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Background Cancer genomes are shaped by mutational processes with complex spatial variation at multiple scales. Entire classes of regulatory elements are affected by local variations in mutation frequency. However, the underlying mechanisms with functional and genetic determinants remain poorly understood. Results We characterise the mutational landscape of 1.3 million gene-regulatory and chromatin architectural elements in 2419 whole cancer genomes with transcriptional and pathway activity, functional conservation and recurrent driver events. We develop RM2, a statistical model that quantifies mutational enrichment or depletion in classes of genomic elements through genetic, trinucleotide and megabase-scale effects. We report a map of localised mutational processes affecting CTCF binding sites, transcription start sites (TSS) and tissue-specific open-chromatin regions. Increased mutation frequency in TSSs associates with mRNA abundance in most cancer types, while open-chromatin regions are generally enriched in mutations. We identify ~ 10,000 CTCF binding sites with core DNA motifs and constitutive binding in 66 cell types that represent focal points of mutagenesis. We detect site-specific mutational signature enrichments, such as SBS40 in open-chromatin regions in prostate cancer and SBS17b in CTCF binding sites in gastrointestinal cancers. Candidate drivers of localised mutagenesis are also apparent: BRAF mutations associate with mutational enrichments at CTCF binding sites in melanoma, and ARID1A mutations with TSS-specific mutagenesis in pancreatic cancer. Conclusions Our method and catalogue of localised mutational processes provide novel perspectives to cancer genome evolution, mutagenesis, DNA repair and driver gene discovery. The functional and genetic correlates of mutational processes suggest mechanistic hypotheses for future studies.

Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4 + T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4 + helper T (T H ) cell responses or the ability to restore CD4 + T H -mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4 + T H 1 cell amplification, prevents CD4 + T H 1 cytokine production and abolishes CD4 + cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating T H 1-like T regulatory cells, demonstrating a system-wide CD4–T H 1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-γ-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1 + follicular helper T cells, despite high PD-1 expression. Thus, CD4 + T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration. Snell et al. examine the heterogeneity of CD4 + T cells in chronic viral infection, showing that PD-L1 blockade enhances a cytotoxic gene program in antigen-specific T H 1 cells and can restore antiviral CD4 + T cell killer function.

Type-II topoisomerases resolve topological stress in DNA through double-strand breaks. While topoisomerases are chemotherapy targets linked to therapy-related genotoxicity, TOP2B is uniquely positioned to influence mutagenesis through its activity in non-dividing cells and sensitivity to topoisomerase poisons. To investigate this, we generated DNA-binding maps of TOP2B, CTCF, and RAD21 in human cancer samples and analyzed these for driver mutations and mutational processes across 6500 whole cancer genomes. TOP2B-CTCF-RAD21 and TOP2B-RAD21 sites are enriched in somatic mutations and structural variants, particularly at sites with evolutionary conservation, high transcription and long-range chromatin interactions. TOP2B binds driver genes such as TP53 , MYC , FOXA1 , and VHL , and many frequently mutated non-coding regions. We show that one non-coding TOP2B-bound element at the non-coding RNA gene RMRP drives tumor initiation and growth in vivo. Our study highlights TOP2B as a safeguard of genome integrity and a marker of mutational processes and hotspots in cancer, underscoring implications for cancer genomics research. Topoisomerases resolve topological DNA stress via double-strand breaks and are established targets of cancer chemotherapies. Here, the authors link genomic binding of TOP2B with localized mutational processes in cancer genomes that include prominent driver genes and translocation hotspots.

Cyclin-dependent kinases (Cdks) are regulatory enzymes with temporal and spatial selectivity for their protein substrates that are governed by cell cycle-regulated cyclin subunits. Specific cyclin–Cdk complexes bind to and phosphorylate target proteins, coupling their activity to cell cycle states. The identification of specific cyclin–Cdk substrates is challenging and so far, has largely been achieved through indirect correlation or use of in vitro techniques. Here, we use a protein-fragment complementation assay based on the optimized yeast cytosine deaminase to systematically identify candidate substrates of budding yeast Saccharomyces cerevisiae Cdk1 and show dependency on one or more regulatory cyclins. We identified known and candidate cyclin dependencies for many predicted protein kinase Cdk1 targets and showed elusory Clb3–Cdk1-specific phosphorylation of γ-tubulin, thus establishing the timing of this event in controlling assembly of the mitotic spindle. Our strategy can be generally applied to identify substrates and accessory subunits of multisubunit protein complexes.

Type I interferons (IFN-Is) are central regulators of anti-tumor immunity and responses to immunotherapy, but they also drive the feedback inhibition underlying therapeutic resistance. In the present study, we developed a mass cytometry approach to quantify IFN-I-stimulated protein expression across immune cells and used multi-omics to uncover pre-therapy cellular states encoding responsiveness to inflammation. Analyzing peripheral blood cells from multiple cancer types revealed that differential responsiveness to IFN-Is before anti-programmed cell death protein 1 (PD1) treatment was highly predictive of long-term survival after therapy. Unexpectedly, IFN-I hyporesponsiveness efficiently predicted long-term survival, whereas high responsiveness to IFN-I was strongly associated with treatment failure and diminished survival time. Peripheral IFN-I responsive states were not associated with tumor inflammation, identifying a disconnect between systemic immune potential and ‘cold’ or ‘hot’ tumor states. Mechanistically, IFN-I responsiveness was epigenetically imprinted before therapy, poising cells for differential inflammatory responses and dysfunctional T cell effector programs. Thus, we identify physiological cell states with clinical importance that can predict success and long-term survival of PD1-blocking immunotherapy.Predicting which patients will respond to checkpoint blocking therapies is a major challenge. Here the authors score the epigenetic imprinting of T cell responsiveness to type 1 interferons and use this information to predict response to anti-PD1 therapy and long-term survival of cancer patients.

Background Kinases and phosphatases (KP) form complex self-regulating networks essential for cellular signal processing. In spite of having a wealth of data about interactions among KPs and their substrates, we have very limited models of the structures of the directed networks they form and consequently our ability to formulate hypotheses about how their structure determines the flow of information in these networks is restricted. Results We assembled and studied the largest bona fide kinase-phosphatase network (KP-Net) known to date for the yeast Saccharomyces cerevisiae . Application of the vertex sort (VS) algorithm on the KP-Net allowed us to elucidate its hierarchical structure in which nodes are sorted into top, core and bottom layers, forming a bow tie structure with a strongly connected core layer. Surprisingly, phosphatases tend to sort into the top layer, implying they are less regulated by phosphorylation than kinases. Superposition of the widest range of KP biological properties over the KP-Net hierarchy shows that core layer KPs: (i), receive the largest number of inputs; (ii), form bottlenecks implicated in multiple pathways and in decision-making; (iii), and are among the most regulated KPs both temporally and spatially. Moreover, top layer KPs are more abundant and less noisy than those in the bottom layer. Finally, we showed that the VS algorithm depends on node degrees without biasing the biological results of the sorted network. The VS algorithm is available as an R package ( https://cran.r-project.org/web/packages/VertexSort/index.html ). Conclusions The KP-Net model we propose possesses a bow tie hierarchical structure in which the top layer appears to ensure highest fidelity and the core layer appears to mediate signal integration and cell state-dependent signal interpretation. Our model of the yeast KP-Net provides both functional insight into its organization as we understand today and a framework for future investigation of information processing in yeast and eukaryotes in general.

Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4.sup.+ T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4.sup.+ helper T (T.sub.H) cell responses or the ability to restore CD4.sup.+ T.sub.H-mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4.sup.+ T.sub.H1 cell amplification, prevents CD4.sup.+ T.sub.H1 cytokine production and abolishes CD4.sup.+ cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating T.sub.H1-like T regulatory cells, demonstrating a system-wide CD4-T.sub.H1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-[gamma]-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1.sup.+ follicular helper T cells, despite high PD-1 expression. Thus, CD4.sup.+ T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration.

CD4 T cells are important effectors of anti-tumor immunity, yet the regulation of CD4 tumor-specific T (T TS ) cells during cancer development is still unclear. We demonstrate that CD4 T TS cells are initially primed in the tumor draining lymph node and begin to divide following tumor initiation. Distinct from CD8 T TS cells and previously defined exhaustion programs, CD4 T TS cell proliferation is rapidly frozen in place and differentiation stunted by a functional interplay of T regulatory cells and both intrinsic and extrinsic CTLA4 signaling. Together these mechanisms paralyze CD4 T TS cell differentiation, redirecting metabolic and cytokine production circuits, and reducing CD4 T TS cell accumulation in the tumor. Paralysis is actively maintained throughout cancer progression and CD4 T TS cells rapidly resume proliferation and functional differentiation when both suppressive reactions are alleviated. Strikingly, Treg depletion alone reciprocally induced CD4 T TS cells to themselves become tumor-specific Tregs, whereas CTLA4 blockade alone failed to promote T helper differentiation. Overcoming their paralysis established long-term tumor control, demonstrating a novel immune evasion mechanism that specifically cripples CD4 T TS cells to favor tumor progression.CD4 T cells are important effectors of anti-tumor immunity, yet the regulation of CD4 tumor-specific T (T TS ) cells during cancer development is still unclear. We demonstrate that CD4 T TS cells are initially primed in the tumor draining lymph node and begin to divide following tumor initiation. Distinct from CD8 T TS cells and previously defined exhaustion programs, CD4 T TS cell proliferation is rapidly frozen in place and differentiation stunted by a functional interplay of T regulatory cells and both intrinsic and extrinsic CTLA4 signaling. Together these mechanisms paralyze CD4 T TS cell differentiation, redirecting metabolic and cytokine production circuits, and reducing CD4 T TS cell accumulation in the tumor. Paralysis is actively maintained throughout cancer progression and CD4 T TS cells rapidly resume proliferation and functional differentiation when both suppressive reactions are alleviated. Strikingly, Treg depletion alone reciprocally induced CD4 T TS cells to themselves become tumor-specific Tregs, whereas CTLA4 blockade alone failed to promote T helper differentiation. Overcoming their paralysis established long-term tumor control, demonstrating a novel immune evasion mechanism that specifically cripples CD4 T TS cells to favor tumor progression.

Glycans are emerging as important regulators of T cell function but remain poorly characterized across the functionally distinct populations that exist . Here, we couple single-cell analysis technologies with soluble lectins and chemical probes to interrogate glycosylation patterns on major T cell populations across multiple mouse and human tissues. Our analysis focused on terminal glycan epitopes with immunomodulatory functions, including sialoglycan ligands for Siglecs. We demonstrate that glycosylation patterns are diverse across the resting murine T cell repertoire and dynamically remodelled in response to antigen-specific stimulation. Surprisingly, we find that human T cell populations do not share the same glycoprofiles or glycan remodelling dynamics as their murine counterparts. We show that these differences can be explained by divergent regulation of glycan biosynthesis pathways between the species. These results highlight fundamental glycophysiological differences between mouse and human T cells and reveal features that are critical to consider for glycan-targeted therapies.

Type I interferon (IFN I) and interferon-gamma are central regulators of antiviral immunity, yet how they cooperatively govern CD8 T cell fate during chronic infection remains unresolved. Here, we uncover a previously unrecognized, spatially encoded interferon circuit that actively constrains progenitor exhausted CD8 T cells (Tpex) during chronic LCMV infection. Persistent IFN I signaling indirectly restricts Tpex expansion by enforcing their sequestration within PDL1 rich B cell niches of lymphoid tissue and by suppressing T cell derived IFN gamma;. Blockade of IFN I signaling enables Tpex migration into T cell zones of splenic follicles driving IFN gamma; production, which in turn sustains PDL1 expression on myeloid cells to re-impose local inhibitory pressure. Combined IFN-I and IFN gamma; blockade disrupts this feedback, promoting coordinated niche redistribution of Tpex and checkpoint remodeling that drives robust Tpex expansion. Single-cell transcriptomics reveal that this layered IFN I with IFN gamma interplay establishes a regulatory balance that constrains Tpex proliferation while preserving effector like transcriptional programs in their progeny effector CD8 T cells, ultimately preventing premature terminal differentiation. Thus, interferons orchestrate the coordinated T cell to myeloid regulatory circuit that integrates tissue organization, cytokine feedback, and checkpoint control to regulate CD8 T cell exhaustion during chronic infection.Competing Interest StatementThe authors have declared no competing interest.Funder Information DeclaredNational Institutes of Health, https://ror.org/01cwqze88Canadian Institutes of Health Research, https://ror.org/01gavpb45