RESUMO
Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade lymphoma with no standard therapy. Nine asymptomatic patients treated with a first-in-human, neoantigen DNA vaccine experienced no dose limiting toxicities (primary endpoint, NCT01209871). All patients achieve stable disease or better, with one minor response, and median time to progression of 72+ months. Post-vaccine single-cell transcriptomics reveal dichotomous antitumor responses, with reduced tumor B-cells (tracked by unique B cell receptor) and their survival pathways, but no change in clonal plasma cells. Downregulation of human leukocyte antigen (HLA) class II molecules and paradoxical upregulation of insulin-like growth factor (IGF) by the latter suggest resistance mechanisms. Vaccine therapy activates and expands bone marrow T-cell clonotypes, and functional neoantigen-specific responses (secondary endpoint), but not co-inhibitory pathways or Treg, and reduces protumoral signaling by myeloid cells, suggesting favorable perturbation of the tumor immune microenvironment. Future strategies may require combinations of vaccines with agents targeting plasma cell subpopulations, or blockade of IGF-1 signaling or myeloid cell checkpoints.
Assuntos
Vacinas Anticâncer , Macroglobulinemia de Waldenstrom , Humanos , Masculino , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Vacinas Anticâncer/administração & dosagem , Pessoa de Meia-Idade , Feminino , Macroglobulinemia de Waldenstrom/imunologia , Macroglobulinemia de Waldenstrom/terapia , Macroglobulinemia de Waldenstrom/genética , Idoso , Microambiente Tumoral/imunologia , Medicina de Precisão/métodos , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologiaRESUMO
Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent antitumor effects in B-cell malignancies including acute lymphoblastic leukemia (ALL), but antigen loss remains the major cause of treatment failure. To mitigate antigen escape and potentially improve the durability of remission, we developed a dual-targeting approach using an optimized, bispecific CAR construct that targets both CD19 and BAFF-R. CD19/BAFF-R dual CAR T cells exhibited antigen-specific cytokine release, degranulation, and cytotoxicity against both CD19-/- and BAFF-R-/- variant human ALL cells in vitro. Immunodeficient mice engrafted with mixed CD19-/- and BAFF-R-/- variant ALL cells and treated with a single dose of CD19/BAFF-R dual CAR T cells experienced complete eradication of both CD19-/- and BAFF-R-/- ALL variants, whereas mice treated with monospecific CD19 or BAFF-R CAR T cells succumbed to outgrowths of CD19-/BAFF-R+ or CD19+/BAFF-R- tumors, respectively. Further, CD19/BAFF-R dual CAR T cells showed prolonged in vivo persistence, raising the possibility that these cells may have the potential to promote durable remissions. Together, our data support clinical translation of BAFF-R/CD19 dual CAR T cells to treat ALL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Animais , Antígenos CD19 , Humanos , Imunoterapia Adotiva , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos Quiméricos/genética , Linfócitos TRESUMO
BACKGROUND: Nuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy. We searched for proteins that modulate the NF-κB response as a prerequisite to identifying novel factors that affect sensitivity to DNA damaging chemotherapy. RESULTS: Using streptavidin-agarose pull-down, we identified the DExD/H-box RNA helicase, DDX39B, as a factor that differentially interacts with κB DNA probes. Subsequently, using both RNA interference and CRISPR/Cas9 technology, we demonstrated that DDX39B inhibits NF-κB activity by a general mechanism involving inhibition of p65 phosphorylation. Mechanistically, DDX39B mediates this effect by interacting with the pattern recognition receptor (PRR), LGP2, a pathway that required the cellular response to cytoplasmic double-stranded RNA (dsRNA). From a functional standpoint, loss of DDX39B promoted resistance to alkylating chemotherapy in glioblastoma cells. Further examination of DDX39B demonstrated that its protein abundance was regulated by site-specific sumoylation that promoted its poly-ubiquitination and degradation. These post-translational modifications required the presence of the SUMO E3 ligase, PIASx-ß. Finally, genome-wide analysis demonstrated that despite the link to the PRR system, DDX39B did not generally inhibit interferon-stimulated gene expression, but rather acted to attenuate expression of factors associated with the extracellular matrix, cellular migration, and angiogenesis. CONCLUSIONS: These results identify DDX39B, a factor with known functions in mRNA splicing and nuclear export, as an RNA-binding protein that blocks a subset of the inflammatory response. While these findings identify a pathway by which DDX39B promotes sensitization to DNA damaging therapy, the data also reveal a mechanism by which this helicase may act to mitigate autoimmune disease.
Assuntos
RNA Helicases DEAD-box/genética , NF-kappa B/metabolismo , Receptores de Reconhecimento de Padrão/genética , Transdução de Sinais , Alquilação , Animais , RNA Helicases DEAD-box/metabolismo , Sondas de DNA , Tratamento Farmacológico , Humanos , Camundongos , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Alkylating chemotherapy is a central component of the management of glioblastoma (GBM). Among the factors that regulate the response to alkylation damage, NF-κB acts to both promote and block cytotoxicity. In this study, we used genome-wide expression analysis in U87 GBM to identify NF-κB-dependent factors altered in response to temozolomide and found the long noncoding RNA (lncRNA) MALAT1 as one of the most significantly upregulated. In addition, we demonstrated that MALAT1 expression was coregulated by p50 (p105) and p53 via novel κB- and p53-binding sites in the proximal MALAT1 coding region. Temozolomide treatment inhibited p50 recruitment to its cognate element as a function of Ser329 phosphorylation while concomitantly increasing p53 recruitment. Moreover, luciferase reporter studies demonstrated that both κB and p53 cis-elements were required for efficient transactivation in response to temozolomide. Depletion of MALAT1 sensitized patient-derived GBM cells to temozolomide cytotoxicity, and in vivo delivery of nanoparticle-encapsulated anti-MALAT1 siRNA increased the efficacy of temozolomide in mice bearing intracranial GBM xenografts. Despite these observations, in situ hybridization of GBM specimens and analysis of publicly available datasets revealed that MALAT1 expression within GBM tissue was not prognostic of overall survival. Together, these findings support MALAT1 as a target for chemosensitization of GBM and identify p50 and p52 as primary regulators of this ncRNA. SIGNIFICANCE: These findings identify NF-κB and p53 as regulators of the lncRNA MALAT1 and suggest MALAT1 as a potential target for the chemosensitization of GBM.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamento farmacológico , NF-kappa B/metabolismo , RNA Longo não Codificante/biossíntese , Temozolomida/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Dano ao DNA/genética , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Prognóstico , RNA Longo não Codificante/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Epithelial-to-mesenchymal transition (EMT) results in changes that promote de-differentiation, migration, and invasion in non-small cell lung cancer (NSCLC). While it is recognized that EMT promotes altered energy utilization, identification of metabolic pathways that link EMT with cancer progression is needed. Work presented here indicates that mesenchymal NSCLC upregulates glutamine-fructose-6-phosphate transaminase 2 (GFPT2). GFPT2 is the rate-limiting enzyme in the synthesis of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is the obligate activator of O-linked N-acetylglucosamine transferase (OGT). METHODS: Analysis of our transcriptomic data indicates that GFPT2 is one of the most significantly upregulated metabolic genes in mesenchymal NSCLC. Ectopic GFPT2 expression, as well as gene silencing strategies were used to determine the importance of this metabolic enzyme in regulating EMT-driven processes of cell motility and invasion. RESULTS: Our work demonstrates that GFPT2 is transcriptionally upregulated by NF-κB and repressed by the NAD+-dependent deacetylase SIRT6. Depletion of GFPT2 expression in NSCLC highlights its importance in regulating cell migration and invasion during EMT. CONCLUSIONS: Consistent with GFPT2 promoting cancer progression, we find that elevated GFPT2 expression correlates with poor clinical outcome in NSCLC. Modulation of GFPT2 activity offers a potentially important therapeutic target to combat NSCLC disease progression.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Neoplasias Pulmonares/patologia , NF-kappa B/metabolismo , Sirtuínas/metabolismo , Células A549 , Carcinoma Pulmonar de Células não Pequenas/genética , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Transdução de Sinais , Ativação TranscricionalRESUMO
Soluble growth factors and cytokines within the tumor microenvironment aid in the induction of the epithelial-to-mesenchymal transition (EMT). Although EMT promotes the development of cancer-initiating cells (CIC), cellular mechanisms by which cancer cells maintain mesenchymal phenotypes remain poorly understood. Work presented here indicates that induction of EMT stimulates non-small cell lung cancer (NSCLC) to secrete soluble factors that function in an autocrine fashion. Using gene expression profiling of all annotated and predicted secreted gene products, we find that NF-κB activity is required to upregulate INHBA/Activin, a morphogen in the TGFß superfamily. INHBA is capable of inducing and maintaining mesenchymal phenotypes, including the expression of EMT master-switch regulators and self-renewal factors that sustain CIC phenotypes and promote lung metastasis. Our work demonstrates that INHBA mRNA and protein expression are commonly elevated in primary human NSCLC and provide evidence that INHBA is a critical autocrine factor that maintains mesenchymal properties of CICs to promote metastasis in NSCLC.