RESUMO
Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta, that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX3CR1+ and CSF1R+ source. These bipotent progenitors are proliferative and vasculogenic, contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally.
Assuntos
Aorta , Macrófagos , Animais , Macrófagos/citologia , Macrófagos/metabolismo , Aorta/citologia , Camundongos , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Diferenciação Celular , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Angiotensina II , Proliferação de Células , Células-Tronco/citologia , Células-Tronco/metabolismo , Camundongos Endogâmicos C57BL , Feminino , Neovascularização Fisiológica , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/genética , Masculino , Hematopoese/fisiologia , Tirosina Quinase 3 Semelhante a fmsRESUMO
FLT3 mutation is one of the most frequent genetic mutations in AML, identified in approximately 30% of patients, and FLT3-ITD mutation is considered a poor prognostic factor. Based on these molecular and clinical backgrounds, FLT3 mutations are considered promising therapeutic targets in AML, and intensive development of targeted therapeutics has been ongoing for more than two decades. Recently, combination of FLT3 inhibitors with intensive chemotherapy for untreated AML patients with FLT3 mutations and FLT3 inhibitor monotherapy for relapsed/refractory patients have been approved. In Japan, the combination of quizartinib and intensive chemotherapy for untreated FLT3-ITD-positive AML was approved in 2023. Clinical use of FLT3 inhibitors shows strong promise for improving the clinical outcomes of these AML patients with an extremely poor prognosis. Meanwhile, various resistance mechanisms to FLT3 inhibitors have been identified, including the emergence of resistance-associated mutations, and attenuated inhibitory effects of FLT3 inhibitors involving the bone marrow microenvironment surrounding AML cells. Thus, future efforts should aim to optimize combination therapy based on the characteristics of each FLT3 inhibitor, develop biomarkers that could inform treatment selection, and to better understand these resistance mechanisms and develop methods for overcoming them.
Assuntos
Leucemia Mieloide Aguda , Mutação , Inibidores de Proteínas Quinases , Tirosina Quinase 3 Semelhante a fms , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Resistencia a Medicamentos AntineoplásicosRESUMO
Conventional dendritic cells subtype 1 (cDC1) play a vital role in the priming and expansion of tumor-specific CD8+ T cells and their recruitment to tumor microenvironment. However, cDC1s are often underrepresented in the microenvironment. Systemic administration of Fms-like tyrosine kinase 3 ligand, a hematopoietic growth factor that binds to FLT3 on myeloid and lymphoid progenitor cells, leads to cDC1 expansion in the periphery and recruitment into the microenvironment. FLT3 pathway stimulation using GS-3583, a novel FLT3 agonistic Fc fusion protein, has the potential to promote T-cell mediated antitumor activity. This was a first-in-human, placebo-controlled study of GS-3583 in healthy participants to evaluate the safety, pharmacokinetics (PK), and pharmacodynamic (PD) of escalating single doses (75-2000 µg) of GS-3583. Each dose cohort enrolled 8-12 healthy participants who received GS-3583 or placebo as single IV infusion at 3:1 ratio. As part of the PD evaluation, the changes in the number of cDC1 cells were investigated. GS-3583 was well-tolerated in healthy participants up to the highest evaluated dose (2000 µg). There have been no serious or grade III or higher adverse events. PK analysis suggested a dose-dependent increase in GS-3583 exposure with target-mediated disposition characteristics at low doses. PD analysis shows that administration of GS-3583 resulted in transient, dose-dependent increases in cDC1 cells that returned to baseline within 3 weeks of drug administration. The pharmacokinetics and pharmacodynamics of GS-3583 following single dosing were characterized in this study which enabled subsequent phase Ib assessments in patients with advanced solid tumors.
Assuntos
Voluntários Saudáveis , Fragmentos Fc das Imunoglobulinas , Proteínas Recombinantes de Fusão , Tirosina Quinase 3 Semelhante a fms , Humanos , Adulto , Masculino , Feminino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Adulto Jovem , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/farmacologia , Fragmentos Fc das Imunoglobulinas/efeitos adversos , Relação Dose-Resposta a Droga , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Método Duplo-Cego , Infusões IntravenosasRESUMO
OBJECTIVE: To investigate the clinical characteristics and influence of co-mutated gene on acute myeloid leukemia patients (AML) with FMS-like tyrosine kinase-3 (FLT3) mutations. METHODS: A total of 273 FLT3+ AML patients were enrolled, and the co-mutation gene data of the patients were collected to further analyze the prognosis of the patients. FLT3 and other common mutations were quantified by PCR amplification products direct sequencing and second-generation sequencing (NGS). RESULTS: When patients were divided into FLT3- ITD +, FLT3- TKD +, FLT3- ITD ++TKD + and FLT3- ITD -+TKD - group according to the type of FLT3 mutations, it was found that the frequencies of TET2, GATA2, NRAS and ASXL1 mutation were significantly different among the 4 groups (all P < 0.05). When patients were divided into allelic ratio (AR) ≥0.5 and <0.5 group, it was found that the frequencies of FLT3- ITD +, FLT3 -ITD - +TKD -, NPM1, NRAS and C-kit were significantly different between the two groups (all P < 0.05). When patients were divided into normal and abnormal karyotype group, it was found that the frequencies of FLT3- ITD +, FLT3- TKD +, NPM1, GATA2 and C-kit were significantly different between the two groups (all P < 0.05). The median overall survival (OS) of AML patients with FLT3 -TKD + (including FLT3- ITD ++TKD +) was longer than that of patients with FLT3- ITD + alone (P < 0.05). The OS and relapse-free survival (RFS) of AML patients with FLT3++TET2+ were both shorter than those of patients with FLT3++TET2- (both P < 0.05). CONCLUSION: The mutation frequencies of co-mutated genes are correlated with subtypes of FLT3, karyotype and AR. AML patients with FLT3 -TKD + have longer OS than patients with FLT3- ITD + alone, and patients with co-mutation of TET2 have shorter median OS and RFS.
Assuntos
Dioxigenases , GTP Fosfo-Hidrolases , Leucemia Mieloide Aguda , Mutação , Nucleofosmina , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Leucemia Mieloide Aguda/genética , Prognóstico , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA2/genética , Proteínas Repressoras/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
OBJECTIVE: To investigate the expression and clinical significance of long noncoding RNA(lncRNA) HEIH in patients with acute myeloid leukemia (AML). METHODS: 50 newly diagnosed AML patients (except M3) admitted to the First Affiliated Hospital of Bengbu Medical College from January 2019 to December 2020 were included in the study, with 30 patients with non-hematological malignancies as controls. The relative expression level of lncRNA HEIH in all patients were detected, the correlation of clinical characteristics, gene mutations, FAB classification, efficacy, prognosis and overall survival (OS) of AML patients with the expression level of lncRNA HEIH were analyzed. RESULTS: The expression level of lncRNA HEIH in AML patients was significantly higher than that in patients with non-hematological malignancies (P <0.01). Moreover, AML patients with high white blood cell count (WBC), CEBPA and FLT3 mutations, poor efficacy, and poor prognosis often showed higher expression of lncRNA HEIH, and patients with high lncRNA HEIH expression showed a shorter overall survival (OS). CONCLUSION: lncRNA HEIH shows an unique molecular biological significance in AML patients, which may provide a new approach for diagnosis, monitoring and targeted therapy of AML.
Assuntos
Leucemia Mieloide Aguda , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Leucemia Mieloide Aguda/genética , Prognóstico , Mutação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Masculino , Relevância ClínicaRESUMO
OBJECTIVE: To observe the inhibitory effect of dobutamine on proliferation of FLT3-ITD mutated acute myeloid leukemia (AML) cells and explore the feasibility of dobutamine as a monotherapy or in combination with quizartinib for the treatment of this type of AML. METHODS: FLT3-ITD mutant cell lines MOLM13 and MV4-11 were cultured in vitro and divided into control group, dobutamine treatment group, quizartinib treatment group, and dobutamine combined with quizartinib treatment group. Cell viability, ROS levels, and apoptosis rate were detected by CCK-8, Flow cytometry, respectively, as well as the expression of YAP1 protein by Western blot. RESULTS: Both dobutamine and quizartinib inhibited the proliferation of FLT3-ITD mutant AML cell lines. Compared with the control group, the dobutamine group exhibited a significant increase in ROS levels (P < 0.01), an increase in apoptosis rates (P < 0.05), and a decrease in YAP1 protein expression (P < 0.01), and decreased YAP1 expression (P < 0.05). CONCLUSION: Dobutamine as a monotherapy can inhibit theproliferation of FLT3-ITD mutated AML cells, inducing apoptosis. Additionally, the combination of quizartinib enhances the targeted inhibitory effect on FLT3-ITD mutated AML. The mechanism may involve the inhibition of YAP1 protein expression in AML cells of this type, leading to an increase in ROS levels and exerting its anti-tumor effects.
Assuntos
Apoptose , Benzotiazóis , Proliferação de Células , Leucemia Mieloide Aguda , Compostos de Fenilureia , Tirosina Quinase 3 Semelhante a fms , Leucemia Mieloide Aguda/tratamento farmacológico , Humanos , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Linhagem Celular Tumoral , Benzotiazóis/farmacologia , Mutação , Fatores de Transcrição , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Sinalização YAP , Proteínas Adaptadoras de Transdução de Sinal , Espécies Reativas de Oxigênio/metabolismoRESUMO
Next-generation sequencing of samples from patients with acute myeloid leukemia (AML) has revealed several driver gene mutations in adult AML. However, unlike other cancers, AML is defined by relatively few mutations per patient, with a median of 4-5 depending on subtype. In this review, we will discuss the most common driver genes found in patients with AML and focus on the most clinically relevant ones that impact treatment strategies. The most common driver gene mutations in AML occur in NPM1 and FLT3, accounting for ~30% each. There are now targeted therapies being tested or already approved for these driver genes. Menin inhibitors, a novel targeted therapy that blocks the function of the menin protein, are in clinical trials for NPM1 driver gene mutant AML after relapse. A number of FLT3 inhibitors are now approved for FLT3 driver gene mutant AML in combination with chemotherapy in the frontline and also as single agent in relapse. Although mutations in IDH1/2 and TP53 only occur in around 10-20% of patients with AML each, they can affect the treatment strategy due to their association with prognosis and availability of targeted agents. While the impact of other driver gene mutations in AML is recognized, there is a lack of data on the actionable impact of those mutations.
Assuntos
Leucemia Mieloide Aguda , Mutação , Nucleofosmina , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação/genética , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/metabolismo , Terapia de Alvo MolecularRESUMO
OBJECTIVE: Patients who carry NUP98::NSD1 or FLT3/ITD mutations are reported to have poor prognosis. Previous studies have confidently reported that the poor outcome in younger AML patients is owning to dual NUP98::NSD1 and FLT3/ITD positivity, with a high overlap for those two genetic lesions. In this study, we assessed the prognostic value of the presence of both NUP98::NSD1 and FLT3/ITD in pediatric AML patients. METHODS: We screened a large cohort of 885 pediatric cases from the COG-National Cancer Institute (NCI) TARGET AML cohort and found 57 AML patients with NUP98 rearrangements. RESULTS: The frequency of NUP98 gene fusion was 10.8% in 529 patients. NUP98::NSD1 fusion was the most common NUP98 rearrangement, with a frequency of 59.6%(34 of 57). NUP98::NSD1 -positive patients who carried FLT3/ITD mutations had a decreased CR1 or CR2 rate than those patients carried FLT3/ITD mutation alone (P = 0.0001). Moreover, patients harboring both NUP98::NSD1 fusion and FLT3/ITD mutation exhibited inferior event-free survival (EFS, P < 0.001) and overall survival (OS, P = 0.004) than patients who were dual negative for these two genetic lesions. The presence of only NUP98::NSD1 fusion had no significant impact on EFS or OS. We also found that cases with high FLT3/ITD AR levels ( > = 0.5) with or without NUP98::NSD1 had inferior prognosis. Multivariate analysis demonstrated that the presence of both NUP98::NSD1 and FLT3/ITD was an independent prognostic factors for EFS (hazard ratio: 3.2, P = 0.001) in patients with pediatric AML. However, there was no obvious correlation with OS (hazard ratio: 1.3, P = 0.618). Stem cell transplantation did not improve the survival rate of cases with NUP98 fusion or NUP98::NSD1 AML in terms of EFS or OS. CONCLUSION: Presence of both NUP98::NSD1 and FLT3/ITD was found to be an independent factor for dismal prognosis in pediatric AML patients. Notably, lack of FLT3/ITD mutations in NUP98::NSD1 -positive patients did not retain its prognostic value.
Assuntos
Leucemia Mieloide Aguda , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Criança , Feminino , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Masculino , Prognóstico , Pré-Escolar , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Adolescente , Lactente , Proteínas de Fusão Oncogênica/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares/genética , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
BACKGROUND: Approximately 25-30% of patients with acute myeloid leukemia (AML) have FMS-like receptor tyrosine kinase-3 (FLT3) mutations that contribute to disease progression and poor prognosis. Prolonged exposure to FLT3 tyrosine kinase inhibitors (TKIs) often results in limited clinical responses due to diverse compensatory survival signals. Therefore, there is an urgent need to elucidate the mechanisms underlying FLT3 TKI resistance. Dysregulated sphingolipid metabolism frequently contributes to cancer progression and a poor therapeutic response. However, its relationship with TKI sensitivity in FLT3-mutated AML remains unknown. Thus, we aimed to assess mechanisms of FLT3 TKI resistance in AML. METHODS: We performed lipidomics profiling, RNA-seq, qRT-PCR, and enzyme-linked immunosorbent assays to determine potential drivers of sorafenib resistance. FLT3 signaling was inhibited by sorafenib or quizartinib, and SPHK1 was inhibited by using an antagonist or via knockdown. Cell growth and apoptosis were assessed in FLT3-mutated and wild-type AML cell lines via Cell counting kit-8, PI staining, and Annexin-V/7AAD assays. Western blotting and immunofluorescence assays were employed to explore the underlying molecular mechanisms through rescue experiments using SPHK1 overexpression and exogenous S1P, as well as inhibitors of S1P2, ß-catenin, PP2A, and GSK3ß. Xenograft murine model, patient samples, and publicly available data were analyzed to corroborate our in vitro results. RESULTS: We demonstrate that long-term sorafenib treatment upregulates SPHK1/sphingosine-1-phosphate (S1P) signaling, which in turn positively modulates ß-catenin signaling to counteract TKI-mediated suppression of FLT3-mutated AML cells via the S1P2 receptor. Genetic or pharmacological inhibition of SPHK1 potently enhanced the TKI-mediated inhibition of proliferation and apoptosis induction in FLT3-mutated AML cells in vitro. SPHK1 knockdown enhanced sorafenib efficacy and improved survival of AML-xenografted mice. Mechanistically, targeting the SPHK1/S1P/S1P2 signaling synergizes with FLT3 TKIs to inhibit ß-catenin activity by activating the protein phosphatase 2 A (PP2A)-glycogen synthase kinase 3ß (GSK3ß) pathway. CONCLUSIONS: These findings establish the sphingolipid metabolic enzyme SPHK1 as a regulator of TKI sensitivity and suggest that combining SPHK1 inhibition with TKIs could be an effective approach for treating FLT3-mutated AML.
Assuntos
Glicogênio Sintase Quinase 3 beta , Leucemia Mieloide Aguda , Fosfotransferases (Aceptor do Grupo Álcool) , Proteína Fosfatase 2 , beta Catenina , Tirosina Quinase 3 Semelhante a fms , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , beta Catenina/metabolismo , beta Catenina/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Animais , Camundongos , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/antagonistas & inibidores , Linhagem Celular Tumoral , Sorafenibe/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
BACKGROUND: Nucleophosmin 1 (NPM1) gene-mutated acute myeloid leukemia (NPM1mut AML) is classified as a subtype with a favorable prognosis. However, some patients fail to achieve a complete remission or relapse after intensified chemotherapy. Genetic abnormalities in concomitant mutations contribute to heterogeneous prognosis of NPM1mut AML patients. METHODS: In this study, 91 NPM1-mutated and FLT3-ITD wild-type (NPM1mut/FLT3-ITDwt) AML patients with intermediate-risk karyotype were enrolled to analyze the impact of common genetic co-mutations on chemotherapeutic outcome. RESULTS: Our data revealed that TET1/2 (52/91, 57.1%) was the most prevalent co-mutation in NPM1mut AML patients, followed by IDH1/2 (36/91, 39.6%), DNMT3A (35/91, 38.5%), myelodysplastic syndrome related genes (MDS-related genes) (ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 genes) (35/91, 38.5%), FLT3-TKD (27/91, 29.7%) and GATA2 (13/91, 14.3%) mutations. Patients with TET1/2mut exhibited significantly worse relapse-free survival (RFS) (median, 28.7 vs. not reached (NR) months; p = 0.0382) compared to patients with TET1/2wt, while no significant difference was observed in overall survival (OS) (median, NR vs. NR; p = 0.3035). GATA2mut subtype was associated with inferior OS (median, 28 vs. NR months; p < 0.0010) and RFS (median, 24 vs. NR months; p = 0.0224) compared to GATA2wt. By multivariate analysis, GATA2mut and MDS-related genesmut were independently associated with worse survival. CONCLUSION: Mutations in TET1/2, GATA2 and MDS-related genes were found to significantly influence the chemotherapeutic outcome of patients with NPM1mut AML. The findings of our study have significant clinical implications for identifying patients who have an adverse response to frontline chemotherapy and provide a novel reference for further prognostic stratification of NPM1mut/FLT3-ITDwt AML patients.
Assuntos
Leucemia Mieloide Aguda , Mutação , Proteínas Nucleares , Nucleofosmina , Tirosina Quinase 3 Semelhante a fms , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Feminino , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Adulto , Tirosina Quinase 3 Semelhante a fms/genética , Idoso , Prognóstico , Adulto Jovem , Resultado do Tratamento , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
Fms-like tyrosine kinase 3 (FLT3) mutations, present in over 30% of acute myeloid leukemia (AML) cases and dominated by FLT3-internal tandem duplication (FLT3-ITD), are associated with poor outcomes in patients with AML. While tyrosine kinase inhibitors (TKIs; e.g., gilteritinib) are effective, they face challenges such as drug resistance, relapse, and high costs. Here, we report that metformin, a cheap, safe, and widely used anti-diabetic agent, exhibits a striking synergistic effect with gilteritinib in treating FLT3-ITD AML. Metformin significantly sensitizes FLT3-ITD AML cells (including TKI-resistant ones) to gilteritinib. Metformin plus gilteritinib (low dose) dramatically suppresses leukemia progression and prolongs survival in FLT3-ITD AML mouse models. Mechanistically, the combinational treatment cooperatively suppresses polo-like kinase 1 (PLK1) expression and phosphorylation of FLT3/STAT5/ERK/mTOR. Clinical analysis also shows improved survival rates in patients with FLT3-ITD AML taking metformin. Thus, the metformin/gilteritinib combination represents a promising and cost-effective treatment for patients with FLT3-mutated AML, particularly for those with low income/affordability.
Assuntos
Compostos de Anilina , Proteínas de Ciclo Celular , Sinergismo Farmacológico , Leucemia Mieloide Aguda , Metformina , Mutação , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Pirazinas , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms , Metformina/farmacologia , Metformina/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Humanos , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Transdução de Sinais/efeitos dos fármacos , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Camundongos , Mutação/genética , Linhagem Celular Tumoral , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT5/genética , Feminino , Ensaios Antitumorais Modelo de Xenoenxerto , Masculino , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a common mutation type in acute myeloid leukemia (AML) and is usually associated with poor patient prognosis. With advancements in molecular diagnostics and the development of tyrosine kinase inhibitors (TKI), the overall survival (OS) of AML patients with FLT3-ITD mutations has been prolonged to some extent, but relapse and drug resistance are still substantial challenges. Ningetinib is a novel TKI against various kinases in relation to tumour pathogenesis and is undergoing clinical trials of lung cancer. In this study, we explored the antitumor activity of ningetinib against AML with FLT3 mutations both in vivo and in vitro. METHODS: Cell proliferation assays were performed in AML cell lines and Ba/F3 cells expressing various FLT3 mutations to validate the antileukemic activity of ningetinib in vitro. Immunoblot assays were used to verify the effect of ningetinib on the FLT3 protein and downstream pathways. Molecular docking and CETSA were used to validate the interaction of ningetinib with target proteins. The survival benefit of ningetinib in vivo was assessed in Ba/F3-FLT3-ITD-, MOLM13, Ba/F3-FLT3-ITD-F691L-, MOLM13-FLT3-ITD-F691L-induced leukemia mouse models. We also used patient-derived primary cells to determine the efficacy of ningetinib. RESULTS: Ningetinib inhibited cell proliferation, blocked the cell cycle, induced apoptosis and bound FLT3 to inhibit its downstream signaling pathways, including the STAT5, AKT and ERK pathways, in FLT3-ITD AML cell lines. In the mouse models with FLT3-ITD and FLT3-ITD-F691L mutation, ningetinib showed superior anti-leukemia activity to existing clinical drugs gilteritinib and quizartinib, significantly prolongating the survival of mice. In addition, ningetinib exhibited activity against patient-derived primary cells harboring FLT3-ITD mutations. CONCLUSION: Overall, our study confirmed the therapeutic role of ningetinib in AML with FLT3-ITD mutations, providing a potential new option for clinically resistant patients.
Assuntos
Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Tirosina Quinase 3 Semelhante a fms , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Humanos , Animais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Proliferação de Células/efeitos dos fármacos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Apoptose/efeitos dos fármacos , Mutação , Transdução de Sinais/efeitos dos fármacosRESUMO
Cluster of differentiation 99 (CD99) is a receptor that is significantly upregulated in acute myeloid leukemia (AML). FMS-like tyrosine kinase 3 internal tandem duplication mutation in AML (FLT3-ITD AML) exhibits even higher levels of CD99 expression. Our group previously employed a novel peptide platform technology called elastin-like polypeptides and fused it with single-chain antibodies capable of binding to FLT3 (FLT3-A192) or CD99 (CD99-A192). Targeting either FLT3 or CD99 using FLT3-A192 or CD99-A192 led to AML cell death and reduced leukemia burden in AML mouse models. Here, we report on the development of a novel Co-Assembled construct that is capable of binding to both CD99 and FLT3 and the antileukemia activity of the bispecific construct in FLT3-ITD AML preclinical models. This dual-targeting Co-Assembled formulation exhibits cytotoxic effects on AML cells (AML cell lines and primary blasts) and reduced leukemia burden and prolonged survival in FLT3-ITD AML mouse models. Altogether, this study demonstrates the potential of an innovative therapeutic strategy that targets both FLT3 and CD99 in FLT3-ITD AML. SIGNIFICANCE: This study investigates a dual-targeting strategy in acute myeloid leukemia (AML), focusing on FLT3 and CD99. The approach demonstrates enhanced therapeutic potential, presenting a novel option for AML treatment.
Assuntos
Antígeno 12E7 , Anticorpos Biespecíficos , Leucemia Mieloide Aguda , Nanopartículas , Tirosina Quinase 3 Semelhante a fms , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Humanos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Camundongos , Nanopartículas/química , Antígeno 12E7/metabolismo , Antígeno 12E7/genética , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , FemininoRESUMO
During the treatment of 89 pediatric patients with Acute Myeloid Leukemia (AML) at the Hematology Department of Kunming Medical University's Children's Hospital from 2020 to 2023, three patients were identified to co-express the NUP98-NSD1, FLT3-ITD, and WT1 gene mutations. The bone marrow of these three patients was screened for high-risk genetic mutations using NGS and qPCR at the time of diagnosis. The treatment was administered following the China Children's Leukemia Group (CCLG)-AML-2019 protocol. All three patients exhibited a fusion of the NUP98 exon 12 with the NSD1 exon 6 and co-expressed the FLT3-ITD and WT1 mutations; two of the patients displayed normal karyotypes, while one presented chromosomal abnormalities. During the induction phase of the CCLG-AML-2019 treatment protocol, the DAH (Daunorubicin, Cytarabine, and Homoharringtonine) and IAH (Idarubicin, Cytarabine, and Homoharringtonine) regimens, in conjunction with targeted drug therapy, did not achieve remission. Subsequently, the patients were shifted to the relapsed/refractory chemotherapy regimen C + HAG (Cladribine, Homoharringtonine, Cytarabine, and G-CSF) for two cycles, which also failed to induce remission. One patient underwent Haploidentical Hematopoietic Stem Cell Transplantation (Haplo-HSCT) and achieved complete molecular remission during a 12-month follow-up period. Regrettably, the other two patients, who did not receive transplantation, passed away. The therapeutic conclusion is that pediatric AML patients with the aforementioned co-expression do not respond to chemotherapy. Non-remission transplantation, supplemented with tailor-made pre- and post-transplant strategies, may enhance treatment outcomes.
Assuntos
Leucemia Mieloide Aguda , Proteínas de Fusão Oncogênica , Proteínas WT1 , Tirosina Quinase 3 Semelhante a fms , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Tirosina Quinase 3 Semelhante a fms/genética , Masculino , Feminino , Criança , Proteínas de Fusão Oncogênica/genética , Proteínas WT1/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Pré-Escolar , Citarabina/uso terapêutico , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Transplante de Células-Tronco Hematopoéticas , Mepesuccinato de Omacetaxina/uso terapêutico , LactenteRESUMO
INTRODUCTION: Acute myeloid leukemia with normal cytogenetics (CN-AML) represents a heterogeneous group having diverse genetic mutations. Understanding the significance of each of these mutations is necessary. In this study, we evaluated the prognostic role of MN1 expression in adult CN-AML patients. METHOD: One hundred and sixty-three de-novo adult AML patients were evaluated for MN1 expression by real-time PCR. MN1 expression was correlated with the clinical characteristics of the patients and their outcomes. RESULTS: Higher MN1 expression was associated with NPM1 wild-type (p<0.0001), CD34 positivity (p=0.006), and lower clinical remission rate (p=0.027). FLT3-ITD and CEBPA mutations had no association with MN1 expression. On survival analysis, a high MN1 expression was associated with poor event-free survival (Hazard Ratio 2.47, 95% Confidence Interval: 1.42-4.3; p<0.0001) and overall survival (Hazard Ratio 4.18, 95% Confidence Interval: 2.17-8.08; p<0.0001). On multivariate analysis, the MN1 copy number emerged as an independent predictor of EFS (p<0.0001) and OS (p<0.0001). CONCLUSION: MN1 expression is an independent predictor of outcome in CN-AML.
Assuntos
Biomarcadores Tumorais , Leucemia Mieloide Aguda , Nucleofosmina , Transativadores , Proteínas Supressoras de Tumor , Humanos , Masculino , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Feminino , Adulto , Pessoa de Meia-Idade , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Prognóstico , Adulto Jovem , Transativadores/genética , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Taxa de Sobrevida , Seguimentos , Adolescente , Mutação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Medição de Risco , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Idoso de 80 Anos ou maisRESUMO
BACKGROUND: FLT3 gene mutations are genetic abnormality that caused leukemogenesis. Furthermore, presence of FLT3 mutations is associated with poor prognosis in AML. This study aimed to identify FLT3 gene mutations so that it can be used as a genetic reference for the AML patients in Indonesian population. METHODS: This cross-sectional study recruited 63 AML de novo patients between August 2021 and July 2023 at Cipto Mangukusumo General Hospital and Dharmais Cancer Hospital. We collected peripheral blood from the patients for DNA isolation. FLT3 gene mutation was detected using PCR method, then followed by the Sanger sequencing. Novel mutation in exon-14 continued to in silico study using SWISS MODEL server for modelling protein and PyMOL2 software for visualizing the protein model. RESULTS: Frequency FLT3-ITD mutation was 22% and 6 (10%) patients had a novel mutation on juxtamembrane domain. The number of FLT3-ITD insertions was 24 bp to 111 bp, with a median of 72 bp. Novel mutation indicated a change in the protein sequence at amino acid number 572 from Tyrosine to Valine and formed a stop codon (UGA) at amino acid position ins572G573. In-silico study from novel mutation showed the receptor FLT3 protein was a loss of most of the juxtamembrane domain and the entire kinase domain. CONCLUSION: A novel FLT3 gene mutation was found in this study in the juxtamembrane domain. Based on the sequencing analysis and in silico studies, this mutation is likely to affect the activity of the FLT3 receptor. Therefore, further studies on this novel mutation are needed.
Assuntos
Leucemia Mieloide Aguda , Mutação , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Leucemia Mieloide Aguda/genética , Masculino , Feminino , Mutação/genética , Pessoa de Meia-Idade , Adulto , Estudos Transversais , Idoso , Indonésia , Domínios Proteicos/genética , Adulto Jovem , Éxons/genética , AdolescenteRESUMO
Hyperactive FMS-like receptor tyrosine kinase-3 mutants with internal tandem duplications (FLT3-ITD) are frequent driver mutations of aggressive acute myeloid leukemia (AML). Inhibitors of FLT3 produce promising results in rationally designed cotreatment schemes. Since FLT3-ITD modulates DNA replication and DNA repair, valid anti-leukemia strategies could rely on a combined inhibition of FLT3-ITD and regulators of cell cycle progression and DNA integrity. These include the WEE1 kinase which controls cell cycle progression, nucleotide synthesis, and DNA replication origin firing. We investigated how pharmacological inhibition of FLT3 and WEE1 affected the survival and genomic integrity of AML cell lines and primary AML cells. We reveal that promising clinical grade and preclinical inhibitors of FLT3 and WEE1 synergistically trigger apoptosis in leukemic cells that express FLT3-ITD. An accumulation of single and double strand DNA damage precedes this process. Mass spectrometry-based proteomic analyses show that FLT3-ITD and WEE1 sustain the expression of the ribonucleotide reductase subunit RRM2, which provides dNTPs for DNA replication. Unlike their strong pro-apoptotic effects on leukemia cells with FLT3-ITD, inhibitors of FLT3 and WEE1 do not damage healthy human blood cells and murine hematopoietic stem cells. Thus, pharmacological inhibition of FLT3-ITD and WEE1 might become an improved, rationally designed therapeutic option.
Assuntos
Apoptose , Proteínas de Ciclo Celular , Dano ao DNA , Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Proteínas Tirosina Quinases , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Animais , Pirazóis , PirimidinonasRESUMO
A pleiotropic immunoregulatory cytokine, TGF-ß, signals via the receptor-regulated SMADs: SMAD2 and SMAD3, which are constitutively expressed in normal cells. Here, we show that selective repression of SMAD3 induces cDC differentiation from the CD115+ common DC progenitor (CDP). SMAD3 was expressed in haematopoietic cells including the macrophage DC progenitor. However, SMAD3 was specifically down-regulated in CD115+ CDPs, SiglecH- pre-DCs, and cDCs, whereas SMAD2 remained constitutive. SMAD3-deficient mice showed a significant increase in cDCs, SiglecH- pre-DCs, and CD115+ CDPs compared with the littermate control. SMAD3 repressed the mRNA expression of FLT3 and the cDC-related genes: IRF4 and ID2. We found that one of the SMAD transcriptional corepressors, c-SKI, cooperated with phosphorylated STAT3 at Y705 and S727 to repress the transcription of SMAD3 to induce cDC differentiation. These data indicate that STAT3 and c-Ski induce cDC differentiation by repressing SMAD3: the repressor of the cDC-related genes during the developmental stage between the macrophage DC progenitor and CD115+ CDP.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA , Células Dendríticas , Proteínas Proto-Oncogênicas , Fator de Transcrição STAT3 , Proteína Smad3 , Animais , Camundongos , Células Dendríticas/metabolismo , Células Dendríticas/citologia , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad2/genética , Proteína Smad3/metabolismo , Proteína Smad3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.