Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response (DDR), detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 minutes) and transiently induced at DNA damage sites in response to UV. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL31 and the demethylase FTO2. In the absence of METTL3 catalytic activity, cells showed delayed repair of UV-induced cyclobutane pyrimidine (CPD) adducts and elevated sensitivity to UV, demonstrating the importance of m6A in the UV-responsive DDR. Multiple DNA polymerases are involved in the UV response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair (NER) pathway3, while others participate in trans-lesion synthesis (TLS) to allow replication past damaged lesions in S phase4. DNA polymerase κ (Pol κ), which has been implicated in both NER and TLS5,6, required the catalytic activity of METTL3 for immediate localization to UV-induced DNA damage sites. Importantly, Pol κ over-expression qualitatively suppressed the CPD removal defect associated with METTL3 loss. Taken together, we have uncovered a novel function for RNA m6A modification in the UV-induced DDR, and our findings collectively support a model whereby m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8 T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.

Highlights d PCIF1 is an evolutionarily conserved mRNA m6Am methyltransferase d Loss of PCIF1 leads to loss of m6Am, but m6A level or distribution is not affected d m6Am decreases cap-dependent translation; no effect on transcription nor mRNA stability d m6Am-Exo-Seq is a robust methodology that enables global m6Am mapping

T helper (TH)-cell subsets, such as TH1 and TH17, mediate inflammation in both peripheral tissues and central nervous system. Here we show that STAT5 is required for T helper-cell pathogenicity in autoimmune neuroinflammation but not in experimental colitis. Although STAT5 promotes regulatory T cell generation and immune suppression, loss of STAT5 in CD4+ T cells resulted in diminished development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Our results showed that loss of encephalitogenic activity of STAT5-deficient autoreactive CD4+ T cells was independent of IFN-γ or interleukin 17 (IL-17) production, but was due to the impaired expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), a crucial mediator of T-cell pathogenicity. We further showed that IL-7-activated STAT5 promotes the generation of GM-CSF-producing CD4+ T cells, which were preferentially able to induce more severe EAE than TH17 or TH1 cells. Consistent with GM-CSF-producing cells being a distinct subset of TH cells, the differentiation program of these cells was distinct from that of TH17 or TH1 cells. We further found that IL-3 was secreted in a similar pattern as GM-CSF in this subset of TH cells. In conclusion, the IL-7-STAT5 axis promotes the generation of GM-CSF/IL-3-producing TH cells. These cells display a distinct transcriptional profile and may represent a novel subset of T helper cells which we designate as TH-GM.

Exhausted CD8+ T cells are key targets of immune checkpoint blockade therapy and their ineffective reinvigoration limits the durable benefit in some cancer patients. Here, we demonstrate that histone demethylase LSD1 acts to enforce an epigenetic program in progenitor exhausted CD8+ T cells to antagonize the TCF1-mediated progenitor maintenance and to promote terminal differentiation. Consequently, genetic perturbation or small molecules targeting LSD1 increases the persistence of the progenitor exhausted CD8+ T cells, which provide a sustained source for the proliferative conversion to numerically larger terminally exhausted T cells with tumor-killing cytotoxicity, thereby leading to effective and durable responses to anti-PD1 therapy. Collectively, our findings provide important insights into epigenetic mechanisms that regulate T cell exhaustion and have important implications for durable immunotherapy.

Highlights 24• PCIF1 is an evolutionarily conserved mRNA m6Am methyltransferase 25 Summary 32 mRNA modifications play an important role in regulating gene expression. One of the 33 most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we 34 demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by 35 the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation 36 on 2-O-methylated adenine located at the 5' ends of mRNAs. Furthermore, PCIF1 37 catalyzes only 5' m6Am methylation of capped mRNAs, but not internal m6A 38 methylation in vitro and in vivo. Our global mRNA methylation analysis revealed that 39 there is no crosstalk between m6Am and m6A mRNA methylation events, suggesting 40 that m6Am is functionally distinct from m6A. Importantly, our data indicate that m6Am 41 negatively impacts translation of methylated mRNAs by antagonizing cap binding 42 protein eIF4E. Together, we identify the first and only human mRNA m6Am 43 methyltransferase and demonstrate a novel mechanism of gene expression regulation 44 through PCIF1-mediated m6Am mRNA methylation in eukaryotes. 45 46 Keywords: 47 mRNA modifications, m6Am, PCIF1, translational regulation, transcriptional regulation, 48 epitranscriptomics, cap-dependent translation, eIF4E, gene expression, m6Am-Exo-Seq 49 439 5B. Boxplots of expression level (log2 TPM) of m6Am mRNAs (n=640) vs. all mRNAs 440 expressed in MEL624 cell line (n=15,258). The observed difference is significant (p-value < 441 2.2e-16) using a Mann-Whitney test. Box plots show the 25th-75th percentiles and error bars 442 depict the 10th-90th percentiles.443 5C. Biological process Gene Ontology (GO) terms that have FDR<0.05 identified by DAVID 6.8 444 5D. Genome browser views of 2 example genes with m6Am enrichment using m6Am-Exo-Seq 445 446 26 447 448 Figure 6. PCIF1-mediated deposition of m6Am does not alter transcription or stability of 449 target genes. 450 451 6A. Venn diagrams displaying overlap between m6Am-enriched genes in MEL624 cells and 452 genes identified as significantly upregulated (left) or downregulated (right) in both PCIF1 KO 453 MEL624 cell lines, by RNA-Seq and PRO-Seq. P-values are calculated with a hypergeometric 454 test. 455 6B. Heatmaps of PRO-Seq signal at genes found to be up-or down-regulated using RNA-Seq in 456 PCIF1 KO cells. Shown are the fold changes in PRO-Seq signal across the bodies of genes up-457 regulated in RNA-Seq (left) or down-regulated in RNA-Seq (right). Genes are ranked from most 458 upregulated to least (at left) and least down-regulated to most (at right), demonstrating that 459 PRO-Seq signal scales with results from RNA-seq.460 6C. RNA-Seq signal (left) and normalized PRO-Seq signal (right) within 640 m6Am-enriched 461 genes in Control MELF624 cells, as compared to PCIF1 KO cells. Box plots show the 25th-75th 462 percentiles and error bars depict the 10th-90th percentiles. Differences are not significant (P > 463 0.01). P-values were calculated using the Mann-Whitney test.464 6D. S...

Epigenetic regulators are a class of promising targets in combination with immune checkpoint inhibitors for cancer treatment, but the impact of the broad effects of perturbing epigenetic regulators on tumor immunotherapy remains to be fully explored. Here we show that ablation of the histone demethylase LSD1 in multiple tumor cells induces TGFβ expression, which exerts an inhibitory effect on T-cell immunity through suppressing the cytotoxicity of intratumoral CD8+ T cells and consequently dampens the antitumor effect of LSD1 ablation–induced T-cell infiltration. Importantly, concurrent depletion of LSD1 and TGFβ in combination with PD-1 blockade significantly increases both CD8+ T-cell infiltration and cytotoxicity, leading to eradication of poorly immunogenic tumors and a long-term protection from tumor rechallenge. Thus, combining LSD1 inhibition with blockade of TGFβ and PD-1 may represent a promising triple combination therapy for treating certain refractory tumors. SignIficance: Cotargeting LSD1 and TGFβ cooperatively elevates intratumoral CD8+ T-cell infiltration and unleashes their cytotoxicity, leading to tumor eradication upon anti–PD-1 treatment. Our findings illustrate a duality of epigenetic perturbations in immunotherapy and implicate the combination of LSD1 inhibition with dual PD-1/TGFβ blockade in treating certain poorly immunogenic tumors. This article is highlighted in the In This Issue feature, p. 1861

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by a variety of cells and plays an important role in the inflammatory response in infection as well as in autoimmunity. Recent progress has indicated that CD4 + T cell-derived GM-CSF has a prominent and non-redundant function in mediating autoimmune neuroinflammation. Thus, there is increased interest in the regulation of GM-CSF production by T helper cells, which could translate to the development of novel therapeutics for autoimmune diseases such as multiple sclerosis. This review focuses on our current understanding of the regulation and function of T cell-derived GM-CSF.