Silencing Mutations in NOTCH1 Activate Calcium Signaling in B Cells

The NOTCH1 pathway is known to be up-regulated in many cancer types including non Hodgkin lymphomas (NHLs), colorectal, breast and pancreatic cancers. The precise downstream effects of NOTCH1 mutations in lymphoma are not known.

We applied high throughput sequencing in three different non Hodgkin lymphomas including diffuse large B cell lymphoma (DLBCL, N=94), mantle cell lymphoma (MCL, N=58) and Burkitt lymphoma (BL, N=59). We also sequenced mutations from 208 patients with immune deficiency. In all, we identified 31 mutations in NOTCH1. Five of these mutations were either nonsense or frameshift mutations. Silencing mutations in the PEST domain have been reported in MCLs, and many other silencing mutations were observed in the other lymphoma cohorts as well as immune deficiency cases. The finding of silencing mutations was unexpected, given that in most cancers, NOTCH1 and its pathway are often constitutively active.

Calcium signaling is an important mechanism for activating B cells. Given the shared mutations between NHLs and immune deficiencies, we investigated the role of silencing NOTCH1 mutations in altering calcium signaling by engineering lymphoma cells with a deletion of the transmembrane region resulting in silencing of NOTCH1 using the CRISPR system.

We designed guide RNAs that spanned a 57 base pair region from exon 28 that encodes the transmembrane domain of the NOTCH1 gene. Each guide RNA was ligated into a pX458 Cas9 GFP expressing plasmid. Namalwa NHL cells were transfected, FACS-sorted for GFP positivity and subcloned. Successful deletion of the target region in Cas9 NOTCH1 cells was confirmed by Sanger sequencing and Western blotting.

Cells that were wild type, Cas9 control, or Cas9 NOTCH1 were incubated in a calcium free buffer containing Fluo-4 for detection of intracellular free calcium. Cells were washed and subjected to flow sorting where baseline calcium was recorded. The cells were stimulated with anti-IgM (Fab’)₂ and recorded again for response. To test extracellular calcium influx, 4mM CaCl₂ was added to the calcium free buffer containing cells and recorded again for response.Upon stimulation with anti-IgM (Fab’)₂, we observed a significant increase (p<10^-6) of free cytosolic Calcium in the Cas9 NOTCH1 cells compared to both wild type and Cas9 control. Similar results were obtained when measuring extracellular Calcium influx upon addition of CaCl₂to the buffered solution of cells.

These findings indicate that silencing of NOTCH1 has an activating role in calcium signaling that could contribute to B cell survival and function in NHLs and immune deficiencies. Similar roles have been reported for activating mutations in PIK3CD and STAT3 that contribute to both immune deficiency and cancer. Our study is the first to identify the role of silencing mutations of NOTCH1 in NHLs and immune deficiencies.