Targeting a Highly Deregulated eIF4F Translation Initiation Complex Sensitises IM-Resistant Cells to Tyrosine Kinase Inhibitors and Effectively Suppresses BCR-ABL1 Protein Expression

Background We previously reported that Abelson helper integration site-1 (AHI-1) is highly deregulated in chronic myeloid leukemia (CML) leukemic stem cells (LSCs) and mediates tyrosine kinase inhibitor (TKI) resistance. AHI-1 physically interacts with BCR-ABL1 and its SH3 domain interacts with BCR-ABL1 substrates, like dynamin-2, which regulate leukemic properties of CML LSCs. However, the molecular and biological roles of AHI-1 and its interacting partners in mediating TKI resistance remain largely unknown.

Objective To investigate the molecular functions of AHI-1 and its SH3 domain in regulation of TKI resistance, using advanced antibody microarray analysis.

Methods A high content antibody microarray was performed in BCR-ABL1⁺ cells co-transduced with wild-type (WT) AHI-1 or the deletion of AHI-1 SH3 domain (SH3^Δ) with or without imatinib (IM). Changes in antibody signals for protein expression or phosphorylation were determined using limma and pathway enrichment analysis by g:Profiler. eIF4G1 genetic inhibition by lentiviral-mediated shRNA or pharmacological inhibition by SBI-756, was performed in TKI-resistant cells to assay translation initiation activity by proximity ligation assay (PLA), o-propargyl-puromycin (OPP), and polysome profiling.

Results The antibody microarray analysis revealed that WT AHI-1 cells have the greatest number of changes in the phospho-proteome and proteome compared to BCR-ABL1⁺ cells and AHI-1 SH3^Δ cells with and without IM. Pathway enrichment analysis identified that the targets with significantly increased differential antibody signal after IM treatment in WT AHI-1 cells were related to the regulation of translation initiation complex (p < 0.0001). Interestingly, our RNA-seq dataset analysis further identified several eukaryotic initiation factor 4F (eIF4F) complex members to be significantly increased in CD34⁺ CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex (2-fold, p = 0.001). WT AHI-1 cells also showed increased expression and phosphorylation of eIF4G1 (>2-fold) and eIF4B (>2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, and cyclin D3 (a downstream protein of eIF4F translational activity) as compared to BCR-ABL1⁺ cells, by immunoblotting. These results were similarly demonstrated in IM-resistant cells as compared to IM-sensitive cells (2-5-fold, p < 0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p < 0.0001) and increased TKI sensitivity in IM-resistant cells (p < 0.0021). These cells showed reduced levels of eIF4F complex formation by PLA assay (p = 0.013) and reduced protein expression of cyclin D3 (40%, p = 0.0036) and BCR-ABL1 (70%). Similarly, IM-resistant cells were more sensitive to SBI-756 treatment, an eIF4G1 inhibitor (50% reduction, p = 0.0001) than IM-sensitive cells (30% reduction); these effects were enhanced by a combination of SBI-756 and IM (80%, p < 0.02). Furthermore, SBI-756 treatment reduced PLA foci formation (p = 0.014) and global protein synthesis rates (p < 0.0001). Polysome profiling demonstrated that IM-resistant cells had higher levels of translation activity, which was inhibited by SBI-756 treatment. Most interestingly, SBI-756 treatment reduced protein expression of BCR-ABL1 (70%) and cyclin D3 (40%) in these cells.

Conclusion We have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells. eIF4G1 inhibitor treatment sensitises IM-resistant cells to TKI and reduces BCR-ABL1 protein expression, providing potential treatment strategies to overcome TKI resistance.

No relevant conflicts of interest to declare.