Rebecca L. Goldstein

Targeting the Hsp90-associated viral oncoproteome in gammaherpesvirus-associated malignancies.

PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV(+) primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.

EZH2 and BCL6 Cooperate to Assemble CBX8-BCOR Complex to Repress Bivalent Promoters, Mediate Germinal Center Formation and Lymphomagenesis

The EZH2 histone methyltransferase mediates the humoral immune response and drives lymphomagenesis through formation of bivalent chromatin domains at critical germinal center (GC) B cell promoters. Herein we show that the actions of EZH2 in driving GC formation and lymphoma precursor lesions require site-specific binding by the BCL6 transcriptional repressor and the presence of a non-canonical PRC1-BCOR-CBX8 complex. The chromodomain protein CBX8 is induced in GC B cells, binds to H3K27me3 at bivalent promoters, and is required for stable association of the complex and the resulting histone modifications. Moreover, oncogenic BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in DLBCLs.

Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma

Diffuse large B cell lymphomas (DLBCLs) arise from proliferating B cells transiting different stages of the germinal center reaction. In activated B cell DLBCLs (ABC-DLBCLs), a class of DLBCLs that respond poorly to current therapies, chromosomal translocations and amplification lead to constitutive expression of the B cell lymphoma 6 (BCL6) oncogene. The role of BCL6 in maintaining these lymphomas has not been investigated. Here, we designed small-molecule inhibitors that display higher affinity for BCL6 than its endogenous corepressor ligands to evaluate their therapeutic efficacy for targeting ABC-DLBCL. We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor called FX1 that has 10-fold greater potency than endogenous corepressors and binds an essential region of the BCL6 lateral groove. FX1 disrupted formation of the BCL6 repression complex, reactivated BCL6 target genes, and mimicked the phenotype of mice engineered to express BCL6 with corepressor binding site mutations. Low doses of FX1 induced regression of established tumors in mice bearing DLBCL xenografts. Furthermore, FX1 suppressed ABC-DLBCL cells in vitro and in vivo, as well as primary human ABC-DLBCL specimens ex vivo. These findings indicate that ABC-DLBCL is a BCL6-dependent disease that can be targeted by rationally designed inhibitors that exceed the binding affinity of natural BCL6 ligands.

Pharmacoproteomics identifies combinatorial therapy targets for diffuse large B cell lymphoma

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.

Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphomas

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.

Effective combination therapies for B cell lymphoma predicted by a virtual disease model

The complexity of cancer signaling networks limits the efficacy of most single-agent treatments and brings about challenges in identifying effective combinatorial therapies. In this study, we used chronic active B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma as a model system to establish a computational framework to optimize combinatorial therapy in silico We constructed a detailed kinetic model of the BCR signaling network, which captured the known complex cross-talk between the NFκB, ERK, and AKT pathways and multiple feedback loops. Combining this signaling model with a data-derived tumor growth model, we predicted viability responses of many single drug and drug combinations in agreement with experimental data. Under this framework, we exhaustively predicted and ranked the efficacy and synergism of all possible combinatorial inhibitions of eleven currently targetable kinases in the BCR signaling network. Ultimately, our work establishes a detailed kinetic model of the core BCR signaling network and provides the means to explore the large space of possible drug combinations. Cancer Res; 77(8); 1818-30. ©2017 AACR.

How Biophysical Forces Regulate Human B Cell Lymphomas

Summary The role of microenvironment-mediated biophysical forces in human lymphomas remains elusive. Diffuse large B cell lymphomas (DLBCLs) are heterogeneous tumors, which originate from highly proliferative germinal center B cells. These tumors, their associated neo-vessels, and lymphatics presumably expose cells to particular fluid flow and survival signals. Here, we show that fluid flow enhances proliferation and modulates response of DLBCLs to specific therapeutic agents. Fluid flow upregulates surface expression of B cell receptors (BCRs) and integrin receptors in subsets of ABC-DLBCLs with either CD79A/B mutations or WT BCRs, similar to what is observed with xenografted human tumors in mice. Fluid flow differentially upregulates signaling targets, such as SYK and p70S6K, in ABC-DLBCLs.By selective knockdown of CD79B and inhibition of signaling targets, we provide mechanistic insights into how fluid flow mechanomodulates BCRs and integrins in ABC-DLBCLs. These findings redefine microenvironment factors that regulate lymphoma-drug interactions and will be critical for testing targeted therapies.

Abstract 2045: Oncoproteomic analysis of diffuse large B cell lymphomas reveals rationale for combination treatments.

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 pathway9s 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 GI 50 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 GI 90 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

Abstract 4829: A purine scaffold Hsp90 inhibitor has antitumor activity in KSHV-associated malignancies by suppressing vFLIP

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Kaposis sarcoma herpesvirus (KSHV/ HHV-8), a member of the α-herpesvirus family of human DNA viruses, is the etiologic agent of several malignancies in immune-compromised individuals, such as Kaposis sarcoma (KS) and primary effusion lymphoma (PEL). Activated NF-κB is a critical mechanism by which KSHV-infected PEL cells are protected from apoptosis. KSHV vFLIP has been identified as a viral oncogene that is responsible for NF-κB-dependent anti-apoptotic gene expression in PEL cells, and also prevents autophagy in KSHV-infected endothelial cells. In particular, the IKK signaling complex consisting of IKKα, IKKα, IKKα, vFLIP and Hsp90 was shown to be essential for survival in PEL cells. The chaperone protein Hsp90 binds client proteins involved in the regulation of cell survival and apoptosis signal transduction, including Akt, IKK complex, and KSHV vFLIP. A lack of Hsp90 causes protein misfolding, ubiquitination and degradation. The Hsp90 inhibitor geldanamycin was previously tested in PEL cells and shown to inhibit activity of the IKK complex in vitro. However, geldanamycin is of limited therapeutic potential due to its undesirable pharmacophysiology. We tested a new purine-scaffold Hsp90 inhibitor with high selectivity for tumor versus normal cell Hsp90, which is water-soluble with high oral bioavailability and excellent therapeutic window. Materials and methods: We evaluated the sensitivity of several KSHV infected and uninfected cell lines to treatment with this inhibitor, designated PU-H71. We performed viability and NF-κB reporter luciferase assays, and immunoblot and qRT-PCR analyses of cellular and viral proteins. Finally, we used a mouse PEL xenograft model and in vivo imaging to assess tumor responses to PU-H71. Results: We found all KSHV-positive PEL cell lines to be exquisitely sensitive when compared to uninfected lymphoma cells, with LC50s in the nanomolar range. PU-H71 was shown to induce PEL cell death by apoptosis and autophagy within 48 hours of treatment. PU-H71 also showed preferential toxicity in an in vitro model of KS. Pulldown demonstrated association of the compound with the active vFLIP-IKKα signalosome. Western blot analysis indicated that the IKK signaling complex was destabilized, and components vFLIP and IKKα were degraded upon PU-H71 treatment, resulting in inhibition of NF-κB signaling, as confirmed by reporter luciferase assay. qRT-PCR and reporter analysis indicated low levels of concomitant lytic reactivation. PU-H71 was further shown to inhibit progression of tumor spread and confer a significant survival advantage (p<0.02) in a mouse xenograft model of PEL, with persistence of IKKα destabilization ex vivo. Conclusions: Our findings demonstrate that Hsp90 inhibition with PU-H71 leads to reduced vFLIP levels in KSHV-infected cells, and to tumor responses. Thus, PU-H71 is a promising targeted approach for the treatment of KS and PEL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4829. doi:1538-7445.AM2012-4829

Abstract B12: EIF4E deregulation drives simultaneous expression of B-cell lymphoma oncogenes.

The eukaryotic translation initiation factor 4E (eIF4E) is an oncogene elevated in a large number of cancers. The oncogenic potential of eIF4E arises from its critical roles in the cytoplasm in the mRNA translation and in the nucleus in the mRNA export of specific subset of transcripts. These transcripts can be regulated at the cytoplasmic, nuclear export or at both levels. We therefore analyzed eIF4E expression in 105 cases of DLBCLs by IHC, and found that is expressed in almost all the tumors (88%) and for the majority of cases (72%), eIF4E was expressed in both cellular compartments. Since elevated eIF4E activity leads to increased protein production that favors a subset of transcripts including prominent DLBCL oncogenes such as MYC and BCL2, we analyzed eIF4E expression in “double-hit” and “triple hit” DLBCLs. All the pts samples and DLBCL cell lines harboring two or more oncogenic mutations expressed eIF4E at high levels, suggesting a potential mechanistic association. Cells engineered to overexpress wild-type eIF4E, or mutants active in translation but not export (S53A) or mutants active in export but not translation (W73A), confirmed that eIF4E regulates the nuclear export and translation of MYC and BCL2, and discovered that this mechanism regulates additional DLBCL oncogenes such as BCL6. EIF4E immunoprecipitation from triple-hit DLBCL nuclear fractions followed by mRNA amplification by QPCR (RIP-QPCR) determined that MYC, BCL2 and BCL6 were direct eIF4E mRNA export targets. Polysomal profiling demonstrated that these oncogenes are also preferentially translated by eIF4E cytosolic activity. Accordingly, treatment of triple-hit DLBCL cell lines with the eIF4E competitive inhibitor ribavirin significantly increased the nuclear entrapment of BCL6, MYC and BCL2 transcripts, decreased their proportion of heavy polysomes and lead to a decrease in their protein abundance. In an extended panel of six double- and triple-hit DLBCL cell lines, ribavirin induced cell killing in all of them at clinically achievable concentrations. To assess the anti-lymphoma effect of ribavirin in a more clinically relevant context, we established a patient derived xenograft (PDX) in NSG mice. The specimen was isolated from a naive treatment patient harboring a triple-hit ABC-type DLBCL featuring BCL6 translocation (3q27), BCL2 translocation and MYC amplification. The patient presented a treatment-refractory disease. We then expanded PDX into 10 NSG mice and when tumors were palpable, mice were randomized to receive vehicle or ribavirin for 10 days. We found a significant reduction in tumor growth in PDX. We also found that ribavirin induced mRNA nuclear entrapment of BCL2 and BCL6 and decrease protein levels of BCL2, BCL6 and MYC by day 10. We demonstrated that Hsp90 activity is required to maintain eIF4E-containing heavy polysomes in these oncogene transcripts. Accordingly, Hsp90 inhibition with PU-H71 rapidly promotes ribosomal disassembly leading to increased 40S, 60S and monosomes and decreased heavy polysomes. EIF4E target transcripts such as MYC, BCL6 and BCL2 were more affected, consequently decreasing the protein levels of these transcripts. The combination of Hsp90 and eIF4E inhibition reduced more efficiently the protein levels of these transcripts. To determine whether this molecular effect translates into an improved anti-lymphoma effect, we xenografted two triple-hit DLBCL cell lines in SCID mice and we treated them with vehicle control, ribavirin, PU-H71 or the combination for 10 days. We found that the combination of ribavirin with PU-H71 suppressed lymphoma growth more profoundly in both models. This effect was associated with a decrease in proliferation rate (measured by Ki67 staining) and reduced BCL6 expression. There were no macroscopic or microscopic evidence of toxicity in these mice. In summary, our data provides a novel and potentially non-toxic mechanistically-based approach to target B-cell lymphomas harboring multiple oncogene activation. Given the prevalence of simultaneous chromosomal aberrations in other hematological malignancies we envision a wider role for this strategy. Citation Format: Biljana Kraljacic-Culkjovic, Tharu Fernando, Rebecca Goldstein, Charles Mctavish, Jayeshkumar Patel, Shaoning Yang, Fabrizio Tabbo, Ari Melnick, Giorgio Inghirami, Katherine LB Borden, Leandro Cerchietti. EIF4E deregulation drives simultaneous expression of B-cell lymphoma oncogenes. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B12.