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Papers by Chitra Venugopal

Neuro-Oncology, 2017

NEURO-ONCOLOGY • NOVEMBER 2017 and for advancing our understanding of molecular dependencies in G... more NEURO-ONCOLOGY • NOVEMBER 2017 and for advancing our understanding of molecular dependencies in GSCs. We therefore performed a CRISPR-Cas9 screen in patient-derived GSC lines to elucidate drivers of GSC viability. To identify candidate genes for the screen, we performed an in silico analysis, interrogating The Cancer Genome Atlas (TCGA). We first stratified by CpG island methylator phenotype (CIMP) status since glioma-CIMP (G-CIMP) tumors appear to have a distinct underlying biology and disease course. We selected genes that were upregulated in either non G-CIMP or G-CIMP tumors vs. normal brain tissue and further filtered this gene set by intersecting it with the list of genes for which higher expression was associated with worse survival in each group. We designed a CRISPR-Cas9 library targeting the selected 536 genes and performed a drop-out screen in five distinct patient-derived GSC lines for genes that are critical for survival and maintenance of GSCs. We then validated top hits by individual CRISPR knockout. Using CRISPR screening technology in patient-derived cell lines, we identified crucial dependencies in patient-derived GSCs. Further experiments will characterize the mechanism of these dependencies and assess their potential therapeutic relevance in pre-clinical models.

Neuro-Oncology Advances, 2019

Brain metastases (BM) are the most common brain tumor in adults, with an incidence ten times grea... more Brain metastases (BM) are the most common brain tumor in adults, with an incidence ten times greater than that of primary brain tumors. The most common sources of BM in adult cancer patients include cancers of the lung, breast and melanoma, which together account for almost 80% of all BM. Current clinical modalities for BM include surgery, whole brain radiation therapy and stereotactic radiosurgery but these therapies still offer limited efficacy and reduced survival of only months in treated patients, emphasizing the need for novel BM research approaches and better therapeutic strategies. Our laboratory recently discovered that stem-like cells exist in patient-derived BM from lung, breast and melanoma cancers, which we termed “brain metastasis-initiating cells” or BMICs. Through clinically relevant human-mouse xenograft models established with these patient-derived BMICs, we captured lung, breast and melanoma BMICs at pre-metastasis – a key stage where circulating metastatic cells ...

Neuro-Oncology, Nov 1, 2017

Human glioblastoma (hGBM) carries a dismal prognosis and inevitably relapses despite aggressive t... more Human glioblastoma (hGBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many of the 14 members of the Eph receptor tyrosine kinase family are expressed in hGBM initiating cells (GICs) and constitute potential molecular targets. We hypothesize that multiple members of the EphR family play a critical role in hGBM recurrence. Using a highly specific human EphR antibody panel, we identified differential expression of EphRs in recurrent hGBM (rGBM). We further characterized EphR co-expression along with multiple GIC markers using mass cytometry (CyTOF). Here we show that EphA2 and EphA3 co-expression marks a highly tumorigenic cell population in rGBM that is enriched in GIC marker expression, and exhibits higher in vitro and in vivo self-renewal and proliferation capacity as compared to EphA2+/EphA3-, EphA2-/EphA3+ or EphA2-/EphA3- cells. Knockdown of EphA2 and EphA3 blocks this self-renewal and proliferation capacity, and is marked by increase in the expression of differentiation marker GFAP. Next, we generated and tested a bispecific antibody (BsAb) that co-targets EphA2 and EphA3. In vitro treatment of rGBM with BsAb led to phosphorylation of EphA2 and EphA3, eventually leading to receptor internalization and degradation. The cellular effect of EphA2/A3 blockade was mediated through the down regulation of Akt and MAPK. Intracranial treatment of immune-deficient mice harboring hGBM with BsAb resulted in non-invasive and significantly smaller tumors. Hence, EphA2 and EphA3 co-expression marks an even more potent GIC population in rGBM, and targeting either single EphA2+ or EphA3+ populations alone will allow the remaining GICs to drive tumor recurrence. For the first time, we show that strategic co-targeting of both EphA2 and EphA3 with a BsAb presents a novel and rational therapeutic approach to recurrent GBM, where multiple GIC populations may be driving the intra-tumoral heterogeneity underlying disease progression.

Cancers

Despite aggressive multimodal therapy, glioblastoma (GBM) remains the most common malignant prima... more Despite aggressive multimodal therapy, glioblastoma (GBM) remains the most common malignant primary brain tumor in adults. With the advent of therapies that revitalize the anti-tumor immune response, several immunotherapeutic modalities have been developed for treatment of GBM. In this review, we summarize recent clinical and preclinical efforts to evaluate vaccination strategies, immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T cells. Although these modalities have shown long-term tumor regression in subsets of treated patients, the underlying biology that may predict efficacy and inform therapy development is being actively investigated. Common to all therapeutic modalities are fundamental mechanisms of therapy evasion by tumor cells, including immense intratumoral heterogeneity, suppression of the tumor immune microenvironment and low mutational burden. These insights have led efforts to design rational combinatorial therapies that can reignite the anti-t...

Molecular & Cellular Oncology

Neuro-Oncology

BACKGROUND The incidence of brain metastases (BM) is tenfold higher than primary brain tumors. BM... more BACKGROUND The incidence of brain metastases (BM) is tenfold higher than primary brain tumors. BM commonly originate from primary lung, breast, and melanoma tumors with a 90% mortality rate within one year of diagnosis. Current standard of care for BM includes surgical resection with concurrent chemoradiation, but does not extend median survival past 16 months, posing a large unmet need to identify novel therapies against BM. METHODS From a large in-house biobank of patient-derived BM cell lines, the Singh Lab has generated murine orthotopic patient-derived xenograft (PDX) models of lung, breast, and melanoma BM that recapitulate the stages of BM progression as seen in humans. Using these three PDX models, we identified a population of “pre-metastatic” brain metastasis-initiating cells (BMICs) that are newly arrived in the brain but have yet to form detectable tumors. Pre-metastatic BMICs are not detectable in human patients but are important therapeutic targets with the potential t...

Nature Communications

Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt... more Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt MB having the most favorable prognosis. Since prior reports have illustrated the antitumorigenic role of Wnt activation in Shh MB, we aimed to assess the effects of activated canonical Wnt signaling in Group 3 and 4 MBs. By using primary patient-derived MB brain tumor-initiating cell (BTIC) lines, we characterize differences in the tumor-initiating capacity of Wnt, Group 3, and Group 4 MB. With single cell RNA-seq technology, we demonstrate the presence of rare Wnt-active cells in non-Wnt MBs, which functionally retain the impaired tumorigenic potential of Wnt MB. In treating MB xenografts with a Wnt agonist, we provide a rational therapeutic option in which the protective effects of Wnt-driven MBs may be augmented in Group 3 and 4 MB and thereby support emerging data for a context-dependent tumor suppressive role for Wnt/β-catenin signaling.

European Oncology & Haematology

The Polycomb group (PcG) genes encode for proteins comprising two multiprotein complexes, Polycom... more The Polycomb group (PcG) genes encode for proteins comprising two multiprotein complexes, Polycomb repressive complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2). Although the initial discovery of PcG genes was made in Drosophila, as transcriptional repressors of homeotic (HOX) genes. Polycomb repressive complexes have been since implicated in regulating a wide range of cellular processes, including differentiation and self-renewal in normal and cancer stem cells. Bmi1, a subunit of PRC1, has been long implicated in driving self-renewal, the key property of stem cells. Subsequent studies showing upregulation of Bmi1 in several cancers correlated with increased aggressiveness, radioresistance and metastatic potential, provided rationale for development of targeted therapies against Bmi1. Although Bmi1 activity can be reduced through transcriptional, post-transcriptional and post-translational regulation, to date, the most promising approach has been through small molecule inhib...

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current clinical trials ... more Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naïve tumors, provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumors. By adapting the existing Children’s Oncology Group treatment protocol for children with newly diagnosed high-risk MB for treatment of mice intracranially engrafted with human MB cells, we have characterized the rare treatment-refractory cell population in Group 3 MBs. MB cell populations recovered separately from brains and spines during the course of tumor development and therapy were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy to relapse. One of the most intriguing observations from our gene expression data was consistent over-expression of proteins belonging to Inh...

Future Neurology

The discovery of the brain tumor initiating cells resulted in a paradigm shift within the cancer ... more The discovery of the brain tumor initiating cells resulted in a paradigm shift within the cancer research community to consider brain tumors as an outcome of developmental mechanisms gone awry. This review will guide the reader through the technological advances that hold the powerful potential to allow brain cancer researchers to develop an intimate understanding of the dynamic and complex mechanism governing brain tumor behavior.

Neuro-Oncology

As the most common primary brain tumor in adults causing death, Glioblastoma (GBM) remains a ther... more As the most common primary brain tumor in adults causing death, Glioblastoma (GBM) remains a therapeutic challenge. Unchanged for almost two decades, standard therapy is ineffective at preventing disease relapse with a median patient survival of < 15 months. Stem cell-like subpopulations of tumor cells, known as brain tumor initiating cells (BTICs), evade standard therapy and lead to relapse. Whereas previous studies largely focus on pre-treatment primary GBM (pGBM), we conducted a panel of genome-wide CRISPR-Cas9 gene knockout screens to determine modulators of treatment resistance and de novo genetic vulnerabilities arising at disease recurrence. Using our in vitro model of conventional therapy, we identified genes modulating sensitivity and resistance to Temozolomide and/or radiation therapy in patient-derived pGBM BTICs. Genes modulating sensitivity belong to Fanconi anaemia nuclear complex, interstrand cross link repair, and regulation of stem cell maintenance and differenti...

Journal of Neuro-Oncology

Neuro-Oncology, 2017

NEURO-ONCOLOGY • NOVEMBER 2017 and for advancing our understanding of molecular dependencies in G... more NEURO-ONCOLOGY • NOVEMBER 2017 and for advancing our understanding of molecular dependencies in GSCs. We therefore performed a CRISPR-Cas9 screen in patient-derived GSC lines to elucidate drivers of GSC viability. To identify candidate genes for the screen, we performed an in silico analysis, interrogating The Cancer Genome Atlas (TCGA). We first stratified by CpG island methylator phenotype (CIMP) status since glioma-CIMP (G-CIMP) tumors appear to have a distinct underlying biology and disease course. We selected genes that were upregulated in either non G-CIMP or G-CIMP tumors vs. normal brain tissue and further filtered this gene set by intersecting it with the list of genes for which higher expression was associated with worse survival in each group. We designed a CRISPR-Cas9 library targeting the selected 536 genes and performed a drop-out screen in five distinct patient-derived GSC lines for genes that are critical for survival and maintenance of GSCs. We then validated top hits by individual CRISPR knockout. Using CRISPR screening technology in patient-derived cell lines, we identified crucial dependencies in patient-derived GSCs. Further experiments will characterize the mechanism of these dependencies and assess their potential therapeutic relevance in pre-clinical models.

Neuro-Oncology Advances, 2019

Brain metastases (BM) are the most common brain tumor in adults, with an incidence ten times grea... more Brain metastases (BM) are the most common brain tumor in adults, with an incidence ten times greater than that of primary brain tumors. The most common sources of BM in adult cancer patients include cancers of the lung, breast and melanoma, which together account for almost 80% of all BM. Current clinical modalities for BM include surgery, whole brain radiation therapy and stereotactic radiosurgery but these therapies still offer limited efficacy and reduced survival of only months in treated patients, emphasizing the need for novel BM research approaches and better therapeutic strategies. Our laboratory recently discovered that stem-like cells exist in patient-derived BM from lung, breast and melanoma cancers, which we termed “brain metastasis-initiating cells” or BMICs. Through clinically relevant human-mouse xenograft models established with these patient-derived BMICs, we captured lung, breast and melanoma BMICs at pre-metastasis – a key stage where circulating metastatic cells ...

Neuro-Oncology, Nov 1, 2017

Human glioblastoma (hGBM) carries a dismal prognosis and inevitably relapses despite aggressive t... more Human glioblastoma (hGBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many of the 14 members of the Eph receptor tyrosine kinase family are expressed in hGBM initiating cells (GICs) and constitute potential molecular targets. We hypothesize that multiple members of the EphR family play a critical role in hGBM recurrence. Using a highly specific human EphR antibody panel, we identified differential expression of EphRs in recurrent hGBM (rGBM). We further characterized EphR co-expression along with multiple GIC markers using mass cytometry (CyTOF). Here we show that EphA2 and EphA3 co-expression marks a highly tumorigenic cell population in rGBM that is enriched in GIC marker expression, and exhibits higher in vitro and in vivo self-renewal and proliferation capacity as compared to EphA2+/EphA3-, EphA2-/EphA3+ or EphA2-/EphA3- cells. Knockdown of EphA2 and EphA3 blocks this self-renewal and proliferation capacity, and is marked by increase in the expression of differentiation marker GFAP. Next, we generated and tested a bispecific antibody (BsAb) that co-targets EphA2 and EphA3. In vitro treatment of rGBM with BsAb led to phosphorylation of EphA2 and EphA3, eventually leading to receptor internalization and degradation. The cellular effect of EphA2/A3 blockade was mediated through the down regulation of Akt and MAPK. Intracranial treatment of immune-deficient mice harboring hGBM with BsAb resulted in non-invasive and significantly smaller tumors. Hence, EphA2 and EphA3 co-expression marks an even more potent GIC population in rGBM, and targeting either single EphA2+ or EphA3+ populations alone will allow the remaining GICs to drive tumor recurrence. For the first time, we show that strategic co-targeting of both EphA2 and EphA3 with a BsAb presents a novel and rational therapeutic approach to recurrent GBM, where multiple GIC populations may be driving the intra-tumoral heterogeneity underlying disease progression.

Cancers

Despite aggressive multimodal therapy, glioblastoma (GBM) remains the most common malignant prima... more Despite aggressive multimodal therapy, glioblastoma (GBM) remains the most common malignant primary brain tumor in adults. With the advent of therapies that revitalize the anti-tumor immune response, several immunotherapeutic modalities have been developed for treatment of GBM. In this review, we summarize recent clinical and preclinical efforts to evaluate vaccination strategies, immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T cells. Although these modalities have shown long-term tumor regression in subsets of treated patients, the underlying biology that may predict efficacy and inform therapy development is being actively investigated. Common to all therapeutic modalities are fundamental mechanisms of therapy evasion by tumor cells, including immense intratumoral heterogeneity, suppression of the tumor immune microenvironment and low mutational burden. These insights have led efforts to design rational combinatorial therapies that can reignite the anti-t...

Molecular & Cellular Oncology

Neuro-Oncology

BACKGROUND The incidence of brain metastases (BM) is tenfold higher than primary brain tumors. BM... more BACKGROUND The incidence of brain metastases (BM) is tenfold higher than primary brain tumors. BM commonly originate from primary lung, breast, and melanoma tumors with a 90% mortality rate within one year of diagnosis. Current standard of care for BM includes surgical resection with concurrent chemoradiation, but does not extend median survival past 16 months, posing a large unmet need to identify novel therapies against BM. METHODS From a large in-house biobank of patient-derived BM cell lines, the Singh Lab has generated murine orthotopic patient-derived xenograft (PDX) models of lung, breast, and melanoma BM that recapitulate the stages of BM progression as seen in humans. Using these three PDX models, we identified a population of “pre-metastatic” brain metastasis-initiating cells (BMICs) that are newly arrived in the brain but have yet to form detectable tumors. Pre-metastatic BMICs are not detectable in human patients but are important therapeutic targets with the potential t...

Nature Communications

Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt... more Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt MB having the most favorable prognosis. Since prior reports have illustrated the antitumorigenic role of Wnt activation in Shh MB, we aimed to assess the effects of activated canonical Wnt signaling in Group 3 and 4 MBs. By using primary patient-derived MB brain tumor-initiating cell (BTIC) lines, we characterize differences in the tumor-initiating capacity of Wnt, Group 3, and Group 4 MB. With single cell RNA-seq technology, we demonstrate the presence of rare Wnt-active cells in non-Wnt MBs, which functionally retain the impaired tumorigenic potential of Wnt MB. In treating MB xenografts with a Wnt agonist, we provide a rational therapeutic option in which the protective effects of Wnt-driven MBs may be augmented in Group 3 and 4 MB and thereby support emerging data for a context-dependent tumor suppressive role for Wnt/β-catenin signaling.

European Oncology & Haematology

The Polycomb group (PcG) genes encode for proteins comprising two multiprotein complexes, Polycom... more The Polycomb group (PcG) genes encode for proteins comprising two multiprotein complexes, Polycomb repressive complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2). Although the initial discovery of PcG genes was made in Drosophila, as transcriptional repressors of homeotic (HOX) genes. Polycomb repressive complexes have been since implicated in regulating a wide range of cellular processes, including differentiation and self-renewal in normal and cancer stem cells. Bmi1, a subunit of PRC1, has been long implicated in driving self-renewal, the key property of stem cells. Subsequent studies showing upregulation of Bmi1 in several cancers correlated with increased aggressiveness, radioresistance and metastatic potential, provided rationale for development of targeted therapies against Bmi1. Although Bmi1 activity can be reduced through transcriptional, post-transcriptional and post-translational regulation, to date, the most promising approach has been through small molecule inhib...

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current clinical trials ... more Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naïve tumors, provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumors. By adapting the existing Children’s Oncology Group treatment protocol for children with newly diagnosed high-risk MB for treatment of mice intracranially engrafted with human MB cells, we have characterized the rare treatment-refractory cell population in Group 3 MBs. MB cell populations recovered separately from brains and spines during the course of tumor development and therapy were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy to relapse. One of the most intriguing observations from our gene expression data was consistent over-expression of proteins belonging to Inh...

Future Neurology

The discovery of the brain tumor initiating cells resulted in a paradigm shift within the cancer ... more The discovery of the brain tumor initiating cells resulted in a paradigm shift within the cancer research community to consider brain tumors as an outcome of developmental mechanisms gone awry. This review will guide the reader through the technological advances that hold the powerful potential to allow brain cancer researchers to develop an intimate understanding of the dynamic and complex mechanism governing brain tumor behavior.

Neuro-Oncology

As the most common primary brain tumor in adults causing death, Glioblastoma (GBM) remains a ther... more As the most common primary brain tumor in adults causing death, Glioblastoma (GBM) remains a therapeutic challenge. Unchanged for almost two decades, standard therapy is ineffective at preventing disease relapse with a median patient survival of < 15 months. Stem cell-like subpopulations of tumor cells, known as brain tumor initiating cells (BTICs), evade standard therapy and lead to relapse. Whereas previous studies largely focus on pre-treatment primary GBM (pGBM), we conducted a panel of genome-wide CRISPR-Cas9 gene knockout screens to determine modulators of treatment resistance and de novo genetic vulnerabilities arising at disease recurrence. Using our in vitro model of conventional therapy, we identified genes modulating sensitivity and resistance to Temozolomide and/or radiation therapy in patient-derived pGBM BTICs. Genes modulating sensitivity belong to Fanconi anaemia nuclear complex, interstrand cross link repair, and regulation of stem cell maintenance and differenti...

Journal of Neuro-Oncology