Differential usage of GOT2 derived metabolites in B cell lymphoma cell lines 82

or when Jak/STAT and NF-κB signaling is activated in a way were global gene epression is affecting both cell cycle regulators but also metabolic enzymes. Therefore, one question was, whether in lymphoma cell lines a comparable dependency between Myc or Jak/STAT and NF-kB , glutamine and proliferation can be observed. CA-46 BL cells, OCI-LY3 DLBCL and L428 HL cells were used.Cells were grown for 48 h in glutamine free media and cell doubling was defined. As shown in Figure 39 the cell doubling (and thus proliferation) in all three cell lines was reduced by 50 % after glutamine withdrawal, confirming the importance of glu-tamine metabolism in B cell proliferation. In previous chapters clear evidence was provided that the inhibitor AOA affecting aminotransferase reduces both, Myc or IL10+CpG

stimu-Figure 39: Proliferation of B cell lymphoma cell lines is glutamine dependent.

Cell number of CA-46, OCI-Ly3 and L-428 cells 48 h after seeding in glutamine media relative to glutamine containing media (+Gln). Mean and SD of three independent experiments are shown.

lation, driven proliferation through interrupting metabolic support of cell cycle progression.

Therefore, CA46, OCI-LY3 and L-428 cells were treaed with AOA (Figure 40). A strong reduction in cell doubling as for Glu deprivation was observed and was also comparable to the reduction of proliferation by AOA treatment in P493-6 cells (Figure 40A). Importantly, when adding aspartate or adenine to AOA treated CA-46 cells a twofold increase in the relative cell number was detected. In contrast in OCI-Ly3 and L-428 cells aspartate and to some extend also adenine was rescuing proliferation of AOA treated cells (Figure 40). 90 % of OCI-Ly3 proliferation could be rescued by aspartate and over 60 % by adenine addition (Figure 40B). L-428 cells could nearly be rescued from AOA induced proliferation block by aspartate or adenine addition (both > 80 %, Figure 40C).

As GOT2 was important for aspartate synthesis and proliferation in P493-6 cells (Figure 34, Figure 36), its role in the other cell lines was further analyzed. The GOT2 knockdown of about 40 % resulted in a reduced proliferaiton in all three lymphoma cells lines at about 20-35 %(Figure 41B). Importantly, in CA-46 cells the GOT2 mediated proliferation was rescued by aKG, while the reduced cell proliferation under lower GOT2 gene expression in L-428 and OCI-LY3 cells was reverted by adding aspartate or adenine.

Figure 41: Pathway dependent usage of GOT2 derived metabolites in lymphoma cells.

(A) Immunoblot of GOT2 in CA-46, OCI-Ly3 and L-428 cells 24 h after siRNA mediated knockdown.

Knockdown efficiencies (GOT2/tubulin) relative to scrb control are shown under the images. (B) Relative cell number of CA-46, OCI-LY3 and L-428 cells after GOT2 knockdown and metabolite treatment. 24 h after transfection cells were seeded in fresh media as indicated 1 mMα-ketogluterate (aKG), 10 mM aspartate (asp) or 100µM nucleotide bases adenine (A) and thymine (T) were added to the media and cell number was calculated after 48 h. Mean and SD of cell numbers relative to non-target siRNA (scrb) are shown. All experiments were performed in triplicates.

Summing these data, a comparable dependency on glutamine for aKG synthesis for pro-liferation could be shown for CA-46 cells as well as P493-6 Myc^highcells. Also OCI-Ly3 cells showed a comparable dependency on glutamine for aspartate and nucleotide synthe-sis as IL10+CpG stimulated P493-6 Myc^lowcells. However, L-428 cells showed an equal dependency on aspartate and nucleotides for proliferation as OCI-Ly3. This could be ex-plained by the well known geregulated Jak/STAT and Nf-kB pathways in both cell lines, although mainly for HL cells a more comprehensive analysis fo involved pathways is nec-cessary. Importantly, GOT2 was identified as a central player also in lymphoma cell lines in vitro. Furthermore, either aKG or aspartate and nucleotides can rescue aminotransferase deficiency in this cells depending on the signaling network active in the corresponding cell line mediated either by Myc or NF-κB/STAT3.

3.7.3 GOT2 expression is upregulated in Burkitt and ABC DLBCL patients

To answer the question about GOT2 gene expression in primary lymphoma samples mi-croarray based GE data from Oncomine were used (Rhodes et al., 2004)(Figure 42). In a dataset published by Basso et al. (2005) increased expression of GOT2 in Burkitt (n = 17, p = 4.9E-9) and DLBCL (n = 32, p = 7.6E-7) samples compared to normal B cell controls was observed (Figure 42A). Interestingly highest expression of GOT2 in normal controls

was detected in proliferting centroblast. A higher expression of GOT2 was also observed in HL, however, in this dataset only data from three different cell lines were included. There-fore, a second dataset from Brune et al.(2008) was analyzed (Figure 42B). In this dataset a higher expression ofGOT2 was observed for in BL and DLBCL in comparison to pre-GC B cells and memory B cells but not normal circulating B cells. Thereby the latter again were characterized by the highest expression in control cells. Notably, expression of GOT2 in both subgroups of HL was less pronounced as in the Basso data set. Therefore, it is stated that GOT2 seems to be highly expressed in proliferating B cells or aggressive NHL but also in subsets of HL which needs to be further defined.

In additon to different expression between normal B cells and lymphoma, it is obvious that the different lymphoma subtypes and most notably DLBCL are heterogenic in their GOT2 expression. Therefore it was suggested that further molecular subgroups with differentGOT2 expression exist. As described in the introduction DLBCL can be disitinguished in so called ABC-like and GCB like DLBCL. Expression analysis of primary lymphoma with ABC-like and GCB-like DLBCL subgrouping but also clinical data for R-CHOP treated DLBCl pa-tients are only available from Lenz et al. (2008) which was analyzed using the LYMMML web based analysis tool (http://lymmml.ur.de/). Within this DLBLC cohort 93 ABC-like and 117 GCB-like DLBCL cases but also 33 unclassified DLBCL were analyzed. A higher expression of GOT2 in ABC patients compared to GCB or unclassified DLBCL was ob-served. Importantly, GOT1 was not differential expressed in any of these datasets (data not shown). The high expression of GOT2 in ABC-DLBCL underlines the important role for GOT2 in OCI-Ly3 cells, as OCI-Ly3 cells are molecular classified as ABC-like DLBCL.

Figure 42: High expression of GOT2 in BL and ABC DLBCL patients.

(A+B) Microarray gene expression data ofGOT2 from Bassoet al.(2005) (A) and Bruneet al.(2008) (B) taken from oncomine.org. Expression of normal B cell controls (1 = naive pre-germinal B cell, 2 = B

lympho-Importantly, it also connects ABC DLBCL to the observed STAT3/NF-κB regulation of GOT2 in IL10+CpG stimulated P493-6 as ABC DLBCL are defined by aberrant activated STAT3 and NF-κB signaling (see introduction).

Next it was analyzed, whether high GOT2 expression is also prognostic in DLBCL. This is most likely as in a number of studies for ABC-like DLBCL are worse clincical outcome has been shown (Lenz et al., 2008).