Imatinib and second generation of TKIs such as dasatinib and nilotinib are effective for controlling CML and Ph+ ALL. However, Ph+ leukemia having T315I mutation of BCR-ABL shows resistance not only to imatinib but also to second generation of TKIs. Thus, it is urgent to clarify new strategies controlling Ph+ leukemia having T315I mutation. For this purpose, we assumed that effecter molecules for cell cycle progression regulated by BCR-ABL could be attractive targets for controlling Ph+ leukemia even with T315I mutant. Thus, we performed screening of cell cycle machineries in Ph+ leukemia cell lines whose expression is downregulated by imatinib, and found that CDK4 expression was downregulated by imatinib in most of Ph+ ALL cell lines. Consistently, PD0332991, a potent CDK4/6 inhibitor that is under phase II clinical study for solid tumor patients, showed significantly higher anti-leukemic activity against Ph+ ALL cell lines with intact Rb expression (n=8) (IC50; < 20 nM) in comparison with Ph− ALL cell lines with intact Rb expression (n=24) (IC50; 100nM). We next tested anti-leukemic activity of PD0332991 against Ph+ ALL cell line that has T315I mutant in BCR-ABL. SU/SR is an imatinib-resistant Ph+ ALL cell with T315I mutant (IC50 for imatinib >5,000 nM), which was established from SU-Ph2, an imatinib-sensitive Ph+ ALL cell line (IC50 for imatinib <100 nM), after long-term culture in the presence of gradually increasing concentration of imatinib. As expected, PD0332991 potently showed anti-leukemic activity to SU/SR (IC50; 30 nM) as well as SU-Ph2 (IC50; <25 nM), suggesting that PD0332991 is an attractive agent for controlling Ph+ ALL even with T315I mutation (Nemoto A and Inukai T et al). Based on these in vitro findings, we next analyzed in vivo activity of PD0332991 against SU-Ph2 and SU/SR in xenograft model using NOD/SCID/γc null (NOG) mice. NOG mice were transplanted with SU-Ph2 or SU/SR cells (1×106cells) through the tail vein. After the flow cytometric confirmation of bone marrow (BM) engraftment on day 14, PD0332991 (150 mg/kg) or vehicle (distilled water) was given for 2 weeks (5 days on, 2 days off) from day 21. The chimerism analyses revealed that PD0332991 prevented leukemia growth compared with vehicle in both SU-Ph2 and SU/SR on Day 35 in BM (0.4% vs. 49%, 37% vs. 89% in SU-Ph2 and SU/SR) and in peripheral blood (PB) (0.05% vs. 19%, 0.7% vs. 44% in SU-Ph2 and SU/SR), respectively. As a result, xenograft NOG mice treated with PD0332991 demonstrated higher overall survival compared with xenograft NOG mice treated with vehicle. Next, we tested whether PD0332991 could induce the dephosphorylation of pRb in vivo. PD0332991 (150 mg/kg) or vehicle were given to xenograft NOG mice transplanted with SU/SR or SU-Ph2 cells two times on day 29 and 4 hours before BM aspiration on day 30. Isolated BM cells were stained with anti-human CD19 plus anti-phospho-pRb antibodies. Flow cytometric analysis showed a significant reduction in pRb phosphorylation of SU/SR cells by PD0332991 in comparison with vehicle (9.7% vs. 57.1%, PD0332991 vs. vehicle) and SU-Ph2 cells (17.1% vs. 74.2%, PD0332991 vs. control), respectively. In summary, CDK4/6 inhibitor PD0332991 prevented the growth of Ph+ leukemia with T315I mutation in BCR-ABL in vivo and improved overall survival dramatically. We also demonstrated that PD0332991 showed a significant reduction in pRb phosphorylation in vivo. Our findings provide a rationale for efficacy of CDK4/6 inhibitor in the context of anti-leukemic therapy for Ph+ leukemia patients with T315I mutation in BCR-ABL.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.