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

Abstract

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.

Fig. 1. Identification of IL4 regulated genes in normal human lymph node derived B cells, FL B cells with WT STAT6 and FL B cells with MUT STAT6 (RNAseq results; heatmaps).

A Heatmap displays genes that are ≥1.5-fold upregulated or ≥1.5-fold downregulated in the normal human LN derived B cells (NBC), sorted by fold-change, with a mean TPM value ≥ 1 (for IL4 treated replicates in upregulated) or ≥1 (for CTL replicates in downregulated), pAdj (q) ≤ 0.1 and compared to the other sets. NBC N = 6, FL B cells with WT STAT6 N = 11; FL B cells with MUT STAT6 N = 4. Heatmap shows row wise scaled normalized counts. B Heatmap (core IL4-induced gene program) shows genes that are ≥2-fold upregulated or ≥2-fold downregulated in the NBC, FL B cells with WT STAT6 and FL B cells with MUT STAT6. Heatmap shows row wise scaled normalized counts. STAT6 Signal Transducer and Activator of Transcription 6, IL4 interleukin 4, IL4 interleukin 4, FL follicular lymphoma, RNAseq ribonucleic acid sequencing, WT wild type, MUT mutated, NBC normal B cells.

Fig. 2. Elevated baseline and IL4-induced expression of the majority of IL4 inducible genes in normal human lymph node derived B cells (NBC) or FL B cells carrying MUT STAT6 when compared with the expression in FL B cells carrying WT STAT6.

A Baseline or CTL: no IL4 treatment, IL4: ex vivo IL4 treatment. Box plots. Y-axis: mean TPM on a log2-scale. Each dot and line represent one gene. P-values computed via Wilcox test. B–D Stacked histograms: In each stacked histogram the fraction of genes expressed higher in the indicated sample set is colored in blue. IL4 inducible genes were defined with a fold change ≥2-fold in each of the three data sets independently (NBC: normal B cells, WT: FL B STAT6 WT, Mutant: FL B STAT6 MUT). STAT6 Signal Transducer and Activator of Transcription 6, IL4 interleukin 4, FL follicular lymphoma, CTL control, log logarithmic scale, WT wild type, MUT mutated, NBC normal B cells, TPM transcripts per million.

Fig. 3. CREBBP is required for the induction of IL4 inducible genes.

A The CREBBP gene or AAVS (control) was targeted with pooled CRISPR-Cas9 guide pools in four lymphoma cell lines (SU-DHL4, OCI-LY7, SU-DHL10, DOHH-2) and the pools were analyzed for CREBBP protein expression by immunoblotting (S.E. short exposure; L.E. long exposure). Total ribo depleted RNA from cells pools that were untreated or ex vivo IL4 treated were subjected to RNA-seq. Venn diagrams show the number of genes that are >2-fold upregulated by IL4 in AAVS targeted cell line pools (controls), CREBBP targeted cell line pools (CREBBP −/−), and in normal human B cells (B), or FL B cells with WT STAT6 (C) or FL B cells with Mutant STAT6 (D). IL4 interleukin 4, CREBBP CREB binding protein, AAVS adeno-associated virus integration site, qRT-PCR quantitative reverse transcriptase polymerase chain reaction, FL follicular lymphoma, WT wild type, MUT mutated.

Fig. 4. STAT6 mutations rescue the expression of IL4 inducible genes in CREBBP −/− lymphoma cells.

A RNA seq based expression of selected IL4 responsive genes grouped by four FL genotypes (CREBBP and STAT6) based on the data published by Dreval et al., Blood [48]. Please also see Supplementary Fig. 3. B The expression of six IL4 inducible genes in three CREBBP −/− lymphoma cell lines expressing lentivirally transduced HA-tagged STAT6 WT or STAT MUT (p.377 K and p.419 G). Displayed is the difference of delta Ct values (CT mean gene – Ct mean PGK1) from samples with or without IL4 stimulation. STAT6 Signal Transducer and Activator of Transcription 6, IL4 interleukin 4, FL follicular lymphoma, Ct cycle threshold.

Fig. 5. IL4 induces RRAGD expression and combined IL4 treatment and BCR crosslinking hyperactivates mTOR signaling.

A–C Lymphoma cell line pools transduced with lentiviral CRISPR-Cas9 guide pools targeting AAVS (control) or RRAGD, were treated ex vivo with IL4 for 0–6 h (time course). Cell lysates were made and prepared for immunoblotting using the indicated epitopes. D Non-malignant normal human LN derived B cells were purified via Miltenyi columns and depletion of CD3 T cells. Purified B cells were treated ex vivo with IL4 for 0–6 h (time course). Cell lysates were made and prepared for immunoblotting using the indicated epitopes. E, F Non-malignant normal human LN derived B cells (N = 4) were purified via Miltenyi columns and depletion of CD3 T cells. Purified B cells were left untreated or treated ex vivo with IL4, anti-IgM or both as indicated. Cell lysates were prepared for immunoblotting using the indicated epitopes. G densitometry results for p-p70-S6K and RRAGD normalized to actin for all conditions for four primary human LN-derived B lymphocyte samples (# 111, 89, 94, and 95) combined. Left panel: One-way Anova with post hoc Tukey’s test. Right panel: paired nonparametric Wilcoxon test. *p < 0.05, ***p < 0.001. IL4 interleukin 4, BCR B cell receptor, AAVS adeno-associated virus integration site, CRISPR-Cas9 clustered regularly interspaced short palindromic repeats CRISPR associated protein 9, IgM immunoglobulin M, ANOVA analysis of variance.

Fig. 6. RRAGD is required for S6 Kinase phosphorylation in lymphoma.

A The RRAGD gene or AAVS (control) was targeted with lentivirus carrying pooled CRISPR-Cas9 guides in four lymphoma cell lines and following puromycin selection the pools were analyzed for RRAGD protein expression by immunoblotting. B, C RRAGD −/− or AAVS targeted lymphoma cell line pools were treated with anti-IgM or anti-IgG for 10’, cell lysates were made, and protein prepared for immunoblotting using the indicated epitopes. Densitometry data for mean p-p70-S6K:HSP90 for AAVS control cells are shown, while signals for RRAGD −/− cells were close to background. One-Way ANOVA with post hoc Tukey’s test. Densitometry based on three cell lines and N = 2 repeats *p < 0.05, **p < 0.01. D–F RRAGD −/− or AAVS targeted lymphoma cell line pools were treated +/− IL4 for 6 h and +/− anti-IgM for 10’, cell lysates were made and protein prepared for immunoblotting using the indicated epitopes. D A representative blot for OCI-LY7. E Densitometry of mean p-4E-BP1:total 4E-BP1 for AAVS control cells +/− IL4 for 6 h and +/− anti-IgM/G. One-Way ANOVA with post hoc Dunn’s test. F Densitometry of comparative data for AAVS and RRAGD −/− cells; Mann–Whitney test. Densitometry based on three cell lines and N = 2 repeats. ns, not significant, *p < 0.05, **p < 0.01. AAVS adeno-associated virus integration site, CRISPR-Cas9 clustered regularly interspaced short palindromic repeats CRISPR associated protein 9, IgM immunoglobulin M, IgG immunoglobulin G, IL4 interleukin 4, ANOVA analysis of variance.

Fig. 7. STAT6 mutations hyperinduce RRAGD expression and IL4 and BCR induced mTOR signaling.

Three lymphoma cell lines were lentivirally transduced with cDNA/ORFs for WT or MUT STAT6 and cells sorted via GFP fluorescence. Cells were stimulated with IL4 and BCR crosslinking as indicated or left untreated and detergent lysates prepared for immunoblotting using the indicated epitopes. A–C Representative immunoblotting results for N = 2 independent experiments per cell pool. D Results from densitometry for phospho-S6K normalized to HSP90 for indicated conditions across three cell lines (N = 6). Two-way ANOVA with post hoc Holm Šidák test *p < 0.05. E Results from densitometry for RRAGD normalized to HSP90 for indicated conditions for N = 2 independent experiments across three cell lines (N = 6 measurements). Two-way ANOVA with post hoc Holm Šidák test *p < 0.05; **p < 0.01. STAT6 Signal Transducer and Activator of Transcription 6, IL4 interleukin 4, WT wild type, MUT mutated, GFP green fluorescence protein, cDNA complementary DNA, ORFs open reading frames, IL4 interleukin 4, BCR B cell receptor, ANOVA analysis of variance.