TMOD-14. CREATION OF A GENETICALLY ENGINEERED MOUSE MODEL OF ANAPLASTIC ASTROCYTOMA DRIVEN BY THE IDH1-R132H ONCOGENE

Abstract Despite the high prevalence of IDH1-R132H mutations in lower grade gliomas, the ability to study this mutation in vivo has been hampered by a lack of faithful mouse models. Therefore, we used a CRISPR/Cas9- and AAV-based strategy to create a genetically engineered mouse model (GEMM) of astrocytoma driven by IDH1-R132H that recreates the genetic landscape of human IDH1 mutant astrocytoma. IDH1 mutations in astrocytomas often co-occur with mutations in TP53, ATRX, and either PIK3R1 or PIK3CA. Using human astrocytes immortalized via expression of telomerase (which phenocopies ATRX loss) and HPV E6 and E7 oncoproteins (which phenocopy p53 and pRb loss, respectively), we found that PIK3R1 and IDH1 oncogenes cooperate to promote anchorage-independent cell growth in vitro and orthotopic brain tumor formation in vivo. These data identified a combination of clinically relevant mutations that we hypothesized could be leveraged to cause spontaneous astrocytoma formation in mice. To simultaneously engineer Idh1, Pik3ca, Tp53, and Atrx mutations in mouse brain tissue, we intracranially injected adeno-associated virus (AAV) expressing Cre recombinase and sgRNAs targeting murine Atrx and Tp53 genes into four mouse strains with the following conditional alleles: 1) LSL-Cas9; 2) LSL-Cas9; LSL-Pik3caH1047R, 3) LSL-Cas9; LSL-Idh1R132H, and 4) LSL-Cas9; LSL-Idh1R132H; LSL-Pik3caH1047R. Grade III anaplastic astrocytomas preferentially formed 9-14 months after injecting the mice carrying both the Idh1 and Pik3ca conditional alleles. These astrocytomas harbored all intended mutations, expressed astrocytoma lineage markers, and displayed elevated (R)-2-hydroxyglutarate, the oncometabolite produced by mutant Idh1. To create an additional model with shorter tumor latency, we transplanted glioma stem-like cells derived from our GEMM into recipient mice to produce Idh1 mutant astrocytoma allografts. These allografts provide a tractable platform for preclinical therapeutic studies. Taken together, our findings show that IDH1 and PI3K oncoproteins cooperate to promote gliomagenesis and unveil new genetically faithful mouse models of mutant IDH1-driven astrocytoma.

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IMMU-10. ESTABLISHING EFFECTIVE MODELS FOR IMMUNOTHERAPY IN GBM

Neuro-Oncology ◽

10.1093/neuonc/noz175.504 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi121-vi121

Author(s):

Daniel Zamler ◽

Er-Yen Yen ◽

Takashi Shingu ◽

Jiangong Ren ◽

Cynthia Kassab ◽

...

Keyword(s):

Mouse Model ◽

Cell Function ◽

Genetically Engineered ◽

Death Sentence ◽

Immune Microenvironment ◽

Knockout Mouse Model ◽

Arginase 1 ◽

Genetically Engineered Mouse Model ◽

Human Gbm

Abstract The introduction of immunotherapies has been paradigm shifting for cancers that were previously a death sentence. However, preclinical/clinical studies on glioblastoma (GBM) have generated mixed outcomes in patients, likely due to its great heterogeneity of immune microenvironment, particularly the myeloid cell populations. Primary patient studies have been limited by a difficulty in performing longitudinal studies, uncontrolled environmental conditions, and genetic variability. There is also, unfortunately, a paucity of mouse models that effectively re-capitulate the immune microenvironment of the human disease. To address these difficulties, we have established the Qk/p53/Pten (QPP) triple knockout mouse model established in our lab. The QPP model uses a cre-lox system to induce Qk deletion on a Pten−/−; p53−/− background which helps NSCs maintain their stemness outside the SVZ in Nes-CreERT2;QkiL/L PtenL/L p53L/L mice, which develops glioblastoma with survival of ~105 days. We have preliminarily assessed the QPP tumors as a faithful model to study the immune response to GBM and found them to recapitulate human GBM with respect to differential response to checkpoint blockade therapy and myeloid and T-cells histopathologically, particularly regarding upregulation of Arginase-1 (Arg1). Arg1 is the canonical marker for tumor-associated macrophages (TAMs), which is a major population of myeloid cells that greatly infiltrate in human GBM, sometimes making up more than ~30% of all GBM cells. Given TAMs’ prevalence in the tumor microenvironment and their upregulation of Arg1 in both human GBM and our QPP model, we are testing whether manipulation of Arg1 will impact TAM function and influence GBM growth. We are also evaluating arginine metabolism in TAMs effect on T cell function in GBM. Lastly, we have developed a genetically engineered mouse model to study the role of Arg1 knockout in a GBM context in-vivo. Our studies suggest that Arg1 plays an important role in GBM immune interaction.

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Nme1 and Nme2 genes exert metastasis-suppressor activities in a genetically engineered mouse model of UV-induced melanoma

British Journal of Cancer ◽

10.1038/s41416-020-01096-w ◽

2020 ◽

Vol 124 (1) ◽

pp. 161-165

Author(s):

Nidhi Pamidimukkala ◽

Gemma S. Puts ◽

M. Kathryn Leonard ◽

Devin Snyder ◽

Sandrine Dabernat ◽

...

Keyword(s):

Mouse Model ◽

Suppressor Gene ◽

Genetically Engineered ◽

In Vivo Model ◽

Metastasis Suppressor ◽

Specific Context ◽

Genetically Engineered Mouse Model ◽

Metastatic Activity ◽

First Time

AbstractNME1 is a metastasis-suppressor gene (MSG), capable of suppressing metastatic activity in cell lines of melanoma, breast carcinoma and other cancer origins without affecting their growth in culture or as primary tumours. Herein, we selectively ablated the tandemly arranged Nme1 and Nme2 genes to assess their individual impacts on metastatic activity in a mouse model (HGF:p16−/−) of ultraviolet radiation (UVR)-induced melanoma. Metastatic activity was strongly enhanced in both genders of Nme1- and Nme2-null mice, with stronger activity in females across all genotypes. The study ascribes MSG activity to Nme2 for the first time in an in vivo model of spontaneous cancer, as well as a novel metastasis-suppressor function to Nme1 in the specific context of UVR-induced melanoma.

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Simulation of COVID-19 Symptoms in a Genetically Engineered Mouse Model

10.21203/rs.3.rs-1236117/v1 ◽

2022 ◽

Author(s):

Mahavir Singh ◽

Sathnur Pushpakumar ◽

Nia Bard ◽

Yuting Zheng ◽

Rubens P. Homme ◽

...

Keyword(s):

Mouse Model ◽

Viral Disease ◽

Organ Damage ◽

Genetically Engineered ◽

Host Cells ◽

Suitable Model ◽

Viral Agent ◽

Human Beings ◽

Genetically Engineered Mouse Model

Abstract The ongoing infectious viral disease pandemic (also known as the coronavirus disease-19; COVID-19) by a constantly emerging viral agent commonly referred as the severe acute respiratory syndrome corona virus 2 or SARS-CoV-2 has revealed unique pathological findings from infected human beings, and the postmortem observations. The list of disease symptoms, and post-mortem observations is too long to mention; however, a few notable ones are worth mentioning to put into a perspective in understanding the malignity of this pandemic starting with respiratory distress or dyspnea, chest congestion, muscle or body aches, malaise, fever, chills, etc. We opine that further improvement for delivering highly effective treatment, and preventive strategies would be benefited from validated animal disease models. In this context, we designed a study and show that a genetically engineered mouse expressing the human angiotensin converting enzyme 2; hACE2 (the receptor used by SARS-CoV-2 agent to enter host cells) represents an excellent investigative resource in simulating important clinical features of the COVID-19 infection. The hACE2 mouse model (which is susceptible to SARS-CoV-2) when administered with a recombinant SARS-CoV-2 spike (S) protein intranasally exhibited a profound cytokine storm capable of altering the physiological parameters including significant changes in in vivo cardiac function along with multi-organ damage that was further confirmed via histological findings. More importantly, visceral organs from SARS-CoV-2 spike (S) treated mice revealed thrombotic blood clots as seen during postmortem examination of the mice. Thus, the hACE2 engineered mouse appears to be a suitable model for studying intimate viral pathogenesis paving the way for further identification, and characterization of appropriate prophylactics as well as therapeutics for COVID-19 management.

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Disrupting Ectopic Super-Enhancers to Treat Multiple Myeloma

Blood ◽

10.1182/blood-2021-147551 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1593-1593

Author(s):

Seth Welsh ◽

Daniel Riggs ◽

Erin Meermeier ◽

Chang-Xin Shi ◽

Victoria Garbitt ◽

...

Keyword(s):

Multiple Myeloma ◽

Transcription Factor ◽

Mouse Model ◽

Therapeutic Response ◽

Genetically Engineered ◽

Therapeutic Window ◽

Super Enhancer ◽

Genetically Engineered Mouse Model

Abstract Multiple myeloma (MM) is an incurable form of plasma cell cancer in which primary and secondary chromosomal translocations routinely juxtapose oncogenes to plasma cell-specific super-enhancers. Coincidentally, drugs which target super-enhancers have had success clinically. For example, immunomodulatory imide drugs (IMiDs) degrade super-enhancer-binding pioneer factors IKAROS and AIOLOS, while glucocorticoids (Dexamethasone) and proteasome inhibitors (Bortezomib) have the ability to transrepress or block the processing of super-enhancer-forming NF-κB proteins, respectively. Currently, alternative enhancer-targeting drugs are also in clinical development, like p300 inhibitors which target the acetyl-binding bromodomains and/or histone acetyl transferase activity of the chromatin-regulating coactivator homologs CBP and EP300. Despite showing therapeutic promise, our understanding of how these drugs function, alone or together, remains incomplete. Case in point, we find that IMiD-induced degradation of its target proteins IKAROS and AIOLOS does not guarantee a therapeutic response in vitro, and patients successfully treated with IMiDs eventually relapse; meanwhile, coactivator-targeting therapies like p300 inhibitors are often too toxic in vivo, and lack a therapeutic window. To improve the outcomes of MM patients we need to understand the heterogeneous genetics and transcription-factor milieus of the myeloma enhancer landscape, as well as how to increase the precision of enhancer-disrupting drugs. To accomplish this, our lab utilizes more than 60 human myeloma cell lines that have been extensively characterized at the genetic, proteomic, and drug-therapeutic-response levels. Additionally, we have generated a highly-predictive immunocompetent mouse model (Vk*MYC hCRBN+) that develops human-like MM and is sensitive to both IMiDs and a new class of therapeutics termed "degronimids" (normal mice do not respond to IMiDs or degronimids). Our central hypothesis is that combining a broad coactivator-targeting drug (e.g., the p300 inhibitor GNE-781), with a MM-specific transcription factor-targeting drug (e.g., IMiDs) restricts toxicities to myeloma cells and thus improves the therapeutic window. Currently, we are testing a variety of coactivator-targeting compounds alongside traditional IMiD therapies and other preclinical transcription factor-targeting drugs both in vivo and in vitro. We show that Vk*MYC hCRBN+ mice are exquisitely sensitive to GNE-781, requiring one fourth of the dose needed to treat other cancers and therefore avoiding the neutropenia and thrombocytopenia seen at higher doses. Second, we show that although IMiDs and GNE-781 induce an effective but transient response in vivo as single agents, the combination of the two drugs proved curative, with a progressive deepening of the anti-tumor response occurring even after therapy is discontinued. Ongoing experiments aim to determine how this drug combination, and other coactivator + transcription factor-targeting combinations, permanently disrupt myeloma-specific super-enhancers. Disclosures Neri: BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genentech: Consultancy. Boise: AstraZeneca: Honoraria, Research Funding; AbbVie/Genentech: Membership on an entity's Board of Directors or advisory committees. Chesi: Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma.

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Wild-Type Hras Suppresses the Earliest Stages of Tumorigenesis in a Genetically Engineered Mouse Model of Pancreatic Cancer

PLoS ONE ◽

10.1371/journal.pone.0140253 ◽

2015 ◽

Vol 10 (10) ◽

pp. e0140253 ◽

Cited By ~ 4

Author(s):

Jamie D. Weyandt ◽

Benjamin L. Lampson ◽

Sherry Tang ◽

Matthew Mastrodomenico ◽

Diana M. Cardona ◽

...

Keyword(s):

Pancreatic Cancer ◽

Mouse Model ◽

Genetically Engineered ◽

Wild Type ◽

Genetically Engineered Mouse Model

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P2-066 A genetically engineered mouse model with an enhanced beta-secretase substrate exhibits increased amyloid generation

Neurobiology of Aging ◽

10.1016/s0197-4580(04)80813-3 ◽

2004 ◽

Vol 25 ◽

pp. S242 ◽

Cited By ~ 1

Author(s):

Adam J. Simon ◽

Lin Chen ◽

Eric A. Price ◽

Min Xu ◽

Adam Lucka ◽

...

Keyword(s):

Mouse Model ◽

Genetically Engineered ◽

Beta Secretase ◽

Genetically Engineered Mouse Model

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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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Abstract 215: The Role of Titin in Cardiac Function: Studies in Two Genetically Engineered Mouse Models With Disparate Titin’s Size

Circulation Research ◽

10.1161/res.117.suppl_1.215 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Mei Methawasin ◽

Kirk R Hutchinson ◽

John E Smith ◽

Henk L Granzier

Keyword(s):

Mouse Model ◽

Diastolic Dysfunction ◽

Diastolic Function ◽

Exercise Capacity ◽

Mouse Models ◽

Wheel Running ◽

Left Ventricular ◽

Genetically Engineered ◽

Passive Stiffness ◽

Genetically Engineered Mouse Models

Titin, a myofilament that acts as a molecular spring in the sarcomere, is considered the main contributor to passive stiffness of cardiomyocytes and is responsible for cardiac diastolic function. Increased titin stiffness is related to diastolic dysfunction and HFpEF (Heart Failure with preserved Ejection Fraction). Alteration in size of titin’s spring region leads to changes in cardiomyocyte and left ventricular (LV) chamber stiffness. We tested the effect of alteration in titin’s size in two genetically engineered mouse models. We investigated the effect of shortening titin’s spring region in a mouse model in which I-band/A-band region of titin’s spring has been deleted (TtnΔIAjxn ), in comparison to the effect of lengthening titin’s spring region in a mouse model deficient in titin splicing factor (Rbm20ΔRRM). Integrative approaches were used from single cardiomyocyte mechanics to pressure-volume analysis and exercise study. Study of skinned LV cardiomyocytes revealed that cellular passive stiffness was inversely related to the size of titin. Cellular passive stiffness was increased in TtnΔIAjxn homozygous (-/-) (~ 110 % higher than wildtype (WT)) and was reduced in a graded manner in Rbm20ΔRRM heterozygous (+/-) and -/- cardiomyocytes (~61% and ~87% less than WT). This effect was carried through at the LV chamber level which could be demonstrated in pressure volume (PV) analysis as an increased end-diastolic pressure-volume relationship (EDPVR) in TtnΔIAjxn -/- (~110% higher than WT’s hearts) and reduced EDPVR in Rbm20ΔRRM +/- and -/- (~57% and ~48% less than WT’s hearts). Free-wheel running studies revealed a running deficiency in TtnΔIAjxn -/- mice but an increase in exercise capacity in Rbm20ΔRRM +/– mice. Conclusions: Functional studies from the cellular to in-vivo LV chamber levels showed that mice with shortening of titin’s spring region had increased LV stiffness, diastolic dysfunction and reduced exercise capacity, while mice with lengthening titin’s spring region had compliant LV and increased exercise capacity. Thus, our work supports titin’s important roles in LV diastolic function and suggests that modification of the size of titin’s spring region could be a potential therapeutic strategy for HFpEF.

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TMOD-08A MOUSE MODEL OF YOUNG ADULT GLIOBLASTOMA: IN VIVO EXPRESSION OF MUTATED IDH1 (R132H) GENE USING THE SLEEPING BEAUTY TRANSPOSASE SYSTEM

Neuro-Oncology ◽

10.1093/neuonc/nov237.08 ◽

2015 ◽

Vol 17 (suppl 5) ◽

pp. v227.4-v227

Author(s):

Felipe J. Nunez ◽

Flor Mendez ◽

Carl Koshmann ◽

Alexandra Calinescu ◽

Marta Dzaman ◽

...

Keyword(s):

Young Adult ◽

Mouse Model ◽

Sleeping Beauty ◽

Idh1 R132h ◽

In Vivo Expression

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Inhibition of 2-Hydroxyglutrate Elicits Metabolic-reprograming and Mutant IDH1 Glioma Immunity

10.1101/2020.05.11.086371 ◽

2020 ◽

Author(s):

Padma Kadiyala ◽

Stephen V. Carney ◽

Jessica C. Gauss ◽

Maria B. Garcia-Fabiani ◽

Felipe J. Núñez ◽

...

Keyword(s):

Tumor Regression ◽

Immunological Memory ◽

Immune Checkpoint Blockade ◽

Metabolic Reprogramming ◽

Lower Grade ◽

Combination Strategy ◽

Isocitrate Dehydrogenase 1 ◽

Idh1 R132h ◽

Mutant Idh1 ◽

Promoter Mutations

AbstractMutant isocitrate-dehydrogenase-1 (IDH1-R132H; mIDH1) is a hallmark of adult gliomas. Lower grade mIDH1 gliomas are classified into two molecular subgroups: (i) 1p/19q co-deletion/TERT-promoter mutations or (ii) inactivating mutations in α-thalassemia/mental retardation syndrome X-linked (ATRX) and TP53. This work, relates to the gliomas’ subtype harboring mIDH1, TP53 and ATRX inactivation. IDH1-R132H is a gain-of-function mutation that converts α-ketoglutarate into 2-hydroxyglutarate (D-2HG). The role of D-2HG within the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. Inhibition of 2HG, when used as monotherapy or in combination with radiation and temozolomide (IR/TMZ), led to increased median survival (MS) of mIDH1 glioma bearing mice. Also, 2HG inhibition elicited anti-mIDH1 glioma immunological memory. In response to 2HG inhibition, PD-L1 expression levels on mIDH1-glioma cells increased to similar levels as observed in wild-type-IDH1 gliomas. Thus, we combined 2HG inhibition/IR/TMZ with anti-PDL1 immune checkpoint-blockade and observed complete tumor regression in 60% of mIDH1 glioma bearing mice. This combination strategy reduced T-cell exhaustion and favored the generation of memory CD8+T-cells. Our findings demonstrate that metabolic reprogramming elicits anti-mIDH1 glioma immunity, leading to increased MS and immunological memory. Our preclinical data supports the testing of IDH-R132H inhibitors in combination with IR/TMZ and anti-PDL1 as targeted therapy for mIDH1/mATRX/mTP53 glioma patients.Brief SummaryInhibition of 2-Hydroxyglutrate in mutant-IDH1 glioma in the genetic context of ATRX and TP53 inactivation elicits metabolic-reprograming and anti-glioma immunity.

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