Fig. 6: Notch pathway allelic series highlights driver mechanism in a NOTCH3-rearranged PDX line.

a Fitness posterior distribution of guides targeting gamma secretase complex component APH1A in 15 PDX lines. PDX lines C0331, C1368 and C2271, further assayed in vivo (d–f), shown in colour. b Exon diagram of NOTCH3, showing position of translocation breakpoint with MEMO1P4 in PDX line C1368. Arrows show position of 4 guides targeting NOTCH3 body, and 4 guides targeting the PEST3 domain not present in the translocation, labeled with the in vivo fitness of each guide. c Canonical Notch signaling pathway. The genes shown are included in a Notch-focused sgRNA library, and coloured by functional group. d Fitness of in vivo Notch pathway and NOTCH3 allelic series in C1368 (4 PDX tumours). Guide distributions coloured red (low fitness) and blue (high fitness) indicate estimates that differ from neutrality (fitness = 1) with false discovery rate <0.05 (Benjamini-Hochberg method). The driving function of the NOTCH3 translocation is illustrated by reduced fitness with guides targeting NOTCH3 body but not the missing NOTCH3 PEST domain. Reduced fitness is also seen in multiple cleavage complex components, Notch pathway transcription factors and coactivators. e Fitness (mean + /− SEM) of four guides targeting 5’ NOTCH3 regions other than the PEST domain and four guides targeting the PEST domain. C1368 shows differential sensitivity to NOTCH3 targeting outside the PEST domain, which is missing in this PDX line due to translocation. (PDX tumours: C1368 4, C2271 4, C0331 4.) f Pairwise comparison of guide fitness (median, bars = 95% CI) in C1368 and two other TNBC lines, C0331 and C2271. C1368 shows lower fitness in NOTCH3 compared with both other TNBCs, as well as in the canonical transcription factor HES1. C2271 and C0331 show differential fitness in Notch ligands DLL1 and JAG1/2, and transcriptional coactivator MAML2. (PDX tumours: C1368 4, C2271 4, C0331 4.) Source data are provided as Source Data files.