Marler, Ronald J.

Abstract Multiple myeloma tumors universally target one of the three human cyclin D genes (CCND1, CCND2 or CCND3) for dysregulation (Bergsagel et al., 2005, Blood;106:296). To identify novel pharmaceutical inhibitors of cyclin D2 (CCND2) transactivation we therefore screened the Lopac, Prestwick and Spectrum libraries of drugs and natural compounds (n>4000) using NIH 3T3 cells stably expressing the CCND2 promoter driving a luciferase reporter gene. From this library screen and subsequent validation experiments we identified Kinetin riboside, a nucleoside analogue and plant cytokinin hormone, as a novel inhibitor of CCND1+CCND2 transactivation. By immunoblotting, Kinetin riboside induced rapid suppression of cyclin D1 and D2 proteins (<6 hours) in H929, JJN3, Kms11 and U266 human myeloma cell lines (HMCL) that all over-express either cyclin D1 or D2 due to a range of transforming events relevant to myeloma (including deregulation of FGFR3, MMSET or c-Maf oncogenes, or translocation of CCND1 to the IgH locus). Similar results were obtained in primary CD138+ purified myeloma cells from 5/6 patients. To verify that cyclin D1 and D2 suppression induced by Kinetin riboside is a direct effect and does not occur secondary to cellular arrest in S-phase (when cyclin D protein levels decline) we examined the effects of Kinetin riboside on the cell cycle profile of HMCL. Within 20 hours Kinetin riboside caused the proportion of cells entering S-phase to fall by 50–70% in all HMCL tested, consistent with primary cyclin D suppression and secondary G0/G1 arrest. By MTT assay, Kinetin riboside is cytotoxic to HMCL with an IC50 of <1.7 mg/L (5uM) in 8/12 lines and <5mg/L (15uM) in 11/12 HMCL. By comparison, toxicity studies in Balb/c mice confirm that Kinetin riboside is tolerated in vivo at a dose of 80–100mg/kg i.p. or 25mg/kg i.v. Importantly, Kinetin riboside shows potent synergy with dexamethasone in HMCL that are poorly responsive to one or other agent and Kinetin riboside activity persists during co-culture with myeloma growth factors IL-6, IGF-1 and Baff. Moreover, when tested against unsorted patient bone marrow samples, Kinetin riboside preferentially killed CD138+ myeloma cells at up to 5–8 fold greater rate than normal marrow progenitors. Kinetin riboside induced myeloma cell death is mediated by apoptosis and is associated with caspase 9 cleavage and annexin V binding. Mechanistically, we show that kinetin riboside blocks CCND2 promoter transactivation induced by cAMP or by regulatory phosphoproteins (activated by Forskolin or the PP2A inhibitor, Cantharadin, respectively) and also blocks cis-activation of CCND1 induced by translocation to the IgH enhancer and trans-activation of CCND2 induced by cMaf or FGFR3 over-expression, indicating that kinetin riboside acts at a distal level to block transactivation of CCND1+CCND2 induced by multiple factors. Gene expression profiling reveals that KinR causes rapid induction of the transcriptional repressor, cAMP Response Element Modifier (CREM), which has been reported to bind the cyclin D2 promoter to regulate cyclin D2 expression (Muniz, 2006, Biol Reprod.), providing a putative mechanism for targeted suppression of CCND1+CCND2. Together these studies demonstrate a novel targeted mechanism and substantial pre-clinical activity for kinetin riboside and provide a rationale for clinical evaluation of this drug to improve the outcome of multiple myeloma.

Background Though the importance of the transmembrane mucin MUC1 in mammary oncogenesis has long been recognized, the relative contributions of the cytoplasmic tail and tandem repeat domains are poorly understood. Methods To address this, mouse models of mammary carcinogenesis were created expressing full-length cytoplasmic tail-deleted, or tandem repeat-deleted MUC1 constructs. Results Overexpression of full-length MUC1 resulted in tumor formation in young mice (≤ 12 months); however, loss of either the cytoplasmic tail or the tandem repeat domain abrogated this oncogenic capacity. Aged mice in all strains developed late-onset mammary tumors similar to those previously described for the FVB background. Conclusions This study is the first spontaneous cancer model to address the relative importance of the cytoplasmic tail and tandem repeat domains to MUC1-driven mammary oncogenesis, and suggests that both of these domains are essential for tumor formation.

Transplantation of gene-corrected autologous hepatocytes can cure metabolic disease in a preclinical pig model of hereditary tyrosinemia type 1.

Abstract The role of the transcription factor GLI1 in pancreatic tumor initiation promoting the progression of preneoplastic lesions into tumors is well established. However, its function at later stages of pancreatic carcinogenesis remains poorly understood. To address this issue, we crossed the GLI1 knockout animal (GKO) with cre-dependent pancreatic activation of oncogenic KRAS concomitant with loss of the tumor suppressor TP53 (KPC). Interestingly, in this model, GLI1 played a tumor protective function, where survival of GKO/KPC mice was reduced compared to KPC littermates. Both cohorts developed pancreatic cancer without significant histopathological differences in survival studies. However, analysis of mice using ultrasound-based imaging at earlier time-points showed increased tumor burden in GKO/KPC mice. These animals have larger tumors, decreased body weight, increased lactate dehydrogenase production, and leukopenia. Expression studies identified FAS and FAS ligand as potential mediators of this phenomenon. The FAS /FASL axis, an apoptotic inducer, plays a role in the progression of pancreatic cancer, where its expression is usually lost or significantly reduced in advanced stages of the disease. Chromatin immunoprecipitation assays identified FAS and FASL as direct targets of GLI1 where GKO/KPC mice showed lower levels of this ligand compared to KPC animals. Furthermore, GLI1 overexpression and knockdown studies demonstrate a key regulatory role for GLI1 in both FAS and FASL expression. Finally, decreased levels of apoptosis were detected in the tumor tissue in the absence of GLI1 by TUNEL staining. Together, these findings define a novel pathway regulated by GLI1 controlling pancreatic tumor progression and provide a new theoretical framework to help the design and analysis of trials targeting GLI1-related pathways. Citation Format: Lisa D. Mills, Lizhi Zhang, Ronald J. Marler, Phyllis A. Svingen, Maite Fernandez-Barena, Maneesh Dave, William Bamlet, Robert McWilliams, William A. Faubion, Martin E. Fernandez-Zapico. Loss of GLI1 accelerates pancreatic cancer progression in the KPC mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A76.

Abstract Lymphocyte differentiation from naive CD4+ T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DCXAb) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DCXAb interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFβ, fail to suppress T cell responses, and gain the ability to produce IFN-γ, IL-17, and TNF-α. The ability of IL-6+ DCXAb and the inability of IL-6−/− DCXAb vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DCXAb into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25− T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.

Abstract 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 downregulation 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: To mechanistically confirm the linkage between Muc1 downregulation and MDSC expansion, we generated chimeric mice that did not express Muc1 in the hematopoietic compartment (KO→WT). These mice were used in two models of colitis and colitis-associated cancer (CAC) and their responses were compared with wild-type (WT) chimeras (WT→WT). Results: KO→WT mice show increased levels of MDSCs during colitis and increased protumorigenic signaling in the colon during CAC, resulting in larger colon tumors. RNA and protein analysis show increased upregulation of metalloproteinases, collagenases, defensins, complements, growth factors, cytokines, and chemokines in KO→WT mice as compared with 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. In addition, 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. Clin Cancer Res; 19(18); 5039–52. ©2013 AACR.

Abstract Recent molecular and pathologic evidence indicate that high-grade serous carcinomas of the ovary, which are the most common type of epithelial carcinomas, may arise from the distal part of fallopian tubes or tubal carcinoma implants. Oviductal glycoprotein 1 (Ovgp1), a glycosylated protein found in the epithelial cells in the oviduct, is the only specific tubal marker so far. Ovgp1 promoter-driven SV40 large T-antigen transgenic mice develop tumors in the female reproductive organs. To characterize these tumors, we performed immunohistochemical analysis in paraffin-embedded sections. Mice were sacrificed at 8 or 13 weeks old. Macroscopically we observed large tumors in the uterus and cervix and markedly enlarged region in the oviduct. Due to the large tumor in the uterus and cervix, we could not keep the mice longer than 13 weeks. Histologically, there were no major differences in these affected regions between 8 weeks old and 13 weeks old mice. We observed, (i) relatively small ovaries and ovarian follicular hyperplasia, (ii) intact ovarian surface epithelium showing no atypical histology, (iii) atypical epithelial hyperplasia within the oviduct (with endometrioid architectural and cytologic features), (iv) leiomyosarcomatous proliferation in the myometrium, and (v) squamous carcinoma in situ in the cervix or early invasive cervical cancer with extensive papillary architecture. We did not detect metastasis from the tumor to other organs. The atypical abnormalities of the oviduct were more significant in the proximal site to the uterus than the distal site. The proximal oviduct demonstrated weak and spotty p53 expression, beneath the epithelium, similar to p53 signature in the human oviduct. We hypothesized that stem cell components may contribute to the tumor development and that putative cancer stem cells could be identified by stem cell markers. In the oviduct epithelium, Bmi1 expression was not detectable. Cytoplasmic expression of Sox2 was observed in all epithelial cells of the oviduct. There was little correlation between Sox2/Bmi1 staining and p53 staining in the oviduct. Interestingly, Sox2 and Bmi1 staining in the wild-type control mice showed immunoreactive cells interspersed between glandular structures, a pattern similar to previously described label-retaining cells in the endometrium. The expression pattern in Ovgp1-SV40 mice, Bmi1 and Sox2 expression were more obvious in the endometrial stromal area but not in the leiomyosarcoma region, which implies that stem cell components in the endometrial region may support the leiomyosarcoma progression in this mouse model. In conclusion, Ovgp1-SV40 transgenic mouse is a useful model for studies involving fallopian and lower genital tract carcinogenesis. 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 3272. doi:1538-7445.AM2012-3272