predictor in PDAC using existing gene expression series. Kmt2d depletion in KC/KPC cell lines also led to an increased response to the nucleoside analogue 5-fluorouracil, suggesting that lower levels of this methyltransferase may mediate the sensitivity of PDAC to particular treatments. Therefore, it may also be therapeutically beneficial to target these methyltransferases in PDAC, especially in those patients demonstrating higher KTM2C/D expression. Cancer Res; 76(16); 4861-71. ©2016 AACR.

Identification of KCa3.1 Channel as a Novel Regulator of Oxidative Phosphorylation in a Subset of Pancreatic Carcinoma Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries and overall 5-year survival rates of less than 5%. The most frequent mutations in PDAC are gain-of-function mutations in KRAS as well as loss-of-function mutations in p53. Both mutations have severe impacts on the metabolism of tumor cells. Many of these metabolic changes are mediated by transporters or channels that regulate the exchange of metabolites and ions between the intracellular compartment and the tumor microenvironment. In the study presented here, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well as novel regulators of OxPhos. The siRNA that resulted in the greatest change in cellular oxygen consumption was targeting the KCNN4 gene, which encodes for the Ca2+-sensitive K+ channel KCa3.1. This channel has not previously been reported to regulate OxPhos. Knock-down experiments as well as the use of a small molecule inhibitor confirmed its role in regulating oxygen consumption, ATP production and cellular proliferation. Furthermore, PDAC cell lines sensitive to KCa3.1 inhibition were shown to express the channel protein in the plasma membrane as well as in the mitochondria. These differences in the localization of KCa3.1 channels as well as differences in the regulation of cellular metabolism might offer opportunities for targeted therapy in subsets of PDAC.

Infiltrating immune cells and gene mutations in pancreatic ductal adenocarcinoma.

The aim of this study was to assess the immune profile within the microenvironment of pancreatic ductal adenocarcinoma (PDAC), and to investigate the prognostic value of intratumoral infiltrating immune/inflammatory cells (IICs) in patients after surgery. Eighteen phenotypic markers representing 11 types of IIC and the protein products of genes TP53, CDKN2A/p16 and SMAD4/DPC4 were assessed by immunohistochemistry of specimens from patients with pancreatic cancer. The expression of IICs and the mutational status of the genes were correlated with tumour recurrence and survival, and results were validated in an independent cohort. CD15+ neutrophils, CD20+ B cells and CD206+ tumour-associated macrophages were seen frequently in tumours, and their presence was associated with reduced survival in a cohort of 79 patients. Expression of CD4+ T helper cells, CD8+ cytotoxic T lymphocytes and CD117+ mast cells was associated with a favourable prognosis. A weighted Cox regression recurrence-predictive model was constructed that showed good correlation of IICs and gene mutations. A combination of CD15, CD206, CD117 and Smad4 expression was independently associated with overall (hazard ratio (HR) 3·63, 95 per cent c.i. 2·18 to 6·04; P < 0·001) and recurrence-free (HR 2·93, 1·81 to 4·75; P < 0·001) survival. These findings were validated in an independent cohort (151 patients) and in 54 tissue samples obtained by preoperative endoscopic ultrasound-guided fine-needle aspiration. PDAC has a unique immunosuppressive phenotype that is associated with characteristic gene mutations, disease recurrence and survival after pancreatectomy. Surgical relevance The immune microenvironment plays a critical role in the development of pancreatic ductal adenocarcinoma (PDAC). PDAC is associated with mutations in major driver genes, including KRAS, TP53, CDKN2A/p16 and SMAD4/DPC4. This study shows that the microenvironment of PDAC has a unique immunosuppressive phenotype, which may be driven by oncogene mutations. Patients with PDAC with a highly immunosuppressive profile tended to have poor postoperative survival. A model including three intratumoral infiltrating immune markers (CD15+, CD206+ and CD117+) and a SMAD4 mutation can be used to predict recurrence and survival in patients after surgery for PDAC.

Abstract PR03: Exploring the link between Kras and histone acetylation

Abstract KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) have been shown to extensively rewire cellular metabolism to promote macromolecular biosynthesis and maintain redox homeostasis. Recently, compelling evidence has emerged demonstrating that many epigenetic modifications are sensitive to the availability of cellular metabolites. For example, acetyl-CoA is the donor substrate for lysine acetylation, and histone acetylation is responsive to levels of acetyl-CoA, which is produced largely from glucose metabolism. We thus hypothesized that oncogenic metabolic reprogramming alters metabolite levels in a way that impacts the epigenome and could thus contribute to tumorigenesis. Indeed, we find that oncogenic Akt activation promotes elevated histone acetylation, and that this effect is mediated by ATP-citrate-lyase (ACLY), a nucleocytoplasmic enzyme that converts glucose-derived citrate into acetyl-CoA. In order to understand what cellular processes are affected by such AKT-ACLY-mediated increase in histone acetylation, we employed a mouse model of pancreatic ductal adenocarcinoma, driven by oncogenic Kras (Pdx1-Cre; LSL-KrasG12D; TP53L/+). In this model, the PI3K/Akt pathway is activated downstream of Kras. By immunohistochemistry we found that, in mice with WT Kras, pancreatic acinar cells exhibit very low levels of histone H4 (K5/8/12/16) acetylation (AcH4), although AcH4 is clearly detectable in ductal epithelial cells and islets. By contrast, in mice expressing oncogenic Kras in the pancreas (LSL-KrasG12D; p53L/+; Pdx1-Cre), acinar H4 acetylation is dramatically increased. Remarkably, this overt increase in histone acetylation precedes the appearance of histological abnormalities and persists during several steps in tumor progression. Moreover, Kras and Akt-dependent reuglation of histone acetylation levels can be recapitulated in ex vivo acinar cell culture, suggesting that this is a primary effect of oncogene activation in these cells. Akt inhibition also significantly reduced glucose consumption and phosphorylation of ACLY and ultimately decreased acetyl-CoA levels. Akt inhibition in vitro reduces Kras-induced acinar cell hyperacetylation and block acinar-to-ductal metaplasia, an initiating event of pancreatic carcinoma. Our data indicate that Akt orchestrates a metabolic rewiring in pancreatic tumorigenesis, which promotes histone hyperacetylation, a phenotype evident before the manifestation of the disease and that might conceivably contribute to cell plasticity and tumor progression. Citation Format: Alessandro Carrer, Joyce V. Lee, Supriya Shah, Nicole M. Aiello, Nathaniel W. Snyder, Andrew J. Worth, Ian A. Blair, Ben Z. Stanger, Kathryn E. Wellen. Exploring the link between Kras and histone acetylation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR03.

Abstract PR09: Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression

Abstract A) Pancreatic ductal adenocarcinoma (PDA) depends on a marked reprogramming of metabolic pathways, including the acquisition of autophagy dependence, for survival and growth. How common mutations in PDA cause autophagy dependence as well as the timing of autophagy activation in the course of cancer progression, have not been established. Here we show how Plac8, a gene synergistically up-regulated in response to the common cooperating oncogenic mutations found in PDA (RAS activation and functional loss of p53), is critical to the growth of PDA by sustaining autophagy via facititating autophagosome-lysosome fusion. Furthermore, we delineate that Kras and p53 mutations cooperate to induce autophagic flux. B) To establish Plac8’s role in PDA growth and autophagy we use murine cell lines and human PDA cell lines to determine 1) the lysosomal localization of Plac8, 2) its role in regulating autophagy using both loss of function and gain of function approaches, 3) the impact on lysosomal biology, and 4) the relationship between Plac8 and other genetic pathways governing autophagy. Using genetically engineered models of PDA we determine the timing of autophagy activation in PDA progression and the impact of Plac8 mutation. C) We identify Plac8 as a novel regulator of autophagosome-lysosome fusion required for PDA growth, thus providing a mechanistic link between oncogenic mutations and the activation of autophagy in cancer. Plac8 expression is required for growth of human PDA cells as xenografts in mice, as well as activation of autophagy. We find that concurrent mutation of KRAS and p53 is critical for maximal induction of autophagy in vitro. Correspondingly, using genetically engineered mouse models of PDA (Pdx1-Cre; LSL-KrasG12D; p53L/+), in which loss of p53 function occurs in a step-wise manner relying on the spontaneous loss of a heterozygous WT p53 allele, we see a step-wise incremental increase in LC3 puncta in vivo with each histological stage through the course of PDA progression. Thus, we find that the cooperative effects of KRAS and p53 drive activation of autophagy rather than either mutation alone. The overall survival of a Pdx1-Cre; LSL-KrasG12D; p53L/+; Plac8null murine cohort (OS 27.9 wks) was significantly longer than a Pdx1-Cre; LSL-KrasG12D; p53L/+; Plac8wt cohort (OS 17.0 wks, p=0.0006) demonstrating in vivo that genetic inactivation of Plac8 impedes cancer progression and resulting death. Our data suggest that the role of Plac8 in facilitating autophagy is critical to cancer, as the requirement of Plac8 for both tumorigenicity and autophagy can be compensated by over-expression of Atg12, a gene critical for autophagosome formation or by constitutively activated Rab7, a gene encoding a GTP-binding protein stimulating autophagosome-lysosome fusion. D) We conclude that Plac8 may offer a potential therapeutic window and point of intervention, as Plac8 mutation in the engineered PDA model inhibits cancer progression and significantly improves survival while having a minimal impact on the overall fitness of the animals. In fact, Plac8, and regulation of autophagosome-lysosome fusion, has specific relevance to regulation of autophagy during malignant cell transformation as Plac8 and the processes it regulates, appear to be largely dispensable to many normal physiologic processes. This abstract is also presented as Poster B36. Citation Format: Vijaya Balakrishnan, Kinsey Conan, Michael O'Dell, Jing Li Huang, Laurel Newman, Christa Whitney-Miller, Hartmut Land, Aram Hezel. Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR09.