Abstract
Background: The treatment of blast crisis of chronic myeloid leukaemia (BC-CML) remains difficult and the outcome of patients is poor. A dysregulated apoptosis leads to survival of malignant cells. BH3-mimetics inhibit antiapoptotic proteins of BCL-2 family. These drugs have been recently introduced in the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. This work assumed that these drugs may have a treatment potential for myeloid and/or lymphoid BC-CML. Aims: The aim is to test the sensitivity of imatinib-resistant (IR) clones of KCL-22, model of BC-CML, and primary blast cells to BH3-mimetics and describe a mechanism of sensitivity/resistance using protein analysis of apoptotic pathways. Methods: Isolated IR-clones (n=10) of KCL-22 were characterized by NGS and cultivated with 4µM imatinib (IM) and dilution series of BH3-mimetics (venetoclax – anti-BCL-2, S63845 – anti-MCL-1, A-1155463 - anti-BCL-XL) for 72 hours. IC50 was determined based on proliferation. Protein expression of BCL-2 family (n=10) was conducted in 4 IR-clones cultivated with IM and with/without BH3-mimetics. For in vivo experiments NOD-SCID-gamma mice (n=16) were subcutaneously injected with 106 cells of BCR::ABL1-T315I clone and divided into control group and treated groups. Primary cells of patients with BC-CML (n=4) were exposed to IM and BH3-mimetics for 7 days and LC50 was estimated. Results: IR-clones of KCL-22 differ in their sensitivity to BH3-mimetics (Table 1). The majority of clones (8/10) including 4 clones with T315I were sensitive to MCL-1 inhibitor. A reduced sensitivity was observed in the T315I clone carrying mutated GATA2 and other clone with mutation Y253H and mutated BCOR. Moreover, two T315I clones were sensitive to venetoclax, but other T315I clones with mutations in other cancer related genes showed insensitivity (n=2) or decreased sensitivity (n=1). Interestingly, two T315I clones with mutations in other cancer related genes insensitive to venetoclax were sensitive to BCL-XL inhibitor and in general, it seems that clones insensitive to venetoclax were sensitive to BCL-XL inhibitor and vice versa. Protein analysis of parental KCL-22IR cells and 4 clones showed a high expression of BCL-2 and BCL-XL and undetectable MCL-1 expression. Except for the T315I clone (B8, Table 1), other analysed clones expressed MCL-1 after exposure to BH3-mimetics. The sensitivity of T315I clone to anti-MCL-1 can be explained by detection of cBAX (an active form of apoptotic effector) after exposure to the inhibitor. The parental cell line showed dissociation of antiapoptotic complex MCL-1/BIM after exposure to MCL-1 inhibitor. The released BIM accelerates the apoptosis as an apoptotic activator. In vivo analysis revealed a decreased tumour growth of xenografted T315I clone (B8) in IM, venetoclax or combined treatment compared to control. An additive effect of dual therapy against monotherapy was not observed. This can be explained by IM inhibition of non-mutated BCR::ABL1 in KCL-22IR carrying 2 Ph chromosomes. LC50 analysis of primary blasts (4 patients) showed anti-MCL-1 to be the most potent inhibitor. Mutations in other cancer related genes and/or changes in karyotype increased LC50 for venetoclax and BCL-XL inhibitor or venetoclax, respectively. Image:Summary/Conclusion: The preliminary data of this study showed that the MCL-1 inhibitor S63845 seems to be the most potent BH3-mimetic to induce apoptosis in BC-CML. The combined therapy of TKI and BH3-mimetics should reflect mutation status of BCR::ABL1 and other cancer related genes.
Published Version
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