Improving response to immunotherapy by genistein and antiestrogens

Cancer immunotherapies have been remarkably successful in treating many previously incurable cancers. However, their utility in advanced breast cancer has been limited. For example, immune checkpoint blockers (ICBs) show little to no efficacy as monotherapies in recurrent or metastatic breast cancer. High estrogen levels in the breast tumor microenvironment (TME) may impair the response to ICBs, as 17?-estradiol (E2), by activating estrogen receptor ? (ER?/ESR1), activates immunosuppressive myeloid derived suppressor cells (MDSCs) which impair response to ICBs. There also is a growing evidence showing that the gut microbiome, modified by diet, plays a critical role in the response to ICB. In our pilot study, growth of allografted ESR1 negative E0771 murine mammary tumors was significantly inhibited by anti-PD1 ICB therapy when mice also were treated with ESR1 inhibiting antiestrogen tamoxifen. This effect, however, was strongly dependent on the diet fed to mice. The effect was seen only in mice which consumed 5058 rodent laboratory diet, but not in mice fed 5V5M or AIN93G rodent diet. The key difference between 5058 and 5V5M diets is that only 5058 contains soybeans which are high in isoflavone genistein (GEN). 5V5M diet contains low levels of isoflavones, and AIN93G does not contain any GEN. Although GEN has a chemical structure similar to that of E2, it preferentially binds to the other estrogen receptor: ER?/ESR2. ER? has opposite effects from ER? and its activation augments immunity and inhibits inflammation, including in the gut. Since GEN modifies the gut microbiome, we will investigate here if the diet-dependent effects of anti-PD1+antiestrogen in promoting elimination of mammary tumors are caused by GEN?s effect on the gut microbiome. This will be done by transplanting fecal microbiome from GEN fed donor mice to germ-free recipient mice, and determining if the recipient mice kept on GEN-free diet will respond equally well to anti-PD1 + fulvestrant as mice also fed GEN. We will then study if GEN?s effects on the gut microbiome are mediated by GEN binding and activating ER?/ESR2 in the gut by using conditional knockout mice which do not express ESR2 in the intestine. Finally, we will compare the effects of GEN to that of ESR2 agonist LY500307, currently in clinical trials, in modifying response to PD1 blockage and fulvestrant. If we show the potential for supplementation with GEN or LY500307 to increase responsiveness of mammary tumors to anti-PD1 therapy with antiestrogen treatment, this might allow breast cancer patients with advanced disease to harness the power of immunotherapy.