Plasma cell-free DNA hydroxymethylation profiling reveals anti-PD-1 treatment response and resistance biology in non-small cell lung cancer.

BACKGROUND: Treatment with immune checkpoint inhibitors (ICIs) targeting programmed death-1 (PD-1) can yield durable antitumor responses, yet not all patients respond to ICIs. Current approaches to select patients who may benefit from anti-PD-1 treatment are insufficient. 5-hydroxymethylation (5hmC) analysis of plasma-derived cell-free DNA (cfDNA) presents a novel non-invasive approach for identification of therapy response biomarkers which can tackle challenges associated with tumor biopsies such as tumor heterogeneity and serial sample collection. METHODS: 151 blood samples were collected from 31 patients with non-small cell lung cancer (NSCLC) before therapy started and at multiple time points while on therapy. Blood samples were processed to obtain plasma-derived cfDNA, followed by enrichment of 5hmC-containing cfDNA fragments through biotinylation via a two-step chemistry and binding to streptavidin coated beads. 5hmC-enriched cfDNA and whole genome libraries were prepared in parallel and sequenced to obtain whole hydroxymethylome and whole genome plasma profiles, respectively. RESULTS: Comparison of on-treatment time point to matched pretreatment samples from same patients revealed that anti-PD-1 treatment induced distinct changes in plasma cfDNA 5hmC profiles of responding patients, as judged by Response evaluation criteria in solid tumors, relative to non-responders. In responders, 5hmC accumulated over genes involved in immune activation such as inteferon (IFN)-γ and IFN-α response, inflammatory response and tumor necrosis factor (TNF)-α signaling, whereas in non-responders 5hmC increased over epithelial to mesenchymal transition genes. Molecular response to anti-PD-1 treatment, as measured by 5hmC changes in plasma cfDNA profiles were observed early on, starting with the first cycle of treatment. Comparison of pretreatment plasma samples revealed that anti-PD-1 treatment response and resistance associated genes can be captured by 5hmC profiling of plasma-derived cfDNA. Furthermore, 5hmC profiling of pretreatment plasma samples was able to distinguish responders from non-responders using T cell-inflamed gene expression profile, which was previously identified by tissue RNA analysis. CONCLUSIONS: These results demonstrate that 5hmC profiling can identify response and resistance associated biological pathways in plasma-derived cfDNA, offering a novel approach for non-invasive prediction and monitoring of immunotherapy response in NSCLC.

Targeting Ca2+ and Mitochondrial Homeostasis by Antipsychotic Thioridazine in Leukemia Cells.

Mitochondria have pivotal roles in cellular physiology including energy metabolism, reactive oxygen species production, Ca2+ homeostasis, and apoptosis. Altered mitochondrial morphology and function is a common feature of cancer cells and the regulation of mitochondrial homeostasis has been identified as a key to the response to chemotherapeutic agents in human leukemias. Here, we explore the mechanistic aspects of cytotoxicity produced by thioridazine (TR), an antipsychotic drug that has been investigated for its anticancer potential in human leukemia cellular models. TR exerts selective cytotoxicity against human leukemia cells in vitro. A PCR array provided a general view of the expression of genes involved in cell death pathways. TR immediately produced a pulse of cytosolic Ca2+, followed by mitochondrial uptake, resulting in mitochondrial permeabilization, caspase 9/3 activation, endoplasmic reticulum stress, and apoptosis. Ca2+ chelators, thiol reducer dithiothreitol, or CHOP knockdown prevented TR-induced cell death. TR also exhibited potent cytotoxicity against BCL-2/BCL-xL-overexpressing leukemia cells. Additionally, previous studies have shown that TR exhibits potent antitumor activity in vivo in different solid tumor models. These findings show that TR induces a Ca2+-mediated apoptosis with involvement of mitochondrial permeabilization and ER stress in leukemia and it emphasizes the pharmacological potential of TR as an adjuvant in antitumor chemotherapy.

Targeting the NFAT:AP-1 transcriptional complex on DNA with a small-molecule inhibitor.

The transcription factor nuclear factor of activated T cells (NFAT) has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of activator protein-1 (AP-1), and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened ∼202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite antigen-receptor response element-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other cyclosporin A-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.

Exhaustion-associated regulatory regions in CD8+ tumor-infiltrating T cells.

T-cell exhaustion is a progressive loss of effector function and memory potential due to persistent antigen exposure, which occurs in chronic viral infections and cancer. Here we investigate the relation between gene expression and chromatin accessibility in CD8+ tumor-infiltrating lymphocytes (TILs) that recognize a model tumor antigen and have features of both activation and functional exhaustion. By filtering out accessible regions observed in bystander, nonexhausted TILs and in acutely restimulated CD8+ T cells, we define a pattern of chromatin accessibility specific for T-cell exhaustion, characterized by enrichment for consensus binding motifs for Nr4a and NFAT transcription factors. Anti-PD-L1 treatment of tumor-bearing mice results in cessation of tumor growth and partial rescue of cytokine production by the dysfunctional TILs, with only limited changes in gene expression and chromatin accessibility. Our studies provide a valuable resource for the molecular understanding of T-cell exhaustion in cancer and other inflammatory settings.

Doxorubicin induces cell death in breast cancer cells regardless of Survivin and XIAP expression levels.

Breast cancer is the leading cause of deaths in women around the world. Resistance to therapy is the main cause of treatment failure and still little is known about predictive biomarkers for response to systemic therapy. Increasing evidence show that Survivin and XIAP overexpression is closely associated with chemoresistance and poor prognosis in breast cancer. However, their impact on resistance to doxorubicin (dox), a chemotherapeutic agent widely used to treat breast cancer, is poorly understood. Here, we demonstrated that dox inhibited cell viability and induced DNA fragmentation and activation of caspases-3, -7 and -9 in the breast cancer-derived cell lines MCF7 and MDA-MB-231, regardless of different p53 status. Dox exposure resulted in reduction of Survivin and XIAP mRNA and protein levels. However, when we transfected cells with a Survivin-encoding plasmid, we did not observe a cell death-resistant phenotype. XIAP and Survivin silencing, either alone or in combination, had no effect on breast cancer cells sensitivity towards dox. Altogether, we demonstrated that breast cancer cells are sensitive to the chemotherapeutic agent dox irrespective of Survivin and XIAP expression levels. Also, our findings suggest that dox-mediated modulation of Survivin and XIAP might sensitize cells to taxanes when used in a sequential regimen.

Transcriptional regulation of the c-Myc promoter by NFAT1 involves negative and positive NFAT-responsive elements.

A number of physiological processes in both normal and cancer cells are regulated by the proto-oncogene c-Myc. Among them, processes such as cell cycle regulation, apoptosis, angiogenesis and metastasis are also controlled by the nuclear factor of activated T cells (NFAT) family of transcription factors. It is already known that NFAT upregulates c-Myc expression by binding to an element located in the minimal c-Myc promoter. However, the importance of other NFAT sites in the context of the full promoter has not been evaluated. In this work, we demonstrate that the regulation of c-Myc by NFAT1 is more complex than previously conceived. In addition to the proximal site, NFAT1 directly binds to distal sites in the c-Myc promoter with different affinities. Promoter deletions and site-directed mutagenesis of NFAT binding sites in HEK293T cells suggest that in NFAT1-mediated transactivation, some NFAT elements are negative and dominant and others are positive and recessive. Furthermore, we demonstrate that cooperation with partner proteins, such as p300, enhances NFAT1-mediated transactivation of the c-Myc promoter. At last, the newly identified sites are also responsive to NFAT2 in HEK293T cells. However, in NIH3T3 cells, the regulation mediated by NFAT proteins is not dependent on the known NFAT sites, including the site previously described. Thus, our data suggest that the contribution of NFAT to the regulation of c-Myc expression may depend on a balance between the binding to positive and negative NFAT-responsive elements and cooperation with transcriptional cofactors, which may differ according to the context and/or cell type.

Regulação da expressão gênica pelo NFAT1: o proto-oncogene c-myc e o papel da proteína IRF-2BP2 / [Regulation of gene expression by NFAT1: the proto-oncogene c-myc and the role of the IRF-2BP2 protein]

Durante a transcrição gênica em eucariotos, a produção de níveis significativos de mRNA é conferida pela presença de diversos sítios de ligação para fatores de transcrição específicos, localizados dentro e fora do promotor basal. Dentre os fatores de transcrição que se ligam a estes sítios estão as proteínas da família NFAT (nuclear factor of activated T cells), composta pelos NFAT1-5, sendo o NFAT1 o alvo desse estudo. O NFAT pode regular seus genes alvo direta ou indiretamente e, neste processo, a ativação ou a repressão da transcrição pode ser alterada pela interação com diferentes parceiros proteicos. Esses dois pontos da regulação transcricional mediados pelo NFAT foram avaliados em duas etapas distintas. Na primeira, avaliamos se a regulação do proto-oncogene c-Myc pelo NFAT1 ocorria de forma direta. Tanto NFAT quanto c-Myc estão envolvidos na regulação de genes de ciclo celular, apoptose e angiogênese. Já tinha sido mostrado que as vias de sinalização que ativam NFAT induzem a expressão de c-Myc e que o NFAT1 se liga a um elemento localizado no promotor mínimo de c-Myc, apesar da importância desse elemento não ter sido avaliada no contexto do promotor completo de c-Myc. Nós mostramos que a regulação desse promotor pelo NFAT1 é mais complexa do que se acreditava. Além desse sítio proximal, o NFAT1 se liga a sítios distais com diferentes afinidades. Nossos resultados sugerem que alguns elementos NFAT são negativos e dominantes, enquanto outros são positivos e recessivos. Além disso, demonstramos que a cooperação com p300 aumenta a transativação do promotor de c-Myc mediada pelo NFAT1. Por último, mostramos que os novos sítios identificados também são responsivos ao NFAT2, outro membro da família, que pode tanto ter funções opostas quanto redundantes com o NFAT1. Assim, sugerimos que a contribuição do NFAT para a regulação da expressão de c-Myc é direta e depende do balanço entre a ligação do NFAT aos sítios positivos e negativos e da cooperação com cofatores transcricionais. Na segunda parte do trabalho, focamos nas implicações da interação recém-identificada entre NFAT1 e a proteína IRF-2BP2. A IRF-2BP2 foi pescada num ensaio de duplo híbrido com a região TAD-C do NFAT1, região esta que é menos conservada entre as proteínas NFAT. Mostramos que a IRF- 2BP2 reprime a expressão de genes de citocina induzida pelo NFAT1 e que ela age somente sobre a função do NFAT1 dentro da família NFAT. Nossos dados também sugerem que a IRF-2BP2 não se ligue ao DNA e, portanto, funcione como um correpressor transcricional. Ainda mostramos que a superexpressão de IRF-2BP2 leva ao atraso da progressão das fases G0/G1 para a fase S do ciclo celular e à alteração da expressão de genes de ciclo celular, como de c-Myc, que também é regulado por NFAT1. Esses dados sugerem que a IRF-2BP2 possa ter um papel bem mais amplo na regulação do NFAT1, não se restringindo apenas à modulação de genes de citocinas. Se isso se confirmar, a interação entre NFAT1 e IRF-2BP2 poderá contribuir para explicar as diferenças fenotípicas exercidas pelos diferentes membros da família NFAT.

During gene transcription in eukaryotes, the production of significant mRNA levels is conferred by the presence of several binding sites for specific transcription factors, located inside and outside of the basal promoter. Among the transcription factors that bind to these sites are proteins of the NFAT family (nuclear factor of activated T cells), comprising the NFAT1-5, being the NFAT1 the target of this study. The NFAT can regulate their target genes directly or indirectly and in this process, the activation or repression of transcription can be altered by interaction with different partner proteins. These two points of NFAT-mediated transcriptional regulation were assessed here in two stages. In the first one, we evaluated whether the regulation of the proto-oncogene c-Myc by NFAT1 occurred directly. Both c-Myc as NFAT regulate genes involved in cell cycle, apoptosis and angiogenesis. It has already been shown that the signaling pathways that activate NFAT induce the c-Myc expression and that NFAT1 binds to an element located at the minimal c-Myc promoter, although the importance of this element has not been assessed in the full c-Myc promoter context. We demonstrated that the regulation of this promoter by NFAT1 is more complex than previously conceived. In addition to this proximal site, NFAT1 binds to distal sites with different affinities. Our results suggest that some NFAT elements are negative and dominant, while others are positive and recessive. Furthermore, we demonstrated that cooperation with p300 enhances NFAT1-mediated transactivation of the c-Myc promoter. Finally, we show that the newly identified sites are also responsive to NFAT2, another member of NFAT family, which can both have opposite as well as redundant functions with NFAT1. We suggest that NFAT1 the contribution of NFAT to the regulation of c-Myc expression is direct and may depend on a balance between the binding to positive and negative NFAT-responsive elements, and cooperation with transcriptional cofactors. In the second part of this work, we focused on the implications of the recently identified interaction between NFAT1 and the protein IRF-2BP2. The IRF-2BP2 was identified in a two-hybrid assay with the TAD-C region of the NFAT1, a region which is less conserved among the NFAT proteins. We showed that IRF-2BP2 represses the expression of cytokine genes induced by NFAT1 and regulates specifically the function of NFAT1 among the NFAT family members. Our data also suggest that the IRF- 2BP2 does not bind to DNA and therefore functions as a transcriptional correpressor. Therefore, the overexpression of IRF-2BP2 leads to a delayed transition from G0/G1 to S phase of cell cycle and also alters the expression of many cell cycle genes, such as c-Myc, which is also regulated by NFAT1. These data suggest that the IRF-2BP2 may have a much greater role regulating NFAT1, not being restricted to the modulation of cytokine genes. If this is confirmed, the interaction between NFAT1 and IRF-2BP2 may help to explain the phenotypic differences exerted by different NFAT family members.

Interferon regulatory factor 2 binding protein 2 is a new NFAT1 partner and represses its transcriptional activity.

The nuclear factor of activated T cells (NFAT) family of transcription factors is expressed in a wide range of cell types and regulates genes involved in cell cycle, differentiation, and apoptosis. NFAT proteins share two well-conserved regions, the regulatory domain and the DNA binding domain. The N- and C-terminal ends are transactivation sites and show less sequence similarity, whereas their molecular functions remain poorly understood. Here, we identified a transcriptional repressor, interferon regulatory factor 2 binding protein 2 (IRF-2BP2), which specifically interacts with the C-terminal domain of NFAT1 among the NFAT family members. IRF-2BP2 was described as a corepressor by inhibiting both enhancer-activated and basal transcription. Gene reporter assays demonstrated that IRF-2BP2 represses the NFAT1-dependent transactivation of NFAT-responsive promoters. The ectopic expression of IRF-2BP2 in CD4 T cells resulted in decreased interleukin-2 (IL-2) and IL-4 production, supporting a repressive function of IRF-2BP2 for NFAT target genes. Furthermore, NFAT1 and IRF-2BP2 colocalized in the nucleus in activated cells, and the mutation of a newly identified nuclear localization signal in the IRF-2BP2 rendered it cytoplasmic, abolishing its repressive effect on NFAT1 activity. Collectively, our data demonstrate that IRF-2BP2 is a negative regulator of the NFAT1 transcription factor and suggest that NFAT1 repression occurs at the transcriptional level.

Melatonin protects CD4+ T cells from activation-induced cell death by blocking NFAT-mediated CD95 ligand upregulation.

Over the past 20 y, the hormone melatonin was found to be produced in extrapineal sites, including cells of the immune system. Despite the increasing data regarding the biological effects of melatonin on the regulation of the immune system, the effect of this molecule on T cell survival remains largely unknown. Activation-induced cell death plays a critical role in the maintenance of the homeostasis of the immune system by eliminating self-reactive or chronically stimulated T cells. Because activated T cells not only synthesize melatonin but also respond to it, we investigated whether melatonin could modulate activation-induced cell death. We found that melatonin protects human and murine CD4(+) T cells from apoptosis by inhibiting CD95 ligand mRNA and protein upregulation in response to TCR/CD3 stimulation. This inhibition is a result of the interference with calmodulin/calcineurin activation of NFAT that prevents the translocation of NFAT to the nucleus. Accordingly, melatonin has no effect on T cells transfected with a constitutively active form of NFAT capable of migrating to the nucleus and transactivating target genes in the absence of calcineurin activity. Our results revealed a novel biochemical pathway that regulates the expression of CD95 ligand and potentially other downstream targets of NFAT activation.

Dual roles for NFAT transcription factor genes as oncogenes and tumor suppressors.

Nuclear factor of activated T cells (NFAT) was first described as an activation and differentiation transcription factor in lymphocytes. Several in vitro studies suggest that NFAT family members are redundant proteins. However, analysis of mice deficient for NFAT proteins suggested different roles for the NFAT family of transcription factors in the regulation of cell proliferation and apoptosis. NFAT may also regulate several cell cycle and survival factors influencing tumor growth and survival. Here, we demonstrate that two constitutively active forms of NFAT proteins (CA-NFAT1 and CA-NFAT2 short isoform) induce distinct phenotypes in NIH 3T3 cells. Whereas CA-NFAT1 expression induces cell cycle arrest and apoptosis in NIH 3T3 fibroblasts, CA-NFAT2 short isoform leads to increased proliferation capacity and induction of cell transformation. Furthermore, NFAT1-deficient mice showed an increased propensity for chemical carcinogen-induced tumor formation, and CA-NFAT1 expression subverted the transformation of NIH 3T3 cells induced by the H-rasV12 oncogene. The differential roles for NFAT1 are at least partially due to the protein C-terminal domain. These results suggest that the NFAT1 gene acts as a tumor suppressor gene and the NFAT2 short isoform acts gene as an oncogene, supporting different roles for the two transcription factors in tumor development.

Regulação do Proto-oncogene c-myc pelo Fator de Transcrição NFAT1

A família de fatores de transcrição NFAT (Fator Nuclear de Células T ativadas) tem diferentes funções regulatórias no ciclo celular, apoptose, diferenciação celular e angiogênese. O proto-oncogene c-myc, que está envolvido em todos estes mecanismos, é reprimido em alguns modelos, em resposta à Ciclosporina A, que inibe a ativação das proteínas NFAT. Dados anteriores de nosso laboratório mostraram que os linfócitos de camundongos NFAT1-/- sensibilizados com ovalbumina, apresentaram níveis aumentados de c-MYC quando comparados com os linfócitos dos camundongos NFAT1+/+. Desta forma, o objetivo deste trabalho foi avaliar se o NFAT1 regula diretamente a expressão de c-MYC. Este estudo mostrou que linfócitos T de camundongos NFAT1-/- naives superexpressam c-MYC em relação aos linfócitos NFAT1+/+, através de ensaios de PCR em tempo real. Uma análise de bioinformática encontrou sete supostos sítios de ligação para NFAT no promotor de c-myc, conservados em humanos e camundongos. Três desses sítios foram confirmados por um ensaio de mudança de mobilidade eletroforética, incluindo o sítio proximal, que é regulado por NFAT2. Além disso, um ensaio de imunoprecipitação de cromatina com linfócitos murinos demonstrou que o NFAT1 se liga diretamente ao promotor de c-myc in vivo. Estes resultados sugerem que o NFAT1 tem um importante papel na regulação do promotor de c-myc, aparentemente regulando negativamente sua expressão.

The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors has different regulatory functions in the cell cycle, apoptosis, cell differentiation and angiogenesis. The c-myc proto-oncogene, which is also involved in those mechanisms, is repressed, in some models, in response to Cyclosporine A that inhibits NFAT activation. Previous data of our laboratory found that lymphocytes from NFAT1-/- mice sensitized with ovalbumin presented higher levels of c-MYC mRNA when compared with the NFAT1+/+. Hence, the aim of this work was to evaluate whether the NFAT1 directly regulates the c-MYC expression. This study showed that T lymphocytes from NFAT1-/- naive mice overexpress c-MYC mRNA when compared with the NFAT1+/+ mice assessed by Real Time PCR. A bioinformatic analysis found seven putative NFAT binding sites in the c-myc promoter, conserved in human and mouse. Three of them were confirmed by an Eletrophoretic Mobility Shift Assay, including the proximal site, which is upregulated by NFAT2. Additionally, a Chromatin Immunoprecipitation Assay with mouse T lymphocytes demonstrated that NFAT1 directly binds to c-myc promoter in vivo. These findings suggest that NFAT1 plays an important role in the regulation of c-myc promoter apparently through the inhibition of this expression.