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NOTCH1 and the pathobiology of ALK-positive ALCL
A
D
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Figure 2. Analysis of whole exome sequencing data yields insights into the ALK+ anaplastic large cell lymphoma genomic landscape. (A) Proportion of variant type for each patient. (B) Mean and standard deviation of the proportion of variant types for patients who did (n=9) or did not (n=9) relapse within 5 years after diagnosis (***P<0.001). (C) Mutational signatures20 derived from the variant profiles of each of the 11 patients for whom we hold sequenced matched peripheral blood. (D, E) Scatter plots of the pathways (D) or domains (E) found to be enriched in our dataset, displaying the number of databases in which each hit was found to be enriched, along with the (-)log10 of the statistical enrichment P-value of the software in which each hit was found to be most enriched, and the corresponding number of genes involved. SNV: single nucleotide variant.
although these were only detected in one patient each and so were not studied further. Of note, there was no significant difference in prognosis for patients presenting with NOTCH1 T349P and/or T311P over those with wild-type (WT) NOTCH1 when considering the whole cohort of patients (Online Supplementary Table S9, Online Supplementary Figure S3J). However, if adult patients were considered in isolation, there was a significant reduction in overall survival for those with NOTCH1 mutations (P<0.05) but these data are based on just three patients and so should be interpreted with caution.
Variant T349P, at position 1045, is within the sixth of 34 exons of NOTCH1, which encodes one of the numer- ous EGF-like domains that make up the extracellular domain of NOTCH1 (EGF-like domain 9 of 36, which is a calcium-binding domain). NOTCH1 T349P was pre- dicted to be a function-altering mutation by variant effect prediction software including SIFT (score=0.01) and PolyPhen (score=0.999), among others.26-30 Furthermore, the COSMIC database shows that T311 and T349 are the two most frequently reported mutated amino acids at the presumed NOTCH1/JAG1 interface across a range of
cancers (including chronic myelomonocytic leukemia,31 chronic lymphocytic leukemia,32 T-cell acute lymphoblas- tic leukemia,33 rhabdomyoscaroma34 and squamous cell carcinoma35) (Figure 3A).
To determine the impact of the detected NOTCH1 mutants on cell proliferation, WT, T349P or T311P mutants of NOTCH1 were expressed in HEK293FT cells (Figure 3B and C). These cells were chosen because of their low levels of endogenous NOTCH1 expression. In comparison, ALCL cell lines express high levels of WT NOTCH1. A significant increase in ATP production, sug- gestive of enhanced cell proliferation (as determined by a RealTime-Glo assay), was observed at 72 h for cells expressing the NOTCH1 T349P mutant as compared to WT NOTCH1 in the absence of exogenously applied lig- and (Figure 3D). The T311P mutant also led to an increase in proliferation, although this was not statistically signif- icant when compared to WT NOTCH1. However, a sig- nificant increase in proliferation was detected when using an MTT assay (Online Supplementary Figure S3I). In addi- tion, the transcriptional targets of NOTCH1 activity, HES1 and HEY1, were expressed at higher levels in the
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