Combination of panobinostat with ponatinib synergistically overcomes imatinib‐resistant CML cells

Abstract

The major mechanism of imatinib (IM) resistance of CML is the reactivation of ABL kinase either through BCR‐ABL gene amplification or mutation. We investigated the cytotoxicity of a pan‐ABL tyrosine kinase inhibitor, ponatinib, and a pan‐histone deacetylase inhibitor, panobinostat, against IM‐resistant CML cells in vitro. Two different IM‐resistant cell lines, K562/IM‐R1 and Ba/F3/T315I were evaluated in comparison with their respective, parental cell lines, K562 and Ba/F3. K562/IM‐R1 overexpressed BCR‐ABL due to gene amplification. Ba/F3/T315I was transfected with a BCR‐ABL gene encoding T315I‐mutated BCR‐ABL. Ponatinib inhibited the growth of both K562/IM‐R1 and Ba/F3/T315I as potently as it inhibited their parental cells with an IC 50 of 2–30 nM. Panobinostat also similarly inhibited the growth of all of the cell lines with an IC 50 of 40–51 nM. This was accompanied by reduced histone deacetylase activity, induced histone H3 acetylation, and an increased protein level of heat shock protein 70, which suggested disruption of heat shock protein 90 chaperone function for BCR‐ABL and its degradation. Importantly, the combination of ponatinib with panobinostat showed synergistic growth inhibition and induced a higher level of apoptosis than the sum of the apoptosis induced by each agent alone in all of the cell lines. Ponatinib inhibited phosphorylation not only of BCR‐ABL but also of downstream signal transducer and activator of transcription 5, protein kinase B, and ERK1/2 in both K562/IM‐R1 and Ba/F3/T315I, and the addition of panobinostat to ponatinib further inhibited these phosphorylations. In conclusion, panobinostat enhanced the cytotoxicity of ponatinib towards IM‐resistant CML cells including those with T315I‐mutated BCR‐ABL.

Role of HDACs in normal and malignant hematopoiesis

“…Hence, combining HDACis with other chemotherapeutic agents is considered to be an effective way to enhance tumor drug sensitivity by improving the cellular efficacy and toxicity of HDACis to tumor cells [116][117][118][119][120][121][122][123][124][125] (Table 3 and Table 4). To date, the different mechanisms of HDACis combined with chemotherapeutic agents such as topoisomerase inhibitors, platinumbased chemotherapeutics, proteasome inhibitors, tyrosine kinase pathway inhibitors and epigenetic modifiers for advanced or drug-resistant hematological malignancies include (1) acetylating histones and inducing p21-CDK-mediated cell cycle arrest; (2) inducing apoptosis by regulating the expression of pro-and antiapoptotic genes through the intrinsic or extrinsic pathway; (3) inducing DNA damage and oxidative stress; (4) activating BTK (in CLL) or inhibiting ERK (in MM) and AKT (in CML) signaling pathways; and (5) regulating the expression of drug resistance-related molecules, such as downregulating BCR-ABL and upregulating Bim in hematological malignancies and downregulating CD44 in multiple myeloma (MM), NF-κB in ALL, γ-catenin in CML, and BRCA1, CHK1 and RAD51 in AML [126][127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143][144][145]. Specific combination strategies and their corresponding mechanisms are summarized in Table 3 [146][147][148]…”

CAY10683 and imatinib have synergistic effects in overcoming imatinib resistance via HDAC2 inhibition in chronic myeloid leukemia

“…Recently, it was suggested that the IM resistance in CML cells should be handled through the combined treatment of BCR-ABL kinase inhibitor with histone deacetylase (HDAC) inhibitors. [4][5][6] Histone deacetylase (HDAC) mediates non-histone and histone deacetylation and regulates transcriptional factor activities participating in tumor progression and initiation, and in regulating many critical proteins (like tumor suppressor gene) at the post-transcription level. 7,8 The HDAC inhibitors (HDACi) are promising for use in treating some cancer types.…”

Characterization of Newly Established Pralatrexate-resistant Cell Lines and the Mechanisms of Resistance

“…The IC 50 value of each drug was calculated from an analysis of growth inhibition. To evaluate the proliferative activity of each cell line, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2 h-tetrazolium-5carboxanilide (XTT) assays were performed according to the manufacturer's instructions (Roche, Indianapolis, IN, USA) [10,[14][15][16][17][18]. Cross-resistance to other antileukemic agents was also assessed.…”

“…Western blotting analysis was performed [10,14]. Anti-FPGS, anti-DHFR, anti-DNMT3B, and anti-β-actin antibodies were used as primary antibodies, and anti-rabbit polyclonal antibodies were used as secondary antibodies.…”