Page 152 - 2022_03-Haematologica-web

A.N. Bastidas Torres et al.
Figure 4. Distribution of recurrent chromosomal rearrangements, copy number alterations and deleterious indels/single nucleotide variants in pcAECyTCL. (A) First panel: recurrent chromosomal rearrangements impacting cancer genes. Second panel: recurrent large-scale copy number alterations (CNA) (>3 Mb). Third panel: focal minimal common regions (MCR) (≤ 3 Mb) shared by CNA; bona fide cancer genes residing within focal MCR are specified. Fourth panel: Indels and single nucleotide variants (SNV) in cancer genes leading to protein truncations, reported as pathogenic in literature or predicted as disease-causing (SIFT and PolyPhen-2). Only genes altered in more than one patient are indicated. CTX: interchromosomal rearrangement; ITX: intrachromosomal rearrangement. (B) Circos plots showing genetic alterations in patients AEC6 and AEC9. Despite inter-patient heterogeneity, molecular abnormalities affecting genes with roles in the cell cycle, chromatin regulation and JAK2 signaling (genes in light purple) were recurrent in primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (pcAECyTCL).
these chimeric proteins were expected to increase survival of Ba/F3 cells in the absence of IL3. Seven days after IL3 withdrawal, survival of Ba/F3 cells expressing each of the three engineered JAK2 fusions was noticeably higher (P<0.05, student’s t-test) than survival of the parental Ba/F3 cells (wild-type control) and Ba/F3 cells expressing eGFP (negative control) (Figure 7B).
We next evaluated the effect of FDA-approved JAK1/2 inhibitor ruxolitinib on each of the three IL3-independent cell lines carrying JAK2 fusions. Ruxolitinib inhibited the growth of all cell lines in a dose-dependent manner (Figure 7C and D) with half maximal inhibitory concentration (IC50) values in the low nanomolar range (9–15 nM), in con- cordance with the reported inhibitory activity of this drug.19 Since fusion partners PCM1 and TFG have exten- sively been proven by others to confer chimeric kinases (including JAK2) the ability to trans-autophosphorylate via self-oligo/dimerization,20-25 we carried on further vali- dation with JAK2 fusion containing novel kinase fusion partner KHDRBS1. For extra verification, we treated Ba/F3 cells expressing KHDRBS1-JAK2 with inhibitor AZD1480, which has higher specificity for JAK2 than ruxolitinib,26 and confirmed that cytokine-independent survival of
these cells depends on JAK2 signaling (Figure 7D). Finally, we corroborated by western blotting that growth inhibi- tion exerted by ruxolitinib and AZD1480 was accompa- nied by a dose-dependent inhibition of JAK2 and STAT5 phosphorylation in Ba/F3 cells driven by KHDRBS1-JAK2 (Figure 7D).
Discussion
This study describes the first high-resolution genetic profiling of pcAECyTCL using next-generation sequenc- ing. The landscape of structural genomic alterations of pcAECyTCL was characterized by considerable genomic instability and inter-patient heterogeneity. Most rearrangements (328 of 426) identified in pcAECyTCL dis- rupted annotated genes, and approximately one-third of all rearranged genes (91 of 305) were found to play roles in signal transduction and transcriptional regulation. In addi- tion, four of 12 patients experienced chromothripsis/chro- moplexy-like events which mediated the deletion of rele- vant tumor suppressors (e.g., CDKN2C, CHD5, FAS, PTEN, etc.). In full agreement with previously published
708
haematologica | 2022; 107(3)
AB