scholarly journals 47 Genes Define Myeloma Cell Acquired Resistance to Bortezomib and Have Profound Prognostic Implications in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 499-499
Author(s):  
Xenofon Papanikolaou ◽  
Caleb K. Stein ◽  
Ricky D Edmondson ◽  
Veronica Macleod ◽  
Ruslana Tytarenko ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Advisory Board ◽  
University Of Arkansas ◽  
Newly Diagnosed ◽  
Medical Sciences ◽  
Gene Probes ◽  
The Common

Abstract The proteasome inhibitor Bortezomib (Bz), the first agent of a new class of drugs in Multiple Myeloma (MM), has shown remarkable activity and forms an integral part of modern MM treatment. Nevertheless, resistance to Bz eventually develops in a significant proportion of patients, with adverse effects on survival. Numerous publications have addressed this issue through in vitro developed models of acquired Bz resistance (BzR). However the results were quite different in each publication, none of the produced Bz myeloma cell lines was provably stable, no common mechanism of resistance could be demonstrated, and hence were of minimal relevance to the clinical setting. In order to address these issues an effort was made for the development of an in vitro model of acquired BzR that would resemble the clinical reality in the most accurate way. Two myeloma cell lines were used, one resembling a multisensitive (JJN3) and the other a multiresistant (U266) drug behavior, that were both sensitive to Bz. An at least 20 fold increase in the 48h Bz IC50 was noted for both cell lines. The increase in the IC50 was able to be verified a year after culturing the cell lines in normal medium thus ensuring a stable resistance phenotype. To delineate the molecular mechanisms that underlie the development of BzR a combined genetic/Gene Expression Profile (GEP) and functional/Proteomics approach was used with emphasis in the common elements of both cell lines. The hypothesis was that if certain pathways are activated in the cells that actually produce the phenotype of BzR they must fulfil two important criteria: 1) They must be present in all the levels of the BzR, 2) The gene changes have to be verified in the level of the gene encoded proteins thus securing their functional importance. GEP of the naïve cell lines along with the GEP of the Bz resistant cells at different levels of BzR (5-fold, 10-fold, 20-fold) were used. The statistical analysis revealed 100 gene probes common in both cell lines that achieved their highest change as soon as BzR was established and remained stable at that level for all later versions (P<0.1, q<0.1) and 115 gene probes common in both cell lines that their change was proportional to the level of BzR (P<0.001, q <0.005). The proteomics analysis of the Bz resistant cell lines at their latest level of resistance (20-fold) revealed 262 proteins common in both cell lines that were up-regulated and 263 common in both cell lines that were down-regulated (change >10% to be considered significant). The intersection of the list of the common genes with the list of the common proteins revealed 47 gene-proteins all but one novel in MM. They can be grouped in distinct biological categories with the most prominent ones being the ROS/Mitochondrial Factor category comprising of 10 gene-proteins, the E3 Ubiquitin Pathway 6 genes-proteins and Translation Regulation 5 genes-proteins. Even more importantly 30 of them have profound survival implications in MM -all of them novel in MM- both for Overall Survival (OS) and Progression Free Survival (PFS) in both Bz (TT3) and non Bz (TT2) containing protocols implying that myeloma cells apply both Bz specific and non-specific mechanisms to acquire BzR. Based on these 30 genes-proteins a GEP risk score (GEP-30) was constructed that was able to achieve remarkable statistical power in both Bz containing and non containing trials of both newly diagnosed (TT2 with and without thalidomide i.e. TT2+ and TT2-, TT3a, TT3b, HOVON, MRC IX, Figure 1A,B,C) and relapsed MM (TT6 , OS: NR vs 1.52 yr P<0.00001, PFS: NR vs 1.13 yr P<0.00001 for low and high risk) Figure 1. KM plots for OS and PFS of GEP-30 for newly diagnosed MM Figure 1. KM plots for OS and PFS of GEP-30 for newly diagnosed MM Figure 1B. Figure 1B. Figure 1C. Figure 1C. Disclosures Stein: University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Epstein:University of Arkansas for Medical Sciences: Employment. Heuck:Janssen: Other: Advisory Board; Celgene: Consultancy; Millenium: Other: Advisory Board; Foundation Medicine: Honoraria; University of Arkansas for Medical Sciences: Employment.

High GRP78 (78-kDa Glucose-Regulated Protein) Expression Predicts for a Favorable Clinical Outcome in Patients with Multiple Myeloma and May be a Potentially Useful Therapeutic Target in the Treatment of Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4206-4206 ◽  
Author(s):  
Hang Quach ◽  
Daniel North ◽  
Susanna Freddi ◽  
Shuh Y Tan ◽  
Lenny Straszkowski ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Clinical Outcome ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Newly Diagnosed ◽  
Favorable Clinical Outcome ◽  
Glucose Regulated Protein

Abstract Background: GRP78 (78-kDa glucose-regulated protein) is a molecular chaperone that is upregulated during cellular stress. It has been well demonstrated that GRP78 upregulation is associated with chemoresistance and metastasis in solid tumours. GRP78 has not been widely explored in multiple myeloma (MM), however, we and others have shown that GRP78 is much more overexpressed in myeloma cell lines compared to other cell lines. To assess the clinical relevance of GRP78 overexpression in MM, we investigated the association of plasma cell GRP78 expression on primary bone marrow (BM) trephines to clinical outcome in patients with MM, and correlate this finding to concurrent in vitro studies to investigate the potential usefulness of targeting GRP78 for the treatment of MM. Method: The degree of GRP78 expression within CD138+ plasma cells was assessed by immunohistochemistry (IHC) on archived bone marrow trephines of patients with newly diagnosed MM, who underwent autologous stem cell transplant (ASCT) at St.Vincent's Hospital Melbourne. Independent assessment of GRP78 was performed by 3 hematopathologists, who underwent initial calibration. The degree of GRP78 expression within plasma cells was assigned as low, medium or high. Clinical data was abstracted from medical records of the corresponding patients with respect to baseline demographics, treatment-response, progression free survival (PFS), time to next treatment (TTNT) and overall survival (OS). The association GRP78 expression to each of these clinical parameters was assessed using Kaplan-Meier product limit method and the Mantel-Cox logrank test. In vitro, GRP78 expression was also quantified in various myeloma cell lines by RT-PCR and western blot. The association of GRP78 expression to MM-cell survival and drug resistance was assessed in vitro. The impact of GRP78 inhibition on reversal of drug resistance and myeloma-cell viability was investigated. Result: Between the years 2000 to 2014, a total of 243 patients with newly diagnosed MM underwent ASCT as part of initial therapy, and were included in the study. Baseline bone marrow trephine was available for CD138 and GRP78 staining for 91 patients. Of these, 20, 42 and 34% of patients had low, medium and high expression of GRP78 within BM plasma cells, respectively. Low GRP78 expression was associated with a shorter PFS (HR 2.4, p=0.0006) and shorter TTNT (HR 2.5, p=0.008) compared to intermediate or high GRP78 expression. No significant difference was seen in OS. High GRP78 correlated with a higher probability of achieving CR (p=0.03). In vitro, inhibition of GRP78 resulted in decreased myeloma cell viability, and sensitized myeloma cells to various antimyeloma agents. As a result, synergistic anti-myeloma activity was seen when GRP78 inhibition was combined with melphalan (synergy quotient (SQ) 1.2), dexamethasone (SQ 1.97) and especially bortezomib (SQ 2.06). Conclusion: In contrast to what is reported for solid tumours in the literature, higher GRP78 expression appeared to predict for a more favorable clinical outcome in patients with MM. In vitro, GRP78 inhibition resulted in significant anti-myeloma effects and increased the antimyeloma activity of various agents especially bortezomib. Together, these findings suggest that GRP78 is potentially a useful biomarker and therapeutic target that warrants further investigation in patients with MM. Disclosures Quach: Celgene Corp, ONYX, Janssen, Takeda, Novartis, BMS: Honoraria, Research Funding.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

scholarly journals Global Genomic Analysis of Newly Diagnosed t(4 ;14) Multiple Myeloma Reveals a Specific Mutational Spectrum and Identifies PKD2 As a Potential Therapeutic Target

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4462-4462
Author(s):  
Xiu Ly Song ◽  
Raphaël Szalat ◽  
Alexis Talbot ◽  
HaiVu Nguyen ◽  
Mehmet K. Samur ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Plasma Cells ◽  
Genomic Analysis ◽  
Sequencing Analysis ◽  
Newly Diagnosed ◽  
Potential Therapeutic Target ◽  
Mutational Landscape

Abstract In Multiple Myeloma (MM), the t(4;14) translocation is associated with a poor outcome. However, beside this translocation, the genetic events which determine the adverse evolution of the disease and the resistance to treatments remain elusive. In this study we performed whole exome or RNA sequencing analysis of samples from 65 newly diagnosed t(4;14) MM. We found that NRAS, KRAS, MAPK and FGFR3 are frequently mutated (12%, 9%, 13.8%, and 20% respectively). Overall, the FGFR3/RAS/BRAF/MAPK genes were mutated in 36 cases (54%). There was a negative correlation between mutations in FGFR3 and those occurring in NRAS, KRAS and BRAF as expected from the mutually exclusive occurrence of mutations in these genes. In addition to alterations in TP53 and DIS3, we found marked elevated frequency of mutations in PRKD2 (10.7%), ATM/ATR (10.7%) and MYCBP2 (7.6%), reduced frequency in FAM46C (1.5%) and no mutation in TRAF3 and CCND1. Mutations in ATM/ATR were strongly associated with the MB4-2 breakpoint (Bp) (p = 1.62 10-4) and significantly correlated with mutations affecting genes coding for members of the MAPK family. We observed a positive correlation between non-silent mutations in PRKD2 and the MB4-1 or MB4-3 Bp (p = 1.3 10-2). Of note, PRKD2 mutations are exclusively found in 3 t(4;14) MM cell lines and among the 84 MM sequenced by Bolli et al. (1), none of the non t(4;14) patient were mutated in PRKD2, indicating that this genetic lesion is associated with t(4;14) MM. In the NCI-H929 t(4;14) MM cell line, which is mutated for PRKD2, encoding the PKD2 serine/threonine kinase, we observed elevated levels of phosphorylated PKD2. Furthermore, inhibition of PKD, decreased PKD2 phosphorylation and triggered reduced proliferation and apoptosis of MM cell lines and fresh plasma cells from patients in vitro. These results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Altogether, these results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Reference 1. Bolli, N., Avet-Loiseau, H., Wedge, D.C., Van Loo, P., Alexandrov, L.B., Martincorena, I., Dawson, K.J., Iorio, F., Nik-Zainal, S., Bignell, G.R., et al. (2014). Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nat Commun 5, 2997. Disclosures Munshi: Janssen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties.

scholarly journals Pixantrone demonstrates significant in vitro activity against multiple myeloma and plasma cell leukemia

2019 ◽  
Vol 98 (11) ◽  
pp. 2569-2578
Author(s):  
Ella Willenbacher ◽  
Karin Jöhrer ◽  
Wolfgang Willenbacher ◽  
Brigitte Flögel ◽  
Richard Greil ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cell ◽  
Myeloma Cell ◽  
Plasma Cell Leukemia ◽  
Phase Iii ◽  
In Vivo Model ◽  
Cell Leukemia ◽  
Cam Assay

Abstract Treatment results for multiple myeloma and plasma cell leukemia have considerably improved, but cure remains elusive and establishing new therapeutic approaches constitutes a major unmet clinical need. We analyzed the anti-myeloma properties of the aza-anthracenedione pixantrone which has been successfully used in a phase III study for the treatment of patients with aggressive non-Hodgkin’s lymphoma as monotherapy as well as in combination regimes in vitro and in an adapted in vivo model (ex ovo chicken chorioallantoic membrane (CAM) assay). Pixantrone significantly inhibited proliferation and metabolic activity of all investigated myeloma cell lines. Importantly, anti-myeloma effects were more pronounced in tumor cell lines than in stromal cells, mesenchymal stem cells, and peripheral blood mononuclear cells of healthy controls. Apoptosis of myeloma cell lines was observed only after a 7-day incubation period, indicating a fast cytostatic and a slower cytotoxic effect of this drug. Pixantrone reduced the viability of primary plasma cells of patients and induced downregulation of myeloma-cell growth in the CAM assay. Additionally, we demonstrate in vitro synergism between pixantrone and the histone deacetylase inhibitor panobinostat with respect to its anti-proliferative features. From these data, we conclude that systematic investigations of the clinical usefulness of pixantrone in the framework of controlled clinical trials are clearly indicated (e.g., in penta-refractory patients).

Inhibition of HIF1A Signaling by a Novel Class of Sulfonanilides for Targeted Treatment of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2856-2856 ◽  
Author(s):  
Dirk Hose ◽  
Anja Seckinger ◽  
Hartmut Goldschmidt ◽  
Tobias Meiβner ◽  
Blanka Rebacz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Chromosomal Aberrations ◽  
Myeloma Cell ◽  
Proliferation Index ◽  
The Novel ◽  
Myeloma Cells ◽  
Signaling Inhibitors ◽  
Exposure Levels

Abstract Abstract 2856 Poster Board II-832 BACKGROUND. Molecular profiling of multiple myeloma allows the identification of novel targets, including HIF1A, and evaluation of their expression within large cohorts of patients. We report here the expression of HIF1A in myeloma and for the first time the preclinical testing of 4 members of a novel class of sulfonanilide HIF1A signaling inhibitors. PATIENTS AND METHODS. Expression of HIF1A was assessed using Affymetrix DNA-microarrays in 329 samples of CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). Proliferation of primary myeloma cells (n=67) was determined by propidium iodine staining. The effect of the novel HIF1A signaling inhibitors ELR510490, ELR510454, ELR510444 and ELR105813 on the proliferation of 12 human myeloma cell lines and the first three on the survival of 5 primary myeloma cell-samples cultured within their microenvironment was tested, and their ability to inhibit HIF1A signaling was examined using a cell-based reporter assay. Studies were also conducted to determine in vitro stability (in plasma and microsomes), as well as single-dose PK (SDPK) parameters and maximum tolerated dose (MTD) levels after dosing in mice. RESULTS. We found (i) HIF1A to be expressed by 95.4% of CD138-purified primary myeloma cell samples from previously untreated patients. (ii) HIF1A expression shows a weak but significant correlation (r=0.3, p<0.001) with a gene expression based proliferation index. (iii) Of the chromosomal aberrations tested, myeloma cells of patients with presence of a translocation t(4,14) show a significantly higher expression of HIF1A (p<0.001) vs. patients without. Myeloma cells of hyperdiploid patients show a significantly lower expression of HIF1A (p=0.02) vs. non hyperdiploid patients. (iii) HIF1A expression does not show a correlation with event-free or overall survival. (iv) The sulfonanilides ELR510490, ELR510444, ELR510454 and ELR105813 completely inhibit proliferation of all tested myeloma cell lines at nM concentrations. (v) The compounds tested, i.e. ELR510490, ELR510444, ELR510454, are active on all primary myeloma cell-samples tested. (vi) The compounds show a pronounced effect on the HIF1A signaling pathway at EC50s of 1-25nM. (vii) Pre-clinical pharmacology data for the compounds ELR510444 and ELR510490 in mice indicate favorable absorption, distribution, metabolism, and excretion (ADME) profiles as well as exposure levels upon dosing at well-tolerated levels that are significantly above the in vitro EC50 in all the cell lines tested. CONCLUSION. HIF1A is expressed in almost all primary myeloma cells. The novel HIF1A signaling inhibitors tested are very active on myeloma cell lines as well as primary myeloma cells and show favorable in vivo profiles with exposure levels in mice significantly higher than the concentrations required for the inhibition of cell proliferation or apoptosis induction in vitro. This class of compounds thus represents a promising weapon in the therapeutic arsenal against multiple myeloma. Disclosures: Rebacz: ELARA Pharmaceuticals: Employment. Lewis:ELARA Pharmaceuticals: Employment. Schultes:ELARA Pharmaceuticals: Employment.

Constitutive and Immunoproteasome Inhibitors Increase IκB and Decrease NF-κB Expression In Dendritic Cell

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3493-3493
Author(s):  
Ahmad-Samer Samer Al-Homsi ◽  
Zhongbin Lai ◽  
Tara Sabrina Roy ◽  
Niholas Kouttab
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Peripheral Blood ◽  
Research Funding ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell

Abstract Introduction Constitutive and immunoproteasome inhibitors (C&IPI) were thought to suppress nuclear factor-κB (NF-κB) pathway by preventing IκB degradation, which prevents NF-κB translocation into the nucleus. This mechanism of action has since been questioned by a number of studies. First, bortezomib promoted constitutive NF-κB activity in endothelial cell carcinoma. Second, NF-κB constitutive activity was resistant to bortezomib in multiple myeloma cell lines. Third, bortezomib increased IκB mRNA but post-transcriptionally downregulated IκB in normal cells and in multiple myeloma cell lines resulting in induced canonical NF-κB activation. Lastly, bortezomib increased nuclear levels of IκB as opposed to lowering cytoplasmic levels in cutaneous T cell lymphoma cell line suggesting that nuclear translocation of IκB was possibly responsible for NF-κB inhibition. The inhibitory activity of C&IPI on dendritic cells (DC) is of interest in the prevention of graft versus host disease (GvHD). It has been shown that different C&IPI impede DC maturation and T cell priming both in vitro and in vivo. Herein we sought to understand the mechanism of action of proteasome and immunoproteasome inhibitors on DC and to test their effect on IκB and NF-IκB expression. Materials and Methods We first performed RT PCR on lysates of DC obtained from the peripheral blood of 7 patients who received post-transplant cyclophosphamide and bortezomib as prevention of GvHD on a phase I clinical trial. Patients received allogeneic transplantation from matched-related or unrelated donors. Patients received no other immunosuppressive therapy except for rabbit anti-thymocyte globulin for those receiving graft from unrelated donor. Steroids were not allowed on the study. Samples were obtained on days +1, +4, and +7. The results were analyzed in comparison to samples obtained on day 0 before stem cell infusion. We then performed the same experiment on lysates of DC obtained from the peripheral blood of healthy volunteer donors. DC were untreated or incubated with bortezomib (10 nM for 4 h), carfilzomib (30 nM for 1 h), oprozomib (100 nM and 300 nM for 4 h), ONX 0914 (200 nM for 1 h), PR-825 (125 nM for 1 h), or PR-924 (1000 nM for 1 h). The drug concentration and duration of exposure were chosen based on the IC50 on proteasome activity and to reproduce in vivo conditions. We also performed IκB western blot on DC isolated from peripheral blood of healthy volunteers, untreated or incubated with bortezomib (10 nM for 4 h) or oprozomib (300 nM for 4 h). Each experiment was performed at least in triplicate. Results We found that the combination of cyclophosphamide and bortezomib significantly and progressively increased IκB mRNA while decreasing NF-κB mRNA in DC studied ex vivo. We also found that all studied C&IPI increased IκB mRNA to a variable degree while only oprozomib (300 nM) decreased NF-κB mRNA in DC in vitro. Finally, both bortezomib and oprozomib increased IκB protein level in DC in vitro (figure). Conclusion Our data suggest that C&IPI increase IκB expression in DC. As opposed to the previously reported data in other cell types, the effect is not associated with post-transcriptional downregulation. Cyclophosphamide and bortezomib also decrease NF-κB expression in DC in vivo while only oprozomib had the same effect in vitro. The effect of C&IPI on IκB and NF-κB expression may represent a new mechanism of action and suggests their effect may be cell-type dependent. Disclosures: Al-Homsi: Millennium Pharmaceuticals: Research Funding. Off Label Use: The use of cyclophosphamide and bortezomib for GvHD prevention. Lai:Millennium Pharmaceuticals: Research Funding.

High Trip13 and Low P31 Comet Cause Drug Resistance and Poor Prognosis In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3820-3820
Author(s):  
Yi Tao ◽  
Zhimin Gu ◽  
Ye Yang ◽  
Hongwei Xu ◽  
Xiaojing Hu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Caspase 3 ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Newly Diagnosed ◽  
Caspase 9 ◽  
Over Expression

Abstract Background We have recently established that increased chromosomal instability (CIN) signature is linked to drug resistance and poor outcome in multiple myeloma (MM) and other cancers. Thyroid Hormone Receptor Interactor 13 (Trip13), one of the 56 drug-resistant genes, plays a key role in chromosomal recombination and structure development during meiosis and has been reported to be increased in some malignancies including lung cancer, prostate cancer and breast cancer. In this study, we investigated how important Trip13 is in myelomagenesis and progression. Materials and Methods Gene expression profiling (GEP) was analyzed on plasma cells from 22 healthy donors, 44 patients with monoclonal gammopathy of undetermined significance (MGUS), 351 patients with newly diagnosed multiple myeloma, and 9 human myeloma cell lines, as well as on 36 sequential samples at diagnosis, pre-1st, pre-2nd and post-2nd autologous stem cell transplantation (ASCT). Over-expression and knock-down experiments of Trip13 were performed on myeloma cell lines by lentivirus transfection. Cell viability was assessed by trypan exclusion assay. Western blots were used to detect the expression of Trip13, P31 comet, caspase-8, caspase-9, caspase-3 and PARP, and checkpoint related proteins MAD2 and CDC20 in Trip13 overexpressed or Trip13 shRNA-transfected myeloma cells. Results Sequential GEP samples showed that Trip13 expression increased in 8 of 9 patients after chemotherapy and ASCT compared to the samples at diagnosis strongly suggesting that increased Trip13 is associated with drug resistance. Trip13 was already significantly increased in MGUS patients, newly diagnosed MM patients and MM cell lines compared with normal plasma cells. Furthermore, Trip13 was significantly higher in high-risk MMs than in low-risk MMs and increased Trip13 was linked to an inferior event-free survival (EFS) (p<0.01) and overall survival (OS) (p<0.01) in 351 newly diagnosed MMs. In contrast, the Trip13-interacting gene P31 comet was down-regulated in high-risk MMs and high expression of P31 was associated with good outcome. Interestingly, patients with high Trip13 and low P31 comet have the worst outcome compared to patients with only one of these, suggesting the interaction of Trip 13 and p31 has a synergistic effect on MM progression. Transfection of Trip13 into ARP1 and OCI-My5 cells significantly increased cell proliferation, while knock-down Trip13 in OCI-My5, H929, RPMI8226 cells inhibited cell growth and induced MM cell apoptosis with increases of cleaved caspase-8, caspase-9, caspase-3 and PARP. Mechanistic studies showed that Trip13 over-expression decreased P31comet and MAD2 expression by western blotting, but increased CDC20. Conclusions The association of increased Trip13 and decreased p31 is a good biomarker for MM drug resistance and poor prognosis. Our results also show Trip13 and P31 comet could be potential targets to overcome drug resistance in MM. Disclosures: No relevant conflicts of interest to declare.

Gene Expression Profiling of Extramedullary Disease-Related Toward Identification of a Terminal Disease Pathway in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1777-1777
Author(s):  
Sarah Waheed ◽  
Hongwei Wang ◽  
Pingping Qu ◽  
Christoph Heuck ◽  
Aasiya Matin ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Advisory Board ◽  
University Of Arkansas ◽  
Q Value ◽  
Medical Sciences ◽  
Training Set ◽  
Optimal Cutoff ◽  
Advisory Committees ◽  
Gene Probes ◽  
Test Sets

Abstract Introduction Extramedullary disease (EMD) is a primary disease manifestation of MM, which while not seen frequently at presentation increases in incidence at relapse where its incidence seems to be increasing following the introduction of novel agents. Patients with EMD have a shorter overall survival as well as an increased incidence of anemia, thrombocytopenia, elevated serum lactate dehydrogenase, cytogenetic abnormalities, and high-risk features as determined by gene expression profiling. There is also an increased incidence of the high risk MAF subtypes t (14:16 or 14; 20). Understanding the biology of EMD and identifying its present could give important information about how to improve the outcome of this group. In this work we have used GEP analysis of bone marrow derived plasma cells to predict the presence of EMD so that we can identify the genomic risk factors that define the features of a plasma cell clone, which can develop the capacity to metastasize outside the BM. Materials and Methods We focused on patients treated on TT protocols, at the UAMS, Myeloma Institute between 1989 - 2010, a total of 1154 patients, of which 46 developed EMD before the start of therapy (EMD-1), and 91 developed EMD after registration to UAMS for MM treatment EMD-2. Results We show that most EMD2 cases (57.14%) develop within 3 years after initiation of therapy at the UAMS with few cases developing after this time. Predicting the risk of EMD Combining patients with EMD1 and EMD2 diagnosis within 3 years gave a total of 98 EMD cases. We used 824 samples from 1017 myeloma patients who never developed EMD and had follow up at least 3 years as a comparator group. The data were divided into training (n=619 with 66 EMD cases and 553 controls) and test sets (n=303 with 32 EMD cases and 271 controls). Using the training set, we identified 5 significant gene probes (with a q value < 0.001) and made a score to predict cases and controls. The sensitivity and specificity turned out to be 74.24% and 77.40% in the training set, and 56.25% and 76.75% in the test set, respectively. Predicting the time to EMD2 We tested whether we could predict time to EMD2 based on using baseline GEP samples. In this analysis, all EMD2 cases and controls were included. We divided the data into training (n=743 with 61 EMD2 and 682 controls) and test sets (n=365 with 30 EMD2 and 335 controls). By fitting a uniform Cox regression model to each gene in the training set, we identified 68 gene probes that are associated with time to EMD2 (with a q-value <0.1). We then created a score based on the 68 gene probes and identified an optimal cutoff based on the training set. Applying the optimal cutoff to both training and test sets, we found that the new 68-gene high/low risk model is a good predictor on the cumulative incidence of EMD2 (p value < 0.0001). Conclusion We show that EMD2 cases mostly occur within 3 years of diagnosis and a 68 gene based risk score that can predict a cumulative incidence of EMD. Of the 68 genes that are used to develop the prognostic score for EMD, 6 genes are also part of the 70-gene risk score developed by our group. GEP studies can help us identify EMD-specific gene signature that can further help develop target agents. Figure 1. Figure 1. Disclosures Waheed: University of Arkansas for Medical Sciences: Employment. Wang:Cancer Research and Biostatistics: Employment. Qu:Cancer Research and Biostatistics: Employment. Heuck:Millenium: Other: Advisory Board; Foundation Medicine: Honoraria; Janssen: Other: Advisory Board; University of Arkansas for Medical Sciences: Employment; Celgene: Consultancy. Matin:University of Arkansas for Medical Sciences: Employment. Jethava:University of Arkansas for Medical Sciences: Employment. van Rhee:University of Arkansa for Medical Sciences: Employment. Hoering:Cancer Research and Biostatistics: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Davies:University of Arkansas for Medical Sciences: Employment; Millenium: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Morgan:Weismann Institute: Honoraria; University of Arkansas for Medical Sciences: Employment; CancerNet: Honoraria; MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals The Serine Protease Matriptase Acts As a Tumour Suppressor in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-14
Author(s):  
Ida Steiro ◽  
Pegah Abdollahi ◽  
Magne Børset ◽  
Tobias S. Slørdahl
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Serine Protease ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Tumour Suppressor ◽  
Hepatocyte Growth

Both in newly diagnosed multiple myeloma (MM) and during progression of the disease, malignant plasma cells are found circulating in peripheral blood as well as in the bone marrow (BM). The disseminated nature of MM is strongly dependent on the interplay between the cancer cells and the BM microenvironment, promoting myeloma cell migration in the BM. Matriptase (ST14), a type-II transmembrane serine protease primarily found in epithelial tissues, is overexpressed in a variety of human malignancies and is sufficient to induce tumour formation in mice. Frequently, a concomitant reduction in the levels of its cognate inhibitor hepatocyte growth factor activator inhibitor (HAI)-1 (SPINT1) is observed in carcinomas, while expression and function of the related inhibitor HAI-2 (SPINT2) is yet to be clarified. Dysregulated expression causing increased matriptase proteolytic activity has been associated with cancer growth, survival and metastasis. Here, we show for the first time a role of matriptase as a possible tumour suppressor in myeloma pathogenesis. Gene expression analysis of primary cells from MM patients (n=24) and human myeloma cell lines (n=8) revealed highly variable levels of matriptase, HAI-1 and HAI-2. This observation prompted us to investigate the functional role of matriptase in vitro. We showed that stable overexpression of matriptase in INA-6, a MM cell line with no endogenous ST14 expression, reduced migration by more than 50% in response to the combination of the pro-migratory cytokines stromal cell-derived factor-1 alpha (SDF-1α) and hepatocyte growth factor (HGF, Fig. 1A). Conversely, stable knockdown of matriptase in two MM cell lines with high endogenous matriptase expression (RPMI-8226 and JJN-3) significantly enhanced migration in vitro. Mechanistically, matriptase overexpression blocked activation of Src kinase (Fig. 1B), well-known as a critical player in metastasis formation promoting cancer cell motility, invasiveness and angiogenesis. In agreement with our result, previous studies have demonstrated the activation of Src family kinases (SFK) downstream SDF-1/CXCR4-signaling. Finally, we performed survival analyses in the public available MMRF CoMMpass trial database (release version IA14). Low ST14 expression was associated with significant worse overall survival (P=0.05, Fig. 1C) and progression-free survival (P=0.02, Fig. 1D). Altogether, our data are in marked contrast to the role ascribed to matriptase in epithelial and certain non-epithelial tumours as an oncogenic protein and an unfavourable prognostic marker. In conclusion, these findings suggest a novel role of matriptase as a tumour suppressor in MM pathogenesis. Disclosures Slørdahl: Celgene: Consultancy; Janssen and Celgene: Honoraria.

scholarly journals REGN5458, a Bispecific BCMAxCD3 T Cell Engaging Antibody, Demonstrates Robust In Vitro and In Vivo Anti-Tumor Efficacy in Multiple Myeloma Models, Comparable to That of BCMA CAR T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1944-1944 ◽  
Author(s):  
David J Dilillo ◽  
Kara Olson ◽  
Katja Mohrs ◽  
T. Craig Meagher ◽  
Kevin Bray ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

Abstract Improving therapies for multiple myeloma (MM) remains a high medical need because of the significant morbidity and mortality of the disease. Targeted immunotherapies represent a promising opportunity to fill this clinical need. B cell maturation antigen (BCMA) is an attractive cell-surface target for MM due to its consistent expression on MM patient malignant plasma cells and expression limited in normal tissue primarily to plasma cells. Redirection of a patient's T cells to recognize tumors by CD3-binding bispecific molecules or through the generation of chimeric antigen receptor (CAR) T cells, has shown preliminary evidence of clinical activity. Bispecific antibodies concurrently engage a tumor antigen on cancer cells and the CD3 signaling machinery on T cells, bringing the tumor cell and T cell into proximity and facilitating T cell activation and tumor cell killing. By contrast, CAR T cell therapy involves re-infusion of the patient's own T cells after ex vivo engineering to express CARs targeting tumor antigens and triggering T cell signaling. Here we describe the generation of REGN5458, a human bispecific antibody that binds to BCMA and CD3. In vitro, REGN5458 efficiently activates T cells and induces polyclonal T cell killing of myeloma cell lines with a range of BCMA cell-surface densities, and also induces cytotoxicity of primary human plasma cells. Similar to gamma-sectretase inhibitors, incubation of myeloma cell lines with REGN5458 increased surface levels of BCMA. In xenogenic studies, after BCMAhigh NCI-H929 and BCMAlow MOLP-8 MM cells were co-implanted with PBMC and grown subcutaneously in immunodeficient NOD/SCID/L2Rgamma-deficient (NSG) mice, REGN5458 doses as low as 0.4 mg/kg significantly suppressed the growth of both tumors. Using aggressive, systemic xenogenic tumor models, in which NSG mice were engrafted with PBMC and intravenously injected with BCMAhigh OPM-2 cells or BCMAlow MOLP-8 cells expressing luciferase, REGN5458 reduced tumor burden and suppressed tumor growth at doses as low as 0.4 mg/kg. In immunocompetent mice genetically engineered to express human CD3, REGN5458 inhibited the growth of syngeneic murine tumors expressing human BCMA at doses as low as 0.04 mg/kg. Finally, as REGN5458 binds to cynomolgus CD3 and BCMA and mediates cytotoxicity of primary cynomolgus plasma cells, the pharmacology of REGN5458 was evaluated in cynomolgus monkeys. REGN5458 administration was well-tolerated, resulting in a mild inflammatory response characterized by transiently increased CRP and serum cytokines. Importantly, REGN5458 treatment led to the depletion of BCMA+ plasma cells in the bone marrow, demonstrating cytotoxic activity in non-human primates. The anti-tumor efficacy of REGN5458 was compared to BCMA-specific CAR T cells using 2nd generation CAR lentiviral constructs containing a single-chain variable fragment binding domain from REGN5458's BCMA binding arm and 4-1BB and CD3z signaling domains. Human PBMC-derived T cells were transduced to express this CAR and expanded. Both REGN5458 and the BCMA CAR T cells demonstrated similar targeted cytotoxicity of myeloma cell lines and primary patient blasts in vitro, and were capable of clearing established systemic OPM-2-luciferase myeloma tumors in NSG mice, but with different kinetics: treatment with REGN5458 resulted in rapid clearance of tumors within 4 days, whereas treatment with BCMA CAR T cells allowed tumors to continue to grow for 10-14 days following injection before rapidly inducing tumor clearance. Thus, REGN5458 exerts its therapeutic effect rapidly after injection, using effector T cells that are already in place. In contrast, BCMA CAR T cells require time to traffic to the tumor site and expand, before exerting anti-tumor effects. Collectively, these data demonstrate the potent pre-clinical anti-tumor activity of REGN5458 that is comparable to that of CAR T cells, and provide a strong rationale for clinical testing of REGN5458 in patients with MM. Disclosures Dilillo: Regeneron Pharmaceuticals: Employment. Olson:Regeneron Pharmaceuticals: Employment. Mohrs:Regeneron Pharmaceuticals: Employment. Meagher:Regeneron Pharmaceuticals: Employment. Bray:Regeneron Pharmaceuticals: Employment. Sineshchekova:Regeneron Pharmaceuticals: Employment. Startz:Regeneron Pharmaceuticals: Employment. Retter:Regeneron Pharmaceuticals: Employment. Godin:Regeneron Pharmaceuticals: Employment. Delfino:Regeneron Pharmaceuticals: Employment. Lin:Regeneron Pharmaceuticals: Employment. Smith:Regeneron Pharmaceuticals: Employment. Thurston:Regeneron Pharmaceuticals: Employment. Kirshner:Regeneron Pharmaceuticals: Employment.

Sign in / Sign up

Export Citation Format

Share Document