ALBERT

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.

Description: Mantle cell lymphoma (MCL) is a distinct lymphoma subtype representing 6-8% of non-Hodgkin's lymphoma (NHL). Although with current standard therapy high initial response rates can be achieved, early relapses and rapid disease progression determine the clinical course of most MCL patients. Recently, Bruton´s tyrosine Kinase (BTK) inhibitors have been introduced with highly promising clinical activity. Nevertheless, interindividual responsiveness is heterogenous and primary and secondary resistance has been reported. However, molecular mechanisms driving resistance to BTK inhibition are not well understood yet. Among other factors, interactions between the tumor and its microenvironment have been proposed to play an important role in response to targeted therapy. In this study, we investigated the influence of tumor cell interaction with its microenvironment on sensitivity to the BTK inhibitor CC292 in vitro. MCL cell lines JeKo-1, Z-138 and Granta-519 were treated with 5 µM of CC292 alone or in co-culture with human bone marrow stromal cells (HS-5) and cell death induction and proliferation were assessed. Expression of proteins involved in BCR signaling and other tumor-promoting pathways was analyzed by Western Blot. Co-cultured MCL cells settled within the stromal cell layer were separated using MACS Feeder removal microbeads prior to Western Blot analysis. In all cell lines, direct interaction with the microenvironment markedly reduced sensitivity towards CC292 treatment (by 22% (JeKo-1), 33% (Granta) and 64 % (Z-138)). Importantly, cell-cell contact was shown to play a crucial role for mediating resistance to CC292 as only those MCL cells settled within the stromal cell layer proved to be significantly less vulnerable to the inhibitor compared to MCL cells co-cultured with HS-5 but separated by a transwell insert. Western Blot analysis showed a reduction of protein levels of phBTK upon treatment with CC292 in both, mono- and co-cultured cells. Interestingly, direct interaction of MCL cells with the microenvironment strongly induced protein expression of phAkt. Accordingly, phosphorylation (inactivation) of the pro-apoptotic FoxO1, a downstream-target of phAkt, was increased and its translocation to the nucleus was decreased in those cells. We could show that the effect of microenvironment interaction on sensitivity towards CC292 is mediated by Akt as knockdown of Akt using siRNA restored sensitivity to the drug. Furthermore, co-treatment of MCL cells with CC292 and the specific Akt inhibitor MK-2206 hampered upregluation of phAkt in co-cultivated cells and prevented Akt-mediated sequestration of FoxO1 in the cytoplasm, resulting in translocation of FoxO1 to the nucleus. Thus, combination with MK-2206 could significantly overcome microenvironment-mediated protection from growth inhibition and apoptosis induction upon CC292 treatment. Moreover, combination of the BTK inhibitor CC292 and the Akt inhibitor MK-2206 proved to act synergistically in MCL cells in all dose combinations tested (Combination index 0,73-0,93 in Z-138; 0,47-0,78 in JeKo-1). Taken together, cell-cell-interaction of MCL cells with their microenvironment protected them from CC292-induced cell death. This effect was mediated by increased phAkt expression resulting in inhibition of pro-apoptotic signaling and could effectively be overcome by combination with the specific Akt inhibitor MK-2206. Furthermore, CC292 and MK-2206 acted synergistically in MCL cells. Our results indicate that co-targeting the PI3K/Akt-pathway might be a promising strategy to overcome resistance to BTK inhibition mediated by interaction with the microenvironment. Disclosures Dreyling: Sandoz: Other: Scientific advisory board; Roche: Other: Scientific advisory board, Research Funding, Speakers Bureau; Novartis: Other: Scientific advisory board; Mundipharma: Other: Scientific advisory board, Research Funding; Janssen: Other: Scientific advisory board, Research Funding, Speakers Bureau; Gilead: Other: Scientific advisory board, Speakers Bureau; Celgene: Other: Scientific advisory board, Research Funding, Speakers Bureau; Bayer: Other: Scientific advisory board, Speakers Bureau; Acerta: Other: Scientific advisory board.