RESUMEN
Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.
Asunto(s)
Inmunoterapia , Neoplasias , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 2 , Animales , Humanos , Ratones , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Inhibidores de Puntos de Control Inmunológico , Inmunoterapia/métodos , Interferones/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 2/antagonistas & inhibidores , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Overexpression of the histone methyltransferase MMSET in t(4;14)+ multiple myeloma patients is believed to be the driving factor in the pathogenesis of this subtype of myeloma. MMSET catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2), and its overexpression causes a global increase in H3K36me2, redistributing this mark in a broad, elevated level across the genome. Here, we demonstrate that an increased level of MMSET also induces a global reduction of lysine 27 trimethylation on histone H3 (H3K27me3). Despite the net decrease in H3K27 methylation, specific genomic loci exhibit enhanced recruitment of the EZH2 histone methyltransferase and become hypermethylated on this residue. These effects likely contribute to the myeloma phenotype since MMSET-overexpressing cells displayed increased sensitivity to EZH2 inhibition. Furthermore, we demonstrate that such MMSET-mediated epigenetic changes require a number of functional domains within the protein, including PHD domains that mediate MMSET recruitment to chromatin. In vivo, targeting of MMSET by an inducible shRNA reversed histone methylation changes and led to regression of established tumors in athymic mice. Together, our work elucidates previously unrecognized interplay between MMSET and EZH2 in myeloma oncogenesis and identifies domains to be considered when designing inhibitors of MMSET function.
Asunto(s)
Metilación de ADN/genética , Epigénesis Genética/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Mieloma Múltiple/genética , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica/genética , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Cromatina/genética , Femenino , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Humanos , Lisina/genética , Ratones , Ratones Endogámicos C57BL , Mieloma Múltiple/metabolismo , Complejo Represivo Polycomb 2/genética , ARN Interferente Pequeño/genéticaRESUMEN
Interferons (IFNs) have important antiviral and antineoplastic properties, but the precise mechanisms required for generation of these responses remain to be defined. We provide evidence that during engagement of the Type I IFN receptor (IFNR), there is up-regulation of expression of Sprouty (Spry) proteins 1, 2, and 4. Our studies demonstrate that IFN-inducible up-regulation of Spry proteins is Mnk kinase-dependent and results in suppressive effects on the IFN-activated p38 MAP kinase (MAPK), the function of which is required for transcription of interferon-stimulated genes (ISGs). Our data establish that ISG15 mRNA expression and IFN-dependent antiviral responses are enhanced in Spry1,2,4 triple knock-out mouse embryonic fibroblasts, consistent with negative feedback regulatory roles for Spry proteins in IFN-mediated signaling. In other studies, we found that siRNA-mediated knockdown of Spry1, Spry2, or Spry4 promotes IFN-inducible antileukemic effects in vitro and results in enhanced suppressive effects on malignant hematopoietic progenitors from patients with polycythemia vera. Altogether, our findings demonstrate that Spry proteins are potent regulators of Type I IFN signaling and negatively control induction of Type I IFN-mediated biological responses.
Asunto(s)
Interferón Tipo I/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Receptor de Interferón alfa y beta/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Interferón Tipo I/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Fosfoproteínas/genética , Policitemia Vera/genética , Policitemia Vera/metabolismo , Policitemia Vera/patología , Proteínas Serina-Treonina Quinasas , Receptor de Interferón alfa y beta/genética , Células U937 , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. SIGNIFICANCE: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression.This article is highlighted in the In This Issue feature, p. 1307.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Genes Supresores de Tumor , Leucemia Mieloide Aguda/genética , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ratones , Ratones Endogámicos C57BL , Mutación , Trastornos Mieloproliferativos/genéticaRESUMEN
We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 mum diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover.
RESUMEN
NSD2, a histone methyltransferase specific for methylation of histone 3 lysine 36 (H3K36), exhibits a glutamic acid to lysine mutation at residue 1099 (E1099K) in childhood acute lymphocytic leukemia (ALL), and cells harboring this mutation can become the predominant clone in relapsing disease. We studied the effects of this mutant enzyme in silico, in vitro, and in vivo using gene edited cell lines. The E1099K mutation altered enzyme/substrate binding and enhanced the rate of H3K36 methylation. As a result, cell lines harboring E1099K exhibit increased H3K36 dimethylation and reduced H3K27 trimethylation, particularly on nucleosomes containing histone H3.1. Mutant NSD2 cells exhibit reduced apoptosis and enhanced proliferation, clonogenicity, adhesion, and migration. In mouse xenografts, mutant NSD2 cells are more lethal and brain invasive than wildtype cells. Transcriptional profiling demonstrates that mutant NSD2 aberrantly activates factors commonly associated with neural and stromal lineages in addition to signaling and adhesion genes. Identification of these pathways provides new avenues for therapeutic interventions in NSD2 dysregulated malignancies.
Asunto(s)
Reprogramación Celular , N-Metiltransferasa de Histona-Lisina , Mutación Missense , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Represoras , Sustitución de Aminoácidos , Células HeLa , Xenoinjertos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
PURPOSE: The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. EXPERIMENTAL DESIGN: We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage-committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. RESULTS: JAK2V617F -mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. CONCLUSIONS: Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.
Asunto(s)
Janus Quinasa 2/metabolismo , Megacariocitos/metabolismo , Megacariocitos/patología , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Factor de Transcripción STAT5/metabolismo , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Proliferación Celular/fisiología , Femenino , Humanos , Janus Quinasa 2/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trastornos Mieloproliferativos/genética , Mutación Puntual , Factor de Transcripción STAT5/genética , Transducción de SeñalRESUMEN
CHAF1B is the p60 subunit of the chromatin assembly factor (CAF1) complex, which is responsible for assembly of histones H3.1/H4 heterodimers at the replication fork during S phase. Here we report that CHAF1B is required for normal hematopoiesis while its overexpression promotes leukemia. CHAF1B has a pro-leukemia effect by binding chromatin at discrete sites and interfering with occupancy of transcription factors that promote myeloid differentiation, such as CEBPA. Reducing Chaf1b activity by either heterozygous deletion or overexpression of a CAF1 dominant negative allele is sufficient to suppress leukemogenesis in vivo without impairing normal hematopoiesis.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Cromatina/metabolismo , Hematopoyesis/fisiología , Leucemia Mieloide Aguda/patología , Nucleosomas/metabolismo , Proteínas/metabolismo , Adulto , Animales , Sitios de Unión/fisiología , Diferenciación Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/genética , Factor 1 de Ensamblaje de la Cromatina/genética , Exorribonucleasas , Femenino , Hematopoyesis/genética , Humanos , Células Jurkat , Leucemia Mieloide Aguda/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/fisiología , Proteínas/genética , Proteínas Represoras , RibonucleasasRESUMEN
The ROCK-II isoform of Rho's downstream effector, Rho kinase, has been linked with greater invasion and metastasis in solid tumors. We have previously shown that ROCK-II is overexpressed at the advancing edge of colon cancers. The mechanism whereby ROCK-II contributes invasion, particularly in the setting of colon cancer, remains to be elucidated fully. To better understand its contribution, we evaluated ROCK-II expression in both non-malignant (NCM460 and IEC-6) and malignant (Caco-2 E, SW620, and HCT-116) intestinal epithelial cell lines grown in type I collagen scaffolds. Using multiphoton microscopy, we observed that ROCK-II localized to the actin cytoskeleton in non-malignant cells but localized to the cell periphery as focal collections with an absence of adjacent collagen in all colon cancer cell lines. By transmission electron microscopy, these collections corresponded with finger-like projections previously described as invadopodia. Immunogold staining with cortactin, matrix metalloprotease (MMP)-2, -9, and -13 confirmed that these were indeed invadopodia. To further link ROCK-II to colon cancer invasion, we treated non-malignant and malignant intestinal epithelial cell lines with ROCK-II siRNA and evaluated depth of invasion, proliferation, and MMP-2, -9, and -13 activities. The most striking effect was seen in the highly tumorigenic cell lines, SW620 and HCT-116, wherein ROCK-II knockdown resulted in a two-fold or more reduction in invasion. This reduction in invasion was not due to a decrease in cell proliferation, as a significant reduction in proliferation was only observed in the two non-malignant intestinal cell lines. Finally, both MMP-2 and -13 activities were significantly decreased in all colon cancer cell lines. Taken together, these data suggest for the first time that ROCK-II is a critical mediator of colon cancer cell invasion through its modulation of MMP-2 and -13 at the site of invadopodia but regulates proliferation in non-malignant intestinal cells.
Asunto(s)
Neoplasias del Colon/enzimología , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Seudópodos/metabolismo , Quinasas Asociadas a rho/metabolismo , Citoesqueleto de Actina/metabolismo , Línea Celular , Proliferación Celular , Colágeno Tipo I/metabolismo , Neoplasias del Colon/patología , Activación Enzimática , Células Epiteliales/enzimología , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Microscopía de Fluorescencia por Excitación Multifotónica , Invasividad NeoplásicaRESUMEN
Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Histona Demetilasas/deficiencia , Mieloma Múltiple/patología , Proteínas Nucleares/deficiencia , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/patología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Desdiferenciación Celular/efectos de los fármacos , Desdiferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Clonales , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Demetilasas/metabolismo , Histonas/metabolismo , Indazoles/farmacología , Factores Reguladores del Interferón/metabolismo , Lisina/metabolismo , Metilación , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/genética , Mutación/genética , Proteínas Nucleares/metabolismo , Fenotipo , Piridonas/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Two of the most common myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), are associated with exceedingly low survival rates despite recent therapeutic advances. While their etiology is not completely understood, evidence suggests that certain chromosomal abnormalities contribute to MDS and AML progression. Among the most frequent chromosomal abnormalities in these disorders are alterations of chromosome 7: either complete loss of one copy of chromosome 7 (-7) or partial deletion of 7q (del(7q)), both of which increase the risk of progression from MDS to AML and are associated with chemoresistance. Notably, 7q36.1, a critical minimally deleted region in 7q, includes the gene encoding the histone methyltransferase mixed-lineage leukemia 3 (MLL3), which is also mutated in a small percentage of AML patients. However, the mechanisms by which MLL3 loss contributes to malignancy are unknown. Using an engineered mouse model expressing a catalytically inactive form of Mll3, we found a significant shift in hematopoiesis toward the granulocyte/macrophage lineage, correlating with myeloid infiltration and enlargement of secondary lymphoid organs. Therefore, we propose that MLL3 loss in patients may contribute to the progression of MDS and AML by promoting myelopoiesis.