BackgroundImatinib mesylate (IM) induces clinical remission of chronic myeloid leukemia (CML). The Abelson helper integration site 1 (AHI-1) oncoprotein interacts with BCR-ABL and Janus kinase 2 (JAK2) to mediate IM response of primitive CML cells, but the effect of the interaction complex on the response to ABL and JAK2 inhibitors is unknown.MethodsThe AHI-1–BCR-ABL–JAK2 interaction complex was analyzed by mutational analysis and coimmunoprecipitation. Roles of the complex in regulation of response or resistance to ABL and JAK2 inhibitors were investigated in BCR-ABL + cells and primary CML stem/progenitor cells and in immunodeficient NSG mice. All statistical tests were two-sided.ResultsThe WD40-repeat domain of AHI-1 interacts with BCR-ABL, whereas the N-terminal region interacts with JAK2; loss of these interactions statistically significantly increased the IM sensitivity of CML cells. Disrupting this complex with a combination of IM and an orally bioavailable selective JAK2 inhibitor (TG101209 [TG]) statistically significantly induced death of AHI-1–overexpressing and IM-resistant cells in vitro and enhanced survival of leukemic mice, compared with single agents (combination vs TG alone: 63 vs 53 days, ratio = 0.84, 95% confidence interval [CI] = 0.6 to 1.1, P = .004; vs IM: 57 days, ratio = 0.9, 95% CI = 0.61 to 1.2, P = .003). Combination treatment also statistically significantly enhanced apoptosis of CD34+ leukemic stem/progenitor cells and eliminated their long-term leukemia-initiating activity in NSG mice. Importantly, this approach was effective against treatment-naive CML stem cells from patients who subsequently proved to be resistant to IM therapy.ConclusionsSimultaneously targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may improve outcomes in patients destined to develop IM resistance.
Overcoming drug resistance and targeting leukemic stem cells (LSCs) remain major challenges in curing BCR-ABL human leukemia. Using an advanced drug/proliferation screen, we have uncovered a prosurvival role for protein phosphatase 2A (PP2A) in tyrosine kinase inhibitor (TKI)-insensitive leukemic cells, regulated by an Abelson helper integration site-1-mediated PP2A-β-catenin-BCR-ABL-JAK2 protein complex. Genetic and pharmacological inhibition of PP2A impairs survival of TKI nonresponder cells and sensitizes them to TKIs in vitro, inducing a dramatic loss of several key proteins, including β-catenin. We also demonstrate that the clinically validated PP2A inhibitors LB100 and LB102, in combination with TKIs, selectively eliminate treatment-naïve TKI-insensitive stem and progenitor cells, while sparing healthy counterparts. In addition, PP2A inhibitors and TKIs act synergistically to inhibit the growth of TKI-insensitive cells, as assessed by combination index analysis. The combination eliminates infiltrated BCR-ABL blast cells and drug-insensitive LSCs and confers a survival advantage in preclinical xenotransplant models. Thus, dual PP2A and BCR-ABL inhibition may be a valuable therapeutic strategy to synergistically target drug-insensitive LSCs that maintain minimal residual disease in patients.
LB100 sensitizes resistant chronic phase CML stem and progenitor cells to TKIs and spares healthy bone marrow cells.
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.
Tyrosine kinase inhibitor (TKI) therapies 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. Particularly, leukemic stem cells (LSCs) are less responsive to TKIs and are a critical target population for TKI resistance. We previously demonstrated that Abelson helper integration site-1 (AHI-1) is highly upregulated in LSCs and interacts with multiple kinases, including BCR-ABL and JAK2. We further showed that AHI-1-mediated complexes contribute to enhanced transforming activity of BCR-ABL and drug resistance of LSCs, suggesting AHI-1 as a new therapeutic target in CML. 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). PP2A is a family of serine/threonine phosphatases that regulate cell signaling cascades involved in proliferation and cell cycle control of cancer cells. Evidence suggests the dysfunction of specific PP2A protein complexes is primarily responsible for the changes that lead to cell transformation. It was also 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 CAN inhibits the growth of AHI-1-transduced BCR-ABL+ cells by about 30% compared to control cells, but this effect was significantly enhanced to 93% with the addition of imatinib (IM). As well, AHI-1-suppressed cells were more sensitive to CAN treatment. PP2A is highly expressed in K562 and IM-resistant K562 cells and combination treatment with CAN and TKI prevents growth and induces apoptosis in these cells more effectively than 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 have recently evaluated new, pre-clinically validated PP2A inhibitors, LB100 and LB102. These specifically suppress PP2A activity up to 75% in CML cells, as determined by immunoprecipitation (IP) phosphatase assays, and inhibit growth of these cells. Importantly, PP2A inhibitors plus TKIs significantly reduced the yield of colonies from CD34+ CML stem/progenitor cells compared to any single agent or combination of TKIs and this enhanced effect was most pronounced on cells from IM nonresponders (p<0.05). New PP2A inhibitors are also much less toxic to CD34+ normal BM cells compared to CAN (2-3 fold, p<0.001). Long-term culture-initiating cell assays showed that more primitive cells were eliminated to a greater extent by combination treatments. Moreover, the combination resulted in strong synergy in IM-insensitive cells (CI value <0.5). Notably, cell cycle analysis showed that treatment with LB100/LB102 alone induces a shift from G1 to G2/M phase (3-fold, p<0.05). A similar shift in the cell population was observed after combination treatment with IM, suggesting that the G2/M phase arrest is solely due to PP2A inhibition. Confocal microscopy showed the G2/M arrest, leading to mitotic catastrophe. Mechanistically, we have identified the PP2A subunit B (PR55a) as an AHI-1 interacting protein using IP/mass spectrometry; this was further confirmed in AHI-1-transduced cells by IP-Western analysis. Most interestingly, Western blot analysis showed that the combination treatment not only inhibits tyrosine phosphorylation of BCR-ABL, JAK2, STAT5, AKT and P38, but also significantly inhibits protein expression of these proteins compared to single agents. Inhibition of protein expression of AHI-1 and β-catenin by the combination was also observed and a new protein interaction between AHI-1 and β-catenin was identified. Moreover, the combination directly affects PP2A-mediated β-catenin dephosphorylation and BCR-ABL-mediated phosphorylation of β-catenin in CML cells, which leads to protein degradation of multiple proteins. In summary, we have uncovered new AHI-1-PP2A (PR55a) and AHI-1- β-catenin interactions and simultaneously targeting both BCR-ABL and PP2A activities in CML LSCs may offer an important new therapeutic possibility, through destabilization of the protein-protein interactions mediated by AHI-1. Disclosures No relevant conflicts of interest to declare.
4181 Ahi-1 (Abelson helper integration site-1) is an oncogene that was initially identified by provirus insertional mutagenesis in v-abl-induced murine pre-B cell lymphoma. The Ahi-1/AHI-1 protein contains an SH3 domain, multiple SH3 binding sites and a WD40-repeat domain, all known to be important mediators of protein-protein interactions. Human AHI-1 is highly deregulated in human leukemic cells, particularly in BCR-ABL+ leukemic stem cells from patients with chronic myeloid leukemia (CML). We have demonstrated that overexpression of Ahi-1 in primitive hematopoietic cells confers a growth advantage in vitro and induces leukemia in vivo; these effects can be enhanced by BCR-ABL, a fusion oncogene that plays a major role in the genesis of CML. AHI-1 can physically interact with BCR-ABL and JAK2 in CML cells and this interaction complex further mediates tyrosine kinase inhibitor (TKI) response/resistance of CML stem/progenitor cells. Despite its importance, the mechanism by which this complex affects cell proliferation and survival and regulates sensitivity of CML cells to TKIs remains unknown. To identify and characterize which functional domain(s) of Ahi-1 is critical for its interaction with BCR-ABL and/or Jak2, full length Ahi-1 and several mutant forms, including N-terminal deletion (N-terΔ, containing both SH3 and WD40-repeat domains), SH3 deletion (SH3Δ) and double WD40-repeat domain and SH3 domain deletions (SH3WD40Δ) were generated and stably transduced into BCR-ABL inducible BaF3 cells, in which the level of expression of BCR-ABL can be down-regulated by exposure to doxycycline. Epitope-tagged full length and mutant Ahi-1 constructs were also transiently expressed in 293T cells co-expressed with either BCR-ABL or Jak2. Co-IP experiments showed that Ahi-1 is highly expressed and stably associated with BCR-ABL-Jak2 complex in BCR-ABL inducible cells co-transduced with full-length Ahi-1 and that it can directly interact with Jak2; Ahi-1 was detected in both Ahi-1-transduced BaF3 cells (without BCR-ABL) and BCR-ABL inducible cells after IP with a Jak2 antibody. Interestingly, N-terminal region of Ahi-1 was required for Ahi-1-Jak2 interaction as a predicted 70 kD product was not detectable in the same cells transduced with the Ahi-1N-terΔ mutant. In contrast, N-terminal region of Ahi-1 was not associated with Ahi-1-BCR-ABL interaction since BCR-ABL was still detectable in BCR-ABL inducible cells co-transduced with the Ahi-1N-terΔ mutant after IP with the anti-ABL antibody. The deletion of either the SH3 domain or both the SH3 and WD40-repeat domains did not interfere with their interaction with Jak2. Ahi-1 lacking both the SH3 and WD40-repeat domains lost its ability to interact with BCR-ABL when the SH3WD40Δ mutant and BCR-ABL were co-expressed in 293T cells and the SH3 deletion did not affect Ahi-1's interaction with BCR-ABL, indicating that the WD40-repeart domain is required for a direct Ahi-1-BCR-ABL interaction. As expected, overexpression of full-length Ahi-1 in BCR-ABL inducible cells resulted in fewer Annexin V+ apoptotic cells after imatinib (IM) treatment compared to BCR-ABL inducible cells (5.9% and 8% v.s.26% and 34% with either 2 or 5 μM IM) after 24 hours. Interestingly, cells expressing the SH3WD40Δ mutant displayed dramatically increased cell sensitivity to IM with increased Annexin V+ cells (62% and 69% v.s.5.9% and 8%), while cells expressing the SH3Δ and the N-terΔ mutants had similar numbers of Annexin V+ cells as compared to BCR-ABL inducible cells (32% and 16% v.s.26% with 2 μM IM). Colony forming cell assays (CFC) further showed significantly reduced growth factor independent CFC numbers generated from the SH3WD40Δ mutant compared to BCR-ABL inducible cells co-expressed with the full-length Ahi-1 (4% v.s.46% with 5μM IM compared to untreated cells). Interestingly, a gene expression study comparing full-length Ahi-1-transduced BCR-ABL cells with its mutants further demonstrated that these changes were associated with deregulated expression of Bcl-2 and Cis (Cytokine inducible SH2 protein) involved in apoptosis. These results suggest that the WD40-repeat domain is important for Ahi-1's role in mediating IM-induced apoptosis and that targeting this critical domain of Ahi-1may provide a novel therapeutic option for treatment of CML. Disclosures: No relevant conflicts of interest to declare.
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