Acitretin Reduces L-Selectin Expression and Tumour Cell Homing in Chronic Lymphocytic Leukaemia (CLL)

Introduction:

The microenvironment plays a major role in the survival of Chronic Lymphocytic Leukaemia (CLL) cells by promoting CLL cell growth and drug resistance. The traffic of CLL cells that involve migration from the blood and adhesion to the high endothelial venules (HEVs) facilitates the homing of lymphocytes in the lymph nodes to protect CLL cells and offer them the necessary survival signals. L- Selectin mediates adhesion of CLL cells to LN-microenvironment through a multistep process that entails rolling, sticking, and crawling. Acitretin is a retinoid that used in the treatment of severe psoriasis and less frequently in the control of squamous skin cancer. Acitretin activates nuclear retinoic acid receptors (RAR), resulting in induction of cell differentiation, inhibition of cell proliferation, and inhibition of tissue infiltration by inflammatory cells. It is reported that retinoids induce apoptosis in CLL and improve the sensitivity of CLL cells to fludarabine.

Method:

We used MEC-1 "CLL like" cell line and CLL cells from known CLL patients to investigate the effect of acitretin on the expression of the major markers that control CLL cells traffic including L-selectin (CD62L), CD49d and CXCR4 (CD184) . We treated CLL cells with increasing doses of acitretin (10 nM up to 10 mM). After treatment for 48 to 72 hours, L-selectin, CD49d and CXCR4 expression was measured using flowcytometry. Dimethyl sulfoxide (DMSO) treated cells were used as a negative control. A Leukocyte adhesion assay was done using CytoSelect Leukocyte-endothelium adhesion assay according to the protocol provided. To assess the mechanism through which L-Selectin reduces the adhesion of CLL cells to the endothelium, we used western blot to measure the effect of acitretin on PI3k-p85.

Results:

Acitretin in a dose as low as 10 nM significantly reduced the expression of L-selectin in primary CLL cells after treatment for 48 hours (P= 0.0475) and 72 hours (P= 0.0048). On the contrary, acitretin did not change the expression of CD49d or CXCR4. Similar significant reduction in L-selectin expression was recognized when MEC-1 cells were treated with 10 nM of acitretin for 48 hours (P= 0.0179) and 72 hours (P= 0.0159).

To test the effect of acitretin on the adhesion of MEC-1 cells to the endothelium; we used CytoSelect adhesion assay that showed significant reduction in the adhesion of MEC-1 cells to human umbilical vein endothelial cells (HUVEC) after treatment with acitretin 10 mM for 48 hours (P= 0.0048). Ibrutinib treatment has also significantly reduced the adhesion of MEC-1 cells to HUVEC cells (P= 0.0045) and when combined with acitretin the reduction in adhesion was more pronounced (P= 0.0003).

Finally, to understand the mechanism through which acitretin down regulates the expression of L-selectin and reduces the adhesion of MEC-1 cells, we tested the effect of acitretin on the PI3K pathway using western blot. PI3K is required for the cytoskeleton changes during neutrophil rolling on E‐selectin and we investigated if similar interaction takes place between PI3K and L-selectin in CLL cells. Treatment of MEC-1 cells with acitretin 10 mM for 48 hours resulted in significant down regulation of the PI3K-p85 (P= 0.0007). Treatment with Ibrutinib alone or combined with acitretin did not result in significant change in PI3K- p85. This may explain the mechanism through which acitretin reduces the expression of L-selectin and compromises CLL adhesion to the endothelium.

Conclusion:

We showed that the retinoid acitretin significantly reduced the expression of L-selectin in CLL cells and MEC-1 "CLL like" cells in vitro. The reduction in L-selectin resulted in reduced adhesion of CLL cells to the endothelium hampering homing of CLL cells to lymph nodes. The mechanism through which acitretin exerts this effect can be explained by down regulation of PI3K-p85. The use of acitretin as an adjunct treatment in CLL can be beneficial, however further research including in vivo studies is required.

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