Search

Glioblastoma (GBM) is uniformly lethal due to profound treatment resistance. Altered cellular metabolism is a key mediator of GBM treatment resistance. Uptake of the essential sulfur-containing amino acid methionine is drastically elevated in GBMs compared to normal cells, however, it is not known how this methionine is utilized or whether it relates to GBM treatment resistance. Here, we find that radiation acutely increases the levels of methionine-related metabolites in a variety of treatment-resistant GBM models. Stable isotope tracing studies further revealed that radiation acutely activates methionine to S-adenosyl methionine (SAM) conversion through an active signaling event mediated by the kinases of the DNA damage response. In vivo tumor SAM synthesis increases after radiation, while normal brain SAM production remains unchanged, indicating a tumor- specific metabolic alteration to radiation. Pharmacological and dietary strategies to block methionine to SAM conversion slowed DNA damage response and increased cell death following radiation in vitro. Mechanistically, these effects are due to depletion of DNA repair proteins and are reversed by SAM supplementation. These effects are selective to GBMs lacking the methionine salvage enzyme methylthioadenosine phosphorylase. Pharmacological inhibition of SAM synthesis hindered tumor growth in flank and orthotopic in vivo GBM models when combined with radiation. By contrast, methionine depletion does not reduce tumor SAM levels and fails to radiosensitize intracranial models, indicating depleting SAM, as opposed to simply lowering methionine, is critical for hindering tumor growth in intracranial models of GBM. These results highlight a new signaling link between DNA damage and SAM synthesis and define the metabolic fates of methionine in GBM in vivo . Inhibiting radiation-induced SAM synthesis slows DNA repair and augments radiation efficacy in GBM. Using MAT2A inhibitors to deplete SAM may selectively overcome treatment resistance in GBMs with defective methionine salvage while sparing normal brain.

Background: Hyperactivated protein arginine methyltransferases (PRMTs) are implicated in human cancers. Inhibiting tumor intrinsic PRMT5 was reported to potentiate antitumor immune responses, highlighting the possibility of combining PRMT5 inhibitors (PRMT5i) with cancer immunotherapy. However, global suppression of PRMT5 activity impairs the effector functions of immune cells. Here, we sought to identify strategies to specifically inhibit PRMT5 activity in tumor tissues and develop effective PRMT5i-based immuno-oncology (IO) combinations for cancer treatment, particularly for methylthioadenosine phosphorylase (MTAP)-loss cancer., Methods: Isogeneic tumor lines with and without MTAP loss were generated by CRISPR/Cas9 knockout. The effects of two PRMT5 inhibitors (GSK3326595 and MRTX1719) were evaluated in these isogenic tumor lines and T cells in vitro and in vivo . Transcriptomic and proteomic changes in tumors and T cells were characterized in response to PRMT5i treatment. Furthermore, the efficacy of MRTX1719 in combination with immune checkpoint blockade was assessed in two syngeneic murine models with MTAP-loss tumor., Results: GSK3326595 significantly suppresses PRMT5 activity in tumors and T cells regardless of the MTAP status. However, MRTX1719, a methylthioadenosine-cooperative PRMT5 inhibitor, exhibits tumor-specific PRMT5 inhibition in MTAP-loss tumors with limited immunosuppressive effects. Mechanistically, transcriptomic and proteomic profiling analysis reveals that MRTX1719 successfully reduces the activation of the PI3K pathway, a well-documented immune-resistant pathway. It highlights the potential of MRTX1719 to overcome immune resistance in MTAP-loss tumors. In addition, MRTX1719 sensitizes MTAP-loss tumor cells to the killing of tumor-reactive T cells. Combining MRTX1719 and anti-PD-1 leads to superior antitumor activity in mice bearing MTAP-loss tumors., Conclusion: Collectively, our results provide a strong rationale and mechanistic insights for the clinical development of MRTX1719-based IO combinations in MTAP-loss tumors., Competing Interests: Competing interests: JRA reports non-financial support and reasonable reimbursement for travel from European Society for Medical Oncology and Loxo Oncology; receiving consulting and travel fees from Ellipses Pharma, Molecular Partners, IONCTURA, Sardona, Mekanistic, Amgen, Merus, MonteRosa, Aadi and Bridgebio (including serving on the scientific advisory board); consulting fees from Vall d’Hebron Institute of Oncology/Ministero De Empleo Y Seguridad Social, Chinese University of Hong Kong, Boxer Capital, LLC, Tang Advisors, LLC and Guidepoint, receiving research funding from Blueprint Medicines, Merck Sharp and serving as investigator in clinical trials with Cancer Core Europe, Symphogen, BioAlta, Pfizer, Kelun-Biotech, GlaxoSmithKline, Taiho, Roche Pharmaceuticals, Hummingbird, Yingli, Bicycle Therapeutics, Merus, Aadi Bioscience, ForeBio, Loxo Oncology, Hutchison MediPharma, Ideaya, Amgen, Tango Therapeutics, Mirati Therapeutics, Linnaeus Therapeutics, MonteRosa, Kinnate, Yingli, Debio, BioTheryX, Storm Therapeutics, Beigene, MapKure, Relay, Novartis, FusionPharma, C4 Therapeutics, Scorpion Therapeutics, Incyte, Fog Pharmaceuticals, Tyra, Nuvectis Pharma. MTB is a co-founder of EpiCypher. No potential conflicts of interest were disclosed by other authors., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

There is significant interest in engineering human amino acid degrading enzymes as non-immunogenic chemotherapeutic agents. We describe a high-throughput fluorescence activated cell sorting (FACS) assay for detecting the catalytic activity of amino acid degrading enzymes in bacteria, at the single cell level. This assay relies on coupling the synthesis of the GFP reporter to the catalytic activity of the desired amino acid degrading enzyme in an appropriate E. coli genetic background. The method described here allows facile screening of much larger libraries (10(6)-10(7)) than was previously possible. We demonstrate the application of this technique in the screening of libraries of bacterial and human asparaginases and also for the catalytic optimization of an engineered human methionine gamma lyase.

Cancer has become the leading cause of death in the developed world and has remained one of the most difficult diseases to treat. One of the difficulties in treating cancer is that conventional chemotherapies often have unacceptable toxicities toward normal cells at the doses required to kill tumor cells. Thus, the demand for new and improved tumor specific therapeutics for the treatment of cancer remains high. Alterations to cellular metabolism constitute a nearly universal feature of many types of cancer cells. In particular, many tumors exhibit deficiencies in one or more amino acid synthesis or salvage pathways forcing a reliance on the extracellular pool of these amino acids to satisfy protein biosynthesis demands. Therefore, one treatment modality that satisfies the objective of developing cancer cell-selective therapeutics is the systemic depletion of that tumor-essential amino acid, which can result in tumor apoptosis with minimal side effects to normal cells. While this strategy was initially suggested over 50 years ago, it has been recently experiencing a renaissance owing to advances in protein engineering technology, and more sophisticated approaches to studying the metabolic differences between tumorigenic and normal cells. Dietary restriction is typically not sufficient to achieve a therapeutically relevant level of amino acid depletion for cancer treatment. Therefore, intravenous administration of enzymes is used to mediate the degradation of such amino acids for therapeutic purposes. Unfortunately, the human genome does not encode enzymes with the requisite catalytic or pharmacological properties necessary for therapeutic purposes. The use of heterologous enzymes has been explored extensively both in animal studies and in clinical trials. However, heterologous enzymes are immunogenic and elicit adverse responses ranging from anaphylactic shock to antibody-mediated enzyme inactivation, and therefore have had limited utility. The one notable exception is Escherichia colil-asparaginase II (EcAII), which has been FDA-approved for the treatment of childhood acute lymphoblastic leukemia. The use of engineered human enzymes, to which natural tolerance is likely to prevent recognition by the adaptive immune system, offers a novel approach for capitalizing on the promising strategy of systemic depletion of tumor-essential amino acids. In this work, we review several strategies that we have developed to: (i) reduce the immunogenicity of a nonhuman enzyme, (ii) engineer human enzymes for novel catalytic specificities, and (iii) improve the pharmacological characteristics of a human enzyme that exhibits the requisite substrate specificity for amino acid degradation but exhibits low activity and stability under physiological conditions., (Copyright © 2012 Elsevier Inc. All rights reserved.)