RESUMO
The multiplexed cancer cell line screening platform PRISM demonstrated its utility in testing hundreds of cell lines in a single run, possessing the potential to speed up anti-cancer drug discovery, validation and optimization. Here we described the development and implementation of a next-generation PRISM platform combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing, cell line DNA barcoding and next-generation sequencing to enable genetic and/or pharmacological assessment of target addiction in hundreds of cell lines simultaneously. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with a public database.
Assuntos
Antineoplásicos/farmacologia , Sistemas CRISPR-Cas , Detecção Precoce de Câncer/métodos , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células HEK293 , Humanos , Neoplasias/diagnósticoRESUMO
Thalidomide analogs exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2-FGFR1 and ZMYM2-FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment.
Assuntos
Neoplasias Hematológicas , Talidomida , Proteínas de Ligação a DNA , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Lenalidomida/farmacologia , Proteínas Oncogênicas , Fatores de Transcrição/metabolismoRESUMO
CC-122 is a next-generation cereblon E3 ligase-modulating agent that has demonstrated promising clinical efficacy in patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Mechanistically, CC-122 induces the degradation of IKZF1/3, leading to T-cell activation and robust cell-autonomous killing in DLBCL. We report a genome-wide CRISPR/Cas9 screening for CC-122 in a DLBCL cell line SU-DHL-4 with follow-up mechanistic characterization in 6 DLBCL cell lines to identify genes regulating the response to CC-122. Top-ranked CC-122 resistance genes encode, not only well-defined members or regulators of the CUL4/DDB1/RBX1/CRBN E3 ubiquitin ligase complex, but also key components of signaling and transcriptional networks that have not been shown to modulate the response to cereblon modulators. Ablation of CYLD, NFKBIA, TRAF2, or TRAF3 induces hyperactivation of the canonical and/or noncanonical NF-κB pathways and subsequently diminishes CC-122-induced apoptosis in 5 of 6 DLBCL cell lines. Depletion of KCTD5, the substrate adaptor of the CUL3/RBX1/KCTD5 ubiquitin ligase complex, promotes the stabilization of its cognate substrate, GNG5, resulting in CC-122 resistance in HT, SU-DHL-4, and WSU-DLCL2. Furthermore, knockout of AMBRA1 renders resistance to CC-122 in SU-DHL-4 and U-2932, whereas knockout of RFX7 leads to resistance specifically in SU-DHL-4. The ubiquitous and cell line-specific mechanisms of CC-122 resistance in DLBCL cell lines revealed in this work pinpoint genetic alternations that are potentially associated with clinical resistance in patients and facilitate the development of biomarker strategies for patient stratification, which may improve clinical outcomes of patients with R/R DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B , Piperidonas , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Canais de Potássio , Quinazolinonas , Ubiquitina-Proteína LigasesRESUMO
A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).
Assuntos
Acetamidas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Isoindóis/farmacologia , Leucemia Mieloide Aguda/patologia , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Piperidonas/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Acetamidas/uso terapêutico , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Humanos , Isoindóis/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Células-Tronco Neoplásicas/enzimologia , Proteína do Fator Nuclear 45/fisiologia , Proteínas do Fator Nuclear 90/fisiologia , Fatores de Terminação de Peptídeos/metabolismo , Piperidonas/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise , Bibliotecas de Moléculas Pequenas , Estresse Fisiológico , Serina-Treonina Quinases TOR/fisiologia , Células U937 , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Gaucher disease (GD) manifests heterogeneously and other conditions are often misdiagnosed in its place, leading to diagnostic delays. The Gaucher Earlier Diagnosis Consensus (GED-C) initiative proposed a point-scoring system (PSS) based on the signs and covariables that are most indicative of GD to help clinicians identify which individuals to test for GD. AIMS: To validate the PSS retrospectively in a test population including patients with GD and other conditions with overlapping manifestations. METHODS: Four cohorts of adults with GD, liver disease, haematological malignancy or immune thrombocytopenia were identified from hospital records. Clinical data were audited for GED-C factors identified as potentially indicative of GD and aggregate scores calculated (sum of scores/number of factors) based on published PSS weightings. Threshold discriminatory PSS scores, sensitivity and specificity were determined by receiver-operating characteristic analysis. RESULTS: Among 100 patients (GD, n = 25; non-GD, n = 75), analyses based on 11 possible factors estimated group mean (standard deviation) PSS scores of: GD (n = 14), 1.08 (0.25); non-GD (n = 38), 0.58 (0.31). Mean between-group difference (95% confidence interval) was -0.49 (-0.68, -0.31) and area under the receiver-operating characteristic analysis curve (95% confidence interval) was 0.88 (0.78, 0.97). A threshold PSS score of 0.82 identified all 14 patients with GD in the analysis set (100% sensitivity) and 27 of 38 patients in the non-GD group (71% specificity). Patients with liver disease and haematological malignancy were most likely to have manifestations overlapping GD. CONCLUSIONS: Preliminary validation of the GED-C PSS discriminated effectively between patients with GD and those with overlapping signs.
Assuntos
Doença de Gaucher , Adulto , Diagnóstico Precoce , Doença de Gaucher/diagnóstico , Doença de Gaucher/epidemiologia , Humanos , Curva ROC , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: The PRoposing Early Disease Indicators for Clinical Tracking in Fabry Disease (PREDICT-FD) initiative aimed to reach consensus among a panel of global experts on early indicators of disease progression that may justify FD-specific treatment initiation. DESIGN AND SETTING: Anonymous feedback from panellists via online questionnaires was analysed using a modified Delphi consensus technique. Questionnaires and data were managed by an independent administrator directed by two non-voting cochairs. First, possible early indicators of renal, cardiac and central/peripheral nervous system (CNS/PNS) damage, and other disease and patient-reported indicators assessable in routine clinical practice were compiled by the cochairs and administrator from panellists' free-text responses. Second, the panel scored indicators for importance (5-point scale: 1=not important; 5=extremely important); indicators scoring ≥3 among >75% of panellists were then rated for agreement (5-point scale: 1=strongly disagree; 5=strongly agree). Indicators awarded an agreement score ≥4 by >67% of panellists achieved consensus. Finally, any panel-proposed refinements to consensus indicator definitions were adopted if >75% of panellists agreed. RESULTS: A panel of 21 expert clinicians from 15 countries provided information from which 83 possible current indicators of damage (kidney, 15; cardiac, 15; CNS/PNS, 13; other, 16; patient reported, 24) were compiled. Of 45 indicators meeting the importance criteria, consensus was reached for 29 and consolidated as 27 indicators (kidney, 6; cardiac, 10; CNS/PNS, 2; other, 6; patient reported, 3) including: (kidney) elevated albumin:creatinine ratio, histological damage, microalbuminuria; (cardiac) markers of early systolic/diastolic dysfunction, elevated serum cardiac troponin; (CNS/PNS) neuropathic pain, gastrointestinal symptoms suggestive of gastrointestinal neuropathy; (other) pain in extremities/neuropathy, angiokeratoma; (patient-reported) febrile crises, progression of symptoms/signs. Panellists revised and approved proposed chronologies of when the consensus indicators manifest. The panel response rate was >95% at all stages. CONCLUSIONS: PREDICT-FD captured global opinion regarding current clinical indicators that could prompt FD-specific treatment initiation earlier than is currently practised.
Assuntos
Doença de Fabry , Consenso , Técnica Delphi , Progressão da Doença , Doença de Fabry/diagnóstico , Doença de Fabry/tratamento farmacológico , Humanos , Inquéritos e QuestionáriosRESUMO
Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4CRBN E3 ubiquitin ligase is a plausible major driver of thalidomide teratogenicity. The structure of the second zinc finger of SALL4 in complex with pomalidomide, cereblon and DDB1 reveals the molecular details of recruitment. Sequence differences and a shifted binding position relative to Ikaros offer a path to the rational design of cereblon-binding drugs with reduced teratogenic risk.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ligação a DNA/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Fatores de Transcrição/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , Proteólise/efeitos dos fármacos , Especificidade por Substrato , Talidomida/análogos & derivados , Talidomida/química , Talidomida/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação/genéticaRESUMO
OBJECTIVES: Management of pain is suboptimal in many prehospital and emergency department settings, and European guidelines are lacking. We carried out the Consensus On Management of PAin Caused by Trauma (COMPACT) Delphi initiative to gain insights into the factors physicians consider important when selecting analgesics for trauma pain. PATIENTS AND METHODS: A pan-European panel of experts in emergency medicine or pain (N = 31) was recruited to participate in the COMPACT Delphi initiative. In round 1, panelists supplied free-text responses to an open question about the attributes of analgesics for emergency pain relief favored by physicians. Common themes were consolidated into factors. In round 2, factors rated important by more than 75% of the panel were taken forward into round 3. In round 3, the point at which the consensus was achieved was defined a priori as at least 75% of panelists agreeing or strongly agreeing that a factor was important. RESULTS: Twenty-nine experts participated, representing 12 European countries and with a mean (SD) of 20 (8.6) years of clinical experience. Most worked in an emergency department (79.3%). The consensus was achieved for 10 factors that were important to consider when selecting analgesics for trauma pain relief. The highest level of consensus was achieved for 'efficacy' (100%), followed by 'safety and tolerability' (96.6%), and 'ease of use' (93.1%). CONCLUSION: These findings may facilitate the development of evidence-based guidelines supporting the provision of pain management in prehospital, emergency department, and critical care settings.
Assuntos
Analgésicos/uso terapêutico , Serviço Hospitalar de Emergência , Manejo da Dor , Dor/etiologia , Ferimentos e Lesões/complicações , Técnica Delphi , Humanos , Dor/tratamento farmacológico , Manejo da Dor/métodos , Manejo da Dor/normasRESUMO
RUVBL1 and RUVBL2 are ATPases associated with diverse cellular activities (AAAs) that form a complex involved in a variety of cellular processes, including chromatin remodeling and regulation of gene expression. RUVBLs have a strong link to oncogenesis, where overexpression is correlated with tumor growth and poor prognosis in several cancer types. CB-6644, an allosteric small-molecule inhibitor of the ATPase activity of the RUVBL1/2 complex, interacts specifically with RUVBL1/2 in cancer cells, leading to cell death. Importantly, drug-acquired-resistant cell clones have amino acid mutations in either RUVBL1 or RUVBL2, suggesting that cell killing is an on-target consequence of RUVBL1/2 engagement. In xenograft models of acute myeloid leukemia and multiple myeloma, CB-6644 significantly reduced tumor growth without obvious toxicity. This work demonstrates the therapeutic potential of targeting RUVBLs in the treatment of cancer and establishes a chemical entity for probing the many facets of RUVBL biology.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , Antineoplásicos/farmacologia , Azepinas/farmacologia , Benzamidas/farmacologia , Proteínas de Transporte/antagonistas & inibidores , DNA Helicases/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Células HCT116 , Humanos , Mutação , Ligação ProteicaRESUMO
Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.
Assuntos
Regulação da Expressão Gênica , Peptídeo Hidrolases/química , Teratogênicos/química , Talidomida/química , Fatores de Transcrição/química , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Homozigoto , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas , Ligantes , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Peptídeo Hidrolases/genética , Proteólise , Coelhos , Testículo/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo , Dedos de ZincoRESUMO
The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the K48-linked polyubiquitination of CRL4CRBN neomorphic substrates via a sequential ubiquitination mechanism. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, it will be of fundamental interest to explore if loss of UBE2G1 activity is linked to clinical resistance to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins.
Assuntos
Peptídeo Hidrolases/metabolismo , Proteólise , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Fator de Transcrição Ikaros/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Talidomida/análogos & derivados , Talidomida/farmacologia , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375-86. ©2017 AACR.
Assuntos
Adenosina Trifosfatases/genética , Indóis/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/genética , Fator 1 Nuclear Respiratório/genética , Inibidores de Proteassoma/administração & dosagem , Pirimidinas/administração & dosagem , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas Nucleares/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Ubiquitina/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
B acute lymphoblastic leukemia (B-ALL) cells are distinctively vulnerable to endoplasmic reticulum (ER) stress. Recently, inhibition of p97 was shown to induce ER stress and subsequently cell death in solid tumors and in multiple myeloma. We investigated the role of a novel, orally available, p97 inhibitor (CB-5083; Cleave Biosciences) in B-ALL. CB-5083 induced a significant reduction in viability in 10 human B-ALL cell lines, harboring the most common fusion-genes involved in pediatric and adult B-ALL, with IC50s ranging from 0.34 to 0.76 µM. Moreover, CB-5083 significantly reduced the colony formation of OP1 and NALM6 cells. Early and strong induction of apoptosis was demonstrated in BALL1 and OP1 cells, together with a robust cleavage of PARP. CB-5083 induced ER stress, as documented through: 1) prominent expression of chaperones (GRP78, GRP94, PDI, DNAJC3, and DNAJB9); 2) increased activation of IRE1-alpha, as demonstrated by the splicing of XBP1; and 3) activation of PERK, which resulted in a significant overexpression of CHOP, and its downstream genes. CB-5083 reduced the viability also in GRP78-/-, GRP94-/-, and XBP1-/- cells, suggesting that none of these proteins alone was strictly required for CB-5083 activity. Moreover, we showed that the absence of XBP1 (XBP1-/-) increased the sensitivity to CB-5083, leading to the hypothesis that XBP1 splicing counteracts the activity of CB-5083, probably mitigating ER stress. Finally, vincristine was synergistic with CB-5083 in both BALL1 and OP1 cells. In summary, the targeting of p97 with CB-5083 is a novel promising therapeutic approach that should be further evaluated in B-ALL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteína com Valosina/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Sinergismo Farmacológico , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas , Proteína com Valosina/antagonistas & inibidores , Proteína com Valosina/genéticaRESUMO
Proteasome inhibitors have revolutionized the treatment of multiple myeloma but have had disappointing results when treating solid tumors. The work of Weyburne et al. (2017), published in this issue of Cell Chemical Biology, sheds some light on this conundrum and suggests a novel approach to achieve solid tumor efficacy.
Assuntos
Mieloma Múltiplo , Neoplasias de Mama Triplo Negativas , Humanos , Complexo de Endopeptidases do Proteassoma , Inibidores de ProteassomaRESUMO
Lobar torsion is well documented after pneumonectomy, but is very rare after lung transplant. To the best of our knowledge, this is the twelfth reported case of lobar torsion after lung transplant. In our case, bronchoscopies and chest radiographs were inconclusive; however, CT scan clearly demonstrated findings consistent with right middle lobe torsion. We review the literature and discuss the epidemiology, clinical presentation, imaging features, and treatment considerations for this condition. We also propose that if a clinical picture could be secondary to torsion and bronchoscopies and chest x ray are inconclusive that a CT scan should be obtained as soon as possible since early recognition increases the likelihood of being able to successfully detorse the lung and avoid lobectomy.
Assuntos
Pneumopatias/diagnóstico por imagem , Transplante de Pulmão , Complicações Pós-Operatórias/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Anormalidade Torcional/diagnóstico por imagem , Idoso , Humanos , Pneumopatias/cirurgia , Masculino , Pneumonectomia , Anormalidade Torcional/cirurgiaRESUMO
p97 is a AAA-ATPase with multiple cellular functions, one of which is critical regulation of protein homeostasis pathways. We describe the characterization of CB-5083, a potent, selective, and orally bioavailable inhibitor of p97. Treatment of tumor cells with CB-5083 leads to accumulation of poly-ubiquitinated proteins, retention of endoplasmic reticulum-associated degradation (ERAD) substrates, and generation of irresolvable proteotoxic stress, leading to activation of the apoptotic arm of the unfolded protein response. In xenograft models, CB-5083 causes modulation of key p97-related pathways, induces apoptosis, and has antitumor activity in a broad range of both hematological and solid tumor models. Molecular determinants of CB-5083 activity include expression of genes in the ERAD pathway, providing a potential strategy for patient selection.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Homeostase/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HEK293 , Humanos , Indóis/química , Indóis/farmacologia , Células K562 , Camundongos Nus , Camundongos SCID , Estrutura Molecular , Terapia de Alvo Molecular/métodos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pirimidinas/química , Pirimidinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ubiquitinadas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The AAA-ATPase p97 plays vital roles in mechanisms of protein homeostasis, including ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Herein we describe our lead optimization efforts focused on in vitro potency, ADME, and pharmaceutical properties that led to the discovery of a potent, ATP-competitive, D2-selective, and orally bioavailable p97 inhibitor 71, CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers associated with inhibition of UPS and ERAD functions, which induces irresolvable proteotoxic stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid accumulation of markers of the unfolded protein response (UPR) and subsequently induces apoptosis leading to sustained antitumor activity in in vivo xenograft models of both solid and hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple myeloma and solid tumors.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/química , Indóis/química , Proteínas Nucleares/antagonistas & inibidores , Pirimidinas/química , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Apoptose , Disponibilidade Biológica , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Xenoenxertos , Humanos , Indóis/farmacocinética , Indóis/farmacologia , Camundongos Nus , Simulação de Acoplamento Molecular , Transplante de Neoplasias , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Ubiquitina/metabolismo , Resposta a Proteínas não DobradasRESUMO
Anomalous pulmonary vein anatomy is infrequently encountered during reconstructive lung surgery, especially lung transplantation. Complications of pulmonary venous anastomosis carry high morbidity and mortality. We report a case of anomalous pulmonary vein reconstruction with a decellularized porcine small intestinal submucosa-derived extracellular matrix during bilateral lung transplantation in an 18-year-old woman with cystic fibrosis.
Assuntos
Bioprótese , Implante de Prótese Vascular/instrumentação , Prótese Vascular , Fibrose Cística/cirurgia , Intestino Delgado/transplante , Transplante de Pulmão/métodos , Procedimentos de Cirurgia Plástica/instrumentação , Veias Pulmonares/transplante , Adolescente , Fibrose Cística/diagnóstico , Feminino , Humanos , Flebografia/métodos , Veias Pulmonares/anormalidades , Veias Pulmonares/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
RATIONALE: Fosfomycin/tobramycin for inhalation (FTI), a unique, broad-spectrum antibiotic combination, may have therapeutic potential for patients with cystic fibrosis (CF). OBJECTIVES: To evaluate safety and efficacy of FTI (160/40 mg or 80/20 mg), administered twice daily for 28 days versus placebo, in patients greater than or equal to 18 years of age, with CF, chronic Pseudomonas aeruginosa (PA) airway infection, and FEV(1) greater than or equal to 25% and less than or equal to 75% predicted. METHODS: This double-blind, placebo-controlled, multicenter study assessed whether FTI/placebo maintained FEV(1) % predicted improvements achieved following a 28-day, open-label, run-in course of aztreonam for inhalation solution (AZLI). MEASUREMENTS AND MAIN RESULTS: A total of 119 patients were randomized to FTI (160/40 mg: n = 41; 80/20 mg: n = 38) or placebo (n = 40). Mean age was 32 years and mean FEV(1) was 49% predicted at screening. Relative improvements in FEV(1) % predicted achieved by the AZLI run-in were maintained in FTI groups compared with placebo (160/40 mg vs. placebo: 6.2% treatment difference favoring FTI, P = 0.002 [primary endpoint]; 80/20 mg vs. placebo: 7.5% treatment difference favoring FTI, P < 0.001). The treatment effect on mean PA sputum density was statistically significant for the FTI 80/20 mg group versus placebo (-1.04 log(10) PA colony-forming units/g sputum difference, favoring FTI; P = 0.01). Adverse events, primarily cough, were consistent with CF disease. Respiratory events, including dyspnea and wheezing, were less common with FTI 80/20 mg than FTI 160/40 mg. No clinically significant differences between groups were reported for laboratory values. CONCLUSIONS: FTI maintained the substantial improvements in FEV(1) % predicted achieved during the AZLI run-in and was well tolerated. FTI is a promising antipseudomonal therapy for patients with CF.
Assuntos
Antibacterianos/administração & dosagem , Fibrose Cística/complicações , Fosfomicina/administração & dosagem , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa , Tobramicina/administração & dosagem , Administração por Inalação , Adulto , Antibacterianos/efeitos adversos , Tosse/induzido quimicamente , Fibrose Cística/fisiopatologia , Método Duplo-Cego , Combinação de Medicamentos , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Fosfomicina/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Pseudomonas/fisiopatologia , Pseudomonas aeruginosa/efeitos dos fármacos , Testes de Função Respiratória , Tobramicina/efeitos adversos , Resultado do Tratamento , Estados UnidosRESUMO
The mammalian target of rapamycin complex 1 (mTORC1) links the control of mRNA translation, cell growth, and metabolism to diverse stimuli. Inappropriate activation of mTORC1 can lead to cancer. Phorbol esters are naturally occurring products that act as potent tumor promoters. They activate isoforms of protein kinase C (PKCs) and stimulate the oncogenic MEK/ERK signaling cascade. They also activate mTORC1 signaling. Previous work indicated that mTORC1 activation by the phorbol ester PMA (phorbol 12-myristate 13-acetate) depends upon PKCs and may involve MEK. However, the precise mechanism(s) through which they activate mTORC1 remains unclear. Recent studies have implicated both the ERKs and the ERK-activated 90-kDa ribosomal S6 kinases (p90(RSK)) in activating mTORC1 signaling via phosphorylation of TSC2 (a regulator of mTORC1) and/or the mTORC1 component raptor. However, the relative importance of each of these kinases and phosphorylation events for the activation of mTORC1 signaling is unknown. The recent availability of MEK (PD184352) and p90(RSK) (BI-D1870) inhibitors of improved specificity allowed us to address the roles of these protein kinases in controlling mTORC1 in a variety of human and rodent cell types. In parallel, we used specific shRNAs against p90(RSK1) and p90(RSK2) to further test their roles in regulating mTORC1 signaling. Our data indicate that p90(RSKs) are dispensable for the activation of mTORC1 signaling by phorbol esters in all cell types tested. Our data also reveal striking diversity in the requirements for MEK/ERK in the control of mTORC1 between different cell types, pointing to additional signaling connections between phorbol esters and mTORC1, which do not involve MEK/ERK. This study provides important information for the design of efficient strategies to combat the hyperactivation of mTORC1 signaling by oncogenic pathways.