*2.3. Inhibition of AKT Signaling Attenuates Repair of Dox-Induced DNA Damage*

Next, we performed alkaline-based DNA comet assay to examine whether enhanced apoptosis of cancer cells treated with Dox and MK-2206 was due to DNA damage which remained unrepairable in presence of AKT inhibitor. For this purpose, STS or GIST cells were initially treated with Dox for 2 h to induce DNA damage, and followed by the wash-out of chemotherapeutic agent and further cultured for 8 h in absence or presence of MK-2206. Strikingly, we observed a substantial increase of tail moment (TM) and olive tail moment (OTM) in GIST cells, thereby illustrating unrepairable DNA damage in AKT-inhibited tumor cells (Figure 3A,B). Similar data was obtained in GIST cells that were not washed from Dox. Again, we observed that MK-2206 substantially attenuated DNA damage repair which was evidenced by increased TM (Figure S5A,B). As expected, we observed a substantial increase of TM in Dox-treated cells when compared to the previous experimental settings, thereby illustrating the substantial DNA damage due to the permanent exposure to Dox. Given that alkaline-based DNA comet assay aimed to detect all the types of DNA lesions, we performed neutral version of single-cell electrophoresis to examine whether inhibition of AKT-signaling attenuates repair of Dox-induced DNA DSBs. Similar to alkaline version, we observed an increase of both TM and OTM in GIST cells treated with Dox in presence of MK-2206, thereby revealing that AKT-signaling plays an important role in regulating of repair of Dox-induced DNA lesions, including DNA DSBs (Figure S5C,D).

*2.2. Inhibition of AKT-Signaling Enhances Doxorubicin-Induced Apoptosis of STS and GIST* 

cells was specific and was not due to the changes in expression of the total AKT.

To further corroborate these findings, we examined whether inhibition of AKT signaling can potentiate pro-apoptotic effect of Dox in STS and GIST. For this purpose, cancer cell lines were treated with the low dose of Dox (0.25 g/mL) in absence (control) or presence of MK-2206 (5 M) and further subjected for western blotting to examine expression of apoptotic markers (cleaved forms of PARP and caspase-3). Strikingly, a substantial increase of cleaved caspase-3 and PARP was observed in the majority of STS and GIST cells treated with combination of AKT and Topo II inhibitors (Figure 2). As expected, MK-2206 used alone has no cytotoxic effect on the tumor cells, whereas pro-apoptotic effect of Dox used alone was much less when compared to the effect of combination of these inhibitors. The most sensitive for AKT inhibition were Dox-treated RD rhabdomyosarcoma cells (Figure 2A), U2-OS osteosarcoma (Figure 2B), GIST T-1R (Figure 2D) and 430 (Figure 2E) which was evidenced by a substantial cell death after the treatment with Dox and MK-2206. Quantification by mean pixel density revealed that PARP and caspase-3 cleavage was substantially increased in all types of cancer cell lines treated with Dox in combination of MK-2206 (Figure S3.1). The inhibitory effect of MK-2206 on AKT pathway in tumor cells was confirmed by a substantial decrease of AKT phosphorylation (Figure 2). Similarly, changes in phospho-AKT (Ser473) expression were quantified and normalized to actin (Figure S3.1), thereby illustrating that decreased AKT phosphorylation in MK-2206-inhibited

In addition to increased levels of cleaved caspase-3 and PARP in cancer cells treated with Dox in presence of AKT inhibitor, we also observed the increased numbers of apoptotic (e.g., Annexin Vpositive cells) cells in RD rhabdomyosarcoma and GIST T-1R cells treated with combination of Dox and MK-2206 when compared to non-treated cells or cells treated with Dox alone (Figure S4), thereby revealing that AKT inhibition effectively sensitized STS and GIST cells to Dox treatment and induced

**Figure 2.** AKT inhibition potentiates pro-apoptotic activity of Dox in STS and GIST. Immunoblot analysis for apoptosis markers (cleaved forms of poly-(ADP)-ribose-polymerase (PARP) and caspase-3) in RD rhabdomyosarcoma (**A**), U2-OS osteosarcoma (**B**), HT-1080 fibrosarcoma (**C**), GIST T-1R (**D**), GIST 430 (**E**) and SK-LMS-1 leiomyosarcoma (**F**) cells after treatment with DMSO (control), Dox (0.25 g/mL) and MK-2206 (5 M) alone and in combination (e.g., Dox + MK-2206) for 72 h. The lysates were also probed for total and phosphorylated forms of AKT to illustrate AKT inhibition by MK-2206. pAKT expression is shown by arrows. Actin was used as a loading control.

Unrepairable DNA damage in Dox-treated tumor cells cultured in presence of AKT inhibitor was also confirmed by quantitative analysis of γ-H2AX-expression, a well-known marker of the DNA DSBs. As expected, MK-2206 used alone has no impact on γ-H2AX-expression, thereby revealing absence of DNA DSBs in GIST T-1R cells treated with AKT inhibitor (Figure 4B). In contrast, Dox-treated cells exhibited an increased pattern of γ-H2AX-expression. Of note, Dox was washed out from the cell culture 2 h post treatment to exclude permanent DNA damage, and hereby allowing to repair Dox-induced DNA lesions (Figure 4C). Strikingly, we observed a retention of γ-H2AX-expression after AKT inhibition in vast majority of Dox-treated GIST cells (Figure 4D). Similar findings were observed for various STS cell lines, including HT-1080 fibrosarcoma cells, as shown in Figure S6.1. In contrast to the AKT inhibitor, inhibition of MAPK-signaling pathway by selective inhibitor U0126, did not have the similar effects on DNA repair in Dox-treated GIST (Figure S6.2). WB data also revealed a substantial increase of γ-H2AX-expression in all tumor cell lines treated with combination of Dox and MK-2206 when compared to the cells treated with Dox alone (Figure 5). Strikingly, our WB data also illustrated a substantial decrease of Rad51 expression in most of Dox-treated cells (Figure 4).

All together this data illustrates that inhibition of AKT-signaling in STS and GIST sensitizes them to Dox due to the inhibition of DNA damage repair.
