High-mobility group box Maestre, Lorena; García-García, Juan Fernando; Jiménez, Scherezade ...
PloS one,
02/2020, Letnik:
15, Številka:
2
Journal Article
Recenzirano
Thymocyte selection-associated high-mobility group box (TOX) is a DNA-binding factor that is able to regulate transcription by modifying local chromatin structure and modulating the formation of ...multi-protein complexes. TOX has multiple roles in the development of the adaptive immune system including development of CD4 T cells, NK cells and lymph node organogenesis. However very few antibodies recognizing this molecule have been reported and no extensive study of the expression of TOX in reactive and neoplastic lymphoid tissue has been performed to date. In the present study, we have investigated TOX expression in normal and neoplastic lymphoid tissues using a novel rat monoclonal antibody that recognizes its target molecule in paraffin-embedded tissue sections. A large series of normal tissues and B- and T-cell lymphomas was studied, using whole sections and tissue microarrays. We found that the majority of precursor B/T lymphoblastic, follicular and diffuse large B-cell lymphomas, nodular lymphocyte-predominant Hodgkin lymphomas and angioimmunoblastic T-cell lymphomas strongly expressed the TOX protein. Burkitt and mantle cell lymphomas showed TOX expression in a small percentage of cases. TOX was not found in the majority of chronic lymphocytic leukemia, myelomas, marginal zone lymphomas and classical Hodgkin lymphomas. In conclusion, we describe for the first time the expression of TOX in normal and neoplastic lymphoid tissues. The co-expression of TOX and PD-1 identified in normal and neoplastic T cells is consistent with recent studies identifying TOX as a critical regulator of T-cell exhaustion and a potential immunotherapy target. Its differential expression may be of diagnostic relevance in the differential diagnosis of follicular lymphoma, the identification of the phenotype of diffuse large B-cell lymphoma and the recognition of peripheral T-cell lymphoma with a follicular helper T phenotype.
Background. PI3Kδ is expressed in B-cells and has a central role in the B-cell receptor signaling in B-cell derived malignancies. Idelalisib was the first-in-class PI3Kδ inhibitors and several ...second-generation compounds are undergoing clinical investigation as single agents and in combinations. To identify modalities to overcome the resistance that develops to this class of agents, we have developed two idelalisib-resistant models derived from splenic marginal zone lymphoma (SMZL) cell lines.
Materials and Methods. Cells were kept under idelalisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. Cells underwent transcriptome and miRNA profiling by RNA-Seq, whole exome sequencing (WES), lipidomics profiling, pharmacological screening (348 compounds), and FACS analysis. Cytokines and growth factor secretion was performed by ELISA.
Results. Two RES models were obtained from VL51 and Karpas1718 with 7-10 fold times higher IC50s than PAR counterparts. In both models, conditioned media from RES cells transferred the resistance in the PAR cells. While WES did not identify somatic mutations associated with resistance, RNA-Seq and lipidomics analyses showed that the two cell lines had developed resistance activating different modalities.
The VL51 RES model showed an enrichment in BCR-TLR-NFkB (TLR4, CD19, SYK), IL6-STAT3 (IL6, CD44), chemokines (CXCL10, CXCR4, CXCR3) and PDGFR (PDGFRA, PRKCE) signatures, paired with increased p-AKT and p-BTK levels, decreased cardiolipins and sphingomyelins levels, and increased levels of specific triacylglycerols and glycerophosphocholines. In particular, there was an over-expression of surface expression of PDGFRA and secretion of IL6 in the medium. Silencing of both IL6and PDGFRA by siRNAs reverted the resistance, while the silencing of the individual genes had only a partial effect. These data were paired with the acquired sensitivity to the PDGFR inhibitor masitinib, identified in the pharmacologic screening.
In the Karpas1718 model, we observed an increased p-AKT activity with an enrichment for B-cell activation signatures (RAG1, RAG2, TCL1A), proliferation (E2F2, MKI67), ERBB signaling (HBEGF, NRG2, ERRB4), increased levels of some triacylglycerols and repressed levels for specific glycerophosphocholines. HBEGF secretion was confirmed by ELISA. The addition of recombinant HBEGF to the medium induced resistance in the PAR cells. Combination with the pan ERBB inhibitor lapatinib was beneficial in the K1718 RES. Recombinant HBEGF also induced resistance to the BTK inhibitor ibrutinib in the PAR cells and in the mantle cell lymphoma SP-53 cell line.
Specific members of the let-7 family of miRNAs were repressed in the RES lines derived from both cell lines, indicating the involvement of miRNA deregulation in the mechanism of resistance. Indeed, let-7 members are known to directly target IL6-STAT3 and cytokine signaling cascade, as well PI3K-AKT network. In solid tumors, let-7 members are also expressed at low levels in tumors with constitutive active ERBB signaling, in accordance with the activation of ERBB pathway and p-AKT we observed in our Karpas1718model. Experiments with a LIN28B inhibitor are now on-going.
Finally, we validated the findings across a panel of 34 B-cell lymphoma cell lines, in which IL6, PDGFRA, HBEGF and LIN28 expression levels were negatively correlated with idelalisib sensitivity, while the latter was positively correlated with let-7 levels (P <0.05).
Conclusions. We developed two distinct models derived from MZL of secondary resistance to the PI3Kδ inhibitor idelalisib. We identified treatments that might overcome resistance to idelalisib and are worth of further investigations. The two models, driven by different biologic processes, will allow the evaluation of further alternative therapeutic approaches.
Stathis:PharmaMar: Other: Renumeration; ADC Therapeutics: Other: Institutional research funding; Abbvie: Other: Renumeration; Bayer: Other: Institutional research funding; Novartis: Other: Institutional research funding; MEI-Pharma: Other: Institutional research funding; Roche: Other: Institutional research funding; Pfizer: Other: Institutional research funding; Merck: Other: Institutional research funding. Stuessi:Gilead: Speakers Bureau. Zucca:Gilead: Honoraria, Other: travel grant. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; Jazz Pharmaceuticals: Other: travel grants; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants.
Abstract
Introduction: Gains affecting chromosome 11 are recurrent events in lymphomas. The 11q24.3 gain occurs in 25% of diffuse large B-cell lymphoma (DLBCL), and it is associated with the ...overexpression of two ETS transcription factors, ETS1 and FLI1 (Blood 2013). Here, we have focused on the latter to identify the network of FLI1 regulated genes in GCB DLBCL.
Methods: GCB and ABC cell lines. Gene expression data were obtained from public datasets GSE98588, phs001444.v2.p1, GSE95013, GSE10846, and EGAS00001002606. Anti-FLI1 antibody - ChIP Grade (ab15289). ChIP-Seq for FLI1 paired with transcriptome analysis (RNA-Seq) after FLI1 silencing (siRNA) was performed. Sequencing was carried out using the NextSeq 500 (Illumina). Detection of peaks was analyzed using HOMER (v2.6); differential expressed genes were identified using moderated t-test (limma R-package) and functionally annotated with g:Profiler.
Results: The analysis of DLBCL cell lines showed that FLI1 protein levels were higher in GCB (n=12) than ABC (n=8) cell lines and was more commonly expressed at high levels in GCB (n= 414) than ABC (n= 518) DLBCL clinical specimens. Integration of identified binding sites from ChIP-Seq with RNA-Seq from GCB DLBCL cell lines (OCI-Ly1 and VAL) with genetically silenced FLI1 allowed the identification of putative FLI1 direct targets. The FLI1 negatively regulated genes included tumor-suppressor genes involved in negative regulation of cell cycle and p53 cascade. Among the FLI1 positively regulated targets we found genes annotated for immune response, MYC targets and B cell receptor, TNF-alpha and IL2 signaling pathways. Of note, direct targets of FLI1 overlapped with those genes regulated by ETS1, the other transcription factor co-gained in DLBCL, suggesting a functional convergence within the ETS family. ASB2 was downregulated after FLI1 silencing and had FLI1 binding sites in both promoter region and distal enhancer regions. Furthermore, ASB2 is known to promote NF-kB activation in T-cell acute lymphoblastic leukemia and might be an essential gene in DLBCL cells according to a genetic screening. Consistently, ASB2 gene silencing was toxic in GCB DLBCL lines. We observed inhibition of NF-kB pathway by a strong protein downregulation of RELB, along with increased IκBα upon ASB2 and FLI1 silencing, although with no differences in NF-kB2 levels. Only FLI1 silencing caused downregulation of NF-kB1 and RELA protein levels, but no effect on these two proteins was observed upon ASB2 silencing. These results indicate that FLI1 regulates either the classic NF-kB pathway at transcriptional level or the alternative pathway, via ASB2, in GCB DLBCL.
Conclusions: FLI1 is expressed at higher levels in GCB than ABC DLBCL and directly regulates a network of biologically crucial genes and processes in DLBCL, contributing to the regulation of NF-kB pathway in GCB DLBCL. ASB2, a subunit of a multimeric E3 ubiquitin ligase complex, is a novel FLI1 direct target, and its inhibition might represent a therapeutic approach for GCB DLBCL.
Citation Format: Giulio Sartori, Sara Napoli, Luciano Cascione, Elaine Y.L. Chung, Valdemar Priebe, Alberto J. Arribas, Andrea Rinaldi, Michela Dall'Angelo, Mattia Forcato, Silvio Bicciato, Margot Thome, Francesco Bertoni. The FLI1 direct target ASB2 promotes NF-KB pathway activation in diffuse large B-cell lymphoma of the germinal center B-cell type abstract. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-07.
PI3KPPinhibitors are active in patients with lymphoid neoplasms and a first series of them have been approved for the treatment of multiple types of B-cell lymphoid tumors, including marginal zone ...lymphoma (MZL). The identification of the mechanisms underlying either primary or secondary resistance is fundamental to optimize the use of novel drugs. Here, we present a model of secondary resistance to PI3Kffinhibitors obtained by prolonged exposure of a splenic MZL cell line to idelalisib. The VL51 cell line was kept under continuous exposure to idelalisib. The study included detailed characterization of the model, pharmacological screens, silencing experiments, validation experiments on multiple cell lines and on clinical specimens. VL51 developed resistance to idelalisib, copanlisib, duvelisib, and umbralisib. An integrative analysis of transcriptome and methylation underlined an enrichment of up-regulated transcripts and lowmethylated promoters in resistant cells, including IL-6/STAT3 and PDGFRA related genes and surface CD19 expression, alongside the repression of the let-7 family miRNAs, of miR-125, miR-130, miR-193 and miR-20. The use of the IL-6R blocking antibody tocilizumab, the STAT3 inhibitor stattic, the LIN28 inhibitor LIN1632, the PDGFR inhibitor masitinib and the anti-CD19 antibody drug conjugate loncastuximab tesirine were active compounds in the resistant cells as single agents and/or in combination with PI3K//inhibition. Findings were validated on additional in vitro lymphoma models and on clinical specimens. A novel model of resistance obtained from splenic MZL allowed the identification of therapeutic approaches able to improve the anti-tumor activity of PI3Kttinhibitors in B-cell lymphoid tumors.
Abstract
Background: PI3Kδ is expressed in B cells and has a central role in the B-cell receptor signaling. Copanlisib is a highly selective PI3Kδ and PI3Kα inhibitor, and it is currently under ...clinical development in indolent lymphomas including marginal zone lymphoma (MZL). Copanlisib is Food and Drug Administration (FDA) approved for the treatment of patients with relapsed or refractory follicular lymphoma. Nevertheless, a subset of patients can eventually relapse due to acquired resistance. A better understanding of resistance mechanisms could help to design improved therapies; hence, we generated MZL cell lines resistant to copanlisib.
Materials and Methods: Cells were kept on copanlisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 3 weeks of drug-free culture. Multidrug resistance phenotype was ruled out by confirming sensitivity to vincristine. Cells underwent transcriptome profiling (RNA-Seq) and immunophenotypic analysis.
Results: The RES models were obtained from VL51 cell line with over 50-fold times higher IC50s than PAR counterparts. Of note, the copanlisib-resistant lines showed decreased sensitivity to other PI3K inhibitors such as duvelisib (50-fold) and idelalisib (5-fold) and to the BTK inhibitor ibrutinib (15-fold), suggesting that the mechanism observed here might drive resistance to other downstream B-cell receptor inhibitors. Gene expression profiles of RES showed the upregulation of cytokine signaling (IL1A, IL1B, CXCR4), NFkB (LTA, TNF), MAPK (RASGRP4, RASGRP2), and JAK-STAT (STAT3, JAK3) signaling pathways and negative regulators of apoptosis (CD44, JUN). Conversely, repressed genes in RES were involved in cell adhesion (ITGA4, ITGB1), antigen presentation (HLAs), and IFN response (PARP12, GBP6). Consistent with the overexpression of antiapoptotic signaling genes, RES cells exhibited also resistance to the BCL2-inhibitor venetoclax, either as a single as in combination with copanlisib. Flow cytometry confirmed the CXCR4 upregulation and the downregulation of CD49d (ITGA4), paired with reduced CD20 and CD81 surface expression. In accordance, addition of a CXCR4 inhibitor overcame resistance to copanlisib.
Conclusions: We created a model of secondary resistance to the PI3K inhibitor copanlisib, derived from an MZL cell line. This model will help in clarifying mechanisms of resistance to the drug and to evaluate alternative therapeutic approaches. Indeed, we already identified novel potential targets, such as IL1 and CXCR4, that might be exploited in overcoming resistance to copanlisib and are worthy of further investigation.
Citation Format: Alberto J. Arribas, Sara Napoli, Luciano Cascione, Eugenio Gaudio, Roberta Bordone-Pittau, Marilia Barreca, Giulio Sartori, Chiara Tarantelli, Filippo Spriano, Andrea Rinaldi, Anastasios Stathis, Georg Stussi, Davide Rossi, Emanuele Zucca, Francesco Bertoni. Mechanisms of resistance to the PI3K inhibitor copanlisib in marginal zone lymphoma abstract. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-46.
SMZL usually has an indolent clinical course. Transformation to aggressive lymphoma occurs in only 4-5% of cases. Tools to identify patients facing a more aggressive clinical course and targeted ...therapies are needed. In the last years, we have characterized SMZL by DNA profiling (Blood 2011) and RNA/miRNA expression profiling (Mod Path 2013), and shown that somatic mutations of NOTCH2 and other genes involved in the pathway are frequent and mutually exclusive with mutations affecting NKFB pathway (JEM 2012). Here, we report genome-wide DNA promoter methylation profiling of 137 SMZL samples with its clinical and biologic implications.
A test series of 101 samples, including 3 SMZL cell lines (Karpas1718, VL51, SSK41) and 98 clinical SMZL cases from 10 Centers and, a validation set of 36 SMZL cases from 2 additional Centers were analyzed with the Illumina Infinium HumanMethylation27 arrays, as described (BJH 2013). Gene GEP was available for a subset of 11/101 cases. Methylation profiles were correlated with the presence of the copy number aberrations (CNAs) and somatic mutations recurrent in SMZL, IGHV mutational status, HCV infection and clinical data. Probes mapped outside CpG islands were discarded. Supervised analyses were performed using a t-test on the continuous beta values, followed by multiple test correction. For Fisher's test, probes were classified into unmethylated (beta-values <0.3) or methylated (beta-value >0.3).
Unsupervised clustering analysis of 101 SMZL samples identified 2 clusters.
Cluster one (23/101, 23%) comprised the 3 cell lines and included cases characterized by a generalized higher degree of promoter methylation (High-M), and had a significantly poorer overall survival (OS) than the remaining 79/101 (77%) cases (Low-M) (P=0.048; HR 2.4; 95% CI, 1-5.8).
To better understand the biology underlying this clustering, supervised analysis comparing High-M to Low-M cases was performed. By combining t- and Fisher's tests, 3200 gene probes showed differential methylation status between High-M and Low-M lymphomas. This probes signature was significantly enriched of promoters of promoter regions of PRC2-complex targets, genes involved in chromatin remodeling, and HOX and SOX family genes, and genes down-regulated by CDH1. Genes coding for subunits of the PRC2-complex (EZH2, EED, SUZ12) appeared unmethylated in their promoter regions and over-expressed by GEP in the High-M set, while EZH2-target genes and genes harboring the H3K27me3 mark had methylated promoters and down-regulated expression. A number of reported tumor-suppressor genes were highly methylated in High-M cases, including KLF4, DAPK1, CDKN2A/B, CDKN1C, CDH1, CDH2 and TIMP3. Known pro-survival lymphoma genes, such as SYK, MCL1, CARD11, BIRC5, BTK, BCL10, FOXP1, CD40, TACI and TCL1A/B, were un-methylated and over-expressed in High-M cases.
The prognostic relevance of the High-M phenotype was confirmed in an independent validation set. An unsupervised clustering using the top-100 differentially methylated probes obtained by t-test in the test series identified two clusters with one (11/36, 30%) bearing methylation profile similar to High-M and a poorer OS (P=0.038).
All the samples were then pooled to improve the statistical power of further analyses regarding the impact of methylation on the clinical course and on the association with biologic variables. High-M was significantly associated with poorer OS (P=0.008; HR 2.58; 95% CI, 1.2-5.4), transformation into aggressive lymphoma (33% vs 0.02%, P<0.001) and with 7q31-33 loss (38% vs 16%, P=0.026). In addition, High-M showed a higher frequency of NOTCH-pathway somatic mutations (39% vs 16%, P=0.063). HCV was more frequent in Low-M (20% vs 4%, P=0.058).
In a Cox regression model adjusted for the variables significantly associated with outcome at univariate analysis High-M, age>60, Interguppo Italiano Linfomi (IIL) SMZL score, HCV infection, High-M (HR 16; 95% CI, 1.3-201) and IIL SMZL score (HR 4.5; 95% CI, 1.3-15) retained their prognostic significance on OS (P<0.001).
Genome-wide promoter-methylation profiling in splenic MZL identified a subset of patients with poorer OS, characterized by a high promoter methylation and by specific genetic and biologic features. These data may provide the rational to explore epigenetic drugs in SMZL. *, AJA and AR equally contributed.
No relevant conflicts of interest to declare.
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