STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma

Activating mutations in STAT6 are common in Follicular Lymphoma (FL) and transformed FL and various other B cell lymphomas. Here, we report RNA-seq based gene expression data on normal human lymph node derived B lymphocytes (NBC; N = 6), and primary human FL WT (N = 11) or mutant (N = 4) for STAT6 before and after ex vivo stimulation with IL4. We found that STAT6 mutants result in broad based augmentation of IL4-induced gene expression. Unexpectedly, in FL with WT STAT6 we measured reduced baseline and IL4-induced gene expression levels when compared with NBC lymphocytes or FL with STAT6 mutations. We tracked the attenuated IL4/JAK/STAT6 response to co-existing CREBBP mutations and experimentally verified that intact CREBBP is required for the induction of many IL4-induced genes. One of the IL4-induced genes here identified is RRAGD, a small G-protein involved in lysosomal mTOR activation. We show that IL4 treatment induced RRAGD expression, that RRAGD is required for mTOR activation in lymphoma cells and that IL4-enhanced BCR signaling induced mTOR activation. The IL4 and BCR-induced mTOR activation was reduced by CREBBP mutants and augmented by mutant STAT6, establishing a link between STAT6 mutations and mTOR regulated pro-growth pathways in lymphoma. Cryopreserved single-cell suspensions from human non-malignant lymph node (LN) biopsies or lymphomatous LNs were thawed, washed and depleted of CD3+ T cells and CD14+ macrophages using Miltenyi beads and columns (Miltenyi, 130-050-101 and 130-050-201). The B lymphocytes were cultured for 4 h in the presence or absence of 10 ng/ml of IL4. Total RNA was purified using the Trizol reagent followed by RNAeasy column purification (Qiagen). RNA was assessed using the Bioanalyzer (Agilent #5067-1513) and a Qubit RNA High Sensitivity assay (Thermofisher # Q32855), and 2ng of RNA input was processed for sequencing using the Takara SMART-Seq stranded kit (#634444).