Stefano Baldoni

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype.

Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.

NOTCH1 PEST domain mutation is an adverse prognostic factor in B‐CLL

Prosper, J.Y., Campbell, K., Sutherland, D.R., Metcalfe, P., Horsfall, W. & Ouwehand, W.H. (2002) A tyrosine703serine polymorphism of CD109 defines the Gov platelet alloantigens. Blood, 99, 1692– 1698. Smith, J.W., Hayward, C.P., Horsewood, P., Warkentin, T.E., Denomme, G.A. & Kelton, J.G. (1995) Characterization and localization of the Gova/b alloantigens to the glycosylphosphatidylinositol-anchored protein CDw109 on human platelets. Blood, 86, 2807–2814.

A new genetic lesion in B‐CLL: a NOTCH1 PEST domain mutation

et al, 2007). In conclusion, our data show an association between PAX5 aberrant splicing and BCP-ALL. We also provide, for the first time, evidence of imbalance between the full-length and PAX5D2 isoforms as a common event in BCP-ALL. Given the well-documented role of this gene in the development of BCP, the cumulative effect of point mutations and post-transcriptional regulation of alternative splicing of PAX5 pre-mRNA may suggest its primary pathogenic role in BCP-ALL of both children and adults.

Notch1 modulates mesenchymal stem cells mediated regulatory T-cell induction.

Notch1 signaling is involved in regulatory T (Treg)‐cell differentiation. We previously demonstrated that, when cocultured with CD3+ cells, mesenchymal stem cells (MSCs) induced a T‐cell population with a regulatory phenotype. Here, we investigated the molecular mechanism underlying MSC induction of human Treg cells. We show that the Notch1 pathway is activated in CD4+ T cells cocultured with MSCs. Inhibition of Notch1 signaling through GSI‐I or the Notch1 neutralizing antibody reduced expression of HES1 (the Notch1 downstream target) and the percentage of MSC‐induced CD4+CD25highFOXP3+ cells in vitro. Moreover, we demonstrate that FOXP3 is a downstream target of Notch signaling in human cells. No crosstalk between Notch1 and TGF‐β signaling pathways was observed in our experimental system. Together, these findings indicate that activation of the Notch1 pathway is a novel mechanism in the human Treg‐cell induction mediated by MSCs.

T regulatory cell separation for clinical application

We selected T regulatory cells (Tregs) from standard leukapheresis using double-negative selection (anti-CD8 and anti-CD19) followed by positive selection (anti-CD25) and 72 procedures were performed. A median of 263×10(6)cells (range 143-470×10(6)) were recovered with a mean of CD4(+)/CD25(+) cells of 94.5±2.4% (36.5±18.6% CD4(+)/CD25(+hi)). FoxP3(+) cells were equal to 79.8%±22.2. CD127(+) cells were 12.5%±8.2. The inhibition assay showed an inhibition rate of 67±22. Cells isolated by means of this approach can be used in allogeneic hematopoietic stem cell transplantation to reduce the incidence and severity of GvHD without bystander inhibition of general immunity.

A revised NOTCH1 mutation frequency still impacts survival while the allele burden predicts early progression in chronic lymphocytic leukemia

A revised NOTCH1 mutation frequency still impacts survival while the allele burden predicts early progression in chronic lymphocytic leukemia

NOTCH and NF-κB interplay in chronic lymphocytic leukemia is independent of genetic lesion

The NOTCH and nuclear factor kappa B (NF-κB) pathways are both constitutively activated in Chronic Lymphocytic Leukemia (CLL). We first described the NOTCH1 PEST domain mutation in a CLL subgroup, but the activation of the NOTCH pathway in NOTCH1-unmutated cases remains unexplained. Here, we investigated whether genetic lesions in the NF-κB/NOTCH loop might support the NOTCH activation status by sequencing negative (TNFAIP3/A20) and positive (TRAF2, TRAF5, TNFRSF11A/RANK, MAP3K7/TAK1, and CARD11) regulators of NF-κB together with NF-κB targets on the NOTCH pathway, the NOTCH ligands Jagged1 and Jagged2, in CLL patients. The sequence analysis revealed four missense mutations for A20, TRAF2, TRAF5 and RANK1 genes, all causing a change in amino acid group from polar to non-polar, but functional domains were not involved. Specific predictive software analyses confirmed that the amino acid changes have a low-functional impact on the protein. Our results show that in CLL, NF-κB regulators and Jagged are both unmutated, suggesting that the Jagged-mediated interplay between NF-κB and NOTCH is independent of genetic lesions.

The NOTCH1/CD39 axis: a Treg trip-switch for GvHD.

Regulatory T cells (Tregs) suppress alloimmune reaction such as graft versus host disease (GvHD)1 and promote tolerance induction to allogeneic organ transplants.2 In high-risk acute leukaemia patients undergoing full-haplotype mismatched transplantation we demonstrated that adoptive immunotherapy with Tregs conventional T cells (Tcons) almost completely prevented acute and chronic GvHD, favoured post-transplant immunological reconstitution and was associated with a powerful graft-versus-leukaemia (GvL) effect.3, 4, 5 Interestingly, GvHD severity and mortality was markedly reduced by inactivation of NOTCH signalling in donor T cells by means of humanised antibodies and conditional genetic models.6, 7, 8 The present study attempted to unravel the connection between Tregs and NOTCH signalling in Tcons for GvHD prevention. We discovered that NOTCH1 downregulation on Tcons is a new Treg mechanism of action and showed that Tregs use the CD39 pathway to modulate NOTCH1 expression on Tcons.

Constitutive phosphorylation of the active Notch1 intracellular domain in chronic lymphocytic leukemia cells with NOTCH1 mutation

Alterations in Notch signaling are involved in chronic lymphocytic leukemia (CLL) pathogenesis, a hematological disease characterized by the accumulation of CD19+/CD5+ B cells resistant to apoptosis. We previously reported that constitutive Notch1/2 activation contributes to apoptosis resistance of CLL cells.1 Furthermore, a NOTCH1 PEST domain mutation, resulting in a truncated protein more stable and active than wild-type (WT) protein, has recently emerged as a recurrent genetic lesion in CLL patients with adverse prognosis and poor outcome.2, 3 Despite the progress achieved on the role of NOTCH1 mutations in CLL outcome, little is known regarding their role in CLL cell biology. There is evidence that NOTCH1 mutation stabilizes Notch1 signaling in CLL cells,4 but the molecular mechanisms underlying this effect remain to be defined.

Bepridil exhibits anti-leukemic activity associated with NOTCH1 pathway inhibition in chronic lymphocytic leukemia: Activity of bepridil against CLL

Dysregulated NOTCH1 signaling, by either gene mutations or microenvironment interactions, has been increasingly linked to chronic lymphocytic leukemia (CLL). Thus, inhibiting NOTCH1 activity represents a potential therapeutic opportunity for this disease. Using gene expression‐based screening, we identified the calcium channel modulator bepridil as a new NOTCH1 pathway inhibitor. In primary CLL cells, bepridil induced selective apoptosis even in the presence of the protective stroma. Cytotoxic effects of bepridil were independent of NOTCH1 mutation and other prognostic markers. The antitumor efficacy of bepridil was associated with inhibition of NOTCH1 activity through a decrement in trans‐membrane and activated NOTCH1 protein levels with unchanged NOTCH2 protein levels. In a CLL xenotransplant model, bepridil significantly reduced the percentage of leukemic cells infiltrating the spleen via enhanced apoptosis and decreased NOTCH1 activation. In conclusion, we report in vitro and in vivo anti‐leukemic activity of bepridil associated with inhibition of the NOTCH1 pathway in CLL. These data provide a rationale for the clinical development of bepridil as anti‐NOTCH1 targeted therapy for CLL patients.