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

Abstract

Purpose: 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 ³H-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+HU´ and ´ponatinib+palbociclib´ combinations in advanced CML.

Funding: This project was supported by the Austrian Science Funds (FWF) projects F4701-B20, F4704-B20 and P30625.

Keywords: BCR-ABL1 mutations; CDK4/CDK6 – Palbociclib; CML; Hydroxyurea; TKI resistance.

Fig. 1

Hydroxyurea (HU) induces molecular response and suppresses the T315I-positive sub-clone(s) in advanced CML. Four heavily pretreated CML patients (#1–#4) were treated with HU, BCR-ABL1 tyrosine kinase inhibitors (imatinib, 400 mg/day; dasatinib, 100 mg/day; nilotinib, 2 × 400 mg/day per os), polychemotherapy, or hematopoietic stem cell transplantation (HSCT) as indicated. The percentage of BCR-ABL1 mRNA relative to ABL1 mRNA (according to the international scale) as well as the percentage of BCR-ABL1^T315I mRNA relative to BCR-ABL1 mRNA (BCR-ABL1^T315I/BCR-ABL1, determined by ligase-dependent PCR) are shown in the upper panels. The white blood count (WBC) of the same patients are shown in the lower panels. 3 + 7: combined chemotherapy following the 3 + 7-protocol consisting of daunorubicine (60 mg/m² per day, days 1–3) and cytosine arabinoside (200 mg/m² per day, days 1–7).

Fig. 2

Hydroxyurea (HU) inhibits the proliferation of primary CML cells and TKI-resistant cell lines harboring T315I-mutated BCR-ABL1. Primary leukemic cells obtained from CML patients (A), normal BM cells (B) and KCL22 cells expressing BCR-ABL1^WT (C, left upper image, dotted line), KCL22 cells expressing BCR-ABL1^T315I (C, left upper image, black line), untransfected (BCR-ABL1-negative) Ba/F3 cells (kept in 0.1 ng/ml IL-3) (C, upper right, middle and lower panels, stippled lines) and Ba/F3 cells expressing BCR-ABL1^WT (C, dotted lines) or various mutant forms of BCR-ABL1 (B, black lines) were incubated in control medium (Co) or medium containing various concentrations of HU at 37 °C for 48 h. Thereafter, ³H-thymidine-uptake was measured. (A and B) Results are expressed as percent of control and represent the mean±S.D. from triplicates. Patients´ numbers refer to Table 2. TKI-res., TKI-resistant. (C) Results are expressed as percent of control and represent the mean±S.D. from 3 independent experiments.

Fig. 3

Hydroxyurea (HU) synergizes with ponatinib in inducing growth inhibition in BCR-ABL1 positive cell lines and suppresses out-growth of cells harboring T315I-inculding BCR-ABL1 mutations. (A-C) Human CML cell lines (A), Ba/F3 cells expressing various mutant forms of BCR/ABL1 (B), and primary leukemic cells obtained from CML patients (C), were incubated in control medium (Co) or in various concentrations of HU, ponatinib or the combination of both drugs as indicated at 37 °C for 48 h. Thereafter, ³H-thymidine-uptake was measured. Results are expressed as percent of control and represent the mean±S.D. from triplicates. Patients' numbers in (C) refer to Table 2. (D) Human CML cell lines were incubated with control medium (Co) or medium containing various concentrations of HU, ABL001 or the combination of both drugs as indicated at 37 °C for 48 h. Thereafter, ³H-thymidine-uptake was measured Results are expressed as percent of control and represent the mean±S.D. of triplicates. (E) Ba/F3p210^WT (labeled by Venus), Ba/F3p210^T315I (labeled by GFP) and Ba/F3p210^T315I/E255V (labeled by tdTomato) were mixed in a 1:1:1 ratio and incubated together in control medium or in the presence of HU (100 µM), ponatinib (10 nM) or the combination of both drugs for 72 h (h). Thereafter, the ratio between clones in each condition was measured by flow cytometry. The percentage of non-viable cells was determined by trypan blue staining. Results show one typical experiment. Almost identical data were obtained in 2 other independent experiments.

Fig. 4

Synergistic effects between HU and ponatinib are mediated by suppression of CDK4/CDK6. (A) KCL22 and KCL22^T315I cells were kept in control medium (Co) or incubated in various concentrations of hydroxyurea (HU) or palbociclib as indicated for 24 h. Thereafter, PI was added and cell cycle distribution was determined by flow cytometry. The percentage of cells in G1-phase, G2/M-phase and S-phase in each condition are shown. Results represent the mean of 3 independent experiments. (B) K562, KU812, KCL22 and KCL22^T315I cells were kept in RPMI medium supplemented with 1% fetal calf serum (FCS) in the absence (“0”) or presence of 500 µM HU for 24 or 48 h as indicated. Thereafter, cells were subjected to Western blot analysis using antibodies against, PARP, CDK4, CDK6 or β-tubulin as indicated. (C) Primary CML cells were kept in control medium (Co) or in various concentrations of palbociclib (as indicated) for 48 h before ³H-thymidine-uptake was measured. Results are expressed as percent of control and represent the mean±S.D. from triplicates. Patients' numbers refer to Table 2. res., resistant. (D) KU812, K562, KCL22 and KCL22^T315I were incubated in control medium (Co) or in various concentrations of palbociclib, ponatinib or the combination of both drugs (as indicated) at 37 °C for 48 h. Then, ³H-thymidine-uptake was measured. Results are expressed as percent of control and represent the mean±S.D. from triplicates.