Inhibition of protein de-novo synthesis does not affect the MIR23A activation upon BCR stimulationupon BCR stimulation

To test whether the MIR23A cluster is induced by direct downstream signaling, the U2932 R1 cells were treated with cyclohexamide (CHX) simultaneously to BCR stimulation. CHX inhibits protein de-novo synthesis by blocking translation elongation of the ribosome. Under this conditions only transcription factors, that were activated by direct upstream signaling, but not by secondary effects, are translocated into the nucleus and can induce MIR23A cluster expression. In order to control effective CHX treatment, the c-MYC protein levels were an-alyzed. C-MYC has a high protein turnover rate, being rapidly degraded by the proteasome (Gregory and Hann, 2000). Thus, it functions perfectly as an indicator for effective ribosomal inhibition.

As depicted in fig. 3.7 a) the MIR23A levels (pri-miR-23a, pre-miR-23a and pre-miR-27a) can still be activated upon BCR stimulation with anti-IgM F(ab)2-fragment, when protein de-novo synthesis is effectively blocked by CHX treatment. Notably, c-MYC accumulates upon BCR stimulation in U2932 R1 fig. 3.7 b), hence it was further analyzed as a potential transcription factor responsible for MIR23A regulation in section 3.1.3.6.

In summary, no protein de-novo synthesis is required for BCR dependent activation of the MIR23A cluster, indicating a direct activation of MIR23A promoter.

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Figure 3.7.: Inhibition of protein de-novo synthesis does not interfere with MIR23A activation upon BCR cross-link

U2932 R1 was pre-treated for 1 h with 100 μg/mL cyclohexamide (CHX) before stimulation with 13 μg/mL anti-IgM F(ab)2-fragment for 2 h. (a) qRT-PCR analyses of pri-miR-23a, pre-miR-23a and pre-miR-27a (b) Western blot analyses of c-MYC. GAPDH served as loading control. Mean with 95% CI, endogenous control: SNORD48for pre-miRNAs andGAPDHfor pri-miR, one representative experiment of three is shown.

3.1. IDENTIFICATION OF SIGNALING PATHWAYS REGULATING THE MIR23A CLUSTER 61 3.1.3.5. BTK/MEK/ERK signaling activates the MIR23A cluster

In order to narrow down which signaling cascade downstream of the BCR is responsible for activation of the MIR23A cluster in U2932 R1, the key enzymes BTK, MEK, PI3K and AKT, which mediate the downstream effects of BCR (section 1.2.1), were systematically inhibited by treatment with chemical inhibitors. These pre-treated cells were then stimulated with anti-IgM F(ab)2-fragment followed by MIR23A cluster expression analyses. NFκB signaling was excluded, because previous experiments showed no effect on MIR23A cluster expression when stimulating this pathway in BL (BL-2) or DLBCL (U2932 R1) cell lines (fig. 3.1.1 and 3.1.2).

Ibrutinib irreversibly inhibits the Burton tyrosine kinase (BTK) by covalent binding to Cys-481 in the ATP binding domain (Davids and Brown, 2014). BTK is one of the first proteins that is phos-phorylated upon antigen binding. Importantly, BTK plays a key role in BCR signaling, because it spreads the message by phosphorylation of PLCγ2 onto different downstream cascades: the ERK, NFκB and PI3/AKT signaling. As a consequence, BTK inhibition should impair all sig-naling cascades downstream of the B cell receptor. As a proof of principle it should no longer be possible to activate the MIR23A cluster, when treating the cells with Ibrutinib and anti-IgM F(ab)2-fragment simultaneously. This is exactly what could be observed when performing this experiment with U2932 R1 (fig. 3.8 a): The pre-miR-23a and pre-miR-27a levels are acti-vated upon anti-IgM F(ab)2-fragment stimulation as already previously observed. Treatment with Ibrutinib alone does not alter pre-miR-23a and pre-miR-27a expression levels. Further-more, the used ibrutinib concentration was not toxic to the cells, as shown in MTT assays (fig.

A.3). As expected, the MIR23A cluster is not activated upon double treatment with Ibrutinib and anti-IgM F(ab)2-fragment. As shown in fig. 3.8 b) and 3.9 b) Ibrutinib effectively inhibited phosphorylation of BTK and further downstream of P-ERK.

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Figure 3.8.: MIR23A cluster expression upon inhibition of key enzymes of BCR signaling U2932 R1 was pre-treated for 2 hours with the respective inhibitor (1 μM Ibrutinib, 200 nM BEZ235, 3 μM MK2206) before simultaneous stimulation with 13 μg/mL anti-IgM F(ab)2-fragment for 2h. Relative expression levels of (a) pre-miR-23a and pre-miR-27a were detected by qRT-PCR analyses. (Mean with 95% CI, endogenous control:

SNORD48) (b-c) Western blot analyses of BCR downstream kinases P-ERK/ERK and P-AKT/AKT. GAPDH served as loading control. One representative experiments of three is shown.

BEZ235 is an ATP-competitive PI3-kinase (p110α/γ/δ/β) and mTOR (p70S6K) inhibitor. PI3K phosphorylates PIP2 to make PIP3, which recruits PDK1 and AKT to the cell membrane, where AKT is activated by phosphorylation of mTORC2 and PDK1. Treatment with BEZ235 inhibits therefore effectively the PI3K/mTOR/AKT signaling (Barrettet al., 2012). Indeed, Western blot analyses show effective inhibition of P-AKT in U2932 R1 (fig. 3.8 c). The pre-miR-23a and pre-miR-27a levels are slightly increased upon treatment of U2932 R1 with BEZ235 alone, but can not be further increased when treated together with anti-IgM F(ab)2-fragment (fig. 3.8 a).

However, the error bars are high, making interpretations difficult. In order to interpret these data

3.1. IDENTIFICATION OF SIGNALING PATHWAYS REGULATING THE MIR23A CLUSTER 63

correctly, in a next experiment the cells were treated with an additional inhibitor specific for AKT:

MK2206. MK2206 allosterically inhibits auto-phosphorylation of T308 and S473 of AKT1, 2 and 3. It thereby prevents the AKT-dependent phosphorylation of numerous downstream targets.

Indeed, the control Western blot shows an effective inhibition of AKT phosphorylation (fig. 3.8 c). In contrast to BEZ235, MK2206 itself does not increase the pre-miR-23a or pre-miR-27a levels (fig. 3.8 a). However, double treatment with MK2206 and anti-IgM F(ab)2-fragment lead to an activation of the MIR23A cluster. Therefore, AKT signaling is not responsible for MIR23A cluster activation upon BCR stimulation in U2932 R1.

Next, the MAPK/ERK signaling was studied by inhibition of MEK1/2 with Trametinib in U2932 R1. Trametinib is a high specific inhibitor acting allosterically and reversible (Lugowskaet al., 2015). Non-toxic concentrations of trametinib were used for the following experiments (see MTT assays fig. A.3) Interestingly, Trametinib alone slightly downregulated pri-miR-23a, pre-miR-23a and pre-miR-27a levels (fig. 3.9 a). Furthermore, cells that were pre-treated with Trametinib were not able to activate the MIR23A cluster when simultaneously stimulated with anti-IgM F(ab)2-fragment. As a control, Western blot analyses (fig. 3.9 b) show that Trametinib effectively inhibited downstream ERK phosphorylation.

These observations indicate that BTK/MEK/ERK signaling is the BCR downstream cascade that is predominantly responsible for the MIR23A cluster activation in DLBCL model cell line U2932 R1.

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Figure 3.9.: Inhibition of BTK and MEK1/2 prevents MIR23A activation upon BCR stimulation in U2932 R1

DLBCL cell line U2932 R1 was pretreated with Ibrutinib (1 μM) and Trametinib (125 nM) for 2 hours, before BCR stimulation by 13 μg/mL anti-IgM F(ab)2-fragment. Protein samples were taken after 30 min and RNA samples after 2 h of stimulation. (a) qRT-PCR analyses of pri-miR-23a, pre-miR-23a and pre-miR-27a expression upon inhibitor/anti-IgM F(ab)2-fragment double treatment. (Mean with 95% CI, endogenous control:SNORD48for miR-NAs,GAPDHfor pri-miR-23a) (b and c) Western blot analyses verifying effective inhibition of P-BTK by Ibrutinib and P-ERK by Trametinib. GAPDH served as loading control. (Ibrutinib n=3, Trametinib n=2)