ABSTRACT
To investigate chronic lymphocytic leukemia (CLL)-initiating cells, we assessed NOTCH1 mutation/expression in hematopoietic stem cells (HSCs). In NOTCH1-mutated CLL, we detected subclonal mutations in 57% CD34+/CD38- HSCs. NOTCH1 mutation was present in 66% CD34+/CD38+ progenitor cells displaying an increased mutational burden compared to HSCs. Flow cytometric analysis revealed significantly higher NOTCH1 activation in CD34+/CD38- and CD34+/CD38+ cells from CLL patients, regardless NOTCH1 mutation compared to healthy donors. Activated NOTCH1 resulted in overexpression of the NOTCH1 target c-MYC. We conclude that activated NOTCH1 is an early event in CLL that may contribute to aberrant HSCs in this disease.
ABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Bepridil/pharmacology , Calcium Channel Blockers/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Receptor, Notch1/antagonists & inhibitors , Receptor, Notch1/metabolism , Animals , Apoptosis/drug effects , Biomarkers, Tumor/metabolism , Chemotaxis/drug effects , Drug Screening Assays, Antitumor , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mice , Mutation , Prognosis , Receptor, Notch1/genetics , Tumor Microenvironment , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Richter syndrome (RS) is a rare event in chronic lymphocytic leukemia (CLL) that is influenced by biological factors and prior CLL treatments. Ibrutinib is a Bruton tyrosine kinase inhibitor that has shown remarkable efficacy in CLL; however, little is known about its relationship to RS. We report a case of ibrutinib efficacy against CLL in a patient with prolonged remission of RS. METHODS: The patient was diagnosed with CLL in 2003. Biological findings at onset included absent ZAP70 expression, mutated IGVH, and NOTCH1 mutation. He was treated with FCR with partial response. In 2013, he progressed to RS, not clonally related to the underlying CLL. The patient was treated with anthracycline- and platinum-based regimens, obtaining a complete remission. After 3 years, he presented a CLL progression with worsening lymphocytosis, anemia, thrombocytopenia, increased splenomegaly, and lymphadenopathies. Positron emission tomography-computed tomography scan excluded pathologic uptake. Thus, he was started on ibrutinib. RESULTS: At 12 months' follow-up, we observed white blood cell normalization, increased hemoglobin and platelet levels, disappearance of lymphadenopathy, and spleen size reduction. Therapy was well-tolerated with no evidence of RS. CONCLUSION: This case demonstrates sustained RS remission in a patient with CLL under ibrutinib therapy, thus improving our knowledge on the use of this new drug in CLL and beyond.
Subject(s)
Cell Transformation, Neoplastic/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Neoplasm Recurrence, Local/drug therapy , Protein Kinase Inhibitors/therapeutic use , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Adenine/analogs & derivatives , Aged , Cell Transformation, Neoplastic/pathology , Disease Progression , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/complications , Male , Neoplasm Recurrence, Local/complications , Piperidines , Remission Induction , SyndromeABSTRACT
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.