E35, a Novel Analog of Emodin, Selectively Eliminates Acute Leukemia Stem and Progenitor Cells

Leukemia stem cells (LSCs) play central roles in the pathogenesis of acute leukemia and contribute to both disease initiation and relapse. Thus, identification of agents to eradicate LSCs is an important priority. Our previous studies have demonstrated that emodin, an extracted natural compound, might exert anti-leukemic effects in vitro. To improvethe bioavailability and solubility of emodin, we designed and synthesized a family of novel emodin analogs. Among them, E35 has demonstrated greater bioactivity. In this study, we investigated whether E35 may selectively eliminate acute leukemia stem and progenitor cellsand the underlying mechanism involved. We isolated CD34⁺ cells from leukemic stem-like cell line KG1a and resistant myeloid cell line HARs. Results demonstrated that E35 affected the viability of CD34⁺ leukemia cells with the average IC50 value of 15.33 ± 3.88 μM in KG1a cells and 9.12 ± 0.08 μM in HARs, respectively. We then performed the biological studies in primary leukemia specimens from 33 acute leukemia patients. All of the primary leukemia cells were sensitive to E35 with the average IC50 value of 13.86 ± 9.35 μM. Treatment with 16 μM and 32 μM E35 resulted in viability of 41.68 ± 3.90% (n = 33) and 16.63 ± 3.09% (n = 21) for overall primary leukemia cells, and 29.68 ± 4.18% (n = 17) and 15.98 ± 6.71% (n = 7) for CD34⁺ leukemia cells, respectively. However, both normal mononuclear cells and CD34⁺ cells obtained from healthy donors were not significantly affected by E35. The viability at 16 μM was 79.43 ± 3.73% (n = 6) for overall healthy cells and 88.64 ± 5.00% (n = 4) for CD34⁺ hematopoieticcells. Around 65% normal hematopoieticcells remained alive even though treated with E35 as high as 32 μM. E35 strongly induced robust apoptosis of LSCs from KG1a and HARs. After exposure to 16 μM E35 for 24 hours, the percentages of apoptotic LSCs in KG1a and HARs significantly increased up to 4.7 and 2.0 folds compared with those of without treatment groups (P < 0.01, n = 3), respectively. The percentage of CD34⁺ Annexin V⁺ cells from primary leukemia specimens following E35 treatment was also significantly greater than that in untreated group (27.29 ± 6.29% vs 10.08 ± 2.15%; P < 0.05, n = 15). Consistently, E35 stimuli raised the percentage of CD34⁺ CD38^- LSCs undergoing apoptosis from 19.79 ± 4.91% to 28.36 ± 7.14% (n = 14). Notably, E35 did not trigger apoptosis in normal hematopoietic stemcells on comparison with control group (P > 0.05, n = 5). Western Blotting results showed that activation of Caspase-family proteins (eg, pro-caspase and PARP cleavage) and inhibiton of Akt/mTOR signaling pathway (eg, p-Akt, p-4E-BP1 and p-p-70S6K blockage) might be key steps toward LSC-specific apoptosis by E35. To determine whether E35 targets functionally defined leukemia stem and progenitor cells, colony assays and NOD/SCID xenotransplantation were performed. Primary AML cells versus normal cells were treated with E35 for 18 hours, followed by plating in methylcellulose culture or transplanting into Busulfan preconditioned NOD/SCID mice. Normal hematopoietic cells with E35 treatment did not affect myeloid colony formation relative to untreated control (n = 6). In contrast, E35 treatment strongly inhibited the ability of leukemia cells to form colonies (P < 0 .0001, n = 7). Similarly, primary leukemic cells with with E35 treatment led to a significant decrease in stem cell activity, with reduction engraftment levels from 19.47 ± 4.77 % to 2.92 ± 0.34 % (P < 0.01, n = 7). In contrast, the engraftment levels of E35 preconditioned normal cells were not statistical significant different from those of healthy cells without treatment (28.10 ± 5.30% vs 31.10 ± 6.88%, n = 4). These data demonstrate that E35 specifically ablates leukemic but not normal hematopoietic progenitor- and stem-cell function. In conclusion, E35 has the potential to selectively eradicate primitive leukemic cells, indicating it is a novel therapeutic candidate against hematologic cancers.