P656: NOVEL THIAZOLIDINEDIONE WITH HDAC INHIBITING PROPERTIES OPTIMIZED FOR CHRONIC MYELOID LEUKEMIA TREATMENT

Background: Clinical success of tyrosine kinase inhibitors (TKIs) has transformed chronic myeloid leukemia (CML) into treatable but not curable, truly chronic disease and established treatment-free remission as a new goal for CML therapy. TKI discontinuation is feasible in approximately half of CML patients with stable deep molecular response, but due to the persistence of quiescent leukemic stem cells (LSC) the risk of relapse remains. The observation that peroxisome proliferator-activated receptor γ (PPARγ) agonist, thiazolidinedione (TZD) family, pioglitazone, targets CML-LSCs in vitro has initiated several clinical trials that showed the potential of the combined treatment to allow safe discontinuation of imatinib after concomitant use of pioglitazone. The treatment is well tolerated, its efficacy, however, remains ambiguous. Since it was shown that histone deacetylase inhibitor (HDACi) treatment may also target CML-LSCs in patients receiving tyrosine kinase inhibitors, there is a strong rationale to combine PPARγ agonist and HDACi activities to effectively deplete CML-LSCs and allow for safe treatment discontinuation. To achieve that goal dual PPARγ agonist and HDACi has been designed and synthetized. Here, we present in vitro data on its potential to treat CML. Aims: Evaluation of anti-leukaemic properties of PPARγ agonist/ HDACi dual TZD in combination with TKIs for CML treatment. Methods: A series of double activity TZDs has been designed and synthetized. MTS and XTT cytotoxicity assays in K-562 cell line were used to determine IC50 of all compounds and select a compound with the highest anti-leukaemic activity and the highest potential for combination therapy with TKIs (imatinib and dasatinib). All studies were performed using the selected novel compound (7l) in comparison to pioglitazone. PPARγ activation was measured using Transcription Factor Assay Kit (Abcam), while HDAC activity using Western blot for total H3ac. To elucidate the mechanism of action of the new compound cell cycle analysis, apoptosis assay, growth curve, and clonogenic assay were performed. In addition, high-throughput functional kinase assay using PamGene technology was performed to evaluate kinase activation profile after 7l administration. The results were confirmed with phosphoflow. Results: Addition of novel, double-acting TZD 7l to TKIs significantly decreased clonogenic potential of K-562 cells affecting not only the number but also the size and morphology of the colonies suggesting strong effect on stem-like cells. Similar increase in antileukemic efficacy of TKIs was observed in cytotoxic assays. Moreover, 7l showed stronger synergistic effect with TKIs compared to pioglitazone. Cell cycle arrest in G1/G0 phase was observed, that is concordant with the effects of pioglitazone treatment. Apoptosis assays and growth analysis showed that 7l inhibits proliferation of CML cells without significant increase in cell death. High-throughput kinase activity analysis showed mild inhibition of BCR-ABL signaling in 7l-treated cells (not observed in pioglitazone-treated cells) and inhibition of MAPK, Ras and PI3K-Akt signaling pathways. Summary/Conclusion: Double acting thiazolidinedione 7l with PPARγ ligand and HDACi properties in combination with TKIs significantly reduces clonogenic potential of CML cells and exerts higher antileukemic potential than pioglitazone. Further studies are warranted. Keywords: HDAC inhibitor, Leukemic stem cell, Tyrosine kinase inhibitor, Chronic myeloid leukemia