RESUMO
Unlike genomic alterations, gene expression profiles have not been widely used to refine cancer therapies. We analyzed transcriptional changes in acute myeloid leukemia (AML) cell lines in response to standard first-line AML drugs cytarabine and daunorubicin by means of RNA sequencing. Those changes were highly cell- and treatment-specific. By comparing the changes unique to treatment-sensitive and treatment-resistant AML cells, we enriched for treatment-relevant genes. Those genes were associated with drug response-specific pathways, including calcium ion-dependent exocytosis and chromatin remodeling. Pharmacological mimicking of those changes using EGFR and MEK inhibitors enhanced the response to daunorubicin with minimum standalone cytotoxicity. The synergistic response was observed even in the cell lines beyond those used for the discovery, including a primary AML sample. Additionally, publicly available cytotoxicity data confirmed the synergistic effect of EGFR inhibitors in combination with daunorubicin in all 60 investigated cancer cell lines. In conclusion, we demonstrate the utility of treatment-evoked gene expression changes to formulate rational drug combinations. This approach could improve the standard AML therapy, especially in older patients.
Assuntos
Leucemia Mieloide Aguda , Humanos , Idoso , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Daunorrubicina/farmacologia , Linhagem Celular , Montagem e Desmontagem da Cromatina , Receptores ErbBRESUMO
Activating KRAS mutations are detected in a substantial number of hematologic malignancies. In a murine T-cell acute lymphoblastic leukemia (T-ALL) model, we previously showed that expression of oncogenic Kras induced a premalignant state accompanied with an arrest in T-cell differentiation and acquisition of somatic Notch1 mutations. These findings prompted us to investigate whether the expression of oncogenic KRAS directly affects DNA damage repair. Applying divergent, but complementary, genetic approaches, we demonstrate that the expression of KRAS mutants is associated with increased expression of DNA ligase 3α, poly(ADP-ribose) polymerase 1 (PARP1), and X-ray repair cross-complementing protein 1 (XRCC1), all essential components of the error-prone, alternative nonhomologous end-joining (alt-NHEJ) pathway. Functional studies revealed delayed repair kinetics, increased misrepair of DNA double-strand breaks, and the preferential use of microhomologous DNA sequences for end joining. Similar effects were observed in primary murine T-ALL blasts. We further show that KRAS-mutated cells, but not KRAS wild-type cells, rely on the alt-NHEJ repair pathway on genotoxic stress. RNA interference-mediated knockdown of DNA ligase 3α abolished resistance to apoptotic cell death in KRAS-mutated cells. Our data indicate that targeting components of the alt-NHEJ pathway sensitizes KRAS-mutated leukemic cells to standard chemotherapeutics and represents a promising approach for inducing synthetic lethal vulnerability in cells harboring otherwise nondruggable KRAS mutations.
Assuntos
Antineoplásicos/farmacologia , Reparo do DNA/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ensaio Cometa , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Transdução GenéticaRESUMO
SIRT1 is an important regulator of cellular stress response and genomic integrity. Its role in tumorigenesis is controversial. Whereas sirtuin 1 (SIRT1) can act as a tumor suppressor in some solid tumors, increased expression has been demonstrated in many cancers, including hematologic malignancies. In chronic myeloid leukemia, SIRT1 promoted leukemia development, and targeting SIRT1 sensitized chronic myeloid leukemia progenitors to tyrosine kinase inhibitor treatment. In this study, we investigated the role of SIRT1 in acute myeloid leukemia (AML). We show that SIRT1 protein, but not RNA levels, is overexpressed in AML samples harboring activating mutations in signaling pathways. In FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)(+)-cells protein, expression of SIRT1 is regulated by FLT3 kinase activity. In addition, SIRT1 function is modulated via the ATM-DBC1-SIRT1 axis in a FLT3-ITD-dependent manner. In murine leukemia models driven by MLL-AF9 or AML1-ETO coexpressing FLT3-ITD, SIRT1 acts as a safeguard to counteract oncogene-induced stress, and leukemic blasts become dependent on SIRT1 activity. Pharmacologic targeting or RNAi-mediated knockdown of SIRT1 inhibited cell growth and sensitized AML cells to tyrosine kinase inhibitor treatment and chemotherapy. This effect was a result of the restoration of p53 activity. Our data suggest that targeting SIRT1 represents an attractive therapeutic strategy to overcome primary resistance in defined subsets of patients with AML.
Assuntos
Dano ao DNA/fisiologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Ativação Enzimática/fisiologia , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Sirtuína 1/genéticaRESUMO
The chimeric monoclonal antibody rituximab is the standard of care for patients with B-cell non-Hodgkin lymphoma (B-NHL). Rituximab mediates complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity of CD20-positive human B cells. In addition, rituximab sensitizes B-NHL cells to cytotoxic chemotherapy and has direct apoptotic and antiproliferative effects. Whereas expression of the CD20 antigen is a natural prerequisite for rituximab sensitivity, cell-autonomous factors determining the response of B-NHL to rituximab are less defined. To this end, we have studied rituximab-induced apoptosis in human B-NHL models. We find that rituximab directly triggers apoptosis via the mitochondrial pathway of caspase activation. Expression of antiapoptotic Bcl-xL confers resistance against rituximab-induced apoptosis in vitro and rituximab treatment of xenografted B-NHL in vivo. B-NHL cells insensitive to rituximab-induced apoptosis exhibit increased endogenous expression of multiple antiapoptotic Bcl-2 family proteins, or activation of phosphatidylinositol-3-kinase signaling resulting in up-regulation of Mcl-1. The former resistance pattern is overcome by treatment with the BH3-mimetic ABT-737, the latter by combining rituximab with pharmacologic phosphatidylinositol-3-kinase inhibitors. In conclusion, sensitivity of B-NHL cells to rituximab-induced apoptosis is determined at the level of mitochondria. Pharmacologic modulation of Bcl-2 family proteins or their upstream regulators is a promising strategy to overcome rituximab resistance.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD20/biossíntese , Apoptose , Regulação Neoplásica da Expressão Gênica , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Anticorpos Monoclonais Murinos , Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides , Transplante de Neoplasias , Nitrofenóis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Piperazinas/farmacologia , Rituximab , Transdução de Sinais , Sulfonamidas/farmacologiaRESUMO
MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essential for MOZ-TIF2-induced leukemic transformation itself. Lack of STAT5 in fetal liver cells caused rapid differentiation and loss of replating capacity of MOZ-TIF2-transduced cells enriched for LSCs. Furthermore, mice serially transplanted with Stat5(-/-) MOZ-TIF2 leukemic cells develop AML with longer disease latency and finally incomplete penetrance when compared with mice transplanted with Stat5(+/+) MOZ-TIF2 leukemic cells. These data suggest that STAT5AB is required for the self-renewal of LSCs and represents a combined signaling node of FLT3-ITD and MOZ-TIF2 driven leukemogenesis. Therefore, targeting aberrantly activated STAT5 or rewired downstream signaling pathways may be a promising therapeutic option.
Assuntos
Transformação Celular Neoplásica/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição STAT5/metabolismo , Animais , Southern Blotting , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais/fisiologiaRESUMO
PURPOSE: Pemetrexed, an inhibitor of thymidylate synthase (TS) and additional folate-dependent enzymes, is clinically active in patients suffering from "non-squamous" non-small cell lung cancer (NSCLC). High expression of TS has been implied as biomarker predictive of resistance to pemetrexed. Against this background, we studied whether inhibition of mTOR could lower expression of TS and thus sensitize NSCLC cells to pemetrexed. METHODS AND RESULTS: Using squamous cell carcinoma and adenocarcinoma NSCLC cell lines, we observed that constitutive TS expression levels failed to correlate with sensitivity to growth inhibition or apoptosis imposed by pemetrexed in vitro. Interestingly, pemetrexed strongly induced TS RNA and protein expression in all cell lines. The allosteric "rapalogue" mTOR inhibitor everolimus suppressed constitutive, but not pemetrexed-induced TS expression. Surprisingly, cotreatment with everolimus protected NSCLC cells against pemetrexed-induced apoptosis. This resulted in increased long-term clonogenic survival of NSCLC cells treated with pemetrexed plus everolimus as compared to pemetrexed alone. No such negative interaction was observed when everolimus was combined with recombinant TRAIL, a proliferation-independent proapoptotic agent. CONCLUSIONS: Rapalogues may suppress the antitumor activity of pemetrexed by slowing cell cycle progression. This should be considered when combining pemetrexed and mTOR inhibitors in NSCLC treatment.
Assuntos
Glutamatos/farmacologia , Guanina/análogos & derivados , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Everolimo , Citometria de Fluxo , Fluoruracila/farmacologia , Guanina/farmacologia , Humanos , Imunossupressores/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/patologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Pemetrexede , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo , Fatores de TempoRESUMO
INTRODUCTION: The oxidoreductase WWOX was initially described as a putative tumor suppressor in breast cancer. Non-small cell lung cancers (NSCLCs) frequently show aberrant WWOX expression. Herein, we characterized WWOX at a functional level in preclinical NSCLC models and in primary NSCLC biopsies. METHODS: The human wild-type (wt) WWOX complementary DNA and a mutant WWOX with structurally disrupted short-chain dehydrogenase/reductase domain were conditionally expressed at physiological levels in several human NSCLC models. Resulting transgenic cell populations were analyzed with respect to clonogenic survival and apoptosis sensitivity in vitro and tumor growth in immune-deficient mice. Tissue microarrays prepared from surgically resected primary human NSCLC tumors were studied to correlate intratumoral WWOX expression with patient outcomes. RESULTS: Conditional expression of wt WWOX, but not mutant WWOX, suppressed clonogenic survival of NSCLC cells in vitro and tumor growth in vivo. In addition, preserved intratumoral WWOX expression was associated with improved outcome in a cohort of 85 patients with surgically resected NSCLC. Unexpectedly, wt WWOX failed to sensitize NSCLC cells to various apoptotic stimuli but robustly protected against apoptosis induced by inhibitors of growth factor signal transduction. CONCLUSIONS: WWOX acts as a tumor suppressor in human NSCLC models in a short-chain dehydrogenase/reductase domain-dependent manner. This activity is independent of sensitization to apoptotic cell death. WWOX expression as detected by immunohistochemistry may be a prognostic biomarker in surgically resected, early-stage NSCLC.
Assuntos
Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/enzimologia , Oxirredutases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/cirurgia , Camundongos , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Análise Serial de Tecidos , Oxidorredutase com Domínios WWRESUMO
BACKGROUND: Patients with inflammatory bowel disease (IBD) have a markedly increased risk to develop colon cancer, but there are only limited data about the host antitumor response in such colitis-associated cancer. In the present study we aimed at assessing the role of perforin-dependent effector mechanisms in the immune response in a murine model of colitis-associated colon cancer. METHODS: Wildtype and perforin-deficient mice were analyzed in a mouse model of colitis-associated colon cancer using azoxymethane (AOM) and dextran sodium sulfate (DSS). RESULTS: Tumors of wildtype mice showed infiltration of CD4+, CD8+ T cells, natural killer (NK) cells, high numbers of apoptotic cells, and expression of the transcription factor eomesodermin and cytotoxic effector proteins, suggesting a potential role of the antitumor immune response in AOM/DSS tumorigenesis. Furthermore, perforin deficiency resulted in reduced apoptosis of epithelial cells as compared to wildtype mice, whereas tumor infiltration by NK cells, CD8+, and CD4+ T cells was unchanged. However, perforin-deficient mice surprisingly developed significantly fewer tumors than wildtype mice. Subsequent studies identified an important role of perforin in regulating colitis activity, as perforin deficiency caused a significant reduction of DSS colitis activity and proinflammatory cytokine production as compared to wildtype controls. CONCLUSIONS: Perforin is involved in both the antitumor immune response and the regulation of activity of mucosal inflammation in colitis-associated cancer. Our data emphasize the possible consequences for therapeutic strategies targeting colitis-associated colon cancer.
Assuntos
Colite/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Proteínas Citotóxicas Formadoras de Poros/deficiência , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença Crônica , Colite/induzido quimicamente , Colite/patologia , Neoplasias do Colo/etiologia , Citotoxicidade Imunológica , Sulfato de Dextrana/toxicidade , Mucosa Intestinal/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Citotóxicas Formadoras de Poros/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , Linfócitos T/imunologiaRESUMO
Promoter hypermethylation preventing expression of the RAS association domain family 1 isoform A (RASSF1A) gene product is among the most abundant epigenetic deregulations in human cancer. Restoration of RASSF1A inhibits tumor cell growth in vitro and in murine xenograft models. Rassf1a-deficient mice feature increased spontaneous and carcinogen-induced tumor formation. Mechanistically, RASSF1A affects several cellular functions, such as microtubule dynamics, migration, proliferation, and apoptosis; however, its tumor-suppressive mechanism is incompletely understood. To study the functional consequences of RASSF1A expression in human cancer cells, we made use of a doxycycline-inducible expression system and a RASSF1A-deficient lung cancer cell line. We observed that RASSF1A induces cell cycle arrest in G(1) phase and senescence in vitro and in tumors established in immunodeficient mice. RASSF1A-mediated growth inhibition was accompanied by the up-regulation of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) and proceeded independently of p53, p14(Arf), and p16(Ink4a). Loss of p21(Cip1/Waf1) or coexpression of the human papilloma virus 16 oncoprotein E7 was found to override RASSF1A-induced cell cycle arrest and senescence. Conditional RASSF1A affected mitogen-activated protein kinase and protein kinase B/Akt signaling to up-regulate p21(Cip1/Waf1) and to facilitate its nuclear localization. In summary, RASSF1A can mediate cell cycle arrest and senescence in human cancer cells by p53-independent regulation of p21(Cip1/Waf1).
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Supressoras de Tumor/fisiologia , Quinases raf/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Ciclo Celular , Senescência Celular , Histonas/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Oncogênicas Virais/genética , Proteínas E7 de PapillomavirusRESUMO
Adoptive cellular immunotherapy inducing a graft-versus-tumor (GVT) effect is the therapeutic mainstay of allogeneic hematopoietic stem cell transplantation (ASCT) for high-risk leukemias. Autologous immunotherapies using vaccines or adoptive transfer of ex vivo-manipulated lymphocytes are clinically explored in patients with various cancer entities. Main reason for failure of ASCT and cancer immunotherapy is progression of the underlying malignancy, which is more prevalent in patients with advanced disease. Elucidating the molecular mechanisms contributing to immune escape will help to develop strategies for the improvement of immunologic cancer treatment. To this end, we have undertaken functional screening and expression cloning of factors mediating resistance to antigen-specific cytotoxic T lymphocytes (CTLs). We have identified Cdc42, a GTPase regulating actin dynamics and growth factor signaling that is highly expressed in invasive cancers, as determinator of cancer cell susceptibility to antigen-specific CTLs in vitro and adoptively transferred immune effectors in vivo. Cdc42 prevents CTL-induced apoptosis via mitogen-activated protein kinase (MAPK) signaling and posttranscriptional stabilization of Bcl-2. Pharmacologic inhibition of MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) overcomes Cdc42-mediated immunoresistance and activation of Bcl-2 in vivo. In conclusion, Cdc42 signaling contributes to immune escape of cancer. Targeting Cdc42 may improve the efficacy of cancer immunotherapies.
Assuntos
Linfócitos/imunologia , Linfócitos/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Proteína cdc42 de Ligação ao GTP/imunologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Apoptose , Sequência de Bases , Células Cultivadas , Citotoxicidade Imunológica/imunologia , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/patologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Linfócitos/citologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Transplante de Neoplasias , Neoplasias/genética , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transcrição Gênica/genética , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
The promise of cancer immunotherapy is long-term disease control with high specificity and low toxicity. However, many cancers fail immune interventions, and secretion of immunosuppressive factors, defective antigen presentation, and expression of death ligands or serpins are regarded as main escape mechanisms. Here, we study whether deregulation of growth and survival factor signaling, which is encountered in most human cancers, provides another level of protection against immunologic tumor eradication. We show in two models that activated cell autonomous protein kinase B (PKB)/AKT signaling mediates resistance against tumor suppression by antigen-specific CTLs in vitro and adoptively transferred cellular immune effectors in vivo. PKB/AKT-dependent immunoresistance of established tumors is reversed by genetic suppression of endogenous Mcl-1, an antiapoptotic member of the Bcl-2 family. Mechanistically, deregulated PKB/AKT stabilizes Mcl-1 expression in a mammalian target of rapamycin (mTOR)-dependent pathway. Treatment with the mTOR inhibitor rapamycin effectively sensitizes established cancers to adoptive immunotherapy in vivo. In conclusion, cancer cell-intrinsic PKB/AKT signaling regulates the susceptibility to immune-mediated cytotoxicity. Combined targeting of signal transduction pathways may be critical for improvement of cancer immunotherapies.