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H. Larose et al.
across several concentrations (Figure 6D, Online Supplementary Figure S6B). Karpas 299 cells have different genetic defects, which may explain the antagonistic results in this cell line.42 Indeed, a significant increase in apoptotic cells was observed following 48 h of treatment with a combination of 50 nM crizotinib and 2 mM PF- 03084014 (Figure 6E, Online Supplementary Figure S6C).
Ideally, a single-agent ALK inhibitor would provide a less toxic frontline treatment approach in the future, although resistance would be expected to develop. Therefore, crizotinib-resistant ALCL cell lines were assessed for their sensitivity to GSI which led to a signifi- cant decrease in cell proliferation (Figure 6F, Online Supplementary Figure S6D-F). These data suggest the potential use of GSI as either second-line treatment for ALK inhibitor-resistant disease, or frontline therapy in combination with ALK tyrosine kinase inhibitors.
NOTCH1 is a potential biomarker predictive of relapse in ALK+ anaplastic large cell lymphoma
A clinically annotated FFPE tissue microarray of 89 ALK+ ALCL patient samples, biopsied at the time of the patients’ initial presentation, was analyzed for cleaved NOTCH1 protein expression (Online Supplementary Figure S6G). Of the 89 patient tumors assessed, 88.8% showed high cleaved NOTCH1 staining (moderate and strong staining cate- gories), in keeping with previously published results.7,8,43 Interestingly, patients with low cleaved NOTCH1 staining (negative and weak staining categories) are more likely to relapse (based on the 10-year event-free survival) and have a significantly worse prognosis (P<0.05) (Figure 6G).
Discussion
The genetics underlying ALK+ ALCL at the level of somatic mutations remains largely unknown. Being a rela- tively rare cancer with a common, well-characterized driv- ing oncogenic event, more could be done to uncover other genetic alterations. We therefore sequenced 18 ALK+ ALCL tumors and analyzed their genome together with seven previously reported ALK+ cases9 in order to uncover patho- genic mechanisms and novel therapeutic targets. Mutational signature analysis showed signatures 1, 3 and 26 in all our patient samples. The latter two signatures have their roots in homologous recombination DNA dou- ble-strand-break repair and mismatch DNA repair deficien- cy.20 This suggests that DNA damage repair mechanisms might be impaired in these patients, predisposing them to ALCL, perhaps through germline mutations in DNA repair proteins (such as a BRCA2 variant K3326X, COSM4984873, found in germline sample 67B44). In con- trast, signature 1, accounting for the majority of the total contribution, is associated with a prevalence of C>T tran- sitions at NpCpG trinucleotides and spontaneous deami- nation of 5-methyl-cytosine, considered an age-related phenomenon due to endogenous mutational processes.21 In general, 5-methyl cytosine residues are unstable within DNA and are prone to mutation, representing hotspots for this activity.45 This is paradoxical with the young age range of ALK+ ALCL patients, with the majority being young adults, and suggests that the mechanism(s) leading to such mutations may be ‘speeded up’. Indeed, CpG transitions may be a consequence of secondary factors that promote deamination, such as exogenous mutagens and carcino- gens, for example polycyclic aromatic hydrocarbons.45
To identify pathways that are key to ALCL biology, GSEA was employed and revealed a number of pathways commonly affected by mutations in ALK+ ALCL. Interestingly, the TCR signaling pathway was prominent in our analysis. It has previously been shown that NPM- ALK can substitute for key TCR-induced distal signaling pathways and silencing of proximal proteins has been shown in ALCL.46 Another key pathway identified was NOTCH1; gain-of-function mutations in NOTCH1 have previously been identified in a number of other cancers, most notably in approximately 50 to 60% of T-cell acute lymphoblastic leukemia.47,48 However, most of these muta- tions are in the intracellular domains of the protein, with few reported in the extracellular domains.49-51 In contrast, novel mutations in the EGF-like domain of extracellular NOTCH1 were detected in 9.3% (T349P) and 10.2% (T311P) of ALK+ ALCL patients analyzed in this study. Predicted to be deleterious, the functional significance of these mutations was investigated using bioinformatics analysis. It has previously been shown that EGF-like domains 8 to 12 are important for NOTCH1 binding to its ligands.36,52 Specifically, threonine bases, lost in T349P and T311P, within the EGF-like domains are post-translational- ly modified by O-linked glycosylation which is necessary for ligand-engaged NOTCH1 signaling.53-55 Mutations to proline, a rigid and bulky amino acid, result in a change in tertiary structure, often forcing a change in b-sheet confor- mation (EGF-like domains are made of b-sheets among others). We demonstrated the positive impact of the T349P mutant on NOTCH1 activity, as shown by enhanced cell proliferation when expressed in HEK293 cells. We theorize that NOTCH1 T349P could modulate ligand binding (either directly or through modulation of calcium binding, particularly as calcium signaling is thought to be dysregu- lated in ALCL and calcium ions play an important role in NOTCH1 ligand binding56,57).
Regardless of the presence of NOTCH1 mutations, NOTCH1 constitutes a therapeutic target in ALCL inde- pendently of ALK status; suppression of NOTCH1 expres- sion or activity led to an increase in apoptosis, in keeping with previous reports.7,8,58 Intriguingly, NPM-ALK has pre- viously been shown to be sufficient to induce NOTCH1 expression.25 Not only did we confirm this by silencing NPM-ALK in ALCL, but we also showed that NPM-ALK acts through STAT3, which binds to the NOTCH1 pro- moter. This could explain why we observed synergistic effects between crizotinib and GSI in inducing cell death despite our evidence that NPM-ALK promotes NOTCH1 expression. Indeed, studies have shown that crizotinib synergizes with brentuximab vedotin, which targets CD30-expressing cells, despite NPM-ALK having been shown to drive CD30 expression.59 ALK inhibitors are now being added to frontline therapy60 (e.g., in trial NCT01979536) although this combination has led to some unexpected toxicity.11 NOTCH1 inhibition may therefore serve as a second-line treatment. Indeed, although GSI have suffered from gastrointestinal-related toxicity, isoform-specific GSI or antibody-based treat- ments that target NOTCH1 directly (NCT03422679) have shown more promise.61 Published studies variously describe that GSI-1 (both as a single treatment and in com- bination) and PF-03084014 are relatively well tolerated.62-64
Ultimately, our data show that GSI and ALK inhibitors act additively/synergistically and induce apoptosis of ALCL cell lines, and furthermore that ALK inhibitor-resis-
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