RESUMO
Dose-limiting toxicity poses a major limitation to the clinical utility of targeted cancer therapies, often arising from target engagement in nonmalignant tissues. This obstacle can be minimized by targeting cancer dependencies driven by proteins with tissue-restricted and/or tumor-restricted expression. In line with another recent report, we show here that, in acute myeloid leukemia (AML), suppression of the myeloid-restricted PIK3CG/p110γ-PIK3R5/p101 axis inhibits protein kinase B/Akt signaling and compromises AML cell fitness. Furthermore, silencing the genes encoding PIK3CG/p110γ or PIK3R5/p101 sensitizes AML cells to established AML therapies. Importantly, we find that existing small-molecule inhibitors against PIK3CG are insufficient to achieve a sustained long-term antileukemic effect. To address this concern, we developed a proteolysis-targeting chimera (PROTAC) heterobifunctional molecule that specifically degrades PIK3CG and potently suppresses AML progression alone and in combination with venetoclax in human AML cell lines, primary samples from patients with AML and syngeneic mouse models.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase , Leucemia Mieloide Aguda , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Humanos , Animais , Camundongos , Transdução de Sinais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Proteólise/efeitos dos fármacos , Feminino , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêuticoRESUMO
Recent advancements in shRNA and Cas protein technologies have enabled functional screening methods targeting genes or non-coding regions using single or pooled shRNA and sgRNA. CRISPR-based systems have also been developed for modulating DNA accessibility, resulting in CRISPR-mediated interference (CRISPRi) or activation (CRISPRa) of targeted genes or genomic DNA elements. However, there is still a lack of software tools for integrating diverse array of functional genomics screening outputs that could offer a cohesive framework for comprehensive data integration. Here, we developed PitViper, a flexible and interactive open-source software designed to fill this gap, providing reliable results for the type of elements being screened. It is an end-to-end automated and reproducible bioinformatics pipeline integrating gold-standard methods for functional screening analysis. Our sensitivity analyses demonstrate that PitViper is a useful tool for identifying potential super-enhancer liabilities in a leukemia cell line through genome-wide CRISPRi-based screening. It offers a robust, flexible, and interactive solution for integrating data analysis and reanalysis from functional screening methods, making it a valuable resource for researchers in the field.
RESUMO
PURPOSE: Outcomes for Richter transformation (RT) are poor with current therapies. The efficacy and safety of anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T) for RT are not established. METHODS: We performed an international multicenter retrospective study of patients with RT who received CAR-T. Patient, disease, and treatment characteristics were summarized using descriptive statistics, and modeling analyses were used to determine association with progression-free survival (PFS) and overall survival (OS). PFS and OS were estimated from the date of CAR-T infusion. RESULTS: Sixty-nine patients were identified. The median age at CAR-T infusion was 64 years (range, 27-80). Patients had a median of four (range, 1-15) previous lines of therapy for CLL and/or RT, including previous Bruton tyrosine kinase inhibitor and/or BCL2 inhibitor therapy in 58 (84%) patients. The CAR-T product administered was axicabtagene ciloleucel in 44 patients (64%), tisagenlecleucel in 17 patients (25%), lisocabtagene maraleucel in seven patients (10%), and brexucabtagene autoleucel in one patient (1%). Eleven patients (16%) and 25 patients (37%) experienced grade ≥3 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, respectively. The overall response rate was 63%, with 46% attaining a complete response (CR). After a median follow-up of 24 months, the median PFS was 4.7 months (95% CI, 2.0 to 6.9); the 2-year PFS was 29% (95% CI, 18 to 41). The median OS was 8.5 months (95% CI, 5.1 to 25.4); the 2-year OS was 38% (95% CI, 26 to 50). The median duration of response was 27.6 months (95% CI, 14.5 to not reached) for patients achieving CR. CONCLUSION: CAR-T demonstrates clinical efficacy for patients with RT.
Assuntos
Antígenos CD19 , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Estudos Retrospectivos , Masculino , Pessoa de Meia-Idade , Idoso , Adulto , Feminino , Antígenos CD19/uso terapêutico , Antígenos CD19/imunologia , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Idoso de 80 Anos ou mais , Receptores de Antígenos Quiméricos/uso terapêutico , Receptores de Antígenos Quiméricos/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/mortalidade , Intervalo Livre de ProgressãoRESUMO
The PI3K/AKT/mTOR (PAM) signaling pathway is a highly conserved signal transduction network in eukaryotic cells that promotes cell survival, cell growth, and cell cycle progression. Growth factor signalling to transcription factors in the PAM axis is highly regulated by multiple cross-interactions with several other signaling pathways, and dysregulation of signal transduction can predispose to cancer development. The PAM axis is the most frequently activated signaling pathway in human cancer and is often implicated in resistance to anticancer therapies. Dysfunction of components of this pathway such as hyperactivity of PI3K, loss of function of PTEN, and gain-of-function of AKT, are notorious drivers of treatment resistance and disease progression in cancer. In this review we highlight the major dysregulations in the PAM signaling pathway in cancer, and discuss the results of PI3K, AKT and mTOR inhibitors as monotherapy and in co-administation with other antineoplastic agents in clinical trials as a strategy for overcoming treatment resistance. Finally, the major mechanisms of resistance to PAM signaling targeted therapies, including PAM signaling in immunology and immunotherapies are also discussed.
Assuntos
Neoplasias , Fosfatidilinositol 3-Quinases , Humanos , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
During apoptosis, mitochondrial outer membrane permeabilization (MOMP) enables certain mitochondrial matrix macromolecules to escape into the cytosol. However, the fate of mitochondrial RNA (mtRNA) during apoptosis is unknown. Here, we demonstrate that MOMP results in the cytoplasmic release of mtRNA and that executioner caspases-3 and -7 (casp3/7) prevent cytoplasmic mtRNA from triggering inflammatory signaling. In the setting of genetic or pharmacological casp3/7 inhibition, apoptotic insults result in mtRNA activation of the MDA5/MAVS/IRF3 pathway to drive Type I interferon (IFN) signaling. This pathway is sufficient to activate tumor-intrinsic Type I IFN signaling in immunologically cold cancer models that lack an intact cGAS/STING signaling pathway, promote CD8+ T-cell-dependent anti-tumor immunity, and overcome anti-PD1 refractoriness in vivo. Thus, a key function of casp3/7 is to inhibit inflammation caused by the cytoplasmic release of mtRNA, and pharmacological modulation of this pathway increases the immunogenicity of chemotherapy-induced apoptosis.
Assuntos
Antineoplásicos , Interferon Tipo I , Caspases/metabolismo , RNA Mitocondrial , Caspase 3/metabolismo , Apoptose , Interferon Tipo I/metabolismo , Antineoplásicos/farmacologia , Nucleotidiltransferases/metabolismoRESUMO
Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.
Assuntos
Leucemia Mieloide Aguda , Proteínas Proto-Oncogênicas c-akt , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Carioferinas/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Estados Unidos , Proteína Exportina 1RESUMO
Targeting mitochondrial metabolism has emerged as a treatment option for cancer patients. The ABL tyrosine kinases promote metastasis, and enhanced ABL signaling is associated with a poor prognosis in lung adenocarcinoma patients. Here we show that ABL kinase allosteric inhibitors impair mitochondrial integrity and decrease oxidative phosphorylation. To identify metabolic vulnerabilities that enhance this phenotype, we utilized a CRISPR/Cas9 loss-of-function screen and identified HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway and target of statin therapies, as a top-scoring sensitizer to ABL inhibition. Combination treatment with ABL allosteric inhibitors and statins decreases metastatic lung cancer cell survival in vitro in a synergistic manner. Notably, combination therapy in mouse models of lung cancer brain metastasis and therapy resistance impairs metastatic colonization with a concomitant increase in animal survival. Thus, metabolic combination therapy might be effective to decrease metastatic outgrowth, leading to increased survival for lung cancer patients with advanced disease.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Oncogênicas v-abl/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteínas Oncogênicas v-abl/genética , Proteínas Oncogênicas v-abl/metabolismo , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deciphering the impact of metabolic intervention on response to anticancer therapy may elucidate a path toward improved clinical responses. Here, we identify amino acid-related pathways connected to the folate cycle whose activation predicts sensitivity to MYC-targeting therapies in acute myeloid leukemia (AML). We establish that folate restriction and deficiency of the rate-limiting folate cycle enzyme MTHFR, which exhibits reduced-function polymorphisms in about 10% of Caucasians, induce resistance to MYC targeting by BET and CDK7 inhibitors in cell lines, primary patient samples, and syngeneic mouse models of AML. Furthermore, this effect is abrogated by supplementation with the MTHFR enzymatic product CH3-THF. Mechanistically, folate cycle disturbance reduces H3K27/K9 histone methylation and activates a SPI1 transcriptional program counteracting the effect of BET inhibition. Our data provide a rationale for screening MTHFR polymorphisms and folate cycle status to nominate patients most likely to benefit from MYC-targeting therapies. SIGNIFICANCE: Although MYC-targeting therapies represent a promising strategy for cancer treatment, evidence of predictors of sensitivity to these agents is limited. We pinpoint that folate cycle disturbance and frequent polymorphisms associated with reduced MTHFR activity promote resistance to BET inhibitors. CH3-THF supplementation thus represents a low-risk intervention to enhance their effects.See related commentary by Marando and Huntly, p. 1791.This article is highlighted in the In This Issue feature, p. 1775.
Assuntos
Ácido Fólico/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resistencia a Medicamentos Antineoplásicos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-myc/biossíntese , Células U937RESUMO
We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.
Assuntos
Apoptose/genética , Carcinoma Ductal Pancreático/genética , Sistema de Sinalização das MAP Quinases/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias PancreáticasRESUMO
Local adaptation directs populations towards environment-specific fitness maxima through acquisition of positively selected traits. However, rapid environmental changes can identify hidden fitness trade-offs that turn adaptation into maladaptation, resulting in evolutionary traps. Cancer, a disease that is prone to drug resistance, is in principle susceptible to such traps. We therefore performed pooled CRISPR-Cas9 knockout screens in acute myeloid leukemia (AML) cells treated with various chemotherapies to map the drug-dependent genetic basis of fitness trade-offs, a concept known as antagonistic pleiotropy (AP). We identified a PRC2-NSD2/3-mediated MYC regulatory axis as a drug-induced AP pathway whose ability to confer resistance to bromodomain inhibition and sensitivity to BCL-2 inhibition templates an evolutionary trap. Across diverse AML cell-line and patient-derived xenograft models, we find that acquisition of resistance to bromodomain inhibition through this pathway exposes coincident hypersensitivity to BCL-2 inhibition. Thus, drug-induced AP can be leveraged to design evolutionary traps that selectively target drug resistance in cancer.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Pleiotropia Genética/genética , Neoplasias/genética , Adaptação Fisiológica/genética , Animais , Evolução Biológica , Sistemas CRISPR-Cas/genética , Linhagem Celular , Linhagem Celular Tumoral , Meio Ambiente , Aptidão Genética/genética , Células HEK293 , Células HL-60 , Humanos , Camundongos , Proteínas Nucleares/genética , Fenótipo , Locos de Características Quantitativas/genética , Fatores de Transcrição/genéticaRESUMO
Aortic root size and cusp fusion pattern have been related to disease outcomes in bicuspid aortic valve (BAV). This study seeks to characterize symmetry of the aortic sinuses in adult and pediatric BAV patients and its relationship to valvulopathy and root aortopathy. Aortic sinus-to-commissure (S-C) lengths were measured on cardiac MRI of adult and pediatric BAV patients with right-and-left coronary (RL) or right-and-non-coronary (RN) leaflet fusion and tricuspid aortic valve (TAV) controls. Coefficient of variance (CoV) of S-C lengths was calculated to quantify sinus asymmetry, or eccentricity. BAV cohort included 149 adults (48 ± 15 years) and 51 children (15 ± 5 years). TAV cohort included 40 adults (60 ± 13 years) and 20 children (15 ± 5 years). In adult and pediatric BAV patients, the non-fused aortic sinus was larger than either fused sinus. In RL fusion, the non-coronary S-C distance was larger than right or left S-C distances in adults (n = 121, p < 0.001) and larger than the right S-C distance in children (n = 41, p = 0.013). Sinus eccentricity (CoV) in BAV patients was higher than in TAV patients (p < 0.001) and did not correlate with age (p = 0.12). CoV trended higher in RL adults with aortic regurgitation (AR) compared to those without AR (p = 0.081), but was lower in RN adults with AR than without AR (p = 0.006). CoV did not correlate to root Z scores (p = 0.06-0.55) or ascending aortic (AAo) Z scores in adults (p = 0.45-0.55) but correlated negatively to AAo Z score in children (p = 0.005-0.03). Most adult and pediatric BAV patients with RL and RN leaflet fusion demonstrate eccentric dominance of the non-fused aortic sinus irrespective of age. The degree of eccentricity varies with valve dysfunction and BAV phenotype but does not relate to the degree of aortic root dilatation, nor does eccentricity correlate with ascending aorta dilatation in adults.
Assuntos
Valva Aórtica/anormalidades , Doenças das Valvas Cardíacas/patologia , Seio Aórtico/patologia , Adolescente , Adulto , Aorta/diagnóstico por imagem , Aorta/patologia , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/patologia , Doença da Válvula Aórtica Bicúspide , Criança , Feminino , Doenças das Valvas Cardíacas/etiologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Seio Aórtico/diagnóstico por imagem , Adulto JovemRESUMO
Extrinsic pathway agonists have failed repeatedly in the clinic for three core reasons: Inefficient ligand-induced receptor multimerization, poor pharmacokinetic properties, and tumor intrinsic resistance. Here, we address these factors by (i) using a highly potent death receptor agonist (DRA), (ii) developing an injectable depot for sustained DRA delivery, and (iii) leveraging a CRISPR-Cas9 knockout screen in DRA-resistant colorectal cancer (CRC) cells to identify functional drivers of resistance. Pharmacological blockade of XIAP and BCL-XL by targeted small-molecule drugs strongly enhanced the antitumor activity of DRA in CRC cell lines. Recombinant fusion of the DRA to a thermally responsive elastin-like polypeptide (ELP) creates a gel-like depot upon subcutaneous injection that abolishes tumors in DRA-sensitive Colo205 mouse xenografts. Combination of ELPdepot-DRA with BCL-XL and/or XIAP inhibitors led to tumor growth inhibition and extended survival in DRA-resistant patient-derived xenografts. This strategy provides a precision medicine approach to overcome similar challenges with other protein-based cancer therapies.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteína bcl-X/antagonistas & inibidores , Animais , Antineoplásicos/química , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Células HCT116 , Células HT29 , Humanos , Camundongos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Crosstalk between metabolic and survival pathways is critical for cellular homeostasis, but the connectivity between these processes remains poorly defined. We used loss-of-function CRISPR/Cas9 knockout screening to identify metabolic genes capable of influencing cellular commitment to apoptosis, using sensitization to the BCL-2 inhibitor ABT-199 in BCL-2-dependent acute myeloid leukemia (AML) cell lines as a proxy for apoptotic disposition. This analysis revealed metabolic pathways that specifically cooperate with BCL-2 to sustain survival. In particular, our analysis singled out heme biosynthesis as an unappreciated apoptosis-modifying pathway. Although heme is broadly incorporated into the proteome, reduction of heme biosynthesis potentiates apoptosis through the loss of ETC activity, resulting in baseline depolarization of the mitochondrial membrane and an increased propensity to undergo apoptosis. Collectively, our findings chart the first apoptotic map of metabolism, motivating the design of metabolically engaged combination chemotherapies and nominating heme biosynthesis as an apoptotic modulator in AML.
Assuntos
Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Heme/biossíntese , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Antineoplásicos/farmacologia , Apoptose/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Leucemia Mieloide Aguda/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Células THP-1 , Transdução GenéticaRESUMO
Intrinsic resistance to anti-HER2 therapy in breast cancer remains an obstacle in the clinic, limiting its efficacy. However, the biological basis for intrinsic resistance is poorly understood. Here we performed a CRISPR/Cas9-mediated loss-of-function genetic profiling and identified TALDO1, which encodes the rate-limiting transaldolase (TA) enzyme in the non-oxidative pentose phosphate pathway, as essential for cellular survival following pharmacological HER2 blockade. Suppression of TA increases cell susceptibility to HER2 inhibition in two intrinsically resistant breast cancer cell lines with HER2 amplification. Mechanistically, TA depletion combined with HER2 inhibition significantly reduces cellular NADPH levels, resulting in excessive ROS production and deficient lipid and nucleotide synthesis. Importantly, higher TA expression correlates with poor response to HER2 inhibition in a breast cancer patient cohort. Together, these results pinpoint TA as a novel metabolic enzyme possessing synthetic lethality with HER2 inhibition that can potentially be exploited as a biomarker or target for combination therapy.
Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptor ErbB-2/genética , Mutações Sintéticas Letais/genética , Transaldolase/genética , Neoplasias da Mama/metabolismo , Sistemas CRISPR-Cas , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Testes Genéticos , Células HEK293 , Humanos , Lapatinib/farmacologia , Análise do Fluxo Metabólico , NADP/metabolismo , Via de Pentose FosfatoRESUMO
While inhibitors of BCL-2 family proteins (BH3 mimetics) have shown promise as anti-cancer agents, the various dependencies or co-dependencies of diverse cancers on BCL-2 genes remain poorly understood. Here we develop a drug screening approach to define the sensitivity of cancer cells from ten tissue types to all possible combinations of selective BCL-2, BCL-XL, and MCL-1 inhibitors and discover that most cell lines depend on at least one combination for survival. We demonstrate that expression levels of BCL-2 genes predict single mimetic sensitivity, whereas EMT status predicts synergistic dependence on BCL-XL+MCL-1. Lastly, we use a CRISPR/Cas9 screen to discover that BFL-1 and BCL-w promote resistance to all tested combinations of BCL-2, BCL-XL, and MCL-1 inhibitors. Together, these results provide a roadmap for rationally targeting BCL-2 family dependencies in diverse human cancers and motivate the development of selective BFL-1 and BCL-w inhibitors to overcome intrinsic resistance to BH3 mimetics.
Assuntos
Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Masculino , Camundongos , RNA Mensageiro/metabolismo , Sulfonamidas/farmacologia , Proteína bcl-X/metabolismoRESUMO
We demonstrate a hybrid microfluidic system that combines fluidic trapping and acoustic switching to organize an array of single cells at high density. The fluidic trapping step is achieved by balancing the hydrodynamic resistances of three parallel channel segments forming a microfluidic trifurcation, the purpose of which was to capture single cells in a high-density array. Next, the cells were transferred into adjacent larger compartments by generating an array of streaming micro-vortices to move the cells to the desired streamlines in a massively parallel format. This approach can compartmentalize single cells with efficiencies of ≈67% in compartments that have diameters on the order of â¼100 um, which is an appropriate size for single cell proliferation studies and other single cell biochemical measurements.
Assuntos
Acústica , Dispositivos Lab-On-A-Chip , Análise de Célula Única/instrumentação , Análise Serial de Tecidos/instrumentação , Linhagem Celular Tumoral , Humanos , HidrodinâmicaRESUMO
The notochord gives rise to spinal segments during development, and it becomes embedded within the nucleus pulposus of the intervertebral disc (IVD) during maturation. The disruption of the notochord band has been observed with IVD degeneration. Since the mechanical competence of the IVD relies on its structural constituents, defining the structure of the notochord during aging is critical for investigations relating to IVD function and homeostasis. The assessment and imaging of the notochord has classically relied on histological techniques, which introduces sectioning artifacts during preparation and spatial biases. Magnetic resonance imaging (MRI) does not offer sufficient resolution to discriminate the notochord from the surrounding the nucleus pulposus, especially in murine models. Current X-ray based computed tomography systems provide imaging resolutions down to the single- and sub- micron scales, and when coupled with contrast-enhancing agents, enable the high-resolution three-dimensional imaging of relatively small features. Utilizing phosphomolybdic acid to preferentially bind to collagen cationic domains, we describe the structure of the notochord remnants with aging in the lumbar IVDs of BALB/c mice. These results provide a highly quantitative and sensitive approach to monitoring the IVD during postnatal development.
Assuntos
Notocorda/diagnóstico por imagem , Notocorda/crescimento & desenvolvimento , Intensificação de Imagem Radiográfica , Microtomografia por Raio-X , Animais , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Notocorda/ultraestrutura , Intensificação de Imagem Radiográfica/métodos , Microtomografia por Raio-X/métodosRESUMO
The intervertebral disc (IVD) is susceptible to degenerative changes that are associated with low back pain. Murine models are often used to investigate the mechanistic changes in the development, aging, and diseased states of the IVD, yet the detection of early degenerative changes in structure is challenging because of the minute size of the murine IVDs. Histology is the gold standard for examining the IVD structure, but it is susceptible to sectioning artifacts, spatial biases, and requires the destructive preparation of the sample. We have previously demonstrated the feasibility of using Ioversol for the contrast-enhanced micro-computed tomography (microCT) to visualize and quantitate the intact healthy murine IVD. In this work, we demonstrate utility of this approach to monitor the longitudinal changes of in vitro nucleolytic- and mechanical injury- degeneration models of the murine discs and introduce novel quantitative metrics to characterize the structure and composition of the IVD. Moreover, we compared the imaging quality and quantitation of these in vitro models to magnetic resonance imaging (MRI) and histology. Stab puncture, trypsin injection, and collagenase injection all induced detectable and significant changes in structure and composition of the discs overtime. Compared to MRI and histology, contrast-enhanced microCT produced superior images that capture the degenerative progression in these models. Contrast-enhanced microCT was also capable of monitoring the structural deteriorations via the changes in disc height and volume, and novel the nucleus pulposus intensity/disc intensity (NI/DI) parameter provides a surrogate measure of proteoglycan composition (R = 0.96). Overall, this approach allows for the nondestructive monitoring of the structure and composition of the IVD at very high resolutions.
Assuntos
Degeneração do Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/diagnóstico por imagem , Animais , Imageamento por Ressonância Magnética , Proteoglicanas , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
Combinatorial inhibition of effector and feedback pathways is a promising treatment strategy for KRAS mutant cancers. However, the particular pathways that should be targeted to optimize therapeutic responses are unclear. Using CRISPR/Cas9, we systematically mapped the pathways whose inhibition cooperates with drugs targeting the KRAS effectors MEK, ERK, and PI3K. By performing 70 screens in models of KRAS mutant colorectal, lung, ovarian, and pancreas cancers, we uncovered universal and tissue-specific sensitizing combinations involving inhibitors of cell cycle, metabolism, growth signaling, chromatin regulation, and transcription. Furthermore, these screens revealed secondary genetic modifiers of sensitivity, yielding a SRC inhibitor-based combination therapy for KRAS/PIK3CA double-mutant colorectal cancers (CRCs) with clinical potential. Surprisingly, acquired resistance to combinations of growth signaling pathway inhibitors develops rapidly following treatment, but by targeting signaling feedback or apoptotic priming, it is possible to construct three-drug combinations that greatly delay its emergence.