TMOD-08. DEVELOPING DERIVATIVE GBM PDX, IN VIVO, FROM TREATMENT NAÏVE SOURCES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi217-vi217
Author(s):  
David James ◽  
Craig Horbinski ◽  
Roger Stupp ◽  
Atique Ahmed
Keyword(s):  
Mutagenic Effect ◽  
Recurrent Glioblastoma ◽  
Response To Treatment ◽  
Mismatch Repair Gene ◽  
Repair Gene ◽  
Msh6 Mutation ◽  
Treatment Naïve ◽  
Post Therapy ◽  
Recurrent Gbm

Abstract PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

scholarly journals TMOD-35. DEVELOPING RECURRENT GBM PDX, IN VIVO, FROM TREATMENT NAÏVE SOURCES

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii235-ii235
Author(s):  
Charles James ◽  
Shi-Yuan Cheng ◽  
Craig Horbinski ◽  
Jann Sarkaria ◽  
Roger Stupp ◽  
...  
Keyword(s):  
Mutagenic Effect ◽  
Recurrent Glioblastoma ◽  
Response To Treatment ◽  
Mismatch Repair Gene ◽  
Repair Gene ◽  
Msh6 Mutation ◽  
Treatment Naïve ◽  
Post Therapy ◽  
Recurrent Gbm

Abstract PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

Phase II trial of the phosphatidyinositol-3 kinase (PI3K) inhibitor BKM120 in recurrent glioblastoma (GBM).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 2015-2015 ◽  
Author(s):  
Patrick Y. Wen ◽  
W. K. Alfred Yung ◽  
Ingo K. Mellinghoff ◽  
Kathleen Lamborn ◽  
Shakti Ramkissoon ◽  
...  
Keyword(s):  
Phase Ii ◽  
Pi3k Pathway ◽  
Homozygous Deletion ◽  
Recurrent Glioblastoma ◽  
Pi3k Inhibitor ◽  
Eligibility Criteria ◽  
Drug Concentrations ◽  
Recurrent Gbm

2015 Background: The PI3K pathway is activated in most GBMs and represents a potential therapeutic target. BKM120 is an oral, pan-Class I PI3K inhibitor that enters the brain at therapeutic concentrations demonstrated to inhibit PI3K pathway, and potently inhibits the growth of U87 GBM tumors and human glioma tumor spheres in vitro and in vivo. Methods: The Ivy Foundation Early Phase Clinical Trials Consortium is conducting a phase II study of BKM120 in recurrent GBM patients with activation of the PI3K pathway (mutation, homozygous deletion or loss of IHC of PTEN, PIK3CA or PIK3RI mutations, or detectable pAKT). Additional eligibility criteria included radiologic progression, 1st or 2nd relapse, > 18 yrs, KPS > 60, adequate bone marrow and organ function, controlled blood glucose, and no enzyme-inducing antiepileptic drugs. Patients received BKM120 100mg daily. The study consisted of 2 parts conducted concurrently. Part 1 involved up to 15 patients who received BKM120 daily for 8-12 days prior to surgery for recurrent disease. Patients underwent FDG PET, pharmacokinetic (PK) studies, and tumor was obtained for drug concentrations and pharmacodynamic effects. Part 2 consisted of up to 50 patients with unresectable GBM treated with BKM120. The primary endpoint for Part 2 was 6-month progression-free survival (p0 =15%; p1= 32%). Results: To date 7 patients have been enrolled into Part 1, 33 into part 2. There were 5 women and 35 men. Median age was 54 yrs (29-68). Treatment was fairly well-tolerated. Major grades 3/4 toxicities were asymptomatic lipase elevation (5), fatigue (3), hyperglycemia (3), rash (3) elevated AST (1), and depression (1). Analysis of tumor from Part 1 showed reduction of pAkt by IHC. Genotyping of tumor specimens is ongoing. To date 33 patients had positive pAkt, 21 had PTEN loss by IHC. Of the first 19 patients who underwent whole exome sequencing, 3 had PIK3Ca mutations and 6 had PTEN mutations. Conclusions: BKM120 is generally well tolerated in patients with recurrent GBM and achieves adequate tumor concentration to inhibit pAkt. Updated PK and efficacy data and correlation of the latter with tumor genotype will be presented. Clinical trial information: NCT01339052.

scholarly journals A DNA Polymerase ε Mutant That Specifically Causes +1 Frameshift Mutations Within Homonucleotide Runs in Yeast

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1623-1632 ◽  
Author(s):  
J M Kirchner ◽  
H Tran ◽  
M A Resnick
Keyword(s):  
Mismatch Repair ◽  
Frameshift Mutation ◽  
Mismatch Repair Gene ◽  
Repair Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Exonuclease I ◽  
Important Residue ◽  
Replicative Polymerases ◽  
Synergistic Increase

Abstract The DNA polymerases δ and ε are the major replicative polymerases in the yeast Saccharomyces cerevisiae that possess 3′ → 5′ exonuclease proofreading activity. Many errors arising during replication are corrected by these exonuclease activities. We have investigated the contributions of regions of Polε other than the proofreading motifs to replication accuracy. An allele, pol2-C1089Y, was identified in a screen of Polε mutants that in combination with an exonuclease I (exo1) mutation could cause a synergistic increase in mutations within homonucleotide runs. In contrast to other polymerase mutators, this allele specifically results in insertion frameshifts. When pol2-C1089Y was combined with deletions of EXO1 or RAD27 (homologue of human FEN1), mutation rates were increased for +1 frameshifts while there was almost no effect on −1 frameshifts. On the basis of genetic analysis, the pol2-C1089Y mutation did not cause a defect in proofreading. In combination with a deletion of the mismatch repair gene MSH2, the +1 frameshift mutation rate for a short homonucleotide run was increased nearly 100-fold whereas the −1 frameshift rate was unchanged. This suggests that the Pol2-C1089Y protein makes +1 frameshift errors during replication of homonucleotide runs and that these errors can be corrected by either mismatch repair (MMR) or proofreading (in short runs). This is the first report of a +1-specific mutator for homonucleotide runs in vivo. The pol2-C1089Y mutation defines a functionally important residue in Polε.

EXTH-62. COMBINATORIAL THERAPY TARGETING TRANSCIPTION AND IMMUNO-METABOLISM IN RECURRENT GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi177-vi177
Author(s):  
Pratiksha Dighe ◽  
Rinette Woo ◽  
Nathan Salomonis ◽  
Anu Bhattacharjee ◽  
Mehdi Nosrati ◽  
...  
Keyword(s):  
Effective Treatment ◽  
Recurrent Glioblastoma ◽  
Metabolic Reprogramming ◽  
Metabolic Inhibitor ◽  
Rna Seq ◽  
Human Gbm ◽  
Almost All ◽  
Recurrent Gbm

Abstract Recurrent glioblastomas (GBM) are notoriously difficult to treat, and in spite of aggressive chemo- and radiation therapy they inevitably recur in almost all GBM patients within 14 months following initial diagnosis. To interrogate pathways driving therapeutic resistance, we compared matched primary and recurrent IDH wild type glioblastoma samples from a cohort of patients, using RNA-Seq. Our analyses showed that pathways involved with tumor immune and metabolic reprogramming were up-regulated in recurrent GBMs compared to untreated, primary samples. Based on these findings, we tested the anti-tumor efficacy of over 20 rationally selected targeted therapeutic agents alone and in combination in a high-throughput drug screen assay designed to measure long term cell viability of primary tumorspheres from patients who underwent surgery for recurrent GBM. Top performing drug combinations from these screens were validated in vivo using patient-derived intracranial mouse models of glioma. Our data demonstrated that the combination of the transcriptional/metabolic inhibitor TG02 and the dual PI3K/mTOR inhibitor GDC-0084 significantly prolonged in vivo survival of tumor bearing mice, performing better than either drug alone. Notably, both inhibitors are undergoing human clinical trials as single agents with positive initial safety profiles and superior blood brain barrier penetrance. RNA Seq and functional assays of tumor samples from treated mice showed that only the combination treatment (TG02 + GDC-0084) significantly inhibited expression of immunomodulatory cytokines driving tumor progression, including those identified by us to be overexpressed in recurrent human GBM samples. Overall, these data suggest that combining TG02 and GDC-0084 is an effective treatment for recurrent GBMs. Furthermore, our results support the use of a translational research platform consisting of a personalized pharmaco-genomics testing, using patient-derived tumor samples towards designing more effective treatment for recurrent GBM patients.

scholarly journals CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

2022 ◽  
Vol 10 (1) ◽  
pp. e003289
Author(s):  
Mathieu Seyfrid ◽  
William Thomas Maich ◽  
Vaseem Muhammad Shaikh ◽  
Nazanin Tatari ◽  
Deepak Upreti ◽  
...  
Keyword(s):  
Standard Of Care ◽  
Recurrent Glioblastoma ◽  
Preclinical Model ◽  
Dismal Prognosis ◽  
Car T ◽  
Cell Assays ◽  
Human Gbm ◽  
Recurrent Gbm

PurposeGlioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs.Experimental designIn the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70’s role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples.ResultsCD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells.ConclusionCD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.

scholarly journals OS12.6 Intracranial GBM PDX Mutations Induced by Temozolomide Treatment

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii23-iii23
Author(s):  
C James ◽  
C Horbinski ◽  
E Bartom ◽  
K Burdett ◽  
D Scholtens ◽  
...  
Keyword(s):  
Sequence Data ◽  
In Vivo Models ◽  
Subcutaneous Tumor ◽  
Exome Sequence Data ◽  
Study Results ◽  
Treatment Naïve ◽  
Animal Hosts ◽  
Exome Sequence ◽  
Recurrent Gbm

Abstract BACKGROUND Temozolomide (TMZ) is routinely used in the post-surgical treatment of patients with newly diagnosed glioblastoma (GBM). Nearly all GBM recur following TMZ treatment, and there is an increasing body of data that detail TMZ-associated mutations in recurrent GBM. In vivo models of recurrent GBM are scarce in comparison to models of treatment-naïve GBM. MATERIAL AND METHODS Mice with intracranial treatment naïve GBM patient derived xenograft (PDX) were treated with radiotherapy (RT) only, TMZ only, or RT + TMZ. Tumors were monitored for recovery from treatment by bioluminescence imaging (BLI), and upon animal subject decline due to growing tumor, subjects were euthanized, with viable intracranial tumor dissected and used to establish subcutaneous tumor in new animal hosts that received no treatment. Subcutaneous tumors were sequentially passaged through two additional animal hosts. DNA was extracted from third passage subcutaneous tumors and subjected to full exome sequencing. PDX exome sequence data were examined for genes with nonsynonymous frameshift and nonsense variants (mutations). RESULTS Exome sequence analysis showed striking differences between PDX receiving RT only vs. TMZ with or without RT. Most notable is the substantially greater incidence of G-C to A-T transition mutations in TMZ-treated PDX, which are a signature of TMZ treatment. All TMZ-treated PDX also showed substantially more genes being mutated than PDX receiving RT only: one cycle RT only = 88; one cycle TMZ only = 339; one cycle of concurrent RT + TMZ = 531; one cycle of concurrent RT + TMZ followed by two additional cycles of TMZ only = 1502. The PDX subjected to multiple cycles of TMZ treatment prior to establishment as a subcutaneous tumor has shown stable resistance to TMZ treatment in subsequent experiments. Importantly, TMZ-treated PDX had mutations in LTBP4, MSH6, and PREX1 genes that are enriched in patient GBM following recurrence after initial TMZ treatment (Nat Genet 2016;48:768–76); these genes were not mutated in PDX receiving RT only. CONCLUSION Our study results support the following: 1) Intracranial GBM PDX treated with TMZ develop a global genomic treatment signature, as well as specific mutations that are observed in recurrent patient GBMs; 2) Mutations detected in TMZ-treated intracranial tumors are stable, persisting after repeated subcutaneous passages without further treatment; 3) Paired naïve and TMZ-treated GBM PDX will facilitate studies aimed at increasing our understanding of TMZ resistance, and help identify new therapies for more effective treatment of recurrent GBM.

scholarly journals Hitting more birds with one stone: CD70 as an actionable immunotherapeutic target in recurrent glioblastoma

2021 ◽  
Author(s):  
Mathieu Seyfrid ◽  
William Maich ◽  
Vaseem M Shaikh ◽  
Nazanin Tatari ◽  
Deepak Upreti ◽  
...  
Keyword(s):  
Standard Of Care ◽  
Recurrent Glioblastoma ◽  
Preclinical Model ◽  
Tumor Initiating Cells ◽  
Dismal Prognosis ◽  
Car T ◽  
Cell Assays ◽  
Recurrent Gbm

Purpose: Glioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of brain tumor initiating cells (BTICs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here we identify CD70 as a potential therapeutic target for recurrent GBM BTICs. Experimental Design: In the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We utilize CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate the role of CD70 in GBM. Next, we developed and tested an anti-CD70 CAR-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples. Results: CD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations. Conclusion: CD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable poly-therapeutic avenue to co-target both GBM and its TIME.

scholarly journals Nitrogen-Regulated Hypermutator Strain of Synechococcus sp. for Use in In Vivo Artificial Evolution

2003 ◽  
Vol 69 (11) ◽  
pp. 6427-6433 ◽  
Author(s):  
Daniel Emlyn-Jones ◽  
G. Dean Price ◽  
T. John Andrews
Keyword(s):  
Transcriptional Control ◽  
Fold Increase ◽  
Cellular Damage ◽  
Mismatch Repair Gene ◽  
Resistance Mutations ◽  
Repair Gene ◽  
Dna Mismatch ◽  
Photosynthetic Organism

ABSTRACT Artificially evolved variants of proteins with roles in photosynthesis may be selected most conveniently by using a photosynthetic organism, such as a cyanobacterium, whose growth depends on the function of the target protein. However, the limited transformation efficiency of even the most transformable cyanobacteria wastes much of the diversity of mutant libraries of genes produced in vitro, impairing the coverage of sequence space. This highlights the advantages of an in vivo approach for generating diversity in the selection organism itself. We constructed two different hypermutator strains of Synechococcus sp. strain PCC 7942 by insertionally inactivating or nutritionally repressing the DNA mismatch repair gene, mutS. Inactivation of mutS greatly increases the mutation rate of the cyanobacterium's genes, leading to an up-to-300-fold increase in the frequency of resistance to the antibiotics rifampin and spectinomycin. In order to control the rate of mutation and to limit cellular damage resulting from prolonged hypermutation, we placed the uninterrupted mutS gene in the cyanobacterial chromosome under the transcriptional control of the cyanobacterial nirA promoter, which is repressed in the presence of NH4 + as an N source and derepressed in its absence. By removing or adding this substrate, hypermutation was activated or repressed as required. As expected, hypermutation caused by repression in PnirA-mutS transformants led to an accumulation of spectinomycin resistance mutations during growth.

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