Targeting TKI-Insensitive CML Stem/Progenitor Cells By Effective Inhibition of a Novel PP2A-AHI-1-BCR-ABL-JAK2 Complex

Abstract Imatinib Mesylate (IM) and other ABL tyrosine kinase inhibitors (TKIs) have had a major impact on treatment of early phase CML patients. However, TKI monotherapies are not curative and initial and acquired resistance remain challenges. We demonstrated that CML stem cells are less responsive to TKIs and are a critical target population for TKI resistance. To prevent the development of resistant subclones, improved treatment approaches that target other elements active in CML stem cells are needed. One candidate is Abelson helper integration site-1 (AHI-1), an oncogene we identified that is upregulated in CML stem cells and interacts with multiple kinases, including BCR-ABL and JAK2. These complexes initiate BCR-ABL-transforming activity and mediate TKI response/resistance of CML stem/progenitor cells. Loss of these interactions significantly increases IM-sensitivity of CML stem/progenitor cells. These findings indicate that AHI-1 is a new therapeutic target in CML stem cells, but there are no specific small molecule inhibitors available that target AHI-1. By screening the Prestwick Chemical Library, we have recently identified a specific growth inhibitory compound that potentially targets AHI-1: Cantharidin (CAN), an inhibitor of protein phosphatase 2A (PP2A). CAN inhibited the growth of AHI-1-transduced cells by about 30% compared to control cells, but this effect was significantly enhanced to 93% with the addition of IM. As well, AHI-1-suppressed cells were more sensitive to CAN treatment, suggesting specific targeting of AHI-1 by a PP2A inhibitor. PP2A is a family of serine/threonine phosphatases that regulate numerous cell signaling cascades involved in proliferation and cell cycle control of cancer cells. It has been reported that PP2A activity can be upregulated or downregulated in cancer cells and can play either positive or negative roles in signaling pathways, suggesting that activation/suppression of PP2A activity and its specific pathways is differentially regulated in cancer cells. It has also been reported that PP2A activity is downregulated in CML cells, particularly in blast crisis CML, due to overexpression of SET, and that the use of a PP2A activator inhibits the growth of CML cells. Interestingly, we have now demonstrated that combination treatment with IM and CAN significantly prevents growth and induces apoptosis in CML K562 and IM-resistant K562 cells compared to single treatments (2-3 fold, p<0.001). The combination also greatly reduced colony formation (CFC) of CD34+ CML cells, but CAN also inhibited CFCs of CD34+ normal bone marrow (BM) cells. To overcome toxicity issues, we recently obtained new, pre-clinically validated PP2A inhibitors, LB1.0 and LB1.2. The combination of LB1.0 or LB1.2 (5µM) with IM (5µM) is more effective at selectively reducing CFCs generated from CD34+ treatment-naïve IM-nonresponder cells than single agents (56% vs. 13%), and they are also much less toxic to CD34+ normal BM cells compared to CAN (2-3 fold, p<0.005). Long-term culture-initiating cell assays also showed that more primitive cells were eliminated to a greater extent by combination treatments. Moreover, cell cycle analysis in CML cells showed that treatment with LB1.0/LB1.2 alone was able to induce a shift from G1 to G2/M phase (three fold, p<0.05). A similar shift in the cell population was also observed after combination treatment with IM, suggesting that the G2/M phase arrest is solely due to PP2A inhibition. Confocal microscopy confirmed the G2/M arrest, leading to mitotic catastrophe in the treated cells. Mechanistically, we have further identified the PP2A subunit B (PR55a) as a potential AHI-1 interacting protein using immunoprecipitation/mass spectrometry. Western blot analysis showed that the combination treatment significantly suppresses protein expression of AHI-1, BCR-ABL, JAK2, STAT5, AKT and β-catenin compared to single agents and that the combination directly affects PP2A-mediated β-catenin dephosphorylation and BCR-ABL-mediated phosphorylation of β-catenin in CML cells, which may lead to protein degradation of several key proteins. These results indicate that we have uncovered a new AHI-1-PP2A (PR55a) interaction and that simultaneously targeting both BCR-ABL and PP2A activities in CML stem/progenitor cells may provide a more effective treatment option for CML patients. Disclosures No relevant conflicts of interest to declare.

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