Bone, Nancy D.

AbstractWe recently reported that chronic lymphocytic leukemia (CLL) cells synthesize and release vascular endothelial growth factor (VEGF) under normoxic and hypoxic conditions. CLL B cells also express VEGF membrane receptors (VEGF-R1 and VEGF-R2), suggesting that they use VEGF as a survival factor. To assess the mechanism of apoptosis resistance related to VEGF, we determined the impact of VEGF on CLL B cells, and we studied the impact of epigallocatechin-3-gallate (EGCG), a known receptor tyrosine kinase (RTK) inhibitor, on VEGF receptor status and viability of CLL B cells. VEGF165 significantly increased apoptotic resistance of CLL B cells, and immunoblotting revealed that VEGF-R1 and VEGF-R2 are spontaneously phosphorylated on CLL B cells. EGCG significantly increased apoptosis/cell death in 8 of 10 CLL samples measured by annexin V/propidium iodide (PI) staining. The increase in annexin V/PI staining was accompanied by caspase-3 activation and poly–adenosine diphosphate ribose polymerase (PARP) cleavage at low concentrations of EGCG (3 μg/mL). Moreover, EGCG suppressed the proteins B-cell leukemia/lymphoma-2 protein (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and myeloid cell leukemia-1 (Mcl-1) in CLL B cells. Finally, EGCG (3-25 μg/mL) suppressed VEGF-R1 and VEGF-R2 phosphorylation, albeit incompletely. Thus, these results suggest that VEGF signaling regulates survival signals in CLL cells and that interruption of this autocrine pathway results in caspase activation and subsequent leukemic cell death.

Abstract Adaphostin (NSC 680410), an analog of the tyrphostin AG957, was previously shown to induce Bcr/abl down-regulation followed by loss of clonogenic survival in chronic myelogenous leukemia (CML) cell lines and clinical samples. Adaphostin demonstrated selectivity for CML myeloid progenitors in vitro and remained active in K562 cells selected for imatinib mesylate resistance. In the present study, the mechanism of action of adaphostin was investigated in greater detail in vitro. Initial studies demonstrated that adaphostin induced apoptosis in a variety of Bcr/abl- cells, including acute myelogenous leukemia (AML) blasts and cell lines as well as chronic lymphocytic leukemia (CLL) samples. Further study demonstrated that adaphostin caused intracellular peroxide production followed by DNA strand breaks and, in cells containing wild-type p53, a typical DNA damage response consisting of p53 phosphorylation and up-regulation. Importantly, the antioxidant N-acetylcysteine (NAC) blunted these events, whereas glutathione depletion with buthionine sulfoximine (BSO) augmented them. Collectively, these results not only outline a mechanism by which adaphostin can damage both myeloid and lymphoid leukemia cells, but also indicate that this novel agent might have a broader spectrum of activity than originally envisioned. (Blood. 2003;102:4512-4519)

AbstractPreviously, it was reported that patients with multiple myeloma (MM) who have higher baseline levels of blood CD4+or CD19+ cells have longer survival. This article extends the analysis of immune cell levels and survival in a large cohort (N = 504) of patients with MM entered on Eastern Cooperative Oncology Group (ECOG) phase 3 trial (9486). Newly diagnosed patients with MM received 2 cycles of vincristine, bischloroethylnitrosourea, melphalan, cytoxan, prednisone (VBMCP) and were treated on one of 3 randomized arms: VBMCP with either interferon or high-dose cyclophosphamide, or VBMCP alone. Blood immune cell levels were studied at trial entry (baseline), after 2 cycles of chemotherapy, after 2 years of therapy, and at relapse. Baseline CD3+, CD4+, CD8+, CD19+, and CD4+ subset cell levels were all positively associated with survival (P = .0087 toP < .0001). A multivariate analysis incorporating CD4+ and CD19+ cell levels defined 3 separate groups of patients with MM to survival outcome. Higher CD19+ blood levels were positively associated with MM-patient survival at entry to the study, at year 2, and at relapse (P < .0001 at all 3 timepoints). Patients with MM had evidence of immune cell reconstitution after 2 years of therapy, but the rate and extent of recovery was greater for CD8+, which was greater than CD4+, which was greater than CD19+. This latter data affirms the positive relationship between the quantitative status of the blood immune system in MM and survival. In addition, the importance of the CD19+ blood cells to survival is evident throughout the course of MM. Therapeutic efforts to maintain an intact immune system may be crucial in maximizing chemotherapeutic and/or immunotherapy efforts in this disease.