Xiaoxian Zhao

Protein Kinase Cδ Is Required for p47phox Phosphorylation and Translocation in Activated Human Monocytes

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCδ, -ε, -θ, and -η) and conventional PKC (including PKCα and -β), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCε, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCδ, -θ, or -η is required. Furthermore, rottlerin (at doses that inhibit PKCδ activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O⨪2 production at similar doses. In addition to pharmacological inhibitors, PKCδ-specific antisense oligodeoxyribonucleotides were used. PKCδ antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCδ in vitro. Furthermore, lysate-derived PKCδ from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCδ plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.

Lysophosphatidic Acid Is Constitutively Produced by Human Peritoneal Mesothelial Cells and Enhances Adhesion, Migration, and Invasion of Ovarian Cancer Cells

Lysophosphatidic acid (LPA) is both a potential marker and a therapeutic target for ovarian cancer. It is critical to identify the sources of elevated LPA levels in ascites and blood of patients with ovarian cancer. We show here that human peritoneal mesothelial cells constitutively produce LPA, which accounts for a significant portion of the chemotactic activity of the conditioned medium from peritoneal mesothelial cells to ovarian cancer cells. Both production of LPA by peritoneal mesothelial cells and the chemotactic activity in the conditioned medium can be blocked by HELSS [an inhibitor of the calcium-independent phospholipase A(2) (iPLA(2))] and AACOCF(3) [an inhibitor of both cytosolic PLA(2) (cPLA(2)) and iPLA(2)]. Moreover, cell-based enzymatic activity assays for PLA(2) indicate that peritoneal mesothelial cells have strong constitutive PLA(2) activity. Receptors for LPA, LPA(2), and LPA(3) are involved in the conditioned medium-induced chemotactic activity. Invasion of ovarian cancer cells into peritoneal mesothelial cells has also been analyzed and shown to require PLA(2), LPA receptors, and the mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase signaling pathway. Thus, we show here, for the first time, that human peritoneal mesothelial cells constitutively produce bioactive lipid signaling molecules, such as LPA, via iPLA(2) and/or cPLA(2) activities. Conditioned medium from peritoneal mesothelial cells stimulate migration, adhesion, and invasion of ovarian cancer cells, and may play similar roles in vivo.

Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex.

Phagocyte NADPH oxidase is critical for defense against pathogens and contributes to inflammatory tissue injury. One component of the NADPH oxidase complex is the small GTP-binding protein Rac. There are two isoforms of Rac, and Rac2 is the predominant isoform in neutrophils and has been shown to be essential for NADPH oxidase activity. In primary human monocytes we report that in contrast to neutrophils, Rac1 is the predominantly expressed isoform. Upon monocyte activation by a variety of agents, we found that Rac1 dissociates from Rho GDP dissociation inhibitor (RhoGDI) and translocates to the membrane. We also found that Rac1 interacts with two other NADPH oxidase components, p67phox and p47phox, upon monocyte activation. These data indicate that Rac1, and not Rac2, is a component of the activated NADPH oxidase in monocytes. This finding suggests that it may be possible to selectively interfere with either monocyte or neutrophil NADPH oxidase activity, thereby selectively targeting chronic versus acute inflammatory processes.

Caspase-3-dependent Activation of Calcium-independent Phospholipase A2 Enhances Cell Migration in Non-apoptotic Ovarian Cancer Cells

Calcium-independent phospholipase A2 (iPLA2) plays a pivotal role in phospholipid remodeling and many other biological processes, including inflammation and cancer development. iPLA2 can be activated by caspase-3 via a proteolytic process in apoptotic cells. In this study we identify novel signaling and functional loops of iPLA2 activation leading to migration of non-apoptotic human ovarian cancer cells. The extracellular matrix protein, laminin-10/11, but not collagen I, induces integrin- and caspase-3-dependent cleavage and activation of overexpressed and endogenous iPLA2. The truncated iPLA2 (amino acids 514-806) generates lysophosphatidic acid and arachidonic acid. Arachidonic acid is important for enhancing cell migration toward laminin-10/11. Lysophosphatidic acid activates Akt that in turn acts in a feedback loop to block the cleavage of poly-(ADP-ribose) polymerase and DNA fragmentation factor as well as prevent apoptosis. By using pharmacological inhibitors, blocking antibodies, and genetic approaches (such as point mutations, dominant negative forms of genes, and siRNAs against specific targets), we show that β1, but not β4, integrin is involved in iPLA2 activation and cell migration to laminin-10/11. The role of caspase-3 in iPLA2 activation and cell migration are supported by several lines of evidence. 1) Point mutation of Asp513 (a cleavage site of caspase-3 in iPLA2) to Ala blocks laminin-10/11-induced cleavage and activation of overexpressed iPLA2, whereas mutation of Asp733 to Ala has no such effect, 2) treatment of inhibitors or a small interfering RNA against caspase-3 results in decreased cell migration toward laminin-10/11, and 3) selective caspase-3 inhibitor blocks cleavage of endogenous iPLA2 induced by laminin-10/11. Importantly, small interfering RNA-mediated down-regulation of endogenous iPLA2 expression in ovarian carcinoma HEY cells results in decreased migration toward laminin, suggesting that our findings are pathophysiologically important.

Targeting C-type lectin-like molecule-1 for antibody-mediated immunotherapy in acute myeloid leukemia

Background C-type lectin-like molecule-1 is a transmembrane receptor expressed on myeloid cells, acute myeloid leukemia blasts and leukemic stem cells. To validate the potential of this receptor as a therapeutic target in acute myeloid leukemia, we generated a series of monoclonal antibodies against the extracellular domain of C-type lectin-like molecule-1 and used them to extend the expression profile analysis of acute myeloid leukemia cells and to select cytotoxic monoclonal antibodies against acute myeloid leukemia cells in preclinical models. Design and Methods C-type lectin-like molecule-1 expression was analyzed in acute myeloid leukemia cell lines, and in myeloid derived cells from patients with acute myeloid leukemia and healthy donors. Anti-C-type lectin-like molecule-1 antibody-mediated in vitro cytotoxic activity against acute myeloid leukemia blasts/cell lines and in vivo anti-cancer activity in a mouse xenograft model were assessed. Internalization of C-type lectin-like molecule-1 monoclonal antibodies upon receptor ligation was also investigated. Results C-type lectin-like molecule-1 was expressed in 86.5% (45/52) of cases of acute myeloid leukemia, in 54.5% (12/22) of acute myeloid leukemia CD34+/CD38− stem cells, but not in acute lymphoblastic leukemia blasts (n=5). Selected anti-C-type lectin-like molecule-1 monoclonal antibodies mediated dose-dependent complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity specifically against acute myeloid leukemia-derived cell lines. Exogenous expression of the transmembrane receptor in HEK293 cells rendered the cells susceptible to antibody-mediated killing by monoclonal antibodies to the receptor. Furthermore, these monoclonal antibodies demonstrated strong complement-dependent cytotoxicity against freshly isolated acute myeloid leukemia blasts (15/16 cases; 94%). The monoclonal antibodies were efficiently internalized upon binding to C-type lectin-like molecule-1 in HL-60 cells. Moreover, a lead chimeric C-type lectin-like molecule-1 monoclonal antibody reduced the tumor size in xenograft mice implanted with HL-60 cells. Conclusions Our results demonstrate that targeting C-type lectin-like molecule-1 with specific cytotoxic monoclonal antibodies is an attractive approach which could lead to novel therapies for acute myeloid leukemia.

Protein kinase Cδ regulates p67phox phosphorylation in human monocytes

Phosphorylation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components p67phox and p47phox accompanies the assembly and activation of this enzyme complex. We have previously reported that activation of human monocytes with opsonized zymosan (ZOP), a potent stimulator of NADPH oxidase activity, results in the phosphorylation of p67phox and p47phox. In this study, we investigated the regulation of p67phox phosphorylation. Although protein kinase C (PKC)α has previously been shown to regulate NADPH oxidase activity, we found that inhibition of PKCα had no effect on p67phox phosphorylation. Our studies demonstrate that pretreatment of monocytes with antisense oligodeoxyribonucleotides specific for PKCδ or rottlerin, a selective inhibitor for PKCδ, inhibited the phosphorylation of p67phox in monocytes, and Go6976, a specific inhibitor for conventional PKCs, PKCα and PKCβ, had no such inhibitory effect. Additional studies indicate that ZOP stimulation of monocytes induces PKCδ and p67phox to form a complex. We also demonstrate that lysates from activated monocytes as well as PKCδ immunoprecipitates from activated monocytes can phosphorylate p67phox in vitro and that pretreatment of monocytes with rottlerin blocked the phosphorylation in each case. We further show that recombinant PKCδ can phosphorylate p67phox in vitro. Finally, we show that PKCδ‐deficient monocytes produce significantly less superoxide anion in response to ZOP stimulation, thus emphasizing the functional significance of the PKCδ regulation of p67phox phosphorylation. Taken together, this is the first report to describe the requirement of PKCδ in regulating the phosphorylation of p67phox and the related NADPH oxidase activity in primary human monocytes.

Combination of ibrutinib with ABT-199: synergistic effects on proliferation inhibition and apoptosis in mantle cell lymphoma cells through perturbation of BTK, AKT and BCL2 pathways

Mantle cell lymphoma (MCL) is considered incurable with a shorter survival than indolent lymphomas. Current standard therapeutic regimens have high initial response rates, however, even with high dose therapy, drug resistance is common and recurrence is anticipated (Garraway & Janne, 2012). Adaptive resistance often results in progression to more clinically aggressive disease. Most patients eventually relapse with a median overall survival of 4–6 years. Thus, more effective therapies are needed. Abnormal activation of Bruton tyrosine kinase (BTK) mediated B-cell-receptor (BCR) signalling pathway contributes to the pathogenesis of B-cell malignancies (Davis et al, 2010). Signals mediated by BTK trigger cell survival pathways such as NFjB, RAS/RAF/MEK/ERK and PI3K/AKT. BTK is therefore an attractive target for inhibition of B-cell growth. A clinical trial suggested Ibrutinib is a promising agent in patients with relapsed or refractory MCL (Wang et al, 2013). However, considering that genetic mutations cause resistance to ibrutinib in chronic lymphocytic leukaemia (CLL) patients and altered signalling pathways are common mechanisms of resistance to single agents, Ibrutinib monotherapy is not expected to cure MCL (Zucca & Bertoni, 2013). The BCL2 family proteins play a critical role in regulating apoptosis and lymphoid malignancies frequently have high BCL2 expression. The BH3-only mimetic ABT-199 selectively inactivates BCL2 and is a promising drug for treatment of BCL2-dependent cancers. However, acquired mutations could cause resistance to ABT-199 in lymphoma cells (Fresquet et al, 2014). These observations indicate potential clinical challenges of applying ABT-199 as a single agent. Combination of ABT199 with other agents may be a strategy to overcome acquired resistance. The effectiveness of ABT-199 as a single agent or in combination is largely unexplored in MCL. Given the presumed non-overlapping pathways, we hypothesized that ibrutinib/ABT-199 combination may overcome the resistance observed with single agents alone. To test this, a new MCL cell line CCMCL1 (Zhao et al, 2013) was examined for the response to these agents. Synergistic inhibition of proliferation was confirmed in 22 of 24 tested combinations, based on combination index (CI) values <1, in which 18 combinations had CI values <0 7, suggesting strong synergy (Fig 1A). Synergistic apoptosis induction was observed in all combinations (Fig 1B). The same assay with four additional MCL cell lines, Jeko-1, Mino, JVM2 and Rec-1, showed strong synergistic effects, as evidenced by CI values <0 7 with a majority being <0 2 (Fig 1C, D). Testing with primary cells from two cases of recurrent MCL also displayed robust synergy of apoptosis induction (Figure S1). Although these two cases displayed different degrees of response to single agents, synergy was observed for both. Another noteworthy finding of this study is molecular mechanisms underlying the interaction of ibrutinib/ABT-199 in MCL cells. In CCMCL1 cells, ibrutinib caused dephosphorylation of BTK(Y223). ABT-199 had no detectable effect on this target but there was a larger decrease of p-BTK(Y223) upon cells co-treated with ibrutinib/ABT-199. A similar effect was observed on p-AKT(S473) (Fig 2A). Immunoblotting of four other MCL cell lines confirmed that this combination enhanced dephosphorylation of the above signalling molecules (Figure S2), which was associated with survival/proliferation of malignant B-cells. The impact of ibrutinib and/or ABT-199 on BCL2 family proteins (BCL2, MCL1 and BCL2L1) was more cell-line dependent. In CCMCL1 cells, ibrutinib alone downregulated MCL1. Each single agent had little effect on BCL2 and BCL2L1, but the combination down-regulated both of these proteins (Fig 2B). Co-treatment of other MCL cell lines with ibrutinib/ABT-199 resulted in decrease of at least one BCL2 family protein (Figure S3). Ibrutinib/ABT-199 co-treatment also more effectively triggered reduced mitochondrial membrane potential compared to single agent, and more poly (ADP-ribose) polymerase (PARP) cleavage (Fig 2C, D) suggested caspase activation. In summary, we report the therapeutic potential of ibrutinib/ABT-199 combination in MCL cells. This combination displayed strongly synergistic effects in all tested cell lines and primary cells from recurrent MCL patients. These cell lines represent different types of MCL, including aggressive MYCtranslocated CCMCL1 (X. Zhao, S. Shetty, M. R. Smith, J. Bodo, E. D. Hsi unpublished data). Mechanistically, ibrutinib/ ABT-199 interaction caused synergistic effects in MCL cells through perturbation of p-BTK and p-AKT mediated survival signals and of BCL2 family proteins (Fig 2E). Ibrutinib was developed as a BTK inhibitor to block BCR signalling. To date, few studies described alterations of BCR signalling causing any effects on expression or function of BCL2 family proteins. However, BTK is necessary for BCR-induced phosphorylation of cAMP-response elementbinding protein (CREB) (Blois et al, 2004). In mature B cells,

Acquired resistance to venetoclax (ABT-199) in t(14;18) positive lymphoma cells

The chromosomal translocation t(14;18) in follicular lymphoma (FL) is a primary oncogenic event resulting in BCL-2 over-expression. This study investigates activity of the BH3 mimetic venetoclax (ABT-199), which targets BCL-2, and mechanisms of acquired resistance in FL. The sensitivity of FL cells to venetoclax treatment correlated with BCL-2/BIM ratio. Cells with similar expression of anti-apoptotic proteins, but with higher levels of BIM were more sensitive to the treatment. Venetoclax induced dissociation of BCL-2/BIM complex and a decrease in mitochondrial potential. Interestingly the population of cells that survived venetoclax treatment showed increased p-ERK1/2 and p-BIM (S69), as well as a decrease in total BIM levels. Venetoclax resistant cells initially showed elevated levels of p-AKT and p-Foxo1/3a, a dissociation of BIM/BCL-2/BECLIN1 complex, and a decrease in SQSTM1/p62 level (indicating increased autophagy) together with a slight decline in BIM expression. After stable resistant cell lines were established, a significant reduction of BCL-2 levels and almost total absence of BIM was observed. The acquisition of these resistance phenotypes could be prevented via selective ERK/AKT inhibition or anti-CD20 antibody treatment, thus highlighting possible combination therapies for FL patients.

Aberrant activation-induced cytidine deaminase expression in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia ☆

Activation-induced cytidine deaminase (AID) is expressed in germinal center B cells and plays a critical role in somatic hypermutation and class-switch recombination of immunoglobulin genes. Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) carries a poor prognosis and is specifically treated with tyrosine kinase inhibitors. Interestingly, AID has been shown to be aberrantly expressed and functional in Ph+ ALL and is thought to contribute to genetic instability. We hypothesized that AID might be detectable in routinely processed bone marrow biopsies by immunohistochemistry (IHC) and assist in identifying Ph+ ALL. We found that AID was expressed in 26 (70%) of 37 cases of Ph+ ALL but only 1 (2.9%) of 38 cases of Ph- ALL cases. There was a significant difference in AID expression between these 2 ALL groups (P < .001, Fisher exact test). The expression of AID was confirmed by RT-PCR (reverse-transcriptase polymerase chain reaction) and correlated with IHC scoring. AID protein is expressed in a large proportion of Ph+ ALL cases at levels detectable by IHC in clinical samples and might be useful to rapidly identify cases likely to have a BCR/ABL1 fusion.

SLAMF7 (CD319/CS1) is expressed in plasmablastic lymphoma and is a potential diagnostic marker and therapeutic target

The plasmablastic lymphomas (PBLs) are a group of aggressive B cell neoplasms that share pathobiological features with large B cell lymphoma (LBCL) and plasma cell myeloma. They are characterized by a diffuse proliferation of large immunoblastic cells with an immunophenotype resembling plasma cells (Colomo et al, 2004; Swerdlow et al, 2016). First described in the oral cavity of human immunodeficiency virus (HIV)-positive patients, PBL is now recognized to occur in other anatomic sites and in other immunosuppressed states, including among the elderly (Hsi et al, 2011).