Pascal St-Onge

Genomic and genealogical investigation of the French Canadian founder population structure.

Characterizing the genetic structure of worldwide populations is important for understanding human history and is essential to the design and analysis of genetic epidemiological studies. In this study, we examined genetic structure and distant relatedness and their effect on the extent of linkage disequilibrium (LD) and homozygosity in the founder population of Quebec (Canada). In the French Canadian founder population, such analysis can be performed using both genomic and genealogical data. We investigated genetic differences, extent of LD, and homozygosity in 140 individuals from seven sub-populations of Quebec characterized by different demographic histories reflecting complex founder events. Genetic findings from genome-wide single nucleotide polymorphism data were correlated with genealogical information on each of these sub-populations. Our genomic data showed significant population structure and relatedness present in the contemporary Quebec population, also reflected in LD and homozygosity levels. Our extended genealogical data corroborated these findings and indicated that this structure is consistent with the settlement patterns involving several founder events. This provides an independent and complementary validation of genomic-based studies of population structure. Combined genomic and genealogical data in the Quebec founder population provide insights into the effects of the interplay of two important sources of bias in genetic epidemiological studies, unrecognized genetic structure and cryptic relatedness.

A childhood acute lymphoblastic leukemia-specific lncRNA implicated in prednisolone resistance, cell proliferation, and migration

Childhood acute lymphoblastic leukemia (cALL) is the most common pediatric cancer and, despite an 85% cure rate, still represents a major cause of disease-related death in children. Recent studies have implicated long non-coding RNAs (lncRNAs) in cALL etiology, progression, and treatment response. However, barring some exceptions little is known about the functional impact of lncRNAs on cancer biology, which limits their potential as potential therapeutic targets. We wanted to investigate the functional role of lncRNAs identified as specifically overexpressed in pre-B cALL by whole-transcriptome sequencing. Here we report five lncRNAs specifically upregulated in pre-B cALL that had significant impacts on cancer hallmark traits such as cell proliferation, migration, apoptosis, and treatment response. In particular, silencing of the RP11-137H2.4 lncRNA effectively restored normal glucocorticoid (GC) response in a GC-resistant pre-B cALL cell line and specifically modulated expression of members of both the NRAS/BRAF/NF-?B MAPK cascade and cell cycle pathways. Since GC form the cornerstone of cALL chemotherapy and resistance in cALL confers a dismal prognosis, characterizing RP11-137H2.4sexact role and function in this process will be critical to the development of new therapeutic approaches to overcome GC resistance in children treated for cALL.

Genomic characterization of pediatric T-cell acute lymphoblastic leukemia reveals novel recurrent driver mutations

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with variable prognosis. It represents 15% of diagnosed pediatric ALL cases and has a threefold higher incidence among males. Many recurrent alterations have been identified and help define molecular subgroups of T-ALL, however the full range of events involved in driving transformation remain to be defined. Using an integrative approach combining genomic and transcriptomic data, we molecularly characterized 30 pediatric T-ALLs and identified common recurrent T-ALL targets such as FBXW7, JAK1, JAK3, PHF6, KDM6A and NOTCH1 as well as novel candidate T-ALL driver mutations including the p.R35L missense mutation in splicesome factor U2AF1 found in 3 patients and loss of function mutations in the X-linked tumor suppressor genes MED12 (frameshit mutation p.V167fs, splice site mutation g.chrX:70339329T>C, missense mutation p.R1989H) and USP9X (nonsense mutation p.Q117*). In vitro functional studies further supported the putative role of these novel T-ALL genes in driving transformation. U2AF1 p.R35L was shown to induce aberrant splicing of downstream target genes, and shRNA knockdown of MED12 and USP9X was shown to confer resistance to apoptosis following T-ALL relevant chemotherapy drug treatment in Jurkat leukemia cells. Interestingly, nearly 60% of novel candidate driver events were identified among immature T-ALL cases, highlighting the underlying genomic complexity of pediatric T-ALL, and the need for larger integrative studies to decipher the mechanisms that contribute to its various subtypes and provide opportunities to refine patient stratification and treatment.

Characterization of the microDNA through the response to chemotherapeutics in lymphoblastoid cell lines

Recently, a new class of extrachromosomal circular DNA, called microDNA, was identified. They are on average 100 to 400 bp long and are derived from unique non-repetitive genomic regions with high gene density. MicroDNAs are thought to arise from DNA breaks associated with RNA metabolism or replication slippage. Given the paucity of information on this entirely novel phenomenon, we aimed to get an additional insight into microDNA features by performing the microDNA analysis in 20 independent human lymphoblastoid cell lines (LCLs) prior and after treatment with chemotherapeutic drugs. The results showed non-random genesis of microDNA clusters from the active regions of the genome. The size periodicity of 190 bp was observed, which matches DNA fragmentation typical for apoptotic cells. The chemotherapeutic drug-induced apoptosis of LCLs increased both number and size of clusters further suggesting that part of microDNAs could result from the programmed cell death. Interestingly, proportion of identified microDNA sequences has common loci of origin when compared between cell line experiments. While compatible with the original observation that microDNAs originate from a normal physiological process, obtained results imply complementary source of its production. Furthermore, non-random genesis of microDNAs depicted by redundancy between samples makes these entities possible candidates for new biomarker generation.

Specific expression of novel long non-coding RNAs in high-hyperdiploid childhood acute lymphoblastic leukemia

Pre-B cell childhood acute lymphoblastic leukemia (pre-B cALL) is a heterogeneous disease involving many subtypes typically stratified using a combination of cytogenetic and molecular-based assays. These methods, although widely used, rely on the presence of known chromosomal translocations, which is a limiting factor. There is therefore a need for robust, sensitive, and specific molecular biomarkers unaffected by such limitations that would allow better risk stratification and consequently better clinical outcome. In this study we performed a transcriptome analysis of 56 pre-B cALL patients to identify expression signatures in different subtypes. In both protein-coding and long non-coding RNAs (lncRNA), we identified subtype-specific gene signatures distinguishing pre-B cALL subtypes, particularly in t(12;21) and hyperdiploid cases. The genes up-regulated in pre-B cALL subtypes were enriched in bivalent chromatin marks in their promoters. LncRNAs is a new and under-studied class of transcripts. The subtype-specific nature of lncRNAs suggests they may be suitable clinical biomarkers to guide risk stratification and targeted therapies in pre-B cALL patients.

LncRNAs downregulated in childhood acute lymphoblastic leukemia modulate apoptosis, cell migration, and DNA damage response

Childhood acute lymphoblastic leukemia (cALL) accounts for 25% of pediatric cancers and is one of the leading causes of disease-related death in children. Although long non-coding RNAs (lncRNAs) have been implicated in cALL etiology, progression, and treatment response, little is known about their exact functional role. We had previously sequenced the whole transcriptome of 56 cALL patients and identified lncRNA transcripts specifically silenced in tumoral cells. Here we investigated the impact of restoring the expression of three of these (RP11-624C23.1, RP11-203E8, and RP11-446E9) in leukemic cell lines had dramatic impact on cancer hallmark cellular phenotypes such as apoptosis, cell proliferation and migration, and DNA damage response. Interestingly, both RP11-624C23.1 and RP11-203E8 had very similar impacts on DNA damage response, specifically displaying lower γ-H2A.X and higher apoptosis levels than control cells in response to genotoxic stress. These results indicate that silencing RP11-624C23.1 or RP11-203E8 could provide a selective advantage to leukemic cells by increasing resistance to genotoxic stress, possibly by modulating the DDR pathway. Since genotoxic agents are fundamental parts of antineoplastic treatment, further investigation of the mechanisms these lncRNAs impact would provide novel and interesting avenues for overcoming treatment resistance.

Genomic determinants of long-term cardiometabolic complications in childhood acute lymphoblastic leukemia survivors

BackgroundWhile cure rates for childhood acute lymphoblastic leukemia (cALL) now exceed 80%, over 60% of survivors will face treatment-related long-term sequelae, including cardiometabolic complications such as obesity, insulin resistance, dyslipidemia and hypertension. Although genetic susceptibility contributes to the development of these problems, there are very few studies that have so far addressed this issue in a cALL survivorship context.MethodsIn this study, we aimed at evaluating the associations between common and rare genetic variants and long-term cardiometabolic complications in survivors of cALL. We examined the cardiometabolic profile and performed whole-exome sequencing in 209 cALL survivors from the PETALE cohort. Variants associated with cardiometabolic outcomes were identified using PLINK (common) or SKAT (common and rare) and a logistic regression was used to evaluate their impact in multivariate models.ResultsOur results showed that rare and common variants in the BAD and FCRL3 genes were associated (p<0.05) with an extreme cardiometabolic phenotype (3 or more cardiometabolic risk factors). Common variants in OGFOD3 and APOB as well as rare and common BAD variants were significantly (p<0.05) associated with dyslipidemia. Common BAD and SERPINA6 variants were associated (p<0.05) with obesity and insulin resistance, respectively.ConclusionsIn summary, we identified genetic susceptibility loci as contributing factors to the development of late treatment-related cardiometabolic complications in cALL survivors. These biomarkers could be used as early detection strategies to identify susceptible individuals and implement appropriate measures and follow-up to prevent the development of risk factors in this high-risk population.

Targeting MYC Overexpressing Leukemia with Cardiac Glycoside Proscillaridin Through Downregulation of Histone Acetyltransferases

Targeting MYC oncogene remains a major therapeutic goal in cancer chemotherapy. Here, we demonstrate that proscillaridin, a cardiac glycoside approved for heart failure treatment, causing Na+/K+ pump inhibition, targets efficiently MYC overexpressing cancer cells. At clinically relevant doses, proscillaridin induced rapid downregulation of MYC protein level, and produced growth inhibition preferentially against MYC overexpressing leukemic cell lines including lymphoid and myeloid stem cell populations. Transcriptomic profile of leukemic cells after treatment showed a downregulation of gene sets involved in MYC pathways, cell replication and an upregulation of genes involved in hematopoietic differentiation. Gene expression changes were associated with an epigenetic remodeling of chromatin active marks. Proscillaridin induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27). In addition, loss of lysine acetylation was observed also in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferase proteins (such as CBP and P300) involved in histone and MYC acetylation. Overall, these results strongly support the repurposing of proscillaridin in MYC overexpressing leukemia and suggest a novel strategy to target MYC by inducing the downregulation of histone acetyltransferases involved in its stability.

TRPV4 and KRAS and FGFR1 gain-of-function mutations drive giant cell lesions of the jaw

Giant cell lesions of the jaw (GCLJ) are debilitating tumors of unknown origin with limited available therapies. Here, we analyze 58 sporadic samples using next generation or targeted sequencing and report somatic, heterozygous, gain-of-function mutations in KRAS, FGFR1, and p.M713V/I-TRPV4 in 72% (42/58) of GCLJ. TRPV4 p.M713V/I mutations are exclusive to central GCLJ and occur at a critical position adjacent to the cation permeable pore of the channel. Expression of TRPV4 mutants in HEK293 cells leads to increased cell death, as well as increased constitutive and stimulated channel activity, both of which can be prevented using TRPV4 antagonists. Furthermore, these mutations induce sustained activation of ERK1/2, indicating that their effects converge with that of KRAS and FGFR1 mutations on the activation of the MAPK pathway in GCLJ. Our data extend the spectrum of TRPV4 channelopathies and provide rationale for the use of TRPV4 and RAS/MAPK antagonists at the bedside in GCLJ.Giant cell lesions of the jaw (GCLJ) are debilitating benign tumors of unclear origin. The authors identify driver recurrent somatic mutations in TRPV4, KRAS and FGFR1 and show they converge on aberrant activation of the MAPK pathway. Their findings extend the spectrum of TRPV4 channelopathies and provide rationale for targeted therapies at the bedside in GCLJ.

Abstract 4885: Identification of actionable targets for refractory/relapsed childhood cancer leading to personalized targeted therapy (TRICEPS Study)

Childhood cancer is a group of heterogeneous complex diseases. Although 80% of these children are cured with conventional therapies, it remains the first cause of death among children in Western countries. A significant number of refractory/relapse patients will eventually succumb to their disease and the lack of therapeutic advances for these patients is even more worrisome. Indeed, no significant progress has been noted over the last decade for these patients, urging the need for new and more effective therapeutic approaches. Precision medicine and more effective personalized targeted therapies (PTT) are a major breakthrough leading to increased cure rates and decreased treatment-related morbidity and mortality for the patients with refractory or relapsed tumors. To address this challenge, the TRICEPS study was initiated on April 2014 at the Sainte-Justine UHC (Montreal, Canada) with an overreaching goal to explore the feasibility of performing genomic-driven targeted therapy in pediatric and adolescent (aged 0-21 years) patients with relapsed or refractory childhood cancer. This study offers in-depth genomic and transcriptomic investigation of patient’s tumoral material to identify patient-specific alterations and actionable driver mutation(s) that can be targeted with approved targeted drug and within a reasonable clinically relevant timeframe to assess the feasibility of going from biopsy to a detailed tumor analysis report. Over a period of 30 months, 44 relapsed/refractory cancer patients were recruited. Twenty-two of them underwent extensive genomic investigation (exomic and transcriptomic sequencing) within a median timeframe of 9.7 weeks from patient enrolment to return of results. Patient screen failures occurred due to benign/necrotic tumor biopsies or low tumor purity resulting in suboptimal DNA/RNA quantity or quality for genomic analysis. In all 22 patients, we have identified clinically relevant genomic alterations (SNVs, indels, fusions, CNAs) and relapse-specific mutations influencing patient management and providing options for personalized interventions. We assessed the functional impact of some of these cancer-specific alterations. This was the case of a novel relapse-specific rearrangement, identified on relapsed childhood ETP-ALL, and leading to asparagine synthetase (ASNS) up-regulation through a promoter exchange. The expression of this fusion was associated with reduced apoptosis following l-asparaginase treatment. This study shows that PPT based on next generation sequencing technology is a powerful approach that could be implemented in the clinic within a foreseeable future to guide treatment of hard-to-treat childhood cancers and to further improve patient care and outcomes. Citation Format: Fida Khater, Stephanie Vairy, Sylvie Langlois, Jasmine Healy, Sophie Dumoucel, Mathieu Lajoie, Thomas Sontag, Pascal St-Onge, Henrique Bittencourt, Dorothee Dal Soglio, Anne-Sophie Carret, Sonia Cellot, Josette Champagne, Michel Duval, Maja Krajinovic, Jean-Marie Leclerc, Valerie Larouche, Natalie Patey, Sebastien Perreault, Nelson Piche, Yvan Samson, Pierre Teira, Monia Marzouki, Daniel Sinnett. Identification of actionable targets for refractory/relapsed childhood cancer leading to personalized targeted therapy (TRICEPS Study) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4885. doi:10.1158/1538-7445.AM2017-4885