Extended Data Fig. 8: Novel TSG candidates and validation of CHST11 IPA as cancer-promoting isoform.
From: Widespread intronic polyadenylation inactivates tumour suppressor genes in leukaemia
a, As in Fig. 3c, but shown are known (red gene names) and novel TSG candidates (black gene names) among the abundant CLL-IPAs. CLL-IPAs seem to inactivate these genes as they mostly occur upstream or within 10% (of overall amino acid length) of the mutations. P = 1 × 10−8, two-sided Wilcoxon rank-sum test performed on all 136 TSGs; P = 1 × 10−8, two-sided Wilcoxon rank-sum test performed on the novel TSGs, n = 119. Position of the TR mutation was determined using the data obtained from the MSK cbio portal and indicates the hot spot mutation. Right, the fraction of CLL samples affected represents the fraction of CLL samples (out of 59) with significant expression of the IPA isoform. Genes were included if they were affected in at least 20% of samples investigated either by 3′-seq or RNA-seq. b, Contingency table for enrichment of novel TSGs among highly recurrent CLL-IPAs. P value was obtained from two-sided Fisher’s exact test. c, TSGs have larger protein sizes. Box plots are as in Fig. 1e. **P = 0.005, two-sided Mann–Whitney U-test. The increased overall mutation rate of known TSGs correlates with larger protein size. P = 1 × 10−6, Spearman’s correlation coefficient, r = 0.74. d, CHST11 IPA generates 18 new amino acids (grey) downstream of exon 1. e, Experimental set-up to measure paracrine WNT activity produced by MEC1 B cells either expressing GFP, GFP–CHST11 or GFP–CHST11 IPA and using a WNT reporter expressed in HEK293T cells. Primary CLL cells and the CLL cell line MEC1 express several WNTs, including WNT5B. In the presence of CHST11 WNT (red dots) binds to sulfated proteins on the surface of WNT producing cells, whereas WNT is secreted into the medium in the presence of CHST11 IPA. WNT-conditioned medium activates a WNT reporter in HEK293T cells. This set-up refers to Fig. 4f, g. f, Western blot, performed once, for WNT5 shown as in Fig. 4f, but including HeLa cells as positive control for WNT5 expression. Actin was used as loading control on the same blot.