RESUMEN
Tumor reliance on glycolysis is a hallmark of cancer. Immunotherapy is more effective in controlling glycolysis-low tumors lacking lactate dehydrogenase (LDH) due to reduced tumor lactate efflux and enhanced glucose availability within the tumor microenvironment (TME). LDH inhibitors (LDHi) reduce glucose uptake and tumor growth in preclinical models, but their impact on tumor-infiltrating T cells is not fully elucidated. Tumor cells have higher basal LDH expression and glycolysis levels compared with infiltrating T cells, creating a therapeutic opportunity for tumor-specific targeting of glycolysis. We demonstrate that LDHi treatment (a) decreases tumor cell glucose uptake, expression of the glucose transporter GLUT1, and tumor cell proliferation while (b) increasing glucose uptake, GLUT1 expression, and proliferation of tumor-infiltrating T cells. Accordingly, increasing glucose availability in the microenvironment via LDH inhibition leads to improved tumor-killing T cell function and impaired Treg immunosuppressive activity in vitro. Moreover, combining LDH inhibition with immune checkpoint blockade therapy effectively controls murine melanoma and colon cancer progression by promoting effector T cell infiltration and activation while destabilizing Tregs. Our results establish LDH inhibition as an effective strategy for rebalancing glucose availability for T cells within the TME, which can enhance T cell function and antitumor immunity.
Asunto(s)
Glucosa , L-Lactato Deshidrogenasa , Microambiente Tumoral , Animales , Ratones , Glucosa/metabolismo , Microambiente Tumoral/inmunología , Microambiente Tumoral/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/inmunología , Humanos , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 1/antagonistas & inhibidores , Transportador de Glucosa de Tipo 1/inmunología , Transportador de Glucosa de Tipo 1/genética , Línea Celular Tumoral , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Glucólisis/efectos de los fármacos , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Neoplasias del Colon/inmunología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Neoplasias del Colon/metabolismo , Inhibidores Enzimáticos/farmacología , Inmunoterapia , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéuticoRESUMEN
MEK inhibitors (MEKis) have shown limited success as a treatment for MAPK/ERK pathway-dependent cancers due to various resistance mechanisms tumor cells can employ. CH5126766 (CKI27) is an inhibitor that binds to MEK and prevents release of RAF, reducing the relief of negative feedback commonly observed with other MEKis. We observed that CKI27 increased MHC expression on tumor cells and improved T cell-mediated killing. Yet, CKI27 also decreased T-cell proliferation, activation, and cytolytic activity by inhibiting the MAPK/ERK pathway that is activated downstream of T cell-receptor signaling. Therefore, we aimed to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. Intermittent administration of CKI27 allowed T cells to partially recover and co-stimulation via GITR and OX-40 agonist antibodies completely alleviated inhibition of function. In Kras mutant lung and colon tumor mouse models, intermittent CKI27 and anti-GITR significantly decreased tumor growth and prolonged survival when further combined with CTLA-4 immune checkpoint blockade. Moreover, this triple combination increased CD8+ and CD4+ T-cell proliferation, activation, and effector/memory subsets in the tumor draining lymph nodes and tumors and led to intratumoral regulatory T cell (Treg) destabilization. These data, collectively, will allow for more informed decisions when optimizing combination regimens by overcoming resistance, reducing toxicity, and generating long-term immune responses.
RESUMEN
In addition to playing a major role in tumor cell biology, p53 generates a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages. We examined whether increasing p53 signaling in the tumor microenvironment influences antitumor T cell immunity. Our findings indicate that increased p53 signaling induced either pharmacologically with APR-246 (eprenetapopt) or in p53-overexpressing transgenic mice can disinhibit antitumor T cell immunity and augment the efficacy of immune checkpoint blockade. We demonstrated that increased p53 expression in tumor-associated macrophages induces canonical p53-associated functions such as senescence and activation of a p53-dependent senescence-associated secretory phenotype. This was linked with decreased expression of proteins associated with M2 polarization by tumor-associated macrophages. Our preclinical data led to the development of a clinical trial in patients with solid tumors combining APR-246 with pembrolizumab. Biospecimens from select patients participating in this ongoing trial showed that there was a suppression of M2-polarized myeloid cells and increase in T cell proliferation with therapy in those who responded to the therapy. Our findings, based on both genetic and a small molecule-based pharmacological approach, suggest that increasing p53 expression in tumor-associated macrophages reprograms the tumor microenvironment to augment the response to immune checkpoint blockade.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Macrófagos Asociados a Tumores , Animales , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones , Quinuclidinas , Microambiente Tumoral , Proteína p53 Supresora de Tumor/genéticaRESUMEN
KRAS is one of the driver oncogenes in non-small-cell lung cancer (NSCLC) but remains refractory to current modalities of targeted pathway inhibition, which include inhibiting downstream kinase MEK to circumvent KRAS activation. Here, we show that pulsatile, rather than continuous, treatment with MEK inhibitors (MEKis) maintains T cell activation and enables their proliferation. Two MEKis, selumetinib and trametinib, induce T cell activation with increased CTLA-4 expression and, to a lesser extent, PD-1 expression on T cells in vivo after cyclical pulsatile MEKi treatment. In addition, the pulsatile dosing schedule alone shows superior anti-tumor effects and delays the emergence of drug resistance. Furthermore, pulsatile MEKi treatment combined with CTLA-4 blockade prolongs survival in mice bearing tumors with mutant Kras. Our results set the foundation and show the importance of a combinatorial therapeutic strategy using pulsatile targeted therapy together with immunotherapy to optimally enhance tumor delay and promote long-term anti-tumor immunity.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Linfocitos T/metabolismo , Animales , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Piridonas/farmacología , Piridonas/uso terapéutico , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , Tasa de Supervivencia , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
Genetic and functional studies underscore the central role of JAK/STAT signaling in myeloproliferative neoplasms (MPNs). However, the mechanisms that mediate transformation in MPNs are not fully delineated, and clinically utilized JAK inhibitors have limited ability to reduce disease burden or reverse myelofibrosis. Here we show that MPN progenitor cells are characterized by marked alterations in gene regulation through differential enhancer utilization, and identify nuclear factor κB (NF-κB) signaling as a key pathway activated in malignant and non-malignant cells in MPN. Inhibition of BET bromodomain proteins attenuated NF-κB signaling and reduced cytokine production in vivo. Most importantly, combined JAK/BET inhibition resulted in a marked reduction in the serum levels of inflammatory cytokines, reduced disease burden, and reversed bone marrow fibrosis in vivo.
Asunto(s)
Citocinas/metabolismo , Inflamación/tratamiento farmacológico , Trastornos Mieloproliferativos/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Janus Quinasa 2/genética , Ratones Transgénicos , Mutación/efectos de los fármacos , FN-kappa B/metabolismo , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacosRESUMEN
JAK1 is a critical effector of pro-inflammatory cytokine signaling and plays important roles in immune function, while abnormal JAK1 activity has been linked to immunological and neoplastic diseases. Specific functions of JAK1 in the context of hematopoiesis, and specifically within hematopoietic stem cells (HSCs), have not clearly been delineated. Here, we show that conditional Jak1 loss in HSCs reduces their self-renewal and markedly alters lymphoid/myeloid differentiation in vivo. Jak1-deficient HSCs exhibit decreased competitiveness in vivo and are unable to rescue hematopoiesis in the setting of myelosuppression. They exhibit increased quiescence, an inability to enter the cell cycle in response to hematopoietic stress, and a marked reduction in cytokine sensing, including in response to type I interferons and IL-3. Moreover, Jak1 loss is not fully rescued by expression of a constitutively active Jak2 allele. Together, these data highlight an essential role for Jak1 in HSC homeostasis and stress responses.
Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Interleucina-3/metabolismo , Janus Quinasa 1/metabolismo , Estrés Fisiológico , Alelos , Animales , Trasplante de Médula Ósea , Ciclo Celular , Diferenciación Celular , Activación Enzimática , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Terapia de Inmunosupresión , Interferón Tipo I/metabolismo , Ratones Noqueados , Células Mieloides/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: When the glial fibrillary acidic protein (GFAP) promoter is used to express cellular toxins that eliminate glia in mice, intestinal epithelial permeability and proliferation increase; this led to the concept that glia are required for maintenance of the gastrointestinal epithelium. Many enteric glia, however, particularly in the mucosa, do not express GFAP. In contrast, virtually all enteric glia express proteolipid protein 1 (PLP1). We investigated whether elimination of PLP1-expressing cells compromises epithelial maintenance or gastrointestinal motility. METHODS: We generated mice that express tamoxifen-inducible Cre recombinase under control of the Plp1 promoter and carry the diptheria toxin subunit A (DTA) transgene in the Rosa26 locus (Plp1CreER;Rosa26DTA mice). In these mice, PLP1-expressing glia are selectively eliminated without affecting neighboring cells. We measured epithelial barrier function and gastrointestinal motility in these mice and littermate controls, and analyzed epithelial cell proliferation and ultrastructure from their intestinal tissues. To compare our findings with those from previous studies, we also eliminated glia with ganciclovir in GfapHSV-TK mice. RESULTS: Expression of DTA in PLP1-expressing cells selectively eliminated enteric glia from the small and large intestines, but caused no defects in epithelial proliferation, barrier integrity, or ultrastructure. In contrast, administration of ganciclovir to GfapHSV-TK mice eliminated fewer glia but caused considerable non-glial toxicity and epithelial cell death. Elimination of PLP1-expressing cells did not reduce survival of neurons in the intestine, but altered gastrointestinal motility in female, but not male, mice. CONCLUSIONS: Using the Plp1 promoter to selectively eliminate glia in mice, we found that enteric glia are not required for maintenance of the intestinal epithelium, but are required for regulation of intestinal motility in females. Previous observations supporting the concept that maintenance of the intestinal epithelium requires enteric glia can be attributed to non-glial toxicity in GfapHSV-TK mice and epithelial-cell expression of GFAP. Contrary to widespread notions, enteric glia are therefore not required for epithelial homeostasis. However, they regulate intestinal motility in a sex-dependent manner.
Asunto(s)
Sistema Nervioso Entérico/fisiología , Motilidad Gastrointestinal , Mucosa Intestinal/fisiología , Intestinos/inervación , Neuroglía/fisiología , Animales , Proliferación Celular , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/ultraestructura , Femenino , Ganciclovir/toxicidad , Genotipo , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Homeostasis , Integrasas/genética , Integrasas/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestructura , Intestinos/efectos de los fármacos , Intestinos/ultraestructura , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Proteolipídica de la Mielina/genética , Neuroglía/metabolismo , Neuroglía/ultraestructura , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fenotipo , Regiones Promotoras Genéticas , ARN no Traducido/genética , Factores Sexuales , Factores de TiempoRESUMEN
The development of the dual Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib for the treatment of myeloproliferative neoplasms (MPNs) has led to studies of ruxolitinib in other clinical contexts, including JAK-mutated acute lymphoblastic leukemia (ALL). However, the limited ability of JAK inhibition to induce molecular or clinicopathological responses in MPNs suggests a need for development of better therapies for JAK kinase-dependent malignancies. Here, we demonstrate that heat shock protein 90 (HSP90) inhibition using a purine-scaffold HSP90 inhibitor in early clinical development is an effective therapeutic approach in JAK-dependent ALL and can overcome persistence to JAK-inhibitor therapy in ALL cells.
Asunto(s)
Benzodioxoles/farmacología , Proteínas HSP90 de Choque Térmico , Janus Quinasa 1 , Janus Quinasa 2 , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras , Purinas/farmacología , Animales , Femenino , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Masculino , Ratones , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the enteric nervous system (ENS), glia outnumber neurons by 4-fold and form an extensive network throughout the gastrointestinal tract. Growing evidence for the essential role of enteric glia in bowel function makes it imperative to understand better their molecular marker expression and how they relate to glia in the rest of the nervous system. We analyzed expression of markers of astrocytes and oligodendrocytes in the ENS and found, unexpectedly, that proteolipid protein 1 (PLP1) is specifically expressed by glia in adult mouse intestine. PLP1 and S100ß are the markers most widely expressed by enteric glia, while glial fibrillary acidic protein expression is more restricted. Marker expression in addition to cellular location and morphology distinguishes a specific subpopulation of intramuscular enteric glia, suggesting that a combinatorial code of molecular markers can be used to identify distinct subtypes. To assess the similarity between enteric and extraenteric glia, we performed RNA sequencing analysis on PLP1-expressing cells in the mouse intestine and compared their gene expression pattern to that of other types of glia. This analysis shows that enteric glia are transcriptionally unique and distinct from other cell types in the nervous system. Enteric glia express many genes characteristic of the myelinating glia, Schwann cells and oligodendrocytes, although there is no evidence of myelination in the murine ENS. GLIA 2015;63:2040-2057.
RESUMEN
Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.