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RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins.
RUNX1 (also called AML1) is a member of the RUNX transcription factor family and plays an essential role in the development of normal hematopoiesis (1, 2). RUNX1 forms a core-binding factor (CBF) complex with its cofactor, CBFB. RUNX1 and CBFB are the most frequent targets of chromosomal translocations in acute myeloid leukemia (AML), generating the leukemogenic fusion proteins AML1-ETO and CBFB-MYH11. In these CBF leukemias, the dominant inhibition of RUNX1 function by these fusion proteins has been considered a critical pathway for leukemia development (3). In MLL fusion leukemia, RUNX1 expression is downregulated through degradation by MLL fusion proteins (ref. 4 and G. Huang, unpublished observations). Furthermore, inactivating RUNX1 mutations have been frequently found in a variety of myeloid neoplasms, including myelodysplastic syndrome (MDS) and cytogenetically normal AML (5–8). Therefore, RUNX1 has been regarded as a beneficial tumor suppressor for myeloid leukemogenesis. The tumor suppressor activity of RUNX1 has also been demonstrated in several mouse AML models. Runx1-deficient cells showed increased susceptibility to AML development in collaboration with MLL-ENL, NRASG12S, and EVI5 (9–11).
However, human genetic evidence also raises the possibility that RUNX1 activity is required for optimal growth of AML cells. RUNX1 mutation is usually heterozygous, and complete loss of RUNX1 is rarely seen in human AMLs. In particular, no RUNX1 mutation was found in AMLs with leukemogenic fusion proteins, such as CBF and MLL fusion leukemias (7, 8, 12). Experimentally, it was shown that Cbfb-Myh11 knockin mice developed leukemia partly through RUNX1 repression–independent activities, and a mutant CBFB-MYH11 lacking the RUNX1 binding domain induced leukemia quickly despite its inefficient suppression of RUNX1 function (13, 14).
We have developed an experimental system to model myeloid leukemogenesis using primary human cord blood (CB) cells (15, 16). The CBF fusion proteins AML1-ETO and CBFB-MYH11 promote self-renewal and long-term proliferation of human CB CD34+ cells in vitro (17–19). The MLL fusion protein MLL-AF9 immortalizes CB cells in vitro and produces human leukemia in immunodeficient mice (20). These engineered pre-leukemic and leukemic cells recapitulate many features of the clinical diseases and have been useful in testing different therapeutic strategies (21–25).
Using these human cell-based models, we identified a context-specific, dual role for RUNX1 in human myeloid neoplasms. Consistent with the general assumption, RUNX1 induces myeloid differentiation in normal CB cells, thereby working as a tumor suppressor. Unexpectedly, we also found that RUNX1 plays a critical role in the growth and survival of human AML cells. We used a mouse model of AML, which demonstrated that the combined deletion of Runx1/Cbfb diminishes the leukemogenic activity of murine MLL-AF9 cells. Our study uncovers a prosurvival role for RUNX1 in myeloid leukemia and identifies RUNX1 as a potential therapeutic target beyond CBF leukemia.
We first examined the effect of forced expression of RUNX1 and its mutants in human CB CD34+ cells. R139G and D171N have a point mutation in the RUNT domain. S291fsX9 (S291fs) is a C-terminal truncation mutant lacking an activation domain. L378fsX196ΔEx7 (L378fs) is a C-terminal elongation mutant and lacks sequences of exon 7. These mutations were identified exclusively in MDS/AML patients and were shown to have a dominant-negative effect over wild-type RUNX1 (5, 26, 27). L29S was also detected in 5% of healthy people and may not be relevant to leukemogenesis (ref. 7 and Figure Figure1A).1A). We confirmed the expression of each protein by immunoblotting in CB cells (Figure (Figure1B).1B). RUNX1 and L29S inhibited the growth of CB cells, as evidenced by the loss of GFP-expressing cells in culture. In contrast, the frequency of the cells expressing other mutants gradually increased at the late phase of culture, suggesting enhanced self-renewal of these cells (Figure (Figure1C).1C). We found that RUNX1-expressing cells showed decreased CD34 (a stem cell marker) and increased CSF2RA (a marker for myeloid differentiation) expression, suggesting that RUNX1 induces myeloid differentiation of CD34+ cells. Leukemogenic RUNX1 mutants lost this activity (Figure (Figure1D).1D). In particular, S291fs-expressing cells eventually dominated the culture and grew for more than 3 months (Supplemental Discussion and Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI68557DS1). We also assessed cell cycle status and apoptosis on days 5 through 7 of culture in CB cells expressing RUNX1, D171N, S291fs, or vector. D171N decreased the frequency of CB cells in the S/G2/M phases, consistent with its transient inhibitory effect during the early phase of culture. Wild-type RUNX1 slightly increased the frequency of annexin V+ apoptotic cells, which may also contribute to the growth-inhibitory effect of RUNX1 in CB cells (Figure (Figure1E).1E). Thus, RUNX1 acts as a tumor suppressor in CB cells mainly by inducing myeloid differentiation, and loss of this differentiation-inducing activity is a common feature of leukemogenic RUNX1 mutants.
Next, we transduced RUNX1 and its mutants (D171N and S291fs) into the engineered human AML cells generated by expressing AML1-ETO or MLL-AF9 in human CB cells (18–20). Interestingly, both mutants inhibited the growth of these engineered AML cells (Figure (Figure2,2, A and B). RUNX1 overexpression also inhibited the growth of AML1-ETO cells by inducing myeloid maturation (Figure (Figure2,2, A and C), but the growth-inhibitory effect of RUNX1 was not seen in MLL-AF9 cells (Figure (Figure2B).2B). Similar to the results seen in CB cells, the mutants did not decrease CD34 expression in AML1-ETO cells (Figure (Figure2C).2C). Instead, the mutant-expressing AML cells showed a decrease in the proportion of S/G2/M phase cells and an increase in the proportion of annexin V+ cells compared with vector-transduced cells. In particular, the mutants, but not wild-type RUNX1, induced cell cycle arrest in AML1-ETO cells and apoptosis in MLL-AF9 cells (Figure (Figure2,2, D and E). Because the mutant overexpression perturbs RUNX1-mediated gene regulation (Supplemental Figure 2 and Supplemental Discussion), these results indicate that proper RUNX1 function is necessary for efficient growth of these AML cells and could explain why no RUNX1 mutation was found in CBF and MLL fusion leukemias.
RUNX1 was abundantly expressed and phosphorylated in all the engineered human AML cells, with the highest expression in AML1-ETO cells (Figure (Figure3,3, A and B). To assess the role of endogenous RUNX1 in human cells, we knocked down RUNX1 expression in CB, in the engineered AML cells, and in myeloid cell lines using two independent RUNX1-specific shRNA lentiviruses that showed modest (sh-1) and strong (sh-2) knockdown efficiency (Figure (Figure3C3C and Supplemental Figure 3A, B). We found that the reduction of RUNX1 expression resulted in substantial growth inhibition in both CB and AML cells, but the growth-inhibitory effect was relatively weak in CB cells (Figure (Figure3D3D and Supplemental Figure 3C). The growth of BCR-ABL–driven leukemia cells (K562) was unaffected by RUNX1 reduction (Supplemental Figure 3D). Cell cycle and apoptosis analysis revealed a substantial decrease in the proportion of S/G2/M phase cells and an increase in the percentage of annexin V+ cells in RUNX1-depleted AML1-ETO and MLL-AF9 cells (Figure (Figure3,3, E and F). Consistent with the results of mutant overexpression, RUNX1 depletion blocked myeloid differentiation in CB and AML1-ETO cells, as evidenced by an increased expression of the stem cell marker CD34 (Supplemental Figure 3E). Thus, RUNX1 promotes cell cycle progression and inhibits apoptosis in AML cells, thereby supporting their growth.
It has been shown that some shRNAs have off-target or nonspecific toxicity (28). We therefore examined whether the reintroduction of RUNX1 could reverse the growth-inhibitory effect of each shRNA. We cotransduced vector or RUNX1 (coexpressing GFP) with each shRNA (coexpressing venus) in MLL-AF9 cells and monitored changes in GFP/venus frequency in culture. High expression of RUNX1 in GFP+ and GFP/venus double-positive (DP) populations was confirmed by immunoblotting (Figure (Figure4A).4A). In the vector-transduced cultures, the frequency of both venus+ and DP cells decreased on day 11 because of the growth-inhibitory effect of RUNX1 shRNAs. In contrast, RUNX1 reintroduction in the DP cells prevented this negative effect, as evidenced by the abundant DP cells in the cultures on day 11 (Figure (Figure4B).4B). The shRNA-resistant versions of RUNX1 also reversed the negative effect of each shRNA (Supplemental Figure 4). Furthermore, RUNX1-5A, a nonfunctional RUNX1 mutant carrying five phospho-deficient mutations (29), did not reverse the negative effect of sh-2, whereas all functional RUNX1 (wild-type, RUNX1-2A, and RUNX1-4A) did (Figure (Figure4,4, C and D, Supplemental Figure 5, and Supplemental Discussion). Taken together, we conclude that the growth-inhibitory effect of the RUNX1 shRNAs is in fact due to RUNX1 downregulation.
Using MLL-AF9–expressing CB cells as a model for human AML (20), we further assessed the role for RUNX1 in the generation of AML. We cotransduced MLL-AF9 (coexpressing GFP) with a nontargeting (NT) control shRNA or RUNX1 shRNA (coexpressing venus) into CD34+ cells and cultured the cells in vitro or directly transplanted them into the NOD/SCID mice in which several human cytokine genes (SCF, GMCSF, and IL3) are transgenically expressed (NS-S/GM/3: NSS) (ref. 30 and Figure Figure5A).5A). In these mice, MLL-AF9–expressing CB cells produced human AML within 4 to 5 weeks (Figure (Figure5B).5B). Consistent with earlier results, RUNX1 depletion inhibited the MLL-AF9–driven long-term proliferation of CB cells (Figure (Figure5C).5C). Furthermore, MLL-AF9 cells transduced with RUNX1 shRNAs were outcompeted by nontransduced cells during leukemia development in vivo, as indicated by the depletion of venus+ cells in leukemic mice (Figure (Figure5D5D and Supplemental Figure 6A).
Next, we assessed the effect of a recently reported RUNX1 inhibitor, Ro5-3335, on the growth of MLL-AF9 cells (31). We confirmed an inhibitory effect of Ro5-3335 on human RUNX1 and RUNX2 using a CB cell culture assay (Supplemental Figure 6, B and C). We observed that the proliferation of MLL-AF9 cells was more sensitive to Ro5-3335, as compared with the proliferation of CB and K562 cells, correlating with the relative impact of RUNX1 shRNAs on the proliferation of these cells (Figure (Figure5E).5E). Ro5-3335 treatment induced cell cycle arrest and apoptosis in MLL-AF9 cells, similar to the effects of shRNA-mediated RUNX1 depletion (Figure (Figure5,5, F and G).
We then examined the effect of RUNX1 depletion in primary leukemia cells derived from patients with AML harboring the MLL fusions MLL-SEPT6 and MLL-ENL (Figure (Figure6A).6A). The AML cells with MLL-SEPT6 showed good engraftment and myeloid cell expansion in NOD/SCID/IL2rg–/– mice expressing human SCF, GMCSF, and IL3 (NSG-S/GM/3: NSGS) (ref. 32 and Figure Figure6B).6B). Those AML cells were transduced with NT or RUNX1 shRNAs and were then cultured on a stromal cell line to assist in the in vitro expansion of these primary leukemia cells. As expected, RUNX1 depletion inhibited the growth of the primary AML cells in vitro (Figure (Figure6,6, C and D). We also transplanted the shRNA-transduced patient sample with MLL-SEPT6 directly into the NSGS mice. RUNX1-depleted AML cells showed a reduced engraftment capacity, as indicated by the reduction of the venus+ fraction in human myeloid (hCD45+/hCD33+) cells (Figure (Figure6D).6D). Thus, RUNX1 supports the maintenance of human MLL fusion leukemia both in vitro and in vivo.
In addition to RUNX1, there are two other mammalian RUNX transcription factors, RUNX2 and RUNX3, which have redundant functions in murine hematopoiesis (33, 34). These RUNX proteins had similar effects on human AML cells (Supplemental Figure 7). Interestingly, the rescue experiment showed that RUNX2, but not RUNX3, restored the efficient growth of RUNX1-depleted human MLL-AF9 cells, suggesting that RUNX2 has the capacity to substitute for RUNX1 in MLL fusion leukemia (Figure (Figure77A).
Importantly, we recently showed that loss of Runx1 accelerates the development of murine MLL-ENL leukemia using Runx1 knockout mice (11). The “growth-inhibitory” role of RUNX1 in murine MLL-ENL cells sharply contrasts with the “growth-promoting” role of RUNX1 in human MLL-AF9 cells. This discrepancy probably indicates the compensatory effect of other RUNX proteins in Runx1-deficient murine cells. Indeed, microarray data revealed an upregulation of Runx2 mRNA in Runx1-deficient murine MLL-ENL leukemia cells in vivo (Figure (Figure7B,7B, Supplemental Figure 8, A and B, and Supplemental Table 5). We also found an upregulation of RUNX2 protein in Runx1-deleted murine and human MLL-AF9 cells in vitro (Figure (Figure7C7C and Supplemental Figure 8C). To assess the dosage-dependent function of RUNX in murine leukemia, we established an inducible gene knockout system for Runx1 and its cofactor Cbfb in MLL-AF9 cells (Figure (Figure7D).7D). CBFB is an important cofactor for all RUNX proteins, therefore, depletion of CBFB along with RUNX1 will result in a further reduction of RUNX activity. The deletion of Runx1/Cbfb in MLL-AF9 cells resulted in a substantial reduction of colony numbers, while single deletion of Runx1 showed little effect (Figure (Figure7E).7E). Furthermore, Runx1/Cbfb-deleted cells were gradually outcompeted by nonexcised cells, while Runx1-deleted MLL-AF9 cells continued to grow even after four rounds of plating (Figure (Figure77F).
We also performed similar experiments using a Cre-YFP–expressing retrovirus and bone marrow progenitors derived from mice with wild-type alleles, heterozygous floxed alleles of Runx1/Cbfb (Runx1/Cbfbf/wt), or homozygous floxed alleles of Cbfb or Runx1/Cbfb (Cbfbf/f or Runx1/Cbfbf/f) (Figure (Figure8A).8A). Again, the combined deletion of Runx1/Cbfb in MLL-AF9 cells caused a marked reduction in colony numbers, whereas Cre expression showed marginal effects in wild-type and Runx1/Cbfbf/wt progenitors expressing MLL-AF9 (Figure (Figure8B).8B). Runx1/Cbfb-depleted MLL-AF9 cells lost the characteristic leukemia cell morphology with prominent granules (Figure (Figure8C)8C) and contained fewer GR-1/c-KIT double-positive cells than the controls (Figure (Figure8D).8D). Single deletion of Cbfb also moderately reduced clonogenic activity and GR-1 expression in MLL-AF9 cells (Figure (Figure8,8, B and D). However, the effect of single loss of Cbfb was not as strong as double deletion of Runx1/Cbfb, indicating a CBFB-independent function of RUNX1. The colony-forming activity recovered after three rounds of plating in Runx1/Cbfb- and Cbfb-depleted cultures, however; those colonies contained primarily nonexcised Runx1- and/or Cbfb-floxed alleles (Supplemental Figure 9).
Finally, to address whether RUNX1/CBFB are essential to maintain MLL-AF9–induced murine AML in vivo, we used a bone marrow transplant assay (Supplemental Figure 10A). Mice treated with pIpC to induce Runx1/Cbfb deletion showed less GFP+ MLL-AF9 leukemia cells in peripheral blood on day 25 (Supplemental Figure 10B) and died with significantly longer latencies (Figure (Figure8E).8E). In contrast, pIpC did not affect the development of leukemia in mice transplanted with control MLL-AF9 cells (Supplemental Figure 10C). We confirmed the excised allele of Runx1 in GFP+ MLL-AF9 cells immediately after pIpC injection, but leukemia cells harvested from moribund mice primarily harbored a nonexcised Runx1 allele (Figure (Figure8F).8F). Taken together, these results demonstrate the requirement of a certain level of RUNX activity, even in murine leukemia cells.
To identify downstream RUNX targets, we performed global gene expression analysis in murine Runx1/Cbfbf/f MLL-AF9/CreER cells after treatment with 4OHT or EtOH for 24 hours (Supplemental Figure 11). This relatively short-term experiment allowed us to identify early responsive genes upon Runx1/Cbfb depletion. We confirmed the absence of RUNX1 and CBFB protein expression at 24 hours. Of note, RUNX2, which was upregulated in single Runx1-deleted cells (Figure (Figure7C),7C), was downregulated in Runx1/Cbfb-deleted cells (Figure (Figure9A).9A). This was probably due to the loss of CBFB, which protects RUNX proteins from ubiquitin-dependent degradation (35). The dysregulated genes in Runx1/Cbfb-deleted cells contained several known RUNX1 targets, including RUNX1 itself and C/EBPα (Supplemental Tables 3 and 4).
Among these candidate Runx target genes, we focused on Bcl2, which was shown to be important for the pathogenesis of MLL fusion leukemia (22, 36). It was also shown that RUNX1 binds to the TGTGGT sequence in the BCL2 promoter (37). BCL2 was downregulated not only by Runx1/Cbfb double deletion, but also by single Runx1 deletion in murine MLL-AF9 cells (Figure (Figure9B).9B). Additional microarray data using in vivo murine MLL-ENL leukemia cells also showed substantial downregulation of BCL2 in Runx1-deficient MLL-ENL leukemia cells (Figure (Figure9C,9C, Supplemental Figure 12, and Supplemental Table 6). In human MLL-AF9 cells, both shRNA-mediated RUNX1 knockdown and Ro5-3333 treatment decreased BCL2 expression at mRNA and protein levels (Figure (Figure9,9, D and E). To understand the role for BCL2 in RUNX1-mediated cell survival, we first engineered vector or BCL2-overexpressing human MLL-AF9 cells and then assessed the effects of shRNA-mediated RUNX1 depletion in these cells (Figure (Figure9F).9F). BCL2-transduced MLL-AF9 cells grew better than vector-transduced cells upon RUNX1 knockdown, although BCL2-transduced cells were not fully rescued from the effects of RUNX1 depletion (Figure (Figure9G9G and Supplemental Figure 13A). BCL2-transduced MLL-AF9 cells were relatively resistant to apoptotic cell death induced by RUNX1 depletion compared with control cells. However, cell cycle arrest induced by RUNX1 depletion became more apparent in BCL2-transduced cells (Figure (Figure9H9H and Supplemental Figure 13, B–D). As a possible explanation for this effect on the cell cycle, we found that CDKN1A was significantly upregulated in RUNX1-depleted human MLL-AF9 cells and Runx1/Cbfb-deleted mouse MLL-AF9 cells. However, CDKN1A knockdown did not rescue the growth defect of RUNX1-depleted human MLL-AF9 cells, suggesting the presence of other factors regulating RUNX1-mediated cell cycle progression (Supplemental Figure 14).
Primary human cells offer a potentially ideal target for the study of oncogene function. Using human cell–based models, we demonstrated dual functions of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibits the growth of CB cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity is required for the growth of AML cells. Human AML cells are more sensitive to RUNX1 inhibition probably because their RUNX activity is already reduced. We also used a mouse AML model and showed that the combined loss of RUNX1/CBFB inhibits the development of murine MLL fusion leukemia, while single loss of RUNX1 accelerates it (11). Based on these results, we propose a model to explain the dosage-dependent function of RUNX in myeloid leukemogenesis (Figure (Figure10).10). Partial loss of RUNX activity blocks myeloid maturation and supports AML development, while a further reduction of RUNX activity results in cell cycle arrest and cell death.
In the past few decades, the scientific community has primarily focused on “oncogenes” that are overexpressed in tumor cells as ideal therapeutic targets. However, such oncogenes are usually also important for the maintenance of normal stem cells, and therefore it remains unclear whether inhibiting such oncogenes would favor normal cells over tumor cells. Instead, genes that are underexpressed in cancer cells but are essential for their survival will be promising targets to selectively eradicate cancer cells while preserving normal stem cells. Importantly, many so-called “tumor suppressors” appear to have such features. In addition to RUNX1, two other well-known tumor suppressors, PML and FOXOs, were shown to have survival roles in leukemia stem cells (38, 39). Interestingly, recent genome-wide sequence studies in myeloid neoplasms found inactivating mutations in genes that were shown to have oncogenic properties, including GATA2, EZH2, and NOTCH pathway genes (40–44). Although these findings reveal a potential tumor suppressor role for these oncogenes, it is highly likely that these genes still retain some growth-promoting function. Indeed, several recent reports showed an important role for EZH2 in the development of MLL fusion leukemia (45–47). Dosage-dependent functions of these genes in myeloid leukemogenesis need to be clarified in future studies.
Mouse models have proven to be invaluable tools for the understanding of human cancer. Nevertheless, significant differences exist between mouse models and clinical diseases. The current study highlights the potential influence on tumor development of a compensatory mechanism in mice lacking individual genes. Our data, together with a recent report showing that RUNX2 is upregulated by MLL-AF9 in mouse bone marrow cells (48), strongly indicate the functional redundancy between RUNX1 and RUNX2 in MLL fusion leukemia. The upregulated RUNX2 probably provides sufficient RUNX activity for Runx1-deficient murine MLL-ENL cells to develop leukemia. It is not clear why we did not see the compensatory effects in human cell–based models. Given that RUNX1 is not mutated in patients with MLL fusion leukemia, human MLL fusion AML cells may require stronger RUNX activity than the corresponding mouse cells for optimal growth. Alternatively, adverse effects of shRNAs used in human cells and/or different experimental assays could affect the results. While this manuscript was under review, Wilkinson et al. showed that RUNX1 is a transcriptional target of MLL-AF4 and supports the growth of MLL-AF4 cells (49). Although they argued that RUNX1 is specifically important for the growth of MLL-AF4 cells, our study clearly demonstrates that other types of MLL fusion leukemia also require a certain level of RUNX activity. Compounds targeting RUNX/CBFB function will show substantial efficacy in MLL fusion leukemia, and these compounds can be expected to exhibit a reasonable therapeutic window relative to normal blood stem cells.
We identified BCL2 as an important mediator for the survival effect of RUNX1 in human MLL fusion leukemia. However, BCL2 did not rescue the cell cycle arrest induced by RUNX1 depletion. Furthermore, Runx1-deficient murine MLL-AF9 cells still developed leukemia despite reduced BCL2 expression. Therefore, other factors must also contribute to the RUNX1-mediated leukemia cell growth. The mechanisms for RUNX1-mediated cell cycle progression remain elusive, but CDKN1A appears to be at least involved in this process, given its consistent upregulation in RUNX1-inhibited leukemia cells. Further characterization of RUNX/CBFB-mediated gene regulation may reveal the way to specifically block the survival function of RUNX without preventing its tumor suppressor role in promoting myeloid differentiation.
In summary, we found an unexpected survival- and growth-promoting role for RUNX1 in the development and maintenance of AML with leukemogenic fusion proteins. Recent efforts to target this transcription factor complex in CBF leukemia (31) should be extended to AML associated with MLL rearrangements and potentially to other myeloid neoplasms with RUNX1 dysfunction.
CD34+ cells were separated using an EasySep CD34 selection kit (STEMCELL Technologies). CB cells and CB cells transduced with leukemia fusion genes were cultured in Iscove’s modified Dulbecco’s media (IMDM) containing 20% BIT9500 (STEMCELL Technologies) and 10 ng/ml SCF, megakaryocyte growth and development factor (TPO), FLT3 ligand (FLT3L), IL-3, and IL-6, as described previously (15, 16). Primary AML cells derived from patients were cultured on a stromal cell line, OP9-DL1, with the same culture media.
The retroviral constructs pMYs-IRES-EGFP (pMYs-IG), pMYs-RUNX1b-IG, pMYs-RUNX1b/D171N-IG, and pMYs-RUNX1b/S291fs-IG were provided by T. Kitamura (Institute of Medical Science, University of Tokyo, Tokyo, Japan). RUNX1b/R139G, RUNX2, and RUNX3 cDNAs were provided by M. Kurokawa (University of Tokyo, Tokyo, Japan), and we cloned them into pMYs-IG. These cDNAs were fused with a FLAG epitope tag at the N terminus. RUNX1b-L378fs and RUNX1b-L29S were obtained using mRNA prepared from a myeloid leukemia cell line, CG-SH (27), and were inserted into the pMYs-IG. RUNX1c and its phospho-deficient mutants (2A, 4A, and 5A) were provided by A.S. Kraft (The Medical University of South Carolina, Charleston, South Carolina, USA) and cloned into pMYs-IG. For MLL-AF9 expression, we used pMSCV-MLL-AF9-pgk-EGFP or pMSCV-MLL-AF9-pgk-puro. The pBabe-IRES-puro, pBabe-BCL2-IRES-puro, and pMSCV-CreERT2-puro constructs were from Addgene ( http://www.addgene.org). Lentiviral vector MISSION pLKO.1-shRNA-puro constructs targeting human RUNX1 (TRCN0000013659 [sh-1] and TRCN0000013660 [sh-2]) (Supplemental Figure 3) were obtained from Sigma-Aldrich. The venus marker was excised from the pLKO.1-venus construct with BAMH1 and KPN1 and was subcloned into the corresponding sites on the RUNX1 shRNAs to replace the puromycin-resistant gene. Viral production was performed by transfecting viral plasmids along with gag-, pol-, and env-expressing plasmids into 293T cells, as described previously (17, 24).
Cells were analyzed on a FACSCanto I, II, or III flow cytometer (BD). Antibodies for flow cytometric analysis were all purchased from BD Biosciences (Supplemental Table 1). Cells were stained with fluorochrome-conjugated antibodies incubated for 30 minutes at 4°C and were washed with 2% FBS in PBS prior to analysis. Cell cycle analysis (Vybrant DyeCycle Violet stain; Invitrogen) and apoptosis analysis (Annexin V-APC kit; BD Biosciences) were performed according to the manufacturer’s recommendations.
Western blot analysis was performed as described previously (21). Detection was performed using SuperSignal West Pico or Femto Chemiluminescent Substrate (Pierce, Thermo Scientific). For immunofluorescence, cytospins were fixed with paraformaldehyde and permeabilized with Triton X-100 and then incubated with primary antibodies, followed by labeling with Alexa Fluor 568–conjugated anti-rabbit antibody (Molecular Probes). Nuclei were visualized with DAPI (SlowFade Gold Antifade; Invitrogen). Cells were visualized on a Zeiss Axiovert 200 M fluorescent microscope with a Zeiss Plan-Apochromat 63×/1.4 objective (Carl Zeiss). Data were recorded with an ORCA-ER digital camera (Hamamatsu). Deconvolution was performed with OpenLab 4.0.3 software (Improvisions). The antibodies used are shown in Supplemental Table 1.
RNA was isolated with the RNeasy kit (QIAGEN). RNA was reversed transcribed using MuLV RT and random hexamers (Applied Biosystems). cDNA was then subjected to quantitative PCR using SYBR Green or TaqMan technology (Applied Biosystems). The primers used are shown in Supplemental Table 2.
Human MLL-AF9 cells, CD34+ CB, and K562 cells were plated with titrating doses of Ro5-3335 (ranging from 0 μM to 100 μM) in triplicate. After 72 hours, 10 μl WST-1 cell proliferation assay premix (MK 400; Takara Bio Inc.) was added to each well. Plates were read at 450 to 560 nm to measure optical density. Cells treated with 10 μM Ro5-3335 for 72 hours were analyzed for cell cycle status and apoptosis using flow cytometry, as described above.
Human CB CD34+ cells were transduced with MLL-AF9 (coexpressing GFP) and shRNAs (coexpressing venus), and were injected intrafemorally into sublethally irradiated (250 cGy from a cesium source) 6- to 8-week-old NSS mice immediately after transduction. Approximately 3 × 105 CB cells were injected into a recipient mouse. Primary AML cells harboring MLL-SEPT6 were transduced with venus-expressing shRNAs and were injected into NSGS mice using the same method. Approximately 1 × 106 AML cells were injected into a recipient mouse. The immunodeficient mice were placed on doxycycline chow (TestDiet; Modified Prolab RMH-1500 with 0.0625% doxycycline) prior to, during, and for several weeks after irradiation.
The mice harboring a Runx1 allele with exon 4 flanked by loxP sites (Runx1f/f) (50) and the mice harboring a Cbfb allele with exon 5 flanked by loxP sites (Cbfbf/f) (51) were crossed to generate Runx1/Cbfbf/f mice. Another Runx1 mutant mouse strain with loxP-flanked exon 5 (52) was used to generate in vivo MLL-ENL leukemia (11).
Murine myeloid colony assays were plated in M3434 cytokine-enriched methylcellulose according to the manufacturer’s instructions (STEMCELL Technologies). CreER-expressing cells were selected in M3434 containing 1 μg/ml puromycin. Cells (5 × 103) were plated for each round of plating. Colony counting and replating were performed every 4 or 5 days.
Bone marrow progenitors (c-KIT+ cells) were transduced with MLL-AF9 and were injected into sublethally irradiated (700 Gy) recipient mice via the tail vein. Secondary transplantation was performed in the same way using 1 × 106 spleen cells isolated from leukemic mice.
Total RNA was prepared using an RNeasy Mini Kit (QIAGEN). For microarray analysis with MLL-AF9/CreER-expressing Runx1/Cbfbf/f cells, the quality of the total RNA was checked by an Agilent Bioanalyzer 2100 (Hewlett Packard) using the RNA 6000 Nano Assay. For each sample, the Ambion WT Expression Kit (Life Technologies) synthesized the cDNA target from 300 nanograms of total RNA. Then the GeneChip WT Terminal Labeling Kit (Affymetrix) was used to both chemically fragment and biotin label the cDNA target. Labeled probes were hybridized to the GeneChip Mouse Gene 1.0 ST Array (Affymetrix) and were scanned with the Affymetrix GeneChip Scanner 3000 7G. Raw data files were created by Command Console, the Affymetrix operating software program. All samples were normalized using the robust multichip average (RMA). Upregulated or downregulated genes in Runx1/Cbfb-deleted cells were identified using Qlucore Omics Explorer ( http://www.qlucore.com). For microarray analysis with Runx1f/f- and Runx1Δ/Δ-MLL-ENL in vivo leukemia cells, labeling of fragmented cDNA was done using the NuGen Ovation Biotin labeling system (NuGEN Technologies). Labeled probes were hybridized to the Mouse Genome 430 2.0 Array (Affymetrix) and scanned with a GeneChip Scanner 3000 7G (Affymetrix). The expression data were extracted from the image files produced with GeneChip 1.0 Operating software (Affymetrix). Normalization and expression value calculations were performed using the DNA Chip Analyzer ( www.dchip.org) (53). Microarray data were deposited in the NCBI’s Gene Expression Omnibus (GEO accession numbers GSE47350 and GSE47402).
The survival distributions (Figure (Figure8E)8E) were compared using a log-rank test. A P value of less than 0.05 was considered statistically significant.
Human umbilical CB cells and patient samples were obtained from the Translational Trials Development and Support Laboratory at Cincinnati Children’s Hospital Medical Center (Cincinnati, Ohio, USA) in accordance with IRB-approved protocols. Written informed consent was obtained in accordance with the Declaration of Helsinki. All mouse experiments were performed under an IACUC-approved protocol of the Cincinnati Children’s Hospital in accordance with accepted national standards and guidelines.
We thank the Flow Cytometry Core, the Mouse Core, and the Microarray Core at Cincinnati Children’s Hospital Medical Center for their help. This work was supported by an Institutional Clinical and Translational Science Award; an NIH/NCRR grant (1UL1RR026314-01); a Translational Trials Development and Support Laboratory award (USPHS, MO1 RR 08084); a Center of Excellence in Molecular Hematology P30 award (DK090971); a grant from the CancerFree Kids Foundation for Cancer Research (to J.C. Mulloy and S. Goyama); a grant from the Intramural Research Program of the National Human Genome Research Institute, NIH (to P.P. Liu); and a JSPS Postdoctoral Fellowship for Research Abroad (to S. Goyama). J.C. Mulloy is a Leukemia and Lymphoma Society Scholar. The contents of the manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2013;123(9):3876–3888. doi:10.1172/JCI68557.