Biology subjects

Yegnasubramanian, S.

Publications and source records attributed to Yegnasubramanian, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Clear cell renal cell carcinoma consensus transcriptomic programs reveal converging trajectories towards aggressive disease

Clear cell renal cell carcinoma (ccRCC) is characterized by a branching genomic trajectory in which early biallelic VHL inactivation splits into PBRM1- and BAP1-mutant lineages. However, driver mutations alone do not account for the molecular and phenotypic heterogeneity. To dissect this heterogeneity, we developed a non-negative matrix factorization (NMF)-based gene expression analytical framework for identifying recurrent transcriptional programs across factorization dimensionalities and across datasets. We applied it to three curated datasets (IMmotion151, n = 823; JAVELIN Renal 101, n = 726; TCGA, n = 614) to define 17 consensus transcriptomic programs (CTPs). Mapping CTPs onto single-cell RNAseq (scRNAseq) of human ccRCC tumors and patient-derived tumorgraft models linked these programs to their cellular sources, distinguishing RCC-intrinsic, tumor-cell-extrinsic, and mixed programs. RCC-intrinsic CTPs associated with canonical drivers, including VHL (R1), PBRM1 (R2), BAP1 (R4), PTEN/TSC1 (R3), TFE3/TFEB fusions (R5), NF2 (R6), and CDKN2A/TP53 (MP-Prolif). Additional CTPs captured tumor microenvironment (TME) composition (TME-Tcell, TME-Myelo, TME-Endo, TME-Stroma) and biological processes active across multiple cellular compartments, including proliferation, Y-chromosome-linked expression in male tumors, ciliary biology, and translation. Trajectory inference methods revealed PBRM1-like and BAP1-like branches that converged on a shared aggressive late transcriptomic stage (TS) associated with higher nuclear grade, additional driver alterations, myeloid/stromal infiltration, and poor clinical outcomes. Spatial transcriptomics (and multiregional sequencing) of paired conventional ccRCC and sarcomatoid regions linked TS advancement with morphological progression and clonal evolution. After adjusting for stage, grade, and BAP1/PBRM1 status, TS remained independently prognostic. Furthermore, our data suggest that immune checkpoint inhibitor combinations are particularly beneficial for MP-Prolif and not R1 utilizing specimens. In summary, we present an atlas of recurring transcriptomic programs in RCC and an ontological framework bridging genotype, tumor-cell-intrinsic gene expression, and microenvironment remodeling, with implications for risk stratification and treatment selection in ccRCC.

Cancer Biology↗

bioRxiv2025

A transition zone enriched WIF1⁺ basal cell subtype is associated with benign prostatic hyperplasia

ABSTRACT/SUMMARYThe cellular composition and disease susceptibilities of the distinct zones of the human prostate remain incompletely understood. Through extensive single-cell RNA sequencing (scRNA-seq) of benign regions from prostatectomy specimens, we identified a basal cell population expressing WIF1, VCAN, and NRG1, among other genes, that was significantly enriched in the transition zone (TZ). Benign prostatic hyperplasia (BPH) is a common condition that causes widespread morbidity and is nearly exclusively localized to the TZ. Analysis of previously published scRNA-seq datasets further confirmed that WIF1+ basal cells were significantly enriched in BPH compared to normal prostate. Pathway and cell-cell communication analyses revealed that this basal subtype is associated with programs related to cell proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, and hormone response. Together, the molecular signature, zonal distribution, and pathway enrichment suggest that TZ-enriched WIF1+ basal cells may contribute to BPH pathogenesis by promoting epithelial and stromal remodeling.

cell biology↗

bioRxiv2025

Cell Type-Specific Effects of miR-21 Loss Attenuate Tumor Progression in MYC-Driven Prostate Cancer

Aberrant microRNA expression is common in cancer, yet cell-type-specific microRNA activity in the tumor microenvironment (TME) remains poorly understood. Here, we show that germline deletion of miR-21 significantly attenuated the progression of MYC-driven prostate cancer (PCa), reducing prostate weight, tumor burden, and proliferation index in Hi-Myc mice. In situ hybridization revealed elevated miR-21 expression in multiple cell types during disease progression. Inflammatory and premalignant lesions in mouse and human prostate showed increased miR-21 in both stroma and epithelium, with further enrichment in the stroma of invasive adenocarcinoma. In Hi-Myc mice, single cell RNA-sequencing revealed miR-21 gene regulation in neoplastic, stromal, and immune cells in a cell-type-specific manner, impacting both direct and indirect targets. Notably, miR-21 deletion reduced immune infiltration into the prostate TME, particularly Trem2-expressing macrophages and regulatory T cells. The Timp1-fibroblast gene signature in MYC-driven PCa was suppressed in miR-21 knockout prostates. Cell-cell communication analysis showed that miR-21 suppressed TGF-beta signaling in the TME, partially through Ski and Smad7 suppression in cancer-associated fibroblasts. These findings underscore the crucial role of miR-21 in PCa and provide some of the first in situ insights into cell-type-specific miRNA activity in solid tumors.

cancer biology↗

bioRxiv2024

Triptolide sensitizes cancer cells to nucleoside DNA methyltransferase inhibitors through inhibition of DCTPP1-mediated cell-intrinsic resistance

While nucleoside DNA methyltransferase inhibitors (DNMTi) such as decitabine and azacitidine are effective in treating myelodysplatic syndrome (MDS)/leukemia, they have had limited utility for the majority of other cancers. Through a chemical library screen, we identified that triptolide, a diterpenoid epoxide from Tripterygium wilfordii, or analogs significantly augmented the epigenetic and anti-cancer effects of decitabine in vitro and in vivo. These effects were attributable to inhibition of DCTPP1-mediated cleavage of 5-aza-deoxycytidine triphosphate, the convergent activated metabolite of nucleoside DNMTi, leading to enhanced drug incorporation into genomic DNA, increased DNMT degradation, enhanced global DNA demethylation and associated transcriptional reprogramming. We show that high DCTPP1 expression was associated with cell-intrinsic resistance to nucleoside DNMTi, and that triptolide and its analogs could overcome this resistance. SIGNIFICANCEWe screened a library of existing drugs to identify those capable of enhancing the anti-cancer effects of the nucleoside DNMTi decitabine. The combination of triptolide and decitabine synergistically inhibited cancer cell growth and survival in vitro, and was highly effective in inhibiting xenograft growth in vivo. Biochemical, genetic and structural biology studies with triptolide and its analogs revealed that this synergy was due to their inhibition of DCTPP1-mediated pyrophosphate cleavage from 5-aza-deoxycytidine triphosphate, the active metabolite of DNMTi. The genomic incorporation and efficacy of decitabine in cancer cell lines were significantly correlated with DCTPP1 expression more so than those of other nucleoside metabolizing genes. Triptolide and its analogs comprise rational adjuncts to nucleoside DNMTi ripe for further pre-clinical/clinical translation. HIGHLIGHTSO_LITriptolide synergistically sensitizes cancer cells to DNMTi in vitro. C_LIO_LITriptolide and decitabine combination shows favorable efficacy and safety in vivo. C_LIO_LISynergy of triptolide and decitabine is mediated through inhibition of DCTPP1. C_LIO_LIHigh DCTPP1 expression confers cell intrinsic resistance to DNMTi. C_LI

cancer biology↗

bioRxiv2023

Arrested Agonist Paradigm For Selective Radiosensitization of Prostate Cancer

As a prototypical nuclear hormone receptor, the androgen receptor (AR) signals via a sequential cascade triggered by binding to androgenic ligands such as testosterone and dihydrotestosterone (DHT). This cascade includes dimerization of the ligand-receptor complex, nuclear translocation, chromatin binding to response elements, recruitment of TOP2B and co-activator complexes, and induction of an effector transcriptional program. In prostate cancers, this AR signaling cascade is an essential driver of growth and survival, yet its activity confers potential vulnerabilities through transient TOP2B-mediated DNA double strand breaks. We investigated the ability of non-steroidal AR ligands to activate initial steps of the AR signaling cascade up to the point of AR- and TOP2B-mediated double strand breaks, with subsequent arrest of the signaling cascade to prevent induction of pro-growth/survival transcriptional programs in prostate cancer cells. We identified hydroxyflutamide (FLU) as such an androgen receptor arrested agonist; in androgen-deprived conditions, FLU induced AR nuclear translocation, chromatin binding, and TOP2B-mediated double strand breaks, but failed to induce AR target gene expression and prostate cancer cell growth. The FLU-mediated arrest in the signaling cascade could be attributed to the inability of FLU to allow association of AR with SMARCD2, a critical component of the BAF chromatin remodeling complex required for androgen induced AR co-activation. Interestingly, the FLU-induced, AR- and TOP2B-mediated double strand breaks could be used to selectively sensitize AR-positive prostate cancer cells to ionizing radiation in vitro and in vivo. These findings support a novel arrested agonist paradigm for selective radiosensitization of prostate cancer cells without inducing AR-mediated pro-growth and survival transcriptional programs.

cancer biology↗

bioRxiv2023

Convergent alterations in the tumor microenvironment of MYC-driven human and murine prostate cancer

The tissue microenvironment in prostate cancer is profoundly altered. While such alterations have been implicated in driving prostate cancer initiation and progression to aggressive disease, how prostate cancer cells and their precursors mediate those changes is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we performed extensive single-cell RNA-sequencing (scRNA-seq) and rigorous molecular pathology of the comparative biology between human prostate cancer and key time points in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues, with validation in a large external data set, revealed that cancer cell-intrinsic activation of MYC signaling was the top up-regulated pathway in human cancers, representing a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Likewise, numerous non-malignant cell states in the tumor microenvironment (TME), including non-cancerous epithelial, immune, and fibroblast cell compartments, were conserved across individuals, raising the possibility that these cell types may be a sequelae of the convergent MYC activation in the cancer cells. To test this hypothesis, we employed a GEMM of prostate epithelial cell-specific MYC activation in two mouse strains. Cell communication network and pathway analyses suggested that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogrammed the TME during carcinogenesis, leading to the emergence of cascading cell state alterations in neighboring epithelial, immune, and fibroblast cell types that paralleled key findings in human prostate cancer. Importantly, among these changes, the progression from a precursor-enriched to invasive-cancer-enriched state was accompanied by a cell-intrinsic switch from pro-immunogenic to immunosuppressive transcriptional programs with coinciding enrichment of immunosuppressive myeloid and Treg cells in the immune microenvironment. These findings implicate activation of MYC signaling in reshaping convergent aspects of the TME of prostate cancer as a common denominator across the otherwise well-documented molecular heterogeneity of human prostate cancer.

cancer biology↗

bioRxiv2023

Distinct Myeloid Derived Suppressor Cell Populations Promote Tumor Aggression in Glioblastoma

The diversity of genetic programs and cellular plasticity of glioma-associated myeloid cells, and thus their contribution to tumor growth and immune evasion, is poorly understood. We performed single cell RNA-sequencing of immune and tumor cells from 33 glioma patients of varying tumor grades. We identified two populations characteristic of myeloid derived suppressor cells (MDSC), unique to glioblastoma (GBM) and absent in grades II and III tumors: i) an early progenitor population (E-MDSC) characterized by strong upregulation of multiple catabolic, anabolic, oxidative stress, and hypoxia pathways typically observed within tumor cells themselves, and ii) a monocytic MDSC (M-MDSC) population. The E-MDSCs geographically co-localize with a subset of highly metabolic glioma stem-like tumor cells with a mesenchymal program in the pseudopalisading region, a pathognomonic feature of GBMs associated with poor prognosis. Ligand-receptor interaction analysis revealed symbiotic cross-talk between the stemlike tumor cells and E-MDSCs in GBM, whereby glioma stem cells produce chemokines attracting E-MDSCs, which in turn produce growth and survival factors for the tumor cells. Our large-scale single-cell analysis elucidated unique MDSC populations as key facilitators of GBM progression and mediators of tumor immunosuppression, suggesting that targeting these specific myeloid compartments, including their metabolic programs, may be a promising therapeutic intervention in this deadly cancer. One-Sentence SummaryAggressive glioblastoma harbors two unique myeloid populations capable of promoting stem-like properties of tumor cells and suppressing T cell function in the tumor microenvironment.

immunology↗

bioRxiv2023

MYC-driven increases in mitochondrial DNA copy number occur early and persist throughout prostatic cancer progression

Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type-specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplex in situ method to spatially quantify cell type specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by two orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Our in situ approach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.

cancer biology↗

bioRxiv2023

RepairSwitch: simultaneous functional assessment of homologous recombination and end joining DNA repair pathways in living cells

DNA repair pathways are frequently defective in human cancers. DNA double strand breaks (DSBs) are most often repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ). Alterations in repair pathways can indicate sensitivity to therapeutic agents such as PARP inhibitors, cisplatin, and immunotherapy. Thus, functional assays to measure rates of HR and NHEJ are of significant interest. Several methods have been developed to measure rates of HR or NHEJ; however, there is a need for functional cell-based assays that can measure rates by both major DNA DSB pathways simultaneously. Here, we describe the RepairSwitch assay, a flow cytometry assay to assess rates of HR and NHEJ mediated repair of Cas9 programmed DSB simultaneously using a novel fluorescence switching reporter system. The assay exhibits low background signal and is capable of detecting rare repair events in the 1 in 10,000 range. We demonstrate the utility of RepairSwitch by measuring the potency of inhibitors of ATM (KU-60019, KU-55933), DNA-PK (NU7441), and PARP (Olaparib) on modulating DSB repair rates in HEK293FT cells. The selective ATM inhibitor KU-60019 inhibited HR rates with IC50 of 915 nM. Interestingly, KU-60019 exposure led to a dose responsive increase in rates of NHEJ. In contrast, the less selective ATM inhibitor KU-55933, which also has activity on DNA-PK, showed inhibition of both HR and NHEJ. The selective DNA-PK inhibitor NU7441 inhibited NHEJ efficiency with an IC50 of 299 nM, and showed a dose responsive increase in HR. The PARP inhibitor Olaparib showed lower potency in modulating HR and NHEJ. We next used the RepairSwitch assay to assess how pharmacological and genetic inhibition of DNA methyltransferases (DNMT) impacted rates of HR and NHEJ. The DNMT inhibitor decitabine reduced HR, but increased rates of NHEJ, both in a dose responsive manner, in both HEK293FT and HCT116 cells (IC50 for HR of 187 nM and 1.4 uM respectively). Knockout of DNMT1 and DNMT3B increased NHEJ, while knockout of DNMT3B, but not DNMT1, reduced HR. These results illustrate the utility of RepairSwitch as a functional assay for measuring changes in rates of DSB repair induced by pharmacological or genetic perturbation. Furthermore, the findings illustrate the potential for one DNA repair mechanism to compensate in part for loss of another. Finally, we showed that inhibition of DNMT can lead to reduction of HR and increase in NHEJ, providing some additional insight into recently observed synergy of DNMT inhibitors with PARP inhibitors for cancer treatment.

cell biology↗

bioRxiv2022

Progressive spreading of DNA methylation in the GSTP1 promoter CpG island across transitions from precursors to invasive prostate cancer

Glutathione S-transferase pi 1 (GSTP1) is lowly expressed in normal prostate luminal cells and becomes induced in most proliferative inflammatory atrophy lesions (PIA). GSTP1 becomes silenced in prostatic intraepithelial neoplasia (PIN) and prostate adenocarcinoma (CaP) via cytosine-phospho-guanine (CpG) island promoter hypermethylation. However, GSTP1 methylation patterns in PIA and PIN, and their relationship to patterns in CaP are poorly understood. We used bisulfite genomic sequencing to examine patterns of GSTP1 promoter CpG island methylation in laser capture microdissected benign, PIA, PIN, and CaP regions from 32 subjects that underwent radical prostatectomy. We analyzed 908 sequence clones across 24 normal epithelium, 37 PIA, 18 PIN, and 23 CaP regions, allowing assessment of 34,863 CpG sites with allelic phasing. Normal and PIA lesions were mostly unmethylated with 0.52 and 1.3% of total CpG sites methylated, respectively. PIN and CaP lesions had greater methylation with 24% and 51% of total CpG sites methylated, respectively. The degree of GSTP1 methylation showed progression from PIA << PIN < CaP. PIN lesions showed more partial methylation compared to CaP lesions. Partially methylated lesions were enriched for methylation changes at AP1 and SP1 transcription factor binding sites. These results demonstrate that methylation density in the GSTP1 CpG island in PIN was intermediate relative to that in normal prostate epithelium/PIA and CaP lesions. These results are consistent with gradual spreading of DNA methylation centered at the SP1/AP1 transcription factor binding sites in precursor lesions, with subsequent spreading of methylation across the entire CpG island in transition to CaP.

cancer biology↗

bioRxiv2022

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response

Regulatory T cells (Treg) are conventionally viewed to suppress endogenous and therapyinduced anti-tumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNAseq/TCRseq of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment naive non-small cell lung cancers (NSCLC) with single cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Notably, one subset selectively expresses high levels of OX40 and GITR, whose engangement by cognate ligand mediated proliferative programs and NF-kB activation, as well as multiple genes involved in Treg suppression, in particular LAG3. Functionally, the OX40hiGITRhi subset in the most highly suppressive ex vivo and Treg expression of OX40, GITR and LAG3, correlated with resistance to PD-1 blockade. Surprisingly, in the murine tumor model, we found that virtually all TIL-Treg expressing T cell receptors that are specific for TAA fully develop a distinct Th1-like signature over a two-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFN{gamma} and certain pro-inflammatory granzymes. Application of a gene score from the murine TAA-specific Th1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1 responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally-defined subsets with potentially opposing effects on ICB-induced anti-tumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to anti-tumor responses. One-Sentence SummaryWe define 10 subsets of lung cancer-infiltrating regulatory T cells, one of which is highly suppressive and enriched in anti-PD-1 non-responders and the other is Th1-like and is enriched in PD-1 responders.

immunology↗

bioRxiv2022

The TERT promoter is polycomb-repressed in neuroblastoma cells with long telomeres

Acquiring a telomere maintenance mechanism is one of the hallmarks of high-risk neuroblastoma and commonly occurs by expressing telomerase (TERT). Telomerase-negative neuroblastoma has, characteristically, long telomeres and most utilize the telomerase-independent alternative lengthening of telomeres (ALT) mechanism. Conversely, no discernable telomere maintenance mechanism is detected in a fraction of neuroblastoma with long telomeres, representing a phenomenon referred to as ever-shorter telomeres. Here, we show that, unlike most cancers, DNA of the TERT promoter is broadly hypomethylated in neuroblastoma. In telomerase-positive neuroblastoma cells, the hypomethylated DNA promoter is approximately 1.5-kb in length and is bound by hypermethylated TERT gene body and upstream intergenic sequences. The TERT locus shows active chromatin marks including H3K4me3, H3K27Ac, H3K14Ac, RNA PolII and BRD4 with low enrichment for the repressive mark, H3K27me3. Strikingly, in neuroblastoma with long telomeres, the hypomethylated region spans the entire TERT locus, including multiple nearby genes with enrichment for the repressive H3K27me3 chromatin mark. Furthermore, subtelomeric regions showed enrichment of repressive chromatin marks in neuroblastomas with long telomeres relative to those with short telomeres. These repressive marks were even more evident at the genic loci, suggesting a telomere position effect. Inhibiting H3K27 methylation by the EZH2 inhibitor, Tazemetostat, induced the expression of TERT, particularly in cell lines with long telomeres and H3K27me3 marks in the promoter region. Taken together, these data suggest that epigenetic regulation of TERT expression differs in neuroblastoma depending on the telomere maintenance status, and H3K27 methylation is important in repressing TERT expression in neuroblastoma with long telomeres.

cancer biology↗

bioRxiv2022

Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation

COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Guerin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

immunology↗

bioRxiv2022

Single-cell atlas of epithelial and stromal cell heterogeneity by lobe and strain in the mouse prostate

Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered mouse models in different mouse strains and prostate lobes within each mouse strain, yet, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. To address this, we examined the single-cell transcriptomes of individual mouse prostate lobes from two commonly used mouse strains, FVB/NJ and C57BL/6J. Data dimensionality reduction and clustering analysis revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

cell biology↗

bioRxiv2021

Phased Mutations and Complex Rearrangements in Human Prostate Cancer Cell Lines through Linked-Read Whole Genome Sequencing

A limited number of cell lines have fueled the majority of preclinical Prostate cancer (PCa) research. Despite tremendous effort in characterizing their molecular profiles, comprehensive whole genome sequencing with allelic phasing of somatic genome alterations has not been undertaken to date. Here, we utilized whole genome Linked-read sequencing to obtain haplotype information from the seven most commonly used PCa cell lines (PC3, LNCaP, DU145, CWR22Rv1, VCaP, LAPC4, MDA-PCa-2b), four castrate resistant (CR) subclones (LNCaP_Abl, LNCaP_C42b, VCaP-CR, LAPC4-CR), and an immortalized prostate epithelial line RWPE-1. Phasing of mutations allowed derivation of "Gene-level Haplotype" to assess whether a gene harbored heterozygous mutations in one or both alleles, providing a comprehensive catalogue of mono or bi-allelically inactivated genes. Phased structural variant analysis allowed identification of complex rearrangement chains consistent with chromothripsis and chromoplexy, with breakpoints occurred across a single allele, providing further evidence that complex SVs occurred in a concerted event, rather than through accumulation of multiple independent rearrangements. Additionally, comparison of parental and CR subclones revealed previously known and novel genomic alterations associated with the CR clones. This study therefore comprehensively characterized phased genomic alterations in the commonly used PCa cell lines and provided a useful resource for future cancer research.

cancer biology↗

bioRxiv2021

PHENOTYPIC CHARACTERIZATION OF TWO NOVEL CELL LINE MODELS OF CASTRATION RESISTANT PROSTATE CANCER.

BACKGROUNDResistance to androgen deprivation therapies is a major driver of mortality in advanced prostate cancer. Therefore, there is a need to develop new pre-clinical models that allow the investigation of resistance mechanisms and the assessment of drugs for the treatment of castration resistant prostate cancer. METHODSWe generated two novel cell line models (LAPC4-CR and VCaP-CR) which were derived by passaging LAPC4 and VCaP cells in vivo and in vitro under castrate conditions. We performed detailed transcriptomic (RNA-seq) and proteomic analyses (SWATH-MS) to delineate expression differences between castration-sensitive and castration-resistant cell lines. Furthermore, we characterized the in vivo and in vitro growth characteristics of the novel cell line models. RESULTSThe two cell line derivatives LAPC4-CR and VCaP-CR showed castration resistant growth in vitro and in vivo which was only minimally inhibited by AR antagonists, enzalutamide and bicalutamide. High-dose androgen treatment resulted in significant growth arrest of VCaP-CR but not in LAPC4-CR cells. Both cell lines maintained AR expression, but exhibited distinct expression changes on the mRNA and protein level. Integrated analyses including data from LNCaP and the previously described castration resistant LNCaP-abl cells revealed an expression signature of castration resistance. CONCLUSIONSThe two novel cell line models LAPC4-CR and VCaP-CR and their comprehensive characterization on the RNA and protein level represent important resources to study the molecular mechanisms of castration resistance.

cancer biology↗

bioRxiv2021

Phenotypic shifts of tumor associated macrophages and STAT3 mediated suppression of myeloid derived suppressor cells drive sensitization of HER2+ tumor immunity.

Understanding how novel therapeutic combinations alter solid tumor microenvironments (TME) in immunosuppressive tumors such as breast cancer is essential to improve their responses to immune checkpoint inhibitors (ICIs). Entinostat, an oral histone deacetylase inhibitor (HDACi), has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs, but the precise alterations induced by entinostat and mechanisms of synergy with ICIs remain unknown. Here, we employ single-cell RNA-sequencing on HER2 overexpressing breast tumors from mice treated with entinostat + ICIs to characterize these changes across cell types in the TME. This analysis demonstrates that treatment with entinostat induces a shift from a pro-tumor to an anti-tumor TME signature characterized predominantly by changes in the myeloid cells. Notably, myeloid-derived suppressor cells (MDSCs) are shifted toward the less suppressive granulocytic phenotype in association with reduced signaling through the STAT3 pathway. In addition, tumor-associated macrophages are shifted toward an anti-tumor M1 phenotype by epigenetic reprogramming. Overall, these entinostat-induced TME changes reduce immunosuppression and increase mechanisms of tumor cell killing to improve ICI responses and broaden the population of patients who could potentially benefit from immunotherapy.

immunology↗

bioRxiv2020

Pervasive promoter hypermethylation of silenced TERT alleles in human cancers

In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of telomerase gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating telomerase gene expression in cancer cells is not fully understood. Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally observe hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to the attenuation of TERT activation in cancer cells. SIGNIFICANCEHypermethylation of the TERT promoter alleles to attenuate TERT activation in cancer cells may account for the modest activation of TERT expression in most cancers.

cancer biology↗