Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells

Alena Malyukova,Harsha S Madapura,Kourosh Lotfi,Ana Zovko,Marton Keszei,Dorina Ujvari,Jonas Wallvik,Niclas Björn,Thao T T Nguyen,Noemi Nagy,Daniel Salamon,Elham Yektaei-Karin,Koshi Akahane,Minori Tamai,Takeshi Inukai,Leif Stenke

doi:10.1038/s41419-021-04154-0

Abstract

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.

Highlights

Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 fusion oncoprotein, a constitutively active tyrosine kinase [1, 2]
Combination of small-molecule Myeloid cell leukemia 1 (MCL1) inhibition and Tyrosine kinase inhibitor (TKI) treatment synergistically induces concurrent strong apoptotic and variable levels of gasdermin E (GSDME)-dependent pyroptotic cell death in CML cell lines To analyze the potential interaction between imatinib and S63845 in CML cells, we first tested the effects of different concentrations of the two drugs and their combinations on the viability of the well-characterized, imatinib-sensitive CML lines K562 and JURLMK1, using the alamarBlue and SYTOX Green assays, respectively (Fig. 1)
To study whether S63845 might exert its antiviability effect through the enhancement of imatinib-induced apoptosis of CML cells, we tested the impact of the two drugs, alone or in combination on caspase-3/7 activation and caspase-specific cleavage of poly(ADP-ribose) polymerase (PARP) [21,22,23] in K562 and TCCS cells by western blot (Fig. 2)

Summary

INTRODUCTION

Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 fusion oncoprotein, a constitutively active tyrosine kinase [1, 2]. Several highly potent and selective small-molecule MCL1 inhibitors have recently been discovered and advanced to clinical trials [7, 8] One of these novel inhibitors, S63845, is effective in the low nanomolar range as a single agent or in combination with other anticancer drugs on most myeloma, lymphoma and acute myeloid leukemia derived cell lines in vitro and in vivo, without affecting normal hematopoietic progenitor cells [10,11,12]. It is tolerated in mice at efficacious doses [10] and its derivative MIK665/S64315 has already entered clinical trials in these hematological malignancies and in myelodysplastic syndrome. Primary human CD34+ CML stem/progenitor cells seeded in StemSpan SFEM medium supplemented with 0.5 ng/ml each of SCF, FL and TPO in addition to either 250 nM SYTOX Green Nucleic Acid Stain (in duplicate) or 2.5 μM IncuCyte Caspase-3/7 Green Reagent for Apoptosis (in triplicate) at 3 × 104 cells/well, in 96-well plates were left untreated or treated as indicated, followed by the quantification of the number of SYTOX Green negative, or the ratio of active caspase-3/7 positive cells, using

MATERIALS AND METHODS

RESULTS

Malyukova et al 3

DISCUSSION

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