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Deregulation of JAK2 signaling underlies pcAECyTCL
cally deregulated in pcAECyTCL. Upregulated genes included among others JAK2 itself, components of type I and II cytokine receptors that signal predominantly via JAK2 (i.e., IFNGR2, IL12RB2) and established enhancers of JAK2 signaling (i.e., PTPN11, SH2B1). In contrast, down- regulated JAK-STAT pathway genes included PTPRC and genes encoding receptors exclusively associated with sig- nal transduction via JAK1, JAK3 or TYK2 (Figure 6C).
In order to validate JAK-STAT pathway activation in pcAECyTCL, we investigated the presence of activated STAT proteins (pSTAT3 and pSTAT5) by immunohisto- chemistry (IHC) in eight sequenced patients with avail- able tumor tissue. Robust activation of JAK-STAT signal-
ing (via STAT3, STAT5 or both) was confirmed in all eval- uated patients (Figure 6D).
JAK2 fusions identified in pcAECyTCL confer cytokine- independent survival ability to cells
In order to validate the predicted effects of the JAK2 fusions found in pcAECyTCL (i.e., PCM1-JAK2, KHDRBS1-JAK2, TFG-JAK2) on cell survival, we engi- neered these fusion genes into murine pro-B Ba/F3 cells which die in the absence of exogenous IL3 (Figure 7A). Because self-oligo/dimerizing JAK2 fusions were predict- ed to activate downstream STAT proteins without the need of upstream cues elicited by cytokine stimulation,
Figure 3. Landscape of copy number alterations reveals focal SH2B3 inactivation in pcAECyTCL. (A) Human chromosome ideograms showing regions of gain and loss detected through whole-genome sequencing (WGS) in twelve primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (pcAECyTCL) genomes. Blue bars to the right of the chromosomes depict regions of loss whereas red bars to the left of the chromosomes depict regions of gain. (B) Deletions at 12q24.12 (blue bars), where SH2B3 resides, were the most focal (<500 Kb) copy number alteration (CNA) events in pcAECyTCL. Inactivation of SH2B3 was mediated by interstitial deletions and unbalanced rearrangements. Breakpoints of structural alterations at 12q24.12 in all affected patients were validated by Sanger sequenc- ing. Genomic coordinates of breakpoints according to reference genome GRCh38. Arrows indicate the direction towards which genomic coordinate numbers increase. Plus (+) and minus (-) signs specify strand polarity. CTX: interchromosomal rearrangement; ITX: intrachromosomal rearrangement; iDel: interstitial deletion. (C) Copy number losses involving SH2B3 in patients with pcAECyTCL were validated by droplet digital polymerase chain reaction. Ctrl: control CD8+ T cells.
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