Structure-based design and discovery of potent and selective lysine-specific demethylase 1 (LSD1) inhibitors.

The histone demethylase LSD1 is a key enzyme in the epigenetic regulation of gene transcription. Here we present our efforts to discover small molecule reversible inhibitors of LSD1 as an attractive approach to treat hematologic malignancies and certain solid tumors. Using structure-based drug design, we designed and synthesized a novel series of heteroaromatic imidazole inhibitors that demonstrate potent inhibition of the demethylase activity and low nanomolar cell-based activity. This novel LSD1 inhibitor series was further optimized by attenuating the hERG inhibition and improving oral bioavailability.

Structure-based design and discovery of potent and selective KDM5 inhibitors.

Histone lysine demethylases (KDMs) play a key role in epigenetic regulation and KDM5A and KDM5B have been identified as potential anti-cancer drug targets. Using structural information from known KDM4 and KDM5 inhibitors, a potent series of pyrazolylpyridines was designed. Structure-activity relationship (SAR) exploration resulted in the identification of compound 33, an orally available, potent inhibitor of KDM5A/5B with promising selectivity. Potent cellular inhibition as measured by levels of tri-methylated H3K4 was demonstrated with compound 33 in the breast cancer cell line ZR-75-1.

Design, synthesis and biological evaluation of novel 4-phenylisoquinolinone BET bromodomain inhibitors.

The bromodomain and extra-terminal (BET) family of epigenetic proteins has attracted considerable attention in drug discovery given its involvement in regulating gene transcription. Screening a focused small molecule library based on the bromodomain pharmacophore resulted in the identification of 2-methylisoquinoline-1-one as a novel BET bromodomain-binding motif. Structure guided SAR exploration resulted in >10,000-fold potency improvement for the BRD4-BD1 bromodomain. Lead compounds exhibited excellent potencies in both biochemical and cellular assays in MYC-dependent cell lines. Compound 36 demonstrated good physicochemical properties and promising exposure levels in exploratory PK studies.

Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Genomic landscape of copy number aberrations enables the identification of oncogenic drivers in hepatocellular carcinoma.

UNLABELLED: Cancer is a genetic disease with frequent somatic DNA alterations. Studying recurrent copy number aberrations (CNAs) in human cancers would enable the elucidation of disease mechanisms and the prioritization of candidate oncogenic drivers with causal roles in oncogenesis. We have comprehensively and systematically characterized CNAs and the accompanying gene expression changes in tumors and matched nontumor liver tissues from 286 hepatocellular carcinoma (HCC) patients. Our analysis identified 29 recurrently amplified and 22 recurrently deleted regions with a high level of copy number changes. These regions harbor established oncogenes and tumor suppressors, including CCND1 (cyclin D1), MET (hepatocyte growth factor receptor), CDKN2A (cyclin-dependent kinase inhibitor 2A) and CDKN2B (cyclin-dependent kinase inhibitor 2B), as well as many other genes not previously reported to be involved in liver carcinogenesis. Pathway analysis of cis-acting genes in the amplification and deletion peaks implicates alterations of core cancer pathways, including cell-cycle, p53 signaling, phosphoinositide 3-kinase signaling, mitogen-activated protein kinase signaling, Wnt signaling, and transforming growth factor beta signaling, in a large proportion of HCC patients. We further credentialed two candidate driver genes (BCL9 and MTDH) from the recurrent focal amplification peaks and showed that they play a significant role in HCC growth and survival. CONCLUSION: We have demonstrated that characterizing the CNA landscape in HCC will facilitate the understanding of disease mechanisms and the identification of oncogenic drivers that may serve as potential therapeutic targets for the treatment of this devastating disease.

Prediction of disease-free survival in hepatocellular carcinoma by gene expression profiling.

BACKGROUND: Progression of hepatocellular carcinoma (HCC) often leads to vascular invasion and intrahepatic metastasis, which correlate with recurrence after surgical treatment and poor prognosis. The molecular prognostic model that could be applied to the HCC patient population in general is needed for effectively predicting disease-free survival (DFS). METHODS: A cohort of 286 HCC patients from South Korea and a second cohort of 83 patients from Hong Kong, China, were used as training and validation sets, respectively. RNA extracted from both tumor and adjacent nontumor liver tissues was subjected to microarray gene expression profiling. DFS was the primary clinical end point. Gradient lasso algorithm was used to build prognostic signatures. RESULTS: High-quality gene expression profiles were obtained from 240 tumors and 193 adjacent nontumor liver tissues from the training set. Sets of 30 and 23 gene-based DFS signatures were developed from gene expression profiles of tumor and adjacent nontumor liver, respectively. DFS gene signature of tumor was significantly associated with DFS in an independent validation set of 83 tumors (P = 0.002). DFS gene signature of nontumor liver was not significantly associated with DFS in the validation set (P = 0.827). Multivariate analysis in the validation set showed that DFS gene signature of tumor was an independent predictor of shorter DFS (P = 0.018). CONCLUSIONS: We developed and validated survival gene signatures of tumor to successfully predict the length of DFS in HCC patients after surgical resection.

Liverome: a curated database of liver cancer-related gene signatures with self-contained context information.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. A number of molecular profiling studies have investigated the changes in gene and protein expression that are associated with various clinicopathological characteristics of HCC and generated a wealth of scattered information, usually in the form of gene signature tables. A database of the published HCC gene signatures would be useful to liver cancer researchers seeking to retrieve existing differential expression information on a candidate gene and to make comparisons between signatures for prioritization of common genes. A challenge in constructing such database is that a direct import of the signatures as appeared in articles would lead to a loss or ambiguity of their context information that is essential for a correct biological interpretation of a gene's expression change. This challenge arises because designation of compared sample groups is most often abbreviated, ad hoc, or even missing from published signature tables. Without manual curation, the context information becomes lost, leading to uninformative database contents. Although several databases of gene signatures are available, none of them contains informative form of signatures nor shows comprehensive coverage on liver cancer. Thus we constructed Liverome, a curated database of liver cancer-related gene signatures with self-contained context information. DESCRIPTION: Liverome's data coverage is more than three times larger than any other signature database, consisting of 143 signatures taken from 98 HCC studies, mostly microarray and proteome, and involving 6,927 genes. The signatures were post-processed into an informative and uniform representation and annotated with an itemized summary so that all context information is unambiguously self-contained within the database. The signatures were further informatively named and meaningfully organized according to ten functional categories for guided browsing. Its web interface enables a straightforward retrieval of known differential expression information on a query gene and a comparison of signatures to prioritize common genes. The utility of Liverome-collected data is shown by case studies in which useful biological insights on HCC are produced. CONCLUSION: Liverome database provides a comprehensive collection of well-curated HCC gene signatures and straightforward interfaces for gene search and signature comparison as well. Liverome is available at http://liverome.kobic.re.kr.

Discovery of CC-90011: A Potent and Selective Reversible Inhibitor of Lysine Specific Demethylase 1 (LSD1).

Histone demethylase LSDl (KDMlA) belongs to the flavin adenine dinucleotide (FAD) dependent family of monoamine oxidases and is vital in regulation of mammalian biology. Dysregulation and overexpression of LSD1 are hallmarks of a number of human diseases, particularly cancers that are characterized as morphologically poorly differentiated. As such, inhibitors of LSD1 have potential to be beneficial as a cancer therapy. The most clinically advanced inhibitors of LSDl are covalent inhibitors derived from tranylcypromine (TCP). Herein, we report the discovery of a novel series of reversible and selective LSDl inhibitors. Exploration of structure-activity relationships (SARs) and optimization of ADME properties resulted in the identification of clinical candidate CC-90011. CC-90011 exhibits potent on-target induction of cellular differentiation in acute myeloid leukemia (AML) and small cell lung cancer (SCLC) cell lines, and antitumor efficacy in patient-derived xenograft (PDX) SCLC models. CC-90011 is currently in phase 2 trials in patients with first line, extensive stage SCLC (ClinicalTrials.gov identifier: NCT03850067).

KDM4 Inhibition Targets Breast Cancer Stem-like Cells.

Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here, we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSCs regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation, and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer. Cancer Res; 77(21); 5900-12. ©2017 AACR.

Discovery of differentially expressed genes in human breast cancer using subtracted cDNA libraries and cDNA microarrays.

Identifying novel and known genes that are differentially expressed in breast cancer has important implications in understanding the biology of breast tumorigenesis and developing new diagnostic and therapeutic agents. In this study we have combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarray, as a high throughput methodology designed to identify cDNA clones that are breast tumor- and tissue-specific and are overexpressed in breast tumors. Approximately 2000 cDNA clones generated from the subtracted breast tumor library were arrayed on the microarray chips. The arrayed target cDNAs were then hybridized with 30 pairs of fluorescent-labeled cDNA probes generated from breast tumors and normal tissues to determine the tissue distribution and tumor specificity. cDNA clones showing overexpression in breast tumors by microarray were further analysed by DNA sequencing, GenBank and EST database searches, and quantitative real time PCR. We identified several known genes, including mammaglobin, cytokeratin 19, fibronectin, and hair-specific type II keratin, which have previously been shown to be overexpressed in breast tumors and may play an important role in the malignance of breast. We also discovered B726P which appears to be an isoform of NY-BR-1, a breast tissue-specific gene. Two additional clones discovered, B709P and GABA(A) receptor pi subunit, were not previously described for their overexpression profile in breast tumors. Thus, combining PCR-based cDNA subtraction and cDNA microarray allowed for an efficient way to identify and validate genes with elevated mRNA expression levels in breast cancer that may potentially be involved in breast cancer progression. These differentially expressed genes may be of potential utility as therapeutic and diagnostic targets for breast cancer.

P504S/alpha-methylacyl-CoA racemase: a useful marker for diagnosis of small foci of prostatic carcinoma on needle biopsy.

Establishing a definitive diagnosis of malignancy in prostate needle biopsies with very small foci of adenocarcinoma is a major diagnostic challenge for surgical pathologists. A positive diagnostic marker specific for prostatic adenocarcinoma may enhance our ability to detect limited prostate cancer and reduce errors in diagnosis. P504S, also known as alpha-methylacyl-CoA racemase, recently identified by cDNA subtraction and microarray technology, might serve as such a specific marker because it has been demonstrated to be highly expressed in prostatic adenocarcinoma, but not in benign prostatic glands. However, whether small foci of carcinoma can be reliably detected by this marker is a crucial question for its clinical application. The aim of this study was to assess the utility of P504S immunohistochemistry in detecting small amounts of prostate cancer in prostate needle biopsies. A total of 142 prostate needle biopsies, including 73 cases with a small focus of prostatic adenocarcinoma (

Expression of alpha-methylacyl-CoA racemase (P504s) in various malignant neoplasms and normal tissues: astudy of 761 cases.

Alpha-methylacyl CoA racemase (AMACR), also known as P504S, plays an important role in peroxisomal beta-oxidation of branched-chain fatty acids. It has recently been shown that AMACR is highly expressed in prostate cancer and that it may be an important diagnostic marker for prostate carcinoma. However, little is known about expression of AMACR in normal tissues and other malignant tumors. In this study, we investigated expression of AMACR in 539 malignant tumors and 222 normal human tissues of various types by immunohistochemical analysis. mRNA levels of AMACR in normal organs and in selected tumors were assessed by real time PCR. In normal tissue, high expression of AMACR mRNA was identified in liver, kidney and salivary gland, while AMACR protein was detected in liver (hepatocytes), kidney (tubular epithelial cells), lung (only bronchial epithelial cells), and gallbladder (only mucosal epithelial cells). High expression of AMACR mRNA was found in prostate, liver, and kidney cancers but rarely in stomach and bladder cancers. A high percent of adenocarcinomas arising from these organs express AMACR, including 17 of 21 (81%) of hepatocellular carcinomas and 18 of 24 (75%) of renal cell carcinomas. In addition, carcinomas arising from tissues normally not expressing AMACR were also positive for the antigen, including 17 of 18 (94%) prostate carcinomas, 9 of 29 (31%) of urothelial carcinomas, and 4 of 15 (27%) of gastric adenocarcinomas. Two hundred and fifty cases of adenocarcinomas from lung, breast, pancreas, bile duct, adrenal gland, salivary gland, ovary, thyroid and endometrium were negative or rarely positive for AMACR. Neuroendocrine carcinomas rarely expressed AMACR. Melanomas, squamous cell carcinomas, basal cell carcinomas, soft tissue tumors (including epithelioid sarcomas and synovial sarcoma), thymomas, and germ cell tumors were negative for AMACR. Our data provide important baseline information for using AMACR in clinical practice and also are valuable in furthering understanding of the pathogenic role of AMACR in malignant neoplasms.

Whole-genome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer.

Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.

Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma.

To survey hepatitis B virus (HBV) integration in liver cancer genomes, we conducted massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues. We found that HBV integration is observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%). Copy-number variations (CNVs) were significantly increased at HBV breakpoint locations where chromosomal instability was likely induced. Approximately 40% of HBV breakpoints within the HBV genome were located within a 1,800-bp region where the viral enhancer, X gene and core gene are located. We also identified recurrent HBV integration events (in ≥ 4 HCCs) that were validated by RNA sequencing (RNA-seq) and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue. We also report evidence that suggests that the number of HBV integrations is associated with patient survival.

Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer.

Gastric cancer is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account for only small subsets of the disease. We performed exome sequencing of 22 gastric cancer samples and identified previously unreported mutated genes and pathway alterations; in particular, we found genes involved in chromatin modification to be commonly mutated. A downstream validation study confirmed frequent inactivating mutations or protein deficiency of ARID1A, which encodes a member of the SWI-SNF chromatin remodeling family, in 83% of gastric cancers with microsatellite instability (MSI), 73% of those with Epstein-Barr virus (EBV) infection and 11% of those that were not infected with EBV and microsatellite stable (MSS). The mutation spectrum for ARID1A differs between molecular subtypes of gastric cancer, and mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results reveal the genomic landscape, and highlight the importance of chromatin remodeling, in the molecular taxonomy of gastric cancer.

Detection of alpha-methylacyl-coenzyme-A racemase transcripts in blood and urine samples of prostate cancer patients.

BACKGROUND: Alpha-methylacyl-coenzyme-A racemase (AMACR) has been shown to be a highly specific marker for prostate cancer cells, even in the earliest stages of malignant progression. It is expressed at much higher levels than prostate-specific antigen (PSA) in malignant tissues, and is not expressed at appreciable levels in normal prostatic epithelium. In this study, we demonstrate the quantitative detection of AMACR transcripts in peripheral blood of prostate cancer patients using real-time RT-PCR. In addition, we have undertaken a pilot study to demonstrate the potential application of this technique for the detection of prostate tumor cells in urine samples from patients with prostate cancer. METHODS: A real-time RT-PCR assay was developed for detection of the expression of AMACR in prostate cancer patients. Blood samples from 163 patients were tested at various stages of disease progression, with or without therapy. Blood specimens from patients with benign prostate disorders and other types of cancer were also evaluated. RESULTS: In 28 of 58 samples from patients with known metastatic disease who were undergoing treatment, an AMACR expression signal above the cut-off value was detected, consistent with the presence of circulating tumor cells. In 39 of 88 patients with presumptive organ-confined disease, there was evidence of low levels of circulating tumor cells. Comparison of AMACR RT-PCR with known serum PSA values indicated that a combination of these parameters significantly increased the sensitivity for detection of progressive disease. In a pilot study analyzing urine samples from seven prostate cancer patients, elevated AMACR expression levels were detected in the urine sediments of four of six stage-T1 prostate cancer patients and in the one patient with stage-T2 prostate cancer. CONCLUSION: The data presented in this study indicates that AMACR real-time RT-PCR may aid in the detection and staging of prostate cancer.

VSGP/F-spondin: a new ovarian cancer marker.

The discovery of genes that are overexpressed in ovarian cancers provides valuable insight into ovarian cancer biology and will lead to the development of more effective treatment strategies for combating this disease. To identify genes exhibiting ovarian- and ovarian cancer-specific expression, we generated four subtracted cDNA libraries from primary and metastatic ovarian adenocarcinoma tissues. 3,400 cDNA clones from these libraries were analyzed by microarray for tissue distribution and tumor specificity using 32 pairs of fluorophore-labeled cDNA samples from a variety of normal tissues and ovarian tumor tissues. cDNA clones showing elevated expression in ovarian tumors were identified by DNA sequencing with comparison to public databases, and the most promising candidates were further analyzed by quantitative real-time polymerase chain reaction and Northern blot. This systematic approach led to the identification of a number of genes including vascular smooth muscle growth-promoting factor (VSGP/F-spondin), a secreted protein previously identified and cloned from bovine and human ovary. VSGP/F-spondin protein was observed in ovarian carcinomas but not in normal ovarian epithelium by immunohistochemistry with a VSGP/F-spondin antibody. The expression profile of VSGP/F-spondin identifies this molecule as a potential diagnostic marker or target for developing therapeutic strategies to treat ovarian carcinoma.

Application of a multigene reverse transcription-PCR assay for detection of mammaglobin and complementary transcribed genes in breast cancer lymph nodes.

BACKGROUND: Mammaglobin mRNA expression is found in 70-80% of primary and metastatic breast tumor biopsies. The potential breast tumor markers B305D, B726P, and gamma-aminobutyrate type A receptor pi subunit (GABApi) complement the expression of mammaglobin. Collectively the expression profile of these four genes could be used as a diagnostic and prognostic indicator for breast cancer. METHODS: A multigene reverse transcription-PCR (RT-PCR) assay was established to detect the expression of mammaglobin, GABApi, B305D, and B726P simultaneously. Specific primers and TaqMan probes were used to analyze combined mRNA expression profiles in primary breast tumors and metastatic lymph node specimens. RESULTS: The multigene RT-PCR assay detected substantial expression signals in 27 of 27 primary tumor and 50 of 50 metastatic breast lymph node samples. Specificity studies demonstrated no significant expression signal in 27 non-breast cancer lymph nodes, in 22 various healthy tissue samples, or in 14 colon tumor samples. CONCLUSION: The novel RT-PCR-based assay described here provides a sensitive detection system for disseminated breast tumor cells in lymph nodes. In addition, this multigene assay could also be used to test peripheral blood and bone marrow samples.

P704P, P712P, and P775P: A genomic cluster of prostate-specific genes.

BACKGROUND: Discovery of prostate cancer- and tissue-specific genes will lead to an increased understanding of the molecular events associated with the malignant transformation and tumorigenesis of prostate cells. Such understanding will likely result in the development of promising new markers for screening, diagnosis, and prognosis, as well as potential therapeutic approaches for combating this disease. METHODS: A PCR-based subtraction method was combined with a high-throughput microarray screening approach to identify prostate tissue- and/or cancer-specific genes. Northern blot and quantitative real-time PCR were used to confirm prostate specificity. Bioinformatics analysis was performed to determine gene localization and to identify the open reading frame of novel genes. RESULTS: Three novel cDNA clones, P704P, P712P, and P775P, were identified and characterized to be specific for normal and malignant prostate tissues. Furthermore, P712P mRNA expression was found to be androgen responsive in LNCaP cells. Sequences for all three cDNAs were localized to an 80 kb genomic region on chromosome 22. Attempts to identify full-length transcripts did not reveal any apparent open reading frames, indicating that P704P, P712P, and P775P may belong to a novel class of transcripts with specific patterns of gene expression that do not code for translated proteins. CONCLUSIONS: A genomic cluster of prostate-specific genes with no apparent open reading frame has been discovered using a high-throughput approach combining subtraction with microarray. This may represent an important genomic region having possible connections to prostate biology with potential applications in prostate diagnostics and therapy.

A dietary enzyme: alpha-methylacyl-CoA racemase/P504S is overexpressed in colon carcinoma.

Epidemiological studies have shown that consumption of red meat increases the risk of developing colon cancer. An enzyme, alpha-methylacyl CoA racemase (AMACR), also known as P504S, plays an important role in peroxisomal beta-oxidation of branched-chain fatty acids from red meat and dairy products. High expression of AMACR was recently found in prostate cancer. In this study, we investigated expression of AMACR in 242 cases of colonic tumors including 176 colorectal carcinomas, 38 colon adenomas and 28 hyperplastic (non-neoplastic) polyps by immunohistochemical analysis. The mRNA levels of AMACR expression in normal and colon cancer tissues were assessed by real-time PCR. Significant up-regulation of AMACR mRNA was found in colon carcinomas compared to normal tissue. There was very low or no expression of AMACR protein in normal colon, but AMACR was highly expressed in 76 and 75% of well and moderately differentiated colon carcinomas, respectively, and in 79% of adenomas. In contrast, only 4% of hyperplastic polyps expressed AMACR. Since this enzyme is involved in the metabolism of branched-chain fatty acids from beef, milk and dairy products, our results provide important molecular information regarding a possible link between diet and development of colon cancer. AMACR may also serve as a molecular marker for colon cancers and its precursor lesions.