Therapeutically Rewiring Kinase Pathways in BCR-ABL⁺ Leukemias

The central leukemogenic lesion in chronic myeloid leukemia (CML) is the bcr-abl fusion gene, arising from the reciprocal chromosomal translocation t(9;22)(q34;q11) and resulting in the chimeric Bcr-Abl protein with constitutively activate Abl tyrosine kinase activity. Bcr-Abl is also present in 20–40% of adult acute lymphoblastic leukemia (ALL), and represents poor prognostic factor. The tyrosine kinase inhibitor (TKI) imatinib specifically inhibits tyrosine kinase activity of Bcr-Abl and selectively suppresses the growth and induces apoptosis in CML blasts – and has become the paradigm for inhibiting oncogenic signal transduction as a means of altering leukemic blast (and other transformed cells) biology. However, imatinib and other TKIs have been much less effective in accelerated phase/blast crisis CML and BCR-Abl⁺ ALL due to mutated TKI-resistant enzymes, BCR-Abl overexpression or cellular independence from the pro-survival signals transduced by the oncogenic kinase. We have developed a previously unexplored strategy that utilizes BCR-Abl kinase activity (instead of inhibiting it), in which the oncogenic kinase specifically activates a “prodrug” that subsequently acts as a “suicide” agent, causing leukemic cell differentiation and death. We and others have shown that pharmacologic activation of the serine-threonine kinase protein kinase C (PKC) induces growth arrest, differentiation and apoptosis in CML and AML blasts, and also kills ALL blasts. However, PKC are not tyrosine kinase substrates and are not directly activated by BCR-Abl. However, substitution of a negatively charged “phospho-mimetic” glutamic acid for Ala²⁵ in the pseudo-substrate domain of PKC results in a constitutively active enzyme. We now show that reengineering PKC by “swapping in” the Abl kinase target motif (Ala-X-X-Ile-Tyr²⁵-X-X-Phe/Pro) into the pseudosubstrate domain (A25Y PKC constructs) allows BCR-ABL to phosphorylate the tyrosine at position 25. This phosphorylation activates A25Y PKC specifically in the BCR-Abl⁺ K562 CML cell line, but not BCR-Abl negative AML KG1 cell line, as detected by PKC translocation from the cytoplasm to the plasma membrane. At a cellular level, this BCR-Abl-mediated activation of A25Y PKC results in a 6 fold reduction in cell growth and 6 fold increase in apoptosis as measured by annexin V staining (wild type PKC 2.9% apoptotic vs. A25Y 17.8% apoptotic). These findings demonstrate that the novel strategy of “rewiring” the pro-survival BCR-Abl signaling pathway into the “pro-death” PKC pathway via kinase activated suicide agents results in PKC-mediated growth arrest/apoptosis in BCR-Abl⁺ leukemic cells. More broadly, this therapeutic approach using oncogenic signaling pathways to activate prodrug effector molecules specifically within cancer cells may be particularly useful in TKI-resistant malignancies (especially where resistance to tyrosine kinase inhibitors is due to an amplified signal) or particularly in malignancies that may be less dependent on the oncogenic signal for survival (i.e. BCR-Abl⁺ ALL, EGFR signaling in lung cancer), where the signaling pathway is “on” but blocking it has little effect.