Targeting the Tetramerization by a Specific Peptide Abrogates the Transformation Potential of BCR/ABL and Some of Its Imatinib-Resistant Mutants.

Abstract

As a result of the t(9;22), more than 95% of CMLs and 20–25% of adult ALLs express the p210(BCR-ABL) or the p185(BCR-ABL) fusion protein respectively. The BCR portion of the fusion protein harbors an N-terminal coiled-coil (CC) domain. The CC contains two alpha helical motifs and assembles to dimers with antiparallel orientation. Two of these dimers associate and lead to the tetramerization of BCR. The second alpha-Helix (Helix-2) contributes the majority of the dimer and tetramer interface. In the case of the BCR/ABL fusion protein, the BCR mediated tetramerization of ABL leads to the constitutive activation of the ABL kinase domain. The subsequent permanent activation of multiple downstream signaling pathways induces the leukemic phenotype. Targeted inhibition of BCR/ABL by the ABL kinase inhibitor Imatinib leads to complete remission in CML and ALL patients. However, a large portion of ALL and CML patients in blast crisis become resistant to Imatinib mainly by the acquisition of point mutations in BCR/ABL. Imatinib binds to the inactive conformation of the ABL-kinase which in case of the BCR/ABL fusion protein is present in monomers. We have previously shown that CC-derived peptides that interfere with BCR/ABL tetramer formation reduce the kinase activity of BCR/ABL in vivo and increase the sensitivity of BCR/ABL expressing cells towards Imatinib. Here we investigated the effects of peptides derived from the CC subdomain Helix-2 on BCR/ABL oligomerization and on the biology of cells expressing Wt BCR/ABL and Imatinib-resistant mutants. First we confirmed the interaction between Helix-2 and BCR/ABL by GST-pull-down and co-immunoprecipitation assays. The influence of Helix 2 peptides on the BCR/ABL oligomerization and its capacity to form high-molecular weight (HMW) complexes in vivo was analyzed by size-exclusion HPLC. The effect of Helix 2 on the leukemogenic potential Helix of BCR/ABL was investigated in retrovirally transduced murine IL-3 dependent Ba/F3 cells and human Philadelphia Chromosome (Ph) negative ALL-cell line Nalm-6 and Ph-positve SupB15 cells. Ba/F3 cells transduced with Imatinib-resistant BCR/ABL point mutations were used to investigate the inhibitory potential of CC specific peptides on mutant BCR/ABL in presence and absence of Imatinib.Here we report that CC-specific Helix-2 peptidesinteract with Wt and mutant BCR/ABL;decrease the autophosphorylation of BCR/ABL in transduced Ba/F3 cells;disrupt the HMW-complexes of Wt and mutant BCR/ABL;abrogate the growth of human Ph-positive expressing SupB15 cells without affecting Ph-negative Nalm-6 cells; v) increase the Imatinib sensitivity of IL-3 independent Ba/F3 cells expressing BCR/ABL;reduce the Imatinib resistance of Ba/F3-cells expressing BCR/ABL P-loop mutants Y253F and E255K.Taken together these results show that the disruption of BCR/ABL tetramerization has strong inhibitory effects on BCR/ABL transformed cells. Furthermore our results indicate that disruption of BCR/ABL tetramerization is a therapeutic option for the treatment of patients harboring Imatinib-resistant BCR/ABL mutations.