ABSTRACT
ABSTRACT: To establish a strict p53-dependent gene-expression profile, TP53-/- clones were derived from TP53+/+ and TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53-/- clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation-coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.
Subject(s)
Antineoplastic Agents , Multiple Myeloma , Pyrimidines , Thiophenes , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , CRISPR-Cas Systems , Cell Line , Proto-Oncogene Proteins c-bcl-2/metabolism , Cell Line, Tumor , Apoptosis , Antineoplastic Agents/therapeutic useABSTRACT
Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.
Subject(s)
Endoribonucleases/genetics , Multiple Myeloma/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Aged , Animals , Bortezomib/pharmacology , Endoplasmic Reticulum Stress/genetics , Endoribonucleases/antagonists & inhibitors , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lenalidomide/pharmacology , Male , Mice , Middle Aged , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Unfolded Protein Response/genetics , X-Box Binding Protein 1/genetics , Xenograft Model Antitumor AssaysABSTRACT
BH3 mimetics are promising drugs for hematologic malignancies that trigger cell death by promoting the release of proapoptotic BCL2 family members from antiapoptotic proteins. Multiple myeloma is considered to be a disease dependent mainly on MCL1 for survival, based mostly on studies using cell lines. We used a BH3-mimetic toolkit to study the dependency on BCL2, BCLXL, or MCL1 in malignant plasma cells from 60 patients. Dependencies were analyzed using an unbiased BH3 mimetics cell-death clustering by k-means. In the whole cohort of patients, BCL2 dependency was mostly found in the CCND1 subgroup (83%). Of note, MCL1 dependence significantly increased from 33% at diagnosis to 69% at relapse, suggesting a plasticity of the cellular dependency favoring MCL1 dependencies at relapse. In addition, 35% of overall patient samples showed codependencies on either BCL2/MCL1 or BCLXL/MCL1. Finally, we identified a group of patients not targeted by any of the BH3 mimetics, predominantly at diagnosis in patients not presenting the common recurrent translocations. Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection from cell death observed by BAK knock-down, as well as the complete and early disruption of MCL1/BAK complexes on A1210477 treatment. Interestingly, this complex was also dissociated in A1210477-resistant cells, but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. In conclusion, our study opens the way to rationally use venetoclax and/or MCL1 BH3 mimetics for clinical evaluation in myeloma at both diagnosis and relapse.
Subject(s)
Antineoplastic Agents , Biomimetic Materials , Multiple Myeloma , Myeloid Cell Leukemia Sequence 1 Protein , Peptide Fragments , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Cell Line, Tumor , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
Venetoclax is a selective, orally bioavailable BCL-2 inhibitor that induces cell death in multiple myeloma (MM) cells, particularly in those harboring t(11;14), which express high levels of BCL-2 relative to BCL-XL and MCL-1. In this phase 1 study, patients with relapsed/refractory MM received venetoclax monotherapy. After a 2-week lead-in with weekly dose escalation, daily venetoclax was given at 300, 600, 900, or 1200 mg in dose-escalation cohorts and 1200 mg in the safety expansion. Dexamethasone could be added on progression during treatment. Sixty-six patients were enrolled (30, dose-escalation cohorts; 36, safety expansion). Patients received a median of 5 prior therapies (range, 1-15); 61% were bortezomib and lenalidomide double refractory, and 46% had t(11;14). Venetoclax was generally well tolerated. Most common adverse events included mild gastrointestinal symptoms (nausea [47%], diarrhea [36%], vomiting [21%]). Cytopenias were the most common grade 3/4 events, with thrombocytopenia (32%), neutropenia (27%), anemia (23%), and leukopenia (23%) reported. The overall response rate (ORR) was 21% (14/66), and 15% achieved very good partial response or better (≥VGPR). Most responses (12/14 [86%]) were reported in patients with t(11;14). In this group, ORR was 40%, with 27% of patients achieving ≥VGPR. Biomarker analysis confirmed that response to venetoclax correlated with higher BCL2:BCL2L1 and BCL2:MCL1 mRNA expression ratios. Venetoclax monotherapy at a daily dose up to 1200 mg has an acceptable safety profile and evidence of single-agent antimyeloma activity in patients with relapsed/refractory MM, predominantly in patients with t(11;14) abnormality and those with a favorable BCL2 family profile. Registered at www.clinicaltrials.gov: #NCT01794520.
Subject(s)
Antineoplastic Agents/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Multiple Myeloma/drug therapy , Sulfonamides/therapeutic use , Adult , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Bridged Bicyclo Compounds, Heterocyclic/adverse effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Female , Humans , Male , Middle Aged , Molecular Targeted Therapy , Neoplasm Recurrence, Local/drug therapy , Sulfonamides/adverse effects , Sulfonamides/pharmacokineticsABSTRACT
BACKGROUND: Despite recent advances in the treatment of multiple myeloma, the disease constantly relapses and is still considered as incurable. The current knowledge about the biological mechanisms underlying resistance to the different class of drugs in multiple myeloma remains poor. The primary objective of the MYRACLE (Myeloma Resistance And Clonal Evolution) cohort, a multicenter prospective cohort of patients with multiple myeloma, is to address this limitation. We here describe the study background, design and methods used for this cohort. METHODS/DESIGN: All patients (> 18 year old) diagnosed with de novo or relapsed multiple myeloma and treated in two hematology department from west of France are included in the MYRACLE cohort. Patients provide a signed informed to be included in the study. All subjects are followed until refusal to participate in the study or death. The MYRACLE cohort prospectively collects data on socio-economic status, medical status, imaging, prognosis factors, MM therapies and associated events (resistance, safety issues). Patients also complete standardized quality of life questionnaires. In addition, bone marrow samples will be collected at time of diagnosis and relapses to perform biomarkers analysis and functional assays exploring mechanisms underlying drug resistance. DISCUSSION: The "real-life" MYRACLE cohort offers the opportunity to prospectively collect epidemiological, medical, QoL and biological data from MM patients during the course of the disease (at time of diagnosis and subsequent relapses). At mid-tem, this integrative cohort will be unique at producing a large variety of data that can be used to conceive the most effective personalized therapy for MM patients. Additionally, the MYRACLE cohort will allow integrating the medical care of MM patients in a health and pharmacoeconomic perspective.
Subject(s)
Multiple Myeloma/drug therapy , Neoplasm Recurrence, Local/drug therapy , Female , France , Humans , Male , Prognosis , Prospective Studies , Quality of Life , Surveys and Questionnaires , Survival Analysis , Treatment OutcomeABSTRACT
Mantle cell lymphoma (MCL) accumulates in lymphoid organs, but disseminates early on in extranodal tissues. Although proliferation remains located in lymphoid organs only, suggesting a major role of the tumor ecosystem, few studies have assessed MCL microenvironment. We therefore cocultured primary circulating MCL cells from 21 patients several weeks ex vivo with stromal or lymphoid-like (CD40L) cells to determine which interactions could support their proliferation. We showed that coculture with lymphoid-like cells, but not stromal cells, induced cell-cycle progression, which was amplified by MCL-specific cytokines (insulin-like growth factor-1, B-cell activating factor, interleukin-6, interleukin-10). Of interest, we showed that our model recapitulated the MCL in situ molecular signatures (ie, proliferation, NF-κB, and survival signatures). We further demonstrated that proliferating MCL harbored an imbalance in Bcl-2 family expression, leading to a consequent loss of mitochondrial priming. Of interest, this loss of priming was overcome by the type II anti-CD20 antibody obinutuzumab, which counteracted Bcl-xL induction through NF-κB inhibition. Finally, we showed that the mitochondrial priming directly correlated with the sensitivity toward venetoclax and alkylating drugs. By identifying the microenvironment as the major support for proliferation and drug resistance in MCL, our results highlight a selective approach to target the lymphoma niche.
Subject(s)
Lymphoma, Mantle-Cell/pathology , Lymphoma, Mantle-Cell/therapy , Molecular Targeted Therapy , Tumor Microenvironment , Aged , Aged, 80 and over , Antibodies, Monoclonal, Humanized/pharmacology , Antigens, CD20/immunology , CD40 Ligand/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Coculture Techniques , Down-Regulation/drug effects , Drug Resistance, Neoplasm/drug effects , Female , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Lymphoid Tissue/pathology , Male , Mesoderm/pathology , Middle Aged , Mitochondria/drug effects , Mitochondria/metabolism , NF-kappa B/metabolism , Tumor Microenvironment/drug effects , Up-Regulation/drug effects , bcl-X Protein/metabolismABSTRACT
(1) Background: TP53 deficiency remains a major adverse event in Multiple Myeloma (MM) despite therapeutic progresses. As it is not possible to target TP53 deficiency with pharmacological agents, we explored the possibility of activating another p53 family member, p73, which has not been well studied in myeloma. (2) Methods: Using human myeloma cell lines (HMCLs) with normal or abnormal TP53 status, we assessed TP73 methylation and expression. (3) Results: Using microarray data, we reported that TP73 is weakly expressed in 47 HMCLs and mostly in TP53 wild type (TP53wt) HMCLs (p = 0.0029). Q-RT-PCR assays showed that TP73 was expressed in 57% of TP53wt HMCLs (4 out of 7) and 11% of TP53 abnormal (TP53abn) HMCLs (2 out of 18) (p = 0.0463). We showed that TP73 is silenced by methylation in TP53abn HMCLs and that decitabine increased its expression, which, however, remained insufficient for significant protein expression. Alkylating drugs increased expression of TP73 only in TP53wt HMCLs but failed to synergize with decitabine in TP53abn HMCLs. (4) Conclusions: Decitabine and melphalan does not appear as a promising combination for inducing p73 and bypassing p53 deficiency in myeloma cells.
Subject(s)
Azacitidine/analogs & derivatives , Melphalan/pharmacology , Multiple Myeloma/metabolism , Tumor Protein p73/metabolism , Tumor Suppressor Protein p53/deficiency , Azacitidine/pharmacology , Base Sequence , Cell Line, Tumor , Cisplatin/pharmacology , CpG Islands/genetics , DNA Methylation/drug effects , Decitabine , Gene Expression Regulation, Neoplastic/drug effects , Humans , Tumor Protein p73/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The aim of this study was to assess the efficiency of p53 reactivation and induction of massive apoptosis (PRIMA-1(Met)) in inducing myeloma cell death, using 27 human myeloma cell lines (HMCLs) and 23 primary samples. Measuring the lethal dose (LD50) of HMCLs revealed that HMCLs displayed heterogeneous sensitivity, with an LD50 ranging from 4 µM to more than 200 µM. The sensitivity of HMCLs did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1(Met) did not induce or increase expression of the p53 target genes CDKN1A or TNFRSF10B/DR5. However, PRIMA-1(Met) increased expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1(Met)-induced cell death. PRIMA-1(Met) depleted glutathione (GSH) content and induced reactive oxygen species production. The expression of GSH synthetase correlated with PRIMA-1(Met) LD50 values, and we showed that a GSH decrease mediated by GSH synthetase silencing or by and L-buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, increased PRIMA-1(Met)-induced cell death and overcame PRIMA-1(Met) resistance. PRIMA-1(Met) (10 µM) induced cell death in 65% of primary cells independent of the presence of del17p; did not increase DR5 expression, arguing against an activation of p53 pathway; and synergized with L-buthionine sulphoximine in all samples. Finally, we showed in mouse TP53(neg) JJN3-xenograft model that PRIMA-1(Met) inhibited myeloma growth and synergized with L-buthionine sulphoximine in vivo.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Glutathione/metabolism , Multiple Myeloma/pathology , Quinuclidines/pharmacology , Reactive Oxygen Species/metabolism , Animals , Antineoplastic Agents/therapeutic use , Cell Death/drug effects , Cells, Cultured , Female , Humans , Mice , Mice, SCID , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Quinuclidines/therapeutic use , Signal Transduction/drug effects , Tumor Suppressor Protein p53/physiology , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Mantle Cell Lymphoma (MCL) is often associated with progression, temporary response to therapy and a high relapse rate over time resulting in a poor long-term prognosis. Because MCL is classified as an incurable disease, therapeutic resistance is of great interest. However, knowledge about the biological mechanisms underlying resistance associated with MCL therapies and about associated predictors remains poor. The REFRACT-LYMA Cohort, a multicenter prospective cohort of patients with MCL, is set up to address this limitation. We here describe the study background, design and methods used for this cohort. METHODS/DESIGN: The REFRACT-LYMA Cohort Study aims at including all patients (>18 years old) who are diagnosed with MCL in any stage of the disease and treated in specialized oncology centers in three public hospitals in Northwestern France. Any such patient providing a signed informed consent is included. All subjects are followed up indefinitely, until refusal to participate in the study, emigration or death. The REFRACT-LYMA follow-up is continuous and collects data on socio-economic status, medical status, MCL therapies and associated events (resistance, side effects). Participants also complete standardized quality of life (QOL) questionnaires. In addition, participants are asked to donate blood samples that will support ex vivo analysis of expression and functional assays required to uncover predictive biomarkers and companion diagnostics. If diagnostic biopsies are performed during the course of the disease, extracted biological samples are kept in a dedicated biobank. DISCUSSION: To our knowledge, the REFRACT-LYMA Cohort Study is the first prospective cohort of patients with MCL for whom "real-life" medical, epidemiological and QOL data is repeatedly collected together with biological samples during the course of the disease. The integrative cohort at mid-term will be unique at producing a large variety of data that can be used to conceive the most effective personalized therapy for MCL patients. Additionally, the REFRACT-LYMA Cohort puts the medical care of MCL patients in a health and pharmacoeconomic perspective.
Subject(s)
Lymphoma, Mantle-Cell/therapy , Quality of Life , Research Design , Adolescent , Adult , Aged , Aged, 80 and over , Combined Modality Therapy , Female , Follow-Up Studies , France , Humans , Male , Middle Aged , Neoplasm Grading , Neoplasm Staging , Prognosis , Prospective Studies , Survival Rate , Young AdultABSTRACT
BACKGROUND: The aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells. METHODS: A large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines. RESULTS: Both drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53(mutated) cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (≥ 1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (≤ 19%). CONCLUSION: These data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.
Subject(s)
Antineoplastic Agents/pharmacology , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Signal Transduction/drug effects , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Silencing , Humans , Inhibitory Concentration 50 , Mitochondria/metabolism , Protein Transport , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Transcriptional Activation/drug effects , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismSubject(s)
Apoptosis/drug effects , Multiple Myeloma/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Protein Biosynthesis/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyrroles/pharmacology , Apoptosis/genetics , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Gene Expression , Humans , Multiple Myeloma/genetics , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Proto-Oncogene Proteins c-bcl-2/geneticsABSTRACT
Multiple myeloma is a plasma cell malignancy that is heterogeneous with respect to its causative molecular abnormalities and the treatment response of patients. The Bcl-2 protein family is critical for myeloma cell survival. ABT-737 is a cell-permeant compound that binds to Bcl-2 and Bcl-x(L) but not to Mcl-1. Using a myeloma cell line collection (n = 25) representative of different molecular translocations, we showed that ABT-737 effectively kills a subset of cell lines (n = 6), with a median lethal dose ranging from 7 ± 0.4 nM to 150 ± 7.5 nM. Of interest, all sensitive cell lines harbored a t(11;14). We demonstrated that ABT-737-sensitive and ABT-737-resistant cell lines could be differentiated by the BCL2/MCL1 expression ratio. A screen of a public expression database of myeloma patients indicates that the BCL2/MCL1 ratio of t(11;14) and hyperdiploid patients was significantly higher than in all other groups (P < .001). ABT-737 first induced the disruption of Bcl-2/Bax, Bcl-2/Bik, or Bcl-2/Puma complexes, followed by the disruption of Bcl-2 heterodimers with Bak and Bim. Altogether, the identification of a subset of cell lines and primary cells effectively killed by ABT-737 alone supported the evaluation of ABT-263, an orally active counterpart to ABT-737, for the treatment of t(11;14) and hyperdiploid groups of myeloma harboring a Bcl-2(high)/Mcl-1(low) profile.
Subject(s)
Antineoplastic Agents/therapeutic use , Biphenyl Compounds/therapeutic use , Gene Expression Regulation, Neoplastic , Multiple Myeloma/drug therapy , Nitrophenols/therapeutic use , Proto-Oncogene Proteins c-bcl-2/genetics , Sulfonamides/therapeutic use , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Biphenyl Compounds/pharmacology , Cell Line, Tumor , Cyclin D1/metabolism , Humans , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Nitrophenols/pharmacology , Piperazines/pharmacology , Piperazines/therapeutic use , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfonamides/pharmacology , Tumor Cells, CulturedABSTRACT
Secondary plasma cell leukemia (sPCL) is a rare form of aggressive plasma cell malignancy arising mostly at end-stage refractory multiple myeloma and consequently presenting limited therapeutic options. We analyzed 13 sPCL for their sensitivity to BH3 mimetics targeting either BCL2 (venetoclax) or BCLXL (A1155463) and showed that 3 sPCL were efficiently killed by venetoclax and 3 sPCL by A1155463. Accordingly, BH3 profiling of 2 sPCL sensitive to BCLXL inhibition confirmed their high BCLXL primed profile. While targeting BCLXL using BH3 mimetics induces platelets on-target drug toxicity, the recent development of DT2216, a clinical-stage BCLXL proteolysis targeting chimera PROTAC compound, provides an alternative strategy to target BCLXL. Indeed, DT2216 specifically degrades BCLXL via VHL E3 ligase, without inducing thrombocytopenia. We demonstrated in human myeloma cell lines and sPCL that sensitivity to DT2216 strongly correlated with the sensitivity to A1155463. Interestingly, we showed that low doses of DT2216 (nM range) were sufficient to specifically degrade BCLXL after 48 hours of treatment, consistent with VHL expression, in all cell lines but irrespectively to DT2216 sensitivity. In myeloma cells, DT2216 induced apoptotic cell death and triggered BAX and BAK activation. In conclusion, our study demonstrated that patients with sPCL addicted to BCLXL, a small but a very challenging group, could potentially receive therapeutic benefit from DT2216. Clinical trials of DT2216 in this subset of sPCL patients are warranted.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Drug Resistance, Neoplasm/genetics , Gene Expression Profiling , Melanoma/genetics , Molecular Mimicry , Protein Interaction Domains and Motifs , Sulfonamides/pharmacology , Antineoplastic Agents/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Humans , Melanoma/diagnosis , Melanoma/drug therapy , Sulfonamides/therapeutic useABSTRACT
The purpose of this study was to identify the pathways associated with the ability of CD138(+) human myeloma cells to form colonies in a serum-free semi-solid human collagen-based assay. Only 26% (7 of 27) of human myeloma cell lines were able to spontaneously form colonies. This spontaneous clonogenic growth correlated with the expression of the NOTCH ligand JAG2 (p<0.001). Blocking JAG-NOTCH interactions with NOTCH-Fc chimeric molecules impaired self-colony formation, indicating a role for JAG-NOTCH pathway in colony formation. In two cell lines, silencing of JAG2 blocked both colony formation and in vivo tumor formation in immunocompromised mice. RT-PCR and flow cytometry analysis revealed that JAG2 is often expressed by CD138(+) primary cells. Our results indicate that spontaneous clonogenic growth of myeloma cells requires the expression of JAG2.
Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Multiple Myeloma/metabolism , Receptors, Notch/metabolism , Animals , Cell Line, Tumor , Female , Gene Expression , Gene Silencing , Humans , Intercellular Signaling Peptides and Proteins/genetics , Jagged-2 Protein , Ligands , Membrane Proteins/genetics , Mice , Mice, SCID , Multiple Myeloma/genetics , Signal Transduction , Syndecan-1/metabolismSubject(s)
Antineoplastic Agents/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Compassionate Use Trials , Multiple Myeloma/drug therapy , Sulfonamides/therapeutic use , Translocation, Genetic , Adult , Cell Death/drug effects , Chromosome Deletion , Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 14 , Chromosomes, Human, Pair 17 , Disease Progression , Drug Resistance, Neoplasm , Female , Humans , Leukocytes, Mononuclear/drug effects , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Treatment OutcomeABSTRACT
The level of the Mcl-1 pro-survival protein is highly regulated, and the down-regulation of Mcl-1 expression favors the apoptotic process. Mcl-1 physically interacts with different BH3-only proteins; particularly, Noxa is involved in the modulation of Mcl-1 expression. In this study, we demonstrated that Noxa triggers the degradation of Mcl-1 at the mitochondria according to the exclusive location of Noxa at this compartment. The Noxa-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Because the USP9X deubiquitinase was recently demonstrated to be involved in Mcl-1 protein turnover by preventing its degradation through the removal of conjugated ubiquitin, we investigated whether Noxa affected the deubiquitination process. Interestingly, Noxa over-expression caused a decrease in the USP9X/Mcl-1 interaction associated with an increase in the Mcl-1 polyubiquitinated forms. Additionally, Noxa over-expression triggered an increase in the Mule/Mcl-1 interaction in parallel with the decrease in Mule/USP9X complex formation. Taken together, these modifications result in the degradation of Mcl-1 by the proteasome machinery. The implication of Noxa in the regulation of Mcl-1 proteasomal degradation adds complexity to this process, which is governed by multiple interactions.
Subject(s)
Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , HEK293 Cells , Humans , Mitochondria/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins c-bcl-2/genetics , Tumor Suppressor ProteinsSubject(s)
Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Immunologic Factors/pharmacology , Multiple Myeloma/therapy , Plasma Cells/drug effects , Thalidomide/analogs & derivatives , Animals , Caspase 3/genetics , Caspase 3/immunology , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/immunology , Disease Models, Animal , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/immunology , Ikaros Transcription Factor/genetics , Ikaros Transcription Factor/immunology , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/immunology , Lenalidomide , Mice , Mice, Inbred NOD , Mice, SCID , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/immunology , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/immunology , Multiple Myeloma/genetics , Multiple Myeloma/immunology , Multiple Myeloma/pathology , Plasma Cells/immunology , Plasma Cells/pathology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/immunology , Signal Transduction , Thalidomide/pharmacology , Trans-Activators/genetics , Trans-Activators/immunologyABSTRACT
BACKGROUND: Multiple myeloma is a plasma-cell tumor with heterogeneity in molecular abnormalities and treatment response. DESIGN AND METHODS: We have assessed whether human myeloma cell lines have kept patients' heterogeneity using Affymetrix gene expression profiling of 40 human myeloma cell lines obtained with or without IL6 addition and could provide a signature for stratification of patient risk. RESULTS: Human myeloma cell lines, especially those derived in the presence of IL6, displayed a heterogeneity that overlaps that of the patients with multiple myeloma. Human myeloma cell lines segregated into 6 groups marked by overexpression of MAF, MMSET, CCND1, FRZB with or without overexpression of cancer testis antigens (CTA). Cell lines of CTA/MAF and MAF groups have a translocation involving C-MAF or MAFB, cell lines of groups CCND1-1 and CCND1-2like have a t(11;14) and cell lines of group MMSET have a t(4;14). The CTA/FRZB group comprises cell lines that had no or no recurrent 14q32 translocation. Expression of 248 genes accounted for human myeloma cell line molecular heterogeneity. Human myeloma cell line heterogeneity genes comprise genes with prognostic value for survival of patients making it possible to build a powerful prognostic score involving a total of 13 genes. CONCLUSIONS: Human myeloma cell lines derived in the presence of IL6 recapitulate the molecular diversity of multiple myeloma that made it possible to design, using human myeloma cell line heterogeneity genes, a high-risk signature for patients at diagnosis. We propose this classification to be used when addressing the physiopathology of multiple myeloma with human myeloma cell lines.
Subject(s)
Gene Expression Profiling , Multiple Myeloma/diagnosis , Cell Line, Tumor , Cluster Analysis , Female , Gene Expression Regulation, Neoplastic , Genetic Heterogeneity , Genetic Markers , Humans , Interleukin-6/metabolism , Male , Multiple Myeloma/genetics , Prognosis , Risk Factors , Survival AnalysisABSTRACT
Multiple myeloma (MM) cells express TLR. It has been shown that TLR ligands induce the proliferation, survival, and immune surveillance escape of MM cells through MyD88-TLR pathways. Deciphering TLR function in MM cells will help in understanding the mechanisms of tumor cell growth. In this study, we examined the response of MM cells to the MyD88-independent/TIR-domain-containing adapter-inducing IFN-beta-dependent TLR3. Deregulation of NF-kappaB pathway is a feature of MM cells, and we wondered whether TLR3 activation could mobilize the NF-kappaB pathway. We show that five of seven human myeloma cell line (HMCL) cells expressed TLR3. In the presence of the synthetic TLR3 ligand (poly(I:C)), activation of NF-kappaB pathway was observed in three of five selected TLR3(+) HMCL, NCI-H929, RPMI 8226, and KMM1. In agreement with NF-kappaB activation, only these three HMCL responded to poly(I:C), although by either an increase (KMM1) or a decrease (NCI-H929, RPMI 8226) of proliferation. We show that KMM1 increase of proliferation was prevented by NF-kappaB inhibitor. In contrast, inhibition of proliferation in both NCI-H929 and RPMI 8226 was due to IFN-alpha-induced apoptosis. We next demonstrated that p38 MAPK pathway controlled both IFN-alpha secretion and IFN-alpha-mediated cell death. Moreover, cell death also involved activation of ERK1/2 pathway. In conclusion, our results show that TLR3 ligand induces NF-kappaB pathway activation in MM and support a switching function of type I IFN in the functional outcome of TLR3 triggering in tumor cells.