PD0332991, a CDK4/6 Inhibitor, Has An Anti-Leukemic Activity Against Lymphoid Crisis of CML and Ph+ALL Even with T315I Mutation in BCR-Abl; in Vitro Analysis

Abstract 1511 Since BCR-ABL plays a central role in cell cycle progression of Philadelphia-chromosome positive (Ph+) leukemia cells and CDK4/6 critically involves in G1-progression of cell cycle, we analyzed sensitivity of Ph+ leukemia cell lines to compounds that act as specific CDK4/6 inhibitors. H3-thymidine uptake assay showed that both PD183812 and CBC219476 significantly inhibited cell growth of Ph+ lymphoid leukemia cell lines (n=9) in comparison with Ph+ myeloid leukemia cell lines (n=7) and Ph- ALL cell lines (n=26). Thus, we next tested the anti-leukemic activity of PD0332991, a potent CDK4/6 inhibitor that is under phase II clinical study for solid tumor patients, and found that 8 of 9 Ph+ lymphoid leukemia cell lines showed extremely higher sensitivity to PD0332991; median IC50 was <25 nM. IC50 of Ph+ lymphoid leukemia cell lines was significantly lower than that of Ph+ myeloid cell lines (200 nM, n=7) and Ph-ALL cell lines (100nM, n=25). PD0332991 effectively dephosphorylated Rb protein (pRb), and subsequently induced G1 arrest on all of Ph+ lymphoid leukemia cell lines. Moreover, PD0332991 gradually induced cell death in 4 Ph+ lymphoid leukemia cell lines. Since CDK4/6 inhibitor acts depending on intact pRb, we analyzed protein and gene expression status of Rb. Of note, all Ph+ lymphoid leukemia cell lines expressed intact pRb except for one cell line that showed relative resistance to PD0332991. In contrast, pRb was almost undetectable in Ph+ myeloid cell lines in spite of comparable level of Rb gene expression, which might be mechanism for resistance to PD0332991. However, most of Ph- ALL cell lines had intact pRb expression in spite of their relative resistance to PD0332991, indicating that Rb status alone did not explain higher PD0332991-sensitivity of Ph+ lymphoid leukemia cell lines. Thus, we assumed that Ph+ lymphoid leukemia cells showed higher PD0332991-sensitivity probably because BCR-ABL regulates CDK4/6 expression for cell cycle progression. To clarify this assumption, we treated Ph+ lymphoid leukemia cell lines with imatinib and performed immunoblot analysis of cell cycle machineries such as CDKs, cyclines, and CDK inhibitors. Of note, CDK4 expression level was frequently downregulated by imatinib in Ph+ lymphoid leukemia cell lines. Moreover, imatinib-induced downregulation of CDK4 in Ph+ lymphoid leukemia cell line was abrogated by the addition of IL-7 and FLT3 ligand, which stimulated cell cycle progression of imatinib-treated Ph+ ALL cell line. LY294002, a PI3K inhibitor, but not U0126, a MAPK inhibitor, and AG490, an inhibitor for JAK/STAT pathway, efficiently downregulated CDK4 expression in Ph+ lymphoid leukemia cell lines. Gene expression level of CDK4 in Ph+ lymphoid leukemia cell lines was downregulated by imatinib, and lactastatin, an inhibitor of protein degradation, partially inhibited imatinib-induced downregulation of CDK4 protein in Ph+ lymphoid leukemia cell lines, indicating that BCR-ABL regulates CDK4 expression both in gene expression level and in protein degradation level. These findings indicated that Ph+ lymphoid leukemia cell lines showed higher sensitivity to PD0332991 since BCR-ABL induces cell cycle progression of Ph+ lymphoid leukemia cells by regulating CDK4 as one of downstream pathways. Accordingly, we tested if PD0332991 shows anti-leukemic activity in Ph+ lymphoid leukemia cells that have a T315I mutation of BCR-ABL. SU/SR is an imatinib-resistant Ph+ ALL cell line with T315I mutation (IC50 for imatinib >10 mM), which was established from SU-Ph2, an imatinib-sensitive Ph+ ALL cell line (IC50 for imatinib <0.1 mM), after long-term culture in the presence of gradually increasing concentration of imatinib. Of note, PD0332991 effectively dephosphorylated pRb and inhibited cell growth of both SU/SR and SU-Ph2. Our findings provide a rationale for efficacy of PD0332991 in the context of anti-leukemic therapy for lymphoid crisis of CML and Ph+ ALL patients even with T315I mutation in BCR-ABL. Disclosures: No relevant conflicts of interest to declare.