Marcin Cieślik

Integrative clinical genomics of metastatic cancer

Metastasis is the primary cause of cancer-related deaths. Although The Cancer Genome Atlas has sequenced primary tumour types obtained from surgical resections, much less comprehensive molecular analysis is available from clinically acquired metastatic cancers. Here we perform whole-exome and -transcriptome sequencing of 500 adult patients with metastatic solid tumours of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53, CDKN2A, PTEN, PIK3CA, and RB1. Putative pathogenic germline variants were present in 12.2% of cases of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing to identify gene fusions, pathway activation, and immune profiling. Our results show that integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers.

Targeting the MLL complex in castration resistant prostate cancer

Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration-resistant prostate cancer (CRPC). Although prior work has focused on targeting AR directly, co-activators of AR signaling, which may represent new therapeutic targets, are relatively underexplored. Here we demonstrate that the mixed-lineage leukemia protein (MLL) complex, a well-known driver of MLL fusion–positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via the menin–MLL subunit. Menin expression is higher in CRPC than in both hormone-naive prostate cancer and benign prostate tissue, and high menin expression correlates with poor overall survival of individuals diagnosed with prostate cancer. Treatment with a small-molecule inhibitor of menin–MLL interaction blocks AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice. Taken together, this work identifies the MLL complex as a crucial co-activator of AR and a potential therapeutic target in advanced prostate cancer.

Development of Peptidomimetic Inhibitors of the ERG Gene Fusion Product in Prostate Cancer

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.

MicroRNA-101 regulated transcriptional modulator SUB1 plays a role in prostate cancer

MicroRNA-101, a tumor suppressor microRNA (miR), is often downregulated in cancer and is known to target multiple oncogenes. Some of the genes that are negatively regulated by miR-101 expression include histone methyltransferase EZH2 (enhancer of zeste homolog 2), COX2 (cyclooxygenase-2), POMP (proteasome maturation protein), CERS6, STMN1, MCL-1 and ROCK2, among others. In the present study, we show that miR-101 targets transcriptional coactivator SUB1 homolog (Saccharomyces cerevisiae)/PC4 (positive cofactor 4) and regulates its expression. SUB1 is known to have diverse role in vital cell processes such as DNA replication, repair and heterochromatinization. SUB1 is known to modulate transcription and acts as a mediator between the upstream activators and general transcription machinery. Expression profiling in several cancers revealed SUB1 overexpression, suggesting a potential role in tumorigenesis. However, detailed regulation and function of SUB1 has not been elucidated. In this study, we show elevated expression of SUB1 in aggressive prostate cancer. Knockdown of SUB1 in prostate cancer cells resulted in reduced cell proliferation, invasion and migration in vitro, and tumor growth and metastasis in vivo. Gene expression analyses coupled with chromatin immunoprecipitation revealed that SUB1 binds to the promoter regions of several oncogenes such as PLK1 (Polo-like kinase 1), C-MYC, serine-threonine kinase BUB1B and regulates their expression. Additionally, we observed SUB1 downregulated CDKN1B expression. PLK1 knockdown or use of PLK1 inhibitor can mitigate oncogenic function of SUB1 in benign prostate cancer cells. Thus, our study suggests that miR-101 loss results in increased SUB1 expression and subsequent activation of known oncogenes driving prostate cancer progression and metastasis. This study therefore demonstrates functional role of SUB1 in prostate cancer, and identifies its regulation and potential downstream therapeutic targets of SUB1 in prostate cancer.

Identification and Validation of PCAT14 as Prognostic Biomarker in Prostate Cancer

Rapid advances in the discovery of long noncoding RNAs (lncRNAs) have identified lineage- and cancer-specific biomarkers that may be relevant in the clinical management of prostate cancer (PCa). Here we assembled and analyzed a large RNA-seq dataset, from 585 patient samples, including benign prostate tissue and both localized and metastatic PCa to discover and validate differentially expressed genes associated with disease aggressiveness. We performed Sample Set Enrichment Analysis (SSEA) and identified genes associated with low versus high Gleason score in the RNA-seq database. Comparing Gleason 6 versus 9+ PCa samples, we identified 99 differentially expressed genes with variable association to Gleason grade as well as robust expression in prostate cancer. The top-ranked novel lncRNA PCAT14, exhibits both cancer and lineage specificity. On multivariate analysis, low PCAT14 expression independently predicts for BPFS (P = .00126), PSS (P = .0385), and MFS (P = .000609), with trends for OS as well (P = .056). An RNA in-situ hybridization (ISH) assay for PCAT14 distinguished benign vs malignant cases, as well as high vs low Gleason disease. PCAT14 is transcriptionally regulated by AR, and endogenous PCAT14 overexpression suppresses cell invasion. Thus, Using RNA-sequencing data we identify PCAT14, a novel prostate cancer and lineage-specific lncRNA. PCAT14 is highly expressed in low grade disease and loss of PCAT14 predicts for disease aggressiveness and recurrence.

Cancer transcriptome profiling at the juncture of clinical translation

Methodological breakthroughs over the past four decades have repeatedly revolutionized transcriptome profiling. Using RNA sequencing (RNA-seq), it has now become possible to sequence and quantify the transcriptional outputs of individual cells or thousands of samples. These transcriptomes provide a link between cellular phenotypes and their molecular underpinnings, such as mutations. In the context of cancer, this link represents an opportunity to dissect the complexity and heterogeneity of tumours and to discover new biomarkers or therapeutic strategies. Here, we review the rationale, methodology and translational impact of transcriptome profiling in cancer.

Erratum: Development of Peptidomimetic Inhibitors of the ERG Gene Fusion Product in Prostate Cancer (Cancer Cell (2017) 31(4) (532–548.e7)(S1535610817300600)(10.1016/j.ccell.2017.02.017))

Xiaoju Wang, Yuanyuan Qiao, Irfan A. Asangani, Bushra Ateeq, Anton Poliakov, Marcin Cie slik, Sethuramasundaram Pitchiaya, Balabhadrapatruni V.S.K. Chakravarthi, Xuhong Cao, Xiaojun Jing, Cynthia X. Wang, Ingrid J. Apel, Rui Wang, Jean Ching-Yi Tien, Kristin M. Juckette, Wei Yan, Hui Jiang, Shaomeng Wang, Sooryanarayana Varambally, and Arul M. Chinnaiyan* *Correspondence: arul@umich.edu http://dx.doi.org/10.1016/j.ccell.2017.05.001

Analysis of the androgen receptor–regulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression

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.ARLNC1 is a newly discovered lncRNA that is induced by androgen receptor (AR) and maintains AR signaling by stabilizing the AR transcript. Knockdown of ARLNC1 suppresses AR expression, AR signaling and prostate cancer growth in vitro and in vivo.

Competing for enhancers: PVT1 fine-tunes MYC expression

Cho and colleagues uncover a novel mechanism of transcriptional repression of MYC which attributes a tumor-suppressive role to the neighboring PVT1 promoter, and shed light on recurrent chromosomal rearrangements within the MYC locus. In human cells, chromatin is three-dimensionally organized into discrete self-interacting units called topologically associating domains (TADs). TADs are generally preserved across cell lineages and their boundaries are enriched for binding sites of ubiquitous architectural proteins (e.g., CTCF, cohesins, etc.). These domain boundaries are referred to as chromatin “insulators” as they confine enhancer activity within specified TADs. Dynamic looping interactions within a TAD scaffold, or intra-TAD loops, enable finer regulation of enhancer-promoter pairs in different tissue types. Thus, TADs partition chromatin into functionally interacting enhancer-promoter units and link genome architecture to transcriptional regulation. Disruption and reorganization of TADs provide a putative mechanism to support cancer-specific gene expression programs. A growing number of studies support this model, highlighting recurrent structural rearrangements or large-scale epigenetic reorganization of insulated domains as drivers of oncogene expression. However, the exact mechanisms of transcriptional de-regulation through intra-TAD looping and structural aberrations are still a matter of active research. Adding a new perspective to this body of knowledge, a recent article published in Cell described in extensive details a regulatory mechanism operating within the MYC locus, and attributed an unexpected insulator function to the PVT1 gene promoter. Interestingly, PVT1 and MYC are encoded 53 kb apart within the 8q24 chromosomal region, which is recurrently disrupted through complex rearrangements, focal amplifications, translocations or viral integrations, in a variety of human tumors. This region harbors a number of enhancer elements that establish long-range interactions with MYC, and single nucleotide polymorphisms within these regulatory elements are associated with cancer risk. Previous studies have also revealed that long non-coding RNA (lncRNA) PVT1 is oncogenic and its ablation diminishes MYC-driven tumorigenesis. However, surprisingly, Cho and colleagues found that silencing of the PVT1 promoter instead conferred competitive growth advantage to cancer cells. These observations led the authors on a tour de force to interrogate the functional consequences of PVT1 promoter inactivation. First, the authors found that MYC was the most consistently upregulated transcript upon silencing of the PVT1 promoter through heterochromatinization (using the recently developed CRISPRKRAB technique). Next, using multiple complementary techniques, the authors demonstrated that the PVT1 lncRNA itself was entirely dispensable for transcriptional inhibition of MYC by the PVT1 promoter. This tendered an intriguing possibility that PVT1 promoter inherently harbors tumor-suppressive ability, in turn raising a more fundamental molecular question: How does a few hundred bases long, distally coded regulatory element dynamically control gene expression? To address this question, the authors performed the HiChIP assay (recently developed for protein-directed detection of chromatin conformation) targeting the active enhancer mark histone H3 lysine 27 acetylation (H3K27Ac). This allowed them to delineate changes in the enhancer interactome within the MYC-PVT1 locus in response to PVT1 promoter inactivation. Interestingly, the authors discovered four intragenic enhancer elements within the PVT1 gene that made stronger contacts with the MYC locus upon repression of the PVT1 promoter. Notably, there was concurrent decrease in the interaction of these enhancer sites with the PVT1 promoter. Consistent changes in cis-interactions were detected using an orthogonal conformation capture technique, and direct disruption of individual enhancer elements reversed overexpression of MYC detected in response to silencing of the PVT1 promoter. Furthermore, the authors showed that the epigenetic reader protein, BRD4, primarily mediated rewiring of the intra-TAD loops and facilitated pause release of the RNA polymerase transcriptional complex to drive nascent MYC expression. Thus, the authors proposed a unifying model wherein, PVT1 competes with MYC for access to a common set of enhancers, and thus inactivation/disruption of the PVT1 promoter reapportions greater enhancer activity to MYC leading to increase in expression. In other words, PVT1 promoter partially insulates downstream enhancer elements from regulating MYC expression, and thus functions as an intra-TAD boundary element (Fig. 1). Notably, this study is among the first to demonstrate an insulator-like function for an “active” regulatory region. This opens a new research avenue for future studies to interrogate “enhancer competition” as a regulatory mechanism of lncRNAs in normal or diseased states. Perhaps, the most compelling evidence in support of the proposed model came from allele-specific assessment of Myc expression. In embryonic stem cells derived from a hybrid mouse model, as expected, Pvt1 expression was biallelic. However, upon differentiation into neuronal progenitors, random clones displayed allele-specific expression of Pvt1. Most remarkably, in these clones, expression of Myc was increased only from the allele encoded on the same chromosome as the silenced Pvt1 promoter. In other words, activation of Pvt1 promoter repressed Myc expression only in cis. Interestingly, in human cancers, the PVT1 promoter is recurrently rearranged. Cho and colleagues showed