Integrative Transcriptome and Quantitative Proteome Analyses Identify METTL3 As a Key Regulator for Aberrant RNA Processing in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12
Author(s):  
Yiming Wu ◽  
Meiling Jin ◽  
Kevyn Hart ◽  
Aijun Liao ◽  
Stacey M. Fernandes ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Stop Codon ◽  
Protein Translation ◽  
Rna Metabolism ◽  
Mrna Processing ◽  
Lymphocytic Leukemia ◽  
Rna Seq ◽  
Healthy Donors

Aberrant mRNA processing is known to drive the pathogenesis of chronic lymphocytic leukemia (CLL). Recurrent gene mutations in the RNA splicing factor SF3B1 and widespread RNA intronic polyadenylation impact genome-wide gene expression and inactivate tumor suppressors, respectively. Nevertheless, how mRNA processing is regulated and exerts its function in CLL remain elusive. To comprehensively characterize the role of mRNA processing in CLL, we performed RNA sequencing (RNA-seq) and Tandem Mass Tag (TMT) proteomics using normal and CLL B cells derived from healthy donors (n=5) and untreated CLL patients (n=22). We detected 328 proteins differentially expressed between normal and CLL B cells (|Log2FC|>0.58, q<0.05). Gene set enrichment analysis (GSEA) revealed that proteins involved in RNA metabolism (transcription, splicing, modification, 3'end processing, nuclear export, decay) were upregulated in CLL, while those impacting translation were downregulated. These findings were validated by immunoblotting in an independent set of samples (n=10). However, we observed no significant gene expression changes of RNA metabolism at the transcript level, indicating that regulation of these proteins occurred post-transcriptionally. Since N6-methyladenosine (m6A) is the most abundant RNA internal modification and has emerged as a key regulator for RNA metabolism, we sought to determine whether m6A is dysregulated in CLL cells. With an m6A dotblot assay and HPLC-MS, we consistently detected increased level of m6A in mRNA from CLL cells compared with normal B cells. As one of the most upregulated proteins in CLL, METTL3 writes m6A and promotes translation efficiency through its writer and reader functions, respectively. When we knocked down (KD) METTL3 in CLL cell lines (HG3, MEC1) as well as in primary CLL cells, we observed significant cell death and growth disadvantage in CLL compared to control cells, highlighting METTL3 is essential for CLL survival. We next examined whether KD of METTL3 affects m6A and RNA translation using m6A dotblot and O-propargyl-puromycin run on assays. Loss of METTL3 had subtle impact on m6A levels but it significantly decreased protein translation (t test, p<0.01) in all the cell lines tested (HG3, MEC1, JeKo-1, Mino). To define the target protein that METTL3 affects, we performed an integrated Ribosome profiling and RNA-seq analysis using HG3 and Mino cells with or without METTL3. At both transcriptome and translatome levels, loss of METTL3 significantly decreased genes enriched in the mTORC1 pathway, which has an essential role in translation (Metascape, hypergeometric test, q<0.05). Furthermore, it also decreased the translation efficiencies of genes involved in mRNA processing, DNA synthesis, and cell cycle pathways. This observation suggests that upregulation of METTL3 in CLL cells may regulate protein translation of the RNA metabolism pathway. Since m6A at the stop codon region is critical for METTL3 regulating protein translation, we performed MAZTER sequencing to determine m6A modification sites in normal and CLL B cells derived from healthy donors (n=5) and untreated CLL patients (n=11). We identified 214 genes with significant differential m6A modification at the stop codon region (delta cleavage efficiency>0.1, Wilcoxon rank-sum test, p<0.1, within DRACA motif) between normal and CLL B cells. These genes were highly enriched for mRNA processing (Metascape, q=0.017), supporting our notion that METTL3 may modulate protein expression of mRNA processing genes by recognizing m6A modification via its reader function in CLL. Consistent with its role in regulating protein expression, we detected downregulation of splicing factors (SF3A1, SF3A2, SF3B1, U2AF1) in various METTL3 KD cell lines (HG3, MEC1, JeKo-1, Mino) at only protein level but not transcription level. These data link METTL3 upregulation with RNA metabolism protein enrichment in CLL. Altogether, our integrated analysis uncovered a novel regulatory axis of METTL3 in CLL biology. We demonstrated that CLL cells have an increased m6A modification and upregulation of METTL3 at the protein level, resulting in translation of RNA metabolism related genes through its reader function by the recognition of m6A modification. Our results collectively suggest METTL3 as a central regulator for mRNA processing in CLL and provide a rationale for targeting METTL3 in this disease. Disclosures Brown: Janssen, Teva: Speakers Bureau; Gilead, Loxo, Sun, Verastem: Research Funding; Abbvie, Acerta, AstraZeneca, Beigene, Invectys, Juno/Celgene, Kite, Morphosys, Novartis, Octapharma, Pharmacyclics, Sunesis, TG Therapeutics, Verastem: Consultancy. Rosen:Seattle Genetics: Consultancy; NeoGenomics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; Pebromene: Consultancy; Celgene: Speakers Bureau; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau. Siddiqi:TG Therapeutics: Research Funding; Janssen: Speakers Bureau; Seattle Genetics: Speakers Bureau; Oncternal: Research Funding; BeiGene: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Juno: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Research Funding, Speakers Bureau.

scholarly journals A Novel Spirocyclic Dimer (36-286) Targeting the NF-Kappa B Pathway Displays Potent Anti-Tumor Properties in Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1186-1186
Author(s):  
Alexandria P Eiken ◽  
Audrey L Smith ◽  
Sarbjit Singh ◽  
Sandeep Rana ◽  
Sunandini Sharma ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Growth ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Protein Translation ◽  
Lymphocytic Leukemia ◽  
Rna Seq ◽  
Resistant Cells

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is an incurable, heterogenetic disease dependent on B cell receptor (BCR) signaling with subsequent nuclear factor-kappa B (NF-κB) activation resulting in the evasion of apoptosis and enhanced malignant B cell growth. Targeted therapies such as ibrutinib (IBR; BTK inhibitor) and venetoclax (VEN; BCL2 antagonist) have revolutionized the management of CLL, however ~20% of patients relapse, signifying the urgent need for novel therapeutics for CLL patients especially those with refractory/relapse (ref/rel) disease. Additionally, various tumor microenvironment (TME) stimuli fuel CLL growth and contribute to drug resistance through the activation of numerous signaling pathways (BCR, CD40R, TLR, BAFFR) and consequential sustained NF-κB activation. Currently, there are no FDA approved drugs that effectively target the NF-κB protein family. Herein we introduce 36-286 (N3), a novel spirocyclic dimer which displays NF-κB inhibitory activity and elicits potent anti-leukemic properties. N3 is a dimer of a spirocyclic α-methylene-γ-butyrolactone analog that covalently binds to surface exposed cysteine residues on NF-κB proteins (IKKβ and P65) (Rana S et al, 2016). Our study aims to investigate N3's mode of action (MOA) and to establish its anti-leukemic effects in CLL including drug-resistant disease, thereby introducing a novel therapeutic option for rel/ref disease. Methods: Cell growth via MTS proliferation assay was determined following treatment with N3 (0.125 - 2 μM) in a panel of malignant B cell lines [CLL (HG3, MEC1, OSUCLL), diffuse large B cell lymphoma (Pfeiffer, RC, RIVA), mantle cell lymphoma (Jeko1)], and in patient derived CLL cells stimulated with CpG ODN 2006 (CpG; 3.2 μM). Viability testing of normal B cells isolated from healthy donors was conducted following N3 treatment. Anti-tumor properties of N3 (1 - 2 μM; 4h) in the HG3 and OSUCLL cell lines were further confirmed under conditions mimicking different TME stimuli such as α-IgM (10 μg/mL), CD40L (100 ng/mL), BAFF (50 ng/mL) or CpG (3.2 μM). Protein expression of oncogenic MYC, select NF-κB pathway proteins (IKKα, IKKβ, P65, IκBα, RelB) and the anti-apoptotic protein MCL1 was determined following treatment with N3 (0.25 - 2 μM; 4h) by immunoblot (IB). Next, we induced IBR resistance in HG3 cells by prolonged exposure to increasing IBR concentrations (~10-15 fold its IC 50 in parental cells). Cell proliferation via MTS was determined following treatment with N3 on these resistant cells. To gain insight on the potential MOA of N3 in CLL, we adapted a proteomics-based approach (TMT labeled mass spectrometry) and conducted RNA-seq in OSUCLL cells treated with N3 (1 - 2 μM) for up to 24 h. Subsequent pathway analysis was performed to identify the top factors modulated by N3. Results: N3 showed remarkable efficacy (IC 50 < 0.6 μM) across all the malignant B cell lines evaluated while sparing normal B cells. In CpG stimulated primary CLL, N3 resulted in marked anti-leukemic effects (0.125 μM) comparable to IBR (1 μM). N3 induced cell apoptosis in CLL cell lines in a dose-dependent manner with marked PARP cleavage. Furthermore, our IB analyses of N3 treated CLL cell lines showed reduced levels of NF-κB pathway proteins, MYC and MCL1. Notably, N3 was effective in reducing levels of the above-mentioned proteins in the presence of the various TME stimuli. Strikingly, N3 maintained its cytotoxic effects in ibrutinib resistant HG3 cells. Studies to confirm N3's cytotoxicity in VEN resistant CLL cells are ongoing. Top ten pathways from both proteomics and RNA-seq analyses revealed an upregulation of the unfolded protein response (UPR) and inhibition of cap-dependent protein translation. IB analyses of select factors related to UPR (CHOP, XBP1, PERK, IRE1) and protein translation (eIF2α, 4E-BP1, PDCD4) in N3 treated CLL cells validated our omics' findings. Efforts to identify the proteome wide direct targets of N3 in CLL cells are currently underway. Conclusion: N3 is a novel pre-therapeutic lead that targets multiple survival and proliferation pathways through the inhibition of NF-κB activity and upregulation of UPR. We show that its highly cytotoxic in tumor B cells while sparing normal B cells. Moreover, N3 sustained its anti-tumor properties under different TME stimuli and in IBR resistant cells, indicating the potential use of this compound in rel/ref patients following evaluation in murine CLL models. Disclosures No relevant conflicts of interest to declare.

BI 836826, a Novel Fc-Engineered Antibody in Combination with Phosphoinositide-3-Kinase Inhibitor for Treatment of High Risk Chronic Lymphocytic Leukemia and Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2767-2767
Author(s):  
Deborah M Stephens ◽  
Kyle A. Beckwith ◽  
Priscilla Do ◽  
Carolyn Cheney ◽  
Xiaokui Mo ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Viability ◽  
B Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Advisory Committees

Abstract Background Targeting new antigens in chronic lymphocytic leukemia (CLL) and lymphoma may increase flexibility in the clinic and help circumvent resistance. The tetraspanin CD37 domain mediates transduction of survival and apoptotic signals (Lapalombella et al.,Cancer Cell, 2014), and has been clinically validated by recent trials of otlertuzumab (TRU-016) in CLL and Non-Hodgkin Lymphoma . Ligation of CD37 by this reagent simultaneously induced pro-apoptotic signaling and inhibited pro-survival signaling of phosphoinositide 3-kinase δ (PI3Kδ), which introduces a unique opportunity to use combination strategies employing activation of CD37 and inhibition of PI3Kδ. A new agent BI 836826 is an Fc-engineered anti-CD37 IgG1 that displays improved effector activities as well as crosslinker-independent direct cytotoxicity. We have evaluated the efficacy of BI 836826 combined with the PI3Kδ-selective inhibitor idelalisib in diffuse large B-cell lymphoma (DLBCL) cell lines and primary human CLL B-cells in the University and then by industry to validate the synergistic finding initially reported. Methods Cell viability assays usedCellTiterGlo to measure inhibition of antibody, isotype control, idelalisib or a combination of antibody and compound over 72h in culture. The cell viability of vehicle is measured at the time of dosing (T0) and after seventy-two hours (T72). A GI reading of 0% represents no growth inhibition, GI 100% represents complete growth inhibition, and a GI 200% represents complete death of all cells in the culture well. Annexin V-FITC and propidium iodide measure by flow cytometry was used to assess enhanced killing of primary CLL cells, with incubation of BI 836826 (0.1 µg/mL) and/or idelalisib (1 µM) at 37°C for 24 hours. Trastuzumab included as a non-specific IgG1 control. Data was reported as percentage of viable cells (Annexin V negative, PI negative) normalized to untreated control. Results DLBCL cell lines were variably sensitive to single agent BI 836826. In most of the cell lines tested, the cell viability was inhibited by 40%-50% with BI 836826 in the concentration range of 1-1000 ng/mL (Figure 1A). A synergistic effect was noted in several DLBCL cell lines when BI 836826 was combined with idelalisib. When the maximal effect of BI 836826 was greater than isotype control (GI% > 12, dotted line) and the effect of idelalisib showed a GI50 < 1uM, 3/5 cell lines showed synergy in combination (red dot, Figure 1B). A shift in the EC50of idelalisib can be seen with the addition of increasing amounts of BI 836826 (Figure 1C). In primary CLL B-cell cultures, 1 µM idelalisib displayed weak single agent activity following 24-hour incubation. The cytotoxicity of BI 836826 at 0.1 µg/mL was more variable, although treatment of samples from most CLL patients resulted in 20-50% B-cell death. The combination of these 2 agents resulted in enhanced cytotoxic activity (Figure 2A), and this effect was not attenuated by the presence of del(17)(p13.1), as there was no significant difference in cytotoxicity against these cells compared to those with lower risk cytogenetics (Figure 2B,C). Additionally, the combination was beneficial in CLL B-cells isolated from patients who were refractory to ibrutinib (Figure 2D). Conclusions This collaborative industry and academic endeavor with cross validation of initial mechanistic studies of synergy between CD37 and idelalisib demonstrates that addition of idelalisib to BI 836826 augments cytotoxicity against DLBCL cell lines and primary human CLL B-cells in an additive-to-synergistic manner. In addition, it maintains efficacy against CLL B-cells with del(17)(p13.1) and those from ibrutinib-refractory patients. Further exploration of this therapeutic strategy in clinical trials is strongly warranted. Disclosures Jones: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Grosmaire:Gilead: Employment. Jones:Gilead: Employment. DiPaolo:Gilead: Employment. Tannheimer:Gilead Sciences: Employment. Heider:4Boehringer Ingelheim RCV: Employment.

scholarly journals Transcriptomic Signitures of Azacitidine (AZA) and Decitabine (DAC) Resistance in MDS and CMML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4652-4652
Author(s):  
Faezeh Darbaniyan ◽  
Caleb Class ◽  
Guillermo Montalban-Bravo ◽  
Rashmi Kanagal-Shamanna ◽  
Marcos Estecio ◽  
...  
Keyword(s):  
B Cells ◽  
Signaling Pathways ◽  
Board Of Directors ◽  
Cell Lines ◽  
Respiratory Chain ◽  
Ribosomal Proteins ◽  
Research Funding ◽  
Rna Seq ◽  
Advisory Committees ◽  
Golgi Transport

Abstract INTRODUCTION: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are heterogeneous myeloid neoplastic disorders characterized by ineffective hematopoiesis leading to cytopenias and increased risk of transformation to acute myeloid leukemia (AML) . The hypomethylating agents (HMA) azacitidine (AZA) and decitabine (DAC) improve the natural history of MDS and CMML patients (Fenaux et al 2009). However, over half of the cases experience primary failure defined by a lack of response to HMA treatment which is associated with poor prognosis and a median survival of 4-6 months (Garcia-Manero et al. 2016, Jabbour et al. 2010). The highly heterogeneous pathophysiology of myeloid neoplasms and poorly understood mechanisms underlying therapeutic action of HMAs pose substantial challenges in understanding the biology of HMA failure in MDS and CMML. METHODS: We established a cohort of baseline bone marrow (BM) cells that were collected from 17 CMML and 34 MDS patients with excessive blasts (MDS-EB) prior to their HMA based therapies (Figure 1). RNA-Seq based transcriptomic analysis was performed in CD34+ BM hematopoietic stem and progenitor cells (HSPCs) of patients, known to be the cellular origin of these diseases, to identify biological signatures of primary HMA resistance. RESULTS: Similar to the recent reports regarding the lack of common gene expression signatures in HMA resistant AML cell lines (Leung et al. 2019) , RNA-Seq in patient cohort detected fewer than 0.5% of the total number of differentially expressed genes in non-responders in common for both AZA and DAC. Of note, all the AZA and a large portion of DAC associated genes with down-regulations in non-responders encode immunoglobulins, which is consistent with several recent findings indicating impaired differentiation B cells in association with unfavorable outcomes in MDS and CMML (Ribeiro et al. 2006 and Kahn et al. 2015). We performed flow cytometry analysis in BM cells available for 13 patients prior to start of HMA treatments (5 non-responders and 8 responders), and detected that HMA non-responders had a strong tendency (P=0.07) of decreased baseline frequencies of B cells in their BM than HMA responders. GSEA analysis based on leading edge genes identified over 200 and 300 biological signaling pathways to be associated with AZA and DAC failure respectively, with 78 pathways commonly correlated with treatment failure of both drugs (28 up-regulated and 60 down-regulated, Table 1). Clustering of these commonly altered pathways based on biological functions revealed that most of them are known to have a role in MDS and CMML pathogenesis and/ or drug resistance. For instance, neurotransmitter, olfactory pathways, and associated G-protein coupled receptor signals was recently reported to play a role in regulating the maintenance and differentiation of BM HSPCs (Shao et al. 2021 and Shim et al. 2013), whereas cell junction signaling that involves integrins and increased MAP2K-MAPK signaling were also reported to be associated with AZA resistance in MDS and CMML (Unnikrishnan et al. 2017). Among commonly down-regulated biological pathways in non-responders of AZA and DAC, there were clusters of immunoglobulin-associated signals, protein translation regulatory pathways that involve ribosomal proteins, cell cycle signaling, respiratory chain, and Golgi transport signals. Decreased expression of ribosomal proteins and related impairment of ribosomal functions were known mechanism in MDS and CMML development (Ebert et al. 2008 and Schneider et al. 2016). Furthermore, decreased respiratory chain and Golgi transport signals were also identified by transcriptomic investigation in the DAC resistant TF1-RES cell lines. In addition to the commonly altered signals for both AZA and DAC resistance, innate immune signaling pathways including interferon and toll-like receptor signals were significantly up-regulated in DAC non-responders but down-regulated in AZA non-responders. CONCLUSIONS: In this study, the transcriptomic data between responders and non-responders to AZA and DAC were investigated separately, thereby drug-specific as well as the common biomarkers associated with treatment failures of both drugs could be identified. The relatively low proportion of genes and pathways shared by AZA and DAC non-responders suggest the difference underlying biological mechanisms of AZA and DAC failure. Figure 1 Figure 1. Disclosures Sasaki: Novartis: Consultancy, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees. Wei: Daiichi Sanko: Research Funding.

Dishevelled Proteins of Wnt Signaling Pathways Are Highly Expressed in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4583-4583
Author(s):  
Abdul Salam Khan ◽  
Åsa Sandin ◽  
Anders Österborg ◽  
Mohammad Hojjat-Farsangi ◽  
Håkan Mellstedt ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Wnt Signaling ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Healthy Donors ◽  
Significant Difference ◽  
Gsk 3Β ◽  
Progressive Accumulation

Abstract Abstract 4583 Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world and is characterized by progressive accumulation of CD5+, CD19+, and CD23+ B cells in the blood, bone marrow, and secondary lymphoid organs. The progressive accumulation of malignant B cells is partly due to defective apoptosis. An important constitutively activated signaling pathway in CLL cells might be the Wnt signaling cascades that include the Wnt/b-catenin pathway and the non-canonical Wnt pathway. These pathways might be activated downstream of the constitutively phosphorylated ROR1. Dishevelled family proteins (Dvl1, Dvl2, and Dvl3) are important cytoplasmic mediators of Wnt signaling and have recently been shown to be expressed in many cancer types. The expression and precise roles of individual Dvl proteins are however not clear. The aim of the study was to characterize the expression of Dvl1, Dvl2 and Dvl3 proteins in progressive and non-progressive CLL and compare to normal white blood cells (PBMC) and to assess the expression of other important proteins of the canonical pathway as b-Catenin and GSK-3β as well as the non-canonical pathways as GSK-3α, PKC, ERK and AKT respectively. Leukemic cells from progressive (n=9) and non-progressive (n=9) CLL patients were isolated by Ficoll gradient centrifugation as well as PBMC from healthy donors (n=9). Six cell lines including four CLL cell lines EHEH, CII, I83, 232 B4, and the Jurkat and Lucas cell lines were also used. Dvl 1, Dvl 2, Dvl 3 proteins were analyzed by Western blot and densitometric calculations as well as PKC, GSK-3α, AKT and ERK using antibodies against the total protein and the phosphorylated protein respectively. Immunoprecipitation was performed to check the phosphorylation status of Dvl proteins. Overexpression of Dvl1, Dvl2, and Dvl3 was detected in all progressive and non-progressive CLL patients and the six cell lines. There was a significantly higher expression of Dvl1 and Dvl3 in progressive compared to non-progressive CLL patients (p<0.01 and p<0.01 respectively). In contrast to Dvl1 and Dvl3, Dvl2 was highly expressed in non-progressive as compared to progressive CLL patients. Dvl1 was phosphorylated at serine residues while Dvl2 was phosphorylated at tyrosine residues. b-Catenin followed the same expression pattern as Dvl2 being highly expressed in non-progressive CLL as compared to progressive CLL patients (p<0.001) whereas GSK-3β phosphorylation was significantly higher in progressive than non-progressive CLL patients (p<0.01). There was no significant difference in the degree phosphorylation of PKC and GSK-3α comparing non-progressive CLL, progressive CLL and healthy donors. In summary Dvl1, Dvl2 and Dvl3 are highly upregulated in CLL patients and CLL cell lines but the expression in normal PBMC is almost negligible. Dvl1, Dvl2 and Dvl3 may have a key role in the transduction of Wnt mediated signals in CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Computational Systems Biology Models for the Identification of Metabolic Vulnerabilities in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3084-3084
Author(s):  
Luis Vitores V. Valcárcel ◽  
Raquel Ordoñez ◽  
Iñigo Apaolaza ◽  
Ana Valcárcel ◽  
Leire Garate ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cancer Research ◽  
Systems Biology ◽  
B Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Essential Genes ◽  
Rna Seq ◽  
Metabolic Genes

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal accumulation of clonal plasma cells in the bone marrow. MM heterogeneity is associated to the presence of different genomic and transcriptomic profiles that have a clear impact on the prognosis of the disease. Metabolism has been deeply studied in cancer research, unveiling several vulnerabilities in different tumors. However, information regarding the role of metabolism in the pathogenesis of MM has not been explored in detail. Previous studies from our group using Systems Biology approach, explained the essentiality of metabolic gene RRM1 in several MM cell lines. The aim of our current study was to identify metabolic vulnerabilities in MM based on the application of a system biology approach focus on metabolic networks and trascriptomic data from MM patients. Our hypothesis being that changes in the metabolic landscape of MM could be exploited to uncover novel targets for prognosis and treatment in MM patients. We have analyzed the strand specific RNA-seq data from 35 samples from different subpopulations of B cells (Naïve, Centroblast, Centrocyte, Memory, Tonsilar and Bone Marrow Plasma cells (PCs)) and PCs of 37 MM patient samples. Using only the expression of 3287 metabolic genes included in Recon3D, the latest human metabolic reconstruction, we identified metabolic transcription patterns that clearly differentiate the different B cells from MM plasma cells (Figure 1A). MM samples were more similar to the normal PCs than to other B cells, which suggest that they maintain part of the metabolic pattern of the PCs, but in turn showed significant differences in the expression of metabolic genes. Interestingly, differential expression analysis of metabolic genes in MM PCs revealed a decrease in the expression of genes involved in mitochondrial activity and an increase in those that participate in metabolic proliferation, indicating that these alterations could probably play an important role in the development of this tumor. Using our novel systems biology approach, based on Recon3D and transcriptomic profiles, we predicted essential genes and synthetic lethals (involving two or more genes) that were specific for MM patients and not for the rest of the B cell subpopulations. Our approach makes use of the concept of genetic Minimal Cut Sets, previously introduced by our group in cancer research, which defines subsets of genes that if knocked out at the same time, induce a blockage of cellular proliferation. A metabolic vulnerability is found when only one gene is highly expressed in one of these gMCSs. We also analyzed the essentiality of these genes in more than 500 MM patients samples included in CoMMpass project and MM cell lines analyzed in Cancer Cell Line encyclopedia (CCLE). Using this computational strategy, we detected 8 essential genes involved in 42 gMCS specific for MM patients. From those candidates, GNPAT was our most promising target gene, as it was predicted to be essential for more than 40% of MM patients in our group, approximately 10% of patients of CoMMpass and in the majority of MM cell lines (Figure 1B). Validation of GNPAT expression and other 18 genes involved in the GNPAT gMCS was carried out by RT-qPCR showing similar results that were obtained with our RNA-seq data. Interestingly, the expression of the partner genes included in the GNPAT gMCS, such as DGK gene family, could also be indicators of the effectiveness of knocking-out, where their high expression is an indicator of resistance and their low expression an indicator of sensitivity. In conclusion, our findings suggest that our systems biology computational approach, driven by RNA-seq data, identifies metabolic vulnerabilities (defined as essential genes or synthetic lethal genes) providing pairs of new targets (essential gene) and their associated biomarkers (gMCS) in patients with MM. Figure 1: Metabolic genes expression analysis in human humoral immune response and MM patient samples. A) Standardized PCA result using the expression of metabolic genes included in Recon3D, in distinct subpopulations of B cells and MM samples. B) GNPAT gMCS showing the expression (in TPM) of GNPAT and associated 18 genes in MM cell lines. Figure 1 Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. Melnick:Epizyme: Consultancy; KDAc Therapeutics: Membership on an entity's Board of Directors or advisory committees; Constellation Pharmaceuticals: Consultancy; Janssenn: Research Funding. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.

scholarly journals Pharmacologic Targeting MCL1 with AZD5991 Induces Apoptosis and Mitochondrial Dysfunction in Non-Hodgkin Lymphoma (NHL) Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-33
Author(s):  
Tingting Liu ◽  
Fei Xu ◽  
Elana Thieme ◽  
Vi Lam ◽  
Guang Fan ◽  
...  
Keyword(s):  
B Cells ◽  
Mitochondrial Dysfunction ◽  
B Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Synthetic Lethality ◽  
Lymphocytic Leukemia ◽  
Dependent Manner ◽  
Oncology Research

BCL2 family proteins determine cell fate and comprise pro-apoptotic "initiators" (NOXA, BIM, PUMA), anti-apoptotic "guardians" (BCL2, MCL1, BCLX) and pro-apoptotic effectors (BAX/BAK). Venetoclax, a BCL2 inhibitor, received regulatory approval in therapy of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia. However, venetoclax is modestly effective in NHL. MCL1 is a short-lived pro-survival protein that is frequently overexpressed in NHL, leading to increased cell survival and drug resistance. MCL1 transgenic mice develop lymphoma, mostly of the diffuse large B-cell (DLBCL) type. Thus, selective targeting MCL1 represents a promising pharmacologic strategy in NHL. AZD5991 is a small molecule inhibitor highly selective towards MCL1 (Tron et al, 2018) in clinical trials. Here we explored pre-clinical activity of AZ5591 in NHL. Experiments were conducted in activated B-cell (ABC)-like (OCI-LY3, U-2932) and germinal center (GC)-like (OCI-LY18/19, SUDHL4/6/10, VAL) DLBCL cell lines; parental and ibrutinib-resistant Mino mantle cell lymphoma (MCL) cell lines. Peripheral blood mononuclear cells were isolated from patients with CLL and MCL and co-cultured with BAFF- or CD40L-expressing stroma. AZD5991 was obtained from Activ Biochem Ltd. MCL1 expression was detected in 10 tested DLBCL cell lines, highly expressed in 7/10. Meanwhile, BCLX was upregulated in eight, and BCL2 in 5/10 lines. Four cell lines expressed all three proteins. To confirm relevance of this data, we conducted analysis of primary DLBCL lymph nodes (n=30). MCL1 and BCLX were expressed in 50% and 80% of GC-like, and 10% and 25% of non-GC tumors, respectively. Treatment with AZD5991 restricted growth of DLBCL cells in a dose-dependent manner. GC-like cell lines VAL, SU-DHL4 and SU-DHL6 were highly susceptible with IC50~0.2 µM. Interestingly, they expressed relatively low levels of MCL1. Similarly, parental and ibrutinib-resistant Mino MCL cells were susceptible to MCL1 inhibition, with IC50 of 0.1 µM and 0.5 µM, respectively. Meanwhile, SU-DHL10, OCI-LY19 (GC-like), OCI-LY3 and U-2932 (ABC-like) cells were resistant to AZD5591. Consistent with its mechanism of action, immunoprecipitation assays showed that AZD5591 displaced BIM from MCL1 in NHL cells. We then evaluated MCL1 inhibition in primary neoplastic B-cells. CLL and MCL cells from patients were co-cultured with either CD40L- or BAFF-expressing stroma for 24 h. While CD40L predominantly induced BCLX, BAFF upregulated MCL1 in those cells. Consistent with this, BAFF-stimulated cells were highly sensitive to AZD5991, while CD40L-stimulated cells exhibited resistance. Since MCL1 acts in balance with pro-apoptotic effectors BAX and BAK at the mitochondrial membrane, we assessed the physiological effects of MCL1 inhibition on the mitochondrial function. Treatment with AZD5991 induced mitochondrial depolarization and led to a reduction in mitochondrial mass as well as increased generation of reactive oxygen species. This was accompanied by a decrease in cellular maximal respiratory capacity in both DLBCL and parental/ibrutinib-resistant MCL cells. Meanwhile, the rate of glycolysis was not significantly impacted. Interestingly, MCL1 inhibition induced mitophagy in sensitive (VAL) but not resistant cells (OCI-LY3). Next, we evaluated AZD5991 for synthetic lethality in a functional MTS-based screening assay using a panel of 189 small molecule inhibitors that target a variety of distinct signaling pathways activated in cancer (Tyner et al, 2018). AZD5991 demonstrated synergy with other BH3-mimetics. Co-treatment of DLBCL cells with BCL2/X inhibitors AZ4320, venetoclax and navitoclax overcame resistance to AZD5991. In summary, MCL1 inhibition using selective BH3-mimetic AZD5991 restricts cell proliferation and induces apoptosis in a subset of DLBCL, ibrutinib-resistant MCL cells and primary neoplastic B cells. MCL1 inhibition leads to mitochondrial dysfunction and mitophagy. Resistance to MCL1 inhibition may be overcome by concurrent targeting of alternative anti-apoptotic proteins (BCL2/X) in NHL. Disclosures Tyner: Syros: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Aptose: Research Funding; Constellation: Research Funding; AstraZeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Petra: Research Funding; Agios: Research Funding. Danilov:Abbvie: Consultancy; Celgene: Consultancy; Rigel Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Research Funding; Aptose Biosciences: Research Funding; Astra Zeneca: Consultancy, Research Funding; Verastem Oncology: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; TG Therapeutics: Consultancy; Nurix: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Karyopharm: Consultancy.

scholarly journals The U1 Spliceosomal RNA: A Novel Non-Coding Hotspot Driver Mutation Independently Associated with Clinical Outcome in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 847-847
Author(s):  
Ferran Nadeu ◽  
Shimin Shuai ◽  
Ander Diaz-Navarro ◽  
Irene López ◽  
Silvia Martín ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Expression Patterns ◽  
Lymphocytic Leukemia ◽  
Small Nuclear Rna ◽  
Rna Seq ◽  
Wild Type ◽  
Base Pairing ◽  
Mutational Status

Introduction: Genomic studies of chronic lymphocytic leukemia (CLL) have uncovered &gt;80 potential driver mutations. The vast majority of these mutations affect coding regions, and just two potential drivers have been identified in non-coding elements. Aim: To describe the biological and clinical impact of a recurrent A&gt;C mutation at the third base of the small nuclear RNA U1, the non-coding component of the spliceosome involved in the recognition of the 5' splice site (5'SS). Methods: Whole-genome sequencing (WGS) and RNA-seq from 318 CLL patients were used to identify and characterize a highly recurrent A&gt;C point mutation occurring at position 3 of the U1 snRNA gene (g.3A&gt;C mutation). The U1 wild-type and mutant forms were introduced into three CLL cell lines (JVM3, HG3, MEC1) to validate in vitro the predicted effect of this alteration. We screened two independent cohorts including a total of 1,314 CLL patients for the presence of the mutation using the rhAmp SNP genotyping assay, and integrated the U1 mutational status with well-known driver alterations, IGHV and epigenetic subgroups, and clinical parameters. Results: The U1 mutation was found in 8/78 (10.3%) CLL cases analyzed by WGS. Given its role in 5'SS recognition by base-pairing, we reasoned that this mutation was likely to alter the splicing and expression patterns of CLL. We were able to confirm widespread specific alterations in the transcriptome by comparing RNA-seq data between wild-type and g.3A&gt;C mutated samples. Applying this knowledge to an algorithm aimed to infer the U1 mutational status from expression data, we were able to identify 4 mutated cases among 240 additional cases that had RNA-seq but no WGS. In total, 12/318 (3.8%) CLL patients analyzed by WGS and/or RNA-seq harbored this mutation. This g.3A&gt;C U1 mutation changes the preferential A-U base-pairing between U1 and 5'SS to C-G base-pairing, creating novel splice junctions and altering the splicing pattern of 3,193 introns in 1,519 genes. In addition to altered splicing, 869 genes were differentially expressed between mutated and wild-type cases. We identified specific cancer genes (e.g. MSI2, POLD1, or CD44) and pathways (B-cell receptor signaling, promotion of apoptosis, telomere maintenance, among others) altered by the U1 mutation. To confirm a causal link between this mutation and splicing changes, we introduced exogenous U1 genes with or without the mutation into three cell lines. Subsequent RNA-seq of these cell lines recapitulated the altered splicing and expression patterns observed in CLL patients. We next screened for the presence of the U1 mutation 1,057 patients (cohort 1) using the rhAmp assay and it was found in 30 (2.8%) cases. The distribution of the mutation was similar in Binet stages and CLL vs monoclonal B-cell lymphocytosis. However, the U1 mutation was almost always found in IGHV unmutated CLL (29/30, p=9.0e-11) and within the naïve-like CLL epigenetic subgroup (p=3.7e-7). None of the U1 mutated cases had mutations in the SF3B1 splicing factor. Considering only pre-treatment CLL samples, U1 mutation was associated with a shorter time to first treatment independently of the Binet stage, IGHV mutational status, epigenetic subgroups, and mutations in the well-known CLL drivers SF3B1, NOTCH1, ATMor TP53. In cohort 2 (n=257), this mutation was found in 13 (5.1%) patients, confirming its enrichment in IGHV unmutated cases, naïve-like epigenetic subgroup, and splicing modulation. Despite the relatively small number of pre-treatment samples carrying the U1 mutation (7/178) and short follow-up of the patients (median 2.6 years), the effect of this mutation on time to first treatment in cohort 2 was compatible with the one observed in cohort 1. Finally, we screened for the U1 mutation a cohort of diffuse large B-cell lymphoma (n=108), mantle cell lymphoma (n=101), follicular lymphoma (n=87), splenic marginal zone lymphoma (n=12), acute myeloid leukemia (n=52), and myelodysplastic syndrome (n=67). The mutation was not present in any of the samples analyzed. Conclusions: Here we have reported that the third base of the small nuclear RNA U1 is recurrently mutated in CLL, proved its effect in splicing and gene expression, and shown that this mutation is independently associated with faster disease progression. The g.3A&gt;C U1 mutation represents a novel non-coding driver alteration in CLL with potential clinical and therapeutic implications. Disclosures Ramirez Payer: GILEAD SCIENCES: Research Funding. Terol:Astra Zeneca: Consultancy; Gilead: Research Funding; Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Roche: Consultancy. Lopez-Guillermo:Celgene: Consultancy, Research Funding; Janssen: Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding.

Generation of a New Chimeric Antigen Receptor (CAR) to Target CD23 Expressed on Chronic Lymphocytic Leukemia (B-CLL) Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3538-3538
Author(s):  
Greta Maria Paola Giordano Attianese ◽  
Valentina Hoyos ◽  
Barbara Savoldo ◽  
Virna Marin ◽  
Malika Brandi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Cell Activation ◽  
Lymphocytic Leukemia ◽  
Cytokine Release ◽  
Healthy Donors

Abstract B-Chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of mature B-lymphocytes expressing CD19, CD20dim and aberrantly expressing the CD5 T-cell marker. Moreover, they over-express the B-cell activation marker CD23. Chimeric Antigen Receptors (CAR) are engineered molecules able to redirect T-cell killing/effector activity towards a selected target in a non MHC-restricted manner. First trials targeting B-CLL were based on both monoclonal antibodies and anti-CD19/anti-CD20 CAR-transduced T cells. However, this approach causes the elimination of normal B-lymphocytes and B-precursors with consequent impairment of humoral responses. Selective CD23 expression on B-CLL cells renders it an optimal target to design a specific CAR. A new CD23-targeting CAR to redirect T cells against CD23+ B-CLL has been generated. After transduction, modified T cells were tested for cytotoxicity against different CD23+-targets, using a classic chromium release assay and for specific cytokine release by multiplex flow cytomix assay. The anti-CD23 CAR was stably expressed by healthy donor-derived primary T cells after transduction (average expression,20%;range,10%–60%;n=10) and conferred them a strong cytotoxicity against CD23+ tumor cell lines: Epstein Barr Virus transformed lymphoblastoid cell line (EBV-LCL) (average lysis, 50%; range 15%–70%, at 40:1 Effector:Target (E:T) ratio; n=5); Bjab and Jeko cell lines transduced with human CD23 antigen (average lysis, 60%; range, 20%–75%, at 40:1 E:T ratio; n=3). On the contrary, anti-CD23 transduced T-cells displayed no relevant killing versus normal B cells (average lysis, 8%; range, 1%–15% at 40:1 E:T ratio; n=3), differently from anti-CD19 CAR redirected T-cells, which killed tumor and normal B cells in an indistinct manner. T cells from B-CLL patients were also efficiently transduced with the anti-CD23 CAR (average expression, 80%; range, 70%–90%; n=3) and redirected specifically toward autologous blasts (average lysis, 29%; range, 21%–35% at 20:1 E:T ratio; n=3), without being inhibited by soluble CD23-enriched autologous plasma. Moreover, we demonstrated that expression of the anti-CD23 CAR caused a significant increase in cytokine release from transduced in vitro activated T cells after 48h stimulation with irradiated EBV-LCL at 1:1 ratio, both in healthy donors (n=3) and B-CLL patients (n=2). Anti-CD23 CAR expressing T cells from healthy donors secreted 5.5-fold more INF-gamma (3079 pg/ml vs 561pg/mL, p=0.05) and 11-fold more TNF-alpha (187.17 pg/ml vs 16.53 pg/mL, p=0.05), 147-fold more IL-5 (147 pg/ml vs 0 pg/mL, p=0.05) and 13-fold more IL-8 (590 pg/ml vs 43.24pg/mL, p=0.05), compared to non transduced T cells (n=3). In line with these findings, T cells expressing anti-CD23 CAR from B-CLL donors secreted 8.8-fold more INF-gamma (2988 pg/ml vs 337pg/mL, p=0.05) and 17-fold more TNF-gamma (187.17 pg/ml vs 17.34 pg/mL, p=0.05); 25.8-fold more IL-5 (3483.14 pg/ml vs 134.785 pg/mL, p=0.05), 173-fold more IL-8 (2154 pg/ml vs 12.415 pg/mL, p=0.05), compared to non transduced T cells. Altogether these results suggest that for the potentiality to get selective and potent killing of tumor cells, while sparing normal B cells, and for the capability to induce the selective release of immunostimulatory cytokines, CD23-targeting through a specific CAR holds great promises for adoptive immunotherapy of B-CLL.

Prognostic Implications of PIM-2 Expression in Samples from Patients with Chronic Lymphocytic Leukemia and Impact in the Sensitivity to the Pan-PIM Kinase Inhibitor PIM447

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2923-2923
Author(s):  
Ana Alicia López-Iglesias ◽  
Irena Misiewicz-Krzeminska ◽  
Ignacio Criado ◽  
Miguel Alcoceba ◽  
Susana Hernández-García ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Protein Levels ◽  
Pim Kinases

Abstract Background and objectives. PIM kinases (PIM1, PIM2, PIM3) are proteins known to be overexpressed in several hematological malignancies. In particular, in chronic lymphocytic leukemia (CLL) they are involved in cell survival, resistance to apoptosis (especially PIM2 and PIM3) and interactions with the microenvironment (PIM1). The aim of this study was dual: I) to evaluate the preclinical efficacy of PIM447, a pan PIM kinase inhibitor, in CLL and to study potential synergies with other drugs; and II) to evaluate the expression of PIM-kinases in different stages of the disease and correlate it with the prognosis and the sensitivity to the drug. Methods. Peripheral blood samples from untreated patients with different stages of the disease (monoclonal B lymphocytosis (MBL), stable CLL not requiring treatment (sCLL), and active CLL requiring treatment (aCLL)) were collected after informed consent. The ex vivo efficacy of PIM447 was analyzed by flow cytometry with annexin V in these samples. Moreover, PIM447 efficacy was also analyzed in two cell lines (MEC-1 and JVM-2) by MTT assay. Synergy with other drugs effective in CLL (bendamustine and fludarabine) was evluated with the calcusyn software. Protein levels of PIM Kinase proteins were evaluated by capillary electrophoresis immunoassay (WESTM ProteinSimple) in monoclonal B cells purified by CD19 selection with anti-CD19 magnetic microbeads and the autoMacs Cell separator (both from Miltenyi Biotec) from a subset of patients. Results. The pan PIM inhibitor, PIM447 was active in both cell lines tested, MEC-1 (IC50 5μM) and JVM2 (IC50 7μM), and also in monoclonal B cells from freshly isolated patients samples (sCLL=11; aCLL=5), with no difference in sensitivity between the different stages of the disease (IC50 of 4,8 μM and 4,7 μM for sCLL and aCLL respectively). There was a clear therapeutic window as treatment with PIM447 at doses toxic for monoclonal B cells, preserved T lymphocytes (figure 1) (median % of apoptosis for B cells and T lymphocytes respectively of 23 vs 20 at 5μM and 87 vs 35 at 10 μM). Moreover, PIM447 demonstrated to potentiate the activity of both bendamustine and fludarabine, being especially synergistic with this last one (combination index 0.1-0.6). A second objective was to analyze PIM2 protein expression by western blot in monoclonal B cells from these samples and correlate it with clinical and biological features. Up to now, it has been evaluated in 18 samples (MBL=4; sCLL=8; aCLL=6,). All of them expressed PIM-2. Expression levels of this protein were significantly higher in active CLL as compared with indolent stages of the disease (p=0,012). Patients with an unmutated IGHV status also displayed higher levels of PIM2 (p=0,01). Finally, samples with high PIM2 levels were slightly more resistant to PIM447 as compared with samples with lower protein levels (IC50 of 7,7 μM vs 5 μM, respectively). We are currently completing the analysis of the PIM2 levels of remaining samples and we are also measuring the levels of PIM1 protein, what will be available at the meeting. Conclusions: PIM-Kinase inhibition with PIM447 is effective in vitro in CLL cell lines and ex vivo in samples from patients. It synergizes with other agents especially fludarabine. PIM2 protein levels correlated with the clinical activity of CLL and with the mutational state of IGHV. Although all patients appear sensitive ex vivo to PIM447, further work is required to define PIM2 expression as a marker of sensitivity. Figure 1. Figure 1. Disclosures Ocio: Array BioPharma: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; MSD: Research Funding; Pharmamar: Consultancy, Research Funding; Jassen: Honoraria.

scholarly journals Targeting Inhibition of N6-Methyladenosine Demethylase Fto Displays Potent Anti-Tumor Activities in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-36
Author(s):  
Ya Zhang ◽  
Xinting Hu ◽  
Yang Han ◽  
Xiangxiang Zhou ◽  
Huimin Zhang ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Survival ◽  
Western Blotting ◽  
De Novo ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Rna Seq ◽  
Healthy Donors ◽  
Western Blotting Assay

Introduction Accumulating evidence indicates that Fat mass and obesity-associated protein (FTO), a N6-methyladenosine (m6A) RNA demethylase exerts crucial roles in oncogenesis. FB23-2, a novel inhibitor selectively targeting FTO m6A demethylase activity displayed promising potency in acute myeloid leukemia. Yet, no literature has been reported regarding the effects of FTO and FB23-2 in the tumorigenesis and development of chronic lymphocytic leukemia (CLL). Hence, the aim of this study was to investigate the clinical significance and mechanisms of FTO regulation in CLL. Methods Peripheral blood samples from 55 de novo CLL patients (36 males and 19 females; age range 32-82 years, median 62 years) were collected with informed consents in Shandong Provincial Hospital. CD19+ B cells were isolated with informed consents from healthy donors. Expression levels of FTO mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-seq) were conduct to profile RNA m6A methylation and expression of CLL cells. Lentiviral vectors were constructed to stably silence and overexpress FTO in CLL cells. Besides, cell viability, apoptosis and cell cycle were assessed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. Results Aberrantly increased expression of FTO was observed in CLL patients and CLL cell lines at mRNA and protein level compared with normal B cells from healthy donors (Figure 1A-B). Clinical correlation analyses suggested FTO high expression was significantly associated with 11q23 deletion (p=0.012; Figure 1C). Furthermore, Keplan-Meier plot indicated that elevated FTO expression predicted adverse outcome in CLL patients (HR=1.758, p=0.019; Figure 1D). ROC curve confirmed the prognostic value of FTO in survival of CLL patients (AUC=0.600, p=0.018; Figure 1E). To explore the potential role of FTO in CLL tumorigenesis, CLL cells were transfected with lentiviral vectors to stably silence and overexpress FTO. CLL primary cells and MEC1 cells with silence of FTO exhibited attenuated cell proliferation, increased fast-onset apoptosis (Figure 2A-D). Western blotting assay suggested significant down-regulated Bcl-2, enhanced cleaved-PARP and BAX expression in FTO-deficient CLL cells. Whereas, gain-of-function assay showed promoted cell survival in FTO-overexpressed CLL cells (Figure 2F-G). Additionally, serial dilution of FTO inhibitor FB23-2 decreased viability of MEC1 and primary CLL cells in time-dependent manner, and displayed rare cytotoxicity in normal B cells (Figure 3A-B). Besides, annexin V-PE/7AAD and western blotting assay indicated obvious apoptosis was induced with treatment of FB23-2 in CLL primary cells from 9 de novo CLL patients (Figure 3C-D). Importantly, obvious G2/M phase arrest and enhanced sensitivity to Venetoclax were also detected in FTO-reduced CLL cells. Furthermore, interactive MERIP-seq and RNA-seq of CLL cells with control and deleted FTO expression were performed to investigate the m6A Methylation-Mediated mechanism of FTO regulation of CLL pathogenesis. A total of 573 significantly changed peaks, of which 301 were significantly up-regulated and 272 peaks were significantly down-regulated (Figure 4A). Differentiated peaks were located in 3'UTR (42.58%) and 5'UTR (24.43%). Annotations of bioinformatics analyses indicated that FTO was functionally enriched in cell apoptosis in CLL progression (Figure 4B). Western blotting assay suggested significant down-regulated p-CHK2, c-myc, p-p53, cyclinD1 and enhanced p-H2AX expression in FTO-deficient CLL cells, indicating FTO accelerated CLL cell survival via DNA damage pathway (Figure 4C). Conclusion Taken together, our investigations identified for the first time the oncogenic role of FTO in CLL tumorigenesis and regulatory mechanism of FTO inhibitor FB23-2 in CLL cells by MERIP sequencing and ex vivo evaluation. Expression of FTO was upregulated and associated with inferior prognosis of CLL patients. FB23-2 exerted potent therapeutic potential in abrogating cell survival and inducing cell cycle arrest via m6A methylation. This study provides a rationale on evaluation of FTO-targeted intervention formulating a novel treatment paradigm in progressed CLL that warrants clinical investigation. Disclosures No relevant conflicts of interest to declare.

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