Induction of synthetic lethality in USP22-deficient CRC cells

Based on these findings we assumed that the residual HSP90 levels are essential for the survival and stress-resistance of siUsp22 cells. We therefore hypothesized that diminishing HSP90 completely would result in decreased cell survival. Moreover, since targeting USP22 directly could have tumor-promoting outcomes, our proposed strategy would reflect an attractive targeting approach based on the concept of synthetic lethality. Thus, HCT116 cells were treated with the HSP90 inhibitor Ganetespib under wild type and USP22 knockdown conditions 48 h after siRNA transfection. To determine the general sensitivity towards this inhibitor, cells were treated with increasing Ganetespib concentrations for 48 h.

Figure 55: USP22-depleted cells are more sensitive to the HSP90 inhibitor Ganetespib.

48 h after siRNA transfection, siControl and siUsp22 HCT116 cells were treated with increasing concentrations of the HSP90 inhibitor Ganetespib for 48 h. Surviving cells were visualized by crystal violet staining. USP22-depleted cells showed increased sensitivity towards this drug.

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Subsequently, surviving cells were stained with crystal violet. Indeed, we observed that USP22-depleted cells were more sensitive towards this inhibitor (Figure 55). While only few siUsp22 cells could withstand treatment with 333 nM Ganetespib, siControl cells could tolerate 10-fold higher concentrations.

Consequently, we aimed to confirm our findings by testing for apoptosis markers on protein levels upon USP22 loss and/or HSP90 inhibition. USP22 wild type and -depleted cells were treated with 100 nM Ganetespib or DMSO for 48 h. Protein lysates were subjected to western blot analysis. In conformity with our hypothesis, in siUsp22 cells treated with Ganetespib increased levels of the apoptosis markers cleaved PARP and BIM were observed (Figure 56).

In summary, these findings strongly support the potential of USP22-deficient cells to be targeted by HSP90 inhibitors based on the concept of synthetic lethality.

Figure 56: Ganetespib treatment increases apoptosis levels in USP22-depleted cells. 48 h after knockdown, HCT116 cells (siControl and siUsp22) have been treated either with DMSO or 100 nM Ganetespib for 48 h. Protein lysates were evaluated by western blot in triplicates.

USP22-depleted cells show an increased abundance of the apoptosis markers cleaved PARP (lower PARP band) and BIM upon treatment with the HSP90 inhibitor Ganetespib.

We further assumed that HSP90 inhibition interferes with its function in facilitating P-TEFb complex formation which therefore cannot be recruited by BRD4 to phosphorylate the RNA Pol

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II. To target another player in this process we treated cells with the BET inhibitor JQ1 which targets among others BRD4. Similar to the previous approach, 48 h after siRNA transfection, we treated siControl and siUsp22 HCT116 cells with increasing concentrations of the inhibitor for 48 h. Indeed, control cells could tolerate 250 nM JQ1 while most of the USP22-depleted cells could maximally survive a dose of 100 nM (Figure 57A).

Figure 57: Loss of USP22 elevates sensitivity of CRC cells towards JQ1. 48 h after HCT116 cells were transfected with control or anti-USP22 siRNAs, cells were treated with increasing concentrations of JQ1 for 48 h. (A) A crystal violet-based assay revealed increased sensitivity towards JQ1 upon knockdown of USP22. (B) Using the Celigo^® device the IC50 was determined in these cells. USP22-depleted cells were characterized by a lower IC50 (47 nM) than the controls (224 nM).

To obtain further insights into the sensitivity of siUsp22 cells towards JQ1 treatment, we calculated the IC50 based on Celigo^® proliferation measurements. This experiment revealed

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that the IC50 of cells with USP22 wild type levels was 224 nM and of USP22-depleted cells only 47 nM (Figure 57B). Taken together, CRC cells with low USP22 levels can be specifically targeted by treatment with Ganetespib or the BET inhibitor JQ1.

In our aforementioned approaches, we tested the effects of transient USP22 loss in vitro in several human CRC cell lines by siRNA-mediated knockdown. However, this silencing is only transient and therefore only insufficiently reflects the in vivo situation of USP22 depletion. Thus, we decided to take advantage of the CRISPR/Cas9 gene editing technology to permanently delete the USP22 gene in HTC116 cells. The use of this construct allowed expression of a sgRNA sequence by the U6-promoter and at the same time expression of the Cas9 enzyme necessary for gene editing. For rapid selection of positively transfected cells, the plasmid contained a coding sequence for the Green Fluorescent Protein (GFP).We simultaneously transfected HCT116 cells with two constructs targeting intronic sequences flanking exon 3 to exon 5 of USP22, thereby allowing the excision of this segment. This generated a frameshift with as consequence deletion of functional USP22 gene (Figure 58).

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After 48 h, we evaluated the transfection efficiency by detecting GFP-mediated fluorescence emitted by positively transfected cells (Figure 59A). We selected GFP-positive cells by fluorescence activated cell sorting (FACS) with the help of Sabrina Becker (Cell-sorting technology platform, Department of Haematology and Medical Oncology, UMG). Remarkably, with 32% fluorescent and 3% highly fluorescent cells, the transfection efficiency of HTC116 cells was relatively high (Figure 59B). We decided to culture only highly GFP-positive cells (channel P4) as single cells in 96-well plates.

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Figure 59: Fluorescent cells indicate the presence of the GFP-containing CRISPR/Cas9 constructs targeting USP22. (A) 48 hours after transfection GFP-positive cells were detected.

Scale bar: 1,000 µm. (B) Highly fluorescent cells (gate P4) were sorted as single cells by FACS.

After approximately four weeks of culturing and propagating single cells, several clones were tested for their USP22 levels. Two cell clones (#1, #2) did not show any residual USP22 protein (Figure 60A). As observed after siRNA-mediated knockdown, the loss of USP22 did not result in morphological alterations in HCT116 cells (Figure 60B). In addition, proliferation rates were assessed and again we could confirm previously obtained results in which reduced USP22 expression leads to accelerated cell growth in HCT116 cells (Figure 60C). Finally, we

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confirmed the sensitivity of USP22-depleted cells towards the HSP90 inhibitor Ganetespib (Figure 60D). Together, the effects of USP22 loss were reproduced in another cell system which represents a powerful tool to further investigate the consequences of a permanent USP22 depletion in human CRC cells.

Figure 60: CRISPR/Cas9-mediated USP22 knockout leads to increased proliferation in HCT116 cells. (A) The loss of USP22 on protein level was confirmed in CRISPR/Cas9 clones

#1 and #2. (B) As observed after siRNA-mediated knockdown the loss of USP22 does not affect the morphology of HCT116 cells. Scale bar: 100 µm. (C) Reduced USP22 expression results in increased proliferation compared to USP22 wild type levels (parental and CRISPR/Cas9 clone #3 cells). Mean ± SD, Student's t-test, n=3. (D) The sensitivity towards Ganetespib was confirmed in these USP22-depleted cells.

Together, in this project we demonstrated higher inflammation and tumor burden in mice with an intestinal deletion of Usp22. These findings were supported by data available in public databases in which a heterogeneous expression of USP22 was revealed in CRC patients.

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Accordingly, human CRC cell lines showed heterogeneous USP22 levels and reacted differently after USP22 knockdown with regards to proliferation and migration properties.

mRNA-seq analyses indicated an involvement of USP22-regulated genes in proliferation- and differentiation-related processes as well as response to extracellular stimuli. Accordingly, we detected the downregulation of the heat shock protein HSP90AB1 upon USP22 depletion in vitro. Interestingly, these cells showed increased sensitivity towards temperature changes and could be targeted by HSP90 and BET inhibitors based in the concept of synthetic lethality.

Moreover, CRISPR/Cas9 cells with a USP22 knockout were generated which displayed high sensitivity towards HSP90 inhibition as well. These findings suggest a tumor suppressive function of USP22 and that low USP22 levels in CRC cells could be exploited by targeting these cells with specific inhibitors.

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