Targeting the m6A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication.

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.

Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia.

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

MALT1 inhibition suppresses antigen-specific T cell responses.

The aim of this study was to assess the potential use of a selective small molecule MALT1 inhibitor in solid tumor treatment as an immunotherapy targeting regulatory T-cells (Tregs). In vitro, MALT1 inhibition suppressed the proteolytic cleavage of the MALT1-substrate HOIL1 and blocked IL-2 secretion in Jurkat cells. It selectively suppressed the proliferation of PBMC-derived Tregs, with no effect on conventional CD4+T-cells. In vivo, however, no evident anti-tumor effect was achieved by MALT1 inhibition monotherapy or in combination with anti-CTLA4 in the MB49 cancer model. Despite decreased Treg-frequencies in lymph nodes of tumor-bearing animals, intratumoral Treg depletion was not observed. We also showed that MALT1-inhibition caused a reduction of antigen-specific CD8+T-cells in an adoptive T-cell transfer model. Thus, selective targeting of Tregs would be required to improve the immunotherapeutic effect of MALT1-inhibition. Also, various dosing schedules and combination therapy strategies should be carefully designed and evaluated further.

The thymidine dideoxynucleoside analog, alovudine, inhibits the mitochondrial DNA polymerase γ, impairs oxidative phosphorylation and promotes monocytic differentiation in acute myeloid leukemia.

Mitochondrial DNA encodes 13 proteins that comprise components of the respiratory chain that maintain oxidative phosphorylation. The replication of mitochondrial DNA is performed by the sole mitochondrial DNA polymerase γ. As acute myeloid leukemia (AML) cells and stem cells have an increased reliance on oxidative phosphorylation, we sought to evaluate polymerase γ inhibitors in AML. The thymidine dideoxynucleoside analog, alovudine, is an inhibitor of polymerase γ. In AML cells, alovudine depleted mitochondrial DNA, reduced mitochondrial encoded proteins, decreased basal oxygen consumption, and decreased cell proliferation and viability. To evaluate the effects of polymerase γ inhibition with alovudine in vivo, mice were xenografted with OCI-AML2 cells and then treated with alovudine. Systemic administration of alovudine reduced leukemic growth without evidence of toxicity and decreased levels of mitochondrial DNA in the leukemic cells. We also showed that alovudine increased the monocytic differentiation of AML cells. Genetic knockdown and other chemical inhibitors of polymerase γ also promoted AML differentiation, but the effects on AML differentiation were independent of reductions in oxidative phosphorylation or respiratory chain proteins. Thus, we have identified a novel mechanism by which mitochondria regulate AML fate and differentiation independent of oxidative phosphorylation. Moreover, we highlight polymerase γ inhibitors, such as alovudine, as novel therapeutic agents for AML.

Discovery of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1: optimization of kinase selectivity and pharmacokinetics.

The kinase selectivity and pharmacokinetic optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. The intersection of insights from molecular modeling, computational prediction of metabolic sites, and in vitro metabolite identification studies resulted in a simple and unique solution to both of these problems. These efforts culminated in the discovery of compound 13a, a potent, relatively selective inhibitor of TAK1 with good pharmacokinetic properties in mice, which was active in an in vivo model of ovarian cancer.

Discovery and optimization of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1.

The discovery and potency optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. Micromolar hits taken from high-throughput screening were optimized for biochemical and cellular mechanistic potency to ~10nM, as exemplified by compound 12az. Application of structure-based drug design aided by co-crystal structures of TAK1 with inhibitors significantly shortened the number of iterations required for the optimization.

Novel 6-aminofuro[3,2-c]pyridines as potent, orally efficacious inhibitors of cMET and RON kinases.

A series of novel 6-aminofuro[3,2-c]pyridines as kinase inhibitors is described, most notably, OSI-296 (6). We discuss our exploration of structure-activity relationships and optimization leading to OSI-296 and disclose its pharmacological activity against cMET and RON in cellular assays. OSI-296 is a potent and selective inhibitor of cMET and RON kinases that shows in vivo efficacy in tumor xenografts models upon oral dosing and is well tolerated.

The PARP1 Inhibitor AZD5305 Impairs Ovarian Adenocarcinoma Progression and Visceral Metastases in Patient-derived Xenografts Alone and in Combination with Carboplatin.

PARP inhibitors (PARPi) have changed the management of patients with ovarian cancer and their effectiveness has been demonstrated especially in homologous recombination repair-deficient tumors. These first-generation drugs target PARP1, but also PARP2 and other family members potentially responsible for adverse effects that limit their therapeutic potential and restrict their use in combination with chemotherapeutic agents. We investigated ovarian cancer patient-derived xenografts (OC-PDXs) to assess whether malignant progression could be impaired by a novel inhibitor selective for PARP1 (AZD5305) and to assess the potential of its combination with carboplatin (CPT), the standard-of-care for patients with ovarian cancer. In BRCA-mutated OC-PDXs, AZD5305 achieved greater tumor regressions and longer duration of response as well as a superior impairment of visceral metastasis and improved survival benefit compared with the first-generation dual PARP1/2 inhibitors. The combination of AZD5305 plus CPT was more efficacious than single agents. Subcutaneously growing tumors experienced regression that persisted after therapy stopped. Combination efficacy was greater against tumors that did not respond well to platinum, even at a dose at which AZD5305 monotherapy was ineffective. The combination therapy impaired metastatic dissemination and significantly prolonged the lifespan of mice bearing OC-PDXs in their abdomen. This combination benefit was evident even when CPT was used at suboptimal doses, and was superior to full-dose platinum treatment. These preclinical studies demonstrate that the PARP1-selective inhibitor AZD5305 retains and improves the therapeutic benefit of the first-generation PARPi, providing an opportunity to maximize benefits for this class of anticancer agents. Significance: Selective PARP1i AZD5305 can exceed the efficacy of first-generation PARPi, which target both PARP1 and PARP2, and potentiates the efficacy of CPT when given in combination. AZD5305 alone or in combination with platinum delayed visceral metastasis, ultimately extending the lifespan of OC-PDX-bearing mice. These preclinical models mimic the progression of the disease occurring in patients after debulking surgery, and are translationally relevant.

Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity.

Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.

Design and Preclinical Evaluation of a Novel B7-H4-Directed Antibody-Drug Conjugate, AZD8205, Alone and in Combination with the PARP1-Selective Inhibitor AZD5305.

PURPOSE: We evaluated the activity of AZD8205, a B7-H4-directed antibody-drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models. EXPERIMENTAL DESIGN: IHC and deep-learning-based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly-Gly-Phe-Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models. RESULTS: Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala-PEG8-TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker-payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4-expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance. CONCLUSIONS: These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4-expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482). See related commentary by Pommier and Thomas, p. 991.

Hypoxia-selective targeting by the bioreductive prodrug AQ4N in patients with solid tumors: results of a phase I study.

PURPOSE: AQ4N is a novel bioreductive prodrug under clinical investigation. Preclinical evidence shows that AQ4N penetrates deeply within tumors and undergoes selective activation to form AQ4, a potent topoisomerase II inhibitor, in hypoxic regions of solid tumors. This proof-of-principle, phase I study evaluated the activation, hypoxic selectivity, and safety of AQ4N in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Thirty-two patients with cancer (8 glioblastoma, 9 bladder, 8 head and neck, 6 breast, and 1 cervix) received a single 200 mg/m(2) dose of AQ4N before elective surgery. AQ4 and AQ4N levels in 95 tissues (tumor, healthy tissue) were assessed by liquid chromatography-tandem mass spectrometry. Tissue sections were also analyzed for AQ4 fluorescence using confocal microscopy, and for expression of the hypoxia-regulated glucose transporter, Glut-1. RESULTS: Activated AQ4 was detected in all tumor samples with highest levels present in glioblastoma (mean 1.2 microg/g) and head and neck (mean 0.65 microg/g) tumors; 22 of 32 patients had tumor AQ4 concentrations > or = 0.2 microg/g, levels previously shown to be active in preclinical studies. In 24 of 30 tumor samples, AQ4 was detected at higher concentrations than in adjacent normal tissue (tumor to normal ratio range 1.1-63.6); distant skin samples contained very low concentrations of AQ4 (mean 0.037 microg/g). Microscopic evaluation of tumor sections revealed that AQ4 colocalized within regions of Glut-1+ hypoxic cells. CONCLUSIONS: AQ4N was activated selectively in hypoxic regions in human solid tumors. Intratumoral concentrations of AQ4 exceeded those required for activity in animal models and support the evaluation of AQ4N as a novel tumor-targeting agent in future clinical studies.

Selective tumor targeting by the hypoxia-activated prodrug AQ4N blocks tumor growth and metastasis in preclinical models of pancreatic cancer.

PURPOSE: The antitumor activities and pharmacokinetics of the hypoxia-activated cytotoxin AQ4N and its metabolites were assessed in several preclinical models of pancreatic cancers. EXPERIMENTAL DESIGN: The cytotoxic effects of AQ4N prodrug and its bioreduced form, AQ4, were tested against multiple human tumor cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Nude mice bearing s.c. or orthotopically implanted human BxPC-3 or Panc-1 tumor cells were treated with AQ4N. Tumor growth inhibition, time to progression/end point, and liver metastasis were evaluated in treatment versus control groups. Plasma and tumor levels of AQ4N and its metabolites were quantitated by liquid chromatography-tandem mass spectrometry. RESULTS: In contrast to AQ4N, the bioreduced AQ4 metabolite displayed potent cytotoxicity in many human tumor lines, including those derived from human pancreatic adenocarcinomas. Single-agent administration of AQ4N significantly delayed tumor growth, progression, and survival in a manner comparable with gemcitabine in multiple pancreatic tumor models in vivo. Survival increases were accompanied by a reduction in incidence and spread of liver metastasis. Quantitation of AQ4N and its metabolites in tumor-bearing mice showed that the prodrug is rapidly cleared from the circulation by 24 h and neither of the bioreduced metabolites was detected in plasma. In contrast, AQ4N readily penetrated BxPC-3 tumors and the cytotoxic AQ4 metabolite rapidly accumulated in tumor tissues at high levels in a dose-dependent fashion. CONCLUSION: AQ4N undergoes rapid and selective conversion into the potent antineoplastic metabolite AQ4 in tumors in vivo and provides proof of principle for the use of hypoxia-activated prodrugs in the treatment against pancreatic cancers.

A role for polymerase eta in the cellular tolerance to cisplatin-induced damage.

Mutation of the POLH gene encoding DNA polymerase eta (pol eta) causes the UV-sensitivity syndrome xeroderma pigmentosum-variant (XP-V) which is linked to the ability of pol eta to accurately bypass UV-induced cyclobutane pyrimidine dimers during a process termed translesion synthesis. Pol eta can also bypass other DNA damage adducts in vitro, including cisplatin-induced intrastrand adducts, although the physiological relevance of this is unknown. Here, we show that independent XP-V cell lines are dramatically more sensitive to cisplatin than the same cells complemented with functional pol eta. Similar results were obtained with the chemotherapeutic agents, carboplatin and oxaliplatin, thus revealing a general requirement for pol eta expression in providing tolerance to these platinum-based drugs. The level of sensitization observed was comparable to that of XP-A cells deficient in nucleotide excision repair, a recognized and important mechanism for repair of cisplatin adducts. However, unlike in XP-A cells, the absence of pol eta expression resulted in a reduced ability to overcome cisplatin-induced S phase arrest, suggesting that pol eta is involved in translesion synthesis past these replication-blocking adducts. Subcellular localization studies also highlighted an accumulation of nuclei with pol eta foci that correlated with the formation of monoubiquitinated proliferating cell nuclear antigen following treatment with cisplatin, reminiscent of the response to UV irradiation and further indicating a role for pol eta in dealing with cisplatin-induced damage. Together, these data show that pol eta represents an important determinant of cellular responses to cisplatin, which could have implications for acquired or intrinsic resistance to this key chemotherapeutic agent.

The overexpression of specialized DNA polymerases in cancer.

Specialized DNA polymerases are required to bypass DNA damage lesions that would otherwise cause replication arrest and cell death. When operating on non-canonical templates, such as undamaged DNA or on non-cognate lesions, these polymerases exhibit considerably reduced fidelity, resulting in the generation of mutations. Ectopic overexpression of these polymerases can also lead to an increased mutation rate and an enhanced capability of DNA repair, suggesting that they could potentially act as oncogenes if they were overexpressed in cancers. Here, we examine expression patterns of DNA polymerases in matched normal and tumor samples from a diverse range of tissues. As well as investigating the specialized polymerases beta, lambda, iota and kappa, we also investigate the expression of the replicative polymerases alpha, delta and epsilon. The data presented provide evidence for the overexpression of specialized polymerases in tumors, with more than 45% of the 68 tumor samples studied demonstrating greater than two-fold enhanced expression of at least one specialized polymerase. Of particular note, DNA polymerase beta (pol beta) was found to be overexpressed at both the mRNA and protein level in approximately one third of all tumor types studied, with overexpression being particularly frequent in uterus, ovary, prostate and stomach samples. Pols lambda, and iota were also found to be overexpressed to a significant extent in a range of tumor types, albeit less frequently than pol beta. In contrast, pol kappa was rarely found to be overexpressed in tumors but was found to be commonly underexpressed in many samples. Downregulation of pol beta expression by siRNA resulted in an increased sensitivity to the chemotherapeutic agent cisplatin, suggesting a role for this polymerase in providing tolerance to cisplatin-induced damage. These observations suggest that specialised DNA polymerases, and particularly pol beta, could be considered both as caretaker genes altered during tumorigenesis, and as potential drug targets to sensitise tumors to chemotherapy.

The RON receptor tyrosine kinase promotes metastasis by triggering MBD4-dependent DNA methylation reprogramming.

Metastasis is the major cause of death in cancer patients, yet the genetic and epigenetic programs that drive metastasis are poorly understood. Here, we report an epigenetic reprogramming pathway that is required for breast cancer metastasis. Concerted differential DNA methylation is initiated by the activation of the RON receptor tyrosine kinase by its ligand, macrophage stimulating protein (MSP). Through PI3K signaling, RON/MSP promotes expression of the G:T mismatch-specific thymine glycosylase MBD4. RON/MSP and MBD4-dependent aberrant DNA methylation results in the misregulation of a specific set of genes. Knockdown of MBD4 reverses methylation at these specific loci and blocks metastasis. We also show that the MBD4 glycosylase catalytic residue is required for RON/MSP-driven metastasis. Analysis of human breast cancers revealed that this epigenetic program is significantly associated with poor clinical outcome. Furthermore, inhibition of Ron kinase activity with a pharmacological agent blocks metastasis of patient-derived breast tumor grafts in vivo.

In vivo activation of the hypoxia-targeted cytotoxin AQ4N in human tumor xenografts.

AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.