Purpose: Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC. Experimental Design: Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms. Results: Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance. Conclusions: CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1–NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.

Myelodysplastic syndrome and acute myeloid leukemia (AML) belong to a continuous disease spectrum of myeloid malignancies with poor prognosis in the relapsed/refractory setting necessitating novel therapies. Natural killer (NK) cells from patients with myeloid malignancies display global dysfunction with impaired killing capacity, altered metabolism, and an exhausted phenotype at the single-cell transcriptomic and proteomic levels. In this study, we identified that this dysfunction was mediated through a cross-talk between NK cells and myeloid blasts necessitating cell-cell contact. NK cell dysfunction could be prevented by targeting the αvβ-integrin/TGF-β/SMAD pathway but, once established, was persistent because of profound epigenetic reprogramming. We identified BATF as a core transcription factor and the main mediator of this NK cell dysfunction in AML. Mechanistically, we found that BATF was directly regulated and induced by SMAD2/3 and, in turn, bound to key genes related to NK cell exhaustion, such as HAVCR2 , LAG3 , TIGIT , and CTLA4 . BATF deletion enhanced NK cell function against AML in vitro and in vivo. Collectively, our findings reveal a previously unidentified mechanism of NK immune evasion in AML manifested by epigenetic rewiring and inactivation of NK cells by myeloid blasts. This work highlights the importance of using healthy allogeneic NK cells as an adoptive cell therapy to treat patients with myeloid malignancies combined with strategies aimed at preventing the dysfunction by targeting the TGF-β pathway or BATF.

Immunotherapy has brought new hope for cancer patients in recent times. However, despite the promising success of immunotherapy, there is still a need to address major challenges including heterogeneity in response among patients, the reoccurrence of the disease, and iRAEs (immune-related adverse effects). The first critical step towards solving these issues is understanding the epigenomic events that play a significant role in the regulation of specific biomolecules in the context of the immune population present in the tumor immune microenvironment (TIME) during various treatments and responses. A prominent advantage of this step is that it would enable researchers to harness the reversibility of epigenetic modifications for their druggability. Therefore, we reviewed the crucial studies in which varying epigenomic events were captured with immuno-oncology set-ups. Finally, we discuss the therapeutic possibilities of their utilization for the betterment of immunotherapy in terms of diagnosis, progression, and cure for cancer patients.

Purpose: BRAFV600E-mutated colorectal cancer exhibits a strong correlation with DNA hypermethylation, suggesting that this subgroup of tumors presents unique epigenomic phenotypes. Nonetheless, 5-azacitidine, which inhibits DNA methyltransferase activity, is not efficacious in BRAFV600E colorectal cancer in vivo. Experimental Design: We randomized and treated mice implanted with patient-derived tumor xenografts harboring BRAFV600E mutation with control, 5-azacitidine, vemurafenib (BRAF inhibitor), or the combination. Comprehensive epigenomic profiling was conducted on control and 5-azacitidine–treated tumor samples, including DNA methylation, histone modifications, chromatin accessibility, and gene expression. Combinations of epigenetic agents were explored in preclinical BRAFV600E colorectal cancer models. Results: A profound reduction of DNA methylation levels upon 5-azacitidine treatment was confirmed, however, transcriptional repression was not relieved. This study unbiasedly explored the adaptive engagement of other epigenomic modifications upon 5-azacitidine treatment. A loss of histone acetylation and a gain of histone methylations, including H3K27 and H3K4 trimethylation, were observed around these hypomethylated regions, suggesting the involvement of polycomb repressive complex (PRC) activity around the genome with loss of DNA methylation, therefore maintaining the repression of key tumor-suppressor genes. Combined inhibition of PRC activity through EZH2 inhibition with 5-azacitidine treatment additively improved efficacies in BRAFV600E colorectal cancer cells. Conclusions: In conclusion, DNA hypomethylation by 5-azacitidine exhibits a close association with H3K27me3 and PRC activity in BRAFV600E colorectal cancer, and simultaneous blockade of DNA methyltransferase and EZH2 holds promise as a potential therapeutic strategy for patients with BRAFV600E-mutated colorectal cancer.

Tools for genome-wide rapid identification of peptide–major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell–APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515.

Purpose: Aberrant enhancer dynamics play a critical role in the initiation and progression of colorectal cancer, particularly in the BRAFV600E-mutated metastatic subtype, which uniquely exhibits a strong epigenetic phenotype. Building on this epigenetic vulnerability, bromodomain 2, a reader of H3K27ac-marked enhancers, was found to be synthetically lethal with BRAF + EGFR inhibition. Experimental Design: We evaluated the effectiveness of targeting aberrant enhancers with bromodomain and extraterminal (BET) + MAPK pathway inhibitors in patient-derived xenograft models of metastatic colorectal cancer, followed by comprehensive transcriptomic and chromatin profiling. Results: BET plus standard MAPK inhibitors demonstrated improved efficacy against BRAFV600E colorectal cancer and selective improvements against RAS-mutant colorectal cancer in vivo. This combination induced a more profound downregulation of the MAPK signaling pathway than MAPK inhibition alone. The loss of activation signals on H3K27ac-marked enhancers led to the dysregulation of core regulatory circuitries, especially the MAPK downstream E26 transformation–specific transcription factor (TF) family and MYC. Single-nucleus RNA sequencing + Assay for Transposase-Accessible Chromatin using sequencing distinguished differential transcriptomic and chromatin dynamics at the cell-type level. Profound downregulation of well-differentiated cell types confirmed deep inhibition of MAPK signaling and downstream TF. Conversely, an abundance of dedifferentiated cell populations emerged after MAPK or combination inhibition, suggesting therapy-induced cell-state switching and adaptation. Conclusions: Our work demonstrates that BET inhibition improves MAPK signaling blockade through profound epigenetic reprogramming of core TF circuits. These findings provide a preclinical rationale for the evaluation of BET + BRAF + EGFR inhibition in patients with treatment-refractory BRAFV600E metastatic colorectal cancer (NCT06102902).