Rosalind and Morris Goodman Cancer Research Centre Annual Symposium | May 6-7, 2021

The Rosalind and Morris Goodman Cancer Research Centre (GCRC)

Published online: 19 May 2021

Identifying Circulating Clonal T Cells in a Relapsed Hodgkin Lymphoma Patient Treated with Immune Check Point Inhibitor Pembrolizumab using Single-cell RNA Sequencing.

Matthew Salaciak¹, Samantha Worme¹, Tho-Alfakar Al-Aubodah², Laura Widawski², Ciriaco Piccirillo², Claudia Kleinman³, Ioannis Ragoussis⁴, Nathalie A. Johnson⁵ and Francois Mercier⁵

¹Department of Experimental Medicine, McGill University
²Department of Microbiology and Immunology, McGill University
³Department of Human Genetics, McGill University
⁴McGill Genome Center and Department of Human Genetics
⁵Department of Medicine, McGill University
Corresponding author: Matthew Salaciak, email: matthew.salaciak@mail.mcgill.ca

Abstract

Introduction:
Hodgkin’s Lymphoma is the most common cancer in young adults that is initially treated with chemotherapy. PD1 inhibitors are effective at relapse but not all patients sustain response. Identification of circulating clonal T cells across treatment might elucidate the mechanisms behind resistant T cells which may lead to more effective immunotherapy treatments.
Methods:
We performed single-cell RNA sequencing using 10xGenomics of six samples of peripheral blood mononuclear cells taken from one relapsed Hodgkin’s Lymphoma patient treated with PD-1 inhibitor pembrolizumab. Each sample corresponds to a timepoint marked by a specific disease state. Using Seurat, 24,576 cells were clustered. From this we subset CD4+/CD8+ T cells and Natural Killer T cells to obtain a total of 10,601 cells for further T-cell receptor analysis.
Results:
Using canonical markers for T cell helper, memory, effector and exhausted states, we identified 17 clusters. Scirpy, a tool to analyse T-cell receptors from Single-cell RNA sequencing data was used to identify and track T cell clones across timepoints. Within the CD8+ effector memory cluster, we observed a particular clonotype expanded at relapse and sustained at progression timepoints. Comparing the phenotypic profile of this cluster between treatment naïve and relapse timepoints revealed a dynamic change in gene expression suggesting T cell activation at relapse from a quiescent state.
Conclusions:
Single-cell RNA sequencing may be useful to identify and track specific clonotypes present in the peripheral blood of patients treated with PD1 inhibitors, providing insights into the functional changes of T cells during response, relapse and progression.

Evolution of large copy number variants in breast cancer through genetic network rewiring

Elena Kuzmin^1,2, Jean Monlong³, Mathieu Bourgey⁴, Jarry Barber⁵, Genevieve Morin², Dongmei Zou², Michael Schwartz^1,2, Yang Yang³, Constanza R. Martinez², Hellen Kuasne², Anne-Marie Fortier², Rui Li³, Claudia Kleinman⁶, Sidong Huang^1,2, Quaid Morris⁶, Jiannis Ragoussis³, Guillaume Bourque^3,4, Morag Park^1,2

¹Biochemistry Department, McGill University, Montreal, Canada
²Goodman Cancer Research Centre, Montreal, Canada
³Human Genetics Department, McGill University, Montreal, Canada
⁴McGill University Canadian Centre for Computational Genomics (C3G)
⁵Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, USA
⁶Lady Davis Institute for Medical Research, Montreal, Canada
Corresponding author: Elena Kuzmin, email: elena.kuzmin@mcgill.ca

Abstract

Large chromosomal alterations are common in cancer and often show preferential gain or loss across many cancer types indicating their selective advantage. Triple negative breast cancer (TNBC) exhibits complex mutational spectrum without common oncogenic drivers yet displays consistent loss of large chromosomal regions. Here, we characterize selection pressures that maintain a recurrently deleted region of chromosome 4p in TNBC. We used scDNAseq of a TNBC patient derived xenograft (PDX) model to show that the chr4p deletion is an early event in tumor evolution. We used scRNAseq gene expression and inferred copy number analysis to show that chr4p loss is associated with a proliferative state. This finding was confirmed by a combination of RNA in situ hybridization and immunofluorescence. We then tested the dosage sensitivity of genes residing within this region by individual and dual overexpression in the TNBC PDX-derived cell lines and control normal cell line by assessing their effect on cell proliferation. The overexpression of genes within chr4p elicited a strong cell proliferation defect in cancer but not normal cell line models. We also characterized an unknown gene within chr4p region as a novel member of the STRIPAK complex. Genome-wide pooled ORFeome library screens identified a global pattern of background-specific dosage sensitive regions. Our study shows that large chromosomal deletions are maintained due to evolutionary early genetic network rewiring rendering multiple genes within such regions to be dosage sensitive. Ultimately, this work enhances our understanding of genetic events that modulate TNBC.

Neutrophil oxidative stress mediates obesity-associated vascular inflammation and metastatic transmigration

Sheri A C McDowell^1,2, Robin Luo^1,3, Azadeh Arabzadeh¹, Samuel Dore^1,3, Nicolas Bennett^1,2, Elham Karimi¹, Ryan Yang^1,4, Valérie Breton¹, Katherine Lach⁵, Marianne Issac⁵, Bozena Samborska⁶, Lucas Perus^1,2, Dan Moldoveanu^1,6, Yuhong Wei¹, Benoit Fiset¹, Roni Rayes^1,7, Ian Watson^1,6, Lawrence Kazak^1,6, Marie-Christine Guiot^5,8, Pierre O Fiset⁵, Jonathan D Spicer^1,7,9, Andrew J Dannenberg¹⁰, Logan A Walsh^1,3*, and Daniela F Quail^1,2,7*

¹Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada
²Department of Physiology, Faculty of Medicine, McGill University, Montreal, QC, Canada
³Department of Human Genetics, McGill University, Montreal, QC, Canada
⁴Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada
⁵Department of Pathology, Faculty of Medicine, McGill University, Montreal, QC, Canada
⁶Department of Biochemistry, McGill University, Montreal, QC, Canada
⁷Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, QC, Canada
⁸Montreal Neurological Institute, Montreal University Health Centre, Montreal, QC, Canada
⁹Department of Surgery, McGill University Health Centre, Montreal, QC, Canada
¹⁰Department of Medicine, Weill Cornell Medicine College, New York, NY, USA
Corresponding author: Logan A Walsh and Daniela F Quail, email: daniela.quail@mcgill.ca, logan.walsh@mcgill.ca

Abstract

Approximately two thirds of patients with non-small cell lung cancer (NSCLC) will present with surgically unresectable disease and those with “curable” disease frequently develop loco-regional and/or distant metastasis, ultimately leading to death. Our lab focuses on lung adenocarcinoma (LUAD), a histological subtype that compromises 40% of all NSCLC cases. Using RNA-sequencing data consisting of 517 LUAD patients from The Cancer Genome Atlas, we identified the heparan sulfate proteoglycan (HSPG) axis strongly correlated with overall survival outcome. The expression of two genes in the HSPG axis, heparanase (HPSE) and N-deacetylase/N-sulfotransferase 2 (NDST2) significantly correlated with patient survival. Importantly, our data suggests that these genes may have a synergistic effect on LUAD progression. We hypothesized that these two genes could be novel therapeutic targets to treat lung cancer patients in the future. To address this hypothesis, we engineered lung cancer cells lines to modulate the expression of these genes and performed a series of in vitro and in vivo experiments to assess the effect of these perturbations on lung cancer progression. Our preliminary data suggests that altered expression of HSPE or NDST2 results in significantly reduced migration and invasion of lung cancer cell lines in vitro and a striking reduction of tumor growth (>95%) in vivo. The objective of my research is to define the mechanisms underlying the role of the HPSG signaling axis in lung cancer progression, and to test the therapeutic efficacy of heparanase inhibitory monoclonal antibodies (developed by our lab) using preclinical murine models of lung cancer.

Investigating Intra-Tumour Metabolic Heterogeneity in Triple-Negative Breast Cancer

Marina Fukano^1,2,6, Constanza Martinez^1,3, Myriame St-Arnaud^6,7, Hellen Kuasne¹, Evelyn Zavacky⁵, Dongmei Zuo¹, Anie Monast¹, Yasser Riazalhosseini⁵, Genevieve Deblois^1,6,7, Morag Park^1-4

¹Rosalind & Morris Goodman Cancer Research Centre, McGill University;
²Department of Biochemistry, McGill University;
³Department of Pathology, McGill University;
⁴Department of Oncology, McGill University;
⁵Department of Human Genetics, McGill University;
⁶Institut de recherche en immunologie et en cancérologie, Université de Montréal;
⁷Faculty of Pharmacy, University of Montréal, Montréal, Quebec, Canada
Corresponding author: Marina Fukano, email: marina.fukano@mail.mcgill.ca

Abstract

Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer. Intra-tumour heterogeneity of TNBC poses an unmet challenge in cancer treatment, contributing to the poor outcome of TNBC patients. Tumour progression is accompanied by metabolic and epigenetic reprogramming that support tumour proliferation and adaptation to stress. We hypothesize that TNBC develops intra-tumour metabolic heterogeneity through dynamic reprogramming of epigenetic landscapes in response to oxygen and nutrient availabilities to provide metabolic flexibility to support their growth and adaptation. Through multiplex immunofluorescence staining followed by spatial transcriptomic analysis of TNBC patient-derived xenografts (PDXs) and matched patient samples, we have identified two cancer cell populations within TNBC, respectively characterized by increased glucose and nitrogen metabolism. These two zones exhibit mutually exclusive spatial localization patterns and different distances to tumour vasculatures in multiple TNBC PDXs and matched patient samples, supporting that the zones are driven by adaptive response to hypoxia and nutrient limitations. A repressive heterochromatin modification tri-methylation of lysine 27 on histone 3 (H3K27me3) shows heterogeneous levels within TNBC in vivo, suggesting distinct epigenetic landscapes of these two zones and their roles in defining the metabolic heterogeneity. Importantly, our data propose that these zones might cooperate by producing and using different nutrients, such as lactate and glutamate, which can be advantageous to TNBC growth and adaptation. This project will decipher the role and the regulatory mechanisms of this metabolic heterogeneity within TNBC and investigate the use of this metabolic heterogeneity as a novel target for poor outcome TNBC patients.

The roles of 4E-BP1 in breast cancer metastasis

Predrag Jovanovic^1,2, Sophie Guenin^1,3, Valerie Sabourin¹, Stephanie Totten^1,2, Josie Ursini-Siegel^1,2,3,4, Ivan Topisirovic^1,2,3,4

¹Jewish General Hospital, Lady Davis Institute, Montreal, Canada
²McGill University, Experimental Medicine, Montreal, Canada
³McGill University, Biochemistry, Montreal, Canada
⁴McGill University, Oncology, Montreal, Canada
Corresponding author: Predrag Jovanovic, email: predrag.jovanovic@mail.mcgill.ca

Abstract

Metastasis is the leading cause of death among breast cancer patients. The multi-step process of metastasis is intricate and still poorly understood. However, it is recognized to be a highly inefficient and strenuous process for a cancer cell. mRNA translation, which rapidly and reversibly perturbs the proteome, is thought to play a major role in response to cellular stress during metastatic dissemination. Specifically, eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs) play a major role in translational regulation by impeding the assembly of the eIF4F complex that recruits mRNA to the ribosome. In mammals, 4E-BPs are represented by a family of three members, 4E-BP1, 2, and 3. Although literature has supported the role of eIF4E in promoting breast cancer progression and metastasis, the role of the 4E-BPs in this process is less understood. We demonstrate that the germline loss of 4E-BP1/2 significantly reduces lung metastases in a mouse model of metastatic breast cancer, without impacting the growth of the primary tumors. To dissect the mechanism, we next reconstituted the expression of wild-type 4E-BP1, 4Ala 4E-BP1 and Δ4EBS 4E-BP1 mutants in 4E-BP1/2- deficient breast cancer cells. The 4Ala 4E-BP1 is always bound to eIF4E while the Δ4EBS 4E-BP1 lacks the eIF4E binding site. Remarkably, restoring Δ4EBS 4E-BP1 promotes spontaneous metastasis to the lung from the primary site. This suggests a previously unknown role of 4E-BP1 that may be independent of eIF4E binding and implicates it in the early steps of breast cancer metastasis. We are currently investigating the underlying molecular mechanism.

Investigation of the sexual dimorphic tumor suppressor role of DDX3X in melanoma

M. Lingrand, R. Alkallas, M. Lajoie, I. R. Watson
Goodman Cancer Research Centre, Department of Biochemistry, McGill University, Montreal, Quebec, Canada
Corresponding author: Marine Lingrand, email: marine.lingrand@mail.mcgill.ca

Abstract

Patient sex remains a poorly understood prognostic factor in melanoma. At all melanoma stages, men have higher incidence rates and poorer prognosis (Joosse, A. et al., J Clin Oncol 2012 ; Joosse, A. et al., J Clin Oncol 2013). Recently, our group performed a mutational meta-analysis of cutaneous melanoma combining 1,014 exomes from five studies, which identified new significantly mutated genes (Alkallas, Lajoie et al., Nature Cancer, 2020). Interestingly, we found that loss-of-function (LoF) mutations in X-linked DEAD-box RNA helicase, DDX3X, are solely found in male patients. Additionally, we demonstrated that DDX3X can escape from X-inactivation, which would protect females from complete DDX3X loss in the case of a single mutational event. To date, the reported functions of DDX3X include RNA metabolism, regulation of translation and mediators of important cancer signaling pathways. However, the role of DDX3X in melanoma is not entirely clear. We hypothesize that DDX3X is a sexual dimorphic tumor suppressor gene that plays a role in mediating the observed differences in incidence and outcome observed between female and male melanoma patients. To address this, we generated stable CRISPR/Cas9-mediated DDX3X knock-out (KO) in male human melanocyte and melanoma lines. Moreover, we identified DDX3X-null human lines to carryout DDX3X gain of function studies. In these models, we determined DDX3X plays a role in proliferation, migration and invasion in melanoma providing supports for its tumor suppressor role in this malignancy.

Increasing tumor specific antigens (TSA) immunogenicity with phosphatases inhibitors for the potential treatment of acute myeloid leukemia (AML)

Poirier Alexandre¹, Penafuerte Claudia¹, Perez-Quintero Alberto-Luis¹, Tremblay Michel. L^1,2

¹Goodman Cancer Research Centre, McGill University, Montreal, Canada;
²Department of Biochemistry, McGill University, Montreal, Canada
Corresponding author: Alexandre Poirier, email: alexandre.poirier@mail.mcgill.ca

Abstract

Although recent medical advances in the field leukemias, prognosis remains unfavorable for patients suffering from Acute Myeloid leukemia (AML). Discoveries surrounding tumor- specific antigens (TSAs) are now providing us with a new immune-based approach to treat AML. Dendritic cell (DCs) based vaccines have now become a promising new therapeutic option in the field of cancer immunotherapy. Our aim is to generate mature DCs, from peripheral blood mononuclear cells of leukemic patients, capable of activating a T-cell-response against TSA-bearing leukemic blasts. To further potentiate the immune response toward TSAs, we inhibit the function of protein-tyrosine phosphatases PTPN1 & 2. Here, we demonstrated that PTPN1/2 inhibition in DCs significantly increases T-cell activation and differentiation upon stimulation. The combination of PTPN1/2 inhibitors and TSAs would lead to the development of a potent DC-based vaccine to treat AML. We are now proceeding to evaluate the efficiency of this combination in-vivo in transgenic humanized mouse models. To accompany the DC-based vaccine, we also plan to generate a high-throughput assay capable of identifying the best TSA candidate in each AML victim, thus providing the most optimal vaccine to each patient. Through these outcomes, the project will benefit our ability to provide efficient and personalized therapeutics to treat AML. In light of positive pre-clinical results, we plan on moving the development of this vaccine to human trials. Once the proof of concept is demonstrated in acute leukemias, the next step will be to adapt this strategy to treat other types of malignancies, such as solid tumors.

The Functional Role of DNA Methylation in Human Trophoblastic Stem Cells

Deepak Saini¹, and William A. Pastor^1,2

¹Department of Biochemistry, McGill University, Montreal, Quebec, Canada
²Rosalind and Goodman Cancer Research Center, McGill University, Montreal, Quebec, Canada
Corresponding author: Deepak Saini, email: deepak.saini@mail.mcgill.ca

Abstract

DNA methylation, an epigenetic mark mediated by DNA methyltransferases (DNMTs), is required for regulating gene expression and proper embryonic development. De novo methylation occurs in post-implantation blastocyst, generating unique methylation profiles in both the inner cell mass (ICM), which forms embryonic tissue, and the trophoblast, which contributes to the placenta. Murine trophoblast stem cells (mTSCs) lacking DNA methylation survive normally and can contribute to the placenta, indicating that DNA methylation is not essential for the generation of the mouse placenta. To determine the role of DNA methylation in human placental development, we used nucleofection-based delivery of CRISPR/Cas9 to target DNMT1, an enzyme responsible for DNA methylation maintenance, in bulk populations of human trophoblast stem cells (hTSCs). Here, we observe that the DNMT1 Knockout (DNMT1 KO) allele is gradually decreased in bulk populations of DNMT1 KO hTSCs and their DNA methylation profile show a reduction of DNA methylation by day 7 and quickly return to wild-type levels by day 19. These results indicate reduced cell viability in DNMT1 KO hTSCs. Loss of DNA methylation also led to a loss of nuclear localization of the trophoblast stem cell marker TEAD4, while the majority of upregulated genes are related to germ cell development. We also observed an upregulation of HERV-Fc1 endogenous retrotransposon activity in DNMT1KO hTSCs. In future work, we hope to identify which upregulated genes may be influenced by HERV-Fc1 activity and which contribute to the reduced cell viability observed in DNMT1 KO hTSCs.

The Role of mTOR in Epigenetic Regulation in Cancer

HaEun Kim^1,2, David Papadopoli^1,2, Michael Witcher^1,2, Ivan Topisirovic^1,2

¹Department of Experimental Medicine, Montreal, Quebec, Canada.
²Lady Davis Institute, SMBD JGH, McGill University, Montreal, Quebec, Canada.
Corresponding author: HaEun Kim, email: haeun.kim3@mail.mcgill.ca

Abstract

Changes in gene expression, including those caused by epigenetic dysregulation, represent a hallmark of cancer. mTOR coordinates nutrient availability to the regulation of cell growth and metabolism, which is frequently perturbed in cancer. Furthermore, mTOR is reported to regulate the epigenome via metabolism (i.e. one-carbon metabolism) and alteration of epigenetic modifiers (i.e. EZH2). However, how mTOR signaling controls epigenetic dynamics remains largely unknown. To address this important gap in knowledge, we treated cancer cells that harbor PI3K mutation (MCF7 and HCT116) with mTOR inhibitors (INK128 and rapamycin) to study how mTOR influences global histone methylation. We found that mTOR inhibitors significantly increased H3K9me3, H3K27me3. Next, metabolomic tools (LC-MS and GC-MS) were employed and we discovered that the SAM/methionine ratio, which indicates the capacity of methylation, was significantly decreased in mTOR inhibition. This proposes that alterations in SAM levels are not implicated in the induction of histone methylation in mTOR inhibition. Also, the α-KG level was decreased in mTOR inhibition and its role in the induction of histone methylation will be assessed. Lastly, to identify how mTOR inhibition induced histone methylation marks, we used MEFs in which 2 major mTORC1 (4EBP 1/2 and S6K 1/2) were genetically ablated. We found that S6Ks and 4EBPs may in part mediate the alteration in H3K27me3, but not H3K4me3 and H3K9me3. A better understanding of the role of alterations in mTOR signaling on epigenetic programs may help identify new therapeutic targets to improve current cancer treatments.

c-Src Kinase Drives Luminal B Breast Cancer Progression Through Regulation of FOXM1

Nandi, I.^1,2, Smith, H.W.^1,2, Sanguin-Gendreau, V.^1,2, Papavasiliou, V.^1,2, Lavoie, C.^1,2, Pacis, A.^1,3, Muller, W.J.^1,2

¹Goodman Cancer Research Centre, McGill University, Montreal QC, H3A 1A3, Canada
²Department of Biochemistry, McGill University, Montreal, QC, H3A 1A3, Canada
³Canadian Centre for Computational Genomics, McGill University and Genome Quebec Innovation Centre, Montreal, QC, H3A 0G1, Canada
Corresponding author: Ipshita Nandi, email: ipshita.nandi@mail.mcgill.ca

Abstract

Breast cancer is the most common cancer in Canadian woman. One of the most aggressive subtypes of breast cancer is Luminal B, which occurs in 40% of patients. Although targeted therapies are available, a significant fraction of these patients develop resistance. As such, finding alternative targets that regulate signaling could help improve therapies for Luminal B breast cancer patients.

Clinical data suggests that c-Src kinase activation is a crucial event in human breast cancer progression. c-Src is frequently overexpressed and activated in Luminal B breast cancers and is also elevated in transgenic mouse models of breast cancer, including the commonly used polyomavirus middle-T antigen (PyVmT) model. PyVmT tumors classify with the Luminal B sybtype by genetic analysis and closely mimic human disease progression. Using the PyVmT model to target the deletion of c-Src in the mammary epithelium, we observed that c-Src ablation impairs tumour initiation and progression via down-regulation of FOXM1, a critical proliferation-associated transcription factor widely expressed during the cell cycle.

Collectively, this data illustrates the central role of c-Src in tumour initiation through cell cycle control, potentially suggesting new therapeutic approaches for patients with Luminal B breast cancer.

The Cdc42/Rac1 regulator CdGAP is a molecular target of the TGFβ/Smad signaling pathway and required for Her2-positive breast cancer growth and metastasis

Yi He^1,2, Marie-Anne Goyette³, Jennifer Chapelle^1,2, Jalal Al Rahbani^1,2, Maribel Schönewolff ^1,2, Nadia Boufaied^1,4, William J. Muller⁵, Jean-François Côté^2,3, David P. Labbé^1,2,4, Nathalie Lamarche-Vane^1,2

¹Research Institute of the McGill University Health Centre (RI-MUHC), Cancer Research Program, Montréal, Québec, Canada
²Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada
³Institut de recherches cliniques de Montréal, Montréal, Québec, Canada.
⁴Department of Surgery, Division of Urology, Faculty of Medicine, McGill University, Montréal, Québec, Canada
⁵Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montréal, Québec, Canada
Corresponding author: Jennifer Chapelle, email: jennifer.chapelle@mail.mcgill.ca

Abstract

Metastasis is the leading cause of death in breast cancer patients. The epithelial-to-mesenchymal transition (EMT) has a crucial role in metastasis and is highly critical for tumor cell dissemination. The Cdc42/Rac1 regulator CdGAP (Cdc42 GTPase-activating protein) is highly expressed in breast cancer tissues and is associated with poor clinical outcome in breast cancer patients. CdGAP cooperates in a GAP-independent manner with the transcriptional repressor Zeb2 to function as a critical modulator of breast cancer through repression of E-cadherin transcription. In this study, we found that CdGAP was essential for tumor formation and metastasis to the lungs in the Her2+ mouse breast cancer model. We demonstrated that CdGAP was required for intravasation and growth at the metastatic sites. By using global gene expression approaches, CdGAP depletion was associated with an EMT signature in Her2+ primary tumors, including a decreased expression of the metastatic factor claudin-2 and an increase in E-cadherin expression. In Her2+ breast cancer cells, CdGAP expression was positively regulated by the TGFβ canonical pathway in a Smad-dependent manner and regulates cell proliferation, migration, invasion, and adhesion. Using a proteomic approach, we uncovered the focal adhesion protein Talin as a novel CdGAP protein complex that modulates focal adhesion dynamics in breast cancer cells. Collectively, CdGAP appears as a potential anti-metastatic target for the treatment of Her2+ breast cancer.

The Rac1/Cdc42 regulator CdGAP promotes prostate cancer metastasis by regulating epithelial-to-mesenchymal transition, cell cycle progression, and apoptosis

Chahat Mehra^1,2, Ji-Hyun Chung^1,2, Yi He^1,2, Mónica Lara Márquez^1,2, Marie-Anne Goyette³, Nadia Boufaied¹, Véronique Barrès⁴, Véronique Ouellet⁴, Karl-Phillippe Guérard¹, Carine Delliaux³, Fred Saad^4,5, Jacques Lapointe^1,6, Jean-François Côté^2,3, David P. Labbé^1,2,6*, Nathalie Lamarche-Vane^1,2*

¹Research Institute of the McGill University Health Centre, Cancer Research Program, Montréal, Québec, Canada.
²Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada.
³Institut de recherches cliniques de Montréal, Université de Montréal, Montréal, Québec, Canada.
⁴Centre de recherche du Centre hospitalier de l’Université de Montréal et Institut du cancer de Montréal, Montréal, QC, Canada.
⁵Department of Surgery, Université de Montréal, Montréal, QC, Canada.
⁶Division of Urology, Department of Surgery, McGill University, Montréal, Québec, Canada.
Corresponding author: David P. Labbé, Nathalie Lamarche-Vane, email: david.labbe@mcgill.ca, nathalie.lamarche@mcgill.ca

Abstract

The high mortality of prostate cancer patients is primarily due to metastasis. Therefore, understanding the mechanisms controlling metastatic processes remains essential to develop novel therapies designed to prevent the progression from localized disease to metastasis. The Rac1/Cdc42 regulator CdGAP (Cdc42 GTPase-activating protein) plays important roles in the control of cell adhesion, migration, invasion, and proliferation, which are central to cancer progression. Here, we demonstrate that elevated CdGAP expression is associated with early biochemical recurrence and bone metastasis in prostate cancer patients. CdGAP protein and mRNA levels were elevated in highly metastatic PC-3 cell line. Knockdown of CdGAP in PC-3 cells reduced cell motility, invasion, proliferation and colony-formation ability while inducing an increase in cell apoptosis. Conversely, overexpression of CdGAP in DU-145 cells increased cell migration and invasion. Using global gene expression approaches, we found that CdGAP regulates the expression of genes involved in epithelial-to-mesenchymal transition, apoptosis and cell cycle progression. Subcutaneous injection of CdGAP-depleted PC-3 cells into mice showed a delayed tumor initiation and attenuated tumor growth. Orthotopic injection of CdGAP-depleted PC-3 cells reduced distant metastasic burden. Collectively, these findings support a pro-oncogenic role of CdGAP in prostate tumorigenesis and unveil CdGAP as a potential biomarker and target for prostate cancer treatments.

Determining mechanisms of resistance to Eribulin in TNBC using novel patient-derived models

Kathryn Bozek^1,2, Marguerite Buchanan¹, Cathy Lan¹, Cédric Darini¹, Adriana Aguilar¹, Mark Basik^1,2,3,4

¹Cancer Genomics and Translational Research Laboratory, Lady Davis Institute/Segal Cancer Centre;
²Division of Experimental Medicine, McGill University, Montréal, QC
³Department of Medicine, McGill University, Montréal, QC
⁴Department of Oncology, McGill University, Montréal, QC
Corresponding author: Kathryn Bozek, email: kathryn.bozek@mail.mcgill.ca

Abstract

Eribulin is a chemotherapeutic agent approved for patients with advanced or metastatic breast cancer who have received taxane- and anthracycline-based chemotherapy. Unfortunately, many patients do not respond initially or inevitably develop resistance to Eribulin. We hypothesize that we can determine mechanisms of resistance and identify potential therapeutic targets for Eribulin-resistant Triple-Negative Breast Cancer (TNBC) using patient-derived xenografts (PDXs). We have developed 6 models of acquired-resistant TNBC via the treatment of PDX-bearing NSG mice with Eribulin until the emergence of resistance. For one PDX, we have a matched patient recurrent tumour-derived PDX which became resistant to Eribulin after the patient received carbo/taxol. We also have 3 models of intrinsic resistance and several sensitive models. All models are undergoing RNA-Seq and Copy Number Analysis. By RNA-Seq, we observed the loss of expression of the lncRNA XIST, the main player in X-chromosome inactivation, in two acquired resistant models. Transcript analysis revealed that XIST exon4 expression was also lost in three of our acquired resistant models, suggesting a potential link between XIST inhibition and Eribulin resistance. We are generating conditionally reprogrammed cells for 3 of these matched sensitive-resistant PDXs for validation studies. In our resistant cells, we will re-express XIST via both genetic introduction of the lncRNA and by targeting XIST negative-regulators to determine if XIST re-expression re-sensitizes cells to Eribulin. We will likewise validate these results in the matched PDXs. This study should uncover mechanisms of resistance to Eribulin and has the potential to identify novel therapeutic avenues for patients with Eribulin-resistant TNBC.

Anatomical and cellular heterogeneity in the mouse oviduct-- its potential roles in reproduction and preimplantation development

Keerthana Harwalkar*¹, Matthew J Ford¹, Katie Teng*¹, Nobuko Yamanaka¹, Brenna Yang¹, Ingo Burtscher^2-5, Heiko Lickert^2-5, Yojiro Yamanaka*¹
*Department of Human Genetics, McGill University
¹Rosalind and Morris Goodman Cancer Research Centre, McGill University
²Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Center Munich
³Institute of Stem Cell Research, Helmholtz Center, Munich
⁴German Center for Diabetes Research (DZD), Technical University of Munich, Munich, Germany
⁵Department of Medicine, Technical University of Munich, Munich, Germany
Corresponding author: Keerthana Harwalkar, email: keerthana.harwalkar@mail.mcgill.ca

Abstract

The oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive tissue that provides an environment for internal fertilization and preimplantation development. Interestingly, precancerous lesions of HGSOCs are restricted to the distal fallopian tube, although most of the secretory cell population is in the proximal region. This suggests that the distal FTE is more susceptible to cancerous transformation than the proximal FTE. However, our knowledge of its regional and cellular heterogeneity is still limited. We examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines and found that there are basic coiling patterns and turning points in the coiled mouse oviduct can serve as reliable landmarks for luminal morphological regionalities. We identified anatomical structures in the isthmus (ISM) and uterotubal junction (UTJ) that likely play important roles in reproduction. Interestingly, during ovulation, the isthmus was transiently sealed, keeping the oocytes within the ampulla. Preimplantation embryos travelled along the oviduct and formed a queue within small compartments of the UTJ before uterine entry. Further, the AMP-ISM junction (AIJ) was a unique regionality different from the AMP and ISM in its multi-ciliated cell distribution pattern, luminal morphology, transcriptional factor expression, and acidic mucin secretion. Taken together, our results revealed anatomical and cellular heterogeneity in the mouse oviduct luminal epithelium and suggested functional diversity in each morphologically distinct region.

Obesity-associated inflammation enhances immune checkpoint inhibitor efficacy in lung cancer

Lysanne Desharnais^1,2, Daniela Quail^1,3,4, Logan Walsh^1,2

¹Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada
²Department of Human Genetics, McGill University, Montreal, QC, Canada
³Department of Physiology, Faculty of Medicine, McGill University, Montreal, QC, Canada
⁴Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, QC, Canada
Corresponding author: Lysanne Desharnais, email: lysanne.desharnais@mail.mcgill.ca

Abstract

Lung cancer is the leading cause of cancer mortality, but recent advances in cancer immunotherapy, including immune checkpoint inhibitors (ICIs), have revolutionized patient care and prognosis. Recent retrospective clinical data have shown that obesity is associated with enhanced response to ICIs in lung cancer patients. Given the challenges in predicting which patients will benefit from immunotherapy, the enhanced efficacy in patients with obesity raises is an intriguing observation. The objective of this project is to identify the mechanism driving enhanced response to ICIs in patients with obesity. We are investigating obesity-associated inflammation in two ways. Firstly, we are studying how the systemic inflammatory effects of metabolic syndrome contribute to enhanced ICI efficacy. We have generated a subcutaneous syngeneic model of lung adenocarcinoma in diet-induced obese and lean mice to show that the degree of response to ICIs is greater in the obese mice. Using this mouse model and patient-derived lung tumor organoids, we are analyzing immune cell dynamics and tumor intrinsic factors that differ between lean and obese hosts. In parallel, we are investigating how dietary patterns influence ICI response in lung cancer. Given the importance of the gut microbiome on ICI efficacy and its link to obesity and inflammation, we are studying how different diets modulate the gut microbiota to enhance ICI efficacy. Improving our understanding of the immunological landscape of cancer and obesity will provide insight to optimize ICI responses across a broader patient population.

3D Chromatin Remodeling Potentiates Transcriptional Programs Driving Cell Invasion

Benjamin Lebeau¹, Maïka Jangal¹, Tiejun Zao¹, Chenxi Zheng¹, Michael Witcher¹

¹McGill University and Lady Davis Institute
Corresponding author: Michael Witcher, email: benjamin.lebeau@mail.mcgill.ca

Abstract

Introduction: CTCF is a protein whose normal function is commonly disrupted in a wide array of cancer, including loss of heterozygosity in 50% of breast cancers. Substantial evidence demonstrates that CTCF acts as a tumor suppressor gene. However, the role of CTCF deletion in cancer has not been clearly elucidated. Our novel investigation will uncover the mechanism of action of CTCF in cancer progression by studying CTCF single allele deletion.
Methods and Results: As a model for CTCF LOH, we used CRISPR/Cas9 to knockdown WT CTCF from a single allele in a non-transformed breast epithelial cell line, MCF10A. CTCF +/- cells showed increased invasiveness and mammosphere growth in vitro. RNA-seq indicated that the classical oncogene SNAI1 and the PI3K pathway were strongly upregulated in CTCF+/- cells, which was subsequently validated by Western Blot. ChIP-Seq revealed that CTCF binding was lost around key PI3K genes and from the SNAI1 promoter. Therefore, we demonstrated that the invasiveness of CTCF+/- cells was highly sensitive to Snail knockdown, CTCF addback or inhibition of downstream PI3K effectors, using mTor inhibitors. Similarly, the invasiveness of CTCF +/- PDX derived cells lines was also restored by CTCF addback. At the epigenetic level, CTCF loss leads to aberrant accumulation of H3K4me3 or H3K27ac at key oncogenes: promoting their upregulation. Using Hi-C, we showed that these epigenetic changes are driven by the loss of insulation of sub-megabase chromatin domains at lost sites of CTCF.
Conclusion: These studies reveal mechanistically how CTCF LOH drives oncogenic progression and provide insight into therapeutic avenues targeting CTCF +/-tumors.

Understanding p66ShcA-dependent Regulation of GPNMB in Response to Oxidative Stress in Triple-Negative Breast Cancer

Clark Thomson^1,2, Eduardo Cepeda Cañedo^4,5, Josie Ursini-Siegel^2,4,5,6, Peter M. Siegel^1,2,3

¹Goodman Cancer Research Centre,
²Department of Biochemistry, McGill University
³Department of Medicine, McGill University,
⁴Lady Davis Institute for Medical Research
⁵Division of Experimental Medicine,
⁶Gerald Bronfman Department of Oncology, Montréal, Québec, Canada
Corresponding author: Clark Thomson, email: clark.thomson@mail.mcgill.ca

Abstract

Triple-negative breast cancer (TNBC) is a particularly aggressive and difficult to treat subtype as it lacks specific molecular targets that can be exploited therapeutically. Data generated in our lab has identified GPNMB, a cell surface protein over-expressed in TNBC, as a mediator of pro-metastatic phenotypes that correlates with poor prognosis. In response to several cellular stressors, GPNMB is upregulated following nuclear translocation of the MITF/TFE family of transcription factors (TFE3 and TFEB), which induce GPNMB expression.
One class of stressors that we have shown upregulates GPNMB are compounds that increase the levels of reactive oxygen species (ROS). Interestingly, our data suggests that GPNMB upregulation in response to elevated ROS levels is dependent on the redox protein p66ShcA. ShcA encodes 3 isoforms (p46, p52 and p66) of which p66ShcA is the longest. Importantly, p66ShcA has been shown to promote the formation of intracellular ROS. We have shown that p66ShcA null cells are unable to induce GPNMB expression following a ROS-inducing stimulus. Expression of wildtype p66ShcA can rescue GPNMB expression. Unexpectedly, expression of a mutant forms of p66ShcA that cannot generate mitochondrial ROS also rescue GPNMB expression. Furthermore, we demonstrate that the mechanism through which p66ShcA modulates GPNMB expression is TFE3/TFEB dependent, as depleting TFE3/TFEB attenuates GPNMB upregulation following oxidative stress.
We have identified a non-mitochondrial role for p66ShcA in GPNMB induction following cellular stressors that induce ROS. Further experiments will be designed to characterize the p66ShcA-ROS-GPNMB cascade, with the goal of identifying new targets/therapies for TNBC management.

Pre-operative immune checkpoint inhibition +/- chemotherapy for patients with operable stage IA3-IIA non-small cell lung cancer: a randomized trial, from conception to implementation

Caroline Huynh^1,2, Roni Rayes^1,2, Pierre-Olivier Gaudreau², Vera Hirsh³, Linda Ofiara³, Scott Owen³, Benjamin Shieh³, Logan A. Walsh^1,4, Jonathan D. Spicer^1,2,5

¹Rosalind and Morris Goodman Cancer Research Centre, McGill University
²Research Institute of the McGill University Health Centre
³Division of Medical Oncology, Department of Medicine, McGill University Health Centre
⁴Department of Human Genetics, McGill University
⁵Division of Thoracic and Upper Gastrointestinal Surgery, Department of Surgery, McGill University
Corresponding author: Caroline Huynh, email: caroline.huynh@mail.mcgill.ca

Abstract

Surgery remains a cornerstone of cure for early stage non-small cell lung cancer (NSCLC). The value of systemic therapy in patients without nodal disease is unclear, yet recurrence rates remain high after surgery. With better side effect profiles and improved outcomes, immune checkpoint inhibition (ICI) has become a first-line therapy in advanced NSCLC. For operable NSCLC, previous studies have demonstrated safety, tolerability and unprecedented major pathological response (MPR) rates (20-75%) with pre-operative ICI. However, the ideal timing and regimen, and how pathological response should impact the extent of resection remain unclear. This study aims to evaluate the use of clinically available biomarkers to predict the likelihood of pathological complete response (pCR) with neoadjuvant therapy for patients with operable NSCLC.

In a randomized phase II trial, 44 patients with untreated, operable and histologically-confirmed stage IA3-IIA NSCLC will be enrolled and randomly assigned between neoadjuvant pembrolizumab vs. neoadjuvant pembrolizumab with chemotherapy. Randomization will be stratified by PD-L1. An anatomical lung resection with complete mediastinal lymph node dissection will be required at surgery. All participants will undergo longitudinal ctDNA measurement. Response will also be assessed with imaging (diffusion-weighted MRI, CT, PET). Primary endpoint is ctDNA resolution during neoadjuvant therapy. Secondary endpoints are MPR, pCR, safety and imaging scores. Exploratory endpoints are overall and disease-free survival. Predictive data from single-cell RNA sequencing and imaging mass cytometry will also be correlated to ctDNA resolution and pCR. Together, these strategies may allow for more limited surgical resections, and greatly inform the care of patients with NSCLC.

eIF4E phosphorylation drives the production and spatial organisation of collagen type I in the mammary gland

Samuel Preston^1,2, Christophe Gonçalves², Vincent Richard³, René Zehadi^3,4, Christoph Borchers³, Wilson Miller^1,2,4, Sonia del Rincón^1,2,4

¹Division of Experimental Medicine, McGill University, Montréal, QC, Canada.
²Department of Oncology, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, QC, Canada.
³Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, Montréal, QC, Canada.
⁴McGill Centre for Translational Research in Cancer (MCTRC), McGill University, Montréal, QC, Canada.
Corresponding author: Samuel Preston, email: samuel.preston@mail.mcgill.ca

Abstract

Background: The ECM is a highly dynamic component of the tumor microenvironment (TME) that adapts to reinforce neoplastic progression. The MNK1/2-eIF4E axis exemplifies how translation initiation can act abnormally to promote cancer. eIF4E phosphorylation, uniquely by MNK1/2, induces the translation of a subset of mRNA involved in invasion and metastasis, some of which are known matrisomal proteins. To date, no one has reported on how translational control impacts ECM homeostasis.
Methods: We aim to define ECM signatures that are regulated by the MNK1/2-eIF4E axis. Primary ECM samples were isolated from the mammary glands (MGs) of wild-type and phospho-eIF4E-deficient female mice and subjected to proteomic analysis. In tandem, ECM preparations were used to treat breast cancer cells and primary MG fibroblasts in vitro for immunoblot, qPCR, transwell migration-invasion and functional assays. Tail vein injection and orthotopic mammary fat pad injection models were also used, as well as IHC staining on patient-derived samples.
Results: Matrisomal profiling highlights several proteins that are differentially expressed between conditions. This includes collagen-I which is downregulated in phospho-eIF4E-deficient MG ECM. Phospho-eIF4E-deficient MG fibroblasts produce less collagen-I and produce a less organized 3D matrix as compared to wild-type. Breast cancer cells treated with phospho-eIF4E-deficient MG ECM have suppressed invasive capacity in vitro and in vivo, when compared to wild-type. Breast cancer patients also show a positive correlation between phospho-eIF4E and collagen-I.
Conclusion: We have shown that ECM from a phospho-eIF4E deficient MG fosters a less invasive TME, through its modulation of collagen I production and topology.

Identifying genetic vulnerabilities of chromosome 4p large copy number variants in triple negative breast cancer

Michael Schwartz^1,2, Elena Kuzmin^1,2, Alain Pacis⁴, Hellen Kuasne², Sidong Huang^1,2, Guillaume Bourque^3,4, Morag Park^1,2

¹Biochemistry Department, McGill University, Montreal, Canada
²Goodman Cancer Research Centre, Montreal, Canada
³Human Genetics Department, McGill University, Montreal, Canada
⁴McGill University Canadian Centre for Computational Genomics (C3G), Genome Centre
Corresponding author: Michael Schwartz, email: michael.schwartz4@mail.mcgill.ca

Abstract

Triple Negative Breast Cancer (TNBC) is characterized by a complex mutational spectrum with no common drivers, limiting its treatment options; however, it exhibits recurrent large chromosomal deletions. We previously showed that the chromosome 4p (chr4p) loss is a frequently observed chromosome aberration in TNBC and is associated with a poor prognosis. We also showed that chr4p deletion is an early event in tumor evolution and confers on cells a proliferative advantage. Here, we set out to uncover the genetic vulnerabilities associated with chr4p deletion in TNBC to identify novel therapeutic avenues for TNBC and enhance our understanding of the genetic mechanisms that buffer chr4p deletion. We analyzed whole genome sequencing data of our TNBC Patient-Derived Xenograft (PDX) panel to identify cell models, which harbour chr4p loss. We demonstrated the functional significance of chr4p deletion using RNAseq data, showing a significant reduction in gene expression within the chr4p-deletion region. To identify genes that may be driving the chr4p deletion we delineated the minimal deleted region. We showed that genes with strongest reduction in gene expression have been previously reported to exhibit tumor suppressive capacity. We assessed the infectivity of the PDX cell models using lentivirus expressing GFP. We will test the CRISPR-Cas9 editing efficiency of the PDX cell models using a control essential gene set. We will then use a pooled CRISPR-Cas9 approach to systematically screen for genetic vulnerabilities in TNBC PDX cell models, which harbour copy neutral or deletion state of chr4p to reveal possible therapeutic avenues for precision oncology.

The role of Hippo signaling in stromal-epithelial interactions in acinar-to-ductal metaplasia and pancreatic cancer initiation

Julia Messina-Pacheco¹, Alex Gregorieff¹

¹Department of Pathology, McGill University, Montreal, QC, Canada
Corresponding author: Julia Messina-Pacheco, email: julia.messina-pacheco@mail.mcgill.ca

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths with a 5-year survival rate of approximately 7%. PDAC may originate from acinar cell trans-differentiation into ductal-like cells, termed acinar-to-ductal metaplasia (ADM), triggered by chronic pancreatitis and/or mutations in K-Ras. The progression to PDAC is associated with a dense fibrotic stroma, including cancer-associated fibroblasts (CAFs). YAP is a tension-stimulated CAF activator that promotes ECM stiffening, creating a permissive microenvironment for cancer progression.

Hypothesis: The Hippo pathway may coordinate fibroinflammatory signals emanating from the stromal compartment during regenerative responses to acinar cell injury and progression towards PDAC.

Methods and Results: To resolve the transcriptional changes occurring during the transition from normal parenchyma to ADM to PDAC, we mapped the in situ expression of 1800 RNA targets in patient-derived tissues using Digital Spatial Profiling (DSP). This revealed genes implicated in fibroblast activation, epithelial-to-mesenchymal transition and IFNɣ signaling as potential drivers of ADM. I will evaluate the expression of candidate genes and survey Yap expression at the single cell level in human ADM tissue by multiplexed RNAscope in situ hybridization.
To study the in vivo role of Hippo signaling in stromal cells, we conditionally deleted Yap/Taz in Collagen1a2-producing cells in a murine model of caerulein-induced pancreatitis. I will analyze the resulting phenotype by immunostaining for metaplastic, proliferative, immune and stromal markers.

Conclusion: This work will provide an in-depth understanding of epithelial-stroma crosstalk in ADM, providing a foundation for the development of new therapeutic strategies for treating non-invasive precursor lesion, thereby preventing pancreatic cancer progression.

Investigating the role of cell division orientation in maintaining and breaking epithelial architecture

Mara Whitford^1,2, Ruba Halaoui^1,3, Christina Kalos^1,3, Luke McCaffrey1,2,3

¹Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, QC
²Department of Biochemistry, McGill University, Montreal, QC
³Department of Experimental Medicine, McGill University, Montreal, QC
Corresponding author: Mara Whitford, email: mara.whitford@mail.mcgill.ca

Abstract

Over 80% of cancers are derived from epithelial cells, however, the molecular mechanisms underlying carcinoma initiation and development remain poorly understood. Breast cancer is one of the most frequently diagnosed epithelial cancers. During the early stages of cancer development within the breast duct, luminal cells become stratified, however, how this process occurs remains unclear. Previous studies have shown a correlation between increased perpendicular divisions and stratification during the development of a variety of epithelial cancers, including breast cancer. I hypothesize that cell division orientation induces epithelial stratification as a necessary step for tumour initiation. Our initial findings suggest that spindle orientation in the breast ducts becomes disrupted in the inducible polyoma middle T (PyMT) mouse model of breast cancer, while knock-out of Src in this model corrects spindle orientation and inhibits both stratification and overt tumour development. Knock-out of the spindle orientation regulatory proteins, LGN and NuMA, specifically in the breast ducts of PyMT-Src-/- mice promotes stratification and early invasion, suggesting an important role for disrupted spindle orientation in stratification in this model. Live-imaging of epithelial cysts to track cells, following divisions in vitro, suggests that epithelial cells have the ability to resolve out-of-plane divisions to maintain tissue architecture, while expression of oncogenes, such as KRAS, impair this ability, thus promoting stratification.

Mechanical Control of Epithelial Tissue Morphogenesis by Apoptosis and Non-Professional Efferocytosis

You Chi (Emily) Tang¹, Khoren Ponsin², Adda-Lee Graham-Paquin¹, Magdalena Schindler¹, Anmar Khadra2 and Maxime Bouchard¹

¹- Goodman Cancer Research Centre, Department of Biochmistry, McGill University.
²- Department of Physiology, McGill University
Corresponding author: You Chi (Emily) Tang, email: you.tang@mail.mcgill.ca

Abstract

Apoptosis is a crucial and tightly regulated process during embryonic development for tissue morphogenesis. Apoptotic cell death has been shown not only to sculpt the tissue, but also to act as active driving forces to shape organisms during development.
Our lab studies a system of apoptosis involved in early urogenital system (UGS) maturation, where the elimination of common nephric duct (CND) results in ureter-bladder connection. It was shown that progressive CND elimination is strictly dependent on regulated apoptosis to bring the ureter in contact with the bladder. If this apoptotic rate is altered, diseases such as ureter obstruction and reflux will be resulted.

We observed that apoptotic cell removal in the CND is proceeded by non-professional efferocytosis by surrounding duct epithelial cells, and act as morphogenetic motor to shape the UGS by bringing the ureter closer to the bladder to allow normal genital tract development.

To further investigate in the details of non-professional efferocytosis and how it plays a role in force generation, we developed a mathematical model. It was found that the non-professional efferocytosis rate is about 9 hours in vivo.

In order to have a thorough view of the driving factors that contribute to active UGS tissue morphogenesis, we investigated the role of actomyosin in force generation. Using both ex vivo experiments and mathematical model simulations, they suggested that actomyosin acts as a secondary support of the duct infrastructure that allows apoptosis and non-professional efferocytosis to actively shape the tissue.

Together these results identify that apoptosis and non-professional efferocytosis are drivers for apoptosis-mediated morphogenesis involved in embryonic development.

Predicting COVID-19 Outcomes Using Proteomic Data

Chen-Yang Su^1,3, Sirui Zhou^1,2, Wonseok Jeon³, Vincenzo Forgetta¹, Joelle Pineau³, J Brent Richards^1,2,4

¹Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, Québec, Canada.
²Department of Human Genetics, McGill University, Montréal, Québec, Canada.
³Department of Computer Science, McGill University, Montréal, Québec, Canada.
⁴Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montréal, Québec, Canada.
Corresponding author: Chen-Yang Su, email: chen-yang.su@mail.mcgill.ca

Abstract

Background: Currently, few disease-specific therapies exist for treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and the prediction of which individuals will experience severe COVID-19 is difficult. Determining proteins that play key roles during viral infection can provide useful information to identify individuals who are at risk for severe COVID-19 outcomes, such as the need for respiratory support or death. Analyses of large-scale aptamer-based blood proteomic data in COVID-19 patients may help to predict the course and outcome of the disease, and to identify potential biomarkers for drug candidates.
Methods: In this study, we utilized a dataset composed of one of the largest groups of circulating protein biomarkers for COVID-19 patients collected over the course of disease progression to design an accurate, clinically relevant multiprotein machine learning model for the prediction of COVID-19 adverse outcomes. We categorize COVID-19 outcomes into two groups based on severity level (severe, very severe) and use measured protein levels to predict these outcomes. We then leverage the ability of L1-norm regularization combined with logistic regression (LASSO) for selecting predictive proteins. We then externally validate the robustness of our model in an external dataset with the same proteomic profiling and clinical outcomes.
Results: Our model achieved an area under the receiver operating characteristic (AUROC) score of 0.843 and 0.796 for predicting severe COVID-19 and very severe COVID-19, respectively, on the external test set.
Conclusion: Our results suggest that circulating proteins can improve prediction accuracy of adverse COVID-19 outcomes.

Investigating the Interactome of RASSF Proteins in the Context of RAS Signaling

Maya Nikolova¹, Dhanaraman Thillaivillalan¹, Matthew J. Smith¹

¹Institute for Research in Immunology and Cancer (IRIC), Université de Montréal.
Corresponding author: Maya Nikolova, email: maya.nikolova@umontreal.ca

Abstract

The oncogene RAS is mutated in approximate 20% of all human cancers. Cancers which harbor RAS mutations are refractory and largely resistant to treatment. This small GTPase transduces signals from membrane-bound receptors to intracellular pathways, by signaling to numerous downstream effectors. One intriguing example of RAS effectors is the RAS association domain family (RASSF), a family of ten tumor suppressors. The RASSFs can activate the Hippo pathway, a key regulatory pathway for tissue growth. RAS has been shown to activate Hippo signaling via the RASSFs, however this activation is most likely complex and contextual. This project aims to elucidate this activation by investigating the interactomes of the RAS, RASSF1, and RASSF5 proteins. To accomplish this, I am performing BioID, a proteomic technique which relies on proximity biotinylation, allowing for identification of all cellular proteins which come in proximity to a protein of interest. Using lentiviral infection, I have generated eight stable U2OS cell lines, where expression of the protein of interest fused to a biotin protein ligase can be induced by addition of tetracycline. In addition to mapping the interactome of the RAS and the RASSF proteins alone, I will also be performing BioID of the RASSFs in the presence of RAS, and vice versa, in the hope of identifying other proteins involved in the RAS-mediated activation of Hippo signaling. Understanding how RAS signals to less studied effectors and pathways, particularly those with tumor suppressive functions has significant implications for understanding RAS-driven oncogenesis, and for the development of new therapies.

Molecular mechanisms underlying CdGAP regulation during epithelial-to-mesenchymal transition (EMT), migration, and invasion of human breast cancer cells.

Lara-Márquez, Mónica¹, Yi He¹, and Lamarche-Vane, Nathalie¹

¹Cancer Research Program, Research Institute of the McGill University Health Center, Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
Corresponding author: Lara-Márquez, Mónica, email: monica.laramarquez@mail.mcgill.ca

Abstract

Cdc42 GTPase-activating protein (CdGAP or ARHGAP31) negatively regulates the small GTPases Rac1 and Cdc42. Recently, a novel nuclear function for CdGAP was identified, where it cooperates in a GAP-independent manner with transcriptional repressors to regulate E-cadherin transcription in breast cancer cells. This function correlates with the ability of CdGAP to promote ErbB2-mediated tumor growth and metastasis in vivo. In addition, it was reported that a truncated CdGAP (Q683X) protein, which is expressed in patients with the rare developmental disorder Adams-Oliver Syndrome, is unable to repress E-cadherin transcription. Overall, these data indicate that CdGAP acts as an oncogene through its transcriptional regulatory activity. We hypothesize that high levels of CdGAP in breast cancer cells and the transcriptional activity of CdGAP mediate its oncogenic properties in promoting breast tumorigenesis and metastasis. We aim to dissect the role of the transcriptional versus GAP activity of CdGAP during EMT, migration, invasion, mammary tumor growth and metastasis in triple negative breast cancer cells. We abolished the expression of CdGAP in MDA-MB-231 breast cancer cells and generated clonal MDA-MB-231 knock-in cell lines expressing the CdGAP(Q683X) mutant protein using CRISPR/CAS9 strategy. CdGAP depletion and CdGAP(Q683X) expression resulted in increased E-cadherin expression as well as impaired cell migration. Additionally, CdGAP loss showed changes in cell morphology, including cell spreading and larger cell size. CdGAP(Q683X) induced the formation of F-actin based protrusions. Therefore, this work in progress highlights the important role of CdGAP as a regulator of actin cytoskeletal dynamics and E-cadherin expression in MDA-MB-231 breast cancer cells.

Characterization of micro-peritoneal metastasis in the mouse ovarian cancer models

YuQi Li^1,2, Yojiro Yamanaka^1,2

¹Human Genetics, McGill, Montreal, QC, Canada;
²Rosalind & Morris Goodman Cancer Research Centre, Montreal, QC, Canada
Corresponding author: YuQi Li, email: yuqi.li2@mail.mcgill.ca

Abstract

Introduction: High grade serous ovarian cancers (HGSCs), the most common ovarian malignancy, are rarely curable. This high mortality rate is due to late diagnosis in advanced stages with metastasis throughout the peritoneum, which leads to poor survival, while patients with localized HGSCs have higher survival rates. Therefore, it is essential to understand the mechanism of HGSCs dissemination and the interactions between the peritoneum microenvironment with micrometastases in order to develop novel therapeutic approaches that target this crucial step to improve long-term survival.
Methods: To address these challenges, we develop a novel strategy to derive HGSCs in mice with CRISPR-mediated genome modification, Cre-induced fluorescence tracing, and in vivo electroporation. This strategy possesses the capability of tracing clonal expansion and metastasis via fluorescence and the advantages of testing various mutation combinations in a limited region. After HGSCs in mice have metastasized, we extract different parts of the peritoneum (omentum and mesentery) and stain with various immune and extracellular markers to investigate interactions between cancer cells and mesothelial microenvironment in the peritoneum.
Results: We generated two mouse HGSC models by targeting three or four tumor suppressor genes, TP53, BRCA1 and PTEN with/without LKB1. Within these two mouse models, we observed various unique patterns of how fluorescent-labeled HGSCs micrometastases land onto the mesothelial layer of peritoneum and recruitment of immune cells such as the LYVE1+ macrophages towards the micrometastases.
Conclusions: With this unique mouse model, we can gain more understanding of the key cellular interactions between HGSCs and the local peritoneal environment and the crucial events such as recruiting various immune cells during metastasis. My study can provide insights into the pathways critical for HGSCs metastasis which could serve as therapeutical targets.

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