Nanoformulation of BRD4-Degrading PROTAC: Improving Druggability To Target the ‘Undruggable’ MYC in Pancreatic Cancer
Tamara Minko
Abstract
In a recent study, Saraswat and colleagues identified a novel proteolysis targeting chimera (PROTAC), ARV-825 (ARV), that efficiently degrades bromodomain-containing protein 4 (BRD4) to drug the ‘undruggable’ MYC in pancreatic cancer. ARV-loaded polyethylene glycol–poly lactic acid-co-glycolic acid (PLGA–PEG) polymeric nanoparticles (ARV-NPs) showed promising anticancer activity in both 2D cell culture and 3D multicellular tumor spheroid models of pancreatic cancer. This study demonstrates a unique therapeutic strategy in which targeting BRD4 for degradation via the E3 ubiquitin ligase cereblon (CRBN) pathway leads to sustained inhibition of oncogenic MYC expression for effective treatment of pancreatic cancer. In a recent study, Saraswat and colleagues identified a novel proteolysis targeting chimera (PROTAC), ARV-825 (ARV), that efficiently degrades bromodomain-containing protein 4 (BRD4) to drug the ‘undruggable’ MYC in pancreatic cancer. ARV-loaded polyethylene glycol–poly lactic acid-co-glycolic acid (PLGA–PEG) polymeric nanoparticles (ARV-NPs) showed promising anticancer activity in both 2D cell culture and 3D multicellular tumor spheroid models of pancreatic cancer. This study demonstrates a unique therapeutic strategy in which targeting BRD4 for degradation via the E3 ubiquitin ligase cereblon (CRBN) pathway leads to sustained inhibition of oncogenic MYC expression for effective treatment of pancreatic cancer. Pancreatic cancer is a highly fatal human cancer. Mutation of the KRAS oncogene is the signature genetic event in pancreatic cancer that deregulates cell proliferation as well as apoptosis by activating several signaling pathways [1.Haeberle L. Esposito I. Pathology of pancreatic cancer.Transl. Gastroenterol. Hepatol. 2019; 4: 50Crossref PubMed Scopus (51) Google Scholar]. The prevalence of ‘undruggable’ KRAS drivers and signaling pathway activation contributes to KRAS-mediated drug resistance in pancreatic cancer [2.Fan Z. et al.Critical role of KRAS mutation in pancreatic ductal adenocarcinoma.Translat. Cancer Res. 2018; 7: 1728-1736Crossref Scopus (7) Google Scholar]. This eventually leads to expression of the oncogenic MYC gene, an essential downstream effector of KRAS in pancreatic cancer. The direct link between oncogenic MYC and KRAS drivers raises the possibility that inhibition of MYC expression could be an effective therapeutic strategy for KRAS-mutant tumors such as pancreatic cancer [3.Korc M. Beyond Kras: MYC rules in pancreatic cancer.Cell. Mol. Gastroenterol. Hepatol. 2018; 6: 223-224Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar]. Transcriptional coactivator BRD4 is a member of the bromodomain and extra-terminal domain (BET) family, and a driver of oncogenesis that activates MYC transcription; by destabilizing the MYC protein via phosphorylation, subsequently leading to ubiquitination and degradation, homeostatic levels of c-MYC protein are maintained. Small-molecule BET inhibitors such as JQ1 reduce MYC transcription but have no effect on MYC protein stability [4.Devaiah B.N. et al.MYC protein stability is negatively regulated by BRD4.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 13457-13467Crossref PubMed Scopus (25) Google Scholar]. Owing to the reversible nature of binding of JQ1 to BET, as well as its short half-life, there is a need to identify molecules that would directly degrade the target BRD4 protein instead of merely inhibiting it [5.Wroblewski M. et al.BET-inhibition by JQ1 promotes proliferation and self-renewal capacity of hematopoietic stem cells.Haematologica. 2018; 103: 939-948Crossref PubMed Scopus (15) Google Scholar,6.Maggisano V. et al.Nanoparticles loaded with the BET inhibitor JQ1 block the growth of triple negative breast cancer cells in vitro and in vivo.Cancers. 2019; 12: 91Crossref Scopus (11) Google Scholar]. PROTACs are engineered bifunctional molecules composed of a target-binding ligand joined via a linker to an effector ligand, pomalidomide, that binds to E3 ubiquitin ligase to trigger proteasomal degradation of the target protein [7.Sun X. et al.PROTACs: great opportunities for academia and industry.Signal Transduct. Target. Ther. 2019; 4: 64Crossref PubMed Scopus (162) Google Scholar,8.Otto C. et al.Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells.Neoplasia. 2019; 21: 1110-1120Crossref PubMed Scopus (15) Google Scholar]. The novel PROTAC ARV-825 (ARV) contains two ligands linked by an ethoxy spacer – (i) OTX015 (a thienodiazepine molecule and BRD4 ligand) that targets BRD4 protein, and (ii) phthalimide or pomalidomide that targets the E3 ubiquitin ligase cereblon (CRBN) (Figure 1, left panel). These dual ligands recruit BRD4 to CRBN, leading to rapid and prolonged BRD4 degradation, and therefore to sustained inhibition of MYC expression [9.Li X. Song Y. Proteolysis-targeting chimera (PROTAC) for targeted protein degradation and cancer therapy.J. Hematol. Oncol. 2020; 13: 50Crossref PubMed Scopus (51) Google Scholar]. A previous study by Rathod et al. reported encouraging anticancer activity of ARV in terms of its cytotoxicity, apoptotic, and antimetastatic activity against vemurafenib-resistant melanoma [10.Rathod D. et al.BRD4 PROTAC as a novel therapeutic approach for the treatment of vemurafenib resistant melanoma: preformulation studies, formulation development and in vitro evaluation.Eur. J. Pharm. Sci. 2019; 138105039Crossref PubMed Scopus (18) Google Scholar]. Thus, ARV represents a next-generation extension to JQ1-mediated BRD4 inhibition that acts as a powerful BRD4 protein degrader to drug the ‘undruggable’ MYC (Figure 1, left panel). Saraswat and colleagues recently reported that ARV-mediated degradation of BRD4 could potentially be used to treat pancreatic cancer. This was demonstrated in various 2D and 3D cell culture assays [11.Saraswat A. et al.Nanoformulation of proteolysis targeting chimera targeting 'undruggable' c-Myc for the treatment of pancreatic cancer.Nanomedicine. 2020; 15: 1761-1777Crossref PubMed Scopus (14) Google Scholar] where ARV showed substantial cytotoxic, apoptotic, and anticlonogenic activity in pancreatic cancer cells. Nevertheless, although such a molecule may show exceptional anticancer activity in vitro, for clinical success it must be deliverable to the tumor site. Because PROTACs are composed of two molecules (one for binding to the target protein and a second that recruits the E3 ubiquitin ligase machinery), their molecular weights and other physical properties differ from those of traditional protein-targeting small molecules. Indeed, PROTACs face problems of aqueous solubility, permeability, drug metabolism, and pharmacokinetics (Figure 1, right panel) [12.Wang P. Zhou J. Proteolysis targeting chimera (PROTAC): a paradigm-shifting approach in small molecule drug discovery.Curr. Top. Med. Chem. 2018; 18: 1354-1356Crossref PubMed Scopus (26) Google Scholar,13.Cantrill C. et al.Fundamental aspects of DMPK optimization of targeted protein degraders.Drug Discov. Today. 2020; 25: 969-982Crossref PubMed Scopus (26) Google Scholar], raising concerns regarding drug delivery. Indeed, the extremely poor aqueous solubility of ARV poses a significant challenge to the development of an intravenous formulation [10.Rathod D. et al.BRD4 PROTAC as a novel therapeutic approach for the treatment of vemurafenib resistant melanoma: preformulation studies, formulation development and in vitro evaluation.Eur. J. Pharm. Sci. 2019; 138105039Crossref PubMed Scopus (18) Google Scholar]. Moreover, Rathod et al. [10.Rathod D. et al.BRD4 PROTAC as a novel therapeutic approach for the treatment of vemurafenib resistant melanoma: preformulation studies, formulation development and in vitro evaluation.Eur. J. Pharm. Sci. 2019; 138105039Crossref PubMed Scopus (18) Google Scholar] and Saraswat et al. [11.Saraswat A. et al.Nanoformulation of proteolysis targeting chimera targeting 'undruggable' c-Myc for the treatment of pancreatic cancer.Nanomedicine. 2020; 15: 1761-1777Crossref PubMed Scopus (14) Google Scholar] demonstrated that ARV is rapidly metabolized by the drug-metabolizing enzyme CYP34A. Encapsulation of drug molecules within a polymeric matrix can result in an extended half-life compared with the free drug [14.Fam S.Y. et al.Stealth coating of nanoparticles in drug-delivery systems.Nanomaterials. 2020; 10: 787Crossref Scopus (84) Google Scholar], which would in turn increase their therapeutic efficacy. To avoid the delivery issues associated with ARV, Saraswat and colleagues formulated polymeric nanoparticles (ARV-NPs) by nanoprecipitation using a biodegradable PLGA–PEG matrix (Figure 1, right panel). This formulation would promote drug accumulation in the solid tumor via the enhanced permeability and retention (EPR) effect, and the PEG-rich surface would aid in bypassing the reticuloendothelial system (RES), thereby prolonging the half-life. In addition, encapsulation in PLGA–PEG nanoparticles was shown to prevent ARV degradation by microsomal enzymes in vitro and significantly prolonged its half-life. ARV-NPs also displayed physical stability, controlled in vitro drug release, and negligible in vitro hemolysis, supporting the authors’ hypothesis that, on intravenous administration, ARV-NPs are likely to reach the target tumor site where they would exert their anticancer effects. In support, in vitro cytotoxicity, migration, clonogenic, and apoptosis studies demonstrated that encapsulation in nanoparticles enhanced the anticancer activity of ARV. To further elucidate the mechanism underlying the activity of ARV against pancreatic cancer cells, Saraswat and colleagues performed western blot analysis and found that ARV and ARV-NPs significantly reduced the levels of target proteins including BRD4, MYC, and the anti-apoptotic protein B cell lymphoma 2 (BCL2), while increasing the levels of a proapoptotic marker, cleaved caspase-3. This suggested that ARV-NPs inhibit the proliferation of pancreatic cancer cells via BRD4 degradation, leading to inhibition of MYC expression. This finding is similar to a previous study by Piya et al. in which ARV reduced the levels of BRD4, MYC, and BCL2 proteins for the treatment of leukemia [15.Piya S. et al.BRD4 proteolysis targeting chimera (PROTAC) ARV-825 causes sustained degradation of BRD4 and modulation of chemokine receptors, cell adhesion and metabolic targets in leukemia resulting in profound anti-leukemic effects.Blood. 2016; 128748–748Crossref Google Scholar]. To mimic the cellular morphology and extracellular matrix interactions in the in vivo tumor microenvironment, Saraswat and colleagues developed 3D multicellular tumor spheroids (MCTS) of pancreatic tumor cells. Treatment of MCTSs with ARV and ARV-NPs induced significant apoptosis, leading to marked reduction in spheroid size, confirming their anticancer potential. Although the anticancer effects of ARV and ARV-NPs are likely to be mediated by BRD4 degradation and inhibition of MYC transcription, other pathways may also be targeted. Future investigations should study the effects of ARV on other cellular signaling pathways, including mitogen-activated protein kinases/extracellular signal-regulated kinases (MEKs/ERKs), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), phospholipase C (PLC), and Ras-like (RAL), that are affected in diverse types of cancers. In addition, it would be interesting to study the anticancer effects of ARV and ARV-NPs in KRAS-mutant cancers including pancreatic cancer. Given the promising anticancer activity of ARV-NPs in 2D and 3D cell culture models of pancreatic cancer in vitro, further studies will be necessary to evaluate their anticancer activity in vivo in genetically engineered mouse models (GEMMs) of pancreatic cancer. Overall, this study highlights the potential use of an innovative and selective BRD4 protein degrader molecule, ARV, which was designed using PROTAC technology to degrade BRD4 protein and thereby drug the ‘undruggable’ MYC oncogene in the treatment of pancreatic cancer. The ARV-loaded nanoformulation that was developed to improve the solubility, permeability, pharmacokinetics, and delivery of ARV has great translational potential for the treatment of drug-resistant and KRAS-mutant pancreatic cancers. T.M. is supported by National Institutes of Health (NIH) National Cancer Institute (NCI) grants R01 CA238871 and R01 CA209818 .