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1.
Eur J Haematol ; 105(3): 247-254, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32311143

RESUMO

BACKGROUND: Androgens function through DNA and non-DNA binding-dependent signalling of the androgen receptor (AR). How androgens promote erythropoiesis is not fully understood. DESIGN AND METHODS: To identify the androgen signalling pathway, we treated male mice lacking the second zinc finger of the DNA-binding domain of the AR (ARΔZF2 ) with non-aromatizable 5α-dihydrotestosterone (5α-DHT) or aromatizable testosterone. To distinguish direct hematopoietic and non-hematopoietic mechanisms, we performed bone marrow reconstitution experiments. RESULTS: In wild-type mice, 5α-DHT had greater erythroid activity than testosterone, which can be aromatized to estradiol. The erythroid response in wild-type mice following 5α-DHT treatment was associated with increased serum erythropoietin (EPO) and its downstream target erythroferrone, and hepcidin suppression. 5α-DHT had no erythroid activity in ARΔZF2 mice, proving the importance of DNA binding by the AR. Paradoxically, testosterone, but not 5α-DHT, suppressed EPO levels in ARΔZF2 mice, suggesting testosterone following aromatization may oppose the erythroid-stimulating effects of androgens. Female wild-type mice reconstituted with ARΔZF2 bone marrow cells remained responsive to 5α-DHT. In contrast, ARΔZF2 mice reconstituted with female wild-type bone marrow cells showed no response to 5α-DHT. CONCLUSION: Erythroid promoting effects of androgens are mediated through DNA binding-dependent actions of the AR in non-hematopoietic cells, including stimulating EPO expression.


Assuntos
Androgênios/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eritropoese , Receptores Androgênicos/metabolismo , Androgênios/farmacologia , Animais , Biomarcadores , Eritropoese/efeitos dos fármacos , Eritropoetina/sangue , Feminino , Regulação da Expressão Gênica , Ferro/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Ligação Proteica , Receptores Androgênicos/genética , Transdução de Sinais
2.
Blood ; 129(21): 2882-2895, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28283481

RESUMO

Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs.


Assuntos
Benzotiazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Naftiridinas/farmacologia , Células-Tronco Neoplásicas/enzimologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Fase G2/efeitos dos fármacos , Fase G2/genética , Humanos , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Mutantes , Células-Tronco Neoplásicas/patologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
3.
Blood ; 125(18): 2815-24, 2015 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-25736313

RESUMO

Phosphoinositide signaling regulates diverse cellular functions. Phosphoinositide-3 kinase (PI3K) generates PtdIns(3,4,5)P3 and PtdIns(3,4)P2, leading to the activation of proliferative and anti-apoptotic signaling pathways. Termination of phosphoinositide signaling requires hydrolysis of inositol ring phosphate groups through the actions of PtdIns(3,4,5)P3 3-phosphatase (PTEN), PtdIns(3,4,5)P3 5-phosphatases (eg, SHIP), and PtdIns(3,4)P2 4-phosphatases (eg, INPP4B). The biological relevance of most of these phosphoinositide phosphatases in acute myeloid leukemia (AML) remains poorly understood. Mass spectrometry-based gene expression profiling of 3-, 4- and 5-phosphatases in human AML revealed significant overexpression of INPP4B. Analysis of an expanded panel of 205 AML cases at diagnosis revealed INPP4B overexpression in association with reduced responses to chemotherapy, early relapse, and poor overall survival, independent of other risk factors. Ectopic overexpression of INPP4B conferred leukemic resistance to cytosine arabinoside (ara-C), daunorubicin, and etoposide. Expression of a phosphatase inert variant (INPP4B C842A) failed to abrogate resistance of AML cells to chemotherapy in vitro or in vivo. In contrast, targeted suppression of endogenously overexpressed INPP4B by RNA interference sensitized AML cell lines and primary AML to chemotherapy. These findings demonstrate a previously unsuspected and clinically relevant role for INPP4B gain of function as a mediator of chemoresistance and poor survival outcome in AML independent of its phosphoinositide phosphatase function.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Monoéster Fosfórico Hidrolases/fisiologia , Adolescente , Adulto , Idoso , Regulação Leucêmica da Expressão Gênica , Estudos de Associação Genética , Humanos , Leucemia Mieloide Aguda/mortalidade , Pessoa de Meia-Idade , Monoéster Fosfórico Hidrolases/genética , Polimorfismo de Nucleotídeo Único , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Transcriptoma , Resultado do Tratamento , Adulto Jovem
4.
Blood ; 122(5): 738-48, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23775716

RESUMO

Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds that downregulate Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked proapoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, rapid loss of Mcl-1 protein, and alleviation of its inhibition of proapoptotic Bak. PIK-75 also targets the p110α isoform of PI3K, which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75-mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa, or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to cotarget PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted.


Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células Cultivadas , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HL-60 , Humanos , Hidrazonas/uso terapêutico , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulfonamidas/uso terapêutico , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Blood ; 117(20): 5362-71, 2011 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-21421839

RESUMO

In a recessive ENU mutagenesis screen for embryonic lethality, we identified a mouse pedigree with a missense mutation of SHIP1 (SHIP1(el20)) leading to an amino acid substitution I641T in the inositol-5'-phosphatase domain that represses phosphatidylinositol-3-kinase signaling. Despite detectable expression of functional SHIP1 protein, the phenotype of homozygous SHIP1(el20/el20) mice was more severe than gene-targeted SHIP1-null (SHIP1(-/-)) mice. Compared with age-matched SHIP1(-/-) mice, 5-week-old SHIP1(el20/el20) mice had increased myeloid cells, serum IL-6 levels, marked reductions in lymphoid cells, and died by 7 weeks of age with infiltration of the lungs by activated macrophages. Bone marrow transplantation demonstrated that these defects were hematopoietic-cell-autonomous. We show that the el20 mutation reduces expression in SHIP1(el20/el20) macrophages of both SHIP1 and s-SHIP, an isoform of SHIP1 generated by an internal promoter. In contrast, SHIP1(-/-) macrophages express normal levels of s-SHIP. Compound heterozygous mice (SHIP1(-/el20)) had the same phenotype as SHIP1(-/-) mice, thus providing genetic proof that the more severe phenotype of SHIP1(el20/el20) mice is probably the result of concomitant loss of SHIP1 and s-SHIP. Our results suggest that s-SHIP synergizes with SHIP1 for suppression of macrophage activation, thus providing the first evidence for a role of s-SHIP in adult hematopoiesis.


Assuntos
Ativação de Macrófagos/genética , Ativação de Macrófagos/fisiologia , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/fisiologia , Substituição de Aminoácidos , Animais , Sequência de Bases , Transplante de Medula Óssea , Primers do DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Etilnitrosoureia , Feminino , Genes Recessivos , Hematopoese/genética , Hematopoese/fisiologia , Homozigoto , Inositol Polifosfato 5-Fosfatases , Interleucina-6/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Mutagênese , Mutação de Sentido Incorreto , Fenótipo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/deficiência , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Transdução de Sinais
6.
Blood ; 114(5): 995-1004, 2009 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-19483124

RESUMO

Hedgehog (Hh) ligands bind to the Patched1 (Ptch1) receptor, relieving repression of Smoothened, which leads to activation of the Hh signaling pathway. Using conditional Ptch1 knockout mice, the aim of this study was to determine the effects of activating the Hh signaling pathway in hematopoiesis. Surprisingly, hematopoietic-specific deletion of Ptch1 did not lead to activation of the Hh signaling pathway and, consequently, had no phenotypic effect. In contrast, deletion of Ptch1 in nonhematopoietic cells produced 2 distinct hematopoietic phenotypes. First, activation of Hh signaling in epithelial cells led to apoptosis of lymphoid progenitors associated with markedly elevated levels of circulating thymic stromal lymphopoietin. Second, activation of Hh signaling in the bone marrow cell niche led to increased numbers of lineage-negative c-kit(+) Sca-1(+) bone marrow cells and mobilization of myeloid progenitors associated with a marked loss of osteoblasts. Thus, deletion of Ptch1 leads to hematopoietic effects by distinct cell-extrinsic mechanisms rather than by direct activation of the Hh signaling pathway in hematopoietic cells. These findings have important implications for therapeutics designed to activate the Hh signaling pathway in hematopoietic cells including hematopoietic stem cells.


Assuntos
Proteínas Hedgehog/fisiologia , Linfócitos/citologia , Linfopoese/fisiologia , Células Mieloides/citologia , Mielopoese/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Transplante de Medula Óssea , Linhagem da Célula , Citocinas/sangue , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Linfócitos/metabolismo , Linfopoese/genética , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/metabolismo , Mielopoese/genética , Osteoblastos/citologia , Receptores Patched , Receptor Patched-1 , Quimera por Radiação , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened , Linfopoietina do Estroma do Timo
7.
Mol Ther Oncolytics ; 20: 325-341, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33614914

RESUMO

Chimeric antigen receptor (CAR) T cells have revolutionized blood cancer immunotherapy; however, their efficacy against solid tumors has been limited. A common mechanism of tumor escape from single target therapies is downregulation or mutational loss of the nominal epitope. Targeting multiple antigens may thus improve the effectiveness of CAR immunotherapies. We generated dual CAR-T cells targeting two tumor antigens: TAG-72 (tumor-associated glycoprotein 72) and CD47. TAG-72 is a pan-adenocarcinoma oncofetal antigen, highly expressed in ovarian cancers, with increased expression linked to disease progression. CD47 is ubiquitously overexpressed in multiple tumor types, including ovarian cancer; it is a macrophage "don't eat me" signal. However, CD47 is also expressed on many normal cells. To avoid this component of the dual CAR-T cells killing healthy tissue, we designed a truncated CD47 CAR devoid of intracellular signaling domains. The CD47 CAR facilitates binding to CD47+ cells, increasing the prospect of TAG-72+ cell elimination via the TAG-72 CAR. Furthermore, we could reduce the damage to normal tissue by monomerizing the CD47 CAR. Our results indicate that the co-expression of the TAG-72 CAR and the CD47-truncated monomer CAR on T cells could be an effective, dual CAR-T cell strategy for ovarian cancer, also applicable to other adenocarcinomas.

8.
Cancer Cell ; 29(2): 145-58, 2016 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-26859455

RESUMO

Birinapant is a smac-mimetic (SM) in clinical trials for treating cancer. SM antagonize inhibitor of apoptosis (IAP) proteins and simultaneously induce tumor necrosis factor (TNF) secretion to render cancers sensitive to TNF-induced killing. To enhance SM efficacy, we screened kinase inhibitors for their ability to increase TNF production of SM-treated cells. We showed that p38 inhibitors increased TNF induced by SM. Unexpectedly, even though p38 is required for Toll-like receptors to induce TNF, loss of p38 or its downstream kinase MK2 increased induction of TNF by SM. Hence, we show that the p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Importantly, clinical p38 inhibitors overcame resistance of primary acute myeloid leukemia to birinapant.


Assuntos
Antineoplásicos/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Leucemia/tratamento farmacológico , Proteínas Mitocondriais/fisiologia , Mimetismo Molecular , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose , Humanos , Camundongos , Fator de Necrose Tumoral alfa/biossíntese
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