The phosphatidylinositol 3-kinase pathway (PI3K) is known to play an active role in many malignancies. The role of PI3K inhibition in the treatment of lymphomas has not been fully delineated. We sought to identify a role for therapeutic PI3K inhibition across a range of B cell lymphomas.
We selected three small molecule inhibitors to test in a panel of 60 cell lines that comprised diverse lymphoma types. We tested the selective PI3K inhibitor BKM120 and the dual PI3K/MTOR inhibitors BEZ235 and BGT226 in these cell lines. We applied gene expression profiling to better understand the molecular mechanisms associated with responsiveness to these drugs.
We found that higher expression of the PAK1 gene was significantly associated with resistance to all three PI3K inhibitors. Through RNA-interference mediated knock-down of the PAK1 gene, we demonstrated a dramatic increase in the sensitivity to PI3K inhibition. We further tested a small molecule inhibitor of PAK1 and found significant synergy between PI3K inhibition and PAK1 inhibition.
Thus we demonstrate that PI3K inhibition is broadly effective in lymphomas and PAK1 is a key modulator of resistance to PI3K inhibition.
Leukemias and lymphomas; kinase and phosphatase inhibitors; diffuse large B cell lymphoma; DLBCL; Hodgkin lymphoma; Burkitt lymphoma; primary mediastinal B cell lymphoma; phosphatidylinositol 3-kinase pathway; PI3 Kinase; PI3K; PAK1; drug resistance
The human gut mucosa is a major site of HIV infection and infection-associated pathogenesis. Increasing evidence shows that natural killer (NK) cells play an important role in control of HIV infection but the mechanism(s) by which they mediate antiviral activity in the gut is unclear. Here we show two distinct subsets of NK cells exist in the gut, one localized to intraepithelial spaces (IEL) and the other to the lamina propria (LP). The frequency of both subsets of NK cells was reduced in chronic infection, whereas IEL NK cells remained stable in spontaneous controllers with protective KIR/HLA genotypes. Both IEL and LP NK cells were significantly expanded in immunologic non-responsive (INR) patients, who incompletely recovered CD4+ T cells on HAART. These data suggest that both IEL and LP NK cells may expand in the gut in an effort to compensate for compromised CD4+ T cell recovery, but that only IEL NK cells may be involved in providing durable control of HIV in the gut,
The zinc finger transcriptional repressor Gfi-1 has been shown to play a critical role in early granulopoiesis; however, its role in late neutrophilic development is poorly understood. We report here that forced expression of a dominant negative Gfi-1 mutant, N382S, resulted in augmented mRNA levels of eosinophil major basic protein (MBP) in myeloid cells induced with G-CSF to undergo terminal neutrophilic differentiation. MBP is a cytotoxic protein that is abundantly expressed in eosinophils, but not in neutrophils. Ectopic expression of MBP inhibited the proliferation and survival of differentiating myeloid cells in response to G-CSF. Significantly, while GFI-1 is upregulated during neutrophilic differentiation, it is rapidly downregulated upon induction of eosinophilic differentiation, which was associated with increased MBP expression. Knockdown of GFI-1 in eosinophilic cells also led to increased level of MBP mRNA. These results indicate that Gfi-1 functions to inhibit the expression of MBP and aberrant expression of MBP as a result of loss of Gfi-1 function may cause premature apoptosis of differentiating neutrophils. In contrast, the rapid downregulation of Gfi-1 during eosinophilic development may allow for abundant expression of MBP, a hallmark of eosinophilic differentiation.
Granulopoiesis; Apoptosis; Gfi-1; Eosinophil major basic protein; G-CSF
In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular-phagocytosis (ADCP), antibody dependent cellular-cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection.
Innate Immunity; Fc-receptors; Antibody dependent cellular phagocytosis; HIV-1
Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors—allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function.
Phagocytosis; Antibody; ADCC; antibody-dependent phagocytosis; monocytes; Fc receptor; effector function
The normal counterparts of mantle cell lymphoma (MCL) are naïve quiescent B-cells that have not been processed through the germinal center (GC). For this reason, while lymphomas arising from GC or post-GC B-cells often exhibit plasmacytic differentiation, MCL rarely presents with plasmacytic features. Seven cases of MCL with a monotypic plasma cell (PC) population were collected from six centers and studied by immunohistochemistry, FICTION (Fluorescence immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms), capillary gel electrophoresis, and restriction fragment length polymorphism of immunoglobulin heavy chain analysis (RFLP/IgH) of microdissections of each of the MCL and PC populations to assess their clonal relationship. Clinical presentation was rather unusual compared to typical MCL, with two cases arising from extranodal soft-tissues of the head. All MCL cases were morphologically and immunohistochemically typical, bearing the t(11;14)(q13;q32). In all cases PC populations were clonal. In 5 of the 7 cases, the MCL and PC clones showed identical restriction fragments, indicating a common clonal origin of the neoplastic populations. The two cases with clonal diversity denoted the coexistence of two different tumors in a composite lymphoma/plasma cell neoplasm. Our findings suggest that MCL can present with a PC component that is often clonally related to the lymphoma, representing a rare but unique biological variant of this tumor.
Mantle cell lymphoma; Plasma cells; Monotypic plasma cells; Plasmacytic differentiation; Cyclin D1
In the budding yeast Saccharomyces cerevisiae, mother cells switch mating types between a and α forms, whereas daughter cells do not. This developmental asymmetry arises because the expression of the HO endonuclease, which initiates the interconversion of a and α mating type cassettes, is extinguished by the daughter-specific Ash1 transcriptional repressor. When daughters become mothers in the subsequent cell cycle, Ash1 must be eliminated to enable a new developmental state. Here, we report that the ubiquitin ligase SCFCdc4 mediates the phosphorylation-dependent elimination of Ash1. The inactivation of SCFCdc4 stabilizes Ash1 in vivo, and consistently, Ash1 binds to and is ubiquitinated by SCFCdc4 in a phosphorylation-dependent manner in vitro. The mutation of a critical in vivo cyclin-dependent kinase (CDK) phosphorylation site (Thr290) on Ash1 reduces its ubiquitination and rate of degradation in vivo and decreases the frequency of mating type switching. Ash1 associates with active Cdc28 kinase in vivo and is targeted to SCFCdc4 in a Cdc28-dependent fashion in vivo and in vitro. Ash1 recognition by Cdc4 appears to be mediated by at least three phosphorylation sites that form two redundant diphosphorylated degrons. The phosphorylation-dependent elimination of Ash1 by the ubiquitin-proteasome system thus underpins developmental asymmetry in budding yeast.
We have used the Xenopus laevis egg extract system to study the roles of vertebrate Dna2 in DNA replication and double-strand-break (DSB) repair. We first establish that Xenopus Dna2 is a helicase, as well as a nuclease. We further show that Dna2 is a nuclear protein that is actively recruited to DNA only after replication origin licensing. Dna2 co-localizes in foci with RPA and is found in a complex with replication fork components And-1 and Mcm10. Dna2 interacts with the DSB repair and checkpoint proteins Nbs1 and ATM. We also determine the order of arrival of ATM, MRN, Dna2, TopBP1 and RPA to duplex DNA ends and show that it is the same both in S phase and M phase extracts. Interestingly, Dna2 can bind to DNA ends independently of MRN, but efficient nucleolytic resection, as measured by RPA recruitment, requires both MRN and Dna2. The nuclease activity of Mre11 is required, since its inhibition delays both full Dna2 recruitment and resection. Dna2 depletion inhibits but does not block resection, and Chk1 and Chk2 induction occurs in the absence of Dna2.
And-1; Ctf4; Mcm10; ATM; MRN; Mre11; homologous recombination; lagging strand
Zinc-finger (ZF) transcriptional repressor Gfi-1 plays an important role in hematopoiesis and inner ear development, and also functions as an oncoprotein that cooperates with c-Myc in lymphomagenesis. Gfi-1 represses transcription by directly binding to conserved sequences in the promoters of its target genes. CDKN1A encoding p21Cip1 has been identified as a Gfi-1 target gene and shown to contain Gfi-1 binding sites in the upstream promoter region. We show here that Gfi-1 represses CDKN1A in a manner that is independent of its DNA binding activity. Gfi-1 interacts with POZ-ZF transcription factor Miz-1, originally shown to be a c-Myc interacting partner, and via Miz-1 binds to CDKN1A core promoter. Interestingly, Gfi-1 and c-Myc, through Miz-1, form a ternary complex on the CDKN1A promoter, and act in collaboration to repress CDKN1A. Gfi-1 knockdown results in enhanced levels of p21Cip1 and attenuated cell proliferation. Notably, similar to c-Myc, the expression of Gfi-1 is downregulated by TGFβ and the level of Gfi-1 influences the response of cell to the cytostatic effect of TGFβ. Our data reveal an important mechanism by which Gfi-1 regulates cell proliferation and may also have implications for understanding the role of Gfi-1 in lymphomagenesis.
Gfi-1; Miz-1; CKD inhibitors; proliferation
Increasing evidence suggests that NK cells not only are critical in the initial host defense against pathogens but also may contribute to continued protection from human immunodeficiency virus type 1 (HIV-1) disease progression. NK cell cytolysis can be induced directly through diverse receptor families or can be induced indirectly through Fc receptors by antibodies mediating antibody-dependent cellular cytotoxicity (ADCC). ADCC has been implicated in both protection from simian immunodeficiency virus infection and slower progression of HIV-1 disease. ADCC activity declines with advancing infection, and yet the underlying mechanism for this dysfunction has not been defined, nor has it been determined whether the activity can be reconstituted. Here we demonstrate that NK cell-mediated ADCC is severely compromised in chronic HIV infection. The potency of ADCC function was directly correlated with baseline FcγRIIIa receptor (CD16) expression on NK cells. CD16 expression was negatively influenced by elevated expression of a group of enzymes, the matrix metalloproteinases (MMPs), normally involved in tissue/receptor remodeling. Inhibition of MMPs resulted in increased CD16 expression and augmented ADCC activity in response to antibody-coated target cells. These data suggest that MMP inhibitors may improve NK cell-mediated ADCC, which may provide subjects with an opportunity to harness the cytolytic power of NK cells through naturally occurring nonneutralizing HIV-specific antibodies.