ALBERT

Description: Follicular Lymphoma (FL) is the second most common non-Hodgkin lymphoma and remains an incurable disease for the majority of patients who present with advanced stage disease. Virtually all patients subsequently acquire treatment resistance and a third of patients ultimately develop histologically transformed disease with aggressive clinical course and poor prognosis. Thus, there is an unmet clinical need to decipher the underlying biology of this disease, and identify dysregulated pathways that may serve as therapeutic targets. In a cohort of 258 FL specimens, we identified 33 cases with STAT6 mutations (12.8%), including 15 mutations at D419. Overall, 34 out of 35 (97.1%) of STAT6 mutations were within the DNA-binding domain. MutSigCV analysis (Lawrence, Nature 2013) indicated that STAT6 was significantly mutated in FL, i.e., more commonly mutated than expected based on background mutation rate. Previously, STAT6 mutations, including D419 hotspot mutations, have been reported in other lymphomas, including primary mediastinal B-cell lymphoma (Ritz et al., Blood 2009) and FL (Yildiz et al., Blood 2015). STAT6 proteins are transcription factors that are mainly activated by interleukin-4 (IL-4). A recent study identified a subset of IL-4 producing follicular helper T cells to be involved in the survival of FL B cells (Ame-Thomas et al., Blood 2015). We compared the expression levels of selected STAT6 target genes in FL patient samples with or without STAT6 mutations. Gene expression data (nCounter, Nanostring) was available for 138 patient samples with known STAT6 mutation status. Thereof, 13 cases had STAT6 mutations, including six at D419. Among the STAT6 target genes were CD23 (and its soluble form, sCD23) and SOCS1. Both showed significantly higher expression (P

Description: The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. The underlying molecular mechanisms and therapeutic vulnerabilities are not well understood. IL-4 producing follicular helper T cells (TFH) have been identified as a key component of the malignant B-cell niche. IL-4 activates paracrine signaling via STAT6. In a cohort of 258 patients with advanced stage FL, we identified STAT6 mutations in 13% of diagnostic biopsies (n=33). All mutations were clustered within the DNA binding domain, mostly at D419, and included a polymorphic variant (rs11172102). Gene set enrichment analysis (GSEA) revealed that STAT6 mutant cases were significantly enriched for two distinct IL-4 gene expression signatures. Gene expression data and immunohistochemistry of primary FL samples showed significant up-regulation of IL-4/STAT6 target genes in STAT6 mutant cases, including FCER2, which encodes for CD23. We stably expressed wild type STAT6 or mutant STAT6 (D419G, N421K, and D519V) in two B-cell lymphoma lines (OCI-Ly1, OCI-Ly8), both harboring the FL hallmark translocation t(14;18). Upon IL-4 stimulation, cells expressing mutant STAT6 had significantly increased FCER2 transcript levels. Similarly, IL-4 induced expression of membrane-bound as well as soluble CD23 was significantly increased in STAT6 mutant cells. Cells expressing mutant STAT6 showed significantly increased nuclear accumulation of pSTAT6 following IL-4 stimulation. Of note, we did not observe any effect of STAT6 mutations in the absence of IL-4. RNA sequencing of IL-4-stimulated lymphoma cell lines (STAT6 mutant versus wild type) identified PARP14 -a known transcriptional co-activator of STAT6- among the top differentially expressed genes. Bioinformatics and functional experiments demonstrated that PARP14 per se is a novel STAT6 target gene. Furthermore, reporter assays showed increased transactivation activity of mutant STAT6 at the PARP14 promotor, suggesting a regulatory feed-forward loop. Pharmacological inhibition of PARP and knock-down of PARP14 completely abrogated the mutant STAT6 gain-of-function phenotype. In summary, our results suggest that PARP14 is a novel target in STAT6 mutant FL. Our data also imply that the biological and clinical impact of STAT6 mutations will heavily depend on the (targetable) upstream activation of the IL-4 signaling cascade, including the abundance of IL-4 / TFH cells in the microenvironment of FL. Disclosures Richter: HTG Molecular Diagnostics, Inc.: Research Funding. Klapper:Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Hiddemann:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.