The variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of tumor cells and complex interactions within the tumor microenvironment (TME). IL-4 producing follicular helper T cells (TFH) are critical components of the FL TME. Binding of IL-4 to IL-4R on FL cells activates JAK/STAT signaling. We identified STAT6 mutations (STAT6MUT) in 13% of FL (N = 33/258), all clustered within the DNA binding domain. Gene expression data and immunohistochemistry showed upregulation of IL-4/STAT6 target genes in STAT6MUT FL, including CCL17, CCL22, and FCER2 (CD23). Functionally, STAT6MUT was gain-of-function by serial replating phenotype in pre-B CFU assays. Expression of STAT6MUT enhanced IL-4 induced FCER2/CD23, CCL17 and CCL22 expression and was associated with nuclear accumulation of pSTAT6. RNA sequencing identified PARP14 -a transcriptional switch and co-activator of STAT6- among the top differentially upregulated genes in IL-4 stimulated STAT6MUT lymphoma cells and in STAT6MUT primary FL cells. Quantitative chromatin immunoprecipitation (qChIP) demonstrated binding of STAT6MUT but not STAT6WT to the PARP14 promotor. Reporter assays showed increased IL-4 induced transactivation activity of STAT6MUT at the PARP14 promotor, suggesting a self-reinforcing regulatory circuit. Knock-down of PARP14 or PARP-inhibition abrogated the STAT6MUT gain-of-function phenotype. Thus, our results identify PARP14 as a novel therapeutic target in STAT6MUT FL.
Chronic liver diseases are associated with excessive deposition of extracellular matrix proteins. This so-called fibrosis can progress to cirrhosis and impair vital functions of the liver. We examined whether the receptor tyrosine kinase (RTK) class III inhibitor Crenolanib affects the behavior of hepatic stellate cells (HSC) involved in fibrogenesis. Rats were treated with thioacetamide (TAA) for 18 weeks to trigger fibrosis. After TAA treatment, the animals received Crenolanib for two weeks, which significantly improved recovery from liver fibrosis. Because Crenolanib predominantly inhibits the RTK platelet-derived growth factor receptor-β, impaired HSC proliferation might be responsible for this beneficial effect. Interestingly, blocking of RTK signaling by Crenolanib not only hindered HSC proliferation but also triggered their specification into hepatic endoderm. Endodermal specification was mediated by p38 mitogen-activated kinase (p38 MAPK) and c-Jun-activated kinase (JNK) signaling; however, this process remained incomplete, and the HSC accumulated lipids. JNK activation was induced by stress response-associated inositol-requiring enzyme-1α (IRE1α) in response to Crenolanib treatment, whereas β-catenin-dependent WNT signaling was able to counteract this process. In conclusion, the Crenolanib-mediated inhibition of RTK impeded HSC proliferation and triggered stress responses, initiating developmental processes in HSC that might have contributed to improved recovery from liver fibrosis in TAA-treated rats.
1.The steady state kinetics of the Mgz+-dependent ATPase of beef brain microsomes are assayed by means of a rapid photometric method. Omitting K+ from the reaction medium, the Na+,K+-Mga+-dependent ATPase is not active.2. The MgZ+-dependent ATPase is also active with Ca2+ and inactive in the absence of Mg2+ or Ca2+ when 40 mM Na+ is present. Hence the name "Mg2+-(Ca2+)-ATPase" is proposed.3. The complexes MgATP2-and CaATP2-are evidently the true substrates of the ATPase. MgATP2-is split with a 2.2-fold higher velocity than CaATP2-.4. The MgZ+-(Caz+)-ATPase is inhibited competitively by an excess of free Mg2+-or Ca2+-ions and probably uncompetitively by an excess of free ATP4-.5. The reaction rate of the Mg2+-(Ca2+)-ATPase as a function of the concentrations of substrate, Mg2+-resp. CaZ+-ions and ATP4-can be expressed as On account of the magnitude of the reaction velocity constants k,, k-, and k,, the Michaelisconstant seems to approximate Iczlk, for both substrates.
Chemotherapy combined with anti-CD20 antibodies is the standard treatment for patients with symptomatic advanced stage follicular lymphoma (FL). We have previously shown that EZH2 gene mutations were predictive of differential efficacy of the chemotherapy backbone within the GALLIUM trial (NCT01332968; Jurinovic, ASH 2019). Specifically, patients with EZH2 mutant FL had significantly longer progression free survival with CHOP-based immunochemotherapies as compared to patients with EZH2 wild type FL. In contrast, the EZH2 mutation status was not predictive of treatment outcome in patients receiving bendamustine-based regimens. The underlying biology is unclear. Mutations in EZH2, a histone methyltransferase, occur in 25-30% of FL and mostly affect the Y641 residue within the catalytic domain, resulting in more efficient conversion of H3K27me2 to H3K27me3. First, we tested differences in chemosensitivity in lymphoma cell lines that harbor the FL-hallmark translocation t(14;18)[BCL2/IGH] and intrinsic EZH2 mutations (Karpas422, OCI-Ly1, SU-DHL4, DB) or no EZH2 mutation (SU-DHL16, WSU-FSCCL, OCI-Ly8, OCI-Ly19). Treatment with CHOP (4-hydroperoxycyclophosphamide (4-HC), doxorubicin, vincristine, and prednisone), alone or in combination, or bendamustine revealed marked differences in IC50 for cell viability (CellTiter Glo) and apoptosis (Annexin V) between cell lines, but no correlation with EZH2 mutation status. To better control for cell line specific effects, we stably expressed EZH2 Y641N or wild type (WT) in the EZH2 WT cell line SU-DHL16. EZH2 mutant cells indeed showed significantly increased H3K27me3 levels compared to EZH2 WT cells. However, we did not observe differences in global cellular phenotypes, including cell proliferation and cell cycle phases. Furthermore, IC50 for cell viability and apoptosis with CHOP and bendamustine treatment were not significantly different. As we could not identify differences in direct cytotoxic responses, we hypothesized that EZH2 mutations might indirectly affect treatment efficacy, by altering the interaction of FL cells with their tumor microenvironment (TME) in response to chemotherapy. In a mouse model, Ennishi et al. had previously shown that Ezh2 mutant lymphomas have reduced MHC expression and T-cell infiltrates (Cancer Discovery, 2019). Here, we could show that expression of mutant EZH2 in human SU-DHL16 cells also leads to almost complete MHC-I loss by flow cytometry (Fig A). MHC-I loss was fully reversible when cells were treated with increasing doses of tazemetostat, a specific EZH2 inhibitor, or interferon-gamma. Interestingly, treatment with 4-HC, prednisone, doxorubicin and CHOP also resulted in increasing restoration of MHC-I expression in EZH2 mutant cells, while bendamustine (and vincristine alone) had no impact on MHC expression (Fig B,C). Next, we used a fully human B-cell co-culture model (modified from Caeser et al., Nat Comm 2019) for validation and functional studies. Mirroring the TME-dependence of FL, germinal-center (GC) B-cells from human tonsils immortalized by transduction with BCL2 and MYC absolutely require follicular dendritic cell (FDC) support plus IL21 and CD40L for sustained growth. Stable expression of EZH2 Y641N in these GC-B-cells led to increased H3K27me3 levels as compared to EZH2 WT and empty vector (ev) controls, but did not result in FDC+IL21/CD40L independent growth. Again, we observed significantly lower MHC-I/II expression on EZH2 mutant cells (Fig D). Importantly, we show that EZH2 mutation-induced MHC loss resulted in reduced CytoStim-stimulated conjugate formation when cells were co-cultured with autologous CD4 T-cells isolated from the same tonsils (Fig E). Finally, treatment with doxorubicin but not bendamustine resulted in significant upregulation of MHC-I/II on EZH2 mutant GC-B cells (Fig F). Co-culture experiments with autologous CD4 and CD8 T-cells with and without doxorubicin, CHOP and bendamustine treatment are ongoing to analyze the EZH2 mutation-specific chemotherapy effects on T-cell activation, recruitment, and T-cell mediated killing. In conclusion, our data indicates that the particular chemosensitivity of EZH2 mutant FL to CHOP is not the result of differences in direct cytotoxicity, but -unlike bendamustine- is rather mediated by restoring EZH2 mutation-induced MHC-I/II loss, thereby potentially promoting cytotoxic T-cell responses. Figure 1 Figure 1. Disclosures Hodson: Astra Zeneca: Research Funding. von Bergwelt: Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau. Subklewe: MorphoSys: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Janssen: Consultancy; Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau. Weigert: Janssen: Speakers Bureau; Roche: Research Funding; Epizyme: Membership on an entity's Board of Directors or advisory committees.
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