Aim 1. To test the effect of NTX-301 on resensitizing chemo-resistant ovarian cancer cells to chemotherapy in vitro and in vivo. DNMT inhibitors have been demonstrated to resensitize platinum resistant ovarian cancer (OC) cells to chemotherapy (1). We hypothesize that NTX-301, a novel and potent DNMT1 inhibitor, can be used in a similar fashion to resensitize chemo-resistant ovarian cancer cells to platinum treatment. To test this hypothesis, we propose the following experiments: 1. Effects on cell viability. We will determine the half maximum inhibitory concentration (IC50) of NTX-301 on cell viability of platinum sensitive and resistant variants of the ovarian cancer cell lines OVCAR5, OVCAR3, SKOV3, and COV362, and cells derived from platinum naïve and platinum resistant OC tumors. The resistant OC cells have been developed by treating parental cells with cisplatin or carboplatin at the IC50, followed by recovery of the cells. The procedure was repeated three times. The resistant cells have an IC50 to platinum 2-4X higher compared to parental cells (Wang, Cancer Research, 2020, in press). Cells will be treated with serial doses of NTX-301, and cell viability will be assessed using a Cell Counting Kit-8 (CCK-8). Decitabine will be used as a control for these experiments. 2. DNMT expression and activity. To determine the lowest dose and duration of NTX-301 treatment necessary to inhibit DNMT1, DNMT expression and activity, we will perform time-course experiments using isogenic platinum resistant and sensitive OC cells described above treated with doses of NTX-301 that do not decrease cell viability (from the IC50 Exp.). Levels of DNMT1, 3A and 3B will be measured by western blot and DNMT activity will be assessed with a kit. The lowest dosage of NTX-301, which is associated with maximum inhibition of DNMT expression and activity will be used in the follow-up study to resensitize OC cells to chemotherapy. Decitabine will be used as a control for these experiments. 3. Platinum resensitization. To test the effects of NTX-301 on resensitization of OC cells to platinum treatment in vitro, a panel of platinum resistant and sensitive OC cells described above, will be primed with/without a low dose of NTX-301, and then treated with a range of concentrations of carboplatin and/or cisplatin. Cell viability will be measured with a CCK-8 kit, and IC50 values will be calculated and compared between cisplatin alone vs NTX-301 plus cisplatin combination. Similar experiments will be performed to confirm platinum resensitization by colony formation assay, apoptosis assay, and cell cycle analysis. Decitabine will be used as a control for these experiments. 4. In vivo Experiments. To test the priming effect of NTX-301 on resensitizing platinum resistant OC cells in vivo, we will use a platinum resistant OC xenograft and a HGSOC PDX model. NGS mice bearing platinum resistant OVCAR5 xenografts will be treated with PBS, carboplatin alone (once a week 20mg/Kg ip for 3 weeks; one week rest), NTX301 alone (0/6mg/kg ip daily for 5 days, 2 days off for 3 weeks, one week rest) and the combination (NTX301 0/6mg/kg ip daily for 5 days, 2 days off for 2 weeks, followed by Carboplatin 20mg/Kg ip weekly for 2-3 weeks) (n= 8 mice per group). Decitabine will be used as a control for these experiments. The dose and schedule of administration will be refined based on findings of in vitro experiments. Tumor growth will be monitored twice-a-week. Tumor will be collected at euthanasia to determine the effects of the treatments on tumor size and weig
|Effective start/end date||2/16/21 → 7/31/22|
- PinotBio, Inc. (Matei AGMT 2/18/21)
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