Preclinical Studies of Flavopiridol (FP) in Childhood Acute Lymphoblastic Leukemia (ALL).

Abstract

While approximately 80% of children and adolescents with ALL are cured with frontline therapy, patients that experience a marrow relapse have a poor outcome. Only 10–20% of those that relapse within the first 18–36 months of treatment are long term survivors, even with intensive chemotherapy and/or stem cell transplant. Thus, there is an urgent need to develop novel agents and new combination chemotherapy regimens for children with relapsed ALL. Our group and others have reported that p16 gene deletions occur commonly in relapsed ALL, and that the deletions are frequently acquired in the interval between initial diagnosis and relapse. The p16 protein regulates G1-S transition by controlling cyclin dependent kinase (CDK) 4 and 6 phosphorylation of retinoblastoma protein (pRb). These observations provide a strong rationale to test agents that target the cell cycle regulatory network in relapsed ALL. FP is a semi-synthetic flavonoid that has several possible anti-neoplastic mechanisms of action including CDK inhibition. We tested the activity of FP in vitro using a panel of ALL cell lines (RCH-ACV, Nalm-6, Molt-4 and Jurkat) and K562 (chronic myelogenous leukemia blast crisis). Each of these cell lines lacked detectable p16 protein on Western blot, including RCH-ACV, which has an intact p16 gene. We used WST-1 (a modified MTT assay) to determine the IC50 (concentration producing 50% inhibition in viable cell number) following 96 hours of exposure to FP, doxorubin (DOX), and dexamethasone (DEX). Each cell line was highly resistant to DEX (IC50 >10 ug/mL) and variably sensitive to DOX (IC50 9.67–39.17 ng/mL ). FP IC50s ranged from 99 nM (Molt-4) to 312.5 nM (K562), concentrations achieved in vivo in phase I trials. Detailed analysis of cell cycle kinetics and apoptosis were evaluated in RCH-ACV and Nalm-6 via flow cytometry using propidium iodide (PI) and annexin V/PI respectively. At concentrations approximating the FP IC50, we observed a transient G1-S arrest at ~24 hours with only modest amounts (15–25%) of apoptosis seen at 72 hours. In contrast, all of the decrease in cell proliferation at the DOX IC50 can be attributed to induction of apoptosis. However, at FP concentrations above the IC50, we observed substantial induction of apoptosis in a concentration dependent manner, with approximately 90% apoptosis induced following 72 hours exposure at 300 nM. Western blot analyses demonstrate that FP inhibits phosphorylation of pRb at serine 795, a site mainly phosphorylated by CDK 4, and that this inhibition corresponds with G1-S arrest in RCH-ACV cells. Both inhibition of serine 795 phosphorylation and G1-S arrest are transient. They are observed following 24 hrs exposure to FP, but return to baseline after 48 hrs of exposure. These data indicate a bifunctional potential role of FP in ALL therapy. At lower concentrations it induces transient G1-S arrest, while at higher doses it directly induces apoptosis, suggesting that FP may have potential utility in the treatment of ALL. Our next steps include further study into the mechanism of action of FP in ALL cell lines, as well as study into the possibility of utilizing FP in combination with other agents.