Investigating the role of TAM family kinases and small molecular inhibitors in Resistant Chronic Myeloid Leukemia.

Abstract

Chronic myeloid leukemia (CML) is marked by uncontrolled myeloid cell proliferation and differentiation, driven by the BCR/ABL 1 translocation, which forms the aberrant Philadelphia chromosome. Current treatments using tyrosine kinase inhibitors (TKIs) face limitations such as relapse due to drug tolerance, kinase domain mutations and alternative signaling pathways leads sensitive CML acquire resistance and transform into resistant CML with persistent leukemic stem cells (LSCs). TAM family kinases are often overexpressed in cancers, including CML. This study involves the development of resistant CML model, explores the expression of TAM kinases in drug-resistant cells and the potential and mechanism of TAM family, imidazole and phenanthroline based small molecular inhibitors as therapeutic agents. By using various biochemical and pharmacological approaches our results showed overexpression of AXL (<0.0001), TYRO3, and MERTK (0.0033) in (K562-R) cells compared to (K562-S). Pharmacological targeting of TAM family kinases and use of imidazole and phenanthroline derivatives strongly interfered with proliferation potential of K562-S and K562-R cells, as LDC1267 (<0.0001), R428 (<0.0001) and UNC2250 (<0.0001), imidazole derived inhibitor L7 (0.0021), phenanthroline derived Gd Phen (0.0007) and Bi Phen (0.0028). TAM kinase inhibition also reduces cell proliferation in K562-S (0.0008, <0.0001, <0.0001) respectively. TAM kinase inhibitors also reduced clonogenic potential in K562-S and K562-R cells (<0.0001). We have observed an additive effect by co-targeting TAM kinases with imatinib on K562-S (0.008, <0.0001, <0.0001) and synergistic effect on K562-R (<0.0001). Mechanistically, cell proliferation by small molecular inhibitors is found to be related to the induction of Apoptosis in K562-S and K562-R cells. Furthermore, we showed that TAM family inhibitors and imidazole and phenanthroline derived small molecular inhibitors interfere with Wnt/β-catenin pathway by downregulation of downstream targets c-Myc, Axin2, Eya3 and their activators AXL-RTK and TYRO3 as (<0.0001, <0.0001, <0.0001and 0.0005) on K562-S as well as (0.0002,0.0016, 0.0001,0.0001, 0.0001, 0.0001, 0.0001) on K562-R respectively. The cell cycle analysis revealed cell cycle arrest by upregulation of p21 (<0.0001) and p27 (0.002) by Gd and Bi, p21(0.0117) and p27(0.0022) by LDC1267, p16(0.033), p21(0.016) and p27(0.002) by R428, p21(0.0016) by UNC on K562-R and p16(0.02), p21(0.0152) and p27(<0.0001) by LDC1267, p21(0.0078) and p27(0.004) by R428 in K562-S. Moreover, a significant downregulation of p53 (<0.0001) on both K562-R and K562-S was observed by all three TAM kinase inhibitors. Overall, our findings suggest that molecular targeting of TAM family kinases and use of imidazole and phenenthroline derivatives strongly interfered with proliferation potential of CML cell line K562-S and K562-R by inducing cell cycle arrest and apoptosis.