Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity currently in clinical trials for relapsed multiple myeloma.Here we show that VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Exposure of multiple myeloma cells to VLX1570 resulted in thermostabilization of USP14 at therapeutically relevant concentrations. Transient knockdown of USP14 or UCHL5 expression by electroporation of siRNA reduced the viability of multiple myeloma cells. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response. Sensitivity to VLX1570 was moderately affected by altered drug uptake, but was unaffected by overexpression of BCL2-family proteins or inhibitors of caspase activity. Finally, treatment with VLX1570 was found to lead to extended survival in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.A diverse set of cellular processes such as cell cycle progression, DNA repair, metabolism and cell survival are dynamically controlled by the synthesis and degradation of protein regulators. In eukaryotic cells the regulated degradation of proteins is controlled mainly by the ubiquitin proteasome system (UPS)1 . The UPS is composed of a destruction tag in the form of the small protein ubiquitin and the 26S proteasome, a large multi-subunit proteolytic complex that specifically degrades ubiquitin tagged proteins into small peptides. The proteolytic activities of the proteasome reside within the 20S core particle (20S CP), a barrel like structure composed of 4 stacked heptameric rings (α 7 β 7 β 7 α 7 ) associated with one or two 19S regulatory particles (19S RP) 2,3 . Protein degradation begins with the covalent tagging of substrates with multi-ubiquitin chains, an event that initiates traffic to the proteasome and subsequent capture by highly specific ubiquitin receptors located within the 19S RP. Once bound, substrates undergo a sequence of modifications including de-ubiquitination by proteasome associated deubiquitinases (DUBs), unwinding by the 19S RP ATPases and finally translocation into the 20S CP where they are degraded 4 . Several roles for proteasome DUBs have been proposed including a rescue mechanism for improperly or poorly ubiquitinated substrates, maintenance of ubiquitin homeostasis by ubiquitin

Summary Deubiquitinase enzymes (DUBs) of the proteasomal 19S regulatory particle are emerging as important therapeutic targets in several malignancies. Here we demonstrate that inhibition of two proteasome-associated DUBs (USP14 and UCHL5) with the small molecule DUB inhibitor b-AP15, results in apoptosis of human Waldenström macroglobulinaemia (WM) cell lines and primary patient-derived WM tumour cells. Importantly, b-AP15 produced proteotoxic stress and apoptosis in WM cells that have acquired resistance to the proteasome inhibitor bortezomib. In silico modelling identified protein residues that were critical for the binding of b-AP15 with USP14 or UCHL5 and proteasome enzyme activity assays confirmed that b-AP15 does not affect the proteolytic capabilities of the 20S proteasome β-subunits. In vitro toxicity from b-AP15 appeared to result from a build-up of ubiquitinated proteins and activation of the endoplasmic reticulum stress response in WM cells, an effect that also disrupted the mitochondria. Focused transcriptome profiling of b-AP15-treated WM cells revealed modulation of several genes regulating cell stress and NF-κB signalling, the latter whose protein translocation and downstream target activation was reduced by b-AP15 in vitro. This is the first report to define the effects and underlying mechanisms associated with inhibition of USP14 and UCHL5 DUB activity in WM tumour cells.

Neem leaf extract (NLE) has medicinal properties, which have been attributed to its limonoid content. We identified the NLE tetranorterpenoid, nimbolide, as being the key limonoid responsible for the cytotoxicity of NLE in various preclinical models of human B-lymphocyte cancer. Of the models tested, Waldenströms macroglobulinemia (WM) cells were most sensitive to nimbolide, undergoing significant mitochondrial mediated apoptosis. Notably, nimbolide toxicity was also observed in drug-resistant (bortezomib or ibrutinib) WM cells. To identify putative targets of nimbolide, relevant in WM, we used chemoinformatics-based approaches comprised of virtual in silico screening, molecular modeling and target–ligand reverse docking. In silico analysis revealed the antiapoptotic protein BCL2 was the preferential binding partner of nimbolide. The significance of this finding was further tested in vitro in RS4;11 (BCL2-dependent) tumor cells, in which nimbolide induced significantly more apoptosis compared with BCL2 mutated (Jurkat BCL2Ser70-Ala) cells. Lastly, intraperitoneal administration of nimbolide in WM tumor xenografted mice, significantly reduced tumor growth and IgM secretion in vivo, while modulating the expression of several proteins as seen on immunohistochemistry. Overall, our data demonstrate that nimbolide is highly active in WM cells, as well as other B-cell cancers, and engages BCL2 to exert its cytotoxic activity.

In a continuing search for curcuminoid (CUR) compounds with antitumor activity, a novel series of heterocyclic CUR-BF adducts and CUR compounds based on indole, benzothiophene, and benzofuran along with their aryl pyrazoles were synthesized. Computational docking studies were performed to compare binding efficiency to target proteins involved in specific cancers, namely HER2, proteasome, VEGFR, BRAF, and Bcl-2, versus known inhibitor drugs. The majority presented very good binding affinities, similar to, and even more favorable than those of known inhibitors. The indole-based CUR-BF and CUR compounds and their bis-thiocyanato derivatives exhibited high anti-proliferative and apoptotic activity by in vitro bioassays against a panel of 60 cancer cell lines, more specifically against multiple myeloma (MM) cell lines (KMS11, MM1.S, and RPMI-8226) with significantly lower IC values versus healthy PBMC cells; they also exhibited higher anti-proliferative activity in human colorectal cancer cells (HCT116, HT29, DLD-1, RKO, SW837, and Caco2) than the parent curcumin, while showing notably lower cytotoxicity in normal colon cells (CCD112CoN and CCD841CoN).

Summary Multiple myeloma, the second most common haematological malignancy in the U.S., is currently incurable. Disruption of the intrinsic apoptotic pathway by BCL2 and MCL1 upregulation is observed in >80% of myeloma cases and is associated with an aggressive clinical course. Remarkably, there is no approved drug with the ability to target BCL2 or MCL1. Thus, we investigated the anti-tumour effects of a pan-BCL2 inhibitor, AT-101, which has high binding specificity for BCL2 and MCL1 in preclinical models of plasma cell cancers (Multiple myeloma and Waldenström macroglobulinaemia). Gene expression and immunoblot analysis of six plasma cell cancer models showed upregulation of BCL2 family members. AT-101 was able to downregulate BCL2 and MCL1 in all plasma cell cancer models and induced apoptotic cell death in a caspase-dependent manner by altering mitochondrial membrane permeability. This cytotoxic effect and BCL2 downregulation were further potentiated when AT-101 was combined with lenalidomide/dexamethasone (LDA). NanoString nCounter mRNA quantification and Ingenuity Pathways Analysis revealed differential changes in the CCNA2, FRZB, FYN, IRF1, PTPN11 genes in LDA-treated cells. In summary, we describe for the first time the cellular and molecular events associated with the use of AT-101 in combination with lenalidomide/dexamethasone in preclinical models of plasma cell malignancy.

Although ibrutinib is highly effective in Waldenstrom macroglobulinemia (WM), no complete remissions in WM patients treated with ibrutinib have been reported to date. Moreover, ibrutinib-resistant disease is being steadily reported and is associated with dismal clinical outcome (overall survival of 2.9–3.1 months). To understand mechanisms of ibrutinib resistance in WM, we established ibrutinib-resistant in vitro models using validated WM cell lines. Characterization of these models revealed the absence of BTKC481S and CXCR4WHIM-like mutations. BTK-mediated signaling was found to be highly attenuated accompanied by a shift in PI3K/AKT and apoptosis regulation-associated genes/proteins. Cytotoxicity studies using the AKT inhibitor, MK2206±ibrutinib, and the Bcl-2-specific inhibitor, venetoclax±ibrutinib, demonstrated synergistic loss of cell viability when either MK22016 or venetoclax were used in combination with ibrutinib. Our findings demonstrate that induction of ibrutinib resistance in WM cells can arise independent of BTKC481S and CXCR4WHIM-like mutations and sustained pressure from ibrutinib appears to activate compensatory AKT signaling as well as reshuffling of Bcl-2 family proteins for maintenance of cell survival. Combination treatment demonstrated greater (and synergistic) antitumor effect and provides rationale for development of therapeutic strategies encompassing venetoclax+ibrutinib or PI3K/AKT inhibitors+ibrutinib in ibrutinib-resistant WM.

The survival of Waldenstrom macroglobulinemia (WM) tumor cells hinges on aberrant B-cell receptor (BCR) and MYD88 signaling. WM cells upregulate the proteasome function to sustain the BCR-driven growth while maintaining homeostasis. Clinically, two treatment strategies are used to disrupt these complementary yet mutually exclusive WM survival pathways via ibrutinib (targets BTK/MYD88 node) and bortezomib (targets 20 S proteasome). Despite the success of both agents, WM patients eventually become refractory to treatment, highlighting the adaptive plasticity of WM cells and underscoring the need for development of new therapeutics. Here we provide a comprehensive preclinical report on the anti-WM activity of VLX1570, a novel small-molecule inhibitor of the deubiquitinating enzymes (DUBs), ubiquitin-specific protease 14 (USP14) and ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5). Both DUBs reside in the 19 S proteasome cap and their inhibition by VLX1570 results in rapid and tumor-specific apoptosis in bortezomib- or ibrutinib-resistant WM cells. Notably, treatment of WM cells with VLX1570 downregulated BCR-associated elements BTK, MYD88, NFATC, NF-κB and CXCR4, the latter whose dysregulated function is linked to ibrutinib resistance. VLX1570 administered to WM-xenografted mice resulted in decreased tumor burden and prolonged survival (P=0.0008) compared with vehicle-treated mice. Overall, our report demonstrates significant value in targeting USP14/UCHL5 with VLX1570 in drug-resistant WM and carries a high potential for clinical translation.

Purpose: CD38 has emerged as a high-impact therapeutic target in multiple myeloma, with the approval of daratumumab (anti-CD38 monoclonal antibody). The clinical importance of CD38 in chronic lymphocytic leukemia (CLL) patients has been known for over two decades, though it’s relevance as a therapeutic target in CLL remains understudied. Experimental Design: We investigated the biological effects and anti-tumor mechanisms engaged by daratumumab in primary CLL cells. Besides its known immune-effector mechanisms (ADCC, CDC and ADCP), we also measured direct apoptotic effects of daratumumab alone or in combination with ibrutinib. In vivo anti-leukemic activity was assessed in a partially-humanized xenograft model. The influence of CD38 on BCR signaling was measured via immunoblotting of Lyn, Syk, BTK, PLCγ2, ERK1/2 and AKT. Results: In addition to immune-effector mechanisms; daratumumab also induced direct apoptosis of primary CLL cells, which was partially dependent on FcγR crosslinking. For the first time, we demonstrated the influence of CD38 on BCR signaling where interference of CD38 downregulated Syk, BTK, PLCγ2, ERK1/2 and AKT; effects that were further enhanced by addition of ibrutinib. In comparison to single agent treatment, the combination of ibrutinib and daratumumab resulted in significantly enhanced anti-CLL activity in vitro and significantly decreased tumor growth and prolonged survival in the in vivo CLL xenograft model. Conclusions: Overall, our data demonstrate the anti-tumor mechanisms of daratumumab in CLL; furthermore, we show how co-targeting BTK and CD38 lead to a robust anti-CLL effect, which has clinical implications.