Bradley, Judy Martina

Abstract Recent guidelines by the American Society of Clinical Oncologists recommend that obese individuals receive chemotherapy based on their actual body weight (ABW) without dose reduction. Contrary to prior reports, our own data demonstrate that ABW-dosed chemotherapy or immunotherapy is as effective and well tolerated by obese mice as by lean mice. Nonetheless, obese mice develop significantly greater weight loss in a dose-dependent manner during the entire period that they receive therapy, with corresponding quantifiable reductions in visceral fat depots. This disparity is not explained by altered food intake, as both lean and obese mice experience less than a week of anorexia which does not recur after Cycle 1 of treatment. Furthermore, malabsorption is absent. These observations are relevant to multiple classes of chemo agents. Altered pharmacokinetics of chemotherapy agents in adipose tissue have been reported, yet the pharmacodynamics of obesity remain speculative. Fasting blood glucose monitoring demonstrates glucose level variances which are temporally linked to treatment, rendering it likely that chemotherapy and immunotherapy provoke adipose tissue lipolysis, or alternatively cytoreduction or immunomodulation of adipocyte progenitor cells. Understanding these effects could contribute to improved treatment regimens in the setting of obesity.

A strategically designed sialyl-Tn building block was synthesized in a stereospecific manner for the rapid linear construction of a glycolipopeptide cancer vaccine.

Abstract We report that administration of celecoxib, a specific cyclooxygenase-2 (COX-2) inhibitor, in combination with a dendritic cell-based cancer vaccine significantly augments vaccine efficacy in reducing primary tumor burden, preventing metastasis, and increasing survival. This combination treatment was tested in MMTV-PyV MT mice that develop spontaneous mammary gland tumors with metastasis to the lungs and bone marrow. Improved vaccine potency was associated with an increase in tumor-specific CTLs. Enhanced CTL activity was attributed to a significant decrease in levels of tumor-associated IDO, a negative regulator of T cell activity. We present data suggesting that inhibiting COX-2 activity in vivo regulates IDO expression within the tumor microenvironment; this is further corroborated in the MDA-MB-231 human breast cancer cell line. Thus, a novel mechanism of COX-2-induced immunosuppression via regulation of IDO has emerged that may have implications in designing future cancer vaccines.

Abstract MUC1, a membrane tethered mucin glycoprotein, is overexpressed and aberrantly glycosylated in >80% of human ductal pancreatic adenocarcinoma. However, the role of MUC1 in pancreatic cancer has been elusive, partly due to the lack of an appropriate model. We report the characterization of a novel mouse model that expresses human MUC1 as a self molecule (PDA.MUC1 mice). Pancreatic tumors arise in an appropriate MUC1-tolerant background within an immune-competent host. Significant enhancement in the development of pancreatic intraepithelial preneoplastic lesions and progression to adenocarcinoma is observed in PDA.MUC1 mice, possibly due to increased proliferation. Tumors from PDA.MUC1 mice express higher levels of cyclooxygenase-2 and IDO compared with PDA mice lacking MUC1, especially during early stages of tumor development. The increased proinflammatory milieu correlates with an increased percentage of regulatory T cells and myeloid suppressor cells in the pancreatic tumor and tumor draining lymph nodes. Data shows that during pancreatic cancer progression, MUC1-mediated mechanisms enhance the onset and progression of the disease, which in turn regulate the immune responses. Thus, the mouse model is ideally suited for testing novel chemopreventive and therapeutic strategies against pancreatic cancer.

Abstract With a 5-year survival rate of <5%, pancreatic cancer is one of the most rapidly fatal malignancies. Current protocols for the treatment of pancreas cancer are not as effective as we desire. In this study, we show that a novel Mucin-1 (MUC1)-based vaccine in combination with a cyclooxygenase-2 inhibitor (celecoxib), and low-dose chemotherapy (gemcitabine) was effective in preventing the progression of preneoplastic intraepithelial lesions to invasive pancreatic ductal adenocarcinomas. The study was conducted in an appropriate triple transgenic model of spontaneous pancreatic cancer induced by the KRASG12D mutation and that expresses human MUC1 as a self molecule. The combination treatment elicited robust antitumor cellular and humoral immune responses and was associated with increased apoptosis in the tumor. The mechanism for the increased immune response was attributed to the down-regulation of circulating prostaglandin E2 and indoleamine 2, 3,-dioxygenase enzymatic activity, as well as decreased levels of T regulatory and myeloid suppressor cells within the tumor microenvironment. The preclinical data provide the rationale to design clinical trials with a combination of MUC1-based vaccine, celecoxib, and gemcitabine for the treatment of pancreatic cancer.

The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O -linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam 3 CysSK 4 , a peptide T helper epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.

Abstract MUC1 is an excellent target for immunotherapeutic strategies for cancers expressing Mucin 1 (breast, pancreas etc). MUC1 was recently selected by the NCI working group as the number 2 cancer vaccine target antigen, based on its immunogenicity, oncogenicity and wide-spread cell-surface expression. More than 90% of breast cancers over express MUC1, it stimulates both humoral and cellular immunity, its normal apical distribution is lost in cancer cells and aberrant glycosylation exposes peptide epitopes and novel carbohydrate antigens. Clinical trials using unglycosylated MUC1 epitopes have resulted in MUC1 specific cytotoxic T lymphocytes (CTLs) with limited effects on clinical response. Patients with breast tumors have exhibited spontaneous immune responses to MUC1 with the presence of T cells and antibodies specific for glycosylated MUC1. Because these natural immune responses are often not efficient in eradicating the tumor, it is necessary to specifically modulate these immune responses. Hence it is possible that better immunogenicity would be obtained using peptides more representative of the novel (glycosylated) forms of MUC1 as seen in cancer to which individuals may be less tolerant. The presentation of antigenic peptides by MHC class I molecules is crucial for the development of vaccines. MUC1 peptides from the VNTR region, which has five potential glycosylation sites, can be presented by a variety of class I molecules and can generate MUC1 specific CTLs. But these epitopes lack definite anchors and bind with low affinity compared to high affinity peptides. Hence, we developed several peptides from the MUC1 H-2Kb peptide (SAPDTRPAP) epitope with all possible modifications to improve their MHC and TCR binding efficiency. Preclinical studies were performed using the modified H-2Kb-restricted peptides along with the DC stimulants (CpG-ODN and GM-CSF) and foreign helper epitope (hepatitis B virus optimized I-Ab T helper peptide) in incomplete Freund's adjuvant in C57BL/6 mice transgenic for human MUC1. Peptides that were glycosylated at threonine and had methionine or leucine as the terminal amino acid induced a strong CTL response and were able to overcome tolerance in MUC1.Tg.mice. In the breast cancer model with the MUC1- expressing MMT cell line, the onset of tumor was delayed with peptide immunization and there was significant reduction in the tumor weight. Increased lytic activity of CD8+ CTLs and infiltration of T lymphocytes to the tumor site were observed with modified peptides as compared to the effect of MHC class I Kb and Db restricted native MUC1 peptides. Interestingly, the CTLs specific for glycosylated peptides were able to recognize both the Tn (GalNAc) and peptide epitopes. Thus, MUC1 peptides with carbohydrate at the appropriate position may be an optimal vaccine candidate because they can generate CTLs of dual specificity and high affinity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2403.

Abstract 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 β-catenin to promote oncogenesis. Given the importance of β-catenin in hematopoiesis, we hypothesized that the MUC1 regulation of β-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 β-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 MDSC-mediated 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]

Abstract Much of the ability of the MUC1 oncoprotein to foster tumorigenesis and tumor progression likely originates from the interaction of its cytoplasmic tail with proteins involved in oncogenic signaling. Many of these interactions are regulated by phosphorylation, as the cytoplasmic tail contains seven highly conserved tyrosines and several serine/threonine phosphorylation sites. We have developed a cell line–based model system to study the effects of tyrosine phosphorylation on MUC1 signaling, with particular emphasis on its effects on gene transcription. COS-7 cells, which lack endogenous MUC1, were stably infected with wild-type MUC1 or a MUC1 construct lacking all seven tyrosines (MUC1 Y0) and analyzed for effects on transcription mediated by the extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) pathways. COS.MUC1 Y0 cells showed heightened active ERK1/2 with increased activator protein-1 (AP-1) and signal transducer and activator of transcription 3 (STAT3) transcriptional activity; there was also a simultaneous decrease in NF-κB transcriptional activity and nuclear localization. These changes altered the phenotype of COS.MUC1 Y0 cells, as this line displayed increased invasion and enhanced [3H]thymidine incorporation. Analysis of the three lines also showed significant differences in their cell cycle profile and bromodeoxyuridine incorporation when the cells were serum starved. These data support the growing evidence that MUC1 is involved in transcriptional regulation and link MUC1 for the first time to the NF-κB pathway. (Mol Cancer Res 2006;4(7):489–97)