RESUMO
CRISPR Cas9-based screening is a powerful approach for identifying and characterizing novel drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacologic inhibition of USP1 leads to decreased DNA synthesis concomitant with S-phase-specific DNA damage. Genome-wide CRISPR-Cas9 screens identify RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition is associated with reduced PCNA protein levels. Ectopic expression of WT or ubiquitin-dead K164R PCNA reverses USP1 inhibitor sensitivity. Our results show, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to selected BRCA1/2 WT cancer cell lines enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant tumors. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to posttranslational modifications of PCNA. Taken together, USP1 inhibition may represent a novel therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.
Assuntos
Neoplasias , Mutações Sintéticas Letais , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina/genética , Ubiquitinação , Dano ao DNA , Neoplasias/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
We investigated adhesion pathways that contribute to engraftment of breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1)-induced chronic myelogenous leukemia (CML)-like myeloproliferative neoplasia in a mouse retroviral transduction/transplantation model. Compared with normal stem/progenitor cells, BCR-ABL1(+) progenitors had similar expression of very late antigen-4 (VLA4), VLA5, leukocyte functional antigen-1, and CXCR4 but lower expression of P-selectin glycoprotein ligand-1 (PSGL-1) and of L-selectin. Whereas vascular cell adhesion molecule-1 and P-selectin were not required, deficiency of E-selectin in the recipient bone marrow endothelium significantly reduced engraftment by BCR-ABL1-expressing stem cells following intravenous injection, with leukemogenesis restored by direct intrafemoral injection. BCR-ABL1-expressing cells deficient for PSGL-1 or the selectin ligand-synthesizing enzymes core-2 ß1,6-N-acetylglucosaminyltransferase or fucosyltransferases IV/VII were impaired for engraftment, and destruction of selectin ligands on leukemic progenitors by neuraminidase reduced engraftment. BCR-ABL1-expressing L-selectin-deficient progenitors were also defective in homing and engraftment, with leukemogenesis rescued by coexpression of chimeric E/L-selectin. Antibody to L-selectin decreased the engraftment of BCR-ABL1-transduced stem cells. These results establish that BCR-ABL1(+) leukemic stem cells rely to a greater extent on selectins and their ligands for homing and engraftment than do normal stem cells. Selectin blockade is a novel strategy to exploit differences between normal and leukemic stem cells that may be beneficial in autologous transplantation for CML and perhaps other leukemias.
Assuntos
Medula Óssea/patologia , Movimento Celular/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/fisiologia , Selectinas/fisiologia , Nicho de Células-Tronco , Animais , Células da Medula Óssea/patologia , Células Cultivadas , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Células HEK293 , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the Stat5a/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2(V617F) in retroviral transplantation models of CML and PV. Loss of one Stat5a/b allele resulted in a decrease in BCR-ABL1-induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of Stat5a/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2(V617F) failed to induce polycythemia in recipients after deletion of Stat5a/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5a/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2(V617F), and validate STAT5a/b and the genes they regulate as targets for therapy in these MPNs.
Assuntos
Genes abl/fisiologia , Janus Quinase 2/fisiologia , Transtornos Mieloproliferativos/genética , Fator de Transcrição STAT5/fisiologia , Substituição de Aminoácidos , Animais , Neoplasias da Medula Óssea/genética , Neoplasias da Medula Óssea/metabolismo , Neoplasias da Medula Óssea/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Genes abl/genética , Células HEK293 , Humanos , Janus Quinase 2/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Mutação de Sentido Incorreto/fisiologia , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Células NIH 3T3 , Fenilalanina/genética , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Valina/genéticaRESUMO
Among hematologic neoplasms, chronic myeloid leukemia (CML) is exquisitely sensitive to graft-versus-leukemia (GVL) because patients relapsing after allogeneic hematopoietic stem-cell transplantation (alloHSCT) can be cured by donor leukocyte infusion (DLI); however, the cellular mechanisms and strategies to separate GVL from GVHD are unclear. We used a BCR-ABL1 transduction/transplantation mouse model to study the mechanisms of DLI in MHC-matched, minor histocompatibility antigen-mismatched allogeneic chimeras with CML-like leukemia, in which DLI can be administered at the time of transplantation (early) or after recovery of hematopoiesis (delayed). After early DLI, CML-like leukemia cannot be transferred into immunocompetent secondary recipients as soon as 4 days after primary transplantation, demonstrating that cotransplantation of T lymphocytes blocks the engraftment of BCR-ABL1-transduced stem cells. In contrast, in allogeneic chimeras with established CML-like leukemia, combined treatment with delayed DLI and the kinase inhibitor imatinib eradicates leukemia with minimal GVHD. The GVL effect is directed against minor histocompatibility antigens shared by normal and leukemic stem cells, and is mediated predominantly by CD8+ T cells, with minor contributions from CD5- splenocytes, including natural killer cells. These results define a physiologic model of adoptive immunotherapy of CML that will be useful for investigating the cellular and molecular mechanisms of GVL.
Assuntos
Transplante de Medula Óssea , Modelos Animais de Doenças , Efeito Enxerto vs Leucemia/imunologia , Imunoterapia Adotiva , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Transfusão de Leucócitos , Animais , Southern Blotting , DNA/genética , Proteínas de Ligação a DNA/fisiologia , Feminino , Citometria de Fluxo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Técnicas In Vitro , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Knockout , Reação em Cadeia da Polimerase , Taxa de Sobrevida , Linfócitos T/imunologia , Doadores de Tecidos , Transplante HomólogoRESUMO
Protease-activated receptor-2 (PAR2), a cell surface receptor for trypsin-like proteases, plays a key role in a number of acute and chronic inflammatory diseases of the joints, lungs, brain, gastrointestinal tract, and vascular systems. Despite considerable effort by the pharmaceutical industry, PAR2 has proven recalcitrant to targeting by small molecule inhibitors, which have been unable to effectively prevent the interaction of the protease-generated tethered ligand with the body of the receptor. Here, we report the development of first-in-class cell-penetrating lipopeptide "pepducin" antagonists of PAR2. The design of the third intracellular (i3) loop pepducins were based on a structural model of a PAR2 dimer and by mutating key pharmacophores in the receptor intracellular loops and analogous pepducins. Individual pharmacophores were identified, which controlled constitutive, agonist, and antagonist activities. This approach culminated in the identification of the P2pal-18S pepducin which completely suppressed trypsin and mast cell tryptase signaling through PAR2 in neutrophils and colon cancer cells. The PAR2 pepducin was highly efficacious in blocking PAR2-dependent inflammatory responses in mouse models. These effects were lost in PAR2-deficient and mast-cell-deficient mice, thereby validating the specificity of the pepducin in vivo. These data provide proof of concept that PAR2 pepducin antagonists may afford effective treatments of potentially debilitating inflammatory diseases and serve as a blueprint for developing highly potent and specific i3-loop-based pepducins for other G protein-coupled receptors (GPCRs).
Assuntos
Inflamação/tratamento farmacológico , Lipopeptídeos/farmacologia , Receptor PAR-2/antagonistas & inibidores , Animais , Neoplasias do Colo/metabolismo , Inflamação/etiologia , Inflamação/prevenção & controle , Lipopeptídeos/síntese química , Lipopeptídeos/uso terapêutico , Mastócitos , Camundongos , Neutrófilos , Receptor PAR-2/fisiologia , Receptores Acoplados a Proteínas G , Transdução de Sinais/efeitos dos fármacos , Tripsina/efeitos dos fármacos , Triptases/antagonistas & inibidores , Triptases/efeitos dos fármacosRESUMO
Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for patients with chronic myeloid leukemia (CML). Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead "switch-control" inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML-resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1(T315I)-expressing cell lines, prolongs survival in mouse models of T315I mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph(+) leukemia.
Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Proteínas de Fusão bcr-abl/química , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Proteínas Tirosina Quinases/químicaRESUMO
Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer.
Assuntos
Ascite/enzimologia , Metaloproteases/antagonistas & inibidores , Neovascularização Patológica/enzimologia , Oligopeptídeos/farmacologia , Neoplasias Ovarianas/patologia , Receptor PAR-1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Líquidos Corporais/enzimologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Docetaxel , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Invasividade Neoplásica , Oligopeptídeos/administração & dosagem , Oligopeptídeos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Peritônio/enzimologia , Peritônio/patologia , Taxoides/administração & dosagem , Taxoides/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: A somatic activating mutation (V617F) in the JAK2 tyrosine kinase was recently discovered in the majority of patients with polycythemia vera (PV), and some with essential thrombocythemia (ET) and chronic idiopathic myelofibrosis. However, the role of mutant JAK2 in disease pathogenesis is unclear. METHODS AND FINDINGS: We expressed murine JAK2 WT or V617F via retroviral bone marrow transduction/transplantation in the hematopoietic system of two different inbred mouse strains, Balb/c and C57Bl/6 (B6). In both strains, JAK2 V617F, but not JAK2 WT, induced non-fatal polycythemia characterized by increased hematocrit and hemoglobin, reticulocytosis, splenomegaly, low plasma erythropoietin (Epo), and Epo-independent erythroid colonies. JAK2 V617F also induced leukocytosis and neutrophilia that was much more prominent in Balb/c mice than in B6. Platelet counts were not affected in either strain despite expression of JAK2 V617F in megakaryocytes and markedly prolonged tail bleeding times. The polycythemia tended to resolve after several months, coincident with increased spleen and marrow fibrosis, but was resurrected by transplantation to secondary recipients. Using donor mice with mutations in Lyn, Hck, and Fgr, we demonstrated that the polycythemia was independent of Src kinases. Polycythemia and reticulocytosis responded to treatment with imatinib or a JAK2 inhibitor, but were unresponsive to the Src inhibitor dasatinib. CONCLUSIONS: These findings demonstrate that JAK2 V617F induces Epo-independent expansion of the erythroid lineage in vivo. The fact that the central erythroid features of PV are recapitulated by expression of JAK2 V617F argues that it is the primary and direct cause of human PV. The lack of thrombocytosis suggests that additional events may be required for JAK2 V617F to cause ET, but qualitative platelet abnormalities induced by JAK2 V617F may contribute to the hemostatic complications of PV. Despite the role of Src kinases in Epo signaling, our studies predict that Src inhibitors will be ineffective for therapy of PV. However, we provide proof-of-principle that a JAK2 inhibitor should have therapeutic effects on the polycythemia, and perhaps myelofibrosis and hemostatic abnormalities, suffered by MPD patients carrying the JAK2 V617F mutation.
Assuntos
Janus Quinase 2/genética , Mutação Puntual , Policitemia Vera/enzimologia , Policitemia Vera/genética , Substituição de Aminoácidos , Animais , Transplante de Medula Óssea , Modelos Animais de Doenças , Ativação Enzimática , Eritropoese , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Leucocitose/sangue , Leucocitose/etiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contagem de Plaquetas , Policitemia Vera/sangue , Policitemia Vera/tratamento farmacológico , Mielofibrose Primária/etiologia , Mielofibrose Primária/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução GenéticaRESUMO
In individuals with chronic myeloid leukemia (CML) treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft contribute to leukemic relapse, but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Here we show that CD44 expression is increased on mouse stem-progenitor cells expressing BCR-ABL and that CD44 contributes functional E-selectin ligands. In a mouse retroviral transplantation model of CML, BCR-ABL1-transduced progenitors from CD44-mutant donors are defective in homing to recipient marrow, resulting in decreased engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engraft as efficiently as do wild-type HSCs. CD44 is dispensable for induction of acute B-lymphoblastic leukemia by BCR-ABL, indicating that CD44 is specifically required on leukemic cells that initiate CML. The requirement for donor CD44 is bypassed by direct intrafemoral injection of BCR-ABL1-transduced CD44-deficient stem cells or by coexpression of human CD44. Antibody to CD44 attenuates induction of CML-like leukemia in recipients. These results show that BCR-ABL-expressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML.