ABSTRACT
Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with 5-year overall survival of less than 10% in patients over the age of 65. Limited progress has been made in the patient outcome because of the inability to selectively eradicate the leukemic stem cells (LSC) driving the refractory and relapsed disease. Herein, we investigated the role of the reprogramming factor KLF4 in AML because of its critical role in the self-renewal and stemness of embryonic and cancer stem cells. Using a conditional Cre-lox Klf4 deletion system and the MLL-AF9 retroviral mouse model, we demonstrated that loss-of-KLF4 does not significantly affect the induction of leukemia but markedly decreased the frequency of LSCs evaluated in limiting-dose transplantation studies. Loss of KLF4 in leukemic granulocyte-macrophage progenitors (L-GMP), a population enriched for AML LSCs, showed lessened clonogenicity and percentage in the G2/M phase of the cell cycle. RNAseq analysis of purified L-GMPs revealed decreased expression of stemness genes and MLL-target genes and upregulation of the RNA sensing helicase DDX58. However, silencing of DDX58 in KLF4 knockout leukemia indicated that DDX58 is not mediating this phenotype. CRISPR/Cas9 deletion of KLF4 in MOLM13 cell line and AML patient-derived xenograft cells showed impaired expansion in vitro and in vivo associated with a defective G2/M checkpoint. Collectively, our data suggest a mechanism in which KLF4 promotes leukemia progression by establishing a gene expression profile in AML LSCs supporting cell division and stemness.
Subject(s)
Kruppel-Like Factor 4 , Leukemia, Myeloid, Acute , Animals , Bone Marrow/pathology , Disease Models, Animal , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Myeloid-Lymphoid Leukemia Protein/metabolism , Neoplastic Stem Cells/metabolism , Oncogene Proteins, Fusion/metabolismABSTRACT
Transcription factors that regulate the quiescence, proliferation and homing of lymphocytes are critical for effective immune system function. Here we demonstrate that the transcription factor ELF4 directly activated the tumor suppressor KLF4 'downstream' of T cell antigen receptor signaling to induce cell cycle arrest in naive CD8(+) T cells. Elf4- and Klf4-deficient mice accumulated CD8(+)CD44(hi) T cells during steady-state conditions and generated more memory T cells after immunization. The homeostatic population expansion of CD8(+)CD44(hi) T cells in Elf4-null mice resulted in a redistribution of cells to nonlymphoid tissue because of lower expression of the transcription factor KLF2 and the surface proteins CCR7 and CD62L. Our work describes the combinatorial effect of lymphocyte-intrinsic factors on the homeostasis, activation and homing of T cells.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation , DNA-Binding Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , Transcription Factors/metabolism , Adoptive Transfer , Animals , CD8-Positive T-Lymphocytes/cytology , Cell Cycle , Cell Movement , Gene Expression Regulation , Immunologic Memory , Kruppel-Like Factor 4 , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, TransgenicABSTRACT
Leukemia stem cells are a rare population with a primitive progenitor phenotype that can initiate, sustain, and recapitulate leukemia through a poorly understood mechanism of self-renewal. Here, we report that Krüppel-like factor 4 (KLF4) promotes disease progression in a murine model of chronic myeloid leukemia (CML)-like myeloproliferative neoplasia by repressing an inhibitory mechanism of preservation in leukemia stem/progenitor cells with leukemia-initiating capacity. Deletion of the Klf4 gene severely abrogated the maintenance of BCR-ABL1(p210)-induced CML by impairing survival and self-renewal in BCR-ABL1+ CD150+ lineage-negative Sca-1+ c-Kit+ leukemic cells. Mechanistically, KLF4 repressed the Dyrk2 gene in leukemic stem/progenitor cells; thus, loss of KLF4 resulted in elevated levels of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2), which were associated with inhibition of survival and self-renewal via depletion of c-Myc protein and p53 activation. In addition to transcriptional regulation, stabilization of DYRK2 protein by inhibiting ubiquitin E3 ligase SIAH2 with vitamin K3 promoted apoptosis and abrogated self-renewal in murine and human CML stem/progenitor cells. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling p53- and c-Myc-mediated regulation of survival and self-renewal in CML cells with leukemic-initiating capacity that can be targeted with small molecules.
Subject(s)
Kruppel-Like Transcription Factors/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Neoplastic Stem Cells/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Animals , Cell Survival/drug effects , Cell Survival/genetics , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Deletion , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mice, Knockout , Neoplastic Stem Cells/pathology , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Vitamin K 3/pharmacology , Dyrk KinasesABSTRACT
Dietary influences may affect microbiome composition and host immune responses, thereby modulating propensity toward inflammatory bowel diseases (IBDs): Crohn disease (CD) and ulcerative colitis (UC). Dietary n-6 fatty acids have been associated with UC in prospective studies. However, the critical developmental period when (n-6) consumption may induce UC is not known. We examined the effects of transiently increased n-6 consumption during pediatric development on subsequent dextran-sulfate-sodium (DSS)-induced acute murine colitis. The animals transiently became obese then rapidly lost this phenotype. Interestingly, mice were protected against DSS colitis 40 days after n-6 consumption. The transient high n-6-induced protection against colitis was fat type- and dietary reversal-dependent and could be transferred to germ-free mice by fecal microbiota transplantation. We also detected decreased numbers of chemokine receptor (Cxcr)5(+) CD4(+) T cells in the mesenteric lymph nodes (MLNs) of transiently n-6-fed mice. Further experiments revealed that anti-chemokine ligand (Cxcl)13 (the ligand of Cxcr5) antibody treatment decreased DSS colitis severity, implicating the importance of the Cxcr5-Cxcl13 pathway in mammalian colitis. Consecutively, we found elevated CXCL13 concentrations (CD: 1.8-fold, P = 0.0077; UC: 1.9-fold, P = 0.056) in the serum of untreated pediatric IBD patients. The human serologic observations supported the translational relevance of our findings.
Subject(s)
Colitis/metabolism , Fatty Acids, Omega-6/metabolism , Pediatric Obesity/metabolism , Animals , Colon/metabolism , Diet , Intestinal Mucosa/metabolism , Male , Mice , Mice, Inbred C57BL , Prospective StudiesABSTRACT
The differentiation of CD4(+) T cells into different Th lineages is driven by cytokine milieu in the priming site and the underlying transcriptional circuitry. Even though many positive regulators have been identified, it is not clear how this process is inhibited at transcriptional level. In this study, we report that the E-twenty six (ETS) transcription factor E74-like factor 4 (ELF4) suppresses the differentiation of Th17 cells both in vitro and in vivo. Culture of naive Elf4(-/-) CD4(+) T cells in the presence of IL-6 and TGF-ß (or IL-6, IL-23, and IL-1ß) resulted in increased numbers of IL-17A-positive cells compared with wild-type controls. In contrast, the differentiation to Th1, Th2, or regulatory T cells was largely unaffected by loss of ELF4. The increased expression of genes involved in Th17 differentiation observed in Elf4(-/-) CD4(+) T cells suggested that ELF4 controls their programming into the Th17 lineage rather than only IL-17A gene expression. Despite normal proliferation of naive CD4(+) T cells, loss of ELF4 lowered the requirement of IL-6 and TGF-ß signaling for IL-17A induction in each cell division. ELF4 did not inhibit Th17 differentiation by promoting IL-2 production as proposed for another ETS transcription factor, ETS1. Elf4(-/-) mice showed increased numbers of Th17 cells in the lamina propria at steady state, in lymph nodes after immunization, and, most importantly, in the CNS following experimental autoimmune encephalomyelitis induction, contributing to the increased disease severity. Collectively, our findings suggest that ELF4 restrains Th17 differentiation in dividing CD4(+) T cells by regulating commitment to the Th17 differentiation program.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation/genetics , DNA-Binding Proteins/genetics , Th17 Cells/cytology , Th17 Cells/metabolism , Transcription Factors/genetics , Animals , CD4-Positive T-Lymphocytes/immunology , Cell Line , Cell Survival/genetics , Cytokines/metabolism , Humans , Lymphocyte Activation/immunology , Mice , Mice, Knockout , Th17 Cells/immunologyABSTRACT
Most differentiated CD8(+) T cells die off at the end of an infection, revealing two main subsets of memory T cells - central and effector memory - which can be found in lymphoid tissues or circulating through nonlymphoid organs, respectively. The cell intrinsic regulation of the differentiation of CD8(+) T cells to effector and central memory remains poorly studied. Herein, we describe a novel role of the ETS transcription factor ELF4 in the development and function of memory CD8(+) T cells following infection with Listeria monocytogenes. Adoptively transferred Elf4(-/-) naïve CD8(+) T cells produced lower numbers of effector memory CD8(+) T cells despite a normal pool of central memory. This was caused by suboptimal priming and decreased survival of CD8(+) T cells at the peak of response while enhanced Notch1 signaling and upregulation of eomesodermin correlated with "normal" development of Elf4(-/-) central memory. Finally, loss of ELF4 impaired the expansion of both central and effector memory CD8(+) T cells in a recall response by also activating Notch1 signaling. Altogether, ELF4 emerges as a novel transcriptional regulator of CD8(+) T-cell differentiation in response to infection.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , DNA-Binding Proteins/genetics , Immunologic Memory/genetics , Listeria monocytogenes/immunology , Listeriosis/genetics , Listeriosis/immunology , Transcription Factors/genetics , Animals , Cell Survival/immunology , DNA-Binding Proteins/deficiency , Disease Models, Animal , Gene Expression Regulation , Listeriosis/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Knockout , Ovalbumin/immunology , Receptor, Notch1/metabolism , Signal Transduction , Transcription Factors/deficiencyABSTRACT
Since its discovery, diverse functions have been attributed to the G0/G1 switch gene 2 (G0S2), from lipid metabolism to control of cell proliferation. Our group showed for the first time that G0S2 promotes quiescence in hematopoietic stem cells by interacting with and retaining nucleolin around the nucleus. Herein, we report the role of G0S2 in the differentiation and function of CD8(+) T cells examined in mice with an embryonic deletion of the G0s2 gene. G0S2 expression in naïve CD8(+) T cells decreased immediately after T-cell receptor activation downstream of the mitogen-activated protein kinase, calcium/calmodulin, phosphatidylinositol 3'-kinase and mammalian target of rapamycin pathways. Surprisingly, G0S2-null naïve CD8(+) T cells displayed increased basal and spare respiratory capacity that was not associated with increased mitochondrial biogenesis but with increased phosphorylation of AMP-activated protein kinase α. Naïve CD8(+) T cells showed increased proliferation in response to in vitro activation and in vivo lymphopenia; however, naïve CD8(+) T cells expressing the OT-1 transgene exhibited normal differentiation of naïve cells to effector and memory CD8(+) T cells upon infection with Listeria monocytogenes in a wild-type or a G0s2-null environment, with increased circulating levels of free fatty acids. Collectively, our results suggest that G0S2 inhibits energy production by oxidative phosphorylation to fine-tune proliferation in homeostatic conditions.
Subject(s)
CD8-Positive T-Lymphocytes/cytology , Cell Cycle Proteins/physiology , Mitochondria/metabolism , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Cycle Proteins/deficiency , Cell Cycle Proteins/genetics , Cell Division , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/metabolism , Fatty Acids, Nonesterified/blood , Female , Genes, Reporter , Homeostasis , Listeria monocytogenes , Listeriosis/immunology , Listeriosis/metabolism , Lymphocyte Activation , Lymphopenia/immunology , Lymphopenia/metabolism , Lymphopoiesis , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oxidative Phosphorylation , Phosphorylation , Protein Processing, Post-Translational , Receptors, Antigen, T-Cell/immunology , Signal Transduction , Specific Pathogen-Free OrganismsABSTRACT
The transcriptional circuitry that regulates the quiescence of hematopoietic stem cells is largely unknown. We report that the transcription factor known as MEF (or ELF4), which is targeted by the t(X;21)(q26;q22) in acute myelogenous leukemia, regulates the proliferation of primitive hematopoietic progenitor cells at steady state, controlling their quiescence. Mef null HSCs display increased residence in G0 with reduced 5-bromodeoxyuridine incorporation in vivo and impaired cytokine-driven proliferation in vitro. Due to their increased HSC quiescence, Mef null mice are relatively resistant to the myelosuppressive effects of chemotherapy and radiation. Thus, MEF plays an important role in the decision of stem/primitive progenitor cells to divide or remain quiescent by regulating their entry to the cell cycle.
Subject(s)
DNA-Binding Proteins/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Transcription Factors/metabolism , Animals , Antimetabolites/pharmacology , Bone Marrow Transplantation , Bone and Bones/cytology , Bone and Bones/drug effects , Bone and Bones/physiology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Proliferation , Cells, Cultured , Fluorouracil/pharmacology , Hematopoietic Stem Cells/drug effects , MiceABSTRACT
Novel drugs are needed to increase treatment response in children with high-risk T-cell acute lymphoblastic leukemia (T-ALL). Following up on our previous report on the activation of the MAP2K7-JNK pathway in pediatric T-ALL, here we demonstrate that OTSSP167, recently shown to inhibit MAP2K7, has antileukemic capacity in T-ALL. OTSSP167 exhibited dose-dependent cytotoxicity against a panel of T-ALL cell lines with IC50 in the nanomolar range (10-50 nM). OTSSP167 induces apoptosis and cell cycle arrest in T-ALL cell lines, associated at least partially with the inhibition of MAP2K7 kinase activity and lower activation of its downstream substrate, JNK. Other leukemic T-cell survival pathways, such as mTOR and NOTCH1 were also inhibited. Daily intraperitoneal administration of 10 mg/kg OTSSP167 was well tolerated, with mice showing no hematological toxicity, and effective at reducing the expansion of human T-ALL cells in a cell-based xenograft model. The same dosage of OTSSP167 efficiently controlled the leukemia burden in the blood, bone marrow, and spleen of 3 patient-derived xenografts, which resulted in prolonged survival. OTSSP167 exhibited synergistic interactions when combined with dexamethasone, L-asparaginase, vincristine, and etoposide. Our findings reveal novel antileukemic properties of OTSSP167 in T-ALL and support the use of OTSSP167 as an adjuvant drug to increase treatment response and reduce relapses in pediatric T-ALL.
Subject(s)
Antineoplastic Agents , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Humans , Mice , Animals , Child , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Naphthyridines/pharmacology , Naphthyridines/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , T-Lymphocytes/metabolismABSTRACT
The mitogen-activated protein kinase signaling cascade is conserved across eukaryotes, where it plays a critical role in the regulation of activities including proliferation, differentiation, and stress responses. This pathway propagates external stimuli through a series of phosphorylation events, which allows external signals to influence metabolic and transcriptional activities. Within the cascade, MEK, or MAP2K, enzymes occupy a molecular crossroads immediately upstream to significant signal divergence and cross-talk. One such kinase, MAP2K7, also known as MEK7 and MKK7, is a protein of great interest in the molecular pathophysiology underlying pediatric T cell acute lymphoblastic leukemia (T-ALL). Herein, we describe the rational design, synthesis, evaluation, and optimization of a novel class of irreversible MAP2K7 inhibitors. With a streamlined one-pot synthesis, favorable in vitro potency and selectivity, and promising cellular activity, this novel class of compounds wields promise as a powerful tool in the study of pediatric T-ALL.
ABSTRACT
In acute myeloid leukemia (AML), SWI/SNF chromatin remodeling complexes sustain leukemic identity by driving high levels of MYC. Previous studies have implicated the hematopoietic transcription factor PU.1 (SPI1) as an important target of SWI/SNF inhibition, but PU.1 is widely regarded to have pioneer-like activity. As a result, many questions have remained regarding the interplay between PU.1 and SWI/SNF in AML as well as normal hematopoiesis. Here we found that PU.1 binds to most of its targets in a SWI/SNF-independent manner and recruits SWI/SNF to promote accessibility for other AML core regulatory factors, including RUNX1, LMO2, and MEIS1. SWI/SNF inhibition in AML cells reduced DNA accessibility and binding of these factors at PU.1 sites and redistributed PU.1 to promoters. Analysis of nontumor hematopoietic cells revealed that similar effects also impair PU.1-dependent B-cell and monocyte populations. Nevertheless, SWI/SNF inhibition induced profound therapeutic response in an immunocompetent AML mouse model as well as in primary human AML samples. In vivo, SWI/SNF inhibition promoted leukemic differentiation and reduced the leukemic stem cell burden in bone marrow but also induced leukopenia. These results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. SIGNIFICANCE: Disruption of PU.1-directed enhancer programs upon SWI/SNF inhibition causes differentiation of AML cells and induces leukopenia of PU.1-dependent B cells and monocytes, revealing the on- and off-tumor effects of SWI/SNF blockade.
Subject(s)
Leukemia, Myeloid, Acute , Leukopenia , Animals , Mice , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Bone Marrow/pathology , Promoter Regions, Genetic , Cell Differentiation , Leukopenia/geneticsABSTRACT
OBJECTIVE: The regeneration of the hematopoietic system in bone marrow after chemotherapy depends on a balance between the quiescence and proliferation of lineage-specific progenitor cells. Even though the vascular network in bone is damaged by cytoablation, the transcriptional control of quiescence in endothelial cells is not well known. In this study, we investigated the role of the transcription factor E74-like factor (ELF4) in the proliferation of endothelial cells in bone marrow. METHODS AND RESULTS: Loss-of-function models were used to study the role of ELF4 in human and murine endothelial cells. ELF4 promotes cell cycle entry by activating cyclin-dependent kinase-4 in human umbilical vein endothelial cells. Elf4-null mice exhibited enhanced recovery of bone marrow CD45- CD31+ endothelial cells and sinusoidal blood vessels following administration of 5-fluorouracil. CONCLUSIONS: Loss of ELF4 leads to increased quiescence in bone marrow endothelial cells by the deregulation of cyclin-dependent kinase-4 expression and to enhanced regeneration of sinusoidal blood vessels.
Subject(s)
Bone Marrow Cells/drug effects , Cellular Senescence/drug effects , DNA-Binding Proteins/metabolism , Drug Resistance , Endothelial Cells/drug effects , Fluorouracil/pharmacology , Myeloablative Agonists/pharmacology , Transcription Factors/metabolism , Animals , Bone Marrow Cells/metabolism , COS Cells , Cell Cycle/drug effects , Cell Proliferation/drug effects , Chlorocebus aethiops , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Endothelial Cells/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , NIH 3T3 Cells , Neovascularization, Physiologic/drug effects , Promoter Regions, Genetic , RNA Interference , Time Factors , Transcription Factors/deficiency , Transcription Factors/genetics , TransfectionABSTRACT
T cell receptor activation inhibits expression of the E74-like factor (ELF) 4 and Krüppel-like factor 4 genes to release naive CD8(+) T cells from their quiescent state. In this study, we show that ELF4 controls the ERK-mediated proliferative response by maintaining normal levels of dual-specificity phosphatases 1 and 5 in CD8(+) T cells. In activated CD8(+) T cells, the mammalian target of rapamycin pathway inhibits ELF4 and Krüppel-like factor 4 expression downstream of ERK and PI3K signaling. Our findings demonstrate that rapamycin could be used to modulate expression of this transcriptional network involved in cell-cycle regulation.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Activation/immunology , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/immunology , Transcription Factors/metabolism , Animals , CD8-Positive T-Lymphocytes/immunology , DNA-Binding Proteins/immunology , Gene Expression , Immunoblotting , Intracellular Signaling Peptides and Proteins/immunology , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/immunology , Kruppel-Like Transcription Factors/metabolism , Mice , Protein Serine-Threonine Kinases/immunology , Reverse Transcriptase Polymerase Chain Reaction , TOR Serine-Threonine Kinases , Transcription Factors/immunologyABSTRACT
Children with Down syndrome (DS) are 10-fold more likely to develop B-cell acute lymphoblastic leukemia (B-ALL), with a higher frequency of rearrangements resulting in overexpression of cytokine receptor-like factor 2 (CRLF2). Here, we investigated the impact of CRLF2 overexpression on B-cell progenitor proliferation, immunophenotype, and gene expression profile in the Dp(16)1Yey (Dp16) mouse model of DS compared with wild-type (WT) mice. CRLF2 overexpression enhanced immature B-lymphoid colony development and increased the proportion of less differentiated pre-pro-B cells, with a greater effect in Dp16 versus WT. In CRLF2-rearranged (CRLF2-R) B-ALL patient samples, cells with higher CRLF2 expression exhibited a less differentiated B-cell immunophenotype. CRLF2 overexpression resulted in a gene expression signature associated with E2F signaling both in Dp16 B-progenitors and in DS-ALL patient samples, and PI3K/mTOR and pan-CDK inhibitors, which reduce E2F-mediated signaling, exhibited cytotoxicity in CRLF2-R B-ALL cell lines and patient samples. CRLF2 overexpression alone in Dp16 stem and progenitor cells did not result in leukemic transformation in recipient mice. Thus, CRLF2 overexpression results in reduced B-cell differentiation and enhanced E2F signaling in Dp16 B-progenitor cells and DS-ALL patient samples. These findings suggest a functional basis for the high frequency of CRLF2-R in DS-ALL as well as a potential therapeutically targetable pathway.
Subject(s)
Down Syndrome , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Animals , Cell Differentiation/genetics , Disease Models, Animal , Down Syndrome/complications , Down Syndrome/genetics , Humans , Mice , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Receptors, Cytokine/genetics , Signal TransductionABSTRACT
NK cell deficiencies (NKD) are a type of primary immune deficiency in which the major immunologic abnormality affects NK cell number, maturity, or function. Since NK cells contribute to immune defense against virally infected cells, patients with NKD experience higher susceptibility to chronic, recurrent, and fatal viral infections. An individual with recurrent viral infections and mild hypogammaglobulinemia was identified to have an X-linked damaging variant in the transcription factor gene ELF4. The variant does not decrease expression but disrupts ELF4 protein interactions and DNA binding, reducing transcriptional activation of target genes and selectively impairing ELF4 function. Corroborating previous murine models of ELF4 deficiency (Elf4-/-) and using a knockdown human NK cell line, we determined that ELF4 is necessary for normal NK cell development, terminal maturation, and function. Through characterization of the NK cells of the proband, expression of the proband's variant in Elf4-/- mouse hematopoietic precursor cells, and a human in vitro NK cell maturation model, we established this ELF4 variant as a potentially novel cause of NKD.
Subject(s)
Transcription Factors , Animals , Humans , Mice , DNA-Binding Proteins/genetics , Killer Cells, Natural , Transcription Factors/geneticsABSTRACT
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric leukemia with a worse prognosis than most frequent B-cell ALL due to a high incidence of treatment failures and relapse. Our previous work showed that loss of the pioneer factor KLF4 in a NOTCH1-induced T-ALL mouse model accelerated the development of leukemia through expansion of leukemia-initiating cells and activation of the MAP2K7 pathway. Similarly, epigenetic silencing of the KLF4 gene in children with T-ALL was associated with MAP2K7 activation. Here, we showed the small molecule 5Z-7-oxozeaenol (5Z7O) induces dose-dependent cytotoxicity in a panel of T-ALL cell lines mainly through inhibition of the MAP2K7-JNK pathway, which further validates MAP2K7 as a therapeutic target. Mechanistically, 5Z7O-mediated apoptosis was caused by the downregulation of regulators of the G2/M checkpoint and the inhibition of survival pathways. The anti-leukemic capacity of 5Z7O was evaluated using leukemic cells from two mouse models of T-ALL and patient-derived xenograft cells generated using lymphoblasts from pediatric T-ALL patients. Finally, a combination of 5Z7O with dexamethasone, a drug used in frontline therapy, showed synergistic induction of cytotoxicity. In sum, we report here that MAP2K7 inhibition thwarts survival mechanisms in T-ALL cells and warrants future pre-clinical studies for high-risk and relapsed patients.
ABSTRACT
Acute myeloid leukemia (AML) is an aggressive hematological malignancy of the bone marrow that affects mostly elderly adults. Alternative therapies are needed for AML patients because the overall prognosis with current standard of care, high dose chemotherapy and allogeneic transplantation, remains poor due to the emergence of refractory and relapsed disease. Here, we found expression of the transcription factor KLF4 in AML cell lines is not silenced through KLF4 gene methylation nor via proteasomal degradation. The deletion of KLF4 by CRISPR-CAS9 technology reduced cell growth and increased apoptosis in both NB4 and MonoMac-6 cell lines. Chemical induced differentiation of gene edited NB4 and MonoMac6 cells with ATRA and PMA respectively increased apoptosis and altered expression of differentiating markers CD11b and CD14. Transplantation of NB4 and MonoMac-6 cells lacking KLF4 into NSG mice resulted in improved overall survival compared to the transplantation of parental cell lines. Finally, loss-of-KLF4 did not alter sensitivity of leukemic cells to the chemotherapeutic drugs daunorubicin and cytarabine. These results suggest that KLF4 expression supports AML cell growth and survival, and the identification and disruption of KLF4-regulated pathways could represent an adjuvant therapeutic approach to increase response.
ABSTRACT
Chronic myeloid leukemia is a hematological cancer driven by the oncoprotein BCR-ABL1, and lifelong treatment with tyrosine kinase inhibitors extends patient survival to nearly the life expectancy of the general population. Despite advances in the development of more potent tyrosine kinase inhibitors to induce a durable deep molecular response, more than half of patients relapse upon treatment discontinuation. This clinical finding supports the paradigm that leukemia stem cells feed the neoplasm, resist tyrosine kinase inhibition, and reactivate upon drug withdrawal depending on the fitness of the patient's immune surveillance. This concept lends support to the idea that treatment-free remission is not achieved solely with tyrosine kinase inhibitors and that new molecular targets independent of BCR-ABL1 signaling are needed in order to develop adjuvant therapy to more efficiently eradicate the leukemia stem cell population responsible for chemoresistance and relapse. Future efforts must focus on the identification of new targets to support the discovery of potent and safe small molecules able to specifically eradicate the leukemic stem cell population. In this review, we briefly discuss molecular maintenance in leukemia stem cells in chronic myeloid leukemia and provide a more in-depth discussion of the dual-specificity kinase DYRK2, which has been identified as a novel actionable checkpoint in a critical leukemic network. DYRK2 controls the activation of p53 and proteasomal degradation of c-MYC, leading to impaired survival and self-renewal of leukemia stem cells; thus, pharmacological activation of DYRK2 as an adjuvant to standard therapy has the potential to induce treatment-free remission.
Subject(s)
Gene Expression Regulation, Leukemic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/etiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Neoplastic Stem Cells/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Animals , Carrier Proteins/metabolism , Cell Self Renewal/genetics , Disease Susceptibility , Energy Metabolism , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Neoplastic Stem Cells/pathology , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Dyrk KinasesABSTRACT
Hematopoietic stem cells (HSCs) represent an important target cell population in bone marrow transplantation, cell and gene therapy applications, and the development of leukemia models for research. Because the hematopoietic progeny carries the genetic information of HSCs and replenishes the blood and immune system, corrective gene transfer into HSCs provides an ideal therapeutic approach for many monogenic hematological diseases and a useful tool for studies of HSC function and blood formation in normal and malignant hematopoiesis. However, the efficiency of gene transfer into HSCs has been limited by several features of viral vectors, viral titer, methods of viral transduction, and the property of stem cell quiescence. In this chapter, we describe the production of retrovirus using murine stem cell virus (MSCV)-based retroviral vectors and purification and transduction of murine HSCs.
Subject(s)
Hematopoietic Stem Cells/metabolism , Resting Phase, Cell Cycle , Retroviridae/genetics , Transduction, Genetic , Animals , Antigens, CD34/metabolism , Cells, Cultured , Flow Cytometry , Genetic Vectors , MiceABSTRACT
Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in pediatric patients. Despite advances in the treatment of this disease, many children with T-cell ALL (T-ALL) die from disease relapse due to low responses to standard chemotherapy and the lack of a targeted therapy that selectively eradicates the chemoresistant leukemia-initiating cells (LICs) responsible for disease recurrence. We reported recently that the reprogramming factor Krüppel-like factor 4 (KLF4) has a tumor-suppressive function in children with T-ALL. KLF4 silencing by promoter deoxyribonucleic acid (DNA) methylation in patients with T-ALL leads to aberrant activation of the mitogen-activated protein kinase kinase MAP2K7 and the downstream c-Jun NH2-terminal kinase (JNK) pathway that controls the expansion of leukemia cells via c-Jun and activating transcription factor 2. This pathway can be inhibited with small molecules and therefore has the potential to eliminate LICs and eradicate disease in combination with standard therapy for patients with refractory and relapsed disease. The present review summarizes the role of the KLF4-MAP2K7 pathway in T-ALL pathogenesis and the function of JNK and MAP2K7 in carcinogenesis and therapy.