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.

Inhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses.

There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.

Selective inhibition of cancer cell self-renewal through a Quisinostat-histone H1.0 axis.

Continuous cancer growth is driven by subsets of self-renewing malignant cells. Targeting of uncontrolled self-renewal through inhibition of stem cell-related signaling pathways has proven challenging. Here, we show that cancer cells can be selectively deprived of self-renewal ability by interfering with their epigenetic state. Re-expression of histone H1.0, a tumor-suppressive factor that inhibits cancer cell self-renewal in many cancer types, can be broadly induced by the clinically well-tolerated compound Quisinostat. Through H1.0, Quisinostat inhibits cancer cell self-renewal and halts tumor maintenance without affecting normal stem cell function. Quisinostat also hinders expansion of cells surviving targeted therapy, independently of the cancer types and the resistance mechanism, and inhibits disease relapse in mouse models of lung cancer. Our results identify H1.0 as a major mediator of Quisinostat's antitumor effect and suggest that sequential administration of targeted therapy and Quisinostat may be a broadly applicable strategy to induce a prolonged response in patients.

Redistribution of EZH2 promotes malignant phenotypes by rewiring developmental programmes.

Epigenetic regulators are often hijacked by cancer cells to sustain malignant phenotypes. How cells repurpose key regulators of cell identity as tumour-promoting factors is unclear. The antithetic role of the Polycomb component EZH2 in normal brain and glioma provides a paradigm to dissect how wild-type chromatin modifiers gain a pathological function in cancer. Here, we show that oncogenic signalling induces redistribution of EZH2 across the genome, and through misregulation of homeotic genes corrupts the identity of neural cells. Characterisation of EZH2 targets in de novo transformed cells, combined with analysis of glioma patient datasets and cell lines, reveals that acquisition of tumorigenic potential is accompanied by a transcriptional switch involving de-repression of spinal cord-specifying HOX genes and concomitant silencing of the empty spiracles homologue EMX2, a critical regulator of neurogenesis in the forebrain. Maintenance of tumorigenic potential by glioblastoma cells requires EMX2 repression, since forced EMX2 expression prevents tumour formation. Thus, by redistributing EZH2 across the genome, cancer cells subvert developmental transcriptional programmes that specify normal cell identity and remove physiological breaks that restrain cell proliferation.

Epigenetics and Cancer Stem Cells: Unleashing, Hijacking, and Restricting Cellular Plasticity.

Epigenetic mechanisms have emerged as key players in cancer development which affect cellular states at multiple stages of the disease. During carcinogenesis, alterations in chromatin and DNA methylation resulting from genetic lesions unleash cellular plasticity and favor oncogenic cellular reprogramming. At later stages, during cancer growth and progression, additional epigenetic changes triggered by interaction with the microenvironment modulate cancer cell phenotypes and properties, and shape tumor architecture. We review here recent advances highlighting the interplay between epigenetics, genetics, and cell-to-cell signaling in cancer, with particular emphasis on mechanisms relevant for cancer stem cell formation (CSC) and function.

ROBO2 restricts the nephrogenic field and regulates Wolffian duct-nephrogenic cord separation.

ROBO2 plays a key role in regulating ureteric bud (UB) formation in the embryo, with mutations in humans and mice leading to supernumerary kidneys. Previous studies have established that the number and position of UB outgrowths is determined by the domain of metanephric mesenchymal Gdnf expression, which is expanded anteriorly in Robo2 mouse mutants. To clarify how this phenotype arises, we used high-resolution 3D imaging to reveal an increase in the number of nephrogenic cord cells, leading to extension of the metanephric mesenchyme field in Robo2-null mouse embryos. Ex vivo experiments suggested a dependence of this effect on proliferative signals from the Wolffian duct. Loss of Robo2 resulted in a failure of the normal separation of the mesenchyme from the Wolffian duct/ureteric epithelium, suggesting that aberrant juxtaposition of these two compartments in Robo2-null mice exposes the mesenchyme to abnormally high levels of proliferative stimuli. Our data suggest a new model in which SLIT-ROBO signalling acts not by attenuating Gdnf expression or activity, but instead by limiting epithelial/mesenchymal interactions in the nascent metanephros and restricting the extent of the nephrogenic field. These insights illuminate the aetiology of multiplex kidney formation in human individuals with ROBO2 mutations.

Rapid screening of gene function by systemic delivery of morpholino oligonucleotides to live mouse embryos.

Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis.