scholarly journals CRISPR-Cas9 Screen Identifies Me2, a Mitochondrial Malic Enzyme, As a Molecule Relevant for MCL1 Inhibitor Resistance in AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 912-912
Author(s):  
Fumihiko Nakao ◽  
Takuji Yamauchi ◽  
Yuichiro Semba ◽  
Jumpei Nogami ◽  
Koichi Akashi ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Line ◽  
Cell Lines ◽  
Malic Enzyme ◽  
Molecular Mechanisms ◽  
Small Scale ◽  
Second Screen ◽  
Genome Wide ◽  
Inhibitor Resistance ◽  
Mitochondrial Malic Enzyme

The BCL-2 protein family plays a key role in leukemogenesis via counteracting proapoptotic signals, thereby enhancing leukemia cell survival. Venetoclax, a selective BCL-2 inhibitor, has been proven effective against AML in combination with hypomethylating agents. Since MCL1, another anti-apoptotic protein, is reportedly more abundant than BCL-2 in AML cells and associated with resistance to chemotherapy, development of clinical-grade MCL1 inhibitors has been highly regarded. In fact, a series of MCL1 inhibitors are currently being tested in clinical trials. While relationships between metabolic condition in mitochondria and sensitivity to Venetoclax have been proposed in recent studies, molecular mechanisms underlying MCL1 inhibitor resistance are not well understood. To identify genes/pathways whose loss induce MCL1 inhibitor resistance in AML cells, we performed genome-wide CRISPR-Cas9 screens using a mouse AML cell line in the presence or absence of s63845, a MCL1 inhibitor (Kotschy et al. Nature 2016). To establish AML cell lines with a relatively clean genetic background, we first established AML in mice by transducing the MLL/AF9 leukemia oncogene into mouse bone marrow stem/progenitor cells ex vivo, followed by serial transplants (Yamauchi et al. Cancer Cell 2018). We then harvested AML cells from leukemic mice and established AML cell lines with normal karyotype and intact Trp53 activity. We then performed genome-wide CRISPR-Cas9 screens in the presence or absence of s63845, followed by a second screen with a small-scale library targeting genes that were negatively- or positively-selected upon s63845 treatment in the primary screen. Genes relevant for S63845 resistance were identified using MAGeK MLE (Li et al. Genome Biology 2015) and DrugZ (Colic et al. BioRxiv 2019) programs. Single-guide RNAs (sgRNAs) targeting intrinsic pro-apoptotic genes, such as Bak1, Casp9 and Apaf1, were enriched exclusively in the presence of s63845, attesting to the validity of our experiment. To identify genes relevant to s63845 resistance, we focused on the genes whose sgRNAs were enriched exclusively upon s63845 treatment. We found that loss of Me2, which encodes a mitochondrial malic enzyme that catalyzes the oxidative decarboxylation of malate to pyruvate, promotes AML cell survival only in the presence of s63845, but not in vehicle- or Venetoclax-treated cells. This finding was validated in the second screen, in which 8 independent sgRNAs targeting Me2 were tested. We next generated two independent Me2-null mouse AML cell lines (MLL/AF9 and CALM/AF10) using sgRNAs targeting Me2. Me2-null cells exhibited survival advantage over control cells upon s63845 treatment, revealed by cell proliferation assay. To determine at which step of the apoptotic pathway Me2 deficiency exerts s63845 resistance, we assessed MOMP (mitochondrial outer membrane permeabilization) levels by FACS with or without s63845 treatment. Me2-null cells released less cytochrome C than Me2-wild type (WT) cells upon s63845 treatment. As expected, Me2-null cells exhibited less Annexin-positivity than WT cells upon MCL1 inhibition. Importantly, both mRNA and protein levels of BCL-2 family members, including Bcl-2, Mcl1, Bcl2L1, Bax and Bak1, were comparable regardless of Me2 status. We next performed CRISPR-Cas9 saturation mutagenesis scan of all ME2 exons in the presence or absence of s63845 using Molm-13, a human AML cell line. sgRNAs targeting ME2 coding exons were enriched only in the presence of s63845, while those targeting ME2 3'UTR were unchanged/neutral regardless of experimental conditions. In conclusion, using an unbiased, genome-wide CRISPR/Cas9 screens, we identified Me2, a mitochondrial metabolic enzyme, as a factor relevant for MCL1 inhibitor resistance. Our study may facilitate the understanding of molecular mechanisms underlying acquired resistance to MCL1 inhibitors in AML. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

scholarly journals A novel transposable element based authentication protocol for Drosophila cell lines

2021 ◽  
Author(s):  
Daniel Mariyappa ◽  
Douglas B. Rusch ◽  
Shunhua Han ◽  
Arthur Luhur ◽  
Danielle Overton ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Wide Distribution ◽  
Small Scale ◽  
Double Blind ◽  
Cell Line Authentication ◽  
Drosophila Cell ◽  
Genomic Changes ◽  
Genome Wide

Drosophila cell lines are used by researchers to investigate various cell biological phenomena. It is crucial to exercise good cell culture practice. Poor handling can lead to both inter- and intraspecies cross-contamination. Prolonged culturing can lead to introduction of large- and small-scale genomic changes. These factors, therefore, make it imperative that methods to authenticate Drosophila cell lines are developed to ensure reproducibility. Mammalian cell line authentication is reliant on short tandem repeat (STR) profiling, however the relatively low STR mutation rate in D. melanogaster at the individual level is likely to preclude the value of this technique. In contrast, transposable elements (TE) are highly polymorphic among individual flies and abundant in Drosophila cell lines. Therefore, we investigated the utility of TE insertions as markers to discriminate Drosophila cell lines derived from the same or different donor genotypes, divergent sub-lines of the same cell line, and from other insect cell lines. We developed a PCR-based next-generation sequencing protocol to cluster cell lines based on the genome-wide distribution of a limited number of diagnostic TE families. We determined the distribution of five TE families in S2R+, S2-DRSC, S2-DGRC, Kc167, ML-DmBG3-c2, mbn2, CME W1 Cl.8+, and OSS Drosophila cell lines. Two independent downstream analyses of the NGS data yielded similar clustering of these cell lines. Double-blind testing of the protocol reliably identified various Drosophila cell lines. In addition, our data indicate minimal changes with respect to the genome-wide distribution of these five TE families when cells are passaged for at least 50 times. The protocol developed can accurately identify and distinguish the numerous Drosophila cell lines available to the research community, thereby aiding reproducible Drosophila cell culture research.

scholarly journals A novel transposable element based authentication protocol for Drosophila cell lines

2021 ◽  
Author(s):  
Daniel Mariyappa ◽  
Douglas B Rusch ◽  
Shunhua Han ◽  
Arthur Luhur ◽  
Danielle Overton ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Wide Distribution ◽  
Small Scale ◽  
Double Blind ◽  
Cell Line Authentication ◽  
Drosophila Cell ◽  
Genomic Changes ◽  
Genome Wide

Abstract Drosophila cell lines are used by researchers to investigate various cell biological phenomena. It is crucial to exercise good cell culture practice. Poor handling can lead to both inter- and intraspecies cross-contamination. Prolonged culturing can lead to introduction of large- and small-scale genomic changes. These factors, therefore, make it imperative that methods to authenticate Drosophila cell lines are developed to ensure reproducibility. Mammalian cell line authentication is reliant on short tandem repeat (STR) profiling, however the relatively low STR mutation rate in D. melanogaster at the individual level is likely to preclude the value of this technique. In contrast, transposable elements (TE) are highly polymorphic among individual flies and abundant in Drosophila cell lines. Therefore, we investigated the utility of TE insertions as markers to discriminate Drosophila cell lines derived from the same or different donor genotypes, divergent sub-lines of the same cell line, and from other insect cell lines. We developed a PCR-based next-generation sequencing protocol to cluster cell lines based on the genome-wide distribution of a limited number of diagnostic TE families. We determined the distribution of five TE families in S2R+, S2-DRSC, S2-DGRC, Kc167, ML-DmBG3-c2, mbn2, CME W1 Cl.8+, and OSS Drosophila cell lines. Two independent downstream analyses of the NGS data yielded similar clustering of these cell lines. Double-blind testing of the protocol reliably identified various Drosophila cell lines. In addition, our data indicate minimal changes with respect to the genome-wide distribution of these five TE families when cells are passaged for at least 50 times. The protocol developed can accurately identify and distinguish the numerous Drosophila cell lines available to the research community, thereby aiding reproducible Drosophila cell culture research.

scholarly journals Genome-Wide Role of HSF1 in Transcriptional Regulation of Desiccation Tolerance in the Anhydrobiotic Cell Line, Pv11

2021 ◽  
Vol 22 (11) ◽  
pp. 5798
Author(s):  
Shoko Tokumoto ◽  
Yugo Miyata ◽  
Ruslan Deviatiiarov ◽  
Takahiro G. Yamada ◽  
Yusuke Hiki ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Desiccation Tolerance ◽  
Expression Profiles ◽  
Insect Cell Line ◽  
Gene Expression Profiles ◽  
Late Embryogenesis Abundant ◽  
Heat Shock Factor 1 ◽  
Genome Wide ◽  
A Genome

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

scholarly journals Proton-irradiated breast cells: molecular points of view

2019 ◽  
Vol 60 (4) ◽  
pp. 451-465 ◽  
Author(s):  
Valentina Bravatà ◽  
Francesco P Cammarata ◽  
Luigi Minafra ◽  
Pietro Pisciotta ◽  
Concetta Scazzone ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Fate ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Proton Irradiation ◽  
Expression Profiles ◽  
Treatment Plan ◽  
Mcf10a Cell ◽  
Specific Cell ◽  
Dose Dependent

Abstract Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome

Raman Spectroscopy: An Exploratory Study to Identify Post-Radiation Cell Survival

2020 ◽  
Vol 74 (5) ◽  
pp. 553-562
Author(s):  
Kshama Pansare ◽  
Saurav Raj Singh ◽  
Venkatavaradhan Chakravarthy ◽  
Neha Gupta ◽  
Arti Hole ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Cell Viability ◽  
Cell Survival ◽  
Cell Line ◽  
Cell Lines ◽  
Clonogenic Assay ◽  
Early Stage ◽  
Raman Band ◽  
Breast Adenocarcinoma ◽  
Linear Discriminant

Resistance to radiotherapy has been an impediment in the treatment of cancer, and the inability to detect it at an early stage further exacerbates the prognosis. We have assessed the feasibility of Raman spectroscopy as a rapid assay for predicting radiosensitivity of cancer cells in comparison to the conventional biological assays. Cell lines derived from breast adenocarcinoma (MCF7), gingivobuccal squamous cell carcinoma (ITOC-03), and human embryonic kidney (HEK293) were subjected to varying doses of ionizing radiation. Cell viability of irradiated cells was assessed at different time points using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Raman spectroscopy, and colony-forming capability was evaluated by clonogenic assay. Radiosensitivity observed using MTT assay was limited by the finding of similar cell viability in all the three cell lines 24 h post-irradiation. However, cell survival assessed using clonogenic assay and principal component linear discriminant analysis (PC-LDA) classification of Raman spectra showed correlating patterns. Irradiated cells showed loss of nucleic acid features and enhancement of 750 cm−1 peak probably attributing to resonance Raman band of cytochromes in all three cell lines. PC-LDA analysis affirmed MCF7 to be a radioresistant cell line as compared to ITOC-03 and HEK293 to be the most radiosensitive cell line. Raman spectroscopy is shown to be a rapid and alternative assay for identification of radiosensitivity as compared to the gold standard clonogenic assay.

Targeting Myeloproliferative Diseases with JAK2 Inhibitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3550-3550
Author(s):  
Vadim Markovtsov ◽  
Diane Yu ◽  
Marina Gelman ◽  
Wayne Lang ◽  
Vanessa C. Taylor ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Mutant Cell ◽  
Control Cell ◽  
Mast Cell Activation ◽  
Kinase Gene ◽  
Myeloproliferative Diseases ◽  
Jak2 Inhibitors

Abstract Limited options provided by the current standard of care for the patients suffering from myeloproliferative diseases (MPDs) prompted an extensive search for the underlying molecular mechanisms of these disorders. Recent discovery of a single activating mutation (V617F) in JAK2 kinase gene associated with the development of the polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) opened up a possibility to develop highly targeted therapies against these debilitating ailments. To that end, we engineered cytokine-independent Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F -dependent proliferation. We confirmed the ability of hit compounds to inhibit proliferation of JAK2-dependent tumor cell lines using UKE-1 and SET-2 cells carrying the V617F JAK2 mutation. A FACS-based phosphoSTAT5 assay was then used to demonstrate that the hits directly targeted mutant JAK2. JAK3 activity of each compound was evaluated in IL-2-dependent CTLL-2 cell line using phosphoSTAT5 FACS and proliferation assays. To avoid hits with nonspecific antiproliferative activity, the hits were tested in JAK2-independent MOLT4, A549 and H1299 cell lines. Compound hits with the desirable properties were further evaluated for their ability to inhibit JAK2, JAK3 and other kinases in the context of T cell, B cell, or mast cell activation using a variety of cell-based assays as well as in the in vitro biochemical assays. We identified a number of compounds that potently inhibit growth of the two V617F mutant cell lines with EC50s varying from 20 to 500 nM, but do not affect proliferation of control cell lines MOLT4, A549 and H1299 to the same degree. These compounds induce strong and highly specific suppression of STAT5 phosphorylation with IC50s of 10 to 200 nM in SET-2 and V617F JAK2 expressing Ba/F3 cells. One of the hits with the desirable biological and pharmacokinetic profiles was further evaluated in V617F JAK2 Ba/F3 engraftment mouse model where it demonstrated significant extension of survival at 150 and 200 mg/kg bid. Such potent JAK2 inhibitors could become the basis for the next generation of compounds targeting JAK2-dependent myeloproliferative diseases.

Biological and Molecular Characterization by Integrative Genomics Approach of CMA-03/06, a Newly Established Interleukin-6 Independent Variant of the CMA-03 Human Myeloma Cell Line.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1814-1814
Author(s):  
Donata Verdelli ◽  
Lucia Nobili ◽  
Katia Todoerti ◽  
Laura Mosca ◽  
Sonia Fabris ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Molecular Characterization ◽  
Cell Lines ◽  
Stromal Cells ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Growth And Survival ◽  
Genome Wide

Abstract Abstract 1814 Poster Board I-840 Background The growth and survival of multiple myeloma (MM) cells in the bone marrow microenvironment is regulated by functional complex interactions between the tumor cells and the surrounding bone marrow stromal cells mediated by adhesion molecules and the production of several cytokines of which interleukin-6 (IL-6) has been identified as the most important. Major advances in the investigation of MM biology were made possible by the availability of human myeloma cell lines (HMCLs). The IL-6-dependent CMA-03 cell line was established in our laboratory from a peritoneal effusion of a refractory relapsed MM patient. By gradually decreasing the IL-6 added to the culture, an IL-6-independent variant, CMA-03/06, could be obtained. Aims. To perform a biological and molecular characterization of this novel cell line, and to provide insights into the signaling pathways and target genes involved in the growth and survival of CMA-03/06. Methods. The growth, immunophenotypic, cytogenetic and fluorescence in situ hybridization (FISH) characterization of CMA-03/06 cell line was performed by means of standard procedures. IL-6 production into the culture media was determined using a high sensitivity IL-6 specific ELISA. Genome-wide profiling data were generated by means of Affymetrix GeneChip® Human Mapping 250K Nsp arrays; copy number (CN) alterations were calculated using the DNAcopy Bioconductor package, based on circular binary segmentation method. Global gene expression profiling (GEP) was performed by means of the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix); the supervised analyses were done using the SAM software version 3.0. Results Unlike CMA-03, the addition of IL-6 to the culture medium of CMA-03/06 cells or co-culture with multipotent mesenchymal stromal cells did not induce an increase in CMA-03/06 proliferation. IL-6 was not detected in the supernatants from either CMA-03 or CMA-03/06 cell lines within 48 h, suggesting that the IL-6 independence of CMA03/06 cells is not a result of the development of an autocrine IL-6 loop. Nevertheless, IL-6 induced the activation of STAT3 and STAT1 in both cell lines, even if a slight constitutive STAT3 phosphorylation was found in CMA-03/06. The immunophenotypic analysis showed a significant difference in the expression of three antigens in the 2 cell lines: CD45 was considerably reduced in CMA-03/06 cells, whereas they were found positive for both chains of IL-6 receptor, CD126 and CD130, almost undetectable in CMA-03. Conventional cytogenetic and FISH analyses did not reveal differences between the 2 HMCLs. The genome-wide analysis allowed the identification of about 100 altered chromosomal regions common to both HMCLs, mostly DNA gains. Comparison of CMA-03/06 and CMA-03 cells evidenced a different CN in only 15 small chromosomal regions, 8 of which did not contain any transcript, whereas few genes were located on the other ones. GEP analysis of CMA-03/06 compared with CMA-03 identified 21 upregulated and 47 downregulated genes, many of which particularly relevant for MM biology, mainly involved in cellular signaling, cell cycle, cell adhesion, cell development, regulation of transcription, immunologic, inflammatory or defense activity, apoptosis. None of the genes differentially expressed in CMA-03/06 compared with CMA-03 except 1 were positioned on the chromosomal regions showing a different CN. Finally, CMA-03/06 cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA-03 cells. Conclusions Our data show the IL-6 independence of CMA-03/06 cell line in the absence of an autocrine IL-6 loop; the cells, however, maintain the IL-6 signaling pathway responsiveness. A consistent number of genes particularly relevant for MM biology were found deregulated in CMA-03/06 cell line compared with CMA-03. Furthermore, CMA-03/06 cell line shows an increased resistance to apoptosis. The novel CMA03/06 cell line may thus represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells. Disclosures No relevant conflicts of interest to declare.

CD28 and CD86 Are Necessary for Myeloma Cell Survival.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2946-2946
Author(s):  
Catherine M Gavile ◽  
Jayakumar R Nair ◽  
Kelvin P Lee ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
T Cells ◽  
Growth Factor ◽  
Cell Survival ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival

Abstract Abstract 2946 Multiple myeloma (MM) is a hematologic malignancy characterized by the aberrant proliferation of plasma cells. Myeloma cells retain most of the physiological characteristics of their normal counterpart – the long-lived plasma cell. Myeloma cells secrete immunoglobulin and reside in the bone marrow, where they rely heavily on interactions with the stroma for survival signals. While recent advances in therapeutics have led to an increase in median survival post-diagnosis, the disease remains incurable. Understanding the pathways which mediate growth and survival of these cells will help in identifying new targets that can potentially further improve patient outcomes. CD28 is a receptor better known for its role in T-cell signaling through interaction with its ligands, CD80 or CD86. Interaction between CD28 on T-cells and CD80/86 on antigen-presenting cells leads to survival and proliferation of T-cells. Recent work has shown that the CD80/86-CD28 pathway also plays an important role in normal plasma cell generation and survival. Interestingly, high expression of CD28 and CD86 are poor prognostic markers for myeloma patients. Previous work has shown that CD28 activation provides survival signals for myeloma cells in growth-factor deficient conditions. It has also been shown that CD28 on the myeloma cell interacts with CD80/86 on the dendritic cell, which induces secretion of IL-6 (by the DC), an important myeloma growth factor. However, it is not known if CD28 or CD86 play a role in steady state growth and survival of myeloma cells. In order to determine the role of each of these 2 molecules in myeloma physiology, we knocked-down either CD28 or CD86 on the myeloma cell via lentivirus-mediated shRNAs. We found that knockdown of CD86 leads to apoptosis in 3 myeloma cell lines (RPMI8226, MM1.s, and KMS18). Four days after infection with the lentivirus containing shCD86, 45.7±4.9 and 60.3±4.6 percent control apoptosis was observed in RPMI8226 and MM1.s respectively, while less death was observed in KMS18 (17.6±1.6). CD28-knockdown resulted in apoptosis as well (24.9±4.3 for RPMI8226, 26.8±4.1 for MM1s, 21.8±3.8 for KMS18, percent control apoptosis). Consistent with these findings, we were unable to establish a myeloma cell line with stable knockdown of either CD28 or CD86. Additionally, RPMI8226 cells stably transfected to over-express either Bcl-2, Bcl-xL, or Mcl-1 are protected from cell death induced by CD86 or CD28 silencing. These data suggest that CD28 and CD86 are essential to prevent apoptosis of myeloma cells in vitro. To confirm these findings we determined the effects of CTLA4-Ig on myeloma survival. CTLA4-Ig inhibits CD86-CD28 signaling by binding to CD86, blocking its interaction with CD28. We found that treatment of RPMI8226 and MM1.s cells with CTLA4-Ig caused apoptosis in the myeloma cells after 2 days (23.9±3.9 for RPMI8226 and 20.4±6.2 for MM1.s, percent control apoptosis). Thus like normal plasma cells, CD28 and CD86 are required for the survival of myeloma cells. To determine why silencing of CD86 has a more potent effect than CD28 silencing on myeloma cell survival in 2 out of 3 cell lines, we investigated the effects of silencing on cell surface expression of each of these proteins. CD28 and CD86 mRNA and protein levels were silenced to similar levels by their cognate hairpins. However, in MM.1s and RPMI8226 we found that silencing of CD28 resulted in an increase in CD86 surface expression. This increase was also observed at the mRNA level and in the cells over-expressing Bcl-2 family members, indicating that this is not simply due to the selection of the highest expressing cells. These data suggest a feedback loop exists to regulate CD28-CD86 signaling in myeloma cells. Surprisingly, in the KMS18 cell line, we observe the converse effect, where silencing of CD86 resulted in upregulation of CD28. This provides a likely explanation for why these cells are less susceptible to CD86 silencing than the other two lines. Interestingly, blocking CD86 with CTLA4-Ig treatment also resulted in a modest upregulation in CD28 surface expression of MM.1s and RPMI8226, which suggests that silencing CD86 and binding of CD86 with a soluble receptor are not equivalent, and that multiple signaling feedback pathways exist to regulate the expression of this receptor-ligand pair that is necessary for myeloma cell survival. Disclosures: No relevant conflicts of interest to declare.

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