| dc.description.abstracteng | The interplay of different signaling pathways activated by factors of the cellular environment
and expression the proto-oncogene c-Myc (MYC) defines B cell fate in the germinal center
(GC). Importantly, the same signaling networks are chronically active in different types of
B-cell lymphoma. While the effects of aberrantly expressed MYC on cell proliferation and
metabolism were intensively studied, less is known about the cross talk of pathways, like
NF-kB and JAK/STAT signaling, in the context of B cell proliferation and metabolic reprogramming.
Therefore, the aim of the study was to analyze (i) how chronic active factors
of the GC microenvironment and their interaction networks can reprogram global gene expression
(gGE) and metabolism of resting B cells to support B cell proliferation, (ii) what is
common with MYC overexpression and (iii) whether this is important for lymphoma. Thus
the P493-6 B cell line, carrying a conditional MYC expression construct, was stimulated
with random combinations of GC derived factors as -IgM, CD40L, IGF1, CpG and IL10
to model different signaling pathway and Myc activation on the same genetic background.
Linear regression analysis of gGE and metabolome changes in stimulated Myc depleted cells
revealed qualitative similar but quantitative different changes on these parameters by the
different stimulations. Thereby, greatest changes where observed after IL10+CpG stimulation
due to a strong synergy on gGE and metabolome associated with sustained cell cycle
entry and cell proliferation. Using small molecule inhibitors and RNAi mediated knockdown
a dependency of IL10+CpG induced proliferation on NF-kB and JAK/STAT3 signaling was
revealed. Furthermore, simultaneous binding of STAT3 and p65 to proximal promoter of the
cell cycle regulator CDK4 but also the aspartate amino transaminase GOT2 was detected by
chromatin immunoprecipitation. The increase in gGE mediated by IL10+CpG stimulation
resembled Myc induced gene expression changes accompanied by a comparable cell proliferation
but different glutamine metabolism. Herein, GOT2 was essential for cell proliferation of
both conditions. However, glutamine tracing, respiration analysis and rescue experiments revealed
distinct dependencies of proliferation on glutamine derived metabolites: In IL10+CpG
treated cells a strong dependency of cell proliferation on glutamine derived aspartate and
nucleotides was observed, whereas in MYC overexpressing cells a-ketoglutarate was most
important for respiration and proliferation. Using CA-46, OCI-LY3 and L-428 lymphoma
cell lines the important role of GOT2 for Myc and STAT3/NF-kB dependent proliferation
but also the differences in glutamine usage in context of these pathways could be confirmed.
Last of all, a high expression of GOT2 in proliferating centroblasts but also primary B cell
lymphoma could be shown. Thereby, activated B cell like diffuse large B cell lymphoma
(DLBCL) were characterized by higher GOT2 expression as germinal center like DLBCL.
More importantly high GOT2 expression was associated with a worse clinical outcome of RCHOP
treated DLBCL patients. Overall, a new role for IL10R and TLR9 signaling and the
interaction of STAT3 and NF-kB signaling in gGE, proliferation and metabolism of B cells
was identified that might also be important for lymphomagenesis. | de |