22nd Annual McGill Biomedical Graduate Conference | March 22th, 2022
Experimental Medicine Graduate Students’ Society (EMGSS)
Published online: April 20, 2022Thioredoxin-interacting protein (TXNIP) deficiency promotes cellular senescence via Akt activation
2Faculty of Medicine and Health Sciences, Thamar University, Dhamar, Yemen
Corresponding Author: Mohammed Abubaker , email mohammed.abubaker@mail.mcgill.ca
Abstract
Thioredoxin-interacting protein (TXNIP) is a ubiquitously expressed protein that belongs to the α-arrestin protein family. TXNIP interacts with and inhibits the function of the endogenous antioxidant, Thioredoxin (Trx). Age-dependent dysregulation of the Trx/TXNIP redox system has been implicated in cellular senescence and aging. However, the mechanisms are not fully understood. While Trx overexpression was reported to protect against age-related diseases, TXNIP KO mice had reduced survival in response to paraquat mediated oxidative stress. To investigate the potential effects of TXNIP in cellular senescence we used primary mouse renal glomerular mesangial cells (MC) from WT and TXNIP KO mice. Passaging of cells leads to replicative senescence. In WT MC this was associated with a significant reduction in TXNIP expression in late passage (LP-passage 25) compared to early passage (EP-passage 5-7) (p<0.05), and this was accompanied by appearance of the senescence phenotype determined by significant increases in expression of the cellular senescence markers, p53, p16, p21, phosphorylation of histone H2AX (p<0.05) and senescence associated beta-galactosidase staining (p<0.01). To determine whether TXNIP downregulation played a causal role, primary MC from TXNIP KO were compared to WT at EP 5. TXNIP KO showed significantly higher expression of senescence markers P53 and P16 (p<0.05), and beta-galactosidase staining. AKT activation has been implicated in replicative senescence. Here, increased phosphorylation of AKT and its substrate FOXO were found in LP versus EP WT, and EP KO versus WT MC (p<0.05). These studies indicate that TXNIP downregulation is a mediator of replicative senescence associated with increased AKT signaling.
Accuracy of Native Myocardial T1 and T2 mapping for Quantifying Ischemic Myocardial Injury
2Department of Cardiology, Mcgill University Health Center, Montreal, QC, Canada
3Department of Cardiac Sciences and Radiology, University of Calgary, Calgary, Canada
4Department of Medicine, Alberta University, Edmonton, Alb, Canada
5Department of Internal Medicine, Geisinger School of Medicine, Pennsylvania, USA
Corresponding Author: Leila Haririsanati, email Leila.haririsanati@mail.mcgill.ca
Abstract
Background: Late gadolinium enhancement (LGE) cardiac magnetic resonance imaging is the clinical gold standard method to evaluate fibrosis in ischemic cardiomyopathy (ICMP) patients. LGE is time-consuming, costly and may result in possible side effects. Native (contrast-free) T1/T2 mapping have the potential to assess myocardial injury faster and more safely. This study evaluated the agreement between the presence and extent of myocardial injury measured with T1/T2 mapping and LGE in an ICMP patient population. Method: In this retrospective study, positive LGE was defined as an area of the myocardium > 5 standard deviations (SD) away from the reference myocardium. Elevated T1/T2 times were defined as an area of the myocardium ≥ 2 SD away from reference T1/T2 values. Results: A total of 32 patients (mean age: 64.6 years old, 9.37 % female) were enrolled. Kappa agreement analysis demonstrated moderate to strong agreement between all slices (apex= 0.66, mid= 0.93, basal= 0.87) in identifying the presence of myocardial injury in both T1 maps and LGE. The extent of the myocardial injury area in chronic cases was not significantly different between LGE and T1 maps (p value= 0.31). In acute cases, T2 maps and LGE presented similar extent of myocardial injury. Conclusion: Our study shows a moderate to strong agreement between T1/T2 maps and LGE in identifying the area of ischemic myocardial injury. This finding suggests that multiple native parameters may be used in combination to predict myocardial injury in an ICMP population.
The role of mTOR pathway in the combination therapy of belvarafenib and cobimetinib in melanoma
2Segal Cancer Centre, Lady Davis Institute and Jewish General Hospital, Montréal, QC, Canada.
3Montreal Rossy Cancer Network, Montréal, QC, Canada.
Corresponding Author: Feiyang Cai, email feiyang.cai@mail.mcgill.ca
Abstract
Melanoma is a type of skin cancer. Targeted therapies work in patients with BRAF mutated melanomas, which occupy about 50% of patients, but these same therapies do not work for other melanoma subtypes, which have mutations in NRAS and NF1. In addition, most patients receiving targeted therapy eventually progress because melanoma cells can adapt to the presence of the drug. One of the main interests of our lab is to identify novel drug combinations for use in melanoma patients for whom there are no existing effective therapies. Researchers have found that the combination of a RAF dimer inhibitor (belvarafenib) and a MEK inhibitor (cobimetinib) is effective to treat non-BRAF-mutant melanoma, but resistance still occurs. We hypothesize that inhibitors of protein synthesis will work to potentiate the combination therapy of belvarafenib and cobimetinib in non-BRAF-mutant melanomas. My data thus far, suggests that inhibitors of mTOR signaling robustly induce apoptosis when combined with belvarafenib and cobimetinib, particularly in the hard-to-treat melanomas with NRAS mutations. I will not only continue to employ cell culture experiments, but also test this novel triple combination therapy in immune competent mouse models of NRAS mutated melanoma. We will perform proteomic analysis to determine how the triple combination therapy works to kill melanoma cells. This study is expected to assist in the design of novel therapeutic options for patients with non-BRAF-mutant melanoma.
THX-B, A p75NTR Antagonist, Increases NGF Expression in Bladders Cells In Vitro
2Urology Department, Jewish General Hospital, Montreal, QC, Canada
Corresponding Author: Aya Hajj, email aya99.hajj@gmail.com
Abstract
Urine of patients with overactive bladder syndrome displays low levels of NGF and high activity of the metalloproteinase 9 (MMP-9). Rodents with diabetic voiding dysfunction present the same features and after chronic treatment with the p75NTR antagonist THX-B, NGF levels are restored to normal and bladder function normalized. The aim of this research is to determine the relation of NGF and MMP-9 with THX-B in bladder cells. Primary culture of urothelial and smooth muscle cells was seeded from rat bladder. Expression of NGF and MMP-9 were analyzed by RT-qPCRs, immunoblotting and immunohistochemistry. NGF and MMP-9 are expressed in urothelial and smooth muscle cells. Both proteins could be localized in the cytoplasm of cells using immunohistochemistry. Crispr-Cas9 efficiently inhibited MMP-9 expression, resulting in a significant drop in MMP-9 activity and a substantial increase in NGF secretion without changing proNGF levels. THX-B bolstered NGF secretion by urothelial cells, increasing the NGF/proNGF ratio, by significantly decreasing MMP-9 synthesis and release. THX-B had no effect in smooth muscle cells. NGF, proNGF, and MMP-9 are expressed and secreted by bladder cells. The amount of NGF released by cells is inversely correlated to MMP-9 activity. THX-B promotes NGF release from urothelial cells through lowering MMP-9 activity. These results suggest that THX-B could be a therapeutic tool to improve OAB by targeting the urothelium.
Increased urinary ratio BDNF/proBDNF in a female population with overactive bladder syndrome
2Lady Davis Institute for Medical Research, Urology Department, Jewish General Hospital, Montreal, QC, Canada
Corresponding Author: Claudia Covarrubias, email claudia.covarrubias@mail.mcgill.ca
Abstract
Urine storage and voiding by the bladder are controlled by the peripheral and central nervous systems. Neurotrophins, such as brain-derived neurotrophic factor (BDNF) controls neuroregeneration while its precursor BDNF triggers inflammation and apoptosis, both of which have been proposed to be markers for overactive bladder syndrome (OAB). We herein examine the levels of proBDNF, BDNF and associated proteins and microRNAs in the urine of a female aging population. Urine and blood samples from 20 controls and 20 OAB patients were obtained with validated questionnaires. ProBDNF, BDNF,p75ECD, sortilin and cortisol were measured using specific ELISA kits (Biosensis). MicroRNAs involved in the control of proBDNF translation were measured. BDNF/creatinine levels were not statistically different between the urine of controls versus OAB patients. ProBDNF/creatinine measures were statistically lower in the OAB population. The ratio BDNF/proBDNF was higher in the OAB population. MicroRNAs known to control the translation of proBDNF mRNA by binding its 3’UTR sequence were expressed at similar levels between groups. On the other hand, enzymatic activity of MMP-9 was higher in the OAB group while MMP-3 activity was similar. MicroRNA MiR-491-5p was decreased in the OAB group. On the other hand, p75ECD was increased by OAB but there was no statistical significance between the urinary levels of sortilin or cortisol of controls when compared to OAB patients. These results suggest that the ratio BDNF/proBDNF might be a better indicator of OAB than BDNF or proBDNF alone.The decrease in proBDNF levels together with increased p75NTR could constitute a protective response to OAB.
Investigating mTOR-AR-NuRD Crosstalk on Gene Regulation in Prostate Cancer
2Department of Biochemistry, School of Biomedical Sciences, Faculty of Medicine, McGill University, Montréal, QC, Canada
Corresponding Author: Stephanie Lingwei Han, email lingwei.han@mail.mcgill.ca
Abstract
Prostate cancer (PCa) progression is largely regulated through androgen acting on the androgen receptor (AR), leading to a global change in gene transcription. Mammalian target of rapamycin (mTOR) is another key factor in PCa progression and emerging studies revealed that mTOR directly regulates gene expression by associating with chromatin in the nucleus (nmTOR). However, the detailed mechanism on how nmTOR regulates gene expression with AR in PCa remains unclear. Recently our group has uncovered a nmTOR-AR functional crosstalk in PCa cells where upon androgen stimulation, nmTOR associates with chromatin and activates an oncogenic metabolic gene program in an AR-dependent manner. Using a proteomic approach referred to as rapid immunoprecipitation mass spectrometry of endogenous protein (RIME), we previously identified the nucleosome remodeling and deacetylase (NuRD) complex as a partner of nmTOR-AR bound on chromatin in PCa cells. Here we demonstrate a mTOR-AR-NuRD functional transcriptional complex through ChIP-qPCR analysis, RNA RT-qPCR analysis, and immunoblotting approaches. We find that nmTOR, AR, and the catalytic subunits of NuRD, HDAC1/2 and CHD4, associate and dissociate with their common target genes at the same timepoints, subsequently affecting transcription of these genes. shRNA mediated knockdown of HDAC2 resulted in a delay of chromatin binding of mTOR, AR, and other NuRD components and concomitant decrease in target gene transcription. In the near future, we will perform ChIP-seq and RNA-seq analyses in PCa cells to investigate in greater details the underlying molecular mechanisms implicated in direct gene regulation by nmTOR and its transcriptional partners.
Mouse Model for the Study of Spinal Cord Injury-Induced Heterotopic Ossification
2Faculty of Medicine, University of Sherbrooke, QC, Canada,
3Division of Orthopedic Surgery, Department of Surgery, McGill University, Montreal, QC, Canada,
4Division of Physical Medicine & Rehabilitation, Department of Medicine, McGill University, Montreal, QC, Canada
Corresponding Author: Rachad Aita, email rachad.aita@mail.mcgill.ca
Abstract
Heterotopic ossification (HO) is the debilitating formation of abnormal bone in soft tissues reported in up to half of polytrauma patients with spinal cord injury (SCI) and limb damage. HO pathogenesis remains largely unknown; thus, recurrence prevention and chronic pain relief is suboptimal. In the absence of pertinent animal models of HO, we created a clinically relevant HO model using simultaneous SCI and musculotendinous injury (MTI). Wild type C57BL/6J mice were subjected to spinal cord transection between T10 and T11, followed with MTI by crushing the quadriceps tendon in the LEFT hindlimb. The RIGHT hindlimb remained intact as internal procedural control. Mice were euthanized on postoperative Day 21 when both hindlegs were harvested. High-resolution micro-CT scans were acquired on Day 24 to quantify the volume of ectopic bone around the knee joint. Undecalcified samples were then embedded in methyl methacrylate and consecutive 5µm sections were obtained and stained with Von Kossa to locate the ossification of peri-articular soft tissues, alkaline phosphatase (ALP) to stain osteoblasts, and tartrate-resistant acid phosphatase (TRAP) to stain osteoclasts. A Mann-Whitney U test was used for statistical analysis of HO volumes. Quantitative micro-CT analysis and histology showed ectopic bone deposits in the peri-articular region of all LEFT knees, with consistently higher HO volumes, but none in the RIGHT knees (Q1 – Q3: 0.03 – 0.51 vs 0.00 – 0.00, P=0.144). In conclusion, this mouse model represents a novel, reproducible and clinically relevant preclinical model of SCI-induced HO to investigate pathogenesis and assess novel therapeutics for this condition.
Characterization of KMT2A rearrangement acute myeloid leukemia biomarkers
Corresponding Author: Sarah Denford, email sarah.denford@umontreal.ca
Abstract
Following the development of acute myeloid leukemia driven by KMT2A-MLLT3 (KM3) fusions in murine models and patient samples, a number of genes were found to be biasedly expressed in these cells compared to healthy and other leukemic cells. In an attempt to determine if and how these genes influence leukemogenesis, we are characterizing selected putative biomarkers by identifying their modes of regulation and cellular functions in AML cell lines. METTL7B, one of our genes of interest, has also been identified as a gene of interest in several solid cancers. As of yet, its function and targets is still uncharacterized, although it is believed to have methyltransferase activity. In order to characterize METTL7B, we worked on answering four questions: where is METTL7B localized, is METTL7B’s expression necessary for cell survival, with what proteins does METTL7B interact, and finally, how is METTL7B transcriptionally regulated? By looking into these questions, we have found that METTL7B is a cytoplasmic protein with a likely role in proliferation and RNA metabolism, yet is not essential for cell survival in KM3-AML. By BioID, we have also found that it interacts with AHNAK, a scaffold protein often involved in neuroblastoma. The answer to its method of regulation is still elusive but we have proposed the use of a luciferase assay to identify important sequences. Since METTL7B is a biomarker for KM3-AML, analysis of its interactome and regulatory mechanism should identify the changes in cellular properties associated within the translocation. Therefore, with answers to the questions posed, I should be able to determine the potential functions of METTL7B and its role in KM3-AML.
Identification of SHOC2 as a critical modulator of KRAS dimerization
2Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, QC, Canada
3Department of Oncology, McGill University, Montreal, QC, Canada
Corresponding Author: Huei-Yu (Trista) Chen, email huei-yu.chen@mail.mcgill.ca
Abstract
Background and Objective: Oncogenic KRAS is a crucial driver of three lethal cancer: pancreatic cancer, colon cancer, and lung cancer. Suppressing oncogenic KRAS signaling in cancer has been under intense study for decades. Most efforts have been directed towards developing chemical inhibitors to target the downstream KRAS signaling. However, these inhibitors only have limited success due to the emergence of intrinsic and acquired resistance. Thus, it is urgently needed to develop a new strategy directly inhibiting KRAS. Inspired by recent findings that KRAS dimerization is critical for oncogenic KRAS signaling, the purpose of this study is to characterize SHOC2 as a key modulator of KRAS dimerization and the effects on KRAS oncogenic signaling by disrupting KRAS-SHOC2 interaction. Methodology: The SHOC2-KRAS interaction was analyzed using co-immunoprecipitations, and signaling pathways were investigated by immunoblotting. Furthermore, KRAS dimerization was evaluated through BRET assays. Results: We demonstrated that SHOC2 directly interacts with active KRAS (dimer) instead of inactive KRAS (monomer). In addition, SHOC2 knockout inhibits KRAS dimerization but can be rescued through expressing SHOC2 to the knockout cells. Next, we uncovered an essential role of KRAS P34 residue in KRAS/SHOC2 interaction. Disruption of KRAS/SHOC2 interaction through P34G mutation prevents KRAS dimerization and attenuates the MAPK pathway. Conclusion: Our study successfully identified SHOC2 plays an essential role in regulating KRAS dimerization and oncogenic signaling through interacting with the Switch I domain on KRAS. Moreover, these results open up a future direction for a novel strategy of developing therapeutic methods in KRAS-driven cancers.
Errant epitopes from arginine methylated SARS-CoV-2 proteins predisposing to autoimmune diseases
Corresponding Author: Sarah Khan, email sarah.khan6@mail.mcgill.ca
Abstract
The SARS-CoV-2 nucleocapsid (N) protein is the most abundant protein expressed in virions and infected cells. It contains five RGG/RG motifs, preferential sites for protein arginine methyltransferases (PRMTs). Notably, R95 and R177 are sites of PRMT1 methylation previously identified by our lab (Cai et al., 2021) and are also predicted B and T cell epitopes. Autoantibodies have been reported in SARS-CoV-2-infected patients, and autoantibodies against arginine methylated residues have been isolated from patients with systemic lupus erythematosus (SLE). We propose that SARS-CoV-2-infected patients may develop antibodies against arginine methylated N protein that overlap with self-arginine methylated proteins and lead to autoimmune disease. To test this, we propose performing a series of ELISAs using differentially methylated peptides and plasma from SARS-CoV-2-infected patients. Western blot and immunofluorescence will be used to verify methyl specificity, and FACS will be used to analyze PBMC responsiveness at later time points. We also plan to perform a series of in vitro methylation assays and mass spectrometry to identify other SARS-CoV-2 proteins modified by PRMTs. These findings will have an impact on why certain patients, post-SARS-CoV-2 infection, develop persistent illnesses, especially autoimmune diseases.
Nitric Oxide secretion regulates levels of Neurotrophin Growth Factor in bladder cells
2Urology Department, Jewish General Hospital, Montreal, QC, Canada Lady Davis Institute for Medical Research, 3755 Chemin de la Côte-Sainte-Catherine, Montreal, QC, Canada.
Corresponding Author: Stephanie Sirmakesyan, email stephanie.sirmakesyan@mail.mcgill.ca
Abstract
INTRODUCTION: In the urine samples of patients with overactive bladder syndrome (OAB), low levels of the neurotrophin NGF, and stable concentrations of its precursor proNGF were found, linked to hyperglycemia and elevated levels of nitric oxide (NO). We investigated how NO might control the ratio NGF/proNGF in bladder cells in culture. METHODS: We obtained primary cultures of urothelial (UROs) and smooth muscle cells (SMCs) from rat bladders. Using the Griess reaction, NO levels were measured. ELISA kits were used to measure NGF, proNGF, and cyclic GMP (cGMP). Protease activities were measured using enzymatic kits. Genomic MMP-9 deletion was done by Crispr-Cas9. RESULTS: Hyperglycemic medium led to increased NO secretion, decreased NGF and stable proNGF levels in both cell types. The NO generator sodium nitroprusside (SNP)(300µM) mimicked these effects on NGF and proNGF. SNP potently decreased cGMP levels in UROs and increased them in SMCs. Stable permeable analogs of cGMP, 8-(4-Chlorophenylthio)-cGMP (3mM) and N2,2'-O-Dibutyryl-cGMP (1mM) confirmed the role of cGMP on NGF and proNGF. In MMP-9 KO SMCs, SNP’s effect on NGF was intact, however, in UROs, this effect was partially inhibited. MMP-7, the enzyme converting proNGF into NGF was enhanced by SNP in UROs and decreased in SMCs. CONCLUSIONS: Release of NO is increased by hyperglycemia and leads to decreased NGF through a cGMP pathway. MMP-9 and MMP-7 are central in the control of NGF secretion. This data is consistent with our clinical results, suggesting that hyperglycemia, through enhanced synthesis of NO, could be part of OAB pathology.
Evaluation of the Potency and Efficacy of Selective Estrogen Receptor Down Regulator (SERD) molecules in SUMOylation of Mutant Estrogen Receptors
Corresponding Author: Bahareh Heidari, email bahareh.heidari@umontreal.ca
Abstract
Background: Around 70% of breast tumors express estrogen receptor alpha (ERα) which is the main indicator of potential response to endocrine therapy. The role of ERα in breast cancer progression has led to the development of antiestrogens which are steroidal or non-steroidal molecules designed to compete with estrogens and antagonize ERα. Unfortunately, many human breast tumors become resistant to endocrine therapies and persist due to ERα mutations that result in ligand independent activity. Here, we examined the impact of the two most frequent ERα mutations, D538G and Y537S, on the potency and efficacy of selective estrogen receptor down regulators (SERDs), including fulvestrant, the reference molecule, and new generation SERDs GDC-0927, giredestrant, and AZD-9833, in inducing estrogen receptor SUMOylation, a SERD-specific property. Methodology: To monitor ERα SUMOylation, we used bioluminescence resonance energy transfer (BRET) assays in transfected HEK293 cells with expressing ERα fused to Renilla Luciferase and SUMO3 fused to a yellow fluorescent protein. Results: We observed different impacts of ERα mutations on the potency and efficacy of these SERDs. While mutation Y537S lowered SUMOylation potency with all antiestrogens, mutation D538G lowered SUMOylation efficacy in an antiestrogen-selective manner. Efficacy was more reduced with fulvestrant, which induced the highest levels of SUMOylation with the wild type receptor, than with new generation SERDs, giredestrant and AZD-9833, which both have shorter side chain lengths. Conclusion: These results suggest that patients with tumors progressing after hormonal therapies due to mutations in ERα may benefit differentially from treatment with SERDs depending on the nature of their mutation.
Inactivation of Estrogen Receptor alpha by targeted SUMOylation: a biochemical proof of concept
Corresponding Author: Anais Vivet, email anais.jeannine.irene.vivet@umontreal.ca
Abstract
Estrogen receptor alpha (ERa) positive breast cancer is one of the most prevalent cancers in women worldwide. Therapies targeting this oncogenic transcription factor (TF) include selective down regulators or SERDs, like Fulvestrant. This pure antiestrogen works by triggering chromatin closure, ERa ubiquitination and sumoylation which loses DNA binding and undergoes proteasome degradation(1). Both modifications are generated by similar cascades gathering E1 activating, E2 conjugating and E3 ligating enzymes. According to preliminary data, E3 sumo ligase Protein Inhibitor of Activated STATs (PIAS) 1 participates in ERa sumoylation in presence of fulvestrant. We then hypothesize that the recruitment of E3 sumo activity onto ERa could be sufficient to promote sumoylation and further transcriptional inactivation.
To find out, we fused PIAS1 to ERa and transiently expressed chimaeras in ERa negative HEK293 cells. We assessed protein expression and sumoylation by Western analysis and tested PIAS1’s influence on ERa activity by luciferase assay. Our results show that chimaeras are sumoylated in presence of fulvestrant and suggest they are also transcriptionally inactive. We will verify that the lack of activity is related to ERa inactivation by sumoylation rather than steric hindrance. We will then test chromatin compaction levels at ERa target genes by ChIP, and repercussion on transcriptional activity using RNAseq.
With this proof of concept, new therapeutic strategies could be envisioned for the treatment of ERa positive breast cancer by targeting specific actors of the sumoylation cascade. This strategy could later be extended to other TFs negatively affected by sumoylation like cJun, P53 or STATs(2,3).
1. Traboulsi T, El Ezzy M, Dumeaux V, Audemard E, Mader S. Role of SUMOylation in differential ERα transcriptional repression by tamoxifen and fulvestrant in breast cancer cells. Oncogene. 2019;38(7):1019-1037. doi:10.1038/s41388-018-0468-9
2. Rabellino A, Andreani C, Scaglioni PP. The Role of PIAS SUMO E3-Ligases in Cancer. Cancer Res. 2017;77(7):1542-1547. doi:10.1158/0008-5472.CAN-16-2958
3. Rosonina E, Akhter A, Dou Y, Babu J, Sri Theivakadadcham VS. Regulation of transcription factors by sumoylation. Transcription. 2017;8(4):220-231. doi:10.1080/21541264.2017.1311829
The p75NTR antagonist THX-B improves voiding behaviour and reduces bladder contractility in aging mice
2Urology Department, Jewish General Hospital, Montreal, QC, Canada
Corresponding Author: Aalya Hamouda, email aalya.hamouda@mail.mcgill.ca
Abstract
Introduction: Nerve Growth Factor (NGF) provides homeostatic functions and promotes tissue health. Low levels of urine NGF was observed in aging patients with overactive bladder (OAB). This decrease was linked to high activity of metalloproteinase-9 (MMP-9), which digests NGF. We previously found that THX-B, an antagonist of the proinflammatory receptor p75NTR, increased NGF levels by decreasing MMP-9 activity in urothelial cell culture. Here, aging mice were treated with THX-B to evaluate the in vivo functional benefit of p75NTR antagonism on the aging bladder. Methods: Male C57BL/6J mice of 6-, 12- and 18-months were injected with either PBS (control) or THX-B (50 µg) once weekly for four weeks. Voiding behaviours and patterns, notably, total urine volume, volume of urine, and frequency of urination were assessed using voiding spot assay. Organ baths evaluated bladder contractility. Results: Compared to controls, voiding behaviour and bladder contractility were improved only in the 12-month-old mice treated with THX-B. Specifically, total urine volume and volume per micturition were reduced following four weeks of treatment whereas voiding frequency was reduced following two and four weeks of treatment. Bladder contractility was reduced in the same mice following four weeks of treatment. Conclusion: Our results illustrate an age-specific effect of THX-B. Improvements in bladder behaviour and activity were observed only in 12-month-old treated mice. These results are in accordance with our previous studies and suggest that THX-B could be a new pharmacological tool for improving OAB.
RECEPTOR- AND LIGAND- SPECIFIC DETERMINANTS OF SUMOYLATION OF ESTROGEN RECEPTORS BY ANTIESTROGENS
2Department of Biochemistry, Faculty of Medicine, University of Montreal, QC, Canada
Corresponding Author: Amandine Vallet, email amandine.vallet@umontreal.ca
Abstract
More than 70% of mammary tumors express estrogen receptor alpha (ERα). This estrogen-dependent transcription factor induces tumor cell proliferation and survival, but can be blocked, in the clinic, by antiestrogens. Two types have been described: Selective Estrogen Receptor Modulators (SERMs) or Selective Estrogen Receptor Degraders (SERDs), including fulvestrant (Fulv). SERDs induce the degradation of ERα via the ubiquitin-proteasome pathway. Our group has also observed that ERα is SUMOylated in presence of SERDs. The SUMOylation process consists in the conjugation of SUMO1/2/3 proteins on lysine residues of a target protein, thanks to an activating (E1), a conjugating (E2) and a ligase (E3) enzymes. To identify the structural determinants of ERα required for Fulv-induced SUMOylation, we used the fact that Fulv induces SUMOylation of ERα, but not of its paralog ERβ. We constructed ERα/β chimeras and characterized their SUMOylation in the presence of Fulv by Western blotting and Bioluminescence Resonance Energy Transfer. Our results indicate that a region of the ligand binding domain of ERα that forms the outer rim of the coactivator binding groove is crucial for the Fulv-induced SUMOylation. In this region, amino acids specific to ERα do not include potential substrates of modification (lysine residues). These results suggest that diverging amino acid contribute to the differential recruitment of E3 SUMO ligases by ERα/β. This study should lead to a better understanding of the mechanism of action of antiestrogens on ERs. This will also facilitate the design and testing of novel antiestrogens in their capacity to induce SUMOylation of ERα.
CAXII IS A CELL SURFACE MARKER FOR LUMINAL TUMOR CELLS
Corresponding Author: Lucas Porras, email lucas.porras@umontreal.ca
Abstract
Background: Luminal cancers, defined clinically by expression of the estrogen receptor (ER), represent ~73% of breast tumors, but present a spectrum of ER expression levels. Tumors with heterogeneous ER expression are associated with a greater risk of relapse. We set out to identify surface markers whose expression correlates with that of ER and its upstream regulators genes FOXA1 and GATA3 in order to implement a sorting approach for ER-positive versus ER-negative (ER–/+) cells in heterogenous breast tumors and explore causes of heterogeneous ER expression. Methodology and results: Analysis of transcriptomic profiles in the TCGA BC dataset revealed that mRNA levels of the surface protein carbonic anhydrase XII (CAXII) are highly correlated with those of luminal transcription factors ER, FOXA1 and GATA3. CA12 is regulated by treatment with estradiol through recruitment by all three factors to its enhancers in breast cancer cell models. Immunohistochemistry verified the correlation between CAXII and the three luminal factors in an array of 138 tumors. Further, co-immunofluorescence supported the co-expression of CAXII and ER at the cellular level in in situ and invasive tumors. Fluorescence activated cell sorting enabled discrimination between mixed ER–/+ breast cancer cells, suggesting that CAXII is a useful marker to explore heterogeneous ER expression in dissociated epithelial cells from primary tumors. Conclusion: CAXII is a luminal marker strongly correlated with luminal TFs at the protein as well as mRNA level and should prove useful to detect and purify mixed luminal/basal cell populations within heterogeneous luminal tumors.
Identifying novel roles for MNK1 and MNK2 in the melanoma microenvironment
2Division of Experimental Medicine, McGill University, Montréal, QC, Canada.
3McGill Centre for Translational Research in Cancer, McGill University, Montréal, QC, Canada.
Corresponding Author: Mengqi Li, email mengqi.li@mail.mcgill.ca
Abstract
Melanoma is the most lethal form of skin cancer due to its increased tendency to spread to other organs. Specific proteins, such as the kinases MNK1/2, can function abnormally and promote melanoma metastasis. Clinically, MNK1/2 expression/activity are increased in cancer and associated with poor patient outcome. MNK1/2 have emerged as druggable targets in cancer, with inhibitors blocking MNK1 and MNK2 activity. We have shown that tumors cells lacking MNK1/2 proteins are less likely to grow and metastasize, but whether the MNK1/2 proteins expressed by tumor supportive cells could establish an environment that favors melanoma growth and metastasis remains unknown. We used genetically engineered mice lacking either MNK1, MNK2, or both MNK1/2, and injected these with the same tumor cells to assess the impact of stromal MNK1/2 on melanoma outgrowth. Our results showed that melanomas grown in MNK1 deficient mice grow poorly, while the same melanomas grown in MNK2 null mice grow as well as in WT mice. Moreover, melanomas grown in MNK1/2 double knockout mice have a similar growth defect as MNK1 deficient mice. As melanoma can affect both males and females, we have done these pre-clinical studies in both male and female mice, with both showing similar effects. Our future work seeks to identify potential mediators of the melanoma-promoting action of stromal MNK1. We hypothesize that MNK1 signaling in specific immune cells contributes to melanoma outgrowth and response to therapy.
The Role of The Community Pharmacists in The Management of Acute Pain in Adults: A Scoping Review
2Faculty of Science , Ryerson University, Toronto, ON, Canada
3Research Institute of the McGill University Health Centre, Montreal, QC, Canada
4Department of Surgery, Faculty of Medicine and Health Sciences, McGill University, Montreal, QC, Canada
5Department of Medicine, Faculty of Medicine and Health Sciences, McGill University, Montreal, QC, Canada
6Faculté de pharmacie, Université de Montréal, QC, Canada
7Research Center De L’Institut Universitaire de Gériatrie de Montréal, QC, Canada
Corresponding Author: Khiran Arumugam, email khiran.arumugam@mail.mcgill.ca
Abstract
Background: Acute pain is often under-treated and results in negative health outcomes in adults. A link to community experts, community pharmacists (CP), could support self-management of acute pain. Knowledge on CP practices in the management of acute pain in adults is lacking. We conducted a scoping review to describe CPs’ practices/interventions in acute pain management in adults and to identify barriers and facilitators in CPs’ engagement in relation to adults’ self-management of acute pain. Methodology: We searched the literature in five bibliographic databases for eligible studies published after 1990. Search results were independently screened for inclusion criteria by 2 reviewers. Study design, participants’, CP engagement characteristics were extracted, and the results were synthesized and organized thematically. Results: The appropriate research question was attained after five iterations. We identified 2419 studies that met the inclusion criteria and we retained 39 studies for extraction. Findings suggest that CPs intervene mostly in the domains of low back pain (57.1%), postoperative pain (20.0%), toothache (20.0%), and musculoskeletal injuries (11.4%). CP interventions designed to manage these acute conditions include patient- and CP- specific educational pamphlets and workshops (n = 20), patient counselling (n = 7), opioids stewardship (n = 5), order sets for opioid alternatives (n = 4), and referrals. Additionally, recurrent barriers were centered on limited scope of practice, time-associated constraints, and communication between healthcare professionals. Conclusion: The knowledge gained from this work will provide the foundation for the development of tools to support older adults in better management of acute pain within their community circle of care.
Inhibition of the MNK1/2-eIF4E axis enhances the anti-tumor effects of palbociclib
2Lady Davis Institute of Medical Research, Montreal, QC, Canada,
3University Health Network, Toronto, ON, Canada,
4University of Toronto, Toronto, ON, Canada.
Corresponding Author: Judith LaPierre, email judith.lapierre@mail.mcgill.ca
Abstract
Dysregulation of the cell cycle is a hallmark of cancer and has resulted in the development of drugs targeting regulators of cell cycle progression. Palbociclib is a CDK4/6 inhibitor which has been proven to be effective in hormone receptor-positive breast cancers and is being studied for its efficacy in other cancers such as melanoma. The MAPK and PI3K/AKT/mTOR signalling pathways are the most frequently dysregulated pathways in melanoma. Signalling down these pathways converges at the MAP Kinase Interacting Proteins 1 and 2 (MNK1/2)—eukaryotic initiation factor 4E (eIF4E) axis. The MNK kinases are the only known kinases of eIF4E; the phosphorylation of which results in increased translation of a subset of pro-oncogenic mRNA and is associated with poorer prognosis in a variety of cancers. We have found that treatment with palbociclib results in increased levels of phosphorylated eIF4E and thus hypothesized that the addition of selective MNK1/2 inhibition would enhance the antitumour effects of palbociclib by reducing phospho-eIF4E. We show that the combination of MNK1/2 and CDK4/6 inhibitors results in greater reduction of clonogenic outgrowth compared to either single-agent treatment in melanoma and breast cancer cell lines. We also find reduced expression of mitotic regulators, resulting in a G1 arrest in the combination compared to single-agent treated cells as well as increased overall survival in murine models. Together, our data highlights the promise of the combination of MNK1/2 and CDK4/6 inhibition in breast cancer and melanoma models as a new avenue for treatment of these diseases.
The Efficacy of Machine Learning Based Risk Stratification Tools for Type 2 Diabetes Mellitus: A Systematic Review Protocol
2DREAM-CV Lab, McGill University Health Centre, McGill University, Montreal, QC, Canada
3Division of Cardiology, McGill University Health Centre, McGill University, Montreal, QC, Canada
Corresponding Author: Mariam Jabara, email mariam.jabara@hotmail.com
Abstract
Background: Type 2 Diabetes Mellitus (T2DM) is the most common form of diabetes, accounting for 90% of cases world-wide. As many as one third to one half of patients are undiagnosed. Machine Learning (ML), a subset of Artificial Intelligence (AI), has the potential for great utility in screening and identifying patients with T2DM early-on through its ability to distinguish biological and physiological patterns in the diseased population. Through this review, we seek to address the gap in knowledge around the clinical utility of ML-based screening tools for T2DM. Methods and Analysis: We will conduct a systematic review to assess studies that leverage ML in stratifying risk for developing T2DM. We seek to synthesize and evaluate these tools for their clinical utility by examining their discriminative ability, cost-effectiveness, and impact on disease progression. Electronic records in Embase, MEDLINE, Web of Science Core Collection, and Google Scholar will be searched. Two independent reviewers will screen titles and abstracts based on our defined inclusion/exclusion criteria, and deviations from a consensus will be settled by a third reviewer if necessary. The review will follow the PRISMA-DTA guidelines and study quality will be assessed using the ROBIS tool. Qualitatively, studies will be assessed for their machine learning approach and generalizability. When possible, a meta-analysis will be conducted on studies with commonly reported measures of model performance. Conclusion: This review will address the question of how ML-based tools can be best utilized to identify T2DM patients early-on to enable early intervention strategies.
Investigation of the sexual dimorphic tumor suppressor role of DDX3X in melanoma
Corresponding Author: Marine Lingrand, email marine.lingrand@mail.mcgill.ca
Abstract
Patient sex remains a poorly understood prognostic factor in melanoma. Men have higher incidence rates of melanoma, and at all stages, have poorer prognosis (Joosse, A. et al., J Clin Oncol 2012 ; Joosse, A. et al., J Clin Oncol 2013). Recently, our group identified new significantly mutated genes by performing a mutational meta-analysis of 1,014 melanoma exomes from five studies (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-outs (KOs) in male and female melanoma and melanocyte lines. Moreover, we identified DDX3X-null male human lines to carryout DDX3X gain of function studies. From our studies, we determined that DDX3X loss increases proliferation, migration and invasion only in male melanoma and melanocyte lines supporting for its sexual dimorphic tumor suppressor role in this malignancy.
D-mannose supplementation decreases atherosclerotic lesions in ApoE-/- mice through regulation of monocytosis and intestinal inflammation
Corresponding Author: Jonathan O'Connor, email jonathan.oconnormiranda@mail.mcgill.ca
Abstract
Background: D-mannose, a C-2 epimer of glucose, is an important monosaccharide for protein glycosylation. D-mannose alters gut microbiota preventing high fat diet (HFD)-induced obesity, and exhibits potent anti-inflammatory properties. Whether D-mannose regulates atherosclerosis, a chronic inflammatory disease; remains undefined. We hypothesized that D-mannose would alleviate the pro-atherogenic effects of HFD by regulating gut microbiota and inflammation. Methods & results: ApoE knockout atheroprone mice, received HFD supplemented orally with 0, 5 or 20% D-mannose. Body weights and plasma lipids increased equally in all groups. Plasma mannose levels increased by ~68% in mice treated with 20% D-mannose (51±2 vs 30±5 ng/ml). Importantly, atherosclerotic plaque development was significantly abated in mice receiving 5 and 20% D-mannose compared with 0% (0.23±0.03 (5%), 0.20±0.05 (20%) vs 0.46±0.04mm2), as determined by Oil red O staining. Furthermore, plaques of 20% D-mannose mice displayed a significant 50% increase in α-smooth muscle actin content vs 0% mouse lesions, suggesting that D-mannose stabilizes the atherosclerotic plaques. Flow cytometry of blood and peritoneal cells revealed a 40 and 50% decrease of pro-inflammatory Ly6Chi monocytes, and small peritoneal macrophages respectively, in 20% oral D-mannose mice vs 0%, P<0.05. Suggesting a reduction of HFD-induced monocytosis by D-mannose. Interestingly, D-mannose also reduced by 50% the expression of lipopolysaccharide receptor, Toll-like receptor 4 on macrophages in the lamina propria compared with controls. Conclusion: Our results show that oral D-mannose supplementation reduces atherosclerotic lesions and increases plaque stability. These protective effects could possibly occur through regulation of monocytosis following reduced gut microbiota dysbiosis and intestinal inflammation.
Towards Structure-Guided Design of Aminoglycoside Phosphotransferase Inhibitors
Corresponding Author: Mark Hemmings, email mark.hemmings@mail.mcgill.ca
Abstract
Aminoglycosides are a broad-spectrum class of antibiotics used to treat serious gram-negative and gram-positive bacterial infections. However, widespread resistance has led to a decrease in aminoglycoside clinical use. This resistance can be established through aminoglycoside-modifying enzymes such as APH(2’’)-Ia, which inactivates aminoglycosides through phosphorylation of hydroxyl groups. One strategy to circumvent aminoglycoside resistance is by inhibiting APH’s activity. The production of inhibitors for this class of enzymes is exceedingly challenging, as APHs are structurally similar to eukaryotic protein kinases. We hypothesize that through fragment-based drug-discovery techniques, differences between human and bacterial homologues can be exploited to generate inhibitors specific to APHs. Through saturation transfer difference and waterLOGSY nuclear magnetic resonance experiments, we have screened and identified fragment molecules which bind to APH(2’’)-Ia. X-ray crystallography will be used to determine the position and orientation of fragment binding. Based on structural data, successful fragments can be further expanded to form a drug candidate capable of binding to APH(2’’)-Ia with high affinity and specificity without disrupting the functions of mammalian protein kinases. Such an inhibitor could allow for continued use of aminoglycoside antibiotics in the treatment of otherwise resistant bacterial infections.
The non-uniform actin cytoskeleton of magnocellular neurosecretory cells (MNCs) of the rat supraoptic nucleus (SON).
Corresponding Author: Anzala Murtaz, email anzala.murtaz@mail.mcgill.ca
Abstract
Previous experiments have shown that osmosensory transduction (OT) in rat MNCs is mediated by an N-terminal variant of the transient receptor potential vanilloid 1 (dn-Trpv1) channel. Activation of this channel is mediated by microtubules (MTs) that apply a “push” force to the channel during hypertonicity-induced shrinking. MNCs also feature a thick (~1 µm) layer of actin filaments (f-actin) beneath the plasma membrane, which is also essential for OT, but how f-actin contributes to this process remains unknown. Specifically, it is not known if f-actin interacts with dn-Trpv1 channels and if the actin cortex has specific features that are important for OT. We used immunocytochemistry, proximity ligation assay (PLA) and super-resolution imaging to examine the actin cytoskeleton of isolated MNCs of the rat SON. PLA confirmed previously known MT-Trpv1 channel interactions (Prager-Khoutorsky et al. 2014). Specifically, we observed ~10-100 interaction sites per cell (n=30; 6 preparations), mainly discrete puncta scattered on the cell surface. However, no interaction sites were detected when PLA was performed using antibodies against actin and trpv1 (n=20, 2 preparations). We used super-resolution fluorescence microscopy with a FV 3000 Olympus confocal microscope and image deconvolution (CellSense software, Olympus Canada Ltd) to obtain image stacks of the MNC actin cytoskeleton. These images revealed that the submembrane actin cortex is fenestrated, with small regions (~1-5 µm) of lowered fluorescence (n=50; 2 preparations). To conclude, this study reveals that the submembrane actin cortex of MNCs is non-uniform and does not interact with dn-Trpv1 channels.
Ectopic Expression of PRAME Attenuates Retinoid Response and Supports Proliferation in Squamous Cell Carcinoma Cells
2Division of Experimental Medicine, Faculty of Medicine, McGill University, Montreal, QC, Canada.
3Division of Dermatology, Department of Medicine, McGill University Health Center, Montreal, QC, Canada.
Corresponding Author: Brandon Ramchatesingh, email brandon.ramchatesingh@mail.mcgill.ca
Abstract
Background: Retinoids are anti-neoplastic vitamin A derivatives that activate transcription of terminal differentiation programs. These compounds exhibit efficacy for preventing and treating squamous cell carcinomas (SCCs). Cutaneous SCC (cSCC), head and neck SCC (HNSCC) and their precursor lesions ectopically express Preferentially Expressed Antigen in Melanoma (PRAME), a gamete-restricted gene. In gametes, PRAME represses retinoid signaling and promotes proliferation. The functions, prognostic and therapeutic significance of PRAME expression in benign keratinocytes, cSCC and HNSCC cells have not been investigated. We hypothesize that PRAME confers retinoid-resistance and supports proliferation in benign keratinocytes and SCC cells. Methodology: PRAME expression was evaluated in human epidermal tumors, immortalized keratinocytes and SCC cell lines by immunohistochemistry, immunoblotting and RT-PCR. PRAME-overexpressing and shRNA knockdown cell lines were generated. Cells were treated with retinoids for 24, 48 or 72 hours. Expression of differentiation markers was assessed using immunoblotting, immunocytochemistry (ICC) and RT-PCR. Cell proliferation was assessed using Ki-67 ICC, cell counting assays, and immunoblot analysis of cell cycle proteins. Results: PRAME expression was detected in subsets of epidermal tumors and SCC cell lines. Overexpression of PRAME reduced expression of select tumor suppressors and enhanced cell proliferation. Retinoid treatment was incapable of promoting differentiation when PRAME was overexpressed in SCC cells. Furthermore, PRAME overexpression attenuated retinoid-induced anti-proliferative response. Conclusion: PRAME expression augments proliferation of malignant keratinocytes in vitro and confers resistance to retinoid-induced differentiation and proliferation arrest. Evaluating the prognostic and therapeutic significance of PRAME expression in SCC and precursor lesions is warranted to optimize therapeutic strategies.
The P66ShcA redox protein accelerates breast cancer progression
2McGill University, Montreal, QC, Canada
Corresponding Author: Elias Maldonado, email elias.maldonado@mail.mcgill.ca
Abstract
While cancer treatment has improved in recent years to become more personalized and effective, aggressive breast cancers often adapt to oncogenic or therapeutic stress, posing a major clinical problem. Oxidative stress, an imbalance in the production of reactive oxygen species (ROS), has been shown to promote adaptive responses in breast cancers which increases their aggressive properties. One protein of interest is the p66ShcA redox protein, known for triggering ROS production in the mitochondria in response to stress stimuli. Under moderate and chronic oxidative stress, breast tumours can hijack the tumorigenic properties that are potentiated both by p66ShcA signaling complexes and moderately elevated ROS. Our group has developed the first transgenic mouse model that overexpresses p66ShcA, specifically in the mammary epithelium (MMTV/p66ShcA). We bred these mice to an established transgenic mouse model of metastatic breast cancer that is driven by polyomavirus middle T antigen (MT). We show that p66ShcA overexpression in the mammary epithelium significantly accelerates breast tumour onset and increases breast tumour growth. RNA sequencing results highlighted a transcriptional response indicative of immune cell activation, specifically in p66ShcA overexpressing tumours. Indeed, increased ROS levels have been shown to increase inflammation, which itself functions a tumour promoter. Multiplex chemokine and cytokine assays suggest there are increased levels of inflammation in p66ShcA overexpressing tumours and in circulation, and blood counts show a significant increase in granulocytes. Future experiments will examine the tumorigenic effect of increased ROS and inflammation to understand adaptations that breast tumours undergo to thrive in this inflammatory environment.
Characterization of Phenotype Plasticity in Uveal melanoma
2Division of Experimental Medicine, McGill University, Montréal, QC,Canada
3McGill Centre for Translational Research in Cancer, McGill University, Montréal, QC, Canada.
Corresponding Author: Heejin (Hayley) Shin, email heejin.shin@mail.mcgill.ca
Abstract
Uveal melanoma (UM) is the most-prevalent intraocular cancer type in adults. Unlike cutaneous melanoma, UM does not respond to current MAPK-targeted therapies. Moreover, approximately 50% of patients will develop metastasis. Phenotypic plasticity refers to the ability of a cancer cell to switch from one state to another, and is a proposed mechanism underlying the ability of cancer cells to metastasize. Despite the characterization of 4 phenotypic states in cutaneous melanoma, little is known about differeing cell states in UM. The goal of this project is to examine whether, as observed for cutaneous melanoma, UM cell lines can be characterized into different cell states. We therefore first designed a novel FACS based assay to simultaneously detect the expression of well characterized markers of plasticity: AXL, Melan-A, GFRA2, NGFR, CD166, CD36, Nestin. We next analyzed a broad panel of UM cell lines by FACS to detect the relative expression of the aforementioned markers. To date, we have observed that different UM cell lines express different cell state markers. Having optimized this plasticity panel, we next determined whether UM cell lines treated with anti-cancer agents would result in a change in the expression of phenotypic markers. Most of the UM cell lines showed changes in the expression levels of specific cell state markers with drug treatment. With these results, with the right inhibitor options, aggressive and unresponsive tumor types may be shifted to a state where currently available therapies are able to target and prevent metastasis.
Evolutionary divergence in a disordered linker tunes condensate properties of Polyhomeotic
2Division of Experimental Medicine, McGill University, Montreal, QC, Canada,
3Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX, USA,
4Department of Biochemistry and Molecular Genetics, Midwestern University, Glendale, AZ, USA,
5Department of Biochemistry and Molecular Medicine, University of Montreal, Montreal, QC, Canada
Corresponding Author: Tim Gemeinhardt, email tim.gemeinhardt@mail.mcgill.ca
Abstract
Large-scale chromatin organization is tightly linked to the regulation of gene expression. Polyhomeotic (Ph), a Polycomb Group protein, is implicated in organizing chromatin via the formation of protein condensates. Condensate formation in vivo and in vitro depends on the C terminal Sterile Alpha Motif (SAM) of Ph. Ph SAM can assemble into helical polymers, an activity regulated by the disordered linker connecting SAM to the rest of Ph. A minimal condensate forming Ph protein (mini-Ph) contains the SAM, linker, and two other conserved domains (FCS and HD1). Strikingly, the linker sequence and its effect on SAM polymerization have diverged between Drosophila and mammals. To understand how and why regulation of Ph SAM polymerization and protein condensate formation has evolved, we are dissecting the biochemical and biophysical effects of the linker. Using single-chain molecular dynamics (MD) simulations, we identified linker-SAM interactions mediated by charged residues between the human linker and SAM that do not form with the Drosophila linker. Linker regulation of SAM polymerization is disrupted by changing the surface charge of SAM, implicating linker-SAM electrostatic interactions in regulating polymerization. Multichain simulations also identified linker-linker interactions and predicted that human versus Drosophila linkers have altered phase separation propensity. In vitro, protein-DNA condensates of mini Ph with Drosophila versus human linkers differ in size, morphology, and the mobility of DNA inside them. Our data suggest a model in which linkers have evolved to change the properties and dynamics of Ph condensates through linker-linker and linker-SAM interactions while maintaining the overall conservation of Ph domains.
5-HT2AR/AMPAR Heteroreceptor Complex As A Mediator Of LSD Action.
Corresponding Author: Etienne Billard, email etienne.billard@mail.mcgill.ca
Abstract
The administration of psychedelic compounds in controlled clinical settings leads to antidepressant and anxiolytic effects, accompanied by changes in brain activity and connectivity. Lysergic acid diethylamide (LSD), a potent biased agonist of the G protein-coupled receptor (GPCR) serotonin 2A (5-HT2A) also modulates the glutamatergic system, especially through the receptor AMPAR. LSD enhances social behavior and has anxiolytic effects in rodents by promoting synaptic plasticity via an AMPAR-dependent mechanisms, while in layer V pyramidal neurons psychedelics induce phosphorylation of AMPAR GluA2 subunit and promote its internalization. As several GPCRs (dopamine D2 and β2-adrenergic) can form supramolecular complexes with AMPAR, our hypothesis is that LSD-mediated AMPAR activation can occur via an oligomeric AMPAR/5-HT2AR complex. To demonstrate physical interaction between AMPAR and 5-HT2AR we used co-immunoprecipitation in HEK-293 cells and proximity ligation assays in rat primary cortical neurons. To characterise the unique signalling fingerprint of this heteroreceptor complex we used RET-based biosensors. We have demonstrated that 5-HT2AR and AMPAR can be co-immunoprecipitated, thus being part of a complex. Proximity ligation assays confirmed such physical interaction in neuron. Co-transfection of AMPAR with 5-HT2AR did not impact Gq activation following treatment with LSD, but decreased efficacy for β-arrestin2 recruitment, demonstrating the impact of AMPAR in biasing 5-HT2AR signaling through lateral allostery. Our in vitro data support the existence of a 5-HT2AR/AMPAR hetero-oligomers and suggests further research in primary neuronal culture and in vivo is required. We believe that the 5-HT2AR/AMPAR could be regarded as a new therapeutic target for treating mental illness.
Improving lung cancer outcomes: risk prediction of postoperative readmission following lobectomy in lung cancer
2Department of Medicine, Faculty of Medicine and Health Sciences, McGill University, Montréal, QC, Canada
3Department of Medicine, Division of Respiratory Medicine, McGill University Health Centre, Montréal, QC, Canada
Corresponding Author: Ankita Ghatak, email ankita.ghatak@mail.mcgill.ca
Abstract
Introduction: Postoperative readmission is a strong negative predictor of survival. Traditional models assessing preoperative risk have significant limitations with accuracy when applied to lung cancer patients. Our objective was to use large clinical data to assess the predictive accuracy of a random forests model compared to a traditional model such as logistic regression which uses conventional variables, to predict 90-day postoperative readmission in lung cancer patients resected by lobectomy. Methods: Data was extracted from NSQIP, the MUHC Datawarehouse EHR and Pulmonary Function database and included demographics, comorbidities, preoperative labs, pulmonary function data and admission records. Our primary outcome was 90-day postoperative readmission. The predictive power of a Random Forests using all predictors was compared to a logistic regression with only demographics and comorbidities. Results: The logistic regression showed the following as the most important predictors: gender (OR 2.38, p<0.05), history of severe COPD (OR 2.1, p<0.05) and Charlson comorbidity index (per unit increment) (OR 1.29, p>0.05). The random forests ranked features predictors by importance (in descending order): age, WBC, hematocrit, albumin, sodium, creatinine, BMI, sex, hypertension, Charlson comorbidity index, active smoking within 1 year of surgery and PFT availability. The random forests was visualized via decision trees and had better model performance on all assessed metrics, most significantly the AUC (0.72) compared to the logistic regression (AUC 0.59). Conclusions: Random forests shows better predictive performance than logistic regression and may improve preoperative risk stratification for lung cancer patients. Random forests may have several clinical decision-making applications, such as with prehabilitation.
Defining the role of arginine methylation in R-loop resolution in cancer
Corresponding Author: Samantha Daley, email samantha.daley@mail.mcgill.ca
Abstract
R-loops are three stranded structures formed when RNA hybridizes with the template DNA strand, creating an RNA:DNA hybrid and a portion of ssDNA. Evidence has shown R-loops have some functional importance for cell processes, like regulating gene expression, but they are mainly associated with DNA damage and genomic instability. The most abundant RNA modification, N6-methyladenosine (m6A), has been implicated in the resolution and stability of R-loops, with previous studies showing m6A modifications can affect the levels of R-loops and functional DNA damage repair. The METTL3/METTL14/WTAP complex is responsible for the addition of the m6A modification and METTL14 is known to be arginine methylated by the protein methyltransferase PRMT1. DHX9, an R-loop resolving helicase, associates with METTL14 and is similarly methylated by PRMT1. Using in vitro helicase assays and R-loop pull down experiments, the effect of m6A modifications in R-loops will be examined. Our lab has identified DDX5, a helicase methylated by PRMT5, to regulate the resolution of R-loops through interaction with the exoribonuclease XRN2. DNA:RNA immunoprecipitation (DRIP) followed by sequencing revealed that there are more R-loop loci affected by PRMT5 deficiency than knockdown of DDX5, suggesting that PRMT5 is altering R-loop levels in other ways, possibly through the regulation of other DNA:RNA helicases. It is hypothesized that DHX9 is similarly involved in resolving R-loops, mediated by PRMT1 methylation and by interacting with m6A-reader proteins, it can recruit DNA damage response elements. Upon the knockdown of METTL3 in U2OS cells, an increase in R-loops has been detected through MapR and DRIP-qPCR.
The Effect of Alarmins on Human Airway Smooth Muscle Function
2Meakins-Christie Laboratories, Research Institute of the McGill University Health Center, Montreal, QC, Canada
Corresponding Author: Dora Xiong, email dora.xiong@mail.mcgill.ca
Abstract
One consistently observed feature in asthma is airway hyperresponsiveness (AHR) – an exaggerated airway smooth muscle (ASM) response to various stimuli leading to excessive airway narrowing. While the cause of AHR remains unclear, it has been suggested that alarmins (IL-33, TSLP, and IL-25) interact with ASM and alter its intrinsic contractile properties. These compounds are increased in asthma and may interact with ASM cells through receptors that are upregulated during inflammatory episodes. Recent therapeutic strategies targeting the alarmins have demonstrated potential benefits on the inflammatory response in asthma but did not address their effects on downstream effectors such as ASM. This study aims to determine the effects on ASM contractility produced by exposure to alarmins. Intrapulmonary ASM tissue strips were dissected from post-mortem asthmatic human lungs and incubated with IL-33, TSLP, and IL-25 for up to 48 hours. In vitro mechanics were measured at 24 and 48h to assess changes in contractile force (Fmax), velocity of shortening (Vmax), and responses to various contractile/relaxant agonists. Exposure to alarmins did not affect F¬max or Vmax. Contrary to our expectations, our data showed that exposure to IL-25 increased the amplitude of relaxation induced by the β2-agonist isoproterenol (at 24h: control: 8.9 ± 3.4% relaxation vs IL-25: 25.1 ± 4.6%; p<0.05 and at 48h: control: 9.1 ± 14.8% relaxation vs IL-25: 33.8 ± 11.1%; p<0.05). These results suggest that IL-25 may play a protective role against AHR in asthma. Ongoing studies aim to elucidate the mechanism of action of IL-25 on ASM mechanics in asthma.
Retrofitting the COVID-Alert App for Additional Infectious Disease Exposures
Corresponding Author: Ryan Hanula, email ryan.hanula@mail.mcgill.ca
Abstract
Background: Amidst the SARS-CoV-2 pandemic, the Canadian government released a COVID Alert app to help track and slow the spread of the virus. At a cost of $21 million, approximately 24.1% of eligible Canadians downloaded the application to be notified in the event of a positive contact. Given the considerable price and societal uptake, considerations should be made about the possibility of extracting further use from it for other public health services. Therefore, we propose the application be retrofitted to serve as a notification system for two additional infectious disease scenarios; reported student cases of communicable infections and sexual transmitted infections (STIs) among previous partners. Methods: For both, a 3-point contact relay system via the pre-existing temporary exposure key infrastructure will serve as the foundation. Initiation of the chain will follow the documentation of any human papillomavirus, chlamydia, gonorrhea, syphilis or herpes case as well as measles, pertussis (whooping cough), scarlet fever, meningococcal, or chicken pox infection within a school setting. Furthermore, as originally with the app, anonymity will be preserved throughout the entire chain and potential secondary contacts will only be notified once the initial patient confirms consent. Predicted Results and Conclusion: These added modifications will enhance our ability to oversee a greater number of preventable infectious diseases while minimizing intermediaries to streamline the process. As a result, the time from first prognosis to exposure management will be significantly reduced, likely leading to a reduction in the over 150 000 new STIs and numerous school outbreaks each year.
An Integrative Systematic Review of Anticholinergic Risk Measurement Scales (ARMS) – Can “ARMS” Keep Patients Out of Harm’s Way?
2Center for Outcomes Research and Evaluation (CORE), Research Institute of the McGill University Health Center, Montreal, QC, Canada
3Department of Pharmacy, General Hospital, Aboh, Delta State, Nigeria.
4Department of Psychiatry, Faculty of Medicine, McGill University, Montreal, QC, Canada
5Chronic Viral Illness Service, McGill University Health Centre (MUHC), Montreal, QC, Canada
6Infectious Diseases and Immunity in Global Health Program, MUHC-RI, Montreal, QC, Canada.
7Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada
8School of Physical and Occupational Therapy, Faculty of Medicine and Health Sciences, McGill University, Montreal, QC, Canada
Corresponding Author: Henry Michael, email henry.michael@mail.mcgill.ca
Abstract
Background: Anticholinergic burden refers to the cumulative effect of using multiple medications with anticholinergic properties concomitantly. It is measured using several scales termed Anticholinergic risk measurement scales (ARMS) and has been associated with adverse clinical outcomes. The objectives of this integrative review were specifically to understand to what extent the development of ARMS followed recommended guidelines for methodological rigor, to know to what extent the data interpret ARMS to predict adverse outcomes (cognitive impairment, functional impairment, and frailty), and to know the level of evidence that ARMS as clinical decision tools improve patient outcomes. Methods: A comprehensive search of six databases (MEDLINE, EMBASE, PsychInfo, CINAHL, CENTRAL, and Web of Science) was conducted for relevant studies published from inception until August 2021. Two reviewers screened the primary studies for inclusion and performed the critical appraisal. Results were synthesized using a five-stage process of data reduction, data display, data comparison, conclusion drawing, and verification. Results: 135 studies met the inclusion criteria. 23 scales were identified, with the majority developed via literature review and expert consensus. The Anticholinergic Cognitive Burden (ACB), Anticholinergic Risk Score (ARS), and Drug Burden Index (DBI) scores were more consistent in predicting incident dementia, functional impairment, and frailty. However, there was a lack of evidence of the improvement in clinical outcomes with decreased anticholinergic burden scores. Conclusion: To improve the clinical prognostic utility of ARMS, there is a need to integrate patient factors, improve the interpretability of these scales, and conduct high-quality deprescribing intervention studies.
Targeting mRNA translation to effectively treat uveal melanoma
2Segal Cancer Center, Lady Davis Institute & Jewish General Hospital, Montreal, QC, Canada
3McGill University-Gerald Bronfman Department of Oncology, Montreal, QC, Canada,
4Centre de Recherche du CHU de Québec-Université Laval, QC, Canada
Corresponding Author: Raul Ernesto Flores Gonzalez, email raul.floresgonzalez2@mail.mcgill.ca
Abstract
Uveal Melanoma (UM) is a highly metastatic subtype of melanoma that arises in the eye. UM differs from other types of melanoma with a propensity to metastasize to the liver. Specifically, two mutually exclusive mutations in GNAQ/GNA11 are present in more than 85% of patient tumors, leading to the constitutive activation of the PI3K/mTOR and MAPK signaling pathways. These pathways ultimately converge to activate the eIF4F complex through the regulation of the mRNA cap-binding protein eIF4E. Regulating the levels and availability of eIF4E is a critical step in maintaining cell homeostasis. Phosphorylation of eIF4E (Ser209) is also important as increased levels of phospho-eIF4E have been reported in several cancers. The phosphorylation of eIF4E by its kinases, MNK1/2, leads to an increase in the translation of a subset of mRNAs that encode for proteins with roles in cell survival and invasion. We have screened a panel of UM-derived cell lines to determine the expression status of the two axes that regulate translation: mTOR/4EBP-1 and MNK1/2-eIF4E. Some UM cell lines had increased levels of total/p-eIF4E, MNK1, and hyperphosphorylated 4E-BP1. Our data lead us to hypothesize that uveal melanomas are driven, at least in part, by dysregulated mRNA translation. We evaluated the pharmacological inhibition of MNK1/2 and mTOR with the inhibitors SEL201 and INK128, respectively, in terms of clonogenicity and invasion on our panel of cell lines. As a matter of fact, the pharmacological inhibition of both axes decreased the proliferative and invasive phenotype of some UM cell lines, and the effect was recapitulated under MNK1 deficient cell lines. Moreover, we determined that the integrity of the 4F complex was abrogated under dual MNK and mTOR inhibition by performing a cap-binding assay. We posit that targeting the availability and phosphorylation of eIF4E will lead to increased clinical benefit in metastatic UM.
Mast Cell Homing for Enhanced Fracture Healing
2Regenerative Orthopaedics and Innovation Laboratory, Injury, Repair, & Recovery Program, Research Institute-McGill University Health Centre, Montréal, QC, Canada
3Faculty of Pharmacy, Université de Montréal, QC, Canada
Corresponding Author: Rayan Ben Letaifa, email rayan.benletaifa@mail.mcgill.ca
Abstract
Bone fractures are among the most common musculoskeletal injuries, and approximately 5-10% of patients experience complications such as delayed unions and non-unions. Consequently, such complications may require invasive revision surgeries, which are not always successful. Mast cells, commonly thought of as immunological villains, play a critical role in the inflammatory phase of bone healing and their granules have the greatest range of pro-osteogenic growth factors and cytokines among immunocompetent cells. Inorganic polyphosphates (polyPs), which are key regulatory molecules in coagulation and bone repair, play a key role in modulating immune cell activity during initial stages of bone healing and recruit immune cells to the fracture site. However, the role of PolyPs in the recruitment and modulation of mast cells at the fracture site has yet to be elucidated. Accordingly, we are developing a thermosensitive poloxamer-chitosan composite hydrogel doped with polyPs, which will release polyPs at a controlled rate to attract mast cells at the site of its implantation. In subsequent experiments, the in-vivo effect of polyP-releasing hydrogels on mast cell homing, maturation, and degranulation profiles will be evaluated in a mouse-model of fracture repair developed by our group. Based on preliminary polyP release profile results, we expect the PolyP-doped hydrogel to effectively enhance fracture healing via enhanced mast cell migration, and thus increased release of pro-osteogenic growth factors and cytokines at the fracture site. This research will generate a novel drug delivery system modulating the body’s own immune system to enhance fracture healing, and circumventing fracture complications.
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