Functional impact of NOTCH1 mutations in chronic lymphocytic leukemia

Abstract

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.

Common nonmutationalNOTCH1activation in chronic lymphocytic leukemia

“…Discussion CLL cells in the lymph node are known to display frequent NOTCH1 activation independent of mutation, as documented by their frequent immunohistochemical positivity for ICN1 in lymph node sections of both NOTCH1-mutated and wild-type cases (19,20). Conversely, few reports have shown activation of NOTCH1 in PB CLL samples, and the results involved a relatively low number of cases and a possibly low sensitivity of detection, leading to a significant underestimation of the ICN1 + cases (4,7,18,22). Our finding that ∼50% of PB CLL cases lacking NOTCH1 mutations express ICN1 strongly suggests that the activation of this pathway is more common than what is predicted by the frequency of classic NOTCH1 PEST-truncations.…”

“…Additional ligand-independent mechanisms include activation by other signaling pathways, as reported for the T-cell receptor pathway in T cells (60), or aberrant vesicle trafficking of the NOTCH1 receptor (61). Ligand-dependent mechanisms include binding to ligands expressed by endothelial cells in the blood vessels or by the CLL cells themselves (18,22 HES4 1 2 1 2 3 SE1 SE2 HES4 1 2 1 2 3 SE1 SE2 The enrichment of our NOTCH1 CLL signature in these normal subpopulations compared with GC B cells suggests that the biological programs orchestrated by NOTCH1 in CLL are similar to those already active in the putative normal B-cell counterparts of the disease. Thus, the finding of NOTCH1 activation in CLL cells reflects the constitutive, dysregulated expression of a physiologic signal and its corresponding gene-expression program rather than an ectopic program associated with transformation.…”

“…Although few NOTCH1 targets have been shown to be overexpressed in NOTCH1-mutated cases compared with wild-type CLL (4,17,18), the full spectrum of genes controlled by NOTCH1 and their contribution to the disease pathogenesis have not been identified. Moreover, recent reports documented that CLL cells in the lymph node frequently express ICN1, independent of NOTCH1 PEST-truncation (19,20), especially within the proliferation centers, which represent the key microanatomical sites of interaction of CLL cells with accessory cells and proliferation (21).…”

“…Finally, our results identify the NOTCH1-dependent transcriptional program in CLL cells, thus providing direct insights into the pathogenesis of a large fraction of CLL cases. activation in the peripheral blood (PB) compartment of CLL patients is less clear (4,7,18,22).…”

Lenalidomide interferes with tumor-promoting properties of nurse-like cells in chronic lymphocytic leukemia

“…In a separate set of experiments, NLCs were generated on coverslips with lenalidomide or vehicle and FITC-dextran particle engulfment was quantified as previously described. 23 …”