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Background: Several fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia (AML) are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, associated with dismal prognosis., Methods: We created novel ETO2::GLIS2 models of leukemogenesis through lentiviral transduction and CRISPR-Cas9 gene editing of human fetal and post-natal hematopoietic stem/progenitor cells (HSPCs), performed in-depth characterization of ETO2::GLIS2 transformed cells through multiple omics and compared them to patient samples. This led to a preclinical assay using patient-derived-xenograft models to test a combination of two clinically-relevant molecules., Results: We showed that ETO2::GLIS2 expression in primary human fetal CD34 + hematopoietic cells led to more efficient in vivo leukemia development than expression in post-natal cells. Moreover, cord blood-derived leukemogenesis has a major dependency on the presence of human cytokines, including IL3 and SCF. Single cell transcriptomes revealed that this cytokine environment controlled two ETO2::GLIS2-transformed states that were also observed in primary patient cells. Importantly, this cytokine sensitivity may be therapeutically-exploited as combined MEK and BCL2 inhibition showed higher efficiency than individual molecules to reduce leukemia progression in vivo., Conclusions: Our study uncovers an interplay between the cytokine milieu and transcriptional programs that extends a developmental window of permissiveness to transformation by the ETO2::GLIS2 AML fusion oncogene, controls the intratumoral cellular heterogeneity, and offers a ground-breaking therapeutical opportunity by a targeted combination strategy., (© 2024. The Author(s).)

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.

In the latest World Health Organization classification (WHO), eosinophilic disorders represent a group of rare pathologic conditions with highly heterogeneous pathophysiology. In this report, we describe a case of myeloid neoplasm associated with eosinophilia and rearrangement of PDGFRB gene in a 67-year-old-male patient hospitalized with cerebellous ataxia. Initial investigations showed a bicytopenia with hypereosinophilia varying from 1.1 to 1.6×10 9 /L. Bone marrow aspiration was rich and showed a heterogeneous distribution of myeloid cells with clusters of promyelocytes and proerythroblasts associated with numerous eosinophils and spindle-shaped mast cells but without excess of blasts, dysplasia nor maturation skewing. These aspects suggested an atypical myeloproliferative neoplasm. Bone marrow biopsy was performed showing also a very high cellularity with area of myeloid and erythroid precursors associated with numerous spindle-shaped mast cells. Diagnoses of unclassified myeloid neoplasm and/or systemic mastocytosis were then proposed. Further chromosome analysis showed a t(5;8) translocation with PDGFRB rearrangement revealed in fluorescent in situ hybridization. Patient was treated with imatinib and intravenous immunoglobulin therapy allowing a significant improvement in neurological symptoms and biological results. Patient condition is currently stable after six lines of treatment. This rare hematopoietic neoplasm displays unusual histological and cytological features and can mimic other myeloproliferative neoplasm. Specific cytogenetics analysis should be considered for such cases with hypereosinophilia to select patients that may benefit from targeted therapy., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

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., Competing Interests: AE: Advisory board (with honorarium): Bayer, Seattle Genetics, Affimed, Verastem, Pharmacyclics, Research to Practice, and Physician Education Resource. Research support: Takeda, Seattle Genetics, Merck, NIH/ NCI, Leukemia and Lymphoma Society, and ORIEN. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ravi, Beheshti, Abermil, Lansigan, Kinlaw, Matthan, Mokhtar, Passero, Puliti, David, Dolnikowski, Su, Chen, Bijan, Varshney, Kim, Dave, Rudolph and Evens.)

Isolated trisomy 8 (+8) is a frequent cytogenetic abnormality in the myelodysplastic syndromes (MDS), but its characteristics are poorly reported. We performed a retrospective study of 138 MDS patients with isolated +8, classified or reclassified as MDS (excluding MDS/myeloproliferative neoplasm). Myeloproliferative (MP) features were defined by the repeated presence of one of the following: white blood cell count >10 × 10 9 /l, myelemia (presence of circulating immature granulocytes with a predominance of more mature forms) >2%, palpable splenomegaly. Fifty-four patients (39·1%) had MP features: 28 at diagnosis, 26 were acquired during evolution. MP forms had more EZH2 (33·3% vs. 12·0% in non-MP, P = 0·047), ASXL1 (66·7% vs. 42·3%, P = 0·048) and STAG2 mutations (77·8% vs. 21·7%, P = 0·006). Median event-free survival (EFS) and overall survival (OS) were 25 and 27 months for patients with MP features at diagnosis, versus 28 (P = 0·15) and 39 months (P = 0·085) for those without MP features, respectively. Among the 57 patients who received hypomethylating agent (HMA), OS was lower in MP cases (13 months vs. 23 months in non-MP cases, P = 0.02). In conclusion, MP features are frequent in MDS with isolated +8. MP forms had more EZH2, ASXL1 and STAG2 mutations, responded poorly to HMA, and tended to have poorer survival than non-MP forms., (© 2018 British Society for Haematology and John Wiley & Sons Ltd.)

Phosphoribosyltransferases (PRTs) bind 5'-phospho-α-d-ribosyl-1'-pyrophosphate (PRPP) and transfer its phosphoribosyl group (PRib) to specific nucleophiles. Anthranilate PRT (AnPRT) is a promiscuous PRT that can phosphoribosylate both anthranilate and alternative substrates, and is the only example of a type III PRT. Comparison of the PRPP binding mode in type I, II and III PRTs indicates that AnPRT does not bind PRPP, or nearby metals, in the same conformation as other PRTs. A structure with a stereoisomer of PRPP bound to AnPRT from Mycobacterium tuberculosis (Mtb) suggests a catalytic or post-catalytic state that links PRib movement to metal movement. Crystal structures of Mtb-AnPRT in complex with PRPP and with varying occupancies of the two metal binding sites, complemented by activity assay data, indicate that this type III PRT binds a single metal-coordinated species of PRPP, while an adjacent second metal site can be occupied due to a separate binding event. A series of compounds were synthesized that included a phosphonate group to probe PRPP binding site. Compounds containing a "bianthranilate"-like moiety are inhibitors with IC 50 values of 10-60μM, and K i values of 1.3-15μM. Structures of Mtb-AnPRT in complex with these compounds indicate that their phosphonate moieties are unable to mimic the binding modes of the PRib or pyrophosphate moieties of PRPP. The AnPRT structures presented herein indicated that PRPP binds a surface cleft and becomes enclosed due to re-positioning of two mobile loops., (Copyright © 2017 Elsevier B.V. All rights reserved.)

There are twenty-five published structures of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase ( Mtb -AnPRT) that use the same crystallization protocol. The structures include protein complexed with natural and alternative substrates, protein:inhibitor complexes, and variants with mutations of substrate-binding residues. Amongst these are varying space groups ( i.e. P 2 1 , C 2, P 2 1 2 1 2, P 2 1 2 1 2 1 ). This article outlines experimental details for 3 additional Mtb -AnPRT:inhibitor structures. For one protein:inhibitor complex, two datasets are presented - one generated by crystallization of protein in the presence of the inhibitor and another where a protein crystal was soaked with the inhibitor. Automatic and manual processing of these datasets indicated the same space group for both datasets and thus indicate that the space group differences between structures of Mtb -AnPRT:ligand complexes are not related to the method used to introduce the ligand.

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 in situ 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., (Copyright© 2017 Ferrata Storti Foundation.)

Proteasome-regulated NF-κB has been shown to be important for cell survival in T-cell lymphoma and Hodgkin lymphoma models. Several new small-molecule proteasome inhibitors are under various stages of active preclinical and clinical development. We completed a comprehensive preclinical examination of the efficacy and associated biologic effects of a second-generation proteasome inhibitor, ixazomib, in T-cell lymphoma and Hodgkin lymphoma cells and in vivo SCID mouse models. We demonstrated that ixazomib induced potent cell death in all cell lines at clinically achievable concentrations. In addition, it significantly inhibited tumor growth and improved survival in T-cell lymphoma and Hodgkin lymphoma human lymphoma xenograft models. Through global transcriptome analyses, proteasomal inhibition showed conserved overlap in downregulation of cell cycle, chromatin modification, and DNA repair processes in ixazomib-sensitive lymphoma cells. The predicted activity for tumor suppressors and oncogenes, the impact on "hallmarks of cancer," and the analysis of key significant genes from global transcriptome analysis for ixazomib strongly favored tumor inhibition via downregulation of MYC and CHK1, its target genes. Furthermore, in ixazomib-treated lymphoma cells, we identified that CHK1 was involved in the regulation of MYC expression through chromatin modification involving histone H3 acetylation via chromatin immunoprecipitation. Finally, using pharmacologic and RNA silencing of CHK1 or the associated MYC-related mechanism, we demonstrated synergistic cell death in combination with antiproteasome therapy. Altogether, ixazomib significantly downregulates MYC and induces potent cell death in T-cell lymphoma and Hodgkin lymphoma, and we identified that combinatorial therapy with anti-CHK1 treatment represents a rational and novel therapeutic approach. Cancer Res; 76(11); 3319-31. ©2016 AACR., (©2016 American Association for Cancer Research.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Purpose: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest-derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL., Design: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics., Results: Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine., Conclusions: Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients., (©2012 AACR.)

Context: TMEM127 is a novel pheochromocytoma (PCC) susceptibility gene., Objectives: Our aim was to clearly determine the indications for TMEM127 genetic testing in patients with PCC and/or paraganglioma (PGL)., Patients and Methods: Germline DNA from 642 unrelated patients who did not carry mutations in major PCC susceptibility genes was analyzed. Five hundred fifty-nine patients were affected by PCC, 72 by PGL (22 with head and neck and 50 with thoracic or abdominal location), and 11 by both PCC and PGL. Analysis of the TMEM127 gene was performed by direct sequencing and quantitative multiplex PCR of short fluorescent fragments., Results: In our cohort six mutations (0.9%) were identified. Three of them (p.Ala47Asp, p.Gln64HisfsX18, p.Tyr164X) were found in patients exhibiting clinical criteria for a hereditary disease (young age at diagnosis, bilateral PCC, or family history). The three others (p.Gln157X, p.Val68SerfsX13, p.Val90Met) were detected in patients with an apparently sporadic presentation. No mutation was found among patients with PGL, and no large chromosomal rearrangement spanning the TMEM127 gene was detected., Conclusions: Our results combined with the two previous studies suggest that direct sequencing of TMEM127 should be considered, after a negative screening of VHL, RET, SDHB, and SDHD genes, in patients with PCC.

Identification of the etiologic agent responsible for sinus fungal ball (SFB) is rarely obtained due to either the culture of patient specimens not being ordered or if cultures were inoculated they proved to be negative. Obviously, this has a significant impact on the design of appropriate therapeutic strategies. We investigated whether paraffin-embedded (PE) tissues, the only materials often available, were suitable for the correct identification of the responsible fungi. We obtained PE tissues of SFB from 16 different patients who had risk factors for invasive fungal infections. DNA was extracted using an automated extractor and the internal transcribed spacer (ITS) sequenced following amplification with two sets of primers designed to amplify >300 bp fragments. This was attempted in parallel with a real-time quantitative PCR assay targeting Aspergillus spp. mitochondrial DNA designed to amplify <150 bp fragments. ITS sequencing succeeded in appropriately identifying the etiologic agents in 10 of the 16 samples (nine Aspergillus fumigatus, one Lewia spp.). In contrast, the <150 bp PCR assay amplified all specimens correctly except the one involving Lewia spp. If fungal identification is warranted to understand the pathophysiology of SFB and guide clinicians, we cannot rely only on ITS sequencing of the DNA obtained from PE tissues. The main reason is probably due to the fact that formalin prevents amplification of long DNA fragments and consequently, frozen or fresh tissues should be employed.

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 of 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.

The beta-ICD (1a) or beta-ICD-amide (1e)-catalyzed aza-Morita-Baylis-Hillman reaction between N-sulfonylimines 3 and alkyl vinyl ketones 4 produced the (R)-enriched adducts 5. By adding a catalytic amount of beta-naphthol (2a), the enantioselectivity of the same reaction was inversed leading to (S)-5 in excellent yields and enantioselectivities. Both aromatic and aliphatic imines are accepted as substrates for this reaction.

The aza-MBH reaction of imines 1 and beta-naphthyl acrylate 2 in the presence of C-6' modified beta-isocupreidine derivative 1c (0.1 equiv) and beta-naphthol 5 (0.1 equiv) afforded the corresponding (3S)-aza-MBH adducts 4 in high yield and excellent enantiomeric excess. These catalytic conditions allowed the aliphatic imines to be employed for the first time as electrophilic partners of the aza-MBH reaction. The coexistence of two H-bond donors with different acidic strengths was found to be crucial for the observed high enantioselectivity.