The phosphoinositide 3-kinase (PI3K)-Akt pathway is constitutively active in many tumors, and inhibitors of this prosurvival network, such as LY294002, have been shown to sensitize tumor cells to death stimuli. Here, we report a novel, PI3K-independent mechanism of LY-mediated sensitization of LNCaP prostate carcinoma cells to drug-induced apoptosis. Preincubation of tumor cells to LY294002 or its inactive analogue LY303511 resulted in a significant increase in intracellular hydrogen peroxide (H 2 O 2 ) production and enhanced sensitivity to nonapoptotic concentrations of the chemotherapeutic agent vincristine. The critical role of intracellular H 2 O 2 in LY-induced death sensitization is corroborated by transient transfection of cells with a vector containing human catalase gene. Indeed, overexpression of catalase significantly blocked the amplifying effect of LY pretreatment on caspase-2 and caspase-3 activation and cell death triggered by vincristine. Furthermore, the inability of wortmannin, another inhibitor of PI3K, to induce an increase in H 2 O 2 production at doses that effectively blocked Akt phosphorylation provides strong evidence to unlink inhibition of PI3K from intracellular H 2 O 2 production. These data strongly support death-sensitizing effect of LY compounds independent of the PI3K pathway and underscore the critical role of H 2 O 2 in creating a permissive intracellular milieu for efficient drug-induced execution of tumor cells. (Cancer Res 2005; 65(14): 6264-74)

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells that inhibit T-cell activity and contribute to the immune suppression characteristic of most tumors. We discovered that bone marrow (BM) progenitor cells from the Muc1 knockout (KO) mice differentiated into CD11b + Gr1 + MDSCs in vitro under granulocyte macrophage colony-stimulating factor and interleukin-4 signaling. MUC1 is a tumor-associated mucin and its cytoplasmic tail (MUC1-CT) can regulate B-catenin to promote oncogenesis. Given the importance of B-catenin in hematopoiesis, we hypothesized that the MUC1 regulation of B-catenin is important for MDSC development. Our current study shows that the aberrant development of BM progenitors into CD11b + Gr1 + MDSCs is dependent on the down-regulation of B-catenin levels that occurs in the absence of Muc1. In light of this, KO mice showed enhanced EL4 tumor growth and were able to better tolerate allogeneic BM185 tumor growth, with an accumulation of CD11b + Gr1 + cells in the blood and tumor-draining lymph nodes. WT mice were able to similarly tolerate allogeneic tumor growth when they were injected with CD11b + Gr1 + cells from tumor-bearing KO mice, suggesting that tolerance of allogeneic tumors is dependent on MDSCmediated immune suppression. This further delineates the ability of Muc1 to control MDSC development, which could directly affect tumorigenesis. Knowledge of the biology by which Muc1 regulates the development of myeloid progenitors into MDSCs would also be very useful in enhancing the efficacy of cancer vaccines in the face of tumor immune suppression. [Cancer Res 2009;69(8):3554-62]

Certain classes of tumor cells respond favorably to TRAIL due to the presence of cell surface death receptors DR4 and DR5. Despite this preferential sensitivity, resistance to TRAIL remains a clinical problem and therefore the heightened interest in identifying compounds to revert tumor sensitivity to TRAIL. We recently demonstrated that the phosphatidylinositide-3-kinase (PI3K) inhibitor, LY294002, and its inactive analog LY303511, sensitized tumor cells to vincristine-induced apoptosis, independent of PI3K/Akt pathway. Intrigued by these findings, we investigated the effect of LY303511 on TRAIL-induced apoptosis in HeLa cells. Preincubation of cells with LY30 significantly amplified TRAIL signaling as evidenced by enhanced DNA fragmentation, caspases 2, 3, 8, and 9 activation, and reduction in the tumor colony formation. This increase in TRAIL sensitivity involved mitochondrial membrane permeabilization resulting in the egress of cytochrome c and second mitochondrial activator of caspase/direct IAP-binding protein with low PI, cleavage of X-linked inhibitor of apoptosis protein, and activation of caspase 9. We link this execution signal to the ability of LY30 to downregulate cFLIP S and oligomerize DR5, thus facilitating the signaling of the death initiating signaling complex. The subsequent exposure to TRAIL resulted in processing/activation of caspase 8 and cleavage of its substrate, the BH3 protein Bid. These data provide a novel mechanism of action of this small molecule with the potential for use in TRAIL-resistant tumors. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis preferentially in tumor cells. 1,2 The selective responsiveness of TRAIL is attributed to the increased surface expression of TRAIL receptors (DR4 and DR5) on tumor cells. [3][4][5][6] In contrast, nontransformed cells express decoy receptors that provide a mechanism for evading apoptotic signaling initiated by TRAIL, and hence its tumor selectivity. 7,8 Unfortunately, as with most chemotherapeutic compounds, TRAIL-responsive tumors acquire a resistant phenotype which renders TRAIL therapy ineffective. 9,10 This has stimulated an enormous interest in identifying small molecule compounds that, when used in combination with TRAIL could sensitize tumor cells to TRAILinduced apoptosis. The desired consequence would be the need for a much lower therapeutic dose of TRAIL and at the same time an increase in the efficacy of the sensitizing drug. To that end, various groups have demonstrated that a variety of compounds and proteins (either upon silencing or upregulation) sensitize several classes of tumor cells to TRAIL-induced apoptosis. 11,12 LY303511 (LY30) is an inactive analog of LY294002 (LY29), a widely used inhibitor of the phosphatidylinositide-3-kinase (PI3K)/Akt survival pathway. 13 Previously, LY30 has been purported to have no effect on cells in contrast to its active counterpart, LY29. However, recent studies from our laboratory and other groups have demonstrated that LY30 does have a...

Introduction: Gastric cancer is a leading cause of global cancer mortality with an overall 5-year survival rate of approximately 20% and is particularly prevalent in many Asian countries. ASC amino acid transporter 2 (ASCT2), also known as SLC1A5, is a Na+-dependent glutamine/neutral amino acid transporter. ASCT2 acts as a high-affinity transporter of L-glutamine (Gln) and has been reported to be upregulated in a variety of cancerous tissues, including stomach. Several studies have shown that glutamine is a major nutrient contributing to cancer cell growth, so the glutamine metabolism pathway is an attractive target for gastric cancer treatment. In this study, we evaluated the efficacy of a novel anti-ASCT2 humanized monoclonal antibody, KM8094, as a therapeutic agent against gastric cancer and explored predictive biomarker candidates using patient-derived xenograft (PDX) mouse models. Materials and Methods: The tumor tissues from which the PDX models were generated were provided by Dr. Chan Shing Leng (NUS, Singapore). For the glutamine uptake assay, PDX-tumor cells were incubated with 3H-glutamine in the presence of KM8094. Intracellular 3H-glutamine was measured by liquid scintillation spectrophotometry. In vitro ADCC activity against human gastric cancer cells (SNU-16) was evaluated using human peripheral blood mononuclear cells (PBMC) from either gastric cancer patients or healthy controls as effector cells. In vivo antitumor activity of KM8094 was examined using PDX models. Mice were treated with KM8094 at 10 mg/kg i.v. or phosphate-buffered saline once weekly for 2-3 weeks. Finally, omics analyses of PDX tumor tissues were performed via a gene expression array analysis, DNA methylation array analysis, and metabolomics analysis. Results: All five gastric cancer PDX models histologically expressed ASCT2. KM8094 significantly inhibited the 3H-glutamine uptake in vitro and showed in vivo antitumor efficacy in the gastric cancer PDX models. In addition, KM8094 showed in vitro ADCC activity against gastric cancer cells using PBMCs from gastric cancer patients. These results indicated that KM8094 is a potential new therapeutic agent for gastric cancer treatment. Finally, we explored predictive biomarker candidates of KM8094 by a multi-omics analysis on the PDX models, and a few candidates were selected by genomic analyses. In addition, a metabolomics analysis revealed clear differences in the intracellular energy and redox status between responsive and nonresponsive PDX models. Conclusions: These results demonstrated the therapeutic potential of KM8094 for the treatment of gastric cancer and provide some insight into predictive biomarker candidates for antitumor efficacy. Citation Format: Noriyuki Kasai, Aya Sasakawa, Kenta Hosomi, Tze Wei Poh, Bernadette Lynn Chua, Wei Peng Yong, Jimmy So, Shing Leng Chan, Richie Soong, Koji Kono, Toshihiko Ishii, Kazuya Yamano. Evaluation of a novel monoclonal antibody targeting ASC amino acid transporter 2 using patient-derived xenograft mouse models of gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B035.

Purpose MUC1 is a tumor-associated antigen that is aberrantly expressed in cancer and inflammatory bowel disease (IBD). Even though immune cells express low MUC1 levels, their modulations of MUC1 are important in tumor progression. Consistent with previous clinical data that show increased myeloid-derived suppressor cells (MDSCs) in IBD, we now show that down-regulation of MUC1 on hematopoietic cells increases MDSCs in IBD, similar to our data in tumor bearing mice. We hypothesize that MDSC expansion in IBD is critical for tumor progression. Experimental Design In order to mechanistically confirm the linkage between Muc1 down-regulation and MDSC expansion, we generated chimeric mice that did not express Muc1 in the hematopoietic compartment (KO→WT). These mice were used in 2 models of colitis and colitis associated cancer (CAC) and their responses were compared to wildtype chimeras (WT→WT). Results KO→WT mice show increased levels of MDSCs during colitis and increased pro-tumorigenic signaling in the colon during CAC, resulting in larger colon tumors. RNA and protein analysis demonstrate increased up-regulation of metalloproteinases, collagenases, defensins, complements, growth factors, cytokines and chemokines in KO→WT mice as compared to WT→WT mice. Antibody-mediated depletion of MDSCs in mice during colitis reduced colon tumor formation during CAC. Conclusion Development of CAC is a serious complication of colitis and our data highlight MDSCs as a targetable link between inflammation and cancer. Additionally, the lack of MUC1 expression on MDSCs can be a novel marker for MDSCs, given that MDSCs are still not well characterized in human cancers.