RESUMO
The urokinase-type plasminogen activator receptor (uPAR) is an essential regulator for cell signaling in tumor cell proliferation, adhesion, and metastasis. The ubiquitous nature of uPAR in many aggressive cancer types makes uPAR an attractive target for immunotherapy. Here, we present a rapid and successful workflow for developing cross-reactive anti-uPAR recombinant antibodies (rAbs) using high-throughput optofluidic screening of single B-cells from human uPAR-immunized mice. A total of 80 human and cynomolgus uPAR cross-reactive plasma cells were identified, and selected mouse VH/VL domains were linked to the trastuzumab (Herceptin®) constant domains for the expression of mouse-human chimeric antibodies. The resulting rAbs were characterized by their tumor-cell recognition, binding activity, and cell adhesion inhibition on triple-negative breast cancer cells. In addition, the rAbs were shown to enact antibody-dependent cellular cytotoxicity (ADCC) in the presence of either human natural killer cells or peripheral blood mononuclear cells, and were evaluated for the potential use of uPAR-targeting antibody-drug conjugates (ADCs). Three lead antibodies (11857, 8163, and 3159) were evaluated for their therapeutic efficacy in vivo and were shown to suppress tumor growth. Finally, the binding epitopes of the lead antibodies were characterized, providing information on their unique binding modes to uPAR. Altogether, the strategy identified unique cross-reactive antibodies with ADCC, ADC, and functional inhibitory effects by targeting cell-surface uPAR, that can be tested in safety studies and serve as potential immunotherapeutics.
Assuntos
Leucócitos Mononucleares , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Humanos , Animais , Camundongos , Anticorpos , Transdução de Sinais , Linfócitos BRESUMO
BRAFV600E mutation confers a poor prognosis in metastatic colorectal cancer (CRC) despite combinatorial targeted therapies based on the latest understanding of signaling circuitry. To identify parallel resistance mechanisms induced by BRAF-MEK-EGFR co-targeting, we used a high-throughput kinase activity mapping platform. Here we show that SRC kinases are systematically activated in BRAFV600E CRC following targeted inhibition of BRAF ± EGFR and that coordinated targeting of SRC with BRAF ± EGFR increases treatment efficacy in vitro and in vivo. SRC drives resistance to BRAF ± EGFR targeted therapy independently of ERK signaling by inducing transcriptional reprogramming through ß-catenin (CTNNB1). The EGFR-independent compensatory activation of SRC kinases is mediated by an autocrine prostaglandin E2 loop that can be blocked with cyclooxygenase-2 (COX2) inhibitors. Co-targeting of COX2 with BRAF + EGFR promotes durable suppression of tumor growth in patient-derived tumor xenograft models. COX2 inhibition represents a drug-repurposing strategy to overcome therapeutic resistance in BRAFV600E CRC.
Assuntos
Neoplasias Colorretais , Proteínas Proto-Oncogênicas B-raf , Humanos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptores ErbB/genética , Quinases da Família src/genética , Quinases da Família src/uso terapêuticoRESUMO
Immunotargeting of tumor-specific antigens is a powerful therapeutic strategy. Immunotherapies directed at MHC-I complexes have expanded the scope of antigens and enabled the direct targeting of intracellular oncoproteins at the cell surface. We asked whether covalent drugs that alkylate mutated residues on oncoproteins could act as haptens to generate unique MHC-I-restricted neoantigens. Here, we report that KRAS G12C mutant cells treated with the covalent inhibitor ARS1620 present ARS1620-modified peptides in MHC-I complexes. Using ARS1620-specific antibodies identified by phage display, we show that these haptenated MHC-I complexes can serve as tumor-specific neoantigens and that a bispecific T cell engager construct based on a hapten-specific antibody elicits a cytotoxic T cell response against KRAS G12C cells, including those resistant to direct KRAS G12C inhibition. With multiple K-RAS G12C inhibitors in clinical use or undergoing clinical trials, our results present a strategy to enhance their efficacy and overcome the rapidly arising tumor resistance.
Assuntos
Antineoplásicos , Antígenos de Histocompatibilidade Classe I/imunologia , Neoplasias , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Anticorpos , Antineoplásicos/farmacologia , Humanos , Fatores Imunológicos , Imunoterapia , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , ProteostaseRESUMO
KRAS mutations drive a quarter of cancer mortality, and most are undruggable. Several inhibitors of the MAPK pathway are FDA approved but poorly tolerated at the doses needed to adequately extinguish RAS/RAF/MAPK signaling in the tumor cell. We found that oncogenic KRAS signaling induced ferrous iron (Fe2+) accumulation early in and throughout mutant KRAS-mediated transformation. We converted an FDA-approved MEK inhibitor into a ferrous iron-activatable drug conjugate (FeADC) and achieved potent MAPK blockade in tumor cells while sparing normal tissues. This innovation allowed sustainable, effective treatment of tumor-bearing animals, with tumor-selective drug activation, producing superior systemic tolerability. Ferrous iron accumulation is an exploitable feature of KRAS transformation, and FeADCs hold promise for improving the treatment of KRAS-driven solid tumors.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Animais , Linhagem Celular Tumoral , Ferro/farmacologia , Mutação/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de SinaisRESUMO
Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.
Assuntos
Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/imunologia , Epitopos/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Pancreáticas/imunologia , Proteólise , Animais , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Epitopos/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Pancreáticas/genéticaRESUMO
The androgen receptor (AR) is a central driver of aggressive prostate cancer. After initial treatment with androgen receptor signaling inhibitors (ARSi), reactivation of AR signaling leads to resistance. Alternative splicing of AR mRNA yields the AR-V7 splice variant, which is currently an undruggable mechanism of ARSi resistance: AR-V7 lacks a ligand binding domain, where hormones and anti-androgen antagonists act, but still activates AR signaling. We reveal PKCß as a druggable regulator of transcription and splicing at the AR genomic locus. We identify a clinical PKCß inhibitor in combination with an FDA-approved anti-androgen as an approach for repressing AR genomic locus expression, including expression of AR-V7, while antagonizing full-length AR. PKCß inhibition reduces total AR gene expression, thus reducing AR-V7 protein levels and sensitizing prostate cancer cells to current anti-androgen therapies. We demonstrate that this combination may be a viable therapeutic strategy for AR-V7-positive prostate cancer.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Proteína Quinase C beta/metabolismo , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Resistência a Medicamentos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Splicing de RNA/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
Monoclonal antibodies (mAbs) are a central component of therapy for hematologic malignancies. Widely used mAb agents in multiple myeloma (MM) include daratumumab and elotuzumab. However, not all patients respond to these agents, and resistance is a significant clinical issue. A recently discovered subset of human natural killer (NK) cells lacking expression of FcεRIγ (g-NK cells) was found to have a multifold increase in antibody-dependent effector functions after CD16 crosslinking. In this study, we tested the capacity of g-NK cells to enhance the efficacy of therapeutic mAbs against MM. In vitro, we found that g-NK cells have strikingly superior anti-myeloma cytotoxicity compared with conventional NK (cNK) cells when combined with daratumumab or elotuzumab (â¼sixfold; P < .001). In addition, g-NK cells naturally expressed minimal surface CD38 and SLAMF7, which reduced the incidence of therapeutic fratricide. In tumor-naïve murine models, the persistence of g-NK cells in blood and spleen was >10 times higher than that of cNK cells over 31 days (P < .001). In vivo efficacy studies showed that the combination of daratumumab and g-NK cells led to a >99.9% tumor reduction (by flow cytometry analysis) compared with the combination of daratumumab and cNK cells (P < .001). Moreover, treatment with daratumumab and g-NK cells led to complete elimination of myeloma burden in 5 of 7 mice. Collectively, these results underscore the unique ability of g-NK cells to potentiate the activity of therapeutic mAbs and overcome limitations of current off-the-shelf NK cell therapies without the need for cellular irradiation or genetic engineering.
Assuntos
Antineoplásicos Imunológicos , Mieloma Múltiplo , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Citometria de Fluxo , Humanos , Células Matadoras Naturais , Camundongos , Mieloma Múltiplo/tratamento farmacológicoRESUMO
αvß8 integrin, a key activator of transforming growth factor ß (TGF-ß), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvß8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvß8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of ß8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-ß and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvß8 integrin as a promising target for cancer immunotherapy.
Assuntos
Imunidade , Imunoterapia , Integrinas/metabolismo , Modelos Biológicos , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Animais , Anticorpos Antineoplásicos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Granzimas/metabolismo , Interferon gama/metabolismo , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Transdução de Sinais , Proteína Smad3/metabolismo , Análise de Sobrevida , Linfócitos T Citotóxicos/imunologia , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully in vitro system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL. SIGNIFICANCE: Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully in vitro nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development.This article is highlighted in the In This Issue feature, p. 1861.
Assuntos
Antígenos CD19/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Antígenos Quiméricos/imunologia , Humanos , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , ProteômicaRESUMO
Renewing tissues have the remarkable ability to continually produce both proliferative progenitor and specialized differentiated cell types. How are complex milieus of microenvironmental signals interpreted to coordinate tissue-cell-type composition? Here, we investigate the responses of intestinal epithelium to individual and paired perturbations across eight epithelial signaling pathways. Using a high-throughput approach that combines enteroid monolayers and quantitative imaging, we identified conditions that enrich for specific cell types as well as interactions between pathways. Importantly, we found that modulation of transit-amplifying cell proliferation changes the ratio of differentiated secretory to absorptive cell types. These observations highlight an underappreciated role for transit-amplifying cells in the tuning of differentiated cell-type composition.
Assuntos
Células Epiteliais/citologia , Intestinos/citologia , Animais , Proliferação de Células , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Humanos , Interleucina-4/metabolismo , Absorção Intestinal , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Organoides/citologia , Mapeamento de Interação de Proteínas , Transdução de SinaisRESUMO
Alternative splicing of the androgen receptor (AR) is frequently observed in castration resistant prostate cancer (CRPC). One AR isoform, the AR-V7 splice variant, is a constitutively active transcription factor which lacks a ligand binding domain and is therefore undruggable. AR-V7 expression correlates with resistance to androgen receptor signaling inhibitors (ARSi) and poor clinical prognoses. The occurrence of the AR-V7 splice variant is driven by alternative splicing of AR pre-mRNA by the spliceosome, however the mechanistic details are poorly understood. We demonstrate that the splicing factor RBM39 is critical for alternative splicing of the AR-V7 splice variant mRNA transcripts from AR pre-mRNA, and that the anti-cancer drug, indisulam, reduces AR-V7 mRNA levels by degrading RBM39. We report that indisulam effectively reduces AR-V7 in in vitro and in vivo models.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/genética , Sulfonamidas/farmacologia , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Células Tumorais CultivadasRESUMO
PURPOSE: New therapies have changed the outlook for patients with multiple myeloma, but novel agents are needed for patients who are refractory or relapsed on currently approved drug classes. Novel targets other than CD38 and BCMA are needed for new immunotherapy development, as resistance to daratumumab and emerging anti-BCMA approaches appears inevitable. One potential target of interest in myeloma is ICAM1. Naked anti-ICAM1 antibodies were active in preclinical models of myeloma and safe in patients, but showed limited clinical efficacy. Here, we sought to achieve improved targeting of multiple myeloma with an anti-ICAM1 antibody-drug conjugate (ADC). EXPERIMENTAL DESIGN: Our anti-ICAM1 human mAb was conjugated to an auristatin derivative, and tested against multiple myeloma cell lines in vitro, orthotopic xenografts in vivo, and patient samples ex vivo. The expression of ICAM1 was also measured by quantitative flow cytometry in patients spanning from diagnosis to the daratumumab-refractory state. RESULTS: The anti-ICAM1 ADC displayed potent anti-myeloma cytotoxicity in vitro and in vivo. In addition, we have verified that ICAM1 is highly expressed on myeloma cells and shown that its expression is further accentuated by the presence of bone marrow microenvironmental factors. In primary samples, ICAM1 is differentially overexpressed on multiple myeloma cells compared with normal cells, including daratumumab-refractory patients with decreased CD38. In addition, ICAM1-ADC showed selective cytotoxicity in multiple myeloma primary samples. CONCLUSIONS: We propose that anti-ICAM1 ADC should be further studied for toxicity, and if safe, tested for clinical efficacy in patients with relapsed or refractory multiple myeloma.
Assuntos
Anticorpos Monoclonais/farmacologia , Imunoconjugados/farmacologia , Molécula 1 de Adesão Intercelular/genética , Mieloma Múltiplo/tratamento farmacológico , ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribosil Ciclase 1/imunologia , Adulto , Idoso , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Citometria de Fluxo , Xenoenxertos , Humanos , Imunoconjugados/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologiaRESUMO
Enhancing the efficacy of proteasome inhibitors (PI) is a central goal in myeloma therapy. We proposed that signaling-level responses after PI may reveal new mechanisms of action that can be therapeutically exploited. Unbiased phosphoproteomics after treatment with the PI carfilzomib surprisingly demonstrates the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation is invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrates broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings lead us to evaluate direct spliceosome inhibition in myeloma, which synergizes with carfilzomib and shows potent anti-tumor activity. Functional genomics and exome sequencing further support the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/administração & dosagem , Spliceossomos/efeitos dos fármacos , Animais , Antineoplásicos/administração & dosagem , Feminino , Humanos , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Oligopeptídeos/administração & dosagem , Splicing de RNA/efeitos dos fármacos , Spliceossomos/genética , Spliceossomos/metabolismo , Spliceossomos/microbiologiaRESUMO
The field of chemical modification of proteins has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently, we have developed oxaziridine chemistry for highly selective modification of methionine called redox-activated chemical tagging (ReACT) but have not broadly tested the molecular parameters for efficient and stable protein modification. Here we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, used for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and excellent reaction efficiencies with a panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than 10-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, presumably because of their reduced access to water. There was also a 10-fold variation in stability depending on the nature of the oxaziridine, which was determined to be inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs were sufficient to support their use as antibody drug conjugates and potent in a breast cancer mouse xenograft model over a month. These studies provide key parameters for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation to native or engineered methionines.
Assuntos
Aziridinas/análise , Imunoconjugados/química , Metionina/análise , Animais , Antineoplásicos/normas , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Feminino , Humanos , Imunoconjugados/genética , Imunoconjugados/imunologia , Camundongos , Camundongos Nus , Engenharia de Proteínas/métodos , Estabilidade ProteicaRESUMO
Multiple myeloma (MM) cell lines are routinely used to model the disease. However, a long-standing question is how well these cell lines truly represent tumor cells in patients. Here, we employ a recently described method of transcriptional correlation profiling to compare similarity of 66 MM cell lines to 779 newly diagnosed MM patient tumors. We found that individual MM lines differ significantly with respect to patient tumor representation, with median R ranging from 0.35 to 0.54. ANBL-6 was the "best" line, markedly exceeding all others (p < 2.2e-16). Notably, some widely used cell lines (RPMI-8226, U-266) scored poorly in our patient similarity ranking (48 and 52 of 66, respectively). Lines cultured with interleukin-6 showed significantly improved correlations with patient tumor (p = 9.5e-4). When common MM genomic features were matched between cell lines and patients, only t(4;14) and t(14;16) led to increased transcriptional correlation. To demonstrate the utility of our top-ranked line for preclinical studies, we showed that intravenously implanted ANBL-6 proliferates in hematopoietic organs in immunocompromised mice. Overall, our large-scale quantitative correlation analysis, utilizing emerging datasets, provides a resource informing the MM community of cell lines that may be most reliable for modeling patient disease while also elucidating biological differences between cell lines and tumors.
Assuntos
Perfilação da Expressão Gênica , Mieloma Múltiplo/genética , Transcriptoma , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Humanos , Imunofenotipagem , Camundongos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Mutação , Prognóstico , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The role of the soluble urokinase plasminogen activator receptor (suPAR) in focal segmental glomerulosclerosis (FSGS) as the circulating factor or as a predictor of recurrence after transplantation remains controversial. Previously published studies in mice and isolated podocytes produced conflicting results on the effect of suPAR on podocyte injury, effacement of foot processes, and proteinuria. These discordant results were in part due to diverse experimental designs and different strains of mice. The aim of our study was to determine the reasons for the inconsistencies of the previous studies results with suPAR by using uniform methods and studies in different strains of mice. METHODS: We utilized a primary culture of human podocytes and 2 mouse models, the wild type (WT) and the urokinase plasminogen activator receptor (uPAR) KO (uPAR), in an attempt to resolve the reported conflicting results. RESULTS: In both WT and uPAR mouse models, injection of recombinant uPAR, even at a high dose (100 µg), did not induce proteinuria, effacement of podocytes, or disruption of the cytoskeleton. Injection of suPAR resulted in its deposition exclusively in the glomerular endothelial cells and not in the podocytes of WT mice and was not detected at the uPAR KO mice. Kidneys from patients with recurrent FSGS had negative immunostaining for uPAR. We also evaluated the effect of recombinant uPAR on primary culture of human podocytes. uPAR did not result in podocytes damage. CONCLUSIONS: suPAR by itself is not the cause for direct podocyte injury, in vitro or in vivo. These findings suggest a more complex and still poorly understood role of suPAR in FSGS.
Assuntos
Glomerulosclerose Segmentar e Focal/imunologia , Podócitos/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Animais , Autoanticorpos/administração & dosagem , Autoanticorpos/imunologia , Biópsia , Antígenos CD40/imunologia , Células Cultivadas , Glomerulosclerose Segmentar e Focal/sangue , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Rim/citologia , Rim/patologia , Camundongos , Camundongos Knockout , Podócitos/imunologia , Cultura Primária de Células , Receptores de Ativador de Plasminogênio Tipo Uroquinase/administração & dosagem , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
Patient-derived xenografts (PDXs) are an essential pre-clinical resource for investigating tumor biology. However, cellular heterogeneity within and across PDX tumors can strongly impact the interpretation of PDX studies. Here, we generated a multi-modal, large-scale dataset to investigate PDX heterogeneity in metastatic colorectal cancer (CRC) across tumor models, spatial scales and genomic, transcriptomic, proteomic and imaging assay modalities. To showcase this dataset, we present analysis to assess sources of PDX variation, including anatomical orientation within the implanted tumor, mouse contribution, and differences between replicate PDX tumors. A unique aspect of our dataset is deep characterization of intra-tumor heterogeneity via immunofluorescence imaging, which enables investigation of variation across multiple spatial scales, from subcellular to whole tumor levels. Our study provides a benchmark data resource to investigate PDX models of metastatic CRC and serves as a template for future, quantitative investigations of spatial heterogeneity within and across PDX tumor models.
Assuntos
Neoplasias do Colo/patologia , Modelos Animais de Doenças , Xenoenxertos/patologia , Animais , Genômica , Humanos , Camundongos , Metástase Neoplásica , Proteômica , TranscriptomaRESUMO
A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily through endocytosis showed efficacy in a mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) of 2G10 were designed and produced bearing tubulin inhibitor payloads ligated through seven different linkers. Aldehyde tag technology was employed for linking, and either one or two tags were inserted into the antibody heavy chain, to produce site-specifically conjugated ADCs with drug-to-antibody ratios of either two or four. Both cleavable and non-cleavable linkers were combined with two different antimitotic toxins-MMAE (monomethylauristatin E) and maytansine. Nine different 2G10 ADCs were produced and tested for their ability to target uPAR in cell-based assays and a mouse model. The anti-uPAR ADC that resulted in tumor regression comprised an MMAE payload with a cathepsin B cleavable linker, 2G10-RED-244-MMAE. This work demonstrates in vitro activity of the 2G10-RED-244-MMAE in TNBC cell lines and validates uPAR as a therapeutic target for TNBC.
RESUMO
PURPOSE: Optimal efficacy of a macromolecular prodrug requires balancing the rate of drug release with the rate of prodrug elimination. Since circulating macromolecules have different elimination rates in different species, a prodrug optimal for one species will likely not be for another. The objectives of this work were (a) to develop an approach to optimize pharmacokinetics of a PEG~SN-38 prodrug in a particular species, (b) to use the approach to predict the pharmacokinetics of various prodrugs of SN-38 in the mouse and human, and (c) to develop a PEG~SN-38 conjugate that is optimized for mouse tumor models. METHODS: We developed models that describe the pharmacokinetics of a drug released from a prodrug by the relationship between the rates of drug release and elimination of the prodrug. We tested the model by varying the release rate of SN-38 from PEG~SN-38 conjugates in the setting of a constant prodrug elimination rate in the mouse. Finally, we tested the antitumor efficacy of a PEG~SN-38 optimized for the mouse. RESULTS: Optimization of a PEG~SN-38 prodrug was achieved by adjusting the rate of SN-38 release such that the ratio of t1/2,ß of released SN-38 to the t1/2 of prodrug elimination was 0.2-0.8. Using this approach, we could rationalize the efficacy of previous PEGylated SN-38 prodrugs in the mouse and human. Finally, a mouse-optimized PEG~SN-38 showed remarkable antitumor activity in BRCA1-deficient MX-1 xenografts; a single dose gave tumor regression, suppression, and shrinkage of massive tumors. CONCLUSIONS: The efficacy of a macromolecular prodrug can be optimized for a given species by balancing the rate of drug release from the carrier with the rate of prodrug elimination.