RESUMO
Chromosome copy number variations (CNVs) are a near-universal feature of cancer; however, their individual effects on cellular function are often incompletely understood. Single-cell ribonucleic acid (RNA) sequencing (scRNA-seq) might be leveraged to reveal the function of intra-clonal CNVs; however, it cannot directly link cellular gene expression to CNVs. Here, we report a high-throughput scRNA-seq analysis pipeline that provides paired CNV profiles and transcriptomes for single cells, enabling exploration of the effects of CNVs on cellular programs. RTAM1 and -2 normalization methods are described, and are shown to improve transcriptome alignment between cells, increasing the sensitivity of scRNA-seq for CNV detection. We also report single-cell inferred chromosomal copy number variation (sciCNV), a tool for inferring single-cell CNVs from scRNA-seq at 19-46 Mb resolution. Comparison of sciCNV with existing RNA-based CNV methods reveals useful advances in sensitivity and specificity. Using sciCNV, we demonstrate that scRNA-seq can be used to examine the cellular effects of cancer CNVs. As an example, sciCNV is used to identify subclonal multiple myeloma (MM) cells with +8q22-24. Studies of the gene expression of intra-clonal MM cells with and without the CNV demonstrate that +8q22-24 upregulates MYC and MYC-target genes, messenger RNA processing and protein synthesis, which is consistent with established models. In conclusion, we provide new tools for scRNA-seq that enable paired profiling of the CNVs and transcriptomes of single cells, facilitating rapid and accurate deconstruction of the effects of cancer CNVs on cellular programming.
Assuntos
Variações do Número de Cópias de DNA , Transcriptoma , Cromossomos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA MensageiroRESUMO
Both previous and additional genetic knockdown studies reported herein implicate G protein-coupled receptor kinase 6 (GRK6) as a critical kinase required for the survival of multiple myeloma (MM) cells. Therefore, we sought to develop a small molecule GRK6 inhibitor as an MM therapeutic. From a focused library of known kinase inhibitors, we identified two hits with moderate biochemical potencies against GRK6. From these hits, we developed potent (IC50 < 10 nM) analogues with selectivity against off-target kinases. Further optimization led to the discovery of an analogue (18) with an IC50 value of 6 nM against GRK6 and selectivity against a panel of 85 kinases. Compound 18 has potent cellular target engagement and antiproliferative activity against MM cells and is synergistic with bortezomib. In summary, we demonstrate that targeting GRK6 with small molecule inhibitors represents a promising approach for MM and identify 18 as a novel, potent, and selective GRK6 inhibitor.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Quinases de Receptores Acoplados a Proteína G/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Quinases de Receptores Acoplados a Proteína G/metabolismo , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Quinazolinas/síntese química , Quinazolinas/química , Relação Estrutura-AtividadeRESUMO
Bone morphogenetic protein 7 (BMP7) stimulates renal branching morphogenesis via p38 mitogen-activated protein kinase (p38(MAPK)) and activating transcription factor 2 (ATF-2) (M. C. Hu, D. Wasserman, S. Hartwig, and N. D. Rosenblum, J. Biol. Chem. 279:12051-12059, 2004). Here, we demonstrate a novel role for integrin-linked kinase (ILK) in mediating renal epithelial cell morphogenesis in embryonic kidney explants and identify p38(MAPK) as a target of ILK signaling in a cell culture model of renal epithelial morphogenesis. The spatial and temporal expression of ILK in embryonic mouse kidney cells suggested a role in branching morphogenesis. Adenovirus-mediated expression of ILK stimulated and expression of a dominant negative ILK mutant inhibited ureteric bud branching in embryonic mouse kidney explants. BMP7 increased ILK kinase activity in inner medullary collecting duct 3 (IMCD-3) cells, and adenovirus-mediated expression of ILK increased IMCD-3 cell morphogenesis in a three-dimensional culture model. In contrast, treatment with a small molecule ILK inhibitor or expression of a dominant negative-acting ILK (ILK(E359K)) inhibited epithelial cell morphogenesis. Further, expression of ILK(E359K) abrogated BMP7-dependent stimulation. To investigate the role of ILK in BMP7 signaling, we showed that ILK overexpression increased basal and BMP7-induced levels of phospho-p38(MAPK) and phospho-ATF-2. Consistent with its inhibitory effects on IMCD-3 cell morphogenesis, expression of ILK(E359K) blocked BMP7-dependent increases in phospho-p38(MAPK) and phospho-ATF-2. Inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, failed to inhibit BMP7-dependent stimulation of ILK activity, suggesting that ILK functions upstream of p38(MAPK) during BMP7 signaling. We conclude that ILK functions in a BMP7/p38(MAPK)/ATF-2 signaling pathway and stimulates epithelial cell morphogenesis.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Rim/embriologia , Morfogênese , Proteínas Serina-Treonina Quinases/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Fator 2 Ativador da Transcrição , Animais , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/farmacologia , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/embriologia , Epitélio/metabolismo , Imidazóis/farmacologia , Rim/citologia , Camundongos , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Morfogênese/fisiologia , Mutação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
ILKAP is a protein phosphatase 2C that selectively associates with integrin linked kinase, ILK, to modulate cell adhesion and growth factor signaling. We investigated the role of endogenous cellular ILKAP in antagonizing ILK signaling of two key targets, PKB and GSK3beta. Silencing of endogenous ILKAP by short interfering RNA (siRNA) stimulated GSK3beta phosphorylation at S9, with no effect on PKB S473 phosphorylation. In LNCaP prostate carcinoma cells, transient or stable expression of ILKAP suppressed ILK immune complex kinase activity, demonstrating an interaction between ILKAP and ILK. Consistent with the silencing data, ILKAP inhibition of ILK selectively inhibited S9 phosphorylation of GSK3beta without affecting S473 phosphorylation of PKB. The ILKAP-mediated inhibition of S9 phosphorylation was rescued by overexpression of ILK, but not by a dominant-negative ILK mutant. The expression level of cyclin D1, a target of ILK-GSK3beta signaling, was inversely correlated with ILKAP protein levels, suggesting that antagonism of ILK modulates cell cycle progression. ILKAP expression increased the proportion of LNCaP cells in G1, relative to vector control cells, and siRNA suppression of ILKAP increased entry of cells into the S phase, consistent with ILK antagonism. Anchorage-independent growth of LNCaP cells was inhibited by ILKAP, suggesting a critical role in the suppression of cellular transformation. Taken together, our results indicate that endogenous ILKAP activity inhibits the ILK-GSK3beta signaling axis, and suggest that ILKAP activity plays an important role in inhibiting oncogenic transformation.Oncogene (2004) 23, 3454-3461. doi:10.1038/sj.onc.1207473 Published online 1 March 2004
Assuntos
Transformação Celular Neoplásica , Fosfoproteínas Fosfatases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais , Ciclo Celular , Divisão Celular , Células Cultivadas , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Fosforilação , RNA Interferente Pequeno/farmacologiaRESUMO
We analysed breast tumors and breast cancer cell lines for the expression of beta-parvin (ParvB), an adaptor protein that binds to the integrin-linked kinase (ILK). Quantitative RT-PCR indicated that ParvB mRNA was downregulated, by at least 60%, in four of nine breast tumors, relative to patient-matched normal mammary gland tissue. We also found that ParvB protein levels were reduced by > or =90% in five of seven advanced tumors, relative to matched normal breast tissue. Conversely, ILK protein and kinase activity levels were elevated in these tumors, suggesting that downregulation of ParvB stimulates ILK signaling. Western blot analyses indicated very low levels of ParvB protein in MDA-MB-231 and MCF7 breast cancer cells, facilitating functional studies of the effects of ParvB on ILK signaling. Expression of ParvB in MDA-MB-231 and MCF7 cells increased cell adhesion to collagen. ParvB inhibited ILK kinase activity, anchorage-independent cell growth and in vitro matrigel invasion by MDA-MB-231 cells. EGF-induced phosphorylation of two ILK targets, PKB (Ser473) and glycogen synthase kinase 3beta (Ser9), was also inhibited by ParvB. These results indicated that ParvB inhibits ILK signaling downstream of receptor tyrosine kinases. Our results suggest that loss of ParvB expression is a novel mechanism for upregulating ILK activity in tumors.
Assuntos
Actinina/metabolismo , Neoplasias da Mama/metabolismo , Regulação para Baixo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Adenoviridae/genética , Sequência de Aminoácidos , Anticorpos/química , Western Blotting , Neoplasias da Mama/patologia , Adesão Celular , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Colágeno/química , Colágeno/metabolismo , Colágeno/farmacologia , Corantes/farmacologia , DNA Complementar/metabolismo , Combinação de Medicamentos , Fator de Crescimento Epidérmico/metabolismo , Genes Reporter , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Laminina/química , Laminina/farmacologia , Modelos Genéticos , Dados de Sequência Molecular , Fosforilação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteoglicanas/química , Proteoglicanas/farmacologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Regulação para CimaRESUMO
Proteasome inhibitor (PI) resistance mechanisms in multiple myeloma (MM) remain controversial. We report the existence of a progenitor organization in primary MM that recapitulates maturation stages between B cells and plasma cells and that contributes to clinical PI resistance. Xbp1s(-) tumor B cells and pre-plasmablasts survive therapeutic PI, preventing cure, while maturation arrest of MM before the plasmablast stage enables progressive disease on PI treatment. Mechanistically, suppression of Xbp1s in MM is shown to induce bortezomib resistance via de-commitment to plasma cell maturation and immunoglobulin production, diminishing endoplasmic reticulum (ER) front-loading and cytotoxic susceptibility to PI-induced inhibition of ER-associated degradation. These results reveal the tumor progenitor structure in MM and highlight its role in therapeutic failure.
Assuntos
Proteínas de Ligação a DNA/deficiência , Resistencia a Medicamentos Antineoplásicos/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Inibidores de Proteassoma/uso terapêutico , Fatores de Transcrição/deficiência , Fator 6 Ativador da Transcrição/metabolismo , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Bortezomib , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Humanos , Imunofenotipagem , Proteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Mutação , Plasmócitos/metabolismo , Plasmócitos/patologia , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Inibidores de Proteassoma/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-Box , eIF-2 Quinase/metabolismoRESUMO
PURPOSE: Lenalidomide is an oral immunomodulatory drug with multiple effects on the immune system and tumor cell microenvironment leading to inhibition of malignant cell growth. Based on encouraging reports of lenalidomide in relapsed and refractory chronic lymphocytic leukemia (CLL), we investigated the first-line use of single-agent lenalidomide in CLL. PATIENTS AND METHODS: Using a starting dose of lenalidomide 10 mg/d for 21 days of a 28-day cycle and weekly 5-mg dose escalations to a target of 25 mg, we encountered severe toxicities (tumor lysis, fatal sepsis) in the first two patients enrolled. The study was halted and the protocol amended to a more conservative regimen: starting dose of lenalidomide 2.5 mg with monthly escalations to a target dose of 10 mg, and extended tumor lysis prophylaxis and monitoring. Gene expression profiles from patient samples before and after 7 days of lenalidomide were performed. RESULTS: Twenty-five patients were enrolled on the amended protocol. No further tumor lysis events were reported. Tumor flare was common (88%) but mild. Grade 3 to 4 neutropenia occurred in 72% of patients, with only five episodes of febrile neutropenia. The overall response rate was 56% (no complete responses). Although rapid peripheral lymphocyte reductions were observed, rebound lymphocytoses during the week off-therapy were common. Lenalidomide-induced molecular changes enriched for cytoskeletal and immune-related genes were identified. CONCLUSION: Lenalidomide is clinically active as first-line CLL therapy and is well-tolerated if a conservative approach with slow dose escalation is used. A lenalidomide-induced molecular signature provides insights into its immunomodulatory mechanisms of action in CLL.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Fatores Imunológicos/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Talidomida/análogos & derivados , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Relação Dose-Resposta a Droga , Feminino , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos/metabolismo , Lenalidomida , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Indução de Remissão , Taxa de Sobrevida , Talidomida/uso terapêutico , Resultado do TratamentoRESUMO
PURPOSE: Therapeutic strategies that target insulin-like growth factor 1 receptor (IGF-1R) hold promise in a wide variety of cancers including multiple myeloma (MM). In this study, we describe GTx-134, a novel small-molecule inhibitor of IGF-1R and insulin receptor (IR) and characterized its antitumor activity in preclinical models of MM. EXPERIMENTAL DESIGN: The activity of GTx-134 as a single agent and in combination was tested in MM cell lines and primary patient samples. Downstream effector proteins and correlation with apoptosis was evaluated. Cytotoxcity in bone marrow stroma coculture experiments was assessed. Finally, the in vivo efficacy was evaluated in a human myeloma xenograft model. RESULTS: GTx-134 inhibited the growth of 10 of 14 myeloma cell lines (<5 µmol/L) and induced apoptosis. Sensitivity to GTx-134 correlated with IGF-1R signal inhibition. Expression of MDR-1 and CD45 were associated with resistance to GTx-134. Coculture with insulin-growth factor-1 (IGF-1) or adherence to bone marrow stroma conferred modest resistance, but did not overcome GTx-134-induced cytotoxicity. GTx-134 showed in vitro synergies when combined with dexamethasone or lenalidomide. Further, GTx-134 enhanced the activity of PD173074, a fibroblast growth factor receptor 3 (FGFR3) inhibitor, against t(4;14) myeloma cells. Therapeutic efficacy of GTx-134 was shown against primary cells and xenograft tumors. Although dysregulation of glucose homeostasis was observed in GTx-134-treated mice, impairment of glucose tolerance was modest. CONCLUSIONS: These studies support the potential therapeutic efficacy of GTx-134 in MM. Further, they provide a rationale for clinical application in combination with established antimyeloma treatments and novel targeted therapies.