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INTRODUCTION: Despite the great promise for therapies using antisense oligonucleotides (ASOs), their adverse effects, which include pro-inflammatory effects and thrombocytopenia, have limited their use. Previously, these effects have been linked to the phosphorothioate (PS) backbone necessary to prevent rapid ASO degradation in plasma. The main aim of this study was to assess the impact of the nucleic acid portion of an ASO-type drug on platelets and determine if it may contribute to thrombosis or thrombocytopenia. METHODS: Platelets were isolated from healthy donors and men with advanced prostate cancer. Effects of antisense oligonucleotides (ASO), oligonucleotides, gDNA, and microRNA on platelet activation and aggregation were evaluated. A mouse model of lung thrombosis was used to confirm the effects of PS-modified oligonucleotides in vivo. RESULTS: Platelet exposure to gDNA, miRNA, and oligonucleotides longer than 16-mer at a concentration above 8 mM resulted in the formation of hypersensitive platelets, characterized by an increased sensitivity to low-dose thrombin (0.1 nM) and increase in p-Selectin expression (6–8 fold greater than control; p < 0.001). The observed nucleic acid (NA) effects on platelets were toll-like receptor (TLR) -7 subfamily dependent. Injection of a p-Selectin inhibitor significantly (p = 0.02) reduced the formation of oligonucleotide-associated pulmonary microthrombosis in vivo. CONCLUSION: Our results suggest that platelet exposure to nucleic acids independent of the presence of a PS modification leads to a generation of hypersensitive platelets and requires TLR-7 subfamily receptors. ASO studies conducted in cancer patients may benefit from testing the ASO effects on platelets ex vivo before initiation of patient treatment.

Significance Renal cell carcinomas (RCCs) are heterogeneous malignancies thought to arise from kidney tubular epithelial cells, and clear cell RCC is the most common entity. This study demonstrates that cell atlases generated from benign kidney and two common RCCs using single-cell RNA sequencing can predict putative cells of origin for more than 10 RCC subtypes. A focused analysis of distinct cell-type compartments reveals the potential role of tumor epithelia in promoting immune infiltration and other molecular attributes of the tumor microenvironment. Finally, an observed association between the lack of immunotherapy response and endothelial cell fraction has important clinical implications. The current study, therefore, significantly contributes toward understanding disease ontogenies and the molecular dynamics of tumor epithelia and the microenvironment., Diverse subtypes of renal cell carcinomas (RCCs) display a wide spectrum of histomorphologies, proteogenomic alterations, immune cell infiltration patterns, and clinical behavior. Delineating the cells of origin for different RCC subtypes will provide mechanistic insights into their diverse pathobiology. Here, we employed single-cell RNA sequencing (scRNA-seq) to develop benign and malignant renal cell atlases. Using a random forest model trained on this cell atlas, we predicted the putative cell of origin for more than 10 RCC subtypes. scRNA-seq also revealed several attributes of the tumor microenvironment in the most common subtype of kidney cancer, clear cell RCC (ccRCC). We elucidated an active role for tumor epithelia in promoting immune cell infiltration, potentially explaining why ccRCC responds to immune checkpoint inhibitors, despite having a low neoantigen burden. In addition, we characterized an association between high endothelial cell types and lack of response to immunotherapy in ccRCC. Taken together, these single-cell analyses of benign kidney and RCC provide insight into the putative cell of origin for RCC subtypes and highlight the important role of the tumor microenvironment in influencing ccRCC biology and response to therapy.

Mechanisms governing allogeneic T-cell responses after allogeneic hematopoietic stem cell (HSC) and solid organ transplantation are incompletely understood. To identify lncRNAs involved in regulation of human donor T cells after clinical HSCT, we performed RNA-seq on T cells from healthy subjects as well as on donor T cells from three different groups of HSCT recipients that differed in their degree of histocompatibility mismatch. We found that lncRNA differential expression was greatest in T cells following major histocompatibility complex (MHC)–mismatched HSCT relative to T cells following either MHC-matched or autologous HSCT. The differential expression was validated in an independent patient cohort and in mixed lymphocyte reactions using ex vivo healthy human T-cells. Linc00402, an uncharacterized lncRNA, was identified among the differentially expressed lncRNAs between the mismatched unrelated and matched unrelated donor T cells. We nominated it for further characterization because it was classified as conserved using genomic evolutionary rate profiling and exhibited an 88-fold increase in human T cells relative to all other samples in the FANTOM5 Consortium database. Linc00402 was also increased in donor T cells from patients who underwent allogeneic cardiac transplantation, and in murine T cells. Lower amounts of Linc00402 were found in patients who subsequently developed acute graft-versus-host disease (GVHD). Linc00402 enhanced the activity of the kinases ERK1 and ERK2, increased FOS nuclear accumulation, and augmented the expression of interleukin-2 (IL-2) and Egr-1 after T cell receptor engagement. Functionally, Linc00402 augmented the T cell proliferative response to an allogeneic stimulus but not to a nominal ovalbumin peptide antigen or polyclonal anti-CD3/CD28 stimulus. Thus, our studies identified Linc00402 as a regulator of allogeneic T cell function.

Significance New therapeutic targets are urgently needed against SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic. Results in this study show that targeting the transcriptional regulation of host entry factors TMPRSS2 and ACE2 is a viable treatment strategy to prevent SARS-CoV-2 infection. In particular, inhibitors of androgen receptor (AR) or bromodomain and extraterminal domain (BET) proteins are effective against SARS-CoV-2 infection. AR inhibitors are already approved in the clinic for treatment of prostate cancer and are under investigation in COVID-19 patients; BET inhibitors are also in clinical development for other indications and could be rapidly repurposed for COVID-19., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2). TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity. Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2, TMPRSS2, and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 y of age. In males greater than 70 y old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells. Transcriptional repression of the AR enhanceosome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19.

Both KRAS and EGFR are essential mediators of pancreatic cancer development and interact with Argonaute 2 (AGO2) to perturb its function. Here, in a mouse model of mutant KRAS-driven pancreatic cancer, loss of AGO2 allows precursor lesion (PanIN) formation yet prevents progression to pancreatic ductal adenocarcinoma (PDAC). Precursor lesions with AGO2 ablation undergo oncogene-induced senescence with altered microRNA expression and EGFR/RAS signaling, bypassed by loss of p53. In mouse and human pancreatic tissues, PDAC progression is associated with increased plasma membrane localization of RAS/AGO2. Furthermore, phosphorylation of AGO2Y393 disrupts both the wild-type and oncogenic KRAS-AGO2 interaction, albeit under different conditions. ARS-1620 (G12C-specific inhibitor) disrupts the KRASG12C-AGO2 interaction, suggesting that the interaction is targetable. Altogether, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression., Argonaute 2 (AGO2) binds RAS and is required for cellular transformation. Here, the authors establish a KRAS-driven mouse model of pancreatic cancer with conditional loss of AGO2 and show that the early phase of neoplastic lesion initiation is dependent on EGFR/RAS but not AGO2, while AGO2 is required for pancreatic ductal adenocarcinoma progression and metastasis.

The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long noncoding RNA 1) as an important long noncoding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by the AR protein, but ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression and identify ARLNC1 as a novel therapeutic target.

Transcription factors play a key role in the development of diverse cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the gene encoding the transcription factor ERG is recurrently rearranged and plays a critical role in prostate oncogenesis. Here, we identified a series of peptides that interact specifically with the DNA binding domain of ERG. ERG inhibitory peptides (EIPs) and derived peptidomimetics bound ERG with high affinity and specificity, leading to proteolytic degradation of the ERG protein. The EIPs attenuated ERG-mediated transcription, chromatin recruitment, protein-protein interactions, cell invasion and proliferation, and tumor growth. Thus, peptidomimetic targeting of transcription factor fusion products may provide a promising therapeutic strategy for prostate cancer as well as other malignancies.

Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland1,2. FOXA1 is frequently mutated in hormone-receptor-driven prostate, breast, bladder and salivary-gland tumours3–8. However, it is unclear how FOXA1 alterations affect the development of cancer, and FOXA1 has previously been ascribed both tumour-suppressive9–11 and oncogenic12–14 roles. Here we assemble an aggregate cohort of 1,546 prostate cancers and show that FOXA1 alterations fall into three structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class-1 activating mutations originate in early prostate cancer without alterations in ETS or SPOP, selectively recur within the wing-2 region of the DNA-binding forkhead domain, enable enhanced chromatin mobility and binding frequency, and strongly transactivate a luminal androgen-receptor program of prostate oncogenesis. By contrast, class-2 activating mutations are acquired in metastatic prostate cancers, truncate the C-terminal domain of FOXA1, enable dominant chromatin binding by increasing DNA affinity and—through TLE3 inactivation—promote metastasis driven by the WNT pathway. Finally, class-3 genomic rearrangements are enriched in metastatic prostate cancers, consist of duplications and translocations within the FOXA1 locus, and structurally reposition a conserved regulatory element—herein denoted FOXA1 mastermind (FOXMIND)—to drive overexpression of FOXA1 or other oncogenes. Our study reaffirms the central role of FOXA1 in mediating oncogenesis driven by the androgen receptor, and provides mechanistic insights into how the classes of FOXA1 alteration promote the initiation and/or metastatic progression of prostate cancer. These results have direct implications for understanding the pathobiology of other hormone-receptor-driven cancers and rationalize the co-targeting of FOXA1 activity in therapeutic strategies. Comprehensive genomic analyses and mechanistic studies uncover three structural, functional and clinical classes of activating FOXA1 mutations and locus rearrangements in prostate cancer.

SUMMARYProcessing bodies (PBs) and stress granules (SGs) are prominent examples of sub-cellular, membrane-less compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal impact on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Together, our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration that adapts the cell to volume change.GRAPHICAL ABSTRACTIN BRIEFCells constantly experience osmotic variation. These external changes lead to changes in cell volume, and consequently the internal state of molecular crowding. Here, Jalihal and Pitchiaya et al. show that multimeric proteins respond rapidly to such cellular changes by undergoing rapid and reversible phase separation.HIGHLIGHTSDCP1A undergoes rapid and reversible hyperosmotic phase separation (HOPS)HOPS of DCP1A depends on its trimerization domainSelf-interacting multivalent proteins (valency ≥ 2) undergo HOPSHOPS of CPSF6 explains transcription termination defects during osmotic stress

Molecular classification of cancers into subtypes has resulted in an advance in our understanding of tumour biology and treatment response across multiple tumour types. However, to date, cancer profiling has largely focused on protein-coding genes, which comprise, LncRNAs have been associated with cancer. Here, the authors carry out a systematic review of lncRNAs in breast cancer and show that DSCAM-AS1 is highly expressed in oestrogen receptor positive tumours and enhances cancer through an interaction with hnRNPL; and is also associated with tamoxifen resistance.

The androgen receptor (AR) signaling plays a key role in the development of the normal prostate as well as prostate cancer. While substantial efforts have been undertaken to study AR-regulated protein-coding genes in primary prostate cancer and castration-resistant prostate cancer, few studies have investigated the role of long noncoding RNAs. In this study, we employed transcriptome sequencing to delineate long noncoding RNAs (lncRNAs) associated with AR signaling in prostate cancer progression. ARlnc1 (AR-regulated lncRNA 1) was identified as being the top AR-induced, cancer-associated lncRNA in an integrative analysis of prostate cancer cell lines and tissues. Not only was ARlnc1 induced by AR, but ARlnc1 also was shown to sustain AR signaling by stabilizing the AR transcript via interaction with the AR 3' UTR. Knockdown of ARlnc1 suppressed AR expression, global AR signaling, and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARlnc1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression, and identifies ARlnc1 as a novel therapeutic target. Citation Format: Yajia Zhang, Sethuramasundaram Pitchiaya, Marcin Cieślik, Yashar S. Niknafs, Jean C. Tien, Yasuyuki Hosono, Lisha Wang, Yuanyuan Qiao, Xuhong Cao, Mats Ljungman, Hui Jiang, Rohit Mehra, Shuling Guo, Rohit Malik, Arul M. Chinnaiyan. The androgen receptor-regulated lncRNA landscape reveals a role for ARlnc1 in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2458.

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

The pervasive expression of long non-coding RNAs (lncRNAs) and their roles in a plethora of cellular processes has revolutionized our understanding of functional genetic elements. Emerging evidence suggest that lncRNAs are promising cancer biomarkers and dysregulation of lncRNAs results in attenuated or accelerated oncogenic phenotypes. In support of these findings, recent reports from the Chinnaiyan lab suggest that lncRNAs can potentially drive and act as independent predictors of aggressive prostate cancer (PCa). Central to PCa progression is the androgen receptor (AR), a nuclear hormone receptor, and the dysregulated transcription program it mediates. Consequently, androgen deprivation therapy (ADT) supplemented with anti-androgens is the initial standard-of-care in treating advanced PCa. Yet, the disease often manifests as a lethal, hormone-refractory castration-resistant prostate cancer (CRPC) after initial ADT and this phenomenon is linked to the resumption of AR activity. Therefore, we sought to identify novel and therapeutically actionable targets of AR that can detect the onset of PCa or stratify PCa variants. To this end, our lab recently characterized the transcriptional landscape of cancer and discovered > 50,000 novel transcripts, a significant fraction of which were tissue- and cancer-specific lncRNAs. Using this updated transcriptome as a reference and performing RNA-seq on PCa cells stimulated with dihydrotestosterone (an androgen), we identified Androgen Receptor regulated lncRNA-1 (ARlnc1) as an important PCa-specific, AR-regulated lncRNA that reciprocally regulates AR. By employing single-molecule fluorescence in situ hybridization (smFISH), we find that ARlnc1 and AR transcripts colocalize within the nucleus of PCa cells and that ARlnc1 promotes transcription of AR. Moreover, we found that ARlnc1 interacts with anti-apoptotic, stress-granule related proteins and regulates PCa cell proliferation in vitro and in mouse xenografts. We have additionally found that anti-sense oligonucleotides (ASOs) that target this lncRNA can reduce tumor growth in xenograft models. Taken together, our data suggests that ARlnc1 and AR reciprocally and positively modulate each other to promote PCa progression and that ARlnc1 may serve as an enhancer RNA that regulates AR transcription. Our data suggests that ARlnc1 can be therapeutically targeted to orthogonally modulate the AR signaling axis for PCa treatment. Citation Format: Sethuramasundaram Pitchiaya, Rohit Malik, Marcin Cieslik, Yajia Zhang, Xia Jiang, Arul M. Chinnaiyan. A long non-coding RNA regulates the androgen receptor and mediates prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2549. doi:10.1158/1538-7445.AM2017-2549

The flow of genetic information is regulated by selective nucleocytoplasmic transport of messenger RNA:protein complexes (mRNPs) through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the three-dimensional (3D) pathway taken by mRNPs as they transit through the NPC, and the kinetics and selectivity of transport, remain obscure. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal accuracy of 8 nm and 2 ms to provide new insights into the mechanism of nuclear mRNP export in live human cells. We find that mRNPs exiting the nucleus are decelerated and selected at the centre of the NPC, and adopt a fast-slow-fast diffusion pattern during their brief, ~12 ms, interaction with the NPC. A 3D reconstruction of the export route indicates that mRNPs primarily interact with the periphery on the nucleoplasmic side and in the centre of the NPC, without entering the central axial conduit utilized for passive diffusion of small molecules, and eventually dissociate on the cytoplasmic side.

Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Approximately 30% of all cancers harbor activating mutations in the RAS family of small GTPase proteins, making it one of the most common oncogenic aberrations in humans. Normal RAS proteins (H, K or N-RAS) localize to the inner cell membrane and transduce extracellular growth signals by cycling between an “active” GTP-bound state and “inactive” GDP-bound state. Our understanding of RAS biology is primarily from RAS protein-effector interactions that activate a variety of effectors at the plasma membrane like RAF/PI3K/RalGDS. Yet, targeting mutant RAS proteins or its effectors / pathways remains a challenging endeavor for treating RAS driven cancers. For a comprehensive identification of RAS interactors, we recently performed co-immunoprecipitation (Co-IP) Mass Spectrometric analysis of RAS immunoprecipitates from multiple cancer cell lines with differing KRAS mutation status. In all the cell lines studies, we uncovered an interaction between RAS and the core component of the RNA silencing machinery, Argonaute 2 (AGO2). Endogenously expressed RAS and AGO2 co-sediment and co-localize in intracellular membrane bound endoplasmic reticulum. AGO2 binds the Switch II region in KRAS, irrespective of GDP/GTP bound to RAS. Both endogenous and overexpressed mutant forms of KRAS, attenuate AGO2 related gene silencing function. Using NIH3T3 AGO2-/- cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Overall, our studies suggest that through its interaction with AGO2, RAS function extends well beyond its canonical role in intracellular signaling. We will present detailed characterization of the RAS-AGO2 interaction and its functional aspects that we have discovered so far. Citation Format: Sunita Shankar, Sethuramasundaram Pitchiaya, Rohit Malik, Vishal Kothari, Yasuyuki Hosono, Anastasia K. Yocum, Harika Gundlapalli, Yasmine White, Ari Firestone, Xuhong Cao, Saravana M. Dhanasekaran, Jeanne Stuckey, Gideon Bollag, Kevin Shannon, Nils Walter, Chandan Kumar-Sinha, Arul M. Chinnaiyan. KRAS engages AGO2 to enhance cellular transformation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-008.

Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer. AR regulates a large repertoire of genes; however, the effect of androgen signaling on the regulation of long non-coding RNAs (lncRNA) remains incompletely understood. Using transcriptome sequencing (RNA-seq) of AR-positive cell lines VCaP and LNCaP treated with dihydroxytestosterone (DHT), we identified genes, including lncRNAs, which were strongly regulated by AR. To confirm direct regulation by AR, we interrogated AR-ChIP-seq data from VCaP and LNCaP cells, identifying the lncRNAs with direct AR binding. Existence of these lncRNAs in prostate cancer tissue samples was confirmed by analysis of RNA-seq data from prostate tumors, and the degree of differential expression in prostate tumors (localized and castration resistant metastases) versus benign was determined. The most highly overexpressed lncRNA in this analysis was a 2.7kb multi-exonic transcript present on chromosome 16 called ARlnc1. RACE was utilized to determine the exact exon structure of this gene, and its expression levels in various prostate cancer cell lines as well as independent prostate cancer tissue cohorts was assessed. Further, knockdown of ARlnc1 in AR dependent cell lines inhibited cell proliferation and induced apoptosis. Knockdown of ARlnc1 affected molecular signatures related to cell cycle, mitosis and DNA damage. Interestingly, ARlnc1 knockdown also suppressed global androgen signaling as determined by Gene set enrichment analysis using AR gene signature. Upon investigation of the mechanism through which PRCAT47 regulate AR signaling, we discovered that ARlnc1 regulates AR at the level of translation. Taken together, our data suggests that many lncRNAs are regulated by androgen signaling, and we identify one such lncRNA that is involved in a protein-lncRNA positive feedback loop. Citation Format: Rohit Malik, Yajia Zhang, Marcin Cieslik, Yashar S. Niknafs, Sethuramasundaram Pitchiaya, Yasuyuki Hosono, Shruthi Subramaniam, Sahr Yazdani, Xuhong Cao, Dan Robinson, Arul Chinnaiyan. Integrative analysis of androgen receptor regulated long non-coding RNA in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 983.