Bz Resistance Research Articles

The Antimalarial Agent Artesunate Overcomes Bortezomib Resistance in Myeloma Cell Lines Through Non-Caspase Mediated Apoptosis

AbstractAbstract 4015During terminal differentiation of a B-lymphocyte to a mature plasma cell, the protein load increases considerably while the proteasome capacity decreases. The fact that plasma cells operate close to the limit of their proteasomal capacity is being exploited therapeutically and proteasome inhibition has become one of the pillars of myeloma treatment. The importance of the proteasome in Multiple Myeloma (MM) has also been established in prognostic terms (1). De novo or acquired resistance to proteasome inhibitors (PI) confers poor prognosis in MM. In vitro bortezomib resistance is accompanied by cross-resistance to other known PIs, thus highlighting (2), the need for treatment alternatives.Based on reports that various antimalarial drugs exhibit in vitro anti-MM activity through either α-ring proteasome inhibition (mefloquine) or aggresome formation inhibition (quinine), we examined whether Artesunate (ART), a new highly effective antimalarial drug with a known excellent safety profile, could overcome resistance to PI’s currently in clinical use.Bortezomib (BZ)-resistant sublines of the BZ naïve and sensitive JJN3 and U266 cell lines were developed by exposure to progressively increasing BZ concentrations. Eventually the JJN3BR and U266BR sublines were developed with a 20- and 25-fold IC50 increase, respectively, compared to their BZ naïve counterparts. ART was able to inhibit viability in clinically achievable dosages and in a time-dependent manner in IL-6-independent and -dependent MM cell lines (Table 1). JJN3BR and U266BR showed no evidence of cross-resistance compared to their parental cell lines. The RPMI 8226 cell line known to exhibit one of the highest BZ IC50s among MM cell lines proved to be among the most sensitive to ART. The in-vitro antiMM effectiveness of ART was evident also in primary CD-138+ MM cells from six (6) patients with Relapsed/Refractory MM (Table 2). ART in-vitro anti MM activity was also verified by Annexin-V and 7-AAD flow cytometry for its ability to induce both early and late apoptosis.To elucidate ART's mode of action, we measured the sequence and level of caspase activation through the luminescence assay Caspase-Glo. ART exposure initiated the extrinsinc pathway of apoptosis with a brisk increase in activated caspase-8 levels and effector caspase3/7 levels within 3h, followed by caspase 9 activation at 6h. However, actual apoptosis by ART was not accompanied by a marked caspase activity especially when compared to other potent anti-MM agents. The pan-caspase inhibitor Z-VAD-MFK effectively inhibited caspase activation by ART but failed to inhibit ART's effect on viability, as at least 70% of ART's effect was retained in JJN3, U266 and RPMI 8226 cells. Similar results were obtained in flow cytometry-based apoptosis assays. Immunoblotting of the mitochondrial derived non-caspase mediating apoptosis factors AIF, EndoG and HtrA2 in ART treated JJN3 cells revealed that ART-mediated apoptosis was related to the cytoplasmic and subsequently nuclear translocation of AIF. Conclusion:ART shows marked anti-MM activity and overcomes BZ resistance in vitro. Its benign side effect profile support its potential for myeloma therapy. Most anti-cancer agents induce apoptosis in a caspase-dependent manner. ART's mode of action is mainly caspase-independent, indicating that its role in overcoming drug resistance, could also extend to other anti-MM agents. Experiments aimed at further elucidating the mechanism of action of ART and the evaluation of drug combinations including ART are currently under way.Table 1ART IC50 on various MM cell linesCell LineIC50μM (48h)IC50μM (72h)JJN320.716.4JJN3BR19.515.2U26628.218.6U266BR29.719.5KMS-1130.124.3RPMI-82268.76.7INA-62718.9ARH-7736.732.3Table 2ART IC50 on MM primary cellsMM primary cellsIC50μM (48h)Pt165.9Pt256.2Pt345Pt430.4Pt530.4Pt620.1 Disclosures:Tian:University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties.

Modeling Proteasome Inhibition in Lymphoma

Abstract 4946The proteasome inhibitor Bortezomib (Bz) has been widely used to treat multiple myeloma, relapsed mantle cell lymphoma and is undergoing clinical evaluation for other B cell malignancies including non-Hodgkin lymphoma. Despite its initial success, patients treated with Bz eventually relapse due to the development of drug resistance. Therefore, understanding the basis of drug resistance is a critical component for improved therapy. The acquisition of Bz resistance in lymphomas, particularly those with constitutive expression of the B cell-specific DNA mutator, activation-induced cytidine deaminase (AID), has not been previously characterized.We have utilized the AID-expressing human non-Hodgkin Burkitt’s lymphoma as a model system for this study. Burkitt’s lymphoma lines (Ramos and BL-2) are suitable because they are highly sensitive to Bz induced apoptosis with an IC50 of approximately 11 nM after 48 hrs of treatment. In order to generate Bz resistant Burkitt’s lymphoma lines, Ramos cells were treated weekly with increasing concentrations of Bz for 3 months. Compared to the parental line, this newly formed line displayed an approximately 2.5–3-fold increase in IC50 to Bz as well as to three other proteasome inhibitors (next-generation proteasome inhibitor, MLN 2238, epoxomicin and carfilzomib), while maintaining sensitivity to different chemotherapeutic agents (PD 0332991 cyclin 4/6 dependent kinase inhibitor and melphalan). In this model system, resistance to Bz conferred a general cross-resistance to other proteasome inhibitors, a phenotype that has been stably maintained for 5 months.We next asked whether AID plays a role in the acquisition of Bz resistance in Ramos cells by promoting hypermutation and genomic instability. In support of this hypothesis, the G322A and C326T mutations in the gene encoding the proteasome subunit and target of Bz, psmb5, occur within AID hotspots raising the possibility that AID could directly mutate psmb5. Furthermore, like many proteins, AID is degraded by the proteasome arguing that proteasome inhibition further stabilizes AID protein resulting in aberrant hypermutation. Consistent with this, we detected by flow cytometry (intracellular staining) a 2-fold increase in AID protein following a 24-hour, 20 nM Bz treatment of Ramos cells. AID normally mutates immunoglobulin (Ig) genes and these mutations are necessary for the production of protective antibodies, while aberrant AID activity leads to mutations in non-Ig genes. Surprisingly, despite the increase in AID protein, we observe reduced mutation frequency within the functional IgH gene following 3-month Bz treatment compared to untreated controls. Despite this reduction, array comparative genomic hybridization (a-CGH) studies indicate copy number abnormalities in Bz resistant cells and details of the chromosomal abnormalities and target genes deregulated will be presented. These data demonstrate that Burkitt's lymphoma cells are sensitive to Bz and drug resistance can be readily achieved in vitro. Furthermore, Bz treatment stabilizes AID protein and promotes increased genomic instability. Disclosures:Stessman:Millennium: The Takeda Oncology Company: Research Funding. Mansoor:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

Expression of Germinal Center B Cell Markers in Bortezomib-Resistant Multiple Myeloma Cells

Abstract 129Multiple myeloma (MM), the second most common hematopoietic malignancy, is an incurable plasma cell neoplasm representing approximately 2% of all cancer deaths. The proteasome inhibitor, bortezomib (Bz), has been widely used to treat MM, and emerging next-generation proteasome inhibitors are being evaluated. Despite advances in therapies, patients treated with Bz eventually relapse due to the development of drug resistance. Thus, identifying signatures that distinguish Bz sensitive from resistant MM cells will more accurately predict Bz sensitivity and will aid in the development of novel approaches to reverse resistance.MM populations are characterized by phenotypic heterogeneity suggesting the possibility that certain MM subpopulations are associated with Bz resistance. In order to characterize these populations in greater detail, we have utilized isogenic pairs of Bz sensitive and resistant MM lines derived from the genetically engineered iMycCa/Bcl-xl mouse model of plasma cell malignancy. The iMycCa/Bcl-xl mouse is a highly robust model that very closely resembles human MM based on gene expression profiling (GEP), chromosomal abnormalities and progression of disease and response to therapy (Boylan, et al. Cancer Res (2007) 67: 4069). We have employed this mouse model because of its striking similarity to human MM, and MM cells isolated from these mice can be readily infected by lentivirus as well as adoptively transferred into syngeneic, immunocompetent recipients establishing a powerful system to directly test biologically relevant hypotheses relating to Bz resistance.Isogenic pairs of Bz sensitive and resistant lines were extensively characterized using GEP, quantitative PCR and flow cytometry. We observed increased expression of germinal B cell markers Pax5, AID and Bcl-6 and reduced expression of the plasma cell marker CD93 in Bz resistant lines compared to Bz sensitive pairs suggesting that Bz resistance is associated with germinal center-like B cell characteristics. Remarkably, we detect within the same cell co-expression of both CD138/Pax5 and CD138/AID demonstrating the tremendous plasticity of these cells and their intermediate B cell differentiation phenotype. Recently, both AID and Bcl-6 have been identified as crucial mediators of resistance to tyrosine kinase inhibitors in leukemia cells (Duy, et al. Nature (2011) 473: 384; Klemm, et al. Cancer Cell (2009) 16: 232) raising an intriguing possibility that these proteins thought to be restricted to germinal center B cells could play a role in Bz resistance in MM. These observations have led us to more closely characterize the apparent shift in developmental stage resulting from Bz-driven expression changes or selection. Time course analysis of gene and protein expression following low-dose Bz treatment in the absence of cell death indicates that Bz increases the expression and promotes the stabilization of Pax5, AID and Bcl-6. This demonstrates a drug-induced change in markers associated with B cell differentiation relating to the ultimate emergence of the resistance phenotype. We are now determining whether similar B cell differentiation phenotypes emerge in a recently published human drug trial where GEP was completed prior to and 48 hours after Bz treatment in patients (Shaughnessy, et al. Blood (2011), ahead of print). Thus, we will present phenotypic signatures correlating to Bz resistance in cell lines derived from our mouse model of MM and suggest that targeting B cell differentiation may represent a rational therapeutic approach to overcoming Bz resistance. Disclosures:Stessman:Millennium: The Takeda Oncology Company: Research Funding. Mansoor:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

Markers for benzimidazole resistance in human parasitic nematodes?

Benzimidazole (BZ) resistance is widespread and appears to be readily selected in a variety of nematode parasites of animals. There have been reports of a lack of efficacy of BZ anthelmintics against soil transmitted nematode parasites of humans. However, resistance to BZs in nematodes of humans has not been confirmed. It is difficult to perform tests to confirm anthelmintic resistance in humans for a variety of technical and ethical reasons. The use of anthelmintic drugs for the control of helminth parasites in people is increasing massively as a result of numerous programmes to control gastrointestinal nematode parasites in children, the Global Program for the Elimination of Lymphatic Filariasis and other programmes. Many of these programmes are dependent on BZ anthelmintics and this will increase the pressure for resistance development to BZ anthelmintics in nematode parasites of people. We need to perform monitoring for anthelmintic resistance in these programmes and we need new tools to make that monitoring sensitive, inexpensive and practical. There is a real need for DNA-based markers for BZ resistance in nematode parasites of humans. We have a reasonable understanding of the molecular mechanisms and genetics of BZ resistance in some nematode parasites of animals and similar mechanisms are likely to prevail in nematodes of humans. Based on the likelihood that similar single nucleotide polymorphisms (SNPs) will be involved in BZ resistance in human, as in animal nematode parasites, rapid SNP assays have been developed for possible BZ resistance development in Wuchereria bancrofti.

Single nucleotide polymorphism (SNP) markers for benzimidazole resistance in veterinary nematodes

Resistance to the benzimidazole class of anthelmintics in nematodes of veterinary importance has a long history. Research into the mechanisms responsible for this resistance is subsequently at a more advanced stage than for other classes of anthelmintics. The principal mechanism of resistance to benzimidazoles is likely to involve changes in the primary structure of beta-tubulins, the building blocks of microtubules. Specifically, point mutations in the beta-tubulin isotype 1 gene leading to amino acid substitutions in codons 167, 198, and 200 of the protein have been associated with resistance in nematodes. These single nucleotide polymorphisms offer a means of detecting the presence of resistance within populations. In this mini-review, we focus on the prevalence and importance of these polymorphisms in three groups of nematodes: trichostrongylids, cyathostomins, and hookworms. A brief overview of existing strategies for genotyping single nucleotide polymorphisms is also presented. The CARS initiative hopes to exploit these known polymorphisms to further our understanding of the phenomenon of BZ resistance.

Comparing different formulae to test for gastrointestinal nematode resistance to benzimidazoles in smallholder goat farms in Mexico

The objective was to examine the coincidence in the prevalence of benzimidazole (Bz) resistance in smallholder goat herds, as determined by three average-based and two individually-based faecal egg count reduction (FECR) tests. Nineteen smallholder goat herds with more than 30 animals were selected from 84 herds in Yucatan. Animals shedding 150 eggs/g of faeces (EPG) on day zero were randomly divided into two groups. The control group did not receive treatment and the treated group received fenbendazole (10 mg/kg body weight per os). Feed was withdrawn for 16 h before treatment. Ten days after treatment, both groups were sampled to determine their FEC. Faecal cultures and identification of infective larvae were performed for estimating the proportions of genera of gastrointestinal nematodes (GIN) present. Presence of resistant GIN was determined with three different average-based FECR (FECR 1, FECR 2 and FECR 3) and two individually-based FECR ( iFECR 1 and iFECR 2). The prevalence of herds with Bz resistant nematodes (and 95% confidence interval (95% CI)) was calculated using each formula. Coincidence among formulae was estimated with Kappa values. The prevalence (±95% CI) of Bz resistance calculated with FECR 1 (57.89 ± 22.20) had a high coincidence with iFECR 1 and iFECR 2 (Kappa values of 0.86 and 0.79, respectively). The prevalence with FECR 2 (31.58 ± 20.90) and FECR 3 (21.05 ± 18.33) had a low coincidence with FECR 1 (Kappa < 0.50). Trichostrongylids found on Bz resistant farms were mainly Haemonchus spp., however, some Trichostrongylus spp. and Oesophagostomum spp. were found too. The high coincidence between the standard average-based FECR 1 and the individually based formulae is encouraging and may suggest that either formula could be applied to smallholder farmers. Further laboratory studies are needed to confirm the resistance status in the herds.

Temperature dependent binding of mebendazole to tubulin in benzimidazole-susceptible and -resistant strains of Trichostrongylus colubriformis and Caenorhabditis elegans

The binding of [ 3H]mebendazole ([ 3H]MBZ) to tubulin in benzimidazole-susceptible (BZ-S) and benzimidazole-resistant (BZ-R) strains of Trichostrongylus colubriformis and Caenorhabditis elegans was examined in order to investigate the biochemical changes to tubulin that result in BZ resistance in parasitic and free-living nematodes. In both species the extent of [ 3H]MBZ binding to tubulin was significantly reduced in the BZ-R strain compared with the BZ-S strain. The decrease in [ 3H]MBZ binding in the BZ-R strain of each species was the result of a significant reduction in the amount of charcoal stable [ 3H]MBZ-tubulin complexes and was not related to a change in the association constant of the [ 3H]MBZ-tubulin interaction. [ 3H]MBZ binding to tubulin was temperature dependent, reaching maximum levels at 37°C in BZ-S T. colubriformis and 10°C in BZ-R T. colubriformis. Both the BZ-S and BZ-R strains of C. elegans displayed maximum [ 3H]MBZ binding at 4°C. Resistance ratios derived from the amount of [ 3H]MBZ binding in the BZ-S and BZ-R strains and in vitro development assays demonstrated that the temperature dependence and extent of drug binding was indicative of BZ resistance status and was species specific in the BZ-S isolates. These results indicate that biochemical differences exist in the binding of benzimidazole carbamates to tubulin in nematode species, and suggest that the susceptibility of the parasitic nematodes to the benzimidazole anthelmintics is the result of a unique high affinity and/or high capacity interaction ofbenzimidazole carbamates with tubulin.