Unfolded Protein Response Regulator Bip/Hspa5 As A Chemoresistance Biomarker And Molecular Target In Acute Lymphoblastic Leukemia

BIP/HSPA5, also known as glucose regulated protein (GRP) 78, is an endoplasmic reticulum (ER)-resident multifunctional molecular chaperone that represents a pivotal component of the pro-survival arm of the unfolded protein response (UPR) signaling pathway. We performed a comprehensive meta-analysis of 17 datasets and 668 leukemia/lymphoma cases using the Oncomine Clinical Research Database. 7 of 10 contrasts for high-grade B-lineage lymphoid malignancies exhibited signigficant up-regulation of BIP/HSPA5, including B-lineage acute lymphoblastic leukemia (ALL) (2-fold, P=5.9×10–10). Notably, the gene expression profiles of primary leukemia cells in matched-pair leukemic specimens obtained from 27 B-lineage ALL patients both at the time of initial diagnosis and at the time of relapse were extracted from the NCBI Gene Expression Omnibus (GSE18497) revealed a significant 1.4-fold up-regulation of the BIP8/HSPA5 gene in relapse clones (P=0.0016). Likewise, anti-BIP/HSPA5 Western blot analysis of whole cell lysates from primary leukemic cells from relapsed and newly diagnosed B-lineage ALL patients revealed higher levels of BIP/HSPA5 protein in relapse specimens. Confocal microscopy revealed abundant expression of BIP/HSPA5 protein in the plasma membrane of chemotherapy-resistant ALL cells. Pep42, a cyclic 13-mer cell-penetrating peptide that was identified by screening a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cells, was found to not only specifically bind to surface expressed BIP/HSPA5 but also enter BIP/HSPA5+ cells by receptor-mediated endocytosis. We examined the ability of a doxorubicin conjugate of Pep42 (PEP-DOX) containing a cathepsin B-cleavable linker to target and induce apoptosis in surface BIP/HSPA5+ chemotherapy-resistant B-lineage ALL cells. By using a quantitative flow cytometric apoptosis assay, we documented that within 48 hours of treatment with 0.1–1.0 μM PEP-DOX (but not unconjugated drug-free Pep42, which was included as a control) apoptosis is induced in ≥99.9% of surface BIP/HSPA5+ ALL cells. As PEP-DOX specifically targets surface expressed BIP/HSPA5, 100-fold molar excess of drug-free Pep42 was able to compete with PEP-DOX for surface BIP/HSPA5 binding sites and thereby prevent PEP-DOX-induced apoptosis. Excess recombinant EGF, that was included as a negative control, did not affect PEP-DOX- induced apoptosis. The identification of the BIP/HSPA5 as a chemoresistance biomarker and molecular target for B-lineage ALL may lead to new anti-leukemic treatment strategies for relapsed disease that are much needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-365. doi:10.1158/1538-7445.AM2011-LB-365