RESUMO
Chronic lymphocytic leukaemia (CLL) is associated with apoptosis resistance and defective control of cell growth. Our study describes for the first time a critical role in CLL for the KRAB-zinc finger protein ZNF224. High ZNF224 transcript levels were detected in CLL patients with respect to control cells. Moreover, ZNF224 expression was significantly lowered after conventional chemotherapy treatment in a subset of CLL patients. By in vitro experiments we confirmed that ZNF224 expression is suppressed by fludarabine and demonstrated that ZNF224 is involved in apoptosis resistance in CLL cells. Moreover, we showed that ZNF224 positively modulates cyclin D3 gene expression. Consistently, we observed that alteration of ZNF224 expression leads to defects in cell cycle control. All together, our results strongly suggest that in CLL cells high expression level of ZNF224 can lead to inappropriate cell growth and apoptosis resistance, thus contributing to CLL progression. Targeting ZNF224 could thus improve CLL response to therapy.
Assuntos
Ciclina D3/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Repressoras/genética , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D3/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Proteínas Repressoras/biossíntese , Vidarabina/administração & dosagem , Vidarabina/análogos & derivadosRESUMO
Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O2 3%) and mesenchymal stromal cell (MSC) co-culture with multiparameter flow cytometry to enumerate lymphocytes and differentiating (CD11/CD14/CD15+) or leukemic stem cell (LSC)-enriched (GPR56+) cells within the leukemic bulk. After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically (p = 0.02) and molecular (NPM1c MRD, p = 0.04) response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model.
Assuntos
Leucemia Mieloide Aguda , Células-Tronco Mesenquimais , Antraciclinas/metabolismo , Antraciclinas/uso terapêutico , Citarabina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neoplásicas/metabolismoRESUMO
By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.
Assuntos
Cistina , Leucemia Mieloide Aguda , Linhagem Celular Tumoral , Cisteína , Cistina/metabolismo , Cistina/uso terapêutico , Daunorrubicina/farmacologia , Daunorrubicina/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas Nucleares , Sulfassalazina/farmacologia , Sulfassalazina/uso terapêuticoRESUMO
Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.
Assuntos
Leucemia Mieloide Aguda , Proteínas Proto-Oncogênicas c-akt , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Carioferinas/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Estados Unidos , Proteína Exportina 1RESUMO
The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Adenosina Trifosfatases/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Reparo do DNA , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Proteína com ValosinaRESUMO
Deciphering the impact of metabolic intervention on response to anticancer therapy may elucidate a path toward improved clinical responses. Here, we identify amino acid-related pathways connected to the folate cycle whose activation predicts sensitivity to MYC-targeting therapies in acute myeloid leukemia (AML). We establish that folate restriction and deficiency of the rate-limiting folate cycle enzyme MTHFR, which exhibits reduced-function polymorphisms in about 10% of Caucasians, induce resistance to MYC targeting by BET and CDK7 inhibitors in cell lines, primary patient samples, and syngeneic mouse models of AML. Furthermore, this effect is abrogated by supplementation with the MTHFR enzymatic product CH3-THF. Mechanistically, folate cycle disturbance reduces H3K27/K9 histone methylation and activates a SPI1 transcriptional program counteracting the effect of BET inhibition. Our data provide a rationale for screening MTHFR polymorphisms and folate cycle status to nominate patients most likely to benefit from MYC-targeting therapies. SIGNIFICANCE: Although MYC-targeting therapies represent a promising strategy for cancer treatment, evidence of predictors of sensitivity to these agents is limited. We pinpoint that folate cycle disturbance and frequent polymorphisms associated with reduced MTHFR activity promote resistance to BET inhibitors. CH3-THF supplementation thus represents a low-risk intervention to enhance their effects.See related commentary by Marando and Huntly, p. 1791.This article is highlighted in the In This Issue feature, p. 1775.
Assuntos
Ácido Fólico/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resistencia a Medicamentos Antineoplásicos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-myc/biossíntese , Células U937RESUMO
ZNF224 is a KRAB-zinc finger transcription factor that exerts a key tumor suppressive role in chronic myelogenous leukemia. In this study, we identify the receptor tyrosine kinase Axl as a novel target of ZNF224 transcriptional repression activity. Axl overexpression is found in many types of cancer and is frequently associated with drug resistance. Interestingly, we also found that sensitivity to imatinib can be partly restored in imatinib-resistant chronic myelogenous leukemia cells by ZNF224 overexpression and the resulting suppression of Axl expression. These results, in accordance with our previous findings, support the role of ZNF224 in imatinib responsiveness and shed new insights into potential therapeutic use of ZNF224 in imatinib-resistant chronic myelogenous leukemia.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases/química , Proteínas Repressoras/química , Humanos , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Receptor Tirosina Quinase AxlRESUMO
The transcription factor ZNF224 plays a key proapoptotic role in chronic myelogenous leukemia (CML), by modulating Wilms Tumor protein 1 (WT1) dependent apoptotic genes transcription. Recently, we demonstrated that Bcr-Abl signaling represses ZNF224 expression in Bcr-Abl positive CML cell lines and in CML patients. Interestingly, Imatinib and second-generation tyrosine kinase inhibitors specifically increase ZNF224 expression. On the other hand, Bcr-Abl positively modulates, via JAK2 activation, the expression of the c-Myc oncogene, which is required for Bcr-Abl oncogenic transformation in CML. Consequently, JAK2 inhibitors represent promising molecular therapeutic tools in CML. In this work, we demonstrate that ZNF224 is a novel transcriptional repressor of c-Myc in CML. We also show that ZNF224 induction by Imatinib and AG490, a specific JAK2 inhibitor, is responsible for the transcriptional repression of c-MYC, thus highlighting the crucial role of the ZNF224/c-Myc axis in Imatinib responsiveness. Interestingly, we also report that ZNF224 is induced by AG490 in Imatinib-resistant CML cells, leading to c-Myc repression and apoptosis induction. These findings suggest that the development of molecular tools able to induce ZNF224 expression could provide promising means to bypass Imatinib resistance in CML.
RESUMO
The transcription factor interferon regulatory factor-8 (IRF8) is highly expressed in myeloid progenitors, while most myeloid leukemias show low or absent expression. Loss of IRF8 in mice leads to a myeloproliferative disorder, indicating a tumor-suppressive role of IRF8. The Wilms tumor gene 1 (WT1) protein represses the IRF8-promoter. The zinc finger protein ZNF224 can act as a transcriptional co-factor of WT1 and potentiate the cytotoxic response to the cytostatic drug cytarabine. We hypothesized that cytarabine upregulates IRF8 and that transcriptional control of IRF8 involves WT1 and ZNF224. Treatment of leukemic K562 cells with cytarabine upregulated IRF8 protein and mRNA, which was correlated to increased expression of ZNF224. Knock down of ZNF224 with shRNA suppressed both basal and cytarabine-induced IRF8 expression. While ZNF224 alone did not affect IRF8 promoter activity, ZNF224 partially reversed the suppressive effect of WT1 on the IRF8 promoter, as judged by luciferase reporter experiments. Coprecipitation revealed nuclear binding of WT1 and ZNF224, and by chromatin immunoprecipitation (ChIP) experiments it was demonstrated that WT1 recruits ZNF224 to the IRF8 promoter. We conclude that cytarabine-induced upregulation of the IRF8 in leukemic cells involves increased levels of ZNF224, which can counteract the repressive activity of WT1 on the IRF8-promoter.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Fatores Reguladores de Interferon/fisiologia , Leucemia/patologia , Regulação para Cima/efeitos dos fármacos , Proteínas WT1/metabolismo , Dedos de Zinco , Linhagem Celular Tumoral , HumanosRESUMO
The Kruppel-like protein ZNF224 is a co-factor of the Wilms' tumor 1 protein, WT1. We have previously shown that ZNF224 exerts a specific proapoptotic role in chronic myelogenous leukemia (CML) K562 cells and contributes to cytosine arabinoside-induced apoptosis, by modulating WT1-dependent transcription of apoptotic genes. Here we demonstrate that ZNF224 gene expression is down-regulated both in BCR-ABL positive cell lines and in primary CML samples and is restored after imatinib and second generation tyrosine kinase inhibitors treatment. We also show that WT1, whose expression is positively regulated by BCR-ABL, represses transcription of the ZNF224 gene. Finally, we report that ZNF224 is significantly down-regulated in patients with BCR-ABL positive chronic phase-CML showing poor response or resistance to imatinib treatment as compared to high-responder patients. Taken as a whole, our data disclose a novel pathway activated by BCR-ABL that leads to inhibition of apoptosis through the ZNF224 repression. ZNF224 could thus represent a novel promising therapeutic target in CML.