Anaplastic lymphoma kinase (ALK) belongs to the family of receptor tyrosine kinases. Recently, the incidence of anaplastic large cell lymphoma (ALCL) with ALK rearrangement has raised considerably. The application of ALK-targeted inhibitors such as ceritinib provides an effective therapy for the treatment of ALK-positive cancers. However, with the prolongation of treatment time, the emergence of resistance is inevitable. We found that 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42), a novel ceritinib derivative, could inhibit the proliferation of ALK-positive ALCL cells, induce the apoptosis of Karpas299 cells through the mitochondrial pathway in a caspase-dependent manner. In addition, ZX-42 could suppress ALK and downstream pathways including PI3K/Akt, Erk and JAK3/STAT3 and reduce the nuclear translocation of NFκB by inhibiting TRAF2/IKK/IκB pathway. Taken together, our findings indicate that ZX-42 shows more effective activity than ceritinib against ALK-positive ALCL. We hope this study can provide a direction for the structural modification of ceritinib and lay the foundation for the further development of clinical research in ALK-positive ALCL.

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene occurs in approximately 5% of non-small-cell lung cancers (NSCLCs). The development of ALK tyrosine kinase inhibitors (ALK-TKIs) is a major advance in treating NSCLC with the ALK fusion gene. Nevertheless, acquired resistance to ALK-TKIs ultimately limits their use. A prevalent mechanism of drug resistance in kinases occurs through the mutation of G1202R in ALK. However, the mechanisms underlying G1202R resistance to ceritinib are not fully understood. Here, we demonstrated that the expression of EML4-ALK G1202R mutation in A549 cells induced an epithelial-mesenchymal transition (EMT) phenotype and significantly increased the migration and invasion abilities. These phenomena may be due to the upregulation of signal transducer and activator of transcription 3 (STAT3), accompanied by the elevated expression of Slug in EML4-ALK G1202R mutant cells. Furthermore, the combination of ALK and STAT3 inhibitors restored the sensitivity of EML4-ALK G1202R mutant cells to ceritinib. In conclusion, these data indicate that the EML4-ALK G1202R mutation mediates the EMT phenotype by activating the STAT3/Slug signaling pathway, resulting in resistance to ceritinib, and that the combination of STAT3 and ALK inhibitors may overcome ALK mutation-driven drug resistance in the clinic.

OBJECTIVES:To examine the antiproliferative effects of 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42) on the echinoderm microtubule-associated protein-4/anaplastic lymphoma kinase fusion gene (EML4-ALK) positive lung cancer cell line H2228 and its underlying mechanism.METHODS:The MTT assay was used to study the effect of ZX-42 on H2228 cell growth. Propidium iodide (PI) staining and Western blotting were used to investigate the cell cycle changes. ZX-42-induced cell apoptosis was determined using the Annexin V-FITC/PI (AV/PI) apoptotic assay kit, acridine orange/ethidium bromide (AO/EB) and Hoechst 33258 staining, Rhodamine 123 (Rh 123) fluorescence assay and Western blotting. ZX-42-induced reactive oxygen species (ROS) production was examined by ROS assay kit. Transmission electron microscope, monodansylcadaverine (MDC) staining and the AV/PI apoptotic assay kit were used to demonstrate the relationship between autophagy and apoptosis.KEY FINDINGS:ZX-42 had good cell viability inhibitory effect on H2228 cells. ZX-42 dramatically inhibited ALK and its downstream pathways. ZX-42 also blocked H2228 cell cycle at G1 phase and then induced apoptosis by activating the mitochondrial pathway. Next, ZX-42 induced the production of ROS, and antioxidant N-acetylcysteine (NAC) reduced ROS production and also decreased apoptotic rates. We also found that ZX-42 induced protective autophagy in H2228 cells.CONCLUSIONS:In summary, ZX-42 is a novel ALK inhibitor that significantly inhibits the cell viability of H2228 cells and ultimately induces apoptosis through the mitochondrial pathway, in which autophagy plays a protective role. Therefore, inhibition of autophagy might enhance the anti-cancer effect of ZX-42.

In recent years, anaplastic lymphoma kinase (ALK) rearrangement-positive anaplastic large cell lymphoma (ALCL) has rising morbidity and mortality. Unfortunately, no ALK inhibitor has been approved by the FDA for single treatment of ALK rearrangement-positive ALCL. In this study, we investigated the antitumor effect of ZYY, a novel ALK inhibitor, showing a strong growth inhibitory effect on Karpas299 cells in vitro and in vivo. Specifically, ZYY significantly reduced the mRNA and protein expression of ALK and its downstream signaling proteins in Karpas299 cells. Furthermore, ZYY induced G1 phase arrest and promoted apoptosis in Karpas299 cells. Furthermore, we demonstrated that ZYY-induced apoptosis was mainly related to the mitochondria-dependent endogenous pathway. In vitro studies further showed that ZYY induced autophagy in Karpas299 cells, along with increased levels of the autophagy-related proteins, including LC3II and Beclin-1. Moreover, knockdown Beclin-1 and application of autophagy inhibitor chloroquine potentiated ZYY-induced cytotoxicity and apoptosis in vitro, indicating that cytoprotective autophagy might be triggered by ZYY in Karpas299 cells. Taken together, the novel ALK inhibitor ZYY has tremendous potential for treating human ALCL, and a combination of autophagy and ALK inhibition could effectively elicit potent antitumor effects.

Two series of diarylpyrazoles were designed as potential microtubule targeting agents. Twenty-eight target compounds were synthesized and exhibited potent antiproliferative activity. Compound 15e, displayed potent antiproliferative activity against SGC-7901, KB and HT-1080 cell lines, respectively, and was comparable to the positive control, CA-4. Tubulin polymerization experiments indicated that 15e effectively inhibited the tubulin polymerization, and immunostaining assay revealed that it significantly disrupted tubulin microtubule dynamics. Moreover, cell cycle studies revealed that compound 15e dramatically arrested cell cycle progression at G2/M phase and caused microtubule destabilization. Molecular modeling studies showed that 15e could bind to the colchicine binding site on microtubules.

A series of (1-aryl-1H-pyrazol-4-y1) (3,4,5-trimethoxyphenyl)methanones (8a-p, 9a-p) and ketoxime (10c) derivatives were designed and synthesized as antitubulin agents. All of the target compounds were evaluated for the in vitro anti-proliferative activities against three tumor cell lines (A549, HT-1080, SGC-7901). The most promising compounds in this class were (1-(p-toly1)-1H-pyrazol-4-y1) (3,4,5-trimethoxyphenyl)methanone (9c) and its ketoxime derivative (10c), which significantly inhibited tumor cells growth with IC50 value of 0.054-0.16 jiM. Meanwhile, compound 9c exhibited effectively inhibitory activity of tubulin polymerization. Consistent with its antitubulin activity, compound 9c could destructively damage microtubule network and arrest SGC-7901 cell cycle at G2/M phase significantly. The structure-activity relationship (SAR) and conformational analysis indicate that methyl group at C4-position of C-ring is critical for the activities and the amino group at the C5-position of B-ring plays a negative role in maintaining bioactivity. Furthermore, a molecular docking study was performed to elucidate its binding mode at the colchicine site in the tubulin heterodimer. (C) 2018 Published by Elsevier Masson SAS.