Gundelach, Justin H.

Abstract Medulloblastoma arising from the cerebellum is the most common pediatric brain malignancy, with leptomeningeal metastases often present at diagnosis and recurrence associated with poor clinical outcome. In this study, we used mouse medulloblastoma models to explore the relationship of tumor pathophysiology and dysregulated expression of the NOTCH pathway transcription factor ATOH1, which is present in aggressive medulloblastoma subtypes driven by aberrant Sonic Hedgehog/Patched (SHH/PTCH) signaling. In experiments with conditional ATOH1 mouse mutants crossed to Ptch1+/− mice, which develop SHH-driven medulloblastoma, animals with Atoh1 transgene expression developed highly penetrant medulloblastoma at a young age with extensive leptomeningeal disease and metastasis to the spinal cord and brain, resembling xenografts of human SHH medulloblastoma. Metastatic tumors retained abnormal SHH signaling like tumor xenografts. Conversely, ATOH1 expression was detected consistently in recurrent and metastatic SHH medulloblastoma. Chromatin immunoprecipitation sequencing and gene expression profiling identified candidate ATOH1 targets in tumor cells involved in development and tumorigenesis. Among these targets specific to metastatic tumors, there was an enrichment in those implicated in extracellular matrix remodeling activity, cytoskeletal network and interaction with microenvironment, indicating a shift in transcriptomic and epigenomic landscapes during metastasis. Treatment with bone morphogenetic protein or SHH pathway inhibitors decreased tumor cell proliferation and suppressed metastatic tumor growth, respectively. Our work reveals a dynamic ATOH1-driven molecular cascade underlying medulloblastoma metastasis that offers possible therapeutic opportunities. Cancer Res; 77(14); 3766–77. ©2017 AACR.

The di‐2‐pyridylketone thiosemicarbazone Dp44mT is a metal‐chelating compound that has been demonstrated to have potent activity as an anticancer agent. Here we report that it also has a dramatic inhibitory effect on T‐cell activation in vitro. We found that 10 nM Dp44mT (IC 50 3.2 nM) prevented the up‐regulation of surface CD25, and completely suppressed the activation and proliferation of splenic T cells isolated from Mus musculus that were stimulated with either T‐cell receptor (TCR) cross‐linking antibodies or phorbol ester plus ionomycin. In contrast, Dp44mT had no adverse effects on the survival of resting T cells. In addition, T cells stimulated in the presence of Dp44mT maintained the ability to upregulate CD69 surface expression and secrete interleu‐kin‐2. Consistent with these observations, Dp44mT did not inhibit multiple canonical signals downstream of the TCR, including the nuclear factor of activated T cells. The effects of Dp44mT were easily mitigated by addition of nontoxic copper chelators or N ‐acetylcysteine, indicating a role for copper and reactive oxygen species in its actions. Together, these findings suggest that Dp44mT may serve as a potent immunosuppressive agent that could complicate its use as a cancer therapeutic agent, but might have utility in the treatment of autoimmunity.—Gundelach, J. H., Madhavan, A. A., Wettstein, P. J., Bram, R. J. The anticancer drug Dp44mT inhibits T‐cell activation and CD25 through a copper‐dependent mechanism. FASEB J. 27, 782–792 (2013). www.fasebj.org

Abstract Calcium-modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein that is important during thymopoiesis. However, whether it serves a function in mature lymphocytes is unknown. In this article, we show that CAML is essential for survival of peripheral follicular (Fo) B cells. Conditional deletion of CAML in CD19-Cre transgenic mice caused a significant reduction in Fo cell numbers and increased rates of homeostatic proliferation. CAML-deficient Fo cells showed increased cellular turnover and normal proliferative ability. Although CAML-deficient Fo cells responded to AgR stimulation and to B cell activating factor, they displayed decreased survival and increased apoptosis following stimulation with LPS and IL-4 in vitro. Failure to survive was not due to aberrant B cell development in the absence of CAML, because induced deletion of the gene in mature cells resulted in a similar phenotype. These data establish an essential and ongoing role for CAML in the long-term survival of mature B cells.

Abstract Constitutively active RAS small GTPases promote the genesis of human cancers. An important goal in cancer biology is to identify means of countervailing activated RAS signaling to reverse malignant transformation. Oncogenic K-RAS mutations are found in virtually all pancreatic adenocarcinomas, making the RAS pathway an ideal target for therapeutic intervention. How to best contravene hyperactivated RAS signaling has remained elusive in human pancreatic cancers. Guided by the Drosophila studies, we reasoned that a downstream mediator of RAS signals might be a suitable anti-RAS target. The E3 ubiquitin ligase seven in absentia (SINA) is an essential downstream component of the Drosophila RAS signal transduction pathway. Thus, we determined the roles of the conserved human homologues of SINA, SIAHs, in mammalian RAS signaling and RAS-mediated tumorigenesis. We report that similar to its Drosophila counterpart, human SIAH is also required for oncogenic RAS signaling in pancreatic cancer. Inhibiting SIAH-dependent proteolysis blocked RAS-mediated focus formation in fibroblasts and abolished the tumor growth of human pancreatic cancer cells in soft agar as well as in athymic nude mice. Given the high level of conservation of RAS and SIAH function, our study provides useful insights into altered proteolysis in the RAS pathway in tumor initiation, progression, and oncogenesis. By targeting SIAH, we have found a novel means to contravene oncogenic RAS signaling and block RAS-mediated transformation/tumorigenesis. Thus, SIAH may offer a novel therapeutic target to halt tumor growth and ameliorate RAS-mediated pancreatic cancer. [Cancer Res 2007;67(24):11798–810]