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 >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.

POU2AF1 As a Master Regulator of Oncogenic Transcription Factor Networks in Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-19
Author(s):  
Ricardo De Matos Simoes ◽  
Ryosuke Shirasaki ◽  
Huihui Tang ◽  
Shizuka Yamano ◽  
Benjamin G Barwick ◽  
...  
Keyword(s):  
Cell Growth ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Rna Seq ◽  
Advisory Committees ◽  
Genome Scale ◽  
Crispr Activation

Background: Functional genomics studies based on CRISPR and shRNA have documented that multiple myeloma (MM) cells are preferentially dependent (compared to other neoplasias) on a series of TFs, including IKZF1 and IKZF3 (which are targeted by thalidomide derivatives) and others that are not amenable to degradation or small molecule inhibition. Transcriptional co-factors have been therapeutically targeted, for example, inhibitors of BRD4, a co-factor for pTEFB, can be used to down-regulate c-myc. Aim: To identify new transcriptional vulnerabilities in MM with an emphasis on transcriptional co-factors Methods: We integrated results from genome-scale studies using the AVANA library for loss-of-function by gene editing (in 19 MM lines) and the Calabrese library for CRISPR-mediated gene activation (in 5 MM lines) to identify critical transcriptional co-factors (co-TFs). RNA-Seq analysis was used to identify critical pathways affected by POU2AF1 activation and existing ChIP-Seq tracks in MM cells were reanalyzed. Results: POU2AF1 (OCA-B) was the most preferentially essential TF co-factor in MM cell lines vs. non-MM and one of top genes which, upon CRISPR activation in genome-scale studies, increased MM cell fitness in vitro. We further confirmed the role of this gene using focused libraries of sgRNAs against POU2AF1 in vitro and in an in vivo model of MM cell growth in bone marrow-like scaffolds "functionalized" with humanized mesenchymal bone marrow stromal cells to simulate the human BM. CRISPR activation of POU2AF1 is associated with increased MM cell growth. RNA-Seq of POU2AF1 activation in LP1 cells a transcriptional program characterized by upregulation of other genes that are preferentially essential for MM including PRDM1, SUPT7L, UBE2G2 and TSC1; broad-spectrum oncogenic dependencies (e.g KRAS) and genes known or proposed to be involved in the pathophysiology of MM or other neoplasias (e.g. RUNX2, FGFR3, SMO, CREB5, TNFRSF13B, MEF2D, PCGF2). POU2AF1 overexpression was also associated with down-regulation of CDKN1C; of MHC class II molecules and their transcriptional activator CIITA, suggesting that POU2AF1 activation could also contribute to increased MM growth in vivo by allowing escape from immune surveillance. ATAC-Seq data and genome-wide ChIPseq for H3K27Ac in MM cell lines indicate that chromatin surrounding the POU2AF1 locus was highly accessible, concordant with the consistent expression of this TF in MM cell lines and patient-derived cells. CoMMpass data showed that POU2AF1 expression was enhanced in a subset of MM patients at relapse compared to diagnosis. Motif analysis of ChIP-seq data for POU2AF1 identified significant overlap with motifs for TFs relevant to the POU family (e.g. Oct11, Oct2, Oct4); members of the ETS family (e.g. ELF1, Elf4, GABPA); and other TFs with roles in MM including c-myc; IRF4; NF-kappaB, PRDM1, RUNX2 and the POU2AF1 target CREB5. These data suggest a functional interaction between POU2AF1 and other MM-relevant TFs. The transcriptional signature of POU2AF1 activation is enriched for genes downregulated by suppression/inhibition of MM-preferential TFs or epigenetic regulators including IRF4, PRDM1, IKZF1/3 and DOT1L. POU2AF1 binding motifs are also enriched in the promoter regions of MM-preferential dependencies including several MM-preferential TFs. Conclusions: POU2AF1 is essential for MM cells in vitro and in vivo; has a significantly more pronounced and recurrent role as a dependency in MM compared to most other neoplasias; and can further drive MM cell growth, through its ability to interact with several TFs critical for MM, forming multi-protein functional complexes. These results establish POU2AF1 as a central component in the regulatory network of oncogenic TFs in MM and highlight the value of further exploring POU2AF1 as a therapeutic target in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Cedilla Therapeutics: Consultancy; Tango Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Karyopharm: Research Funding; TEVA: Research Funding; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Abbvie: Research Funding; EMD Serono: 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.

The Lipid Addiction of Diffuse Large B-Cell Lymphoma (DLBCL) and Potential Treatment Strategies with Novel Fatty Acid Synthase (FASN) Small Molecule Inhibitors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4490-4490 ◽  
Author(s):  
Ravi Dashnamoorthy ◽  
Nassera Abermil ◽  
Afshin Behesti ◽  
Paige Kozlowski ◽  
Frederick Lansigan ◽  
...  
Keyword(s):  
Cell Death ◽  
Fatty Acid ◽  
Lipid Metabolism ◽  
Cell Survival ◽  
Binding Affinity ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Fatty Acid Synthase ◽  
The Impact

Abstract Background: Fatty acid (FA) metabolism is altered in several cancers through increased de novo synthesis of lipids via up-regulation fatty acid synthase (FASN) and increased utilization of lipids via β-oxidation. We investigated the dependence of DLBCL survival on FA metabolism. In addition, we examined novel FASN inhibitors TVB3567 and TVB3166 in comparison with cerulenin for the effects on cell survival and PI3K and MAPK-related biological pathways associated with tumor-related FA metabolism in DLBCL. Methods: FASN inhibitors, TVB3567 and TVB3166 (3V Biosciences, CA), cerulenin (FASN inhibitor), orlistat (anti-lipoprotein lipase (LPL) and FASN), PI3K/mTOR, and MEK small molecule inhibitors were studied in OCI-LY3, OCI-LY19, SUDHL4, SUDHL6, and SUDHL10 DLBCL cell lines for the effects of FA inhibition on lipid metabolism, cell signaling, and cell death. The effects of FASN inhibition on global gene expression profile (GEP) were also determined with Affymetrix Human 2.0 ST Genechip with Gene set enrichment analysis (GSEA). We also utilized short hairpin RNA interference (shRNA) to study interactions between FASN and PI3K/MAPK signaling. Finally, AutoDock Vina software (autodock.scripps.edu) was utilized to analyze drug target (FASN enzyme) binding affinity and assist in the design of FASN inhibitors with higher target binding affinity. Results: DLBCL cell lines OCI-LY3, SUDHL4, and SUDHL6 grown in the presence of lipoprotein-depleted serum showed exquisite sensitivity to lipid deprivation resulting in near complete cytotoxicity by MTT. Lipid deprivation-induced apoptotic cell death, detected as cleaved caspase 3 and PARP and Annexin-V/PI positivity, in these cells. Further, these effects were completely rescued by Very Low Density Lipoprotein (VLDL) supplementation to growth medium in SUDHL4 confirming the high lipid-dependency on cell survival in DLBCL. Treatment with pharmacological inhibitors of FASN (ie, TVB3567, TVB3166, cerulenin, or orlistat) resulted in a dose- and time-dependent reduction in cell viability in all DLBCL cell lines. Ingenuity Pathway Analysis (IPA) from GEP with cerulenin-treated OCI-LY3 showed prominent suppression of CD40, TNF, and NFκB dependent inflammatory responses as well as activation of apoptosis as predominant biological activities including significant down-regulation of genes involved in Krebs cycle and p38 MAPK pathways. Interestingly, upstream regulation by IPA predicted activation of MEK/ERK and MYC-dependent functions; and in OCI-LY3 with shRNA knock down of FASN, we observed constitutive activation of ERK as detected with increased phosphorylation by western blot. Activation of MEK/ERK and MYC is expected in part owing to metabolic stress induced by FASN inhibition. Considering the impact of MEK/ERK pathways on lipid metabolism, we next investigated the impact of MEK/ERK on FA metabolism. FASN was significantly decreased following MEK or ERK shRNA in OCILY-3 and SUDHL10 cells. Similarly, pharmacological inhibition of MEK or PI3K/mTOR (using novel small molecule agents AZD6244 (selumetinib) or BEZ235, respectively) resulted in marked down-regulation of FASN expression. Based on these results, FASN inhibition appears to a promising therapeutic target for the treatment of DLBCL, however attaining clinical efficacy with existing compounds require the effective drug concentration to be within the nanomolar range. Thus, we utilized AutoDock to determine drug docking enzyme inhibition constant (ki). We identified high ki values of 33μM and 180μM for Cerulenin and Orilstat, respectively. Therefore, we have developed/constructed modified novel and potent anti-FA compounds with ki <1μM that are currently being investigated. Conclusions: Collectively, we demonstrated that DLBCL cell survival is highly dependent on FA metabolism and that targeting lipid metabolism may be harnessed as a potential therapeutic strategy. We also showed that MEK/ERK-dependent mechanisms are intimately involved in promoting lipid addiction in DLBCL cells. Further investigation is warranted to delineate the mechanisms through which MEK/ERK regulate FASN expression and to determine in vivo implications of FASN inhibition on DLBCL tumor growth. In addition, continued development, design, and enhancement are needed to construct the most optimal anti-FA therapeutic agents. Disclosures Lansigan: Teva Pharmaceuticals: Research Funding; Spectrum Pharmaceuticals: Research Funding.

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.

RUNX1 Is Required For Maintenance Of Established T-ALL Blasts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3742-3742
Author(s):  
Christopher R Jenkins ◽  
Olena O Shevchuk ◽  
Hongfang Wang ◽  
Vincenzo Giambra ◽  
Samuel D Gusscott ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Dominant Negative ◽  
Brdu Incorporation ◽  
Loss Of Function ◽  
Cell Counts

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a clinically aggressive malignancy of immature T cells. Intensive multiagent chemotherapy achieves cure in 80-90% of pediatric patients, but only 40% of adult patients survive beyond 5 years. Data from recent ChIP-seq studies has shown that RUNX1 binds throughout the T-ALL genome at sites co-occupied by known oncogenic transcription factors including TAL1 and NOTCH1. For this reason, it has been suggested that RUNX1 may be part of a transcriptional activation complex that drives an oncogenic gene expression program in this cell context. In contrast, next generation sequencing studies have recently identified heterozygous point mutations throughout the RUNX1 coding region in T-ALL including some which are predicted to encode truncated polypeptides resembling dominant negative alleles, thus raising the possibility that RUNX1 may function rather as a tumor suppressor in this context. In an effort to explore the functional role of RUNX1 in T-ALL, we examined the effect of RUNX1 knockdown in a broad panel of established human T-ALL cell lines and xenograft-expanded patient biopsy samples. Cells transduced with lentiviral shRNAs targeting coding and 3’ UTR regions of RUNX1 showed a clear growth disadvantage as compared to either non-transduced cells in the same culture or cells transduced with non-silencing shRNAs in parallel cultures. As well, absolute cell counts of cultures containing only shRUNX1-transduced cells demonstrated dramatically reduced growth rates as compared to either non-transduced or non-silencing shRNA-transduced controls. BrdU incorporation and CFSE dye dilution studies showed that most cell lines exhibited reduced proliferation in response to RUNX1 knock-down, while a subset of lines also showed reduced cell viability. These phenotypes were largely consistent across a panel of over 20 T-ALL cell lines and 4 xenograft-expanded patient samples, including several which harbored either nonsense or missense RUNX1 mutations. These results support the notion that established T-ALL cells are generally dependent on RUNX1 for continued cell growth and, in some cases, also for survival. We also explored candidate RUNX1 target genes which might be responsible for mediating the observed growth/survival phenotypes. Assembling a short list of the “usual suspects” including genes known to regulate growth of T-ALL cells generally (c-MYC, PTEN), to possess a substantial RUNX1 ChIP-seq peak in T-ALL cells (IGF1R, IL7R), or to be bona fide RUNX1 targets in other cell contexts (p21/WAF1, p27/KIP1), we performed western blot or flow cytometric analyses of multiple shRUNX1-transduced cell lines. We noted consistent regulation of some, but not all of these in a manner consistent with RUNX1 positively supporting cell growth. From these results, we conclude that RUNX1 plays a pro-oncogenic role in established T-ALL cells. We surmise that the complement of presumed loss-of-function RUNX1 mutations observed in patient T-ALLs may be indicative of its known roles in regulating normal T cell development such that loss-of-function mutations may lead to differentiation arrest and consequently promote tumor initiation. For the majority of T-ALLs that express wild-type RUNX1 proteins, however, our results suggest that RUNX1 acts functionally to support maintenance of the malignant clone by promoting expression of known oncogenic factors and repressing expression known tumor suppressors. Disclosures: Aster: Cell Signaling Technology: Consultancy; Merck, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Genentech, Inc.: Honoraria.

scholarly journals The KDM3A-KLF2-IRF4 Axis Maintains Myeloma Cell Survival

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3633-3633
Author(s):  
Hiroto Ohguchi ◽  
Teru Hideshima ◽  
Manoj Bhasin ◽  
Gullu Gorgun ◽  
Loredana Santo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Cell Survival ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Chip Assay ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Histone methylations are tightly regulated by a balance between methyltransferases and demethylases that mediate the addition and removal of these modifications. Importantly, dysregulation of histone methylation is implicated in pathogenesis of cancers, including multiple myeloma (MM). For example, the t(4;14) (p16;q32) is present in 15 - 20% of MM patients and results in overexpression of WHSC1, a histone H3 lysine 36 (H3K36) methyltransferase. On the other hand, approximately 10% of MM patients without the t(4;14) have inactivating mutations in KDM6A, a H3K27 demethylase. KDM3A is a Jumonji C-domain-containing histone demethylase which catalyzes removal of H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). KDM3A is implicated in pathogenesis of different types of cancers. Here we investigated the biological impact of KDM3A in MM. KDM3A expression was significantly elevated in MM patient samples compared to normal plasma cells in publicly available dataset (GSE5900, GSE6691). To evaluate the functional role of KDM3A, shRNAs targeting KDM3A were transduced into MM cell lines: knockdown of KDM3A significantly inhibited MM cell growth (RPMI8226, MM.1S, U266, H929) in vitro and in xenograft model (MM.1S). Apo2.7 staining showed that apoptotic cells were significantly increased after knockdown of KDM3A. We next examined gene expression profiles after knockdown of KDM3A in RPMI8226 cells. With a cutoff of > 1.5-fold downregulation, a total of 305 probe sets were downregulated in KDM3A-knockdown cells relative to control cells. Among putative KDM3A targets, a gene of particular interest is KLF2 which plays a key role in maintenance of B cell and plasma cell phenotype, and function. Another intriguing gene is IRF4, given its known crucial role in MM cell survival. We confirmed that expression of KLF2 and IRF4 was downregulated after knockdown of KDM3A by quantitative realtime PCR and immunoblots in RPMI82226, MM.1S, and U266 cells. KDM3A binding to KLF2 and IRF4 core promoters was demonstrated by chromatin immunoprecipitation (ChIP) assay in RPMI8226 cells. Moreover, knockdown of KDM3A increased H3K9me1 and me2 levels at both promoter regions, indicating that KDM3A directly regulates KLF2 and IRF4 expression by removing H3K9 methylation marks at their promoters in MM cells. shRNAs targeting KLF2 were next transduced into MM cell lines: silencing of KLF2 significantly reduced cell growth of MM cell lines, associated with decreased IRF4. Promoter reporter assays using human IRF4 promoter showed that KLF2 significantly increased luciferase expression in a dose-dependent manner. Moreover, ChIP assay showed that KLF2 bound to IRF4 promoter in RPMI8226 cells. Since transcription factors could form an autoregulatory feedback loop, we hypothesized that IRF4 might regulate KLF2 expression. As expected, knockdown of IRF4 downregulated KLF2 expression at both the mRNA and protein levels in 3 MM cell lines. In addition, ChIP assays demonstrated that IRF4 bound to KLF2 second intron that contains tandem IRF4 motifs in RPMI8226 cells. Collectively, these results suggest that KLF2 activates IRF4 expression and vice versa, forming an autoregulatory loop in MM cells. KLF2 has been reported to control homing of plasma cells to the bone marrow; we therefore hypothesized that KDM3A-KLF2-IRF4 axis might regulate adhesion and homing of MM cells to the bone marrow. Importantly, knockdown of KDM3A, KLF2, or IRF4 decreased adhesion of 3 MM cell lines to bone marrow stromal cells. Furthermore, bone marrow homing of MM.1S cells was significantly reduced after knockdown of KDM3A, KLF2, or IRF4 in a murine xenograft MM model, indicating that KDM3A-KLF2-IRF4 axis regulates, at least in part, MM cell adhesion and homing to the bone marrow. In conclusion, our study demonstrated that KDM3A is a crucial epigenetic regulator of MM cell survival, and that inhibition of KDM3A represents a novel therapeutic strategy in MM. Disclosures Raje: Amgen: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding; Onyx: Consultancy; AstraZeneca: Research Funding; Millenium: Consultancy. Richardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding. Anderson:Oncopep: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy; Celgene: Consultancy; Acetylon: Equity Ownership.

Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth

1997 ◽  
Vol 273 (5) ◽  
pp. L941-L949 ◽  
Author(s):  
Barbara Driscoll ◽  
Lingtao Wu ◽  
Susan Buckley ◽  
Frederick L. Hall ◽  
Kathryn D. Anderson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cyclin D ◽  
Lung Cancer Cell ◽  
Cancer Cell Growth ◽  
Transfected Cells ◽  
The Impact

To investigate the role of cyclin D1 in the regulation of lung cancer cell growth, we created five stably transfected cell lines carrying a cyclin D1 antisense construct. The transfected cells exhibited a marked decrease in the rate of cell growth, in contrast to the original lines (A549 and NCI-H441). The expression of several cell cycle-regulating proteins, including cyclin A, the cyclin-dependent kinases (cdk) 2 and cdk4, in addition to cyclin D1 itself, was markedly decreased. The expression of one cdk inhibitor, p21WAF1/CIP1, increased in the A549-derived cell lines. A specific target of cyclin D1 activity, the growth-suppressing product of the retinoblastoma gene, pRb, exhibited decreased expression and a decreased level of phosphorylation in the transfected cells. Decreased expression of pRb due to a significant increase in its turnover rate suggested that the stability of the protein may depend on phosphorylation by cyclin D1-dependent cdk activity. In addition to the impact on pRb stability, decreased expression of cyclin D1 induced susceptibility to cell death after withdrawal of exogenous growth factors in the antisense transfected cell lines, a response that was not observed in the original cancer cell lines. We conclude that abrogation of cyclin D1 overexpression in lung cancer cells disrupts several key pathways that are required for uncontrolled cell growth and induces those that lead to cell death after growth factor deprivation. Therefore, we speculate that use of antisense cyclin D1 expression in appropriate gene vectors could be a useful method for retarding lung cancer cell growth in accessible tumors such as those of the lung epithelium.

scholarly journals Fusarubin and Anhydrofusarubin Isolated from A Cladosporium Species Inhibit Cell Growth in Human Cancer Cell Lines

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 503 ◽  
Author(s):  
Sabrina Adorisio ◽  
Alessandra Fierabracci ◽  
Isabella Muscari ◽  
Anna Liberati ◽  
Lorenza Cannarile ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Fas Ligand ◽  
Human Cancer ◽  
Dependent Manner ◽  
Food Contaminants ◽  
Human Cancer Cell Lines ◽  
Hematological Cancers ◽  
The Impact

Cladosporium species are endophytic fungi that grow on organic matter and are considered food contaminants. The anti-microbial and anti-tumor naphthoquinones fusarubin (FUS) and anhydrofusarubin (AFU) were isolated using column chromatography from a Cladosporium species residing inside Rauwolfia leaves. The impact of FUS and AFU on cell growth was assessed in acute myeloid leukemia (OCI-AML3) and other hematologic tumor cell lines (HL-60, U937, and Jurkat). Treatment with FUS or AFU reduced the number of OCI-AML3 cells as evaluated by a hemocytometer. Flow cytometry analyses showed that this effect was accompanied by diverse impairments in cell cycle progression. Specifically, FUS (20 or 10 μg/mL significantly decreased the percentage of cells in S phase and increased the percentage of cells in G2/M phase, whereas AFU increased the percentage of cells in G0/G1 phase (50 and 25 μg/mL) and decreased the percentage of cells in S (50 μg/mL) and G2/M (50 and 25 μg/mL) phases. Both substances significantly increased apoptosis at higher concentrations. The effects of FUS were more potent than those of AFU, with FUS up-regulating p21 expression in a p53-dependent manner, as detected by Western blot analyses, likely the consequence of decreased ERK phosphorylation and increased p38 expression (both of which increase p21 stability). FUS also decreased Akt phosphorylation and resulted in increased Fas ligand production and caspase-8/3-dependent apoptosis. These results suggest that FUS and AFU inhibit proliferation and increase apoptosis in cell lines derived from hematological cancers.

CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4174-4174
Author(s):  
Jiaying Tan ◽  
Jay L. Hess
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Polycomb Group ◽  
Human Leukemia ◽  
Interaction Sites

Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2187-2187
Author(s):  
Xiaomei Yan ◽  
Yoshihiro Hayashi ◽  
Xinghui Zhao ◽  
Aili Chen ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Down Regulation ◽  
Leukemia Cell Lines ◽  
Flanking Regions ◽  
The Impact

Abstract Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

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