SELECTIVE EFFECT OF LENALIDOMIDE ON CELL CYCLE AND INOSITIDE-DEPENDENT ERYTHROID SIGNALLING IN DEL(5Q) CELLS AND MYELODYSPLASTIC SYNDROMES (MDS)

Background: Lenalidomide is currently used in the treatment of del(5q) lowrisk MDS patients, where it may suppress the del(5q) clone and restore a normal erythropoiesis. The exact molecular mechanisms underlying the effect of Lenalidomide in MDS patients are not completely understood, even though it has been demonstrated that in Lenalidomide-sensitive del(5q) cell lines, Akt phosphorylation is inhibited. Interestingly, Akt signalling is activated both in high-risk MDS, where it activates cell proliferation, and in low-risk MDS, where it is associated with response to erythropoietin. Aims: To detect the specific effect of Lenalidomide on clonal del(5q) and normal (5q+) cells, in low-risk MDS patients and in cell lines, mainly focusing on erythropoiesis, cell cycle and signal transduction pathways involved in cell proliferation and differentiation. Methods: This study included 6 patients diagnosed with del(5q) MDS (IPSS: Low or Int-1) treated with Lenalidomide. Given the limited number of cells, we quantified the expression of Akt in bone marrow total mononuclear cells by immunocytochemistry only. In particular, we analyzed Akt and RPS14 co-localization, in order to specifically detect the del(5q) clone. Moreover, by Real-Time PCR analyses, we also assessed the expression of Globin genes, to evaluate the effect of the drug on erythropoiesis. To better discriminate between del(5q) and normal (5q+) cells, we studied the effect of Lenalidomide on Namalwa cells, showing a del(5q) karyotype, and in U937 cells, showing a normal 5q chromosome. In both cell lines, we quantified the expression of Globin genes, cell cycle and inositide signalling molecules, by Real-Time PCR, immunocytochemistry, Western blot and flow cytometry. Results: In our case series, 4/6 del(5q) low-risk MDS patients responded to Lenalidomide, whereas the 2 non responder patients early discontinued Lenalidomide for adverse events, and for these patients neither a clinical assessment of Lenalidomide effect, nor a molecular analysis, were possible. Responder patients showed an activation of erythropoiesis, in that Beta-Globin levels increased, as compared with baseline. Moreover, these subjects displayed a specific phosphorylation of Akt in cells not showing the 5q deletion. As for cell lines, Lenalidomide specifically induced an accumulation of Namalwa cells in G0/G1 phase, which corresponded to a slight decrease of p-Akt, an increase of p27 and a decreased expression of cyclin B1, cyclin E and cyclin D3. On the contrary, in normal (5q+) cells, Lenalidomide did not affect cell cycle, but induced both p-Akt and Gamma-Globin. Summary and Conclusions: Our data show that Lenalidomide can induce a selective arrest of cell cycle in G1 phase of del(5q) cells, therefore slowing the proliferation rate of this clonal cell population. On the contrary, cyclins, Akt and Gamma-Globin are specifically activated in normal (5q+) cells, possibly stimulating a normal cell proliferation and erythroid differentiation. Taken together, these findings could lead to a better comprehension of the effect of Lenalidomide in MDS and pave the way to innovative targeted therapies in low-risk MDS patients without del(5q).