Notch2 controls non-autonomous Wnt-signalling in chronic lymphocytic leukaemia

The Wnt signalling pathway, one of the core de-regulated pathways in chronic lymphocytic leukaemia (CLL), is activated in only a subset of patients through somatic mutations. Here we describe alternative, microenvironment-dependent mechanisms of Wnt activation in malignant B cells. We show that tumour cells specifically induce Notch2 activity in mesenchymal stromal cells (MSCs) required for the transcription of the complement factor C1q. MSC-derived C1q in turn inhibits Gsk3-β mediated degradation of β-catenin in CLL cells. Additionally, stromal Notch2 activity regulates N-cadherin expression in CLL cells, which interacts with and further stabilises β-catenin. Together, these stroma Notch2-dependent mechanisms induce strong activation of canonical Wnt signalling in CLL cells. Pharmacological inhibition of the Wnt pathway impairs microenvironment-mediated survival of tumour cells. Similarly, inhibition of Notch signalling diminishes survival of stroma-protected CLL cells in vitro and disease engraftment in vivo. Notch2 activation in the microenvironment is a pre-requisite for the activation of canonical Wnt signalling in tumour cells.

and altered following CLL-MSCs contact according to Gene set enrichment analysis (GSEA). All probe sets interrogating the respective target genes are given.
CLL cells were cultured in medium only (red circles) or on human BMSCs cells (black squares) for 5 days before analysing apoptotic cells by Annexin-V/DAPI staining. Transwells were used to disrupt direct cell-cell contacts (blue triangles). Error bars show mean ± SEM from 5 patients. **p < 0.01.     Constitutive expression of the Notch-ligands Delta-1,-4, Jagged-1 and Jagged-2 were analysed in primary CLL cells obtained from 3 different patients by immunoblotting. b.
Expression of Notch1-4 in cultured primary MSCs derived from 8 weeks old C57BL/6 mice, assessed by flow cytometry. c.
Expression of Notch1 and Notch2 in primary MSCs derived from the bone marrow of 10-weeks-old C57BL/6 Nestin GFP mice. Nestin-positive cells were identified by staining for GFP, endothelial cells by staining with CD31, osteoblasts were CD51 positive. Analyses of 3 additional mice revealed similar results. Isotype controls for Notch were used as negative control and performed on the bulk population. a. Transmembrane-domain (TMD) and cleaved (ICD) Notch2 levels were analysed in EL08-1D2 cells and EL08-1D2 cells co-cultured with CLL primary cells. b.
Notch2 expression of human BMSC cells mono-cultured or co-cultured with primary CLL cells for 48 hours. One representative experiment out of three is shown. Scale bar = 100µm c.
Notch3-4 were analysed in EL08-1D2 cells and EL08-1D2 co-cultured for 48 hours with CLL primary cells obtained from 3 different patients.  a. Nuclear and cytoplasmic β-catenin levels were assessed in CLL cells derived from co-cultures with EL08-1D2 cells or from mono-cultures by cellular sub-fractioning and immunoblotting. Representative results from 3 different patients are shown. Nuclear expression was confirmed by co-expression of Rb. b.
EL08-1D2 cell proliferation was measured by cell count following CRISPR/Cas9 Notch2 deletion for 72h after cell plating. Results from 3 independent biological repeats are shown. Error bars depict SEM. *p < 0.05. d.
Bright-field image of EL08-1D2 cells transduced with a sgRNA control or a Notch2 sgRNA 48h after plating. e.
Quantification of β-catenin levels in CLL cells, cultured on Notch2 proficient or deficient (using either Cre-mediated or Cas9-mediated deletion of Notch2) stromal cells. Immunoblots were quantified by using Image-J software. Expression was normalised to the expression of housekeeping proteins. 20 individual patients were analysed. f.
β-catenin expression in CLL mono-cultures after 24 hours in media supplemented with C1q (N=4). a. N-cadherin expression in 3 primary CLL cells mono-cultured or co-cultured with human BMSCs for 24 hours. b.
β-catenin was immuno-precipitated from primary CLL lysates derived from mono-culture or from co-culture with EL08-1D2 cells and membranes probed with antibodies against N-cadherin. One representative experiment out of three is shown. c.
E-cadherin expression was evaluated by immunoblotting in EL08-1D2 cells in which N-cadherin expression was ablated using CRISPR/Cas9. Two different guide RNAs were used. d.
Flow cytometry analysis of CD19 + cells contamination after aminooxy-biotin labelling of MSCs. The bulk of CLL cells were removed before labelling of MSCs. e.
Pie chart representing the total protein count and the total protein abundance of plasma membrane proteins following mass spectrometry analysis. f. N-cadherin expression was evaluated by immunoblotting in Notch2 fl/fl stromal cells following in vitro Crerecombination. Two independent experiments are shown. g.
Quantitative reverse-transcription polymerase chain reaction analysis of N-cadherin mRNA expression in primary CLL cells 24 hours after co-culture on EL08-1D2 (black circles) and in presence of 5µM ICG-001 (red squares) normalized to expression in control cells (0µM ICG-001). Shown is the mean ± standard deviation of 3 independent experiments with individual primary CLL cells. ***p < 0.001. a. Viability of EL08-1D2 cells, exposed for 48 hours to the inhibitors as indicated, was assessed by staining cells for 7AAD. Oligomycin treatment was used as positive control. b.

XAV939
Percentage of apoptotic CLL cells co-cultured on human BMSCs and increasing doses of XAV939 were detected by flow cytometry and staining for Annexin-V/DAPI. Error bars show mean ± SEM from 4 patient samples. **p < 0.01.

Supplementary Figure 9
β-catenin was assessed in lymph node sections of CLL patients by immunofluorescence microscopy. An antibody was used to specifically detect the active form of β-catenin. Scale bar=100µm. The white dotted box in the 3 rd panel shows the tissue section enlarged and depicted on the right.   Gating strategy for apoptotic CLL cells in mono-culture or in co-culture. SSC-A and FSC-A were used as the initial gate for cell debris and aggregates. In general, FSC-A/FSC-H followed by SSC-W/SSC-A was used to isolate singlets. DAPI was used to discriminate live from dead cells. Apoptotic cells were detected by staining for Annexin-V. b.
Gating strategy for bone marrow derived MSC subpopulations. SSC-A and FSC-A were used as initial gate for cell debris and aggregates. In general, FSC-A/FSC-H followed by SSC-W/SSC-A was used to isolate singlets. DAPI was used to discriminate live from dead cells. Nestin-positive cells were gated on CD45-negative cells.