Presence Of Bortezomib Research Articles

Abstract B007: Investigation of the mechanism of PADI2 regulation by the FBXW7 tumor suppressor

Abstract Serous endometrial cancer (SEC) is an aggressive form of cancer that frequently exhibits recurrent somatic mutations in FBXW7 at residues 465, 479, and 505. FBXW7 is a tumor suppressor and is part of a Skp1-Cullin-F-box containing (SCF) complex that targets various proteins, including certain oncoproteins, for ubiquitin-mediated proteasomal degradation. FBXW7 interacts with Cdc4 phosphodegron (CPD) motif(s) within substrate proteins. We previously reported upregulation of peptidyl arginine deiminase 2 (PADI2), a potentially druggable protein, in FBXW7-mutant SEC cell lines compared to the isogenic FBXW7-wildtype cells. The purpose of this study is to investigate the mechanism by which FBXW7 regulates PADI2 levels. We searched for the CPD motif in the PADI2 protein sequence. We used co-immunoprecipitation and immunoblotting to study FBXW7-PADI2 interactions after transiently transfecting HEK293 cells with constructs expressing MYC-tagged FBXW7 and/or HA-tagged PADI2, in the presence or absence of bortezomib. We identified three putative CPD motifs in PADI2, leading us to hypothesize that wildtype FBXW7 binds PADI2 and thus targets PADI2 for degradation via a proteasome-dependent mechanism. Our experimental data show that FBXW7 interacts with PADI2, in the presence of bortezomib, a proteasome inhibitor, in transiently transfected HEK293 cells. Additionally, we have shown that mutation of the putative CPD motifs of PADI2 abrogates the PADI2-FBXW7 interaction. Our findings implicate PADI2 as a novel substrate of the FBXW7 tumor suppressor. Citation Format: Chandra Mani Kafle, Daphne W. Bell. Investigation of the mechanism of PADI2 regulation by the FBXW7 tumor suppressor [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B007.

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

FABP4 Suppresses the Progress of Multiple Myeloma through Enhancing Alarming Function of Bone Marrow Resident Memory T Lymphocytes

Multiple myeloma (MM) is a hematological malignancy which characterized by malignant plasma cells resident in the bone marrow (BM). The BM microenvironment plays critical roles in the invasion, proliferation and migration of myeloma cells. Despite the therapies of MM improved in recent years, this disease is still incurable and requires precise treatment. Resident memory (TRM) cells are a distinct tissue-localized T cell lineage that is crucial for protective immunity in peripheral tissues. Recent studies reveal TRM cells as a vital component of the host immune response to cancer. TRM-like tumor-infiltrating lymphocytes (TILs) can be found in a wide range of human cancers, where they portend improved prognosis. However, the precise role and mechanism of bone marrow TRM cells in MM still remain elusive. In present study we first found that the accumulation of T RM cells (CD8 +CD44 +CD69 +CD62L -) in BM newly diagnosed MM (NDMM) patients was significantly higher than that of relapse recurrent MM (RRMM) ( Figure A), which was further confirmed by the immunohistochemistry (IHC) staining analysis ( Figure B). By using Seahorse XF e analyzer we found a decrease of oxygen consumption rate (OCR) of T RM cells in RRMM comparing to that of NDMM ( Figure C). Since fatty acid metabolism is important in β oxidation, we further investigated the expression of fatty acid binding proteins (FABP4/FABP5) in T RM cells and showed that FABP4 was dominant in T RM cells of NDMM patients ( Figure D). FABP4 +/+ mice and their relative controls FABP4 -/- mice were adopted to further explore the role and mechanism of T RM cells in MM progression. The abundance of genes related fatty acids β oxidation was significantly decreased in FABP4 -/- T RM cells ( Figure E). The tumor cell viability was decreased in myeloma cells co-cultured with FABP4 -/- T RM cells in the presence of bortezomib ( Figure F). Moreover, the expression of cytotoxic cytokines including IFN γ, TNFα and CXCL10 was significantly higher in FABP4 -/- T RM cells ( Figure G). The alarming function of T RM cells was further enhanced via recruiting more effector T (CD8 +CXCR3 +) cells ( Figure H), therefore the apoptosis of tumor cells was elevated in BM of MM patients. In summary, T RM cells are necessary and sufficient for long-lived protection against tumors. T RM cells derived FABP4 exerts anti-tumor immunity through prolonging the survival of T RM cells and enhancing the recruitment of more effector T cells to suppress the tumor progression ( Figure I). This study suggests that T RM cells might serve as a novel therapeutic target in MM and treatment specific targeting FABP4 might shed light on the development of potential therapeutic strategies for treating MM especially relapse patients. Acknowledgement This project is supported by the Sun Yat-sen University Hundred Talents Program (PT19200101), Natural Science Foundation of Guangdong Province (2022A1515010290).

Abstract 5279: HSP72 expression and suppression of resistance is associated with survival in EGFR mutated NSCLC

Abstract Background: Patients with non-small cell lung cancer (NSCLC) harboring EGFR activating mutations initially respond favorably to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Acquired resistance develops in most patients, usually due to new DNA mutations. Our previous work has shown that EGFR-TKI therapy blocked phosphorylation of Hsp72, leading to degradation and suppression of polymerase α error editing and base excision repair, causing an increase in the overall mutation rate. This study evaluated baseline Hsp72 expression in EGFR mutated NSCLC and assessed Hsp72 ability to suppress EGFR-TKI resistance and improve overall survival (OS) of patients with EGFR mutated NSCLC. Methods: A retrospective cohort of patients was identified with known EGFR mutation positive NSCLC and available tissue from the City of Hope Comprehensive Cancer Center seen between 2008 and 2016. Patients were classified into two groups based on HSP72 levels: less than the mean HSP72 level (low expression, n = 40) and greater than or equal to the mean HSP72 level (high expression, n = 22), and examined OS. A chi-square test for categorical data and multivariate logistic regression analyses was used to study the relation between HSP72 expression and other clinical and demographic data. In vitro, HSP72 overexpression was induced by BPG-15 and degradation was blocked with bortezomib. We analyzed resistance in EGFR mutant NSCLC with osimertinib alone, and in combination with osimertinib and BPG-15 or bortezomib. Results: Sixty-three patients with EGFR mutated NSCLC and available tissue were identified. Median survival of high and low HSP72 expression were 51.5 and 28.8 months respectively (p = 0.003, log-rank test), with HR of 0.47 (95% CI 0.29-0.77). When stratifying by stage (removal of stage I and II patients), the HR associated with a high HSP72 expression remained 0.47 (95% CI 0.29-0.77). In vitro studies showed that in the absence of bortezomib or BGP-15, time to resistance to osimertinib in HCC827 cells was 13 days. In the presence of bortezomib or BGP-15, time to resistance was 29 and 25 days, respectively. A similar phenomenon was observed with the EGFR-TKI, erlotinib. Conclusions: In EGFR mutated NSCLC, a high level of HSP72 expression is associated with improved survival. Furthermore, HSP72 inducing drugs delays the development of resistance in EGFR-mutated NSCLC cancer cells, and mechanisms are under investigation. Supported by R01 CA073764 (BHS), R50 CA211397 (LZ), the William and Anna Tenenblatt Foundation (BHS, KR) and the STOP Cancer Carrie Scott Seed grant (KR). Citation Format: Li Zheng, Angel R. Baroz, Erminia Massarelli, Joy Huang, Ravi Salgia, Binghui Shen, Karen L. Reckamp. HSP72 expression and suppression of resistance is associated with survival in EGFR mutated NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5279.

Translational and Clinical Evidence of a Differentiated Profile for the Novel CELMoD, Iberdomide (CC-220)

Translational and Clinical Evidence of a Differentiated Profile for the Novel CELMoD, Iberdomide (CC-220)

Percutaneous Exposures of volunteers to polychromatic light (480-3400 nm) trigger systemic mechanism of the human myeloma cells growth delay without any effect on bortezomib cytotoxicity in vitro

Polychromatic light is actively used in medicine. However, its oncological safety and effect against cytotoxic therapy remains poorly studied. Multiple myeloma (MM) develops in the bone marrow and therefore malignant plasma cells are inaccessible to direct exposure to light. The aim of our work was to study the influence of polychromatic visible and infra-red light (pVIS + pIR) on growth and sensitivity of the myeloma cells to the cytotoxic effect of drug bortezomib (BTZ) through systemic mechanism. We explored the effect of volunteers blood sera after their 4-daily irradiations with pVIS + pIR light (480-3400 nm) on growth and viability of the human myeloma RPMI 8226 cells in the presence of BTZ at its application in 2 modes: short-term (1 h, 300 nM) and long-term ( 96 h, 5 nM). Viability and proliferative activity of cells was evaluated by MTT assay. It was found that photomodified blood sera delayed growth of myeloma RPMI 8226 cells (by 25 % in 48 h and 23 % in 96 h) but had no effect on spontaneous and mitogen-induced proliferation of autologous peripheral blood T- and B-lymphocytes. We also revealed that pVIS + pIR did not change RPMI 8226 cells sensitivity to BTZ. The results suggest the systemic mechanism of polychromatic light and argue in favor of its oncological safety during/after BTZ therapy of MM patients without effect on the drug cytotoxicity.

Iberdomide (CC-220) Has Synergistic Anti-Tumor and Immunostimulatory Activity Against Multiple Myeloma in Combination with Both Bortezomib and Dexamethasone, or in Combination with Daratumumab in Vitro

Despite significant progress in the treatment of multiple myeloma (MM), the disease remains incurable. Multiple targeted and biologics-based therapies, including immunomodulatory agent IMiD® compounds (lenalidomide and pomalidomide), proteasome inhibitors (bortezomib and carfilzomib), and monoclonal antibodies (daratumumab and elotuzumab) have shown impressive activity in treating advanced MM. Moreover, triplet regimens combining these agents have consistently proven to be more efficacious than doublets in heavily pretreated patients with limited additional toxic effects. Iberdomide (CC-220) is a novel compound being investigated in a phase I/II study (clinicaltrials.gov NCT02773030) for treatment of lenalidomide- and pomalidomide-relapsed/refractory MM (RRMM) in combination with dexamethasone. Preclinical studies of iberdomide have shown that it more potently binds to cereblon than other cereblon-binding compounds, is more efficient at degrading Aiolos and Ikaros, and has enhanced immunomodulatory activity, inducing greater interleukin-2 secretion and granzyme-b degranulation in immune cells (Matyskiela et al and Bjorklund et al, submitted abstract). Clinical studies of bortezomib and daratumumab in combination with other cereblon-binding agents have demonstrated high tolerability with notable efficacy in the RRMM setting; however, these combinations with iberdomide have not been investigated. Here we show in MM cell lines that iberdomide induces deep Aiolos and Ikaros degradation in the presence of bortezomib at clinically relevant concentrations as determined in healthy volunteers. Furthermore, iberdomide treatment in combination with bortezomib produced synergistic antiproliferative activity and deeper induction of apoptosis than combinations of other clinically approved cereblon-binding compounds with bortezomib across multiple cell lines. In addition, adding dexamethasone resulted in further synergistic antiproliferative activity. In combination with daratumumab, iberdomide also had synergistic anti-MM activity in Complement-Dependent Cytotoxicity (CDC) assays. In co-culture systems using myeloma and immune cells, iberdomide significantly increased the antibody-dependent cellular cytotoxic activity of daratumumab. While iberdomide treatment of MM cell lines resulted in increased CD38 surface expression, combinations were more effective when peripheral blood mononuclear cells (PBMCs) were pretreated, suggesting that iberdomide immunomodulatory activity is a significant contributor to the synergy observed. Interestingly, pretreatment of PBMCs with daratumumab resulted in reduced efficacy of the combination. We observed that the treatment of PBMCs with daratumumab resulted in killing of natural killer (NK) cells in the PBMC culture. In contrast, treatment of PBMCs with iberdomide resulted in proliferation of NK cells, possibly helping to rescue the antagonistic effect of daratumumab on NK cell-mediated antibody‐dependent cellular cytotoxicity. Taken together, these preclinical data support further investigation of iberdomide in combination with both bortezomib/dexamethasone and daratumumab in the clinic. DisclosuresAmatangelo:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corporation: Employment, Equity Ownership. Kang:Celgene Corporation: Employment, Equity Ownership. Polonskaia:Celgene Corporation: Employment, Equity Ownership. Viswanatha:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

Proteasome Inhibition with Bortezomib Induces Apoptosis of Long-Lived Plasma Cells in Steroid-Resistant or Relapsed Immune Thrombocytopaenia.

Primary immune thrombocytopaenia (ITP) is the most common haemorrhagic disease. Although most patients respond initially to mainstream therapies, such as corticosteroids, immunosuppressants or rituximab, a large proportion of patients fail to respond or relapse. These treatments only affect B lymphocytes or short-lived plasma cells, but not already existing long-lived plasma cells (LLPCs) which persistently secrete antibodies. We hypothesized that LLPCs may play a role in the corticosteroid-resistant or relapsed ITP patients, and bortezomib, a proteasome inhibitor, may act on plasma cells and offer a therapeutic effect. Although a significant difference in the proportion of CD19-CD38hiCD138+ total LLPCs was not observed by flow cytometry, a glycoprotein (GP) IIb/IIIa-specific enzyme-linked immunosorbent spot (ELISpot) assay of sorted CD19-CD138+ LLPCs confirmed the existence of anti-platelet antibody-secreting LLPCs in ITP patients in contrast to healthy controls. Moreover, the LLPCs could be eliminated in the presence of bortezomib by ELISpot assay, which was also confirmed by flow cytometry. Accordingly, a modified monoclonal antibody immobilization of platelet antigen assay of sorted CD19-CD138+ LLPCs revealed that the concentration of anti-platelet antibodies decreased remarkably when cultured with 0.25 ng/mL bortezomib for 5 days. The apoptosis assay demonstrated that bortezomib could induce apoptosis of LLPCs in a concentration-dependent manner. The proteasome activity assay showed that bortezomib significantly reduced the proteasome activity in sorted CD19-CD138+ LLPCs. Furthermore, in active ITP murine models, bortezomib eliminated LLPCs in vivo and alleviated thrombocytopaenia. We conclude that LLPCs participate in the pathogenesis of ITP and bortezomib may have potential as a novel therapeutic regimen.

GRP78 inhibition enhances ATF4-induced cell death by the deubiquitination and stabilization of CHOP in human osteosarcoma

New targeted therapies are urgently needed to improve the survival of patients with refractory osteosarcoma (OS). In this study, we show that bortezomib (BTZ), not for OS treatment in the clinic, induces endoplasmic reticulum (ER) stress in U-2 OS cells. Loss of GRP78 sensitizes OS to BTZ with concomitant upregulation of ATF4 and CHOP, which indicates excessive protein synthesis. The relevance of these findings is confirmed in vivo as shown by GRP78 knockdown that delays the growth of U-2 OS xenografts in the presence of BTZ. Here, we demonstrate that MG7, a natural polyyne, can trigger apoptosis. Of note, the apoptotic response to MG7 is dependent on ATF4 but not on the upstream PERK signaling pathway. Interestingly, MG7-induced ATF4 expression does not result in an increase in the levels of CHOP. We demonstrate for the first time that GRP78 physically interacts with the N-terminal domain of CHOP to accelerate its ubiquitination in a p300-dependent manner, which in turn desensitize the tumors to ER stress. Overall, inhibiting GRP78 to strengthen the molecular mechanism of ATF4 via stabilizing CHOP protein may provide a potential vulnerability in OS.

Induction of Apoptosis in U937 Cells by Using a Combination of Bortezomib and Low-Intensity Ultrasound

BackgroundWe scrutinized the feasibility of apoptosis induction in blood cancer cells by means of low-intensity ultrasound and the proteasome inhibitor bortezomib (Velcade).Material/MethodsHuman leukemic monocyte lymphoma U937 cells were subjected to ultrasound in the presence of bortezomib and the echo contrast agent Sonazoid. Two types of acoustic intensity (0.18 W/cm2 and 0.05 W/cm2) were used for the experiments. Treated U937 cells were analyzed for viability and levels of early and late apoptosis. In addition, scanning electron microscopy analysis of treated cells was performed.ResultsThe percentage of cells that underwent early apoptosis in the group treated with ultrasound and Sonazoid was 8.0±1.31% (intensity 0.18 W/cm2) and 7.0±1.69% (0.05 W/cm2). However, coupling of bortezomib and Sonazoid resulted in an increase in the percentage of cells in the early apoptosis phase, up to 32.50±3.59% (intensity 0.18 W/cm2) and 33.0±4.90% (0.05 W/cm2). The percentage of U937 cells in the late apoptosis stage was not significantly different from that in the group treated with bortezomib only.ConclusionsOur findings indicate the feasibility of apoptosis induction in blood cancer cells by using a combination of bortezomib, ultrasound contrast agents, and low-intensity ultrasound.

Abstract 5468: Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance

Abstract Introduction: Multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) are lymphoplasmacytic malignancies which are characterized by continuous spread through cell trafficking in and out of the BM. We have recently shown that the spread process in these diseases is mediated by hypoxia. Moreover, despite the introduction of novel therapies, cancer patients relapse due to the development of a drug-resistant stem cell-like subpopulation, which we recently found to be, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome spread and drug resistance in MM and WM by inhibition of the hypoxic responses in these cells. Tirapazamine (TPZ) is a hypoxia-activated pro-drug causing cell apoptosis, which has been shown to improve cancer patient outcome when combined with radiotherapy. We use TPZ for the first time in hematologic malignancies to target the drug-resistant cancer cells and sensitize them to therapy. Procedures: Cell survival was tested on MM (MM1.s, H929, OPM1, RPMI8226) and WM (BCWM.1, MWCL.1) cell lines exposed to normoxia (21% O2) or hypoxia (1% O2) for 24hrs in the presence of bortezomib and carfilzomib, different concentrations of TPZ (in order to obtain an IC50), or combination of those drugs using MTT assay. For adhesion assay, cancer cells were pre-labeled with calcein-AM, treated with TPZ, applied to unlabeled endothelial cells, non-adherent cells were washed after 1hr, and adherent cells were analyzed by a fluorescent reader. Chemotactic properties of cells were tested using a transwell chamber and the migrated cells were counted by flow cytometry. For in vivo study, 5 × 106 MM1s-Luc-GFP cells were injected i.v. into 6 SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. All mice were treated with (1) high-dose bortezomib (1.5mg/kg) only (n = 3), or (2) bortezomib and TPZ (40mg/kg; n = 3), all administered i.p. sequentially twice a week. Bone marrow and peripheral blood were collected and analyzed for the presence of GFP+ cells by flow cytometry. Summary of data: We found that hypoxia induced drug resistance to bortezomib and carfilzomib of MM and WM cells, and that TPZ was active in a dose-dependent manner only in hypoxic conditions. Combination of TPZ with bortezomib and carfilzomib, resensitized cancer cells to death in hypoxia. Addition of TPZ increased adhesion of cancer cells to endothelial cells, and decreased chemotactic properties of cancer cells in hypoxia in vitro. Moreover, post-treatment residual cancer cells were further decreased using TPZ, preventing recurrence of MM in vivo. Conclusion: We report that TPZ prevents hypoxia-induced metastasis and overcomes hypoxia-induced drug resistance in MM and WM. This is the first study to show the efficacy of TPZ in hematologic malignancies in general and in MM and WM in particular. This data provide a preclinical basis for future clinical trials testing efficacy of TPZ in MM and WM. Citation Format: Barbara Muz, Pilar de la Puente, Feda Azab, Micah Luderer, Abdel Kareem Azab. Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5468. doi:10.1158/1538-7445.AM2015-5468

NIK is required for NF-κB-mediated induction of BAG3 upon inhibition of constitutive protein degradation pathways.

Recently, we reported that induction of the co-chaperone Bcl-2-associated athanogene 3 (BAG3) is critical for recovery of rhabdomyosarcoma (RMS) cells after proteotoxic stress upon inhibition of the two constitutive protein degradation pathways, that is, the ubiquitin-proteasome system by Bortezomib and the aggresome-autophagy system by histone deacetylase 6 (HDAC6) inhibitor ST80. In the present study, we investigated the molecular mechanisms mediating BAG3 induction under these conditions. Here, we identify nuclear factor-kappa B (NF-κB)-inducing kinase (NIK) as a key mediator of ST80/Bortezomib-stimulated NF-κB activation and transcriptional upregulation of BAG3. ST80/Bortezomib cotreatment upregulates mRNA and protein expression of NIK, which is accompanied by an initial increase in histone H3 acetylation. Importantly, NIK silencing by siRNA abolishes NF-κB activation and BAG3 induction by ST80/Bortezomib. Furthermore, ST80/Bortezomib cotreatment stimulates NF-κB transcriptional activity and upregulates NF-κB target genes. Genetic inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) or by knockdown of p65 blocks the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein expression. Interestingly, inhibition of lysosomal activity by Bafilomycin A1 inhibits ST80/Bortezomib-stimulated IκBα degradation, NF-κB activation and BAG3 upregulation, indicating that IκBα is degraded via the lysosome in the presence of Bortezomib. Thus, by demonstrating a critical role of NIK in mediating NF-κB activation and BAG3 induction upon ST80/Bortezomib cotreatment, our study provides novel insights into mechanisms of resistance to proteotoxic stress in RMS.

Bone Marrow Stromal Cells Induce Bortezomib Resistance Partly through a Transcriptome Modulation in Multiple Myeloma Cells

INTRODUCTIONIt has been suggested that environment-mediated drug resistance (EMDR) might account for the intrinsic (de novo) resistance to therapy in some myeloma patients developing in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Understanding the mechanisms exploited by bone marroe stromal cells (BMSCs) to generate such resistance has long been sought. Furthermore, knowledge of the role that changes in expression of various genes could play is scanty. In this study we explored how BMSCs could induce resistance to bortezomib and whether it would change the expression of an array of genes in MM cells in the presence of bortezomib.METHODSUsing patient-derived BMSCs and a normal immortalized human BMSC line (HS-5), we set up a model in which several human myeloma cell lines (HMCLs), RPMI-8226, U266, MM1.S, OPM2 and H929, were co-cultured with stromal cells in the presence or absence of bortezomib. Drug cytotoxicity in HS-5 context was performed using XTT assay. Transwell system, Paraformaldehyde (PFA) fixation of BMSCs and treatment of BMSCs with Brefeldin-A (BFA, an inhibitor of intracellular protein transport) were used as controls to delineate the direct or indirect roles of BMSCs. Percentage of apoptotic cells was determined with anti-CD138/APC, annexin-V/FITC and PI staining in FACS. For gene expression array, co-cultures were applied to magnetic cell separation (EasySep, Stem Cell Technologies), total RNA was isolated from MM cell pellets and synthesized cDNAs were applied to real time PCR gene expression arrays. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in co-cultures compared to MM cell monocultures in the presence of bortezomib using the 2-ΔΔCtalgorithm.RESULTSIn the XTT cytotoxicity assay, all tested HMCLs displayed a higher level of viability in the presence of stroma than in stroma-free conditions (average bortezomib IC50s: stroma 22nM vs no stroma 8.53nM, p<0.02). Moreover, FACS analysis for all HMCLs showed that the percentage of bortezomib-induced apoptosis in stroma context was significantly lower than that in stroma-free conditions (p<0.05). Notably, in BFA and PFA treatment settings the level of bortezomib-induced apoptosis was lower (but not significant) than that in Transwell system suggesting that both soluble factors secreted by BMSCs and direct adhesion contribute to stroma-induced resistance to bortezomib. Gene expression analyses in real time PCR showed that the induced resistance was associated with downregulation of some genes involved in cell cycle and proliferation control (CDKN2A, CDC6, CDK2, EGR1, PTEN), downregulation of some genes involved in p53 signaling pathway (PUMA, P73, P53, MDM2, NOXA, BAX) and downregulation of some genes involved in apoptosis (CASP3, PARP).CONCLUSIONOur findings suggest that BMSCs induce resistance against cytotoxic and apoptotic effects of bortezomib in MM cells. To this end, in our tested HMCLs, the gene expression patterns may imply involvement of multiple pathways in controlling stroma-induced resistance to bortezomib. Although gene expression results were verified real time PCR readouts, changes in genes may not always correlate with protein changes. Hence, our study has given us the rationale to proceed with more in depth exploration as to whether BMSCs may render MM cells resistant to bortezomib by negative modulation of p53 signaling pathway controlling apoptosis and cell cycle. DisclosuresNo relevant conflicts of interest to declare.

Myeloid Derived Suppressor Cell Mediated AMPK Activation Regulates Multiple Myeloma Cell Survival

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that accumulate in different cancer types, including Multiple Myeloma (MM). Besides immune regulation, MDSC promote tumor angiogenesis and tumor growth by secretion of cytokines and growth factors. The presence and activation of MDSC in MM patients has been well-documented, however direct actions of MDSC on cancer cells have been poorly defined. In this study, we investigated MDSC distribution in the immunocompetent 5T33MM murine model and the effects of in vivo MDSC targeting by 5-Fluorouracil (5FU) in the presence of bortezomib. In addition, we determined direct effects of MDSC on MM cells in vitroin the presence of currently used anti-MM drugs (Bortezomib and Melphalan) and analyzed downstream pathways.In a first part, we determined the effects of the MM microenvironment on the total MDSC population using the 5T33MM model. The number of MDSC (defined as CD11b+ cells) was only increased the first week after MM cell inoculation, while circulating myeloid cells increased at end-stage of disease. In addition, we evaluated the effect of 5FU, an MDSC depleting chemotherapy agent, on tumor progression. 5T33MM mice (n=7/group) were treated with 50mg/kg 5FU on day 4, with bortezomib (0.7mg/kg, 2 times a week), with the combination of both or with vehicle and tumor load was assessed at day 17. We observed a significant reduction in serum M-spike and idiotype positive cells in the BM and spleen (30% reduction compared to vehicle, p<0.05). Importantly, 5FU in combination with bortezomib showed an even stronger reduction of tumor load compared to single agent therapy (p<0.01, 65% reduction compared to vehicle).Besides immune suppression, MDSC may exert direct effects on tumor growth. To investigate direct actions on MM cells, we performed co-culture experiments of 5T33MM derived MDSC with 5T33MM cells in vitro, resulting in increased survival and proliferation of MM cells. Also in the presence of Bortezomib (2.5-5nM) and Melphalan (15-30µM) we could observe a protection against induced apoptosis. In addition, transwell experiments demonstrated that both cell-cell contact and soluble factors are involved in the observed MDSC-mediated increased survival of MM cells. To investigate underlying pathways we performed a Pathscan® Intracellular Signaling Array (Cell Signaling) demonstrating an upregulation of pAMPK (phospho Adenosine Monophosphate-activated Protein Kinase) in 5T33MM cells after co-culture with MDSC. AMPK plays a key role in energy homeostasis and has been described to be expressed and activated in MM (Baumann et al., 2007). AMPK activation was confirmed by western blot and we could observe an increase in anti-apoptotic factors Mcl-1 and Bcl-2. We subsequently targeted AMPK by BML-275 (compound C) and observed a decrease of AMPK phosphorylation, Mcl-1 and Bcl-2 expression, and an increase in PARP cleavage and apoptosis of MM cells. Using distinct human myeloma cell lines (LP-1, RPMI-8226, U266) we confirmed the effect of BML-275 to reduce MM viability, indicating a role of AMPK in MM cell survival.In conclusion, our data clearly demonstrate that MDSC directly increase the survival of MM cells, even in the presence of bortezomib or melphalan, mediated by AMPK phosphorylation. In addition, targeting of MDSC in combination with bortezomib showed promising preclinical results for treatment of MM patients.(Reference: Baumann P., Mandl-Weber S., Emmerich B., Straka C., Schmidmaier R. (2007) Inhibition of adenosine monophosphate-activated protein kinase induces apoptosis in multiple myeloma cells. Anticancer Drugs. 18(4):405-10) DisclosuresNo relevant conflicts of interest to declare.

Targeted Inhibition of the MLL Transcriptional Complex By Proteosome Inhibitors Elicits a High Response Rate in Relapsed/Refractory MLL Rearranged Leukemia

Infants with MLL rearranged (MLLr) acute lymphoblastic leukemia (ALL) have a poor prognosis, with an event free survival of only 23-44%. Whole genome sequencing (WGS) of this subtype has revealed a paucity of cooperating mutations, with an average of 2.2 somatic single nucleotide variations and/or insertions/deletions per case. Despite recent progress in defining the epigenetic alterations that result from the expression of the MLL fusion protein, these insights have only recently begun to be extrapolated into the development of new therapeutic approaches whose benefits have yet to be defined. Thus, there remains an urgent need for the development of alternative approaches to improve outcomes in these patients.To identify compounds that are active in MLLr disease, we established in vitro and in vivo assays to evaluate drug sensitivity of primary infant ALL patient samples. 15 infant MLLr leukemia samples that have previously undergone WGS were xenografted into NOD/SCID/IL2Rγnull (NSG) mice. All samples engrafted and expanded in NSG mice, leading to overt leukemia with a latency of 49 to 276 days. Purification of leukemic blasts from a single moribund mouse yielded on average 108 cells, providing sufficient material to screen large numbers of compounds. In vitro conditions were defined that support growth in 40% of the patient specimens, allowing for a more accurate determination of drug sensitivity. Growth in vitro was associated with early onset of disease in NSG xenografts and younger age at presentation, allowing us to evaluate patient samples that represent aggressive high risk disease. Using this system, we tested bortezomib in addition to 28 other drugs, including standard ALL therapeutic agents as well as targeted kinase inhibitors and inhibitors of epigenetic marks. Three classes of agents were active in this system: anthracyclines, histone deacetylase inhibitors (HDACi), and the proteasome inhibitor bortezomib. In contrast to anthracyclines and HDACi, where IC50 values were on par with those reported in the literature for primary childhood ALL samples, MLLr infant samples required 10-100 fold less bortezomib to induce toxicity.Bortezomib has been shown to mediate responses through several mechanisms, including NFKB inhibition, stabilization of cell cycle regulatory proteins, and induction of apoptosis. Recently, proteasome inhibition has been demonstrated to lead to accumulated MLL fusion protein levels, triggering apoptosis and cell cycle arrest in MLLr cell lines. To determine if NFKB inhibition also plays a role, we evaluated cellular concentrations of the activated NFKB transcription factor, but failed to see decreased levels when MLLr cells were treated with bortezomib. Bortezomib has also been shown to deregulate ubiquitin stores and deplete histone H2B ubiquitination (H2Bub), an epigenetic mark that is linked to histone methylation and expression. Recently, several groups have demonstrated that H2Bub is required for DOT1L activity and HOX gene expression. We therefore evaluated H2Bub levels in bortezomib-treated patient samples and confirmed depletion of this epigenetic mark. Furthermore, patient samples treated with bortezomib downregulated both the MLL gene expression signature and signatures of downstream targets, such as cMYC, demonstrating that the MLL transcriptional program is inhibited in the presence of bortezomib. ChIP-seq is underway to map H2Bub and H3K79 methylation changes genome wide in response to treatment with bortezomib.The HDACi vorinostat and bortezomib have both been evaluated in Phase I and II pediatric leukemia clinical trials. Based on the safety and efficacy from these earlier studies, we treated 6 relapsed/refractory MLLr leukemia patients with a chemotherapy regimen that included mitoxantrone, vorinostat, and bortezomib. 4 patients had a complete response (CR), 1 patient had a partial response (PR) and 1 patient had stable disease for an overall response rate of 5/6 (83%). Clinical trials are in development to assess this combination further for both relapsed MLLr disease as well as newly diagnosed infant ALL. Our data suggests that these three classes of drugs, identified in our laboratory assays, are clinically active thus validating our system. We are now using this platform to proceed with a high throughput drug screen to identify additional compounds for future clinical development. DisclosuresOff Label Use: Vorinostat and Bortezomib for the treatment of pediatric leukemia.

Bortezomib-Resistance Is Characterized By Upregulation of E3 Ligase Pirh2 Which Protects Bortezomib Induced Apoptosis in Multiple Myeloma Cells

Background Proteasome inhibitor bortezomib is an effective approach to the treatment of multiple myeloma (MM), but drug resistance often emerges and limits its retreatment. However, the action mechanisms are not fully understood. Several lines of evidence link E3 ligase to myeloma and its drug resistance. Pirh2 (p53-induced RING-H2) E3 ubiquitin ligase with intrinsic ubiquitin-protein ligase activity for polyubiquitination and subsequently proteasomal degradation was predisposed for plasma cell hyperplasia and tumorigenesis as reported. In this study, we examined Pirh2 expression in plasma cells of the bone marrow (CD138+ cells) from MM patients and investigate the effect of Pirh2 on the viability, proliferation, survival and drug resistance of MM cells.Methods Bortezomib-sensitive malignant haematopoetic cells can acquire secondary resistance to Bortezomib in vitro. To identify some of the mechanisms involved, we developed myeloma cell lines OPM-2 and RPMI8226 bortezomib resistant (OPM-2-BR, RPMI8226-BR) derivative lines by continuous culture in sub-lethal concentrations of bortezomib. Clones of OPM-2-BR and RPMI8226-BR were obtained after several months, and acquired resistance to bortezomib remained stable over months with extended stimulus duration in the presence of bortezomib. Pirh2 mRNA and protein expressions were detected by real time quantitative PCR(Q-RT PCR) and Western blotting. We also generated stable Pirh2 knock down cell lines and stable Pirh2 overexpression cell lines.Results We found differential expression of Pirh2 in bortezomib-resistant (BR) cells, compared to wild type (WT) controls by Q-RT PCR and Western blotting. We confirmed generally Pirh2 mRNA and protein expressions in MM cell lines. Besides, Pirh2 mRNA expression was also determined from bone marrow samples of MM patients. In summary, we showed that Pirh2 is more highly expressed in MM cell lines and patient MM cells (from newly diagnosed or refractory/recurrence patients with MM) than in normal bone marrow mononuclear cells from normal donors (P<.0001). Interestingly, We found that Pirh2 overexpression cell lines showed negatively effect on the response to bortezomib treatment. Consistent with this, Pirh2 knock down cell lines can overcome bortezomib resistance.Conclusions Collectively, Our findings demonstrate a novel role for Pirh2, which suggests that Pirh2 may act by an unknown mechanism involved in bortezomib resistance of MM. An improved understanding of it can lead to better therapeutics for bortezomib retreatment which will be a rational strategy for clinical translation. DisclosuresNo relevant conflicts of interest to declare.

Ammonium Production and Glutamine-Addiction of Myeloma Cells: New Attractive Targets in Multiple Myeloma

It is a historical notion that the growth of human myeloma cell lines (HMCLs) was limited by depletion of L-glutamine (Gln), and it has been reported that myeloma cells produce an excess of ammonium (NH4+), as a possible rare clinical manifestation in multiple myeloma (MM) patient. Recently, Gln metabolism has been found of critical importance in several types of cancer cells, which have been defined Gln-addicted. However, the relationship between NH4+ production and Gln-addiction in MM cells, as well as the mechanisms involved therein, are unknown, and were investigated in this study.Firstly, we assessed the NH4+ production by several HMCLs (RPMI-8226, JJN3, KMS12-BM, XG1 and OPM2) and found that all these lines produced an excess of NH4+ only in presence of Gln; conversely, non-MM cell lines, such as the acute lymphoblastic leukemia 697 cells, did not. Then, we screened 13 MM patients, finding that 4 of them (1 newly diagnosis and 3 relapsed MM) had increased peripheral blood NH4+ level and one of them presented relevant clinical signs of encephalopathy. Then, we checked NH4+ production by freshly purified CD138+ cells from bone marrow (BM) aspirates of these MM patients, showing that CD138+ plasma cells (PCs), but not CD138- fraction, from hyperammonemic patients produced, in the presence of Gln, significantly more NH4+, than those of non-hyperammonemic patients (median increase: +236%). We retrospectively assessed BM plasma NH4+ levels in a cohort of 30 patients with monoclonal gammopathies finding significantly increased median levels from MGUS to relapsed MM patients (Kruskal Wallis test P=0.01).To study the molecular mechanisms involved in NH4+ production, the expression of enzymes involved in Gln metabolism (GLS1 and GLS2 glutaminases, glutamine synthetase (GS) and asparagine synthetase (ASNS) was evaluated in HMCLs, through Real Time PCR and Western blot. HMCLs expressed GLS1, ASNS, and, at variable levels, GLS2, while, interestingly, had negligible levels of expression of GS, compared to the ALL cell line 697. These observations were extended in two proprietary (NCBI GEO series accessions: GSE13591 and GSE6205) and two publicly available databases (GSE6477 and GSE6691). 323 global dataset, including 18 healthy donors, 28 MGUS, 19 SMM, 200 newly diagnosed and 26 relapsed MM, 9 plasma cell leukemia (PCL) patients, together with 23 HMCLs, were normalized using custom GeneAnnot-based Chip Annotation Files (v.2.2.0) and Robust Multi-array Average procedure. Kruskal-Wallis and Jonckheere-Tepstra tests were applied to find significant differences and trends, respectively, in gene expression levels between different PC dyscrasias. Benjamini-Hochberg procedure was applied for multiple testing correction. In PCL and HMCLs samples, we identified the highest ASNS and the lowest GLS2 gene expression levels. In addition, several genes for Gln transporters, were highly expressed, showing significant differences in expression levels among MM disease phases: in particular, SLC38A1, SLC7A5 and SLC1A5 showed significant increase of expression level from normal PCs to HMCLs, across the different PC dyscrasias, whereas SLC38A3 gene resulted poorly expressed in PCL and HMCL. Interestingly, the expression of SLC38A1, SLC7A5 and SLC1A5 was positively, and SLC38A3 negatively, correlated with that of MYC.We next investigated the effects of Gln depletion on MM cells. We confirmed that in all the HMCLs tested Gln depletion has marked cytotoxic effects, independently on the presence of the GS inhibitor methionine sulfoximine (MSO), consistently with the lack of GS expression by MM cells. In line with these observations, HMCLs were 10-times more sensitive to E. chrysanthe Asparaginase (ASNase) than to E. coli ASNase characterized by a 10-fold lower glutaminase activity. Interestingly ASNase effects were increased in the presence of Bortezomib (0-10nM). The effect of inhibitors of Gln transporters and Gln-metabolizing enzymes on MM cells survival is under investigation.In conclusion our data suggest that in PCs cells acquire features of Gln-addiction during monoclonal gammopathies progression consisting of (i) lack of GS expression, (ii) altered expression of Gln transporters and, in a subset of patients and in HMCLs, (iii) increased NH4+ production. Our data also suggest that Gln-addiction could be a new attractive therapeutic strategy in MM. DisclosuresGiuliani:Celgene Italy: Research Funding.

Fibroblast Activation Protein Protects Bortezomib Induced Apoptosis In Multiple Myeloma Cells Through β-Catenin Signaling Pathway

BackgroundMultiple myeloma(MM) is a malignant plasma cells proliferative disease which is characterized by increased blood calcium level, renal insufficiency, anemia, and bone lesions(CRAB). The intricate cross-talk with the bone marrow microenvironment plays an important role in facilitating the growth and survival of myeloma cells. Fibroblast activation protein(FAP) is a vital transmembrane protein expressed in 90% epithelial tumor stroma, which is reported to be involved in mediating drug resistance, tumorigenesis, neoplastic progression, angiogenesis, invasion and metastasis of tumor cells. The present study is aimed to investigate the roles of FAP may play in regulation of apoptosis in MM cells induced by bortezomib and the potential signaling pathway that FAP may be participated in. MethodsBone marrow mesenchymal stem cells(hBMMSCs) from MM patients and normal donors and the cell line of hBMMSCs were analyzed for expression of the FAP protein by semiquantitative real time-polymerase chain(qRT-PCR), flow cytometry(FCM) and immunofluorescence(IF). Tumor cell-conditioned medium (TCCM) from supernatant of MM cell lines were added to hBMMSCs to observe the effect of TCCM for the expression of FAP. We further studied the function and mechanism of FAP in bortezomib induced apoptosis of myeloma cells by silencing FAP with small interfering RNA(siRNA). Apoptotic cells of MM cells were detected by APC-CD138/annexin V-FITC using flow cytometry analysis. Western blotting was used to elucidate the signaling pathway that FAP may be involved in mediating apoptosis of MM cells induced by bortezomib. ResultsThere was no significant difference in the expression of FAP in hBMMSCs isolated from MM patients and normal donors(p>0.05) as determined by qRT-PCR, FCM and immunofluorescence. hBMMSCs stimulated by TCCM for 7 days displayed an elevated expression of FAP as detected by qRT-PCR(p<0.05). In the presence of 30nM bortezomib, MM cell lines RPMI8226 or CAG cells co-cultured with hBMMSCs in which FAP was knockdown or not by siRNA for 48h demonstrated that FAP is capable of protecting RPMI8226 and CAG cells induced by bortezomib from apoptosis as determined by FCM(NC siRNA vs FAP siRNA, p<0.05). Further study showed that the activity of β-catenin was significantly elevated in RPMI8226 cells after co-cultured with hBMMSC in the presence of bortezomib. Knockdown FAP can reduce the expression of β-catenin and its downstream target proteins, such as c-myc, survivin, cyclin D1 in RPMI8226 cells detected by western blot. ConclusionsTaken together, our data indicated that the expression level of FAP was no difference between the hBMMSCs isolated from MM patients and normal donors. The expression of FAP can be increased by TCCM stimulation. Further study demonstrated that FAP can protect MM cells from apoptosis induced by bortizomib, which is likely through β-catenin signaling pathway. Disclosures:No relevant conflicts of interest to declare.

Elevated Expression Of CKS1B Inhibits Senescence Thorough Enhanced Degradation Of p21 In Multiple Myeloma

BackgroundHigh expression of CKS1B gene as a result of amplification of region 1q21 identifies a subset of patients with poor prognosis in multiple myeloma (MM). We have previously shown that over-expression of CKS1B enhances degradation of p27 and activates both MEK/ERK and JAK/STAT3 signaling pathways, promotes MM cell drug-resistance. However, other mechanisms of drug resistance induced by high expression of CKS1B remain to be identified. Materials and methodsCKS1B was over-expressed or knocked-down by shRNA in OCI-MY5 and KMS28PE myeloma cell lines. Cell viability was detected by PrestoBlue assay; Annexin-V assay was employed to detect cell apoptosis. Colony formation was assessed in soft agar. The direct dye efflux to assess multidrug resistance was performed using eFluxx-ID Multidrug resistance assay. Senescence was detected by SA-β-galactosidase (β-Gal) staining. Expression of SCF-Skp2 substrates was evaluated by western blots. ResultsWe demonstrated that overexpression of CKS1B significant decreased sensitivity to bortezomib and increased the clonogenic capacity compared to cells transfected with empty vector in the presence of bortezomib (p < 0.05). A prominent mechanism of drug resistance involved upregulation of multidrug resistance associated proteins (MRPs). We investigated whether the level of MDR1, BCRP, and MRP1, mitotic checkpoint protein MAD2 and ABC transporter family members was increased in cells with elevated CKS1B. However, none of drug pump proteins was up-regulated or stabilized by over-expression of CKS1B in OCI-MY5 and KMS28PE. Accordingly, over-expression of CKS1B did not promote efflux of the hydrophilic eFluxx-ID™ gold fluorescent dye from MM cells any greater than control cells, suggesting alternative mechanisms. We therefore tested whether CKS1B can inhibit drug-induced senescence in MM cells after treatment with bortezomib by quantifying the percentage of cells expressing senescence-associated β-Gal. Interestingly, MM cells with elevated CKS1B were significantly less β-Gal positive following treatment with bortezomib compared to control cells (p < 0.05). To explore molecular mechanism underlying CKS1B induced anti-senescence, expression of SCF-Skp2 substrates involved in senescence pathway was examined as CKS1B is an essential cofactor for SCF-Skp2 complex. Over-expression of CKS1B significantly reduced expression of p21, p27, and p57 while knock-down of CKS1B stabilized these proteins. Notably, cells with high expression of CKS1B failed to accumulate p21 protein in response to bortezomib. ConclusionOur findings suggest that anti-senescence is a novel mechanism in CKS1B-induced drug resistance in MM and the turnover of p21 regulated by CKS1B could have important implications not only for bortezomib therapy but also to develop therapies aimed at enhancing p21 function independent of p53. Disclosures:No relevant conflicts of interest to declare.

Carboplatin and oxaliplatin in sequenced combination with bortezomib in ovarian tumour models

BackgroundOvarian cancer remains an on-going challenge mainly due to the development of drug resistance and also because the cancer is likely to have metastasized at the time of diagnosis. Currently, chemotherapy based on platinum drugs such as cisplatin is the primary treatment for the disease. Copper transporter 1 is involved in the transport of cisplatin into the cell, but is also down-regulated by the drug. Bortezomib, a proteasome inhibitor, has been reported to block this platinum-induced down-regulation of CTR1, so that in the presence of bortezomib, the cellular uptake of platinum drugs may be increased. Increased platinum accumulation may result in increased platinum − DNA binding so that the platinum drug in combination with bortezomib may produce enhanced cell kill.MethodsIn this study the efficacy of the sequential combinations of carboplatin, oxaliplatin and a trans-platinum compound coded as CH1 with BORT on the human ovarian A2780, A2780cisR, A2780ZD0473R and SKOV-3 cancer cell lines was evaluated. The levels of cellular platinum accumulation and platinum-DNA binding were determined following the treatment with these combinations. In order to investigate the effect of the combinations of the formation of ROS, the total and oxidized glutathione levels were also determined.ResultsPrevention of copper transporter 1 degradation by bortezomib is found to enhance the cellular accumulation of platinum, the level of Platinum − DNA binding and increases oxidative stress especially in the resistant cell lines.ConclusionsThe results suggest that the prevention of CTR1 degradation by bortezomib may be playing a major role in increasing the cellular uptake of platinum drugs and platinum-DNA binding level. Furthermore, the generation of oxidative stress appears to be a major contributor to the enhanced cell kill.