RESUMO
Interleukin-4 (IL-4) is a signature cytokine pivotal in Type 2 helper T cell (Th2) immune response, particularly in allergy and hypersensitivity. Interestingly, IL-4 increases endogenous levels of prostaglandin D2 (PGD2 ) and its metabolites, Δ12 -prostaglandin J2 (Δ12 -PGJ2 ) and 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ), collectively called cyclopentenone PGs (CyPGs). However, the therapeutic role of IL-4 in hematologic malignancies remains unclear. Here, we employed a murine model of acute myeloid leukemia (AML), where human MLL-AF9 fusion oncoprotein was expressed in hematopoietic progenitor cells, to test the effect of IL-4 treatment in vivo. Daily intraperitoneal treatment with IL-4 at 60 µg/kg/d significantly alleviated the severity of AML, as seen by decreased leukemia-initiating cells (LICs). The effect of IL-4 was mediated, in part, by the enhanced expression of hematopoietic- PGD2 synthase (H-PGDS) to effect endogenous production of CyPGs, through autocrine and paracrine signaling mechanisms. Similar results were seen with patient-derived AML cells cultured ex vivo with IL-4. Use of GW9662, a peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, suggested endogenous CyPGs-PPARγ axis mediated p53-dependent apoptosis of LICs by IL-4. Taken together, our results reveal a beneficial role of IL-4 treatment in AML suggesting a potential therapeutic regimen worthy of clinical trials in patients with AML.
Assuntos
Interleucina-4 , Leucemia Mieloide Aguda , Prostaglandina D2 , Animais , Citocinas , Humanos , Interleucina-4/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Camundongos , PPAR gama/metabolismo , Prostaglandina D2/metabolismoRESUMO
Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ceramidas , Citarabina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , SulfonamidasRESUMO
FLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained subpopulation of drug-sensitive cells, so-called drug-tolerant "persisters" (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA sequencing and drug screening, we find that treatment of FLT3 internal tandem duplication AML cells with quizartinib, a selective FLT3 inhibitor, upregulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids (GCs). Mechanistically, the combination of FLT3 inhibitors and GCs enhances cell death of FLT3 mutant, but not wild-type, cells through GC-receptor-dependent upregulation of the proapoptotic protein BIM and proteasomal degradation of the antiapoptotic protein MCL-1. Moreover, the enhanced antileukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and GCs has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3-mutant AML.
Assuntos
Antineoplásicos/uso terapêutico , Glucocorticoides/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2/biossíntese , Proteína 11 Semelhante a Bcl-2/genética , Benzotiazóis/farmacologia , Benzotiazóis/uso terapêutico , Simulação por Computador , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Inflamação/genética , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Seleção Genética , Transcriptoma , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Leukemic stem cells are multipotent, self-renewing, highly proliferative cells that can withstand drug treatments. Although currently available treatments potentially destroy blast cells, they fail to eradicate leukemic progenitor cells completely. Aldehyde dehydrogenase and STAT3 are frequently up-regulated in pre-leukemic stem cells as well as in acute myeloid leukemia (AML) expressing the CD34+CD38- phenotype. The Isatin analog, KS99 has shown anticancer activity against multiple myeloma which may, in part, be mediated by inhibition of Bruton's tyrosine kinase activation. Here we demonstrate that KS99 selectively targets leukemic stem cells with high aldehyde dehydrogenase activity and inhibits phosphorylation of STAT3. KS99 targeted cells co-expressing CD34, CD38, CD123, TIM-3, or CD96 immunophenotypes in AML, alone or in combination with the standard therapeutic agent cytarabine. AML with myelodysplastic-related changes was more sensitive than de novo AML with or without NPM1 mutation. KS99 treatment reduced the clonogenicity of primary human AML cells as compared to normal cord blood mononuclear cells. Downregulation of phosphorylated Bruton's tyrosine kinase, STAT3, and aldehyde dehydrogenase was observed, suggesting interaction with KS99 as predicted through docking. KS99 with or without cytarabine showed in vivo preclinical efficacy in human and mouse AML animal models and prolonged survival. KS99 was well tolerated with overall negligible adverse effects. In conclusion, KS99 inhibits aldehyde dehydrogenase and STAT3 activities and causes cell death of leukemic stem cells, but not normal hematopoietic stem and progenitor cells.
Assuntos
Isatina , Leucemia Mieloide Aguda , Animais , Antígenos CD34 , Citarabina , Subunidade alfa de Receptor de Interleucina-3 , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Células-Tronco Neoplásicas , NucleofosminaRESUMO
Acute myeloid leukemia (AML) is a devastating blood cancer with poor prognosis. Novel effective treatment is an urgent unmet need. Immunotherapy targeting T cell exhaustion by blocking inhibitory pathways, such as PD-1, is promising in cancer treatment. However, results from clinical studies applying PD-1 blockade to AML patients are largely disappointing. AML is highly heterogeneous. Identification of additional immune regulatory pathways and defining predictive biomarkers for treatment response are crucial to optimize the strategy. CD26 is a marker of T cell activation and involved in multiple immune processes. Here, we performed comprehensive phenotypic and functional analyses on the blood samples collected from AML patients and discovered that CD26lowPD-1+ CD8 T cells were associated with AML progression. Specifically, the percentage of this cell fraction was significantly higher in patients with newly diagnosed AML compared to that in patients achieved completed remission or healthy controls. Our subsequent studies on CD26lowPD-1+ CD8 T cells from AML patients at initial diagnosis demonstrated that this cell population highly expressed inhibitory receptors and displayed impaired cytokine production, indicating an exhaustion status. Importantly, CD26lowPD-1+ CD8 T cells carried features of terminal exhaustion, manifested by higher frequency of TEMRA differentiation, increased expression of transcription factors that are observed in terminally exhausted T cells, and high level of intracellular expression of granzyme B and perforin. Our findings suggest a prognostic and predictive value of CD26 in AML, providing pivotal information to optimize the immunotherapy for this devastating cancer.
Assuntos
Leucemia Mieloide Aguda , Receptor de Morte Celular Programada 1 , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Dipeptidil Peptidase 4/metabolismo , Linfócitos T CD8-Positivos , Resultado do TratamentoRESUMO
Relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia-initiating stem cells (LICs) that are typically not targeted by most existing therapies. Using a murine AML model, human AML cell lines, and patient samples, we show that AML LICs are sensitive to endogenous and exogenous cyclopentenone prostaglandin-J (CyPG), Δ12-PGJ2, and 15d-PGJ2, which are increased upon dietary selenium supplementation via the cyclooxygenase-hematopoietic PGD synthase pathway. CyPGs are endogenous ligands for peroxisome proliferator-activated receptor gamma and GPR44 (CRTH2; PTGDR2). Deletion of GPR44 in a mouse model of AML exacerbated the disease suggesting that GPR44 activation mediates selenium-mediated apoptosis of LICs. Transcriptomic analysis of GPR44-/- LICs indicated that GPR44 activation by CyPGs suppressed KRAS-mediated MAPK and PI3K/AKT/mTOR signaling pathways, to enhance apoptosis. Our studies show the role of GPR44, providing mechanistic underpinnings of the chemopreventive and chemotherapeutic properties of selenium and CyPGs in AML.
Assuntos
Leucemia Mieloide Aguda , Selênio , Humanos , Camundongos , Animais , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Linhagem CelularRESUMO
The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the disease motivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for various actin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not being successfully utilized clinically towards cancer treatment. This work establishes the therapeutic activity of ROCK inhibitor (5Z)-2-5-(1H-pyrrolo[2,3-b]pyridine-3-ylmethylene)-1,3-thiazol-4(5H)-one (DJ4) in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors. Herein, DJ4 induced cytotoxic and proapoptotic effects in a dose-dependent manner in human AML cell lines (IC50: 0.05-1.68 µM) and primary patient cells (IC50: 0.264-13.43 µM); however, normal hematopoietic cells were largely spared. ROCK inhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2) and myosin-binding subunit of MLC phosphatase (MYPT), yielding a potent yet selective treatment response at micromolar concentrations, from 0.02 to 1 µM. Murine models injected with luciferase-expressing leukemia cell lines subcutaneously or intravenously and treated with DJ4 exhibited an increase in overall survival and reduction in disease progression relative to the vehicle-treated control mice. Overall, DJ4 is a promising candidate to utilize in future investigations to advance the current AML therapy.
RESUMO
Acute myeloid leukemia (AML) is a disease characterized by uncontrolled proliferation of immature myeloid cells in the blood and bone marrow. The 5-year survival rate is approximately 25%, and recent therapeutic developments have yielded little survival benefit. Therefore, there is an urgent need to identify novel therapeutic targets. We previously demonstrated that acid ceramidase (ASAH1, referred to as AC) is upregulated in AML and high AC activity correlates with poor patient survival. Here, we characterized a novel AC inhibitor, SACLAC, that significantly reduced the viability of AML cells with an EC50 of approximately 3 µmol/L across 30 human AML cell lines. Treatment of AML cell lines with SACLAC effectively blocked AC activity and induced a decrease in sphingosine 1-phosphate and a 2.5-fold increase in total ceramide levels. Mechanistically, we showed that SACLAC treatment led to reduced levels of splicing factor SF3B1 and alternative MCL-1 mRNA splicing in multiple human AML cell lines. This increased proapoptotic MCL-1S levels and contributed to SACLAC-induced apoptosis in AML cells. The apoptotic effects of SACLAC were attenuated by SF3B1 or MCL-1 overexpression and by selective knockdown of MCL-1S. Furthermore, AC knockdown and exogenous C16-ceramide supplementation induced similar changes in SF3B1 level and MCL-1S/L ratio. Finally, we demonstrated that SACLAC treatment leads to a 37% to 75% reduction in leukemic burden in two human AML xenograft mouse models. IMPLICATIONS: These data further emphasize AC as a therapeutic target in AML and define SACLAC as a potent inhibitor to be further optimized for future clinical development.
Assuntos
Ceramidas/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Esfingolipídeos/metabolismo , Idoso , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Isoformas de Proteínas , Transfecção , Células U937 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acute myeloid leukemia is a heterogeneous disease with a 5-year survival rate of 28.3%, and current treatment options constrained by dose-limiting toxicities. One of the key signaling pathways known to be frequently activated and dysregulated in AML is PI3K/AKT. Its dysregulation is associated with aggressive cell growth and drug resistance. We investigated the activity of Phenybutyl isoselenocyanate (ISC-4) in primary cells obtained from newly diagnosed AML patients, diverse AML cell lines, and normal cord blood cells. ISC-4 significantly inhibited survival and clonogenicity of primary human AML cells without affecting normal cells. We demonstrated that ISC-4-mediated p-Akt inhibition caused apoptosis in primary AML (CD34+) stem cells and enhanced efficacy of cytarabine. ISC-4 impeded leukemia progression with improved overall survival in a syngeneic C1498 mouse model with no obvious toxic effects on normal myelopoiesis. In U937 xenograft model, bone marrow cells exhibited significant reduction in human CD45+ cells in ISC-4 (~87%) or AraC (~89%) monotherapy groups compared to control. Notably, combination treatment suppressed the leukemic infiltration significantly higher than the single-drug treatments (~94%). Together, the present findings suggest that ISC-4 might be a promising agent for AML treatment.
RESUMO
BACKGROUND: Successful treatment for acute myeloid leukemia (AML) remains challenging. Inhibiting immune checkpoint to enhance anti-tumor response is an attractive strategy for effective leukemia therapeutics. CD73 is a recently recognized immune checkpoint mediator that is highly expressed on tumor cells and stromal cells in tumor microenvironment. The ectonucleotidase activity of CD73 catalyzes AMP to adenosine, which subsequently inhibits anti-tumor immune responses. In this study, we aim to explore the effect of CD73 in AML. METHODS: Peripheral blood samples collected from patients with newly diagnosed AML (n = 27) were used in this study. CD73 expression on each immune cell component was examined by flow cytometry. Phenotypic study of CD73-expressing T cells and analysis of the correlation between CD73 and other immune checkpoints were performed using flow cytometry-based assays. Functional status of CD73+ vs. CD73- T cells was assessed in an in vitro cytokine release assay upon CD3/CD28 antibody stimulation. RESULTS: In contrast to the long recognized immune suppressive effect of CD73-adenosine signaling in tumor tissue, we made a striking observation that in AML, CD73 expression on CD8 T cells associates with an increased immune response. CD73+ CD8 T cells are more functional, whereas CD73- CD8 T cells exhibit features of exhaustion manifested by high expression of inhibitory receptors such as PD-1 and TIGIT, increased intracellular expression of Eomes, reduced capacity of cytokine production, and high susceptibility to apoptosis. CONCLUSIONS: Our data highlight the potential of CD73 as a double-edged sword in anti-leukemia immunity and argue strongly for the combinational treatment by adding immune checkpoint inhibitors to the CD73-targeting approaches.