Constant activation of the RAF-MEK-ERK pathway as a diagnostic and therapeutic target in hairy cell leukemia

The BRAF-V600E mutation defines genetically hairy cell leukemia among B-cell leukemias and lymphomas. In solid tumors, BRAF-V600E is known to aberrantly activate the oncogenic MEK-ERK pathway, and targeted BRAF and/or MEK inhibitors have shown remarkable efficacy in clinical trials in melanoma patients. However, the MEK-ERK pathway status in hairy cell leukemia has not been thoroughly investigated. We assessed phospho-ERK expression in 37 patients with hairy cell leukemia and 44 patients with neoplasms mimicking hairy cell leukemia (40 splenic marginal zone lymphoma, 2 hairy cell leukemia-variant and 2 splenic lymphoma/leukemia unclassifiable) using immunohistochemistry on routine biopsies and/or Western blotting on purified leukemic cells, and correlated the phospho-ERK status with the BRAF-V600E mutation status. Besides confirming the constant presence of BRAF-V600E in all patients with hairy cell leukemia, we observed ubiquitous phospho-ERK expression in this malignancy. Conversely, all 44 cases with neoplasms mimicking hairy cell leukemia were devoid of BRAF-V600E and none expressed phospho-ERK. Furthermore, the two exceptionally rare cases of non-hairy cell leukemia unclassifiable chronic B-cell neoplasms previously reported to be BRAF-V600E(+) on allele-specific polymerase chain reaction lacked phospho-ERK expression as well, suggesting the presence of the mutation in only a small part of the leukemic clone in these cases. In conclusion, our findings support the use of phospho-ERK immunohistochemistry in the differential diagnosis between hairy cell leukemia and its mimics, and establish the MEK-ERK pathway as a rational therapeutic target in this malignancy

From cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric HIV-1 ribonuclease H inhibitors

The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibitor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thorough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative 22 resulted the most potent dual inhibitor exhibiting IC50s of 0.53 and 2.90 μM against the RNase H and RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pockets within the RT, one located between the RNase H active site and the primer grip region and the other close to the DNA polymerase catalytic centre.status: publishe

From cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric HIV-1 ribonuclease H inhibitors

The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibitor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thorough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative 22 resulted the most potent dual inhibitor exhibiting IC50s of 0.53 and 2.90 lM against the RNase H and RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pockets within the RT, one located between the RNase H active site and the primer grip region and the other close to the DNA polymerase catalytic centre

NOVEL NPM1 EXON 5 MUTATIONS AND GENE FUSIONS LEADING TO ABERRANT CYTOPLASMIC NUCLEOPHOSMIN IN AML

none32siNucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but sporadically also exon 9 and 11, all causing changes at protein C-terminal end (loss of tryptophans and creation of a nuclear export signal-NES motif) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 AML patients, we found non-exon 12 NPM1 mutations in 5/387 (1.3%) NPM1c+ cases. Besides mutations in exon 9 (n=1) and exon 11 (n=1), novel mutations in exon 5 were discovered (n=3). One more exon 5 mutation was identified in additional 141 AML patients selected for wild-type NPM1 exon 12. Furthermore, 3 NPM1 rearrangements (i.e. NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13,979 AML samples screened by cytogenetic/FISH and RNA sequencing. Functional studies demonstrated that in AML cases the new NPM1 proteins harboured an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting any AML-associated NPM1 genetic lesions. Also, this study highlights the need for developing new specific assays for molecular diagnosis and monitoring of NPM1-mutated AML.noneMartelli, Maria Paola; Rossi, Roberta; Venanzi, Alessandra; Meggendorfer, Manja; Perriello, Vincenzo Maria; Martino, Giovanni; Spinelli, Orietta; Ciurnelli, Raffaella; Varasano, Emanuela; Brunetti, Lorenzo; Ascani, Stefano; Quadalti, Corinne; Cardinali, Valeria; Mezzasoma, Federica; Gionfriddo, Ilaria; Milano, Francesca; Pacini, Roberta; Tabarrini, Alessia; Bigerna, Barbara; Albano, Francesco; Specchia, Giorgina; Vetro, Calogero; Di Raimondo, Francesco; Annibali, Ombretta; Avvisati, Giuseppe; Rambaldi, Alessandro; Falzetti, Franca; Tiacci, Enrico; Sportoletti, Paolo; Haferlach, Torsten; Haferlach, Claudia; Falini, BrunangeloMartelli, Maria Paola; Rossi, Roberta; Venanzi, Alessandra; Meggendorfer, Manja; Perriello, Vincenzo Maria; Martino, Giovanni; Spinelli, Orietta; Ciurnelli, Raffaella; Varasano, Emanuela; Brunetti, Lorenzo; Ascani, Stefano; Quadalti, Corinne; Cardinali, Valeria; Mezzasoma, Federica; Gionfriddo, Ilaria; Milano, Francesca; Pacini, Roberta; Tabarrini, Alessia; Bigerna, Barbara; Albano, Francesco; Specchia, Giorgina; Vetro, Calogero; Di Raimondo, Francesco; Annibali, Ombretta; Avvisati, Giuseppe; Rambaldi, Alessandro; Falzetti, Franca; Tiacci, Enrico; Sportoletti, Paolo; Haferlach, Torsten; Haferlach, Claudia; Falini, Brunangel