NI-004

To investigate the mechanism of action and therapeutic benefits of NSC001, a small molecule anticancer compound, in the treatment of triple negative breast cancer (TNBC), a clinical subtype of breast cancer that is more aggressive, has poor prognosis. In TNBC, patients harbor tumors that lack expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Her2).

We hypothesize that NSC001 will have a beneficial therapeutic effect on TNBC cells through the regulation of specific tumor associated signaling pathways in vitro.

We screened 1360 compounds from the National Cancer Institute (NCI) Diversity and Mechanistic Set library using the cell counting kit‐8 (CCK‐8) reagent from Dojindo Molecular Technologies, Inc. The screening was performed by applying 10µM of each candidate drug upon plates harboring the aggressive TNBC cell line, MDA‐MB‐231. Treatment time for all screening was fixed at 24 hours. The efficacy and potential mechanism of action of one of the potent drug candidates identified (NSC001) was further investigated on three TNBC cell lines (MDA‐MB‐231, MDA231‐LM2‐4175, and MDA‐MB‐468) using mass spectrometry and proteomic analysis. Afterwards, the results from the proteomic analysis were validated using molecular methods including cell viability assays, apoptosis assays, cell cycle assays, and western blotting.

We identified eight compounds that reduced the viability of MDA‐MB‐231 cells by up to 80% following a 24 hour treatment time point. Next, our in vitrodata show that NSC001 reduces the cell viability of TNBC cells in a dose‐and‐time dependent manner. Cell death data show that NSC001 induces potent apoptosis in a dose dependent manner in TNBC cells. Also, the results from colony formation assay show that 100nM [NSC001] is sufficient to inhibit the proliferative capacity of TNBC cells. Mass spectrometry and proteomic data reveals that NSC001 activates the cell cycle G2/M DNA damage checkpoint regulation in all the three TNBC cell lines used. Furthermore, we confirmed this finding in our cell cycle assay where we observed a clear G2/M cell cycle phase arrest. The arrest was potently induced at a dose significantly lower than the IC50 of NSC001. Lastly, we validated our proteomic results using western blotting, which confirmed that this low dose of NSC001 induces expression of p21, a cell cycle checkpoint regulator and tumor suppressor protein which is known to trigger cell cycle arrest in most cancer cell lines.

Overall, our data suggest that NSC001 may be a potent small molecule anticancer compound which should be further investigated for its potential to be used clinically in the treatment of TNBC patients.