Downregulation Of Intracellular Bcrabl Levels

Noxious stimuli to cells activate the synthesis of some proteins while inhibiting the synthesis of others. The heat-shock proteins are molecular chaperones that stabilize the tertiary conformation of key cellular proteins, including proteins involved in signal transduc-tion. Misfolding of proteins can produce inactive aggregated forms. Heat-shock protein 90 (hsp90) is a specific molecular chaperone that affects the stability and function of multiple oncogenic proteins, including BCR/ABL.1213 Geldanamycin is a benzoquinone ansamycin antibiotic that inhibits hsp90 by competitively binding to an ATP-binding pocket in the amino terminus of the hsp90 molecule.12-14 Inactivation of hsp90 causes dysfunction and rapid degradation of their "client proteins" (including Raf-1, v-Src, p185c_erbB~2, and BCR/ABL)131516 via the proteosome pathway. A less toxic analog of geldanamycin, 17-AAG, has been shown in preclinical studies to down regulate BCR/ ABL in Ph-positive cell lines K562 and transfected HL60.13 When the hsp90 protein is inhibited, BCR/ABL is rendered sensitive to cellular physiologic mechanisms and can be degraded by the ubiquitin-proteosome pathway -3 activating the intrinsic apop-totic pathway.13 This appears to be the mechanism of action of this class of drugs. Preclinical laboratory studies have shown that BCR/ABL point mutations isolated from imatinib-resistant CML are degraded in vitro by both geldanamycin and 17-AAG. This effect has been demonstrated in both the T3151 and E255K cell lines.17 In both cell lines, the BCR/ABL protein was depleted at a low concentration of geldanamycin (30 nm).12 Leukemic blasts taken from three patients with CML blast crisis, who progressed while on ima-tinib, have been shown in vitro to be susceptible to 17-AAG-induced apoptosis.18 These laboratory observations have raised the possibility of combining other therapeutic agents with 17-AAG in an effort to reverse the drug-resistant phenotype in patients with advanced stage disease.

Arsenic trioxide (ATO) represents a novel agent that has shown preclinical activity in the treatment of imatinib-resistant CML by inducing alteration of mito-chondrial inner transmembrane potential, leading to the release of cytochrome c with subsequent caspase activation and apoptosis.19 Recently, ATO has also been shown to interfere with the translation of BCR/ABL by inhibition of ribosomal p70S6 kinase activity.20,21 Based on this rationale, the ATO and ima-tinib combination has been studied in numerous Ph-positive cell lines. Studies using the K562 and HL60/BCR/ABL cell lines have shown that the proapoptotic activity of imatinib is enhanced in the presence of ATO.21 Cotreatment of K562 and transfected M07p210 cells with approximately equipotent doses of ATO and imatinib additively inhibited growth proliferation.22 In colony-forming assays using CML patient samples, the combination of ATO and imatinib showed increased antiproliferative activity compared to imatinib alone.22 The additive effect of the ATO in combination with imatinib has initiated several phase I and II clinical trials examining the combination.

Interruption of the messenger RNA (mRNA) of mutant BCR/ABL may also be a target for therapeutic intervention. Antisense oligonucleotides could be designed that are complementary to the sequences on mutant BCR/ABL fusion transcripts. These small molecules would interfere with translation by physically blocking ribosome access to mRNA of the aberrant CML cells without affecting normal cells. Other mechanisms may potentially play a role in the antisense effects on BCR/ABL. DNA—RNA hybrids are more susceptible to RNAse activity, and several studies have shown that this strategy is feasible in CML cells in vitro.23-26 However, these agents have been difficult to study and implement in humans, even though they can be delivered relatively safely. This difficulty may, in part, be due to the long half-life of the BCR/ABL protein in vivo and the inability to deliver the drug in a manner that will allow it to be present in the cell for longer periods of time (i.e., 24-48 h). In addition, poor uptake of these antisense oligonucleotides into cells may prohibit their clinical usefulness. A similar strategy is based on RNA interference (RNAi). Several groups have reported in vitro BCR/ABL inhibitory effects, using small interfering RNA oligonucleotides (siRNA) in CML cell lines.27-29 Ribozymes (RNA) and DNAzymes (DNA) are designed to hybridize to specific RNA molecules and initiate hydrolysis of phosphodi-ester bonds in the target RNA. These strategies have all shown promise in the preclinical setting, but the clinical feasibility in imatinib-resistant patients has not yet to be established.

0 0

Post a comment