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MCL1 is an anti-apoptotic protein that is frequently amplified in cancer and confers resistance to current standard of care. Therefore, MCL1 is an attractive anti-cancer target. Here we describe ANJ810 as a novel, potent, and selective MCL1 inhibitor and its key design principle of a rapid systemic clearance to potentially minimize AUC-driven toxicities associated with MCL1 inhibition. ANJ810 induced rapid cell killing within 4 hours in vitro but in the same 4-hour window had no impact on cell viability or troponin I release in hiPSC-derived cardiomyocytes, even at supra-pharmacologic concentrations. In vivo ANJ810 induced efficacy in xenograft hematological and solid tumor models despite the short residence time of ANJ810 in plasma. In totality our data support the hypothesis that short-term inhibition of MCL1 with ANJ810 can induce cell apoptosis in tumor cells while maintaining an acceptable safety profile. We therefore intend to advance ANJ810 to clinical trials.

Aims Human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs. Methods and results We derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch. Conclusions Our study shows that (i) the tri-cellular cardiac microtissues promote post-natal SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B. Translational perspective The cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications.

MCL1 is an anti-apoptotic protein inhibiting cancer cell death. It is frequently amplified or overexpressed in cancer and confers resistance to relevant standard of care. Therefore, MCL1 is an attractive target to potentially re-sensitize tumors to conventional chemotherapy and targeted agents. Here we describe ANJ810 as a novel, potent, and selective MCL1 inhibitor which specifically induces apoptosis in cancer cells. ANJ810 potently and reversibly binds to the BH3-binding groove of human MCL1 (KD = 0.3 nM), thereby inhibiting the interaction with the BH3-only protein NOXA (IC50 = 0.4 nM). ANJ810 shows a 4-log-fold greater selectivity to MCL1 over other anti-apoptotic proteins like Bcl-xL or Bcl-2. In cells, ANJ810 treatment rapidly (< 15 min) disrupts the MCL1-BAK interaction with an IC50 < 10 nM, resulting in caspase-3 activation and cancer cell death. Knockout of BAK and BAX completely rescues cells from ANJ810 induced killing, indicating that initiation of cell death occurs through the intrinsic apoptotic pathway. Screening over 750 cell lines (PRISM), we found that ANJ810 induced anti-cancer activity in 222 cell lines with an IC50 < 1 μM across multiple solid and hematological cancers, including breast, lung, melanoma, sarcoma, lymphoma, and leukemia. The top genomic feature that correlates with sensitivity to ANJ810 treatment is the ratio of Bcl-xL/BAK expression. A key design principle of ANJ810 is its rapid systemic clearance to potentially minimize exposure-driven toxicities associated with MCL1 inhibition. ANJ810 induces efficient cancer cell killing within 4 hours in vitro but has no impact on cell viability or troponin I release in hiPSC-derived cardiomyocytes at supra-pharmacologic concentrations. In vivo, i.v. bolus injections of ANJ810 lead to short plasma residence time, yet are efficacious in xenograft models of multiple myeloma, DLBCL, NSCLC and HCC. ANJ810 will test the hypothesis in human clinical trials that short-term inhibition of MCL1 can overcome tumor resistance with an acceptable safety profile to improve current standard of care. Citation Format: Ulrike Rauh, Guo Wei, Michael Serrano-Wu, Georgios Kosmidis, Stefan Kaulfuss, Franziska Siegel, Kai Thede, James McFarland, Christopher Lemke, Nicolas Werbeck, Katrin Nowak-Reppel, Sabine Pillari, Stephan Menz, Matthias Ocker, Brian Hubbard, Virendar Kaushik, Karl Ziegelbauer, Todd Golub. ANJ810 is a highly selective novel MCL1 inhibitor with optimized in vivo clearance showing robust efficacy in preclinical solid and hematological tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 506.