BTZ Resistance Research Articles

Vitamin D and K Supplementation Is Associated with Changes in the Methylation Profile of U266-Multiple Myeloma Cells, Influencing the Proliferative Potential and Resistance to Bortezomib.

Multiple myeloma (MM) is a plasma cell malignancy that, despite recent advances in therapy, continues to pose a major challenge to hematologists. Currently, different classes of drugs are applied to treat MM, among others, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies. Most of them participate in an interplay with the immune system, hijacking its effector functions and redirecting them to anti-MM activity. Therefore, adjuvant therapies boosting the immune system may be potentially beneficial in MM therapy. Vitamin D (VD) and vitamin K (VK) have multiple so called "non-classical" actions. They exhibit various anti-inflammatory and anti-cancer properties. In this paper, we investigated the influence of VD and VK on epigenetic alterations associated with the proliferative potential of MM cells and the development of BTZ resistance. Our results showed that the development of BTZ resistance is associated with a global decrease in DNA methylation. On the contrary, both control MM cells and BTZ-resistant MM cells exposed to VD alone and to the combination of VD and VK exhibit a global increase in methylation. In conclusion, VD and VK in vitro have the potential to induce epigenetic changes that reduce the proliferative potential of plasma cells and may at least partially prevent the development of resistance to BTZ. However, further ex vivo and in vivo studies are needed to confirm the results and introduce new supplementation recommendations as part of adjuvant therapy.

CRIP1 INDUCES PIS RESISTANCE BY FORMING A COMPLEX WITH USP7 /PA200 AND ENHANCING PROTEASOME ACTIVITY AND AUTOPHAGY IN MULTIPLE MYELOMA

Introduction: Multiple myeloma (MM) is a plasma malignancy characterized by an accumulation of misfolded immunoglobulins in the cell. Although proteasome inhibitor (PI) can treat MM by inhibiting ubiquitin-proteasome system (UPS), autophagy as a compensatory protein clearance mechanism, it still leads to drug resistance. The discovery of new targets that can simultaneously inhibit UPS and autophagy will provide great potential for the treatment of MM. Methods: The proliferation, invasion, migration, proteasome activity and autophagy were detected in CRIP1- knockdown /overexpression and control MM cells. Co-IP (Co-Immunoprecipitation) with TAP/MS (Tandem affinity purification/Mass spectrum) was performed to detect the binding of USP7, CRIP1 and PA200. Myeloma xenograft model was used to determine the role of CRIP1 to promote proliferation of MM cells and induce BTZ resistance in vivo. Results: The levels of CRIP1 were significantly increased in plasma cells from NDMM (newly diagnosed multiple myeloma) compared to healthy donors, and further increased in patients with RRMM (relapsed/refractory multiple myeloma). Moreover, the high expression of CRIP1 is significantly related to the poor prognosis of MM patients, especially those treated with BTZ. Our results in vitro and in vivo showed that CRIP1 knockdown noteworthy inhibited the cell growth, invasion, migration. MM cells were more sensitive to PIs-induced cell growth arrest when CRIP1 knockdown. The proteasome activity and the protein level of LC3B-Ⅱ of the MM cells were significantly reduced with CRIP1 knockdown. Co-IP analysis showed that CRIP1 formed a complex with USP7/PA200. The protein level of CRIP1 decreased and the ubiquitination CRIP1 increased with USP7 inhibition. These data suggested that CRIP1 is the substrate of USP7.CRIP1 promotes the de-ubiquitination and stabilization of PA200 mediated by USP7 by interacting with USP7 and PA200, which in turn promotes MM cell survival and drug resistant. Functional inhibition of CRIP1 by knocking down of USP7 or PSME4 in CRIP1 OE MM cell line led to a decrease of the activity of proteasome and autophagy, thus enhancing the sensitivity to PIs in intro and in vivo. Conclusion: CRIP1 plays a critical role in MM progression and PIs resistance by formation of CRIP1/USP7/PA200 complex. High level of CRIP1 is a biomarker for high-risk MM patients. CRIP1 is a potential target for MM therapy, can simultaneously inhibit proteasome activity and autophagy. Encore Abstract - previously submitted to EHA 2023 The research was funded by the following foundations:(a) CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-040 and 2022-I2M-1-022), (b) National NaturalScience Foundation of China (82170194, 81920108006, 82270175) and Natural Science Foundation of Fujian Province of China (2021J02040). Keywords: Diagnostic and Prognostic Biomarkers, Multiple Myeloma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

Posttranslational modification of Aurora A-NSD2 loop contributes to drug resistance in t(4;14) multiple myeloma.

Backgroundt(4;14)(p16;q32) cytogenetic abnormality renders high level of histone methyltransferase NSD2 in multiple myeloma (MM) patients, and predicts poor clinical prognosis, but mechanisms of NSD2 in promoting chemoresistance have not been well elucidated.MethodsAn epigenetics compound library containing 181 compounds was used to screen inhibitors possessing a prior synergistic effect with bortezomib (BTZ) in vitro. Molecular biology techniques were applied to uncover underlying mechanisms. Transcriptome profile assay was performed by RNA‐seq. NSG mouse‐based xenograft model and intra‐bone model were applied to qualify the synergistic effect in vivo.ResultsWe identified an Aurora kinase A inhibitor (MLN8237) possessed a significant synergistic effect with BTZ on t(4;14) positive MM cells. Aurora A protein level positively correlated with NSD2 level, and gain‐ and loss‐of‐functions of Aurora A correspondingly altered NSD2 protein and H3K36me2 levels. Mechanistically, Aurora A phosphorylated NSD2 at S56 residue to protect the protein from cleavage and degradation, thus methylation of Aurora A and phosphorylation of NSD2 bilaterally formed a positive regulating loop. Transcriptome profile assay of MM cells with AURKA depletion identified IL6R, STC2 and TCEA2 as the downstream target genes responsible for BTZ‐resistance (BR). Clinically, higher expressions of these genes correlated with poorer outcomes of MM patients. Combined administration of MLN8237 and BTZ significantly suppressed tumour growth in LP‐1 cells derived xenografts, and remarkably alleviated bone lesion in femurs of NSG mice.ConclusionsAurora A phosphorylates NSD2 at S56 residue to enhance NSD2 methyltransferase activity and form a positive regulating loop in promoting MM chemoresistance, thus pharmacologically targeting Aurora A sensitizes t(4;14) positive MM to the proteasome inhibitors treatment. Our study uncovers a previously unknown reason of MM patients with t(4;14) engendering chemoresistance, and provides a theoretical basis for developing new treatment strategy for MM patients with different genomic backgrounds.

P-088: Indirubin-3’-monoxime acts as an alternative proteasome inhibitor and confers new regimens for the treatment of Multiple Myeloma

<h3>Background</h3> Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Despite the therapeutic benefit of the proteasome inhibitor (PI) bortezomib, drug resistance still accounts for majority of tumor relapses and cancer-related death in MM patients. Thus, exploring the mechanism underlying BTZ resistance and investigating a novel treatment strategy are urgent requisites for clinical practise. Indirubin-3'-monoxime (Id-3) is an active ingredient of a traditional Chinese medicine. <h3>Method</h3> In the present study, we firstly investigated Id-3 effects on myeloma treatment in patient primary samples and myeloma cell lines. We also investigated the molecular mechanism of Id-3 treatment on MM cells via in vivo and in vitro study. <h3>Results</h3> We first reported that Id-3 is an effective cytotoxicity agentia against MM cells growth by inducing the MM cells apoptosis. Id-3 treatment also effectively induces the cell death in bortezomib-resistant MM cell lines and primary myeloma patient samples. Stringently, we found there is a synergistic anti-MM effect between Id-3 and bortezomib. The treatment with lower dosage combination of Id-3 and bortezomib exhibited a notably inhibition of the growth of myeloma cells both in xenograft mouse model and MM-PDX model. Mechanism study revealed that Id-3 treatment efficiently inhibited the chymotrypsin-like and caspase-like activity of proteasome in MM cells. The level of the proteasome 11s activator, PA200 (PSME4) and PA28y (PSME3) was significantly down-regulated both in mRNA and protein level. GEO datasets analysis showed that the high levels of PSME4 and PSME3 were over-expressed in relapsed patients and MM cell lines which correlated with the drug-resistance of MM cells. Kaplan-Meier analysis showed that patients with high-levels of PSME3 or PSME4 had the shortened survival. Knocking-down the level of PSME4 or PSME3 by shRNA noteworthy inhibited the cell growth and enhanced the sensitivity to bortezomib of MM cells. In vivo study also found that down-regulated the level of PSME3 or PSME4 can suppress the proliferation of MM cells and prolong the survival of MM-bearing mouse. <h3>Conclusions</h3> Taken together, our study supported that Id-3 is an effective agentia against the survival of MM cells both in bortezomib-sensitive and -resistance ones. We further identified that the 11s proteasome activators, PSME3 and PSME4, were down-regulated effectively by Id-3 treatment. High-levels of PSME3 or PSME4 were correlated with the inferior outcome of MM patients. Down-regulating PSME3 and PSME4 by Id-3 or shRNA could efficiently suppressed the growth of MM cells and improve the survival of MM-bearing mice. Our study provides the rational evidence for the clinical application of Id-3 in the treatment of MM.

Bone-Targeted Bortezomib Inhibits Bortezomib-Resistant Multiple Myeloma in Mice by Providing Higher Levels of Bortezomib in Bone.

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic and blood cancers. The difficulty of eliminating bone-residing cancer, especially drug-resistant cancer, necessitates novel, alternative treatments to manipulate tumor cells and their microenvironment, with minimal off-target effects. To this end, bone-targeted conjugate (BP-Btz) was generated by linking bortezomib (Btz, an anticancer, bone-stimulatory drug) to a bisphosphonate (BP, a targeting ligand) through a cleavable linker that enables spatiotemporally controlled delivery of Btz to bone under acidic conditions for treating multiple myeloma (MM). Three conjugates with different linkers were developed and screened for best efficacy in mouse model of MM. Results demonstrated that the lead candidate BP-Btz with optimal linker could overcome Btz resistance, reduced tumor burden, bone destruction, or tumor metastasis more effectively than BP or free Btz without thrombocytopenia and neurotoxicity in mice bearing myeloma. Furthermore, pharmacokinetic and pharmacodynamic studies showed that BP-Btz bound to bone matrix, released Btz in acidic conditions, and had a higher local concentration and longer half-life than Btz in bone. Our findings suggest the potential of bone-targeted Btz conjugate as an efficacious Btz-resistant MM treatment mechanism. © 2021 American Society for Bone and Mineral Research (ASBMR).

CircRNA ITCH increases bortezomib sensitivity through regulating the miR-615-3p/PRKCD axis in multiple myeloma

AimsBortezomib (BTZ) is described as the first-line agent for multiple myeloma (MM) chemotherapy, but the emergence of BTZ resistance usually results in the failure of chemotherapy in MM. Circular RNA (circRNA) itchy E3 ubiquitin protein ligase (circITCH) is a novel identified circRNA that plays a vital role in the development of human cancers. However, the role of circITCH in the development of BTZ resistance in MM remains elusive. Materials and methodsThe expression of circITCH, miR-615-3p, and protein kinase C, delta (PRKCD) was detected with quantitative reverse transcription PCR and western blot. The effects of circITCH on the sensitivity of MM cells to BTZ were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, flow cytometry, and xenograft tumor assay. The interaction of circITCH, microRNA-615-3p, and PRKCD was explored using luciferase reporter assay and RNA immunoprecipitation assay. Key findingscircITCH was downregulated in MM bone marrow specimens and cell lines, as well as BTZ-resistant MM cells. Reduced expression of circITCH was indicative of poor prognosis in MM patients. Upregulation of circITCH enhanced the sensitivity of BTZ-resistant MM cells to BTZ in vitro and in vivo. Furthermore, circITCH was identified as a sponge for miR-615-3p, and PRKCD is confirmed as a direct target of miR-615-3p. Besides, circITCH overexpression enhanced the sensitivity of MM cells to BTZ through miR-615-3p/PRKCD axis. SignificancecircITCH overexpression enhanced the sensitivity of MM cells to BTZ through miR-615-3p/PRKCD axis, providing a novel potential target for combating BTZ resistance in patients with MM.

IRF4 Is Reciprocally Dysregulated Via NF-Κb-Dependent Expression and Loss-of-Function Mutations in Multiple Myeloma

Introduction: Interferon regulatory factor 4 (IRF4) is a transcription factor involved in B cell activation and differentiation into plasma cells, as well as B-cell lineage neoplasia. Diverse genetic abnormalities and dysregulated expression of IRF4 have been documented in various hematologic malignancies, including multiple myeloma (MM). IRF4 is also identified as a major downstream target of CRBN/IKZF to regulate MYC expression, representing a primary mechanism underlying anti-MM action of IMiDs. However, it remains unclear how the expression and function of IRF4 are regulated in MM cells. Materials and Methods: DNAseq, whole exome RNAseq (WES), and label-free analyses of quantitative proteomics and post-translational modifications (PTMs) mapping were conducted to examine genetic aberrations, transcriptional dysregulation, or protein and PTM alterations in primary patient samples and drug-naive vs -resistant cell lines. Flow cytometry, qPCR, and Western blot analysis were utilized to monitor apoptosis or mRNA and protein levels of target genes. Stable transfection with wild-type or mutated IRF4 was carried out to evaluate its function. Analysis of the MM genome-wide GEP databases (R2: Genomics Analysis and Visualization Platform) was performed to validate the clinical significance of IRF4 and its target genes in MM patients. Results: MM cells that acquired bortezomib (btz) resistance (PS-R) exhibited a marked increase in protein levels of multiple key components of the canonical and non-canonical NF-κB signaling cascades, in association with increased NF-κB transcriptional activity. Pharmacological inhibition of NF-κB using several compounds (e.g., Bay 11-7082, parthenolide, triptolide) significantly increased btz sensitivity in PS-R cells and their parental U266 cells, as well as other MM cell lines. These events were accompanied by down-regulation of various genes, including NF-κB-dependent genes and those whose relationship with NF-κB remains uncertain. The latter included IRF4, MYC, MCL1, BIRC2/cIAP1, SQSTM1/p62, among others. Of note, overexpression of wild-type IRF4 sharply reduced sensitivity of drug-naive cells to btz alone or in combination with NF-κB inhibitors. However, IRF4 overexpression did not prevent inactivation of NF-κB and down-regulation of the NF-κB-dependent genes (e.g., Bcl-xL, TNFAIP3/A20), suggesting that IRF4 acts downstream of NF-κB. Surprisingly, IRF4 overexpression failed to restore expression of MYC, a well-established target of IRF4, while largely reversed expression of cIAP1 and Mcl-1 at both mRNA and protein levels. Moreover, inhibitors of either IAP (birinapant/TL32711) or Mcl-1 (S63845) overcame btz-resistance conferred by IRF4 overexpression. DNAseq unveiled multiple IRF4 SNPs located in introns and exons (both 3'-UTR and coding region) of chromosome 6. WES revealed that IRF4 was highly expressed in primary bone marrow and extramedullary samples of MM patients (2~10 folds over normal donors). But no difference at mRNA and protein levels of IRF4 was observed between drug-naive and -resistant cell lines. The global PTM mapping identified two lysine residues (K59 and K399) that were highly ubiquitinated in PS-R cells (p = 0.0447 and p = 0.0004, vs U266 cells). Interestingly, ectopic expression of IRF4 mutants (e.g., K59N, K123R) lost the cytoprotective capability of their wild-type counterpart, in association with failure to reverse down-regulation of cIAP1 and Mcl-1. This observation argues that these point mutations are loss-of-function, which might explain better prognosis of patients carrying IRF4 mutations (including K59N and K123R) than wild-type IRF4 as reported previously. Last, the MM GEP database analysis validated the correlation between IRF4 and Mcl-1 or cIAP expression, as well as the adverse effect of their dysregulation on overall survival of MM patients. Conclusion: IRF4 is highly expressed in MM cells, likely via an NF-κB-dependent mechanism. IRF4 acts downstream of the NF-κB signaling pathways, which in turn directly targets cIAP1 and Mcl-1, rather than MYC, to confer drug resistance. IRF4 could be mutated in MM patients, while the point mutations, at least at certain lysine sites, seem to be loss-of-function and thus predict better outcome. Together, these findings suggest that the oncogenic function of IRF4 might be governed at genetic, transcriptional, and PTM levels in MM cells. Disclosures Kumar: Takeda: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

Long non-coding RNA ANGPTL1-3 promotes multiple myeloma bortezomib resistance by sponging miR-30a-3p to activate c-Maf expression

Multiple Myeloma (MM) is a malignant hematological disease characterized by monoclonal proliferation of plasma cells in the bone marrow. In recent years, the widespread use of new drugs based on bortezomib (Btz) has significantly improved the remission rate of MM patients. However, drug resistance and disease relapse occur within a few years and MM is still considered to be an incurable disease. The amplification of the long arm of chromosome 1 is one of the most common genetic abnormalities in MM patients. Here, we found that long non-coding RNA ANGPTL1-3 which located in 1q region was overexpressed in MM. Lnc-ANGPTL1-3 expression was correlated with MM International Staging System (ISS) and overall survival. Notably, knockdown of lnc-ANGPTL1-3 increased Btz sensitivity of MM cells. Following exploration revealed that lnc-ANGPTL1-3 competitively interacted with miR-30a-3p to c-Maf, a transcription factor which was reported to be associated with Btz resistance. Taken together, our findings demonstrate that lnc-ANGPTL1-3/miR-30a-3p/c-Maf axis plays a critical role in MM Btz resistance.

Peripheral blood exosomes from patients with multiple myeloma mediate bortezomib resistance in cultured multiple myeloma cells

To investigate the role of exosome in mediating bortezomib (Btz) resistance in multiple myeloma cells in vitro and explore the underlying mechanisms. Peripheral blood samples were collected from 15 patients with multiple myeloma with Btz tolerance, and serum exosomes were isolated by ultracentrifugation and identified with electron microscopy, NTA and Western blotting. In vitro cultured multiple myeloma cells were treated with gradient concentrations of Btz to determine the optimal drug concentration for subsequent experiment. The cells were pretreated with different concentrations of exosomes, and their sensitivity to BTZ was assessed using MTS assay. We searched the exosome database Exocarta and used STRING to generate the network map and the protein interaction graph. The diameters of the vesicles isolated from the peripheral blood of the patients were mostly below 200 nm with a mean particle size of 153 nm and a mode of 140.1 nm. The results of Western blotting showed that the isolated exosomes expressed the marker proteins CD63, Tsg101 and Alix. In cultured multiple myeloma cells, pretreatment with exosomes resulted in a decreased sensitivity of the cells to bortezomib, and longer treatment durations and higher exosome concentrations consistently enhanced the resistance of the cells to the same Btz concentration. Analysis of the Exocarta database identified human serum exosomal proteins ABCB1, ABCB4, PDCD6IP, and EGFR, among which EGFR served as a network node. Exosome within a specific concentration range may serve as a signal carrier to mediate the resistance of multiple myeloma cells to Btz. EGFR likely plays a key role to promote exosome-mediated Btz resistance in myeloma cells.

LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p

Radiotherapy, chemotherapy, autologous/allogeneic stem cell transplantation, and targeted drug therapy are currently available therapeutic options for multiple myeloma (MM), but the clinical outcome remains unsatisfactory owing to frequent occurrence of drug resistance. Anti apoptosis is one of the main mechanisms to mediate drug resistance. Studies have shown that MCL-1 plays a key role in the growth of cancer cells “escaping” drug attacks, but the underlying mechanism remains unclear. Our previous study demonstrated that lncRNA H19 was highly expressed in the serum of MM patients. Bioinformatics predicts that miR-29b-3p is the downstream target gene, and MCL-1 is the downstream target protein of miR-29b-3p. Therefore, we speculated that MCL-1 may be involved in the occurrence of drug resistance through epigenetics. On the basis of these previous findings, the present study was intended to explore the biological function of H19, interactions between the downstream target genes, and the effect of H19 on BTZ resistance of myeloma cells. In addition, in vivo experiments we have also confirmed that H19 promoted tumor growth and may develop resistance to bortezomib partly. It was found that H19 reduced cell sensitivity to the chemotherapeutic drug BTZ by working as a miRNA sponge to inhibit the expression of miR-29b-3p, enhance MCL-1 transcriptional translation and inhibit apoptosis. These findings may help gain insights into the molecular mechanism of acquired BTZ resistance and develop new drug targets for the clinical treatment of MM.

Positive transcription elongation factor b (P-TEFb) is a therapeutic target in human multiple myeloma.

The role of the positive RNA Pol II regulator, P-TEFb (positive transcription elongation factor b), in maintenance of the anti-apoptotic protein Mcl-1 and bortezomib (btz) resistance was investigated in human multiple myeloma (MM) cells. Mcl-1 was up-regulated in all MM lines tested, including bortezomib-resistant lines, human MM xenograft mouse models, and primary CD138+ MM cells. Mcl-1 over-expression significantly reduced bortezomib lethality, indicating a functional role for Mcl-1 in bortezomib resistance. MM cell lines, primary MM specimens, and murine xenografts exhibited constitutive P-TEFb activation, manifested by high CTD (carboxy-terminal domain) S2 phosphorylation, associated with a) P-TEFb subunit up-regulation i.e., CDK9 (42 and 55 kDa isoforms) and cyclin T1; and b) marked CDK9 (42 kDa) T186 phosphorylation. In marked contrast, normal hematopoietic cells failed to exhibit up-regulation of p-CTD, CDK9, cyclin T1, or Mcl-1. CDK9 or cyclin T1 shRNA knock-down dramatically inhibited CTD S2 phosphorylation and down-regulated Mcl-1. Moreover, CRISPR-Cas CDK9 knock-out triggered apoptosis in MM cells and dramatically diminished cell growth. Pan-CDK e.g., dinaciclib or alvocidib and selective CDK9 inhibitors (CDK9i) recapitulated the effects of genetic P-TEFb disruption. CDK9 shRNA or CDK9 inhibitors significantly potentiated the susceptibility of MM cells, including bortezomib-resistant cells, to proteasome inhibitors. Analogously, CDK9 or cyclin T1 knock-down or CDK9 inhibitors markedly increased BH3-mimetic lethality in bortezomib-resistant cells. Finally, pan-CDK inhibition reduced human drug-naïve or bortezomib-resistant CD138+ cells and restored bone marrow architecture in vivo. Collectively, these findings implicate constitutive P-TEFb activation in high Mcl-1 maintenance in MM, and validate targeting the P-TEFb complex to circumvent bortezomib-resistance.

The novel β2-selective proteasome inhibitor LU-102 decreases phosphorylation of I kappa B and induces highly synergistic cytotoxicity in combination with ibrutinib in multiple myeloma cells.

Purpose Proteasome-inhibiting drugs (PI) are gaining importance in hematologic oncology. The proteasome carries three proteolytically active subunits (β1, β2, β5). All established PI (bortezomib and carfilzomib), as well as experimental drugs in the field (dalanzomib, oprozomib, and ixazomib), by design target the rate-limiting β5 subunit. It is unknown whether β2-selective proteasome inhibition can also be exploited toward anticancer treatment. Combining PI with the pan B-cell-directed Bruton tyrosine kinase inhibitor ibrutinib appears a natural option for future improved treatment of multiple myeloma (MM) and B-cell lymphomas. However, bortezomib induces phosphorylation of IκB and activation of NF-κB in MM cells, while ibrutinib inhibits the IκB/NF-κB axis, suggesting antagonistic signaling. A β2-selective proteasome inhibitor may lack such antagonistic signaling effects. MethodsWe recently introduced LU-102, the first β2-selective PI available for preclinical testing. We here compare bortezomib with carfilzomib and LU-102 in MM and MCL in vitro with regard to their effects on pIκB/NF-κB signaling and their cytotoxic activity in combination with ibrutinib.ResultsLU-102 reduced phosphorylation of IκB, in contrast to bortezomib and carfilzomib, and was a superior inhibitor of NF-κB activation in MM cells. This translated into highly synergistic cytotoxicity between LU-102 and ibrutinib, which was able to overcome BTZ resistance and CFZ resistance. By contrast, BTZ lacked consistent synergistic cytotoxicity with ibrutinib.ConclusionIbrutinib is highly synergistic with β2-selective proteasome inhibition against MM and MCL in vitro. Novel β2-selective proteasome inhibitors may be exploited to overcome bortezomib/carfilzomib resistance and boost the activity of BTK inhibitors against B-cell-derived malignancies.

Bim-Targeting Therapy Circumvents Adaptive Bortezomib-Resistance In Myeloma Through a Novel Cross-Link Between Autophagy and Apoptosis

In diverse human cancers, oncogene-driven pathways antagonize the lethal effects of Bim, a major apoptosis executioner implicated in the activity of various anti-cancer agents including bortezomib (btz). As loss of Bim plays an important role in tumor development and growth as well as in acquisition of drug resistance, Bim represents an attractive therapeutic target, particularly for efforts to circumvent resistance to standard chemotherapy and diverse novel therapies. In the present study, the role of Bim in acquired resistance of multiple myeloma (MM) cells to btz was investigated. Bcl-2 family member expression profiles revealed high basal levels of Bim in most MM cell lines (Bimhi) and in primary CD138+ MM samples. However, Bimlow cells (e.g., H929) were highly susceptible to btz, arguing against the possibility that endogenous Bim levels correlate with btz sensitivity. In sharp contrast, shRNA Bim knockdown in Bimhi cells conferred pronounced resistance to btz. Interestingly, btz-resistant cells (PS-R) generated by exposure of U266 cells (Bimhi) to progressively increasing btz concentrations (to 20 nM) displayed markedly diminished Bim expression, a phenomenon validated in btz-resistant cells derived from other Bimhi cell lines (e.g., OPM2, RPMI8226). Notably, Bim up-regulation by histone deacetylase inhibitors (HDACIs e.g., SBHA), when combined with BH3-mimetics (e.g., ABT-737) that release Bim from binding to Bcl-2/Bcl-xL, effectively killed btz-resistant MM cells. Moreover, this strategy was highly active against primary CD138+ cells isolated from relapsed MM patients. In view of recent evidence that Bim negatively regulates autophagy (Luo et al., Mol Cell 47:359-70, 2012), the possibility that autophagy might also play a role in Bim-targeting strategies was investigated. Whereas drug-naïve and btz-resistant cells exhibited little difference in expression of autophagy-inducing proteins or autophagy initiation (e.g., by thapsigargin or tunicamycin), resistant cells displayed deficiencies in autophagosome maturation, reflected by delayed removal of GFP-LC3 puncta. Interestingly, SBHA co-administration largely abrogated ABT-737-induced autophagy and markedly increased the association between Beclin-1 and Bcl-2, accompanied by diminished binding of Bim to Bcl-2. Significantly, shRNA Bim knockdown strikingly increased autophagy in Bimhi cells. Moreover, SBHA was unable to inhibit autophagy in such cells due to failure of Bim up-regulation, resulting in a pronounced attenuation of SBHA/ABT-737-induced apoptosis. In Bimlow H929 cells, SBHA also failed to up-regulate Bim and potentiate ABT-737 lethality. However, disruption of autophagy by chloroquine (CQ) dramatically restored the sensitivity of these cells to this Bim-targeting regimen, an event associated with a striking increase in Bim expression. In contrast, CQ only modestly increased lethality in Bimhi cells in which autophagy was already disabled by SBHA-mediated Bim up-regulation. Moreover, compared to parental drug-naïve cells (Bimhi), btz-resistant cells displaying acquired loss of Bim were significantly more susceptible to potentiation of SBHA/ABT-737 lethality by CQ (e.g., 50% vs 22% increase in apoptosis; P < 0.01). Finally, in bim-/- MEFs derived from gene knockout mice, SBHA/ABT-737 co-treatment, with or without the addition of CQ, was unable to induce apoptosis, arguing that the presence of the bim gene is required for restoration of drug sensitivity by Bim-targeting therapy. Together, these findings argue that Bim deficiency represents a novel mechanism of adaptive (rather than intrinsic) btz-resistance in MM cells, and that simultaneous up-regulation of Bim (e.g., by HDACIs) and unleashing of Bim from binding to anti-apoptotic Bcl-2 family members (e.g., by BH3-mimetics such as ABT-737) can circumvent this form of resistance. They also raise the possibility that in addition to direct induction of apoptosis, Bim-mediated suppression of autophagy contributes to the lethality of Bim-targeting strategies, and that disruption of the latter process (e.g., by CQ) may be particularly beneficial in cells in which Bim up-regulation is disabled. Collectively, these findings suggest that Bim-targeting therapy may represent a novel and effective strategy in overcoming btz resistance in MM. This study was supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Disclosures:Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees.

Abstract 1020: VRC2, a novel bortezomib re-sensitizing compound for the treatment of multiple myeloma.

Abstract The development of novel agents including the proteasome inhibitor bortezomib (Btz) has significantly improved treatment outcomes in multiple myeloma (MM). Despite these advances, resistance to therapy invariably emerges and MM remains an incurable disease. The objective of this study was to identify new therapeutic agents and combinations with the ability to overcome resistance to Btz in MM cells. We developed a high throughput drug screening (HTS) assay system to discover small molecules that selectively target Btz resistant MM cells as single agents and/or re-sensitize resistant cells to Btz. For HTS we used isogenic pairs of Btz sensitive (595 P) and resistant (595 VR) cells derived from iMycCα/Bcl-xL transgenic mouse model of MM. Cell viability was the HTS assay read-out, and 3 treatment groups (595 P, 595 VR, and 595 VR + Btz) were included to identify compounds with selective activity against Btz resistant cells as single agents, and compounds that restore Btz sensitivity in the presence of Btz. Our screening of multiple publically-available compound libraries identified several chemical structures with selective activity against Btz resistant cells. One compound in particular showed modest but significant selectivity for Btz resistant cells as a single agent, and most notably, exhibited potent Btz re-sensitizing activity when the 2 drugs were combined. We named this compound Velcade Re-sensitizing Compound 2, or VRC-2 for short. The Btz re-sensitizing activity of VRC-2 was confirmed using multiple human and mouse cell lines that had been selected for Btz resistance in vitro. IC50s for VRC-2 in normal mouse and human fibroblasts were 100-fold higher than that observed in MM cells, suggesting it may have a low toxicity profile in vivo. We are currently evaluating the activity of VRC-2 in animal models of MM, alone and in combination with Btz, and conducting molecular studies to identify its precise mechanism of action. This study establishes the proof-of-principal for our HTS approach and demonstrates the potential of VRC-2 in Btz refractory MM. Citation Format: Amriti Lulla, Holly Stessman, David Dicker, Brian Van Ness, Wafik El-Deiry, Nathan G. Dolloff. VRC2, a novel bortezomib re-sensitizing compound for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1020. doi:10.1158/1538-7445.AM2013-1020

Abstract 3390: Simultaneous Chk1 and MEK1/2 inhibition circumvents Mcl-1-mediated anti-apoptotic functions and bortezomib resistance in human multiple myeloma cells.

Abstract Mcl-1 is a critical survival factor for human multiple myeloma (MM) cells, and also confers resistance to various anti-MM agents, including bortezomib (btz). We have previously reported that exposure of MM cells to Chk1 inhibitors elicits activation of the MEK1/2/ERK1/2 pathway, and that prevention of that process e.g., by MEK1/2 inhibitors resulted in a dramatic increase in apoptosis (Pei et al., Blood 118:5189, 2011). The goal of the present study was to determine whether a strategy combining Chk1 (e.g., CEP3891; CEP) with MEK1/2 inhibitors (e.g., PD184352; PD) might circumvent Mcl-1 related resistance, and if so, by what mechanism(s). In various MM cell lines (e.g., U266, 8226, MM.1S), blockade of CEP-induced ERK1/2 activation by PD sharply down-regulated Mcl-1 in association with Stat3 inactivation, events accompanied by pronounced apoptosis. Ectopic expression of constitutively active (CA)-Stat3 up-regulated Mcl-1 expression and significantly attenuated PD/CEP lethality. While Mcl-1 over-expression conferred marked btz resistance, no cross resistance to PD/CEP occurred. Interestingly, btz-resistant U266 (PS-R) and OPM-2 (O/V10R) cells displayed both increased Stat3 activation and elevated Mcl-1 levels, but were fully sensitive to PD/CEP. Notably, in Mcl-1-overexpressing cells and drug-naïve or -resistant cells exposed to PD/CEP, co-IP demonstrated a marked increase in Mcl-1 binding to Bim, which was robustly up-regulated, accompanied by release of Bak from Mcl-1 and Bak/Bax activation. IL-6 or IGF-1 exposure up-regulates Mcl-1 via Stat3 activation, and this phenomenon was blocked by PD/CEP, suggesting that this strategy may circumvent microenvironment-mediated drug resistance. Indeed, co-culture of MM cells with stromal HS-5 cells conferred resistance to dexamethasone and melphalan, but not to PD/CEP. Significantly, equivalent results were obtained in btz-resistant PS-R cells as well as other OPM2/btz-resistant MM cells co-cultured with HS-5 cells. Significantly, the PD/CEP regimen was also highly active against primary CD138+ MM samples (n = 8 of 10), while sparing their normal CD138− counterparts as well as normal cord blood CD34+ cells (n = 3). Analogous to mechanisms observed in MM cell lines, PD/CEP inhibited Stat3 activation, induced Mcl-1 downregulation, and triggered Bim up-regulation in primary CD138+ MM but not in normal CD138−, cells. Collectively, these findings identify novel mechanisms by which combined Chk1 and MEK1/2 inhibition targets Mcl-1 in MM cells: a) down-regulation of Mcl-1 through inhibition of Stat3 activation and b) disabling Mcl-1 by increasing its binding to up-regulated Bim, thereby circumventing both Mcl-1-related bortezomib- and microenvironment-mediated resistance. Citation Format: Xin-Yan Pei, Yun Dai, Jessica Felthousen, Shuang Chen, Yukie Takabatake, Leena E. Youssefian, Wesley W. Bodie, Lora B. Kramer, Robert Z. Orlowski, Steven Grant. Simultaneous Chk1 and MEK1/2 inhibition circumvents Mcl-1-mediated anti-apoptotic functions and bortezomib resistance in human multiple myeloma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3390. doi:10.1158/1538-7445.AM2013-3390

Impaired bortezomib binding to mutant β5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells

Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the β5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZ-resistant cells revealed upregulation of mutant β5 subunit of the proteasome. These newly identified β5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the β5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant β5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the α7 subunit of the proteasome can overcome this drug resistance modality.

Abstract 2627: Mechanisms of bortezomib resistance in human NSCLC cell lines; potential role of proteasome beta5-subunits and autophagy

Abstract Non-small cell lung cancer (NSCLC) is a disease with poor prognosis and novel treatments are warranted. The proteasome inhibitor bortezomib (BTZ, Velcade), which is successfully applied for the treatment of primary and refractory Multiple Myeloma, may also elicit activity against solid tumors, including lung cancer. To further explore this, we investigated mechanisms underlying a 12-fold difference in BTZ sensitivity in a panel of 3 human NSCLC cells : H460 (IC50:69 nM), A549 (IC50:22 nM) and SW1573 (IC50:6 nM) after 72 hours drug exposure. We also evaluated the effects of long term BTZ exposure to identify whether BTZ resistance may emerge in these NSCLC cells. For this purpose, H460, A549 and SW1573 cells were exposed to stepwise increasing concentrations of BTZ over a period of 5 months resulting in BTZ-resistance levels of 2.6-, 6.9- and 4.9-fold, respectively, compared to their parental counterparts. Since BTZ primarily targets the beta-5 subunit of the proteasome, we examined cross-resistance to other proteasome inhibitors; 4A6, a 6-mer peptide that specifically targets the beta-5 subunit of the proteasome, and 5-amino-8-hydroxyquinoline (5AHQ), which primarily targets the non-catalytic alpha-7 subunit of the proteasome. Moreover, proteasome catalytic activities were measured by means of an intact cell-based fluorogenic assay. Finally, the potential role of autophagy as a possible cell survival or cell death mechanism after BTZ exposure was examined utilizing immunohistochemistry staining with LC3B as a marker for authophagy. The generated BTZ-resistant NSCLC cells displayed up to 10-fold cross-resistance to 4A6, but retained full sensitivity to 5AHQ, suggesting that quantitative or qualitative alterations of the beta-5 subunit are implicated in BTZ resistance. The basal proteasome catalytic activity demonstrated an increase of 2.9- and 1.4-fold for the chymotrypsin like activity and a 4.5- and 1.5-fold for the caspase-like activity in H460 compared to SW1573 and A549, respectively, indicating a relationship between basal proteasome activity and BTZ sensitivity. Of note, no marked differences in basal proteasome activity levels were observed between parental and BTZ-resistant cells. Nevertheless, to inhibit proteasome activity by &amp;gt; 80%, 2-fold higher BTZ concentrations were required in BTZ-resistant cells. Finally, the role of autophagy as a mechanism contributing to BTZ resistance was examined. We observed a marked induction of BTZ-induced autophagy in H460 and SW1573 cells upon exposure to 100 nM and 10 nM BTZ, respectively. Strikingly, no signs of autophagy were noted in the generated BTZ-resistant H460 and SW1573 cells. In conclusion, these data support the notion that alterations in proteasome beta-5 subunit targeting and loss of autophagy may contribute to a reduced efficacy of BTZ in NSCLC cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2627.

Abstract A27: Chronic exposure of malignant hematological cell lines to bortezomib induces de novo hot spot mutations in the PSMB5 gene

Abstract The proteasome inhibitor Bortezomib (BTZ, Velcade®) specifically inhibits the catalytic beta 5 subunit of the proteasome. The introduction of BTZ has shown promising results in the treatment of Multiple Myeloma (MM), Non-Hodgkin lymphoma and leukemia. Despite these encouraging results, clinical trials in MM also revealed that a significant proportion of patients acquired resistance to BTZ mono-therapy. We have developed a BTZ-resistant cell line model by chronic exposure to stepwise increasing concentrations of BTZ, including the AML THP-1 cell-line (Oerlemans &amp; Franke et al, Blood 2008), the T-ALL CCRF-CEM-C7 cell-line and the MM RPMI-8226 cell line (Franke et al, Blood 2009 vol 114(22), abstr 940). Cells were initially selected for growth at 7 nM BTZ to acquire low levels of BTZ resistance (2–3 fold higher IC50 concentrations) and subsequently challenged to concentrations of BTZ up to 500 nM to provoke higher resistance levels. Abilities to resist a BTZ concentration of 100 nM could be achieved relatively fast in the CEM cell line (within 16 weeks), intermediate in the THP-1 cell line (within 22 weeks) and relatively slowly in the 8226 cell line (within 60 weeks). Subsequent sequencing of the PSMB5 gene, encoding the beta 5 proteasome subunit, revealed a series of mutations in individual BTZ-resistant subclones, all resulting in amino-acid changes residing within the highly conserved BTZ binding pocket. The relatively fast induction of mutations provoked the question whether there might already exist a subclone within the cell line that harbours the mutation. To distinguish between the outgrowth of a pre-existing resistant subclone and the occurrence of de novo mutations, we generated new BTZ resistant CEM and THP-1 cells (CEM-BR2 and THP-1-BR2). Interestingly, the new CEM-BR2 cells had a different nucleotide change (G322A resulting in a Ala49Thr substitution) from the original BTZ resistant CEM-BR1 (C323T and G332T resulting in Ala49Val and Cys52Phe amino acid substitutions, respectively). Of note, this new CEM-BR2 mutation represented the same mutation as seen in the original BTZ resistant THP-1-BR1. In addition, the new THP-1-BR2 cells also showed a different mutation (A309G) in the PSMB5 gene, introducing a Met45Val amino-acid substitution) compared to the original BTZ resistant THP-1 cells that showed a Met45Iso substitution. These results indicate that the mutations are acquired during BTZ exposure and are mainly induced in specific hot-spots (dominantly Ala49) within the PSMB5 gene. To explore whether mutation-induced resistance could be bypassed, a new proteasome inhibitor, 5-amino-8-hydroxyquinole (5AHQ), acting as a non-competitive inhibitor of the non-catalytic alpha-7 subunit of the proteasome (Li et al. ASH 2008) was studied in our model system. Strikingly, all BTZ-resistant selectants retained full sensitivity towards 5AHQ (IC50: 4–7 μM, measured in a 4-day MTT cytotoxicity assay) as compared to parental cells. To determine whether mutation induction also occurs in patients treated with BTZ, screening for their presence in clinical samples of BTZ refractory patients is warranted. The notion that 5AHQ can overcome BTZ resistance related to single and multiple mutations in the PSMB5 gene, supports further research in this drug and its analogues. This study is supported by VUmc - Stichting Translational Research (STR) and The Netherlands Organization for Health Research and Development (ZonMw). Citation Information: Clin Cancer Res 2010;16(7 Suppl):A27

The Novel Proteasome Inhibitor 5-Amino-8-Hydroxyquinole (5AHQ) Overcomes Bortezomib Resistance in Malignant Hematological Cell Line Models Harboring Mutations in the PSMB5 Gene.

Abstract 940The proteasome inhibitor Bortezomib (BTZ, Velcade®) specifically inhibits the catalytic beta 5 subunit of the proteasome, thereby interfering in the degradation of proteins involved in cell-cycling, NF-kB activation, apoptosis induction and micro-environmental interactions. The introduction of BTZ has shown promising results in the treatment of Multiple Myeloma (MM), Non-Hodgkin lymphoma and leukemia. Despite these encouraging results, clinical trials in MM also revealed that a significant proportion of patients acquired resistance to BTZ mono-therapy.Previously we reported the development of three different BTZ-resistant cell line models, including the AML THP-1 cell-line (Oerlemans & Franke et al, Blood 2008), the T-ALL CCRF-CEM-C7 cell-line and the MM RPMI-8226 cell line (Franke et al, ASH 2008) after chronic exposure to stepwise increasing concentrations of BTZ. The aim of our current study was to further elucidate the molecular basis of BTZ resistance in these cell lines and investigate whether the resistant phenotype could be overcome by the second generation proteasome inhibitor 5-amino-8-hydroxyquinole (5AHQ), acting as a non-competitive inhibitor of the non-catalytic alpha-7 subunit of the proteasome (Li et al. ASH 2008). When added to intact cells, cell extracts, or isolated rabbit reticulocyte proteasomes (gift of A Navon, Weizmann Institute of Science, Israel), 5AHQ inhibited the chymotrypsin-like enzymatic activity of the proteasome with IC50's of 6.9 ± 0.05 uM, 4.2 ± 0.6 uM, and 2.1 ± 0.18 μM, respectively.Cells were initially selected for growth at 7 nM BTZ to acquire low levels of BTZ resistance (2-3 fold higher IC50 concentrations) and subsequently challenged to higher concentrations of BTZ (up to 500 nM) to provoke higher resistance levels. Sequencing of the PSMB5 gene, encoding for the beta 5 proteasome subunit, revealed a series of mutations in individual BTZ-resistant subclones. An overview of the mutation site in the PSMB5 gene, the amino acid position at which substitutions take place at the beta 5 subunit protein level, and the functional implication of each mutation are depicted in the table below.Cell lineMutationBTZ-resistant subcloneAmino acid changeFunctional effect on beta 5 subunit proteinTHP-1 (AML)NoneTHP1/BTZ7NoneG322ATHP1/BTZ30-200Ala49ThrBTZ binding pocketG322A G311TTHP1/BTZ500Ala49Thr Met45Ileenzyme-substrate interactions (hydrolysis of hydrophobic peptide bonds)CEM-C7 (ALL)G332TCEM/BTZ7Cys52Phesubstrate/inhibitor binding domainG332T C323TCEM/BTZ 30-200Cys52Phe Ala49ValBTZ binding pocket8226 (MM)None8226/BTZ7NoneA247G8226/BTZ14-30Thr21AlaBTZ binding pocketG322A8226/BTZ100Ala49ThrBTZ binding pocketSubsequently, we determined whether THP-1, CEM and 8226 cells with low and high levels of BTZ resistance would be sensitive towards the new proteasome inhibitor 5AHQ. Strikingly, all BTZ-resistant selectants retained full sensitivity towards 5AHQ (IC50: 4-7 μM, measured in a 4-day MTT cytotoxicity assay) as compared to parental cells.Together, these data indicate that in vitro selection of low and high levels of BTZ resistance in 3 hematological cell lines is accompanied by the introduction of multiple mutations of amino acids, all of which have key positions in BTZ binding within the active site of the proteasome beta 5 subunit. Notably, Ala49 mutations were independently observed in all 3 cell lines selected for BTZ concentrations > 100 nM. Knowledge of specific mutations in the PSMB5 gene that can confer BTZ resistance allows purposive screening for these types of mutations in clinical samples of BTZ refractory patients. In this respect, the notion that 5AHQ can bypass BTZ resistance related to single or multiple mutations in the PSMB5 gene, supports further preclinical development and clinical application of this drug. This study is supported by VUmc - Stichting Translational Research (STR) and The Netherlands Organization for Health Research and Development (ZonMw), The Leukemia and Lymphoma Society and the Ontario Institute for Cancer Research through funding from the Ministry of Research and Innovation, Province of Ontario. Disclosures:No relevant conflicts of interest to declare.

Characterization of Bortezomib (Velcade ™) Resistance in a New Set of Human Cell Lines Obtained by Stepwise Selection.

Abstract 2741Poster Board II-717Bortezomib (Velcade ™, PS-341, BTZ) is a boronate dipeptide that reversibly inhibits the 26S proteasome, which is essential for the breakdown of ubiquitinated proteins and the regulation of normal cellular homeostasis. The activity of BTZ in treatment of newly diagnosed and refractory/relapsed multiple myeloma may be limited by the development of chemoresistance, the mechanisms of which are poorly understood. To investigate the molecular basis of Bortezomib resistance, BTZ-resistant (BTZr) cell lines were generated by stepwise selection procedures from HeLa, CCRF-CEM, and 4 multiple myeloma cells lines (8226S, U266, H929, and MM.1S), respectively. These BTZ-selected cell lines displayed varying degrees of elevated resistance (2 to 50 fold) to clinically relevant concentrations of BTZ. In addition, while most of the BTZr cells showed cross resistance to several other proteasome inhibitors (PIs), including MG-132 and Epoxomicin, they remained as sensitive to other chemotherapeutic drugs, such as anthracyclines, vinkalkaloids and etoposide, as their parental cells. The proteasome activity profiles are distinct among the cell lines. All parental cell lines displayed varying levels of chymotrypsin-like (CT-L) activity, which is the primary target of BTZ. Most BTZr lines showed markedly decreased CT-L activity, with a few exceptions. Moreover, the observed CT-L activity in all cell lines can be inhibited directly by BTZ and other PIs. In contrast, very low levels of caspase-like or post-glutamyl peptide hydrolyzing (PGPH) proteasome activity were detected in all cell lines. BTZ resistance in HeLa/BTZ cells was closely associated with increased resistance to PI-induced apoptosis, as shown by reduced number of Annexin V-stained cells and by delayed activation/cleavage of apoptosis proteins, such as Caspase-3 and Poly(ADP-ribose) Polymerase (PARP). Furthermore, the resistance to BTZ affected the mechanisms of cell stress responses. As for the parental HeLa cells, HeLa/BTZr cells retained the ability to form, in response to PI treatment, pro-survival foci in the cytoplasm known as stress granules (SGs). However, the drug concentrations required to induce SG formation in HeLa/BTZr cells are much higher (∼4 fold) than those for the parental HeLa, suggesting the development of stress-coping mechanisms in these BTZr cells. Gene expression profiling studies are in progress to identify transcriptomes individually or generally associated with BTZ resistance in these cell lines. Further characterization of these phenotypically similar, yet mechanistically distinct BTZr cell lines may elucidate diverse mechanisms of drug resistance to Bortezomib and other proteasome inhibitors. Disclosures:No relevant conflicts of interest to declare.