DIABLO is a potential negative regulator of NK cell-mediated killing

DIABLO is a mitochondrial protein that promotes apoptosis by binding inhibitors of apoptosis proteins (Verhagen et al., 2000). Taken that the expression levels of DIABLO showed highest inverse correlation with NKiToxMel (Table 21), the role of DIABLO in melanoma susceptibility to NK cell killing was investigated in more detail.

To examine the expression of DIABLO in melanoma cell lines, the initial ‘prediction model cell panel’ melanoma cell panel was analysed using immunoblot analyses. In Figure 20, the protein abundance of DIABLO in 8 different melanoma cell lines (training panel) is shown. The expression was normalised to the reference protein calnexin. Similar to the RPPA data, the expression of DIABLO decreased with increasing susceptibility to NK cells. For example, WM88 cells expressed double the amount of DIABLO (0.77) in comparison to 1205Lu (0.38).

Figure 20. DIABLO abundance in melanoma correlates with their susceptibility to NK cells(A) Representative immunoblot. (B) Mean and SEM of five analysed immunoblots under specification of the protein expression normalised to the loading control Calnexin. The coloured dots correspond to each cell line presented in (A) in order of appearance from left to right.

After validation of the endogenous protein abundance, the expression of DIABLO was genetically altered to examine if and how DIABLO abundance affects NKiToxMel. Transient overexpression of a gene of interest with a plasmid construct is an established approach to investigate a functional role of a given protein. The use of a GFP labelled plasmids allows an easy validation of transfection efficiency. Hence, melanoma cells were transfected with pmax GFP, a plasmid with high transfection efficiency and GFP signal intensity. After 24 h, the targets cells showed a strong GFP signal and were used in our future cytotoxic assays. Because it was possible that GFP may interfere with the fluorescence signals from calcein-AM also used in the cytotoxic assay, we tested the possible interference. As seen in Figure 21 (A+B), pmax GFP transfected

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Calnexin DIABLO

R² = 0.57

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DIABLO abundancenormalisedto referenceprotein

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WM88 WM1366 WM9 WM983B WM3734 451Lu WM3918 1205Lu

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and non-transfected WM88 cells (A) and WM47 (B) show almost identical killing curves.

Therefore, the interference of the much weaker GFP signals in the calcein-AM based cytotoxic assay could be excluded.

Figure 21. GFP signal does not interfere with calcein-AM detection. WM88 (A) and WM47 (B) were transfected with pmax GFP. After 24 h transfected as well as melanoma cells were challenged with one primary NK cell donor (A) and NK-92 cell line (B) in a 4 h real-time killing assay.

The 1205Lu line has one of the lowest DIABLO expression and was thus used to test if and how DIABLO-GFP overexpression affects NKiToxMel. The GFP construct allowed monitoring of the DIABLO expression and determination of the harvesting time after transfection. The overexpression of DIABLO after 24 h of the performed transfection showed an almost 30-fold increased expression of DIABLO-GFP (Figure 22 A-B). Notably, in the cells overexpressing DIABLO-GFP, the endogenous expression of DIABLO was reduced by about 20 %. However, despite the validated overexpression, the susceptibility of 1205Lu was not influenced by the higher protein abundance of DIABLO. As seen in Figure 22 C, the killing curve of DIABLO-GFP transfected 1205Lu cells was almost congruent with the killing curve of the peDIABLO-GFPC1 control. Both curves showed an initial killing of ~30 % (Figure 22 D) that increased to a value of about 70 % after 4 hours (Figure 22 E).

Figure 22. Overexpression of DIABLO does not change susceptibility of melanoma towards NK cells. 1205Lu cells were transfected with a DIABLO-GFP construct or with the control plasmid peGFPC1. After 24 h cells were harvested and the DIABLO expression was analysed. (A) Representative immunoblot (B) The band intensities of 3 immunoblots were quantified with Image Studio^TM Lite Software. The band intensities of DIABLO (DIABLO-GFP; endogenous DIABLO) were normalised to the band intensities of the loading control Calnexin. (C-E) The susceptibility of DIABLO overexpressing 1205Lu cells were determined in 4 h real-time killing assays. 1205Lu cells transfected with DIABLO-GFP or with the empty plasmid peGFPC1 were exposed to primary NK cells (n=6) in E:T ratio of 5:1. (C) Killing kinetics of 4 h cytotoxic assay. (D) Killing after 60 min and (E) killing after 240 min. All bars and graphs indicate means ± SEM.

Given the fact that DIABLO interacts with other proteins such as XIAP, increasing levels of DIABLO alone, without changing expression levels of its interaction partners might not affect the NKiToxMel. That is why the downregulation of DIABLO was tested next. For this purpose, the WM3734 cells that showed higher expression of DIABLO were transfected with two different siRNAs (#1, #2). One day after the initial transfection, the transfection procedure was repeated. After additional 24 h, WM3734 cells were harvested and further analysed. As shown in Figure 23 A and B, the protein abundance of DIABLO after 24 h of double transfection was reduced by 22 % (#2) and 41 % (#1). The transfection with DIABLO #2 was not sufficient to alter the NKiToxMel whereas DIABLO #1 siRNA slightly increased the susceptibility of WM3734 to primary NK cells (Figure 23 C). The initial killing (Figure 23 D) could be significantly increased from 47 % to 53 % after DIABLO knockdown. This increased susceptibility to NK cells of about 6 % was also detectable 4 h after the NK cell treatment (68 % to 74 %) (Figure 23 E).

Figure 23. DIABLO knockdown slightly increases susceptibility of melanoma towards NK cells.

WM3734 cells were transfected with non-silencing RNA or with two different siRNA (#1 and #2) targeting DIABLO. The next day, the procedure was repeated and WM3734 cells were harvested and further analysed after additional 24 h. The DIABLO expression of WM3734 after DIABLO knockdown was analysed. (A) Representative immunoblot. (B) The band intensities of 4 immunoblots were quantified with Image Studio^TM Lite Software. The band intensities of DIABLO were normalised to the band intensities of the loading control Calnexin. (C-E) The susceptibility of WM3734 after DIABLO knockdown was determined in 4 h real-time killing assays. Target cells were exposed to primary NK cells (n=4) in E:T ratio of 5:1. (C) Killing kinetics of 4 h cytotoxic assay. (D) Killing after 60 min and (E) killing after 240 min. All bars and graphs indicate means ± SEM. Statistical significance of unpaired (B) or paired (D), two-tailed Student's t‐test is indicated with * for p<0.05 and ** for p<0.01

Although the abundance of DIABLO showed the expected heterogeneity in the melanoma cell lines and was even associated with their susceptibility to NK cells (Figure 20), short-term altered expression level of DIABLO did not resulted in dramatic changes in NKiToxMel. These data do not exclude the potential role of DIABLO in susceptibility of melanoma to NK cell killing.

Longer overexpression times or the stable knockdown of DIABLO might be needed to alter NKiToxMel more efficiently.