Christopher J. Pirozzi

Isocitrate dehydrogenase mutations in gliomas: mechanisms, biomarkers and therapeutic target

PURPOSE OF REVIEW Isocitrate dehydrogenases, IDH1 and IDH2, decarboxylate isocitrate to α-ketoglutarate (α-KG) and reduce NADP to NADPH. Point mutations of IDH1 and IDH2 have been discovered in gliomas. IDH mutations cause loss of native enzymatic activities and confer novel activity of converting α-KG to 2-hydroxyglutarate (2-HG). The mechanisms of IDH mutations in gliomagenesis, and their value as diagnostic, prognostic marker and therapeutic target have been extensively studied. This review is to summarize the findings of these studies. RECENT FINDINGS Crystal structural studies revealed conformation changes in mutant IDHs, which may explain the gain of function by mutant IDHs. The product of mutant IDHs, 2-HG, is an inhibitor of α-KG-dependent dioxygenases, which may cause genome-wide epigenetic changes in human gliomas. IDH mutations are a favorable prognostic factor for human glioma and can be used as biomarker for differential diagnosis and subclassification rather than predictor of response to treatment. Preliminary data suggested that inhibiting production of the substrate of mutant IDH enzymes caused slow-down of glioma cell growth. SUMMARY As valuable diagnostic and prognostic markers of human gliomas, there is still a lack of knowledge on biological functions of mutant IDHs, making targeting IDHs in glioma both difficult and unsecured.

Releasing the Block: Setting Differentiation Free with Mutant IDH Inhibitors

Hotspot mutations in IDH1 and IDH2 cause a differentiation block that can promote tumorigenesis. Two recent papers reported that small molecules targeting mutant IDH1 or mutant IDH2 release this differentiation block and/or impede tumor growth, providing a proof-of-concept that mutant IDHs are therapeutically targetable and that their effects are reversible.

Mutant IDH1 Disrupts the Mouse Subventricular Zone and Alters Brain Tumor Progression

IDH1 mutations occur in the majority of low-grade gliomas and lead to the production of the oncometabolite, D-2-hydroxyglutarate (D-2HG). To understand the effects of tumor-associated mutant IDH1 (IDH1-R132H) on both the neural stem cell (NSC) population and brain tumorigenesis, genetically faithful cell lines and mouse model systems were generated. Here, it is reported that mouse NSCs expressing Idh1-R132H displayed reduced proliferation due to p53-mediated cell-cycle arrest as well as a decreased ability to undergo neuronal differentiation. In vivo, Idh1-R132H expression reduced proliferation of cells within the germinal zone of the subventricular zone (SVZ). The NSCs within this area were dispersed and disorganized in mutant animals, suggesting that Idh1-R132H perturbed the NSCs and the microenvironment from which gliomas arise. In addition, tumor-bearing animals expressing mutant Idh1 displayed a prolonged survival and also overexpressed Olig2, features consistent with IDH1-mutated human gliomas. These data indicate that mutant Idh1 disrupts the NSC microenvironment and the candidate cell-of-origin for glioma; thus, altering the progression of tumorigenesis. In addition, this study provides a mutant Idh1 brain tumor model that genetically recapitulates human disease, laying the foundation for future investigations on mutant IDH1-mediated brain tumorigenesis and targeted therapy. Implications: Through the use of a conditional mutant mouse model that confers a less aggressive tumor phenotype, this study reveals that mutant Idh1 impacts the candidate cell-of-origin for gliomas. Mol Cancer Res; 15(5); 507–20. ©2017 AACR.

Adaptive evolution of the GDH2 allosteric domain promotes gliomagenesis by resolving IDH1R132H induced metabolic liabilities

Hotspot mutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in a number of human cancers and confer a neomorphic enzyme activity that catalyzes the conversion of α-ketoglutarate (αKG) to the oncometabolite D-(2)-hydroxyglutarate (D2HG). In malignant gliomas, IDH1R132H expression induces widespread metabolic reprogramming, possibly requiring compensatory mechanisms to sustain the normal biosynthetic requirements of actively proliferating tumor cells. We used genetically engineered mouse models of glioma and quantitative metabolomics to investigate IDH1R132H-dependent metabolic reprogramming and its potential to induce biosynthetic liabilities that can be exploited for glioma therapy. In gliomagenic neural progenitor cells, IDH1R132H expression increased the abundance of dipeptide metabolites, depleted key tricarboxylic acid cycle metabolites, and slowed progression of murine gliomas. Notably, expression of glutamate dehydrogenase GDH2, a hominoid-specific enzyme with relatively restricted expression to the brain, was critically involved in compensating for IDH1R132H-induced metabolic alterations and promoting IDH1R132H glioma growth. Indeed, we found that recently evolved amino acid substitutions in the GDH2 allosteric domain conferred its nonredundant, glioma-promoting properties in the presence of IDH1 mutation. Our results indicate that among the unique roles for GDH2 in the human forebrain is its ability to limit IDH1R132H-mediated metabolic liabilities, thus promoting glioma growth in this context. Results from this study raise the possibility that GDH2-specific inhibition may be a viable therapeutic strategy for gliomas with IDH mutations.Significance: These findings show that the homonid-specific brain enzyme GDH2 may be essential to mitigate metabolic liabilities created by IDH1 mutations in glioma, with possible implications to leverage its therapeutic management by IDH1 inhibitors. Cancer Res; 78(1); 36-50. ©2017 AACR.

Cic Loss Promotes Gliomagenesis via Aberrant Neural Stem Cell Proliferation and Differentiation

Inactivating mutations in the transcriptional repression factor Capicua (CIC) occur in approximately 50% of human oligodendrogliomas, but mechanistic links to pathogenesis are unclear. To address this question, we generated Cic-deficient mice and human oligodendroglioma cell models. Genetic deficiency in mice resulted in a partially penetrant embryonic or perinatal lethal phenotype, with the production of an aberrant proliferative neural population in surviving animals. In vitro cultured neural stem cells derived from Cic conditional knockout mice bypassed an EGF requirement for proliferation and displayed a defect in their potential for oligodendrocyte differentiation. Cic is known to participate in gene suppression that can be relieved by EGFR signal, but we found that cic also activated expression of a broad range of EGFR-independent genes. In an orthotopic mouse model of glioma, we found that Cic loss potentiated the formation and reduced the latency in tumor development. Collectively, our results define an important role for Cic in regulating neural cell proliferation and lineage specification, and suggest mechanistic explanations for how CIC mutations may impact the pathogenesis and therapeutic targeting of oligodendroglioma. Cancer Res; 77(22); 6097-108. ©2017 AACR.

Improved grading of IDH-mutated astrocytic gliomas

The WHO’s revised classification system for CNS tumours now incorporates genetic features, including the mutation status of isocitrate dehydrogenase (IDH) genes. A new article proposes that mutational status of CDKN2A and CDKN2B should also be included to facilitate grading of IDH-mutated gliomas with both prognostic and clinical relevance.

Tumor-Specific Mutations in Gliomas and Their Implications for Immunotherapy

Gliomas have both an inherent tendency to progress to more advanced stages and a high propensity for recurrence following standard of care therapy. Their diffusely infiltrative nature and an immunosuppressive microenvironment are contributing factors toward the therapeutic difficulties observed in patients with glioma. Advances in sequencing technology have made it possible to sequence the genome of an individual patients tumor, shedding light on the mutation spectrum of tumors and the identification of tumor-specific antigens. These data have and will continue to have significant implications in the field of glioma immunotherapy, which seeks to harness the power of the patients immune system to target tumor-specific mutations, offering an ideal means of treating gliomas with minimal invasiveness and off-target collateral damage. In this chapter, we identify and discuss the immunotherapeutic implications of several tumor-specific mutations, including isocitrate dehydrogenase 1-R132H, epidermal growth factor receptor vIII, and mutations in H3F3A. Additionally, the fundamental preclinical trials and the promising data from completed clinical trials will be addressed.

Preclinical Immunotherapeutic Animal Models for Brain Tumors

Glioblastoma (GBM) is the most malignant primary brain tumor in adults. Even with the current standard of care therapy (i.e., surgery, radiotherapy, and chemotherapy), GBM has a poor prognosis. Innovative immunotherapeutic approaches, which engage a patients immune system to trigger a tumor-specific immune response, have generated modest clinical improvements in selected GBM patient populations. Patients with GBM demonstrate extensive systemic immune defects that deter the efficacy of both conventional and immune-based treatments. Therapeutic strategies that simultaneously activate the immune system and neutralize the immunosuppression are critical for enhancing treatment outcomes in GBM patients. Owing to the essential role of the immune system in tumor eradication, preclinical animal models recapitulating the human disease in immunocompetent hosts are vital for assessing the efficacy of anti-​GBM experimental therapies prior to testing in patients. Different immunocompetent animal models available in the preclinical setting for the evaluation of novel brain tumor therapies are summarized in this chapter.

Abstract A032: Using oncolytic polio expressing tumor as a cancer vaccine

Background: The recent clinical success of immune checkpoint inhibitors in melanoma revealed the therapeutic potential of an anti-tumor T cell response. Previous studies using peptide vaccines to elicit anti tumor T cell response have been limited in efficacy. We have discovered that an oncolytic poliovirus leads to persistent IFN dominant activation of antigen presenting cells. Based upon these findings, we propose to use a recombinant poliovirus (PVS-RIPO) expressing tumor antigens to activate antigen presenting cells and elicit an anti tumor T cell response. PVS-RIPO is the live attenuated vaccine (SABIN) type 1 containing a foreign human rhinovirus IRES. Preliminary studies in our lab showed that PVS-RIPO is a potent human dendritic cell and CD8 T cell activator. In addition, PVS-RIPO has recently achieved breakthrough status following a very promising phase I clinical trial targeting recurrent glioblastoma (GBM). The efficacy of PVS-RIPO against GBM is thought to be mediated mainly by an anti-tumor CD8 T cells response. The potent activation of dendritic cells as well as the promising results of the phase I clinical trial makes PVS-RIPO a prime candidate for use as a therapeutic tumor vaccine. The PVS-RIPO vector technology can be adapted to multiple tumor mutations, we are planning to target 2 glioma mutations at this stage: IDH1R132H and EGFRviii mutations. IDH1R132H is a point mutation in the enzyme isocitrate dehydrogenase 1, the mutation is present at high frequency (>70%) in low grade glioma. EGFRviii is a deletion in exon 2-7 of the EGFR gene, it is present in 67% of patients with GBM. Results: Adjuvancy of PVS-RIPO We first tested the immune adjuvancy of PVS-RIPO in an in vitro human system. PVS-RIPO was found to sub lethally infect and activate dendritic cells leading to pro inflammatory cytokine production. In addition, dendritic cell incubated with tumor cell lysate, PVS-RIPO and autologous T cells produced an anti tumor CD8 T cell response. Designing stable PVS-RIPO Vectors Previous attempts to use poliovirus as a vaccine vector were unsuccessful because of its notorious genetic instability. Our lab has developed a novel strategy to generate stable poliovirus vectors: we use the relative genetic plasticity of the IRES (the dispensable stem-loop Domain VI) to encode tumor antigens. We have successfully made stable PVS-RIPO vectors expressing EGFRviii and IDH1R132H antigens. Mouse models to study efficiency of vaccine in vivo Because PVS-RIPO has a rhinovirus IRES, it is incompetent in mouse cells and first had to be adapted for mouse competency. Through murine cell passaging, we have successfully generated a mouse adapted PVS-RIPO virus (mPVS-RIPO) with 5 point mutations in the IRES. We also generated 3 different transgenic (expressing the human poliovirus receptor, CD155) mouse tumor models: 1. Chemically induced model: CT2A-CD155 (C57BL/6-CD155), 2. Genetic model: Neuro Stem Cell-P53fl/fl-mPDGFB-CD155 (C57BL/6-CD155) and 3. Spontaneous tumor model: SMA560-CD155 (VM/DK-CD155). Conclusion: Here we have put together all the required tools to test the antitumor efficacy of a PVS-RIPO vector vaccine in vivo. Given that PVS-RIPO has some replication competence in muscle cells and also in tumor cells, we are planning to administer the mPVS-RIPO vector intramuscularly as well as intratumorally in mice and monitor its anti tumor efficacy. Citation Format: M Mubeen Mosaheb, Elena Dobrikova, Michael C. Brown, Christopher Pirozzi, Vidyalakshmi Chandramohan, Eda Holl, Smita Nair, Matthias Gromeier. Using oncolytic polio expressing tumor as a cancer vaccine [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A032.

Abstract LB-82: MLL2 maintains neoplastic cell growth and global histone H3 lysine 4 monomethylation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA One striking theme emerging from recent findings in cancer genetics is that chromatin remodeling and histone methylation modifiers are frequently altered in human cancers. Among these newly identified cancer genes, one of the best examples is the gene encoding MLL2 (KMT2D, a.k.a. ALR/MLL4), a histone lysine methyltransferase that plays an important role in regulating gene transcription. Genetic alterations suggestive of a functional deficiency in MLL2 and other genes in the same pathways are common. The identification of alterations in MLL2 suggests potential new opportunities for therapeutics, and highlights an urgent need to understand the underlying tumorigenic mechanism. Filling such a knowledge gap has been challenging, due to the lack of appropriate assays for the gigantic (∼600 kDa), understudied MLL2 protein. To overcome the difficulty, we have used innovative somatic gene editing-based assays to determine the effect of an MLL2 deficiency on neoplastic cells. In particular, we have used homologous recombination- and nuclease-mediated gene editing approaches to generate a panel of isogenic human cancer cell lines that differ with respect to their endogenous MLL2 status. Our studies found that an MLL2 deficiency results in attenuated cancer cell proliferation and defective cell migration. We identified direct transcriptional target genes and revealed the connection of MLL2 to multiple cellular signaling pathways. Analysis of histone H3 modifications revealed that MLL2 is essential for maintaining the level of global histone H3 lysine 4 (H3K4) monomethylation and that its enzymatic SET domain is directly responsible for this function. Furthermore, we found that a majority of MLL2 binding sites are located in regions of potential enhancer elements. The finding concerning enhancer elements is significant, as enhancer elements have increasingly been recognized as critically involved in tumorigenesis. Together, these findings revealed the role of MLL2 in mediating diverse signaling pathways and regulating enhancer elements in human cells, and shed light on the tumorigenic role of its deficiency. Our study supports that MLL2 has distinct roles in neoplastic cells, as opposed to pre-neoplastic cells, and that inhibiting MLL2 may be a viable strategy for cancer therapeutics. Citation Format: Changcun Guo, Lee H. Chen, Yafen Huang, Chun-chi Chang, Ping Wang, Christopher J. Pirozzi, Xiaoxia Qin, Xuhui Bao, Paula K. Greer, Roger E. McLendon, Hai Yan, Stephen T. Keir, Darell D. Bigner, Yiping He. MLL2 maintains neoplastic cell growth and global histone H3 lysine 4 monomethylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-82. doi:10.1158/1538-7445.AM2014-LB-82