Anita Hall

Candidate DNA repair susceptibility genes identified by exome sequencing in high-risk pancreatic cancer

The genetic basis underlying the majority of hereditary pancreatic adenocarcinoma (PC) is unknown. Since DNA repair genes are widely implicated in gastrointestinal malignancies, including PC, we hypothesized that there are novel DNA repair PC susceptibility genes. As germline DNA repair gene mutations may lead to PC subtypes with selective therapeutic responses, we also hypothesized that there is an overall survival (OS) difference in mutation carriers versus non-carriers. We therefore interrogated the germline exomes of 109 high-risk PC cases for rare protein-truncating variants (PTVs) in 513 putative DNA repair genes. We identified PTVs in 41 novel genes among 36 kindred. Additional genetic evidence for causality was obtained for 17 genes, with FAN1, NEK1 and RHNO1 emerging as the strongest candidates. An OS difference was observed for carriers versus non-carriers of PTVs with early stage (≤IIB) disease. This adverse survival trend in carriers with early stage disease was also observed in an independent series of 130 PC cases. We identified candidate DNA repair PC susceptibility genes and suggest that carriers of a germline PTV in a DNA repair gene with early stage disease have worse survival.

Increased in vitro and in vivo sensitivity of BRCA2-associated pancreatic cancer to the poly(ADP-ribose) polymerase-1/2 inhibitor BMN 673.

BRCA2-associated pancreatic ductal adenocarcinoma (PDAC) may be sensitive to agents that target homology-directed DNA repair, such as DNA crosslinking agents (DCLs) and PARP inhibitors (PARPis). Here, we assessed the sensitivities of BRCA2-deficient (Capan-1) and BRCA2-proficient (MIA PaCa-2) PDAC cell lines to a panel of DCLs and PARPis. Compared to MIA PaCa-2, Capan-1 was significantly more sensitive to all tested DCLs and PARPis, with similar increased sensitivities to cisplatin and the PARPi BMN 673 compared to other DCLs and the PARPi veliparib. We provide further support for this observation by showing that shRNA-mediated BRCA2 knockdown in PANC-1, a BRCA2-proficient cell line, induces sensitization to cisplatin and BMN 673 but not to veliparib. These findings were validated in a PDAC murine xenograft model derived from a patient with bi-allelic BRCA2 mutations. We found 64% and 61% tumor growth inhibition of this xenograft with cisplatin and BMN 673 treatments, respectively. Cisplatin and BMN 673 treatments reduced cellular proliferation and induced apoptosis. Our findings support a personalized treatment approach for BRCA2-associated PDAC.

The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity.

A compound that binds to tubulin in an unusual way has superior antitumor efficacy and safety and has a distinctive impact on microtubule curvature and dynamics. Throwing a curve ball to cancer Drugs such as vinca alkaloids, which target tubulin and interfere with microtubule function in mitosis, are commonly used for the treatment of cancer. Unfortunately, they also damage microtubules in normal undividing cells including neurons, resulting in toxicity. Wieczorek et al. identified a drug called DZ-2384, which may offer a safer alternative to the vincas. The authors found that although DZ-2384 is very effective at targeting cancer cells by inhibiting mitosis, it preserves the microtubule network in non-dividing cells and in primary neurons at effective doses and is much safer in mouse models. By analyzing the structure of tubulin with different compounds, the authors determined that DZ-2384 binds at the vinca site but induces a distinctive change in the curvature of growing tubulin protofilaments, which may explain its unusual effects on microtubule dynamics and decreased toxicity. Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.

Establishing a clinic-based pancreatic cancer and periampullary tumour research registry in Quebec

BACKGROUND Enrolling patients in studies of pancreatic ductal adenocarcinoma (pdac) is challenging because of the high fatality of the disease. We hypothesized that a prospective clinic-based study with rapid ascertainment would result in high participation rates. Using that strategy, we established the Quebec Pancreas Cancer Study (qpcs) to investigate the genetics and causes of pdac and other periampullary tumours (pats) that are also rare and underrepresented in research studies. METHODS Patients diagnosed with pdac or pat were introduced to the study at their initial clinical encounter, with a strategy to enrol participants within 2 weeks of diagnosis. Patient self-referrals and referrals of unaffected individuals with an increased risk of pdac were also accepted. Family histories, epidemiologic and clinical data, and biospecimens were collected. Additional relatives were enrolled in families at increased genetic risk. RESULTS The first 346 completed referrals led to 306 probands being enrolled, including 190 probands affected with pdac, who represent the population focus of the qpcs. Participation rates were 88.4% for all referrals and 89.2% for pdac referrals. Family history, epidemiologic and clinical data, and biospecimens were ascertained from 91.9%, 54.6%, and 97.5% respectively of patients with pdac. Although demographics and trends in risk factors in our patients were consistent with published statistics for patients with pdac, the qpcs is enriched for families with French-Canadian ancestry (37.4%), a population with recurrent germ-line mutations in hereditary diseases. CONCLUSIONS Using rapid ascertainment, a pdac and pat research registry with high participation rates can be established. The qpcs is a valuable research resource and its enrichment with patients of French-Canadian ancestry provides a unique opportunity for studies of heredity in these diseases.

Regulation of the Src Kinase-associated Phosphoprotein 55 Homologue by the Protein Tyrosine Phosphatase PTP-PEST in the Control of Cell Motility

Background: PTP-PEST regulates cell migration as part of many protein complexes. Results: SKAP-Hom is a substrate of PTP-PEST that is required for proper fibroblasts migration. Enhanced migration was observed when SKAP-Hom-deficient fibroblasts are rescued with its SH3 domain mutant. Conclusion: As a novel substrate of PTP-PEST, SKAP-Hom is important in cellular migration. Significance: PTP-PEST regulates migration through a new complex involving SKAP-Hom. PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.

Reflex Testing for Germline BRCA1, BRCA2, PALB2, and ATM Mutations in Pancreatic Cancer: Mutation Prevalence and Clinical Outcomes From Two Canadian Research Registries

PurposeWe investigated the translational value of reflex testing for germline mutations in four homology-directed DNA repair predisposition genes (BRCA1, BRCA2, PALB2, and ATM) in consecutive patients with pancreatic adenocarcinoma.MethodsOne hundred fifty patients with French-Canadian (FC) ancestry were evaluated for founder mutations, and 114 patients were subsequently assessed by full gene sequencing and multiplex ligation-dependent probe amplification for nonfounder mutations. Two hundred thirty-six patients unselected for ancestry were also assessed for mutations by full gene sequencing.ResultsThe FC founder mutation prevalence among the 150 patients was 5.3% (95% CI, 2.6% to 10.3%), and the nonfounder mutation prevalence across the four genes among the 114 patients tested was 2.6% (95% CI, 0.6% to 7.8%). In the case series unselected for ancestry, 10.0% (95% CI, 2.7% to 26.4%) of patients reporting Ashkenazi Jewish (AJ) ancestry carried an AJ founder mutation, with no nonfounder mutations identified...

Abstract 1135: DRD2 is critical for pancreatic cancer and promises pharmacological therapy by already established antagonists

Introduction and aims: Although the overall five-year survival of all patients with cancer stands at 63%, for pancreatic cancer patients, it is a disheartening 8% - a number that remains largely unchanged for three decades. Of the patients diagnosed with pancreatic cancer, about 85% exhibit pancreatic ductal adenocarcinoma (PDAC). Most of these patients die within 4 to 6 months after diagnosis. The poor prognosis is caused by the detection at only late stages, and lack of effective options for chemotherapy. The widely used chemotherapeutic agent gemcitabine, confers a median survival advantage of only 6 months, and resistance to therapy develops in the vast majority of patients. Given this poor prognosis of patients with PDAC, there is an urgent need to find more effective therapies. Experimental procedures: Microarrays were used to perform global gene expression profiling in 195 PDAC and 41 normal pancreatic tissue samples. Using these profiling data, we undertook an extensive analysis of PDAC transcriptome by superimposing the pathway context and interaction networks of aberrantly expressed genes to identify factors with central roles in PDAC pathways. Next, tissue microarray analysis (TMA) were used to verify the expression of the candidate target in independent set of 152 samples comprising 40 normal pancreatic tissues, 49 chronic pancreatitis sections (CP) and 63 PDAC samples. We further validated the functional relevance of the candidate molecule through RNA interference (RNAi) and pharmacological inhibition in vitro and in vivo. Results: We identified dopamine receptor D2 (DRD2) as a key modulator of cancer pathways in PDAC. DRD2 up-regulation at the protein level was validated in a large independent sample cohort. Most importantly, we found that blockade of DRD2, through RNAi or pharmacological inhibition using FDA-approved antagonists hampers the proliferative and invasive capacities of pancreatic cancer cells while modulating cAMP and endoplasmic reticulum stress pathways. Also, we observed a potent effect of DRD2 antagonists on inhibition of cancer cell proliferation using different model of primary and metastatic tumor cells derived from spontaneous pancreatic cancer mouse models and patient-derived pancreatic adenocarcinoma mouse xenograft (PDX) models. Conclusions: Our findings demonstrate that inhibiting DRD2 represents a novel therapeutic approach for PDAC. Since DRD2 inhibitors are already in the clinic for the management of schizophrenia, our results from this study could support a drug repurposing strategy to expedite clinical evaluation of these agents as novel therapy against pancreatic cancer. Citation Format: Pouria Jandaghi, Hamed S. Najafabadi, Andrea Bauer, Andreas I. Papadakis, Matteo Fassan, Anita Hall, Anie Monast, Maryam Safisamghabadi, Magnus von Knebel Doeberitz, John P. Neoptolemos, Eithne Costello, William Greenhalf, Aldo Scarpa, Bence Sipos, Daniel Auld, Mark Lathrop, Morag Park, Markus W. Buchler, Oliver Strobel, Thilo Hackert, Nathalia Giese, George Zogopoulos, Veena Sangwan, Sidong Huang, Jorg D. Hoheisel, Yaser Riazalhosseini. DRD2 is critical for pancreatic cancer and promises pharmacological therapy by already established antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1135. doi:10.1158/1538-7445.AM2017-1135

Abstract 09: Contribution of known and novel BRCA-mediated DNA repair pathway genes to pancreatic cancer susceptibility

Although 10% of pancreatic adenocarcinoma (PC) cases cluster in families, the genetic basis underlying most familial PC cases is unknown. Mutations in the BRCA-pathway genes (BRCA1/2, PALB2) are rare causes of PC, but may have a more significant role in French-Canadians (FC), a population enriched with founder mutations in these genes. We have hypothesized that 1) mutations in the BRCA-pathway genes represent 5% of the genetic susceptibility to PC in French-Canadians and, 2) that familial PC cases without mutations in known PC susceptibility genes have causative mutations in other tumor suppressor genes. In a prospective clinic-based study, we screened unselected incident PC cases with FC ancestry (n=52) for the common FC founder mutations (n=20) and uncovered the PALB2:c.2323C>T and BRCA2:c.3170_3174delAGAAA founder mutations in 2 kindreds. Additionally, we identified a novel loss-of-function BRCA2 mutation in a 3rd FC family. Since the role of PALB2 as a PC susceptibility gene is not well established, we provide supporting evidence by confirming mutation segregation with disease, as well as loss of the wild-type allele in the corresponding tumours. Of note, both BRCA2 mutation carriers were treated with platinum-based chemotherapy, targeting DNA repair defects in their tumors, and demonstrated marked tumor responses. To search for novel genetic causes of PC, we identified 99 high-risk families, including cases with 2 or more PC-affected relatives or early onset PC presentations, collected prospectively through the Quebec and Ontario Pancreas Cancer Studies. We employed exome sequencing to interrogate all of the protein-coding regions of the human genome in these 99 high-risk cases, as well as in 13 PC-affected relatives. Although these investigations did not reveal a single gene to explain the unaccounted fraction of familial PC, we identified 9 putative familial PC genes with rare, loss-of-function variants among multiple high-risk families. Two of these candidate familial PC genes are involved in the BRCA-mediated DNA repair pathway. Our data suggest that BRCA-pathway mutations may contribute significantly to PC susceptibility in French-Canadians and that mutation carriers have improved clinical outcomes if treated with agents that target BRCA-deficient cells. As well, we have identified 9 novel familial PC candidate genes, including 2 genes that are involved in the BRCA-pathway. Citation Format: Alyssa Smith, Robert Grant, Anita Hall, Najmeh Alirezaie, Spring Holter, Thomas Whelan, Iris Selander, Treasa McPherson, John McPherson, Atilla Omeroglu, Jacek Majewski, William Foulkes, Steven Gallinger, George Zogopoulos. Contribution of known and novel BRCA-mediated DNA repair pathway genes to pancreatic cancer susceptibility. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 09. doi:10.1158/1538-7445.CANSUSC14-09