CD44-Induced Differentiation of Acute Monoblastic Leukemia Cells Is Mediated by Prolonged Activation of ERK1/2 and by Cytokines.

Abstract 3103

Poster Board III-40

We have previously reported that specific monoclonal antibodies (mAbs) to the cell adhesion molecule CD44, trigger monocytic differentiation of blasts from patients with acute monoblastic leukemia (1) and that cytokines contribute this differentiation (2). To further analyze the mechanisms mediating CD44-induced monocytic differentiation of leukemic cells, we have used as a model the THP-1 leukemia cell line which differentiation has been induced by the Hermes-3 anti-CD44 mAb. Monocytic differentiation has been checked at day 3 by measuring, using flow cytometry, the increase in the expression levels of CD11b (myeloid-specific) and CD14 (monocyte-specific) differentiation antigens. Control THP-1 cells (untreated or treated with isotypic IgG) display only a little CD11b and do not express CD14.

First, using western blot analysis, we show that CD44-ligation with Hermes-3 mAb rapidly triggers ERK1/2 MAP kinase phosphorylation, that persists for about 6-8 hours. This prolonged ERK1/2 phosphorylation is essential for CD44-induced differentiation, since its delayed inhibition (using the specific chemical inhibitor U0126) fully abrogates the differentiation process. Similar results have been obtained in Hermes-3 - treated primary blasts from patients with acute monoblastic leukemia. Interestingly, ERK1/2 signalling mediates normal monocytic differentiation induced by cytokines (3). Accordingly, our present results suggest that CD44-ligation might reactivate this physiological differentiation pathway persisting in monoblastic leukemia cells (THP-1 cells and primary leukemic monoblasts).

Second, using real-time RQ-PCR and ELISA assays, we show that ERK1/2 activation leads to synthesis and secretion of a burst of cytokines, including IL-1b, IL-6, IL-8, GM-CSF and TNF-alpha. Among these cytokines, TNF-alpha- and IL-6, acting through an autocrine-paracrine pathway, play an important part in the CD44-induced differentiation process, since their functional inhibition with specific mAbs dramatically decreases the extent of this differentiation. TNF-alpha has a critical role in the increase of CD11b expression level. In addition, it cooperates with IL-6 for inducing CD14 expression. However, TNF-alpha- and IL-6 inhibition does not totally abrogate CD44-induced differentiation. This suggests that another unidentified cytokine is perhaps involved in the differentiation process. Alternatively, CD44-activated ERK1/2 pathway may directly reprogram the expression of monocytic differentiation genes. Interestingly, addition of exogenous IL-6 enhanced the level of CD14, suggesting that IL-6 and CD44-ligation may cooperate for differentiation therapy of acute monoblastic leukemia.

1. Charrad et al. Nat. Med 5:669,1999 2. Delaunay et al. Leukemia 22:873, 2008