Inhibition of SP1 by the mithramycin analog EC-8042 efficiently targets tumor initiating cells in sarcoma

Abstract

Tumor initiating cells (TICs), responsible for tumor initiation, and cancer stem cells (CSCs), responsible for tumor expansion and propagation, are often resistant to chemotherapeutic agents. To find therapeutic targets against sarcoma initiating and propagating cells we used models of myxoid liposarcoma (MLS) and undifferentiated pleomorphic sarcoma (UPS) developed from human mesenchymal stromal/stem cells (hMSCs), which constitute the most likely cell-of-origin for sarcoma. We found that SP1-mediated transcription was among the most significantly altered signaling. To inhibit SP1 activity, we used EC-8042, a mithramycin (MTM) analog (mithralog) with enhanced anti-tumor activity and highly improved safety. EC-8042 inhibited the growth of TIC cultures, induced cell cycle arrest and apoptosis and upregulated the adipogenic factor CEBPα. SP1 knockdown was able to mimic the anti-proliferative effects induced by EC-8042. Importantly, EC-8042 was not recognized as a substrate by several ABC efflux pumps involved in drug resistance, and, opposite to the chemotherapeutic drug doxorubicin, repressed the expression of many genes responsible for the TIC/CSC phenotype, including SOX2, C-MYC, NOTCH1 and NFκB1. Accordingly, EC-8042, but not doxorubicin, efficiently reduced the survival of CSC-enriched tumorsphere sarcoma cultures. In vivo, EC-8042 induced a profound inhibition of tumor growth associated to a strong reduction of the mitotic index and the induction of adipogenic differentiation and senescence. Finally, EC-8042 reduced the ability of tumor cells to reinitiate tumor growth. These data suggest that EC-8042 could constitute an effective treatment against both TIC and CSC subpopulations in sarcoma.

Trabectedin and Campthotecin Synergistically Eliminate Cancer Stem Cells in Cell-of-Origin Sarcoma Models

“…In other types of tumors it has been described that trabectedin is able to induce apoptosis and G2/M cell cycle arrest and to reduce tumorsphere growth in CSC subpopulations derived from prostate cancer cell lines [43]. We also demonstrate that, unlike what we previously reported for doxorubicin [38], trabectedin represses the expression of multiple genes and pathways involved in the development and maintenance of the CSC phenotype. Despite the differential effects of trabectedin and doxorubicin on the expression of genes related to the CSC phenotype, trabectedin treatment of T-5H-FC#1-generated xenografts did not provide a therapeutic advantage over previously reported doxorubicin treatment of these xenografts [38] (Figure S3), thus reproducing the results observed in phase III clinical trials [44].…”

Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma

“…Mithramycin is an antibiotic isolated from Streptomyces plicatus that binds to specific DNA regions to repress transcription of specific genes. Importantly, this compound was found to downregulate the expression of stemness-related genes (including SOX2 and ZEB1) in sarcoma cells (Tornin et al, 2016), and was also shown to have high efficacy for growth inhibition of a SOX2-positive cell population that propagates medulloblastoma (Vanner et al, 2014). Interestingly, mithramycin has been shown to be therapeutically effective in GBM patients in clinical studies conducted in the 1960s (Ransohoff et al, 1965), however further clinical use of this small molecule was hampered because of an unfavorable toxicity profile.…”

The multikinase inhibitor EC‐70124 synergistically increased the antitumor activity of doxorubicin in sarcomas

“…S1C), although its cytotoxic effect on CSC subpopulations was lower than that previously observed in nonselected adherent cultures (Figs. a and 1 b ) and also much lower than the effect of the mythramicin analog EC‐8042, a drug with reported activity on sarcoma CSCs …”

“…The viability of all cell lines in the presence and absence of drugs was determined using the Cell Proliferation reagent WST-1 (Roche, Mannheim, Germany) as described before. 37 The concentration of half-maximal inhibition of viability (IC 50 ) for each treatment was determined by nonlinear regression using the graphPad Prism software (La Jolla, CA). The existence of synergy in drug combinations was determined by calculating the combination index (CI) as described in Supporting information.…”

“…1a and 1b) and also much lower than the effect of the mythramicin analog EC-8042, a drug with reported activity on sarcoma CSCs. 37 Finally, to compare the cytotoxic effect of EC-70124 with other multikinase inhibitors already approved for cancer treatment, we treated T-5H-FC#1 cells with increasing concentrations of sorafenib, pazopanib and imatinib. All of this drugs were much more inefficient than EC-70124 in reducing cell survival, showing IC 50 concentrations of 20 μM or higher (Supporting Information Fig.…”