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 DIPG-22. DISSECTING THE ONCOGENIC ROLE OF FOXR2 IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii291-iii291
Author(s):  
Jessica W Tsai ◽  
Smruti K Patel ◽  
Heather Bear ◽  
Frank Dubois ◽  
Prasidda Khadka ◽  
...  
Keyword(s):  
Cell Lines ◽  
Therapeutic Target ◽  
Transcriptional Profiling ◽  
Genomic Analysis ◽  
In Utero ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Sequencing Analysis ◽  
Integrative Genomics

Abstract BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) pose particular challenges for treatment. We recently completed a genomic analysis of close to 200 DIPGs and high-grade gliomas. We identified that nearly 10% of all DIPGs have increased expression of the fork head domain transcription factor FOXR2. We hypothesize that FOXR2 accelerates gliomagenesis in histone mutant DIPGs and represents a previously unexplored therapeutic target. METHODS To determine whether FOXR2 is sufficient to mediate gliomagenesis, we applied an integrative genomics approach using both in vitro and in vivo DIPG models: mouse neural stem cell models expressing FOXR2, in vivo mouse models using in utero brainstem electroporation, patient-derived DIPG cell lines, and RNA sequencing analysis of human and mouse tumors expressing FOXR2. RESULTS Our data shows that FOXR2 indeed is an oncogene that rapidly accelerates gliomagenesis using an in vivo brainstem in utero electroporation model of DIPG. In human tumors, increased FOXR2 expression is mutually exclusive with MYC amplification suggesting functional redundancy. In vivo, FOXR2 results in large brainstem gliomas and rapid neurologic decline of animals. Transcriptional profiling of these tumors demonstrates activation of MYC signaling pathways. In vitro, we have further identified patient-derived cell lines with increased expression of FOXR2. CONCLUSION FOXR2 is sufficient to enhance gliomagenesis and represents a previously understudied therapeutic target for patients with the devastating disease DIPG.

scholarly journals A Deptor Inhibitor Induces Its Degradation with Resulting Anti-Myeloma Cytotoxicity in Vitro and In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1821-1821
Author(s):  
Mario I Vega ◽  
Yijiang Shi ◽  
Patrick Frost ◽  
Sara Huerta-Yepez ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Cytotoxic Effect ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Inhibition Of Proliferation ◽  
Induction Of Apoptosis

Multiple myeloma (MM) is a hematological disorder characterized by a proliferation of malignant monoclonal plasma cells in the bone marrow (BM) and / or in extramedullary sites. Despite recent progress in OS rates, MM remains an incurable disease and most patients will relapse and require treatment. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. It is known that the inhibition of Deptor results in the inhibition of the proliferation and induction of apoptosis in MM cells. In addition, high levels of Deptor are predictive of a poor response to conventional therapies, indicating that Deptor expression are important as a prognostic marker for patients with myeloma and is a possible therapeutic target. Our group previously identified a drug which prevents mTOR-Deptor binding (NSC126405) and induces cellular cytotoxicity in MM (Shi Y, et al 2016). In this study, we developed a new related chemical inhibitor (43 M) capable of inducing the inhibition of the mTOR / Deptor interaction and results in the negative regulation of Deptor that leads to the inhibition of proliferation and induces apoptosis in several MM cell lines. The cytotoxic effect of 43 M is not dependent of caspase activation and induces the activation of p70 and AKT (T308). This leads to the induction of apoptosis in MM cell lines and tumor cells derived from MM patients. The degradation of Deptor induced by 43 M is dependent on the proteasome complex since it was prevented in the presence of MG132. In vivo, 43 M prevents the expression of Deptor in a xenograft tumor, and delayed tumor growth and interestingly, induces the eradication of tumors in 40% of mice in a murine model of MM, without significant toxic implications. Recent studies show that Deptor expression protects MM cells against Bortezomib treatment, suggesting that anti-Deptor drugs can synergize with proteasome inhibitors (PIs). However, the combination of 43 M + Bortezomib was not synergistic, and was antagonistic in vitro. These results are probably due to the prevention of the proteasomal degradation of Deptor, suggesting a possible use of the 43 M inhibitor in MM in the absence of the current PIs. This study describes for the first time the possible role of Deptor as a therapeutic target using a chemical inhibitor capable of degrading and inducing a cytotoxic effect in MM cell lines. In addition, Deptor is reported as an important therapeutic target in an in vivo MM model. Shi Y, Daniels-Wells TR, Frost P, Lee J, Finn RS, Bardeleben C, Penichet ML, Jung ME, Gera J, Lichtenstein A. Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells. Cancer Res. 2016 Oct 1;76(19):5822-5831 Disclosures No relevant conflicts of interest to declare.

scholarly journals JAM-A as a Prognostic Factor and New Therapeutic Target in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 307-307 ◽  
Author(s):  
Antonio Solimando ◽  
Andreas Brandl ◽  
Mattenheimer Katharina ◽  
Carolin Graf ◽  
Miriram Ritz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Healthy Subjects ◽  
Plasma Cells ◽  
Cell Membrane Protein ◽  
And Migration ◽  
Rpmi 8226

Abstract Cell adhesion in the multiple myeloma (MM) microenvironment is a mechanism by which MM plasma cells escape the effects of therapy and survive. To improve clinical strategies and overcome drug resistance, approaches directed to both MMPCs and bone marrow microenvironment are under investigation. Here, we examined the cell membrane protein Junctional adhesion molecule-A (JAM-A) as a clinical biomarker and novel therapeutic target for MM. We evaluated JAM-A expression by real time PCR (RT-PCR), flow cytometry and immunofluorescence microscopy in 132 MM patients at different stages and various MM cell lines. Next, we measured the concentrations of soluble JAM-A from MM and healthy subjects sera by enzyme linked immune assay (ELISA). We investigated JAM-A functionally in vitro and in vivo by transient gene silencing (siRNA) and with blocking antibodies. Patient-derived plasma cells (MMPCs) expressed increased JAM-A expression levels when compared to control PC from healthy individuals. Elevated JAM-A expression correlated with poor prognosis (Figure 1A,B). Furthermore, soluble JAM-A was significantly increased in MM patient sera when compared to healthy subjects. Additionally, MM cell lines showed high expression of both membrane and cytoplasmic JAM-A. Consequently, inhibition of JAM-A using specific siRNA treatment resulted in diminished tumorigenic potential, including decreased colony formation, chemotaxis and migration. Importantly, treatment of luciferase+RPMI-8226 MM bearing NSG with a JAM-A blocking monoclonal antibody reduced significantly MM progression and dissemination in vivo when compared to MM bearing mice that received an non-specific isotype control antibody (Figure 1C). Conclusively, our data suggest that JAM-A can serve as a biomarker of malignancy in MM patients. Soluble plasma JAM-A could contribute to serum-based clinical stratification. Furthermore, therapeutic targeting of JAM-A appears attractive for clinical translation. Figure 1 Figure 1. Disclosures Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria.

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.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

Identification Of Novel Alternative Splice Variants Of Sirtuins In Multiple Myeloma: Therapeutic Implications

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3121-3121
Author(s):  
Antonia Cagnetta ◽  
Michele Cea ◽  
Sophia Adamia ◽  
Yu-Tzu Tai ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Splice Variants ◽  
Expression Patterns ◽  
Critical Role ◽  
Sequencing Analysis ◽  
Biological Processes ◽  
Newly Diagnosed ◽  
Cloning And Sequencing ◽  
Aberrant Expression

Abstract Background Alternative splicing (AS) is a normal epigenetic event with a critical role in the regulation of gene expression. Previous studies showed increased AS in Multiple Myeloma (MM) cells, suggesting the need to assess both expression level of genes and post translational modifications, mediating overall gene function. The NAD-dependent deacetylases Sirtuins (SIRTs), mammalian homologues of the yeast Sir2, modulate various biological processes including metabolism, cell survival, development, chromatin dynamics, or DNA repair. Recent microarray profiling data using newly diagnosed patients with MM, suggests clinical relevance of such deacetylases since their level predicts for both progression free and overall survival. Among SIRTs family membersSIRT-5, SIRT-6 and SIRT-7 transcript levels positively correlated with disease progression (from MGUS to active MM). These studies provide the rationale for further examining the biological processes including epigenetic changes, mutations, or AS events that contribute to aberrant expression of SIRTs in MM. Methods Purified RNA from MM cell lines, newly diagnosed MM patient cells, as well as peripheral blood mononuclear cells (PBMCs) from normal healthy donors was subjected to SIRT expression analysis. Specifically, SIRTs-specific primers were developed and optimized using standard RT-PCR conditions. RNA integrity was confirmed using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. Aberrant splicing of SIRT-5, SIRT-6, and SIRT-7 was confirmed by cloning and sequencing, followed by analysis of their expression patterns in MM patients versus normal PBMCs. Results We found that SIRTs genes are frequently miss-spliced in MM patients. To our knowledge, this is the first report showing SIRTs miss-splicing event in MM. Through cloning and sequencing analysis, we identified novel spliced isoforms of SIRT-5, SIRT-6 and SIRT-7; these occurred as a result of aberrant AS within exon-12, exon-4 and and exon-5, respectively. Specifically, exon skipping was noted in SIRT-6 and SIRT-7 variants, via cryptic 5 prime or 3 prime splice sites on exon 12 and/or through partial retention of an intron created SIRT-5 variants. The novel spliced forms were widely expressed in MM cell lines and primary cells, without significance occurrence in normal PBMCs. Our preliminary data show that even though these novel isoforms exhibit reduced deacetylase activity versus full-length variants, this characteristic may impart distinct functional outcome. Finally, in support of above studies, our analysis of MM patient samples suggest that AS among SIRTs is associated with poor clinical outcome in MM patients. Conclusion In the current study, we have identified novel transcript variants of SIRT-5, SIRT-6 and SIRT-7 in MM cells. These aberrant isoforms allow for generating transcripts that encode for dysfunctional proteins, which in turn, may contribute to the genetic heterogeneity in MM. Ongoing studies are delineating the function of these newly identified splice variants of SIRTs and their association with MM progression. Overall, our studies will provide basis for utilizing SIRTs variants as prognostic markers and/or as novel therapeutic targets in MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy.

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.

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.

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.

scholarly journals Sulfiredoxin as a Potential Therapeutic Target for Advanced and Metastatic Prostate Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-12 ◽  
Author(s):  
Caroline N. Barquilha ◽  
Nilton J. Santos ◽  
Caio C. D. Monção ◽  
Isabela C. Barbosa ◽  
Flávio O. Lima ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Genetically Engineered ◽  
High Grade ◽  
Sequencing Data ◽  
Potential Therapeutic Target ◽  
Prostate Tumors ◽  
Mouse Prostate

The incidence of prostate cancer (PCa) is increasing, and it is currently the second most frequent cause of death by cancer in men. Despite advancements in cancer therapies, new therapeutic approaches are still needed for treatment-refractory advanced metastatic PCa. Cross-species analysis presents a robust strategy for the discovery of new potential therapeutic targets. This strategy involves the integration of genomic data from genetically engineered mouse models (GEMMs) and human PCa datasets. Considering the role of antioxidant pathways in tumor initiation and progression, we searched oxidative stress-related genes for a potential therapeutic target for PCa. First, we analyzed RNA-sequencing data from Pb-Cre4; Ptenf/f mice and discovered an increase in sulfiredoxin (Srxn1) mRNA expression in high-grade prostatic intraepithelial neoplasia (PIN), well-differentiated adenocarcinoma (medium-stage tumors), and poor-differentiated adenocarcinoma (advanced-stage prostate tumors). The increase of SRXN1 protein expression was confirmed by immunohistochemistry in mouse prostate tumor paraffin samples. Analyses of human databases and prostate tissue microarrays demonstrated that SRXN1 is overexpressed in a subset of high-grade prostate tumors and correlates with aggressive PCa with worse prognosis and decreased survival. Analyses in vitro showed that SRXN1 expression is also higher in most PCa cell lines compared to normal cell lines. Furthermore, siRNA-mediated downregulation of SRXN1 led to decreased viability of PCa cells LNCaP. In conclusion, we identified the antioxidant enzyme SRXN1 as a potential therapeutic target for PCa. Our results suggest that the use of specific SRXN1 inhibitors may be an effective strategy for the adjuvant treatment of castration-resistant PCa with SRXN1 overexpression.

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