Purpose The ER chaperone GRP78 translocates to the surface of tumor cells and promotes survival, metastasis, and resistance to therapy. An oncogenic function of cell surface GRP78 has been attributed to the activation of phosphoinositide 3-kinase (PI3K) pathway. We intend to use a novel anti-GRP78monoclonal antibody (MAb159) to attenuate PI3K signaling and inhibit tumor growth and metastasis. Experimental Design MAb159 was characterized biochemically. Anti-tumor activity was tested in cancer cell culture, tumor xenograft models, tumor metastasis models, and spontaneous tumor models. Cancer cells and tumor tissues were analyzed for PI3K activity. MAb159 was humanized and validated for diagnostic and therapeutic application. Results MAb159 specifically recognized surface GRP78, triggered GRP78 endocytosis, and localized to tumors but not normal organs in vivo. MAb159 inhibited tumor cell proliferation and enhanced tumor cell death both in vitro and in vivo. In MAb159 treated tumors, PI3K signaling was inhibited without compensatory MAPK pathway activation. Furthermore, MAb159 halted or reversed tumor progression in the spontaneous PTEN loss driven prostate and leukemia tumor models, and inhibited tumor growth and metastasis in xenograft models. Humanized MAb159, which retains high affinity, tumor specific localization, and the anti-tumor activity, was non-toxic in mice and had desirable pharmacokinetics. Conclusions GRP78 specific antibody MAb159 modulates PI3K pathway and inhibits tumor growth and metastasis. Humanized MAb159 will enter human trials shortly.

Axl is an oncogenic receptor tyrosine kinase that plays multiple roles in tumorigenesis and metastasis of many cancers. This study is the first to demonstrate that Axl is induced in Kaposi sarcoma and Kaposi sarcoma herpesvirus (KSHV) transformed endothelial cells. Conditionally, expression of one KSHV latency protein vFLIP induces Axl expression in endothelial cells. This induction can be blocked by nuclear factor-B inhibitor, consistent with the known vFLIP mechanism of action. KS cell lines lacking KSHV also have elevated Axl expression, which probably resulted from hypomethylation of AXL promoter. Axl activation activates downstream phosphoinositol-3 kinase signaling, and Axl knockdown by siRNA impairs phosphoinositol-3 kinase signaling. Furthermore, Axl knockdown inhibits KS cell growth and invasion. To explore the potential for translation of these findings, we generated monoclonal antibodies to block the biologic functions of Axl. MAb173, which induces receptor degradation, showed activity in vitro to inhibit KS cell invasion. Moreover, in vivo xenograft studies with KS cells with or without KSHV infection showed that MAb173 reduced tumor growth, increased tumor cell apoptosis, and markedly decreased Axl protein level in tumors. Axl thus has a potential role in KS pathogenesis and is a candidate for prognostic and therapeutic investigations. (Blood. 2010;116(2): 297-305) IntroductionKaposi sarcoma (KS) is a common cancer in HIV-infected persons. It originates from endothelial cells transformed by KS-associated herpesvirus (KSHV). 1 KS tumor cells have spindle cell morphology and characteristically produce excessive and abnormal vascular structures, which contain red blood cells and pigment from their lysis. 1 Nearly all KS cells carry KSHV with viral gene expression profile of latency. 2 It is thus assumed that latency proteins play an important role in KS pathogenesis.Gene expression analysis in KS cells versus endothelial cells by subtractive hybridization showed induction of Axl receptor kinase in KS cells (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Axl is a member of TAM receptor tyrosine kinases family that also includes Tyro3 and Mer. 3,4 Axl is composed of 2 immunoglobin-like domains and dual fibronectin type III repeats in the extracellular region, a single transmembrane and a cytoplasmic domain with kinase activity. 4 Protein S and growth arrest-specific 6 (Gas6) are the ligands for Axl, whereas only the latter has high affinity to Axl. 5,6 Axl activation and signaling have been implicated in multiple cellular responses, including cell survival, proliferation, migration, and adhesion. 7 In vascular biology, Axl receptor signaling has been shown to regulate vascular smooth muscle homeostasis, endothelial cell migration, and vascular network formation. [8][9][10] The primary downstream Axl signaling pathway appears to be phosphatidylinositol 3-kinase (PI3K) pathway. 9,11 However, the Janus kinase-signal transducers ...

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MicroRNAs (miRNAs) regulate cell proliferation and differentiation by controlling the expression of proteins involved in many signaling pathways. Recent studies have shown that dysregulation of miRNA expression is associated with increased tumorigenicity and a poor prognosis in several types of cancers. The miRNA let-7b is one of the severely downregulated miRNAs in mixed-lineage leukemia (MLL)-rearranged acute lymphoblastic leukemia (ALL) patients. In vitro transfection of leukemogenic MLL fusion genes into human embryonic kidney-293 cells suppressed let-7b expression. In leukemic cells with an MLL fusion gene, the regulatory region for let-7b expression was hypermethylated, and its expression was partially recovered after culturing the cells with the demethylating agent 5-azacitidine. These results suggest that loss of let-7b expression may be one of the consequences of oncogenic MLL fusion proteins, and contributes to leukemogenesis possibly through the upregulation of let-7b-regulated target genes with leukemogenic potential in hematopoietic cells. The enforced expression of let-7b in ALL cell lines with an MLL fusion gene inhibited their growth, indicating the possible use of let-7b as a new therapeutic tool for refractory infant ALL with an MLL fusion gene.

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The roles of age, gender, parental self-control and SES varied in a dual-systems model of self-control: good self-control and poor control. Implications of these results are discussed.

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