Cyclin-Dependent Kinase 7/9 Inhibitor SNS-032 Abrogates FIP1-like-1 Platelet-Derived Growth Factor Receptor α and Bcr-Abl Oncogene Addiction in Malignant Hematologic Cells

Abstract

Purpose: The "gate-keeper" mutations T674I platelet-derived growth factor receptor a (PDGFRa) in hypereosinophilic syndrome (HES) and T315I Bcr-Abl in chronic myeloid leukemia (CML) are resistant to imatinib and the second-generation small-molecule tyrosine kinase inhibitors (TKI). However, to combat acquired resistance to imatinib, an alternative approach is to decrease the expression of the addicted gene to efficiently kill resistant malignant hematologic cells. The purpose of this study was to evaluate the strategy of shutting down the transcription and expression of FIP1-like-1 (FIP1L1)-PDGFRa and Bcr-Abl with SNS-032, an inhibitor of cyclin-dependent kinase 7 (CDK7) and CDK9 in phase I clinical trials.Experimental Design: The effects of SNS-032 on PDGFRa and Bcr-Abl signaling pathways, apoptosis, and cell cycling were analyzed in TKI-resistant cells of HES and CML. The in vivo antitumor activity of SNS-032 was assessed with xenografted BaF3-T674I FIP1L1-PDGFRa and KBM5-T315I Bcr-Abl cells in nude mouse models.Results: SNS-032 inhibited the phosphorylation on Ser5 and Ser2 of RNA polymerase II. SNS-032 decreased both the mRNA and protein levels of FIP1L1-PDGFRa and Bcr-Abl and inhibited the proliferation of malignant cells expressing FIP1L1-PDGFRa or Bcr-Abl. It also decreased the phosphorylation of downstream molecules. It induced apoptosis by triggering both the mitochondrial pathway and the death receptor pathway.Conclusions: This CDK7/9 inhibitor potently inhibits FIP1L1-PDGFRa-positive HES cells and Bcr-Ablpositive CML cells regardless of their sensitivity to imatinib. SNS-032 may have potential in treating hematologic malignancy by abrogating oncogene addiction.

Ponatinib Induces Apoptosis in Imatinib-Resistant Human Mast Cells by Dephosphorylating Mutant D816V KIT and Silencing β-Catenin Signaling

“…Ponatinib (purity > 95%, high-performance liquid chromatography) was synthesized in our laboratory, prepared as a 20 mmol/L stocking solution in dimethyl sulfoxide (DMSO) and stored in aliquots at À20 C. Imatinib mesylate was a product of Novartis Pharmaceuticals (23). Z-DEVD-fmk (caspase-3 inhibitor) was purchased from BD Biosciences.…”

“…For detection of AIF and cytochrome c, cytosolic fraction was prepared with digitonin extraction buffer (28). Extracts of subcellular cytoplasmic, mitochondrial, and nuclear fractions were separated as previously described (23).…”

“…Total mRNA was extracted with TRIzol reagent (Invitrogen), then reverse-transcribed into first-strand complementary DNA (cDNA) with maxima first strand cDNA synthesis kit (Thermo Fisher), followed by real-time quantitative PCR with the SYBR Premix Ex Taq kit (TaKaRa) according the protocol described previously (23).The primers are shown in Supplementary Table S1. Each PCR reaction was in a 20 mL volume on a 96-well plate in Roche LightCycler480.…”

“…The MTS assay (CellTiter 96 Aqueous One Solution reagent; Promega) was used to evaluate cell viability as described previously (23,28). Synergism of combinational drugs was evaluated by the median-effect method of Chou and Talalay (28).…”

“…Apoptosis was measured using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Sigma-Aldrich) and analyzed with a FACScalibur flow cytometer and CellQuestPro software as described (8,23).…”