RESUMO
Patients with small-cell lung cancer (SCLC) are in dire need of more effective therapeutic options. Frequent disruption of the G1 checkpoint in SCLC cells creates a dependency on the G2/M checkpoint to maintain genomic integrity. Indeed, in pre-clinical models, inhibiting the G2/M checkpoint kinase WEE1 shows promise in inhibiting SCLC growth. However, toxicity and acquired resistance limit the clinical effectiveness of this strategy. Here, using CRISPR-Cas9 knockout screens in vitro and in vivo, we identified multiple factors influencing the response of SCLC cells to the WEE1 kinase inhibitor AZD1775, including the GCN2 kinase and other members of its signaling pathway. Rapid activation of GCN2 upon AZD1775 treatment triggers a stress response in SCLC cells. Pharmacological or genetic activation of the GCN2 pathway enhances cancer cell killing by AZD1775. Thus, activation of the GCN2 pathway represents a promising strategy to increase the efficacy of WEE1 inhibitors in SCLC.
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AIMS: To assess US payers' per-patient cost of testing associated with next-generation sequencing (NGS) versus polymerase chain reaction (PCR) biomarker testing strategies among patients with metastatic non-small cell lung cancer (mNSCLC), including costs of testing, delayed care, and suboptimal treatment initiation. METHODS: A decision tree model considered biomarker testing for genomic alterations using either NGS, sequential PCR testing, or hotspot panel PCR testing. Literature-based model inputs included time-to-test results, costs for testing/medical care, costs of delaying care, costs of immunotherapy [IO]/chemotherapy [CTX] initiation prior to receiving test results, and costs of suboptimal treatment initiation after test results (i.e. costs of first-line IO/CTX in patients with actionable mutations that were undetected by PCR that would have been identified with NGS). The proportion of patients testing positive for a targetable alteration, time to appropriate therapy initiation, and per-patient costs were estimated for NGS and PCR strategies combined. RESULTS: In a modeled cohort of 1,000,000 members (25% Medicare, 75% commercial), an estimated 1,119 had mNSCLC and received testing. The proportion of patients testing positive for a targetable alteration was 45.9% for NGS and 40.0% for PCR testing. Mean per-patient costs were lowest for NGS ($8,866) compared to PCR ($18,246), with lower delayed care costs of $1,301 for NGS compared to $3,228 for PCR, and lower costs of IO/CTX initiation prior to receiving test results (NGS: $2,298; PCR:$5,991). Cost savings, reaching $10,496,220 at the 1,000,000-member plan level, were driven by more rapid treatment with appropriate therapy for patients tested with NGS (2.1 weeks) compared to PCR strategies (5.2 weeks). LIMITATIONS: Model inputs/assumptions were based on published literature or expert opinion. CONCLUSIONS: NGS testing was associated with greater cost savings versus PCR, driven by more rapid results, shorter time to appropriate therapy initiation, and minimized use of inappropriate therapies while awaiting and after test results.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Idoso , Humanos , Estados Unidos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Medicare , Testes Genéticos , Genômica , Mutação , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da PolimeraseRESUMO
Small cell lung cancer (SCLC) is a lethal form of lung cancer. Here, we develop a quantitative multiplexed approach on the basis of lentiviral barcoding with somatic CRISPR-Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. We found that naphthalene pre-treatment enhances lentiviral vector-mediated SCLC initiation, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Candidate drivers of SCLC identified from a meta-analysis across multiple human SCLC genomic datasets were tested using this approach, which defines both positive and detrimental impacts of inactivating 40 genes across candidate pathways on SCLC development. This analysis and subsequent validation in human SCLC cells establish TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This approach should illuminate drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify therapeutic targets.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Camundongos , Animais , Humanos , Carcinoma de Pequenas Células do Pulmão/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Pulmonares/patologia , Genes Supressores de Tumor , GenômicaRESUMO
The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.
Assuntos
Neoplasias Ósseas , Antígeno CD47 , Animais , Linhagem Celular Tumoral , Humanos , Imunoterapia , Camundongos , Recidiva Local de Neoplasia , Fagocitose , Microambiente TumoralRESUMO
The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ciclina D/metabolismo , Adenocarcinoma de Pulmão/genética , Animais , Divisão Celular , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Genes Supressores de Tumor , Humanos , Neoplasias Pulmonares/genética , Camundongos , Piperazinas/farmacologia , Piridinas/farmacologia , Células U937 , UbiquitinaçãoRESUMO
Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.
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Cromograninas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Neoplasias Pulmonares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteômica/métodos , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células A549 , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cromograninas/genética , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína Fosfatase 2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The availability of viable human tissues is critical to support translational research focused on personalized care. Most studies have relied on fresh frozen or formalin-fixed paraffin-embedded tissues for histopathology, genomics, and proteomics. Yet, basic, translational, and clinical research downstream assays such as tumor progression/invasion, patient-derived xenograft, organoids, immunoprofiling, and vaccine development still require viable tissue, which are time-sensitive and rare commodities. We describe the generation of two-dimensional (2D) and three-dimensional (3D) cultures to validate a viable freeze cryopreservation technique as a standard method of highest quality specimen preservation. After surgical resection, specimens were minced, placed in CryoStor™ media, and frozen using a slow freezing method (-1°C/min in -80°C) for 24 hours and then stored in liquid nitrogen. After 15-18 months, the tissues were thawed, dissociated into single-cell suspensions, and evaluated for cell viability. To generate primary 2D cultures, cells were plated onto Collagen-/Matrigel-coated plates. To develop 3D cultures (organoids), the cells were plated in reduced serum RPMI media on nonadherent plates or in Matrigel matrix. The epithelial nature of the cells was confirmed by using immunohistochemistry for cytokeratins. DNA and RNA isolation was performed using QIAGEN AllPrep kits. We developed primary lines (2D and 3D) of colon, thyroid, lung, renal, and liver cancers that were positive for cytokeratin staining. 3D lines were developed from the same cohort of tumor types in both suspended media and Matrigel matrix. Multiple freeze-thaw cycles did not significantly alter the viability and growth of 2D and 3D lines. DNA/RNA recovery was similar to its fresh frozen cohort. In this study, we validated 2D and 3D tissue cultures as methods to corroborate the feasibility of viable cryopreservation of tumor tissue. This proof-of-principle study, if more widely implemented, should improve accessibility of human viable tumor tissue/cells in a time-independent manner for many basic, preclinical, and translational assays.
Assuntos
Técnicas de Cultura de Células/métodos , Criopreservação/métodos , Preservação de Tecido/métodos , Biomarcadores/metabolismo , Linhagem Celular/citologia , Linhagem Celular/metabolismo , Sobrevivência Celular , Marcadores Genéticos , Humanos , Organoides/citologia , Organoides/metabolismo , Estudo de Prova de Conceito , Análise de Célula Única , Células Tumorais Cultivadas/citologiaRESUMO
Several intravenous iron complexes are available for the treatment of iron deficiency anemia (IDA). Iron dextran (DEX) is associated with an elevated risk of potentially serious anaphylactic reactions, whereas others must be administered in several small infusions to avoid labile iron reactions. Ferric carboxymaltose (FCM) is a nondextran intravenous iron which can be administered in high single doses. A randomized, open label, and multicenter comparison of FCM to DEX in adults with IDA and baseline hemoglobin of ≤11.0 g/dL was conducted. A total of 160 patients were in the safety population (FCM n = 82; DEX n = 78). Adverse events, including immune system disorders (0% in FCM versus 10.3% in DEX, P = 0.003) and skin disorders (7.3% in FCM versus 24.4% in DEX, P = 0.004), were less frequently observed in the FCM group. A greater portion of patients in the FCM group experienced a transient, asymptomatic decrease in phosphate compared to patients in the DEX group (8.5% in FCM versus 0% in DEX, P = 0.014). In the FCM arm, the change in hemoglobin from baseline to the highest observed level was 2.8 g/dL, whereas the DEX arm displayed a change of 2.4 g/dL (P = 0.20). Treatment of IDA with FCM resulted in fewer hypersensitivity-related reactions than DEX.
RESUMO
BACKGROUND: Iron deficiency anemia (IDA) is a common finding in patients after bariatric surgery. The cause is multifactorial including reduced oral iron intake and malabsorption. While many patients can be managed with oral supplements, parenteral iron may be needed to restore and maintain iron stores. METHODS: Subjects who had previous bariatric surgery and had participated in phase 3 industry-sponsored clinical trials designed to assess the safety and/or efficacy of intravenous (IV) ferric carboxymaltose (FCM) were retrospectively selected from the databases of each of these studies. Demographic data, efficacy measures [hemoglobin, ferritin, and transferrin saturation (TSAT)], and adverse events were compared between FCM and other agents utilized as comparators in the trials. RESULTS: Two hundred eighty-one subjects from the intention to treat (ITT) population were included (mean age 49 years, BMI 33 kg/m(2), including 253 females). FCM had similar or improved efficacy (p < 0.05) in terms of increasing hemoglobin, ferritin, and TSAT values when compared to other iron products used as standard of care for IDA. The incidence of adverse events in the FCM patients (n = 123) versus patients receiving any IV iron (n = 126) was 61 and 56.3 %, respectively. The adverse events were similar in both groups with the exception of a transient decrease in serum phosphate which was observed more frequently in the FCM group. CONCLUSIONS: These data in post-bariatric surgery IDA patients suggest that FCM is a safe and effective alternative to existing iron products permitting higher and thus less frequent individual doses.
Assuntos
Anemia Ferropriva/terapia , Cirurgia Bariátrica/efeitos adversos , Compostos Férricos/uso terapêutico , Hematínicos/uso terapêutico , Síndromes de Malabsorção/terapia , Maltose/análogos & derivados , Obesidade Mórbida/cirurgia , Nutrição Parenteral , Complicações Pós-Operatórias/terapia , Adulto , Anemia Ferropriva/sangue , Anemia Ferropriva/etiologia , Biomarcadores/sangue , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Feminino , Ferritinas/sangue , Hemoglobinas , Humanos , Infusões Intravenosas , Ferro da Dieta/uso terapêutico , Síndromes de Malabsorção/sangue , Síndromes de Malabsorção/etiologia , Masculino , Maltose/uso terapêutico , Pessoa de Meia-Idade , Obesidade Mórbida/sangue , Obesidade Mórbida/complicações , Nutrição Parenteral/métodos , Complicações Pós-Operatórias/sangue , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Retrospectivos , Transferrina , Resultado do Tratamento , Estados Unidos/epidemiologiaRESUMO
Levels of hepcidin, a major regulator of iron homeostasis, may identify patients with iron deficiency anemia (IDA) who will not respond to oral iron therapy. In this study, IDA patients underwent a 14-day trial (run-in) course of ferrous sulfate therapy. Nonresponders (Hgb increase <1 g/dL with 67% compliance rate) were randomized to IV ferric carboxymaltose (FCM; two injections of 750 mg) or further oral iron for 14 days. Screening hepcidin levels were 38.4 versus 11.3 ng/mL, P = 0.0002 in nonresponders versus responders to a trial of oral iron. Hepcidin of > 20 ng/mL, showed sensitivity of 41.3%, specificity of 84.4%, and positive predictive value of 81.6% for predicting nonresponsiveness to oral iron. PPVs for ferritin> 30 ng/mL or transferrin saturation (TSAT)>15% were 59.2 and 55%, respectively. Negative predictive values for hepcidin, ferritin, and TSAT were 46.3, 22.7, and 19.7, respectively. FCM versus oral iron showed Hgb increases of ≥ 1 gm/dL in 65.3% versus 20.8% (P < 0.0001) and Hgb increases of 1.7 ± 1.3 versus 0.6 ± 0.9 g/dL (P = 0.0025), respectively. We conclude that hepcidin predicts nonresponsiveness to oral iron in patients with IDA and is superior to TSAT or ferritin for this purpose. Nonresponse to oral iron therapy does not rule out IDA, since two-thirds of patients subsequently responded to intravenous iron.