The D661Y mutation is recurrent in T/NK-cell neoplasms and was previously shown to be associated with both STAT3 constitutive phosphorylation and increased transcriptional activity

The D661Y mutation is recurrent in T/NK-cell neoplasms and was previously shown to be associated with both STAT3 constitutive phosphorylation and increased transcriptional activity.23 Indeed, western blot analysis confirmed basal STAT3 phosphorylation in this cell line, which was abrogated on enforced expression of exogenous SOCS1 (supplemental Figure 8). Open in a separate window Figure 4. Reconstitution of wild-type SOCS1 causes cell death in cHL lines carrying concurrent mutations of Sand genes. sequencing and digital polymerase chain reaction) used to validate it. Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) for was performed according to standard protocols described in the supplemental Data. Functional experiments in cHL cell lines L1236, HDLM2, L540, and L428 cells were subjected to lentiviral transduction of anti-short-hairpin RNAs (shRNA) or LYPLAL1-IN-1 the coding sequence, followed by monitoring of cell death, as described in the supplemental Data. These data are shown in the main text as raw percentages of viable cells (and in supplementary figures as percentage of viable cells relative to the corresponding infected negative control set at 100%) because cHL cell lines are notoriously difficult to infect and their viability often decreases after infection, which may potentially influence the sensitivity of each cell line to different treatments. Mouse monoclonal to ERBB2 The same 4 cHL cell lines, as well as 2 additional ones (ie, SUPHD1 and UHO1), were also treated with the JAK2 inhibitor fedratinib and/or the XPO1 inhibitor selinexor, and then monitored for apoptosis and/or viability, as detailed in the supplemental Data. The experiments with fedratinib, which were aimed at confirming pharmacologically the apoptosis induction observed on genetic silencing of the JAK-STAT pathway with sh-RNAs, were performed with fedratinib concentrations in the low micromolar range (1.5 and 3 M), based on the drug concentration (1.5 M) previously established to cause 50% of maximal growth inhibition (IC50) in the STAT6 wild-type cHL cell line L428.7 The experiments with selinexor aimed at providing an initial assessment of the potential dependency of HRS cells on XPO1 and were performed at the dose of 100 nM, based on the median IC50 value of 123 nM that was previously established in 23 hematological and solid LYPLAL1-IN-1 tumor cell lines (including the B-cell lymphoma line Ramos, where selinexor IC50 was also 123 nM).8 Western blotting was performed to verify STAT6 downregulation and exogenous SOCS1 expression after lentiviral transduction, as well as to analyze the phosphorylation status of STAT transcription factors basally and after JAK2 inhibition, using the procedures and reagents described in the supplemental Data. All experiments were independently performed at least twice, giving reproducible results. Results The cHL coding genome To define the genetic basis of cHL, we laser-microdissected HRS cells9 (n = 1200-1800 per case), along with a similar number of adjacent nonneoplastic cells, from hematoxylin/eosin-stained frozen lymph node sections of 34 patients with cHL (supplemental Table 1; supplemental Figure 1). DNA from each tumor and matched normal sample was subjected in duplicate to whole-genome amplification (WGA) and independent WES of the duplicates to control LYPLAL1-IN-1 the bias introduced by the WGA reaction through a novel bioinformatics pipeline ad hoc designed (supplemental Data). Unamplified germline DNA from peripheral blood cells was also included as control in 26/34 patients. The median coverage depth in WGA-tumor, WGA-normal, and unamplified normal samples was 99, 114, and 142, respectively (supplemental Table 2; supplemental Figure 2). We identified a median of 47 nonsilent somatic mutations per tumor that were present at 20% variant allele frequency, and hence, presumably in the major tumor clone (median: 43 single-nucleotide variants and 3 short indels per tumor; supplemental Figure 3; supplemental Table 3). Deeper sequencing analysis of 150 candidate tumor-specific changes identified across 26 samples previously subjected to WES confirmed the presence of 139 mutations (93%), including 130/139 (94%) single-nucleotide variants and 9/11 (82%) short indels, validating the high specificity of the approach (supplemental Table 4). Importantly, allele frequency estimates of somatic mutations in the deep LYPLAL1-IN-1 targeted sequencing experiment were highly similar to those obtained in the WES experiment (correlation, 0.88; value 2.2e-16; supplemental Figure 4). Somatic mutations of selected genes were also validated by Sanger sequencing on tumor vs normal WGA-DNA (supplemental Table 5), and somatic variants of the most recurrently targeted gene (and (32% of cases), (24%), (18%), and (16%) (Figure 1; supplemental Table 3). Open.