Inhibitory Effects of Icaritin on TKI-Resistant CML Cells through Disruption of the BCR-ABL-GRB2-RAS-MAPK Signaling Pathway

Abstract

Abstract The ABL tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM) is effective at inducing clinical remission in early phase chronic myeloid leukemia (CML) patients, but is not curative. Early relapses and acquired drug resistance remain some issues in IM-treated patients. In particular, relapses are frequently associated with point mutations in the BCR-ABL tyrosine kinase domain (TK, > 50%). Newer TKIs, dasatinib (DA) and nilotinib, have increased potency over IM and show a broader spectrum of activity against mutant forms of BCR-ABL. However, none of these agents is able to target a critical T315I gatekeeper mutation of BCR-ABL in TKI-resistant patients. We have demonstrated that CML stem cells are genetically unstable and generate many BCR-ABL TK mutations in vitro and in vivo. They are also less responsive to TKIs and are a critical target population for TKI-resistance. Thus improved treatment approaches to specifically target CML stem cells and BCR-ABL-T315I resistant cells are clearly needed. It has recently been reported that estrogen receptor variant ERα36 is highly deregulated not only in breast cancer cells, but also in liver cancer and leukemic cells, and that targeting this specific variant with a small molecular inhibitor (Icaritin, SNG162) inhibits CML cell growth. However, the underlying molecular mechanisms of these observations are not understood. Whether this inhibitor, alone or in combination with new ABL inhibitors, can target primary CML stem/progenitor cells and T315I-resistant cells have also not been investigated. In this study, we utilized two cell line model systems: K562 cells and IM-resistant K562 cells without BCR-ABL TK mutation and human UT7 cells expressing either wild type BCR-ABL or carrying the BCR-ABL-T315I mutation. We have now demonstrated that protein expression of ERα36 is highly upregulated in both IM-resistant and BCR-ABL-T315I mutant cells as compared to control cells. Interestingly, the use of pre-clinically validated ERα36 inhibitors (SNG162 and SNG1153) alone inhibits cell growth and induces apoptosis of these cells. BCR-ABL-T315I cells are more sensitive to ERα36 inhibitors treatment, with twofold increases in Annexin V+ cells detected in BCR-ABL-T315I cells after SNG1153 treatment, compared to those detected in BCR-ABL expressing cells. These effects can be further enhanced by combination treatment with a TKI. Importantly, we have discovered that treatment of IM-resistant and BCR-ABL-T315I mutant cells with SNG162 and SNG1153 inhibitors, alone or with a TKI, significantly reduced phosphorylation of BCR-ABL on tyrosine residue 177 (Tyr177), a residue essential for BCR-ABL induced leukemogenesis through its binding to GRB2 and activation of the downstream RAS-MAPK pathway. This new observation was supported by detection of a significant reduction in phosphorylation of MEK1/2 kinase, an important component of the RAS-MAPK pathway, in these cells. Most importantly, IP-Western analysis further demonstrated that the BCR-ABL-GRB2 protein interaction was markedly interrupted in cells treated with SNG inhibitors plus a TKI, which correlates with reduced phosphorylation of Tyr177 in these cells. Moreover, colony-forming cell (CFC) assays showed that SNG inhibitors (SNG162 and SNG1153) in combination with a TKI are more effective at inhibiting growth of CD34+ treatment-naïve IM-nonresponder cells as compared to single drug treatment (46% vs. 25%). We further demonstrated that SNG162 and SNG1153 (up to 10 μM and 5 uM) are not toxic to CD34+ normal bone marrow cells. Interestingly, a combination treatment of DA plus SNG1153 dramatically reduced the level of engrafted leukemic cells in transplanted immunodeficient NSG mice and prolonged the survival of these mice compared to mice treated with DA alone. Median survival of DA treated mice is 87.5 days, while some DA and SNG1153 treated mice remain alive for more than 102 days after transplantation. ERα36 thus emerges as an attractive druggable target for combination therapies to target TKI-insensitive CML stem/progenitor cells and T315I-resistant cells. Disclosures Zhang: Shenogen Pharma Group Ltd: Employment. Ding:Shenogen Pharma Group Ltd: Employment. Lin:Shenogen Pharma Group Ltd: Employment. Meng:Shenogen Pharma Group Ltd: Employment.