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

Abstract

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.

Generation of CD20-specific TCRs for TCR gene therapy of CD20low B-cell malignancies insusceptible to CD20-targeting antibodies

“…Although CD20 is also expressed on healthy B-cells which are depleted in the course of therapy, long-term B-cell aplasia is well manageable [1, 2]. However, refractory disease to CD20-targeted mAb treatment has been reported with various mechanisms of resistance: downregulation of CD20 expression [3–5], internalization of CD20:mAb complex [6], and inhibition of complement-dependent cytotoxicity (CDC) [7–10]. Therefore, additional therapeutic strategies are required.…”

Common nonmutationalNOTCH1activation in chronic lymphocytic leukemia

“…NOTCH1 mutations in CLL were shown to associate with poor prognosis, including a specific subset of patients carrying trisomy 12, disease progression, transformation to highly aggressive diffuse large B-cell lymphomas, termed Richter syndrome, and immunochemotherapy resistance (3,4,(12)(13)(14)(15)(16).…”

The HDAC inhibitor valproate induces a bivalent status of the CD20 promoter in CLL patients suggesting distinct epigenetic regulation of CD20 expression in CLLin vivo

“…I83-E95 cells carry del13q as reported by the supplier DSMZ. Since mutation of c7541_7542delCT, resulting in a frameshift deletion of NOTCH1, represents more than 80% of NOTCH1 mutations in CLL, and has been shown to affect CD20 expression in CLL [13], the NOTCH1 genomic region between 7270–7680 bp in I83-E95 cells was sequenced. According to our analysis, I83-E95 cells are wild type regarding NOTCH1 c7541_7542delCT.…”

“…Moreover, recent data show evidence for a NOTCH1 c7541_7542delCT mutation-driven epigenetic downregulation of CD20 expression. This downregulation is correlated to a worse response to rituximab-containing therapy in patients with NOTCH1 c7541_7542delCT mutation, but also to sensitivity to valproate-induced upregulation of CD20 in NOTCH1 c7541_7542delCT mutant cells during in vitro treatment of patient cells [9, 13]. …”