Managing tumor recurrence is a major challenge in breast cancer as it is often fatal. Women with triple-negative breast cancer (TNBC) have the highest rate of early recurrence, which is thought to be due to the presence of breast cancer stem cells (BCSCs) in these tumors. BCSCs express the CD44+/CD24-/EpCAMand/or ALDH+ signature and possess the ability to self-renew and initiate tumor formation in mice. While several treatment strategies for TNBC exist, the identification of agents that eliminate BCSCs has not been explored. Radiation and chemotherapy results in an enrichment of this population, making current treatments ineffective. Treatment strategies for TNBC that are currently in practice do not address the problem of BCSCs, and the identification of agents that eliminate BCSCs is essential to achieve long-term survival of TNBC patients. This application addresses the overarching challenge of (1) identifying what drives breast cancer growth and determining how to stop it; (2) revolutionizing treatment regimens by replacing interventions that have life-threatening toxicities with the ones that are safe and effective; and (3) eliminating the mortality associated with breast cancer.

To develop targeted therapies for TNBC, we synthesized a large number (~3000) of novel kinase inhibitors and screened this compound library for compounds that induce the death of CD44high/CD24low cells purified from four different TNBC cell lines, MDA-MB-231, MDA-MB-468, Hs-578T, and BT-20. This strategy identified ON108600, which showed remarkable toxicity towards CD44high/CD24low tumor cells derived from these four cell lines with little toxicity to normal cells. Kinase assays revealed that ON108600 is an inhibitor of CK2 alpha, TNIK, and Dyrk kinases, which have been shown to play critical roles in the survival and metastasis of breast cancer stem cells. Previous studies have shown that CK2 is overexpressed in TNBC and that elevated CK2 activity is associated with poorer prognosis. Downregulation of CK2 results in induction of apoptosis in cultured cell and xenograft cancer models, further suggesting its potential as a therapeutic target. TNIK is a recently identified kinase, which is overexpressed in TNBC and acts as a regulatory component of Wnt/beta-catenin pathway, which has been found to promote tumor cell proliferation, migration, and metastasis. Dyrk1 A&B have been shown to impart on cancer stem cells the ability to remain in a state of quiescence when exposed to therapeutic agents or radiation, which allows tumor cells to escape from the effects of radiation and chemotherapy. By inhibiting the activity of these enzymes, we believe that ON108600 induces the death of breast cancer stem cells and this compound, either alone or in combination with other chemotherapies, has the potential to prevent relapse of TNBC.

From the X-ray crystal structure studies, we demonstrated that ON108600 blocks the binding of both ATP and GTP to the kinase domain of CK2alpha1, which makes this compound a first-in-class inhibitor of GTP-competitive inhibitors. In this proposal, we outline experiments aimed at the development of this small molecule as a cancer therapeutic, with the overall goal of conducting studies aimed at providing insights into the best ways of utilizing ON108600 in the treatment of TNBC. Our proposed studies will not only provide an understanding of the molecular basis for the action of this compound but also will provide information about resistance mechanisms that TNBC might employ to overcome the effects of this compound. Our studies show that ON108600 is not only active as a single agent but also significantly increased apoptosis of TNBCs when combined with paclitaxel or doxorubicin. In this proposal, we propose to explore the effects of ON108600 either as a single agent or in combination with other cytotoxic and targeted therapeutic agents using human xenografts grown in nude mice as well as PDX (patient-derived xenograft) models of TNBC.

Through the completion of the studies proposed herein, our team comprising cancer biologists, structural biologists, pharmacologists, and clinical investigators will develop a small molecule, ON108600, into a cancer therapeutic, one of the latest of a new generation of drugs built upon our understanding of basic cell proliferation mechanisms.