Introduction: Leiomyosarcomas are rare mesenchymal neoplasms characterized by a smooth-muscle differentiation pattern. Patients show poor disease-specific survival due to high recurrence rates and low response to chemotherapy and radiation. Development of novel chemotherapeutic regimens is the need of the hour. Epigenetic mechanisms such as hypermethylation of gene promotor regions lead to abnormal gene silencing and have been found to influence the initiation and progression of cancer. Epigenetic drugs have been approved for treatment of various malignancies such as myelodysplastic syndrome, acute myeloid leukemias, and subsets of lymphomas. This study aims to understand the potential efficacy of epigenetic drugs called DNA methyltransferease inhibitors (DNMTi) in the treatment of leiomyosarcomas.

Methods: DNMTi used in this study include azacytidine (Aza), decitabine (Dac), and guadecitabine. Aza and Dac are antimetabolites, which get incorporated onto RNA and DNA, respectively, leading to inhibition of DNMT. Guadecitabine is a novel prodrug for Dac with a longer half-life. Various leiomyosarcoma cell lines (SK-UT1, SK-LMS, and MES) were treated with varying doses of guadecitabine (0.01-5 ug/ml), decitabine (Dac: 0.1 uM-5uM), or azacitidine (Aza: 0.1 uM-5 uM) and incubated for a maximum of five days. Cell viability was measured daily via MTT assay. To further characterize the mechanism(s) of drug induced cell death, Caspase Glo® Assay (G8090) was utilized as a measure of apoptosis by proxy of Caspase 3 and 7 activity. CellToxTM Green Cytotoxicity Assay was then employed to measure membrane integrity, thus quantifying nonspecific cell death. This would further measure the toxic effect of the demethylating drugs on leiomyosarcoma cell lines. We also studied the effect of guadecitabine (3 mg/kg, 2x/week) treatment in vivo, using subcutaneous xenografts in NOD/SCID mice. Statistical analysis was conducted using GraphPad Prism 6.

Results: Decreased cell viability was observed in all three leiomyosarcoma cell lines upon exposure to demethylating agents. SK-UT1 showed the highest sensitivity to all three epigenetic drugs (IC50 values: guadecitabine-0.3 uM, Aza-2.1 uM, and Dac-2.3 uM), MES was sensitive to Aza and guadecitabine (IC50 values: Aza-3.2 uM, guadecitabine-6.14 uM, and Dac-11.77 uM), and SK-LMS was sensitive only to Aza (IC50 values: Aza-2.6 uM, guadecitabine-9.6 uM, and Dac-10.3 uM). To better understand the effect of guadecitabine and AZA on these cell lines, we measured the Caspase 3/7 activity of SK-UT1 and SK-LMS using a Promega kit as a readout for apoptosis. The Caspase activity measured in SKUT1 was remarkably higher in both drugs when compared to that of SK-LMS in presence of same drugs. The amount of necrosis measured in both cell lines in presence of guadecitabine and Aza was negligible. Considerable tumor growth inhibition was also observed after in vivo treatment with guadecitabine in both SK-UT1 and SK-LMS mice xenografts.

Conclusion: The use of demethylating agents leads to substantial inhibition of tumor growth in leiomyosarcoma cell lines and xenograft models. Further studies are required to explore the potential therapeutic applications and precise mechanism of action of demethylating agents in leiomyosarcoma treatment.

Funding supported by ASTEX Pharmaceuticals.

Note: uM= Micromole

Citation Format: Cynthia De Carvalho Fischer, Yue Hu, Jasvinder Singh, Maya Thakar, Manjusha Thakar, Nita Ahuja. Treatment with demethylating drugs inhibits tumor growth in leiomyosarcoma cell lines and xenograft models [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A35.