RESUMO
Burkitt lymphoma (BL) represents the most aggressive B-cell-lymphoma. Beside the hallmark of IG-MYC-translocation, surface B-cell receptor (BCR) is expressed, and mutations in the BCR pathway are frequent. Coincidental infections in endemic BL, and specific extra-nodal sites suggest antigenic triggers. To explore this hypothesis, BCRs of BL cell lines and cases were screened for reactivities against a panel of bacterial lysates, lysates of Plasmodium falciparum, a custom-made virome array and against self-antigens, including post-translationally modified antigens. An atypically modified, SUMOylated isoform of Bystin, that is, SUMO1-BYSL was identified as the antigen of the BCR of cell line CA46. SUMO1-BYSL was exclusively expressed in CA46 cells with K139 as site of the SUMOylation. Secondly, an atypically acetylated isoform of HSP40 was identified as the antigen of the BCR of cell line BL41. K104 and K179 were the sites of immunogenic acetylation, and the acetylated HSP40 isoform was solely present in BL41 cells. Functionally, addition of SUMO1-BYSL and acetylated HSP40 induced BCR pathway activation in CA46 and BL41 cells, respectively. Accordingly, SUMO1-BYSL-ETA' immunotoxin, produced by a two-step intein-based conjugation, led to the specific killing of CA46 cells. Autoantibodies directed against SUMO1-BYSL were found in 3 of 14 (21.4%), and autoantibodies against acetylated HSP40 in 1/14(7.1%) patients with sporadic Burkitt-lymphoma. No reactivities against antigens of the infectious agent spectrum could be observed. These results indicate a pathogenic role of autoreactivity evoked by immunogenic post-translational modifications in a subgroup of sporadic BL including two EBV-negative BL cell lines.
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Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors.
Assuntos
Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma de Efusão Primária/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imidazóis/farmacologia , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/genética , Linfoma de Efusão Primária/tratamento farmacológico , Linfoma de Efusão Primária/genética , Compostos Macrocíclicos/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinolinas/farmacologiaRESUMO
For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors (CDKN2A, CDKN2B), expression of fusion genes (ETV6-RUNX1, BCR-ABL1, MEF2D-BCL9) or of genes targeted by point mutations (KRAS A146T, NRAS G12C, PAX5 R38H). MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.
Assuntos
Biomarcadores Tumorais , Linhagem Celular Tumoral , Predisposição Genética para Doença , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Alelos , Aberrações Cromossômicas , Análise Mutacional de DNA , Progressão da Doença , Humanos , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , RNA-Seq , Sequenciamento do ExomaRESUMO
Recently, we documented a hematopoietic NKL-code mapping physiological expression patterns of NKL homeobox genes in human myelopoiesis including monocytes and their derived dendritic cells (DCs). Here, we enlarge this map to include normal NKL homeobox gene expressions in progenitor-derived DCs. Analysis of public gene expression profiling and RNA-seq datasets containing plasmacytoid and conventional dendritic cells (pDC and cDC) demonstrated HHEX activity in both entities while cDCs additionally expressed VENTX. The consequent aim of our study was to examine regulation and function of VENTX in DCs. We compared profiling data of VENTX-positive cDC and monocytes with VENTX-negative pDC and common myeloid progenitor entities and revealed several differentially expressed genes encoding transcription factors and pathway components, representing potential VENTX regulators. Screening of RNA-seq data for 100 leukemia/lymphoma cell lines identified prominent VENTX expression in an acute myelomonocytic leukemia cell line, MUTZ-3 containing inv(3)(q21q26) and t(12;22)(p13;q11) and representing a model for DC differentiation studies. Furthermore, extended gene analyses indicated that MUTZ-3 is associated with the subtype cDC2. In addition to analysis of public chromatin immune-precipitation data, subsequent knockdown experiments and modulations of signaling pathways in MUTZ-3 and control cell lines confirmed identified candidate transcription factors CEBPB, ETV6, EVI1, GATA2, IRF2, MN1, SPIB, and SPI1 and the CSF-, NOTCH-, and TNFa-pathways as VENTX regulators. Live-cell imaging analyses of MUTZ-3 cells treated for VENTX knockdown excluded impacts on apoptosis or induced alteration of differentiation-associated cell morphology. In contrast, target gene analysis performed by expression profiling of knockdown-treated MUTZ-3 cells revealed VENTX-mediated activation of several cDC-specific genes including CSFR1, EGR2, and MIR10A and inhibition of pDC-specific genes like RUNX2. Taken together, we added NKL homeobox gene activities for progenitor-derived DCs to the NKL-code, showing that VENTX is expressed in cDCs but not in pDCs and forms part of a cDC-specific gene regulatory network operating in DC differentiation and function.
Assuntos
Células Dendríticas/metabolismo , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Linhagem Celular Tumoral , Biologia Computacional/métodos , Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Genes Homeobox , Humanos , Imunofenotipagem , TranscriptomaRESUMO
Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm that is genetically characterized by the absence of both the Philadelphia chromosome and BCR-ABL1 fusion gene and the high prevalence of mutations in the colony-stimulating factor 3 receptor (CSF3R). Additional disease-modifying mutations have been recognized in CNL samples, portraying a distinct mutational landscape. Despite the growing knowledge base on genomic aberrations, further progress could be gained from the availability of representative models of CNL. To address this gap, we screened a large panel of available leukemia cell lines, followed by a detailed mutational investigation with focus on the CNL-associated candidate driver genes. The sister cell lines CNLBC-1 and MOLM-20 were derived from a patient with CNL and carry CNL-typical molecular hallmarks, namely mutations in several genes, such as CSF3R, ASXL1, EZH2, NRAS, and SETBP1. The use of these validated and comprehensively characterized models will benefit the understanding of the pathobiology of CNL and help inform therapeutic strategies.
Assuntos
Leucemia Neutrofílica Crônica , Leucemia , Linhagem Celular , Humanos , Leucemia Neutrofílica Crônica/genética , Mutação , Receptores de Fator Estimulador de Colônias/genéticaRESUMO
Natural killer T (NKT)-cells with both T- and NK-cell antigens can be classified into αß or γδ type according to the TCR gene expression. The WHO classification of lymphoid neoplasms did not further subdivide the above-mentioned NKT-cell malignancies according to the expression of these TCR types. γδ T-cells can be stimulated and expanded by Zoledronic acid, usually carrying Vγ9 Vδ2 TCR and various NK-associated receptors (NKR) such as CD56, CD94, CD158a, CD158b, CD161, etc. In contrast, αß T-type NKT-cells are positive for Vα24 Vß11 TCR. NKR positive γδ T-cells have clearly different features than the NKT-cells with Vα24 Vß11 TCR type, αß NKT. NKT-cells carrying γδ TCR should be classified and named as γδ NKT-cells to distinguish the cells explicitly from αß NKT-cells.
Assuntos
Linfócitos Intraepiteliais , Células T Matadoras Naturais , Receptores de Antígenos de Linfócitos T gama-delta , Antígenos CD , Expressão Gênica , Humanos , Linfócitos Intraepiteliais/classificação , Linfócitos Intraepiteliais/imunologia , Células T Matadoras Naturais/classificação , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T gama-delta/classificação , Receptores de Antígenos de Linfócitos T gama-delta/imunologiaRESUMO
Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation and are grouped into classes and subclasses according to sequence similarities. Here, we analyzed the activities of the 20 members strong TALE homeobox gene class in early hematopoiesis and in lymphopoiesis including developing and mature B-cells, T-cells, natural killer (NK)-cells and innate lymphoid cells (ILC). The resultant expression pattern comprised eleven genes and which we termed TALE-code enables discrimination of normal and aberrant activities of TALE homeobox genes in lymphoid malignancies. Subsequent expression analysis of TALE homeobox genes in public datasets of Hodgkin lymphoma (HL) patients revealed overexpression of IRX3, IRX4, MEIS1, MEIS3, PBX1, PBX4 and TGIF1. As paradigm we focused on PBX1 which was deregulated in about 17% HL patients. Normal PBX1 expression was restricted to hematopoietic stem cells and progenitors of T-cells and ILCs but absent in B-cells, reflecting its roles in stemness and early differentiation. HL cell line SUP-HD1 expressed enhanced PBX1 levels and served as an in vitro model to identify upstream regulators and downstream targets in this malignancy. Genomic studies of this cell line therein showed a gain of the PBX1 locus at 1q23 which may underlie its aberrant expression. Comparative expression profiling analyses of HL patients and cell lines followed by knockdown experiments revealed NFIB and TLX2 as target genes activated by PBX1. HOX proteins operate as cofactors of PBX1. Accordingly, our data showed that HOXB9 overexpressed in HL coactivated TLX2 but not NFIB while activating TNFRSF9 without PBX1. Further downstream analyses showed that TLX2 activated TBX15 which operated anti-apoptotically. Taken together, we discovered a lymphoid TALE-code and identified an aberrant network around deregulated TALE homeobox gene PBX1 which may disturb B-cell differentiation in HL by reactivation of progenitor-specific genes. These findings may provide the framework for future studies to exploit possible vulnerabilities of malignant cells in therapeutic scenarios.
Assuntos
Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/genética , Doença de Hodgkin/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Linfócitos B/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genes Homeobox , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Linfócitos/metabolismo , Proteína Meis1/genética , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Leukemic stem cells (LSCs) comprise a very rare cell population that results in the development of acute myeloid leukemia. The selective targeting of drivers in LSCs with small molecule inhibitors holds promise for treatment of acute myeloid leukemia. Recently, we reported the identification of inhibitors of the histone lysine demethylase JMJD1C that preferentially kill MLL rearranged acute leukemia cells. Here, we report the identification of jumonji domain modulator #7 (JDM-7). Surface plasmon resonance analysis showed that JDM-7 binds to JMJD1C and its family homolog JMJD1B. JDM-7 did not significantly suppress cell proliferation in liquid cell culture at higher doses, although it led to a significant decrease in semi-solid colony formation experiments at lower concentrations. Moreover, low doses of JDM-7 did not suppress the proliferation of erythroid progenitor cells. We identified that JDM-7 downregulates the LSC self-renewal gene HOXA9 in leukemia cells. We further found that the structure of JDM-7 is similar to that of tadalafil, a drug approved by the US Food and Drug Administration. Molecular docking and surface plasmon resonance analysis showed that tadalafil binds to JMJD1C. Moreover, similar to JDM-7, tadalafil suppressed colony formation of leukemia cells in semi-solid cell culture at a concentration that did not affect primary umbilical cord blood cells. In summary, we have identified JDM-7 and tadalafil as potential JMJD1C modulators that selectively inhibit the growth of LSCs.
Assuntos
Antineoplásicos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Cultura Primária de Células , Tadalafila/farmacologia , Tadalafila/uso terapêuticoRESUMO
The NKL-code describes normal expression patterns of NKL homeobox genes in hematopoiesis. Aberrant expression of NKL homeobox gene subclass members have been reported in several hematopoietic malignancies including acute myeloid leukemia (AML). Here, we analyzed the oncogenic role of the HMX-group of NKL homeobox genes in AML. Public expression profiling data-available for HMX1 and HMX2-indicate aberrant activity of HMX2 in circa 2% AML patients overall, rising to 31% in those with KMT2A/MLL rearrangements whereas HMX1 expression remains inconspicuous. AML cell lines EOL-1, MV4-11 and MOLM-13 expressed both, HMX2 and neighboring HMX3 genes, and harbored KMT2A aberrations, suggesting their potential functional association. Surprisingly, knockdown experiments in these cell lines demonstrated that KMT2A inhibited HMX2/3 which, in turn, did not regulate KMT2A expression. Furthermore, karyotyping and genomic profiling analysis excluded rearrangements of the HMX2/3 locus in these cell lines. However, comparative expression profiling and subsequent functional analyses revealed that IRF8, IL7- and WNT-signalling activated HMX2/3 expression while TNFa/NFkB- signalling proved inhibitory. Whole genome sequencing of EOL-1 identified two mutations in the regulatory upstream regions of HMX2/3 resulting in generation of a consensus ETS-site and transformation of a former NFkB-site into an SP1-site. Reporter-gene assays demonstrated that both mutations contributed to HMX2/3 activation, modifying ETS1/ELK1- and TNFalpha-mediated gene regulation. Moreover, DMSO-induced eosinophilic differentiation of EOL-1 cells coincided with HMX2/3 downregulation while knockdown of HMX2 induced cell differentiation, collectively supporting a fundamental role for these genes in myeloid differentiation arrest. Finally, target genes of HMX2/3 were identified in EOL-1 and included suppression of differentiation gene EPX, and activation of fusion gene FIP1L1-PDGFRA and receptor-encoding gene HTR7, both of which enhanced oncogenic ERK-signalling. Taken together, our study documents a leukemic role for deregulated NKL homeobox genes HMX2 and HMX3 in AML, revealing molecular mechanisms of myeloid differentiation arrest.
Assuntos
Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Leucemia Mieloide Aguda/genética , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/patologia , TranscriptomaRESUMO
NKL homeobox genes encode developmental transcription factors and display an NKL-code according to their physiological expression pattern in hematopoiesis. Here, we analyzed public transcriptome data from primary innate lymphoid cells (ILCs) for NKL homeobox gene activities and found that ILC3 expressed exclusively HHEX while in ILC1 and ILC2 these genes were silenced. Deregulation of the NKL-code promotes hematopoietic malignancies, including anaplastic large cell lymphoma (ALCL) which reportedly may derive from ILC3. Accordingly, we analyzed NKL homeobox gene activities in ALCL cell lines and investigated their role in this malignancy. Transcriptome analyses demonstrated low expression levels of HHEX but powerfully activated HLX. Forced expression of HHEX in ALCL cell lines induced genes involved in apoptosis and ILC3 differentiation, indicating tumor suppressor activity. ALCL associated NPM1-ALK and JAK-STAT3-signalling drove enhanced expression of HLX while discounting HHEX. Genomic profiling revealed copy number gains at the loci of HLX and STAT3 in addition to genes encoding both STAT3 regulators (AURKA, BCL3, JAK3, KPNB1, NAMPT, NFAT5, PIM3, ROCK1, SIX1, TPX2, WWOX) and targets (BATF3, IRF4, miR135b, miR21, RORC). Transcriptome data of ALCL cell lines showed absence of STAT3 mutations while MGA was mutated and downregulated, encoding a novel potential STAT3 repressor. Furthermore, enhanced IL17F-signalling activated HLX while TGFbeta-signalling inhibited HHEX expression. Taken together, our data extend the scope of the NKL-code for ILCs and spotlight aberrant expression of NKL homeobox gene HLX in ALCL. HLX represents a direct target of ALCL hallmark factor STAT3 and deregulates cell survival and differentiation in this malignancy.
RESUMO
Certified cell line models provide ideal experimental platforms to answer countless scientific questions. The LL-100 panel is a cohort of cell lines that are broadly representative of all leukemia-lymphoma entities (including multiple myeloma and related diseases), rigorously authenticated and validated, and comprehensively annotated. The process of the assembly of the LL-100 panel was based on evidence and experience. To expand the genetic characterization across all LL-100 cell lines, we performed whole-exome sequencing and RNA sequencing. Here, we describe the conception of the panel and showcase some exemplary applications with a focus on cancer genomics. Due diligence was paid to exclude cross-contaminated and non-representative cell lines. As the LL-100 cell lines are so well characterized and readily available, the panel will be a valuable resource for identifying cell lines with mutations in cancer genes, providing superior model systems. The data also add to the current knowledge of the molecular pathogenesis of leukemia-lymphoma. Additional efforts to expand the breadth of available high-quality cell lines are clearly warranted.
Assuntos
Pesquisa Biomédica , Leucemia/patologia , Linfoma/patologia , Linhagem Celular Tumoral , Evolução Clonal/genética , Humanos , Leucemia/genética , Linfoma/genética , Mutação/genéticaRESUMO
EZH2 gain of function mutations (EZH2GOFmu) has been implicated in the pathogenesis of B-non-Hodgkin lymphoma. The EZH2-specific inhibitor GSK126 inhibits trimethylation of histone H3K27 and induces target gene expression. However, in 3/4 EZH2GOFmu B-NHL lymphoma cell lines, GSK126 (400 nM) did not induce growth arrest. Only at high doses (10 µM), the inhibitor was effective as antiproliferative agent, comparably in EZH2GOFmu, wild-type, and EZH2-negative cell lines, suggesting that at high concentrations, the antiproliferative effects of GSK126 are off-target effects. In sum, we could not confirm that B-NHL cell lines with EZH2GOFmu show a higher sensitivity to GSK126 than EZH2 wild-type cell lines do. Only 1/4 EZH2GOFmu B-NHL cell lines tested (PFEIFFER) were sensitive to GSK126 (400 nM) inducing growth arrest. If these results can be translated to patients, they raise the question of whether the presence of EZH2 activating mutations alone allows selection for targeted therapy with EZH2 inhibitors.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Complexo Repressor Polycomb 2 , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Humanos , Metilação , Mutação , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismoRESUMO
Patients suffering from chronic lymphocytic leukemia (CLL) display highly diverse clinical courses ranging from indolent cases to aggressive disease, with genetic and epigenetic features resembling this diversity. Here, we developed a comprehensive approach combining a variety of molecular and clinical data to pinpoint translocation events disrupting long-range chromatin interactions and causing cancer-relevant transcriptional deregulation. Thereby, we discovered a B cell specific cis-regulatory element restricting the expression of genes in the associated locus, including PRMT5 and DAD1, two factors with oncogenic potential. Experimental PRMT5 inhibition identified transcriptional programs similar to those in patients with differences in PRMT5 abundance, especially MYC-driven and stress response pathways. In turn, such inhibition impairs factors involved in DNA repair, sensitizing cells for apoptosis. Moreover, we show that artificial deletion of the regulatory element from its endogenous context resulted in upregulation of corresponding genes, including PRMT5. Furthermore, such disruption renders PRMT5 transcription vulnerable to additional stimuli and subsequently alters the expression of downstream PRMT5 targets. These studies provide a mechanism of PRMT5 deregulation in CLL and the molecular dependencies identified might have therapeutic implementations.
Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Estresse Fisiológico , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores Tumorais/genética , Epigenômica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Células Tumorais CultivadasRESUMO
Histone demethylases are promising therapeutic targets as they play fundamental roles for survival of Mixed lineage leukemia rearranged acute leukemia (MLLr AL). Here we focused on the catalytic Jumonji domain of histone H3 lysine 9 (H3K9) demethylase JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. JMJD1C Jumonji domain inhibitor 4 (JDI-4) and JDI-12 that share a common structural backbone were detected within the top 15 compounds. Surface plasmon resonance analysis showed that JDI-4 and JDI-12 bind to JMJD1C and its family homolog KDM3B with modest affinity. In vitro demethylation assays showed that JDI-4 can reverse the H3K9 demethylation conferred by KDM3B. In vivo demethylation assays indicated that JDI-4 and JDI-12 could induce the global increase of H3K9 methylation. Cell proliferation and colony formation assays documented that JDI-4 and JDI-12 kill MLLr AL and other malignant hematopoietic cells, but not leukemia cells resistant to JMJD1C depletion or cord blood cells. Furthermore, JDI-16, among multiple compounds structurally akin to JDI-4/JDI-12, exhibits superior killing activities against malignant hematopoietic cells compared to JDI-4/JDI-12. Mechanistically, JDI-16 not only induces apoptosis but also differentiation of MLLr AL cells. RNA sequencing and quantitative PCR showed that JDI-16 induced gene expression associated with cell metabolism; targeted metabolomics revealed that JDI-16 downregulates lactic acids, NADP+ and other metabolites. Moreover, JDI-16 collaborates with all-trans retinoic acid to repress MLLr AML cells. In summary, we identified bona fide JMJD1C inhibitors that induce preferential death of MLLr AL cells.
Assuntos
Antineoplásicos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Leucemia Aguda Bifenotípica/tratamento farmacológico , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Adulto , Idoso , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Medula Óssea/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desmetilação do DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia Aguda Bifenotípica/patologia , Masculino , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Tretinoína/farmacologia , Tretinoína/uso terapêuticoAssuntos
DNA (Citosina-5-)-Metiltransferases/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação de Sentido Incorreto , Substituição de Aminoácidos/genética , Arginina/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , DNA Metiltransferase 3A , Análise Mutacional de DNA , Histidina/genética , HumanosRESUMO
Recently, we have documented a hematopoietic NKL-code mapping physiological expression patterns of NKL homeobox genes in early hematopoiesis and in lymphopoiesis, which spotlights genes deregulated in lymphoid malignancies. Here, we enlarge this map to include normal NKL homeobox gene expressions in myelopoiesis by analyzing public expression profiling data and primary samples from developing and mature myeloid cells. We thus uncovered differential activities of six NKL homeobox genes, namely DLX2, HHEX, HLX, HMX1, NKX3-1 and VENTX. We further examined public expression profiling data of 251 acute myeloid leukemia (AML) and 183 myelodysplastic syndrome (MDS) patients, thereby identifying 24 deregulated genes. These results revealed frequent deregulation of NKL homeobox genes in myeloid malignancies. For detailed analysis we focused on NKL homeobox gene NANOG, which acts as a stem cell factor and is correspondingly expressed alone in hematopoietic progenitor cells. We detected aberrant expression of NANOG in a small subset of AML patients and in AML cell line NOMO-1, which served as a model. Karyotyping and genomic profiling discounted rearrangements of the NANOG locus at 12p13. But gene expression analyses of AML patients and AML cell lines after knockdown and overexpression of NANOG revealed regulators and target genes. Accordingly, NKL homeobox genes HHEX, DLX5 and DLX6, stem cell factors STAT3 and TET2, and the NOTCH-pathway were located upstream of NANOG while NKL homeobox genes HLX and VENTX, transcription factors KLF4 and MYB, and anti-apoptosis-factor MIR17HG represented target genes. In conclusion, we have extended the NKL-code to the myeloid lineage and thus identified several NKL homeobox genes deregulated in AML and MDS. These data indicate a common oncogenic role of NKL homeobox genes in both lymphoid and myeloid malignancies. For misexpressed NANOG we identified an aberrant regulatory network, which contributes to the understanding of the oncogenic activity of NKL homeobox genes.
Assuntos
Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide/genética , Síndromes Mielodisplásicas/genética , Células Mieloides/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Regulação da Expressão Gênica , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Humanos , Cariótipo , Fator 4 Semelhante a Kruppel , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Células Mieloides/citologia , Proteína Homeobox Nanog/antagonistas & inibidores , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Recently, we have described physiological expression patterns of NKL homeobox genes in early hematopoiesis and in subsequent lymphopoiesis. We identified nine genes which constitute the so-called NKL-code. Aberrant overexpression of code-members or ectopically activated non-code NKL homeobox genes are described in T-cell leukemia and in T- and B-cell lymphoma, highlighting their oncogenic role in lymphoid malignancies. Here, we introduce the NKL-code in normal hematopoiesis and focus on deregulated NKL homeobox genes in B-cell lymphoma, including HLX, MSX1 and NKX2-2 in Hodgkin lymphoma; HLX, NKX2-1 and NKX6-3 in diffuse large B-cell lymphoma; and NKX2-3 in splenic marginal zone lymphoma. Thus, the roles of various members of the NKL homeobox gene subclass are considered in normal and pathological hematopoiesis in detail.
RESUMO
For many years, immortalized cell lines have been used as model systems for cancer research. Cell line panels were established for basic research and drug development, but did not cover the full spectrum of leukemia and lymphoma. Therefore, we now developed a novel panel (LL-100), 100 cell lines covering 22 entities of human leukemia and lymphoma including T-cell, B-cell and myeloid malignancies. Importantly, all cell lines are unequivocally authenticated and assigned to the correct tissue. Cell line samples were proven to be free of mycoplasma and non-inherent virus contamination. Whole exome sequencing and RNA-sequencing of the 100 cell lines were conducted with a uniform methodology to complement existing data on these publicly available cell lines. We show that such comprehensive sequencing data can be used to find lymphoma-subtype-characteristic copy number aberrations, mRNA isoforms, transcription factor activities and expression patterns of NKL homeobox genes. These exemplary studies confirm that the novel LL-100 panel will be useful for understanding the function of oncogenes and tumor suppressor genes and to develop targeted therapies.
Assuntos
Leucemia/patologia , Linfoma/patologia , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA/genética , Exoma/genética , Éxons/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia/genética , Linfoma/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Recently, we have presented a scheme, termed "NKL-code", which describes physiological expression patterns of NKL homeobox genes in early hematopoiesis and in lymphopoiesis including main stages of T-, B- and NK-cell development. Aberrant activity of these genes underlies the generation of hematological malignancies notably T-cell leukemia. Here, we searched for deregulated NKL homeobox genes in main entities of T-cell lymphomas comprising angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL), hepatosplenic T-cell lymphoma (HSTL), NK/T-cell lymphoma (NKTL) and peripheral T-cell lymphoma (PTCL). Our data revealed altogether 19 aberrantly overexpressed genes in these types, demonstrating deregulated NKL homeobox genes involvement in T-cell lymphomas as well. For detailed analysis we focused on NKL homeobox gene MSX1 which is normally expressed in NK-cells. MSX1 was overexpressed in subsets of HSTL patients and HSTL-derived sister cell lines DERL-2 and DERL-7 which served as models to characterize mechanisms of deregulation. We performed karyotyping, genomic and expression profiling, and whole genome sequencing to reveal mutated and deregulated gene candidates, including the fusion gene CD53-PDGFRB. Subsequent knockdown experiments allowed the reconstruction of an aberrant network involved in MSX1 deregulation, including chromatin factors AUTS2 and mutated histone HIST1H3B(K27M). The gene encoding AUTS2 is located at chromosome 7q11 and may represent a basic target of the HSTL hallmark aberration i(7q). Taken together, our findings highlight an oncogenic role for deregulated NKL homeobox genes in T-cell lymphoma and identify MSX1 as a novel player in HSTL, implicated in aberrant NK- and T-cell differentiation.