PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

Sequencing of cancer cell subpopulations identifies micrometastases in a bladder cancer patient.

PURPOSE: Pathologic staging of bladder cancer patients remains a challenge. Standard-of-care histology exhibits limited sensitivity in detection of micrometastases, which can increase risk of cancer progression and delay potential adjuvant therapies. Here, we sought to develop a proof of concept novel molecular approach to improve detection of cancer micrometastasis. EXPERIMENTAL DESIGN: We combined fluorescence activated cell sorting and next-generation sequencing and performed whole-exome sequencing of total cancer cells and cancer cell subpopulations in multiple tumor specimens and regional lymph nodes in a single patient with muscle-invasive urothelial carcinoma of the bladder following radical cystectomy. RESULTS: Mean allele frequency analysis demonstrated a significant correlation between primary tumor cancer cells and cancer cells isolated from the lymph nodes, confirming lymph node disease despite negative pathologic staging. RNA-sequencing revealed intratumoral heterogeneity as well as enrichment for immune system and lipid metabolism gene sets in the micrometastatic cancer cell subpopulations. CONCLUSIONS: Our analysis illustrates how next-generation sequencing of cancer cell subpopulations may be utilized to enrich for cancer cell markers and enhance detection of bladder cancer micrometastases to improve pathologic staging and provide insight into cancer cell biology.

Activation of Notch1 synergizes with multiple pathways in promoting castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer.

Biased Expression of the FOXP3Δ3 Isoform in Aggressive Bladder Cancer Mediates Differentiation and Cisplatin Chemotherapy Resistance.

PURPOSE: The transcriptional regulation mediating cancer cell differentiation into distinct molecular subtypes and modulating sensitivity to existing treatments is an enticing therapeutic target. Our objective was to characterize the ability of the forkhead/winged transcription factor FOXP3 to modulate the differentiation of bladder cancer. EXPERIMENTAL DESIGN: Expression of FOXP3 was analyzed by immunohistochemistry in a tumor microarray of 587 samples and overall survival in a subset of 187 patients following radical cystectomy. Functional assays were performed in SW780 and HT1376 cell lines in vitro and in vivo and gene expression profiling performed by RNA-Seq. Validation was undertaken using gene expression profiles of 131 patients from The Cancer Genome Atlas (TCGA) consortium in bladder cancer. RESULTS: FOXP3 expression correlates with bladder cancer stage and inversely with overall survival, with biased expression of the FOXP3Δ3 isoform. Functional assays of FOXP3Δ3 demonstrated resistance to chemotherapy in vitro, whereas subcutaneous xenografts overexpressing FOXP3Δ3 developed larger and more poorly differentiated bladder cancers. RNA expression profiling revealed a unique FOXP3Δ3 gene signature supporting a role in chemotherapy resistance. Accordingly, knockdown of Foxp3 by siRNA in HT1376 cells conferred sensitivity to cisplatin- and gemcitabine-induced cytotoxicity. Validation in TCGA dataset demonstrated increased expression of FOXP3 in subtypes II to IV and skewing of molecular subtypes based on FOXP3Δ3-specific gene expression. CONCLUSIONS: (i) Biased expression of the FOXP3Δ3 isoform in bladder cancer inversely correlates with overall survival, (ii) FOXP3Δ3 induces a unique gene program that mediates cancer differentiation, and (iii) FOXP3Δ3 may augment chemotherapy resistance. Clin Cancer Res; 22(21); 5349-61. ©2016 AACR.

Molecular profiling of premalignant lesions in lung squamous cell carcinomas identifies mechanisms involved in stepwise carcinogenesis.

Lung squamous cell carcinoma (SCC) is thought to arise from premalignant lesions in the airway epithelium; therefore, studying these lesions is critical for understanding lung carcinogenesis. Previous microarray and sequencing studies designed to discover early biomarkers and therapeutic targets for lung SCC had limited success identifying key driver events in lung carcinogenesis, mostly due to the cellular heterogeneity of patient samples examined and the interindividual variability associated with difficult to obtain airway premalignant lesions and appropriate normal control samples within the same patient. We performed RNA sequencing on laser-microdissected representative cell populations along the SCC pathologic continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. This is the first gene expression profiling study of airway premalignant lesions with patient-matched SCC tumor samples. Our results provide much needed information about the biology of premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of SCC, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients.

The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation.

The generation of research-quality, clinically relevant cell types in vitro from human pluripotent stem cells requires a detailed understanding of the equivalent human cell types. Here we analysed 134 human embryonic and fetal samples from 6 to 20 developmental weeks and identified the stages at which cKIT(+) primordial germ cells (PGCs), the precursors of gametes, undergo whole-genome epigenetic reprogramming with global depletion of 5mC, H3K27me3 and H2A.Z, and the time at which imprint erasure is initiated and 5hmC is present. Using five alternative in vitro differentiation strategies combined with single-cell microfluidic analysis and a bona fide human cKIT(+) PGC signature, we show the stage of cKIT(+) PGC formation in the first 16 days of differentiation. Taken together, our study creates a resource of human germ line ontogeny that is essential for future studies aimed at in vitro differentiation and unveiling the mechanisms necessary to pass human DNA from one generation to the next.