Abstract 2045: Oncoproteomic analysis of diffuse large B cell lymphomas reveals rationale for combination treatments.
Authors: Goldstein Rebecca L. , Cerchietti Leandro C. , Chiosis Gabriela , Melnick Ari 
Published: August 2014

The identification of novel therapeutic targets and drug combinations is critical to improve treatment for the 35% of diffuse large B cell lymphoma (DLBCL) patients who do not respond to standard immuno-chemotherapy. One such target is heat shock protein 90 (Hsp90), a molecular chaperone involved in protein folding and stability. Tumor cells are enriched for a fraction of Hsp90 (teHsp90) found in higher-order multi-chaperone complexes that preferentially chaperone oncogenic client proteins, which are depleted upon exposure to teHsp90 inhibition (teHsp90i). We have shown that PU-H71, a novel, purine-scaffold, highly selective teHsp90 inhibitor has potent anti-tumor activity in DLBCL cell lines and xenografts, and that combined inhibition of teHsp90 and its client protein (BCL6) synergizes to kill DLBCLs. We combined PU-H71 affinity-capture and proteomic analysis to identify the teHsp90 oncoproteome in two DLBCL cell lines OCI-Ly1 and OCI-Ly7, revealing a total of 844 proteins (602 in OCI-Ly1, 684 in OCI-Ly7, 441 (52%) in common). Of note, 19 proteins of the B cell receptor (BCR) pathway were identified. In DLBCLs, several proteins are mutated in this pathway resulting in its chronic activation, implicating the pathway in lymphomagenesis and survival of DLBCL. The interaction of teHsp90 with the identified BCR proteins was validated using PU-H71 affinity-capture followed by immunoblot. BCR proteins, specifically Syk and Btk, two of the pathway's proximal kinases, are depleted following treatment with PU-H71 (24 hours, 1M). Short treatment with PU-H71 (1M, 2 hours) decreases basal levels of activated Btk and Syk and also inhibits the activation of Btk and Syk following BCR stimulation with IgM and IgG. Combinatorial treatment of R406 (Syki) with PU-H71 synergizes to inhibit growth of DLBCL cell lines (CI GI50 0.73-0.97), and combinatorial treatment of PCI-32765 (Btki) with PU-H71 strongly synergizes to inhibit growth of ABC DLBCL cell lines (CI GI90 0.432-0.59). Combinatorial treatment of PCI-32765 (Btki) (12.5 mg/kg, ad libidum) with PU-H71 (75 mg/kg/day, ip injection) significantly decreases tumor growth as compared to PU-H71 alone (p-value = 0.03) in an ABC DLBCL xenograft model. Both subunits of CD79, the transmembrane signaling moiety of the BCR, were identified in the proteomics experiment suggesting a novel localization and function of teHsp90 in regulation of the BCR signalosome at the cell membrane. Total internal reflection fluorescent (TIRF) microscopy images depict the localization of Hsp90 at the cell membrane. Experiments to determine the effect of teHsp90i on IgM clustering and formation of the BCR signalosome are ongoing.

Citation Format: Rebecca L. Goldstein, Leandro C. Cerchietti, Gabriela Chiosis, Ari Melnick. Oncoproteomic analysis of diffuse large B cell lymphomas reveals rationale for combination treatments. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2045. doi:10.1158/1538-7445.AM2013-2045