Identification And Mechanism Study Of Two Small Molecular Compounds With Anti-cancer Activities

A large number of chemotherapeutic drugs are genotoxic agents, which activate the DNA damage response to inhibit cell proliferation and/or to induce cell death. DNA damage is sensed by Atm and Atr, which phosphorylate and activate Chk2/1 and p53 to suppress cell propagation. p53 is an important tumor suppressor and a target for anti-cancer drug development as well. In order to find novel chemotherapeutic drug candidates with genotoxic activity, we screened 80 kinase inhibitors and 33 phosphatase inhibitors with the activation of p53 as an indicator. We identified 5-Iodotubercidin, a purine derivative and an inhibitor for various kinases including adenosine kinase, and Bisindolylmaleimide IX, one of the Bisindolylmaleimide derivatives is a PKC inhibitor, from the screen. We further studied the potential of these two small molecule compounds as chemotherapeutic drugs and the molecular mechanisms by which the two compounds execute their functions.In the first part, we confirmed that 5-Iodotubercidin is a strong p53 activator in several cell lines. We found that 5-Iodotubercidin could cause DNA damage, verified by induction of DNA breaks and nuclear foci positive for γH2AX and TopBP1, activation of Atm and Chk2, and S15 phosphorylation and up-regulation of p53. As such, 5-Iodotubercidin induces G2/M cell cycle arrest in a p53-dependent manner. Itu also induces cell death in p53-dependent and-independent manners. DNA breaks were likely generated by incorporation of 5-Iodotubercidin metabolite into DNA. Moreover, 5-Iodotubercidin showed anti-tumor activity as it could reduce the tumor size in carcinoma xenograft mouse models in p53-dependent and-independent manners. These findings reveal 5-Iodotubercidin as a novel genotoxic drug that has chemotherapeutic potential.In the second part, by comparing the efficacy of Bisindolylmaleimide IX on a variety of cancer cell lines, we found that Bisindolylmaleimide IX has therapeutic potential against CML including drug-resistant CML. Chronic myeloid leukemia treatment with BCR-ABL inhibitors is often hampered by development of drug resistance. We show that Bisindolylmaleimide IX is a DNA Topoisomerase inhibitor that activates DDR. Bisindolylmaleimide IX is highly effective in targeting cells positive for BCR-ABL, a kinase that is known to induce genome instability, compared to other cancer cell lines tested. BCR-ABL positive cells showed enhanced DNA damage attributable to decreased expression of Topoisomerases, increased cell cycle arrest and increased cell death in response to Bisindolylmaleimide IX. In addition, Bisindolylmaleimide IX also inhibits Raf and the downstream oncogene addiction pathway to kill cells positive for BCR-ABL or drug-resistant T315 I BCR-ABL. Mouse cancer model experiments show that Bisindolylmaleimide IX is effective in treating leukemia-like disorders induced by BCR-ABL or T315 I BCR-ABL, and prolongs the lifespan of these model mice. Thus, unlike the current BCR-ABL inhibitor drugs, Bisindolylmaleimide IX presents a potential drug to treat CML including drug-resistant CML via activating BCR-ABL-dependent genotoxic stress response and inhibiting the oncogene addiction pathway of BCR-ABL.