Safety and anti-cancer efficacy of FACT inactivation in mice

Roswell Park Cancer Institute Corp, Buffalo NY

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Abstract

? DESCRIPTION (provided by applicant): Property of cancer cells making them aggressive and difficult to eliminate is infinite self-renewal capability. Studies of artificially generated non-tmor cells with capability to endless self-renewal (ES, iPS cells) revealed that one of the common features between them and tumor cells is specific chromatin state. Modulation of chromatin state associated with self-renewal for cancer treatment was almost unexplored (except HDAC inhibition) due to lack of knowledge which factor(s) is responsible for this state and general view that chromatin remodeling factors lack sufficient specificity to be a good target for cancer therapy. Contrary to this view, we have recently found that chromatin remodeling complex, FACT, is active and essential almost exclusively in ES and tumor cells. FACT, heterodimer of two subunits, SSRP1 and SPT16, is H2A/H2B histone chaperone. Our interest in FACT was commenced by our discovery of small molecules, curaxins, simultaneously able to activate p53 and inhibit NF-?B. We found that these activities of curaxins as well as their toxicity to tumor cells are due to functional inactivation of FACT. We found high levels of FACT in aggressive metastatic poorly differentiated tumors and in cells possessing properties of tumor initiating or cancer stem cells. In line with this, genetic inactivation of FACT is toxic only for ES and tumor cells, but not non-ES normal cells, including some normal adult stem cells. Thus our hypothesis is that inhibition of FACT will have potent anti-cancer effect, with specific activity against cancr stem cells, and reliable safety due to its essentiality only for cells with abnormally active self-renewal program (SRP). However we still do not know if FACT is needed for any normal non-ES cells and this hampers the full scale program to develop specific FACT inhibitors. Since general knockout (KO) of FACT was not possible due to early embryonic lethality, to answer these questions we propose to develop mouse model of conditional FACT KO which will allow switch on and off FACT expression in the context of the whole mammalian organism. Availability of this model will allow answering multiple fundamental questions of how FACT controls chromatin state and SRP in different cells, at different stages of embryonic development and in different tumors and will suggest the most rational way of development of FACT inhibitors. At this stage we propose to: 1. Assess safety and anti-tumor efficacy of FACT inactivation after birth. We will cross mice in which we already flanked critical exons of Ssrp1 with LoxP sites (Ssrp1fl/fl) with mice carrying tamoxifen regulated Cre recombinase (CreERT2). We will observe consequences of FACT inactivation for normal tissues and engrafted syngeneic tumors in these mice upon tamoxifen administration at different moments of life. 2. To compare normal and tumor cell survival upon temporary inactivation of FACT. To test this we plan to generate Ssrp1-ERT2 knock-in mouse model in which presence of SSRP1 in nuclei will be regulated with tamoxifen. Therefore this model will allow FACT to be inhibited and restored multiple times.