Karolina Dzwonek

Activating mutations in ALK kinase domain confer resistance to structurally unrelated ALK inhibitors in NPM‑ALK‑positive anaplastic large‑cell lymphoma

PurposeCrizotinib, the first FDA-approved ALK inhibitor, showed significant antitumor activity in young patients with anaplastic large-cell lymphoma (ALCL) frequently displaying ALK rearrangement. However, long-term therapeutic benefits of crizotinib are limited due to development of drug resistance. CH5424802—more potent and selective ALK inhibitor—comprises a good candidate for second-line treatment in crizotinib-relapsed patients. The aim of this study was to determine possible mechanisms of resistance to ALK inhibitors that can appear in ALCL patients.MethodsALK+ ALCL cell lines resistant to crizotinib (Karpas299CR) and to CH5424802 (Karpas299CHR) were established by long-term exposure of Karpas299 cells to these inhibitors. Next, alterations in their sensitivity to ALK, HSP90 and mTOR inhibitors were investigated by cell viability and BrdU incorporation assays and immunoblot analysis.ResultscDNA sequencing of ALK kinase domain revealed activating mutations—I1171T in Karpas299CR and F1174C in Karpas299CHR. The resistant cells displayed diminished sensitivity to structurally unrelated ALK inhibitors—crizotinib, CH5424802 and TAE684. Nevertheless, CH5424802 and TAE684 were still more potent against the resistant cells than crizotinib. Moreover, Karpas299CR and Karpas299CHR cells remained sensitive to HSP90 or mTOR inhibitors.ConclusionsResistance mediated by activating mutations in ALK kinase domain may emerge in ALCL patients during ALK inhibitors treatment. However, more potent second-generation ALK inhibitors, HSP90 or mTOR inhibitors may represent an effective therapy for relapsed ALK+ ALCL patients.

Differences in gene expression and alterations in cell cycle of acute myeloid leukemia cell lines after treatment with JAK inhibitors

Janus kinase (JAK) inhibitors are a promising treatment strategy in several hematological malignancies and autoimmune diseases. A number of inhibitors are in clinical development, and two have already reached the market. Unfortunately, all of them are burdened with different toxicity profiles. To check if the JAK inhibitors of different selectivity evoke different responses on JAK2-dependent and independent cells, we have used three acute myeloid leukemia cell lines with confirmed JAK2 mutation status. We have found that JAK inhibitors exert distinct effect on the expression of BCLXL, CCND1 and c-MYC genes, regulated by JAK pathway, in JAK2 wild type cells in comparison to JAK2 V617F-positive cell lines. Moreover, cell cycle analysis showed that inhibitors alter the cycle by arresting cells in different phases. Our results suggest that observed effect of JAK2 inhibitors on transcription and cell cycle level in different cell lines are associated not with activity within JAK family, but presumably with other off-target activities.

Discovery of TRAF-2 and NCK-interacting kinase (TNIK) inhibitors by ligand-based virtual screening methods

TRAF-2 and NCK-interacting kinase (TNIK) is a serine–threonine kinase with a proposed role in Wnt/β-catenin and JNK pathways. Due to its implication in Wnt-mediated colorectal carcinogenesis, selective TNIK inhibition has emerged as an attractive anti-cancer therapeutic strategy. So far, only a few TNIK inhibitors have been described and none of them reached advanced preclinical development. In this study, a virtual screening approach was applied for the investigation of novel TNIK inhibitors. The best performing ShaEP methodology for similarity searching was applied for screening of a commercially available small-molecule database. Among several discovered TNIK kinase inhibitors, a compound containing the furan-2-carboxamide scaffold was found to be the most active, with IC50 = 0.85 μM. An advanced substructure search led to the discovery of a more potent and selective compound with IC50 = 258 nM. The most active compounds were tested in vitro for their effect on the Wnt/β-catenin pathway and proliferation of Wnt-active colorectal cancer cells. The compounds identified in this study represent attractive starting points for the development of more potent and selective small-molecule TNIK inhibitors for both therapeutic application and research into TNIKs biological role.

Discovery of selective, orally bioavailable inhibitor of mouse chitotriosidase

This article describes our work towards the identification of a potent and selective inhibitor of mouse chitotriosidase (mCHIT1). A series of small molecule inhibitors of mCHIT1 and mAMCase have been developed from early lead compound 1. Examination of synthetized analogues led to discovery of several novel highly potent compounds. Among them compound 9 (OAT-2068) displays a remarkable 143-fold mCHIT1 vs. mAMCase selectivity. To explain the observed SAR molecular docking experiments were performed, which were in line with the experimental data from the enzymatic assays. Inhibitor 9 (OAT-2068) was found to have an excellent pharmacokinetic profile. This, together with high activity and selectivity, makes the compound an ideal and unique tool for studying the role of CHIT1 in biological models.