Discovery and Optimization of the First ATP Competitive Type-III c-MET Inhibitor.

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.

PRDM15 is a key regulator of metabolism critical to sustain B-cell lymphomagenesis.

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology.

Structural and Molecular Insight into Resistance Mechanisms of First Generation cMET Inhibitors.

Many small molecule inhibitors of the cMET receptor tyrosine kinase have been evaluated in clinical trials for the treatment of cancer and resistance-conferring mutations of cMET are beginning to be reported for a number of such compounds. There is now a need to understand specific cMET mutations at the molecular level, particularly concerning small molecule recognition. Toward this end, we report here the first crystal structures of the recent clinically observed resistance-conferring D1228V cMET mutant in complex with small molecule inhibitors, along with a crystal structure of wild-type cMET bound by the clinical compound savolitinib and supporting cellular, biochemical, and biophysical data. Our findings indicate that the D1228V alteration induces conformational changes in the kinase, which could have implications for small molecule inhibitor design. The data we report here increases our molecular understanding of the D1228V cMET mutation and provides insight for future inhibitor design.

Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation.

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.

GRHL2-miR-200-ZEB1 maintains the epithelial status of ovarian cancer through transcriptional regulation and histone modification.

Epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, has been implicated to contribute to the molecular heterogeneity of epithelial ovarian cancer (EOC). Here we report that a transcription factor--Grainyhead-like 2 (GRHL2) maintains the epithelial phenotype. EOC tumours with lower GRHL2 levels are associated with the Mes/Mesenchymal molecular subtype and a poorer overall survival. shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype results in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, microRNA-200b/a is identified as the direct transcriptional target of GRHL2 and regulates the epithelial status of EOC through ZEB1 and E-cadherin. Our study demonstrates that loss of GRHL2 increases the levels of histone mark H3K27me3 on promoters and GRHL2-binding sites at miR-200b/a and E-cadherin genes. These findings support GRHL2 as a pivotal gatekeeper of EMT in EOC via miR-200-ZEB1.

Targeting MYC in cancer therapy: RNA processing offers new opportunities.

MYC is a transcription factor, which not only directly modulates multiple aspects of transcription and co-transcriptional processing (e.g. RNA-Polymerase II initiation, elongation, and mRNA capping), but also indirectly influences several steps of RNA metabolism, including both constitutive and alternative splicing, mRNA stability, and translation efficiency. As MYC is an oncoprotein whose expression is deregulated in multiple human cancers, identifying its critical downstream activities in tumors is of key importance for designing effective therapeutic strategies. With this knowledge and recent technological advances, we now have multiple angles to reach the goal of targeting MYC in tumors, ranging from the direct reduction of MYC levels, to the dampening of selected house-keeping functions in MYC-overexpressing cells, to more targeted approaches based on MYC-induced secondary effects.

Antisense oligonucleotide-mediated MDM4 exon 6 skipping impairs tumor growth.

MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient-derived xenograft (PDX) mouse models, antisense oligonucleotide-mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target.

MYC regulates the core pre-mRNA splicing machinery as an essential step in lymphomagenesis.

Deregulated expression of the MYC transcription factor occurs in most human cancers and correlates with high proliferation, reprogrammed cellular metabolism and poor prognosis. Overexpressed MYC binds to virtually all active promoters within a cell, although with different binding affinities, and modulates the expression of distinct subsets of genes. However, the critical effectors of MYC in tumorigenesis remain largely unknown. Here we show that during lymphomagenesis in Eµ-myc transgenic mice, MYC directly upregulates the transcription of the core small nuclear ribonucleoprotein particle assembly genes, including Prmt5, an arginine methyltransferase that methylates Sm proteins. This coordinated regulatory effect is critical for the core biogenesis of small nuclear ribonucleoprotein particles, effective pre-messenger-RNA splicing, cell survival and proliferation. Our results demonstrate that MYC maintains the splicing fidelity of exons with a weak 5' donor site. Additionally, we identify pre-messenger-RNAs that are particularly sensitive to the perturbation of the MYC-PRMT5 axis, resulting in either intron retention (for example, Dvl1) or exon skipping (for example, Atr, Ep400). Using antisense oligonucleotides, we demonstrate the contribution of these splicing defects to the anti-proliferative/apoptotic phenotype observed in PRMT5-depleted Eµ-myc B cells. We conclude that, in addition to its well-documented oncogenic functions in transcription and translation, MYC also safeguards proper pre-messenger-RNA splicing as an essential step in lymphomagenesis.

Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity.

Constitutively active MYC and reactivated telomerase often coexist in cancers. While reactivation of telomerase is thought to be essential for replicative immortality, MYC, in conjunction with cofactors, confers several growth advantages to cancer cells. It is known that the reactivation of TERT, the catalytic subunit of telomerase, is limiting for reconstituting telomerase activity in tumors. However, while reactivation of TERT has been functionally linked to the acquisition of several "hallmarks of cancer" in tumors, the molecular mechanisms by which this occurs and whether these mechanisms are distinct from the role of telomerase on telomeres is not clear. Here, we demonstrated that first-generation TERT-null mice, unlike Terc-null mice, show delayed onset of MYC-induced lymphomagenesis. We further determined that TERT is a regulator of MYC stability in cancer. TERT stabilized MYC levels on chromatin, contributing to either activation or repression of its target genes. TERT regulated MYC ubiquitination and proteasomal degradation, and this effect of TERT was independent of its reverse transcriptase activity and role in telomere elongation. Based on these data, we conclude that reactivation of TERT, a direct transcriptional MYC target in tumors, provides a feed-forward mechanism to potentiate MYC-dependent oncogenesis.

Storage of bacteria and yeast.

Yeast and bacteria can be cryopreserved and stored almost indefinitely. It is useful to prepare stocks for archival purposes.

Preparation of cells for microscopy using cytospin.

Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Cells are immobilized on a solid support that is optically suitable for microscopy, and then fixed. When coupled with antibody-based immunofluorescent or immunocytochemical methods, the expression of specific proteins can be quantified and localized. Specialized stains and dyes can also be used to visualize nucleic acids or other cellular structures. See alternative protocols for fixation of suspension cells on Preparation of Cells for Microscopy using 'Cell Blocks' and for adherent cells on Preparation of Cells for Microscopy using Chamber Slides and Coverslips.

Preparation of cells for microscopy using chamber slides and coverslips.

Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Cells are immobilized on a solid support that is optically suitable for microscopy, and then fixed. When coupled with antibody-based immunofluorescent or immunocytochemical methods, the expression of specific proteins can be quantified and localized. Specialized stains and dyes can also be used to visualize nucleic acids or other cellular structures. See alternative protocols for fixation of suspension cells on Preparation of Cells for Microscopy using Cytospin and Preparation of Cells for Microscopy using 'Cell Blocks'.

Preparation of cells for microscopy using 'cell blocks'.

Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Embedding cells in agarose and then in paraffin as 'cell blocks' allows for them to be processed in the same manner that tissue specimens are processed for histology. This method is advantageous because numerous sections can be cut from one cell block. Additionally, sectioning renders antigens within the nucleus and other cell organelles more accessible to antibodies. However, access to specialized equipment for histological tissue processing is required See alternative protocols for fixation of suspension cells on Preparation of Cells for Microscopy using Cytospin and for adherent cells on Preparation of Cells for Microscopy using Chamber Slides and Coverslips.

Isolation of genomic DNA from mammalian cells.

The isolation of genomic DNA from mammalian cells is a routine molecular biology laboratory technique with numerous downstream applications. The isolated DNA can be used as a template for PCR, cloning, and genotyping and to generate genomic DNA libraries. It can also be used for sequencing to detect mutations and other alterations, and for DNA methylation analyses.

Myc enforces overexpression of EZH2 in early prostatic neoplasia via transcriptional and post-transcriptional mechanisms.

EZH2 is part of the PRC2 polycomb repressive complex that is overexpressed in multiple cancer types and has been implicated in prostate cancer initiation and progression. Here, we identify EZH2 as a target of the MYC oncogene in prostate cancer and show that MYC coordinately regulates EZH2 through transcriptional and post-transcriptional means. Although prior studies in prostate cancer have revealed a number of possible mechanisms of EZH2 upregulation, these changes cannot account for the overexpression EZH2 in many primary prostate cancers, nor in most cases of high grade PIN. We report that upregulation of Myc in the mouse prostate results in overexpression of EZH2 mRNA and protein which coincides with reductions in miR-26a and miR-26b, known regulators of EZH2 in some non-prostate cell types, albeit not in others. Further, in human prostate cancer cells, Myc negatively regulates miR-26a and miR-26b via direct binding to their parental Pol II gene promoters, and forced overexpression of miR-26a and miR-26b in prostate cancer cells results in decreased EZH2 levels and suppressed proliferation. In human clinical samples, miR-26a and miR-26b are downregulated in most primary prostate cancers. As a separate mechanism of EZH2 mRNA upregulation, we find that Myc binds directly to and activates the transcription of the EZH2 promoter. These results link two major pathways in prostate cancer by providing two additional and complementary Myc-regulated mechanisms by which EZH2 upregulation occurs and is enforced during prostatic carcinogenesis. Further, the results implicate EZH2-driven mechanisms by which Myc may stimulate prostate tumor initiation and disease progression.

Alterations in nucleolar structure and gene expression programs in prostatic neoplasia are driven by the MYC oncogene.

Increased nucleolar size and number are hallmark features of many cancers. In prostate cancer, nucleolar enlargement and increased numbers are some of the earliest morphological changes associated with development of premalignant prostate intraepithelial neoplasia (PIN) lesions and invasive adenocarcinomas. However, the molecular mechanisms that induce nucleolar alterations in PIN and prostate cancer remain largely unknown. We verify that activation of the MYC oncogene, which is overexpressed in most human PIN and prostatic adenocarcinomas, leads to formation of enlarged nucleoli and increased nucleolar number in prostate luminal epithelial cells in vivo. In prostate cancer cells in vitro, MYC expression is needed for maintenance of nucleolar number, and a nucleolar program of gene expression. To begin to decipher the functional relevance of this transcriptional program in prostate cancer, we examined FBL (encoding fibrillarin), a MYC target gene, and report that fibrillarin is required for proliferation, clonogenic survival, and proper ribosomal RNA accumulation/processing in human prostate cancer cells. Further, fibrillarin is overexpressed in PIN lesions induced by MYC overexpression in the mouse prostate, and in human clinical prostate adenocarcinoma and PIN lesions, where its expression correlates with MYC levels. These studies demonstrate that overexpression of the MYC oncogene increases nucleolar number and size and a nucleolar program of gene expression in prostate epithelial cells, thus providing a molecular mechanism responsible for hallmark nucleolar alterations in prostatic neoplasia.

MYC overexpression induces prostatic intraepithelial neoplasia and loss of Nkx3.1 in mouse luminal epithelial cells.

Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina-features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These "Super-Lo-MYC" mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a "paradoxical" increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas.

MYC and Prostate Cancer.

Prostate cancer, the majority of which is adenocarcinoma, is the most common epithelial cancer affecting a majority of elderly men in Western nations. Its manifestation, however, varies from clinically asymptomatic insidious neoplasms that progress slowly and do not threaten life to one that is highly aggressive with a propensity for metastatic spread and lethality if not treated in time. A number of somatic genetic and epigenetic alterations occur in prostate cancer cells. Some of these changes, such as loss of the tumor suppressors PTEN and p53, are linked to disease progression. Others, such as ETS gene fusions, appear to be linked more with early phases of the disease, such as invasion. Alterations in chromosome 8q24 in the region of MYC have also been linked to disease aggressiveness for many years. However, a number of recent studies in human tissues have indicated that MYC appears to be activated at the earliest phases of prostate cancer (e.g., in tumor-initiating cells) in prostatic intraepithelial neoplasia, a key precursor lesion to invasive prostatic adenocarcinoma. The initiation and early progression of prostate cancer can be recapitulated in genetically engineered mouse models, permitting a richer understanding of the cause and effects of loss of tumor suppressors and activation of MYC. The combination of studies using human tissues and mouse models paints an emerging molecular picture of prostate cancer development and early progression. This picture reveals that MYC contributes to disease initiation and progression by stimulating an embryonic stem cell-like signature characterized by an enrichment of genes involved in ribosome biogenesis and by repressing differentiation. These insights pave the way to potential novel therapeutic concepts based on MYC biology.

Molecular alterations in prostate cancer as diagnostic, prognostic, and therapeutic targets.

Prostatic adenocarcinoma is extremely common in Western nations, representing the second leading cause of cancer death in American men. The recent application of increasingly sophisticated molecular approaches to the study of prostate cancer in this "postgenomic" era has resulted in a rapid increase in the identification of somatic genome alterations and germline heritable risk factors in this disease. These findings are leading to a new understanding of the pathogenesis of prostate cancer and to the generation of new targets for diagnosis, prognosis, and prediction of therapeutic response. Although we are still in the very early phase of clinical development, some of the molecular alterations identified in prostate cancer are being translated into clinical practice. The purpose of this review is to update the practicing surgical pathologist, and residents-in-training in pathology, regarding recent findings in the molecular pathobiology of prostate cancer. We will highlight some of the somatic molecular alterations associated with prostate cancer development and progression, with a focus on newer discoveries. In addition, recent studies in which new molecular diagnostic approaches have been applied in the clinic will be discussed.

Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis.

The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. In contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (eg Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression.