Helper T cell immunity in humans with inherited CD4 deficiency.

CD4+ T cells are vital for host defense and immune regulation. However, the fundamental role of CD4 itself remains enigmatic. We report seven patients aged 5-61 years from five families of four ancestries with autosomal recessive CD4 deficiency and a range of infections, including recalcitrant warts and Whipple's disease. All patients are homozygous for rare deleterious CD4 variants impacting expression of the canonical CD4 isoform. A shorter expressed isoform that interacts with LCK, but not HLA class II, is affected by only one variant. All patients lack CD4+ T cells and have increased numbers of TCRαß+CD4-CD8- T cells, which phenotypically and transcriptionally resemble conventional Th cells. Finally, patient CD4-CD8- αß T cells exhibit intact responses to HLA class II-restricted antigens and promote B cell differentiation in vitro. Thus, compensatory development of Th cells enables patients with inherited CD4 deficiency to acquire effective cellular and humoral immunity against an unexpectedly large range of pathogens. Nevertheless, CD4 is indispensable for protective immunity against at least human papillomaviruses and Trophyrema whipplei.

Blood-based screening for HPV-associated cancers.

Background: HPV-associated oropharyngeal cancer (HPV+OPSCC) is the most common HPV-associated cancer in the United States yet unlike cervical cancer lacks a screening test. HPV+OPSCCs are presumed to start developing 10-15 years prior to clinical diagnosis. Circulating tumor HPV DNA (ctHPVDNA) is a sensitive and specific biomarker for HPV+OPSCC. Taken together, blood-based screening for HPV+OPSCC may be feasible years prior to diagnosis. Methods: We developed an HPV whole genome sequencing assay, HPV-DeepSeek, with 99% sensitivity and specificity at clinical diagnosis. 28 plasma samples from HPV+OPSCC patients collected 1.3-10.8 years prior to diagnosis along with 1:1 age and gender-matched controls were run on HPV-DeepSeek and an HPV serology assay. Results: 22/28 (79%) of cases and 0/28 controls screened positive for HPV+OPSCC with 100% detection within four years of diagnosis and a maximum lead time of 7.8 years. We next applied a machine learning model classifying 27/28 cases (96%) with 100% detection within 10 years. Plasma-based PIK3CA gene mutations, viral genome integration events and HPV serology were used to orthogonally validate cancer detection with 68% (19/28) of the cohort having multiple cancer signals detected. Molecular fingerprinting of HPV genomes was performed across patients demonstrating that each viral genome was unique, ruling out contamination. In patients with tumor blocks from diagnosis (15/28), molecular fingerprinting was performed within patients confirming the same viral genome across time. Conclusions: We demonstrate accurate blood-based detection of HPV-associated cancers with lead times up to 10 years before clinical cancer diagnosis and in doing so, highlight the enormous potential of ctDNA-based cancer screening.

Clonal heterogeneity in ER+ breast cancer reveals the proteasome and PKC as potential therapeutic targets.

Intratumoral heterogeneity impacts the success or failure of anti-cancer therapies. Here, we investigated the evolution and mechanistic heterogeneity in clonal populations of cell models for estrogen receptor positive breast cancer. To this end, we established barcoded models of luminal breast cancer and rendered them resistant to commonly applied first line endocrine therapies. By isolating single clones from the resistant cell pools and characterizing replicates of individual clones we observed inter- (between cell lines) and intra-tumor (between different clones from the same cell line) heterogeneity. Molecular characterization at RNA and phospho-proteomic levels revealed private clonal activation of the unfolded protein response and respective sensitivity to inhibition of the proteasome, and potentially shared sensitivities for repression of protein kinase C. Our in vitro findings are consistent with tumor-heterogeneity that is observed in breast cancer patients thus highlighting the need to uncover heterogeneity at an individual patient level and to adjust therapies accordingly.

Membrane recruitment of the polarity protein Scribble by the cell adhesion receptor TMIGD1.

Scribble (Scrib) is a multidomain polarity protein and member of the leucine-rich repeat and PDZ domain (LAP) protein family. A loss of Scrib expression is associated with disturbed apical-basal polarity and tumor formation. The tumor-suppressive activity of Scrib correlates with its membrane localization. Despite the identification of numerous Scrib-interacting proteins, the mechanisms regulating its membrane recruitment are not fully understood. Here, we identify the cell adhesion receptor TMIGD1 as a membrane anchor of Scrib. TMIGD1 directly interacts with Scrib through a PDZ domain-mediated interaction and recruits Scrib to the lateral membrane domain in epithelial cells. We characterize the association of TMIGD1 with each Scrib PDZ domain and describe the crystal structure of the TMIGD1 C-terminal peptide complexed with PDZ domain 1 of Scrib. Our findings describe a mechanism of Scrib membrane localization and contribute to the understanding of the tumor-suppressive activity of Scrib.

Colorectal Cancer Organoid-Stroma Biobank Allows Subtype-Specific Assessment of Individualized Therapy Responses.

In colorectal cancers, the tumor microenvironment plays a key role in prognosis and therapy efficacy. Patient-derived tumor organoids (PDTO) show enormous potential for preclinical testing; however, cultured tumor cells lose important characteristics, including the consensus molecular subtypes (CMS). To better reflect the cellular heterogeneity, we established the colorectal cancer organoid-stroma biobank of matched PDTOs and cancer-associated fibroblasts (CAF) from 30 patients. Context-specific phenotyping showed that xenotransplantation or coculture with CAFs improves the transcriptomic fidelity and instructs subtype-specific stromal gene expression. Furthermore, functional profiling in coculture exposed CMS4-specific therapeutic resistance to gefitinib and SN-38 and prognostic expression signatures. Chemogenomic library screening identified patient- and therapy-dependent mechanisms of stromal resistance including MET as a common target. Our results demonstrate that colorectal cancer phenotypes are encrypted in the cancer epithelium in a plastic fashion that strongly depends on the context. Consequently, CAFs are essential for a faithful representation of molecular subtypes and therapy responses ex vivo. SIGNIFICANCE: Systematic characterization of the organoid-stroma biobank provides a resource for context dependency in colorectal cancer. We demonstrate a colorectal cancer subtype memory of PDTOs that is independent of specific driver mutations. Our data underscore the importance of functional profiling in cocultures for improved preclinical testing and identification of stromal resistance mechanisms. This article is featured in Selected Articles from This Issue, p. 2109.

Intestinal brush border formation requires a TMIGD1-based intermicrovillar adhesion complex.

Intestinal epithelial cells absorb nutrients through the brush border, composed of dense arrays of highly ordered microvilli at their apical membranes. A protocadherin-based intermicrovillar adhesion complex localized at microvilli tips mediates microvilli packing and organization. Here, we identified a second adhesion complex localized at the proximal base region of microvilli. This complex contained the immunoglobulin superfamily member TMIGD1, which directly interacted with the microvillar scaffolding proteins EBP50 and E3KARP. Complex formation with EBP50 required the activation of EBP50 by the actin-binding protein ezrin and was enhanced by the dephosphorylation of Ser162 in the PDZ2 domain of EBP50 by the phosphatase PP1α. Binding of the EBP50-ezrin complex to TMIGD1 enhanced the dynamic turnover of EBP50 at microvilli. Enterocyte-specific inactivation of Tmigd1 in mice resulted in microvillar blebbing, loss of intermicrovillar adhesion, and perturbed brush border formation. Thus, we identified a second adhesion complex in microvilli and propose a mechanism that promotes microvillar formation and dynamics.

5'isomiR-183-5p|+2 elicits tumor suppressor activity in a negative feedback loop with E2F1.

BACKGROUND: MicroRNAs (miRNAs) and isomiRs play important roles in tumorigenesis as essential regulators of gene expression. 5'isomiRs exhibit a shifted seed sequence compared to the canonical miRNA, resulting in different target spectra and thereby extending the phenotypic impact of the respective common pre-miRNA. However, for most miRNAs, expression and function of 5'isomiRs have not been studied in detail yet. Therefore, this study aims to investigate the functions of miRNAs and their 5'isomiRs. METHODS: The expression of 5'isomiRs was assessed in The Cancer Genome Atlas (TCGA) breast cancer patient dataset. Phenotypic effects of miR-183 overexpression in triple-negative breast cancer (TNBC) cell lines were investigated in vitro and in vivo by quantifying migration, proliferation, tumor growth and metastasis. Direct targeting of E2F1 by miR-183-5p|+2 was validated with a 3'UTR luciferase assay and linked to the phenotypes of isomiR overexpression. RESULTS: TCGA breast cancer patient data indicated that three variants of miR-183-5p are highly expressed and upregulated, namely miR-183-5p|0, miR-183-5p|+1 and miR-183-5p|+2. However, TNBC cell lines displayed reduced proliferation and invasion upon overexpression of pre-miR-183. While invasion was reduced individually by all three isomiRs, proliferation and cell cycle progression were specifically inhibited by overexpression of miR-183-5p|+2. Proteomic analysis revealed reduced expression of E2F target genes upon overexpression of this isomiR, which could be attributed to direct targeting of E2F1, specifically by miR-183-5p|+2. Knockdown of E2F1 partially phenocopied the effect of miR-183-5p|+2 overexpression on cell proliferation and cell cycle. Gene set enrichment analysis of TCGA and METABRIC patient data indicated that the activity of E2F strongly correlated with the expression of miR-183-5p, suggesting transcriptional regulation of the miRNA by a factor of the E2F family. Indeed, in vitro, expression of miR-183-5p was regulated by E2F1. Hence, miR-183-5p|+2 directly targeting E2F1 appears to be part of a negative feedback loop potentially fine-tuning its activity. CONCLUSIONS: This study demonstrates that 5'isomiRs originating from the same arm of the same pre-miRNA (i.e. pre-miR-183-5p) may exhibit different functions and thereby collectively contribute to the same phenotype. Here, one of three isomiRs was shown to counteract expression of the pre-miRNA by negatively regulating a transcriptional activator (i.e. E2F1). We speculate that this might be part of a regulatory mechanism to prevent uncontrolled cell proliferation, which is disabled during cancer progression.

On the impact of batch effect correction in TCGA isomiR expression data.

MicroRNAs (miRNAs) are small non-coding RNAs with diverse functions in post-transcriptional regulation of gene expression. Sequence and length variants of miRNAs are called isomiRs and can exert different functions compared to their canonical counterparts. The Cancer Genome Atlas (TCGA) provides isomiR-level expression data for patients of various cancer entities collected in a multi-center approach over several years. However, the impact of batch effects within individual cohorts has not been systematically investigated and corrected for before. Therefore, the aim of this study was to identify relevant cohort-specific batch variables and generate batch-corrected isomiR expression data for 16 TCGA cohorts. The main batch variables included sequencing platform, plate, sample purity and sequencing depth. Platform bias was related to certain length and sequence features of individual recurrently affected isomiRs. Furthermore, significant downregulation of reported tumor suppressive isomiRs in lung tumor tissue compared to normal samples was only observed after batch correction, highlighting the importance of working with corrected data. Batch-corrected datasets for all cohorts including quality control are provided as supplement. In summary, this study reveals that batch effects present in the TCGA dataset might mask biologically relevant effects and provides a valuable resource for research on isomiRs in cancer (accessible through GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164767).

Stromal NRG1 in luminal breast cancer defines pro-fibrotic and migratory cancer-associated fibroblasts.

HER3 is highly expressed in luminal breast cancer subtypes. Its activation by NRG1 promotes activation of AKT and ERK1/2, contributing to tumour progression and therapy resistance. HER3-targeting agents that block this activation, are currently under phase 1/2 clinical studies, and although they have shown favorable tolerability, their activity as a single agent has proven to be limited. Here we show that phosphorylation and activation of HER3 in luminal breast cancer cells occurs in a paracrine manner and is mediated by NRG1 expressed by cancer-associated fibroblasts (CAFs). Moreover, we uncover a HER3-independent NRG1 signaling in CAFs that results in the induction of a strong migratory and pro-fibrotic phenotype, describing a subtype of CAFs with elevated expression of NRG1 and an associated transcriptomic profile that determines their functional properties. Finally, we identified Hyaluronan Synthase 2 (HAS2), a targetable molecule strongly correlated with NRG1, as an attractive player supporting NRG1 signaling in CAFs.

A Wnt-Induced Phenotypic Switch in Cancer-Associated Fibroblasts Inhibits EMT in Colorectal Cancer.

Tumor progression is recognized as a result of an evolving cross-talk between tumor cells and their surrounding nontransformed stroma. Although Wnt signaling has been intensively studied in colorectal cancer, it remains unclear whether activity in the tumor-associated stroma contributes to malignancy. To specifically interfere with stromal signals, we generated Wnt-independent tumor organoids that secrete the Wnt antagonist Sfrp1. Subcutaneous transplantation into immunocompetent as well as immunodeficient mice resulted in a strong reduction of tumor growth. Histologic and transcriptomic analyses revealed that Sfrp1 induced an epithelial-mesenchymal transition (EMT) phenotype in tumor cells without affecting tumor-intrinsic Wnt signaling, suggesting involvement of nonimmune stromal cells. Blockage of canonical signaling using Sfrp1, Dkk1, or fibroblast-specific genetic ablation of ß-catenin strongly decreased the number of cancer-associated myofibroblasts (myCAF). Wnt activity in CAFs was linked with distinct subtypes, where low and high levels induced an inflammatory-like CAF (iCAF) subtype or contractile myCAFs, respectively. Coculture of tumor organoids with iCAFs resulted in significant upregulation of EMT markers, while myCAFs reverted this phenotype. In summary, we show that tumor growth and malignancy are differentially regulated via distinct fibroblast subtypes under the influence of juxtacrine Wnt signals. SIGNIFICANCE: This study provides evidence for Wnt-induced functional diversity of colorectal cancer-associated fibroblasts, representing a non-cell autonomous mechanism for colon cancer progression. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5569/F1.large.jpg.

AKT-dependent NOTCH3 activation drives tumor progression in a model of mesenchymal colorectal cancer.

Recently, a transcriptome-based consensus molecular subtype (CMS) classification of colorectal cancer (CRC) has been established, which may ultimately help to individualize CRC therapy. However, the lack of animal models that faithfully recapitulate the different molecular subtypes impedes adequate preclinical testing of stratified therapeutic concepts. Here, we demonstrate that constitutive AKT activation in intestinal epithelial cells markedly enhances tumor invasion and metastasis in Trp53ΔIEC mice (Trp53ΔIECAktE17K) upon challenge with the carcinogen azoxymethane. Gene-expression profiling indicates that Trp53ΔIECAktE17K tumors resemble the human mesenchymal colorectal cancer subtype (CMS4), which is characterized by the poorest survival rate among the four CMSs. Trp53ΔIECAktE17K tumor cells are characterized by Notch3 up-regulation, and treatment of Trp53ΔIECAktE17K mice with a NOTCH3-inhibiting antibody reduces invasion and metastasis. In CRC patients, NOTCH3 expression correlates positively with tumor grading and the presence of lymph node as well as distant metastases and is specifically up-regulated in CMS4 tumors. Therefore, we suggest NOTCH3 as a putative target for advanced CMS4 CRC patients.

S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8+ T cell expansion.

Tumor immunosuppression is a limiting factor for successful cancer therapy. The lipid sphingosine-1-phosphate (S1P), which signals through 5 distinct G protein-coupled receptors (S1PR1-5), has emerged as an important regulator of carcinogenesis. However, the utility of targeting S1P in tumors is hindered by S1P's impact on immune cell trafficking. Here, we report that ablation of the immune cell-specific receptor S1PR4, which plays a minor role in immune cell trafficking, delayed tumor development and improved therapy success in murine models of mammary and colitis-associated colorectal cancer through increased CD8+ T cell abundance. Transcriptome analysis revealed that S1PR4 affected proliferation and survival of CD8+ T cells in a cell-intrinsic manner via the expression of Pik3ap1 and Lta4h. Accordingly, PIK3AP1 expression was connected to increased CD8+ T cell proliferation and clinical parameters in human breast and colon cancer. Our data indicate a so-far-unappreciated tumor-promoting role of S1P by restricting CD8+ T cell expansion via S1PR4.

Cellular and Molecular Changes of Brain Metastases-Associated Myeloid Cells during Disease Progression and Therapeutic Response.

Brain-resident microglia and bone marrow-derived macrophages represent the most abundant non-cancerous cells in the brain tumor microenvironment with critical functions in disease progression and therapeutic response. To date little is known about genetic programs that drive disease-associated phenotypes of microglia and macrophages in brain metastases. Here we used cytometric and transcriptomic analyses to define cellular and molecular changes of the myeloid compartment at distinct stages of brain metastasis and in response to radiotherapy. We demonstrate that genetic programming of tumor education in myeloid cells occurs early during metastatic onset and remains stable throughout tumor progression. Bulk and single cell RNA sequencing revealed distinct gene signatures in brain-resident microglia and blood-borne monocytes/macrophages during brain metastasis and in response to therapeutic intervention. Our data provide a framework for understanding the functional heterogeneity of brain metastasis-associated myeloid cells based on their origin.

Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids.

Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor ß (TGF-ß) resistance as a paradigm to establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC-/-;KRASG12D mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) to adjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight a powerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.

3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids.

Immunotherapy using chimeric antigen receptor (CAR)-engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models are needed that allow to test CAR-mediated cytotoxicity in a tissue-like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3-dimensional (3D) patient-derived colon organoids. Luciferase-based measurement served as a quantitative read-out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR-engineered NK-92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen-specific cytotoxicity was studied with CAR-NK-92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor-specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.

Human colon organoids reveal distinct physiologic and oncogenic Wnt responses.

Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.

The transcriptional regulator FUBP1 influences disease outcome in murine and human myeloid leukemia.

The transcriptional regulator far upstream element binding protein 1 (FUBP1) acts as an oncoprotein in solid tumor entities and plays a role in the maintenance of hematopoietic stem cells. However, its potential function in leukemia is unknown. In murine models of chronic (CML) and acute myeloid leukemia (AML) induced by BCR-ABL1 and MLL-AF9, respectively, knockdown of Fubp1 resulted in prolonged survival, decreased numbers of CML progenitor cells, decreased cell cycle activity and increased apoptosis. Knockdown of FUBP1 in CML and AML cell lines recapitulated these findings and revealed enhanced DNA damage compared to leukemia cells expressing wild type FUBP1 levels. FUBP1 was more highly expressed in human CML compared to normal bone marrow cells and its expression correlated with disease progression. In AML, higher FUBP1 expression in patient leukemia cells was observed with a trend toward correlation with shorter overall survival. Treatment of mice with AML with irinotecan, known to inhibit topoisomerase I and FUBP1, significantly prolonged survival alone or in combination with cytarabine. In summary, our data suggest that FUBP1 acts as cell cycle regulator and apoptosis inhibitor in leukemia. We demonstrated that FUBP1 might play a role in DNA repair, and its inhibition may improve outcome in leukemia patients.

Dynamic Formation of Microvillus Inclusions During Enterocyte Differentiation in Munc18-2-Deficient Intestinal Organoids.

Background & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID. Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions. Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin-positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane. Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.