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In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110␦ signaling in multiple myeloma ( IntroductionThe bone marrow (BM) microenvironment plays a crucial role in pathogenesis of multiple myeloma (MM) by promoting cell proliferation, survival, migration, and drug resistance. [1][2][3][4] The PI3K/AKT pathway mediates growth and drug resistance in MM cells and also plays a significant role in autophagy. 5,6 PI3K is activated via upstream tyrosine kinase-associated receptors for growth factors, cytokines, antigens, and costimulatory molecules. It in turn activates AKT, which mediates cell proliferation, cell cycle, apoptosis, and autophagy. 7 Class IA PI3K consists of 5 isoforms of regulatory subunits (p85␣, p50␣, p55␣, p85␤, and p55␥), which interact with class IA isoforms. Class IA PI3K is composed of p110␣, -␤, and -␦ isoforms. 8 Among the 8 distinct mammalian isoforms of PI3K, class I PI3Ks are responsible for Akt activation. Importantly, p110␦ is expressed in many cancers, including colon and bladder carcinoma, glioblastoma, and acute myeloid leukemia blasts. 9,10 In the current study, we demonstrate high expression of p110␦ in patient MM cells. Previous studies have shown that CAL-101, a potent and selective p110␦ inhibitor, has broad antitumor activity against cancer cells of hematologic origin. 11,12 Moreover, inhibition of p110␦ induces cleavage of caspases and LC3, consistent with apoptotic and autophagic cell death, respectively. Here we show that p110␦ blockade with CAL-101, a potent and selective p110␦ inhibitor, inhibits MM cell growth even in the presence of interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or bone marrow stromal cells (BMSCs), associated with decreased phosphorylation of AKT and P70S6k. We also confirmed inhibition of human MM cell growth triggered by p110␦ inhibition in our xenograft mouse models of human MM. These studies therefore show that small molecule inhibitors of p110␦ trigger significant anti-MM cytotoxicity both in vitro and in vivo, providing the framework for their clinical evaluation to improve patient outcome in MM. Methods Materialsp110␦ inhibitor CAL-101 and IC488743 were provided by Calistoga Pharmaceuticals. CAL-101 was dissolved in dimethyl sulphoxide at 10mM and stored at Ϫ20°C for in vitro study. IC488743 was dissolved in 1% carboxyl methylcellulose/0.5% Tween 80 and stored at 4°C for in vivo study. Recombinant human p110␣, ␤, ␥, and ␦ were reconstituted with sterile phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin. Bortezomib was provided by Millennium Pharmaceuticals. 3-Methyladenine was purchased from Sigma-Aldrich. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. Cell culture Dex 1460BLOOD, 2 SEPTEMBER 2010 ⅐ VOLUME 116, NUMBER 9For personal use only. on March 28, 2019. by guest www.bloodjournal.org From Germany). LB human MM ce...

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Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G 2 -M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.The cell surface proteoglycan CD138 (syndecan-1) is an integral membrane protein acting as a receptor for the extracellular matrix. Within the normal human hematopoetic compartment, CD138 is expressed on differentiated plasma cells and is a primary diagnostic marker of multiple myeloma (MM; ref. 1). The large extracellular domain of CD138 binds via its heparin sulfate chains to soluble extracellular molecules, including the growth factors epidermal growth factor, fibroblast growth factor, and hepatocyte growth factor, and to insoluble extracellular molecules, such as collagen and fibronectin (2, 3). CD138 also mediates cell-cell adhesion through interactions with heparinbinding molecules. Studies of plasma cell differentiation show that CD138 is a differentiation antigen (4) and a coreceptor for MM growth factors (5).Several monoclonal antibodies (mAb; i.e.,