Modification of BRCA1-associated breast cancer risk by HMMR overexpression

Breast cancer risk for carriers of BRCA1 pathological variants is modified by genetic factors. Genetic variation in HMMR may contribute to this effect. However, the impact of risk modifiers on cancer biology remains undetermined and the biological basis of increased risk is poorly understood. Here, we depict an interplay of molecular, cellular, and tissue microenvironment alterations that increase BRCA1-associated breast cancer risk. Analysis of genome-wide association results suggests that diverse biological processes, including links to BRCA1-HMMR profiles, influence risk. HMMR overexpression in mouse mammary epithelium increases Brca1-mutant tumorigenesis by modulating the cancer cell phenotype and tumor microenvironment. Elevated HMMR activates AURKA and reduces ARPC2 localization in the mitotic cell cortex, which is correlated with micronucleation and activation of cGAS-STING and non-canonical NF-kappa B signaling. The initial tumorigenic events are genomic instability, epithelial-to-mesenchymal transition, and tissue infiltration of tumor-associated macrophages. The findings reveal a biological foundation for increased risk of BRCA1-associated breast cancer. The effect of hyaluronan-mediated motility receptor (HMMR) expression in BRCA1-associated breast cancer risk remains unknown. Here, HMMR overexpression induces the activation of cGAS-STING and non-canonical NF-kappa B signalling, instigating an immune permissive environment for breast cancer development

Persistence of targetable genomes and proteomes through disease evolution in pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. The five-year event free survival is 85% but the incidence of relapse is high and prognosis after relapse remains poor. To improve outcomes for those patients, precision medicine programs are designed to target specific genomic alterations. Although many biomarkers have been identified, targeted therapies have been less successful than expected. This is primarily a consequence of the aggressiveness of relapse cancers and the limited understanding of functional changes underlying genetic biomarkers. Ideally, the molecular and functional characterization of potential targets could start earlier to prepare for potential relapse. However, there are conflicting reports of how targetable lesions persist through disease progression and little known about progression in ALL proteomes. I hypothesize that the targetable lesions detected in childhood leukemias will be stable through disease progression and the combined genome and proteome analysis will better clarify dysregulated pathways. I first assessed the performance of a recently developed childhood cancer-specific next generation sequencing (NGS) assay in 28 childhood tumour specimens. The childhood cancer-specific assay detected almost 10% more targets than a broad cancer panel and both panels showed high concordance with whole-genome sequencing (WGS). I next investigated genomic stability and persistence of druggable events in paired diagnosis (Dx)-relapse (R) samples from 11 patients treated at BC Children’s Hospital, and whole exome sequencing data from paired Dx-R samples from 69 patients treated at St. Jude’s Hospital. Approximately 64% of patients had at least one druggable target retained between diagnosis and relapse. Six paired specimens were treated in-vitro with variant-matched targeted inhibitors, and although the sensitivity to inhibitors was low, IC50 doses of paired samples were highly correlated (r=0.8486). Similarly, a comprehensive proteome analysis of paired ALL specimens revealed high statistical equivalence (median = 85%) and similar abundance profiles of cancer-associated proteins between diagnosis and relapse. Finally, discovery whole-proteome analysis identified PARP1 as a potential new pan-ALL therapeutic target, and sensitivity to PARP1/2 inhibitors was confirmed via in-vitro drug assays. My thesis indicates that comprehensive interrogation of tumour genomes and proteomes through disease progression may provide support for implementing a prospective precision oncology approach.Medicine, Faculty ofGraduat

Adaptation of the Th-MYCN Mouse Model of Neuroblastoma for Evaluation of Disseminated Disease

High-risk neuroblastoma remains a profound clinical challenge that requires eradication of neuroblastoma cells from a variety of organ sites, including bone marrow, liver, and CNS, to achieve a cure. While preclinical modeling is a powerful tool for the development of novel cancer therapies, the lack of widely available models of metastatic neuroblastoma represents a significant barrier to the development of effective treatment strategies. To address this need, we report a novel luciferase-expressing derivative of the widely used Th-MYCN mouse. While our model recapitulates the non-metastatic neuroblastoma development seen in the parental transgenic strain, transplantation of primary tumor cells from disease-bearing mice enables longitudinal monitoring of neuroblastoma growth at distinct sites in immune-deficient or immune-competent recipients. The transplanted tumors retain GD2 expression through many rounds of serial transplantation and are sensitive to GD2-targeted immune therapy. With more diverse tissue localization than is seen with human cell line-derived xenografts, this novel model for high-risk neuroblastoma could contribute to the optimization of immune-based treatments for this deadly disease.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofPediatrics, Department ofReviewedFacultyResearche

Targetable lesions and proteomes predict therapy sensitivity through disease evolution in pediatric acute lymphoblastic leukemia

Abstract Childhood acute lymphoblastic leukemia (ALL) genomes show that relapses often arise from subclonal outgrowths. However, the impact of clonal evolution on the actionable proteome and response to targeted therapy is not known. Here, we present a comprehensive retrospective analysis of paired ALL diagnosis and relapsed specimen. Targeted next generation sequencing and proteome analysis indicate persistence of actionable genome variants and stable proteomes through disease progression. Paired viably-frozen biopsies show high correlation of drug response to variant-targeted therapies but in vitro selectivity is low. Proteome analysis prioritizes PARP1 as a pan-ALL target candidate needed for survival following cellular stress; diagnostic and relapsed ALL samples demonstrate robust sensitivity to treatment with two PARP1/2 inhibitors. Together, these findings support initiating prospective precision oncology approaches at ALL diagnosis and emphasize the need to incorporate proteome analysis to prospectively determine tumor sensitivities, which are likely to be retained at disease relapse

Pathogenic BRCA1 variants disrupt PLK1-regulation of mitotic spindle orientation

Female carriers of BRCA1 mutations possess high breast cancer risk, which may reflect deficient growth control of mammary progenitor cells. Here, the authors study progenitor-enriched fractions from these carriers and describe a loss of PLK1-mediated mitotic spindle positioning and an inability of the progeny to acquire features of mature luminal cells. Preneoplastic mammary tissues from human female BRCA1 mutation carriers, or Brca1-mutant mice, display unexplained abnormalities in luminal differentiation. We now study the division characteristics of human mammary cells purified from female BRCA1 mutation carriers or non-carrier donors. We show primary BRCA1 mutant/+ cells exhibit defective BRCA1 localization, high radiosensitivity and an accelerated entry into cell division, but fail to orient their cell division axis. We also analyse 15 genetically-edited BRCA1 mutant/+ human mammary cell-lines and find that cells carrying pathogenic BRCA1 mutations acquire an analogous defect in their division axis accompanied by deficient expression of features of mature luminal cells. Importantly, these alterations are independent of accumulated DNA damage, and specifically dependent on elevated PLK1 activity induced by reduced BRCA1 function. This essential PLK1-mediated role of BRCA1 in controlling the cell division axis provides insight into the phenotypes expressed during BRCA1 tumorigenesis

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways

Background: Murine xenografts of pediatric leukemia accurately recapitulate genomic aberrations. How this translates to the functional capacity of cells remains unclear. Here, we studied global protein abundance, phosphorylation, and protein maturation by proteolytic processing in 11 pediatric B- and T- cell ALL patients and 19 corresponding xenografts. Methods: Xenograft models were generated for each pediatric patient leukemia. Mass spectrometry-based methods were used to investigate global protein abundance, protein phosphorylation, and limited proteolysis in paired patient and xenografted pediatric acute B- and T- cell lymphocytic leukemia, as well as in pediatric leukemia cell lines. Targeted next-generation sequencing was utilized to examine genetic abnormalities in patients and in corresponding xenografts. Bioinformatic and statistical analysis were performed to identify functional mechanisms associated with proteins and protein post-translational modifications. Results: Overall, we found xenograft proteomes to be most equivalent with their patient of origin. Protein level differences that stratified disease subtypes at diagnostic and relapse stages were largely recapitulated in xenografts. As expected, PDXs lacked multiple human leukocyte antigens and complement proteins. We found increased expression of cell cycle proteins indicating a high proliferative capacity of xenografted cells. Structural genomic changes and mutations were reflected at the protein level in patients. In contrast, the post-translational modification landscape was shaped by leukemia type and host and only to a limited degree by the patient of origin. Of 201 known pediatric oncogenic drivers and drug-targetable proteins, the KMT2 protein family showed consistently high variability between patient and corresponding xenografts. Comprehensive N terminomics revealed deregulated proteolytic processing in leukemic cells, in particular from caspase-driven cleavages found in patient cells. Conclusion: Genomic and host factors shape protein and post-translational modification landscapes differently. This study highlights select areas of diverging biology while confirming murine patient-derived xenografts as a generally accurate model system.Medicine, Faculty ofNon UBCPathology and Laboratory Medicine, Department ofPediatrics, Department ofReviewedFacult