Mutations in NOTCH1 PEST domain orchestrate CCL19-driven homing of chronic lymphocytic leukemia cells by modulating the tumor suppressor gene DUSP22.

Even if NOTCH1 is commonly mutated in chronic lymphocytic leukemia (CLL), its functional impact in the disease remains unclear. Using CRISPR/Cas9-generated Mec-1 cell line models, we show that NOTCH1 regulates growth and homing of CLL cells by dictating expression levels of the tumor suppressor gene DUSP22. Specifically, NOTCH1 affects the methylation of DUSP22 promoter by modulating a nuclear complex, which tunes the activity of DNA methyltransferase 3A (DNMT3A). These effects are enhanced by PEST-domain mutations, which stabilize the molecule and prolong signaling. CLL patients with a NOTCH1-mutated clone showed low levels of DUSP22 and active chemotaxis to CCL19. Lastly, in xenograft models, NOTCH1-mutated cells displayed a unique homing behavior, localizing preferentially to the spleen and brain. These findings connect NOTCH1, DUSP22, and CCL19-driven chemotaxis within a single functional network, suggesting that modulation of the homing process may provide a relevant contribution to the unfavorable prognosis associated with NOTCH1 mutations in CLL.

NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation.

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression.

Functional impact of NOTCH1 mutations in chronic lymphocytic leukemia.

The purpose of this study was to compare the expression and function of NOTCH1 in chronic lymphocytic leukemia (CLL) patients harboring a wild-type (WT) or mutated NOTCH1 gene. NOTCH1 mRNA and surface protein expression levels were independent of the NOTCH1 gene mutational status, consistent with the requirement for NOTCH1 signaling in this leukemia. However, compared with NOTCH1-WT CLL, mutated cases displayed biochemical and transcriptional evidence of an intense activation of the NOTCH1 pathway. In vivo, expression and activation of NOTCH1 was highest in CLL cells from the lymph nodes as confirmed by immunohistochemistry. In vitro, the NOTCH1 pathway was rapidly downregulated, suggesting that signaling relies upon micro-environmental interactions even in NOTCH1-mutated cases. Accordingly, co-culture of Jagged1(+) (the NOTCH1 ligand) nurse-like cells with autologous CLL cells sustained NOTCH1 activity over time and mediated CLL survival and resistance against pro-apoptotic stimuli, both abrogated when NOTCH1 signaling was pharmacologically switched off. Together, these results show that NOTCH1 mutations have stabilizing effects on the NOTCH1 pathway in CLL. Furthermore, micro-environmental interactions appear critical in activating the NOTCH1 pathway both in WT and mutated patients. Finally, NOTCH1 signals may create conditions that favor drug resistance, thus making NOTCH1 a potential molecular target in CLL.

WITHDRAWN: Proteinase 3 (PR3) gene is highly expressed in CBF leukemias and codes for a protein with abnormal nuclear localization that confers drug sensitivity.

Core-binding factor (CBF) leukemias are characterized by a high degree of sensitivity to high-dose cytarabine (ARA-C) treatment and by a relatively favorable prognosis compared with most other forms of adult acute myeloid leukemia (AML). The molecular basis of the response to chemotherapy is still being analyzed. The proteinase 3 (PR3) gene codes for a serine protease with a broad spectrum of proteolytic activity. PR3 is involved in the control of proliferation of myeloid leukemia cells, and when it is abnormally expressed, it confers factor-independent growth to hematopoietic cells. In this study, we analyzed the expression levels of PR3 in 113 AML patients. PR3 is highly expressed in AML, mainly in CBF leukemias in which PR3 is not only expressed, but also abnormally localized within the nuclear compartment. Nuclear PR3 results in cleavage of nuclear factor (NF)-kappaB p65 into an inactive p56 subunit lacking any transcriptional activity. The nuclear localization of PR3 is responsible for increased proliferation, apoptosis arrest and increased sensitivity to high-dose ARA-C. This study provides a new molecular mechanism that is responsible for NF-kappaB inactivation and increased sensitivity to chemotherapy in CBF leukemias.Leukemia advance online publication, 14 January 2010; doi:10.1038/leu.2009.207.

Different isoforms of the B-cell mutator activation-induced cytidine deaminase are aberrantly expressed in BCR-ABL1-positive acute lymphoblastic leukemia patients.

The main reason for the unfavorable clinical outcome of BCR-ABL1-positive acute lymphoblastic leukemia (ALL) is genetic instability. However, how normal B-cell precursors acquire the genetic changes that lead to transformation has not yet been completely defined. We investigated the expression of the activation-induced cytidine deaminase (AID) and its role in clinical outcome in 61 adult BCR-ABL1-positive ALL patients. AID expression was detected in 36 patients (59%); it correlated with the BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors. Different AID splice variants were identified: full-length isoform; AIDDeltaE4a, with a 30-bp deletion of exon 4; AIDDeltaE4, with the exon 4 deletion; AIDins3, with the retention of intron 3; AIDDeltaE3-E4 isoform without deaminase activity. AID-FL predominantly showed cytoplasmic localization, as did the AID-DeltaE4a and AID-DeltaE3E4 variants, whereas the C-terminal-truncated AID-DeltaE4 showed a slightly increased nuclear localization pattern. AID expression correlated with a higher number of copy number alterations identified in genome-wide analysis using a single-nucleotide polymorphism array. However, the expression of AID at diagnosis was not associated with a worse prognosis. In conclusion, BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that may act as mutators outside the immunoglobulin (Ig) gene loci in promoting genetic instability.

Deferasirox treatment improved the hemoglobin level and decreased transfusion requirements in four patients with the myelodysplastic syndrome and primary myelofibrosis.

Transfusion-induced iron overload is a frequent problem that clinicians have to face in the treatment of patients affected by both myelodysplastic syndrome (MDS) and primary myelofibrosis (PMF). Different options are currently available for chelation therapy, e.g. oral once-daily administration of the iron chelator deferasirox. In 3 patients with MDS and 1 patient with PMF, deferasirox therapy resulted in an improvement in the hemoglobin level and a reduction in transfusion dependence. Our data open new insights regarding the benefit of iron chelation therapy not only for transfusional iron overload of myelodysplastic and myelofibrotic patients but also for the increase in hemoglobin levels. The biological mechanism of action of deferasirox, an effect which is not shared by other iron chelators, is still obscure and requires further investigations.

Increase sensitivity to chemotherapeutical agents and cytoplasmatic interaction between NPM leukemic mutant and NF-kappaB in AML carrying NPM1 mutations.

Mutations in nucleophosmin (NPM) exon 12 and the resulting delocalization of NPM into the cytoplasm are the most specific and frequent cellular events in acute myeloid leukemia patients (AML) with normal karyotype. Cytoplasmatic NPM (NPMc+) is associated with responsiveness to chemotherapy and better prognosis. The activation of nuclear factor-kappaB (NF-kappaB) has been demonstrated to occur in a subset of AML patients and is thought to induce resistance to many chemotherapeutical agents. In this study, we demonstrate the increased in vitro sensitivity of NPMc+ cells to chemotherapeutical agents and their reduced NF-kappaB activity. Furthermore, we provide evidence of the interaction between NPMc+ and NF-kappaB in the cytoplasm, resulting in the sequestration and inactivation of NF-kappaB. The cytosolic localization and consequent inactivation of NF-kappaB justifies the reduced NF-kappaB DNA-binding activity observed in NPMc+ patients. These data, taken together, may provide a possible explanation for the increased rate of chemosensitivity observed among the NPMc+ patients.

WT1 transcript amount discriminates secondary or reactive eosinophilia from idiopathic hypereosinophilic syndrome or chronic eosinophilic leukemia.

Idiopathic hypereosinophilic syndromes (HES) comprise a spectrum of indolent to aggressive diseases characterized by persistent hypereosinophilia. Hypereosinophilia can result from the presence of a defect in the hematopoietic stem cell giving rise to eosinophilia, it can be present in many myeloproliferative disorders or alternatively it may be a reactive form, secondary to many clinical conditions. The hybrid gene FIP1L1-PDGRFalpha was identified in a subset of patients presenting with HES or chronic eosinophilic leukemia (CEL). In spite of this, the majority of HES patients do not present detectable molecular lesions and for many of them the diagnosis is based on exclusion criteria and sometimes it remains doubt. In this study we explored the possibility to distinguish between HES/CEL and reactive hypereosinophilia based on WT1 transcript amount. For this purpose, 312 patients with hypereosinophilia were characterized at the molecular and cytogenetic level and analyzed for WT1 expression at diagnosis and during follow-up. This study clearly demonstrates that WT1 quantitative assessment allows to discriminate between HES/CEL and reactive eosinophilia and represents a useful tool for disease monitoring especially in the patients lacking a marker of clonality.

NF-kB inhibition as a strategy to enhance etoposide-induced apoptosis in K562 cell line.

NF-kB is a transcription factor that mediates antiapoptotic signals in several cancer cell lines. Here we have demonstrated that the cytotoxic drug, Etoposide, activates NF-kB in K562, a chronic myeloid leukemia blast crisis cell line. Treatment with the NF-kB inhibitors MG-132, Bay11-7082, and Resveratrol impedes Etoposide-induced NF-kB activation, rendering K562 sensitive to Etoposide-induced apoptosis. Stable expression of mutant form of IkB-alpha, which retains NF-kB inactive in the cytoplasm of cells, confirmed the data obtained with molecular inhibitors. Both inhibitors and stable expression of SR-IkB are associated with down-modulation of the antiapoptotic protein Bcl-xL, suggesting that the survival pathway activated by Etoposide involves NF-kB-mediated Bcl-xL expression.

The NF-kappaB pathway blockade by the IKK inhibitor PS1145 can overcome imatinib resistance.

Imatinib represents at present the most attractive therapy for BCR-ABL positive leukemias, even though a percentage of CML patients develop resistance to this compound. For these resistant patients a therapeutic approach based on a combination of drugs is more likely to be effective. In the last years, constitutive NF-kappaB/Rel activity has been demonstrated in several hematological malignancies. As a result, NFkB/Rel-blocking approaches have been proposed as antineoplastic strategies. Furthermore, the identification of specific kinases within the NF-kappaB activation pathway offers a selective target to address tailored therapies. In the current study, we show that the IKK inhibitor PS1145 is able to inhibit the proliferation of CML cell lines and primary BM cells. Moreover, the addition of Imatinib increases the effects of PS1145 in resistant cell lines and BM cells from resistant patients, with a further increase of apoptosis and inhibition of proliferation and colony growth. Our data provide the rational for a new therapeutic approach, which combines Imatinib and the IKK inhibitor PS1145 in CML resistant patients.