Citations

Key Points Deep sequencing identifies a significant reservoir of subclonal mutations affecting key genes in CLL pathogenesis. Convergent evolution of genetic lesions in tumor subclonal populations is recurrently found in CLL.

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.

Abstract Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 protein family. Increased Mcl-1 expression is associated with failure to achieve remission after treatment with fludarabine and chlorambucil in patients with chronic lymphocytic leukemia (CLL). However, the influence of Mcl-1 expression has not been examined in CLL trials using chemoimmunotherapy. We investigated Mcl-1 protein expression prospectively as part of a phase 2 study evaluating the efficacy of pentostatin, cyclophosphamide, and rituximab in patients with untreated CLL. No significant difference by Mcl-1 expression was noted in pretreatment or response parameters. However, in patients with higher Mcl-1 expression, both minimal residual disease-negative status and progression-free survival was found to be significantly reduced (57% vs 19%, P = .01; 50.8 vs 18.7 months; P = .02; respectively). Mcl-1 expression may therefore be useful in predicting poor response to chemoimmunotherapy. These findings further support pursuing treatment strategies targeting this important antiapoptotic protein. (Because the trials described were conducted before the requirement to register them was implemented, they are not registered in a clinical trial database.)