Inhibition Of Purine Synthesis Research Articles

Severe distributive shock, neutrophilic dermatosis, and ST-elevation myocardial infarction in the setting of azathioprine hypersensitivity syndrome

BackgroundAzathioprine is a purine synthesis inhibitor used as an immunosuppressive therapy for many immune-mediated diseases. Azathioprine hypersensitivity reaction is a rare, life-threatening adverse reaction characterized by a range of multisystem manifestations including fever, abdominal pain, arthralgias, erythematous cutaneous eruption, acute renal failure, neutrophilia, and more rarely, distributive shock. Although acute heart failure has been rarely described in association with azathioprine hypersensitivity syndrome, myocardial infarction has, to our knowledge, never been associated with this entity.Case PresentationWe describe a case of a 59-year-old male with Crohn’s disease who developed severe azathioprine hypersensitivity syndrome that included distributive shock, neutrophilic dermatosis, and acute coronary syndrome with ST-elevation. Clinical improvement was seen after cessation of azathioprine and administration of glucocorticoid therapy.ConclusionPrompt recognition of azathioprine hypersensitivity syndrome, which can manifest as shock and neutrophilic dermatosis, is key to ensure rapid azathioprine cessation.

Purine salvage promotes treatment resistance in H3K27M-mutant diffuse midline glioma

BackgroundDiffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT) but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain.MethodsWe performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and our models, quantified purine synthesis using stable isotope tracing, and characterized the in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT.ResultsDMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novo synthesis and apparent lower activity of purine salvage demonstrated via stable isotope tracing of key metabolites in purine synthesis and by lower expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the rate-limiting enzyme of purine salvage into IMP and GMP. Inhibition of de novo guanylate synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells upregulated HGPRT expression and hypoxanthine-derived guanylate salvage but maintained high levels of guanine-derived salvage. Exogenous guanine supplementation decreased radiosensitization in cells treated with combination RT and de novo purine synthesis inhibition. Silencing HGPRT combined with RT markedly suppressed DMG-H3K27M tumor growth in vivo.ConclusionsOur results indicate that DMG-H3K27M cells rely on highly active purine synthesis, both from the de novo and salvage synthesis pathways. However, highly active salvage of free purine bases into mature guanylates can bypass inhibition of the de novo synthetic pathway. We conclude that inhibiting purine salvage may be a promising strategy to overcome treatment resistance in DMG-H3K27M tumors.

In Vitro Profiling of Commonly Used Post-transplant Immunosuppressants Reveals Distinct Impact on Antiviral T-cell Immunity Towards CMV.

Infectious complications, including widespread human cytomegalovirus (CMV) disease, frequently occur after hematopoietic stem cell and solid organ transplantation due to immunosuppressive treatment causing impairment of T-cell immunity. Therefore, in-depth analysis of the impact of immunosuppressants on antiviral T cells is needed. We analyzed the impact of mTOR inhibitors sirolimus (SIR/S) and everolimus (EVR/E), calcineurin inhibitor tacrolimus (TAC/T), purine synthesis inhibitor mycophenolic acid (MPA/M), glucocorticoid prednisolone (PRE/P) and common double (T+S/E/M/P) and triple (T+S/E/M+P) combinations on antiviral T-cell functionality. T-cell activation and effector molecule production upon antigenic stimulation was impaired in presence of T+P and triple combinations. SIR, EVR and MPA exclusively inhibited T-cell proliferation, TAC inhibited activation and cytokine production and PRE inhibited various aspects of T-cell functionality including cytotoxicity. This was reflected in an in vitro infection model, where elimination of CMV-infected human fibroblasts by CMV-specific T cells was reduced in presence of PRE and all triple combinations. CMV-specific memory T cells were inhibited by TAC and PRE, which was also reflected with double (T+P) and triple combinations. EBV- and SARS-CoV-2-specific T cells were similarly affected. These results highlight the need to optimize immune monitoring to identify patients who may benefit from individually tailored immunosuppression.

Nucleoside supplements as treatments for mitochondrial DNA depletion syndrome.

Introduction: In mitochondrial DNA (mtDNA) depletion syndrome (MDS), patients cannot maintain sufficient mtDNA for their energy needs. MDS presentations range from infantile encephalopathy with hepatopathy (Alpers syndrome) to adult chronic progressive external ophthalmoplegia. Most are caused by nucleotide imbalance or by defects in the mtDNA replisome. There is currently no curative treatment available. Nucleoside therapy is a promising experimental treatment for TK2 deficiency, where patients are supplemented with exogenous deoxypyrimidines. We aimed to explore the benefits of nucleoside supplementation in POLG and TWNK deficient fibroblasts. Methods: We used high-content fluorescence microscopy with software-based image analysis to assay mtDNA content and membrane potential quantitatively, using vital dyes PicoGreen and MitoTracker Red CMXRos respectively. We tested the effect of 15 combinations (A, T, G, C, AT, AC, AG, CT, CG, GT, ATC, ATG, AGC, TGC, ATGC) of deoxynucleoside supplements on mtDNA content of fibroblasts derived from four patients with MDS (POLG1, POLG2, DGUOK, TWNK) in both a replicating (10% dialysed FCS) and quiescent (0.1% dialysed FCS) state. We used qPCR to measure mtDNA content of supplemented and non-supplemented fibroblasts following mtDNA depletion using 20µM ddC and after 14- and 21-day recovery in a quiescent state. Results: Nucleoside treatments at 200µM that significantly increased mtDNA content also significantly reduced the number of cells remaining in culture after 7days of treatment, as well as mitochondrial membrane potential. These toxic effects were abolished by reducing the concentration of nucleosides to 50µM. In POLG1 and TWNK cells the combination of ATGC treatment increased mtDNA content the most after 7days in non-replicating cells. ATGC nucleoside combination significantly increased the rate of mtDNA recovery in quiescent POLG1 cells following mtDNA depletion by ddC. Conclusion: High-content imaging enabled us to link mtDNA copy number with key read-outs linked to patient wellbeing. Elevated G increased mtDNA copy number but severely impaired fibroblast growth, potentially by inhibiting purine synthesis and/or causing replication stress. Combinations of nucleosides ATGC, T, or TC, benefited growth of cells harbouring POLG mutations. These combinations, one of which reflects a commercially available preparation, could be explored further for treatment of POLG patients.

CTNI-61. MYCOPHENOLATE MOFETIL INHIBITS GLIOBLASTOMA PURINE METABOLISM IN HUMANS: AN INTERIM RESULT OF PHASE 0/1 CLINICAL TRIAL TO OVERCOME TREATMENT RESISTANCE IN NEWLY DIAGNOSED AND RECURRENT GLIOBLASTOMA

Abstract BACKGROUND Mycophenolate mofetil (MMF), a purine synthesis inhibitor, improves radiation and temozolomide efficacy in preclinical glioblastoma models. This phase 0/1 trial (NCT04477200) is the first-in-human study to assess the tolerability of MMF with chemoradiation in glioblastoma, MPA accumulation, and purine synthesis inhibition in tumor. METHODS Adult patients meeting eligibility are given MMF 500-2000 PO BID using TITE-CRM dose assignment. Thirty recurrent glioblastoma patients receive MMF one-week prior to and concurrently with re-irradiation (40.5 Gy), with 28 days of dose-limiting toxicity (DLT) period. Thirty newly-diagnosed glioblastoma patients receive MMF one-week prior to and concurrently with chemoradiation and followed for 28 days for DLT1-period. During the adjuvant temozolomide (days 1-5/28-day cycle) period, MMF is given days 0-5 on a separate dose-escalation (DLT2 period = cycles 1-2). Tissues from phase-0 recurrent glioblastoma patients (N = 8) receiving MMF 500-2000mg BID one-week pre-operatively are analyzed using mass spectrometry. RESULTS Enhancing and non-enhancing tumor from phase 0 subjects yielded above 1 µM active drug metabolite, and the GTP/IMP ratio was decreased by 75% in enhancing tumor in MMF-treated patients compared to untreated controls (p = 0.009), indicating effective target engagement and inhibition of purine synthesis. No DLT has been observed with adjuvant cyclic temozolomide in phase 1 newly-diagnosed group (N = 26), nor in the recurrent-disease group (N = 18) with re-irradiation. In newly diagnosed arm, 3 subjects experienced DLT1 at 2000mg BID; grade-3 hemiparesis and cognitive disturbance, and grade-4 thrombocytopenia. Other main toxicities are mild nausea and fatigue. Interim median overall survival in recurrent phase 1 is 15.6 months, and not reached yet in newly diagnosed phase 1. CONCLUSION MMF combined with chemoradiation has been reasonably well tolerated in glioblastoma patients with preliminary promising evidence of intracranial target engagement of its active metabolite. This study will yield a recommended phase 2 dose and preliminary efficacy estimate for future randomized phase 2/3 trial.

TMET-13. DNA DAMAGE SIGNALING ACTIVATES GTP SYNTHESIS TO PROMOTE GLIOBLASTOMA TREATMENT RESISTANCE

Abstract Glioblastoma (GBM) is the most common type of invasive brain tumor in adults and is uniformly fatal due to inherent resistance to radiation therapy (RT) and chemotherapy. Our group and others have found that metabolites can regulate DNA repair and therapy resistance in brain tumors, but little is known about how DNA damage regulates metabolic pathway activity in cancer. Here we show that following treatment with RT, GBMs increase rates of de novo guanylate synthesis in vitro and in orthotopic patient-derived xenograft models via signaling through the DNA repair protein DNA-PK. To determine if disrupting this regulation can augment GBM treatment efficacy, we combined an FDA-approved inhibitor of purine synthesis (mycophenolate mofetil, MMF) with chemoradiation in a variety of mouse models of GBM. Critically, targeting GTP synthesis improved the efficacy of both RT alone and chemoradiation in multiple patient-derived and syngeneic intracranial models. Our translational studies have shown that mycophenolic acid, the active metabolite of MMF, penetrates the blood-brain barrier at concentrations sufficient to inhibit GTP synthesis in GBMs of human patients. The phase 1 arms of our study, which are ongoing, are testing the safety and efficacy of combining MMF with standard of care chemoradiation for patients with primary and recurrent GBM.

A Phase 0 Study Assessing the Intracranial Activity of a Metabolic Radiosensitizer in Patients with Glioblastoma

Efforts to overcome treatment resistance in glioblastoma (GBM) have been unsuccessful due to tumor heterogeneity and poor intracranial drug penetration. Targeting altered metabolism is a promising approach to improve GBM therapy despite this heterogeneity. Mycophenolate mofetil (MMF) is an inhibitor of purine synthesis that sensitizes GBM to radiation and temozolomide (TMZ) in vitro and in vivo, but its ability to cross the blood brain barrier and inhibit GBM metabolism in patients is unknown. NCT04477200 is a phase 0/1 dose escalation study of MMF combined with radiation and temozolomide in GBM. Here we report the phase 0 results of this study assessing the intracranial activity of MMF. Purine (GTP and IMP) and mycophenolic acid (MPA, the active metabolite of MMF) concentrations were determined using mass spectrometry in flash-frozen tumor (enhancing and non-enhancing) and normal cortex obtained from 8 patients with recurrent GBM who received MMF (500, 1000, 1500 and 2000 mg BID, N = 2 patients each dose level) for 1 week prior to re-resection and 5 control patients who did not receive MMF prior to re-resection. Plasma MPA concentration was similarly quantified to calculate the enhancing tumor, non-enhancing tumor and normal cortex to plasma MPA ratios. Patients who received MMF had a mean MPA concentration of 2.2 ± 0.7 µM in the enhancing tumor samples, 1.2 ± 0.5 µM in the non-enhancing tumor samples and 1.3 ± 0.5 µM in normal cortex. MPA concentration was negligible in control patients. This corresponded to tissue/plasma MPA ratios of 0.31, 0.17 and 0.10 for enhancing tumor, non-enhancing tumor and normal cortex, respectively. The GTP/IMP ratio was decreased by 75% in enhancing tumor in MMF-treated patients compared to untreated controls (p = 0.009), indicating effective target engagement and inhibition of purine synthesis. The GTP/IMP ratio was also decreased in cortex and non-enhancing tumor, though a paucity of control samples prevented statistical analysis. Twice daily MMF treatment yields intracranial drug concentrations above 1 µM and lowers the GTP/IMP ratio in GBMs, consistent with target engagement. As we have previously observed radiosensitization in vitro with MPA concentrations of 1 µM, these data suggest that MMF may achieve adequate CNS penetration for therapeutic benefit. The Phase 1 component of this study to determine the dose limiting toxicity and maximally tolerated dose of MMF when combined with reirradiation in recurrent GBM and radiation and TMZ in newly diagnosed GBM is ongoing.

Spatiotemporal Regulation of De Novo and Salvage Purine Synthesis during Brain Development.

The levels of purines, essential molecules to sustain eukaryotic cell homeostasis, are regulated by the coordination of the de novo and salvage synthesis pathways. In the embryonic central nervous system (CNS), the de novo pathway is considered crucial to meet the requirements for the active proliferation of neural stem/progenitor cells (NSPCs). However, how these two pathways are balanced or separately used during CNS development remains poorly understood. In this study, we showed a dynamic shift in pathway utilization, with greater reliance on the de novo pathway during embryonic stages and on the salvage pathway in postnatal-adult mouse brain. The pharmacological effects of various purine synthesis inhibitors in vitro and the expression profile of purine synthesis enzymes indicated that NSPCs in the embryonic cerebrum mainly use the de novo pathway. Simultaneously, NSPCs in the cerebellum require both the de novo and the salvage pathways. In vivo administration of de novo inhibitors resulted in severe hypoplasia of the forebrain cortical region, indicating a gradient of purine demand along the anteroposterior axis of the embryonic brain, with cortical areas of the dorsal forebrain having higher purine requirements than ventral or posterior areas such as the striatum and thalamus. This histologic defect of the neocortex was accompanied by strong downregulation of the mechanistic target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase (S6K)/S6 signaling cascade, a crucial pathway for cell metabolism, growth, and survival. These findings indicate the importance of the spatiotemporal regulation of both purine pathways for mTORC1 signaling and proper brain development.

Abstract P1124: Immunosuppression Drug Mediated Cardiovascular Effects

Heart transplantation continues to be a life-saving therapy for patients with end-stage heart failure; yet the average long-term graft survival remains at only 12.5 years. Cardiac allograft vasculopathy (CAV) and cardiac graft dysfunction are both key contributors to long-term morbidity and mortality in the heart transplant population. Studies have suggested that immunosuppressive agents may modulate cardiovascular function, yet the mechanisms for this modulation is incompletely understood. The primary classes of immunosuppressive agents utilized post-heart transplantation are mammalian target of rapamycin inhibitors (e.g. sirolimus), calcineurin inhibitors (e.g. tacrolimus), and purine synthesis inhibitors (e.g. mycophenolate mofetil). Our prior findings indicate no discernible change to cell contraction or viability, but increased fibrosis and adverse remodeling in response to tacrolimus treatment. Using cardiac organoids; composed of human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes, endothelial cells, and cardiac fibroblasts; we investigated the effects of these agents on the cardiovascular system using RNA-sequencing and proteomic analysis. Proteomic analysis of cardiac organoids was done using Luminex bead-array assay. Treatment with MPA coincided with a significant reduction of proinflammatory cytokines including the various interleukin-families and TNF-alpha/beta. Tacrolimus yielded mixed results on proteins associated with cardiac remodeling but indicated significant upregulation of proteins that have been implicated in CAV progression. In addition, RNA-sequencing findings revealed significant upregulation of genes associated with cardiac fibrosis and remodeling with tacrolimus treatment in comparison to sirolimus treatment. These findings correlate with functional profile observed on treatment of cardiac organoids. Our results provider further insight into the pathways by which differential immunosuppression drugs influence cardiovascular remodeling.

Therapeutic targeting of metabolic vulnerabilities in cancers with MLL3/4-COMPASS epigenetic regulator mutations.

Epigenetic status-altering mutations in chromatin-modifying enzymes are a feature of human diseases, including many cancers. However, the functional outcomes and cellular dependencies arising from these mutations remain unresolved. In this study, we investigated cellular dependencies, or vulnerabilities, that arise when enhancer function is compromised by loss of the frequently mutated COMPASS family members MLL3 and MLL4. CRISPR dropout screens in MLL3/4-depleted mouse embryonic stem cells (mESCs) revealed synthetic lethality upon suppression of purine and pyrimidine nucleotide synthesis pathways. Consistently, we observed a shift in metabolic activity toward increased purine synthesis in MLL3/4-KO mESCs. These cells also exhibited enhanced sensitivity to the purine synthesis inhibitor lometrexol, which induced a unique gene expression signature. RNA-Seq identified the top MLL3/4 target genes coinciding with suppression of purine metabolism, and tandem mass tag proteomic profiling further confirmed upregulation of purine synthesis in MLL3/4-KO cells. Mechanistically, we demonstrated that compensation by MLL1/COMPASS was underlying these effects. Finally, we demonstrated that tumors with MLL3 and/or MLL4 mutations were highly sensitive to lometrexol in vitro and in vivo, both in culture and in animal models of cancer. Our results depicted a targetable metabolic dependency arising from epigenetic factor deficiency, providing molecular insight to inform therapy for cancers with epigenetic alterations secondary to MLL3/4 COMPASS dysfunction.

HGG-02. METABOLIC TARGETING OF HIGH-GRADE GLIOMAS WITH MTAP LOSS

Abstract Malignant transformation of pediatric glioma is often associated with homozygous deletion of CDKN2A. With CDKN2A loss, MTAP which is in the same chromosomal location, 9p21, is homozygously deleted in a majority of HGGs and some LGGs. The MTAP gene codes for methylthioadenosine phosphorylase (MTAP) protein which plays an important role in methionine salvage pathway. Absence of MTAP results in accumulation of 5’-methylthioadenosine (MTA), de novo purine synthesis, and tumor cell dependency on protein arginine methyltransferase 5 (PRMT5) and its substrate provider, methionine adenosyltransferase II alpha (MAT2A). We hypothesize that drugs targeting PRMT5, MAT2A, and de novo purine synthesis, in combination with drugs targeting hyper-activated RAS/MAPK pathway, will be effective in inhibiting MTAP-null glioma growth. Patient-derived glioma cells were validated for MTAP loss using genome sequencing, RNA sequencing and western blot. MTA accumulation caused by MTAP loss was validated through high-performance liquid chromatography. Cells were subjected to monotherapy and combination drug therapy with PRMT5, MAT2A and de novo purine synthesis inhibitors, in addition to drugs targeting RAS/MAPK signaling pathway. Additionally, MTAP-null (MTAPnull) in vivo models are being developed.We show accumulation of MTA in MTAPnull cells compared to MTAP-wild type (MTAPWT) cells, caused by loss of MTAP that cleaves MTA to adenine and 5-methylthioribose-1-phosphate (MTR). Accumulation of MTA makes MTAPnull glioma cells more sensitive to MAT2A inhibitor AG-270 in comparison to MTAPWT cells. Additionally, dual-drug therapies involving MTA and AG-270 result in additive effect on MTAPnull cells and antagonistic effect on MTAPWT cells. De novo purine synthesis inhibitor and drugs targeting the RAS/MAPK pathways also exhibited cell growth inhibition of MTAPnull cells. Overall, metabolic targeting of HGGs bearing CDKN2A/MTAP loss is indispensable for targeted therapy and for minimizing toxicity towards surrounding normal cells lacking similar mutations.

Methylthioadenosine phosphorylase deficiency in tumors: A compelling therapeutic target.

The methionine salvage pathway is responsible for recycling sulfur-containing metabolites to methionine. This salvage pathway has been found to be implicated in cell apoptosis, proliferation, differentiation and inflammatory response. Methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5'-methylthioadenosine, a by-product produced from polyamine biosynthesis. The MTAP gene is located adjacent to the cyclin-dependent kinase inhibitor 2A gene and co-deletes with CDKN2A in nearly 15% of tumors. Moreover, MTAP-deleted tumor cells exhibit greater sensitivity to methionine depletion and to the inhibitors of purine synthesis. In this review, we first summarized the molecular structure and expression of MTAP in tumors. Furthermore, we discussed PRMT5 and MAT2A as a potential vulnerability for MTAP-deleted tumors. The complex and dynamic role of MTAP in diverse malignancies has also been discussed. Finally, we demonstrated the implications for the treatment of MTAP-deleted tumors.

Abstract 1093: Measuring and inhibiting purine metabolism in patients with glioblastoma

Abstract Introduction: We have previously found that purine metabolism is an important metabolic mediator of treatment resistance in glioblastoma (GBM). Mycophenolate mofetil (MMF), an inhibitor of purine synthesis, synergizes with radiation and temozolomide in mouse models of GBM. No measurements of purine synthesis have been performed in human cancer patients, nor has the purine inhibitor MMF been used clinically for patients with GBM. Methods: We infused clinical-grade 13C6 glucose into patients undergoing craniotomy for presumed GBM and used mass spectrometry to measure carbon incorporation into purines in cortex and disparate tumor regions. We are conducting a phase 0/1 trial (NCT04477200) to determine the safety and potential efficacy of the purine inhibitor MMF in patients with GBM. In the phase 0 arm, patients with recurrent GBM needing tumor re-resection receive MMF for one week prior to surgery and tumor and brain tissue is analyzed by mass spectrometry to determine drug concentrations and target engagement. In the phase 1 portions, patients (age >18, KPS ≥ 60) with recurrent GBM are treated with MMF and a second course of radiation while patients with newly diagnosed GBM are treated with radiation (RT), temozolomide (TMZ) and MMF followed by cyclic MMF and TMZ. A TITE-CRM algorithm is employed to dose-escalate MMF (range 500-2000 mg BID) and determine its maximum tolerated dose in combination with RT and/or TMZ. Results: Stable isotope tracing has been performed on 8 patients with suspected GBM. Purine synthesis was increased in tumor tissue compared to normal cortex in all patients, with most patients having 10-30-fold increased purine labeling in tumor. We have enrolled 8 patients on our phase 0 peri-surgical study and all had active concentrations of mycophenolic acid (the active metabolite of MMF) in enhancing and non-enhancing GBM tissue. We have enrolled 28 evaluable phase 1 patients (13 recurrent, 15 new). No dose-limiting toxicities have been observed with MMF (1000-2000 mg BID) in combination with re-irradiation or cyclic TMZ. In concurrent chemoradiation with MMF, we have observed two dose limiting grade 3 toxicities at 2000 mg, both improved with treatment break. Other main toxicities are mild nausea and fatigue. Interim median overall survival in recurrent phase 1 patients is 15.6 months, which compares favorably with typical survival time of 8-10 months in this population. In the newly diagnosed cohort, median survival is not yet reached, but 14 of 15 patients are alive and without progressive tumor. Conclusions: Glucose-derived purine synthesis is elevated in human GBM compared to cortex, which could provide a favorable therapeutic index for purine synthesis inhibition. The purine synthesis inhibitor MMF achieves active concentrations in human GBM, is reasonably well tolerated in combination with standard GBM treatments, and is associated with promising preliminary clinical outcomes. Citation Format: Yoshie Umemura, Andrew J. Scott, Wajd Al-Holou, Anjali Mittal, Weihua Zhou, Kari Wilder-Romans, Jie Xu, Nathan Qi, Maureen Kachman, Michelle Kim, Larry Junck, Denise Leung, Nathan Clarke, Jason Heth, Bo Wen, Amit Pai, Vijay Tarnal, Deepak Nagrath, Theodore Lawrence, Costas A. Lyssiotis, Daniel R. Wahl. Measuring and inhibiting purine metabolism in patients with glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1093.

Abstract 3677: DNA damage signaling activates de novo GTP synthesis to promote chemoradiation resistance in glioblastoma

Abstract Glioblastoma (GBM) is uniformly fatal due to inherent radiation (RT) and chemotherapy resistance. We have found this therapeutic resistance is mediated by alterations in tumor cellular metabolic activity. Our group and others have found that metabolites can regulate DNA repair and RT resistance in brain tumors, but little is known about how DNA damage regulates metabolic pathway activity in cancer. Here, we show that DNA damage acutely increases guanine-containing purine metabolites in multiple in vitro and intracranial GBM models. By interrogating metabolic fluxes in vitro using a variety of stable isotope tracers, we confirmed RT-induced elevation in guanylates was due to increased de novo purine synthesis (DNPS) rather than activation of purine salvage. By developing and using novel stable isotope tracing methods to directly measure DNPS in awake, unrestrained mice, we confirmed that orthotopic GBMs have higher DNPS rates than adjacent cortical tissue that further increase after treatment with RT. Neither salvage synthesis of purines nor pyrimidine synthesis were impacted by RT in any intracranial tissues. With these findings, we opened a clinical study to directly measure purine synthesis in patients, and we found that human GBMs have similarly high purine synthesis rates compared to normal brain tissue. Because DNA damage activated DNPS without affecting purine salvage or pyrimidine synthesis, we reasoned that active signaling may be involved. Indeed, therapy-induced DNPS increases are lost in vitro and in vivo upon pharmacological or genomic inhibition of the DNA-damage sensing kinase DNA-PK. Moreover, RT and DNA-PK have direct influence over the spatial organization of DNPS enzymes, including IMPDH, the rate-limiting step in guanylate synthesis. Because purines can promote DNA repair, these findings suggest that DNA-PK signaling helps promote DNA repair in part by causing the spatial reorganization of DNPS enzymes, thereby activating purine synthesis. To determine if disrupting this regulation can augment GBM treatment efficacy, we combined an FDA-approved inhibitor of purine synthesis with chemoradiation in a variety of mouse models of GBM. Critically, targeting GTP synthesis improved the efficacy of both RT alone and chemoradiation in multiple patient-derived and syngeneic intracranial models, suggesting a potential therapeutic targeting opportunity in patients. In this study, we have developed novel methodology to directly measure purine synthesis in brain tumors in mice and humans. With these tools, we discovered that after DNA damage, DNA-PK mediates a novel pathway controlling the spatial reorganization of purine synthesis enzymes and subsequent DNPS increases. The resulting elevation of GTP levels promotes therapy resistance in tumors, and we are now directly measuring and inhibiting this molecular activity in patients with GBM in an effort to improve standard therapy. Citation Format: Andrew J. Scott, Alexandra M. O'Brien, Weihua Zhou, Vidhi Pareek, Zhou Sha, Sravya Palavalasa, Ayesha U. Kothari, Kari Wilder-Romans, Li Zhang, Anthony C. Andren, Sriram Chandrasekaran, Jason Heth, Yoshie Umemura, Nathan Qi, John Woulfe, Sriram Venneti, Meredith A. Morgan, Theodore S. Lawrence, Wajd N. Al-Holou, Costas A. Lyssiotis, Daniel R. Wahl. DNA damage signaling activates de novo GTP synthesis to promote chemoradiation resistance in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3677.

CTNI-35. PHASE 0/1 TRIAL OF MYCOPHENOLATE MOFETIL COMBINED WITH CHEMORADIATION TO OVERCOME TREATMENT RESISTANCE IN NEWLY DIAGNOSED AND RECURRENT GLIOBLASTOMA BY TARGETING PURINE METABOLISM

Abstract BACKGROUND Purine metabolism promotes glioblastoma growth, stemness, invasiveness, and treatment resistance. The purine synthesis inhibitor, mycophenolate mofetil (MMF), improves radiation and temozolomide efficacy in preclinical glioblastoma models. This phase 0/1 trial (NCT04477200) aims to determine the maximum-tolerated dose of MMF combined with chemoradiation in glioblastoma. It also aims to quantify the levels of the active metabolite of MMF and the extent of purine synthesis inhibition in enhancing and non-enhancing tumor tissue. METHODS Key eligibility criteria are age ≥ 18 and KPS ≥ 60%. TITE-CRM dose-escalation is used for MMF dosing starting at 1000mg PO BID (range 500-2000mg BID). Thirty recurrent glioblastoma patients receive MMF one-week prior to and concurrently with re-irradiation (40.5 Gy in 15 fractions), with 28 days of dose-limiting toxicity (DLT) period. Thirty newly diagnosed glioblastoma patients receive MMF one-week prior to and concurrently with standard radiation with concurrent temozolomide and followed for 28 days for DLT1-period, then MMF is given days 0-5 of each adjuvant cyclic temozolomide using a separate dose-escalation with the first two cycles as the DLT2-period. In phase 0, eight recurrent glioblastoma patients receive MMF 500-2000mg PO BID one-week pre-operatively and tissues are analyzed using mass spectrometry. RESULTS Eighteen phase 1 subjects (11 recurrent, 7 new) have received study treatment with no DLT to date up to MMF 2000mg BID. Main toxicities are mild nausea, fatigue, and elevated liver enzymes. No notable study-related hematotoxicity nor opportunistic infection have been observed. The active metabolite of MMF accumulated to active concentrations in enhancing and non-enhancing tumor tissue and appeared to inhibit purine synthesis. CONCLUSION MMF combined with chemoradiation has been reasonably well tolerated in glioblastoma patients with preliminary evidence of intracranial target engagement of its active metabolite. This study will yield a recommended phase 2 dose and preliminary efficacy estimate for future randomized phase 2/3 trial.

Cardiovascular effects of immunosuppression agents.

Immunosuppressive medications are widely used to treat patients with neoplasms, autoimmune conditions and solid organ transplants. Key drug classes, namely calcineurin inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and purine synthesis inhibitors, have direct effects on the structure and function of the heart and vascular system. In the heart, immunosuppressive agents modulate cardiac hypertrophy, mitochondrial function, and arrhythmia risk, while in vasculature, they influence vessel remodeling, circulating lipids, and blood pressure. The aim of this review is to present the preclinical and clinical literature examining the cardiovascular effects of immunosuppressive agents, with a specific focus on cyclosporine, tacrolimus, sirolimus, everolimus, mycophenolate, and azathioprine.

Purine Synthesis Inhibitor L-Alanosine Impairs Mitochondrial Function and Stemness of Brain Tumor Initiating Cells.

Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous MTAP deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in purine salvage, we demonstrate that purine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo purine synthesis, attenuates stemness of MTAP-deficient GBM cells. This ALA-induced reduction in stemness is mediated in part by compromised mitochondrial function, highlighted by ALA-induced elimination of mitochondrial spare respiratory capacity. Notably, these effects of ALA are apparent even when the treatment was transient and with a low dose. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify purine supply as an essential component in maintaining mitochondrial function in GBM cells and highlight a critical role of mitochondrial function in sustaining GBM stemness. We propose that purine synthesis inhibition can be beneficial in combination with the standard of care for MTAP-deficient GBMs, and that it may be feasible to achieve this benefit without inflicting major toxicity.

Sensing Methods of Immunosuppressant Drugs: Calcineurin Inhibitors and Purine Synthesis Inhibitor Agents

Immunosuppression is associated as a necessary protocol in post-transplantation follow-up to improve graft survival rate. Two typical classes of immunosuppressants that have been commonly used as oral long-term medication are calcineurin inhibitors, e.g. cyclosporine A (CsA) and tacrolimus (TAC), and purine synthesis inhibitor agents, e.g. azathioprine (AZT) and mycophenolate (MF). As a common feature for all of them, they have a narrow therapeutic index which may lead to toxicity or effectiveness of therapy in super therapeutic and subtherapeutic concentrations, respectively. So, despite the undisputed value of immunosuppressants, therapeutic drug monitoring is a vital necessity for post-renal transplantation follow-up to maintain an appropriate balance between a predefined therapeutic dose and toxicity and likelihood adverse effects. Previously, most of the analytical methods that have been employed for immunosuppressant detection are based on immunoassay and chromatography methods which the drawbacks such as expensiveness, time-consuming responding, lack of stability, and skilled persons to interpretation obtained results make them inappropriate platforms for in-situ applications. In this way, we need facile, fast responding analytical methods as like optical and electrochemical sensors which can be developed to use in point-of-care applications. Here, we summarized the analytical methods for the determination of CsA, TAC, AZT, and MF which are based on fast responding methods.

The Intersection of Purine and Mitochondrial Metabolism in Cancer.

Nucleotides are essential to cell growth and survival, providing cells with building blocks for DNA and RNA, energy carriers, and cofactors. Mitochondria have a critical role in the production of intracellular ATP and participate in the generation of intermediates necessary for biosynthesis of macromolecules such as purines and pyrimidines. In this review, we highlight the role of purine and mitochondrial metabolism in cancer and how their intersection influences cancer progression, especially in ovarian cancer. Additionally, we address the importance of metabolic rewiring in cancer and how the evolving landscape of purine synthesis and mitochondria inhibitors can be potentially exploited for cancer treatment.

MTAP Deficiency-Induced Metabolic Reprogramming Creates a Vulnerability to Cotargeting De Novo Purine Synthesis and Glycolysis in Pancreatic Cancer.

Methylthioadenosine phosphorylase (MTAP) is a key enzyme associated with the salvage of methionine and adenine that is deficient in 20% to 30% of pancreatic cancer. Our previous study revealed that MTAP deficiency indicates a poor prognosis for patients with pancreatic ductal adenocarcinoma (PDAC). In this study, bioinformatics analysis of The Cancer Genome Atlas (TCGA) data indicated that PDACs with MTAP deficiency display a signature of elevated glycolysis. Metabolomics studies showed that that MTAP deletion-mediated metabolic reprogramming enhanced glycolysis and de novo purine synthesis in pancreatic cancer cells. Western blot analysis revealed that MTAP knockout stabilized hypoxia-inducible factor 1α (HIF1α) protein via posttranslational phosphorylation. RIO kinase 1 (RIOK1), a downstream kinase upregulated in MTAP-deficient cells, interacted with and phosphorylated HIF1α to regulate its stability. In vitro experiments demonstrated that the glycolysis inhibitor 2-deoxy-d-glucose (2-DG) and the de novo purine synthesis inhibitor l-alanosine synergized to kill MTAP-deficient pancreatic cancer cells. Collectively, these results reveal that MTAP deficiency drives pancreatic cancer progression by inducing metabolic reprogramming, providing a novel target and therapeutic strategy for treating MTAP-deficient disease. SIGNIFICANCE: This study demonstrates that MTAP status impacts glucose and purine metabolism, thus identifying multiple novel treatment options against MTAP-deficient pancreatic cancer.