The ciliary GTPase Arl3 maintains tissue architecture by directing planar spindle orientation during epidermal morphogenesis

Arl/ARF GTPases regulate ciliary trafficking, but their tissue specific functions are unclear. Here we demonstrate that ciliary GTPase Arl3 is required for mitotic spindle orientation of basal stem cells during skin development. Arl3 loss diminished cell divisions within the plane of the epithelium, leading to increased perpendicular divisions, expansion of progenitor cells, and loss of epithelial integrity. These observations suggest that an Arl3-dependent mechanism maintains cell division polarity along the tissue axis, and disruption of planar spindle orientation has detrimental consequences for epidermal architecture. Defects in planar cell polarity (PCP) can disrupt spindle positioning during tissue morphogenesis. Upon Arl3 loss, PCP signaling molecules Celsr1 and Vangle2 failed to maintain planar polarized distributions, resulting in defective hair follicle angling, a hallmark of disrupted PCP. In the absence of Celsr1 polarity, Frizzled-6 lost its asymmetrical distribution and abnormally segregated to the apical cortex of basal cells. We propose that Arl3 regulates polarized endosomal trafficking of PCP components to compartmentalized membrane domains. Cell-cell communication via ciliary GTPase signaling directs mitotic spindle orientation and PCP signaling, processes critical for the maintenance of epithelial architecture.</jats:p

Abstract A94: Imatinib modulates CD4+ T cells in gastrointestinal stromal tumor (GIST).

Abstract Introduction: Imatinib mesylate partially exerts its antitumor effects in GIST by inhibiting tumor production of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (Ido), thereby activating antitumor CD8+ T cells. The role of conventional CD4+ T cells (CD4+FoxP3-) in imatinib therapy is unknown. We examined the effects of imatinib on CD4+ T cells in GIST. Methods: We injected imatinib or saline intraperitoneally for one week to KitV558-/+ mice that develop spontaneous GISTs. 1-methyl-D-tryptophan (D-1MT), an Ido inhibitor was given via oral gavage. We assessed treatment response by tumor weight and immune cells by flow cytometry. Regulatory T cells (Treg) were defined as CD4+FoxP3+. Matched blood and tumor specimens from GIST patients were freshly analyzed for immune composition. Tumors were classified as untreated, sensitive, or resistant to imatinib based on radiologic assessment just before the time of surgery. Results: Imatinib increased CD4+ number, activation, and degranulation in the draining lymph node (DLN) but not the spleen of GIST mice (p&amp;lt;0.05). In the tumor, imatinib increased CD4+ number, activation, proliferation, and the intratumoral CD4+/Treg ratio (p&amp;lt;0.05). Conversely, Ido inhibition did not alter CD4+ number, activation, proliferation, or the CD4+/Treg ratio in the DLN or tumor. In untreated human GISTs (n=14), CD4+ T cells demonstrated greater activation and memory phenotype compared to autologous blood CD4+ T cells, but secreted Th2 cytokines on in vitro restimulation (p&amp;lt;0.05). Imatinib treatment altered the intratumoral CD4+/Treg ratio, with a higher ratio in sensitive tumors (n=17) compared to resistant tumors (n=12; p&amp;lt;0.05). Conclusion: In mouse GIST, imatinib activates CD4+ T cells independently of Ido and increases the intratumoral CD4+/Treg ratio, a hallmark of immunologic outcome. Similarly, imatinib sensitive human GISTs have a higher CD4+/Treg ratio than resistant tumors. Hence, CD4+ T cells may contribute to the antitumor effects of imatinib in GIST. Combination immunotherapy with imatinib and CD4+ T cell modulating agents may be a promising therapy for GIST. Citation Format: Vinod P. Balachandran, Michael Cavnar, Shan Zeng, Zubin Bamboat, Lee Ocuin, Hebroon Obaid, Eric Sorenson, Teresa Kim, Rachel Popow, Ronald P. DeMatteo. Imatinib modulates CD4+ T cells in gastrointestinal stromal tumor (GIST). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A94.</jats:p

Nilotinib protects the murine liver from ischemia/reperfusion injury.

BACKGROUND & AIMS: The mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK), and p38, mediate liver ischemia/reperfusion (I/R) injury via cell death and inflammatory cytokine expression, respectively. Nilotinib is an orally available receptor tyrosine kinase inhibitor used for chronic myelogenous leukemia that also has in vitro activity against JNK and p38. In this study, we examine its therapeutic potential against hepatic I/R injury. METHODS: The effects of nilotinib on liver I/R injury were tested using a murine model of warm, segmental liver I/R. Serum ALT was measured and livers were analyzed by histology, RT-PCR, Western blot, and flow cytometry. The in vitro effects of nilotinib on hepatocyte and non-parenchymal cell (NPC) MAPK activation and cytokine production were also tested. RESULTS: Mice receiving nilotinib had markedly lower serum ALT levels and less histologic injury and apoptosis following liver I/R. Nilotinib did not inhibit its known receptor tyrosine kinases. Nilotinib lowered intrahepatic expression of IL-1β, IL-6, MCP-1, and MIP-2 and systemic levels of IL-6, MCP-1, and TNF. Nilotinib reduced NPC activation of p38 MAPK signaling and decreased the recruitment of inflammatory monocytes and their production of TNF. Nilotinib attenuated JNK phosphorylation and hepatocellular apoptosis. In vitro, nilotinib demonstrated direct inhibition of JNK activation in isolated hepatocytes cultured under hypoxic conditions, and blocked activation of p38 MAPK and cytokine production by stimulated NPCs. CONCLUSIONS: Nilotinib lowers both liver JNK activation and NPC p38 MAPK activation and may be useful for ameliorating liver I/R injury in humans

Imatinib potentiates antitumor T cell responses in gastrointestinal stromal tumor through the inhibition of Ido.

Imatinib mesylate targets mutated KIT oncoproteins in gastrointestinal stromal tumor (GIST) and produces a clinical response in 80% of patients. The mechanism is believed to depend predominantly on the inhibition of KIT-driven signals for tumor-cell survival and proliferation. Using a mouse model of spontaneous GIST, we found that the immune system contributes substantially to the antitumor effects of imatinib. Imatinib therapy activated CD8(+) T cells and induced regulatory T cell (T(reg) cell) apoptosis within the tumor by reducing tumor-cell expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (Ido). Concurrent immunotherapy augmented the efficacy of imatinib in mouse GIST. In freshly obtained human GIST specimens, the T cell profile correlated with imatinib sensitivity and IDO expression. Thus, T cells are crucial to the antitumor effects of imatinib in GIST, and concomitant immunotherapy may further improve outcomes in human cancers treated with targeted agents

KIT oncogene inhibition drives intratumoral macrophage M2 polarization.

Tumor-associated macrophages (TAMs) are a major component of the cancer microenvironment. Modulation of TAMs is under intense investigation because they are thought to be nearly always of the M2 subtype, which supports tumor growth. Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and typically results from an activating mutation in the KIT oncogene. Using a spontaneous mouse model of GIST and 57 freshly procured human GISTs, we discovered that TAMs displayed an M1-like phenotype and function at baseline. In both mice and humans, the KIT oncoprotein inhibitor imatinib polarized TAMs to become M2-like, a process which involved TAM interaction with apoptotic tumor cells leading to the induction of CCAAT/enhancer binding protein (C/EBP) transcription factors. In human GISTs that eventually developed resistance to imatinib, TAMs reverted to an M1-like phenotype and had a similar gene expression profile as TAMs from untreated human GISTs. Therefore, TAM polarization depends on tumor cell oncogene activity and has important implications for immunotherapeutic strategies in human cancers