CDK4/CDK6 inhibition as a novel strategy to suppress the growth and survival of BCR-ABL1T315I+ clones in TKI-resistant CML

Abstract

Ponatinib is the only approved tyrosine kinase inhibitor (TKI) suppressing BCR-ABL1 T315I-mutated cells in chronic myeloid leukemia (CML). However, due to side effects and resistance, BCR-ABL1 T315I-mutated CML remains a clinical challenge. Hydroxyurea (HU) has been used for cytoreduction in CML for decades. We found that HU suppresses or even eliminates BCR-ABL1 T315I + sub-clones in heavily pretreated CML patients. Based on this observation, we investigated the effects of HU on TKI-resistant CML cells in vitro. Methods: Viability, apoptosis and proliferation of drug-exposed primary CML cells and BCR-ABL1+ cell lines were examined by flow cytometry and 3 H-thymidine-uptake. Expression of drug targets was analyzed by qPCR and Western blotting. Findings: HU was more effective in inhibiting the proliferation of leukemic cells harboring BCR-ABL1 T315I or T315I-including compound-mutations compared to cells expressing wildtype BCR-ABL1. Moreover, HU synergized with ponatinib and ABL001 in inducing growth inhibition in CML cells. Furthermore, HU blocked cell cycle progression in leukemic cells, which was accompanied by decreased expression of CDK4 and CDK6. Palbociclib, a more specific CDK4/CDK6-inhibitor, was also found to suppress proliferation in primary CML cells and to synergize with ponatinib in producing growth inhibition in BCR-ABL1 T315I + cells, suggesting that suppression of CDK4/CDK6 may be a promising concept to overcome BCR-ABL1 T315I-associated TKI resistance. Interpretation: HU and the CDK4/CDK6-blocker palbociclib inhibit growth of CML clones expressing BCR-ABL1 T315I or complex T315I-including compound-mutations. Clinical studies are required to confirm single drug effects and the efficacy of`ponatinib+HUandponatinib+palbociclibcombinations in advanced CML. Funding: This project was supported by the Austrian Science Funds (FWF) projects F4701-B20, F4704-B20 and P30625.

TCF7 knockdown inhibits the imatinib resistance of chronic myeloid leukemia K562/G01 cells by neutralizing the Wnt/β‑catenin/TCF7/ABC transporter signaling axis

“…These results indicate that TCF7 may play a vital role in the development of drug resistance in chronic myeloid leukemia (CML) cells. An increasing number of studies have shown that replacing or combining other targets to conquer leukemia drug resistance has become a feasible strategy ( 48 , 49 ). In the present study, even when BCR-ABL1 activity was inhibited entirely, TCF7 expression was not significantly altered, indicating that TCF7 expression is BCR-ABL1-independent and combined targets of TCF7 and BCR-ABL1 may have a synergistic effect on the inhibition of CML imatinib-resistant cells.…”

Illuminating novel biological aspects and potential new therapeutic approaches for chronic myeloproliferative malignancies

“…Indeed, in vitro testing revealed great efficacy of palbociclib and ribociclib at concentrations readily achievable in the clinical setting. Moreover, synergistic effects of ponatinib in combination with HU or ciclibs against cells carrying highly resistant CMs including the T315I mutation have been demonstrated 16 . It appears therefore that the upregulation of CDK6 documented in BCR‐ABL1‐positive cells displaying the T315I mutation alone or as part of a CM, provides a therapeutically exploitable vulnerability of the mutant cells.…”

Oncogenic Kinase Cascades Induce Molecular Mechanisms That Protect Leukemic Cell Models from Lethal Effects of De Novo dNTP Synthesis Inhibition

“…To achieve these goals, we compared two cellular systems that show very high (NB4 APL cells) or very low (K562 CML cells) sensitivity to hydroxyurea-induced apoptosis [11]. We incubated them with a clinically achievable dose of 0.5 mM hydroxyurea [7] for 24 h and analyzed cell lysates by mass spectrometry (Figure 1a). Proteomics showed that hydroxyurea decreased the levels of RAF1 in NB4 cells but not in K562 cells (Figure 1a, Supplementary Figure S1a).…”

“…Chemotherapeutics eliminate transformed cells through the induction of replication stress and DNA damage [1][2][3]. Hydroxyurea is used as cytoreductive therapy for leukemic disorders and brain tumors [4][5][6][7]. This drug specifically inhibits ribonucleotide reductase (RNR) and thereby depletes the cellular dNTP pool [8].…”

“…Chronic myeloid leukemia (CML) cells that are driven by the tyrosine kinase BCR-ABL1 (encoded by the reciprocal chromosomal translocation t(9;22)) [12,13] display particular robustness to apoptosis upon dNTP depletion by hydroxyurea [11]. Despite the long-standing use of hydroxyurea in the clinic [4][5][6][7], molecular details on specific signaling pathways that determine whether leukemic cells are responsive or resistant to pro-apoptotic effects of this drug are still to be identified. The modulation of such mechanisms in hydroxyurea-treated cells could deliver novel and improved treatment options.…”