Response of cell lines and primary marrow cells to DZNep and 5AZA.

<p>Proliferation study in, <b>A</b>) KG1a cells overexpressing let-7b, <b>B</b>) primary marrow cells from healthy donors and MDS patients with <10% marrow blasts, and <b>C</b>) MDS patients with 10%–20% marrow myeloblasts. Exposure to DZNep, 5AZA or both affected the proliferation in KG1a cells overexpressing let-7b and cells from MDS marrows with 10%–20% but not in cells from patients with <10% marrow blasts (or healthy donors). Exposure to DZNep, 5AZA or combination of both agents (inside of red boxes) affected the proliferation in KG1a cells overexpressing let-7b and cells from MDS marrows with 10–20% but not in cells from patients with <10% marrow blasts or healthy donors. <b>D</b>) and <b>E</b>) show the extent of apoptosis induced by DZNep, 5AZA or both in MDS patients with <10% and 10%–20% marrow blasts, respectively. The extent of apoptosis was significantly greater in MDS marrows with higher myeloblast counts, more so in cells being treated with the drug combination than with single agents (p = 0.04, 0.001). The results are displayed as mean ±SEM for each MDS group (5 samples for <10% marrow blasts and 6 samples for 10–20% marrow blasts) (Student t-test). <b>F</b>) Two different primary BM CD34+ cells specimens showing decreased H3K27m2 and H3K27m3, and decreased EZH2 protein expression upon combined treatment with Dznep+5AZA; Histone H3 served as nuclear loading control. <b>G</b>) Working model: role of KDM2B/let-7b/EZH2 in epigenetic regulation of cell proliferation in MDS. KDM2B represses let-7b and up-regulates EZH2. DZnep treatment inhibits histone: H3K27; methylation and bypasses the KDM2B/let-7b/EZH2 effect leading to reduced cell proliferation.