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. 2021 Aug;100(8):2023-2029.
doi: 10.1007/s00277-020-04357-z. Epub 2021 Jun 10.

Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K

Afsar Ali Mian  1   2 , Isabella Haberbosch  3 , Hazem Khamaisie  4 , Abed Agbarya  5 , Larissa Pietsch  6   7 , Elizabeh Eshel  8 , Dally Najib  8 , Claudia Chiriches  1 , Oliver Gerhard Ottmann  1 , Oliver Hantschel  9   10 , Ricardo M Biondi  6   7   11 , Martin Ruthardt  12 , Jamal Mahajna  13   14
Affiliations

Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K

Afsar Ali Mian et al. Ann Hematol. 2021 Aug.

Abstract

Resistance remains the major clinical challenge for the therapy of Philadelphia chromosome-positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common "gatekeeper" mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph- cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia-like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

Keywords: Allosteric inhibition; BCR-ABL1; Compound mutations; Crizotinib; Philadelphia chromosome–positive leukemia; TKI resistance.

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Conflict of interest statement

Dr. Hantschel reports receiving consulting fees from Intima Biosciences Inc. Dr. Ottmann reports receiving honoraria for AdBoards from Pfizer, Novartis, Celgene, Amgen, Incyte, Takeda, Roche, Fusion Pharma, and Sun Pharma. His research is supported by Incyte, Amgen, and Celgene. Dr. Ruthardt reports having received honoraria from Novartis, Fusion Pharma, and Sun Pharma and his research was supported by Fusion Pharma and Sun Pharma.

All other authors declare to have no conflict of interest.

Figures

Fig. 1
Fig. 1
Inhibition of the ABL1 kinase activity by crizotinib blocks the factor-independent growth of Ba/F3 cells mediated by BCR-ABL1 and BCR-ABL1T315I. a Western blot analysis of lysates of Ba/F3 cells expressing BCR-ABL1 and BCR-ABL1T315I using antibodies directed against the following targets: c-Abl, Abl-Y245 (anti-phospho-ABL1), Crkl, phosphorylated Crkl, Stat5, phosphorylated Stat5 (anti-phospho-STAT5), and β-tubulin (anti-β-tubulin). Molecular mass reference (kDa) values are presented, and c-Abl and β-tubulin were used as loading controls. To avoid bias of stress-induced signaling by factor withdrawal, we performed these experiments in the presence of IL-3. b The effect of crizotinib on the factor-independent growth of Ba/F3 cells expressing BCR-ABL1 or BCR-ABL1T315I was assessed in cells selected by IL-3 withdrawal. These cells were seeded in semi-solid medium and exposed to the indicated concentrations of GNF-2 (allosteric inhibitor of ABL1), imatinib, nilotinib, crizotinib, and dasatinib. At day 14, colonies were stained. c The effect of crizotinib on cell proliferation and viability of factor-independent Ba/F3 cells upon the expression of BCR-ABL1, BCR-ABL1T315I, BCR-ABL1Y253F, and BCR-ABL1F317L was assessed with an XTT assay. The lack of cytotoxic effects was confirmed in empty vector-transduced Ba/F3 cells in the presence of IL-3 at a concentration of less than 1 μM. The mean of three experiments ± SD is given. d Crizotinib inhibits human Ph+ patient-derived cell lines and primary Ph+ ALL PD-LTCs. Proliferation/cytotoxicity assays using XTT were performed on human Ph+ cell lines derived from Ph+ ALL or CML patients. SupB15 (Ph+ ALL) expressing p185BCR-ABL1 or BV-173 cells expressing p210BCR-ABL1 (lymphocytic CML-BC cells) were exposed to increasing concentrations of crizotinib. The means of three experiments ± SD each performed in triplicates are given. e Proliferation of PD-LTCs - PH (sens - TKI sensitive), BV (res - TKI-resistant), and KÖ (expressing BCR-ABL1T315I) - XTT assays upon exposure to increasing concentrations of crizotinib were performed. The Ph− PD-LTCs HP (Ph−) was used as a control. The means ± SD of three experiments each performed in triplicates are given. f Interaction of His-ABL with biotin-myristoyl-peptide (100%) and the displacement of the interaction by GNF2 and crizotinib
Fig. 2
Fig. 2
The efficacy of crizotinib in vivo in models of Ph+ leukemia. a For the induction of CML-like disease, sub-lethally irradiated C57BL/6N mice were transplanted intravenously with 1 × 105 Sca1+-positive BM cells expressing BCR-ABL1 or BCR-ABL1T315I. Eight mice/group were treated orally either with crizotinib (100 mg/kg) or ponatinib (25 mg/kg) once daily for 20 days (treatment). For the design of in vivo experimentation, see the Supplementary Information. b Crizotinib prolongs the survival of mice with BCR-ABL1-derived ALL. 5 × 104 spleen cells from ALL mice (frozen stock in liquid N2) were transplanted into sub-lethally (4.5 Gy) irradiated recipients. The mice were treated with crizotinib (100 mg/kg) or ponatinib (25 mg/kg) by gavage for 20 days. c Response of the compound mutation BCR-ABL1T315I-E255K to crizotinib and ponatinib. The effect of crizotinib on the factor-independent growth of Ba/F3 expressing BCR-ABL1 or BCR-ABL1T315I-E255K was performed on cells selected by IL-3 withdrawal. These cells were exposed to the indicated concentrations of PF-114, ponatinib, and asciminib. Cell proliferation and viability were assessed by XTT assays. The means ± SD of three experiments are given
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