Essential Survival Signals Research Articles

Abstract 4944: Combined treatment of BTK and PI3K inhibitors synergistically disrupts BCR-signaling, overcomes microenviroment-mediated survival and drug resistance in mantle cell lymphoma.

Abstract Mantle cell lymphoma (MCL) is recognized as an aggressive lymphoma characterized by overexpression of cyclin D1 as a result of t (11:14) translocation. It carries poor prognosis due to the emergence of drug resistance and currently no effective cure is available for this disease. Dynamic interactions between the lymphoma cell and its microenvironment play a critical role in lymphoma development and response to therapy. Recently, inhibitors of BCR signaling have become an area of substantial clinical interest. B-cell receptor (BCR) provides essential growth and survival signals to lymphomas. We therefore studied the role of BCR signal pathways in stroma mediated survival and drug resistance of mental cell lymphoma. We demonstrate that all mantle cell lymphoma cell lines and primary MCL cells express key BCR downstream signaling molecules, BTK (Bruton's tyrosine kinase), AKT and ERK, in activated (phosphorylated) form. Phosphorylation statutes of pBTK, pAKT and pERK are further up regulated upon adhesion to lymph node stroma cells. Cell adhesion between lymphoma cells and tumor stroma cells is essential for tumor cell survival and drug resistance. Inhibition of BTK by PCI 32765 or PI3Kδ by CAL101 abolishes intrinsic as well as stroma induced activation of BTK, AKT, ERK. It also significantly disrupts tumor - stromal cell adhesion and induces cell apoptosis. Moreover, combined treatment of PCI 32765 and CAL101 synergetically inhibit BCR signaling and induce cell apoptosis in absence and presence of HK stroma cells. CXCR4 has been reported to play a critical role in lymphoma cell homing, migration, and adhesion. We evaluated CXCR4 expression upon MCL adhesion to stroma cells. We demonstrate that the CXCR4 level was significantly increased in Jeko-1 and HBL2 cell lines and primary MCL cells when co-cultured with HK stroma cells. The stroma cells-induced CXCR4 increase was significantly abolished by BCR signaling inhibitors, PCI 32765 and Cal101. We further explored the effect of those inhibitors on cyclin D1. We show that cyclin D1 is constitutively expressed in MCL cell lines and primary MCL cells and that its expression was further increased when co-cultured with HK stoma cells. Both PCI 32765 and Cal101 decreased intrinsic cyclin D1 expression in the absence and presence of HK cells. Furthermore, combined treatment of PCI 32765 and Cal101 more strikingly attenuated cyclin D1 expression than either agent alone. Collectively, Our study demonstrates (1) the constitutive expression and biological function of BCR signaling in MCL, (2) the essential role of BCR signaling in microenvironment (stroma cell) mediated MCL survival, adhesion and drug resistance, and (3) the therapeutic potential of using novel BCR inhibitors, PCI 32765 and Cal101, in single or combine for MCL therapy. Citation Format: Jiangchuan Tao, Tint Lwin, Xiaohong Zhao, Bijal Shah, Ling Zhang, Lynn Moscinski, William Dalton, Eduardo M. Sotomayor, Jianguo Tao. Combined treatment of BTK and PI3K inhibitors synergistically disrupts BCR-signaling, overcomes microenviroment-mediated survival and drug resistance in mantle cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4944. doi:10.1158/1538-7445.AM2013-4944

Type IV collagen stimulates pancreatic cancer cell proliferation, migration, and inhibits apoptosis through an autocrine loop

BackgroundPancreatic cancer shows a highly aggressive and infiltrative growth pattern and is characterized by an abundant tumor stroma known to interact with the cancer cells, and to influence tumor growth and drug resistance. Cancer cells actively take part in the production of extracellular matrix proteins, which then become deposited into the tumor stroma. Type IV collagen, an important component of the basement membrane, is highly expressed by pancreatic cancer cells both in vivo and in vitro. In this study, the cellular effects of type IV collagen produced by the cancer cells were characterized.MethodsThe expression of type IV collagen and its integrin receptors were examined in vivo in human pancreatic cancer tissue. The cellular effects of type IV collagen were studied in pancreatic cancer cell lines by reducing type IV collagen expression through RNA interference and by functional receptor blocking of integrins and their binding-sites on the type IV collagen molecule.ResultsWe show that type IV collagen is expressed close to the cancer cells in vivo, forming basement membrane like structures on the cancer cell surface that colocalize with the integrin receptors. Furthermore, the interaction between type IV collagen produced by the cancer cell, and integrins on the surface of the cancer cells, are important for continuous cancer cell growth, maintenance of a migratory phenotype, and for avoiding apoptosis.ConclusionWe show that type IV collagen provides essential cell survival signals to the pancreatic cancer cells through an autocrine loop.

Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation

Antigen receptors activate pathways that control cell survival, proliferation, and differentiation. Two important targets of antigen receptors, NF-κB and Jun N-terminal kinase (JNK), are activated downstream of CARMA1, a scaffolding protein that nucleates a complex including BCL10, MALT1, and other IκB kinase (IKK)-signalosome components. Somatic mutations that constitutively activate CARMA1 occur frequently in diffuse large B cell lymphoma (DLBCL) and mediate essential survival signals. Mechanisms that downregulate this pathway might thus yield important therapeutic targets. Stimulation of antigen receptors induces not only BCL10 activation but also its degradation downstream of CARMA1, thereby ultimately limiting signals to its downstream targets. Here, using lymphocyte cell models, we identify a kinase-independent requirement for TAK1 and its adaptor, TAB1, in antigen receptor-induced BCL10 degradation. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 overexpression restrains CARMA1-dependent activation of NF-κB by reducing BCL10 levels. TAK1 also promotes counterselection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counterbalances NF-κB and JNK signals essential for the activation and survival of lymphocytes and CARMA1-addicted lymphoma types.

The Cell Surface Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Contributes to Epidermal Growth Factor Receptor-mediated Cell Migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

The function of FGF signaling in the lens placode

Previous studies suggested that FGF signaling is important for lens formation. However, the times at which FGFs act to promote lens formation, the FGFs that are involved, the cells that secrete them and the mechanisms by which FGF signaling may promote lens formation are not known. We found that transcripts encoding several FGF ligands and the four classical FGF receptors are detectable in the lens-forming ectoderm at the time of lens induction. Conditional deletion of Fgfr1 and Fgfr2 from this tissue resulted in the formation of small lens rudiments that soon degenerated. Lens placodes lacking Fgfr1 and 2 were thinner than in wild-type embryos. Deletion of Fgfr2 increased cell death from the initiation of placode formation and concurrent deletion of Fgfr1 enhanced this phenotype. Fgfr1/2 conditional knockout placode cells expressed lower levels of proteins known to be regulated by FGF receptor signaling, but proteins known to be important for lens formation were present at normal levels in the remaining placode cells, including the transcription factors Pax6, Sox2 and FoxE3 and the lens-preferred protein αA-crystallin. Previous studies identified a genetic interaction between BMP and FGF signaling in lens formation and conditional deletion of Bmpr1a caused increased cell death in the lens placode, resulting in the formation of smaller lenses. In the present study, conditional deletion of both Bmpr1a and Fgfr2 increased cell death beyond that seen in Fgfr2CKO placodes and prevented lens formation. These results suggest that the primary role of autocrine or paracrine FGF signaling is to provide essential survival signals to lens placode cells. Because apoptosis was already increased at the onset of placode formation in Fgfr1/2 conditional knockout placode cells, FGF signaling was functionally absent during the period of lens induction by the optic vesicle. Since the expression of proteins required for lens formation was not altered in the knockout placode cells, we can conclude that FGF signaling from the optic vesicle is not required for lens induction.

The Novel JAK2 Inhibitor NVP-BSK805 Has Cytotoxic Activity on Malignant Plasma Cells

AbstractAbstract 2993In multiple myeloma (MM) and plasma cell leukemia, activation of the JAK/STAT pathway is induced by interleukin (IL)-6, which is produced and secreted into the tumor microenvironment primarily by stromal cells. Upon binding of IL-6 to its specific alpha-chain receptor, dimerization of the gp130 signaling subunits leads to activation of associated JAK kinases and STAT transcription factors. In particular, STAT3 has been shown to be essential for myeloma cell growth and survival. NVP-BSK805 (Novartis) is a novel substituted quinoxaline JAK2 inhibitor tool compound which displays more than 20-fold selectivity for JAK2 over the other JAK family members and more than 100-fold selectivity over a panel of additional kinases (Baffert et al., Mol Cancer Ther 9:1945, 2010). The study presented here aims at growth inhibitory effects of NVP-BSK805 in malignant plasma cells. NVP-BSK805 inhibited the growth of six human myeloma cell lines displaying dose-dependent activity with IC50 concentrations between 2.6 μ mol/L and 6.8 μ mol/L. Among the cell lines, IL-6 dependent INA-6 cells were most sensitive to the inhibitory effects of the compound: both IL-6 and bone marrow stromal cell induced proliferation as measured by [3H]-thymidine uptake was completely inhibited at 4 μ mol/L and IC50 concentrations were less than 1 μ mol/L. Viability of the stromal cells was not significantly affected. NVP-BSK805 concentrations as low as 0.5 μ mol/L were sufficient to yield a marked reduction of IL-6 induced STAT3 phosphorylation and complete abrogation at 2 μ mol/L, thereby blocking essential survival signals. Accordingly, treatment of INA-6 cells with NVP-BSK805 for 48 hours led to significant apoptosis starting at 2 μ mol/L with a 30% increase in annexin V-positive cell numbers compared to DMSO controls. Importantly, NVP-BSK805 showed potent cytotoxic activity on plasma cell-enriched primary tumor samples from patients with extramedullary plasma cell disease that are highly responsive to IL-6: in 3 out of 4 tumor samples the IC50 concentrations were between 0.5 μ mol/L and 0.6 μ mol/L. These studies are extended to combinations of NVP-BSK805 with PI3K, mToR, MAPK, HDAC, and IGF-1R inhibitors in order to optimize targeted therapy strategies facing different pathway alterations in individual myeloma patients. In INA-6 cells, synergistic activity was found combining NVP-BSK805 with rapamycin and the MEK1 inhibitor U0126. Preclinical in vivo studies are ongoing. Our results with NVP-BSK805 substantiate the use of JAK inhibitors as a therapeutic strategy for patients with MM. Since results from studies with pan-JAK inhibitors such as pyridone 6 indicate involvement of additional JAK kinases, the choice of the optimal compound will depend on its JAK family selectivity and the biology of JAK signaling in MM. Disclosures:No relevant conflicts of interest to declare.

Targeting the CD28-B7 Pro-Survival Pathway In Multiple Myeloma

AbstractAbstract 132Multiple myeloma (MM), an incurable neoplasia of terminally differentiated plasma cells, are critically dependent on their interactions with bone marrow stromal cells (BMSC) for essential survival signals, growth and immunosuppressive factors. Very little is known about the specific BM cell type or the molecular elements in these interactions, an understanding of which could provide novel targets that could be interdicted to enhance conventional chemotherapy. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma tumoral expansion. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with BM cells that express B7 (CD80/CD86) such as dendritic cells (DCs, that are known to be closely associated with MM cells in the BM) or with CD86+ MM cells themselves. We had previously shown (ASH2008, #I-769) that blocking CD28-CD86 interactions between myeloma cells with high affinity B7 ligand CTLA4Ig (Abatacept®) sensitized myeloma cells to chemotherapy. Now we show that myeloma cells co-cultured with myeloid DCs in vitro derive both direct and indirect survival signals from DCs, and this can be partially blocked by commercially available reagents. Our data show that flow cytometric analysis of mononuclear cells (MNC) from BM aspirates of myeloma patients with increased CD138+ plasma cell populations (9-58%), show an increased CD11b+ (myeloid) population (20-37%) as well, which is in contrast to healthy transplant donor controls (12-15% CD11b+, 4–6% CD138+). Moreover, a larger fraction (11-47%) of the myeloma CD138+ plasma cells expressed CD28 compared to healthy control (3.3-7.7%). Also, when we analyzed gene expression datasets (NCBI #GSE5900 and GSE4204) from plasma cells (PC) of normal donors, monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SM) and newly diagnosed multiple myeloma (MM), we found a progressive increase in patients showing CD28 expression with increasing severity of disease (normal<MGUS<SM<MM) (Fig 1A). When we sorted the highest scoring MM group (n=538) into 8 genetic subgroups as defined earlier, CD28 expression was found to peak in the MF subgroup (typically associated with poor survival in myeloma patients) (Zhan et al. 2006, Blood 108, pp. 2020) relative to total population (p<0.0001) (Fig 1B). Antibody mediated activation of MM-CD28 over 48 hrs increased viability of myeloma cell line MM.1S cultured under serum starvation (3.7%) or with drugs ATO (1.9%), melphalan (18%) or dexamethasone (3.3%) to 66%, 21%, 33% and 11% respectively. Viability of MM.1S cells or primary CD138+ plasma cells (isolated from myeloma BM aspirates) cultured under serum starvation was enhanced >3 fold (p<0.001) when co-cultured with monocyte derived DCs, and in MM.1S this was partially reversed when either MM-CD28 or DC-B7 was blocked (Fig 2). Similar protection of MM.1S was also observed against a gradient of dexamethasone or melphalan. CD28 activation was accompanied by rapid tyrosine phosphorylation of CD28, association of p85 (PI3K), activation of Vav-1 and increase in CD28 associated tyrosine kinase activity, as shown by immunoprecipitation, western and kinase activity assays. We had previously shown that MM-CD28 interaction drive DC production of pro-survival factor IL-6 and immunosuppressive factor IDO via DC-B7 “backsignaling” (ASH2008 #I-769). Now we show that MM induced DC production of IL-6 (8 ng/ml) was partially inhibited in presence of CD28 blocking αCD28(Fab) fragments (3 ng/ml) or with protein kinase C (PKC) inhibitor Bisindolylmaleimide-I (2.1ng/ml). Activity of the immunosuppressive enzyme IDO in these co-cultures was completely inhibited in the presence of a novel IDO inhibitor from Incyte corporation, and this helped partially reverse IDO mediated suppression of T-cell proliferation in proliferation assays using co-culture supernatants. In conclusion, our data characterizes CD28-B7 pathway and DCs in the BM as vital for myeloma survival and also as possible targets to include in future strategies in the treatment of myeloma. [Display omitted] [Display omitted] Disclosures:Boise:University of Chicago: Patents & Royalties.

Spleen tyrosine kinase inhibition prevents chemokine- and integrin-mediated stromal protective effects in chronic lymphocytic leukemia

AbstractThe microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.

Abstract 701: Elevated insulin-like growth factor 1 receptor provides an essential survival signal via Bcl-xL in rhabdomyosarcoma cells as an alternative to Bcl-2 overexpression

Abstract Insulin-like growth factors and their receptor IGF1R play important roles in growth, development, stress response, aging, and cancer. Many antibodies targeting IGF1R are in varies stages of clinical development. It is important to understand the mechanism of the antibody-mediated cancer cell growth inhibition and death, and to identify biomarkers predictive of responses that can be implemented in clinical investigations. We previously reported that IGF1R mediated cell growth suppression of rhabdomyosarcoma (RMS) in vitro and in vivo, in which the anti-proliferative activity was correlated with IGF1R levels and AKT signaling (Cao, et al., Cancer Research 2008, 68:8039). Here, we show that in some RMS cells, treatment with a humanized IGF1R antibody results in a rapid onset of mitochondrial-dependent apoptosis, including mitochondrial depolarization, cytochrome C release, and the activation of specific caspases. The antibody-induced cell death is mediated in part via AKT-dependent pathway, and both the inhibition of AKT signaling and induction of cell death by anti-IGF1R can be rescued by the introduction of a constitutively active AKT. Further analysis reveals IGF1R antibody-induced hypophosphorylation (activation) of BAD and the activation of downstream BAX. Bcl-xL was previously shown to be a key target of activated BAD. Our data indicate that cells with the transgenic expression of Bcl-xL are completely resistant to antibody-induced cell death, while still susceptible to antibody-mediated growth inhibition. Moreover, siRNA against Bcl-xL enhances anti-IGF1R-induced apoptosis. Interestingly, the examination of RMS cell lines and tumors reveals an inverse correlation between elevated IGF1R and another pro-survival gene Bcl-2 (P=0.033). Thus, our results indicate that RMS pathogenesis may involve two strategies for cell survival: 1) increasing IGF1R expression that enhances Bcl-xL activity; or 2) up-regulating Bcl-2 expression. Our study suggests a particular subgroup of the cancer with elevated IGF1R and low Bcl-2 maybe more susceptible to anti-IGF1R treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 701.

The Apical Complex Couples Cell Fate and Cell Survival to Cerebral Cortical Development

Cortical development depends upon tightly controlled cell fate and cell survival decisions that generate a functional neuronal population, but the coordination of these two processes is poorly understood. Here we show that conditional removal of a key apical complex protein, Pals1, causes premature withdrawal from the cell cycle, inducing excessive generation of early-born postmitotic neurons followed by surprisingly massive and rapid cell death, leading to the abrogation of virtually the entire cortical structure. Pals1 loss shows exquisite dosage sensitivity, so that heterozygote mutants show an intermediate phenotype on cell fate and cell death. Loss of Pals1 blocks essential cell survival signals, including the mammalian target of rapamycin (mTOR) pathway, while mTORC1 activation partially rescues Pals1 deficiency. These data highlight unexpected roles of the apical complex protein Pals1 in cell survival through interactions with mTOR signaling.

Spleen Tyrosine Kinase Inhibition Prevents Chemokine- and Integrin-Mediated Stromal Protective Effects in Chronic Lymphocytic Leukemia.

Abstract 2356Poster Board II-333B cell chronic lymphocytic leukemia (CLL) remains incurable despite recent therapeutic advances, and identification of novel molecular targets in CLL is highly desirable. The microenvironment provides essential growth and survival signals to CLL cells and confers resistance to cytotoxic agents, a phenomenon termed cell adhesion-mediated drug resistance (CAM-DR). The chemokine CXCL12 and the integrin ligand VCAM have been identified as important microenvironmental pathways that mediate CLL cell survival and CAM-DR. We have previously demonstrated that inhibition of spleen tyrosine kinase (SYK), a key component of the BCR signaling pathway, induces apoptosis in primary CLL cells when cultured alone or supported by stroma (Buchner et al., Cancer Res. 2009). In this study, we investigated SYK inhibition with regard to the interaction between CLL cells and the microenvironment.Stromal cell coculture resulted in SYK activation in primary CLL cells as indicated by phospho-protein analysis by flow cytometry (p<0.001) and immunoblotting. In vitro stimulation with 20 ng/ml recombinant CXCL12 or VLA-4 integrin ligation by contact to VCAM-1 precoated wells (10 μg/ml) for 15 sec had equivalent effects. SYK inhibition by the pharmacological SYK inhibitor R406 significantly reduced F-actin polymerization and Akt phosphorylation induced by CXCL12, integrin ligand, or stromal contact. In functional experiments, migration of CLL cells towards CXCL12 was reduced to 77% (p<0.001) by R406 in transwell culture. Likewise, CLL cell adhesion to VCAM was limited by SYK inhibition to 50% (p<0.01) compared to control cells in flow chamber experiments. In response to a number of physiological stimuli, including BCR ligation, integrins are activated through the modulation of their conformation to mediate high-affinity adhesion. Analysis of the expression of a high affinity epitope of CD29, the β1 chain of VLA-4, on CLL cells by flow cytometry with and without SYK inhibition revealed a significant decrease of 31% (p<0.0001), while the expression of total CD29 decreased only by 10% (p<0.01). To verify the specificity of the effects of pharmacological SYK inhibitors, we performed siRNA mediated silencing of SYK in primary CLL cells. 48 h after transfection, we observed a downregulation of SYK protein expression by flow cytometry by 27% (p<0.05), while the viability of the cells was not significantly affected for 72 h. Stimulation of control- and SYK-siRNA treated cells with VCAM significantly increased Akt phosphorylation in control but not in SYK-siRNA transfected cells. Furthermore, SYK-silenced CLL cells adhered significantly less to VCAM-coated surfaces than control-silenced CLL cells.Beyond inhibition of these defined pathways, R406 abrogated the protective effect of stromal coculture on CLL cell survival. The anti-apoptotic factor Mcl-1 was upregulated in CLL cells cultured with stroma compared to those cultured alone (mean MFI ± SEM: 144 ± 21 vs. 182 ± 23, p<0.001). SYK inhibition almost completely abolished the upregulation of Mcl-1 in stroma-cocultured CLL cells (mean MFI ± SEM: 182 ± 23 vs. 130 ± 15, p<0.001). Finally, SYK inhibition also prevented CAM-DR against fludarabine in stroma coculture.Taken together, our data demonstrate direct effects of SYK inhibition on chemokine and integrin-mediated CLL-stroma interactions and establish a novel therapeutic mechanism of action for SYK inhibitors in CLL. Indeed, a promising clinical activity in patients with relapsed/refractory CLL/SLL with an overall response rate of 54% has been reported for the oral prodrug of R406, fostamatinib disodium in CLL patients (Friedmann et al, ASH 2008). Of note, an unexplained transient but dramatic leukocyte flare during lymph node regression was reported in several of these patients. Our results offer a convincing explanation for this phenomenon and may even suggest that SYK blockade in CLL is a promising therapeutic principle not only due to inhibition of BCR signaling, but perhaps even more importantly by disrupting the contact of CLL cells with their protective microenvironment. Disclosures:No relevant conflicts of interest to declare.

CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival

AbstractCX3CR1 is a chemokine receptor with a single ligand, the membrane-tethered chemokine CX3CL1 (fractalkine). All blood monocytes express CX3CR1, but its levels differ between the main 2 subsets, with human CD16+ and murine Gr1low monocytes being CX3CR1hi. Here, we report that absence of either CX3CR1 or CX3CL1 results in a significant reduction of Gr1low blood monocyte levels under both steady-state and inflammatory conditions. Introduction of a Bcl2 transgene restored the wild-type phenotype, suggesting that the CX3C axis provides an essential survival signal. Supporting this notion, we show that CX3CL1 specifically rescues cultured human monocytes from induced cell death. Human CX3CR1 gene polymorphisms are risk factors for atherosclerosis and mice deficient for the CX3C receptor or ligand are relatively protected from atherosclerosis development. However, the mechanistic role of CX3CR1 in atherogenesis remains unclear. Here, we show that enforced survival of monocytes and plaque-resident phagocytes, including foam cells, restored atherogenesis in CX3CR1-deficent mice. The fact that CX3CL1-CX3CR1 interactions confer an essential survival signal, whose absence leads to increased death of monocytes and/or foam cells, might provide a mechanistic explanation for the role of the CX3C chemokine family in atherogenesis.

Stress as an Intercellular Signal: The Emergence of Stress-Associated Molecular Patterns (SAMP)

Abstrct Cells are continuously exposed to stressful situations that generally entail generation of reactive oxygen species and other redox alterations. Low levels of stress are physiological and can transmit essential survival or adaptive signals. At higher levels, however, the responses become maladaptive and cause damage. Frequently, stressful events occurring in a few cells propagate, resulting in tissue or even systemic response. Here we review recent evidence suggesting that stressed cells signal their state by expressing on their surface and secreting suitable molecular clues, which we propose to term Stress-Associated Molecular Patterns (SAMP). A unifying mechanism seems to involve the release of oxidoreductases and redox modifiers into the intercellular space, with structural and functional alterations in key signaling molecules. These observations open the way to novel therapeutic strategies.

Zbtb4 represses transcription of P21CIP1 and controls the cellular response to p53 activation

In response to stimuli that activate p53, cells can undergo either apoptosis or cell cycle arrest, depending on the precise pattern of p53 target genes that is activated. We show here that Zbtb4, a transcriptional repressor protein, associates with the Sin3/histone deacetylase co-repressor and represses expression of P21CIP1 as part of a heterodimeric complex with Miz1. In vivo, expression of ZBTB4 is downregulated in advanced stages of multiple human tumours. In cell culture, depletion of ZBTB4 promotes cell cycle arrest in response to activation of p53 and suppresses apoptosis through regulation of P21CIP1, thereby promoting long-term cell survival. Our data suggest that Zbtb4 is a critical determinant of the cellular response to p53 activation and reinforce the notion that p21Cip1 can provide an essential survival signal in cells with activated p53.

Beta1 integrin as a molecular therapeutic target

Purpose: Radiation therapy (RT) is a commonly used modality to treat human cancer. However, dose-limiting toxicity and resistance are major problems to improving treatment efficacy. Increasing evidence has pointed to the critical role of the tumor microenvironment and cell-extracellular matrix (ECM), specifically via β1 integrins, in modifying response to therapy, including radiation. β1 integrins have been implicated in several key processes in malignant progression and metastasis, and more recently in mediating resistance to cytotoxic chemotherapy and radiation. Evidence is mounting that β1 integrins mediate essential survival signals post-IR, through both canonical and non-canonical integrin signaling pathways that make it a highly promising therapeutic target for several solid malignancies.Conclusions: In this mini-review article, we briefly summarize the most recent emerging findings concerning β1 integrin and radiation survival that indicate the promise of β1 integrin inhibitory agents as radiosensitizers.

VH Replacement Rescues Progenitor B Cells with Two Nonproductive VDJ Alleles

Inaccurate VDJ rearrangements generate a large number of progenitor (pro)-B cells with two nonproductive IgH alleles. Such cells lack essential survival signals mediated by surface IgM heavy chain (muH chain) expression and are normally eliminated. However, secondary rearrangements of upstream VH gene segments into assembled VDJ exons have been described in mice transgenic for productive muH chains, a process known as VH replacement. If VH replacement was independent of muH chain signals, it could also modify nonproductive VDJ exons and thus rescue pro-B cells with unsuccessful rearrangements on both alleles. To test this hypothesis, we homologously replaced the JH cluster of a mouse with a nonproductive VDJ exon. Surprisingly, B cell development in IgHVDJ-/VDJ- mice was only slightly impaired and significant numbers of IgM-positive B cells were produced. DNA sequencing confirmed that all VDJ sequences from muH chain-positive B lymphoid cells were generated by VH replacement in a RAG-dependent manner. Another unique feature of our transgenic mice was the presence of IgH chains with unusually long CDR3-H regions. Such IgH chains were functional and only modestly counter-selected, arguing against a strict length constraint for CDR3-H regions. In conclusion, VH replacement can occur in the absence of a muH chain signal and provides a potential rescue mechanism for pro-B cells with two nonproductive IgH alleles.

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.

AKT1 Provides an Essential Survival Signal Required for Differentiation and Stratification of Primary Human Keratinocytes

Keratinocyte differentiation and stratification are complex processes involving multiple signaling pathways, which convert a basal proliferative cell into an inviable rigid squame. Loss of attachment to the basement membrane triggers keratinocyte differentiation, while in other epithelial cells, detachment from the extracellular matrix leads to rapid programmed cell death or anoikis. The potential role of AKT in providing a survival signal necessary for stratification and differentiation of primary human keratinocytes was investigated. AKT activity increased during keratinocyte differentiation and was attributed to the specific activation of AKT1 and AKT2. Targeted reduction of AKT1 expression, but not AKT2, by RNA interference resulted in an abnormal epidermis in organotypic skin cultures with a thin parabasal region and a pronounced but disorganized cornified layer. This abnormal stratification was due to significant cell death in the suprabasal layers and was alleviated by caspase inhibition. Normal expression patterns of both early and late markers of keratinocyte differentiation were also disrupted, producing a poorly developed stratum corneum.

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1838 Cell-cell contact by cadherins provides an essential survival signal to migrating smooth muscle cells

1838 Cell-cell contact by cadherins provides an essential survival signal to migrating smooth muscle cells