PBX3 and MEIS1 Cooperate in Hematopoietic Cells to Drive Acute Myeloid Leukemias Characterized by a Core Transcriptome of the MLL-Rearranged Disease

Abstract

Overexpression of HOXA/MEIS1/PBX3 homeobox genes is the hallmark of mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML). HOXA9 and MEIS1 are considered to be the most critical targets of MLL fusions and their coexpression rapidly induces AML. MEIS1 and PBX3 are not individually able to transform cells and were therefore hypothesized to function as cofactors of HOXA9. However, in this study, we demonstrate that coexpression of PBX3 and MEIS1 (PBX3/MEIS1), without ectopic expression of a HOX gene, is sufficient for transformation of normal mouse hematopoietic stem/progenitor cells in vitro. Moreover, PBX3/MEIS1 overexpression also caused AML in vivo, with a leukemic latency similar to that caused by forced expression of MLL-AF9, the most common form of MLL fusions. Furthermore, gene expression profiling of hematopoietic cells demonstrated that PBX3/MEIS1 overexpression, but not HOXA9/MEIS1, HOXA9/PBX3, or HOXA9 overexpression, recapitulated the MLL-fusion-mediated core transcriptome, particularly upregulation of the endogenous Hoxa genes. Disruption of the binding between MEIS1 and PBX3 diminished PBX3/MEIS1-mediated cell transformation and HOX gene upregulation. Collectively, our studies strongly implicate the PBX3/MEIS1 interaction as a driver of cell transformation and leukemogenesis, and suggest that this axis may play a critical role in the regulation of the core transcriptional programs activated in MLL-rearranged and HOX-overexpressing AML. Therefore, targeting the MEIS1/PBX3 interaction may represent a promising therapeutic strategy to treat these AML subtypes.

Figure 1. Co-expression of PBX3 and MEIS1 can transform normal mouse bone marrow (BM) progenitor cells in vitro and induce rapid AML in vivo

(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.

Figure 2. PBX3/MEIS1-induced AML is transmissible in secondary transplantation recipients

(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.

Figure 3. Comparison of gene expression profiles between different groups of leukemic or normal control cells

(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 (32). (C) Gene sets that are differentially enriched between Group 2 and Group 3 samples. NSE, normalized enrichment score; FDR, false discovery rate.

Figure 4. Heatmap of expression profiles of the 116 genes that are expressed at a significantly higher level in Group 3 (i.e., PBX3+MEIS1 and MLL-AF9 samples) than in both Groups 1 and 2

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).

Figure 5. Effects of knockdown of PBX3 and/or MEIS1 on the expression of endogenous HOXA genes in MLL-rearranged AML cells

Endogenous expression levels of MEIS1, PBX3, HOXA4, HOXA5, HOXA7 and HOXA9 in MonoMac-6/t(9;11) 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.

Figure 6. The binding between Meis1 and Pbx3 is critical for their synergistic effects on cell transformation and up-regulation of homeobox genes

(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 Figure 6A). Mean±SD values are shown in Figure 6B.