Cyclins are important regulators of cell cycle in neoplastic cells. Regimented fluctuation in their expression is required for orderly cell cycle progression. Cyclins partner and modulate cyclin-dependent kinases (CDK) which are necessary for their activity. Since neoplastic cells are characterized by deregulated cell cycle with excessive proliferation, targeting cell cycle progression has been an attractive therapeutic approach. CDK inhibitors, most notably flavopiridol and dinaciclib, have shown efficacy in early phase clinical trials in CLL. The dominant fraction of the CLL clone is represented by B-cells in a resting (G0) state of the cell cycle. Efficacy of the CDK inhibitors in CLL is attributed to their effect on CDK-7/9 which facilitate initiation and sustenance of RNA polymerase-mediated transcription. P1446A is a novel potent and orally active kinase inhibitor which inhibits CDK's at nanomolar concentrations (CDK-1 IC50 25 nM, CDK-4 IC50 90 nM, CDK-6 IC50 210 nM and CDK-9 IC5022 nM). P1446A has shown clinical activity in Phase I studies in solid tumors and exhibited minimal hematologic toxicity. Here we studied activity of P1446 in primary CLL B-cells and MEC1 cells.
We enrolled 40 patients with median age of 70 years at the CLL clinic at Dartmouth. Median time of follow-up was 4 years and most patient (75%) were untreated. 60% of patients had unmutated IGHV. The majority of patients had normal cytogenetics, trisomy 12 or 13q deletion, and 3 patients (10%) had deletion 11q. Additionally, 10 patients with deletion 17p were enrolled at the CLL Center at Dana Farber Cancer Institute. CLL B cells were isolated from peripheral blood using standard Ficoll-Hypaque technique and cultured in RPMI supplemented with 15% fetal bovine serum. Apoptosis was quantified by means of Annexin V/7-AAD staining and flow cytometry as well as immunoblotting (PARP cleavage). B-cell receptor simulation was performed with 10 mg/mL immobilized anti-IgM. We also determined activity of P1446 in MEC1 cells.
CLL cells showed features of apoptosis (PARP cleavage) after a 2-hour incubation with 1500 nM P1446A. While apoptosis began to occur with lower concentrations of P1446A (0.2 μM), maximal efficacy was reached with 1.5 μM. After 24 hours of incubation with that dose, 84.1±4.7% CLL cells demonstrated Annexin V staining, compared to 30.2±2.5% of cells treated with vehicle control (p<0.001). P1446A-induced cell death did not depend on the IGHV mutational status (p=0.44) or cytogenetic abnormalities. However, samples which carried deletion 17p showed decreased sensitivity towards the CDK inhibitor compared with those which had normal cytogenetics or non-17p abnormality (p=0.003). Still, 37.5±4.4% cells carrying 17p del underwent apoptosis upon 24-hour exposure to 1.5 μM P1446 (normalized to untreated control). P1446A was significantly more toxic to CLL cells compared to normal B-cells (p<0.001). To determine whether P1446A had an impact on BCR signaling-mediated survival, we co-cultured CLL cells with 10 mg/mL immobilized anti-IgM. As expected, BCR engagement resulted in CLL cell rescue from spontaneous apoptosis in a subset of patient samples, while co-incubation with P1446A abrogated this protective effect.
Similarly, P1446 induced significant apoptosis in MEC1 cell line leading to apoptosis induction after 6 hours of incubation with 1.5 μM P1446A. We determined that 48-hour incubation with 3 μM P1446A resulted in a two-fold reduction of MEC1 metabolic activity in an MTT assay.
Our data shows that a novel CDK inhibitor P1446A demonstrates significant pre-clinical activity in CLL.
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Asterisk with author names denotes non-ASH members.