Search

Supplementary Methods and Materials from Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

Supplementary Figure 2 from Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of γ-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way. [Cancer Res 2009;69(19):7672–80]

Myc transcription factors are well-established drivers of human cancers. However, despite being amongst the most frequently mutated, translocated and overexpressed oncogenes, no therapy targeting the Myc family members directly has been developed to date. To sustain uncontrolled cell proliferation and tumor growth, Myc-driven cancers are known to be addicted to protein translation. This addiction creates a dependency on critical components of the translational machinery providing in turn a unique opportunity for therapeutic intervention. We hypothesized that targeting the translational termination factor GSPT1, a key regulator of protein synthesis, would constitute a vulnerability for Myc-driven tumors. GSPT1 contains a well-defined degron allowing for the recruitment of the E3 ligase cereblon (CRBN) and subsequent proteasomal degradation in the presence of molecular glue degraders. Herein we describe a novel orally bioavailable GSPT1-directed small molecule degrader MRT-2359, which has been rationally designed and optimized to selectively induce apoptosis in translationally addicted cells. MRT-2359 promotes complex formation between CRBN and GSPT1 and potently induces GSPT1 degradation in a CRBN- and degron-dependent manner. The high selectivity of MRT-2359 was subsequently demonstrated by the lack of activity in cells expressing a non-degradable GSPT1 mutant. Although MRT-2359 degrades GSPT1 in all the cell lines tested, profiling in a large panel of cancer lines revealed profound and preferential antiproliferative activity in Myc-driven cell lines, such as high N-Myc expressing non-small cell lung cancer (NSCLC) lines and high L-Myc expressing small cell lung cancer (SCLC) lines. In the Myc-driven cells, degradation of GSPT1 led to translational repression as manifested by a global shift from polysomes to monosomes resulting in the reduction of a subset of proteins as assessed by quantitative proteomics. In particular, N- or L-Myc protein levels decreased and as a consequence the known Myc target genes were downregulated at the mRNA level. Despite the robust degradation of GSPT1, no marked effect was observed in low N-Myc lines, confirming the selective activity of our GSPT1 degrader in Myc-driven lung cancers. Finally, oral administration of MRT-2359 in high N-Myc NSCLC xenografts and PDXs led to complete intratumoral GSPT1 degradation and concomitant decrease in N-Myc protein levels, resulting in tumor regression. In contrast, MRT-2359 had limited or no activity in low N-Myc NSCLC models, further corroborating the selective vulnerability of Myc-driven tumors to GSPT1 degradation. Together these data support the therapeutic potential of GSPT1-directed MGDs in Myc-driven solid tumors addicted to the protein translation machinery and warrant rapid evaluation towards the clinic. Citation Format: Gerald Gavory, Mahmoud Ghandi, Anne-Cecile d’Alessandro, Debora Bonenfant, Agustin Chicas, Frederic Delobel, Brad Demarco, Alexander Flohr, Christopher King, Anne-Laure Laine, Vittoria Massafra, Rajiv Narayan, Arnaud Osmont, Giorgio Ottaviani, Dave Peck, Sarah Pessa, Nooreen Rubin, Thomas Ryckmans, Martin Schillo, Ambika Singh, Simone Tortoioli, Dominico Vigil, Vladislav Zarayskiy, John Castle, Filip Janku, Owen Wallace, Silvia Buonamici, Bernhard Fasching. Identification of MRT-2359 a potent, selective and orally bioavailable GSPT1-directed molecular glue degrader (MGD) for the treatment of cancers with Myc-induced translational addiction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3929.

The Myc family of transcription factors is a well-established driver of human cancers. However, despite being amongst the most frequently mutated, translocated and overexpressed oncogenes, no therapy directly targeting the Myc family members has been developed to date. Abnormal activation of Myc results in uncontrolled cell growth that is associated with high translational output and ramp up of the protein translational machinery. This creates a dependency to protein translation and in turn represents a potential therapeutic vulnerability for Myc-driven tumors. Based on these considerations, we hypothesized that targeting the translational termination factor GSPT1, a key player of protein synthesis, may constitute a vulnerability for Myc-driven tumors. Using our proprietary Quantitative and Engineered Elimination of Neosubstrates (QuEENTM) platform we characterized and explored the known G-loop degron in GSPT1 that renders it amenable to cereblon-induced degradation by molecular glue degraders (MGDs). We rationally designed and subsequently screened a proprietary library of cereblon-binding small molecules, including GSPT1-directed MGDs, in human mammary epithelial cells (HMECs) expressing doxycycline-inducible c-Myc. Doxycycline treatment led to sustained c-Myc expression and as a consequence to the induction of key biomarkers of enhanced protein translation, such as phospho 4EBP1 (p4EBP1). We identified MRT-048 as a potent and highly selective GSPT1 degrader and demonstrated its ability to induce cell death in Myc-driven HMEC cells whilst sparing control cells (EC50 0.64 μM vs 30 μM respectively). This confirmed the selective vulnerability of Myc-driven cell growth to GSPT1 degradation. In follow-up studies, we confirmed the correlation between p4EBP1 as biomarker of Myc-activation and sensitivity to MRT-048 in a large panel of breast cancer cell lines. Moreover, MRT-048 treatment of animals xenografted with breast cancer cells induced tumor regression and was associated with complete GSPT1 degradation. Mechanistically, we observed that GSPT1 degradation induced by MRT-048 led to inhibition of genes regulated by Myc and ribosomal stalling at stop codons of several mRNAs. Additionally, polysome profiling of cancer cells treated with MRT-048 was associated with a global reduction of the intensities of the polysome peaks and concomitant increase in the monosome peaks as previously observed in GSPT1 knockdown experiments, suggesting that GSPT1 degradation by our MGD molecules affects both the termination and initiation stages of protein translation. We believe these data support the therapeutic potential of GSPT1-directed MGDs in Myc-driven tumors dependent on protein translation machinery. Citation Format: Gerald Gavory, Bernhard Fasching, Debora Bonenfant, Amine Sadok, Ambika Singh, Martin Schillo, Vittoria Massafra, Anne-Cecile d’Alessandro, John Castle, Mahmoud Ghandi, Agustin Chicas, Frederic Delobel, Alexander Flohr, Giorgio Ottaviani, Thomas Ryckmans, Anne-Laure Laine, Oliv Eidam, Hannah Wang, Ilona Bernett, Laura Chan, Chiara Gorrini, Theo Roumiliotis, Jyoti Choudhary, Yann-Vai LeBihan, Marc Cabry, Mark Stubbs, Rosemary Burke, Rob Van Montfort, John Caldwell, Rajesh Chopra, Ian Collins, Silvia Buonamici. Identification of GSPT1-directed molecular glue degrader (MGD) for the treatment of Myc-driven breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA004.

Oncogenic EGFR mutations occur in approximately 50% of glioblastomas (GBM) and largely reside in the extracellular domain. Prior attempts to reposition current generation EGFR inhibitors to treat GBM likely failed due to poor brain penetration and an inability to potently target the full spectrum of oncogenic mutations. As EGFR oncogenic mutations are found to be co-expressed in many GBMs, it is important that an inhibitor be broadly active against the entire family of relevant EGFR mutants. Additionally, a successful inhibitor would require a pharmacokinetic (PK) profile that allows for sufficient penetration of the blood brain barrier to elicit robust target engagement of the brain tumor. Using these design principles, we designed a series of highly potent molecules exemplified by BDTX-507. This molecule is an irreversible inhibitor of EGFR with antiproliferative IC50's less than 10 nM against the spectrum of GBM-relevant EGFR mutations. PK/PD studies demonstrated sustained pERK suppression exceeding 24 hours following a single QD dose. Furthermore, when dosed in GBM xenografts, including an intracranial Viii PDX (GBM6), robust tumor regressions and improved survival were observed. Emerging from this series were two advanced compounds, BDTX-700 and BDTX-1535, which also demonstrated potent inhibition of the GBM EGFR spectrum, selectivity v. wild-type EGFR, and an excellent CNS PK profile. BDTX-1535 is currently being evaluated in IND-enabling studies for future clinical evaluation in GBM patients. Citation Format: Matthew O'Connor, Matt Lucas, Darlene Romashko, Sara Rasmussen, Tai-An Lin, Nigel Waters, Raffaele Fiorenza, Iwona Wrona, Deborah Chen, Theodore Nicolaides, David R. Raleigh, Tomoko Ozawa, George Trainor, Luca Arista, Alexander Flohr, Giorgio Ottaviani, Chris Roberts, Elizabeth Buck. CNS penetrant, irreversible inhibitors potently inhibit the family of allosteric oncogenic EGFR mutants expressed in GBM and demonstrate efficacy in patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB140.

Allosteric oncogenic mutations occur outside the canonical ATP-binding site of EGFR and HER2, for which there are no approved single agent therapies that target this family. BDTX-189 is a potent, selective, irreversible inhibitor of the family of nearly 50 allosteric EGFR and HER2 mutant variants. The goal of this translational analysis was to predict the clinical pharmacokinetic (PK) profile of BDTX-189 utilizing in vitro data on the absorption, distribution and metabolism of BDTX-189 as well as in vivo PK data in preclinical species. The prospective PK modeling was conducted prior to initiation of a Phase 1/2 study, to provide predictions of clinical exposures and active dose range. A challenge in the design of irreversible inhibitors with optimal PK properties is the lack of reliable methods to predict their disposition and elimination in human. The PK of covalent drugs is often driven by extrahepatic elimination pathways, and therefore conventional approaches to predict human clearance using human hepatocytes or allometric scaling can lead to poor predictive accuracy. We employed a novel physiologically-based PK (PBPK) modeling strategy that accounted for compound-specific determinants of BDTX-189 metabolism and disposition. PK studies following intravenous (IV) and oral (PO) administration were conducted in preclinical species as well as in vitro studies to understand the ADME properties of BDTX-189. These preclinical data formed the basis of a PBPK modeling approach to predict the likely PK profile of BDTX-189 in human. The mechanistic assumptions used in the final models were able to recapitulate the observed animal PK after both IV and PO administration and thus predictions utilizing similar assumptions for human were considered plausible. Taken together with BDTX-189 exposure-response data in mouse models of anti-tumor efficacy, this enabled the prediction of potentially active dose levels in patients. Preclinical PBPK modeling indicated that BDTX-189 would be readily orally absorbed with a short elimination half-life (approximately 2 hours) while maintaining suppression of ErbB pathway biomarkers over the dosing interval, consistent with the irreversible mechanism of action and the desired ‘hit-and-run' PK/pharmacodynamic (PD) profile. Active dose levels in human were projected to be in the 400 - 800 mg QD range, based on the exposure - tumor growth inhibition relationship in multiple mouse PDX models harboring ErbB allosteric mutations. This study demonstrates that a PBPK modeling approach and an understanding of the determinants of clearance can provide an effective framework for preclinical-to-clinical translation. BDTX-189 is currently under clinical evaluation in the ongoing MasterKey-01 trial (NCT04209465), and clinical PK will be reported in due course. Citation Format: Giorgio Ottaviani, Matthew O'Connor, Alexander Flohr, Darlene Romashko, Alexander Mayweg, Elizabeth Buck, Nigel Waters. Prospective preclinical modeling to estimate clinical pharmacokinetics and doses of BDTX-189, an inhibitor of allosteric ErbB mutations in advanced solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB127.

Mutation of either the intracellular catalytic domain or the extracellular domain of the receptor for epidermal growth factor (EGFR) drives oncogenicity. Extracellular domain EGFR mutations are highly expressed in patients with glioblastoma. Despite clinical success with targeting EGFR catalytic site mutants, no drugs have proven effective in glioblastoma patients expressing extracellular EGFR mutations. Herein, we define the molecular mechanism for oncogenic activation of families of extracellular EGFR mutations and reveal how this mechanism renders current generation small molecule ATP-site inhibitors ineffective. We demonstrate that a group of the most commonly expressed extracellular domain EGFR mutants expressed in glioblastomas is activated by disulfide-bond mediated covalent homodimerization, collectively referred to as locked dimerization (LoDi-EGFR oncogenes). Strikingly, current generation small molecules binding to the active kinase conformation potently inhibit catalytic site mutants, but induce covalent dimerization and activate LoDi-EGFR receptors, manifesting in paradoxical acceleration of proliferation. These data demonstrate how the locked-dimer mechanism of EGFR oncogenesis has profound impact on the activity of small molecules acting at the distal catalytic site, providing further evidence for “inside-out” allosteric signaling in EGFR. This provides a mechanistic understanding for the failure of current generation EGFR inhibitors to effectively treat LoDi-EGFR mutants in GBM and sets guidelines for discovery of selective LoDi-EGFR inhibitors. Citation Format: Matthew O'Connor, Theodore Nicolaides, Jie Zhang, Alexander Flohr, Roberto Iacone, Alexander V. Mayweg, David M. Epstein, Elizabeth Buck. Epidermal growth factor receptor oncogenes expressed in glioblastoma are activated as covalent dimers and exhibit unique pharmacology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-111.