The genetic landscape of adult acute myeloid leukemias (AML) has been recently unraveled. However, due to their genetic heterogeneity, only a handful of markers are currently used for the evaluation ...of minimal residual disease (MRD). Recent studies using multi-target strategies indicate that detection of residual mutations in less than 5% of cells in complete remission is associated with a better survival. Here, in a series of 69 AMLs with known clonal architecture, we design a clone-specific strategy based on fluorescent
hybridization and high-sensitivity next generation sequencing to detect chromosomal aberrations and mutations, respectively, in follow-up samples. The combination of these techniques allows tracking chromosomal and genomic lesions down to 0.5-0.4% of the cell population in remission samples. By testing all lesions in follow-up samples from 65 of 69 evaluable patients, we find that initiating events often persist and appear to be, on their own, inappropriate markers to predict short-term relapse. In contrast, the persistence of two or more lesions in more than 0.4% of the cells from remission samples is strongly associated with lower leukemia-free and overall survivals in univariate and multivariate analyses. Although larger prospective studies are needed to extend these results, our data show that a personalized, clone-specific, MRD follow up strategy is feasible in the vast majority of AML cases.
Paragangliomas are neuroendocrine tumors frequently associated with mutations in RET, NF1, VHL, and succinate dehydrogenase (SDHx) genes. Methylome analysis of a large paraganglioma cohort identified ...three stable clusters, associated with distinct clinical features and mutational status. SDHx-related tumors displayed a hypermethylator phenotype, associated with downregulation of key genes involved in neuroendocrine differentiation. Succinate accumulation in SDH-deficient mouse chromaffin cells led to DNA hypermethylation by inhibition of 2-OG-dependent histone and DNA demethylases and established a migratory phenotype reversed by decitabine treatment. Epigenetic silencing was particularly severe in SDHB-mutated tumors, potentially explaining their malignancy. Finally, inactivating FH mutations were identified in the only hypermethylated tumor without SDHx mutations. These findings emphasize the interplay between the Krebs cycle, epigenomic changes, and cancer.
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•SDH and FH mutations establish a hypermethylator phenotype in PGL/PCC•Succinate inhibits DNA and histone demethylases in SDH-deficient chromaffin cells•DNA hypermethylation silences key genes involved in neuroendocrine differentiation•A stronger hypermethylator phenotype may explain malignancy of SDHB-mutated tumors
Germline mutations in the RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, MAX, TMEM127, NF1 or VHL genes are identified in about 30% of patients with pheochromocytoma or paraganglioma and somatic mutations in ...RET, VHL or MAX genes are reported in 17% of sporadic tumors. In the present study, using mutation screening of the NF1 gene, mapping of chromosome aberrations by single nucleotide polymorphism (SNP) array, microarray-based expression profiling and immunohistochemistry (IHC), we addressed the implication of NF1 somatic alterations in pheochromocytomas and paragangliomas. We studied 53 sporadic tumors, selected because of their classification with RET/NF1/TMEM127-related tumors by genome wide expression studies, as well as a second set of 11 independent tumors selected on their low individual levels of NF1 expression evaluated by microarray. Direct sequencing of the NF1 gene in tumor DNA identified the presence of an inactivating NF1 somatic mutation in 41% (25/61) of analyzed sporadic tumors, associated with loss of the wild-type allele in 84% (21/25) of cases. Gene expression signature of NF1-related tumors highlighted the downregulation of NF1 and the major overexpression of SOX9. Among the second set of 11 tumors, two sporadic tumors carried somatic mutations in NF1 as well as in another susceptibility gene. These new findings suggest that NF1 loss of function is a frequent event in the tumorigenesis of sporadic pheochromocytoma and strengthen the new concept of molecular-based targeted therapy for pheochromocytoma or paraganglioma.
Chemotherapy resistance is the main cause of treatment failure in acute myeloid leukemia (AML) and has been related to ATP-binding cassette (ABC) transporter activity. However, the links between ABC ...activity, immunophenotype, and molecular AML parameters have been poorly evaluated. Moreover, the prognostic value of ABC activity, when compared to new molecular markers, is unknown. Here we investigated the links between ABC activity, as evaluated by JC-1 +/- cyclosporine A assay, and immunophenotypic, cytogenetic, molecular, and targeted next-generation sequencing features in 361 AML patients. High ABC activity was found in 164 patients and was significantly associated with less proliferating disease, an immature immunophenotype (expression of CD34, HLA-DR, CD117, CD13), and gene mutations defining AML as belonging to secondary-type ontogenic groups. Low ABC activity was associated with more mature myeloid differentiation (CD34-, cyMPO+, CD15+, CD33+) or monocytic commitment (CD64+, CD4+weak, CD14+), with NPM1 mutations, KMT2A rearrangements, and core-binding factor gene fusions, hallmarks of the de novo-type AML ontogeny. ABC activity was one of the major factors we identified using a random forest model for early prediction of AML ontogeny. In the 230 patients evaluated at diagnosis and intensively treated, high ABC activity was a predictive factor for primary resistance, and in multivariate analysis including full molecular data, an independent factor for event-free survival (P=0.0370). JC-1 +/- cyclosporine A assay could be used at diagnosis to predict AML ontogeny and to complete prognosis evaluation in addition to new molecular markers.
Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and ...de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP–NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin’s lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN–PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.
The generation of specific T lymphocyte subsets is under the strict control of specific transcription factors, as this has been shown by knockout experiments in mice. Here, we show that siRNAs that ...specifically target the transcription factor Gata3 (which is required for the development of T helper 1 cells) or T-Bet (which is required for the development of T helper 2 cells) can be effective in vivo. Thus, the intraperitoneal injection of siRNAs specific for Gata3 or t-Bet leads to the specific depletion of their target gene products in vivo, in the spleen and in the lymph nodes from mice. The immunomodulatory action of these siRNAs was validated in a model of anti-tumor vaccination in which colorectal cancer cells that succumb to anthracyclin-induced immunogenic cell death were injected subcutaneously into one flank, in the absence of any adjuvant, and live tumor cells were injected simultaneously in the opposite flank of immunocompetent mice. In this setting, the siRNA targeting t-Bet was able to accelerate tumor growth while the siRNA targeting Gata3 significantly reduced the proliferation of cancer cells in vivo. These effects were dependent on the immune response elicited by dying tumor cells because both siRNAs failed to modulate the growth of tumors in non-vaccinated mice. The immune response-dependent anticancer effect of the Gata3-specific siRNA was not due to the induction of class I interferons and could be fully abolished by co-injection of t-Bet-specific siRNA. These results demonstrate the possibility to use siRNAs for immunomodulaton in vivo and illustrate the antagonistic implication of distinct T helper populations in anti-cancer immune responses.
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