RESUMEN
The field of cancer immunotherapy has experienced significant progress, resulting in the emergence of numerous biological drug candidates requiring in vivo efficacy testing and a better understanding of their mechanism of action (MOA). Humanized immune system (HIS) models are valuable tools in this regard. However, there is a lack of systematic guidance on HIS modeling. To address this issue, the present study aimed to establish and optimize a variety of HIS models for immune-oncology (IO) study, including genetically engineered mouse models and HIS models with human immune components reconstituted in severely immunocompromised mice. The efficacy and utility of these models were tested with several marketed or investigational IO drugs according to their MOA, followed by immunophenotypic analysis and efficacy evaluation. The results of the present study demonstrated that the HIS models responded to various IO drugs as expected and that each model had unique niches, utilities and limitations. Researchers should carefully choose the appropriate models based on the MOA and the targeted immune cell populations of the investigational drug. The present study provides valuable methodologies and actionable technical guidance on designing, generating or utilizing appropriate HIS models to address specific questions in translational IO.
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Modelos Animales de Enfermedad , Inmunoterapia , Neoplasias , Animales , Humanos , Ratones , Inmunoterapia/métodos , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Ratones TransgénicosRESUMEN
BACKGROUND: Targeted estrogen receptor degradation has been approved to effectively treat ER + breast cancers. Due to the poor bioavailability of fulvestrant, the first generation of SERD, many efforts were made to develop oral SERDs. With the approval of Elacestrant, oral SERDs demonstrated superior efficacy than fulvestrant. However, due to the poor ability of known SERDs to penetrate the blood-brain barrier (BBB), breast cancer patients with brain metastasis cannot benefit from clinical SERDs. METHODS: The ER inhibitory effects were evaluated on ERα protein degradation, and target genes downregulation. And anti-proliferation activities were further determined in a panel of ER + breast cancer cell lines. The subcutaneous and intracranial ER + tumor models were used to evaluate the efficacy of anti-tumor effects. Brain penetrability was determined in multiple animal species. RESULTS: SCR-6852 is a novel SERD and currently is under early clinical evaluation. In vitro studies demonstrated that it strongly induced both wildtype and mutant ERα degradation. It potently inhibited cell proliferation in a panel of ER + breast cancer cell lines, including the cell lines containing ESR1 mutations (Y537 and D538). Furthermore, SCR-6852 exhibited pure antagonistic activities on the ERÉ signal axis identified both in vitro and in vivo. Oral administration of SCR-6852 at 10 mg/kg resulted in tumor shrinkage which was superior to Fulvestrant at 250 mg/kg, notably, in the intracranial tumor model, SCR-6852 effectively inhibited tumor growth and significantly prolonged mice survival, which correlated well with the high exposure in brains. In addition to mice, SCR-6852 also exhibited high brain penetrability in rats and dogs. CONCLUSIONS: SCR-6852 is a novel SERD with high potency in inducing ERα protein degradation and pure antagonistic activity on ERÉ signaling in vitro and in vivo. Due to the high brain penetrability, SCR-6852 could be used to treat breast patients with brain metastasis.
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Neoplasias Encefálicas , Receptores de Estrógenos , Ratas , Ratones , Animales , Perros , Receptores de Estrógenos/metabolismo , Fulvestrant/farmacología , Receptor alfa de Estrógeno/metabolismo , Antagonistas de Estrógenos , Encéfalo , Neoplasias Encefálicas/tratamiento farmacológicoRESUMEN
Malignant ascites (MA) is caused by intraperitoneal spread of solid tumor cells and results in a poor quality of life. Chemotherapy is a common first-line treatment for patients with MA. Taxotere ® (DTX) is widely used in solid tumor therapies. However, the low water solubility and side effects caused by additives in the formulation restrict the clinical application of docetaxel. HT001 is a clinical stage docetaxel micelle developed to overcome the solubility issue with improved safety profiles. To support clinical development and expand clinical application of HT001, this study used in vitro and in vivo approaches to investigate the anti-tumor effects of HT001 when applied as monotherapy or in combination with anti-angiogenic agents. HT001 demonstrated comparable anti-proliferative activities as docetaxel in a broad range of cancer cell lines in vitro. Furthermore, HT001 suppressed tumor growth in a dose-dependent manner in A549, MCF-7, and SKOV-3 xenograft tumor mouse models in vivo. In a hepatocellular carcinoma H22 malignant ascites-bearing mouse model, HT001 presented a dose-dependent inhibition of ascites production, prolonged animal survival, and reduced VEGF levels. When dosed at 20 mg/kg, the HT001-treated group exhibited curative results, with no ascites formation in 80% of mice at the end of the study while all the mice in the vehicle control group succumbed. Similar results were obtained in HT001 treatment of mice bearing malignant ascites produced by human ovarian cancer ES-2 cells. Notably, the combination of HT001 with Endostar not only significantly reduced ascites production but also prolonged survival of H22 ascites-bearing mice. HT001 showed similar PK and tissue distribution profiles as DTX in non-rodent hosts. Collectively, these results demonstrate potent anti-tumor activity of HT001 in multiple solid tumor models or malignant ascites models, and reveal synergistic effects with anti-angiogenic agents, supporting the clinical development and clinical expansion plans for HT001.
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Targeting DNA damage response (DDR) pathway has been proposed as an approach for amplifying tumor-specific replicative lesions. RAD51 plays a central role in the DDR process, and thus represents a promising anti-tumor target. We here report the discovery of a series of next generation RAD51 inhibitors that can prevent RAD51 foci formation. The lead compounds dramatically impaired human cancer cell growth, induced cell cycle arrest in S-phase, and resulted in elevated γH2AX. Furthermore, cancer cells became sensitized to chemotherapy and other DDR inhibitors. Dosed either as a single agent or in combination with cisplatin, the compounds significantly inhibited tumor growth in vivo. By upregulating ATR-CHK1 signaling, the RAD51 inhibitors increased surface PD-L1 levels in various tumor cells, suggesting a potential combination of RAD51 inhibitors with PD-1/PD-L1 blockade. Overall, our findings provide the preclinical rationale to explore RAD51 inhibitors as monotherapy or in combination with chemotherapy, immunotherapy or DDR-targeting therapy in cancer treatment.
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BACKGROUND: The application of RNA-seq technology has become more extensive and the number of analysis procedures available has increased over the past years. Selecting an appropriate workflow has become an important issue for researchers in the field. METHODS: In our study, six popular analytical procedures/pipeline were compared using four RNA-seq datasets from mouse, human, rat, and macaque, respectively. The gene expression value, fold change of gene expression, and statistical significance were evaluated to compare the similarities and differences among the six procedures. qRT-PCR was performed to validate the differentially expressed genes (DEGs) from all six procedures. RESULTS: Cufflinks-Cuffdiff demands the highest computing resources and Kallisto-Sleuth demands the least. Gene expression values, fold change, p and q values of differential expression (DE) analysis are highly correlated among procedures using HTseq for quantification. For genes with medium expression abundance, the expression values determined using the different procedures were similar. Major differences in expression values come from genes with particularly high or low expression levels. HISAT2-StringTie-Ballgown is more sensitive to genes with low expression levels, while Kallisto-Sleuth may only be useful to evaluate genes with medium to high abundance. When the same thresholds for fold change and p value are chosen in DE analysis, StringTie-Ballgown produce the least number of DEGs, while HTseq-DESeq2, -edgeR or -limma generally produces more DEGs. The performance of Cufflinks-Cuffdiff and Kallisto-Sleuth varies in different datasets. For DEGs with medium expression levels, the biological verification rates were similar among all procedures. CONCLUSION: Results are highly correlated among RNA-seq analysis procedures using HTseq for quantification. Difference in gene expression values mainly come from genes with particularly high or low expression levels. Moreover, biological validation rates of DEGs from all six procedures were similar for genes with medium expression levels. Investigators can choose analytical procedures according to their available computer resources, or whether genes of high or low expression levels are of interest. If computer resources are abundant, one can utilize multiple procedures to obtain the intersection of results to get the most reliable DEGs, or to obtain a combination of results to get a more comprehensive DE profile for transcriptomes.
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Perfilación de la Expresión Génica , Transcriptoma , Animales , Perfilación de la Expresión Génica/métodos , Ratones , RNA-Seq , Ratas , Análisis de Secuencia de ARN/métodos , Secuenciación del ExomaRESUMEN
BACKGROUND: Signaling through VEGF/VEGFR induces cancer angiogenesis and affects immune cells. An increasing number of studies have recently focused on combining anti-VEGF/VEGFR agents and immune checkpoint inhibitors (ICIs) to treat cancer in preclinical and clinical settings. BD0801 is a humanized rabbit anti-VEGF monoclonal antibody in the clinical development stage. METHODS: In this study, the anti-cancer activities of BD0801 and its potential synergistic anti-tumor effects when combined with different immunotherapies were assessed by using in vitro assays and in vivo tumor models. Ex vivo studies were conducted to reveal the possible mechanisms of actions (MOA) underlying the tumor microenvironment modification. RESULTS: BD0801 showed more potent antitumor activity than bevacizumab, reflected by stronger blockade of VEGF/VEGFR binding and enhanced inhibitory effects on human umbilical vein endothelial cells (HUVECs). BD0801 exhibited dose-dependent tumor growth inhibitory activities in xenograft and murine syngeneic tumor models. Notably, combining BD0801 with either anti-PD-1 or anti-PD-L1 antibodies showed synergistic antitumor efficacy in both lung and colorectal cancer mouse models. Furthermore, the mechanistic studies suggested that the MOA of the antitumor synergy involves improved tumor vasculature normalization and enhanced T-cell mediated immunity, including increased tumor infiltration of CD8+ and CD4+ T cells and reduced double-positive CD8+PD-1+ T cells. CONCLUSIONS: These data provide a solid rationale for combining antiangiogenic agents with immunotherapy for cancer treatment and support further clinical development of BD0801 in combination with ICIs.
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Inhibidores de la Angiogénesis/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de la Angiogénesis/farmacología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones , Microambiente TumoralRESUMEN
Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFß-SMMHC (core binding factor ß and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFß for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFß-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFß-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers.
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Antineoplásicos/uso terapéutico , Bencimidazoles/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Animales , Antineoplásicos/química , Bencimidazoles/química , Línea Celular Tumoral , Subunidad alfa 2 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas de Fusión Oncogénica/metabolismo , Mapas de Interacción de Proteínas , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances, there is no mutation in BCR-ABL, and the basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed a large-scale RNA interference screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL(+) cells IM-resistant. In these IMSG knockdown cells, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling is sustained after IM treatment because of up-regulation of PRKCH, which encodes the protein kinase C (PKC) family member PKCη, an activator of CRAF. PRKCH is also up-regulated in samples from CML patients with BCR-ABL-independent IM resistance. Combined treatment with IM and trametinib, a U.S. Food and Drug Administration-approved MEK inhibitor, synergistically kills BCR-ABL(+) IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL-independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels of PRKCH, and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent IM resistance in CML and CML stem cells.
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Benzamidas/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Terapia Molecular Dirigida , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Benzamidas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Genes Relacionados con las Neoplasias , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , ARN Interferente Pequeño/metabolismo , Análisis de Supervivencia , Regulación hacia Arriba/efectos de los fármacos , Quinasas raf/metabolismoRESUMEN
Acute myeloid leukemia (AML) results from the activity of driver mutations that deregulate proliferation and survival of hematopoietic stem cells (HSCs). The fusion protein CBFß-SMMHC impairs differentiation in hematopoietic stem and progenitor cells and induces AML in cooperation with other mutations. However, the combined function of CBFß-SMMHC and cooperating mutations in preleukemic expansion is not known. Here, we used Nras(LSL-G12D); Cbfb(56M) knock-in mice to show that allelic expression of oncogenic Nras(G12D) and Cbfß-SMMHC increases survival of preleukemic short-term HSCs and myeloid progenitor cells and maintains the differentiation block induced by the fusion protein. Nras(G12D) and Cbfß-SMMHC synergize to induce leukemia in mice in a cell-autonomous manner, with a shorter median latency and higher leukemia-initiating cell activity than that of mice expressing Cbfß-SMMHC. Furthermore, Nras(LSL-G12D); Cbfb(56M) leukemic cells were sensitive to pharmacologic inhibition of the MEK/ERK signaling pathway, increasing apoptosis and Bim protein levels. These studies demonstrate that Cbfß-SMMHC and Nras(G12D) promote the survival of preleukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define Nras(LSL-G12D); Cbfb(56M) mice as a valuable genetic model for the study of inversion(16) AML-targeted therapies.
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Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Oncogénicas/metabolismo , Preleucemia/metabolismo , Preleucemia/patología , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Supervivencia Celular , Técnicas de Sustitución del Gen , Leucemia Experimental/etiología , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Unión al GTP Monoméricas/genética , Mutación Missense , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas Oncogénicas/genética , Proteínas de Fusión Oncogénica/genética , Preleucemia/genética , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Oncogenic mutations in components of cytokine signaling pathways elicit ligand-independent activation of downstream signaling, enhancing proliferation and survival in acute myeloid leukemia (AML). The myeloproliferative leukemia virus oncogene, MPL, a homodimeric receptor activated by thrombopoietin (THPO), is mutated in myeloproliferative disorders but rarely in AML. Here we show that wild-type MPL expression is increased in a fraction of human AML samples expressing RUNX1-ETO, a fusion protein created by chromosome translocation t(8;21), and that up-regulation of Mpl expression in mice induces AML when coexpressed with RUNX1-ETO. The leukemic cells are sensitive to THPO, activating survival and proliferative responses. Mpl expression is not regulated by RUNX1-ETO in mouse hematopoietic progenitors or leukemic cells. Moreover, we find that activation of PI3K/AKT but not ERK/MEK pathway is a critical mediator of the MPL-directed antiapoptotic function in leukemic cells. Hence, this study provides evidence that up-regulation of wild-type MPL levels promotes leukemia development and maintenance through activation of the PI3K/AKT axis, and suggests that inhibitors of this axis could be effective for treatment of MPL-positive AML.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Proteínas de Fusión Oncogénica/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Trombopoyetina/metabolismo , Trombopoyetina/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Médula Ósea/metabolismo , Médula Ósea/patología , Trasplante de Médula Ósea , Ciclo Celular , Proliferación Celular , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Humanos , Técnicas para Inmunoenzimas , Leucemia Mieloide Aguda/genética , Ratones , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , Proteína 1 Compañera de Translocación de RUNX1 , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Trombopoyetina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Tasa de Supervivencia , Trombopoyetina/genética , Translocación Genética , Células Tumorales CultivadasRESUMEN
The receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is considered a master regulator of adipocyte differentiation and promotes glucose and lipid metabolism in mature adipocytes. We recently identified the yeast Sterile 20 (Ste20) protein kinase ortholog, Map4k4, in an RNA interference-based screen as an inhibitor of PPARgamma expression in cultured adipocytes. Here, we show that RNA interference-mediated silencing of Map4k4 elevates the levels of both PPARgamma1 and PPARgamma2 proteins in 3T3-L1 adipocytes without affecting PPARgamma mRNA levels, suggesting that Map4k4 regulates PPARgamma at a post-transcriptional step. PPARgamma degradation rates are remarkably rapid as measured in the presence of cycloheximide (t(1/2) = 2 h), but silencing Map4k4 had no effect on PPARgamma degradation. However, depletion of Map4k4 significantly enhances [(35)S]methionine/cysteine incorporation into proteins, suggesting that Map4k4 signaling decreases protein translation. We show a function of Map4k4 is to inhibit rapamycin-sensitive mammalian target of rapamycin (mTOR) activity, decreasing 4E-BP1 phosphorylation. In addition, our results show mTOR and 4E-BP1 are required for the increased PPARgamma protein expression upon Map4k4 knockdown. Consistent with this concept, adenovirus-mediated expression of Map4k4 decreased PPARgamma protein levels and mTOR phosphorylation. These data show that Map4k4 negatively regulates PPARgamma post-transcriptionally, by attenuating mTOR signaling and a 4E-BP1-dependent mechanism.