*2.1. Inhibition of AKT-Signaling Enhances Cytotoxicity of Topo II Inhibitors in STS and GIST*

To examine whether inhibition of AKT signaling potentiates the cytotoxic activities of Dox in STS and GIST, we performed MTS-based survival assay with a broad spectrum of cancer cell lines, including SK-LMS-1 leiomyosarcoma, RD rhabdomyosarcoma, HT-1080 fibrosarcoma, A673 Ewing's sarcoma, U2-OS osteosarcoma, IM-sensitive and resistant gastrointestinal stromal tumors (e.g., GIST T-1 vs. GIST T-1R and GIST 430, respectively). Cells indicated above were treated with Dox for 72 h alone or in combination with MK-2206, a selective AKT-inhibitor. We observed that inhibition of AKT signaling potentiated cytotoxic activity of Dox and inhibited growth of the vast majority of cancer cell lines included in present study (Figure 1). The IC50 values for MK-2206, Dox used alone or in combination are shown on Table 1. In particular, we observed an approximately two-fold decrease of IC50 for all types of cancer cell lines treated with combination of Dox and MK-2206, when compared to cells treated with Dox alone. Synergism of Dox and MK-2206 was also calculated by combination index (CI) values for each molar ratio of Dox and MK-2206 (Table 2) and was depicted as a heat-map shown on Figure 1B. In addition, the average synergistic effects of Dox and MK-2206 were calculated by using of R-package of computational tool Synergy Finder. Indeed, we observed a prominent synergism for Dox and MK-2206 in RD and SK-LMS-1 cells (Figure S1) and other STS cell lines, including U2-OS osteosarcoma and HT-1080 fibrosarcoma cells (Figure S2).

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

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

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 V-positive 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 apoptotic cell death.

**2. Results** 

*2.1. Inhibition of AKT-Signaling Enhances Cytotoxicity of Topo II Inhibitors in STS and GIST* 

To examine whether inhibition of AKT signaling potentiates the cytotoxic activities of Dox in STS and GIST, we performed MTS-based survival assay with a broad spectrum of cancer cell lines, including SK-LMS-1 leiomyosarcoma, RD rhabdomyosarcoma, HT-1080 fibrosarcoma, A673 Ewing's sarcoma, U2-OS osteosarcoma, IM-sensitive and resistant gastrointestinal stromal tumors (e.g., GIST T-1 vs. GIST T-1R and GIST 430, respectively). Cells indicated above were treated with Dox for 72 h alone or in combination with MK-2206, a selective AKT-inhibitor. We observed that inhibition of AKT signaling potentiated cytotoxic activity of Dox and inhibited growth of the vast majority of cancer cell lines included in present study (Figure 1). The IC50 values for MK-2206, Dox used alone or in combination are shown on Table 1. In particular, we observed an approximately two-fold decrease of IC50 for all types of cancer cell lines treated with combination of Dox and MK-2206, when compared to cells treated with Dox alone. Synergism of Dox and MK-2206 was also calculated by combination index (CI) values for each molar ratio of Dox and MK-2206 (Table 2) and was depicted as a heat-map shown on Figure 1B. In addition, the average synergistic effects of Dox and MK-2206 were calculated by using of R-package of computational tool Synergy Finder. Indeed, we observed a prominent synergism for Dox and MK-2206 in RD and SK-LMS-1 cells (Figure S1) and other STS cell

**Figure 1.** An impact of AKT inhibition on cytotoxic activity of doxorubicin (Dox) in soft tissue sarcomas (STS) and gastrointestinal stromal tumors (GIST). (**A**) MTS-based viability assay in RD rhabdomyosarcoma, U2-OS osteosarcoma, HT-1080 fibrosarcoma, GIST T-1R, GIST 430 and SK-LMS-1 leiomyosarcoma cells treated with Dox alone (solid lines) or in combination of MK-2206 (dashed **Figure 1.** An impact of AKT inhibition on cytotoxic activity of doxorubicin (Dox) in soft tissue sarcomas (STS) and gastrointestinal stromal tumors (GIST). (**A**) MTS-based viability assay in RD rhabdomyosarcoma, U2-OS osteosarcoma, HT-1080 fibrosarcoma, GIST T-1R, GIST 430 and SK-LMS-1 leiomyosarcoma cells treated with Dox alone (solid lines) or in combination of MK-2206 (dashed lines) for 72 h. The data was normalized to DMSO-treated controls. Values are the means ± standard deviation (*n* = 3). (**B**) the heat-map illustrating the combination index (CI) values for Dox and MK2206 combinations at various molar ratios for cancer cell lines indicated above. CI values were calculated with CompuSyn Software (Version 1.0), based on the Chou–Talalay algorithm. CI values < 1 indicate the synergy between Dox and MK-2206.


**Table 1.** IC50 values for Dox in STS and GIST treated in absence or presence of MK-2206, an AKT inhibitor.


**Table 2.** CI values for each molar ratio of Dox and MK-2206 in STS and GIST.
