Olena Barbash

Smyd3 links lysine methylation of map3k2 to ras-driven cancer

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

BET Inhibition Silences Expression of MYCN and BCL2 and Induces Cytotoxicity in Neuroblastoma Tumor Models

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.

Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762.

The bromodomain and extraterminal (BET) protein BRD2-4 inhibitors hold therapeutic promise in preclinical models of hematologic malignancies. However, translation of these data to molecules suitable for clinical development has yet to be accomplished. Herein we expand the mechanistic understanding of BET inhibitors in multiple myeloma by using the chemical probe molecule I-BET151. I-BET151 induces apoptosis and exerts strong antiproliferative effect in vitro and in vivo. This is associated with contrasting effects on oncogenic MYC and HEXIM1, an inhibitor of the transcriptional activator P-TEFb. I-BET151 causes transcriptional repression of MYC and MYC-dependent programs by abrogating recruitment to the chromatin of the P-TEFb component CDK9 in a BRD2-4-dependent manner. In contrast, transcriptional upregulation of HEXIM1 is BRD2-4 independent. Finally, preclinical studies show that I-BET762 has a favorable pharmacologic profile as an oral agent and that it inhibits myeloma cell proliferation, resulting in survival advantage in a systemic myeloma xenograft model. These data provide a strong rationale for extending the clinical testing of the novel antimyeloma agent I-BET762 and reveal insights into biologic pathways required for myeloma cell proliferation.

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation.

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

PRMT5 Is a Critical Regulator of Breast Cancer Stem Cell Function via Histone Methylation and FOXP1 Expression

Summary Breast cancer progression, treatment resistance, and relapse are thought to originate from a small population of tumor cells, breast cancer stem cells (BCSCs). Identification of factors critical for BCSC function is therefore vital for the development of therapies. Here, we identify the arginine methyltransferase PRMT5 as a key in vitro and in vivo regulator of BCSC proliferation and self-renewal and establish FOXP1, a winged helix/forkhead transcription factor, as a critical effector of PRMT5-induced BCSC function. Mechanistically, PRMT5 recruitment to the FOXP1 promoter facilitates H3R2me2s, SET1 recruitment, H3K4me3, and gene expression. Our findings are clinically significant, as PRMT5 depletion within established tumor xenografts or treatment of patient-derived BCSCs with a pre-clinical PRMT5 inhibitor substantially reduces BCSC numbers. Together, our findings highlight the importance of PRMT5 in BCSC maintenance and suggest that small-molecule inhibitors of PRMT5 or downstream targets could be an effective strategy eliminating this cancer-causing population.

Abstract CT014: GSK525762, a selective bromodomain (BRD) and extra terminal protein (BET) inhibitor: results from part 1 of a phase I/II open-label single-agent study in patients with NUT midline carcinoma (NMC) and other cancers

Background: GSK525762 is a potent, selective pan-BET inhibitor that abrogates binding of BET family proteins (BRD2, BRD3, BRD4 and BRD-T) to acetylated histones. In pre-clinical models this results in suppression of BET target genes that drive oncogenic pathways, resulting in growth inhibition of cancer cell lines (median IC50 of 50-1698 nM for solid tumors and 50 nM for NMC). GSK525762 is being evaluated clinically for treatment of pts with hematologic malignancies and solid tumors including NMC, a rare, aggressive cancer with few treatment options. NMC is characterized by BRD3-/BRD4-NUT fusion oncoproteins that represents a rational therapeutic target for BET inhibitors. Methods: Primary objectives of Part 1 were to determine safety, tolerability, and maximum tolerated dose (MTD) for GSK525762 in solid tumors, using a combined N-CRM and 3+3 dose escalation. Secondary objectives include PK and PD analysis and preliminary evaluation of activity. Oral once daily (qd) and twice daily (bid) schedules have been evaluated. PD activity was assessed with [18F]-deoxy-glucose-PET scans and changes in cytokine release from LPS-stimulated PBMC of treated pts. The dose-limiting toxicity (DLT) observation period was 28 days. Response evaluation was by RECIST 1.1 and pts were followed to disease progression, discontinuation due to adverse events, withdrawal of consent or death. Results: As of 28-Jan 2016, a total of 70 (including 17 NMC) pts have been treated at doses of 2-100 mg qd and 20 and 30 mg bid. The most common adverse events (all grades, ?20%) observed were thrombocytopenia (44%), nausea (40%), vomiting (29%), anemia (26%), fatigue (26%), decreased appetite (24%), diarrhoea (23%) and dysgeusia (20%). DLTs occurred in 5 pts at doses of 60 mg (n = 1, 11%), 80 mg (n = 3, 14%) and 100 mg (n = 1, 11%). The most common AEs leading to dose interruption were thrombocytopenia and hyperbilirubinemia (each n = 7, 10%). Limited data shows similar tolerability at bid doses. PK was dose-proportional after single and repeat dosing with large between patient variability. Dose dependent inhibition of LPS stimulated BRD regulated cytokine (IL-6, TNF-α, MCP-1, IL-8) release suggests target engagement. Of 11 NMC pts treated at 60-100 mg qd, evaluation is awaited on 1 pt. Of 10 pts evaluated, 2 (20%; 95% CI (2.5%, 56%)) had confirmed PR (15 and 23 weeks) and 4 (40%; 95% CI (12%, 74%)) had SD. An 80mg once-daily dose will be evaluated in NMC and other solid tumor expansion cohorts. Conclusion: GSK525762 is an active, orally bio-available BET inhibitor showing dose-proportional PK and good tolerability up to 80 mg qd dosing. Preliminary evidence of clinical activity observed in pts with NMC provides a rational targeted therapy for this aggressive tumor and proof of concept for BET inhibitors in the clinic. Study funded by GSK (NCT01587703). Citation Format: Peter J. O’Dwyer, Sarina A. Piha-Paul, Christopher French, Sara Harward, Gerladine Ferron-Brady, Yuehui Wu, Olena Barbash, Anastasia Wyce, Meg Annan, Thierry Horner, Nigel J. Parr, Rab K. Prinjha, Christopher Carpenter, Geoffrey Shapiro, Arindam Dhar, Christine Hann. GSK525762, a selective bromodomain (BRD) and extra terminal protein (BET) inhibitor: results from part 1 of a phase I/II open-label single-agent study in patients with NUT midline carcinoma (NMC) and other cancers. [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 CT014.

Activation of the p53-MDM4 regulatory axis defines the anti-tumour response to PRMT5 inhibition through its role in regulating cellular splicing

Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.

MEK inhibitors overcome resistance to BET inhibition across a number of solid and hematologic cancers

BET inhibitors exhibit broad activity in cancer models, making predictive biomarkers challenging to define. Here we investigate the biomarkers of activity of the clinical BET inhibitor GSK525762 (I-BET; I-BET762) across cancer cell lines and demonstrate that KRAS mutations are novel resistance biomarkers. This finding led us to combine BET with RAS pathway inhibition using MEK inhibitors to overcome resistance, which resulted in synergistic effects on growth and survival in RAS pathway mutant models as well as a subset of cell lines lacking RAS pathway mutations. GSK525762 treatment up-regulated p-ERK1/2 levels in both RAS pathway wild-type and mutant cell lines, suggesting that MEK/ERK pathway activation may also be a mechanism of adaptive BET inhibitor resistance. Importantly, gene expression studies demonstrated that the BET/MEK combination uniquely sustains down-regulation of genes associated with mitosis, leading to prolonged growth arrest that is not observed with either single agent therapy. These studies highlight a potential to enhance the clinical benefit of BET and MEK inhibitors and provide a strong rationale for clinical evaluation of BET/MEK combination therapies in cancer.

Abstract CT038: A phase I, open-label, dose-escalation study to investigate the safety, pharmacokinetics, pharmacodynamics, and clinical activity of GSK3326595 in subjects with solid tumors and non-Hodgkin's lymphoma

Background Protein arginine methyltransferase 5 (PRMT5) is the primary enzyme responsible for symmetric arginine dimethylation of multiple proteins that impact cell proliferation. Its substrates include histones and proteins involved in signal transduction, gene transcription, DNA repair, and mRNA splicing. PRMT5 overexpression occurs in a number of different cancers, and higher expression is correlated with poor prognosis. Additional published data implicates PRMT5 in tumorigenesis, and as such it represents a novel target for therapeutic intervention in oncology. GSK3326595 is a potent, specific, and reversible inhibitor of PRMT5 that inhibits proliferation and induces cell death in a broad range of solid and hematologic tumor cell lines. It also exhibits potent antitumor activity in vivo in animal models. Methods Study 204653 is a Phase I, two-part, open-label, dose escalation/expansion study assessing the safety and tolerability of GSK3326595 in adult subjects with relapsed/refractory solid tumors and non-Hodgkin’s lymphoma. Dose escalation is being performed in subjects with solid tumors of any histology. An accelerated dose titration is employed with one subject per dose level until the occurrence of a ≥ Grade 2 non-disease related toxicity. Thereafter, subjects are enrolled in cohorts of approximately 3, and a modified toxicity probability interval (mTPI) method is used to guide dose escalation decisions. Dose escalation continues until the maximum tolerated dose (MTD) is identified. All data, including safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity, will be used to identify a recommended Phase 2 dose (RP2D). In Part 1, approximately 42 subjects are to be enrolled (30 subjects in dose escalation and an additional 12 subjects at or about the MTD to collect additional data, including PD and metabolites); no hypothesis will be tested, and all analysis will be descriptive and exploratory. In Part 2, the clinical activity of GSK3326595 will be evaluated in expansion cohorts of subjects with select tumor types. Based on preclinical data, enrollment is initially limited to subjects with triple-negative breast cancer (TNBC), metastatic bladder cancer (mBC), glioblastoma multiforme (GBM), and non-Hodgkin’s lymphoma (NHL); additional cohorts may be added based on emerging preclinical and clinical data. Two cohorts of NHL are scheduled, allocated by TP53 wild type versus mutant status. Up to 138 subjects may be enrolled in Part 2, and cohorts may be closed early for futility. As of 17 January 2017, recruitment is ongoing across four centers (USA, Canada, Netherlands, and France), and four subjects have been enrolled into the dose-escalation cohorts. ClinicalTrials.gov identifier: NCT02783300 Study is funded by GlaxoSmithKline Citation Format: Drew Rasco, Anthony Tolcher, Lillian L. Siu, Kimberley Heinhuis, Sophie Postel-Vinay, Olena Barbash, Jacqueline L. Egger, Shelby Gorman, Thierry Horner, Arindam Dhar, Brandon E. Kremer. A phase I, open-label, dose-escalation study to investigate the safety, pharmacokinetics, pharmacodynamics, and clinical activity of GSK3326595 in subjects with solid tumors and non-Hodgkin9s lymphoma [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 CT038. doi:10.1158/1538-7445.AM2017-CT038

Abstract 4693: Mechanism-based combination strategies for BET inhibitors in NUT midline carcinoma

NUT midline carcinoma (NMC) is a highly aggressive squamous cell cancer that responds poorly to standard chemotherapuetic approaches. NMC is characterized by translocations involving the NUT (nuclear protein in testes) protein, which in a majority of cases is fused to the BET (bromodomain and extra-terminal) protein family members BRD3 or BRD4. BET proteins (BRD2, BRD3, BRD4, BRDT) are epigenetic readers that modulate expression of genes involved in cell growth and oncogenesis. Selective small molecule inhibitors of BET proteins, such as the GSK I-BETs (I-BET762, I-BET151), abrogate binding of BET proteins to acetylated chromatin and inhibit the expression of BET target genes. Here we describe the activity in I-BET762 and other BET inhibitors in pre-clinical models of NMC. Consistent with previous reports, we observe profound growth inhibition and cytotoxicity in NMC cell lines in vitro, as well as significant tumor growth inhibition or tumor regression in cell line xenografts of NMC. I-BET762 treatment in NMC cell lines results in transcriptional changes affecting MYC and other pathways critical for cancer cell growth. We explore the contribution of these changes to the anti-proliferative effects observed in NMC models, and identify rational combinations to improve upon the efficacy of I-BET762 as a monotherapy. Taken together, our data highlight novel mechanisms through which BET inhibitors impact NMC cell growth and survival, and suggest potential treatment strategies to improve response in this highly aggressive disease. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Anastasia Wyce, Peter Soden, Daniel J. Felitsky, Jeanne J. Matteo, Susan Korenchuk, Gary Thripp, Kathryn Keenan, Charles F. McHugh, Rab Prinjha, Christopher Carpenter, Nicholas Smithers, Olena Barbash. Mechanism-based combination strategies for BET inhibitors in NUT midline carcinoma. [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 4693.