Folate receptor (FR)-targeted therapeutics is currently a paradigm for the development of a broad range of experimental drug delivery systems and immuno-therapies in cancer. All of these treatment modalities will greatly benefit from modulators to selectively up-regulate each receptor isoform in the appropriate target cells. This laboratory accomplished the previous goal of elucidating novel mechanisms of tissue specific regulation of human FRs and showed that their expression could be selectively modulated using nuclear receptor ligands. This proposal will continue and expand one major aspect of those studies, the regulation of the FR-beta gene through retinoic acid receptors (RARs) in acute myelogenous leukemia (AML). FR-beta was expressed in >70 percent of AMLs and in the bone marrow engrafting subpopulation of primary AML cells. The receptor was upregulated by all-trans retinoic acid (ATRA) in a manner limited to the receptor-positive cells and independent of the ability of ATRA to cause cell growth inhibition and terminal differentiation. The ATRA effect conferred a substantial therapeutic benefit, on FR-targeted liposomal doxorubicin, in a mouse ascites tumor model of AML. The mechanism of transactivation of the FR-beta promoter appeared to be fundamentally novel, involving both direct and cell-mediated effects to cause de novo association of RARalpha and disassociation of RARbeta/gamma at distinct sites in a Sp1/ets/Ap1 complex and decreased association of transfactors to novel Ap1-like repressor elements. In preliminary studies, ATRA-induction of FR-beta was greatly potentiated by inhibitors of histone deacetylase (HDAC) suggesting the possibility of using clinically well tolerated HDAC inhibitors both to increase the benefit of ATRA treatment in FR-beta-targeted therapies and to expand the spectrum of responsive AMLs. This proposal seeks a further thorough mechanistic understanding of the regulation of FR-beta by retinoids and HDAC inhibitors, together with in vivo data, in order to enable the further development of a new strategy for treating AML and evaluation of the clinical contexts for its application.
Aim 1 will identify key determinants of the ATRA effect from further mechanistic studies and will also test a novel mechanistic model in which RARalpha is a co-activator and RARbeta/gamma are corepressors of the FR-beta promoter.
Aim 2 will explore the mechanistic role of HDAC inhibitors using the hypothesis that HDAC inhibitors will potentiate the retinoid effects by increasing histone acetylation at specific sites/targets of ATRA action.
Aim 3 will test the hypothesis that HDAC inhibitors will potentiate retinoid induction of FRbeta in different AML subtypes in a physiologic milieu.
Aims 1 and 2 will include extensive studies using RAR subtype-specific agonists and antagonists, various HDAC inhibitors, primary AML cells and established cell lines, in vitro transcription assays with reconstituted chromatin, chromatin immunoprecipitation (ChIP) assays, gene knock-down or over-expression and various standard molecular techniques.
Aim 3 will use both a mouse ascites model and the NOD/SCID engraftment model of human AML. The studies should provide a solid foundation for designing clinical trials of new AML therapies and should also elucidate a fundamentally novel mechanism of gene regulation by retinoid receptors.
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