Abstract BACKGROUND Pediatric high-grade gliomas (pHGG) harboring H3.3 K27M/G34R mutations present formidable challenges in treatment due to their aggressiveness and resistance to standard chemotherapy. Our preliminary investigations have shown promising results, indicating that combining 2-deoxyglucose (2DG), a glycolytic inhibitor, with DNA-damaging agents synergistically inhibits pHGG growth. However, prolonged exposure to 2DG induces resistance, necessitating the exploration of alternative therapies. This study aims to investigate the potential of panobinostat, a histone deacetylase inhibitor, in sensitizing 2DG-resistant pHGG with H3.3 K27M/G34R mutations to apoptosis. METHODS Transcriptomic analysis via Illumina NovaSeq 6000 and metabolomic profiling employing Liquid Chromatography-High-Resolution Mass Spectrometry was used to delineate the molecular landscape of naive and 2DG-resistant pHGG cell lines. Functional assays, including the Agilent Seahorse Mito Stress Test and flow cytometry, assessed bioenergetics and mitochondrial dynamics. Intracellular NAD+ and NADH levels were quantified utilizing NAD+/NADH Glo assays, while apoptotic markers and metabolic stress were measured using the Annexin V-FITC Conjugates for Apoptosis Detection kit and western blot. RESULTS Comparative analysis revealed increased transketolase activity in 2DG-resistant cells, augmenting the interaction between the pentose phosphate pathway and glycolysis, resulting in elevated levels of NADPH and ATP. Additionally, these resistant cells displayed enhanced reliance on mitochondrial energy metabolism, as evidenced by substantial increases in mitochondrial membrane potential and mass, coupled with elevated NAD+ levels. Subsequent treatment with panobinostat induced a significant decrease in NAD+ biosynthesis and ATP generation. Furthermore, this intervention triggered the upregulation of pro-apoptotic BCL-2 family proteins, particularly BAX and BAK, concomitant with a notable reduction in mitochondrial membrane potential and the accumulation of reactive oxygen species. Consequently, heightened caspase-3 activity ensued, leading to significantly elevated cell death rates in 2DG-resistant gliomas. CONCLUSION Our findings highlight panobinostat as a promising therapy to resensitize 2DG-resistant pHGG cells to apoptosis, offering hope for treating these aggressive tumors with H3.3 K27M/G34R mutations.

Abstract Background: Human high-grade gliomas are aggressive brain cancers known for their resistance to treatment. Targeted therapies with single agents have shown limited success, and the presence of tumor heterogeneity complicates treatment. Combining multiple chemotherapeutic agents has emerged as a potential strategy to overcome these challenges. 2-deoxy-D-glucose (2-DG) is a glucose analog known for its ability to disrupt glycolysis, protein folding, and the pentose phosphate pathway. This disruption renders cancer cells more susceptible to further damage from reactive oxygen species and DNA damage. Previous studies have demonstrated synergistic effects when combining 2-DG with chemotherapeutic drugs in various cancers. Our study examines combining 2-DG with DNA-damaging agents for high-grade gliomas to uncover potential synergies, theorizing that this combination enhances cell death and metabolic stress, offering insights for better treatments against these tumors. Methods: The cell proliferation, clonogenic growth, and cell migration assays assessed viability, clonogenic formation, and migration. Additionally, the cell cycle and reactive oxygen species analysis were employed to evaluate cell division, oxidative stress, and antioxidant capacity. The Illumina NovaSeq 6000 platform measured transcript levels, and Liquid Chromatography-High-Resolution Mass Spectrometry analyzed metabolite changes in high-grade glioma cell lines following 2-DG treatment. The Agilent Seahorse Mito Stress Test evaluated alterations in cellular bioenergetics. SYNERGYFINDER 3 quantified synergistic effects of 2-DG with DNA-damaging agents, while the Annexin V-FITC Conjugates and a ROS multiplex assay assessed cell death and metabolic stress. Results: Our findings demonstrate that when used as a single agent, 2-DG induces cell senescence and limits cell migration but is insufficient to trigger cell death. Moreover, 2-DG treatment shifts glioma cells towards oxidative phosphorylation for energy production, elevates ROS levels, and hampers DNA synthesis and repair mechanisms. Our synergy study reveals promising synergistic effects when combining 2-DG with DNA-damaging agents, including 5-FU, Methotrexate, Gemcitabine, and Lomustine. This combined therapy leads to significant ROS generation and increased cell death compared to single-agent treatments. Furthermore, prolonged exposure to 2-DG leads to increased levels of both glucogenic and ketogenic amino acids within glioma cells, potentially serving as an alternative energy source. This finding hints at a prospective therapeutic approach for targeting drug-resistant cells. Conclusions: Our results suggest that the anticancer therapeutic effect of 2-DG is enhanced when used in combination with DNA-damaging agents, indicating this strategy as a potential approach for treating human high-grade glioma. Citation Format: Tracy Miller, Esther Jane, Matthew Halbert, Taylor Gatesman, Stacey Wendell, Dinesh Mohanakrishnan, Sameer Agnihotri, Daniel Premkumar, Ian Pollack. Enhanced chemosensitivity to DNA-damaging agents in human high-grade glioma through 2-DG treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2037.

Abstract BACKGROUND Glioblastoma is the most common primary central nervous system tumour with a median survival of less than 15 months. Addressing the underlying redundant, and converging signaling pathways necessitates the utilization of small molecule inhibitors capable of simultaneous targeting and inhibition. In glioma, the aberrant CDK4/6-Rb pathway dysregulation resulting in disruption of cell cycle checkpoint progression can lead to uncontrolled cell division, a hallmark of cancer. The neuro-pharmacokinetics of abemaciclib, a Cyclin-dependent kinase inhibitor, is being currently investigated in phase 1 clinical trials by National Cancer Institue, [NCT05413304]. The importance of panobinostat as a Histone deacetylase inhibitor and potentiating the effects of various small molecular inhibitors in published literature is paramount. This study is based on investigating ways to overcome resistance by exploring the synergistic cytotoxic effects of abemaciclib in combination with panobinostat in glioma. METHODS We investigated the antiproliferative effects of abemaciclib and panobinostat on glioma cells using an MTS cell proliferation assay. Inhibitor combination analysis was done using, SYNERGYFINDER via zip-model. Flow cytometry with Annexin V staining further assessed cell death and apoptosis induced. RESULTS The antiproliferative effects of abemaciclib, alone and in combination with panobinostat, in pediatric lines (KNS 42, SJG2), and adult human glioblastoma line, SF188 exhibited lower IC50 as compared to adult high grade glioma lines (LN 18, LNZ 308). Synergy analysis revealed a strong cytotoxic effect in KNS 42 and SF 188 cells treated with the combination. Flow cytometric analysis indicated that the combination therapy induced significant cell death only in a subset of cell lines- KNS 42 and SF 188, suggesting underlying genetic heterogeneity influences treatment response, warranting further mechanistic studies. CONCLUSION Combination therapies using abemaciclib and panobinostat, targeting CDK4/6 and histone deacetylation pathways, show promise against aggressive and treatment-resistant gliomas. Further investigation into these combination approaches is crucial to understand resistant mechanisms, optimize efficacy, and establish their clinical efficacy for improving glioma patient outcomes.

Abstract Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. However, there remains a knowledge gap in identifying effectors of ERK signaling and their roles in DMG-H3K27a. Leveraging several patient multi-omic datasets, we identified a unique relationship between ERK5 and brain tumor glycolysis. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a, where ERK5 knockdown leads to decreases in glycolysis and increases in mitochondrial metabolism. We demonstrate through loss of function and overexpression studies that ERK5 mediates glycolysis through regulation of glycolytic enzyme Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (PFKFB3). PFKFB3 is a protein that catalyzes the synthesis or degradation of fructose-2,6-bisphosphate (F2,6BP). Furthermore, we establish a novel mechanism, in which ERK5 mediates PFKFB3 expression via activation of MEF2A, an oncogenic transcription factor. DMG-H3K27a cells are sensitive to the loss or inhibition of PFKFB3 both in vitro and in orthotopic mouse models. However, monotherapy is ineffective in heterogenous diseases such as gliomas, including DMG-H3K27a. To combat this, we show multi-targeted therapy against the ERK5-PFKFB3 axis is synergistic in vitro and in vivo. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma. Moreover, we have identified resistance to our dual therapy using several systems biology approaches, which we are currently investigating and validating. Collectively, our study defines a novel, targetable axis in DMG-H3K27a and highlights the importance of tumor cell metabolism in defining therapeutic vulnerabilities.

Supplementary Figures 1-6 from Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF-κB Signaling Pathway

In previous studies, we demonstrated that panobinostat, a histone deacetylase inhibitor, and bortezomib, a proteasomal inhibitor, displayed synergistic therapeutic activity against pediatric and adult high-grade gliomas. Despite the remarkable initial response to this combination, resistance emerged. Here, in this study, we aimed to investigate the molecular mechanisms underlying the anticancer effects of panobinostat and marizomib, a brain-penetrant proteasomal inhibitor, and the potential for exploitable vulnerabilities associated with acquired resistance. RNA sequencing followed by gene set enrichment analysis (GSEA) was employed to compare the molecular signatures enriched in resistant compared with drug-naïve cells. The levels of adenosine 5'-triphosphate (ATP), nicotinamide adenine dinucleotide (NAD)+ content, hexokinase activity, and tricarboxylic acid (TCA) cycle metabolites required for oxidative phosphorylation to meet their bioenergetic needs were analyzed. Here, we report that panobinostat and marizomib significantly depleted ATP and NAD+ content, increased mitochondrial permeability and reactive oxygen species generation, and promoted apoptosis in pediatric and adult glioma cell lines at initial treatment. However, resistant cells exhibited increased levels of TCA cycle metabolites, which required for oxidative phosphorylation to meet their bioenergetic needs. Therefore, we targeted glycolysis and the electron transport chain (ETC) with small molecule inhibitors, which displayed substantial efficacy, suggesting that resistant cell survival is dependent on glycolytic and ETC complexes. To verify these observations in vivo, lonidamine, an inhibitor of glycolysis and mitochondrial function, was chosen. We produced two diffuse intrinsic pontine glioma (DIPG) models, and lonidamine treatment significantly increased median survival in both models, with particularly dramatic effects in panobinostat- and marizomib-resistant cells. These data provide new insights into mechanisms of treatment resistance in gliomas.

Supplemental File 1: Differential Gene Expression between drug naive versus resistant/"recovery" U87 cell line

PDF file - 69K, Effect of YM-155 and ABT-737 on human astrocytes

Abstract Diffuse midline glioma is an aggressive brain tumor with a median age at diagnosis of 6.3 years and 5-year survival rate of 2.2%.The defining histone mutation H3K27M precludes docking of a protein responsible for epigenetic modification resulting in erroneously accessible chromatin and subsequent transcription of oncogenic pathways, including the RAS-MERK5-ERK5 signaling cascade. To determine which oncogenic processes are regulated by extracellular signal-regulated kinase 5 (ERK5), gene-set enrichment analysis of patient-derived datasets demonstrated gene networks involved in glycolysis to be enriched with ERK5 expression. In confirmation, loss of ERK5 via shRNA interference reduced cell proliferation and glycolysis in DIPG IV and SF8628 cells. Reintroduction of ERK5 wildtype (WT) into ERK5 knockdown lines rescued cell proliferation and glycolysis, while the addition of ERK5 kinase dead domain (KDD) only partially rescued these survival and metabolic defects. Targeted evaluation of glycolysis enzyme expression via qRT-PCR revealed a direct relationship between ERK5 and proglycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Mechanistically, ERK5 activation of PFKFB3 was mediated by activation of transcriptional factor myocyte enhancer factor 2A (MEF2A) as demonstrated by coimmunoprecipitation and luciferase promoter assays. Expression of PFKFB3 was elevated at both the mRNA and protein level in DMG patient-derived samples. Genetic knockdown of PFKFB3 via shRNA interference resulted in reduced proliferation and glycolysis in DIPG IV and SF8628 cells. Similarly, pharmacologic inhibition of PFKFB3 with small molecule inhibitor PFK-158 capitulated these results. This inhibitor demonstrated blood brain barrier penetrance in silico and extended survival of in vivo mouse models. Multitargeted drug therapy against both ERK5 and PFKFB3 produced a synergetic in vitro response with increased sensitivity of these cells to apoptosis compared to single treatment alone. In conclusion, these results support ERK5 regulation of glycolysis through the critical metabolic effector PFKFB3. Multitargeted drug therapy against this axis represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A ( MAT2A ) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S -adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

H3K27-mutant diffuse midline gliomas (DMGs) are defined as grade IV tumors by the World Health Organization. DMGs are inoperable and resistant to chemo/radio therapies. Median survival ranges from 8-11 months, with 2% of patients surviving beyond 5 years. H3K27M mutations lead to global epigenetic and transcriptional reprogramming driven by global loss of negative transcriptional regulator H3K27 trimethylation (H3K27me3). Loss of H3K27me3 is an initiating event in gliomagenesis. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model. An unbiased integrated systems biology approach identified that H3K27M but not isogenic controls relied on the amino acid methionine and the enzyme Methionine Adenosyltransferase 2A (MAT2A). MAT2A is a central regulator of one-carbon metabolism by converting methionine to S-adenosylmethionine (SAM), the universal methyl-donor for protein and nucleotide methylation reactions. In complementary genetic approaches, we applied these findings to patient-derived cell lines with the H3K27M mutation. We hypothesize that MAT2A abrogation, genetic/pharmacological, would alter DMG viability by disrupting the methylome. The current MAT2A sensitivity paradigm is based on Methylthioadenosine Phosphorylase (MTAP) deletion through a synthetic lethal mechanism. We provide a novel mechanism whereby H3K27M cells are sensitive to MAT2A loss, independent of MTAP and through Adenosylmethionine Decarboxylase 1 (AMD1) overexpression disrupting MAT2A regulation. This results in H3K27M cells having lower MAT2A protein levels, conferring a sensitivity by inhibiting residual MAT2A. Genetic/pharmacological aberrations to MAT2A resulted in reduced proliferation. Parallel H3K36me3 ChIP and RNA-sequencing identified loss of oncogenic and developmental transcriptional programs associated with MAT2A loss. In vivo syngeneic and patient-derived xenograft models with both inducible MAT2A knockdown or methionine restricted diets showed extended survival. These results suggest novel interactions between methionine metabolism and the epigenome of H3K27M gliomas and provide evidence that MAT2A, presents exploitable therapeutic vulnerabilities in histone mutant gliomas.

Diffuse midline gliomas (DMGs) and other high-grade gliomas (HGG) are the most commonly lethal intrinsic brain tumors of childhood. Accordingly, novel treatment approaches are needed that consider the heterogeneity of these tumors. Using an extensive pharmacological screen, we identified several two-drug combinations that were particularly effective against a range of DMG and HGG cell lines, the most active being the combination of the histone deacetylase inhibitor, panobinostat, and a proteasome inhibitor, marizomib, both in clinical trials, which synergistically induced tumor-specific apoptosis. However, at clinically achievable concentrations, a small population of resistant tumor cells survived and dominated, mimicking the clinical scenario, which prompted our development of drug resistance models as mechanistic tools. Resistance mechanisms were examined using RNA sequencing of drug-resistant versus -naïve cells. NMNAT2, a critical mediator of NAD synthesis, was overexpressed in DIPGs in the context of high baseline QPRT expression, suggesting a critical role of NAD synthesis and utilization pathways in resistance. Gene-set enrichment analysis suggested a key role for glycolytic pathways in the resistance phenotype. In support of this hypothesis, we observed increased levels of mitochondrial mass and NAD and ATP in resistant cells compared to drug-naïve cells, as well as upregulation of glycolytic intermediates by LC/MS. Conversely, inhibition of OXPHOS and glycolysis pathways resensitized resistant glioma cells to apoptosis in vitro. To test this hypothesis in vivo, mice were injected with drug-naïve or resistant DIPG-013 glioma cells and were exposed to an OXPHOS and glycolysis inhibitor, lonidamine. Lonidamine alone extended survival of naïve (18.6 + 1.4 d vs. 25 + 1.6 d, p=0.0003) and resistant (18.7 + 1 d vs. 62 + 1 d, p=0.0002) DIPG13p models (Log-Rank). This supports the application of metabolic therapy as a novel strategy for treating resistant MGs that has activity in a clinically relevant DIPG model. Supported by NIHS10OD023402, the DIPG/DMG Collaborative, the CNI, and Connor’s Cure.

Acquired resistance to conventional chemotherapeutic agents limits their effectiveness and can cause cancer treatment to fail. Because enzymes in the aurora kinase family are vital regulators of several mitotic events, we reasoned that targeting these kinases with tozasertib, a pan‐aurora kinase inhibitor, would not only cause cytokinesis defects, but also induce cell death in high‐grade pediatric and adult glioma cell lines. We found that tozasertib induced cell cycle arrest, increased mitochondrial permeability and reactive oxygen species generation, inhibited cell growth and migration, and promoted cellular senescence and pro‐apoptotic activity. However, sustained exposure to tozasertib at clinically relevant concentrations conferred resistance, which led us to examine the mechanistic basis for the emergence of drug resistance. RNA‐sequence analysis revealed a significant upregulation of the gene encoding pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), a pyruvate dehydrogenase (PDH) inhibitory kinase that plays a crucial role in the control of metabolic flexibility under various physiological conditions. Upregulation of PDK1, PDK2, PDK3, or PDK4 protein levels was positively correlated with tozasertib‐induced resistance through inhibition of PDH activity. Tozasertib‐resistant cells exhibited increased mitochondrial mass as measured by 10‐N‐nonyl‐Acridine Orange. Inhibition of PDK with dichloroacetate resulted in increased mitochondrial permeability and cell death in tozasertib‐resistant glioma cell lines. Based on these results, we believe that PDK is a selective target for the tozasertib resistance phenotype and should be considered for further preclinical evaluations.

Abstract To improve therapeutic responses in patients with glioma, new combination therapies that exploit a mechanistic understanding of the inevitable emergence of drug resistance are needed. Intratumoral heterogeneity enables a low barrier to resistance in individual patients with glioma. We reasoned that targeting two or more fundamental processes that gliomas are particularly dependent upon could result in pleiotropic effects that would reduce the diversity of resistant subpopulations allowing convergence to a more robust therapeutic strategy. In contrast to the cytostatic responses observed with each drug alone, the combination of the histone deacetylase inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induced apoptosis of adult and pediatric glioma cell lines at clinically achievable doses. Resistance that developed was examined using RNA-sequencing and pharmacologic screening of resistant versus drug-naïve cells. Quinolinic acid phosphoribosyltransferase (QPRT), the rate-determining enzyme for de novo synthesis of NAD+ from tryptophan, exhibited particularly high differential gene expression in resistant U87 cells and protein expression in all resistant lines tested. Reducing QPRT expression reversed resistance, suggesting that QPRT is a selective and targetable dependency for the panobinostat–bortezomib resistance phenotype. Pharmacologic inhibition of either NAD+ biosynthesis or processes such as DNA repair that consume NAD+ or their simultaneous inhibition with drug combinations, specifically enhanced apoptosis in treatment-resistant cells. Concomitantly, de novo vulnerabilities to known drugs were observed. Implications: These data provide new insights into mechanisms of treatment resistance in gliomas, hold promise for targeting recurrent disease, and provide a potential strategy for further exploration of next-generation inhibitors.

Resistance of tumor cells to the induction of apoptosis is an important reason for the failure of anticancer treatments in patients with gliomas. Several factors working in concert have been implicated as sources of this treatment resistance; therefore, innovative therapeutic approaches that conspire to attack key tumor vulnerabilities are needed. HDAC and proteasome inhibitors are two classes of agents that have shown some benefit in the clinic, but patients often rapidly manifest intrinsic or acquired resistance mechanisms that limits their individual efficacy. We demonstrate that the combination of the HDAC inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induces apoptosis of human glioma cell lines. However, acquired resistance is observed even with this initially effective combination. To examine the mechanism of this resistance, we performed RNA sequencing and pharmacological screening of resistant compared to sensitive cells. Based on these studies, we present evidence that quinolinic acid phosphoribosyltransferase (QPRT), an enzyme catalyzes a rate determining step in de novo NAD+ biosynthesis, provides a critical adaptive survival mechanism that allows cancer cells to evade an initially effective therapeutic combination. We identified 1004 genes that had a significant change between panobinostat and bortezomib- resistant cells versus inhibitor naïve control cells. Furthermore, pathway analysis revealed that the experimental regimen significantly altered metabolic pathways. By silencing QPRT (using siRNA) we demonstrated that we can overcome resistance, thus suggesting QPRT as a crucial crossroad for cancer cell survival and as a new anticancer target. We also showed that treatment of panobinostat and bortezomib resistant cells with FK866, niraparib, selisistat, epacadostat, gemcitabine, 5-fluoruracil, and methotrexate significantly increased cell death highlighting the importance of NAD+ and folate pathway inhibitors in resensitizing the resistant cells. Together, targeting QPRT or NAD+ consuming enzymes hold promise for eliminating recurrent disease in glioma. Citation Format: Esther P. Jane, Daniel R. Premkumar, Sameer Agnihotri, Max Myers, Ansuman Chattopadhyay, D. Lansing Taylor, Mark Schurdak, Andrew Stern, Ian F. Pollack. Gene signatures identify quinolate phosphoribosyltransferase as a key mediator of acquired resistance to Panobinostat and Bortezomib in glioma, and NAD+ biosynthesis as a targetable pathway to reverse treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3014.

In the present study, we investigated the effect of CDK inhibitors (ribociclib, palbociclib, seliciclib, AZD5438, and dinaciclib) on malignant human glioma cells for cell viability, apoptosis, oxidative stress, and mitochondrial function using various assays. None of the CDK inhibitors induced cell death at a clinically relevant concentration. However, low nanomolar concentrations of dinaciclib showed higher cytotoxic activity against Bcl‐xL silenced cells in a time‐ and concentration‐dependent manner. This effect was not seen with other CDK inhibitors. The apoptosis‐inducing capability of dinaciclib in Bcl‐xL silenced cells was evidenced by cell shrinkage, mitochondrial dysfunction, DNA damage, and increased phosphatidylserine externalization. Dinaciclib was found to disrupt mitochondrial membrane potential, resulting in the release of cytochrome c, AIF, and smac/DIABLO into the cytoplasm. This was accompanied by the downregulation of cyclin‐D1, D3, and total Rb. Dinaciclib caused cell cycle arrest in a time‐ and concentration‐dependent manner and with accumulation of cells in the sub‐G1 phase. Our results also revealed that dinaciclib, but not ribociclib or palbociclib or seliciclib or AZD5438 induced intrinsic apoptosis via upregulation of the levels of pro‐apoptotic proteins (Bax and Bak), resulting in the activation of caspases and cleavage of PARP. We also found an additional mechanism for the dinaciclib‐induced augmentation of apoptosis due to abrogation RAD51‐cyclin D1 interaction, specifically proteolysis of the DNA repair proteins RAD51 and Ku80. Our results suggest that successfully interfering with Bcl‐xL function may restore sensitivity to dinaciclib and could hold the promise for an effective combination therapeutic strategy.