A spatial architecture-embedding HLA signature to predict clinical response to immunotherapy in renal cell carcinoma.

An important challenge in the real-world management of patients with advanced clear-cell renal cell carcinoma (aRCC) is determining who might benefit from immune checkpoint blockade (ICB). Here we performed a comprehensive multiomics mapping of aRCC in the context of ICB treatment, involving discovery analyses in a real-world data cohort followed by validation in independent cohorts. We cross-connected bulk-tumor transcriptomes across >1,000 patients with validations at single-cell and spatial resolutions, revealing a patient-specific crosstalk between proinflammatory tumor-associated macrophages and (pre-)exhausted CD8+ T cells that was distinguished by a human leukocyte antigen repertoire with higher preference for tumoral neoantigens. A cross-omics machine learning pipeline helped derive a new tumor transcriptomic footprint of neoantigen-favoring human leukocyte antigen alleles. This machine learning signature correlated with positive outcome following ICB treatment in both real-world data and independent clinical cohorts. In experiments using the RENCA-tumor mouse model, CD40 agonism combined with PD1 blockade potentiated both proinflammatory tumor-associated macrophages and CD8+ T cells, thereby achieving maximal antitumor efficacy relative to other tested regimens. Thus, we present a new multiomics and spatial map of the immune-community architecture that drives ICB response in patients with aRCC.

PI3K/mTOR inhibition induces tumour microenvironment remodelling and sensitises pS6high uterine leiomyosarcoma to PD-1 blockade.

BACKGROUND: Uterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some 'challenging-to-treat' cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB. METHODS: We performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient-derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single-agent and a combination of PI3K/mTOR inhibitors with PD-1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single-cell RNA/TCR sequencing of serially collected biopsies. RESULTS: PI3K/mTOR over-activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB-resistant humanized uLMS PDX model, fostering adaptive anti-tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti-tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD-1+ T cells displaying an exhausted phenotype. When combined with anti-PD-1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single-agent anti-PD-1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper-expansion of a cytotoxic CD8+ T-cell population supported by a CD4+ Th1 niche. CONCLUSIONS: Our findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.

An autophagy program that promotes T cell egress from the lymph node controls responses to immune checkpoint blockade.

Lymphatic endothelial cells (LECs) of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady-state and tumor-bearing conditions is lacking. Intravital imaging combined with immune phenotyping shows that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs by increasing the availability of the main egress signal sphingosine-1-phosphate. Single-cell RNA sequencing of tumor-draining LNs shows that loss of ATG5 remodels niche-specific LEC phenotypes involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC autophagy prevents recruitment of tumor-infiltrating T and natural killer cells and abrogates response to immunotherapy. Thus, an LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics.

Tumor endothelial cell autophagy is a key vascular-immune checkpoint in melanoma.

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.

Single-cell profiling and zebrafish avatars reveal LGALS1 as immunomodulating target in glioblastoma.

Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs-features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets.

AP-1 and TGFß cooperativity drives non-canonical Hedgehog signaling in resistant basal cell carcinoma.

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.

Loss of Primary Cilia Drives Switching from Hedgehog to Ras/MAPK Pathway in Resistant Basal Cell Carcinoma.

Basal cell carcinomas (BCCs) rely on Hedgehog (HH) pathway growth signal amplification by the microtubule-based organelle, the primary cilium. Despite naive tumor responsiveness to Smoothened inhibitors (Smoi), resistance in advanced tumors remains common. Although the resistant BCCs usually maintain HH pathway activation, squamous cell carcinomas with Ras/MAPK pathway activation also arise, and the molecular basis of tumor type and pathway selection are still obscure. Here, we identify the primary cilium as a critical determinant controlling tumor pathway switching. Strikingly, Smoothened inhibitor-resistant BCCs have an increased mutational load in ciliome genes, resulting in reduced primary cilia and HH pathway activation compared with naive or Gorlin syndrome patient BCCs. Gene set enrichment analysis of resistant BCCs with a low HH pathway signature showed increased Ras/MAPK pathway activation. Tissue analysis confirmed an inverse relationship between primary cilia presence and Ras/MAPK activation, and primary cilia removal in BCCs potentiated Ras/MAPK pathway activation. Moreover, activating Ras in HH-responsive cell lines conferred resistance to both canonical (vismodegib) and noncanonical (atypical protein kinase C and MRTF inhibitors) HH pathway inhibitors and conferred sensitivity to MAPK inhibitors. Our results provide insights into BCC treatment and identify the primary cilium as an important lineage gatekeeper, preventing HH-to-Ras/MAPK pathway switching.

TFAP2C- and p63-Dependent Networks Sequentially Rearrange Chromatin Landscapes to Drive Human Epidermal Lineage Commitment.

Tissue development results from lineage-specific transcription factors (TFs) programming a dynamic chromatin landscape through progressive cell fate transitions. Here, we define epigenomic landscape during epidermal differentiation of human pluripotent stem cells (PSCs) and create inference networks that integrate gene expression, chromatin accessibility, and TF binding to define regulatory mechanisms during keratinocyte specification. We found two critical chromatin networks during surface ectoderm initiation and keratinocyte maturation, which are driven by TFAP2C and p63, respectively. Consistently, TFAP2C, but not p63, is sufficient to initiate surface ectoderm differentiation, and TFAP2C-initiated progenitor cells are capable of maturing into functional keratinocytes. Mechanistically, TFAP2C primes the surface ectoderm chromatin landscape and induces p63 expression and binding sites, thus allowing maturation factor p63 to positively autoregulate its own expression and close a subset of the TFAP2C-initiated surface ectoderm program. Our work provides a general framework to infer TF networks controlling chromatin transitions that will facilitate future regenerative medicine advances.

Honey bee Royalactin unlocks conserved pluripotency pathway in mammals.

Royal jelly is the queen-maker for the honey bee Apis mellifera, and has cross-species effects on longevity, fertility, and regeneration in mammals. Despite this knowledge, how royal jelly or its components exert their myriad effects has remained poorly understood. Using mouse embryonic stem cells as a platform, here we report that through its major protein component Royalactin, royal jelly can maintain pluripotency by activating a ground-state pluripotency-like gene network. We further identify Regina, a mammalian structural analog of Royalactin that also induces a naive-like state in mouse embryonic stem cells. This reveals an important innate program for stem cell self-renewal with broad implications in understanding the molecular regulation of stem cell fate across species.

Retinoic acid and BMP4 cooperate with p63 to alter chromatin dynamics during surface epithelial commitment.

Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1-3, yet conversion of hESCs into transplantable cells or tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 (encoded by TP63)4,5 during surface ectoderm commitment. In contrast to other master regulators6-9, p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 (trimethylated histone H3 lysine 27). Cohesin HiChIP10 visualizations of chromosome conformation show that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated by TFAP2C regulation11. Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by exposure to specific morphogens.

CRISPR-Mediated Reorganization of Chromatin Loop Structure.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Noncanonical hedgehog pathway activation through SRF-MKL1 promotes drug resistance in basal cell carcinomas.

Hedgehog pathway-dependent cancers can escape Smoothened (SMO) inhibition through mutations in genes encoding canonical hedgehog pathway components; however, around 50% of drug-resistant basal cell carcinomas (BCCs) lack additional variants of these genes. Here we use multidimensional genomics analysis of human and mouse drug-resistant BCCs to identify a noncanonical hedgehog activation pathway driven by the transcription factor serum response factor (SRF). Active SRF along with its coactivator megakaryoblastic leukemia 1 (MKL1) binds DNA near hedgehog target genes and forms a previously unknown protein complex with the hedgehog transcription factor glioma-associated oncogene family zinc finger-1 (GLI1), causing amplification of GLI1 transcriptional activity. We show that cytoskeletal activation through Rho and the formin family member Diaphanous (mDia) is required for SRF-MKL-driven GLI1 activation and for tumor cell viability. Remarkably, nuclear MKL1 staining served as a biomarker in tumors from mice and human subjects to predict tumor responsiveness to MKL inhibitors, highlighting the therapeutic potential of targeting this pathway. Thus, our study illuminates, for the first time, cytoskeletal-activation-driven transcription as a personalized therapeutic target for combatting drug-resistant malignancies.