Proteolysis Targeting Chimeric Research Articles

299P - Activity of BET-proteolysis targeting chimeric (PROTAC) compounds in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is an incurable disease, therefore novel therapies are needed. Bromo and extraterminal domain inhibitors (BETi) are currently in clinical development with promising activity in TNBC. However, resistance to these agents will limit their efficacy. Proteolysis targeting chimeric (PROTAC) are novel compounds that by binding to a ubiquitin ligase promotes protein degradation. MethodsIn our study we explore the antitumoral activity of two BET-PROTACs, MZ1 and ARV-825 in TNBC and ovarian cancer. We used MDAMB-231, BT549 and MDAMB-231 JQ1 resistant TNBC cells lines; and OVCAR3 and SKOV3 ovarian cancer cells lines. We evaluated the antiproliferation effect using MTT assay, colony-forming assay and three-dimensional cultures in Matrigel. To explore the molecular mechanism of drugs we used flow cytometry (cell cycle and cell death). The analysis of protein expression was determined by western blot. In vivo studies included BALB/c nu/nu mice that were injected with MDAMB-231 JQ1 resistant cells and later treated with BETi JQ1 and BET-PROTAC MZ1. ResultsBoth PROTACs efficiently and maintained deleted the levels of BRD4 in MDA-MB-231 and in the resistant JQ1 cell line MDA-MB-231R. MZ1 and ARV-825 showed a profound antiproliferative effect in MDA-MD-231 sensitive and resistant cells using 2D and 3D cultures, and had a similar activity in other triple negative (BT549) and ovarian cancer (SKOV3, OVCAR) cell lines. MZ1 slightly arrested cells at G2/M in sensitive and resistant JQ1 MDA-MB-231 cells. In addition, a profound effect on apoptosis was observed, that was more evident in the sensitive cell line, effect that was caspase dependent. Given the potent antiproliferative activity of MZ1 no synergistic activity was observed when combined with docetaxel, cisplation or olaparib. Finally, administration of MZ1 was able to recue tumor growth in nude mice of MDA-MB-231 JQ1 resistant cells, by reducing the expression of BRD4. ConclusionsWe describe the profound activity of BET-PROTACs in TNBC cell lines and in an in vivo BETi resistant model. This data provides options for further clinical development of these agents in TNBC. Legal entity responsible for the studyThe authors. FundingInstituto de Salud Carlos III (PI16/01121), ACEPAIN; Diputación de Albacete, CIBERONC, CRIS Cancer Foundation, implementation research program of the UCLM System of Science, Technology and Innovation-Secti (co-funded by the European Commission/FSE funds) and scientific foundation of the AECC. DisclosureAll authors have declared no conflicts of interest.

Discovery of SIAIS178 as an Effective BCR-ABL Degrader by Recruiting Von Hippel–Lindau (VHL) E3 Ubiquitin Ligase

The oncogenic fusion protein BCR-ABL is the driving force of leukemogenesis in chronic myeloid leukemia (CML). Despite great progress for CML treatment through application of tyrosine kinase inhibitors (TKIs) against BCR-ABL, long-term drug administration and clinical resistance continue to be an issue. Herein, we described the design, synthesis, and evaluation of novel proteolysis-targeting chimeric (PROTAC) small molecules targeting BCR-ABL which connect dasatinib and VHL E3 ubiquitin ligase ligand by extensive optimization of linkers. Our efforts have yielded SIAIS178 (19), which induces proper interaction between BCR-ABL and VHL ligase leading to effective degradation of BCR-ABL protein, achieves significant growth inhibition of BCR-ABL+ leukemic cells in vitro, and induces substantial tumor regression against K562 xenograft tumors in vivo. In addition, SIAIS178 also degrades several clinically relevant resistance-conferring mutations. Our data indicate that SIAIS178 as efficacious BCR-ABL degrader warrants extensive further investigation for the treatment of BCR-ABL+ leukemia.

Activity of BET-proteolysis targeting chimeric (PROTAC) compounds in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is an incurable disease where novel therapeutic strategies are needed. Proteolysis targeting chimeric (PROTAC) are novel compounds that promote protein degradation by binding to an ubiquitin ligase. In this work, we explored the antitumoral activity of two novel BET-PROTACs, MZ1 and ARV-825, in TNBC, ovarian cancer and in a BET inhibitor resistant model.MethodsOVCAR3, SKOV3, BT549, MDA-MB-231 cell lines and the JQ1 resistant cell line MDA-MB-231R were evaluated. MTTs, colony-forming assay, three-dimensional cultures in matrigel, flow cytometry, and western blots were performed to explore the anti-proliferative effect and biochemical mechanism of action of MZ1 and ARV-825. In vivo studies included BALB/c nu/nu mice engrafted with MDA-MB-231R cells.ResultsThe BET-PROTACs MZ1 and ARV-825 efficiently downregulated the protein expression levels of the BET protein BRD4, in MDA-MB-231 and MDA-MB-231R. MZ1 and ARV-825 also showed an antiproliferative effect on sensitive and resistant cells. This effect was corroborated in other triple negative (BT549) and ovarian cancer (SKOV3, OVCAR3) cell lines. MZ1 provoked G2/M arrest in MDA-MB-231. In addition, a profound effect on caspase-dependent apoptosis was observed in both sensitive and resistant cells. No synergistic activity was observed when it was combined with docetaxel, cisplatin or olaparib. Finally, in vivo administration of MZ1 rescued tumor growth in a JQ1-resistant xenograft model, reducing the expression levels of BRD4.ConclusionsUsing both in vitro and in vivo approaches, we describe the profound activity of BET-PROTACs in parental and BETi-resistant TNBC models. This data provides options for further clinical development of these agents in TNBC.

BRD4 PROTAC as a novel therapeutic approach for the treatment of vemurafenib resistant melanoma: Preformulation studies, formulation development and in vitro evaluation

Limited therapeutic interventions and development of resistance to targeted therapy within few months of therapy pose a great challenge in the treatment of melanoma. Current work was aimed to investigate; (a) Anticancer activity of a novel class of compound - Bromodomain and Extra-Terminal motif (BET) protein degrader in sensitive and vemurafenib-resistant melanoma (b) Preformulation studies and formulation development. ARV-825 (ARV), a molecule designed using PROteolysis-TArgeting Chimeric (PROTAC) technology, degrades BRD4 protein instead of merely inhibiting it. Based on extensive preformulation studies, ARV loaded self-nanoemulsifying preconcentrate (ARV-SNEP) was developed and optimized. ARV showed extremely poor aqueous solubility (<7 μg/mL) and pH dependent hydrolytic degradation. CaCO-2 cell uptake assay and human liver microsome studies proved that ARV is a substrate of CYP3A4 but not of P-gp efflux pump. Optimized ARV-SNEP spontaneously formed nanoglobules of 45.02 nm with zeta potential of −3.78 mV and significantly enhanced solubility of ARV in various aqueous and bio-relevant media. Most importantly, ARV showed promising cytotoxicity, anti-migration and apoptotic activity against vemurafenib-resistant melanoma cells. ARV-SNEP could be potentially novel therapeutic approach for the treatment of drug-resistant melanoma. This is the very first paper investigating a PROTAC class of molecule for the treatment of drug resistant cancer, preformulation and formulation studies.

Abstract 1394: Resistance to BET inhibitor leads to new therapeutic vulnerabilities in castration resistant prostate cancer

Abstract BRD4 plays a major role in the transcription networks orchestrated by androgen receptor (AR) in castration resistant prostate cancer (CRPC) cells. Bromodomain and extraterminal protein (BET) inhibitors displace BRD4 protein from chromatin, resulting in the inhibition of oncogenic transcriptional programs. Several BET inhibitors (BETi) are in phase I clinical trials for a variety of malignancies including CRPC. Here we describe a general mechanism of acquired resistance to BETi due to modulation of cellular pathways that are amenable to targeted therapies in CRPC cells. BETi resistant CRPC cells displayed cross resistance to a variety of BETi in the absence of gatekeeper mutations or persistent drug pump activation. Resistant cells exhibited reduced chromatin bound BRD4, and were less sensitive to Proteolysis Targeting Chimeric (PROTAC) -mediated degradation or genetic knockdown, suggesting a BRD4-independent transcription program. Transcriptomic analysis revealed reactivation of AR-signaling due to CDK9-mediated serine-81 phosphorylation of AR, with a consequent increase in sensitivity to CDK9 inhibitors and anti-androgens in BETi resistant cells. Additionally, increased DNA damage associated with PRC2-mediated transcriptional silencing of DNA damage response (DDR) genes was observed due to the loss of BRD4 from their proximal promoter regions in the resistant cells, leading to PARP inhibitor sensitivity. Collectively, our results identify the therapeutic limitation of BETi as a monotherapy in CRPC. However, data showing the reactivation of AR-signaling and increased DNA damage in the BETi resistant cells provide unique opportunities for combination therapies in managing CRPC. Citation Format: Aishwarya S. Pawar, Irfan A. Asangani. Resistance to BET inhibitor leads to new therapeutic vulnerabilities in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1394.

Two-headed PROTAC: an effective new tool for targeted protein degradation.

Two-headed PROTAC: an effective new tool for targeted protein degradation.