PROJECT TITLE: Lead Optimization of c-Myc Inhibitor JY-3-094
c-Myc is a proto-oncogenic transcription factor that belongs to the basic helix-loop-helix leucine zipper (bHLH-ZIP) family, and its dysregulation has been observed in, and directly contributes to the progression of, almost all known cancers and tumors, including breast cancer, lung cancer, pancreatic cancer, colorectal cancer and Burkitt's lymphoma. Importantly, in order to recognize and bind its target DNA, c-Myc must first associate with its obligate bHLH-ZIP partner Max. It is this c-Myc–Max heterodimer that is the functional transcriptional activator, binding the Enhancer box (E-box) sequence 5'-CACGTG-3' and thereby regulating the expression of around 3000 target genes, many of which are crucial for normal cell proliferation, differentiation and apoptosis. Disruption of the c-Myc–Max dimer with small-molecules has recently been validated as a therapeutic approach toward the inhibition of c-Myc activity.
Owing to its intrinsically disordered state, the monomeric form of c-Myc is devoid of obvious binding sites. This renders the design of small-molecule inhibitors of c-Myc incredibly challenging. Consequently, the majority of c-Myc inhibitors reported to date have been identified from screening large chemical libraries. However, these compounds exhibit only low micromolar IC50 values against c-Myc–Max heterodimerization and against the proliferation of c-Myc-overexpressing cancer cells (HL60 and Daudi). It is not surprising, therefore, that there are currently no c-Myc inhibitors in clinical trials. Given that about one-seventh of tumors exhibit changes in the c-myc proto-oncogene or its expression, coupled with an anticipated 577,190 cancer deaths in the United States in 2013, there is an urgent need for more potent and diverse c-Myc inhibitors. A yeast two-hybrid-based approach was recently utilized to screen a library of 10,000 compounds for inhibitors of c-Myc–Max association, which led to the identification of a handful of small-molecules that includes 10074-G5. We have recently determined the pharmacophore of 10074-G5. In the course of this work, we discovered the new c-Myc inhibitor JY-3-094, which inhibits c-Myc–Max dimerization with an IC50 of 33 M corresponding to a five-fold improvement over the lead (IC50 = 146 M), and is equipotent with the best c-Myc inhibitors currently reported in the literature. While the carboxylic acid function of JY-3-094 in the para position of the aniline restricts cell entry, this was remedied by preparing a panel of ester pro-drugs, the most potent of which inhibits the growth of HL60 and Daudi cells with IC50s in the single-digit micromolar range. Interestingly, the phenol ester pro-drug of JY-3-094 itself also inhibits c-Myc–Max dimerization with a comparable IC50 value of 39 M providing a new lead compound for further optimization.
The proposed studies are driven by the overall hypothesis that the development of more potent c-Myc inhibitors will more effectively disrupt the transcriptionally active c-Myc–Max heterodimer, which, in turn, will inhibit the proliferation of c-Myc-overexpressing cancer cell lines.
PROJECT TITLE: Differential effects of PI3K and PTEN on metastatic potential
The acquisition of PIK3CA mutations and PTEN loss are often assumed to be reciprocal mutations since they can be mutually exclusive and each promotes AKT activation. However, recent clinical studies highlight major differences in patient outcomes when PTEN loss/mutation or PIK3CA mutation occurs, where PTEN loss leads to a worse patient prognosis(1-3) , perhaps due to functions of PTEN beyond that of PI3K pathway maintenance. We have recently established that PTEN loss in non-tumorigenic mammary epithelial cells (MECs) leads to the production of long, dynamic, tubulin-based membrane protrusions upon detachment(4). These novel protrusions, termed microtentacles (McTNs), are increased within the PTEN-/- cell population in frequency and length per cell as compared to their isogenic, PTEN-expressing parental counterparts, and aid in cell reattachment. Our findings also indicate that the actin-severing protein, cofilin, is highly activated in the PTEN-/- cells, weakening the actin cortex, thus providing a molecular mechanism for increased McTNs(4). Interestingly, MECs containing knock-in expression of the two most common patient derived PI3K-activating mutations neither produced increased McTNs, nor contained an increase in activated cofilin, illustrating a distict cytoskeletal dissimilarity between PTEN loss and PIK3CA mutations(4). Additionally, inhibition of the dysregulated PI3K in the PTEN-/- cells had no affect on McTN numbers or activated cofilin. Therefore, PTEN regulates the cytoskeleton through a mechanism that is distinct from its lipid phosphatase activity.
We will test the hypothesis that PTEN-mediated regulation of cofilin influences the metastatic potential of breast tumor cells in a manner that is distinct from the activation of PI3K. In the first specific aim, we will identify the functions of PTEN which are responsible for cofilin regulation. In Aim 2, we will define how PTEN-regulated cofilin affects the metastatic potential of breast tumors cells in vivo. Given the differences in cytoskeletal structure and signaling noted between cells with PTEN loss and PIK3CA activation, the current PI3K inhibitor therapies in clinical trials may not be sufficient for patients with PTEN loss. Understanding the molecular targets of PTEN that promote McTNs and reattachment could provide new therapeutic opportunities to reduce metastatic potential. Once substrates of PTEN are known, the kinases responsible for their phosphorylation could be targeted therapeutically. Presumably, the high level of phosphorylation in these PTEN substrates is what drives cytoskeletal alterations in PTEN-/- tumor cells.
PROJECT TITLE: Mechanisms required for epidermal cancer stem cell maintenance
The existence of self-renewing, epidermal cancer stem cells has been suggested by many studies in the mouse system. However, fewer studies have focused on human epidermal cancer stem (ECS) cells. A particular problem has been the lack of a suitable in vitro cell culture system for ECS cell study. This has delayed studies of the impact of diet-derived cancer prevention agents on human ECS cells. To address this issue, we have developed an in vitro cultivation system for propagation of human epidermal squamous cell carcinoma ECS cells. Compared to the bulk of the tumor cells, ECS cells display properties of cancer stem cells including ability to self-renew and high level expression of stem cell marker proteins. We show that the polycomb group (PcG) stem cell maintenance proteins, Bmi-1 and Ezh2, are markedly increased in ECS cells. We further show that treatment with sulforaphane (SFN), an important diet (broccoli)-derived cancer preventive agent, reduces ECS cell survival at concentrations 20-fold lower than non-stem cells. This is associated with loss of expression of polycomb group proteins and other stem cell survival proteins, suggesting that epidermal tumor stem cells may be a preferred target of SFN action. However, these new observations suggest many important questions. First, we do not know if Bmi-1 and Ezh2 mediate enhanced sensitivity of epidermal cancer stem (ECS) cells to SFN. Second, we know nothing about the impact of SFN on the molecular profile of these cells. This is important since we have a limited knowledge of the proteins required for maintenance of ECS cell survival, and the molecular signature may point to key signaling pathways required for ESC maintenance and identify whether they change in response to SFN treatment. Third, we do not know if ECS cells will display a similar enhanced SFN sensitivity in vivo and so it will be important to test this using tumor xenograft models. Fourth, we do not know if ESC cells from patients with epidermal squamous cell carcinoma will display enhanced sensitivity to SFN?
The goal of this proposal is to begin addressing these questions. We propose that epidermal ECS cells survive because they express high levels of stem cell maintenance proteins. We hypothesize that SFN reduces ECS cell survival by reducing the level of these stem cell maintenance proteins and that ECS cells are highly sensitive to the survival-reducing effects of SFN as compared to non-stem cells. Our goal is to characterize the sulforaphane anti-stem cell mechanism of action with an ultimate goal of developing sulforaphane as an epidermal stem cell-directed cancer prevention agent.