Functional loss of a non-canonical BCOR-PRC1.1 complex accelerates SHH-driven medulloblastoma formation

Medulloblastoma is a childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH)-subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs) and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional co-repressor BCOR are recurrent and highly enriched in SHH-medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a germline genetically engineered mouse model to delete exons 9/10 of Bcor (BcorΔE9-10) in GNPs during development. This leads to reduced expression of C-terminally truncated BCOR (BCORΔE9-10). While BcorΔE9-10 alone did not promote tumorigenesis or affect GNP differentiation, BcorΔE9-10 combined with loss of the SHH-receptor gene Ptch1 resulted in highly penetrant medulloblastomas. In Ptch1+/-;BcorΔE9-10 tumors, the growth factor gene Igf2 was aberrantly upregulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/-;BcorΔE9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.

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DIPG-68. ALPHA-THALASSEMIA X-LINKED MENTAL RETARDATION PROTEIN (ATRX) LOSS-OF-FUNCTION IN A MOUSE MODEL OF DIFFUSE INTRINSIC PONTINE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.111 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii300-iii300

Author(s):

Chen Shen ◽

David Picketts ◽

Oren Becher

Keyword(s):

Mouse Model ◽

Pediatric Brain Tumor ◽

High Grade Glioma ◽

Genetic Alterations ◽

Genetically Engineered ◽

Tumor Incidence ◽

Loss Of Function ◽

Nestin Expression ◽

Genetically Engineered Mouse Model ◽

Clinical Cases

Abstract Diffuse Intrinsic Potine Glioma (DIPG) is a rare pediatric brain tumor for which no cure or efficacious therapies exist. Previous discoveries have revealed that, DIPG harbors distinct genetic alterations, when compared with adult high-grade glioma (HGG) or even with non-DIPG pediatric HGGs. ATRX alteration is found in 9% of clinical cases of DIPG, and significantly overlaps with H3.3K27M mutation and p53 loss, the two most common genetic changes in DIPG, found in 80% and 77% clinical cases, respectively. Here we developed genetically engineered mouse model of brainstem glioma using the RCAS-Tv-a system by targeting PDGF-B overexpression, p53 loss, H3.3K27M mutation and ATRX loss-of function to Nestin-expression brainstem progenitor cells of the neonatal mouse. Specifically, we used Nestin-Tv-a; p53 floxed; ATRX heterozygous female and Nestin-Tv-a; p53 floxed; ATRX floxed male breeders, generated offsprings with ATRX loss of function (n=18), ATRX heterozygous females (n=6), and ATRX WT (n=10). Median survial of the three groups are 65 days, 88 days and 51 days, respectively. Also, ATRX null mice is lower in tumor incidence (44.4%), compared with ATRX WT (80%). We evaluated the pathological features of DIPG with or without ATRX alteration, RNA-seq is performed to identify differentially expressed genes between ATRX WT and loss-of-function. In conclution, this study generated the first genetically modified mouse model studying ATRX loss-of-function in DIPG, and suggested that ATRX loss-of-function in DIPG may slow down tumorigenesis and decrease tumor incidence.

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EPEN-20. EZHIP/CATACOMB COOPERATES WITH PDGF-A AND p53 LOSS TO GENERATE A GENETICALLY ENGINEERED MOUSE MODEL FOR POSTERIOR FOSSA A EPENDYMOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.157 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii311-iii311

Author(s):

Emily Kagan ◽

Daniel Brat ◽

Ali Shilatifard ◽

Andrea Piunti ◽

Oren Becher

Keyword(s):

Median Survival ◽

Critical Role ◽

Pediatric Brain Tumor ◽

Genetically Engineered ◽

Rank Test ◽

Loss Of Function ◽

Wild Type ◽

Term Survival ◽

Log Rank Test ◽

Genetically Engineered Mouse Model

Abstract BACKGROUND PFA ependymoma is a pediatric brain tumor with only 30% long-term survival. Recently a gene called CXORF67/EZHIP/CATACOMB (henceforward: CATACOMB) was found to be overexpressed in PFA ependymoma. CATACOMB’s mechanism of action has been found to be analogous to that of the H3K27M mutation as its expression reduces H3K27me3 via inhibition of PRC2 catalytic activity. METHODS We infected NESTIN- or GFAP-expressing neonatal hindbrain progenitors with wild-type CATACOMB or a loss of function (LOF) point mutant (M406K), alone, with PDGFA, and with and without p53 deletion. RESULTS CATACOMB overexpression alone or with p53 loss was insufficient to induce tumorigenesis. CATACOMB overexpression with PDGFA and p53 loss was sufficient to induce tumorigenesis using either the LOF mutant (M406K) or the wild-type CATACOMB in both cells-of-origin. The histology appeared more ependymoma-like when CATACOMB was expressed in GFAP-expressing progenitors. Median survival for the model initiated in NESTIN progenitors was 99.5 days for the CATACOMB mutant (n=26) group and 61 days for the CATACOMB wild-type (n=28; log-rank test p=0.0033). Median survival for the model initiated in GFAP progenitors were 144 days for the CATACOMB mutant (n=19) group and 65 days for the CATACOMB wild-type (n=21; log-rank test is P<0.0013). Immunohistochemistry for H3K27me3 demonstrated that CATACOMB wild-type tumors had reduced H3K27me3 compared to CATACOMB mutant tumors. CONCLUSIONS Disrupting CATACOMB inhibitory activity toward PRC2 significantly increases survival in mice in both models, suggesting this activity plays a critical role in accelerating tumorigenesis. Ependymoma-like histology was more commonly observed in the model initiated in the GFAP-expressing progenitors.

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IDH1-R132H acts as a tumor suppressor in glioma via epigenetic up-regulation of the DNA damage response

Science Translational Medicine ◽

10.1126/scitranslmed.aaq1427 ◽

2019 ◽

Vol 11 (479) ◽

pp. eaaq1427 ◽

Cited By ~ 44

Author(s):

Felipe J. Núñez ◽

Flor M. Mendez ◽

Padma Kadiyala ◽

Mahmoud S. Alghamri ◽

Masha G. Savelieff ◽

...

Keyword(s):

Dna Damage ◽

Tumor Suppressor ◽

Mouse Model ◽

Dna Damage Response ◽

Genetically Engineered ◽

Low Grade ◽

Loss Of Function ◽

Genetically Engineered Mouse Model ◽

Damage Response ◽

Inactivating Mutations

Patients with glioma whose tumors carry a mutation in isocitrate dehydrogenase 1 (IDH1R132H) are younger at diagnosis and live longer. IDH1 mutations co-occur with other molecular lesions, such as 1p/19q codeletion, inactivating mutations in the tumor suppressor protein 53 (TP53) gene, and loss-of-function mutations in alpha thalassemia/mental retardation syndrome X-linked gene (ATRX). All adult low-grade gliomas (LGGs) harboring ATRX loss also express the IDH1R132H mutation. The current molecular classification of LGGs is based, partly, on the distribution of these mutations. We developed a genetically engineered mouse model harboring IDH1R132H, TP53 and ATRX inactivating mutations, and activated NRAS G12V. Previously, we established that ATRX deficiency, in the context of wild-type IDH1, induces genomic instability, impairs nonhomologous end-joining DNA repair, and increases sensitivity to DNA-damaging therapies. In this study, using our mouse model and primary patient-derived glioma cultures with IDH1 mutations, we investigated the function of IDH1R132H in the context of TP53 and ATRX loss. We discovered that IDH1R132H expression in the genetic context of ATRX and TP53 gene inactivation (i) increases median survival in the absence of treatment, (ii) enhances DNA damage response (DDR) via epigenetic up-regulation of the ataxia-telangiectasia–mutated (ATM) signaling pathway, and (iii) elicits tumor radioresistance. Accordingly, pharmacological inhibition of ATM or checkpoint kinases 1 and 2, essential kinases in the DDR, restored the tumors’ radiosensitivity. Translation of these findings to patients with IDH1132H glioma harboring TP53 and ATRX loss could improve the therapeutic efficacy of radiotherapy and, consequently, patient survival.

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Oncogenic KRAS Requires Complete Loss of BAP1 Function for Development of Murine Intrahepatic Cholangiocarcinoma

10.1101/2021.10.12.464103 ◽

2021 ◽

Author(s):

Rebecca K Marcus ◽

Sammy Ferri-Borgogno ◽

Abdel Hosein ◽

Wai Chin Foo ◽

Bidyut Ghosh ◽

...

Keyword(s):

Median Survival ◽

Intrahepatic Cholangiocarcinoma ◽

Genetically Engineered ◽

Primary Hepatocellular Carcinoma ◽

Allelic Loss ◽

Loss Of Function ◽

Hepatic Expression ◽

Dismal Prognosis ◽

Genetically Engineered Mouse Model

Intrahepatic cholangiocarcinoma (ICC) is a primary biliary malignancy that harbors a dismal prognosis. Oncogenic mutations of KRAS and loss of function mutations of BRCA1-associated protein 1 (BAP1) have been identified as recurrent somatic alterations in ICC. However, an autochthonous genetically engineered mouse model of ICC that genocopies the co-occurrence of these mutations has never been developed. By crossing Albumin-Cre mice bearing conditional alleles of mutant Kras and/or floxed Bap1, Cre-mediated recombination within the liver was induced. Mice with hepatic expression of mutant KrasG12D alone (KA), bi-allelic loss of hepatic Bap1 (BhomoA), and heterozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhetKA) developed primary hepatocellular carcinoma (HCC), but no discernible ICC. In contrast, mice with homozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhomoKA) devel-oped discrete foci of HCC and ICC. Further, the median survival of BhomoKA mice was significantly shorter at 24 weeks, when compared to median survival of ≥40 weeks in BhetKA mice and approximately 50 weeks in BhomoA and KA mice (p <0.001). Microarray analysis performed on liver tissue from KA and BhomoKA mice identified differentially expressed genes in the setting of BAP1 loss and suggests that deregulation of ferroptosis might be one mechanism by which loss of BAP1 cooperates with oncogenic Ras in hepato-biliary carcinogenesis. Our autochthonous model provides an in vivo platform to further study this lethal class of neoplasm.

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Oncogenic KRAS Requires Complete Loss of BAP1 Function for Development of Murine Intrahepatic Cholangiocarcinoma

Cancers ◽

10.3390/cancers13225709 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5709

Author(s):

Rebecca Marcus ◽

Sammy Ferri-Borgogno ◽

Abdel Hosein ◽

Wai Chin Foo ◽

Bidyut Ghosh ◽

...

Keyword(s):

Median Survival ◽

Intrahepatic Cholangiocarcinoma ◽

Genetically Engineered ◽

Primary Hepatocellular Carcinoma ◽

Allelic Loss ◽

Loss Of Function ◽

Hepatic Expression ◽

Dismal Prognosis ◽

Genetically Engineered Mouse Model

Intrahepatic cholangiocarcinoma (ICC) is a primary biliary malignancy that harbors a dismal prognosis. Oncogenic mutations of KRAS and loss-of-function mutations of BRCA1-associated protein 1 (BAP1) have been identified as recurrent somatic alterations in ICC. However, an autochthonous genetically engineered mouse model of ICC that genocopies the co-occurrence of these mutations has never been developed. By crossing Albumin-Cre mice bearing conditional alleles of mutant Kras and/or floxed Bap1, Cre-mediated recombination within the liver was induced. Mice with hepatic expression of mutant KrasG12D alone (KA), bi-allelic loss of hepatic Bap1 (BhomoA), and heterozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhetKA) developed primary hepatocellular carcinoma (HCC), but no discernible ICC. In contrast, mice with homozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhomoKA) developed discrete foci of HCC and ICC. Further, the median survival of BhomoKA mice was significantly shorter at 24 weeks when compared to the median survival of ≥40 weeks in BhetKA mice and approximately 50 weeks in BhomoA and KA mice (p < 0.001). Microarray analysis performed on liver tissue from KA and BhomoKA mice identified differentially expressed genes in the setting of BAP1 loss and suggests that deregulation of ferroptosis might be one mechanism by which loss of BAP1 cooperates with oncogenic Ras in hepato-biliary carcinogenesis. Our autochthonous model provides an in vivo platform to further study this lethal class of neoplasm.

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Alterations in ZnT1 expression and function lead to impaired intracellular zinc homeostasis in cancer

Cell Death Discovery ◽

10.1038/s41420-019-0224-0 ◽

2019 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Adrian Israel Lehvy ◽

Guy Horev ◽

Yarden Golan ◽

Fabian Glaser ◽

Yael Shammai ◽

...

Keyword(s):

Malignant Tumors ◽

Zinc Homeostasis ◽

Healthy Population ◽

Missense Mutations ◽

Protein Alignment ◽

Loss Of Function ◽

Intracellular Zinc ◽

Dismal Prognosis ◽

Zinc Levels ◽

And Function

Abstract Zinc is vital for the structure and function of ~3000 human proteins and hence plays key physiological roles. Consequently, impaired zinc homeostasis is associated with various human diseases including cancer. Intracellular zinc levels are tightly regulated by two families of zinc transporters: ZIPs and ZnTs; ZIPs import zinc into the cytosol from the extracellular milieu, or from the lumen of organelles into the cytoplasm. In contrast, the vast majority of ZnTs compartmentalize zinc within organelles, whereas the ubiquitously expressed ZnT1 is the sole zinc exporter. Herein, we explored the hypothesis that qualitative and quantitative alterations in ZnT1 activity impair cellular zinc homeostasis in cancer. Towards this end, we first used bioinformatics to analyze inactivating mutations in ZIPs and ZNTs, catalogued in the COSMIC and gnomAD databases, representing tumor specimens and healthy population controls, respectively. ZnT1, ZnT10, ZIP8, and ZIP10 showed extremely high rates of loss of function mutations in cancer as compared to healthy controls. Analysis of the putative functional impact of missense mutations in ZnT1-ZnT10 and ZIP1-ZIP14, using homologous protein alignment and structural predictions, revealed that ZnT1 displays a markedly increased frequency of predicted functionally deleterious mutations in malignant tumors, as compared to a healthy population. Furthermore, examination of ZnT1 expression in 30 cancer types in the TCGA database revealed five tumor types with significant ZnT1 overexpression, which predicted dismal prognosis for cancer patient survival. Novel functional zinc transport assays, which allowed for the indirect measurement of cytosolic zinc levels, established that wild type ZnT1 overexpression results in low intracellular zinc levels. In contrast, overexpression of predicted deleterious ZnT1 missense mutations did not reduce intracellular zinc levels, validating eight missense mutations as loss of function (LoF) mutations. Thus, alterations in ZnT1 expression and LoF mutations in ZnT1 provide a molecular mechanism for impaired zinc homeostasis in cancer formation and/or progression.

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Photic Regulation of Circadian Rhythms and Voluntary Ethanol Intake: Role of Melanopsin-expressing Intrinsically Photosensitive Retinal Ganglion Cells

Journal of Biological Rhythms ◽

10.1177/0748730420981228 ◽

2020 ◽

pp. 074873042098122

Author(s):

Matthew C. Hartmann ◽

Walter D. McCulley ◽

Samuel T. Johnson ◽

Corey S. Salisbury ◽

Nikhil Vaidya ◽

...

Keyword(s):

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Circadian System ◽

Genetically Engineered ◽

Ethanol Intake ◽

Constant Darkness ◽

Retinal Ganglion ◽

Retinal Photoreceptors ◽

Genetically Engineered Mouse Model ◽

Lighting Regimen

“Non-image-forming” (NIF) effects of light are mediated primarily by a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment, melanopsin (OPN4). These NIF functions include circadian entrainment, pupillary reflexes, and photic effects on sleep, mood, and cognition. We recently reported that mice of multiple genotypes exhibit reduced voluntary ethanol intake under both constant darkness (DD) and constant light (LL) relative to standard light-dark (LD) conditions. In the present study, we sought to determine whether these effects are mediated by melanopsin-expressing ipRGCs and their potential relationship to photic effects on the circadian system. To this end, we examined the effects of environmental lighting regimen on both ethanol intake and circadian activity rhythms in a genetically engineered mouse model ( Opn4aDTA/aDTA) in which melanopsin expression is completely blocked while ipRGCs are progressively ablated due to activation of attenuated diphtheria toxin A (aDTA) transgene under the control of the Opn4 promoter. As expected from previous studies, Opn4aDTA/aDTA mice displayed dramatic attenuation of circadian photosensitivity, but surprisingly, showed identical suppression of ethanol intake under both DD and LL as that seen in controls. These results demonstrate that the effects of lighting regimen on voluntary ethanol intake are independent of melanopsin-expressing ipRGCs and ipRGC-mediated photic effects on the circadian system. Rather, these effects are likely mediated by classical retinal photoreceptors and central pathways.

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ATRT-13. DIFFERENT CELLS OF ORIGIN PAVE THE WAY FOR MOLECULAR HETEROGENEITY IN RHABDOID TUMORS

Neuro-Oncology ◽

10.1093/neuonc/noaa222.012 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii278-iii278

Author(s):

Monika Graf ◽

Marta Interlandi ◽

Natalia Moreno ◽

Dörthe Holdhof ◽

Viktoria Melcher ◽

...

Keyword(s):

Single Cell ◽

Expression Patterns ◽

Time Frame ◽

Precursor Cells ◽

Genetically Engineered ◽

Molecular Heterogeneity ◽

Loss Of Function ◽

Cellular Origin ◽

Cells Of Origin ◽

Rhabdoid Tumors

Abstract Rhabdoid tumors (RT) are rare but highly aggressive pediatric neoplasms. These tumors carry homozygous loss-of-function alterations of SMARCB1 in almost all cases with an otherwise low mutational load. RT arise at different intracranial (ATRT) as well as extracranial (MRT) anatomical sites. Three main molecular subgroups (ATRT-SHH, ATRT-TYR, ATRT-MYC) have been characterized for ATRT which are epigenetically and clinically diverse, while MRT show remarkable similarities with ATRT-MYC distinct from ATRT-SHH and ATRT-TYR. Even though there are hypotheses about various cells of origin among RT subgroups, precursor cells of RT have not yet been identified. Previous studies on the temporal control of SMARCB1 knockout in genetically engineered mouse models have unveiled a tight vulnerable time frame during embryogenesis with regard to the susceptibility of precursor cells to result in RT. In this study, we employed single-cell RNA sequencing to describe the intra- and intertumoral heterogeneity of murine ATRT-SHH and -MYC as well as extracranial MYC tumor cells. We defined subgroup-specific tumor markers for all RT classes but also observed a notable overlap of gene expression patterns in all MYC subgroups. By comparing these single-cell transcriptomes with available single-cell maps of early embryogenesis, we gained first insights into the cellular origin of RT. Finally, unsupervised clustering of published human RT methylation data and healthy control tissues confirmed the existence of different cells of origin for intracranial SHH tumors and MYC tumors independent of their anatomical localizations.

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