The Anti-Inflammatory Investigational Agent LMP-420 Demonstrates in Vitro Cytotoxic Activity against Chronic Lymphocytic Leukemia Cells.

Chronic Lymphocytic Leukemia (CLL) is a common incurable hematologic malignancy. When therapy is required, maximizing durable responses is often at the risk of increasing toxicity. Thus, developing novel therapeutic agents that have minimal overlapping toxicity with currently used chemotherapy would be advantageous. To this end, we investigated LMP-420, a boronic acid containing purine nucleoside analogue, that potently inhibits tumor necrosis factor alpha (TNF) transcription in stimulated peripheral blood mononuclear cells (PBMCs) without affecting cell viability. Since TNF has been implicated in promoting CLL cell viability and can be produced by CLL cells themselves, we hypothesized that LMP-420 would be cytotoxic for CLL lymphocytes, either alone or in combination with fludarabine.

To test the activity of LMP-420, we negatively selected circulating CLL cells from blood collected from patients using the RosetteSep B-cell enrichment cocktail (StemCell Technologies) and a Ficoll-Hypaque gradient. This method yields greater than 95% purity of malignant lymphocytes, determined by immunophenotyping CD5⁺CD19⁺ cells. Prognostic markers such as IgV_H mutation status, CD38 and ZAP70 expression, and interphase cytogenetics were determined as previously described. We assessed the fractional toxicity and 50% effective dose (ED50) of LMP-420, fludarabine, or the combination, with the MTS colorimetric cytotoxicity assay, in which CLL cells were incubated for three days in Hybridoma media + 10% fetal bovine serum with serial dilutions of drug alone or in combination. Apoptosis was measured via annexin V flow cytometry methods and caspase 3/7 activity assays. We determined the effect of LMP-420 compared to fludarabine on the normal hematopoietic system by testing serial dilutions of both agents in killing normal PBMCs and in suppressing erythroid and myeloid colony formation.

The median ED50 of LMP-420 for CLL cells was 423 nM (range 0.01 to 2224 nM, n = 21). Two patients had high-risk cytogenetics (17p or 11q deletions), and their ED50 values for LMP-420 were 691 and 90 nM, respectively. The cytotoxic effect of fludarabine was potentiated on average 80 or 261 fold with the addition of LMP-420 at concentrations of 62 or 250 nM, respectively (ranges 1.14 – 947 and 1.19 – 2754). This agent killed malignant lymphocytes by apoptotic mechanisms in a dose-responsive fashion, as demonstrated by both Annexin V staining and caspase 3/7 activity assays. While LMP-420 has potent anti-CLL activity, it has minimal effects on normal hematologic cells. For example, fludarabine suppresses erythroid and myeloid colony formation by greater than 50% at a concentration of 1 uM, while this level of inhibition is seen for LMP-420 at a concentration of 90 uM. The average cytotoxic ED50 of LMP-420 on normal PBMCs using the MTS assay was greater than 90 uM, whereas for fludarabine, it was 5.3 uM. This finding was confirmed with apoptosis assays.

The results of these experiments demonstrate that LMP-420, a novel inhibitor of TNF expression, has cytotoxic activity against CLL cells, including those with high-risk features. LMP-420 appears to increase the cytotoxic effect of the chemotherapy agent fludarabine, while imparting minimal increase in hematologic toxicity. Thus, LMP-420 is promising new therapeutic agent in CLL.

No relevant conflicts of interest to declare.