The impact of disruption of the PI3K (phosphatidylinositol 3-kinase) pathway on the response of human
leukemia cells to pharmacological
cyclin-dependent kinase (CDK) inhibitors has been examined. Exposure of U937 monocytic
leukemia cells to minimally toxic concentrations of
flavopiridol (FP),
roscovitine, or
CGP74514A for 3 h in conjunction with the PI3K inhibitor
LY294002 (abbreviated LY in the article) resulted in a marked decrease in Akt phosphorylation. Coexposure of cells to LY and CDK inhibitors also resulted in an early (i.e., within 3 h) and striking increase in mitochondrial damage [e.g.,
cytochrome c, second mitochondria-derived activator of
caspases/direct inhibitor of apoptosis (IAP)-
binding protein with low isoelectric point (Smac/DIABLO), and apoptosis-initiating factor (AIF) release],
caspase activation, and apoptosis. Similar interactions were observed in a variety of other
leukemia cell types (e.g., HL-60, Jurkat, Raji, and NB4). Apoptosis, induced by FP/LY, was substantially blocked by ectopic expression of Bcl-2, but to a considerably lesser extent by dominant-negative
caspase-8. FP-induced apoptosis was not enhanced by agents that inhibited
protein kinase (PK) A (
H89), PKC (GFX),
mitogen-activated
protein (MAP)/
extracellular signal-regulated kinase (ERK)
kinase (MEK1/2;
U0126),
p38 MAP kinase (MAPK;
SB202190), m-target of
rapamycin (TOR;
rapamycin), or
ataxia-telangiectasia mutation (ATM;
caffeine), whereas the PI3K inhibitor
wortmannin exerted effects similar to those of LY. The dramatic potentiation of CDK inhibitor-induced apoptosis by LY was accompanied by diminished Bad phosphorylation, induction of Bcl-2 cleavage, and down-regulation of X-linked IAP (XIAP) and Mcl-1. Cells exposed to CDK inhibitors + LY also exhibited reduced phosphorylation of
glycogen synthase kinase (GSK)-3,
forkhead transcription factor (FKHR), p70(S6K), and ERK, but increased activation of p34(cdc2) and
p38 MAPK. LY/CDK inhibitor-treated cells also displayed diminished pRb dephosphorylation on CDK2- and CDK4-specific sites,
retinoblastoma protein cleavage, and down-regulation of
cyclin D(1). Inducible expression of constitutively active (myristolated) Akt significantly, albeit partially, attenuated apoptosis in Jurkat
leukemia cells treated with either FP alone or the combination of FP and LY. Finally, cotreatment with LY and FP resulted in a dramatic increase in apoptosis in primary leukemic blasts obtained from a patient with
acute myeloblastic leukemia. Together, these findings suggest that the PI3K/Akt pathway plays a major role in regulating the apoptotic response of human
leukemia cells to pharmacological CDK inhibitors and raise the possibility that combined interruption of CDK- and PI3K-related pathways may represent a novel therapeutic strategy in
hematological malignancies.