PMC

Endogenous expression levels of MEIS1, PBX3, HOXA4, HOXA5, HOXA7 and HOXA9 in MonoMac-6/t(;) AML cells were detected by qPCR 48 hours post transfection of anti-MEIS1 siRNA oligos (siMEIS1) and/or anti-PBX3 siRNA oligos (siPBX3), or of scrambled control oligos (Ctrl). Mean±SD values are shown. *, p<0.05; **, p<0.01.

Of them, 22 genes are potential direct target genes of MLL-fusion proteins. Expression data was mean centered. Red represents a high expression (scale shown in the upper middle).

(A) Unsupervised hierarchical clustering analysis of the Control (n=5), HOXA9+MEIS1 (n=3), HOXA9+PBX3 (n=3), HOXA9 (n=3), PBX3+MEIS1 (n=3), and MLL-AF9 (n=3) groups. The hierarchical clustering tree is shown. (B) Gene sets that are shared by Group 2 (G2; including HOXA9+MEIS1, HOXA9+PBX3, and HOXA9) and Group 3 (G3; including PBX3+MEIS1 and MLL-AF9 (MA9)) samples, with a significantly different pattern in Group 1 (G1; normal control (NC)) samples, as detected by GSEA (). (C) Gene sets that are differentially enriched between Group 2 and Group 3 samples. NSE, normalized enrichment score; FDR, false discovery rate.

(A) Kaplan-Meier survival curves of secondary transplantation recipient (CD45.2+) mice transplanted with primary leukemic BM cells (CD45.1+) of the HOXA9+MEIS1 (recipient mouse number: n=10), HOXA9+PBX3 (n=11), PBX3+MEIS1 (n=11) and MLL-AF9 (n=10) groups. Primary AML BM cells from two donor mice were used for each group. There is no significant difference (p>0.1) between survival of the PBX3+MEIS1 group and that of any other three groups. (B) Flow cytometry analysis of leukemic BM cells from the above secondary BMT recipient mice. Antibodies against Mac-1 and c-Kit were used. Flow data of leukemic BM samples from one recipient mouse is shown as representative for each group. (C) Kaplan-Meier survival curves of secondary transplantation recipient (CD45.2+) mice transplanted with primary leukemic spleen cells (CD45.1+) of the HOXA9+MEIS1 (n=5), PBX3+MEIS1 (n=5) and MLL-AF9 (n=5) groups. Primary AML spleen cells from one donor mouse were used for each group. There is no significant difference (p>0.1) between survival of the PBX3+MEIS1 group and that of any other two groups.

(A) In vitro colony-forming/replating assays. Briefly, mouse normal BM progenitor (lineage negative; Lin-) cells collected from 4- to 6-week-old B6.SJL (CD45.1) mice were retrovirally co-transduced with MSCVneo+MSCV-PIG (Control), MSCVneo-MLL-AF9+MSCV-PIG (MLL-AF9), MSCVneo-HOXA9+MSCV-PIG-MEIS1 (HOXA9+MEIS1), MSCVneo-MEIS1+MSCV-PIG-PBX3 (PBX3+MEIS1), MSCVneo-Meis1+MSCV-PIG-Pbx3 (Pbx3+Meis1), MSCVneo-Meis1-M2ΔLRF/ΔLLEL+MSCV-PIG-Pbx3 (Pbx3+Meis1-M2ΔLRF/ΔLLEL) or MSCVneo-Meis1-Δ64-202+MSCV-PIG-Pbx3 (Pbx3+ Meis1-Δ64-202). The colony cells were replated every 7 days for up to 4 passages and colony numbers were counted for each passage. Mean±SD values of colony counts are shown (left panel). The domain structure of the wild-type and mutant Meis1 proteins are also shown (right panel). (B,C) qPCR (B) and Western blotting (C) analyses of expression levels of a series of representative Homeobox genes in colony cells from the first passage of four groups (samples generated from colony-forming/replating assays shown in ). Mean±SD values are shown in .

(A) In vitro colony-forming/replating assays. Briefly, mouse normal BM progenitor (lineage negative; Lin-) cells collected from 4- to 6-week-old B6.SJL (CD45.1) mice were retrovirally co-transduced with MSCVneo+MSCV-PIG (Control), MSCVneo-HOXA9+MSCV-PIG (HOXA9), MSCVneo+MSCV-PIG-MEIS1 (MEIS1), MSCVneo+MSCV-PIG-PBX3 (PBX3), MSCVneo-HOXA9+MSCV-PIG-MEIS1 (HOXA9+MEIS1), MSCVneo-HOXA9+MSCV-PIG-PBX3 (HOXA9+PBX3), MSCVneo-MEIS1+MSCV-PIG-PBX3 (PBX3+MEIS1), or MSCVneo-MLL-AF9+MSCV-PIG (MLL-AF9). The colony cells were replated every 7 days for up to 5 passages and colony numbers were counted for each passage. Mean±SD values of colony counts are shown. (B) Mouse BM transplantation (BMT) assays were conducted for the above 8 groups with the first-passage colony cells (CD45.1) as donors, which were transplanted into lethally irradiated 8- to 10-week-old C57BL/6 (CD45.2) recipient mice. Kaplan-Meier curves are shown. Five mice were studied in each group, except for the MLL-AF9 group in which 6 mice were studied. (C) Cell/tissue morphologies of the 8 groups. Peripheral blood (PB) and BM cells were stained with Wright-Giemsa. The spleen and liver tissues were stained with hematoxylin and eosin (H&E). The length of bars represents 10 μm for PB and BM, and 100 μm for spleen and liver.