Ghosh, Asish K.

Abstract Our study in CLL delineated a unique mechanism of how bone marrow stromal cells (BMSCs), may influence CLL B-cell signaling and drug resistance via modifying β-catenin and Axl status in leukemic cells. Purified primary CLL B-cells (n= 31) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=23) or CLL patients (n=12) 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 real-time PCRs. We observed significant increases in expression of Axl at 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 co-cultured CLL B-cells. But, we did not see any significant change in β-catenin or Axl expression in BMSCs co-cultured with CLL B-cells. The CLL B-cells from co-culture had an upregulation in downstream P-ERK-1/2 but no change in P-AKT(Ser473). High nuclear active-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) suggests inactivation of GSK-3β and β-catenin accumulation in co-cultured CLL B-cells. Additionally, we determined the phosphorylation status of Axl in CLL B-cells in co-culture compared with cultured alone. There was no change either at Y702 or total tyrosine phosphorylation 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 Axl inhibitor TP-0903 on CLL B-cells in co-culture vs cultured alone. Interestingly, both Axl and β-catenin expression levels were further upregulated in CLL B-cells exposed to TP-0903 compared to CLL B-cells without TP-0903 in co-culture, indicating possible roles for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance. However, TP-0903 decreased P-Axl(Y702) as well as an Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells even in presence of BMSCs. This was associated with induction of cell death and overrode BMSC mediated protection. Treatments of fludarabine or chlorambucil also led to increase in expressions of both β-catenin and Axl 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. Here we show that stroma mediated increased expression in both β-catenin and Axl likely contribute to the survival and drug resistance of B-cells from patients with CLL. Understanding their role in drug metabolism is underway. Citation Format: Sutapa Sinha, Charla Secreto, Justin Boysen, Steven L. Warner, David J. Bearss, Asish Ghosh, Neil E. Kay. Enhanced expression of β-catenin and Axl receptor tyrosine kinase in chronic lymphocytic leukemia (CLL) B-cells with co-culture on marrow stromal cells: Implications for drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2144.

Abstract 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 CLL plasma contained elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated functional implication of the FGF/FGFR signaling axis in CLL B cell biology. We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor, but not FGFR2/FGFR4, in primary CLL B cells. Interestingly, constitutively phosphorylated FGFR and its downstream signaling adaptor protein fibroblast receptor substrate 2-α were also detected in CLL B cells. However, in vitro stimulation of FGFR with recombinant bFGF was unable to increase endogenous phosphorylation levels of FGFR in CLL B cells from most CLL patients tested, suggesting existence of an autocrine FGF/FGFR signaling loop in the leukemic B cells. We know that Axl a known predominant RTK in CLL B cells seems to act as a docking site for multiple cellular kinases/lipase. Therefore, in an effort to determine whether Axl is functionally associated with FGFR in CLL B cells, we first examined if these two RTKs exist in the same molecular complex. Indeed we found that Axl forms a complex with FGFR3 in CLL B cells and is therefore likely to regulate FGFR signaling. Our hypothesis in this regard was further supported when we inhibited Axl phosphorylation in CLL B cells using a high-affinity Axl inhibitor (TP-0903; Tolero). Axl inhibition resulted in significant reduction of FGFR phosphorylation in CLL B cells. Finally, inhibition of FGFR signaling using a high-affinity FGFR inhibitor induced significant levels of apoptosis in CLL B cells (n=20) with a mean LD50 dose of ∼2.5 μM. Of interest, LD50 dose for CLL B cell apoptosis was much lower for the Axl inhibitor (mean LD50=140nM; n=20) compared to the FGFR inhibitor, which we believe occurs partly because TP-0903 seems to exert its inhibitory effect on P-FGFR likely via P-Axl inhibition in CLL B cells. In conclusion, we have detected expression of constitutively active FGFR in primary CLL B cells. Interestingly, our preliminary findings suggest that Axl forms an active RTK complex with FGFR and thus likely regulates FGFR signaling in these leukemic B cells. This is evidenced by the finding that Axl inhibition significantly reduced FGFR phosphorylation accompanied by massive induction of CLL B cell apoptosis, suggesting that targeting dual RTK signaling pathways (Axl and FGFR) in CLL B cells warrants further investigation as a way to develop a more effective and efficient therapeutic intervention for CLL patients. Citation Format: Sutapa Sinha, Justin Boyson, Charla Secreto, Steven L. Warner, Neil E. Kay, Asish K. Ghosh. Fibroblast growth factor receptor is expressed as a constitutively active receptor tyrosine kinase in chronic lymphocytic leukemia B cells and exists in an active complex with Axl: Dual targeting in CLL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3448. doi:10.1158/1538-7445.AM2014-3448

Abstract Purpose: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite aggressive therapeutic approaches. We previously found that Axl receptor tyrosine kinase (RTK) plays a critical role in CLL B-cell survival. Here, we explored the possibility of using a high-affinity Axl inhibitor as a single agent or in combination with Bruton's tyrosine kinase (BTK) inhibitors for future clinical trial to treat patients with CLL. Experimental Design: Expression/activation status of other members of the TAM (e.g., Tyro3, Axl, and MER) family of RTKs in CLL B cells was evaluated. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 with or without the presence of CLL bone marrow stromal cells (BMSCs). Inhibitory effects of TP-0903 on the Axl signaling pathway were also evaluated in CLL B cells. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination. Results: CLL B cells overexpress Tyro3, but not MER. Of interest, Tyro3 remains as constitutively phosphorylated and forms a complex with Axl in CLL B cells. TP-0903 induces massive apoptosis in CLL B cells with LD50 values of nanomolar ranges. Importantly, CLL BMSCs could not protect the leukemic B cells from TP-0903–induced apoptosis. A marked reduction of the antiapoptotic proteins Mcl-1, Bcl-2, and XIAP and upregulation of the proapoptotic protein BIM in CLL B cells was detected as a result of Axl inhibition. Finally, combination of TP-0903 with BTK inhibitors augments CLL B-cell apoptosis. Conclusions: Administration of TP-0903 either as a single agent or in combination with BTK inhibitors may be effective in treating patients with CLL. Clin Cancer Res; 21(9); 2115–26. ©2015 AACR.

Key Points miR-155 levels are significantly and increasingly overexpressed as normal B cells progress to MBL and to CLL. Plasma levels of miR-155 before treatment were significantly lower in patients with CLL who experienced complete response than in all others.

Abstract Recently, we detected that chronic lymphocytic leukemia (CLL) B-cell–derived microvesicles in CLL plasma carry a constitutively phosphorylated novel receptor tyrosine kinase (RTK), Axl, indicating that Axl was acquired from the leukemic B cells. To examine Axl status in CLL, we determined the expression of phosphorylated-Axl (P-Axl) in freshly isolated CLL B cells by Western blot analysis. We detected differential levels of P-Axl in CLL B cells, and further analysis showed that expression of P-Axl was correlated with the other constitutively phosphorylated kinases, including Lyn, phosphoinositide-3 kinase, SyK/ζ-associated protein of 70 kDa, phospholipase C γ2 in CLL B cells. We found that these intracellular signaling molecules were complexed with P-Axl in primary CLL B cells. When Axl and Src kinases were targeted by a Src/Abl kinase inhibitor, bosutinib (SKI-606), or a specific-inhibitor of Axl (R428), robust induction of CLL B-cell apoptosis was observed in both a dose- and time-dependent manner. Therefore, we have identified a novel RTK in CLL B cells which appears to work as a docking site for multiple non-RTKs and drives leukemic cell survival signals. These findings highlight a unique target for CLL treatment.

Abstract 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.

Purpose To define the optimal dose of Polyphenon E for chronic daily administration and tolerability in patients with chronic lymphocytic leukemia (CLL). Patients and Methods Previously untreated patients with asymptomatic Rai stage 0 to II CLL were eligible for participation. Polyphenon E with a standardized dose of epigallocatechin-3-gallate (EGCG) was administered using the standard phase I design with three to six patients per dose level (range, 400 to 2,000 mg by mouth twice a day). Trough plasma EGCG levels were measured 1 month after initiation of therapy. Response was classified using the National Cancer Institute (NCI) Working Group (WG) Criteria. Results Thirty-three eligible patients were accrued to dose levels 1 to 8. The maximum-tolerated dose was not reached. The most common adverse effects included transaminitis (33%, all grade 1), abdominal pain (30% grade 1, 0% grade 2, and 3% grade 3), and nausea (39% grade 1 and 9% grade 2). One patient experienced an NCI WG partial remission. Other signs of clinical activity were also observed, with 11 patients (33%) having a sustained ≥ 20% reduction in absolute lymphocyte count (ALC) and 11 (92%) of 12 patients with palpable adenopathy experiencing at least a 50% reduction in the sum of the products of all nodal areas during treatment. Trough plasma EGCG levels after 1 month of treatment ranged from 2.9 to 3,974 ng/mL (median, 40.4 ng/mL). Conclusion Daily oral EGCG in the Polyphenon E preparation was well tolerated by CLL patients in this phase I trial. Declines in ALC and/or lymphadenopathy were observed in the majority of patients. A phase II trial to evaluate efficacy using 2,000 mg twice a day began in November 2007.

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.

Abstract Purpose: Chronic lymphocytic leukemia (CLL) is incurable with current chemotherapy treatments. Curcumin (diferuloylmethane), an active ingredient in the spice turmeric, inhibits tumor metastasis, invasion, and angiogenesis in tumor cell lines. We evaluated the effects of curcumin on the viability of primary CLL B cells and its ability to overcome stromal mediated protection. Experimental Design: The in vitro effect of curcumin on primary CLL B cells was evaluated using fluorescence activated cell sorter analysis and Western blotting. For some experiments, CLL B cells were cocultured with human stromal cells to evaluate the effects of curcumin on leukemia cells cultured in their microenvironment. Finally, the effect of curcumin in combination with the green tea extract epigallocatechin-3 gallate (EGCG) was evaluated. Results: Curcumin induced apoptosis in CLL B cells in a dose-dependent (5-20 μmol/L) manner and inhibited constitutively active prosurvival pathways, including signal transducers and activators of transcription 3 (STAT3), AKT, and nuclear factor κB. Moreover, curcumin suppressed expression of the anti-apoptotic proteins Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP), and up-regulated the pro-apoptotic protein BIM. Coculture of CLL B cells with stromal cells resulted in elevated levels of STAT3, increased expression of Mcl-1 and XIAP, and decreased sensitivity to curcumin. When curcumin was administered simultaneously with EGCG, antagonism was observed for most patient samples. In contrast, sequential administration of these agents led to substantial increases in CLL B-cell death and could overcome stromal protection. Conclusions: Curcumin treatment was able to overcome stromal protection of CLL B cells on in vitro testing and to synergize with EGCG when administered in a sequential fashion. Additional evaluation of curcumin as a potential therapeutic agent for treatment of CLL seems warranted.