Discovery of a novel ALK/ROS1/FAK inhibitor, APG-2449, in preclinical non-small cell lung cancer and ovarian cancer models

Abstract

Background: Tyrosine kinase inhibitors (TKIs) are mainstays of cancer treatment. However, their clinical benefits are often constrained by acquired resistance. To overcome such outcomes, we have rationally engineered APG-2449 as a novel multikinase inhibitor that is highly potent against oncogenic alterations of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), and focal adhesion kinase (FAK). Here we present the preclinical evaluation of APG-2449, which exhibits antiproliferative activity in cells carrying ALK fusion or secondary mutations.

Methods: KINOMEscan® and LANCE TR-FRET were used to characterize targets and selectivity of APG-2449. Water-soluble tetrazolium salt (WST-8) viability assay and xenograft tumorigenicity were employed to evaluate therapeutic efficacy of monotherapy or drug combination in preclinical models of solid tumors. Western blot, pharmacokinetic, and flow cytometry analyses, as well as RNA sequencing were used to explore pharmacokinetic-pharmacodynamic correlations and the mechanism of actions driving drug combination synergy.

Results: In mice bearing wild-type or ALK/ROS1-mutant non-small-cell lung cancer (NSCLC), APG-2449 demonstrates potent antitumor activity, with correlations between pharmacokinetics and pharmacodynamics in vivo. Through FAK inhibition, APG-2449 sensitizes ovarian xenograft tumors to paclitaxel by reducing CD44+ and aldehyde dehydrogenase 1-positive (ALDH1+) cancer stem cell populations, including ovarian tumors insensitive to carboplatin. In epidermal growth factor receptor (EGFR)-mutated NSCLC xenograft models, APG-2449 enhances EGFR TKI-induced tumor growth inhibition, while the ternary combination of APG-2449 with EGFR (osimertinib) and mitogen-activated extracellular signal-regulated kinase (MEK; trametinib) inhibitors overcomes osimertinib resistance. Mechanistically, phosphorylation of ALK, ROS1, and FAK, as well as their downstream components, is effectively inhibited by APG-2449.

Conclusions: Taken together, our studies demonstrate that APG-2449 exerts potent and durable antitumor activity in human NSCLC and ovarian tumor models when administered alone or in combination with other therapies. A phase 1 clinical trial has been initiated to evaluate the safety and preliminary efficacy of APG-2449 in patients with advanced solid tumors, including ALK+ NSCLC refractory to earlier-generation ALK inhibitors.

Trial registration: Clinicaltrial.gov registration: NCT03917043 (date of first registration, 16/04/2019) and Chinese clinical trial registration: CTR20190468 (date of first registration, 09/04/2019).

Keywords: Anaplastic lymphoma kinase (ALK); Focal adhesion kinase (FAK); ROS proto-oncogene 1 receptor tyrosine kinase (ROS1); Solid tumors; Targeted therapies.

Conflict of interest statement

D. D. Fang, R. Tao, G. Wang, Y. Li, K. Zhang, C. Xu, G. Zhai, Q. Wang, J. Wang, C. Tang, P. Min, D. Xiong, J. Chen, D. Yang, and Y Zhai are full-time employees of Ascentage Pharma and equity shareholders of Ascentage Pharma Group International, the ultimate parent of Ascentage Pharma. S. Wang is a cofounder of Ascentage Pharma Group International, owns stock in the company, and receives grants and personal fees. He is a member of its board of directors, is its Chief Scientific Advisor, and is also a paid consultant. S. Wang and J. Chen hold an issued and licensed patent (US10709705B2) filed by the University of Michigan on APG-2449 and its analogs and receive royalties from the University of Michigan on this patent. The University of Michigan owns equity in, and has received research contracts from, affiliates of Ascentage Pharma for which S. Wang is the principal investigator. All other authors declare that they have no other competing interests.

© 2022. The Author(s).

Figures

Fig. 1

APG-2449 exhibits potent inhibitory activity against ALK/ROS1/FAK kinases and proliferation of relevant cell lines. A, Chemical structure of APG-2449. B, Dissociation constants (Kd values, nM) of APG-2449 calculated from KINOMEscan®. C, TREEspot interaction map of KINOMEscan selectivity panel for APG-2449 at 100 nM. Red spheres: kinases inhibited to < 35% of controls. Circle size reflects binding affinity. D, Inhibition of wild-type (wt) ALK and ALK mutants by APG-2449 and second-generation ALK inhibitors alectinib and ceritinib, using LANCE TR-FRET assay (IC50). E, Antiproliferative activity of APG-2449 (log IC50) in a panel of cancer cell lines. Data represent the mean of at least 2 independent experiments performed with triplicates. LANCE TR-FRET, lanthanide chelate excite time-resolved fluorescence resonance energy transfer

Fig. 2

APG-2449 demonstrates antitumor activity and pharmacokinetic/pharmacodynamic correlation in ALK- or ROS1-positive xenografts in vivo. Antitumor activity of APG-2449 was evaluated in mice bearing subcutaneous tumors derived from H3122 NSCLC cells carrying EML4-ALK fusion gene (A; treated for 3 weeks, n = 7 per treatment group; T/C values were assessed on Day 21), KARPAS-299 cells carrying NPM-ALK fusion gene (B; treated for 3 weeks, n = 7 per treatment group; T/C values were assessed on Day 13), or Ba/F3 cells carrying CD74-ROS1 fusion gene (C; the control group was treated for 2 weeks and the other groups for 3 weeks, n = 5 per treatment group; T/C values were assessed on Day 15). D, Pharmacokinetics of APG-2449 in plasma (left panel) or tumors (right panel) of mice carrying KARPAS-299-derived CDX tissues (treated once, n = 3 per treatment group). E, Western blot analysis of ALK signaling pathway in tumors collected 24 hours after administration from the same experiment as shown in (D). Each lane represents a tumor collected from an individual animal. F, Western blot analysis of ROS1 signaling pathway in HCC78 cells carrying SLC34A2-ROS1 fusion following treatments in vitro. Dimethyl sulfoxide (DMSO) was included as a control. EML4, echinoderm microtubule-associated protein-like 4; NPM, nucleophosmin; SLC34A2, solute carrier family 34 member 2

Fig. 3

Secondary ALK or ROS1 mutations conferring drug resistance are responsive to APG-2449 in vivo. Antitumor activity of APG-2449 against acquired, drug-resistant mutations was evaluated in subcutaneous xenograft models derived from Ba/F3 cells carrying EML4-ALKL1196M mutation (A; treated for 2 weeks, n = 6 per treatment group), Ba/F3 cells carrying EML4-ALKG1202R mutation (B; treated for 2 weeks, n = 5 per treatment group), Ba/F3 cells carrying CD74-ROS1L2026M mutation (C; treated for 3 weeks while 2 groups were terminated earlier as indicated, n = 4 or 5 per treatment group), NSCLC PDX LD1–0006-390,637 harboring ALKL1196M_G1202R double mutations (D; treated for 3 weeks, n = 5 per treatment group), or NSCLC PDX LU-01-0582R with acquired resistance to crizotinib (E; treated for 4 weeks, n = 3 or 4 per treatment group)

Fig. 4

Combination of APG-2449 and paclitaxel inhibits tumor growth of FAK-expressing ovarian cancer xenografts in mice. Western blotting of FAK signaling pathway in PA-1 ovarian cells treated with APG-2449 or defactinib for 4 hours in vitro (A). Efficacy studies in subcutaneous CDX models derived from PA-1 (B; treated for 3 weeks, n = 5 per treatment group) or OVCAR-3 (C; treated for 4 weeks, n = 5 per treatment group; T/C values assessed on Day 28) ovarian cancer cells. D, A mouse trial experiment was conducted in a panel of 6 ovarian cancer PDX models (treated for 3–6 weeks, n = 2 per treatment group). E, Heatmap of genes significantly changed in ovarian PDX tumors that responded to APG-2449 plus paclitaxel combination (synergy ratio > 2) versus nonresponders (synergy ratio < 2). p < 0.05 (false discovery rate < 0.05). F, Western blot analysis of FAK signaling pathways in tumor samples responsive (OV2018) and nonresponsive (OV1396) to APG-2449 plus paclitaxel treatment; tumor samples collected 4 hours after the last administration from the experimental animals shown in panel D. G, IHC staining of CD44- or ALDH1A1-positive cells in OVCAR-3 tumors collected 4 hours after the last treatment from tumor-bearing mice treated with APG-2449, paclitaxel or the combination for 10 days. H, SKOV-3 cells were treated with increasing doses of APG-2449 for 72 hours. I, SKOV-3 cells were treated with APG-2449 (1 μM), paclitaxel (5 nM), or the combination for 72 hours. CD44+ and ALDH1A+ cells were determined by flow cytometry. ***p < 0.001, **p < 0.01 vs. DMSO controls. Date presented are representative of 2 independent experiments as the mean ± SEM of triplicate biological replicates

Fig. 5

APG-2449 synergizes with EGFR inhibitors and overcomes osimertinib-resistance when combined with osimertinib/trametinib in xenograft models. Combination treatments with APG-2449 and EGFR inhibitors were evaluated in an HCC827 CDX model (A; treated for 3 weeks, n = 5 per treatment group), NCI-H1975 CDX model (B; treated for 3 weeks, n = 5 per treatment group), and NSCLC PDX model LD1–0006-215,676 harboring EGFRL858R_T790M and ROS1 fusion (C; treated for 61 days, n = 6 per treatment group) in mice. D, Tumor growth curve of NCI-H1975 CDX model rechallenged with APG-2449 plus osimertinib (Day 47- Day 67) upon disease progression from 21-day osimertinib treatment (Day 1-Day 21). E, Western blot analysis of EGFR and FAK downstream signaling in NCI-H1975 xenografts collected 4 hours after the last administration from tumor-bearing mice treated with indicated compounds for 2 weeks. Each lane represents an individual animal. F, Cell viability (IC50) of PC-9 (parental) or PC-9/OR (osimertinib-resistant) cells treated with osimertinib at indicated concentrations for 3 days. Data are representative of 3 independent experiments and shown as the mean ± SEM of triplicates. ****p < 0.0001. G, Cell viability of PC-9/OR cells treated with indicated compounds for 72 hours. H, PC-9/OR xenograft models were treated with the indicated compounds for 3 weeks (n = 5 per treatment group)

Fig. 6

Scheme for the mechanisms of action of APG-2449. Created with BioRender (Toronto, Ontario, Canada). CSC, cancer stem cell; EML, echinoderm microtubule-associated protein-like; MEK-ERK, mitogen-activated protein kinase kinase-extracellular signal-regulated kinase; PI3K-AKT, phosphatidylinositol 3-kinase and protein kinase B; SHP, SH2 containing protein tyrosine phosphatase; STAT, signal transducer and activator of transcription