ALBERT

Description: The angiopoietin (ANGPT)–TIE2/TEK signaling pathway is essential for blood and lymphatic vascular homeostasis. ANGPT1 is a potent TIE2 activator, whereas ANGPT2 functions as a context-dependent agonist/antagonist. In disease, ANGPT2-mediated inhibition of TIE2 in blood vessels is linked to vascular leak, inflammation, and metastasis. Using conditional knockout studies in mice, we show TIE2 is predominantly activated by ANGPT1 in the cardiovascular system and by ANGPT2 in the lymphatic vasculature. Mechanisms underlying opposing actions of ANGPT2 in blood vs. lymphatic endothelium are poorly understood. Here we show the endothelial-specific phosphatase VEPTP (vascular endothelial protein tyrosine phosphatase) determines TIE2 response to ANGPT2. VEPTP is absent from lymphatic endothelium in mouse in vivo, permitting ANGPT2/TIE2-mediated lymphangiogenesis. Inhibition of VEPTP converts ANGPT2 into a potent TIE2 activator in blood endothelium. Our data support a model whereby VEPTP functions as a rheostat to modulate ANGPT2 ligand effect on TIE2.

Description: Introduction CLL remains an incurable disease and represents a significant health problem in the western world. Increasing evidence highlights that the impact of marrow stromal cells is a key component influencing CLL B-cell survival. We have utilized an in vitro bone marrow stromal cell (BMSC) model system and found unique alterations in CLL B-cells with BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and drug resistance. Methods Purified primary CLL B-cells (n= 39) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=26) or CLL patients (n=17) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and where needed real time PCR was done to assess the presence of intracellular proteins. In separate experiments to assess CLL B-cell killing, purified CLL B-cells were treated with TP-0903, fludarabine, chlorambucil and ibrutinib as single agents with or without BMSC co-culture. Results We observed significant increases in expression of Axl for both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSC. But, we did not see any significant change in β-catenin or Axl protein expression in BMSCs co-cultured with CLL B-cells. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. The CLL B-cells from co-culture also had upregulation in phosphorylated (P)-ERK-1/2 but no change in P-AKT(Ser473). High nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β (Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β (Ser9) detected in co-cultured CLL B-cells suggests inactivation of GSK-3β and increasing β-catenin accumulation in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. We also determined the phosphorylation status of Axl in CLL B-cells in co-culture with BMSC but found no change either at Y702 (Axl kinase domain) or total tyrosine phosphorylation levels for Axl in CLL B-cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of the highly specific Axl inhibitor TP-0903 on CLL B-cell status of Axl and b-catenin while in BMSC co-culture. Interestingly, both Axl and β-catenin protein expression levels were found to be further upregulated in CLL B-cells exposed to sub-lethal doses of TP-0903 in co-culture with BMSC. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil or ibrutinib) also led to increase in expression levels of both β-catenin and Axl CLL B-cells co-cultured with BMSC. Of interest CLL B-cells were less sensitive to the chemotherapy drugs in presence of BMSCs, suggesting a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. This was not true for the Axl inhibitor as TP-0903 was able to induce robust cell death by targeting P-Axl and overcome BMSC mediated protection even in the presence of increased Axl and b-catenin. We also found that TP-0903 decreased P-Axl as well as the Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells in BMSC co-culture. Conclusions Here we show that marrow stromal cell mediated increased expression in both β-catenin and Axl in CLL B-cells is associated with leukemic B cell survival and drug resistance. The mechanism for this may in part be via activated ERK levels that also occur when CLL B-cells contact BMSC. The BMSC resistance appears to be more profound for chemotherapeutic agents since Axl inhibitor can still induce CLL B-cell killing with BMSC co-culture. These studies suggest that a further understanding of the roles of Axl and β-catenin in the resistance status of CLL B-cells mediated by contact with BMSC are warranted. Disclosures Warner: Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals, Inc: Employment. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

Description: Microvesicles (MVs) released by malignant cancer cells constitute an important part of the tumor microenvironment. They can transfer various messages to target cells and may be critical to disease progression. Here, we demonstrate that MVs circulating in plasma of B-cell chronic lymphocytic leukemia (CLL) patients exhibit a phenotypic shift from predominantly platelet derived in early stage to leukemic B-cell derived at advanced stage. Furthermore, the total MV level in CLL was significantly greater compared with healthy subjects. To understand the functional implication, we examined whether MVs can interact and modulate CLL bone marrow stromal cells (BMSCs) known to provide a “homing and nurturing” environment for CLL B cells. We found that CLL-MV can activate the AKT/mammalian target of rapamycin/p70S6K/hypoxia-inducible factor-1α axis in CLL-BMSCs with production of vascular endothelial growth factor, a survival factor for CLL B cells. Moreover, MV-mediated AKT activation led to modulation of the β-catenin pathway and increased expression of cyclin D1 and c-myc in BMSCs. We found MV delivered phospho-receptor tyrosine kinase Axl directly to the BMSCs in association with AKT activation. This study demonstrates the existence of separate MV phenotypes during leukemic disease progression and underscores the important role of MVs in activation of the tumor microenvironment.

Description: The molecular mechanism of autocrine regulation of vascular endothelial growth factor (VEGF) in chronic lymphocytic leukemia (CLL) B cells is unknown. Here, we report that CLL B cells express constitutive levels of HIF-1α under normoxia. We have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1α degradation and found it to be at a notably low level in CLL B cells compared with normal B cells. We demonstrate that the microRNA, miR-92-1, overexpressed in CLL B cells, can target the VHL transcript to repress its expression. We found that the stabilized HIF-1α can form an active complex with the transcriptional coactivator p300 and phosphorylated-STAT3 at the VEGF promoter and recruit RNA polymerase II. This is initial evidence that pVHL, without any genetic alteration, can be regulated by microRNA and explains the aberrant autocrine VEGF secretion in CLL.

Description: Background: B-Chronic Lymphocytic Leukemia (CLL) is predominantly characterized as a clonal B-cell disorder where a minor fraction of CLL B cells proliferate, but the majority of these cells are non-cycling. Importantly, CLL B cells also exhibit apoptosis resistance that results from a complex set of redundant mechanisms. This latter feature presents a significant problem in relation to disease progression and developing effective therapies in CLL. Previously, we and others have reported that leukemic CLL cells spontaneously secrete pro-angiogenic vascular endothelial growth factor (VEGF) that can significantly enhance CLL leukemic B cell apoptosis resistance via unknown mechanisms. In addition, the molecular mechanism of the constitutive upregulation of VEGF in CLL B cells is unknown. Methods and Results: Here, we report that CLL B cells express a high level of HIF-1α protein under normoxic conditions as compared to that of normal B cells in Western blot analysis using a specific antibody. Accumulation of HIF-1α in CLL B cells resulted in upregulation of its downstream target VEGF. To comprehend this abnormal overexpression of HIF-1α, we have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1α degradation by Western blot analysis and found it to be either absent or notably low level in CLL B cells as compared to that of normal B cells. Furthermore, we have shown by in vitro reporter gene assays that miR-92a, a microRNA known to be overexpressed in CLL B cells, can target the VHL 3′-untranslated region (UTR) and is likely a reason for the depressed levels of pVHL in CLL B cells. To validate our hypothesis, we transiently transfected the human embryonic kidney cell line (293T) with miR-92a and observed a dose-dependent repression of pVHL level. Post-transcriptional regulation of the VHL gene was further confirmed when we observed a subtle but definite increase of the pVHL levels monitored by immunoblot following nucleofection of the antisense oligo targeted to miR-92a into primary CLL B cells. To examine whether HIF-1α is functionally active in CLL B cells, we performed co-immunoprecipitation experiments and found that HIF-1α forms an active complex by physical association with the transcriptional co-activator p300 and phospho-STAT3 in CLL B cells. The latter molecule is known to be constitutively expressed in CLL B cells but its function has been unknown. Subsequently, we have found via chromatin immunoprecipitation analyses in CLL B cells that HIF-1α and STAT3 are bound to the VEGF promoter and also recruit RNA polymerase II, further substantiating that this complex likely functions in the activation of VEGF transcription. Summary: Overall, our data show that CLL B cells express constitutively elevated HIF-1α levels in normoxia due, at least in part, to miR-92a regulated diminished pVHL expression. This is a unique previously undescribed mechanism for enhanced HIF-1α levels in human malignancy. We believe that Increased VEGF expression is associated with the combined effects of HIF-1α, p300 and constitutively active STAT3 as a functional complex and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. These findings now provide additional strategy points for interrupting the VEGF autocrine pathway in CLL B cells.

Description: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease and represents a significant health problem in the western world. We and others have reported that primary CLL B-cells spontaneously produce increased levels of proangiogenic basic fibroblast growth factor (bFGF) in vitro and that most CLL plasma contains elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated the functional implication of the FGF/FGF receptor (FGFR) signaling axis in CLL B-cell biology. We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor tyrosine kinase (RTK), but no or notably low levels of FGFR2/FGFR4, by flow cytometry and Western blot analyses in primary CLL B-cells. This observation was further supported by detection of FGFR1/FGFR3 transcripts in CLL B-cells by semi-quantitative reverse transcriptase polymerase chain reaction. Although both FGFR1 and FGFR3 in CLL B-cells remain as constitutively phosphorylated, we found significantly higher levels of phosphorylation on FGFR3 and thus this latter receptor is likely the predominant RTK of the FGFR family in these leukemic B-cells. Of note, in vitro stimulation of FGFRs with recombinant bFGF was unable to increase total phosphorylation on FGFRs from their constitutive basal levels in CLL B-cells. Further analysis using a bFGF neutralizing antibody suggested that FGFR phosphorylation in CLL B-cells is likely independent of bFGF ligation. We then interrogated the mechanism of how FGFRs were being phosphorylated and/or maintained at the observed constitutive levels of phosphorylation in CLL B-cells. Our previous studies established that Axl is a critical RTK in CLL B-cells since it acts as a docking site for multiple cellular kinases/lipase, an observation supported by earlier literatures in human malignancies. Given this, Axl is likely capable of cross talk with other RTKs including FGFRs to regulate FGFR-signaling in CLL B-cells. Therefore, in an effort to determine whether Axl is functionally associated with FGFR, we examined if these two RTKs exist in the same molecular complex in CLL B-cells. Indeed, immunoprecipitation assays demonstrated that Axl formed a complex with FGFR3 in CLL B-cells, suggesting that Axl is likely functionally linked to the FGFR signaling. In this regard we found that Axl inhibition, using a high-affinity Axl inhibitor (TP-0903; Tolero Pharmaceuticals), resulted in significant reduction of total FGFR phosphorylation in CLL B-cells. Additionally, siRNA-mediated partial depletion of Axl in CLL B-cells reduced total FGFR phosphorylation. In contrast, inhibition of FGFR phosphorylation using a high-affinity FGFR inhibitor could not alter phosphorylation levels on Axl RTK in CLL B-cells. Together, these findings suggest that Axl has a dominant role in the regulation of FGFR signaling in CLL B-cells. To find out if inhibition of FGFR can induce apoptosis in CLL B-cells we used a specific inhibitor for FGFR (TKI-258; Novartis) to treat CLL B-cells. Here we found a substantial level of apoptosis induction in the leukemic B-cells with a mean LD50 dose of ~2.5 μM. Interestingly, Axl inhibition by TP-0903 induced a robust level of apoptosis in CLL B-cells in the nanomolar dose range with a mean LD50 dose of 0.14 mM. Thus Axl inhibition exerts a very robust cytotoxic effect on CLL B-cell survival likely targeting both Axl and FGFR signaling pathways via Axl inhibition. In conclusion, we have detected expression of constitutively active FGFR1 and 3 in primary CLL B-cells and that inhibition of FGFR signaling induces considerable levels of CLL B-cell apoptosis albeit lower than that observed on Axl RTK inhibition. Interestingly, our findings here suggest that Axl forms an active RTK complex with FGFR and that Axl inhibition modifies FGFR phosphorylation levels. Thus it is likely that Axl RTK can regulate FGFR signaling in the CLL B-cells. In total these observations suggest that the finding of robust induction of apoptosis in CLL B-cells is as a result of targeting two signaling pathways with Axl inhibition: Axl and FGFR. These studies further support investigation of Axl inhibition as a way to develop a more effective and efficient therapeutic intervention for CLL patients. Disclosures Warner: Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Description: Background: A challenge for novel therapeutic strategies will be the fine tuning of intracellular reactive oxygen species (ROS) signaling in order to effectively deprive cells from ROS-induced tumor promoting events and subsequent tipping the balance to ROS-induced apoptotic signaling. ROS plays a critical role in regulation of the pro-survival receptor tyrosine kinase (RTK) signaling pathways in human cancers. Studies have identified mitochondrial metabolism as the key source for abundant ROS in chronic lymphocytic leukemia (CLL). Unlike in other malignant cells, increased oxidative phosphorylation but not increased aerobic glycolysis has been found in CLL B-cells. While we have detected constitutively phosphorylated/active RTKs like Axl in CLL B-cells, the mechanism of activation remains largely undefined. Here we report that (i) elevated ROS activates the Axl signaling pathway in CLL B-cells and, (ii) mechanism of increased ROS accumulation in the leukemic B-cells. Methods: CLL B- and normal B-cells were purified from peripheral blood of previously untreated CLL patients and normal healthy individuals, respectively using a RossetteSep B-Cell enrichment kit. CLL B-cells were exposed to H2O2 for 5 min and cell lysates were analyzed for activation of RTKs including Axl by immunoprecipitation/Western blots. We also examined expression status of SIRT3, acetylated-superoxide dismutase (SOD)2 and catalase in CLL B-cells by Western blots. Catalase mRNA levels in CLL B-/normal B-cells were determined by qRT-PCR. In some experiments, genomic DNA was isolated from CLL B-/normal B-cells for catalase promoter methylation studies. Finally, accumulation of O2‾ and H2O2 in CLL B- and normal B-cells were measured by flow cytometry after treating the cells withdihydroethidium (DHE) and dichlorohydrofluorescein diacetate (DCFDA), respectively. Results: We found that enforced induction of ROS significantly increases tyrosine phosphorylation levels on multiple cellular proteins in CLL B-cells (Fig. 1). Further analysis finds that increased ROS activates Axl and its downstream targets AKT/Erk1/2 MAPK and the fibroblast growth factor receptor (FGFR) which we recently defined as a downstream target of Axl; while ROS accumulation did not show any significant effect on other RTKs, cMET or IGFR1, in CLL B-cells. Interestingly, the histone deacetylase SIRT3 which activates mitochondrial SOD2 via deacetylation, we found, was overexpressed in CLL B-cells as compared to normal B-cells indicating more efficient conversion of O2‾ into H2O2 in the leukemic B-cells (Fig. 2). However expression of catalase, which converts H2O2 into O2 and H2O, was reduced significantly in CLL B-cells as compared to normal B-cells both at mRNA and protein levels. To delineate the mechanism of reduced expression of catalase in CLL B-cells we studied the human catalase promoter region for potential methylation sites using the method of HpaII restriction enzyme digestion where HpaII is unable to cut DNA (CCGG site) when the internal cytosine is methylated. Initial findings from this approach suggest that while the catalase gene promoter in CLL B-cells may contain methylated cytosines, the catalase gene in normal B-cells may not confer methylation in the promoter region. Next, genomic DNA from CLL B-cells (n=10) and normal B-cells (n=6) was subjected to bisulfite treatment, followed by PCR amplification and sequencing. This approach revealed no significant promoter methylation of the catalase gene in normal B-cells, while moderate to heavily methylated promoter of the catalase gene was detected in CLL B-cells from majority of CLL patients (9 of 10). Further studies are in progress to examine catalase promoter methylation status in a larger cohort of CLL patients and its association with H2O2 accumulation, RTK phosphorylation and risk-factors. Indeed, flow cytometric analysis finds lower levels of O2‾ but accumulation of H2O2 in CLL B-cells as compared to normal B-cells. Taken together, these findings suggest that although SOD2 remains highly active, increased accumulation of H2O2 may occur in CLL B-cells due to epigenetic silencing of the catalase gene. Conclusion: These observations may explain, at least in part, the expression of constitutively active Axl signaling pathway in CLL B-cells that plays a role in CLL B cell survival and may account for the prolonged survival and/or apoptotic resistance of the leukemic B-cells. Disclosures No relevant conflicts of interest to declare.