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1.
Cell Rep Med ; 5(4): 101484, 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38554704

ABSTRACT

The use of Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib achieves a remarkable clinical response in mantle cell lymphoma (MCL). Acquired drug resistance, however, is significant and affects long-term survival of MCL patients. Here, we demonstrate that DNA methyltransferase 3A (DNMT3A) is involved in ibrutinib resistance. We find that DNMT3A expression is upregulated upon ibrutinib treatment in ibrutinib-resistant MCL cells. Genetic and pharmacological analyses reveal that DNMT3A mediates ibrutinib resistance independent of its DNA-methylation function. Mechanistically, DNMT3A induces the expression of MYC target genes through interaction with the transcription factors MEF2B and MYC, thus mediating metabolic reprogramming to oxidative phosphorylation (OXPHOS). Targeting DNMT3A with low-dose decitabine inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting DNMT3A-mediated metabolic reprogramming to OXPHOS with decitabine provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL.


Subject(s)
Adenine/analogs & derivatives , Lymphoma, Mantle-Cell , Piperidines , Protein-Tyrosine Kinases , Humans , Animals , Mice , Adult , Agammaglobulinaemia Tyrosine Kinase/metabolism , Drug Resistance, Neoplasm/genetics , DNA Methyltransferase 3A , Oxidative Phosphorylation , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/pathology , Decitabine/metabolism , Decitabine/therapeutic use
3.
Blood ; 142(22): 1879-1894, 2023 11 30.
Article in English | MEDLINE | ID: mdl-37738652

ABSTRACT

The use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell-like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.


Subject(s)
Antineoplastic Agents , Lymphoma, Large B-Cell, Diffuse , Lymphoma, Mantle-Cell , Metformin , Humans , Adult , Animals , Mice , Agammaglobulinaemia Tyrosine Kinase/metabolism , Oxidative Phosphorylation , Drug Resistance, Neoplasm , Cell Line, Tumor , Antineoplastic Agents/therapeutic use , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/pathology , Lymphoma, Large B-Cell, Diffuse/pathology , Metformin/pharmacology , Early Growth Response Protein 1/metabolism
4.
Sci Transl Med ; 15(689): eabq8513, 2023 03 29.
Article in English | MEDLINE | ID: mdl-36989375

ABSTRACT

Although the overall survival rate of B cell acute lymphoblastic leukemia (B-ALL) in childhood is more than 80%, it is merely 30% in refractory/relapsed and adult patients with B-ALL. This demonstrates a need for improved therapy targeting this subgroup of B-ALL. Here, we show that the ten-eleven translocation 1 (TET1) protein, a dioxygenase involved in DNA demethylation, is overexpressed and plays a crucial oncogenic role independent of its catalytic activity in B-ALL. Consistent with its oncogenic role in B-ALL, overexpression of TET1 alone in normal precursor B cells is sufficient to transform the cells and cause B-ALL in mice within 3 to 4 months. We found that TET1 protein is stabilized and overexpressed because of its phosphorylation mediated by protein kinase C epsilon (PRKCE) and ATM serine/threonine kinase (ATM), which are also overexpressed in B-ALL. Mechanistically, TET1 recruits STAT5B to the promoters of CD72 and JCHAIN and promotes their transcription, which in turn promotes B-ALL development. Destabilization of TET1 protein by treatment with PKC or ATM inhibitors (staurosporine or AZD0156; both tested in clinical trials), or by pharmacological targeting of STAT5B, greatly decreases B-ALL cell viability and inhibits B-ALL progression in vitro and in vivo. The combination of AZD0156 with staurosporine or vincristine exhibits a synergistic effect on inhibition of refractory/relapsed B-ALL cell survival and leukemia progression in PDX models. Collectively, our study reveals an oncogenic role of the phosphorylated TET1 protein in B-ALL independent of its catalytic activity and highlights the therapeutic potential of targeting TET1 signaling for the treatment of refractory/relapsed B-ALL.


Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma , Proto-Oncogene Proteins , Animals , Mice , Proto-Oncogene Proteins/metabolism , Phosphorylation , Staurosporine , Signal Transduction , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , DNA-Binding Proteins/metabolism
5.
J Clin Invest ; 132(3)2022 02 01.
Article in English | MEDLINE | ID: mdl-34882582

ABSTRACT

Mantle cell lymphoma (MCL) is a phenotypically and genetically heterogeneous malignancy in which the genetic alterations determining clinical indications are not fully understood. Here, we performed a comprehensive whole-exome sequencing analysis of 152 primary samples derived from 134 MCL patients, including longitudinal samples from 16 patients and matched RNA-Seq data from 48 samples. We classified MCL into 4 robust clusters (C1-C4). C1 featured mutated immunoglobulin heavy variable (IGHV), CCND1 mutation, amp(11q13), and active B cell receptor (BCR) signaling. C2 was enriched with del(11q)/ATM mutations and upregulation of NF-κB and DNA repair pathways. C3 was characterized by mutations in SP140, NOTCH1, and NSD2, with downregulation of BCR signaling and MYC targets. C4 harbored del(17p)/TP53 mutations, del(13q), and del(9p), and active MYC pathway and hyperproliferation signatures. Patients in these 4 clusters had distinct outcomes (5-year overall survival [OS] rates for C1-C4 were 100%, 56.7%, 48.7%, and 14.2%, respectively). We also inferred the temporal order of genetic events and studied clonal evolution of 16 patients before treatment and at progression/relapse. Eleven of these samples showed drastic clonal evolution that was associated with inferior survival, while the other samples showed modest or no evolution. Our study thus identifies genetic subsets that clinically define this malignancy and delineates clonal evolution patterns and their impact on clinical outcomes.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Lymphoma, Mantle-Cell , Neoplasm Proteins , Transcriptome , Adult , Aged , Aged, 80 and over , Disease-Free Survival , Female , Humans , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/mortality , Male , Middle Aged , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Survival Rate
6.
Clin Cancer Res ; 26(5): 1034-1044, 2020 03 01.
Article in English | MEDLINE | ID: mdl-31811017

ABSTRACT

PURPOSE: In classical Hodgkin lymphoma, the malignant Reed-Sternberg cells express the cell surface marker CD30. Brentuximab vedotin is an antibody-drug conjugate (ADC) that selectively delivers a potent cytotoxic agent, monomethyl auristatin E (MMAE), to CD30-positive cells. Although brentuximab vedotin elicits a high response rate (75%) in relapsed/refractory Hodgkin lymphoma, most patients who respond to brentuximab vedotin eventually develop resistance. PATIENTS AND METHODS: We developed two brentuximab vedotin-resistant Hodgkin lymphoma cell line models using a pulsatile approach and observed that resistance to brentuximab vedotin is associated with an upregulation of multidrug resistance-1 (MDR1). We then conducted a phase I trial combining brentuximab vedotin and cyclosporine A (CsA) in patients with relapsed/refractory Hodgkin lymphoma. RESULTS: Here, we show that competitive inhibition of MDR1 restored sensitivity to brentuximab vedotin in our brentuximab vedotin-resistant cell lines by increasing intracellular MMAE levels, and potentiated brentuximab vedotin activity in brentuximab vedotin-resistant Hodgkin lymphoma tumors in a human xenograft mouse model. In our phase I trial, the combination of brentuximab vedotin and CsA was tolerable and produced an overall and complete response rate of 75% and 42% in a population of patients who were nearly all refractory to brentuximab vedotin. CONCLUSIONS: This study may provide a new therapeutic strategy to combat brentuximab vedotin resistance in Hodgkin lymphoma. This is the first study reporting an effect of multidrug resistance modulation on the therapeutic activity of an ADC in humans. The expansion phase of the trial is ongoing and enrolling patients who are refractory to brentuximab vedotin to confirm clinical activity in this population with unmet need.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Resistance, Neoplasm/drug effects , Hodgkin Disease/drug therapy , Reed-Sternberg Cells/pathology , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/metabolism , Adult , Aged , Animals , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Brentuximab Vedotin/administration & dosage , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclosporine/administration & dosage , Female , Hodgkin Disease/metabolism , Hodgkin Disease/pathology , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Prospective Studies , Reed-Sternberg Cells/drug effects , Reed-Sternberg Cells/metabolism , Treatment Outcome , Xenograft Model Antitumor Assays , Young Adult
8.
Blood ; 133(4): 306-318, 2019 01 24.
Article in English | MEDLINE | ID: mdl-30530749

ABSTRACT

The neural transcription factor SOX11 is usually highly expressed in typical mantle cell lymphoma (MCL), but it is absent in the more indolent form of MCL. Despite being an important diagnostic marker for this hard-to-treat malignancy, the mechanisms of aberrant SOX11 expression are largely unknown. Herein, we describe 2 modes of SOX11 regulation by the cell-cycle regulator cyclin D1 (CCND1) and the signal transducer and activator of transcription 3 (STAT3). We found that ectopic expression of CCND1 in multiple human MCL cell lines resulted in increased SOX11 transcription, which correlated with increased acetylated histones H3K9 and H3K14 (H3K9/14Ac). Increased H3K9/14Ac and SOX11 expression was also observed after histone deacetylase 1 (HDAC1) or HDAC2 was depleted by RNA interference or inhibited by the HDAC inhibitor vorinostat. Mechanistically, we showed that CCND1 interacted with and sequestered HDAC1 and HDAC2 from the SOX11 locus, leading to SOX11 upregulation. Interestingly, our data revealed a potential inverse relationship between phosphorylated Y705 STAT3 and SOX11 expression in MCL cell lines, primary tumors, and patient-derived xenografts. Functionally, inactivation of STAT3 by inhibiting the upstream Janus kinase (JAK) 1 or JAK2 or by STAT3 knockdown was found to increase SOX11 expression, whereas interleukin-21 (IL-21)-induced STAT3 activation or overexpression of the constitutively active form of STAT3 decreased SOX11 expression. In addition, targeting SOX11 directly by RNA interference or indirectly by IL-21 treatment induced toxicity in SOX11+ MCL cells. Collectively, we demonstrate the involvement of CCND1 and STAT3 in the regulation of SOX11 expression, providing new insights and therapeutic implications in MCL.


Subject(s)
Cyclin D1/metabolism , Lymphoma, Mantle-Cell/genetics , SOXC Transcription Factors/genetics , STAT3 Transcription Factor/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Chromatin/metabolism , Gene Expression Regulation, Neoplastic , Genetic Loci , HEK293 Cells , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/metabolism , Histones/metabolism , Humans , Interleukins/pharmacology , Phosphotyrosine/metabolism , Protein Binding , Protein Processing, Post-Translational , SOXC Transcription Factors/metabolism , Up-Regulation/genetics
9.
Leuk Lymphoma ; 58(3): 676-688, 2017 03.
Article in English | MEDLINE | ID: mdl-27338091

ABSTRACT

Elevated cyclin D1 (CCND1) expression levels in mantle cell lymphoma (MCL) are associated with aggressive clinical manifestations related to chemoresistance, but little is known about how this important proto-oncogene contributes to the resistance of MCL. Here, we showed that RNA interference-mediated depletion of CCND1 increased caspase-3 activities and induced apoptosis in the human MCL lines UPN-1 and JEKO-1. In vitro and xenotransplant studies revealed that the toxic effect of CCND1 depletion in MCL cells was likely due to increase in histone H2AX phosphorylation, a DNA damage marker. DNA fiber analysis suggested deregulated replication initiation after CCND1 depletion as a potential cause of DNA damage. Finally, in contrast to depletion or inhibition of cyclin-dependent kinase 4, CCND1 depletion increased chemosensitivity of MCL cells to replication inhibitors hydroxyurea and cytarabine. Our findings have an important implication for CCND1 as a potential therapeutic target in MCL patients who are refractory to standard chemotherapy.


Subject(s)
Cyclin D1/metabolism , DNA Damage , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/metabolism , Animals , Apoptosis/genetics , Cell Cycle/genetics , Cell Line, Tumor , Cell Survival/genetics , DNA Replication , Disease Models, Animal , Heterografts , Humans , Lymphoma, Mantle-Cell/pathology , Mice , Proto-Oncogene Mas , RNA Interference , RNA, Small Interfering/genetics
10.
Oncotarget ; 7(45): 73558-73572, 2016 Nov 08.
Article in English | MEDLINE | ID: mdl-27713153

ABSTRACT

Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5-10 µM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL.


Subject(s)
Antineoplastic Agents/pharmacology , Cyclin D1/genetics , Drug Resistance, Neoplasm/genetics , Lymphoma, Mantle-Cell/genetics , Mutation , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Adenine/analogs & derivatives , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Cyclin D1/metabolism , Humans , Lymphoma, Mantle-Cell/metabolism , Phosphorylation , Piperidines , Protein Stability , Protein Transport , Proteolysis , Ubiquitination
11.
Cancer Cell ; 23(4): 435-49, 2013 Apr 15.
Article in English | MEDLINE | ID: mdl-23541952

ABSTRACT

We performed a loss-of-function RNA interference screen to define therapeutic targets in multiple myeloma, a genetically diverse plasma cell malignancy. Unexpectedly, we discovered that all myeloma lines require caspase-10 for survival irrespective of their genetic abnormalities. The transcription factor IRF4 induces both caspase-10 and its associated protein cFLIPL in myeloma, generating a protease that does not induce apoptosis but rather blocks an autophagy-dependent cell death pathway. Caspase-10 inhibits autophagy by cleaving the BCL2-interacting protein BCLAF1, itself a strong inducer of autophagy that acts by displacing beclin-1 from BCL2. While myeloma cells require a basal level of autophagy for survival, caspase-10 tempers this response to avoid cell death. Drugs that disrupt this vital balance may have therapeutic potential in myeloma.


Subject(s)
Autophagy/physiology , Caspase 10/genetics , Caspase 10/metabolism , Multiple Myeloma/enzymology , Multiple Myeloma/pathology , Apoptosis/drug effects , Apoptosis/physiology , Autophagy/drug effects , Caspase Inhibitors/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Gene Knockdown Techniques , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , RNA Interference
12.
Cancer Discov ; 3(5): 564-77, 2013 May.
Article in English | MEDLINE | ID: mdl-23471820

ABSTRACT

UNLABELLED: Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the Janus-activated kinase (JAK) tyrosine kinase family, TYK2, and its downstream effector STAT1, in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently showed TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway in T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of interleukin (IL)-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the antiapoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway. SIGNIFICANCE: In recent years, "pathway dependence" has been revealed in specific types of human cancer, which can be important because they pinpoint proteins that are particularly vulnerable to antitumor-targeted inhibition (so-called Achilles' heel proteins). Here, we use RNAi technology to identify a novel oncogenic pathway that involves aberrant activation of the TYK2 tyrosine kinase and its downstream substrate, STAT1, which ultimately promotes T-ALL cell survival through the upregulation of BCL2 expression


Subject(s)
Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , STAT1 Transcription Factor/metabolism , TYK2 Kinase/metabolism , Animals , Antineoplastic Agents/pharmacology , Bone Marrow Cells , Cell Line , Cell Survival/drug effects , Cells, Cultured , Humans , Interleukin-10/metabolism , Janus Kinase 3/antagonists & inhibitors , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Piperidines/pharmacology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , RNA Interference , STAT1 Transcription Factor/genetics , Signal Transduction , TYK2 Kinase/antagonists & inhibitors , TYK2 Kinase/genetics , Tyrphostins/pharmacology
13.
Methods Mol Biol ; 971: 245-63, 2013.
Article in English | MEDLINE | ID: mdl-23296968

ABSTRACT

RNA interference (RNAi) is a conserved posttranscriptional gene silencing mechanism that has recently emerged as a breakthrough genetic tool in functional genomics and drug target discovery. An increasing number of studies applying RNAi in high-throughput screens have begun to unravel complex signaling networks underlying diverse cellular processes. This chapter describes an approach to construct a conditional small-hairpin (sh)RNA library and its application in human lymphoma cell lines. A library cloning procedure outlines the incorporation of shRNA sequences and random 60-mer "bar code" oligonucleotides, enabling rapid identification of the hairpin by microarrays. Lymphoma cell lines are optimized for efficient retroviral transduction and tetracycline inducibility. The shRNA library is suitable for identifying molecular targets in cancer, but also versatile for various screening strategies.


Subject(s)
Gene Library , Genetic Techniques , Lymphoma/genetics , Lymphoma/pathology , RNA, Small Interfering/genetics , Cell Line, Tumor , Cloning, Molecular , Fluorescent Dyes/metabolism , HEK293 Cells , Humans , Nucleic Acid Hybridization/drug effects , Oligodeoxyribonucleotides/genetics , Oligonucleotide Array Sequence Analysis , Phenotype , RNA Interference , RNA Probes/genetics , RNA Probes/metabolism , Retroviridae/genetics , Tetracycline/pharmacology , Transduction, Genetic
14.
Cancer Res ; 72(22): 5889-99, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-23002205

ABSTRACT

We identified Bub1b as an essential element for the growth and survival of rhabdomyosarcoma (RMS) cells using a bar-coded, tetracycline-inducible short hairpin RNA (shRNA) library screen. Knockdown of Bub1b resulted in suppression of tumor growth in vivo, including the regression of established tumors. The mechanism by which this occurs is via postmitotic endoreduplication checkpoint and mitotic catastrophe. Furthermore, using a chromatin immunoprecipitation assay, we found that Bub1b is a direct transcriptional target of Forkhead Box M1 (FoxM1). Suppression of FoxM1 either by shRNA or the inhibitor siomycin A resulted in reduction of Bub1b expression and inhibition of cell growth and survival. These results show the important role of the Bub1b/FoxM1 pathway in RMS and provide potential therapeutic targets.


Subject(s)
Forkhead Transcription Factors/metabolism , Protein Serine-Threonine Kinases/metabolism , Rhabdomyosarcoma/metabolism , Animals , Cell Cycle Proteins , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Survival/physiology , DNA Barcoding, Taxonomic/methods , Female , Forkhead Box Protein M1 , Gene Knockdown Techniques , Humans , MCF-7 Cells , Mice , Mice, Nude , Mice, SCID , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/genetics , RNA, Small Interfering/genetics , Rh-Hr Blood-Group System/biosynthesis , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Signal Transduction , Transplantation, Heterologous
15.
Nature ; 470(7332): 115-9, 2011 Feb 03.
Article in English | MEDLINE | ID: mdl-21179087

ABSTRACT

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-ß. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.


Subject(s)
Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Mutation/genetics , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Oncogenes/genetics , Amino Acid Sequence , Amino Acid Substitution , Burkitt Lymphoma/genetics , Cell Line, Tumor , Cell Survival , Cytokines/metabolism , High-Throughput Nucleotide Sequencing , Humans , Hydrophobic and Hydrophilic Interactions , Interleukin-1 Receptor-Associated Kinases/biosynthesis , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-1 Receptor-Associated Kinases/metabolism , Janus Kinases/metabolism , Lymphoma, B-Cell, Marginal Zone/genetics , Lymphoma, Large B-Cell, Diffuse/classification , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Myeloid Differentiation Factor 88/chemistry , NF-kappa B/metabolism , Phosphorylation , Protein Structure, Tertiary , RNA Interference , Receptors, Interleukin-1/metabolism , STAT3 Transcription Factor/metabolism , Sequence Analysis, RNA , Signal Transduction , Toll-Like Receptors/metabolism
16.
Cancer Cell ; 18(6): 590-605, 2010 Dec 14.
Article in English | MEDLINE | ID: mdl-21156283

ABSTRACT

Chromosome band 9p24 is frequently amplified in primary mediastinal B cell lymphoma (PMBL) and Hodgkin lymphoma (HL). To identify oncogenes in this amplicon, we screened an RNA interference library targeting amplicon genes and thereby identified JAK2 and the histone demethylase JMJD2C as essential genes in these lymphomas. Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas by decreasing tyrosine 41 phosphorylation and increasing lysine 9 trimethylation of histone H3, promoting heterochromatin formation. MYC, a major target of JAK2-mediated histone phosphorylation, was silenced after JAK2 and JMJD2C inhibition, with a corresponding increase in repressive chromatin. Hence, JAK2 and JMJD2C cooperatively remodel the PMBL and HL epigenome, offering a mechanistic rationale for the development of JAK2 and JMJD2C inhibitors in these diseases.


Subject(s)
Epigenesis, Genetic , Hodgkin Disease/genetics , Lymphoma, B-Cell/genetics , Mediastinal Neoplasms/genetics , Cell Line, Tumor , Chromosomes, Human, Pair 9 , Histones/metabolism , Hodgkin Disease/pathology , Humans , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/genetics , Janus Kinase 2/physiology , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/physiology , Lymphoma, B-Cell/pathology , Phosphorylation
17.
Nature ; 463(7277): 88-92, 2010 Jan 07.
Article in English | MEDLINE | ID: mdl-20054396

ABSTRACT

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.


Subject(s)
B-Lymphocytes/metabolism , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Agammaglobulinaemia Tyrosine Kinase , Amino Acid Motifs , B-Lymphocytes/pathology , CARD Signaling Adaptor Proteins/genetics , CARD Signaling Adaptor Proteins/metabolism , CD79 Antigens/chemistry , CD79 Antigens/genetics , CD79 Antigens/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cell Survival , Guanylate Cyclase/genetics , Guanylate Cyclase/metabolism , Humans , Lymphoma, Large B-Cell, Diffuse/genetics , Mutation , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , RNA Interference , Receptors, Antigen, B-Cell/deficiency , Receptors, Antigen, B-Cell/genetics , src-Family Kinases/metabolism
18.
Nature ; 458(7234): 92-6, 2009 Mar 05.
Article in English | MEDLINE | ID: mdl-19118383

ABSTRACT

The transcription factor NF-kappaB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-kappaB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-kappaB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1alpha (CK1alpha) as a bifunctional regulator of NF-kappaB. CK1alpha dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1alpha kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1alpha has thus a dual 'gating' function which first promotes and then terminates receptor-induced NF-kappaB. ABC DLBCL cells required CK1alpha for constitutive NF-kappaB activity, indicating that CK1alpha functions as a conditionally essential malignancy gene-a member of a new class of potential cancer therapeutic targets.


Subject(s)
Casein Kinases/metabolism , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , NF-kappa B/metabolism , Receptors, Antigen/metabolism , Adaptor Proteins, Signal Transducing/metabolism , B-Cell CLL-Lymphoma 10 Protein , CARD Signaling Adaptor Proteins/metabolism , Caspases/metabolism , Cell Proliferation , Cell Survival , Cells, Cultured , Feedback, Physiological , Guanylate Cyclase/metabolism , Humans , I-kappa B Kinase/metabolism , Jurkat Cells , Lymphoma, Large B-Cell, Diffuse/enzymology , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Neoplasm Proteins/metabolism , Protein Binding , Signal Transduction
19.
Proc Natl Acad Sci U S A ; 105(52): 20798-803, 2008 Dec 30.
Article in English | MEDLINE | ID: mdl-19104039

ABSTRACT

A subtype of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like (ABC) DLBCL, depends on constitutive nuclear factor-kappaB (NF-kappaB) signaling for survival. Small molecule inhibitors of IkappaB kinase beta (IKKbeta), a key regulator of the NF-kappaB pathway, kill ABC DLBCL cells and hold promise for the treatment of this lymphoma type. We conducted an RNA interference genetic screen to investigate potential mechanisms of resistance of ABC DLBCL cells to IKKbeta inhibitors. We screened a library of small hairpin RNAs (shRNAs) targeting 500 protein kinases for shRNAs that would increase the killing of an ABC DLBCL cell line in the presence of a small molecule IKKbeta inhibitor. Two independent shRNAs targeting IKKalpha synergized with the IKKbeta inhibitor to kill three different ABC DLBCL cell lines but were not toxic by themselves. Surprisingly, IKKalpha shRNAs blocked the classical rather than the alternative NF-kappaB pathway in ABC DLBCL cells, as judged by inhibition of IkappaBalpha phosphorylation. IKKalpha shRNA toxicity was reversed by coexpression of wild-type but not kinase inactive forms of IKKalpha, suggesting that IKKalpha may directly phosphorylate IkappaBalpha under conditions of IKKbeta inhibition. In models of physiologic NF-kappaB pathway activation by CARD11 or tumor necrosis factor-alpha, compensatory IKKalpha activity was also observed with IKKbeta inhibition. These results suggest that therapy for ABC DLBCL may be improved by targeting both IKKalpha and IKKbeta, possibly through CARD11 inhibition.


Subject(s)
I-kappa B Kinase/antagonists & inhibitors , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/enzymology , NF-kappa B/metabolism , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , CARD Signaling Adaptor Proteins/metabolism , Drug Delivery Systems/methods , Drug Screening Assays, Antitumor/methods , Guanylate Cyclase/metabolism , Humans , I-kappa B Kinase/metabolism , Jurkat Cells , Phosphorylation/drug effects , Protein Kinase Inhibitors/therapeutic use , RNA Interference , Tumor Necrosis Factor-alpha/metabolism
20.
Nature ; 454(7201): 226-31, 2008 Jul 10.
Article in English | MEDLINE | ID: mdl-18568025

ABSTRACT

The transcription factor IRF4 (interferon regulatory factor 4) is required during an immune response for lymphocyte activation and the generation of immunoglobulin-secreting plasma cells. Multiple myeloma, a malignancy of plasma cells, has a complex molecular aetiology with several subgroups defined by gene expression profiling and recurrent chromosomal translocations. Moreover, the malignant clone can sustain multiple oncogenic lesions, accumulating genetic damage as the disease progresses. Current therapies for myeloma can extend survival but are not curative. Hence, new therapeutic strategies are needed that target molecular pathways shared by all subtypes of myeloma. Here we show, using a loss-of-function, RNA-interference-based genetic screen, that IRF4 inhibition is toxic to myeloma cell lines, regardless of transforming oncogenic mechanism. Gene expression profiling and genome-wide chromatin immunoprecipitation analysis uncovered an extensive network of IRF4 target genes and identified MYC as a direct target of IRF4 in activated B cells and myeloma. Unexpectedly, IRF4 was itself a direct target of MYC transactivation, generating an autoregulatory circuit in myeloma cells. Although IRF4 is not genetically altered in most myelomas, they are nonetheless addicted to an aberrant IRF4 regulatory network that fuses the gene expression programmes of normal plasma cells and activated B cells.


Subject(s)
Interferon Regulatory Factors/metabolism , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Animals , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Cell Survival , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Chromatin Immunoprecipitation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genes, myc/genetics , Humans , Interferon Regulatory Factors/deficiency , Interferon Regulatory Factors/genetics , Mice , Multiple Myeloma/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , Transcriptional Activation
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