Poster Session – Epigenetic modulators, Wednesday 29 November 2016 Poster abstracts S89 Results: Resminostat exhibited a dose-dependent inhibition of the viability of MyLa and HuT78 cells. High-throughput bead-based ELISA revealed the hyperacetylation of various histone H3 residues upon treating the SzS cells with resminostat. Moreover, the epigenetic modulating small molecule induced apoptosis while only marginally affecting the cell cycle in these cell lines. Treatment of MyLa cells with resminostat reversed the anomalous STAT expression, namely inducing an increase in STAT4 and a decrease in STAT6 expression. Conclusion: Resminostat displayed conclusive in vitro anti-tumor activities both in MF and SzS cells. The regulation of the aberrant STAT signaling on transcription level suggests a stabilization of the less advanced CTCL stage (Th1) or even a reconversion of the advanced (Th2) to the less advanced Th1 phenotype. Hence, resminostat represents a promising treatment option for indolent as well as aggressive CTCL subtypes, possibly as maintenance therapy. In upcoming functional assays, the impact of resminostat on the immunopathogenesis of CTCL will be analyzed and the anti-tumoral effects confirmed in an in vivo model. Conflict of interest: Other Substantive Relationships: The authors are employees of 4SC and share. D. Vitt is CSO and CDO of 4SC. 266 Poster (Board P092) Phosphorylation regulates EZH2 neoplastic functions in triple-negative breast cancer T. Anwar 1 , C. Kleer 1 . 1 University of Michigan Medical School, Department of Pathology, Ann Arbor, USA Background: Triple negative (estrogen receptor, progesterone receptor, HER2-neu negative) breast cancers (TNBC) comprise 15% of all breast cancers but are responsible for a disproportionately high number of deaths. Overexpression of the histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is an independent prognostic biomarker significantly associated with poorly-differentiated TNBCs and poor patient outcome. We previously identified a novel link between EZH2 and the p38 mitogen-activated protein kinase, an important mediator of progression and metastasis of TNBC. We found that EZH2 binds to p38, and that EZH2 and activated p38 were concordantly expressed in the metastases of breast cancer patients. Based on these data and previous in vitro studies, we hypothesized that p38 MAPK may also regulate EZH2 through phosphorylation in breast cancer. We further hypothesized that this phosphorylation event may be important for EZH2 contribution to malignancy. Methods: In order to test this hypothesis, we performed knockdown rescue experiments in triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-436. Stable knockdown of EZH2 was achieved using shRNA targeting the 3’UTR. Knockdown of EZH2 was rescued by reintroduction of myc-EZH2 (WT) or a T367A-EZH2 phosphorylation-deficient mutant. Cell lines were then used in functional assays of proliferation, migration, and invasion in vitro. Cells were also used in a murine orthotopic xenograft model in vivo and primary tumor and metastatic growth was monitored by bioluminescence. In order to further interrogate the importance of this phosphorylation event, we developed a phospho-specific EZH2 T367 (pEZH2) antibody and examined the utility of this antibody as a biomarker. Results: p38-mediated phosphorylation of EZH2 at T367 contributes to the migratory and invasive properties of TNBC in vitro. Phosphorylation at T367 was also associated with reduced time to metastasis in vivo. Mechanistically, phosphorylation by p38 does not affect binding to other PRC2 members but may affect EZH2 catalytic activity. In clinical specimens, pEZH2 was upregulated in metastases of breast cancer when compared to matched primary tumors from the same patient. Conclusions: p38-mediated phosphorylation of EZH2 at T367 contributes to malignancy of triple-negative breast cancers. Our data suggest a new mechanism by which EZH2 is regulated and may offer an additional mechanism by which EZH2 contributes to TNBC progression. No conflict of interest. 267 Poster (Board P093) Combination of the novel BET inhibitor BI 894999 with CDK9 inhibition suggests a promising regimen for the treatment of AML U. Tontsch-Grunt 1 , F. Savarese 1 , A. Baum 1 , D. Scharn 1 , D. Gerlach 1 , M.H. Hofmann 1 , J. Popow 1 , N. Schweifer 1 , H. Engelhardt 1 , H. Musa 2 , C.P. Lee 3 , G.M. Munzert 4 , N. Kraut 1 . 1 Boehringer Ingelheim RCV GmbH & Co KG, Research Oncology, Vienna, Austria; 2 Boehringer Ingelheim Pharma GmbH & Co KG, Transl. Medicine and Clin. Pharmacology, Biberach, Germany; 3 Boehringer Ingelheim Ltd. Bracknell, Medicine TA Oncology, Berkshire, United Kingdom; 4 Boehringer Ingelheim Pharma GmbH & Co KG, Medicine TA Oncology, Biberach, Germany Background: Bromodomain and extra-terminal (BET) protein inhibitors comprising the family members BRD2, 3, 4 and T are being extensively studied as treatment options in human hematological malignancies as well as in solid cancers. BRD4 is a key epigenetic regulator of transcriptional elongation, particularly of oncogenes including MYC. BI 894999 shows remarkable synergy with CDK9 inhibition in vitro and in xenograft models. BI 894999 is a novel, potent and selective orally bioavailable inhibitor of the BET family. This compound is in clinical trials (NCT02516553). Material and Methods: We studied the effect of BI 894999 in vitro (cell cycle by FACS, proliferation assays and apoptosis by IncuCyte ZOOM ® live cell imaging) and in vivo in xenografts for hematological malignancies. Likewise, combinations with CDK9 inhibitors were investigated in vitro and in vivo. Results: BI 894999 is a highly potent drug, particularly in hematological cell lines. Proliferation of AML, MM, CML, T-ALL, B and T cell lymphoma cell lines are inhibited at an EC 50 <100nM, mostly in the single digit nM range. Compound profiling using the BROMOscan™ assay revealed high selectivity for BRD2, 3, 4 and BRDT. Potent tumor growth inhibition of this compound was demonstrated in xenograft models of AML (MV- 4−11, THP-1, and MOLM13) as monotherapy or in combination with CDK9 inhibitors Alvocidib and LDC000067. The synergistic effect is not limited to AML lines with MLL translocations, but holds true for AML cell lines with other genetic modifications as well. In addition, CDK9 pathway modulation was investigated. Combination of the BET inhibitor BI 894999 and CDK9i results in a massive reduction of MYC expression and is proposed as the main driver for the excellent efficacy of this combination leading to tumor regression. Extended PK/PD analyses verified HEXIM1, a negative regulator of p-TEFb, as an excellent PD biomarker, which is robustly induced by BI 894999 and which is utilized in clinical trials. Conclusions: BET bromodomain inhibitor BI 894999 shows potent antiproliferative effect in hematological cell lines, primary human malignant cells and in in vivo tumor models. Synergy with CDK9i leads to remarkable tumor regressions. HEXIM1 is described as a robust PD biomarker. No conflict of interest. 268 Poster (Board P094) ODM-207, a novel BET-bromodomain inhibitor as a therapeutic approach for the treatment of patients with castration resistant prostate cancer M. Bj¨ orkman 1 , E. Mattila 1 , R. Riikonen 1 , C. Sekhar 2 , M. Jaleel 2 , S. Marappan 2 , T. Ikonen 1 , D. Nicorici 1 , S. Samajdar 2 , M. Ramachandra 2 , P. Kallio 1 , A. Moilanen 1 . 1 Orion Corporation Orion Pharma, Research and Development, Espoo, Finland; 2 Aurigene Discovery Technologies Limited, Discovery, Bangalore, India Background: Acetylation of lysine residues on amino-terminal tails of histones has been described to be a hallmark of open chromatin and transcriptional activation, and deregulation of histone acetylation patterns has been linked to aberrant expression of oncogenes resulting in proliferation and tumourigenesis. BET family proteins (Brd2, Brd3, Brd4, and BrdT) bind to acetylated-lysine residues in histones and facilitate the recruitment of transcriptional proteins to chromatin. Pharmacologic inhibition of BET proteins can block cell proliferation in various types of cancer. In many tumour types, the efficacy of BET inhibitors has been attributed to the transcriptional suppression of genes like the MYC. In this study, we evaluated the prostate cancer antitumor activity of ODM-207, a novel, potent and highly selective BET bromodomain inhibitor usin cell lines. Material and Methods: Prostate cancer cell lines (VCaP, LNCaP, 22Rv1 and enzalutamide-resistant subline of VCaP) were screened for sensitivity to ODM-207 in a 4-day growth inhibition assay. The ability of ODM- 207 to downregulate Myc expression was studied in prostate cell lines by immunolabeling Myc in cells. For gene expression analyses, prostate cancer cells were treated with ODM-207 or vehicle and differentially expressed genes were analysed by RNA-sequencing. In the 22Rv1 prostate cancer xenograft model, tumours were established by s.c. inoculation of 22Rv1 cells in male nude mice. Oral treatments with ODM- 207 or vehicle control were started when mean tumour size was 122 mm 3 . Results: ODM-207 dose-dependently suppressed cell proliferation of prostate cancer cells expressing androgen receptor (AR). Interestingly, ODM-207 also suppresses proliferation enzalutamide-resistant prostate cancer cell lines. Mechanistically, treatment with ODM-207 caused robust transcriptional changes of genes involved in e.g. Myc and AR- dependent transcription without effect on AR protein expression. The potent antiproliferative effects of ODM-207 were associated with cellular senescence. In 22Rv1 prostate cancer xenograft, which expresses both the full-length AR and AR splice variant V7, oral administration of ODM- 207 was efficacious in suppressing tumour growth. In this model, ODM-207 inhibits tumour growth at a dose which also inhibits tumour Myc expression. The observed inhibition of tumour Myc levels correlates with the plasma drug exposures. These results indicate that ODM-207 is able to modulate the expression of BET protein downstream targets in the tumours and that sufficient tumour concentrations can be achieved in vivo.