Ultra-sensitive molecular residual disease detection through whole genome sequencing with single-read error correction.

While whole genome sequencing (WGS) of cell-free DNA (cfDNA) holds enormous promise for molecular residual disease (MRD) detection, its performance is limited by WGS error rate. Here we introduce AccuScan, an efficient cfDNA WGS technology that enables genome-wide error correction at single read level, achieving an error rate of 4.2×10 -7 , which is about two orders of magnitude lower than a read-centric de-noising method. When applied to MRD detection, AccuScan demonstrated analytical sensitivity down to 10 -6 circulating tumor allele fraction at 99% sample level specificity. In colorectal cancer, AccuScan showed 90% landmark sensitivity for predicting relapse. It also showed robust MRD performance with esophageal cancer using samples collected as early as 1 week after surgery, and predictive value for immunotherapy monitoring with melanoma patients. Overall, AccuScan provides a highly accurate WGS solution for MRD, empowering circulating tumor DNA detection at parts per million range without high sample input nor personalized reagents. One Sentence Summary: AccuScan showed remarkable ultra-low limit of detection with a short turnaround time, low sample requirement and a simple workflow for MRD detection.

IL-7R licenses a population of epigenetically poised memory CD8+ T cells with superior antitumor efficacy that are critical for melanoma memory.

Recurrence of advanced melanoma after therapy is a major risk factor for reduced survival, and treatment options are limited. Antitumor immune memory plays a critical role in preventing melanoma recurrence and memory T cells could be a potent cell-based therapy, but the identity, and functional properties of the required immune cells are incompletely understood. Here, we show that an IL-7Rhi tumor-specific CD8+ population is critical for antitumor memory and can be epigenetically augmented to drive powerful antitumor immune responses. Using a model of functional antimelanoma memory, we found that high IL-7R expression selectively marks a CD8+ population in lymphoid organs that plays critical roles in maintaining tumor remission after immunotherapy or surgical resection. This population has intrinsic cytotoxic activity, lacks markers of exhaustion and has superior antitumor efficacy. IL-7Rhi cells have a functionally poised epigenetic landscape regulated by DNA methylation, which can be augmented by hypomethylating agents to confer improved survival and complete melanoma clearance in naive mice. Importantly, greater than 95% of tumor-specific T cells in draining lymph nodes after therapy express high levels of IL-7R. This overlap between IL-7Rhi and antigen-specific T cells allows for enrichment of a potent functional CD8+ population without determining antigen-specificity, which we demonstrate in a melanoma model without a known antigen. We identify that IL-7R expression in human melanoma is an independent prognostic factor of improved survival. These findings advance our basic understanding of antitumor memory and suggest a cell-based therapy using high IL-7R expression to enrich for a lymph node population with superior antitumor activity that can be augmented by hypomethylating agents.

Setdb1 -loss induces type-I interferons and immune clearance of melanoma.

Despite recent advances in the treatment of melanoma, many patients with metastatic disease still succumb to their disease. To identify tumor-intrinsic modulators of immunity to melanoma, we performed a whole-genome CRISPR screen in melanoma and identified multiple components of the HUSH complex, including Setdb1 , as hits. We found that loss of Setdb1 leads to increased immunogenicity and complete tumor clearance in a CD8+ T-cell dependent manner. Mechanistically, loss of Setdb1 causes de-repression of endogenous retroviruses (ERVs) in melanoma cells and triggers tumor-cell intrinsic type-I interferon signaling, upregulation of MHC-I expression, and increased CD8+ T-cell infiltration. Furthermore, spontaneous immune clearance observed in Setdb1 -/- tumors results in subsequent protection from other ERV-expressing tumor lines, supporting the functional anti-tumor role of ERV-specific CD8+ T-cells found in the Setdb1 -/- microenvironment. Blocking the type-I interferon receptor in mice grafted with Setdb1 -/- tumors decreases immunogenicity by decreasing MHC-I expression, leading to decreased T-cell infiltration and increased melanoma growth comparable to Setdb1 wt tumors. Together, these results indicate a critical role for Setdb1 and type-I interferons in generating an inflamed tumor microenvironment, and potentiating tumor-cell intrinsic immunogenicity in melanoma. This study further emphasizes regulators of ERV expression and type-I interferon expression as potential therapeutic targets for augmenting anti-cancer immune responses.

Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity.

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma.

INTRODUCTION: We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. METHODS: Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay). RESULTS: Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). CONCLUSION: Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.

Discovery of decreased ferroptosis in male colorectal cancer patients with KRAS mutations.

Aberrant tumor metabolism is a hallmark of cancer in which metabolic rewiring can support tumor growth under nutrient deficient conditions. KRAS mutations occur in 35-45% of all colorectal cancer (CRC) cases and are difficult to treat. The relationship between mutant KRAS and aberrant metabolism in CRCs has not been fully explored and could be a target for intervention. We previously acquired non-targeted metabolomics data from 161 tumor tissues and 39 normal colon tissues from stage I-III chemotherapy naïve CRC patients. In this study, we revealed that only in male patients, tumors with KRAS mutations had several altered pathways that suppress ferroptosis, including glutathione biosynthesis, transsulfuration activity, and methionine metabolism. To validate this phenotype, MC38 CRC cells (KRASG13R) were treated with a ferroptosis inducer; RAS-selected lethal (RSL3). RSL3 altered metabolic pathways in the opposite direction to that seen in KRAS mutant tumors from male patients confirming a suppressed ferroptosis metabolic phenotype in these patients. We further validated gene expression data from an additional CRC patient cohort (Gene Expression Omnibus (GEO)), and similarly observed differences in ferroptosis-related genes by sex and KRAS status. Further examination of the relationship between these genes and overall survival (OS) in the GEO cohort showed that KRAS mutant tumors are associated with poorer 5-year OS compared to KRAS wild type tumors, and only in male patients. Additionally, high compared to low expression of GPX4, FTH1, FTL, which suppress ferroptosis, were associated with poorer 5-year OS only in KRAS mutant tumors from male CRC patients. Additionally, low compared to high expression of ACSL4 was associated with poorer OS for this group. Our results show that KRAS mutant tumors from male CRC patients have suppressed ferroptosis, and gene expression changes that suppress ferroptosis associate with adverse outcomes for these patients, revealing a novel potential avenue for therapeutic approaches.

Discovery of decreased ferroptosis in male colorectal cancer patients with KRAS mutations.

Aberrant tumor metabolism is a hallmark of cancer in which metabolic rewiring can support tumor growth under nutrient deficient conditions. KRAS mutations occur in 35-45% of all colorectal cancer (CRC) cases and are difficult to treat. The relationship between mutant KRAS and aberrant metabolism in CRCs has not been fully explored and could be a target for intervention. We previously acquired non-targeted metabolomics data from 161 tumor tissues and 39 normal colon tissues from stage I-III chemotherapy naïve CRC patients. In this study, we revealed that tumors from male patients with KRAS mutations only, had several altered pathways that suppress ferroptosis, including glutathione biosynthesis, transsulfuration activity, and methionine metabolism. To validate this phenotype, MC38 CRC cells (KRAS G13R ) were treated with a ferroptosis inducer; RAS-selected lethal (RSL3). RSL3 altered metabolic pathways in the opposite direction to that seen in KRAS mutant tumors from male patients confirming a suppressed ferroptosis metabolic phenotype in these patients. We further validated gene expression data from an additional CRC patient cohort (Gene Expression Omnibus (GEO), and similarly observed differences in ferroptosis-related genes by sex and KRAS status. Further examination of the relationship between these genes and overall survival (OS) in the GEO cohort showed that KRAS mutant tumors are associated with poorer 5-year OS compared to KRAS wild type tumors, and only in male patients. Additionally, high compared to low expression of GPX4, FTH1, FTL , which suppressed ferroptosis, were associated with poorer 5-year OS only in KRAS mutant tumors from male CRC patients. Low compared to high expression of ACSL4 was associated with poorer OS for this group. Our results show that KRAS mutant tumors from male CRC patients have suppressed ferroptosis, and gene expression changes that suppress ferroptosis associate with adverse outcomes for these patients, revealing a novel potential avenue for therapeutic approaches.

The Crossroads of Cancer Epigenetics and Immune Checkpoint Therapy.

Immune checkpoint inhibitors (ICI) have significantly improved treatment outcomes for several types of cancer over the past decade, but significant challenges that limit wider effectiveness of current immunotherapies remain to be addressed. Certain "cold" tumor types, such as pancreatic cancer, exhibit very low response rates to ICI due to intrinsically low immunogenicity. In addition, many patients who initially respond to ICI lack a sustained response due to T-cell exhaustion. Several recent studies show that epigenetic modifiers, such as SETDB1 and LSD1, can play critical roles in regulating both tumor cell-intrinsic immunity and T-cell exhaustion. Here, we review the evidence showing that multiple epigenetic regulators silence the expression of endogenous antigens, and their loss induces viral mimicry responses bolstering the response of "cold" tumors to ICI in preclinical models. Similarly, a previously unappreciated role for epigenetic enzymes is emerging in the establishment and maintenance of stem-like T-cell populations that are critical mediators of response to ICI. Targeting the crossroads of epigenetics and immune checkpoint therapy has tremendous potential to improve antitumor immune responses and herald the next generation of sustained responses in immuno-oncology.

Isotope tracing reveals distinct substrate preference in murine melanoma subtypes with differing anti-tumor immunity.

BACKGROUND: Research about tumor "metabolic flexibility"-the ability of cells to toggle between preferred nutrients depending on the metabolic context-has largely focused on obesity-associated cancers. However, increasing evidence for a key role for nutrient competition in the tumor microenvironment, as well as for substrate regulation of immune function, suggests that substrate metabolism deserves reconsideration in immunogenic tumors that are not strongly associated with obesity. METHODS: We compare two murine models: immunologically cold YUMM1.7 and immunologically-hot YUMMER1.7. We utilize stable isotope and radioisotope tracer-based metabolic flux studies as well as gas and liquid chromatography-based metabolomics analyses to comprehensively probe substrate preference in YUMM1.7 and YUMMER1.7 cells, with a subset of studies on the impact of available metabolites across a panel of five additional melanoma cell lines. We analyze bulk RNA-seq data and identify increased expression of amino acid and glucose metabolism genes in YUMMER1.7. Finally, we analyze melanoma patient RNA-seq data to identify potential prognostic predictors rooted in metabolism. RESULTS: We demonstrate using stable isotope tracer-based metabolic flux studies as well as gas and liquid chromatography-based metabolomics that immunologically-hot melanoma utilizes more glutamine than immunologically-cold melanoma in vivo and in vitro. Analyses of human melanoma RNA-seq data demonstrate that glutamine transporter and other anaplerotic gene expression positively correlates with lymphocyte infiltration and function. CONCLUSIONS: Here, we highlight the importance of understanding metabolism in non-obesity-associated cancers, such as melanoma. This work advances the understanding of the correlation between metabolism and immunogenicity in the tumor microenvironment and provides evidence supporting metabolic gene expression as potential prognostic factors of melanoma progression and may inform investigations of adjunctive metabolic therapy in melanoma. TRIAL REGISTRATION: Deidentified data from The Cancer Genome Atlas were analyzed.

Coupled fibromodulin and SOX2 signaling as a critical regulator of metastatic outgrowth in melanoma.

We aimed to study mechanisms controlling metastatic outgrowth of melanoma into clinically relevant lesions, a critical process responsible for the majority of melanoma deaths. To this end, we developed novel in vivo models and identified molecular events that can be ascribed to their distinct phenotypes, indolent or highly metastatic. Induction of a proliferative state at distant sites was associated with high levels of the stem-like/progenitor marker, SOX2, and required the upregulation of FMOD, an extracellular matrix component, which modulates tumor-stroma interactions. Functional studies revealed a possible link between FMOD and SOX2; dual FMOD and SOX2 silencing nearly abolished brain metastasis and had a similar effect on distant metastasis to other sites. Our in vitro data suggests that FMOD and SOX2 cooperation plays an important role in tumor vasculogenic mimicry. Furthermore, we found that FMOD and SOX2 functional roles might converge at the activation of transcriptional co-factors YAP and TAZ, possibly via crosstalk with the tumor suppressor Hippo pathway. Finally, high expression of both genes in patient specimens predicted early development of brain metastasis. Thus, our study identifies FMOD and SOX2 cooperation as a novel regulatory mechanism that might be linked functionally to melanoma metastatic competence.

Targeting the NANOG/HDAC1 axis reverses resistance to PD-1 blockade by reinvigorating the antitumor immunity cycle.

Immune checkpoint blockade (ICB) therapy has shifted the paradigm for cancer treatment. However, the majority of patients lack effective responses because of the emergence of immune-refractory tumors that disrupt the amplification of antitumor immunity. Therefore, the identification of clinically available targets that restrict antitumor immunity is required to develop potential combination therapies. Here, using transcriptomic data on patients with cancer treated with programmed cell death protein 1 (PD-1) therapy and newly established mouse preclinical anti-PD-1 therapy-refractory models, we identified NANOG as a factor restricting the amplification of the antitumor immunity cycle, thereby contributing to the immune-refractory feature of the tumor microenvironment (TME). Mechanistically, NANOG induced insufficient T cell infiltration and resistance to CTL-mediated killing via the histone deacetylase 1-dependent (HDAC1-dependent) regulation of CXCL10 and MCL1, respectively. Importantly, HDAC1 inhibition using an actionable agent sensitized NANOGhi immune-refractory tumors to PD-1 blockade by reinvigorating the antitumor immunity cycle. Thus, our findings implicate the NANOG/HDAC1 axis as a central molecular target for controlling immune-refractory tumors and provide a rationale for combining HDAC inhibitors to reverse the refractoriness of tumors to ICB therapy.

Physachenolide C induces complete regression of established murine melanoma tumors via apoptosis and cell cycle arrest.

Melanoma is an aggressive skin cancer that metastasizes to other organs. While immune checkpoint blockade with anti-PD-1 has transformed the treatment of advanced melanoma, many melanoma patients fail to respond to anti-PD-1 therapy or develop acquired resistance. Thus, effective treatment of melanoma still represents an unmet clinical need. Our prior studies support the anti-cancer activity of the 17ß-hydroxywithanolide class of natural products, including physachenolide C (PCC). As single agents, PCC and its semi-synthetic analog demonstrated direct cytotoxicity in a panel of murine melanoma cell lines, which share common driver mutations with human melanoma; the IC50 values ranged from 0.19-1.8 µM. PCC treatment induced apoptosis of tumor cells both in vitro and in vivo. In vivo treatment with PCC alone caused the complete regression of established melanoma tumors in all mice, with a durable response in 33% of mice after discontinuation of treatment. T cell-mediated immunity did not contribute to the therapeutic efficacy of PCC or prevent tumor recurrence in YUMM2.1 melanoma model. In addition to apoptosis, PCC treatment induced G0-G1 cell cycle arrest of melanoma cells, which upon removal of PCC, re-entered the cell cycle. PCC-induced cycle cell arrest likely contributed to the in vivo tumor recurrence in a portion of mice after discontinuation of treatment. Thus, 17ß-hydroxywithanolides have the potential to improve the therapeutic outcome for patients with advanced melanoma.

KDM5B promotes immune evasion by recruiting SETDB1 to silence retroelements.

Tumours use various strategies to evade immune surveillance1,2. Immunotherapies targeting tumour immune evasion such as immune checkpoint blockade have shown considerable efficacy on multiple cancers3,4 but are ineffective for most patients due to primary or acquired resistance5-7. Recent studies showed that some epigenetic regulators suppress anti-tumour immunity2,8-12, suggesting that epigenetic therapies could boost anti-tumour immune responses and overcome resistance to current immunotherapies. Here we show that, in mouse melanoma models, depletion of KDM5B-an H3K4 demethylase that is critical for melanoma maintenance and drug resistance13-15-induces robust adaptive immune responses and enhances responses to immune checkpoint blockade. Mechanistically, KDM5B recruits the H3K9 methyltransferase SETDB1 to repress endogenous retroelements such as MMVL30 in a demethylase-independent manner. Derepression of these retroelements activates cytosolic RNA-sensing and DNA-sensing pathways and the subsequent type-I interferon response, leading to tumour rejection and induction of immune memory. Our results demonstrate that KDM5B suppresses anti-tumour immunity by epigenetic silencing of retroelements. We therefore reveal roles of KDM5B in heterochromatin regulation and immune evasion in melanoma, opening new paths for the development of KDM5B-targeting and SETDB1-targeting therapies to enhance tumour immunogenicity and overcome immunotherapy resistance.

3D Model of the Early Melanoma Microenvironment Captures Macrophage Transition into a Tumor-Promoting Phenotype.

Tumor immune response is shaped by the tumor microenvironment (TME), which often evolves to be immunosuppressive, promoting disease progression and metastasis. An important example is melanoma tumors, which display high numbers of tumor-associated macrophages (TAMs) that are immunosuppressive but also have the potential to restore anti-tumor activity. However, to therapeutically target TAMs, there is a need to understand the early events that shape their tumor-promoting profile. To address this, we built and optimized 3D in vitro co-culture systems, composed of a collagen-I matrix scaffolding murine bone-marrow-derived macrophages (BMDMs), YUMM1.7 melanoma cells, and fibroblasts to recreate the early melanoma TME and study how interactions with fibroblasts and tumor cells modulate macrophage immune activity. We monitored BMDM behavior and interactions through time-lapse imaging and characterized their activation and secretion. We found that stromal cells induced a rapid functional activation, with increased motility and response from BMDMs. Over the course of seven days, BMDMs acquired a phenotype and secretion profile that resembled melanoma TAMs in established tumors. Overall, the direct cell-cell interactions with the stromal components in a 3D environment shape BMDM transition to a TAM-like immunosuppressive state. Our systems will enable future studies of changes in macrophage-stromal cross-talk in the melanoma TME.

Anti-PD-1/L1 lead-in before MAPK inhibitor combination maximizes antitumor immunity and efficacy.

Rationally sequencing and combining PD-1/L1-and MAPK-targeted therapies may overcome innate and acquired resistance. Since increased clinical benefit of MAPK inhibitors (MAPKi) is associated with previous immune checkpoint therapy, we compare the efficacies of sequential and/or combinatorial regimens in subcutaneous murine models of melanoma driven by BrafV600, Nras, or Nf1 mutations as well as colorectal and pancreatic carcinoma driven by KrasG12C. Anti-PD-1/L1 lead-in preceding MAPKi combination optimizes response durability by promoting pro-inflammatory polarization of macrophages and clonal expansion of interferon-γhi, and CD8+ cytotoxic and proliferative (versus CD4+ regulatory) T cells that highly express activation genes. Since therapeutic resistance of melanoma brain metastasis (MBM) limits patient survival, we demonstrate that sequencing anti-PD-1/L1 therapy before MAPKi combination suppresses MBM and improves mouse survival with robust T cell clonal expansion in both intracranial and extracranial metastatic sites. We propose clinically testing brief anti-PD-1/L1 (± anti-CTLA-4) dosing before MAPKi co-treatment to suppress therapeutic resistance.

The deacylase SIRT5 supports melanoma viability by influencing chromatin dynamics.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy.

Cytokines were the first modern immunotherapies to produce durable responses in patients with advanced cancer, but they have only modest efficacy and limited tolerability1,2. In an effort to identify alternative cytokine pathways for immunotherapy, we found that components of the interleukin-18 (IL-18) pathway are upregulated on tumour-infiltrating lymphocytes, suggesting that IL-18 therapy could enhance anti-tumour immunity. However, recombinant IL-18 previously did not demonstrate efficacy in clinical trials3. Here we show that IL-18BP, a high-affinity IL-18 decoy receptor, is frequently upregulated in diverse human and mouse tumours and limits the anti-tumour activity of IL-18 in mice. Using directed evolution, we engineered a 'decoy-resistant' IL-18 (DR-18) that maintains signalling potential but is impervious to inhibition by IL-18BP. Unlike wild-type IL-18, DR-18 exerted potent anti-tumour effects in mouse tumour models by promoting the development of poly-functional effector CD8+ T cells, decreasing the prevalence of exhausted CD8+ T cells that express the transcriptional regulator of exhaustion TOX, and expanding the pool of stem-like TCF1+ precursor CD8+ T cells. DR-18 also enhanced the activity and maturation of natural killer cells to effectively treat anti-PD-1 resistant tumours that have lost surface expression of major histocompatibility complex class I molecules. These results highlight the potential of the IL-18 pathway for immunotherapeutic intervention and implicate IL-18BP as a major therapeutic barrier.

Structure-Guided Identification of DNMT3B Inhibitors.

Methyltransferase 3 beta (DNMT3B) inhibitors that interfere with cancer growth are emerging possibilities for treatment of melanoma. Herein we identify small molecule inhibitors of DNMT3B starting from a homology model based on a DNMT3A crystal structure. Virtual screening by docking led to purchase of 15 compounds, among which 5 were found to inhibit the activity of DNMT3B with IC50 values of 13-72 µM in a fluorogenic assay. Eight analogues of 7, 10, and 12 were purchased to provide 2 more active compounds. Compound 11 is particularly notable as it shows good selectivity with no inhibition of DNMT1 and 22 µM potency toward DNMT3B. Following additional de novo design, exploratory synthesis of 17 analogues of 11 delivered 5 additional inhibitors of DNMT3B with the most potent being 33h with an IC50 of 8.0 µM. This result was well confirmed in an ultrahigh-performance liquid chromatography (UHPLC)-based analytical assay, which yielded an IC50 of 4.8 µM. Structure-activity data are rationalized based on computed structures for DNMT3B complexes.