Chenghua Yang

MALT1 Small Molecule Inhibitors Specifically Suppress ABC-DLBCL In Vitro and In Vivo

MALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo.

Structural Architecture of the CARMA1/Bcl10/MALT1 Signalosome: Nucleation-Induced Filamentous Assembly

The CARMA1/Bcl10/MALT1 (CBM) signalosome mediates antigen receptor-induced NF-κB signaling to regulate multiple lymphocyte functions. While CARMA1 and Bcl10 contain caspase recruitment domains (CARDs), MALT1 is a paracaspase with structural similarity to caspases. Here we show that the reconstituted CBM signalosome is a helical filamentous assembly in which substoichiometric CARMA1 nucleates Bcl10 filaments. Bcl10 filament formation is a highly cooperative process whose threshold is sensitized by oligomerized CARMA1 upon receptor activation. In cells, both cotransfected CARMA1/Bcl10 complex and the endogenous CBM signalosome are filamentous morphologically. Combining crystallography, nuclear magnetic resonance, and electron microscopy, we reveal the structure of the Bcl10 CARD filament and the mode of interaction between CARMA1 and Bcl10. Structure-guided mutagenesis confirmed the observed interfaces in Bcl10 filament assembly and MALT1 activation in vitro and NF-κB activation in cells. These data support a paradigm of nucleation-induced signal transduction with threshold response due to cooperativity and signal amplification by polymerization.

Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2

Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90β, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.

Identification of an Allosteric Pocket on Human Hsp70 Reveals a Mode of Inhibition of This Therapeutically Important Protein

Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70.

The epichaperome is an integrated chaperome network that facilitates tumour survival

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes—dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically ‘rewired’ to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.

Affinity Purification Probes of Potential Use To Investigate the Endogenous Hsp70 Interactome in Cancer

Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins.

The CBM signalosome: Potential therapeutic target for aggressive lymphoma?

The CBM signalosome plays a pivotal role in mediating antigen-receptor induced NF-κB signaling to regulate lymphocyte functions. The CBM complex forms filamentous structure and recruits downstream signaling components to activate NF-κB. MALT1, the protease component in the CBM complex, cleaves key proteins in the feedback loop of the NF-κB signaling pathway and enhances NF-κB activation. The aberrant activity of the CBM complex has been linked to aggressive lymphoma. Recent years have witnessed dramatic progresses in understanding the assembly mechanism of the CBM complex, and advances in the development of targeted therapy for aggressive lymphoma. Here, we will highlight these progresses and give an outlook on the potential translation of this knowledge from bench to bedside for aggressive lymphoma patients.

Abstract A19: MALT1 inhibition as an anchor for combinatorial therapy of ABC-DLBCL.

MALT1 (Mucossa Associated Lymphoid tissue Lymphoma Translocated protein 1) is critical for the proliferation and survival of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL), the most chemo-resistant form of DLBCL. MALT1 mediates activation of the B-cell receptor pathway (BCR) downstream of characteristic somatic mutations in CD79, CARD11 or MYD88 that lead to chronically activated NF-κB. MALT1 is a paracaspase and the effector enzyme of the CARD11/Bcl10/MALT1 signalosome, a high order assembly that functions as an amplifier of BCR signaling to NF-κB. MALT1 constitutes a compelling therapeutic target because: i) it is the only paracaspase in humans, ii) MALT1 knockout mice are viable, and iii) ABC-DLBCLs are biologically dependent on MALT1 paracaspase activity. MALT1 is only active when forming multimeric complexes. In order to identify potential MALT1 inhibitors we engineered a leucine zipper-MALT1, obliged and enzymatically active dimer, and established a paracaspase enzymatic assay for high throughput screening. Screening a ~50,000 compound chemical diversity library allowed us the identification and validation of 19 distinct chemical scaffolds that inhibited MALT1 with an IC50 Given that multiple pathways contribute to ABC-DLBCL pathogenesis, we hypothesized that MALT1 inhibitors would be most effective within combinatorial therapy regimens. Along these lines MI-2 strongly enhanced the activity of CHOP chemotherapy drugs against ABC-DLBCL cells. The addition of MI-2 to doxorubicin allowed for 2.5 to 13-fold reduction in the doxorubicin dose as determined by the dose-reduction index that was specific for the doxorubicin resistant cell lines (GI50 > 200 nM). Because the BCR pathway constitutes a complex network of signaling molecules beyond NF-κB activation, we tested combination of MI-2 with inhibitors of other proteins in this pathway affecting other branches of this pathway. Combination of MI-2 with the pan PI3K inhibitor BKM120, that was in our hands the most effective against a broad group of ABC-DLBCL cell lines, resulted in synergistic cell killing of OCI-Ly10 and Rc-K8 and had an additive effect in HBL-1 while it was less than additive for OCI-Ly3 and TMD8. These cell lines harbor mutations in different proteins of the pathway, which may contribute to the differences in response to the combination. Finally MI-2 strongly synergized with BH3 mimetics (most notably ABT-737) that target fundamental complementary survival pathways to BCR signaling in ABC-DLBCLs. Synergistic killing was at least partially due to induction of apoptosis, as concurrent administration of the two drugs induced increased apoptosis assessed by Caspase-7/3 activity and Annexin V+ DAPI- flow cytometry. In summary, we identified the first specific MALT1 inhibitor drug and demonstrated a promising role for MALT1 targeted therapy as an anchor of rational combinatorial therapy against ABC-DLBCL. Citation Format: Lorena Fontan, Chenghua Yang, Himaly Shinglot, Venkataraman Kabaleeswaran, Volpon Laurent, Michael Osborne, Elena Beltran, Monica Rosen, Rita Shaknovich, Shao N. Yang, Randy D. Gascoyne, Leandro Cerchietti, Jose A. MArtinez-Climent, J Fraser Glickman, KAtherine Borden, Hao Wu, Ari Melnick. MALT1 inhibition as an anchor for combinatorial therapy of ABC-DLBCL. [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 A19.

Abstract 1733: Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells

Background: Heat shock protein 70 family members play an important role in cancer. They are up-regulated in wide variety of tumors and the increased Hsp70 protein expression correlates with metastases, resistance to treatment and poor prognosis. Multiple mechanisms explain cancer cells dependence on Hsp70, such as inhibition of apoptosis by Hsp70, induction of autophagy and control of stability of onco-proteins. These Hsp70 activities are mediated in cancer by its ability to chaperone and interact with a large number of proteins in a cell-specific, context dependent manner. Hypothesis: Reagents that enable the capture of tumor-specific Hsp70 complexes facilitate the identification of context-dependent Hsp70 interactomes. Results: Our laboratory recently reported the identification of a novel allosteric site located in the nucleotide binding domain of Hsp70 (Chem Biol 2013). It has also reported the discovery of ligands that bind to the allosteric pocket of Hsp70, inhibit its function in cancer cells and result in anti-cancer activity (J Med Chem 2013). Structure-activity relationship studies in this ligand series gave insights on the attachment of specific linkers for the design of Hsp70-related chemical tools. Here we present the design of Hsp70-directed reagents and use biochemical and cell-based methods to validate Hsp70-directed affinity purification probes. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins. Significance: The knowledge derived from the use of such reagents will be extremely valuable not only to understand tumor-specific roles of Hsp70 and associated mechanisms but also to develop rational strategies for the clinical implementation of these agents to cancer treatment. They may also provide clues on the altered functional proteome in individual tumors, a quest yet elusive by today9s proteomics methods. Citation Format: Anna A. Rodina, Tony Taldone, Yanlong Kang, Pallav Patel, John Koren, Pengrong Yan, Erica DaGama Gomes, Chenghua Yang, Maulik Patel, Liza Shrestha, Stefan Ochiana, Ronnie Maharaj, Alexander Gozman, Marc Cox, Hediye Erdjument-Bromage, Ronald Hendrickson, Leandro Cerchietti, Ari Melnick, Monica Guzman, Gabriela Chiosis. Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2015-1733

Abstract 2693: Harnessing MALT1 inhibition for rational combinatorial therapy of ABC-DLBCL

The MALT1 paracaspase plays a critical role in the proliferation and survival of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL), the most chemo-resistant form of DLBCL. MALT1 mediates activation of the B-cell receptor (BCR) downstream of somatic mutations in signaling components such as: CD79, CARD11, A20 or MYD88, leading to chronically activated NF-κB. MALT1 is the effector enzyme of the CARD11/Bcl10/MALT1 signalosome, a massive, high order structure that functions as an amplifier of BCR signaling to NF-κB. MALT1 is a compelling therapeutic target since: i) it is the only paracaspase in humans, ii) MALT1 knockout mice are viable, and iii) ABC-DLBCLs are biologically dependent on MALT1 activity. MALT1 is only active when forming multimeric complexes. In order to identify potential MALT1 inhibitors we biochemically engineered an obligate dimerized form of MALT1 and an enzymatic assay for high throughput screening. We screened a ∼50,000 compound chemical diversity library and identified and validated 19 distinct chemical scaffolds that inhibited MALT1 with an IC50 Given that multiple pathways contribute to ABC-DLBCL pathogenesis we hypothesized that MALT1 inhibitors would be most effective within combinatorial therapy regimens. Along these lines MI-2 strongly enhanced the activity of CHOP chemotherapy drugs against ABC-DLBCL cells. BCR signaling forms a complex network of signaling molecules beyond NF-κB, and accordingly MALT1 targeted therapy was strongly enhanced with small molecules that affect other branches of this pathway, such as PI3K inhibitors (e.g. BKM120). Finally MI-2 synergized with small molecules such as BH3 mimetics (most notably ABT-737) that target fundamental complementary survival pathways to BCR signaling in ABC-DLBCLs. In summary, we identified the first specific MALT1 inhibitor drug and demonstrated a promising role for MALT1 targeted therapy as an anchor of rational combinatorial therapy against ABC-DLBCL. Citation Format: Lorena Fontan, Chenghua Yang, Venkataraman Kabaleeswaran, Laurent Volpon, Michael Osborne, Elena Beltran, Monica Rosen, Rita Shaknovich, Shao N. Yang, Randy D. Gascoyne, Leandro Cerchietti, Jose A. Martinez-Climent, J. Fraser Glickman, Katherine Borden, Hao Wu, Ari Melnick. Harnessing MALT1 inhibition for rational combinatorial therapy of ABC-DLBCL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2693. doi:10.1158/1538-7445.AM2014-2693