TWIST2 Was Deregulated In Human Acute Myeloid Leukemia and Might Play a Tumor Suppressor Role

Abstract 3141

TWIST encodes a typical basic helix-loop-helix transcription factor. Twist-2, as a member of this family was recently identified as a negative regulator of normal murine myelopoiesis (Sharabi AB. et al. PLoS Biol. 2008, 6:2786-2800), however the expression and functional role of TWIST2 in normal and leukemic human myeloid cells is poorly understood. To address this, we detected the transcript expression of TWIST2 from CD34+ cells of 19 AML patients compared with those of their normal counterparts from 6 healthy donors, and found that the expression of this transcript was 5-fold decreased in the patients. A lentiviral vector, which could only express YFP was used as control; and then the other lentiviral vector to express TWIST2 plus YFP was constructed and validated to efficiently infect various human AML cell lines including THP-1, Dami, HEL, HL60, NB4 and SHI-1. Using all these cell lines, we found that averagely the control cells could expand in the liquid culture 46-fold within 6 days, however the TWIST2 overexpressed cells could only increase 7-fold; moreover in the colony-forming cell (CFC) assays with both Dami and THP-1 cells, the TWIST2 overexpressed cells lost nearly 80% CFC capacities compared with those of control cells (n=3, P<0.05). Next we found that more TWIST2 overexpressed cells could reside in the G0/G1 phase compared with control cells (61% vs. 44%, n=2, P<0.05) and accordingly less TWIST2 overexpressed cells were present in the G2/S/M phase compared with control cells (39% vs. 56%, n=2, P<0.05). In addition, the control and TWIST2 viruses infected Dami (2×10⁶ cells/mouse) and THP-1 cells (4×10⁶ cells/mouse) were subcutaneously injected into nude mice without irradiation respectively, and the control cells could generated tumors (7/7 mice with Dami cells and 2/8 mice with THP-1 cells) but not the TWIST2 overexpressed cells (0/8 mice with Dami cells and 0/6 mice with THP-1 cells) within 4 weeks. To obtain molecular insights of how TWIST2 suppressed the tumourgenesis capacity of these cells, microarray analysis (Agilent 44K) were used to assess the differentially expressed transcripts between control and TWIST2 overexpressed THP-1 cells (from 3 independent experiments). Some examples of the differentially expressed genes included CCND1, RBBP9 and EPHB4, which were down-regulated in the TWIST2 overexpressed cells compared with control cells; in the other hand CDKN1A (p21^Cip1), TP53INP1, DDI1 and AKTIP, which were up-regulated in the TWIST2 overexpressed cells compared with control cells. Taken together, these data demonstrated that the TWIST2 was deregulated in human AML patients, and suggested it might play a tumor suppressor role in these cells.