Abstract

Despite Bcr-Abl tyrosine kinase inhibitors (TKIs) are highly effective in CML, development of TKI-resistance leading to blast crisis (BC) progression is still a challenge in CML therapy. This has motivated the generation of novel agents and combination strategies to overcome the resistance. ABL001 is a small molecule allosteric Bcr-Abl kinase inhibitor that occupies the myristoyl pocket, leading to a kinase autoinhibition conformation. ABL001 has entered phase I clinical trials to treat patients with chronic, accelerated, or blast phase CML that relapsed from TKIs (Hughes et al., ASH 2016). Its activity in BC CML and ability to enhance the traditional TKIs that target ATP binding site are under investigation. TP53, a key tumor suppressor gene plays a crucial role in mediating cell death in part through regulating the expression or activity of Bcl-2 family proteins. Although highly mutated in solid tumors, the mutation rate of TP53 is relatively low in CML. However, p53 antagonist, MDM2 can be regulated by Bcr-Abl signaling and ARF, which inhibits MDM2 is frequently deleted in BC CML suggesting therapeutic potentials of inhibition of MDM2 in BC CML. Indeed, activation of p53 by MDM2 inhibition has been shown to enhance the toxicity of TKIs in BC CML (Carter et al., Oncotarget 2015, 6(31):30487). In this study, we examined the activity of ABL001 in CML cells and investigated if ABL001 enhances the activity of TKIs and synergizes with MDM2 inhibitor CGM097 in samples from heavily treated TKI resistant BC CML patients.

BaF3 cells expressing BCR-ABL with or without mutations, human CML cell lines (KBM5 and KBM5-STI-T315I), and peripheral blood mononuclear cells (PBMC) isolated from primary BC CML patient samples were treated with various doses of ABL001, CGM097 or Nilotinib alone or in combination. Apoptosis was determined by Annexin V/7-AAD staining in cell lines and CD45+ (bulk), CD34+CD38+ and CD34+CD38- cells of CML patient PBMC samples. EC50 was calculated using CompuSyn or Graphpad. Protein levels were examined by Western blotting.

We found that ABL001 induced apoptosis in cell lines expressing BCR-ABL (BaF3p210 and KBM5) or mutant BCR-ABL (BaF3p210E255K and KBM5-STI-T315I) but had limited activity in BaF3p210T315I cells. Importantly, ABL001 induced apoptosis in bulk, CD34+CD38+, or/and CD34+CD38- cell populations at low nM concentrations in 5 out of 6 BC CML patient samples tested. Patient samples that responded to ABL001 had either T315I and/or E255 mutations in BCR-ABL kinase domain. ABL001 inhibited Bcr-Abl signaling as measured by the levels of p-Bcr-Abl/p-Stat5/p-Crkl in BCR-ABL positive cells with or without BCR-ABL mutations. ABL001 showed higher activity than Nilotinib in 4 out of 5 responding patient samples. Nilotinib did not significantly enhance ABL001-induced apoptosis in the tested BC CML patient samples and CML cell lines. Inhibition of MDM2 with CGM097 activated p53 and induced apoptosis in BC CML patient samples. The activity of ABL001 was enhanced by CGM097 in the majority of patient samples which did not have p53 mutations. ABL001 combined with CGM097 was more effective than the combination of nilotinib with CGM097. The combinations of TKI and CGM097 were more effective in killing leukemia cells than each agent alone even when leukemia cells were co-cultured with MSCs. Neither ABL001 or CGM097 alone, nor the combination showed toxicity on normal bone marrow stem/progenitor cells.

In conclusion, our results demonstrate that the allosteric Bcr-Abl Inhibitor ABL001 overcomes TKI-resistance and enhances MDM2 inhibitor CGM097 activity in BC CML cells. The combination of ABL001 and CGM097 warrants clinical evaluation.

Disclosures

Carter: novartis: Research Funding; Daiichi Sankyo: Research Funding.

Author notes

*

Asterisk with author names denotes non-ASH members.