Growth of chronic myeloid leukemia cells is inhibited by infection with Ad-SH2-HA adenovirus that disrupts Grb2-Bcr-Abl complexes

Abstract

Abstract. the persistence of Bcr-Abl-positive cells in patients on imatinib therapy indicates that inhibition of the Bcr-Abl kinase activity alone might not be sufficient to eradicate the leukemia cells. Many downstream effectors of Bcr-Abl have been described, including activation of both the grb2-sos-ras-MApK and grb2-gab2-pI3K-Akt pathways. the Bcr-Abl-grb2 interaction, which is mediated by the direct interaction of the grb2 sH2 domain with the phospho-Bcr-Abl y177, is required for activation of these signaling pathways. therefore, disrupting their interaction represents a potential therapeutic strategy for inhibiting the oncogenic downstream signals of Bcr-Abl. Adenovirus Ad-sH2-HA expressing the grb2 sH2 domain was constructed and applied in this study. As expected, Ad-sH2-HA efficiently infected cMl cells and functioned by binding to the phospho-Bcr-Abl y177 site, competitively disrupting the grb2 sH2-phospho-Bcr-Abl y177 complex. they induced potent anti-proliferation and apoptosis-inducing effects in cMl cell lines. Moreover, the ras, MApK and Akt activities were significantly reduced in the Ad-sH2-HA treated cells. these were not observed with the point-mutated control adenovirus Ad-sm-HA with abolished phospho-Bcr-Abl y177 binding sites. these data indicate that, in addition to the direct targeting of Bcr-Abl, selective inhibition of its downstream signaling pathways may be a therapeutic option for cMl, and the Ad-sH2-HAmediated killing strategy could be explored as a promising anti-leukemia agent in cMl.

Inhibition of BCR/ABL Protein Expression by miR-203 Sensitizes for Imatinib Mesylate

“…At different time points (0 h, 24 h, 48 h, 72 h), cell proliferation assays using the MTT method were performed as described previously [10]. Cells were exposed to different concentrations of imatinib (Novartis) with different time (0 h, 24 h, 48 h, 72 h) and cell survival was measured by MTT assay.…”

“…Cell lysis, SDS-PAGE, and immunoblotting were done as described previously [10]. Antibodies against c-ABL, Bax, STAT5 and phosphorylated-STAT5, PARP, caspase 3 were from Cell Signaling Technology (Beverly, MA).…”

Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells

“…The SH2 domain of Grb2 had a high affinity with the phospho-Bcr-Abl Y177. In addition, we designed a mutant sequence (Sm) with one amino acid mutation (R27K) in the binding domain, which had been reported to block the binding to phospho-Bcr-Abl Y177 [23]. Each PCR fragment was cloned into the pAd5/F35-CMV plasmid.…”

“…Interaction of Bcr-Abl Y177 with Grb2 potentially promotes association of Grb2-SH3 domains with guanine nucleotide exchange factor Sos, which can activate RAS by stimulating exchange of GDP for GTP [26]. Our previous works have demonstrated that the exogenous SH2 can disrupt the interaction between Grb2 and Bcr-Abl Y177, thus reducing MAPK and Akt kinase activities [23]. In this study, we constructed HF2S, which contains exogenous SH2 to specifically bind to the phospho-Y177 site of Bcr-Abl and transferred it into the nucleus by a combination of FN3R and AP21967.…”

“…The SH2 domain of Grb2 binds specifically to phosphorylated Y177 of Bcr/Abl fusion protein with high affinity, which is required for the activation of RAS signaling and the efficient induction of the myeloproliferative disease by Bcr-Abl [22]. Our previous study has demonstrated that an ectopically expressed SH2 peptide disrupts the interaction between Grb2 and Bcr-Abl Y177 and inhibits the oncogenic downstream signals of Bcr-Abl [23]. Heterodimerization was induced between FN3R and HF2S upon AP21967.…”