ABSTRACT
BACKGROUND: Inhibition of kinases is the ever-expanding therapeutic approach to various types of cancer. Typically, assessment of the treatment response is accomplished by standard, volumetric imaging procedures, performed weeks to months after the onset of treatment, given the predominantly cytostatic nature of the kinase inhibitors, at least when used as single agents. Therefore, there is a great clinical need to develop new monitoring approaches to detect the response to kinase inhibition much more promptly. Noninvasive 1H magnetic resonance spectroscopy (MRS) can measure in vitro and in vivo concentration of key metabolites which may potentially serve as biomarkers of response to kinase inhibition. METHODS: We employed mantle cell lymphoma (MCL) cell lines demonstrating markedly diverse sensitivity of inhibition of Bruton's tyrosine kinase (BTK) regarding their growth and studied in-depth effects of the inhibition on various aspects of cell metabolism including metabolite synthesis using metabolomics, glucose and oxidative metabolism by Seahorse XF technology, and concentration of index metabolites lactate, alanine, total choline and taurine by 1H MRS. RESULTS: Effective BTK inhibition profoundly suppressed key cell metabolic pathways, foremost pyrimidine and purine synthesis, the citrate (TCA) cycle, glycolysis, and pyruvate and glutamine/alanine metabolism. It also inhibited glycolysis and amino acid-related oxidative metabolism. Finally, it profoundly and quickly decreased concentration of lactate (a product of mainly glycolysis) and alanine (an indicator of amino acid metabolism) and, less universally total choline both in vitro and in vivo, in the MCL xenotransplant model. The decrease correlated directly with the degree of inhibition of lymphoma cell expansion and tumor growth. CONCLUSIONS: Our results indicate that BTK inhibition exerts a broad and profound suppressive effect on cell metabolism and that the affected index metabolites such as lactate, alanine may serve as early, sensitive, and reliable biomarkers of inhibition in lymphoma patients detectable by noninvasive MRS-based imaging method. This kind of imaging-based detection may also be applicable to other kinase inhibitors, as well as diverse lymphoid and non-lymphoid malignancies.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase , Lymphoma, Mantle-Cell , Protein Kinase Inhibitors , Humans , Cell Line, Tumor , Protein Kinase Inhibitors/pharmacology , Animals , Agammaglobulinaemia Tyrosine Kinase/metabolism , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/pathology , Lymphoma, Mantle-Cell/drug therapy , Signal Transduction/drug effects , Biomarkers, Tumor/metabolism , Cell Proliferation/drug effects , Xenograft Model Antitumor Assays , Mice , Biomarkers/metabolismABSTRACT
This study reports that hairy and enhancer of split homolog-1 (HES1), known to repress gene transcription in progenitor cells of several cell lineages, was strongly expressed in cells and tissues of T-cell lymphoma expressing the oncogenic chimeric tyrosine kinase nucleophosmin (NPM)-anaplastic lymphoma kinase [ALK; ALK+ T-cell lymphoma (TCL)]. The structural analysis of the Orange domain of HES1 indicated that HES1 formed a highly stable homodimer. Of note, repression of HES1 expression led to inhibition of ALK+ TCL cell growth in vivo. The expression of the HES1 gene was induced by NPM-ALK through activation of STAT3, which bound to the gene's promoter and induced the gene's transcription. NPM-ALK also directly phosphorylated HES1 protein. In turn, HES1 up-regulated and down-regulated in ALK+ TCL cells, the expression of numerous genes, protein products of which are involved in key cell functions, such as cell proliferation and viability. Among the genes inhibited by HES1 was thioredoxin-interacting protein (TXNIP), encoding a protein implicated in promotion of cell death in various types of cells. Accordingly, ALK+ TCL cells and tissues lacked expression of TXNIP, and its transcription was co-inhibited by HES1 and STAT3 in an NPM-ALK-dependent manner. Finally, the induced expression of TXNIP induced massive apoptotic cell death of ALK+ TCL cells. The results reveal a novel NPM-ALK-controlled pro-oncogenic regulatory network and document an important role of HES and TXNIP in the NPM-ALK-driven oncogenesis, with the former protein displaying oncogenic and the latter tumor suppressor properties.
Subject(s)
Anaplastic Lymphoma Kinase , Carrier Proteins , Lymphoma, T-Cell , Transcription Factor HES-1 , Anaplastic Lymphoma Kinase/genetics , Carcinogenesis/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Humans , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/metabolism , Lymphoma, T-Cell/pathology , Oncogenes , Phosphorylation , Transcription Factor HES-1/genetics , Transcription Factor HES-1/metabolismABSTRACT
Bonded cumomers are sets of isotopomers of 13 C-labeled metabolites containing a particular sequence of contiguously or singly labeled carbon atoms. Only these isotopomers contribute to multiplet structure in the 13 C NMR spectrum. We discuss the application of this technique to the study of quantitative tumor metabolism, bioenergetics, and the Warburg effect. The advantages and sensitivity of bonded cumomer analysis over positional enrichment analysis are discussed. When sensitivity requirements are met, bonded cumomer analysis enables the extraction of fluxes through specific metabolic pathways with higher precision. In conjunction with isotopomer control analysis, we evaluate the sensitivity of experimentally measurable metabolite multiplets to determine the robustness of flux analysis in 13 C spectra of tumors. This review examines the role of glycolytic and tricarboxylic acid cycle metabolism with special emphasis on flux through the pentose phosphate pathway (PPP). The impact of reversibility of the nonoxidative branch of the PPP with various 13 C glucose tracers on fine-structure multiplets is analyzed.
Subject(s)
Models, Biological , Neoplasms , Humans , Magnetic Resonance Spectroscopy/methods , Energy Metabolism , Citric Acid Cycle , Glucose/metabolism , Carbon Isotopes/metabolismABSTRACT
OBJECTIVE: Targeted therapy is an important part of the treatment of lung adenocarcinoma. Tests for EGFR mutation, ALK, ROS1, RET and NTRK gene fusions are needed to make a treatment decision. These gene fusions are traditionally detected by fluorescence in situ hybridisation (FISH) or immunohistochemistry. In this study, we investigated whether gene fusions in pulmonary adenocarcinoma could be accurately detected by RNA next-generation sequencing (RNA-NGS) and whether cytology cell blocks could be used effectively for this test. METHODS: Archived cytological specimens of lung adenocarcinoma submitted for RNA sequencing between 2019 and 2022 at Fox Chase Cancer Center were retrospectively retrieved. Hybrid capture-based targeted RNA next generation sequencing was used, which covers 507 fusion genes, including ALK, ROS1, RET and NTRKs, irrespective of their partner genes. DNA NGS, FISH and chromosomal microarray analysis were used to confirm the results of the RNA-NGS. RESULTS: A total of 129 lung adenocarcinoma cytology specimens were submitted for molecular testing. Eight of 129 (6.2%) cases were excluded from RNA sequencing as their cell blocks contained inadequate numbers of tumour cells. One case (0.8%) failed to yield adequate RNA. The overall success rate was 93% (120/129). Ten of 120 (8.3%) cytology cases were positive for gene fusions, including 7 ALK, 2 ROS1 fusion genes, and 1 RET fusion gene. Twenty-two cell block cases were also tested for ALK fusion genes using FISH. However, 11 of 22 (50%) failed the testing due to inadequate material. CONCLUSIONS: Cytology cell blocks can be used as the main source of material for molecular testing for lung cancer. Detection of gene fusions by RNA-based NGS on cell blocks is convenient and reliable in daily practice.
Subject(s)
Adenocarcinoma of Lung , Adenocarcinoma , Lung Neoplasms , Humans , High-Throughput Nucleotide Sequencing/methods , Anaplastic Lymphoma Kinase/genetics , Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/genetics , RNA , Retrospective Studies , Adenocarcinoma/diagnosis , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Proto-Oncogene Proteins/genetics , Adenocarcinoma of Lung/diagnosis , Adenocarcinoma of Lung/genetics , Gene Fusion , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Oncogene Proteins, Fusion/geneticsABSTRACT
Given the poor prognosis of MYC-overexpressing diffuse large B cell lymphoma (DLBCL) and B cell lymphoma unclassifiable with features intermediate between DLBCL and Burkitt lymphoma/high grade B cell lymphoma (BCLU/HGBL), and preclinical data suggesting that MYC may regulate the antitumor immune response, we sought to characterize expression of immune checkpoint proteins on tumor tissue from patients diagnosed with these lymphomas. Immunohistochemical staining for immune checkpoint protein expression was applied to 56 cases of MYC-overexpressing DLBCL and BCLU/HGBL, 35 of which also harbored MYC rearrangement (MYC-R). Analysis revealed both frequent overexpression of immune checkpoint proteins as well as differences in overexpression patterns based upon MYC-R status, with MYC-R cases more likely to overexpress PD-L1 and PD-1 in the tumor microenvironment (50 vs. 15%, p = 0.02 and 32 vs. 5%, p = 0.02, respectively) but less likely to overexpress CTLA-4 and CD80 on tumor cells (34 vs. 71%, p = 0.01 and 34 vs. 81%, p = 0.001, respectively), as compared to cases without MYC-R. These data may suggest a biologic rationale for investigation of the effect of checkpoint inhibitor therapies in these subgroups of MYC-overexpressing DLBCL and BCLU/HGBL.
Subject(s)
Biomarkers, Tumor , Gene Expression Regulation, Neoplastic , Genes, myc , Immune Checkpoint Proteins/genetics , Lymphoma, B-Cell/genetics , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cyclophosphamide/adverse effects , Cyclophosphamide/therapeutic use , Doxorubicin/adverse effects , Doxorubicin/therapeutic use , Female , Humans , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/mortality , Lymphoma, B-Cell/pathology , Male , Middle Aged , Neoplasm Staging , Prednisone/adverse effects , Prednisone/therapeutic use , Prognosis , Recurrence , Rituximab/adverse effects , Rituximab/therapeutic use , Survival Analysis , Treatment Outcome , Vincristine/adverse effects , Vincristine/therapeutic useABSTRACT
It has been proposed that CD4 T-cell responses to Staphylococcus aureus (SA) can inadvertently enhance neoplastic progression in models of skin cancer and cutaneous T-cell lymphoma (CTCL). In this prospective study, we explored the effect of transient antibiotic treatment on tumor cells and disease activity in 8 patients with advanced-stage CTCL. All patients experienced significant decrease in clinical symptoms in response to aggressive, transient antibiotic treatment. In some patients, clinical improvements lasted for more than 8 months. In 6 of 8 patients, a malignant T-cell clone could be identified in lesional skin, and a significant decrease in the fraction of malignant T cells was observed following antibiotics but an otherwise unchanged treatment regimen. Immunohistochemistry, global messenger RNA expression, and cell-signaling pathway analysis indicated that transient aggressive antibiotic therapy was associated with decreased expression of interleukin-2 high-affinity receptors (CD25), STAT3 signaling, and cell proliferation in lesional skin. In conclusion, this study provides novel evidence suggesting that aggressive antibiotic treatment inhibits malignant T cells in lesional skin. Thus, we provide a novel rationale for treatment of SA in advanced CTCL.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Lymphoma, T-Cell, Cutaneous/drug therapy , Skin Neoplasms/drug therapy , Aged , Cell Proliferation/drug effects , Female , Humans , Lymphoma, T-Cell, Cutaneous/metabolism , Lymphoma, T-Cell, Cutaneous/pathology , Male , Middle Aged , Prospective Studies , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/pathologyABSTRACT
Inhibitors of Bruton tyrosine kinase (BTK), a kinase downstream of BCR, display remarkable activity in a subset of mantle cell lymphoma (MCL) patients, but the drug resistance remains a considerable challenge. In this study, we demonstrate that aberrant expression of ROR1 (receptor tyrosine kinase-like orphan receptor 1), seen in a large subset of MCL, results in BCR/BTK-independent signaling and growth of MCL cells. ROR1 forms a functional complex with CD19 to persistently activate the key cell signaling pathways PI3K-AKT and MEK-ERK in the BCR/BTK-independent manner. This study demonstrates that ROR1/CD19 complex effectively substitutes for BCR-BTK signaling to promote activation and growth of MCL cells. Therefore, ROR1 expression and activation may represent a novel mechanism of resistance to inhibition of BCR/BTK signaling in MCL. Our results provide a rationale to screen MCL patients for ROR1 expression and to consider new therapies targeting ROR1 and/or CD19 or their downstream signaling pathways for MCL-expressing ROR1.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/metabolism , Lymphoma, Mantle-Cell/pathology , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Humans , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/metabolism , Piperidines , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptor Tyrosine Kinase-like Orphan Receptors/antagonists & inhibitors , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Receptors, Antigen, B-Cell/antagonists & inhibitors , Receptors, Antigen, T-Cell/antagonists & inhibitors , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
Mutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , DNA Repair/drug effects , Leukemia, Myeloid, Acute , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/metabolism , Animals , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Benzothiazoles/pharmacology , Cell Line, Tumor , DNA Ligase ATP/genetics , DNA Ligase ATP/metabolism , Fanconi Anemia Complementation Group N Protein/genetics , Fanconi Anemia Complementation Group N Protein/metabolism , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mutation , Phenylurea Compounds/pharmacology , Phthalazines/pharmacology , Piperazines/pharmacology , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Protein Kinase Inhibitors/pharmacology , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase physiologically expressed by fetal neural cells. However, aberrantly expressed ALK is involved in the pathogenesis of diverse malignancies, including distinct types of lymphoma, lung carcinoma, and neuroblastoma. The aberrant ALK expression in nonneural cells results from chromosomal translocations that create novel fusion proteins. These protein hybrids compose the proximal part of a partner gene, including its promoter region, and the distal part of ALK, including the coding sequence for the entire kinase domain. ALK was first identified in a subset of T-cell lymphomas with anaplastic large cell lymphoma (ALCL) morphology (ALK+ ALCL), the vast majority of which harbor the well-characterized nucleophosmin (NPM)-ALK fusion protein. NPM-ALK co-opts several intracellular signal transduction pathways, foremost being the STAT3 pathway, normally activated by cytokines from the interleukin-2 (IL-2) family to promote cell proliferation and to inhibit apoptosis. Many genes and proteins modulated by NPM-ALK are also involved in evasion of antitumor immune response, protection from hypoxia, angiogenesis, DNA repair, cell migration and invasiveness, and cell metabolism. In addition, NPM-ALK uses epigenetic silencing mechanisms to downregulate tumor suppressor genes to maintain its own expression. Importantly, NPM-ALK is capable of transforming primary human CD4+ T cells into immortalized cell lines indistinguishable from patient-derived ALK+ ALCL. Preliminary clinical studies indicate that inhibition of NPM-ALK induces long-lasting complete remissions in a large subset of heavily pretreated adult patients and the vast majority of children with high-stage ALK+ ALCL. Combining ALK inhibition with other novel therapeutic modalities should prove even more effective.
Subject(s)
Cell Transformation, Neoplastic/genetics , Lymphoma, Large-Cell, Anaplastic/genetics , Lymphoma, T-Cell/genetics , Oncogene Proteins, Fusion/genetics , Protein-Tyrosine Kinases/genetics , Anaplastic Lymphoma Kinase , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cytokines/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lymphoma, Large-Cell, Anaplastic/drug therapy , Lymphoma, Large-Cell, Anaplastic/metabolism , Lymphoma, Large-Cell, Anaplastic/pathology , Lymphoma, T-Cell/drug therapy , Lymphoma, T-Cell/metabolism , Lymphoma, T-Cell/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , Molecular Targeted Therapy/methods , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleophosmin , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effectsABSTRACT
Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.
Subject(s)
Blast Crisis/genetics , Genes, Tumor Suppressor , Genes, abl , Leukemia, Experimental/genetics , Leukemia, Myeloid, Chronic-Phase/genetics , Oncogene Proteins v-abl/physiology , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Proteins c-abl/physiology , Tumor Suppressor Proteins/physiology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Blast Crisis/drug therapy , Blast Crisis/enzymology , Blast Crisis/pathology , Cell Division/drug effects , Cell Line, Tumor , Cytostatic Agents/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Genomic Instability , Humans , Imatinib Mesylate/pharmacology , Imatinib Mesylate/therapeutic use , Imidazoles/pharmacology , Imidazoles/therapeutic use , Leukemia, Experimental/drug therapy , Leukemia, Experimental/enzymology , Leukemia, Experimental/pathology , Leukemia, Myeloid, Chronic-Phase/drug therapy , Leukemia, Myeloid, Chronic-Phase/enzymology , Leukemia, Myeloid, Chronic-Phase/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/enzymology , Oncogene Proteins v-abl/antagonists & inhibitors , Oncogene Proteins v-abl/genetics , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/genetics , Oxidative Stress , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-abl/genetics , Pyridazines/pharmacology , Pyridazines/therapeutic use , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/geneticsABSTRACT
Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region ß chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis.
Subject(s)
Enterotoxins/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Interleukin-17/biosynthesis , Neoplasm Proteins/metabolism , STAT3 Transcription Factor/metabolism , Sezary Syndrome/metabolism , T-Lymphocytes/metabolism , Cell Line, Tumor , Coculture Techniques , Female , Humans , Interleukin Receptor Common gamma Subunit/metabolism , Lymphocyte Activation/drug effects , Male , Piperidines/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Sezary Syndrome/pathology , T-Lymphocytes/pathologyABSTRACT
The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity.
Subject(s)
Adoptive Transfer , Antigens, CD19 , Lymphoma, B-Cell , Plasma Cells , T-Lymphocytes , Adolescent , Adult , Antigens, CD19/blood , Antigens, CD19/immunology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Child , Female , Humans , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Lymphoma, B-Cell/blood , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/pathology , Lymphoma, B-Cell/therapy , Male , Middle Aged , Plasma Cells/immunology , Plasma Cells/metabolism , Plasma Cells/pathology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , T-Lymphocytes/transplantationSubject(s)
Cell Transdifferentiation , Immunotherapy/adverse effects , Lymphoma, Mantle-Cell/therapy , Neoplasms, Second Primary/etiology , Sarcoma/etiology , Adenine/administration & dosage , Adenine/adverse effects , Adenine/analogs & derivatives , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Transdifferentiation/genetics , Cell Transdifferentiation/immunology , Cellular Reprogramming/genetics , Cellular Reprogramming/immunology , Cellular Reprogramming/physiology , Combined Modality Therapy , Epigenesis, Genetic/physiology , Epigenome/immunology , Gene Rearrangement , Genes, Immunoglobulin Heavy Chain/genetics , Humans , Immunotherapy/methods , Lymph Nodes/pathology , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/immunology , Neoplasms, Second Primary/diagnosis , Piperidines/administration & dosage , Piperidines/adverse effects , Receptors, Antigen, T-Cell/therapeutic use , Rituximab/administration & dosage , Rituximab/adverse effects , Sarcoma/genetics , Sarcoma/immunology , Sarcoma/pathology , Transplantation, Autologous/adverse effects , Tumor Cells, CulturedABSTRACT
Patients with cutaneous T-cell lymphoma (CTCL) are frequently colonized with Staphylococcus aureus (SA). Eradication of SA is, importantly, associated with significant clinical improvement, suggesting that SA promotes the disease activity, but the underlying mechanisms remain poorly characterized. Here, we show that SA isolates from involved skin express staphylococcal enterotoxins (SEs) that induce crosstalk between malignant and benign T cells leading to Stat3-mediated interleukin-10 (IL-10) production by the malignant T cells. The SEs did not stimulate the malignant T cells directly. Instead, SEs triggered a cascade of events involving cell-cell and asymmetric cytokine interactions between malignant and benign T cells, which stimulated the malignant T cells to express high levels of IL-10. Much evidence supports that malignant activation of the Stat3/IL-10 axis plays a key role in driving the immune dysregulation and severe immunodeficiency that characteristically develops in CTCL patients. The present findings thereby establish a novel link between SEs and immune dysregulation in CTCL, strengthening the rationale for antibiotic treatment of colonized patients with severe or progressive disease.
Subject(s)
Bacterial Proteins/pharmacology , Enterotoxins/pharmacology , Lymphocyte Activation/drug effects , Lymphoma, T-Cell, Cutaneous/immunology , Staphylococcus aureus , T-Lymphocytes/immunology , Cell Line, Tumor , Female , Humans , Interleukin-10/immunology , Lymphoma, T-Cell, Cutaneous/pathology , Male , STAT3 Transcription Factor/immunology , Staphylococcal Infections/immunology , Staphylococcal Infections/pathology , T-Lymphocytes/pathologyABSTRACT
In this article, we report that cutaneous T cell lymphoma (CTCL) cells and tissues ubiquitously express the immunosuppressive cell surface protein CD80 (B7-1). CD80 expression in CTCL cells is strictly dependent on the expression of both members of the STAT5 family, STAT5a and STAT5b, as well as their joint ability to transcriptionally activate the CD80 gene. In IL-2-dependent CTCL cells, CD80 expression is induced by the cytokine in a Jak1/3- and STAT5a/b-dependent manner, whereas in the CTCL cells with constitutive STAT5 activation, CD80 expression is also STAT5a/b dependent but is independent of Jak activity. Although depletion of CD80 expression does not affect the proliferation rate and viability of CTCL cells, induced expression of the cell-inhibitory receptor of CD80, CD152 (CTLA-4), impairs growth of the cells. Coculture of CTCL cells with normal T lymphocytes consisting of both CD4(+) and CD8(+) populations or the CD4(+) subset alone, transfected with CD152 mRNA, inhibits proliferation of normal T cells in a CD152- and CD80-dependent manner. These data identify a new mechanism of immune evasion in CTCL and suggest that the CD80-CD152 axis may become a therapeutic target in this type of lymphoma.
Subject(s)
B7-1 Antigen/immunology , Lymphoma, T-Cell, Cutaneous/immunology , STAT5 Transcription Factor/immunology , Tumor Suppressor Proteins/immunology , Adult , B7-1 Antigen/genetics , B7-1 Antigen/metabolism , Blotting, Western , CTLA-4 Antigen/immunology , CTLA-4 Antigen/metabolism , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/immunology , Humans , Immunohistochemistry , Interleukin-2/pharmacology , Janus Kinase 1/immunology , Janus Kinase 1/metabolism , Janus Kinase 3/immunology , Janus Kinase 3/metabolism , Lymphoma, T-Cell, Cutaneous/genetics , Lymphoma, T-Cell, Cutaneous/metabolism , Models, Immunological , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Inappropriately regulated expression of interleukin (IL)-17A is associated with the development of inflammatory diseases and cancer. However, little is known about the role of other IL-17 family members in carcinogenesis. Here, we show that a set of malignant T-cell lines established from patients with cutaneous T-cell lymphoma (CTCL) spontaneously secrete IL-17F and that inhibitors of Janus kinases and Signal transducer and activator of transcription 3 are able to block that secretion. Other malignant T-cell lines produce IL-17A but not IL-17F. Upon activation, however, some of the malignant T-cell lines are able to coexpress IL-17A and IL-17F, leading to formation of IL-17A/F heterodimers. Clinically, we demonstrate that IL-17F messenger RNA expression is significantly increased in CTCL skin lesions compared with healthy donors and patients with chronic dermatitis. IL-17A expression is also increased and a significant number of patients express high levels of both IL-17A and IL-17F. Concomitantly, we observed that the expression of the IL-17 receptor is significantly increased in CTCL skin lesions compared with control subjects. Importantly, analysis of a historic cohort of 60 CTCL patients indicates that IL-17F expression is associated with progressive disease. These findings implicate IL-17F in the pathogenesis of CTCL and suggest that IL-17 cytokines and their receptors may serve as therapeutic targets.
Subject(s)
Gene Expression Regulation, Neoplastic , Gene Expression Regulation , Interleukin-17/metabolism , Lymphoma, T-Cell, Cutaneous/metabolism , Biopsy , Cell Line, Tumor , Cytokines/metabolism , Disease Progression , Female , Humans , Janus Kinases/metabolism , Jurkat Cells , Male , Mycosis Fungoides/metabolism , STAT3 Transcription Factor/metabolism , Skin/pathologyABSTRACT
Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.
Subject(s)
Apoptosis , Aptamers, Peptide/pharmacology , Gene Expression Profiling , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mutation/genetics , Rad52 DNA Repair and Recombination Protein/genetics , Recombination, Genetic/genetics , Animals , Aptamers, Peptide/chemistry , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Case-Control Studies , Cell Differentiation , Cell Proliferation , DNA Damage/genetics , DNA Repair/genetics , Epigenomics , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/prevention & control , Mice , Mice, SCID , Models, Molecular , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/prevention & control , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Oligonucleotide Array Sequence Analysis , Peptide Fragments , RNA, Messenger/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Rad52 DNA Repair and Recombination Protein/antagonists & inhibitors , Rad52 DNA Repair and Recombination Protein/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4(+) T lymphocytes. Direct comparison of gene expression by ALK(+) TCL cells treated with an ALK inhibitor and IL-2-dependent ALK(-) TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene-regulation pattern. Depending on the analysis method, up to 67% of the affected genes were modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT- and IL-2-signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes-CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4-was confirmed at the protein level. In both ALK(+) TCL and IL-2-stimulated ALK(-) TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, whereas transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4(+) T lymphocytes, at least in part, by using the pre-existing, IL-2-dependent signaling pathways.
Subject(s)
CD4-Positive T-Lymphocytes/pathology , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , Interleukin-2/physiology , Lymphoma, T-Cell/enzymology , Oncogene Proteins, Fusion/physiology , Protein-Tyrosine Kinases/physiology , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/metabolism , Carbazoles/pharmacology , Cell Line, Tumor , Early Growth Response Protein 1/biosynthesis , Early Growth Response Protein 1/genetics , Enzyme Activation , Humans , Lymphoma, T-Cell/genetics , MAP Kinase Signaling System , Molecular Mimicry , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Oncogene Proteins, Fusion/antagonists & inhibitors , Phenylurea Compounds/pharmacology , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational , Protein-Tyrosine Kinases/antagonists & inhibitors , STAT3 Transcription Factor/physiology , STAT5 Transcription Factor/physiology , Signal Transduction/geneticsABSTRACT
With this study we have demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their malignant transformation. The transformed cells become immortalized and display morphology and immunophenotype characteristic of patient-derived anaplastic large-cell lymphomas. These unique features, which are strictly dependent on NPM-ALK activity and expression, include perpetual cell growth, proliferation, and survival; activation of the key signal transduction pathways STAT3 and mTORC1; and expression of CD30 (the hallmark of anaplastic large-cell lymphoma) and of immunosuppressive cytokine IL-10 and cell-surface protein PD-L1/CD274. Implantation of NPM-ALK-transformed CD4(+) T lymphocytes into immunodeficient mice resulted in formation of tumors indistinguishable from patients' anaplastic large-cell lymphomas. Our findings demonstrate that the key aspects of human carcinogenesis closely recapitulating the features of the native tumors can be faithfully reproduced in vitro when an appropriate oncogene is used to transform its natural target cells; this in turn points to the fundamental role in malignant cell transformation of potent oncogenes expressed in the relevant target cells. Such transformed cells should permit study of the early stages of carcinogenesis, and in particular the initial oncogene-host cell interactions. This experimental design could also be useful for studies of the effects of early therapeutic intervention and likely also the mechanisms of malignant progression.