ERBB Signaling Promotes BCR-Abl Expression and Modulates Multiple Signaling Pathways as Assessed by High-Throughput, Reverse Phase Protein Array.

ERBB family receptor tyrosine kinases, EGFR, Her-2, Her-3 and Her-4, are prognostic and therapeutic targets in multiple carcinomas, including breast, lung, and head and neck tumors. We previously reported aberrant expression and activation of Her-2 and Her-4 in a panel of Ph+ ALL cell lines: Z33, Z119, and Z181. Loss of cell viability and apoptotic DNA fragmentation were observed after treatment with the irreversible pan ERBB inhibitor, CI-1033, in vitro. In the present study, we define the mechanisms of cell death initiated by ERBB blockade. We examined the expression and Tyr1248 phosphorylation of Her-2 in Z119 and Z181 cell lines upon CI-1033 treatment and found a 4-fold decrease in Ph-Y1248 by Western blotting. We then assessed the impact upon downstream signaling pathways using reverse-phase protein microarray (RPPA) - a new high-throughput method to measure and profile the total expression and post-translational phosphorylation of signaling pathway components. Leukemia cells treated with CI-1033 assessed with RPPA using 34 different antibodies showed mTOR expression to be decreased by more than two-fold in Z119 and Z181. Concomitantly, we saw a more than three-fold decrease in phosphorylated S6-S240/244. This suggests that down-regulation of translational control caused by inhibition of activated ERBB-family receptors contributes to the reduced proliferation in these cells. We also observed a 3.5-fold decrease in phosphorylated glycogen synthase kinase (GSK)-3a/b 21/9 in Z119 and a 1.7-fold decrease in Z181. This suggests inactivation of GSK3, which is regulated by Akt/PKB signaling. We also observed a four-fold decrease in Bcr-Abl expression in Z181, identified as p190 by Western. This suggests that ERBB signaling helps maintain expression of Bcr-Abl protein, driving Ph+ lymphomagenesis, perhaps explaining why ERBB blockade induces apoptosis in Ph+ ALL. Elucidating the mechanism of this apoptosis, CI-1033 caused activation of caspase-3 in Z119 and Z181 as measured by DEVD-AMC cleavage, and caused PARP cleavage. Thus multiple, non-redundant signaling pathways downstream of ERBB can be downregulated with a small molecule inhibitor, CI-1033, causing reduced viability and increased apoptosis in ERBB+ Ph+ ALL cell lines. Our data suggest that inhibition of these signals may provide additional therapeutic efficacy to Ph+ ALL patients.

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