Comprehensive Analysis of the Structural, Biochemical and Signaling Differences of the p210 and p185 Isoforms of Bcr-Abl in CML and B-ALL

Abstract

Distinct chromosomal translocation breakpoints result in the expression of two major isoforms of Bcr-Abl - p210 and p185 - from the Philadelphia (Ph)-chromosome. While p210 is the molecular hallmark of Chronic Myeloid Leukemia (CML), in Ph-positive B-cell Acute Lymphoblastic Leukemia (B-ALL), ~1/3 of the patients express p210 and ~2/3 express p185. In mouse models, under certain experimental conditions, expression of p185 causes disease with a shorter latency and more B-ALL than CML-like leukemias as did p210. Ph-positive B-ALL patients show a poorer response and frequent resistance to tyrosine kinase inhibitor (TKI) therapy as compared to CML patients. Specific intrinsic differences in p185 and p210 Bcr-Abl signaling have long been hypothesized, but have never been studied in a comparative, unbiased and comprehensive manner. We have now:(i) Mapped the differences in the p210 and p185 protein interaction network: We performed an unbiased proteomics analysis of the composition of the endogenous p210 and p185 protein complexes in murine cell lines using SILAC (stable isotope labeling with amino acids in cell culture) quantification. The interactome analysis confirmed previously reported p210 interactors in human CML cells and identified unexpectedly many interactors that preferentially associate with either p185 or p210, including an endocytosis-regulating complex (enriched in p185) and a phosphatase complex (enriched in p210). Several differential interactors were validated in a panel of nine human CML and B-ALL cell lines.(ii) Performed a comprehensive phospho-proteomics study in p210 and p185 cell lines: Using phospho-tyrosine, as well as general phospho-peptide enrichment strategies coupled to SILAC quantification, we mapped >800 phosphorylation sites and identified differentially activated signaling by p210 and p185, including differential activation of the STAT5 and STAT3 transcription factors, SRC kinases and ERK1/2 signaling. These findings were validated in a panel of nine human CML and B-ALL cell lines.(iii) Determined the atomic structures of the Dbl-homology (DH) and pleckstrin homology (PH) domains of p210: The DH-PH domain unit of p210 is the only structural difference to p185 and has remained the only structurally uncharacterized region of Bcr-Abl. We solved different structures of these domains by NMR spectroscopy, X-ray crystallography, as well as Small-Angle X-ray scattering, alone and in complex with an engineered monobody inhibitor targeting the DH domain. While the DH-PH structural motif is found in Rho guanine nucleotide exchange factors (Rho GEF), our results have not provided evidence that the p210 DH-PH domain acts as a Rho GEF or binds Rho GTPases, in contrast to previous reports. On the other hand, we found the PH domain to be important for specific membrane binding of p210 and we determined the binding specificity of the PH domain to certain phospho-inositide lipids.Our results provide the first systematic insight showing critical differences in protein-protein interactions, signaling and structure of p210 and p185 Bcr-Abl. This will contribute to a better understanding of Bcr-Abl dependent signaling, mechanisms of oncogenic transformation, resulting disease pathobiology and responses to TKIs. DisclosuresHantschel:Ariad Pharmaceuticals: Honoraria.