RESUMO
Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.
Assuntos
Adenocarcinoma/terapia , Epitopos de Linfócito T/imunologia , Imunoterapia/métodos , Mucina-1/imunologia , Linfócitos T/fisiologia , Adenocarcinoma/imunologia , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Engenharia Genética , Glicosilação , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos , Mucina-1/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Fator 4 Semelhante a Kruppel , Leucemia Mieloide Aguda , Animais , Medula Óssea/patologia , Modelos Animais de Doenças , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas de Fusão Oncogênica/metabolismoRESUMO
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.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Deleção de Genes , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Vitamina K 3/farmacologia , Quinases DyrkRESUMO
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.
Assuntos
Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Camundongos , Animais , Criança , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Naftiridinas/farmacologia , Naftiridinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Linfócitos T/metabolismoRESUMO
Since antibiotic development lags, we search for potential drug targets through directed evolution experiments. A challenge is that many resistance genes hide in a noisy mutational background as mutator clones emerge in the adaptive population. Here, to overcome this noise, we quantify the impact of mutations through evolutionary action (EA). After sequencing ciprofloxacin or colistin resistance strains grown under different mutational regimes, we find that an elevated sum of the evolutionary action of mutations in a gene identifies known resistance drivers. This EA integration approach also suggests new antibiotic resistance genes which are then shown to provide a fitness advantage in competition experiments. Moreover, EA integration analysis of clinical and environmental isolates of antibiotic resistant of E. coli identifies gene drivers of resistance where a standard approach fails. Together these results inform the genetic basis of de novo colistin resistance and support the robust discovery of phenotype-driving genes via the evolutionary action of genetic perturbations in fitness landscapes.
Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Proteínas de Escherichia coli , Escherichia coli , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , MutaçãoRESUMO
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.
RESUMO
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.