A Novel Measure of Chromosome Instability Can Account for Prognostic Difference in Multiple Myeloma

Multiple myeloma (MM) is characterized by complex genetic abnormalities whose complexity signifies varying degree of chromosomal instability (CIN). In this study, we introduced a novel CIN measure, chromosome instability genome event count (CINGEC), which considered both copy number aberrations and interstitial breakpoints from high-resolution genome-wide assays. When assessed in two aCGH MM datasets, higher CINGEC was associated with poor survival. We then derived a CINGEC-associated gene expression profile (GEP) signature, CINGECS, using a dataset that has both aCGH and GEP. Genes in CINGECS were mainly involved in DNA damage responses besides in aneuploidy and other generic oncogenic processes contrary to other CIN associated GEP signatures. Finally, we confirmed its survival association in three GEP datasets that encompassed newly diagnosed patients treated with transplant-based protocol with or without novel agents for induction as well as relapsed patients treated with bortezomib. Furthermore, CINGECS was independent of many GEP-based prognostic signatures. In conclusion, our novel CIN measure has definite biological and clinical significance in myeloma.     

Preclinical screening of histone deacetylase inhibitors combined with ABT-737, rhTRAIL/MD5-1 or 5-azacytidine using syngeneic Vk*MYC multiple myeloma

Multiple myeloma (MM) is an incurable malignancy with an unmet need for innovative treatment options. Histone deacetylase inhibitors (HDACi) are a new class of anticancer agent that have demonstrated activity in hematological malignancies. Here, we investigated the efficacy and safety of HDACi (vorinostat, panobinostat, romidepsin) and novel combination therapies using in vitro human MM cell lines and in vivo preclinical screening utilizing syngeneic transplanted Vk*MYC MM. HDACi were combined with ABT-737, which targets the intrinsic apoptosis pathway, recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL/MD5-1), that activates the extrinsic apoptosis pathway or the DNA methyl transferase inhibitor 5-azacytidine. We demonstrate that in vitro cell line-based studies provide some insight into drug activity and combination therapies that synergistically kill MM cells; however, they do not always predict in vivo preclinical efficacy or toxicity. Importantly, utilizing transplanted Vk*MYC MM, we report that panobinostat and 5-azacytidine synergize to prolong the survival of tumor-bearing mice. In contrast, combined HDACi/rhTRAIL-based strategies, while efficacious, demonstrated on-target dose-limiting toxicities that precluded prolonged treatment. Taken together, our studies provide evidence that the transplanted Vk*MYC model of MM is a useful screening tool for anti-MM drugs and should aid in the prioritization of novel drug testing in the clinic.     

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

Background

Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.

Methods

HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity^® Pathway Analysis.

Results

Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.

Conclusion

This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.     

Histone deacetylase inhibitors: overview and perspectives

Histone deacetylase inhibitors (HDACi) comprise structurally diverse compounds that are a group of targeted anticancer agents. The first of these new HDACi, vorinostat (suberoylanilide hydroxamic acid), has received Food and Drug Administration approval for treating patients with cutaneous T-cell lymphoma. This review focuses on the activities of the 11 zinc-containing HDACs, their histone and nonhistone protein substrates, and the different pathways by which HDACi induce transformed cell death. A hypothesis is presented to explain the relative resistance of normal cells to HDACi-induced cell death.     

A systems biology approach to identify molecular pathways altered by HDAC inhibition in osteosarcoma

Osteosarcoma (OS) is the most common primary tumor in humans and dogs affecting the skeleton, and spontaneously occurring OS in dogs serves as an extremely useful model. Unacceptable toxicities using current treatment protocols prevent further dose-intensification from being a viable option to improve patient survival and thus, novel treatment strategies must be developed. Histone deacetylase inhibitors (HDACi) have recently emerged as a promising class of therapeutics demonstrating an ability to enhance the anti-tumor activity of traditional chemotherapeutics. To date, gene expression analysis of OS cell lines treated with HDACi has not been reported, and evaluation of the resultant gene expression changes may provide insight into the mechanisms that lead to success of HDACi. Canine OS cells, treated with a clinically relevant concentration of the HDACi valproic acid (VPA), were used for expression analysis on the Affymetrix canine v2.0 genechip. Differentially expressed genes were grouped into pathways based upon functional annotation; pathway analysis was performed with MetaCore and Ingenuity Pathways Analysis software. Validation of microarray results was performed by a combination of qRT-PCR and functional/biochemical assays revealing oxidative phosphorylation, cytoskeleton remodeling, cell cycle, and ubiquitin-proteasome among those pathways most affected by HDACi. The mitomycin C-bioactivating enzyme NQ01 also demonstrated upregulation following VPA treatment, leading to synergistic reductions in cell viability. These results provide a better understanding of the mechanisms by which HDACi exert their effect in OS, and have the potential to identify biomarkers that may serve as novel targets and/or predictors of response to HDACi-containing combination therapies in OS.     

Barley MLO modulates actin-dependent and actin-independent antifungal defense pathways at the cell periphery

Cell polarization is a crucial process during plant development, as well as in plant-microbe interactions, and is frequently associated with extensive cytoskeletal rearrangements. In interactions of plants with inappropriate fungal pathogens (so-called non-host interactions), the actin cytoskeleton is thought to contribute to the establishment of effective barriers at the cell periphery against fungal ingress. Here, we impeded actin cytoskeleton function in various types of disease resistance using pharmacological inhibitors and genetic interference via ectopic expression of an actin-depolymerizing factor-encoding gene, ADF. We demonstrate that barley (Hordeum vulgare) epidermal cells require actin cytoskeleton function for basal defense to the appropriate powdery mildew pathogen Blumeria graminis f. sp. hordei and for mlo-mediated resistance at the cell wall, but not for several tested race-specific immune responses. Analysis of non-host resistance to two tested inappropriate powdery mildews, Erysiphe pisi and B. graminis f. sp. tritici, revealed the existence of actin-dependent and actin-independent resistance pathways acting at the cell periphery. These pathways act synergistically and appear to be under negative control by the plasma membrane-resident MLO protein.     

Combinations of EGFR and MET inhibitors reduce proliferation and invasiveness of mucosal melanoma cells

Mucosal melanoma (MM) is a very rare and aggressive type of cancer for which immunotherapy or targeted therapy such as BRAF/MEK inhibitors, used in cutaneous melanoma, usually fail. Due to our earlier experience showing the high effectiveness of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) inhibitors in reducing the activation of the MAPK and PI3K/AKT signalling pathways, we aim to test whether these drugs would also be effective for mucosal melanoma. Cells representing two commercially available mucosal melanoma cell lines (GAK and HMVII) and one cell line obtained from a patient's vaginal melanoma were treated with MET or EGFR inhibitors, or combinations of these agents. The dual-inhibitor treatment strategy resulted in a decrease of cell proliferation, migration and invasion. Moreover, combinations of inhibitors led to reduction of pEGFR/EGFR and pMET/MET ratio and downregulation of PI3K/AKT and MEK/ERK1/2-based signalling pathways. Our findings indicate a potential therapeutic strategy based on EGFR and MET inhibitors in mucosal melanoma, which should be further evaluated in vivo and in clinical experiments. They also suggest that targeting multiple receptor tyrosine kinases may block signalling crosstalk and possibly delay the appearance of resistance to kinase inhibitors in mucosal melanoma cells.     

A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET—a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.Subject terms: Myeloma, Cancer therapeutic resistance     

Dual regulation of glucocorticoid-induced leucine zipper (GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma

Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.     

HDACi: molecular mechanisms and therapeutic implications in the innate immune system

Histone deacetylase inhibitors (HDACi) are an emerging class of novel anti-cancer drugs that cause growth arrest, differentiation and apoptosis of tumor cells. In addition, many advances have been made in understanding the immunoregulation of Toll-like receptors, NOD-like receptors and interferons that have recently generated new momentum for the study of HDACi in immunity as a whole, and in the regulation of these innate signaling pathways specifically. HDACi have shown promise as new anti-inflammatory and immunosuppressant agents. They have also demonstrated great potency and relative selectivity in various human/animal models of inflammatory diseases. This review focuses on recent progress and the current state of HDACi knowledge, as well as the molecular mechanisms and therapeutic potential of HDACi for the treatment of inflammatory diseases and cancers.     

Resistance mechanisms to immune checkpoints blockade by monoclonal antibody drugs in cancer immunotherapy: Focus on myeloma

Multiple myeloma (MM) is a clonal B‐cell malignancy characterized by the accumulation of neoplastic proliferation of a plasma cell in the bone marrow that produces a monoclonal immunoglobulin. The immune checkpoint inhibitors against programmed death‐1/programmed death‐1 ligand and cytotoxic T‐lymphocyte antigen 4 axis have demonstrated appropriate anticancer activity in several solid tumors and liquid cancers, and are rapidly transforming the practice of medical oncology. However, in a high percentage of patients, the efficacy of immune checkpoints blockade remains limited due to innate or primary resistance. Moreover, the malignancies progress in many patients due to acquired or secondary resistance, even after the clinical response to immune checkpoints' blockade. The evidence shows that multiple tumor‐intrinsic and tumor‐extrinsic factors and alterations in signaling pathways are involved in primary and secondary resistance to immune checkpoints blockade. Improved identification of intrinsic and extrinsic factors and mechanisms of resistance or response to immune checkpoints blockade may not only provide novel prognostic or predictive biomarkers but also guide the optimal combination/sequencing of immune checkpoint blockade therapy in the clinic. Here, we review the underlying biology and role of immune checkpoints blockade in patients with MM. Furthermore, we review the host and tumor‐related factor effects on immune checkpoints blockade in MM immunotherapy.     

Histone Deacetylase Inhibitors Selectively Target Homology Dependent DNA Repair Defective Cells and Elevate Non-Homologous Endjoining Activity

Background

We have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi) trichostatin A (TSA), confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity.

Results

HDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR) factor BLM or the non-homologous end-joining (NHEJ) and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency.

Conclusions

HDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks (DSBs), and HDR proficiency is correlated with cell survival.     

Vorinostat-induced autophagy switches from a death-promoting to a cytoprotective signal to drive acquired resistance

Histone deacetylase inhibitors (HDACi) have shown promising activity against hematological malignancies in clinical trials and have led to the approval of vorinostat for the treatment of cutaneous T-cell lymphoma. However, de novo or acquired resistance to HDACi therapy is inevitable, and their molecular mechanisms are still unclear. To gain insight into HDACi resistance, we developed vorinostat-resistant clones from the hematological cell lines U937 and SUDHL6. Although cross-resistant to some but not all HDACi, the resistant cell lines exhibit dramatically increased sensitivity toward chloroquine, an inhibitor of autophagy. Consistent with this, resistant cells growing in vorinostat show increased autophagy. Inhibition of autophagy in vorinostat-resistant U937 cells by knockdown of Beclin-1 or Lamp-2 (lysosome-associated membrane protein 2) restores sensitivity to vorinostat. Interestingly, autophagy is also activated in parental U937 cells by de novo treatment with vorinostat. However, in contrast to the resistant cells, inhibition of autophagy decreases sensitivity to vorinostat. These results indicate that autophagy can switch from a proapoptotic signal to a prosurvival function driving acquired resistance. Moreover, inducers of autophagy (such as mammalian target of rapamycin inhibitors) synergize with vorinostat to induce cell death in parental cells, whereas the resistant cells remain insensitive. These data highlight the complexity of the design of combination strategies using modulators of autophagy and HDACi for the treatment of hematological malignancies.     

Mechanisms of powdery mildew resistance in the Vitaceae family

The cultivated grapevine, Vitis vinifera, is a member of the Vitaceae family, which comprises over 700 species in 14 genera. Vitis vinifera is highly susceptible to the powdery mildew pathogen Erysiphe necator. However, other species within the Vitaceae family have been reported to show resistance to this fungal pathogen, but little is known about the mechanistic basis of this resistance. Therefore, the frequency of successful E. necator penetration events, in addition to programmed cell death (PCD) responses, were investigated in a representative genotype from a range of different species within the Vitaceae family. The results revealed that penetration resistance and PCD-associated responses, or combinations of both, are employed by the different Vitaceae genera to limit E. necator infection. In order to further characterize the cellular processes involved in the observed penetration resistance, specific inhibitors of the actin cytoskeleton and secretory/endocytic vesicle trafficking function were employed. These inhibitors were demonstrated to successfully break the penetration resistance in V. vinifera against the nonadapted powdery mildew E. cichoracearum. However, the use of these inhibitors with the adapted powdery mildew E. necator unexpectedly revealed that, although secretory and endocytic vesicle trafficking pathways play a crucial role in nonhost penetration resistance, the adapted powdery mildew species may actually require these pathways to successfully penetrate the plant host.     

A switch from canonical to noncanonical Wnt signaling mediates drug resistance in colon cancer cells

Butyrate, a fermentation product of fiber in the colon, acts as a histone deacetylase inhibitor (HDACi) and induces apoptosis in colon cancer (CC) cells in vitro. We have reported that the apoptotic effects of butyrate are dependent upon the hyperactivation of the Wnt/beta-catenin pathway. However, prolonged exposure of CC cells to increasing concentrations of butyrate results in the acquisition of resistance to the Wnt/beta-catenin- and apoptosis-inducing effects of this agent, as well as cross-resistance to structurally different HDACis. Here we report that one mechanism whereby HDACi resistance arises is through the increase of beta-catenin-independent (noncanonical) Wnt signaling. Compared to HDACi-sensitive HCT-116 CC cells, HDACi-resistant HCT-R cells exhibit higher levels of AKT/PKB cell survival signaling, which is in part induced by WNT5A and its receptor ROR2. The induction of AKT signaling by HDACis is also detected in other CC cell lines, albeit to a lesser extent than in the drug-resistant HCT-R cells. The observations suggested that the apoptotic effect of butyrate and other HDACis in CC cells can be augmented by inhibitors of pAKT. In agreement with the hypothesis, the combination of MK2206, a pAKT inhibitor, and a HDACi (butyrate or LBH589) induced higher apoptosis in CC cells compared to each agent alone. The exposure to both agents also re-sensitized the HCT-R cells to apoptosis. Finally, the concept of simultaneously inducing canonical Wnt activity and suppressing AKT signaling was translated into a combination of diet-derived agents. Diet-derived pAKT inhibitors (caffeic acid phethyl ester, sulforaphane, dilallyl trisulfide) suppressed the butyrate-induced levels of pAKT, and increased the apoptotic effects of butyrate in both drug-sensitive and drug-resistant CC cells.Our findings can be translated into (a) CC therapy employing combinations of synthetic HDACis and inhibitors of pAKT, as well as (b) CC prevention based upon diets that result in sufficient amounts of butyrate and pAKT inhibitors.     

DNA repair pathways in human multiple myeloma

Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment.     

High-throughput ex vivo drug testing identifies potential drugs and drug combinations for NRAS-positive malignant melanoma

Therapy options for patients with metastatic melanoma (MM) have considerably improved over the past decade. However, many patients still need effective therapy after unsuccessful immunotherapy, especially patients with BRAF-negative tumors who lack the option of targeted treatment second line. Therefore, the elucidation of efficient and personalized therapy options for these patients is required. In this study, three patient-derived cancer cells (PDCs) were established from NRAS Q61-positive MM patients. The response of PDCs and five established melanoma cell lines (two NRAS-positive, one wild type, and two BRAF V600-positive) was evaluated toward a panel of 527 oncology drugs using high-throughput drug sensitivity and resistance testing. The PDCs and cell lines displayed strong responses to MAPK inhibitors, as expected. Additionally, the PDCs and cell lines were responsive to PI3K/mTOR, mTOR, and PLK1 inhibitors among other effective drugs currently undergoing clinical trials. Combinations with a MEK inhibitor were tested with other targeted agents to identify effective synergies. MEK inhibitor showed synergy with multikinase inhibitor ponatinib, ABL inhibitor nilotinib, PI3K/mTOR inhibitor pictilisib, and pan-RAF inhibitor LY3009120. The application of the patients’ cancer cells for functional drug testing ex vivo is one step further in the process of identifying potential agents and agent combinations to personalize treatment for patients with MM. Our preliminary study results suggest that this approach has the potential for larger-scale drug testing and personalized treatment applications in our expansion trial. Our results show that drug sensitivity and resistance testing may be implementable in the treatment planning of patients with MM.