Pharmacoproteomics identifies combinatorial therapy targets for diffuse large B cell lymphoma
Rebecca L. Goldstein, Shao Ning Yang, Tony Taldone, Betty Chang, John Gerecitano, Kojo Elenitoba-Johnson, Rita Shaknovich, Wayne Tam, John P. Leonard, Gabriela Chiosis, Leandro Cerchietti, Ari Melnick
Rebecca L. Goldstein, Shao Ning Yang, Tony Taldone, Betty Chang, John Gerecitano, Kojo Elenitoba-Johnson, Rita Shaknovich, Wayne Tam, John P. Leonard, Gabriela Chiosis, Leandro Cerchietti, Ari Melnick
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Research Article Oncology

Pharmacoproteomics identifies combinatorial therapy targets for diffuse large B cell lymphoma

Abstract

Rationally designed combinations of targeted therapies for refractory cancers, such as activated B cell–like diffuse large B cell lymphoma (ABC DLBCL), are likely required to achieve potent, durable responses. Here, we used a pharmacoproteomics approach to map the interactome of a tumor-enriched isoform of HSP90 (teHSP90). Specifically, we chemically precipitated teHSP90-client complexes from DLBCL cell lines with the small molecule PU-H71 and found that components of the proximal B cell receptor (BCR) signalosome were enriched within teHSP90 complexes. Functional assays revealed that teHSP90 facilitates BCR signaling dynamics by enabling phosphorylation of key BCR signalosome components, including the kinases SYK and BTK. Consequently, treatment of BCR-dependent ABC DLBCL cells with PU-H71 attenuated BCR signaling, calcium flux, and NF-κB signaling, ultimately leading to growth arrest. Combined exposure of ABC DLBCL cell lines to PU-H71 and ibrutinib, a BCR pathway inhibitor, more potently suppressed BCR signaling than either drug alone. Correspondingly, PU-H71 combined with ibrutinib induced synergistic killing of lymphoma cell lines, primary human lymphoma specimens ex vivo, and lymphoma xenografts in vivo, without notable toxicity. Together, our results demonstrate that a pharmacoproteome-driven rational combination therapy has potential to provide more potent BCR-directed therapy for ABC DLCBL patients.

Authors

Rebecca L. Goldstein, Shao Ning Yang, Tony Taldone, Betty Chang, John Gerecitano, Kojo Elenitoba-Johnson, Rita Shaknovich, Wayne Tam, John P. Leonard, Gabriela Chiosis, Leandro Cerchietti, Ari Melnick

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Figure 4

Inhibition of teHSP90 induces broad attenuation of BCR signaling at multiple nodes.

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Inhibition of teHSP90 induces broad attenuation of BCR signaling at mult...
(A) Lysates of HBL-1, TMD8, OCI-Ly1, and U2932 cells exposed to vehicle or PU-H71 (1 μM) were immunoprecipitated with antibodies to CD79A or IgG and immunoblotted with indicated antibodies. (B) HBL-1 and OCI-Ly10 cells treated with vehicle or PU-H71 (1 μM, 1 h) before BCR stimulation (IgM + IgG, 10 μg/ml, 15 min) were fixed, permeabilized, and stained with phospho-antibodies or isotype controls. Phospho-proteins were quantified using flow cytometry in 3 biological replicates using unpaired t test. (C) Lysates of HBL-1, TMD8, OCI-Ly1, and U2932 cells treated with vehicle or PU-H71 (1 μM, 1 h) before BCR stimulation (IgM + IgG, 10 g/ml, 15 min) were subjected to immunoblotting. (D) HBL-1 and TMD8 cells were treated with vehicle or PU-H71 (1 μM, 2 h) then incubated with a fluorescent calcium indicator (Fluo-4 AM, 2 μM, 30 min). Calcium release was measured over time by flow cytometry before and after BCR stimulation (IgM + IgG 10 μg/ml, 120 s). Three biological replicates; unpaired t test. (E) HBL-1 and TMD8 cells were transfected with an NF-κB luciferase reporter and treated with PU-H71 (1 μM) and/or BCR stimulation (IgM + IgG, 10 μg/ml, 16 hours). Luciferase activity was measured and is shown as average ± SEM relative to vehicle (n ≥ 3; unpaired t test). (F) Model of PU-H71 effects on BCR signaling at multiples nodes: BCR signalosome stability, kinase activity, calcium release, and NF-κB activity.