Characterization of ERK Docking Domain Inhibitors that Induce Apoptosis by Targeting Rsk-1 and Caspase-9

<p>Abstract</p> <p>Background</p> <p>The extracellular signal-regulated kinase-1 and 2 (ERK1/2) proteins play an important role in cancer cell proliferation and survival. ERK1/2 proteins also are important for normal cell functions. Thus, anti-cancer therapies that block all ERK1/2 signaling may result in undesirable toxicity to normal cells. As an alternative, we have used computational and biological approaches to identify low-molecular weight compounds that have the potential to interact with unique ERK1/2 docking sites and selectively inhibit interactions with substrates involved in promoting cell proliferation.</p> <p>Methods</p> <p>Colony formation and water soluble tetrazolium salt (WST) assays were used to determine the effects of test compounds on cell proliferation. Changes in phosphorylation and protein expression in response to test compound treatment were examined by immunoblotting and <it>in vitro </it>kinase assays. Apoptosis was determined with immunoblotting and caspase activity assays.</p> <p>Results</p> <p><it>In silico </it>modeling was used to identify compounds that were structurally similar to a previously identified parent compound, called <b>76</b>. From this screen, several compounds, termed <b>76.2</b>, <b>76.3</b>, and <b>76.4 </b>sharing a common thiazolidinedione core with an aminoethyl side group, inhibited proliferation and induced apoptosis of HeLa cells. However, the active compounds were less effective in inhibiting proliferation or inducing apoptosis in non-transformed epithelial cells. Induction of HeLa cell apoptosis appeared to be through intrinsic mechanisms involving caspase-9 activation and decreased phosphorylation of the pro-apoptotic Bad protein. Cell-based and <it>in vitro </it>kinase assays indicated that compounds <b>76.3 </b>and <b>76.4 </b>directly inhibited ERK-mediated phosphorylation of caspase-9 and the p90Rsk-1 kinase, which phosphorylates and inhibits Bad, more effectively than the parent compound <b>76</b>. Further examination of the test compound's mechanism of action showed little effects on related MAP kinases or other cell survival proteins.</p> <p>Conclusion</p> <p>These findings support the identification of a class of ERK-targeted molecules that can induce apoptosis in transformed cells by inhibiting ERK-mediated phosphorylation and inactivation of pro-apoptotic proteins.</p

Abstract PD18-11: Dose-Expansion Study of Trastuzumab Deruxtecan as Monotherapy or Combined With Pertuzumab in Patients With Metastatic Human Epidermal Growth Factor Receptor 2-Positive (HER2+) Breast Cancer in DESTINY-Breast07 (DB-07)

Abstract Background: In trials of HER2+ metastatic breast cancer (mBC), trastuzumab deruxtecan (T-DXd) monotherapy showed durable efficacy (DESTINY-Breast01) and significantly prolonged progression-free survival vs trastuzumab emtansine (DESTINY-Breast03). T-DXd is approved in the US for patients with HER2+ unresectable/mBC who received ≥1 prior anti-HER2–based treatment (tx) in the metastatic or neo-/adjuvant setting and recommended for approval in the EU as 2nd-line tx. Preclinical data suggest that T-DXd used in combination with other anticancer tx may lead to improved efficacy. The purpose of DB-07 is to assess the safety and efficacy of T-DXd alone or with other anticancer tx for patients with HER2+ mBC. Here we report preliminary data from the DB-07 dose-expansion phase for T-DXd monotherapy and T-DXd + pertuzumab (P) as 1st-line (1L) tx in mBC. Methods: DB-07 (NCT04538742) is an ongoing, phase 1b/2, 2-part (part 1: dose finding; part 2: dose expansion), modular, open-label trial of T-DXd alone or with other anticancer tx in patients with HER2+ mBC. In part 2, patients in module (mod) 0 received T-DXd 5.4 mg/kg every 3 weeks (Q3W) and in mod 2, T-DXd 5.4 mg/kg + P 420 mg Q3W (loading dose: 840 mg), the recommended phase 2 dose. Patients in these mods must be mBC tx naive. For part 2, the primary objective is to assess safety and tolerability. A secondary objective is to assess the objective response rate (ORR) per local investigators by Response Evaluation Criteria In Solid Tumors v1.1. We report results for patients randomized before Oct 13, 2021 to mods 0 and 2 of part 2 (data cutoff [DCO]: Mar 4, 2022); recruitment is ongoing. Based on the distinct mechanism of action of T-DXd and P, we conducted preclinical studies with the drugs in HER2-overexpressing cell lines to elucidate their potential synergies. To assess the effects on T-DXd internalization, live cell imaging was performed using pH-dependent fluorescently labeled T-DXd. To assess the effects on HER2 signaling, total and p-HER2 levels and downstream substrates were evaluated by immunoblot. Results: 23 patients were enrolled in the T-DXd monotherapy mod; 20 (87.0%) were receiving tx and 3 (13.0%) discontinued tx (withdrawal by patient, n=2; adverse event [AE], n=1) by DCO. 22 patients were enrolled in the T-DXd + P mod; 20 (90.9%) were receiving tx and 2 (9.1%) discontinued tx (AE, n=1; disease progression, n=1) by DCO. All patients experienced AEs (Table); 1 patient in each mod died. The unconfirmed ORR (80% CI) with T-DXd monotherapy and T-DXd + P was 82.6% (68.2%-92.2%) and 77.3% (61.9%-88.5%), respectively; updated data will be presented. Preclinical studies showed that T-DXd was more rapidly and effectively internalized in combination with P than when administered alone. Immunoblotting of cell lysates showed a greater reduction in total HER2 and HER2 signaling in response to combination tx than with T-DXd or P alone. Discussion: In summary, 1L T-DXd monotherapy and T-DXd + P safety profiles and antitumor activity were consistent with those previously reported for T-DXd. Mature data in these mods are awaited, and other T-DXd combinations are being investigated in additional mods. Preclinical studies showed the potential for P to induce greater internalization of T-DXd and inhibition of HER2-driven signaling. These results support investigation of T-DXd in larger ongoing trials (eg, NCT04784715). Table 1: Summary of treatment duration and safety data Citation Format: Erika Hamilton, Komal Jhaveri, Sherene Loi, Carey Anders, Peter Schmid, Konstantin Penkov, Elena Artamonova, Lyudmila Zhukova, Daniil L. Stroyakovsky, Dinesh Chandra Doval, Rafael Villanueva, Flavia Michelini, Sarat Chandarlapaty, Matt Wilson, Sarice R. Boston, Adam Konpa, Shoubhik Mondal, Fabrice Andre. Dose-Expansion Study of Trastuzumab Deruxtecan as Monotherapy or Combined With Pertuzumab in Patients With Metastatic Human Epidermal Growth Factor Receptor 2-Positive (HER2+) Breast Cancer in DESTINY-Breast07 (DB-07) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD18-11.</jats:p