scholarly journals Functional Genomic Landscape of Genes with Recurrent Mutations in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 189-189 ◽  
Author(s):  
Ricardo De Matos Simoes ◽  
Ryosuke Shirasaki ◽  
Huihui Tang ◽  
Michal Sheffer ◽  
Olga Dashevsky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
Research Funding ◽  
Functional Genomic ◽  
Loss Of Function ◽  
Functional Studies ◽  
Tumor Cell Survival ◽  
Genome Scale

Abstract During the last two decades, cell lines and patient-derived samples from multiple myeloma (MM) have been extensively profiled for alterations in their genome with the anticipation that those genes with the most recurrent lesions could represent attractive novel therapeutic targets or markers for aggressive disease. Yet for many of these genes, their functional significance for MM cells has not been formally evaluated. With the advent of new CRISPR/Cas9-based functional genomics platforms, it is possible to generate in genome- or subgenome-scale direct quantitative information on the impact that perturbation of these genes exerts on tumor cell survival, proliferation or other phenotypes. We therefore examined the landscape of our CRISPR-based functional genomic data for these recurrently dysregulated genes We specifically curated information from the MMRF CoMMpass study and multiple other publicly available studies, to identify genes which are recurrently identified to harbor nonsynonymous mutation (SNV or indel), DNA copy number loss or gain, or participation in chromosomal translocations. We then examined the patterns of results for these genes in our genome scale CRISPR-based gene-editing studies for loss-of-function in n=18 MM cell lines. We identified a subset of genes (e.g. FAM46C, CDKN2C, RASA2) which are considered targets for recurrent loss-of-function events and indeed exhibit, for large fractions of the cell lines tested enrichment, of their sgRNAs in CRISPR knock-out studies, consistent with a role of these genes as suppressors of tumor cell survival or proliferation. CRISPR KO of TP53 leads to increased survival/proliferation of only a small minority (2/18 of cell lines tested thus far), which reflects the fact that the overwhelming majority of MM cell lines already harbor LOF events for this gene. Interestingly, a substantial number of genes which have been considered to harbor recurrent LOF events in MM patient samples (e.g. NF1, NF2, CYLD) do not exhibit sgRNA enrichment in CRISPR KO screens in the MM cell lines tested so far. In addition, several other recurrently mutated genes for which their loss- or gain-of-function status had not been previously evaluated with extensive functional studies in MM (e.g. SP140, LTB, EGR1, ATM, PARK2, PRKD2, RAPGEF5, DOCK5, TGDS, TNFAIP8) exhibit in the majority of cell lines tested in in CRISPR knockout studies no significant enrichment or depletion of their sgRNAs. In contrast, PTPN11, CREBBP, EP300, KMT2B, KMT2C, SETD2, SF3B1 and UBR5, are notable examples of recurrently mutated genes which represent dependencies for large fractions of MM cell lines in vitro. These results highlight the value of interpreting results from next generation sequencing studies in the context of information provided by the genome scale by use of functional genomic characterization of available cell line models. We envision that, similar sub-genome scale assays were performed at the level of patient derived samples will also provide direct information about the relevance of some of these genes. In addition, functional studies conducted with context of tumor-microenvironemtn compartment interactions and tumor interface will be needed to evaluate several genes identified in the study. Disclosures Licht: Celgene: Research Funding. Mitsiades:Takeda: Other: employment of a relative; Janssen/ Johnson & Johnson: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding; TEVA: Research Funding.

scholarly journals CRISPR Activation Screen for Drivers of MM Cell Proliferation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3197-3197
Author(s):  
Huihui Tang ◽  
Ricardo De Matos Simoes ◽  
Ryosuke Shirasaki ◽  
Olga Dashevsky ◽  
Brian Glassner ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Tumor Cell ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Research Funding ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Molecular Alterations ◽  
Genome Scale ◽  
Crispr Activation

Abstract CRISPR/Cas9-based gene editing has become a powerful tool for loss-of-function (LOF) studies and has allowed us to systematically interrogate the function of genes regulating the survival and proliferation of multiple myeloma (MM) cells in vitro, in vivo and in the context of treatment resistance (e.g. De Matos Simoes et al., Shirasaki et al., and Gandolfi et al. ASH 2017). We reasoned, however, that important additional information can be obtained from CRISPR-based gain-of-function (GOF) approaches which can achieve transcriptional activation at endogenous genomic loci. We thus performed genome-scale CRISPR activation studies using the dCas9-P65-HSF transcriptional activation system (in which a Cas9 variant lacking nuclease activity [dCas9] confers P65-HSF-mediated activation of genes recognized by sgRNAs against their promoter regions). Specifically, MM.1S cells were transduced with the dCas9-P65-HSF system and pooled lentiviral particles of the Calabrese CRISPR activation sgRNA library, consisting of 2 pooled sgRNA sub-libraries (total of ~110,000 sgRNAs targeting ~18000 genes, at initial coverage of 800 cells/sgRNA). Cells were cultured for 12 weeks and harvested at baseline and various intervals, e.g. 4 and 12 weeks of culture. Next generation sequencing of genomic DNA quantified the abundance of sgRNAs in the tumor cell population and genes were ranked (with rank aggregation algorithms) in terms of their sgRNA enrichment or depletion. These analyses allowed us to observe a series of genes with statistically significant sgRNA enrichment and known or presumed roles in MM biology, including key MM transcription factors such as IRF4, the thalidomide derivative targets IKZF3 and IKZF1, and the co-factor POU2AF1; known oncogenes, e.g. KRAS and MYC; NF-kappaB pathway members, e.g. RELA; and signal transduction regulators, e.g. IGF1R and its downstream effectors IRS1 and AKT2. These results are consistent with the major depletion of sgRNAs for these genes in loss-of-function (LOF) CRISPR knockout studies. However, several other genes with significant sgRNA enrichment in CRISPR activation studies did not exhibit major sgRNA depletion in CRISPR knockout studies, including the B/plasma cell transcription factor POU2F2 (Oct2), for which high protein expression correlates with reduced survival in MM (Toman I. et al 2011); the transcription factor PAX2, the TRAF interacting protein TIFA, or the Toll-like receptor TLR4. Interestingly, significant depletion of sgRNAs was observed for several genes with known or proposed tumor suppressive properties including YAP1 (an oncogene for solid tumors, but reported as tumor suppressor for MM and other blood cancers); the pro-death TNFRSF10A (TRAIL receptor DR4), TP73, CDKN1A, the negative regulator of c-Myc MXI1, or the pro-apoptotic Bcl2 family member BAK1. Depletion or enrichment of sgRNAs for most of the aforementioned genes was detectable by 4 weeks of culture, while more pronounced changes and detection of additional statistically-significant hits was observed in later time-points. For genes with significant sgRNA enrichment in our CRISPR activation study, we examined a series of molecular alterations, including transcript overexpression in MM cell lines or patient-derived samples vs. normal plasma cells, or relapsed/refractory MM vs. earlier disease MM stages; mutational status; correlation of transcript levels with clinical outcome in MM; and extent of open chromatin (based on H3K27Ac chromatin marks) within or proximal to each gene in MM cell lines. Some "hits" from our screen exhibited at least one of these molecular alterations, but most genes harbored no such alterations or their magnitude or frequency ranked outside the top 50-100 genes. These results suggest that CRISPR activation studies can identify important putative regulators of MM biology, which may not be readily detectable based on known annotations of the MM cell genome, transcriptome, or epigenome. Genome-scale CRISPR-based transcriptional activation are an important gain-of-function system to uncover genes which induce vs. suppress tumor cell survival and proliferation, and provide information orthogonal to those yielded by other CRISPR-based approaches that involve loss-of-function interventions. Our use of CRISPR activation allowed us to both validate previously known genes and identify promising new candidate regulators of MM cell biology. Disclosures Mitsiades: EMD Serono: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/ Johnson & Johnson: Research Funding.

Lin28B/Let-7 Axis Regulates Multiple Myeloma Proliferation By Enhancing c-Myc and Ras Survival Pathways

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 273-273
Author(s):  
Salomon Manier ◽  
John T Powers ◽  
Antonio Sacco ◽  
Michaela R Reagan ◽  
Michele Moschetta ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Board ◽  
Loss Of Function ◽  
Advisory Committees ◽  
Expression Levels ◽  
Gain And Loss

Abstract Background MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. Lin28B is an RNA binding protein that regulates Let-7 miRNA maturation. Lin28B and Let-7 have been described to act as oncogenes or tumor suppressor genes, respectively, as demonstrated both in solid cancer and hematologic malignancies. However, the role of the Lin28B/Let-7 axis in Multiple Myeloma (MM) has not been studied. Method Lin28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Let-7 expression levels were assessed in CD138+ primary MM cells and bone marrow stromal cells (BMSCs) by using PCR, as well as in circulating exosomes using miRNA array (Nanostring® Technology). Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. The knockdown of Lin28B was performed on MM cell lines (U266, MM.1S, MOLP-8) by using a lentiviral Lin28B shRNA. Gain- and loss-of function studies for Let-7 were performed using Let-7 mimic and anti-Let-7 transfection in MM cell lines (MM1S, U266) and primary BMSCs. Cell proliferation has been evaluated by using thymidine assays. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. Results Two independent GEP datasets (GSE16558; GSE2658) were analyzed for Lin28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high Lin28B levels correlated with a shorter overall survival (p = 0.0226). We next found that the Let-7 family members are significantly down-regulated in MM primary cells, particularly Let-7a and b (5 fold change, p < 0.05), as demonstrated by using qRT-PCR. Similarly, miRNA arrays showed a lower expression of Let-7-related miRNAs in circulating exosomes obtained from MM patients compared to healthy individuals. We further dissected the functional relevance of Lin28B in MM cells, by performing Lin28 knockdown (KD) in MM cell lines (U266, MOLP-8). This led to a significant decrease in MM cell proliferation associated with G1 phase cell cycle arrest. This was supported by up-regulation of Let-7 and down-regulation of c-Myc, Ras and Cyclin D1 in Lin28 KD MM cells. To further prove that Lin28B-dependent effects on MM cells are mediated by Let7, we next showed that let-7 gain- and loss-of-function studies regulate MM cell proliferation and Myc expression. Lin28B regulation in MM cells is dependent on Let-7, as demonstrated by an increase of both cell proliferation and c-Myc expression after anti-Let-7 transfection in the Lin28B KD cells. We therefore studied the regulation of Let-7 in MM cells through the interaction with BMSCs. Let-7 expression levels were significantly lower in BMSCs obtained from MM patients compared to healthy donors. Interestingly, the Let-7 expression level in MM cells was increased after co-culture with Let-7 over-expressing BMSCs, associated with a decrease of both cell proliferation and c-Myc expression. This suggests a potential transfer of Let-7 from BMSCs to MM cells. Conclusion This work describes a new signaling pathway involving Lin28B, Let-7, Myc and Ras in MM. Let-7 expression in MM cells is also regulated through the interaction of MM cells with BMSCs, leading to cell proliferation and Myc regulation in MM. Interference with this pathway might offer therapeutic perspectives. Disclosures: Leleu: CELGENE: Honoraria; JANSSEN: Honoraria. Daley:Johnson and Johnson: Consultancy, Membership on an entity’s Board of Directors or advisory committees; MPM Capital: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Verastem: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity’s Board of Directors or advisory committees; iPierian: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Solasia, KK: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

scholarly journals Local Activator and T Cell Engager (αBCMA x αPD-L1 x αCD3) with Enhanced Tumor Killing and Minimal Cytokine Release for the Treatment of Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Melissa Vrohlings ◽  
Stephanie Jungmichel ◽  
Jan Müller ◽  
David Senn ◽  
Thomas Schleier ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Cytokine Release ◽  
Private Company

BCMA-targeting bispecific T-cell engagers in clinical development have demonstrated encouraging preclinical efficacy. The most advanced of these is AMG 420, which showed significantly improved response rates in relapsed/refractory multiple myeloma (MM) patients. Nevertheless, median duration until relapse is currently limited to approximately 12 months, highlighting the need for new drugs with novel MoA. Recently, we reported on a Local Activator and T cell Engager (LocATE) antibody that targets BCMA and selectively blocks programmed death-ligand 1 (PD-L1) on malignant cells (ASCO, June 2019). LocATE induced superior T cell activation and cancer cell killing, in vitro and ex vivo, compared to a BCMAxCD3 BiTE alone or in combination with a PD-L1 inhibitor. Here, we sought to further characterize the novel MoA of our LocATE. To assess the therapeutic potential of the LocATE, we first investigated whether potent cytotoxicity is uncoupled from high levels of cytokine release. We evaluated three LocATE molecules with different PD-L1 affinities (low, medium, high). Using BCMA-expressing MM cell lines (U-266, MM.1S, RPMI-8226 and H929) with distinct PD-L1 surface expression levels (3 - 53%), we determined the cytokine profile (IL-2, IL-6, IFN-γ, TNF-α) and target cell lysis induced by each candidate in the presence of CD3-positive human T cells. All three candidates exhibited comparable killing potency, however, low-affinity PD-L1 LocATE antibodies induced significantly less cytokine release (up to 10-fold) than its higher PD-L1 affinity counterparts across all cell lines investigated. Notably, using the low-affinity PD-L1 LocATE, we observed a 2-fold increase in tumor cell killing compared to bispecific BCMAxCD3 targeting controls in cell lines expressing high PD-L1 levels (53%), underlining the contribution of PD-L1 inhibition. Accordingly, phenotypic profiling of effector cells showed that the LocATE more potently induced dose-dependent upregulation of the activation markers CD69, CD25 and HLA-DR compared to bispecific controls. Importantly, cytotoxic activity, T cell activation and cytokine release were not induced when BCMA-negative cells expressing high levels of PD-L1 were treated with LocATE, underlining the BCMA-selective killing mechanism. Since the superior efficacy of LocATE was found to correlate with the expression level of PD-L1 on MM cell lines and upregulation of PD-L1/PD-1 has been reported as one of the major myeloma cell escape mechanisms during treatment with BiTEs, we subsequently investigated the efficacy of LocATE using primary bone marrow samples and peripheral blood mononuclear cell (PBMCs) obtained from MM patients. Six bone marrow mononuclear cell (BMMC) and eight PBMC samples from MM donors of different disease stages were characterized for PD-1/PD-L1 expression levels; analysis of T cell frequency and level of activation/exhaustion was performed based on CD4, CD8, CD25, CD69, Tim-3, Lag-3 and PD-1 marker expression. Using patient samples with high frequencies of PD-1 expressing T cells prior to treatment, LocATE induced superior MM tumor cell lysis and T cell activation compared to BCMAxCD3 bispecific antibodies. No activity was observed on healthy cells, underlining the safe and selective killing mechanism through tumor-local reactivation of exhausted T cells. Collectively, these findings demonstrate that the simultaneous T cell redirection and tumor-specific checkpoint inhibition with the LocATE leads to an improved therapeutic index with robust tumor cell killing and low levels of cytokine release. Capable of counteracting adaptive immune resistance caused by increased PD-1/PD-L1 signaling, the LocATE antibody has the prospect to significantly improve survival for multiple myeloma patients. Disclosures Vrohlings: CDR-Life: Current Employment. Jungmichel:CDR-Life: Current Employment, Other: current option holder. Senn:CDR-Life: Current Employment. Schleier:CDR-Life: Current Employment, Current equity holder in private company. Scheifele:CDR-Life: Current Employment, Current equity holder in private company. Wendelspiess:CDR-Life: Current Employment. Leisner:CDR-Life: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Jaeger:CDR Life AG: Consultancy, Research Funding; Miltenyi: Consultancy, Honoraria; Karyopharm: Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria; F. Hoffmann-La Roche: Honoraria, Research Funding. Borras:CDR-Life: Current Employment, Current equity holder in private company.

Inhibition of AKT/mTOR Signaling Pathway Induces Cell Cycle Arrest and Apoptosis in Acute Myelogenous Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2355-2355
Author(s):  
Weina Chen ◽  
Ioannis Grammatikakis ◽  
Jiang Li ◽  
Vassiliki Leventaki ◽  
L. Jeffrey Medeiros ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Cell ◽  
Cell Survival ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Mtor Signaling ◽  
Mtor Signaling Pathway ◽  
Mutational Status ◽  
Tumor Cell Survival ◽  
Dose Dependent

Abstract Acute myelogeneous leukemia (AML) is a heterogeneous disease and includes a subset of neoplasms that harbor activating mutations of the fms-like tyrosine kinase-3 (FLT3) gene. Mutated FLT3 has recently been shown to activate downstream oncogenic pathways including the PI3K/AKT pathway (Scheijen, et al. Oncogene. 23:3338–3349, 2004; Choudhary, et al. Blood. 106:265–273, 2005). It is known that activated AKT mediates its effects, at least in part, through activation of mammalian target of rapamycin (mTOR). However, the potential role of PI3K/AKT/mTOR signaling pathway in tumor cell survival in AML remains largely unknown. We hypothesized that the PI3K/AKT signaling pathway is activated in AML and contributes to tumor cell survival through activation (phosphorylation) of mTOR and its downstream effectors 4EBP1, p70S6K, ribosomal protein S6 (rpS6), and eIF-4E. We used 3 AML cell lines, including MV4-11 and MOLM-13, that are homozygous and heterozygous for mutated FLT3, respectively, as well as U937 (wild-type FLT3). All 3 cell lines expressed activated (serine 473-phosphorylated) AKT (Ser473pAKT), and phosphorylated 4EBP1, p70S6K and rpS6 shown by Western Blot analysis. Treatment of AML cell lines with LY294002, an inhibitor of PI3K, resulted in a dose-dependent decrease of phosphorylation of AKT, mTOR, 4EBP1, p70S6K, and rpS6. This was associated with decreased cell viability as assessed by trypan-blue exclusion assay. Cell death following inhibition of the PI3K/AKT pathway was predominantly attributed to apoptosis as shown by increased annexin V staining assessed by flow cytometry. These changes were associated with downregulation of the anti-apoptotic proteins cFLIP, Mcl-1, and Bcl-XL that are involved in the extrinsic and intrinsic apoptosis. Cell cycle analysis using flow cytometry also showed that inhibition of PI3K resulted in decreased S-phase and increased G1-phase fraction. These cell cycle changes were associated with increased levels of the cyclin-dependent kinase inhibitor p27 and underphosphorylated Rb in a dose-dependent manner. Similar biologic effects, although to a lesser degree, were found after treatment of AML cells with rapamycin, an inhibitor of mTOR. In addition, expression of activated AKT, mTOR, 4EBP1, p70S6K and rpS6 was assessed in AML tumors (n=19) using tissue microarrays of bone marrow samples and immunohistochemical methods. These included tumors with (n=14) and without (n=5) FLT3 mutations. Using a 10% cutoff to define positivity, 13/19 (68%) expressed Ser473pAKT, 16/18 (89%) mTOR, 15/19 (79%) p4E-BP1, 18/19 (95%) p-p70S6K, and 15/18 (83%) p-rpS6. However, no association between expression of activated AKT, or mTOR signaling proteins and FLT3 mutational status was observed. Our study provides first evidence that the AKT/mTOR signaling pathway is activated in AML cell lines and tumors regardless of FLT3 mutational status. The AKT/mTOR signaling pathway may contribute to cell cycle progression and tumor cell survival in AML. Inhibition of this oncogenic pathway represents a potential target for therapy in patients with AML.

Abstract B10: HGF enhances tumor cell survival in canertinib-treated medulloblastoma cell lines

2014 ◽  
Author(s):  
Walderik W. Zomerman ◽  
Sabine L.A. Plasschaert ◽  
Sander H. Diks ◽  
Harm Jan Lourens ◽  
Eelco W. Hoving ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
Medulloblastoma Cell ◽  
Tumor Cell Survival ◽  
Medulloblastoma Cell Lines

scholarly journals Serum and Glucocorticoid Inducible Kinase 1-Sensitive Survival, Proliferation and Migration of Rhabdomyosarcoma Cells

2017 ◽  
Vol 43 (3) ◽  
pp. 1301-1308 ◽  
Author(s):  
Evi Schmid ◽  
Matias Julian Stagno ◽  
Jing Yan ◽  
Sabine Schleicher ◽  
Willi Yu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
Therapy Resistance ◽  
Tumor Cell Survival ◽  
Rhabdomyosarcoma Cell ◽  
And Migration ◽  
Tumor Types ◽  
Proliferation And Migration

Background/Aims: Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, may show an intrinsic refractoriness to standard chemotherapy in advanced tumor stages, which is associated with poor prognosis. Cellular mechanisms conferring tumor cell survival and therapy resistance in many tumor types include the serum & glucocorticoid inducible kinase (SGK) 1 pathway, a kinase expressed ubiquitously with particularly strong expression in skeletal muscle and some tumor types. The present study explored whether SGK1 is expressed in rhabdomyosarcoma and, if so, whether this kinase impacts on tumor cell survival, proliferation and migration. Multiple in vitro techniques were used to study the role of SGK1 in rhabdomyosarcoma. Methods: The Gene Chip® Human Genome U133 Plus 2.0 Array were employed to examine SGK1 transcriptional activity in healthy muscle and rhabdomyosarcoma tissue. SGK1 transcript levels were quantified in rhabdomyosarcoma cell lines RD (embryonal subtype) and RH30 (alveolar subtype) by RT-PCR, cell viability was measured using MTT assays. Clonal cell growth was assessed via colony forming assays and migration experiments were performed in a transwell system. Results: SGK1 is expressed in embryonal and alveolar rhabdomyosarcoma tissue samples and in RD and RH30 rhabdomyosarcoma cell lines. Administration of EMD638683 – an inhibitor specific for SGK1 - decreased viability of RD and RH30 cells, enhanced the effects of the cytotoxic drug doxorubicin leading to reduced migration and decreased cell proliferation. Conclusions: SGK1 is expressed in rhabdomyosarcoma cells where it contributes to survival, therapy resistance, cell proliferation and migration. Thus, SGK1 inhibitors may be considered a therapeutic option for the treatment of therapy-resistant rhabdomyosarcoma.

scholarly journals Functional Characterization of Genes Driving Enhanced Biological Aggressiveness of Myeloma Cells: Identification of Novel and Understudied "Drivers" of Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 313-313
Author(s):  
Huihui Tang ◽  
Ryosuke Shirasaki ◽  
Ricardo De Matos Simoes ◽  
Sara Gandolfi ◽  
Olga Dashevsky ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Survival ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Research Funding ◽  
Oncology Research ◽  
Positive Regulators ◽  
Genome Scale ◽  
The Impact ◽  
Crispr Activation

Loss-of-function (LOF) studies (e.g. using RNAi or CRISPR) have historically been the main functional approaches to identify and study genes which drive the biology of Multiple Myeloma(MM) or other neoplasias. We hypothesized though that substantial complementary data can be derived from systematically examining the impact of transcriptional activation of endogenous genomic loci of different genes. To perform such gain-of-function (GOF) studies in an open-ended genome-scale manner, we applied CRISPR activation approaches in 4 MM lines (MM1S, KMS11, LP1, L363) which were transduced with the dCas9-P65-HSF transcriptional activation system and the Calabrese genome-scale sgRNA library (2 pooled sub-libraries; total of ~110,000 sgRNAs targeting promoters of ~18000 genes; initial coverage ~800-1000 cells/sgRNA). Next generation sequencing quantified the sgRNA abundance in MM cells at baseline and various time points (e.g. 2-12 weeks of culture), and rank aggregation algorithms identified genes with statistically significant enrichment or depletion of their sgRNAs, reflecting positive vs. negative, respectively, effects of GOF of these genes on MM cell survival/proliferation. These studies identified distinct groups of genes which serve as positive regulators of MM cell growth in at least 3 of the cell lines tested, including critical transcription factors (TFs)/cofactors, such as POU2F2, POU2AF1, IRF4, MYC; growth factor signaling mediators, such as IGF1R and IRS1; Ras family members (e.g. KRAS in MM1S cells); diverse members of the solute carrier family of transporters for amino acids and other bioactive small molecules. We also observed positive regulators of MM cell growth with distinct roles in certain cell lines, e.g. induction of IKZF3, IKZF1, RELA, CD48, MBNL1, PAX2 exhibited significantly more pronounced role as positive regulators of MM1S cells compared to the other cell lines tested. Notably, several positive regulators of MM growth identified in these CRISPR activation studies are not essential for MM cell survival/proliferation in CRISPR knockout studies in the same MM cell lines or many others. Examples of such genes include the TFs POU2F2 (Oct2) and PAX2, the TRAF interacting protein TIFA or the Toll-like receptor TLR4. We went on to validate these results for several such genes (e.g. POU2F2, POU2AF1) with individual sgRNAs for CRISPR activation and/or cDNA overexpression (vs. isogenic controls) in competition experiments and cell cycle analyses. We focused on further probing the mechanistic basis for the role of POU2AF1 as one of the top positive regulators of MM cell growth and performed RNA sequencing analyses of LP1 cells harboring induction of POU2AF1 expression using CRISPR activation. We observed upregulation of a distinct cluster of &gt;50 genes that are themselves positive regulators of LP1 cell growth in our genome-scale CRISPR activation studies. While some of these are known drivers of tumor cell growth (e.g. AURKB, E2F1, FGFR3), the large majority of these POU2AF1-driven positive regulators of MM cell growth have not been previously studied in MM; and exhibit a distinct enrichment for transcriptional regulators and other genes which are not required for LP1 cell survival/proliferation in CRISPR KO studies of LP1 cells. These results indicate that CRISPR-based activation of endogenous gene expression provides data which complementary to those derived from CRISPR LOF studies. Indeed, we not only validated through our GOF studies the relevance of genes which with prominent essentiality in CRISPR LOF studies (e.g. IRF4, POU2AF1, etc.), but also identified promising new candidate regulators of MM cell biology which are not essential for baseline survival/growth of MM cells, but can induce MM cell growth when further activated. These results provide novel insights into the biology of MM cells and may have therapeutic implications towards suppressing the ability of MM cells to transition to states of advanced biological aggressiveness. Disclosures Mitsiades: Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

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