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Biomedical subjects

Roberto Rosati

Publications and source records attributed to Roberto Rosati.

8 recordsLinked to original sources

Inhibition of the atypical kinase WNK1 as a therapeutic strategy in TAL-related T-cell acute lymphoblastic leukemia.

Driver mutations in T-cell acute lymphoblastic leukemia (T-ALL) rarely affect druggable kinases. However, these kinases can be aberrantly activated or repressed as secondary oncogenic events. Thus, integrating unbiased phosphoproteomics with genomic approaches may offer novel opportunities for target discovery and therapeutic interventions. In our study, we identified WNK1 (with no lysine [K]) as a potential target in T-ALL by pairing a list of vulnerable kinases with data from a phosphoproteomic screen of T-ALL cell lines. We subsequently validated WNK1 by loss-of-function-based studies and tested WNK inhibitors in several in vitro and in vivo T-ALL models and clinical T-ALL samples. We showed that therapeutic WNK1 repression promotes polyploidy, resulting in cell proliferation arrest, and morphometric changes, such as incomplete cell division or chromosome segregation through altered mitotic spindle assembly and abscission defects. Furthermore, we found that WNK1 is overexpressed in the TAL1/2-related subgroup, but not in normal thymus or lymph nodes, suggesting a potential translational area for clinical exploitation in poor-prognosis T-ALL carrying PTEN mutations and del(6q). Our work also reports a functional contribution of WNK1 in the leukemia establishment and progression. Structurally WNK1 is an atypical serine/threonine kinase that diverges from canonical kinases by lacking the conserved lysine in subdomain II, instead featuring a cysteine in subdomain I, which is critical for adenosine triphosphate (ATP) binding. This unusual structural configuration creates a distinct ATP-binding pocket with limited sequence similarity to conventional kinases, offering a unique opportunity to develop highly selective small molecules. Targeting this atypical ATP domain could thus provide a therapeutic advantage and broaden the treatment landscape for T-ALL.

WNK Lysine-Deficient Protein Kinase 1↗

Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia.

Nucleophosmin (NPM) exon-12 mutations occur in 50% to 60% of adult acute myeloid leukemia (AML) with normal karyotype and are predictors of favorable prognosis. We evaluated bone marrow or peripheral blood samples from 450 adult patients with AML of the GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto)/AML12 EORTC (European Organization for Research and Treatment of Cancer) trial to (1) search for new exon-12 NPM mutations; (2) determine whether NPM immunostaining on paraffin-embedded biopsies predicts NPM mutations; and (3) investigate altered nucleocytoplasmic NPM traffic in primary AML cells. Fourteen NPM mutations, including 8 new variants, were identified. All 200 AML cases expressing cytoplasmic NPM (NPMc(+) AML) carried NPM mutations. None of the 250 cases with nucleus-restricted NPM (NPMc(-) AML) was mutated. At the C-terminus, NPM leukemic mutants carried mutations of only tryptophan 290 or of both tryptophans 288 and 290 and a new nuclear export signal (NES) motif, which appear to underlie their nuclear export. The specific Crm1/exportin-1 inhibitor leptomycin-B relocated NPM mutants from cytoplasm to nucleus of primary NPMc(+) AML cells, demonstrating that nuclear export is NES dependent. NPM mutants bound and recruited wild-type NPM into leukemic cell cytoplasm. Because alterations at C-terminus of leukemic NPM mutants are similar, immunohistochemistry detects all exon-12 NPM mutations and is a valuable, inexpensive tool in the diagnostic-prognostic work-up of patients with AML with normal karyotype.

Active Transport, Cell Nucleus↗

Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc+ AML.

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.

Active Transport, Cell Nucleus↗

Denaturing high-performance liquid chromatography: a valid approach for identifying NPM1 mutations in acute myeloid leukemia.

NPM1 gene mutations are the most frequent genetic lesion in the 60% of adult acute myeloid leukemias (AMLs) with normal karyotype and no evidence of typical fusion genes (BCR/ABL1, PML/RARA, AML1/ETO, CBFB/MYH11, DEK/CAN). Using direct sequencing we previously identified six different heterozygous mutants within exon 12 encoding the nucleophosmin C-terminus. Because of these mutations the shuttling protein nucleophosmin is aberrantly delocalized in the cytoplasm of leukemic cells (NPMc+). Here, we designed and tested a denaturing high-performance liquid chromatography (DHPLC) assay to detect NPM1 mutated variants. To assess specificity, sensitivity, reliability, and reproducibility, we analyzed DNA from 120 primary adult AMLs and compared DHPLC results with immunohistochemistry and sequencing. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity. Sequencing categorized mutations A, B, and D, and all mutation A cases gave identical elution profiles. The other mutations showed typical chromatograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.

Amino Acid Sequence↗

Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.

BACKGROUND: Nucleophosmin (NPM), a nucleocytoplasmic shuttling protein with prominent nucleolar localization, regulates the ARF-p53 tumor-suppressor pathway. Translocations involving the NPM gene cause cytoplasmic dislocation of the NPM protein. METHODS: We used immunohistochemical methods to study the subcellular localization of NPM in bone marrow-biopsy specimens from 591 patients with primary acute myelogenous leukemia (AML). We then correlated the presence of cytoplasmic NPM with clinical and biologic features of the disease. RESULTS: Cytoplasmic NPM was detected in 208 (35.2 percent) of the 591 specimens from patients with primary AML but not in 135 secondary AML specimens or in 980 hematopoietic or extrahematopoietic neoplasms other than AML. It was associated with a wide spectrum of morphologic subtypes of the disease, a normal karyotype, and responsiveness to induction chemotherapy, but not with recurrent genetic abnormalities. There was a high frequency of FLT3 internal tandem duplications and absence of CD34 and CD133 in AML specimens with a normal karyotype and cytoplasmic dislocation of NPM, but not in those in which the protein was restricted to the nucleus. AML specimens with cytoplasmic NPM carried mutations of the NPM gene that were predicted to alter the protein at its C-terminal; this mutant gene caused cytoplasmic localization of NPM in transfected cells. CONCLUSIONS: Cytoplasmic NPM is a characteristic feature of a large subgroup of patients with AML who have a normal karyotype, NPM gene mutations, and responsiveness to induction chemotherapy.

Adolescent↗

Cryptic insertion producing two NUP98/NSD1 chimeric transcripts in adult refractory anemia with an excess of blasts.

We performed cytogenetic and molecular studies on an adult patient with refractory anemia with an excess of blasts with an add(11)(p15). Multicolor fluorescence in situ hybridization (FISH) identified the extra material on 11p as belonging to chromosome 15. Metaphase FISH with probes for chromosomes 5, 11, and 15 revealed a complex four-break rearrangement. Clone RP5-1173K1, containing exons 10-20 of the NUP98 gene, gave three fluorescence signals on the normal 11, the der(5), and the der(15). 3'-RACE-PCR identified an in-frame fusion between NUP98 and NSD1, which was confirmed by RT-PCR. Two different spliced forms, that is, NUP98 exon 11/NSD1 exon 6 and NUP98 exon 12/NSD1 exon 6, were detected. The reciprocal NSD1/NUP98 was not found. A dual-color experiment with RP5-1173K1 and CTC-549A4, spanning the entire NSD1 gene, indicated an insertion of NUP98 into the NSD1 locus. This is the first report of an adult with myelodysplastic syndrome (MDS) harboring an NUP98/NSD1 fusion resulting from insertion of 5'-NUP98 into the NSD1/5q35 locus.

Amino Acid Sequence↗

NUP98 is fused to the NSD3 gene in acute myeloid leukemia associated with t(8;11)(p11.2;p15).

Fusion between the NUP98 and NSD3 genes in a patient with acute myeloid leukemia associated with t(8;11)(p11.2;p15), is reported for the first time. The t(8;11)(p11.2;p15) was identified by classical cytogenetics. Fluorescence in situ hybridization (FISH) analysis revealed a split signal with a mix of BAC 118H17 and 290A12, indicating the translocation disrupted NUP98. FISH restriction at 8p11-12 showed a split of BAC 350N15. Molecular investigations into candidate genes in this BAC showed the NUP98 fusion partner at 8p11.2 was the NSD3 gene. To date the NSD3 gene has never been implicated in hematologic malignancies.

Acute Disease↗

Genetic profile of acute myeloid leukemia.

Understanding genomic events and the cascade of their effects in cell function is crucial for identifying distinct subsets of acute myeloid leukemia and developing new therapeutic strategies. Conventional cytogenetics, fluorescence in situ hybridization investigations and molecular studies have provided much information over the past few years. This review will focus on major genomic mechanisms in acute myeloid luekemia and on the genes implicated in the pathogenesis of specific subtypes.

Acute Disease↗