NF-κB dysregulation in multiple myeloma.

The nuclear factor-κB (NF-κB) transcription factor family plays critical roles in the pathophysiology of hematologic neoplasias, including multiple myeloma. The current review examines the roles that this transcription factor system plays in multiple myeloma cells and the nonmalignant accessory cells of the local microenvironment; as well as the evidence indicating that a large proportion of myeloma patients harbor genomic lesions which perturb diverse genes regulating the activity of NF-κB. This article also discusses the therapeutic targeting of the NF-κB pathway using proteasome inhibitors, a pharmacological class that has become a cornerstone in the therapeutic management of myeloma; and reviews some of the future challenges and opportunities for NF-κB-related research in myeloma.

Functional-genetic dissection of HDAC dependencies in mouse lymphoid and myeloid malignancies.

Histone deacetylase (HDAC) inhibitors (HDACis) have demonstrated activity in hematological and solid malignancies. Vorinostat, romidepsin, belinostat, and panobinostat are Food and Drug Administration-approved for hematological malignancies and inhibit class II and/or class I HDACs, including HDAC1, 2, 3, and 6. We combined genetic and pharmacological approaches to investigate whether suppression of individual or multiple Hdacs phenocopied broad-acting HDACis in 3 genetically distinct leukemias and lymphomas. Individual Hdacs were depleted in murine acute myeloid leukemias (MLL-AF9;Nras(G12D); PML-RARα acute promyelocytic leukemia [APL] cells) and Eµ-Myc lymphoma in vitro and in vivo. Strikingly, Hdac3-depleted cells were selected against in competitive assays for all 3 tumor types. Decreased proliferation following Hdac3 knockdown was not prevented by BCL-2 overexpression, caspase inhibition, or knockout of Cdkn1a in Eµ-Myc lymphoma, and depletion of Hdac3 in vivo significantly reduced tumor burden. Interestingly, APL cells depleted of Hdac3 demonstrated a more differentiated phenotype. Consistent with these genetic studies, the HDAC3 inhibitor RGFP966 reduced proliferation of Eµ-Myc lymphoma and induced differentiation in APL. Genetic codepletion of Hdac1 with Hdac2 was pro-apoptotic in Eµ-Myc lymphoma in vitro and in vivo and was phenocopied by the HDAC1/2-specific agent RGFP233. This study demonstrates the importance of HDAC3 for the proliferation of leukemia and lymphoma cells, suggesting that HDAC3-selective inhibitors could prove useful for the treatment of hematological malignancies. Moreover, our results demonstrate that codepletion of Hdac1 with Hdac2 mediates a robust pro-apoptotic response. Our integrated genetic and pharmacological approach provides important insights into the individual or combinations of HDACs that could be prioritized for targeting in a range of hematological malignancies.

A dual role for Hdac1: oncosuppressor in tumorigenesis, oncogene in tumor maintenance.

Aberrant recruitment of histone deacetylases (HDACs) by the oncogenic fusion protein PML-RAR is involved in the pathogenesis of acute promyelocytic leukemia (APL). PML-RAR, however, is not sufficient to induce disease in mice but requires additional oncogenic lesions during the preleukemic phase. Here, we show that knock-down of Hdac1 and Hdac2 dramatically accelerates leukemogenesis in transgenic preleukemic mice. These events are not restricted to APL because lymphomagenesis driven by deletion of p53 or, to a lesser extent, by c-myc overexpression, was also accelerated by Hdac1 knock-down. In the preleukemic phase of APL, Hdac1 counteracts the activity of PML-RAR in (1) blocking differentiation; (2) impairing genomic stability; and (3) increasing self-renewal in hematopoietic progenitors, as all of these events are affected by the reduction in Hdac1 levels. This led to an expansion of a subpopulation of PML-RAR-expressing cells that is the major source of leukemic stem cells in the full leukemic stage. Remarkably, short-term treatment of preleukemic mice with an HDAC inhibitor accelerated leukemogenesis. In contrast, knock-down of Hdac1 in APL mice led to enhanced survival duration of the leukemic animals. Thus, Hdac1 has a dual role in tumorigenesis: oncosuppressive in the early stages, and oncogenic in established tumor cells.

Drug response in a genetically engineered mouse model of multiple myeloma is predictive of clinical efficacy.

The attrition rate for anticancer drugs entering clinical trials is unacceptably high. For multiple myeloma (MM), we postulate that this is because of preclinical models that overemphasize the antiproliferative activity of drugs, and clinical trials performed in refractory end-stage patients. We validate the Vk*MYC transgenic mouse as a faithful model to predict single-agent drug activity in MM with a positive predictive value of 67% (4 of 6) for clinical activity, and a negative predictive value of 86% (6 of 7) for clinical inactivity. We identify 4 novel agents that should be prioritized for evaluation in clinical trials. Transplantation of Vk*MYC tumor cells into congenic mice selected for a more aggressive disease that models end-stage drug-resistant MM and responds only to combinations of drugs with single-agent activity in untreated Vk*MYC MM. We predict that combinations of standard agents, histone deacetylase inhibitors, bromodomain inhibitors, and hypoxia-activated prodrugs will demonstrate efficacy in the treatment of relapsed MM.

NKT cell adjuvant-based tumor vaccine for treatment of myc oncogene-driven mouse B-cell lymphoma.

Immunomodulators are effective in controlling hematologic malignancy by initiating or reactivating host antitumor immunity to otherwise poorly immunogenic and immune suppressive cancers. We aimed to boost antitumor immunity in B-cell lymphoma by developing a tumor cell vaccine incorporating α-galactosylceramide (α-GalCer) that targets the immune adjuvant properties of NKT cells. In the Eµ-myc transgenic mouse model, single therapeutic vaccination of irradiated, α-GalCer-loaded autologous tumor cells was sufficient to significantly inhibit growth of established tumors and prolong survival. Vaccine-induced antilymphoma immunity required NKT cells, NK cells, and CD8 T cells, and early IL-12-dependent production of IFN-γ. CD4 T cells, gamma/delta T cells, and IL-18 were not critical. Vaccine treatment induced a large systemic spike of IFN-γ and transient peripheral expansion of both NKT cells and NK cells, the major sources of IFN-γ. Furthermore, this vaccine approach was assessed in several other hematopoietic tumor models and was also therapeutically effective against AML-ETO9a acute myeloid leukemia. Replacing α-GalCer with ß-mannosylceramide resulted in prolonged protection against Eµ-myc lymphoma. Overall, our results demonstrate a potent immune adjuvant effect of NKT cell ligands in therapeutic anticancer vaccination against oncogene-driven lymphomas, and this work supports clinical investigation of NKT cell-based immunotherapy in patients with hematologic malignancies.

Highly specific intracellular ubiquitination of a small molecule.

Ubiquitin is a small, highly conserved protein that acts as a posttranslational modification in eukaryotes. Ubiquitination of proteins frequently serves as a degradation signal, marking them for disposal by the proteasome. Here, we report a novel small molecule from a diversity-oriented synthesis library, BRD1732, that is directly ubiquitinated in cells, resulting in dramatic accumulation of inactive ubiquitin monomers and polyubiquitin chains causing broad inhibition of the ubiquitin-proteasome system. Ubiquitination of BRD1732 and its associated cytotoxicity are stereospecific and dependent upon two homologous E3 ubiquitin ligases, RNF19A and RNF19B. Our finding opens the possibility for indirect ubiquitination of a target through a ubiquitinated bifunctional small molecule, and more broadly raises the potential for posttranslational modification in trans.

Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias.

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Short-chain fatty acids induce apoptosis in colon cancer cells associated with changes to intracellular redox state and glucose metabolism.

BACKGROUND: Short-chain fatty acids (SCFA) are undergoing increased scrutiny as chemotherapeutics for colon cancer, although a comprehensive understanding of their mode of action is lacking. We investigated candidate SCFA for their capability to modulate apoptosis, cell cycle, intracellular redox state and glucose metabolism in the Caco-2 human colon cancer cell line. METHODS: Caco-2 cells were incubated with butyrate, propionate or a combination of these SCFA (1:1) and assessed by flow cytometry, enzyme activity analysis or by isotope ratio mass spectrometry. RESULTS: Butyrate and the SCFA combination induced apoptosis and G2-M arrest to a greater extent than propionate alone (p < 0.05). SCFA treatment led to time-dependent alterations to the oxidative pentose pathway, reductions in glutathione availability and increases in levels of reactive oxygen species (p < 0.05) compared with untreated controls. The rate of D-glucose metabolism was increased by all SCFA, although to the greatest extent by butyrate (p < 0.05). CONCLUSIONS: These results suggest that butyrate, or the combination of both SCFA, induced rapid and extensive apoptosis and G2-M arrest associated with changes to redox state and D-glucose metabolism. These results support the potential for butyrate and propionate to act as adjuncts to conventional chemotherapy regimens for colon cancer.

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Short-chain fatty acid modulation of apoptosis in the Kato III human gastric carcinoma cell line.

The short-chain fatty acid (SCFA) butyrate is known to induce apoptosis in colon cancer cells in vitro and in vivo, however, its mode of action is poorly defined, whilst less is known regarding the effects of the SCFA propionate. This study investigated the potential for butyrate and propionate to alter cell viability, cell cycle regulation and intracellular protective mechanisms in a human gastric cancer cell line (Kato III). Kato III cells were incubated with butyrate or propionate for 24, 48 and 72 hr. At each time point, cells were assessed for the induction of apoptosis and cell cycle alterations using flow cytometry. Oxidative pentose pathway (OPP) activity and glutathione (GSH) availability were also measured as an index of intracellular protection. Butyrate and propionate differentially induced apoptosis and necrosis in Kato III cells and arrested cells in the G2-M phase. OPP activity was significantly increased by both SCFAs although butyrate induced a 10-fold greater increase than propionate. GSH availability was significantly decreased in Kato III cells by butyrate and propionate. These findings demonstrate that butyrate and propionate induce apoptosis and cell cycle alterations in Kato III gastric cancer cells. Moreover, the effects of butyrate were significantly greater than propionate. We propose that alterations to intracellular redox state and GSH availability play an important role in SCFA-mediated cell death in this cell type. The inclusion of butyrate and propionate as adjunctive cancer therapies has the potential to enhance the efficacy of current chemotherapeutics in the treatment of gastric cancer.

Genomic characterisation of Eµ-Myc mouse lymphomas identifies Bcor as a Myc co-operative tumour-suppressor gene.

The Eµ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eµ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eµ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.

Nutrient and antioxidant modulation of apoptosis in gastric and colon cancer cells.

Gastric cancer and colon cancer are major causes of mortality and morbidity worldwide. Many cancers manifest due to changes in gene expression, particularly those involved in cellular proliferation and apoptosis. Apoptosis is an important process that removes damaged or deleterious cells and contributes to normal cellular and tissue homeostasis. Apoptosis is a tightly regulated process mediated by caspases, and the involvement of the Bcl-2 superfamily of membrane bound proteins, among others. Thus, the therapeutic induction of apoptosis has been proposed as a novel method to eliminate cancer cells. The oxidative pentose pathway (OPP) and the glutathione (GSH) antioxidant defense system play an important role in the regulation of cell growth and apoptosis. The OPP regulates intracellular redox status and provides NADPH for the synthesis of GSH, an important antioxidant. GSH is required to inactivate intracellular reactive oxygen species (ROS) which induce apoptosis and cell injury. Depletion of GSH increases the sensitivity of cells to ROS. Many chemotherapeutic agents induce apoptosis through ROS-mediated cell damage. Therefore, we speculate that the therapeutic inhibition of the OPP and/or the GSH defense system may increase the sensitivity of gastric and colon cancer cells to anti-cancer therapy. Moreover, we hypothesize that the short-chain fatty acid, butyrate, will induce apoptosis in gastric cancer cells and, secondly, that differences in butyrate metabolism will exist between these cancer cell lines.

Targeting p38 or MK2 Enhances the Anti-Leukemic Activity of Smac-Mimetics.

Birinapant is a smac-mimetic (SM) in clinical trials for treating cancer. SM antagonize inhibitor of apoptosis (IAP) proteins and simultaneously induce tumor necrosis factor (TNF) secretion to render cancers sensitive to TNF-induced killing. To enhance SM efficacy, we screened kinase inhibitors for their ability to increase TNF production of SM-treated cells. We showed that p38 inhibitors increased TNF induced by SM. Unexpectedly, even though p38 is required for Toll-like receptors to induce TNF, loss of p38 or its downstream kinase MK2 increased induction of TNF by SM. Hence, we show that the p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Importantly, clinical p38 inhibitors overcame resistance of primary acute myeloid leukemia to birinapant.

Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers.

In order to stimulate antigen presentation and T cell activity against cancer, we treated three different tumor models in mice with the monoclonal antibodies anti-CD40 plus anti-CD137 (BiMab). In a subcutaneous transplantable MC38 colon cancer model, there was significant enhancement in the survival of mice following BiMab treatment. Anti-CD40 has shown considerable success against lymphoma in previous studies by other investigators, and we also showed in this study that, in a model of Eµ-Myc lymphoma, there was a statistically significant enhancement of survival of mice following BiMab treatment. Following the success of the BiMab treatment in the previous two models, we wished to determine if it would be successful in a mouse model of multiple myeloma. Firstly, we tested a transplantable model of disease in which multiple myeloma cells derived from Vk*MYC mice were injected intravenously. A minor proportion of anti-CD137 and BiMab treated mice experienced prolongation of life beyond 250 days. Then we tested the therapy in a spontaneously occurring multiple myeloma model, in Vk*MYC transgenic mice. The majority of mice treated survived longer than control mice, although statistical significance was not demonstrated.

The drug vehicle and solvent N-methylpyrrolidone is an immunomodulator and antimyeloma compound.

N-methyl-2-pyrrolidone (NMP) is a common solvent and drug vehicle. We discovered unexpected antineoplastic and immunomodulatory activity of NMP in a cMYC-driven myeloma model. Coincident to this, NMP was identified as an acetyllysine mimetic and candidate bromodomain ligand. Accordingly, NMP-treated cells demonstrated transcriptional overlap with BET-bromodomain inhibition, including downregulation of cMYC and IRF4. NMP's immunomodulatory activity occurred at sub-BET inhibitory concentrations, and, despite phenotypic similarities to lenalidomide, its antimyeloma activity was independent of the IMiD targets cereblon and Ikaros-1/3. Thus, low-affinity yet broad-spectrum bromodomain inhibition by NMP mediates biologically potent, cereblon-independent immunomodulation and at higher doses targets malignant cells directly via BET antagonism. These data reveal that NMP is a functional acetyllysine mimetic with pleotropic antimyeloma and immunomodulatory activities. Our studies highlight the potential therapeutic benefits of NMP, the consequences of current human NMP exposures, and the need for reassessment of scientific literature where NMP was used as an "inert" drug-delivery vehicle.

Molecular and biologic analysis of histone deacetylase inhibitors with diverse specificities.

Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited.

Intrinsic and extrinsic apoptotic pathway signaling as determinants of histone deacetylase inhibitor antitumor activity.

Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that impede the growth and/or survival of tumor cells. Depending on the physiological context, HDACi can induce apoptosis via two well-defined apoptotic pathways; the intrinsic/mitochondrial pathway and the death receptor (DR)/extrinsic pathway. A number of groups have demonstrated that overexpression of prosurvival Bcl-2 family members significantly reduces HDACi-mediated tumor cell death and therapeutic efficacy in preclinical models. In many cases, HDACi activate the intrinsic pathway via upregulation of a number of proapoptotic BH3-only Bcl-2 family genes including Bim, Bid, and Bmf. Additionally, HDACi can engage the extrinsic pathway through upregulation of DR expression, reductions in c-FLIP, and upregulation of ligands such as TRAIL. Overall, it appears that activation of the intrinsic apoptotic pathway is the predominant mechanism of HDACi-induced tumor cell death; however, the DR pathway may also be engaged, either to amplify the apoptotic signal through the intrinsic pathway or to directly induce cell death.

Cellular mucosal defense during Helicobacter pylori infection: a review of the role of glutathione and the oxidative pentose pathway.

Helicobacter pylori is the primary cause of gastritis and peptic ulcer disease and is known to infect greater than 50% of the world's population. It is also known to lead to the onset of gastric cancer and unless treated, lasts throughout life in most individuals. Mouse models of H. pylori infection have improved our ability to study this organism and can be used to investigate the host mucosal response to the infection, particularly the early events postinoculation. Previous studies have shown that H. pylori infection leads to an increased production of reactive oxygen species within the gastric mucosa which are thought to play a major role in the mediation of associated disease. Recent studies have shown differences in the availability of an important antioxidant, glutathione, during chronic H. pylori infection. The availability of glutathione is primarily controlled by the activity of the oxidative pentose pathway. This review proposes that the severity of inflammation and damage associated with H. pylori infection is dependent on the ability of mucosal cells to counteract the increased load of reactive oxygen species. It is hypothesized that the oxidative pentose pathway and glutathione availability are important factors modulating this response. It is suggested that the therapeutic regulation of glutathione availability could provide a novel method for preventing or reducing the damage caused during H. pylori infection.

13C-urea breath test: reproducibility and association with the severity of Helicobacter pylori-associated antral gastritis.

BACKGROUND: The purpose of the present paper was to assess the reproducibility of the (13)C-urea breath test ((13)C-UBT) and its ability to reflect the level of Helicobacter pylori-associated inflammation. METHODS: Asymptomatic H. pylori-positive subjects (n = 21) performed the (13)C-UBT six times. The H. pylori-positive symptomatic subjects (n = 55) performed the (13)C-UBT and had antral biopsies taken for histopathology, culture, urease activity assay and myeloperoxidase activity assay. RESULTS: No significant intraindividual variation in (13)C-UBT results were observed for the asymptomatic subjects. The (13)C-UBT results were significantly higher in symptomatic subjects with a moderate to severe gastritis compared to a mild gastritis and to no inflammation (34.5 +/- 4.4 vs 17.7 +/- 2.8 vs 1.7 +/- 0.1, respectively, P < 0.01). The (13)C-UBT results significantly correlated with urease (r = 0.55) and myeloperoxidase activity (r = 0.82) but not with bacterial load. conclusion: The (13)C-UBT is a reproducible determinant of H. pylori infection and non-invasively assesses the severity of antral inflammation.