RESUMEN
Current limitations in using chimeric antigen receptor T(CART) cells to treat patients with hematological cancers include limited expansion and persistence in vivo that contribute to cancer relapse. Patients with chronic lymphocytic leukemia (CLL) have terminally differentiated T cells with an exhausted phenotype and experience low complete response rates after autologous CART therapy. Because PI3K inhibitor therapy is associated with the development of T-cell-mediated autoimmunity, we studied the effects of inhibiting the PI3Kδ and PI3Kγ isoforms during the manufacture of CART cells prepared from patients with CLL. Dual PI3Kδ/γ inhibition normalized CD4/CD8 ratios and maximized the number of CD8+ T-stem cell memory, naive, and central memory T-cells with dose-dependent decreases in expression of the TIM-3 exhaustion marker. CART cells manufactured with duvelisib (Duv-CART cells) showed significantly increased in vitro cytotoxicity against CD19+ CLL targets caused by increased frequencies of CD8+ CART cells. Duv-CART cells had increased expression of the mitochondrial fusion protein MFN2, with an associated increase in the relative content of mitochondria. Duv-CART cells exhibited increased SIRT1 and TCF1/7 expression, which correlated with epigenetic reprograming of Duv-CART cells toward stem-like properties. After transfer to NOG mice engrafted with a human CLL cell line, Duv-CART cells expressing either a CD28 or 41BB costimulatory domain demonstrated significantly increased in vivo expansion of CD8+ CART cells, faster elimination of CLL, and longer persistence. Duv-CART cells significantly enhanced survival of CLL-bearing mice compared with conventionally manufactured CART cells. In summary, exposure of CART to a PI3Kδ/γ inhibitor during manufacturing enriched the CART product for CD8+ CART cells with stem-like qualities and enhanced efficacy in eliminating CLL in vivo.
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Inmunoterapia Adoptiva/métodos , Isoquinolinas/uso terapéutico , Leucemia Linfocítica Crónica de Células B/terapia , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Purinas/uso terapéutico , Animales , Células Cultivadas , Técnicas de Reprogramación Celular/métodos , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Epigénesis Genética , Humanos , Leucemia Linfocítica Crónica de Células B/genética , RatonesRESUMEN
OBJECTIVES: Low-grade-serous-ovarian-carcinoma (LGSOC) is characterized by a high recurrence rate and limited therapeutic options. About one-third of LGSOC contains mutations in MAPK pathway genes such as KRAS/NRAS/BRAF. Avutometinib is a dual RAF/MEK inhibitor while defactinib and VS-4718 are focal-adhesion-kinase-inhibitors (FAKi). We determined the preclinical efficacy of avutometinib±VS-4718 in LGSOC patient-derived-tumor-xenografts (PDX). METHODS: Whole-exome-sequencing (WES) was used to evaluate the genetic fingerprint of 3 patient-derived LGSOC (OVA(K)250, PERIT(M)17 and A(PE)148). OVA(K)250 tissue was successfully xenografted as PDX into female CB17/lcrHsd-Prkdc/SCID-mice. Animals were treated with either control, avutometinib, VS-4718, or avutometinib/ VS-4718 once daily five days on and two days off through oral gavage. Mechanistic studies were performed ex vivo using avutometinib±defactinib treated LGSOC tumor samples by western blot. RESULTS: WES results demonstrated wild-type KRAS in all 3 LGSOC. OVA(K)250 PDX showed gain-of-function mutations (GOF) in PTK2 and PTK2B genes, and loss-of-heterozygosity in ADRB2, potentially sensitizing to FAK and RAF/MEK inhibition. The combination of avutometinib/ VS-4718 demonstrated strong tumor-growth inhibition compared to controls starting at day 9 (p < 0.002) in OVA(K)250PDX. By 60 days, mice treated with avutometinib alone and avutometinib/VS-4718 were still alive; compared to median survival of 20 days in control-treated mice and of 35 days in VS-4718-treated mice (p < 0.0001). By western-blot assays exposure of OVA(K)250 to avutometinib, FAKi defactinib and their combination demonstrated decreased phosphorylated FAK (p-FAK) as well as decreased p-ERK. CONCLUSION: Avutometinib, and to a larger extent its combination with FAK inhibitor VS-4718, demonstrated promising in vivo activity against a KRAS wild-type LGSOC-PDX. These data support the ongoing registration-directed study (RAMP201/NCT04625270).
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Quinasa 1 de Adhesión Focal , Neoplasias Ováricas , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Humanos , Animales , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ratones , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/patología , Cistadenocarcinoma Seroso/genética , Inhibidores de Proteínas Quinasas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Secuenciación del Exoma , Benzamidas , Difenilamina/análogos & derivados , Pirazinas , SulfonamidasRESUMEN
OBJECTIVES: Uterine carcinosarcomas (UCS) are rare, biologically aggressive tumors. Since UCS may harbor mutations in RAS/MAPK pathway genes we evaluated the preclinical in vitro and in vivo efficacy of the RAF/MEK clamp avutometinib in combination with the focal adhesion kinase (FAK) inhibitors defactinib or VS-4718 against multiple primary UCS cell lines and xenografts. METHODS: Whole-exome-sequencing (WES) was used to evaluate the genetic landscape of 5 primary UCS cell lines. The in vitro activity of avutometinib ± FAK inhibitor was evaluated using cell viability and cell cycle assays against primary UCS cell lines. Mechanistic studies were performed using western blot assays while in vivo experiments were completed in UCS tumor bearing mice treated with avutometinib ± FAK inhibitor by oral gavage. RESULTS: WES results demonstrated multiple UCS cell lines harbor genetic alterations including KRAS, PTK2, BRAF, MAP2K, and MAP2K1, potentially sensitizing to FAK and RAF/MEK inhibition. Four out of five of the UCS cell lines demonstrated in vitro sensitivity to FAK and/or RAF/MEK inhibition when used alone or in combination. By western blot assays, exposure of UCS cell lines to the combination of defactinib/avutometinib demonstrated decreased phosphorylated (p)-FAK as well as decreased p-ERK. In vivo, the combination of avutometinib/VS-4718 demonstrated superior tumor growth inhibition and longer survival compared to single agent treatment and controls starting at day 10 (p < 0.002) in UCS xenografts. CONCLUSION: The combination of avutometinib and defactinib demonstrates promising in vitro and in vivo anti-tumor activity against primary UCS cell lines and xenografts.
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Carcinosarcoma , Neoplasias Uterinas , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Humanos , Animales , Neoplasias Uterinas/tratamiento farmacológico , Neoplasias Uterinas/patología , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Línea Celular Tumoral , Ratones , Carcinosarcoma/tratamiento farmacológico , Carcinosarcoma/patología , Carcinosarcoma/genética , Carcinosarcoma/metabolismo , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Indoles/farmacología , Quinasas raf/antagonistas & inhibidores , Quinasas raf/metabolismo , Quinasas raf/genética , Secuenciación del Exoma , Ratones Desnudos , Benzamidas , Pirazinas , SulfonamidasRESUMEN
A small subset of cases of chronic lymphocytic leukemia undergoes transformation to diffuse large B-cell lymphoma, Richter syndrome (RS), which is associated with a poor prognosis. Conventional chemotherapy results in limited responses, underlining the need for novel therapeutic strategies. Here, we investigate the ex vivo and in vivo efficacy of the dual phosphatidylinositol 3-kinase-δ/γ (PI3K-δ/γ) inhibitor duvelisib (Duv) and the Bcl-2 inhibitor venetoclax (Ven) using 4 different RS patient-derived xenograft (PDX) models. Ex vivo exposure of RS cells to Duv, Ven, or their combination results in variable apoptotic responses, in line with the expression levels of target proteins. Although RS1316, IP867/17, and RS9737 cells express PI3K-δ, PI3K-γ, and Bcl-2 and respond to the drugs, RS1050 cells, expressing very low levels of PI3K-γ and lacking Bcl-2, are fully resistant. Moreover, the combination of these drugs is more effective than each agent alone. When tested in vivo, RS1316 and IP867/17 show the best tumor growth inhibition responses, with the Duv/Ven combination leading to complete remission at the end of treatment. The synergistic effect of Duv and Ven relies on the crosstalk between PI3K and apoptotic pathways occurring at the GSK3ß level. Indeed, inhibition of PI3K signaling by Duv results in GSK3ß activation, leading to ubiquitination and subsequent degradation of both c-Myc and Mcl-1, making RS cells more sensitive to Bcl-2 inhibition by Ven. This work provides, for the first time, a proof of concept of the efficacy of dual targeting of PI3K-δ/γ and Bcl-2 in RS and providing an opening for a Duv/Ven combination for these patients. Clinical studies in aggressive lymphomas, including RS, are under way. This trial was registered at www.clinicaltrials.gov as #NCT03892044.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase Ib , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Femenino , Humanos , Isoquinolinas/farmacología , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Purinas/farmacología , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lymphatic malformations (LM) are characterized by abnormal formation of lymphatic vessels and tissue overgrowth. The lymphatic vessels present in LM lesions may become blocked and enlarged as lymphatic fluid collects, forming a mass or cyst. Lesions are typically diagnosed during childhood and are often disfiguring and life threatening. Available treatments consist of sclerotherapy, surgical removal and therapies to diminish complications. We isolated lymphatic endothelial cells (LM-LEC) from a surgically removed microcystic LM lesion. LM-LEC and normal human dermal-LEC (HD-LEC) expressed endothelial (CD31, VE-Cadherin) as well as lymphatic endothelial (Podoplanin, PROX1, LYVE1)-specific markers. Targeted gene sequencing analysis in patient-derived LM-LEC revealed the presence of two mutations in class I phosphoinositide 3-kinases (PI3K) genes. One is an inherited, premature stop codon in the PI3K regulatory subunit PIK3R3. The second is a somatic missense mutation in the PI3K catalytic subunit PIK3CA; this mutation has been found in association with overgrowth syndromes and cancer growth. LM-LEC exhibited angiogenic properties: both cellular proliferation and sprouting in collagen were significantly increased compared with HD-LEC. AKT-Thr308 was constitutively hyper-phosphorylated in LM-LEC. Treatment of LM-LEC with PI3-Kinase inhibitors Wortmannin and LY294 decreased cellular proliferation and prevented the phosphorylation of AKT-Thr308 in both HD-LEC and LM-LEC. Treatment with the mTOR inhibitor rapamycin also diminished cellular proliferation, sprouting and AKT phosphorylation, but only in LM-LEC. Our results implicate disrupted PI3K-AKT signaling in LEC isolated from a human lymphatic malformation lesion.
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Endotelio/enzimología , Vasos Linfáticos/anomalías , Mutación , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Endotelio/patología , Femenino , Humanos , Masculino , Fosforilación , Sirolimus/farmacologíaRESUMEN
Glioblastomas are very difficult tumors to treat because they are highly invasive and disseminate within the normal brain, resulting in newly growing tumors. We have identified netrin-1 as a molecule that promotes glioblastoma invasiveness. As evidence, netrin-1 stimulates glioblastoma cell invasion directly through Matrigel-coated transwells, promotes tumor cell sprouting and enhances metastasis to lymph nodes in vivo. Furthermore, netrin-1 regulates angiogenesis as shown in specific angiogenesis assays such as enhanced capillary endothelial cells (EC) sprouting and by increased EC infiltration into Matrigel plugs in vivo, as does VEGF-A. This netrin-1 signaling pathway in glioblastoma cells includes activation of RhoA and cyclic AMP response element-binding protein (CREB). A novel finding is that netrin-1-induced glioblastoma invasiveness and angiogenesis are mediated by activated cathepsin B (CatB), a cysteine protease that translocates to the cell surface as an active enzyme and co-localizes with cell surface annexin A2 (ANXA2). The specific CatB inhibitor CA-074Me inhibits netrin-1-induced cell invasion, sprouting, and Matrigel plug angiogenesis. Silencing of CREB suppresses netrin-1-induced glioblastoma cell invasion, sprouting, and CatB expression. It is concluded that netrin-1 plays an important dual role in glioblastoma progression by promoting both glioblastoma cell invasiveness and angiogenesis in a RhoA-, CREB-, and CatB-dependent manner. Targeting netrin-1 pathways may be a promising strategy for brain cancer therapy.
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Catepsina B/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Neovascularización Patológica , Factores de Crecimiento Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Actinas/metabolismo , Animales , Anexina A2/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Colágeno/química , Combinación de Medicamentos , Femenino , Silenciador del Gen , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Laminina/química , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Modelos Biológicos , Invasividad Neoplásica , Netrina-1 , Unión Proteica , Proteoglicanos/química , Proteínas Recombinantes/metabolismoRESUMEN
KRAS is the most frequently mutated oncogene in human cancer and facilitates uncontrolled growth through hyperactivation of the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) pathway. The Son of Sevenless homolog 1 (SOS1) protein functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases and represents a druggable target in the pathway. Using a structure-based drug discovery approach, MRTX0902 was identified as a selective and potent SOS1 inhibitor that disrupts the KRAS:SOS1 protein-protein interaction to prevent SOS1-mediated nucleotide exchange on KRAS and translates into an anti-proliferative effect in cancer cell lines with genetic alterations of the KRAS-MAPK pathway. MRTX0902 augmented the antitumor activity of the KRAS G12C inhibitor adagrasib when dosed in combination in eight out of 12 KRAS G12C-mutant human non-small cell lung cancer and colorectal cancer xenograft models. Pharmacogenomic profiling in preclinical models identified cell cycle genes and the SOS2 homolog as genetic co-dependencies and implicated tumor suppressor genes (NF1 and PTEN) in resistance following combination treatment. Lastly, combined vertical inhibition of RTK/MAPK pathway signaling by MRTX0902 with inhibitors of EGFR or RAF/MEK led to greater downregulation of pathway signaling and improved antitumor responses in KRAS-MAPK pathway-mutant models. These studies demonstrate the potential clinical application of dual inhibition of SOS1 and KRAS G12C and additional SOS1 combination strategies that will aide in the understanding of SOS1 and RTK/MAPK biology in targeted cancer therapy.
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GATA-binding protein 2 (GATA2) and LIM domain only 2 (Lmo2) form common transcription complexes during hematopoietic differentiation. Here we show that these two transcription factors also play a key role in endothelial cells (EC) and lymphatic EC (LEC) function. Primary EC and tumor-associated blood vessels expressed GATA2 and Lmo2. VEGF-induced sprouting angiogenesis in both differentiating embryonic stem cells (embryoid bodies) and primary EC increased GATA2 and Lmo2 levels. Conversely, silencing of GATA2 and Lmo2 expression in primary EC inhibited VEGF-induced angiogenic activity, including EC migration and sprouting in vitro, two key steps of angiogenesis in vivo. This inhibition of EC function was associated with downregulated expression of neuropilin-2 (NRP2), a co-receptor of VEGFRs for VEGF, at the protein, mRNA and promoter levels. NRP2 overexpression partially rescued the impaired angiogenic sprouting in the GATA2/Lmo2 knockdown EC, confirming that GATA2 and Lmo2 mediated EC function, at least in part, by directly regulating NRP2 gene expression. Furthermore, it was found that primary LEC expressed GATA2 and Lmo2 as well. Silencing of GATA2 and Lmo2 expression in LEC inhibited VEGF-induced LEC sprouting, also in a NRP2-dependent manner. In conclusion, our results demonstrate that GATA2 and Lmo2 cooperatively regulate VEGF-induced angiogenesis and lymphangiogenesis via NRP2.
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Células Endoteliales/fisiología , Factor de Transcripción GATA2/fisiología , Proteínas con Dominio LIM/fisiología , Linfangiogénesis/fisiología , Neovascularización Fisiológica/fisiología , Neuropilina-2/fisiología , Transcripción Genética , Animales , Línea Celular Tumoral , Cuerpos Embrioides , Células Endoteliales/metabolismo , Femenino , Factor de Transcripción GATA2/genética , Regulación de la Expresión Génica , Glioblastoma/irrigación sanguínea , Glioblastoma/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas con Dominio LIM/genética , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/patología , Ratones , Ratones Desnudos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Neuropilina-2/genética , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma that is incurable with existing therapies, and therefore presents a significant unmet clinical need. The ability of this disease to overcome therapy, including those that target the B cell receptor pathway which has a pathogenic role in MCL, highlights the need to develop new treatment strategies. Herein, we demonstrate that a distinguishing feature of lymph node resident MCL cells is the expression of phosphatidylinositol 3-kinase γ (PI3Kγ), a PI3K isoform that is not highly expressed in other B cells or B-cell malignancies. By exploring the role of PI3K in MCL using different PI3K isoform inhibitors, we provide evidence that duvelisib, a dual PI3Kδ/γ inhibitor, has a greater effect than PI3Kδ- and PI3Kγ-selective inhibitors in blocking the proliferation of primary MCL cells and MCL cell lines, and in inhibiting tumour growth in a mouse xenograft model. In addition, we demonstrated that PI3Kδ/γ signalling is critical for migration of primary MCL cells and cell lines. Our data indicates that aberrant expression of PI3Kγ is a critical feature of MCL pathogenesis. Thus, we suggest that the dual PI3Kδ/γ duvelisib would be effective for the treatment of mantle cell lymphoma.
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Linfoma de Células del Manto , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Humanos , Ratones , Proliferación Celular , Modelos Animales de Enfermedad , Linfoma de Células del Manto/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacologíaRESUMEN
Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.
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Melanoma , Neoplasias Cutáneas , Neoplasias de la Úvea , Animales , Ratones , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/uso terapéutico , Evaluación Preclínica de Medicamentos , Neoplasias de la Úvea/tratamiento farmacológico , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Aryl hydrocarbon receptor (AHR) is a transcription factor that regulates the activity of multiple innate and adaptive immune cells subsequent to binding to numerous endogenous and exogenous ligands. For example, AHR is activated by the metabolite kynurenine, which is secreted into the tumor microenvironment by cancer cells leading to broad immunosuppression. Therefore, AHR inhibition provides a novel and ideal approach to stimulate immune-mediated recognition and subsequent eradication of tumor cells. We report here the discovery and characterization of IK-175, a novel, potent and selective AHR antagonist with favorable ADME and pharmacokinetic profiles in preclinical species. IK-175 inhibits AHR activity in experimental systems derived from multiple species including mouse, rat, monkey, and humans. In human primary immune cells, IK-175 decreased AHR target gene expression and anti-inflammatory cytokine release and increased proinflammatory cytokine release. Moreover, IK-175 led to a decrease in suppressive IL17A-, IL-22+ expressing T cells in a Th17 differentiation assay. IK-175 dose dependently blocks ligand-stimulated AHR activation of Cyp1a1 transcription in mouse liver and spleen, demonstrating on-target in vivo activity. IK-175 increases proinflammatory phenotype of the tumor microenvironment in mouse syngeneic tumors and in adjacent tumor-draining lymph nodes. As a monotherapy and combined with an anti-PD-1 antibody, IK-175 demonstrates antitumor activity in syngeneic mouse models of colorectal cancer and melanoma. IK-175 also demonstrates antitumor activity combined with liposomal doxorubicin in syngeneic mouse tumors. These studies provide rationale for targeting AHR in patients with cancer. IK-175 is being evaluated in a phase I clinical trial in patients with advanced solid tumors.
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Neoplasias , Receptores de Hidrocarburo de Aril , Animales , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocinas/metabolismo , Humanos , Terapia de Inmunosupresión , Quinurenina , Ratones , Neoplasias/tratamiento farmacológico , Ratas , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Microambiente TumoralRESUMEN
The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.
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PURPOSE: Uveal melanoma is the most common eye cancer in adults. Approximately 50% of patients with uveal melanoma develop metastatic uveal melanoma (mUM) in the liver, even after successful treatment of the primary lesions. mUM is refractory to current chemo- and immune-therapies, and most mUM patients die within a year. Uveal melanoma is characterized by gain-of-function mutations in GNAQ/GNA11, encoding Gαq proteins. We have recently shown that the Gαq-oncogenic signaling circuitry involves a noncanonical pathway distinct from the classical activation of PLCß and MEK-ERK. GNAQ promotes the activation of YAP1, a key oncogenic driver, through focal adhesion kinase (FAK), thereby identifying FAK as a druggable signaling hub downstream from GNAQ. However, targeted therapies often activate compensatory resistance mechanisms leading to cancer relapse and treatment failure. EXPERIMENTAL DESIGN: We performed a kinome-wide CRISPR-Cas9 sgRNA screen to identify synthetic lethal gene interactions that can be exploited therapeutically. Candidate adaptive resistance mechanisms were investigated by cotargeting strategies in uveal melanoma and mUM in vitro and in vivo experimental systems. RESULTS: sgRNAs targeting the PKC and MEK-ERK signaling pathways were significantly depleted after FAK inhibition, with ERK activation representing a predominant resistance mechanism. Pharmacologic inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in uveal melanoma cells and exerted cytotoxic effects, leading to tumor collapse in uveal melanoma xenograft and liver mUM models in vivo. CONCLUSIONS: Coupling the unique genetic landscape of uveal melanoma with the power of unbiased genetic screens, our studies reveal that FAK and MEK-ERK cotargeting may provide a new network-based precision therapeutic strategy for mUM treatment.See related commentary by Harbour, p. 2967.
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Quinasa 1 de Adhesión Focal/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Mutación con Ganancia de Función , Pruebas Genéticas/métodos , Sistema de Señalización de MAP Quinasas/genética , Melanoma/genética , Melanoma/terapia , Terapia Molecular Dirigida , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/terapia , Animales , Terapia Combinada , Femenino , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones Endogámicos NOD , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.
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Adyuvantes Inmunológicos/uso terapéutico , Hidrolasas/uso terapéutico , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/metabolismo , Neoplasias/tratamiento farmacológico , Animales , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Humanos , Neoplasias/enzimología , Neoplasias/inmunología , Neoplasias/metabolismo , Microambiente TumoralRESUMEN
For appropriate development, tissue and organ system morphogenesis and maturation must occur in synchrony with the overall developmental requirements of the host. Mistiming of such developmental events often results in disease. The hematopoietic system matures from the fetal state, characterized by robust erythrocytic output that supports prenatal growth in the hypoxic intrauterine environment, to the postnatal state wherein granulocytes predominate to provide innate immunity. Regulation of the developmental timing of these myeloerythroid states is not well understood. In this study, we find that expression of the heterochronic factor Lin28b decreases in common myeloid progenitors during hematopoietic maturation to adulthood in mice. This decrease in Lin28b coincides with accumulation of mature let-7 microRNAs, whose biogenesis is regulated by Lin28 proteins. We find that inhibition of let-7 in the adult hematopoietic system recapitulates fetal erythroid-dominant hematopoiesis. Conversely, deletion of Lin28b or ectopic activation of let-7 microRNAs in the fetal state induces a shift toward adult-like myeloid-dominant output. Furthermore, we identify Hmga2 as an effector of this genetic switch. These studies provide the first detailed analysis of the roles of endogenous Lin28b and let-7 in the timing of hematopoietic states during development.
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Proteínas de Unión al ADN/metabolismo , Eritropoyesis/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína HMGA2/biosíntesis , MicroARNs/metabolismo , Células Progenitoras Mieloides/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteína HMGA2/genética , Ratones , Ratones Noqueados , MicroARNs/genética , Células Progenitoras Mieloides/citología , Proteínas de Unión al ARNRESUMEN
We characterize the binding affinity and the thermodynamics of hybridization of triplex-forming antiparallel purine-hairpins composed of two antiparallel purine domains linked by a loop directed toward single-stranded and double-stranded DNA (ssDNA, dsDNA). Gel retardation assays and melting experiments reveal that a 13-mer purine-hairpin binds specifically and with a K ( d ) of 8 x 10(8) M to polypyrimidine ssDNA to form a triple helical structure. Remarkably, we show that purine-hairpins also bind polypurine/polypyrimidine stretches included in a dsDNA of several hundred bp in length. Binding of purine-hairpins to dsDNA occurs by triplex formation with the polypyrimidine strand, causing displacement of the polypurine strand. Because triplex formation is restricted to polypurine/polypyrimidine stretches of dsDNA, we studied the triplex formation between purine-hairpins and polypyrimidine targets containing purine interruptions. We found that an 11-mer purine-hairpin with an adenine opposite to a guanine interruption in the polypyrimidine track binds to ssDNA and dsDNA, allowing expansion of the possible target sites and increase in the length of purine-hairpins. Thus, when using a 20-mer purine-hairpin targeting an interruption-containing polypyrimidine target, the binding affinity is increased compared to its 13-mer antiparallel purine-hairpin counterpart. Surprisingly, this increase is much more pronounced than that observed for a tail-clamp purine-hairpin extended up to 20 nt in the Watson-Crick domain only. Thus, triplexforming antiparallel purine-hairpins can be a potentially useful strategy for both single-strand and double-strand nucleic acid recognition.
Asunto(s)
ADN/química , Purinas/química , Secuencia de Bases , Ensayo de Cambio de Movilidad Electroforética , Conformación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
Semaphorin 3F (SEMA3F) provides neuronal guidance cues via its ability to bind neuropilin 2 (NRP2) and Plexin A family molecules. Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood. Here, we analyze SEMA3F-NRP2 signaling responses in human endothelial, T cell and tumor cells using phosphokinase arrays, immunoprecipitation and Western blot analyses. Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase. We also find that the expression of vascular endothelial growth factor, as well as mTOR-inducible cellular activation responses and cytoskeleton stability are inhibited by SEMA3F-NRP2 interactions in vitro. In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts. Taken together, SEMA3F regulates mTOR signaling in diverse human cell types, suggesting that it has broad therapeutic implications.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-2/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Actinas/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Hipoxia/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Proteínas de la Membrana/genética , Ratones , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica/metabolismo , Proteínas del Tejido Nervioso/genética , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Carga Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The antiapoptotic protein survivin is an attractive target in cancer therapy because it is expressed differently in tumors and normal tissues and it is potentially required for cancer cells to remain viable. Given that survivin is also overexpressed in endothelial cells (ECs) of newly formed blood vessels found in tumors, its RNA targeting might compromise EC viability and interfere with tumor angiogenesis. We used two antisense strategies against survivin expression, antisense oligonucleotides (aODN) and small interfering RNA (siRNA), to study in ECs the contribution of survivin in various steps leading to tumor angiogenesis. A 21-mer phosphorothioate aODN and two siRNA oligonucleotides against survivin mRNA were designed to downregulate survivin expression. Survivin targeting caused (1) a strong growth-inhibitory effect, (2) a 4-fold increase in apoptosis, (3) an accumulation of cells in the S phase and a decrease in G2/M phase, (4) a dose-dependent inhibition of EC migration on Vitronectin, and (5) a decrease in capillary formation. Control oligonucleotides, an unrelated oligonucleotide, and one with four mismatches, had no significant effect. All these results show that survivin is a suitable target in cancer therapy because its inhibition in EC causes both a proapoptotic effect and an interruption of tumor angiogenesis. The two strategies used, classic aODN and siRNA technology, were very effective. Moreover, the latter can be used in the low nanomolar range, thus increasing the sensitivity of the treatment.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Neovascularización Patológica/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , ARN Interferente Pequeño/uso terapéutico , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Apoptosis/efectos de los fármacos , Técnicas de Cultivo de Célula , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/biosíntesis , Oligonucleótidos Antisentido/farmacología , ARN Interferente Pequeño/farmacología , SurvivinRESUMEN
S100A4, a member of the S100 calcium-binding protein family secreted by tumor and stromal cells, supports tumorigenesis by stimulating angiogenesis. We demonstrated that S100A4 synergizes with vascular endothelial growth factor (VEGF), via the RAGE receptor, in promoting endothelial cell migration by increasing KDR expression and MMP-9 activity. In vivo overexpression of S100A4 led to a significant increase in tumor growth and vascularization in a human melanoma xenograft M21 model. Conversely, when silencing S100A4 by shRNA technology, a dramatic decrease in tumor development of the pancreatic MiaPACA-2 cell line was observed. Based on these results we developed 5C3, a neutralizing monoclonal antibody against S100A4. This antibody abolished endothelial cell migration, tumor growth and angiogenesis in immunodeficient mouse xenograft models of MiaPACA-2 and M21-S100A4 cells. It is concluded that extracellular S100A4 inhibition is an attractive approach for the treatment of human cancer.