HSP90 enables the activation of many client proteins of which the most clinically validated is HER2. NVP-AUY922, a potent HSP90 inhibitor, is currently in phase II clinical trials. To explore its potential clinical use in HER2-amplified breast and gastric cancers, we evaluated the effect of AUY922 alone and in combination with trastuzumab in both trastuzumab-sensitive and -resistant models. A panel of 16 human gastric and 45 breast cancer cell lines, including 16 HER2-amplified (3 and 13, respectively) cells, was treated with AUY922 over various concentrations. In both breast and gastric cancer, we used cell lines and xenograft models with conditioned trastuzumab-resistance to investigate the efficacy of AUY922 alongside trastuzumab. Effects of this combination on downstream markers were analyzed via Western blot analysis. AUY922 exhibited potent antiproliferative activity in the low nanomolar range (<40 nmol/L) for 59 of 61 cell lines. In both histologies, HER2-amplified cells expressed greater sensitivity to AUY than HER2-negative cells. In conditioned trastuzumab-resistant models, AUY922 showed a synergistic effect with trastuzumab. In vitro, the combination induced greater decreases in HER2, a G 2 cell-cycle arrest, and increased apoptosis. In a trastuzumab-resistant gastric cancer xenograft model, the combination of AUY922 and trastuzumab showed greater antitumor efficacy than either drug alone. These data suggest that AUY922 in combination with trastuzumab has unique efficacy in trastuzumab-resistant models. The combination of HSP90 inhibition and direct HER2 blockade represents a novel approach to the treatment of HER2-amplified cancers and clinical trials based on the above data are ongoing.

We examine the requirements for converting naive, mature CD4+ cells from an activation-induced death (AID)-resistant to a -sensitive phenotype. Priming for sensitivity to AID can be divided into two steps. The first is a mitogen/CD3-dependent, cyclosporin-sensitive signal and the second a cytokine-dependent, cyclosporin-insensitive one. Under these conditions, interleukin (IL)-2, but not IL-4, IL-7 or IL-15, the receptors of which share a common receptor gamma chain, is capable of providing the cytokine signal for inducing sensitivity to AID. Increased expression of the low-affinity IL-2R alpha chain (CD25) is associated with acquisition of AID sensitivity and antibodies to CD25 block acquisition of AID sensitivity in the presence of IL-2. As with T cell hybridomas, AID is dependent on both CD95 and CD95 ligand (CD95L) expression, but unlike hybridomas, the sensitive and resistant phenotypes of primary CD4+ cells cannot be distinguished by levels of CD95 expression, functional CD95L nor the fraction of cells in cycle. The results suggest that the unique function of IL-2 is to regulate proteins, either not important or constitutively regulated in T cell hybridomas, that are essential for cell-autonomous suicide of activated CD4+ cells. These experiments provide a mechanism for the recent observations of chronic lymphoproliferation and autoimmune disease in mice with null mutations in IL-2 or CD25.

During mammalian pregnancy, one or more semiallogeneic fetuses gestate in direct contact with the maternal circulation and uterine tissue. However, a damaging maternal immune response is not normally provoked. We studied two possible mechanisms for this maternal-fetal tolerance, alone and in combination. First, we directly tested the hypothesis that the striking absence of MHC class I molecules on most placenta trophoblasts protects the fetus from maternal immune attack, by creating transgenic mice which express Ld in giant cell trophoblasts. Second, because Fas ligand (FasL) may contribute to immune privilege, we tested whether functional FasL expression by the fetus, or Fas expression by the mother, contributes to successful reproduction in a fully allogeneic breeding. Our data indicate that neither abnormal expression of MHC class I in giant cells, nor disruption of the Fas-FasL system, nor a combination of these two defects, has an adverse effect on pregnancy outcome. These results suggest that during healthy allogeneic pregnancy, down-regulation of MHC class I and expression of FasL on placenta are not critical events, and other factors must prevent a harmful maternal immune response.

During mammalian pregnancy, one or more semiallogeneic fetuses gestate in direct contact with the maternal circulation and uterine tissue. However, a damaging maternal immune response is not normally provoked. We studied two possible mechanisms for this maternal-fetal tolerance, alone and in combination. First, we directly tested the hypothesis that the striking absence of MHC class I molecules on most placenta trophoblasts protects the fetus from maternal immune attack, by creating transgenic mice which express L d in giant cell tro-phoblasts. Second, because Fas ligand (FasL) may contribute to immune privilege, we tested whether functional FasL expression by the fetus, or Fas expression by the mother, contributes to successful reproduction in a fully allogeneic breeding. Our data indicate that neither abnormal expression of MHC class I in giant cells, nor disruption of the Fas-FasL system, nor a combination of these two defects, has an adverse effect on pregnancy outcome. These results suggest that during healthy allogeneic pregnancy, down-regulation of MHC class I and expression of FasL on placenta are not critical events, and other factors must prevent a harmful maternal immune response.

Background: Cyclin-dependent kinases (CDKs) play a significant role in regulating cell cycle progression through association with cyclins. CDK4 and CDK6 interact with cyclin D1 to mediate hyperphosphorylation of retinoblastoma (Rb) during early G1 phase. Palbociclib is a highly selective inhibitor of CDK4 and CDK6 which functions by blocking pRb phosphorylation resulting in G1 arrest in sensitive cell lines. We evaluated the effect of palbociclib in gastric and colon cancer cell lines to explore potential biomarkers of response and to guide patient selection in colon and gastric cancer. Methods: Panels of 17 gastric and 27 colon cancer cell lines were exposed in vitro to palbociclib over various concentrations to generate dose response curves. Analysis of variance (ANOVA) was used to identify differentially expressed genes between sensitive and resistant cell lines. Genes identified by ANOVA and effects of palbociclib on pRB were analyzed via western blot. Results: Palbociclib was found to have potent anti-proliferative activity in the low nanomolar range (&lt; 150 nM) for 14 of the 44 gastric and colon cancer cell lines tested. In gastric cancer, cyclin D1-amplified cells cells expressed greater sensitivity to the compound when compared to cyclin D1-negative cells. HER2 amplified cell lines were also statistically more sensitive than HER2 negative cells. Combination studies with palbociclib and trastuzumab demonstrated significant synergy in HER2 amplified gastric cancer models. Furthermore, cyclin E emerged as a biomarker for resistance to the compound in gastric cancers. Contrary to observations made in other cancers, expression levels of p16 (CDK4 inhibitor) and p21 (CDK2 inhibitor) in colon cancer indicated that p16 loss and p21 gain predict for resistance rather than sensitivity to CDK4 and CDK6 inhibition. Conclusions: Palbociclib demonstrates anti-proliferative activity in several gastric and colon cancer cell lines. Molecular markers found to predict for sensitivity to this agent enhance patient selection for future clinical studies of palbociclib. Citation Format: Zev A. Wainberg, Ann Yufa, Adrian Anghel, Amy M. Rogers, Tin Manivong, Shahriar Adhami, Habib Hamidi, Dylan Conklin, Richard S. Finn, Dennis J. Slamon. Expression of p16 in colon cancer and cyclin D1 in gastric cancer predicts response to CDK4/6 inhibition in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4557. doi:10.1158/1538-7445.AM2014-4557