Effects of niacin restriction on sirtuin and PARP responses to photodamage in human skin.

Sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs), NAD(+)-dependent enzymes, link cellular energy status with responses to environmental stresses. Skin is frequently exposed to the DNA damaging effects of UV irradiation, a known etiology in skin cancer. Thus, understanding the defense mechanisms in response to UV, including the role of SIRTs and PARPs, may be important in developing skin cancer prevention strategies. Here, we report expression of the seven SIRT family members in human skin. SIRTs gene expressions are progressively upregulated in A431 epidermoid carcinoma cells (SIRTs1 and 3), actinic keratoses (SIRTs 2, 3, 5, 6, and 7) and squamous cell carcinoma (SIRTs 1-7). Photodamage induces dynamic changes in SIRT expression with upregulation of both SIRT1 and SIRT4 mRNAs. Specific losses of SIRT proteins occur early after photodamage followed by accumulation later, especially for SIRT4. Niacin restriction, which decreases NAD(+), the sirtuin substrate, results in an increase in acetylated proteins, upregulation of SIRTs 2 and 4, increased inherent DNA damage, alterations in SIRT responses to photodamage, abrogation of PARP activation following photodamage, and increased sensitivity to photodamage that is completely reversed by repleting niacin. These data support the hypothesis that SIRTs and PARPs play important roles in resistance to photodamage and identify specific SIRTs that respond to photodamage and may be targets for skin cancer prevention

Lineage-specific T-cell responses to cancer mucosa antigen oppose systemic metastases without mucosal inflammatory disease.

Cancer mucosa antigens are emerging as a new category of self-antigens expressed normally in immunologically privileged mucosal compartments and universally by their derivative tumors. These antigens leverage the established immunologic partitioning of systemic and mucosal compartments, limiting tolerance opposing systemic antitumor efficacy. An unresolved issue surrounding self-antigens as immunotherapeutic targets is autoimmunity following systemic immunization. In the context of cancer mucosa antigens, immune effectors to self-antigens risk amplifying mucosal inflammatory disease promoting carcinogenesis. Here, we examined the relationship between immunotherapy for systemic colon cancer metastases targeting the intestinal cancer mucosa antigen guanylyl cyclase C (GCC) and its effect on inflammatory bowel disease and carcinogenesis in mice. Immunization with GCC-expressing viral vectors opposed nascent tumor growth in mouse models of pulmonary metastasis, reflecting systemic lineage-specific tolerance characterized by CD8(+), but not CD4(+), T-cell or antibody responses. Responses protecting against systemic metastases spared intestinal epithelium from autoimmunity, and systemic GCC immunity did not amplify chemically induced inflammatory bowel disease. Moreover, GCC immunization failed to promote intestinal carcinogenesis induced by germ-line mutations or chronic inflammation. The established role of CD8(+) T cells in antitumor efficacy, but CD4(+) T cells in autoimmunity, suggests that lineage-specific responses to GCC are particularly advantageous to protect against systemic metastases without mucosal inflammation. These observations support the utility of GCC-targeted immunotherapy in patients at risk for systemic metastases, including those with inflammatory bowel disease, hereditary colorectal cancer syndromes, and sporadic colorectal cancer

A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia

Efforts to identify and annotate cancer driver genetic lesions have been focused primarily on the analysis of protein-coding genes; however, most genetic abnormalities found in human cancer are located in intergenic regions. Here we identify a new long range-acting MYC enhancer controlled by NOTCH1 that is targeted by recurrent chromosomal duplications in human T cell acute lymphoblastic leukemia (T-ALL). This highly conserved regulatory element, hereby named N-Me for NOTCH MYC enhancer, is located within a broad super-enhancer region +1.47 Mb from the MYC transcription initiating site, interacts with the MYC proximal promoter and induces orientation-independent MYC expression in reporter assays. Moreover, analysis of N-Me knockout mice demonstrates a selective and essential role of this regulatory element during thymocyte development and in NOTCH1-induced T-ALL. Together these results identify N-Me as a long-range oncogenic enhancer implicated directly in the pathogenesis of human leukemia and highlight the importance of the NOTCH1-MYC regulatory axis in T cell transformation and as a therapeutic target in T-ALL. © 2014 Nature America, Inc. All rights reserved. a r t i c l e s advance online publication nature medicine Supplementary Tables 1 and 2). We identified no duplications in this region in 258 non-T-ALL hematologic tumors, and no germline copy number variant polymorphisms encompassing this area have been reported. Moreover, analysis of normal (remission) DNA confirmed the somatic origin of these copy number alterations in all four cases with available material To functionally characterize the potential role of this NOTCH1 binding site in gene regulation, we performed local ChIP analysis of chromatin regulatory factors and epigenetic histone marks in HPB-ALL T-ALL cells. These analyses confirmed high levels of NOTCH1 binding at this site and revealed bona fide active enhancer features associated with this region, including occupancy and high levels of P300 (also called EP300) and histone H3 Lys4 monomethylation (H3K4me1) with low levels of H3K4 trimethylation (H3K4me3) ( On the basis of these results, we proposed that this +1.4 Mb MYC NOTCH1-occupied enhancer-hereby named N-Me for NOTCHbound MYC enhancer-could function as an important regulatory element driving the activation of MYC downstream of NOTCH1 in T-ALL. Consistent with this hypothesis, chromatin configuration 3C (chromosome conformation capture) analysis of the MYC locus demonstrated the association of this enhancer with proximal regulatory sequences in the MYC promoter The N-Me enhancer is required for thymocyte development To test the specificity and functional relevance of the N-Me enhancer in T cell development and transformation, we used homologous recombination in mouse embryonic stem cells to generate N-Me knockout and conditional knockout mice N-Me is required for NOTCH1-induced T cell leukemogenesis Given the important role of NOTCH1-induced MYC upregulation in the pathogenesis of T-ALL, we hypothesized that deletion of the N-Me enhancer could disrupt NOTCH1-induced leukemogenesis. To test this possibility, we transplanted isogenic C57BL/6 mice with wild-type or N-Me heterozygous or homozygous knockout hematopoietic progenitors infected with retroviruses driving the expression of an To explore the pathogenic role of N-Me-mediated Myc expression in NOTCH1-induced leukemia tumor maintenance, we generated ∆E-NOTCH1-induced T-ALL tumors from wild-type (Rosa26TM-Cre N-Me +/+ ) and tamoxifen-inducible conditional heterozygous (Rosa26TM-Cre N-Me flox/+ ) and homozygous (Rosa26TM-Cre N-Me flox/flox ) N-Me knockout hematopoietic progenitors. In these experiments, mice transplanted with ∆E-NOTCH1-expressing wildtype and tamoxifen-inducible heterozygous and homozygous conditional N-Me knockout cells developed NOTCH1-induced T-ALLs with identical kinetics and immunophenotypes ( To better assess the mechanisms mediating the antileukemic effects of N-Me inactivation, we then analyzed the cellular and transcriptional phenotypes of N-Me conditional inducible knockout T-ALL cells after tamoxifen treatment. In this setting, N-Me deletion in T-ALL cells DISCUSSION NOTCH1 has a central role in the pathogenesis of T-ALL 24 and drives an oncogenic transcriptional program that promotes cell growth proliferation and survival in T-ALL lymphoblasts. Importantly, the oncogenic effects of NOTCH1 are closely linked to activation of the MYC oncogene © 2014 Nature America, Inc. All rights reserved. a r t i c l e s nature medicine advance online publication a broad regulatory area of about 100 kb located 1.7 Mb telomeric to the Myc gene, 400 kb downstream of N-Me 35 . This Myc regulatory region contains multiple enhancers that are active in myeloid cells but not the thymus and is duplicated in about 3% of acute myeloid leukemias The requirement for N-Me-mediated upregulation of Myc expression downstream of Notch1 was even more apparent in the context of leukemia initiation, where loss of one and two copies of N-Me delayed and abrogated tumor development by oncogenic NOTCH1, respectively. In addition, N-Me was also required for the maintenance of NOTCH1-induced leukemias, as secondary deletion of one copy of N-Me in established tumors resulted in a marked delay in tumor progression, and loss of two copies effectively abrogated leukemia propagation and the self-renewal capacity of leukemia-initiating cells. These results are consistent with the well-established quantitative effects of MYC expression in other tumor settings. Loss of one copy of Myc has been shown to attenuate intestinal tumorigenesis 37 , and homozygous deletion of Myc completely abrogates tumor development induced by loss of Apc in the gut Several lines of evidence support a role for loss of Myc expression as the primary driver in the developmental and tumor phenotypes associated with N-me loss. In this regard, we observed marked reductions in Myc expression in developing T cells from N-Me knockout mice and in T-ALL lymphoblasts after N-Me inactivation. Moreover, retroviral expression of Myc restored T cell lymphopoiesis from NMe-deficient hematopoietic progenitors and rescued the defects in leukemia cell growth induced by secondary deletion on N-Me in NOTCH1-induced T-ALL cells. In addition, MYC inactivation has been associated with a global decrease in transcriptional activity, with a particularly pronounced downregulation of genes involved in growth, proliferation and metabolism The generation of the N-Me conditional knockout model presented here was also useful in analyzing the specific role of this enhancer in transcriptional control. Thus, even though in some cases enhancerpromoter interactions have been implicated in the regulation of transcription by promoting the release of RNA Pol II pausing 42 , deletion of N-Me in T-ALL lymphoblasts resulted in unloading of RNA Pol II at the Myc transcription initiation site without any apparent increase in RNA Pol II pausing. Overall, our results identify the N-Me regulatory sequence as a critical mediator of NOTCH1-induced MYC expression that is required for T cell development and transformation and substantiates a pathogenic role for chromosomal duplications targeting this enhancer in the pathogenesis of T-ALL. METHODS COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Curr. Top. Microbiol. Immunol. 360, 163-182 (2012 NOTCH1 inhibition. We inhibited NOTCH1 in JURKAT cells with 250 nM DBZ ((S)-2-(2-(3,5-difluorophenyl)acetamido)-N-((S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo [b,d]azepin-7-yl) propanamide) (Syncom) for 48 h as described previously Genomic analysis of primary T-ALL samples. A total of 160 cases of T-ALL from adult and pediatric patients referred to Saint-Louis Hospital, Paris, France were analyzed for copy number abnormalities using array-comparative genomic hybridization with informed consent under the supervision of the Institutional Review Board of the Institut Universitaire d'Hématologie, Université ParisDiderot. Sureprint G3 human CGH 180K, 244K, 400K or 1M arrays (Agilent technologies) were used, and copy number alterations were identified using Genomic Workbench software and the ADM-2 algorithm (Agilent Technologies) as described previously T-ALL oncogenic subtype was determined on the basis of gene expression profiling, as reported previousl

Prognostic evaluation of re-resection for recurrent glioblastoma using the novel RANO classification for extent of resection:A report of the RANO resect group

BACKGROUND: The value of re-resection in recurrent glioblastoma remains controversial as a randomized trial that specifies intentional incomplete resection cannot be justified ethically. Here, we aimed to (1) explore the prognostic role of extent of re-resection using the previously proposed Response Assessment in Neuro-Oncology (RANO) classification (based upon residual contrast-enhancing (CE) and non-CE tumor), and to (2) define factors consolidating the surgical effects on outcome. METHODS: The RANO resect group retrospectively compiled an 8-center cohort of patients with first recurrence from previously resected glioblastomas. The associations of re-resection and other clinical factors with outcome were analyzed. Propensity score-matched analyses were constructed to minimize confounding effects when comparing the different RANO classes. RESULTS: We studied 681 patients with first recurrence of Isocitrate Dehydrogenase (IDH) wild-type glioblastomas, including 310 patients who underwent re-resection. Re-resection was associated with prolonged survival even when stratifying for molecular and clinical confounders on multivariate analysis; ≤1 cm3 residual CE tumor was associated with longer survival than non-surgical management. Accordingly, "maximal resection" (class 2) had superior survival compared to "submaximal resection" (class 3). Administration of (radio-)chemotherapy in the absence of postoperative deficits augmented the survival associations of smaller residual CE tumors. Conversely, "supramaximal resection" of non-CE tumor (class 1) was not associated with prolonged survival but was frequently accompanied by postoperative deficits. The prognostic role of residual CE tumor was confirmed in propensity score analyses. CONCLUSIONS: The RANO resect classification serves to stratify patients with re-resection of glioblastoma. Complete resection according to RANO resect classes 1 and 2 is prognostic.</p

Surgical management and outcome of newly diagnosed glioblastoma without contrast enhancement ('low grade appearance') - a report of the RANO resect group

BACKGROUND: Resection of the contrast-enhancing (CE) tumor represents the standard of care in newly diagnosed glioblastoma. However, some tumors ultimately diagnosed as glioblastoma lack contrast enhancement and have a 'low grade appearance' on imaging (non-CE glioblastoma). We aimed to (I) volumetrically define the value of non-CE tumor resection in the absence of contrast enhancement, and to (II) delineate outcome differences between glioblastoma patients with and without contrast enhancement. METHODS: The RANO resect group retrospectively compiled a global, eight-center cohort of patients with newly diagnosed glioblastoma per WHO 2021 classification. The associations between post-operative tumor volumes and outcome were analyzed. Propensity score-matched analyses were constructed to compare glioblastomas with and without contrast enhancement. RESULTS: Among 1323 newly diagnosed IDH-wildtype glioblastomas, we identified 98 patients (7.4%) without contrast enhancement. In such patients, smaller post-operative tumor volumes were associated with more favourable outcome. There was an exponential increase in risk for death with larger residual non-CE tumor. Accordingly, extensive resection was associated with improved survival compared to lesion biopsy. These findings were retained on a multivariable analysis adjusting for demographic and clinical markers. Compared to CE glioblastoma, patients with non-CE glioblastoma had more favourable clinical profile and superior outcome as confirmed in propensity score analyses by matching the patients with non-CE glioblastoma to patients with CE glioblastoma using a large set of clinical variables. CONCLUSIONS: The absence of contrast enhancement characterizes a less aggressive clinical phenotype of IDH-wildtype glioblastomas. Maximal resection of non-CE tumors has prognostic implications and translates into favourable outcome

Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy

Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials

Therapeutic targeting of Hairy and Enhancer of Split 1 (HES1) transcriptional programs in T-cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological tumor resulting from the malignant transformation of immature T-cell progenitors. Originally associated with a dismal prognosis, the outcome of T-ALL patients has improved remarkably over the last two decades as a result of the introduction of intensified chemotherapy protocols. However, these treatments are associated with significant acute and long-term toxicities, and the treatment of patients presenting with primary resistant disease or those relapsing after a transient response remains challenging. Oncogenic activation of NOTCH1 signaling plays a central role in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), with mutations on this signaling pathway affecting more than 60% of patients at diagnosis. However the transcriptional regulatory circuitries driving T-cell transformation downstream of NOTCH1 remain incompletely understood. Here we identify HES1, a transcriptional repressor controlled by NOTCH1 as a critical mediator of NOTCH1 induced leukemogenesis strictly required for tumor cell survival. Mechanistically, we demonstrate that HES1 inhibits leukemia cell death by repressing BBC3, the gene encoding the PUMA BH3-only proapototic factor. Finally, we identify perhexiline, a small molecule inhibitor of mitochondrial carnitine palmitoyltransferase-1, as a HES1-signature antagonist drug with robust antileukemic activity against NOTCH1 induced leukemias in vitro and in vivo