Abstract 4842: p300 targeting impairs treg function and promotes host anti-tumor immunity

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The long-held view that tumors were not sufficiently immunogenic to promote host immune responses has now been revised to the assessment that at least for some tumors, Foxp3+ T-regulatory (Treg) cells actively suppress anti-tumor immunity. Seeking to build on our ongoing assessment of the regulation of Foxp3+ Treg biology by HDAC and HAT enzymes, we have begun to use combined genetic and pharmacologic approaches to identify and modulate key mechanism within Tregs that limit host immunity. Microarray studies of gene expression in WT, p300-/- and CBP-/- Tregs showed that p300 or CBP deletion led to down-regulation of Foxp3 expression, as well as that of a number of signature Treg genes. Cells co-transfected with Foxp3 and increasing amounts of p300 expression vectors showed that Foxp3 acetylation was enhanced by increasing levels of p300 levels, whereas a p300 small molecule inhibitor, C646 (p300i), impaired Foxp3 acetylation and inhibited Treg function using in vitro suppression assays. In vivo use of C646 showed negligible effects on CD4, CD8 or Treg cell numbers, but qPCR studies showed C646 decreased Treg expression of CTLA-4, GITR, IL-10 and TGF-b, in vitro assays again showed impaired Treg suppression. Additional studies showed, critically, that use of the p300i impaired Treg but not T effector cells in allograft recipients. Thus, parent-to-F1 assays involving alloactivation and proliferation of CD4 an dCD8 T cells were unaffected by C646, and this p300i restored allograft rejection in Treg-dependent models of allograft survival. Turning to tumor models, TC1 lung cancer cells were injected in the flanks of WT B6 mice, B6 mice in which p300 was deleted in CD4+ T cells (Treg plus Teff using CD4-Cre) or just in Tregs (Foxp3-Cre) (n=10/grp), p300 was inhibited using C646 (Alzet pumps, 14 d, 0.9 mg/kg/d) from day 6 post-tumor injection; in both genetic and pharmacologic approaches, p300 targeting suppressed tumor cell growth. qPCR analysis of tumors in C646-treated mice showed increased CD4 and granzyme B mRNA but decreased Foxp3; these data were confirmed by immunohistology, along with increased IFN-g production by purified CD8 cells (ELISPOT), along with reductions in tumor volumes and weights in these C646-treated mice. C646 use did not impair tumor growth in immunodeficient (RAG-/-) mice, in which tumors were exposed to drug in RAG-/- mice as seen by decreased acetylation of histone 3 in tumor extracts (Western blot); no effects on acetylation of certain p300-independent targets within tumor extracts was seen (e.g. acetylation of alpha-tubulin was unimpaired). The p300i also had no direct effect on tumor cell proliferation in vitro. We conclude that our comprehensive genetic and pharmacologic studies show that p300 is an important target for modulation of host Foxp3+ Treg functions, such that Treg suppression can be incrementally reduced and host anti-tumor responses promoted without inducing concomitant autoimmunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4842. doi:1538-7445.AM2012-4842