1. Cancer Biology
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HOXA9 promotes MYC-mediated leukemogenesis by maintaining gene expression for multiple anti-apoptotic pathways

  1. Ryo Miyamoto
  2. Akinori Kanai
  3. Hiroshi Okuda
  4. Yosuke Komata
  5. Satoshi Takahashi
  6. Hirotaka Matsui
  7. Toshiya Inaba
  8. Akihiko Yokoyama  Is a corresponding author
  1. Tsuruoka Metabolomics Laboratory, National Cancer Center, Japan
  2. Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan
  3. Department of Hematology and Oncology, Kyoto University Graduate School of Medicine, Japan
  4. Department of Molecular Laboratory Medicine, Graduate School of Medical Sciences, Kumamoto University, Japan
  5. Division of Hematological Malignancy, National Cancer Center Research Institute, Japan
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Cite this article as: eLife 2021;10:e64148 doi: 10.7554/eLife.64148

Abstract

HOXA9 is often highly expressed in leukemias. However, its precise roles in leukemogenesis remain elusive. Here, we show that HOXA9 maintains gene expression for multiple anti-apoptotic pathways to promote leukemogenesis. In MLL fusion-mediated leukemia, MLL fusion directly activates the expression of MYC and HOXA9. Combined expression of MYC and HOXA9 induced leukemia, whereas single gene transduction of either did not, indicating a synergy between MYC and HOXA9. HOXA9 sustained expression of the genes implicated in the hematopoietic precursor identity when expressed in hematopoietic precursors, but did not reactivate it once silenced. Among the HOXA9 target genes, BCL2 and SOX4 synergistically induced leukemia with MYC. Not only BCL2, but also SOX4 suppressed apoptosis, indicating that multiple anti-apoptotic pathways underlie cooperative leukemogenesis by HOXA9 and MYC. These results demonstrate that HOXA9 is a crucial transcriptional maintenance factor that promotes MYC-mediated leukemogenesis, potentially explaining why HOXA9 is highly expressed in many leukemias.

Introduction

Mutations of transcriptional regulators often cause aberrant gene regulation of hematopoietic cells, which leads to leukemia. Structural alterations of the mixed lineage leukemia gene (KMT2A also known as MLL) by chromosomal translocations cause malignant leukemia that often associates with poor prognosis despite the current intensive treatment regimens (Tamai and Inokuchi, 2010). KMT2A encodes a transcriptional regulator termed MLL that maintains segment-specific expression of homeobox (HOX) genes during embryogenesis (Yu et al., 1998), which determines the positional identity within the body (Luo et al., 2019; Deschamps and van Nes, 2005; Wang et al., 2009). During hematopoiesis, MLL also maintains the expression of posterior HOXA genes and MEIS1 (another homeobox gene), which promote the expansion of hematopoietic stem cells and immature progenitors (Jude et al., 2007; Krivtsov et al., 2006; McMahon et al., 2007; Thorsteinsdottir et al., 2002; Yagi et al., 1998). The oncogenic MLL fusion protein constitutively activates its target genes by constitutively recruiting transcription initiation/elongation factors thereto (Yokoyama et al., 2010; Lin et al., 2010; Okuda et al., 2015). Consequently, HOXA9 and MEIS1 are highly transcribed in MLL fusion-mediated leukemia (Krivtsov et al., 2006). Forced expression of HOXA9 (but not MEIS1) immortalize hematopoietic progenitor cells (HPCs) ex vivo (Schnabel et al., 2000; Kroon et al., 1998). Co-expression of HOXA9 with MEIS1 causes leukemia in mice which recapitulates MLL fusion-mediated leukemia (Kroon et al., 1998). Moreover, overexpression of HOXA9 is observed in many non-MLL fusion-mediated leukemias such as those with NPM1 mutation and NUP98 fusion and is associated with poor prognosis (Collins and Hess, 2016). These findings highlight HOXA9 as a major contributing factor in leukemogenesis. Nevertheless, the mechanism by which HOXA9 promotes oncogenesis remains elusive.

HOXA9 is considered to function as a transcription factor, which retains a sequence-specific DNA binding ability. HOX proteins have an evolutionally conserved homeodomain which possesses strong sequence preferences (Berger et al., 2008). HOXA9 associates with other homeodomain proteins such as PBX and MEIS family proteins (Schnabel et al., 2000; Shen et al., 1999). HOXA9 and those HOXA9 cofactors form a stable complex on a DNA fragment harboring consensus sequences for each homeodomain protein (Shen et al., 1999; Chang et al., 1996), suggesting that they form a complex of different combinations in a locus-specific manner depending on the availability of the binding sites. Recently, it has been reported that HOXA9 specifically associates with enhancer apparatuses (e.g. MLL3/4) to regulate gene expression (Sun et al., 2018; Huang et al., 2012; Zhong et al., 2018). However, the mechanisms by which HOXA9 activates gene expression remain largely unclear.

In this study, we reveal the oncogenic roles for HOXA9 and its target gene products in leukemogenesis and its unique mode of function as a transcriptional maintenance factor that preserves an identity of a hematopoietic precursor.

Results

MLL fusion proteins and HOXA9 sustain MYC expression against differentiation-induced transcriptional suppression

To identify the direct target genes of MLL fusion proteins, we first examined the genome-wide localization pattern of MLL fusion proteins by chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq), using HB1119 cells, a cell line expressing the MLL-ENL fusion protein. We observed MLL ChIP signals on the MYC, HOXA9, HOXA10, and MEIS1 loci (Figure 1AOkuda et al., 2017), which were further confirmed by ChIP-quantitative polymerase chain reaction (qPCR) analysis (Figure 1—figure supplement 1A). These ChIP signals can be mostly attributed to MLL-ENL as the knockdown of wild-type MLL did not affect the MLL ChIP signals (Figure 1A and Figure 1—figure supplement 1B,COkuda et al., 2017). The distribution of MLL-ENL was enriched at transcription start sites in a genome-wide manner, which is similar to that of wild-type MLL observed in non-MLL fusion cell lines including HEK293T and REH (Okuda et al., 2017; Miyamoto et al., 2020).

Figure 1 with 2 supplements see all
MLL fusion proteins and HOXA9 sustain MYC expression against differentiation-induced transcriptional suppression.

(A) Genomic localization of MLL-ENL in HB1119 cells. ChIP signals at the loci of posterior HOXA genes, MEIS1, MYC, and PITX2 (negative control) are shown using the Integrative Genomics Viewer (The Broad Institute). HB1119 cells were transduced with shRNA specific for wild-type (wt) MLL but not MLL-ENL (sh- KMT2A) to deplete wt MLL, as shown in Figure 1—figure supplement 1B. Ab: antibody. (B) Expression of MLL-target genes and Mxd1 (a differentiation marker) during myeloid differentiation. Bone marrow cells at various differentiation stages were obtained by FACS sorting and analyzed by qRT-PCR. Expression levels relative to KSL are shown (Mean with SD, n = 3, PCR replicates). MLL-AF10-LCs and MLL-ENL-ICs were included in the analysis for comparison. KSL: c-Kit+, Sca1+, and Lineage; CMP: common myeloid progenitor; GMP: granulocyte macrophage progenitor; int: intermediate. (C) Transforming potential of MLL target genes. Clonogenic potential of the indicated constructs was analyzed by myeloid progenitor transformation assays. Colony forming unit per 104 cells (CFU) (Mean with SD, n = 3, biological replicates), relative colony size (Mean with SD, n ≥ 100), and relative mRNA levels of indicated genes (Mean with SD, n = 3, PCR replicates) were measured at the indicated time points. #: Both endogenous murine transcripts and exogenous human transcripts were detected by the qPCR primer set used and the columns were shown with faded color. N.A.: not assessed. (D) Morphologies of the colonies and transformed cells. Representative images of bright field (left) and May-Grunwald-Giemsa staining (right) are shown with scale bars. (E) Effects of Myc knockdown on MLL-ENL- and HOXA9-ICs. Relative CFU (Mean with SD, n = 3, biological replicates) and mRNA level of Myc (Mean with SD, n = 3 PCR replicates) are shown. Statistical analysis was performed using ordinary one-way ANOVA with the vector control. ****p < 0.0001.

To characterize the dynamic changes in MLL target gene expression during differentiation, we isolated bone marrow cells from mice at various differentiation stages ranging from the most immature population (c-Kit+, Sca1+, Lineage; KSL) containing hematopoietic stem cells to highly differentiated hematopoietic cells (c-Kitlow/Mac1high) by fluorescence-activated cell sorting (FACS) and performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. For comparison, we analyzed leukemia cells (LCs) harvested from mice suffering with MLL-AF10-induced leukemia (MLL-AF10-LCs) and immortalized cells (ICs) transformed by MLL-ENL ex vivo (MLL-ENL-ICs). In accordance with previous reports (Krivtsov et al., 2006; Somervaille and Cleary, 2006; Yokoyama et al., 2013), the expression levels of Hoxa9, Hoxa10, and Meis1 were downregulated during normal hematopoietic differentiation but remained high in MLL fusion-expressing cells (Figure 1B). Myc was highly expressed in KSL, common myeloid progenitors (CMPs), and granulocyte/macrophage progenitors (GMPs), which contain actively dividing populations (Passegué et al., 2005), but was completely suppressed at highly differentiated c-kitlow/Mac1high stages. In the two MLL fusion-expressing cell lines, Myc was expressed at comparable levels to those in the progenitor fractions (CMP and GMP; Figure 1B). Mxd1, a differentiation marker, was highly expressed in differentiated populations alone. These results indicate that Myc, Hoxa9, Hoxa10, and Meis1 are intrinsically programmed to be silenced in normal hematopoietic differentiation but are aberrantly maintained by MLL fusion proteins.

To assess the oncogenic potential of MLL target genes, we performed myeloid progenitor transformation assays (Lavau et al., 1997; Okuda and Yokoyama, 2017a), wherein HPCs were isolated from mice, retrovirally transduced with each MLL target gene, and cultured in a semi-solid medium supplemented with cytokines promoting myeloid lineage differentiation (Figure 1C,D). HPCs exogenously expressing MLL-ENL or MLL-AF10 produced a large number of colonies in the third and fourth passages with high mRNA levels of Myc, Hoxa9, Hoxa10, and Meis1 in colonies of the first passage, confirming their potent transforming capacities. These cells were considered ‘immortalized’ as they proliferate indefinitely in this ex vivo culture (Lavau et al., 1997; Okuda and Yokoyama, 2017a). Ectopic expression of MYC immortalized HPCs; therefore, MYC expression is sufficient to induce proliferation of HPCs. Endogenous Myc expression was severely diminished in MYC-ICs, demonstrating that Myc expression is programed to be silenced and cannot be sustained by MYC itself. Ectopic expression of HOXA9 and HOXA10 (but not MEIS1) immortalized HPCs. Endogenous Myc expression was maintained in HOXA9/A10-ICs, indicating that HOXA9/A10 can sustain Myc expression against differentiation-induced transcriptional suppression. It should be noted that the colony size of HOXA9/A10-ICs was relatively small compared with MYC- or MLL fusion-ICs, suggesting a weaker proliferative potential. Knockdown of Myc by shRNA completely repressed the colony-forming ability of MLL-ENL-ICs and HOXA9-ICs (Figure 1E and Figure 1—figure supplement 2). Taken together, these results demonstrate that MLL fusion proteins and HOXA9 maintain Myc expression despite of the differentiation-induced transcriptional suppression, and the maintenance of MYC expression is indispensable for immortalization of HPCs ex vivo.

HOXA9 confers the identity of a hematopoietic precursor while MYC drives anabolic pathways

To identify the genes specifically regulated by HOXA9 but not by MYC, we performed RNA-seq analysis of HOXA9-ICs and MYC-ICs which do not express HOXA9 (Figure 1C). Genes highly expressed in HOXA9-ICs but lowly expressed in MYC-ICs (defined as ‘HOXA9 high signature’) were associated with hematopoietic identity/functions. In contrast, genes highly expressed in MYC-ICs and lowly expressed in HOXA9-ICs (defined as ‘MYC high signature’) were associated with anabolic pathways involved in nucleotide/protein production (Figure 2A,B). Pathways involved in lipid metabolism were commonly upregulated in both HOXA9- and MYC-ICs compared with non-immortalized c-kit-positive HPCs (Figure 2—figure supplement 1A,B). MLL-AF10-ICs, which express endogenous Hoxa9 and Myc at high levels (Figure 1C), expressed both HOXA9 high and MYC high signature genes (Figure 2A–C), indicating that MLL-AF10-ICs possess MYC-mediated highly proliferative potential and HOXA9-mediated hematopoietic identity. These differences between the HOXA9 high and MYC high signatures indicate that HOXA9 maintains the identity of a hematopoietic precursor, while MYC promotes proliferation by upregulating anabolic pathways.

Figure 2 with 1 supplement see all
HOXA9 confers the identity of a hematopoietic precursor while MYC drives anabolic pathways.

(A) Relative expression of the top 50 genes categorized as the HOXA9 high signature and the MYC high signature in HOXA9-, MYC-, and MLL-AF10-ICs. The RPKM data are provided in Figure 2—source data 1. (B) Pathways related to the HOXA9 high signature (blue) and the MYC high signature (red). The top 500 genes in the HOXA9 high or MYC high signatures were subjected to KEGG pathway analysis. The summary is provided in Figure 2—source data 1 (C) Gene set enrichment analysis of the HOXA9 high and MYC high signatures in MLL-AF10-ICs compared with MYC-ICs (top) and HOXA9-ICs (bottom), respectively.

Figure 2—source data 1

Gene expression profiles of HOXA9-, MYC-, and MLL-AF10-transformed cells.

https://cdn.elifesciences.org/articles/64148/elife-64148-fig2-data1-v1.xlsx

Apoptosis is induced by MYC, and alleviated by HOXA9 and MLL-AF10

Given that excessive MYC activity promotes apoptosis (McMahon, 2014), we evaluated the apoptotic tendencies in immortalized HPCs. MYC-ICs exhibited increased γH2AX, cleaved poly (ADP) ribose polymerase (PARP), and cleaved caspase 3 levels, indicative of a high degree of replication stress and apoptosis (Figure 3A). FACS analysis with Annexin V also showed that MYC-ICs had more significant apoptotic fraction than that of HOXA9-ICs (Figure 3B). MLL-AF10-ICs exhibited weak apoptotic tendencies similarly to HOXA9-ICs, although their MYC expression tended to be higher than HOXA9-ICs (Figures 1C and 3A). Furthermore, most MYC-ICs underwent massive apoptosis 1 day after cytokine removal, whereas HOXA9-ICs and MLL-AF10-ICs showed relative resistance (Figure 3C) and exhibited successful recovery of the live cell population after cytokine reintroduction (Figure 3—figure supplement 1). These results indicate that HOXA9 confers anti-apoptotic properties.

Figure 3 with 1 supplement see all
Apoptosis is induced by MYC, while is alleviated by HOXA9 and MLL-AF10.

(A) Protein expression of transgenes and apoptotic markers in HOXA9-, MYC-, and MLL-AF10-ICs. (B and C) Apoptotic tendencies of HOXA9-, MYC-, and MLL-AF10-ICs. Representative FACS plots and the summarized data (Mean with SD, n = 3, biological replicates) of Annexin V staining of HOXA9-, MYC-, and MLL-AF10-ICs in the presence of cytokines (B) and 1d after their removal (C) are shown. (D) In vivo leukemogenic potential of MLL target genes and MLL-AF10. Kaplan-Meier curves of mice transplanted with HPCs transduced with the indicated genes and the number of replicates are shown.

Next, we evaluated the leukemogenic potential of HOXA9 and MYC in vivo. Transplantation of HPCs exogenously expressing MLL-AF10 into syngeneic mice induced leukemia with full penetrance (Figure 3D). In contrast, neither MYC nor its homologue MYCN could initiate leukemia within 200 days under these experimental conditions, indicating that activation of the MYC high signature alone is insufficient for leukemogenesis in vivo. Although one recipient mouse of MYC-transduced HPCs became sick and was sacrificed approximately 150 days after transplantation, it did not exhibit any leukemia-associated signs. The mouse Myc gene also failed to induce leukemia. Thus, it is unlikely that the inability of MYC alone to induce leukemia under these experimental conditions is due to immune suppression of the cells expressing human transgenes. HOXA9 did not induce leukemia within 200 days either, suggesting that the HOXA9 high signature alone is also insufficient to induce leukemia. Taken together, these results suggest that both high MYC activity and HOXA9-mediated resistance to apoptosis are necessary for driving leukemogenesis in vivo.

HOXA9 promotes MYC-mediated leukemogenesis

We next examined the gene expression of patients with leukemia using publicly available microarray data of the Microarray Innovations in LEukemia (MILE) study (Haferlach et al., 2010). Of 108 cases with MLL translocation, 38 were acute myelogenous leukemia [AML], and 70 were acute lymphoblastic leukemia [ALL]. Most of the cases (78.7%) were categorized in the HOXA9high/MEIS1high group, while some were in the HOXA9low/MEIS1high (13.0%) or HOXA9high/MEIS1low (8.3%) group (Figure 4A). MYC was expressed at high levels irrespective of HOXA9 or MEIS1 expression. These data indicate variability in transcriptional profiles among MLL fusion-mediated leukemia cases, with MYC expression remaining consistently high. HOXA9low/MEIS1high leukemia was predominantly found in ALL, likely due to the MLL-AF4 cases, some of which do not express HOXA genes (Lin et al., 2016). HOXA9high/MEIS1low leukemia was mainly found in AML (Figure 4B).

Figure 4 with 4 supplements see all
HOXA9 promotes MYC-mediated leukemogenesis.

(A and B) Expression profiles of MLL fusion-mediated leukemia patients reported in the MILE study (Haferlach et al., 2010). Probe intensities of the indicated genes are plotted for all MLL fusion-mediated leukemia patients (A). Patients in the HOXA9low/MEIS1high (blue) and HOXA9high/MEIS1low (red) groups are highlighted. Probe intensities of HOXA9 and MEIS1 are plotted separately by leukemia phenotype (ALL or AML) (B). The expression profiles are provided in Figure 4—source data 1. (C) Transforming potential of various combinations of MLL target genes. CFU (Mean with SD, n = 3, biological replicates) and relative colony size (Mean with SD, n ≥ 100) are shown as in Figure 1C. (D) Morphologies of the colonies and transformed cells. Bright-field (left) and May-Grunwald-Giemsa staining (right) images are shown with scale bars. (E) In vivo leukemogenic potential of various oncogene combinations. Kaplan-Meier curves of mice transplanted with HPCs transduced with the indicated genes are shown as in Figure 3D. Bone marrow cells from moribund mice were harvested and used for secondary transplantation.

Figure 4—source data 1

Gene expression profiles of MLL fusion-mediated leukemia patients in the MILE data.

https://cdn.elifesciences.org/articles/64148/elife-64148-fig4-data1-v1.xlsx

Next, we assessed the effects of the combined expression of MYC, HOXA9, and MEIS1 in mouse leukemia models. The MYC/HOXA9 combination exhibited high clonogenicity ex vivo, with colony-forming capacity and colony size comparable to those of MLL-ENL- and HOXA9/MEIS1-ICs (Figure 4C,D). A weak synergy between MYC and MEIS1 was also observed. HOXA9/MEIS1-ICs showed high Myc expression (Figure 4—figure supplement 1A) and MYC-dependent proliferation (Figure 4—figure supplement 1B), indicating that MYC is essential in HOXA9/MEIS1-mediated leukemic transformation. In the in vivo leukemogenesis assays, MYC, HOXA9, or MEIS1 expression alone did not initiate leukemia, whereas co-expression of HOXA9 and MEIS1 did in all recipient mice as previously reported (Figure 4EKroon et al., 1998). The combined expression of MYC/HOXA9 or MYC/MEIS1 induced leukemia in 38% and 18% of recipient mice, respectively, indicating a synergy of MYC with HOXA9 and MEIS1. qRT-PCR analysis showed that LCs maintained the expression patterns of endogenous Hoxa9, Meis1, and Myc similar to those of the respective ICs (Figure 4—figure supplement 2). We observed rapid onset of leukemia with full penetrance in the secondary transplantation for the three combinations tested, confirming the presence of leukemia-initiating cells (Figure 4E). These results indicate a cooperative role for HOXA9 and MEIS1 in MYC-mediated leukemogenesis with stronger synergy between HOXA9 and MYC.

All the MYC/HOXA9- and HOXA9/MEIS1-induced leukemias were positive to a myeloid marker Mac1 but negative to lymphoid marker B220 or CD3e (Figure 4—figure supplement 3A,B), indicating that both MYC/HOXA9- and HOXA9/MEIS1-combinations induced myeloid leukemia. Gene set enrichment analysis (GSEA) of MYC/HOXA9-LCs and HOXA9/MEIS1-LCs revealed that genes involved in protein synthesis (e.g. ribosome biogenesis, tRNA metabolic process) were enriched in MYC/HOXA9-LCs (Figure 4—figure supplement 4A), while tumour necrosis factor α signaling and KRAS pathways were enriched in HOXA9/MEIS1-LCs (Figure 4—figure supplement 4B). These results suggested that these two combinations promoted leukemogenesis in slightly different ways, wherein MYC-mediated anabolic pathways played more prominent roles in leukemogenesis by MYC/HOXA9 than by HOXA9/MEIS1.

HOXA9 functions as a transcription maintenance factor

Some HOXA9 high signature genes, namely Bcl2, Sox4, and Igf1, have been implicated in leukemogenesis (Zhang et al., 2013; Du et al., 2005; Delbridge et al., 2016; Steger et al., 2015), suggesting that they may be responsible for the synergy between HOXA9 and MYC. Indeed, Bcl2, Sox4, and Igf1 were highly transcribed in HPCs transformed by the combinations containing HOXA9 (i.e. MYC/HOXA9-ICs, HOXA9/vector-ICs, and HOXA9/MEIS1-ICs) but not those lacking it (i.e. MYC/vector-ICs and MYC/MEIS1-ICs) (Figure 5A and Figure 5—figure supplement 1A). Conditional loss of function experiments using HOXA9 conjugated with estrogen receptor (HOXA9-ER) confirmed critical regulation of these genes by HOXA9 (Figure 5B). However, stepwise transduction of MYC followed by HOXA9 failed to upregulate these HOXA9 target genes, indicating that HOXA9 could not reactivate its target genes once silenced (Figure 5C and Figure 5—figure supplement 1B). Conditional inactivation and subsequent reactivation of HOXA9 within the same cell population confirmed that HOXA9 cannot reactivate its target genes once silenced (Figure 5—figure supplement 1C). RNA-seq analysis of various human cell lines showed that HOXA9 was highly expressed in all the MLL fusion cell lines tested (Figure 5—figure supplement 2A, highlighted in blue). BCL2 and SOX4 were also expressed but at different levels depending on the cell lines. In accord, most of the MLL fusion-mediated leukemia patient samples in the MILE study with high HOXA9 expression expressed BCL2 and SOX4 at high levels (Figure 5—figure supplement 2BHaferlach et al., 2010). IGF1 was only expressed in MV4-11 cells despite comparable HOXA9 expression among the MLL fusion cell lines, supporting the notion that HOXA9 expression itself cannot trigger the expression of silenced HOXA9 target genes. These results indicate that HOXA9 is a transcriptional maintenance factor that may be involved in the maintenance of chromatin structure previously activated by other transcriptional/epigenetic factors.

Figure 5 with 2 supplements see all
HOXA9 functions as a transcription maintenance factor.

(A) Gene expression of HPCs immortalized by various transgenes. Relative mRNA levels of HOXA9 target genes (Mean with SD, n = 3, PCR replicates) in myeloid progenitors transformed by various combinations of MLL target genes are shown. Two genes were transduced into HPCs in a simultaneous manner. (B) Gene expression after inactivation of HOXA9. HOXA9-ER and MYC were doubly transduced into HPCs and cultured in the presence of 4-OHT ex vivo. After 4-OHT withdrawal, qRT-PCR analysis was performed for the indicated genes (Mean, n=4, biological replicates). Statistical analysis was performed using unpaired two-tailed Student’s t-test. **p< 0.01, *p < 0.05. (C) Gene expression of HPCs immortalized by step-wise transduction of various transgenes. Relative mRNA levels of HOXA9 target genes in myeloid progenitors transformed by various combinations of MLL target genes are shown as in A. Two genes were transduced into HPCs in a stepwise manner.

BCL2 and SOX4 promote MYC-mediated leukemogenesis by alleviating apoptosis

To identify the roles for BCL2 and SOX4 in leukemic transformation, we evaluated the leukemogenic potential of the combined expression of BCL2 or SOX4 with MYC. In myeloid progenitor transformation assays, SOX4 by itself showed weak immortalization capacity as previously reported (Zhang et al., 2013), while BCL2 did not transform HPCs (Figure 6A). Co-expression of BCL2 or SOX4 with MYC led to a substantial increase in colony-forming capacity. The combined expression of BCL2 with MYC induced leukemia in vivo with a penetrance similar to that with the MYC/HOXA9 combination (Figures 4E and 6B, and Figure 6—figure supplement 1A), in accord with previous reports (Beverly and Varmus, 2009; Luo et al., 2005). SOX4 also promoted MYC-mediated leukemogenesis in vivo (Figure 6B and Figure 6—figure supplement 1A), while neither MYC, BCL2, nor SOX4 alone induced leukemia within 200 days. FACS analysis indicated that all of MYC/SOX4-induced leukemias were of myeloid lineage (Figure 6—figure supplement 1B). In contrast, all MYC/BCL2-induced leukemias were of lymphoid lineage, the half of which was B-cell type, and the other half was T-cell type, consistent with a previous report (Luo et al., 2005). It should be noted that single gene transduction of MYC and SOX4 induced leukemia by others in different settings, albeit with low penetrance (Du et al., 2005; Luo et al., 2005). These differences are possibly due to the differences of virus titers and viral genome integration-related gene activation. Furthermore, the combined expression of HOXA9, BCL2, or SOX4 with MYC alleviated apoptotic tendencies (Figure 6C,D). Although SOX4 is reported to modulate transcription of pro/anti-apoptotic genes (Ramezani-Rad et al., 2013), their expression was not drastically altered by SOX4 in this context (Figure 6E,F). Thus, the mechanism underlying the anti-apoptotic properties of SOX4 in this setting is currently unclear. Taken together, the results indicate that multiple anti-apoptotic pathways mediated by BCL2 and SOX4 promote MYC-mediated leukemic transformation.

Figure 6 with 1 supplement see all
BCL2 and SOX4 promote MYC-mediated leukemogenesis by alleviating apoptosis.

(A) Transforming potential of various combinations of MYC and HOXA9 target genes. CFU (Mean with SD, n = 3, biological replicates) is shown as in Figure 1C. (B) In vivo leukemogenic potential of various combinations of MYC and HOXA9 target genes. Kaplan-Meier curves of mice transplanted with HPCs transduced with the indicated genes are shown as in Figure 3D. (C and D) Apoptotic tendencies of MYC-expressing progenitors co-transduced with HOXA9, BCL2, or SOX4. Representative FACS plots (C) and the summarized data (D) (Mean with SD, n = 3, biological replicates) of Annexin V staining are shown. Statistical analysis was performed using ordinary one-way ANOVA with MYC-ICs. (E) Expression of apoptosis-related proteins in HPCs transformed by various combinations of transgenes. Western blots of HPCs transformed by indicated transgenes are shown (F) Relative expression levels of apoptosis-related genes in MYC/SOX4-ICs and MYC/vector-ICs. Relative mRNA levels of the indicated apoptosis-related genes (Mean, n = 2, biological replicates) are shown.

Endogenous BCL2 and SOX4 support the initiation and maintenance of leukemia

To examine the roles for endogenous BCL2 and SOX4 in the initiation and maintenance of LCs, we conducted myeloid progenitor transformation and in vivo leukemogenesis assays using Bcl2- and Sox4-knockout HPCs. We transduced various oncogenes into HPCs isolated from fetal livers of Bcl2- and Sox4-knockout embryos (Kamada et al., 1995; Schilham et al., 1996) and cultured them ex vivo. Neither Bcl2 nor Sox4 deletion affected the immortalization of HPCs by HOXA9, MYC, or MLL-AF10, indicating that BCL2 and SOX4 are dispensable for proliferation ex vivo (Figure 7—figure supplement 1). On the other hand, Bcl2 deletion delayed the onset of leukemia induced by MLL-AF10 and the HOXA9/MEIS1 combination in vivo (Figure 7A and Figure 7—figure supplement 2A). Sox4 deletion also delayed the onset of HOXA9/MEIS1-induced leukemia, although it did not affect MLL-AF10-induced leukemia (Figure 7B and Figure 7—figure supplement 2A). We also analyzed the steady-state apoptosis of LCs harvested from moribund mice with apoptotic markers (cleaved caspase, γH2AX and Annexin V). HOXA9/MEIS1-LCs were slightly more apoptotic in the absence of Bcl2 and Sox4 (Figure 7—figure supplement 2B,C). However, we did not observe substantial differences in MLL-AF10-LCs between the WT and Bcl2/Sox4 KO genotypes. These results are consistent with the different kinetics of leukemia onset (Figure 7A,B), and the minor differences in apoptotic tendencies in full-blown leukemia cells suggest that these LCs had acquired adequate anti-apoptotic properties at the time of disease presentation. These results indicate that endogenous BCL2 and SOX4 partially contribute to initiating leukemia in vivo.

Figure 7 with 3 supplements see all
Endogenous BCL2 and SOX4 support the initiation and maintenance of leukemia.

(A) Effects of Bcl2-deficiency on the initiation of leukemogenesis in vivo. HPCs were isolated from Bcl2+/+ or Bcl2–/– embryos and transduced with the retroviruses for MLL-AF10 or the HOXA9/MEIS1 combination. Kaplan-Meier curves of mice transplanted with the transduced HPCs are shown. Statistical analysis was performed using the log-rank test and Bonferroni correction with the wildtype control. *p ≤ 0.05. (B) Effects of Sox4-deficiency on the initiation of leukemogenesis in vivo. In vivo leukemogenesis assay was performed on Sox4+/+ or Sox4–/– embryos as in A (C) Effects of Bcl2- or Sox4-deficiency on the maintenance of leukemia initiating cells. Western blots of MLL-ENL leukemia cells transduced with sgRNA for BCL2 or SOX4 are shown on the left. Kaplan-Meier curves of mice transplanted with MLL-ENL leukemia cells transduced with the indicated sgRNAs are shown on the right. Statistical analysis was performed using the log-rank test and Bonferroni correction with the vector control. (D) Rescue of in vivo leukemogenic potential by sgRNA-resistant transgenes. Before transduction of sgRNA, MLL-ENL-LCs were transduced with sgRNA-resistant BCL2 or SOX4. In vivo leukemogenesis assay was performed as described in Figure 3D. (E) A model illustrating HOXA9-mediated pathogenesis in MLL fusion-mediated leukemia.

Next, we examined the roles for endogenous BCL2 and SOX4 in maintaining leukemia-initiating cells using a mouse leukemia cell line that we previously established with MLL-ENL (Okuda et al., 2017). CRISPR/Cas9-mediated sgRNA competition assays indicated the negligible contribution of Bcl2 and Sox4 to MLL-ENL-LC proliferation ex vivo (Figure 7—figure supplement 3). To test their roles in vivo, we transplanted Bcl2- or Sox4-deficient LCs into syngeneic mice, where we observed delayed onset and reduced incidence rate for both Bcl2- and Sox4-knockout LCs (Figure 7C). Forced expression of sgRNA-resistant cDNAs of BCL2 and SOX4 restored leukemogenic potential, validating the on-target effects of sgRNAs (Figure 7D). These results suggest that MLL-ENL-LCs are partially dependent on endogenous BCL2 and SOX4 in vivo. Taken together, our results indicate that MLL fusion-mediated LCs depend on the expression of multiple anti-apoptotic genes via HOXA9 to varying degrees to achieve survival advantages for disease initiation and maintenance (Figure 7E).

Discussion

In this study, we found that HOXA9 regulates a variety of genes to maintain hematopoietic precursor identity and its associated anti-apoptotic properties. In leukemic transformation, MLL fusion proteins exploit both HOXA9 and MYC downstream pathways. Accordingly, HOXA9 and MYC synergistically induce leukemia in mouse models. Thus, we propose that MLL fusion proteins employ two arms to promote oncogenesis: MYC-mediated proliferation and HOXA9-mediated resistance to differentiation/apoptosis.

It is widely accepted that two types of mutations need to occur before leukemia onset; class I mutations that confer proliferative advantages and class II mutations that block differentiation (Gilliland, 2002). However, it was unclear how MLL mutations fit this theory because MLL fusion-mediated leukemia does not require additional mutations in many cases (Andersson et al., 2015). A comparison of gene expression profiles of HOXA9- and MYC-transformed HPCs demonstrated that HOXA9 maintains the expression of a wide range of genes associated with hematopoietic precursor identity. Thus, HOXA9 appears to maintain the intrinsic transcriptional programs of immature HPCs, which are programed to be silenced during differentiation. On the other hand, MYC upregulates genes involved with de novo nucleotide/protein synthesis. This is in line with the known involvement of MYC in proliferation and associated anabolic processes (Ji et al., 2011). Furthermore, MLL-AF10 activated both the HOXA9 high and MYC high signature genes to induce leukemia while ectopic expression of HOXA9 and MYC synergistically induced leukemia. Thus, our findings suggest that MLL fusion proteins activate both HOXA9- and MYC-dependent programs as alternative mechanisms to the combination of class I and II mutations.

Although HOXA9 maintained MYC expression to immortalize myeloid progenitors ex vivo, it did not induce leukemia in vivo. We speculate that HOXA9 alone cannot maintain MYC expression at a level sufficient to confer leukemogenic ability in vivo. MLL fusion- and HOXA9/MEIS1-transduced cells, which are capable of inducing leukemia in vivo, expressed Myc 20–100% more than HOXA9-ICs (Figure 1C and Figure 4—figure supplement 1A). This additional MYC activity may be required to achieve sufficient leukemogenic ability, therefore MYC/HOXA9-transduced cells were able to induce leukemia in vivo (Figure 4E). It should be noted that MYC/HOXA9-ICs and -LCs expressed endogenous Myc at a lower level than other HOXA9-expressing cells (i.e. MLL-AF10- and HOXA9/MEIS1-ICs/LCs) (Figure 4—figure supplement 2), indicating that there is a threshold of MYC expression a cell can endure. We speculate that HOXA9 uplifts the threshold of MYC expression by conferring anti-apoptotic properties but cannot hyper-activate MYC expression by itself. Thus, additional MYC activation mediated by MLL fusions and MEIS1 confers proliferative advantages sufficient to induce leukemia in vivo.

The therapeutic efficacy of BCL2 inhibitor has been reported in AML including MLL fusion-mediated leukemia (Pan et al., 2014). Because there is a correlation between the expression levels of HOX proteins and the sensitivity to BCL2 inhibitor in AML patient samples, the aberrant expression of HOXA9 is the potential mechanism for BCL2-dependence of MLL leukemia (Brumatti et al., 2013; Kontro et al., 2017). Oncogenic MYC expression often leads to apoptosis, which needs to be alleviated by additional genetic events for leukemic cell survival (McMahon, 2014). Indeed, co-expression of HOXA9, BCL2, or SOX4 promoted MYC-mediated leukemogenesis, while MYC alone was insufficient to induce leukemia in vivo. Although the downstream mechanisms could not be addressed, SOX4 also exhibited anti-apoptotic effects on MYC-expressing cells. BCL2 was highly expressed in MLL fusion-mediated leukemia (Figure 5—figure supplement 2A,B), and many human MLL fusion leukemia cell lines were sensitive to a BCL-2 inhibitor (i.e. ABT-199)(Pan et al., 2014). High expression of SOX4 is correlated to poor survival in AML patients (Lu et al., 2017). These results support the key roles of BCL2 and SOX4 in the development of MLL leukemia. Ectopic expression of MYC and BCL2 induced lymphoid leukemia, whereas the MYC/SOX4 combination exclusively induced myeloid leukemia (Figure 6—figure supplement 1B), suggesting that BCL2 confers relatively stronger survival advantages in the lymphoid lineage than in the myeloid lineage in vivo, while SOX4 does in the myeloid lineage. Importantly, there was a partial effect of single-gene knockout of Bcl2 and Sox4 on leukemia initiation and maintenance. This indicates that MLL fusion proteins exploit multiple anti-apoptotic pathways and that blocking a single anti-apoptotic pathway may be insufficient to completely abrogate leukemic potential. Thus, simultaneously blocking multiple anti-apoptotic pathways may be required for efficient molecularly targeted therapy of HOXA9-expressing leukemia.

Our results also provide an insight into the mode of function of HOXA9. HOX genes are known to be expressed in a position-specific manner, conferring a positional identity to a cell (Wang et al., 2009). Our results suggest that HOXA9 unlikely functions as a major upstream factor determining tissue-specific gene expression by turning a silenced chromatin into transcriptionally active chromatin. Rather, HOX proteins likely play a supportive role in maintaining gene expression, which was activated by other transcriptional regulators. Our observation that HOXA9 cannot reactivate gene expression once silenced fits this hypothesis. Accordingly, HOXA9 maintains a subset of genes related to hematopoietic identity when expressed in hematopoietic precursors (Figure 2B). Recently, it has been reported that HOXA9 may recruit enhancer apparatuses, as it colocalizes with active enhancer mark (i.e. acetylated histone H3 lysine 27) (Sun et al., 2018). We speculate that HOXA9 may support the maintenance of an active enhancer, but unlikely establishes it on silenced chromatin. Further functional analysis of HOXA9 is required to understand how HOXA9 regulates gene expression.

In summary, our results describe the oncogenic roles for HOXA9 as a transcriptional maintenance factor for multiple anti-apoptotic genes, which are necessary to promote MYC-mediated leukemogenesis. In the case of MLL fusion-mediated leukemia, MLL fusion proteins directly activate both MYC and HOXA9, while HOXA9 maintains expression of MYC, BCL2, and SOX4, achieving high MYC activity and anti-apoptotic properties simultaneously (Figure 7E). Thus, MLL fusion-mediated LCs possess highly proliferative potentials and survival advantages using HOXA9 as a critical mediator.

Materials and methods

Vector constructs

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For protein expression vectors, cDNAs obtained from Kazusa Genome Technologies Inc (Nagase et al., 2008) were modified by PCR-mediated mutagenesis and cloned into the pMSCV vector (for virus production) or pCMV5 vector (for transient expression) by restriction enzyme digestion and DNA ligation. The MSCV-neo MLL-ENL and MLL-AF10 vectors have been previously described (Okuda et al., 2017). sgRNA-expression vectors were constructed using the pLKO5.sgRNA.EFS.GFP vector (RRID:Addgene_57822) (Heckl et al., 2014). shRNA-expression vectors were purchased from Dharmacon. The target sequences are listed in Supplementary file 1.

Cell lines

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HEK293T cells were a gift from Michael Cleary and were authenticated by the JCRB Cell Bank in 2019 (Key Resources Table). HEK293TN cells were purchased from System Biosciences (RRID:CVCL_UL49). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (PS). The Platinum-E (PLAT-E) ecotropic virus packaging cell line—a gift from Toshio Kitamura (RRID:CVCL_B488)(Morita et al., 2000)—was cultured in DMEM supplemented with 10% FBS, puromycin, blasticidin, and PS. The human leukemia cell lines including HB1119, (RRID:CVCL_8227), K562 (RRID:CVCL_0004), RS4-11 (RRID:CVCL_0093), THP1 (RRID:CVCL_0006), and EOL1 (RRID:CVCL_0258) were gifts from Michael Cleary (Tkachuk et al., 1992; Yokoyama et al., 2004), and were cultured in RPMI 1640 medium supplemented with 10% FBS and PS. The MV4-11 cell line (ATCC, RRID:CVCL_0064) was cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS and PS. The ML-2 cell line (DSMZ, RRID:CVCL_1418) was cultured in RPMI 1640 medium supplemented with 10% FBS and PS. MLL-ENL LCs (MLL-ENLbm0713) were described previously (Okuda et al., 2017). All the cell lines except HEK293TN, PLAT-E, and HB1119 were authenticated using short tandem repeat (STR)-PCR method by the JCRB Cell Bank. Cells were cultured in the incubator at 37°C and 5% CO2, and routinely tested for mycoplasma using the MycoAlert Mycoplasma detection kit (Lonza). 

Animal models

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For the in vivo leukemogenesis assay, 8-week-old female C57BL/6JJcl (C57BL/6J) or C. B-17/Icr-scid/scidJcl (SCID) mice were purchased from CLEA Japan (Tokyo, Japan). Bcl2-knockout mice were a gift from Yoshihide Tsujimoto and provided via RIKEN BRC (Kamada et al., 1995). Sox4-knockout mice were a gift from Hans Clevers and provided via RIKEN BRC (Schilham et al., 1996).

Western blotting

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Western blotting was performed as previously described (Yokoyama et al., 2002). Antibodies used in this study are listed in Key Resources Table.

Virus production

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Ecotropic retrovirus constructed in pMSCV vectors was produced using PLAT-E packaging cells (Morita et al., 2000). Lentiviruses were produced in HEK293TN cells using the pMDLg/pRRE (RRID:Addgene_12251), pRSV-rev (RRID:Addgene_12253), and pMD2.G (RRID:Addgene_12259) vectors, all of which were gifts from Didier Trono (Dull et al., 1998). The virus-containing medium was harvested 24–48 hr following transfection and used for viral transduction.

Myeloid progenitor transformation assay

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The myeloid progenitor transformation assay was conducted as previously described (Lavau et al., 1997; Okuda and Yokoyama, 2017a). Bone marrow cells were harvested from the femurs and tibiae of 5-week-old female C57BL/6J mice. c-Kit+ cells were enriched using magnetic beads conjugated with an anti-c-Kit antibody (Miltenyi Biotec, RRID:AB_2753213), transduced with a recombinant retrovirus by spinoculation, and then plated (4 × 104 cells/ sample) in a methylcellulose medium (IMDM, 20% FBS, 1.6% methylcellulose, and 100 µM β-mercaptoethanol) containing murine stem cell factor (mSCF), interleukin 3 (mIL-3), and granulocyte-macrophage colony-stimulating factor (mGM-CSF; 10 ng/mL each). During the first culture passage, G418 (1 mg/mL) or puromycin (1 μg/mL) was added to the culture medium to select for transduced cells. Hoxa9 expression was quantified by qRT-PCR after the first passage. Cells were then re-plated once every 4–6 days with fresh medium; the number of plated cells for the second, third, and fourth passages was 4 × 104, 2 × 104, and 1 × 104 cells/well, respectively. CFUs were quantified per 104 plated cells at each passage.

In vivo leukemogenesis assay

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In vivo leukemogenesis assays were conducted as previously described (Lavau et al., 1997; Okuda and Yokoyama, 2017b). c-Kit+ cells (2 × 105) prepared from the femurs and tibiae of 5-week-old female C57BL/6J mouse were transduced with retrovirus by spinoculation and intravenously transplanted into sublethally irradiated (5–6 Gy) C57BL/6J mice. For secondary leukemia, LCs (2 × 105) cultured ex vivo for more than three passages were transplanted. As for knockouts of Bcl2 and Sox4, mice heterozygous for Bcl2 or Sox4 were crossed, and c-Kit+ cells were isolated from fetal livers at E14–15 (for Bcl2) or E13 (for Sox4). The next day, cells were transduced with the retroviruses for MLL-AF10 or HOXA9/MEIS1 and transplanted intravenously into sublethally irradiated (2.5 Gy) SCID mice [2 × 105 (for Bcl2) or 1 × 105 cells/mouse (for Sox4)].

qRT-PCR

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Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse-transcribed using the Superscript III First Strand cDNA Synthesis System (Thermo Fisher Scientific) with oligo (dT) primers. Gene expression was analyzed by qPCR using TaqMan probes (Thermo Fisher Scientific). Relative expression levels were normalized to those of GAPDH/Gapdh, ACTB/Actb, or TBP/Tbp and determined using a standard curve and the relative quantification method, according to manufacturer’s instructions (Thermo Fisher Scientific). Commercially available/custom made PCR probes used are listed in Supplementary file 1.

ChIP-qPCR and ChIP-seq

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ChIP was performed as previously described (Okuda et al., 2017), using the fanChIP method (Miyamoto and Yokoyama, 2021). DNA was precipitated with glycogen, dissolved in TE buffer, and analyzed by qPCR (ChIP-qPCR) or deep sequencing (ChIP-seq). The qPCR probe/primer sequences are listed in Supplementary file 1. Deep sequencing was performed using the TruSeq ChIP Sample Prep Kit (Illumina) and HiSeq2500 (Illumina) at the core facility of Hiroshima University and described in our previous publication (Okuda et al., 2017).

RNA-seq

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Total RNA was prepared using the RNeasy Kit (Qiagen) and analyzed using a Bioanalyzer (Agilent Technologies). Deep sequencing was performed using a SureSelect Strand Specific RNA Library Prep Kit (Agilent Technologies) and GAIIx (Illumina) with 36 bp single-end reads or HiSeq2500 (Illumina) with 51 bp single-end reads at the core facility of Hiroshima University. Sequenced reads were mapped to the human genome assembly hg19 or the mouse genome assembly mm9 using CASAVA 1.8.2 (Illumina, RRID:SCR_001802), and read counts were normalized as reads per kilo base of exon per million mapped (RPKM). To define HOXA9 high and MYC high signature genes, data were trimmed by removing lowly expressed genes whose RPKM values were less than 2, and the relative expression between HOXA9-ICs and MYC-ICs was estimated. The top 50 HOXA9 high and MYC high signature genes were visualized as a heatmap using the Complex Heatmap package (RRID:SCR_017270)(Gu et al., 2016). To define HOXA9-MYC common target genes, lowly expressed genes were removed, and genes with more than two-fold expression compared to c-Kit+ cells were determined for each of HOXA9-ICs and MYC-ICs. The overlapping genes were defined as ‘HOXA9-MYC common target genes’. GSEA analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the DAVID (RRID:SCR_001881)(Jiao et al., 2012), GSEA (Subramanian et al., 2005), and Metascape (Zhou et al., 2019).

sgRNA competition assay

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Cas9 was introduced via lentiviral transduction using the pKLV2-EF1a-Cas9Bsd-W vector (RRID:Addgene_68343) (Tzelepis et al., 2016). Cas9-expressing stable lines were established with blasticidin (10–30 µg/mL) selection. The targeting sgRNA was co-expressed with GFP via lentiviral transduction using pLKO5.sgRNA.EFS.GFP vector (RRID:Addgene_57822)(Heckl et al., 2014). Percentages of GFP+ cells were initially determined by FACS analysis at 2 or 3 days after sgRNA transduction and then measured once every 3–5 days.

FACS analysis and sorting

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To detect apoptosis, two to five million cells were suspended in 200 µL of reaction buffer (140 mM NaCl, 10 mM HEPES, 2.5 mM CaCl2, and 0.1% BSA) and incubated with APC-Annexin V (RRID:AB_2868885) for 15 min and Propidium iodide for 5 min at room temperature. The cells were then centrifuged, resuspended in fresh reaction buffer, and analyzed with FACS Melody (BD Bioscience). FACS sorting of mouse bone marrow cells was performed with fluorophore-conjugated antibodies listed in Key Resources Table as previously described (Yokoyama et al., 2013).

Accession numbers

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Deep sequencing data used in this study have been deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive under the accession numbers listed in Supplementary file 1.

Statistics

Statistical analyses were performed using GraphPad Prism seven software (RRID:SCR_002798). Data are presented as the mean with standard deviation (SD). Comparisons between two groups were analyzed by unpaired two-tailed Student’s t-test, while multiple comparisons were performed by ordinary one-way analysis of variance (ANOVA) followed by Dunnett’s test or two-way ANOVA. Mice transplantation experiments were analyzed by the log-rank test and Bonferroni correction was applied for multiple comparisons. p Values < 0.05 were considered statistically significant. n.s.: p>0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, and ****: p ≤ 0.0001.

Study approval

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All animal experimental protocols were approved by the National Cancer Center (Tokyo Japan) Institutional Animal Care and Use Committee.

Appendix 1

Appendix 1—key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)C57BL/6JCLEA Japan
Strain, strain background (Mus musculus)Bcl2 KO (B6.129P2-Bcl2<tm1Tsu>/TsuRbrc)Gift from Yoshihide Tsujimoto (via RIKEN BRC) Kamada et al., 1995
Strain, strain background (Mus musculus)Sox4 KO (STOCK Sox4<tm1Cle>/Mmmh)Gift from Hans Clevers (via RIKEN BRC) Schilham et al., 1996
Cell line (Homo-sapiens)PLAT-EGift from Toshio Kitamura Morita et al., 2000RRID:CVCL_B488
Cell line (Homo-sapiens)HB1119Gift from Michael L. Cleary Tkachuk et al., 1992; Yokoyama et al., 2004The cell line was verified by the expression of MLL-ENL
Cell line (Homo-sapiens)HEK293TNSystem BioscienceCat# LV900A-1
RRID:CVCL_UL49
Cell line (Homo-sapiens)HEK293TGift from Michael L. Cleary Yokoyama et al., 2004authenticated by the JCRB Cell Bank in 2019
Cell line (Homo-sapiens)K562Gift from Michael L. Cleary Yokoyama et al., 2004RRID:CVCL_0004authenticated by the JCRB Cell Bank in 2021
Cell line (Homo-sapiens)MV4-11ATCCCat# CRL-9591
RRID:CVCL_0064
authenticated by the JCRB Cell Bank in 2021
Cell line (Homo-sapiens)RS4-11Gift from Michael L. ClearyRRID:CVCL_0093authenticated by the JCRB Cell Bank in 2021
Cell line (Homo-sapiens)ML-2DSMZCat# ACC15
RRID:CVCL_1418
authenticated by the JCRB Cell Bank in 2021
Cell line (Homo-sapiens)THP1Gift from Michael L. ClearyRRID:CVCL_0006authenticated by the JCRB Cell Bank in 2021
Cell line (Homo-sapiens)EOL-1Gift from Michael L. ClearyRRID:CVCL_0258authenticated by the JCRB Cell Bank in 2021
AntibodyMLLn ab#1
(Rabbit polyclonal)
In-house Yokoyama et al., 2002rpN1ChIP (1:400)
AntibodyMLLn ab#2 (Rabbit monoclonal, D2M7U)Cell Signaling TechnologyCat# 14689
RRID:AB_2688009
ChIP (1:400)
AntibodyMLLc (Rabbit monoclonal, D6G8N)Cell Signaling TechnologyCat# 14197
RRID:AB_2688010
WB (1:1000)
AntibodyMENIN (Rabbit polyclonal)Bethyl LaboratoriesCat# A300-105A
RRID:AB_2143306
ChIP (1:400)
WB (1:1000)
AntibodyMYC (Rabbit monoclonal, D3N8F)Cell Signaling TechnologyCat# 13987
RRID:AB_2631168
WB (1:2000)
AntibodyCaspase3 (Rabbit monoclonal, D3R6Y)Cell Signaling TechnologyCat# 14220
RRID:AB_2798429
WB (1:2000)
AntibodyCleaved Caspase3 (Rabbit monoclonal, 5A1E)Cell Signaling TechnologyCat# 9664
RRID:AB_2070042
WB (1:2000)
AntibodyPARP (Rabbit polyclonal)Cell Signaling TechnologyCat# 9542
RRID:AB_2160739
WB (1:2000)
AntibodyγH2AX (Rabbit polyclonal)Bethyl LaboratoriesCat# A300-081A
RRID:AB_203288
WB (1:1000)
AntibodyBCL2 (Mouse monoclonal, C-2)Santa Cruz BiotechnologyCat# sc-7382
RRID:AB_626736
WB (1:500)
AntibodySOX4 (Mouse monoclonal, B-7)Santa Cruz BiotechnologyCat# sc-518016WB (1:200)
AntibodyHOXA9 (Rabbit polyclonal)MilliporeCat# 07–178 RRID:AB_11210179WB (1:1000)
AntibodyGAPDH (Rabbit polyclonal)Santa Cruz BiotechnologyCat# sc-25778
RRID:AB_10167668
WB (1:2000)
AntibodyBAK (Rabbit monoclonal, D4E4)Cell Signaling TechnologyCat# 12105
RRID:AB_2716685
WB (1:2000)
AntibodyBAD (Rabbit monoclonal, D24A9)Cell Signaling TechnologyCat# 9239
RRID:AB_2062127
WB (1:2000)
AntibodyBCL-XL (Rabbit monoclonal, 54H6)Cell Signaling TechnologyCat# 2764
RRID:AB_2228008
WB (1:2000)
AntibodyMCL1 (Rabbit monoclonal, D35A5)Cell Signaling TechnologyCat# 5453
RRID:AB_10694494
WB (1:2000)
AntibodyAPC Annexin VBD BiosciencesCat# 550475
RRID:AB_2868885
FACS (1:100)
AntibodyPropidium iodideThermo Fisher ScientificP3566FACS (1:2000)
AntibodyGr-1 (Rat monoclonal, RB6-8C5)BD BiosciencesCat# 553127
RRID:AB_394643
FACS (1:100)
AntibodyB220 (Rat monoclonal, RA3-6B2)BD BiosciencesCat#: 553088
RRID:AB_394618
FACS (1:100)
AntibodyTER119 (Rat monoclonal, TER-119)BD BiosciencesCat#: 557915
RRID:AB_396936
FACS (1:100)
AntibodyCD3e (Hamster monoclonal, 145–2 C11)BD BiosciencesCat#: 553062
RRID:AB_394595
FACS (1:100)
AntibodyMac/CD11b (Rat monoclonal, M1/70)BD BiosciencesCat#: 553310
RRID:AB_394774
FACS (1:100)
Antibodyc-Kit (Rat monoclonal, 2B8)BD BiosciencesCat#: 553355
RRID:AB_394806
FACS (1:100)
AntibodySca1 (Rat monoclonal, D7)BD BiosciencesCat#: 553108
RRID:AB_394629
FACS (1:100)
AntibodyCD34 (Rat monoclonal, RAM34)BD BiosciencesCat#: 751621
RRID:AB_2875614
FACS (1:100)
AntibodyCD16/32 (Rat monoclonal, 93)BD BiosciencesCat#: 751690
RRID:AB_2875675
FACS (1:100)
Recombinant DNA reagentpMSCV-neoClontechCat#: 634401Gene expression vector
Recombinant DNA reagentpMSCV-puroClontechCat#: 634401Gene expression vector
Recombinant DNA reagentpMSCV-hygroClontechCat#: 634401Gene expression vector
Recombinant DNA reagentpLKO5.EFS.GFPAddgene (gift from Benjamin Ebert) Heckl et al., 2014Addgene Plasmid #57822
RRID:Addgene_57822
sgRNA backbone
Recombinant DNA reagentpKLV2-Cas9.bsdAddgene (gift from Kosuke Yusa) Tzelepis et al., 2016Addgene Plasmid #68343
RRID:Addgene_68343
Cas9 expression vector
Recombinant DNA reagentpMDLg/pRREAddgene (gift from Didier Trono) Dull et al., 1998Addgene Plasmid #12251
RRID:Addgene_12251
Lentivirus packaging vector
Recombinant DNA reagentpRSV-revAddgene (gift from Didier Trono) Dull et al., 1998Addgene Plasmid #12253
RRID:Addgene_12253
Lentivirus packaging vector
Recombinant DNA reagentpMD2.GAddgene (gift from Didier Trono) Dull et al., 1998Addgene Plasmid #12259
RRID:Addgene_12259
Lentivirus packaging vector
Recombinant DNA reagentpLKO.1-puroAddgene (gift from Bob Weinberg) Stewart et al., 2003Addgene Plasmid #84530
RRID:Addgene_84530
shRNA backbone
Sequence-based reagentsgRNAsThis studySee Supplementary file 1
Sequence-based reagentshRNAThis studySee Supplementary file 1
Sequence-based reagentqPCR primersThermo Fisher ScientificSee Supplementary file 1
Commercial assay or kitRNeasy Mini KitQiagenCat#: 74106
Commercial assay or kitSuper Script III First-Strand Synthesis SystemThermo Fisher ScientificCat# 18080051
Commercial assay or kitTruSeq ChIP Sample Prep Kit SetBIlluminaCat# IP-202–1024
Commercial assay or kitSure Select Strand Specific RNA Library Prep KitAgilent TechnologiesCat#: G9691A
Software, algorithmGraphPad Prism7GraphPad Software IncRRID:SCR_002798Data analysis
Software, algorithmFlowJoBD BiosciencesRRID:SCR_008520FACS data analysis
Software, algorithmIntegrative Genomics ViewerThorvaldsdóttir et al., 2013RRID:SCR_011793Data visualization
Software, algorithmCASAVA 1.8.2IlluminaRRID:SCR_001802RNA-seq data analysis
Software, algorithmDAVIDJiao et al., 2012RRID:SCR_001881RNA-seq data analysis
Software, algorithmComplex heatmapGu et al., 2016RRID:SCR_017270RNA-seq data analysis
Software, algorithmR2: Genome Analysis and Visualization PlatformAMC: Oncogenomicshttp://r2.amc.nl http://r2platform.comRNA-seq data analysis
Software, algorithmGene set enrichment analysisSubramanian et al., 2005https://www.gsea-msigdb.org/gsea/index.jspRNA-seq data analysis
Otherc-Kit magnetic beadsMiltenyi BiotecCat# 130-091-224
RRID:AB_2753213

Data availability

ChIP-seq data have been deposited to the DDBJ archive and have been published (accession number: DRA004871). RNA-seq data have been deposited to the DDBJ archive and have been published (accession number: DRA010090, DRA012078, DRA004874, DRA012079).

The following data sets were generated
    1. HIROSHIMA
    (2020) DDBJ GEA
    ID E-GEAD-360. Expression profiles of murine myeloid progenitors immortalized by various oncogenes.
    1. HIROSHIMA
    (2021) DDBJ GEA
    ID E-GEAD-435. Expression profiles of murine myeloid progenitors immortalized by various oncogenes.
    1. HIROSHIMA
    (2021) DDBJ GEA
    ID E-GEAD-436. Expression profiles of human cell lines.
    1. HIROSHIMA
    (2018) DDBJ DRA
    ID DRA010090. Sequence reads of murine myeloid progenitors immortalized by various oncogenes.
    1. HIROSHIMA
    (2015) DDBJ DRA
    ID DRA012078. Sequence reads of murine myeloid progenitors immortalized by various oncogenes.
    1. HIROSHIMA
    (2016) DDBJ DRA
    ID DRA004874. Sequence reads of various factors/modificaions in HB1119 cells.
    1. HIROSHIMA
    (2015) DDBJ DRA
    ID DRA012079. Sequence reads of human cell lines.
    1. HIROSHIMA
    (2020) DDBJ GEA
    ID E-GEAD-321. Expression profiles of HB1119 and 293T cell lines.
The following previously published data sets were used
    1. HIROSHIMA
    (2016) DDBJ
    ID DRA004871. Genomic localization of various factors/modificaions in HB1119 cells.
    1. HIROSHIMA
    (2020) DDBJ GEA
    ID E-GEAD-319. Genomic localization of various factors/modificaions in HB1119 cells.

References

    1. Kamada S
    2. Shimono A
    3. Shinto Y
    4. Tsujimura T
    5. Takahashi T
    6. Noda T
    7. Kitamura Y
    8. Kondoh H
    9. Tsujimoto Y
    (1995)
    bcl-2 deficiency in mice leads to pleiotropic abnormalities: accelerated lymphoid cell death in Thymus and spleen, polycystic kidney, hair Hypopigmentation, and distorted small intestine
    Cancer Research 55:354–359.

Decision letter

  1. Erica A Golemis
    Senior and Reviewing Editor; Fox Chase Cancer Center, United States

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

The manuscript of Miyamoto et al. describes the synergistic interactions between HOXA9 and MYC induced by MLL-AF10 fusions in myeloid leukemogenesis. Detailed analysis of gene expression profiles resulting from overexpression of MLL-AF10 provide mechanistic insight into how cell death induced by elevated MYC is counted by signaling from BCL2 or SOX4, which are up-regulated in HOXA9, supporting leukemogenesis. This manuscript will be of interest to experimental haematologists studying initiation and maintenance factors in leukaemia.

Decision letter after peer review:

Thank you for submitting your article "HOXA9 promotes MYC-mediated leukemogenesis by maintaining gene expression for multiple anti-apoptotic pathways" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by Erica Golemis as the Senior and Reviewing Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. Presentation of ChIPseq data in Figure 1 could be substantially improved by providing more details of MLL-occupancy patterns. More information (particularly about computational analyses) should also be provided in the methods For example, in Figure 1A, the authors attempt to identify direct target genes of the MLL fusion protein MLL-ENL by performing ChIPseq using an anti-MLL antibody. Whether or not the signal can be attributed to MLL-ENL or wild-type MLL is unclear. Furthermore, genome-wide MLL-occupancy patterns are not shown. It would also be useful to reconcile current data with other publicly available datasets for MLL or MLL-fusion protein occupancy in comparable contexts.

2. It would appear (based on capitalisation), that the authors are over-expressing human transgenes in mouse cells. This is not necessarily a concern, but should be considered when interpreting the data. Likewise, whether the primers used for qPCR are detecting expression of the transgenes, the endogenous genes or both is important (for some of the figures such as Figure 1C there seems to be a mix e.g. Myc vs HoxA9/HOXA9)?

3. Most of the in vivo transplantation experiments have not been performed using fluorescent reporters or congenic recipients that would enable identification of donor-derived cells. Differences between the groups could be attributed to differential engraftment, or potentially even immune rejection (assuming ectopic expression of human transgenes in an immune-competent context). Disease features in recipient mice (beyond survival) are also not shown and expression of transgenes at end-point not confirmed.

4. The authors claim that the data in Figure 5B confirms direct regulation of Bcl2, Sox4 and Igf1 by HOXA9. However, the regulation could also be indirect e.g. HOXA9 could regulate a transcription factor that regulates those genes, or HOXA9 depletion could induce differentiation that may result in downregulation of those genes.

5. More specifically, the authors showed that HOXA9 introduction into MYC-IC failed to show activation of HOXA9 target genes. Although the authors claim that HOXA9 is a transcription maintenance factor with these results (page 14), they did not mention the possible difference of cell-of-origin. A number of oncogenes that function as chromatin remodeling factors failed to show such functions if they are introduced inappropriate target cells. Therefore, the title "HOXA9 functions as a transcription maintenance factor" should be modified. Also, they argue that HOXA9 is not a major upstream factor since the similarly functional fibroblasts express different HOX genes. This notion is too speculative and should be modified or removed.

6. The role of MYC, HOXA9 and BCL2 has been extensively studied in AML including with sophisticated in vivo models utilising conditional alleles. Likewise, many studies have sought to identify essential genes downstream of MLL fusion proteins. A lot of additional data is publicly available (e.g. RNAseq in AML patients) the analysis of which could be used in many different ways to strengthen the manuscript.

Inclusion of analysis based on these data should be considered.

7. The authors showed that Myc expression is comparable between HOXA9-IC and MLL-ENL-IC (Figure 1C), indicating that both HOXA9 and MLL-ENL could support Myc expression as is also argued by authors. This information is inconsistent with the results that co-expression of HOXA9 and MYC induces leukemia whereas HOXA9 single expression does not (Figure 4E). The authors should explain this apparent discrepancy.

8. Moreover, Myc silencing at ~50% expression level of control completely abrogated colony formation in MLL-ENL-IC and HOXA9-IC (Figure 1E). Is there fine-tuning system that requires critical expression level of Myc? Or did the authors obtain much more silencing effect at the protein level? Please clarify this point.

9. In relation to the above questions, given that Myc expression is upregulated by HOXA9 like by MLL-AF10, there should be common genetic pathways regulated both HOXA9 and MYC. I would request the authors to provide the gene list and pathways commonly regulated by HOXA9 and MYC in Figure 2.

10. In discussion (page 21, lines 6-8) the authors claim that MLL fusion proteins promote oncogenesis by activating both MYC- and HOXA9-related pathways. However, the cooperative effect of MYC for HOXA9 is much weaker than that of MEIS1 as is shown in Figure 4E. Given that MEIS1 is the direct target of MLL fusions, I wonder whether MYC is dispensable in the presence of MEIS1. Since there have been several studies on MEIS1 function in leukemogenesis, the authors should show common and/or distinct downstream genes/pathways between MYC- and MEIS1-driven leukemogenesis.

11. BCL2 and SOX4 knockout showed delay in MLL-AF10- and HOXA9/MEIS1-induced leukemogenesis (Figure 7A, B). Please provide the data wheter these delays are caused by increased apoptosis by presenting the Annexin V staining as well as Caspase 3 cleavage and H2AX expression.

https://doi.org/10.7554/eLife.64148.sa1

Author response

Essential revisions:

1. Presentation of ChIPseq data in Figure 1 could be substantially improved by providing more details of MLL-occupancy patterns. More information (particularly about computational analyses) should also be provided in the methods For example, in Figure 1A, the authors attempt to identify direct target genes of the MLL fusion protein MLL-ENL by performing ChIPseq using an anti-MLL antibody. Whether or not the signal can be attributed to MLL-ENL or wild-type MLL is unclear. Furthermore, genome-wide MLL-occupancy patterns are not shown. It would also be useful to reconcile current data with other publicly available datasets for MLL or MLL-fusion protein occupancy in comparable contexts.

We performed ChIP-seq analysis of HB1119 cells in which wildtype MLL, but not MLL-ENL, was specifically knocked down by shRNA (Figure 1A, Figure 1—figure supplement-1B,C), as shown In our previous publication (Okuda et al., 2017). Depletion of wildtype MLL did not affect the ChIP signals. Thus, we concluded that most of the MLL ChIP signals can be attributed to MLL-ENL. These data was presented in our previous report (Okuda et al., 2017) and partially adopted in the revised manuscript. MLL and MLL fusion proteins localize near transcription start sites (TSSs)( Figure 1—figure supplement-1C) because MLL has a CXXC domain that recognizes unmethylated CpGs (Okuda et al., 2014). Such TSS-centric localization of MLL is observed in many other non-MLL-rearranged cell lines such as HEK293T (embryonic kidney) and REH (Leukemia) cells (Miyamoto et al., 2020), in addition to HB1119 cells (MLL-rearranged leukemia cells)(Okuda et al., 2017). We mentioned this in the revised manuscript.

2. It would appear (based on capitalisation), that the authors are over-expressing human transgenes in mouse cells. This is not necessarily a concern, but should be considered when interpreting the data. Likewise, whether the primers used for qPCR are detecting expression of the transgenes, the endogenous genes or both is important (for some of the figures such as Figure 1C there seems to be a mix e.g. Myc vs HoxA9/HOXA9)?

We used human transgenes in the presented experiments. The qPCR probes for mouse Hoxa9 and Meis1 detected human HOXA9 and MEIS1, respectively. Hence, we described HOXA9/Hoxa9 and MEIS1/Meis1 to clearly indicate that these probes detect both human and mouse genes. The qPCR probe for mouse endogenous Myc did not detect the human MYC transgene. The samples producing qPCR signals for both endogenous murine genes and exogenous human transgenes are highlighted by # and faded color (Figure 1C).

3. Most of the in vivo transplantation experiments have not been performed using fluorescent reporters or congenic recipients that would enable identification of donor-derived cells. Differences between the groups could be attributed to differential engraftment, or potentially even immune rejection (assuming ectopic expression of human transgenes in an immune-competent context). Disease features in recipient mice (beyond survival) are also not shown and expression of transgenes at end-point not confirmed.

As for the possibility of immune rejection of the cells expressing human transgenes:

As shown in Figure 3D, the mouse Myc gene was tested in addition to human MYC and did not induce leukemia in vivo, supporting that the enhanced MYC function alone is insufficient to induce leukemia under these experimental conditions. It has been shown that the mouse Hoxa9 gene is also a weak oncogene in vivo by Kroon et al. whereas it induced leukemia as a combination with Meis1(Kroon et al., 1998). The human HOXA9 transgene phenocopied mouse Hoxa9 in our assays. These results did not support the possibility of immune rejection of the human transgene-expressing cells. We mentioned that in the revised manuscript.

As for the possibility of different engraftment:

We did not mean to exclude the possibility of different engraftment as the reason of not inducing leukemia by a certain oncogene. It is likely that HOXA9 promotes engraftment of MYC-transduced cells by conferring survival advantage with BCL2/SOX4-mediated anti-apoptotic properties. It is possible that HOXA9 mediates additional functions to promote engraftment other than providing anti-apoptotic properties. However, we chose to focus on the HOXA9-mediated anti-apoptotic functions in this paper.

As for the disease features:

We have added the expression and immune phenotype data in Figure 4—figure supplement-3B and Figure 6—figure supplement-1B.

In contrast to MLL-AF10 and HOXA9 containing gene sets (HOXA9-MEIS1, HOXA9-MYC), MYC-BCL2 induced lymphoid leukemia in vivo, consistent with the previous report (Luo et al., 2005). We speculate that HOXA9 and SOX4 are more functional in the myeloid lineage, while BCL2 functions more efficiently in the lymphoid lineage than in the myeloid lineage. Consequently, the MYC-BCL2 combination tended to induce lymphoid leukemia.

As for the expression of the transgene at end-point:

Regarding the expression of the transgenes in Figure 3D and 4E, we have provided the RT-qPCR data for the transgenes in Figure 4—figure supplement-3B.

Regarding the expression of the transgenes in Figure 6B, the protein expression of the transgenes is shown in Figure 6—figure supplement-1A.

Regarding the expression of the transgenes in Figure 7A, B, we have provided the RT-qPCR data for the transgenes in Figure 7—figure supplement 2A.

4. The authors claim that the data in Figure 5B confirms direct regulation of Bcl2, Sox4 and Igf1 by HOXA9. However, the regulation could also be indirect e.g. HOXA9 could regulate a transcription factor that regulates those genes, or HOXA9 depletion could induce differentiation that may result in downregulation of those genes.

The regulatory mechanisms by which HOXA9 controls the expression of its target genes are of great interest. Indeed, the expression of BCL2 and/or SOX4 could be regulated indirectly by HOXA9. We changed the wording by removing the word “direct” in the revised manuscript.

5. More specifically, the authors showed that HOXA9 introduction into MYC-IC failed to show activation of HOXA9 target genes. Although the authors claim that HOXA9 is a transcription maintenance factor with these results (page 14), they did not mention the possible difference of cell-of-origin. A number of oncogenes that function as chromatin remodeling factors failed to show such functions if they are introduced inappropriate target cells. Therefore, the title "HOXA9 functions as a transcription maintenance factor" should be modified. Also, they argue that HOXA9 is not a major upstream factor since the similarly functional fibroblasts express different HOX genes. This notion is too speculative and should be modified or removed.

To test whether the inability to activate HOXA9 target genes by reactivating HOXA9, we performed qRT-PCR analysis of the cells wherein HOXA9 is inactivated and later reactivated within the same cell population (Figure 5—figure supplement-1C). Reactivation of HOXA9 by adding back 4OHT did not rescued the expression of HOXA9 target genes. Thus, we think that the reason why HOXA9 did not activate the expression of its target genes in MYC-transformed cells is not due to the difference of cell-of-origin, rather because HOXA9 is a transcription maintenance factor which cannot initiate the expression from the silenced locus.

6. The role of MYC, HOXA9 and BCL2 has been extensively studied in AML including with sophisticated in vivo models utilising conditional alleles. Likewise, many studies have sought to identify essential genes downstream of MLL fusion proteins. A lot of additional data is publicly available (e.g. RNAseq in AML patients) the analysis of which could be used in many different ways to strengthen the manuscript.

Inclusion of analysis based on these data should be considered.

As advised by the reviewer, we added RNA-seq data of MLL-rearranged leukemia cell lines in Figure 5—figure supplement 2A and publicly available MILE study data in Figure 5—figure supplement 2B. MLL- rearranged leukemia cells (i.e., HB1119, MV4-11, RS4-11, ML-2, THP1, and EOL-1) expressed HOXA9 and MYC. Although MEIS1 is a well-known MLL target gene, some MLL-rearranged leukemia cell lines did not express MEIS1 (Figure 5—figure supplement 2A), consistent with the data shown in Figure 4A, B. BCL2 and SOX4 are expressed in all the MLL- rearranged leukemia cell lines tested (Figure 5—figure supplement 2B). However, their expression levels are not consistent among the cell lines. Analysis of publicly available data (i.e., the MILE study) showed most of the HOXA9 high leukemia samples expressed SOX4 and BCL2 at high levels (Figure 5—figure supplement 2B). IGF1 was expressed only in MV4-11 cells among the MLL- rearranged leukemia cell lines tested (Figure 5—figure supplement 2A). These notions support our conclusion that HOXA9 is a transcription maintenance factor, but not a transcription initiation factor. The expression of HOXA9 does not initiate the expression of all the HOXA9-target genes. It perhaps maintains the expression profile of the cell-of-origin where MLL gene rearrangement occurred. Some HOXA9-target genes such as BCL2 and SOX4 promote leukemogenesis, therefore their expression tends to be maintained at high levels. Pan et al. showed that BCL2 was highly expressed in MLL-rearranged leukemia patients and many MLL- rearranged leukemia cell lines such as MOLM-13 and THP1 were sensitive to BCL2 inhibitor (Pan et al., 2014). High SOX4 expression was also shown to be correlated to poor prognosis of AML(Lu et al., 2017). Those studies were mentioned in the discussion in the revised manuscript.

7. The authors showed that Myc expression is comparable between HOXA9-IC and MLL-ENL-IC (Figure 1C), indicating that both HOXA9 and MLL-ENL could support Myc expression as is also argued by authors. This information is inconsistent with the results that co-expression of HOXA9 and MYC induces leukemia whereas HOXA9 single expression does not (Figure 4E). The authors should explain this apparent discrepancy.

In MLL-ENL or MLL-AF10-ICs, endogenous Myc is roughly 20% more expressed at the RNA levels compared to HOXA9-ICs (Figure 1C). MYC proteins are also roughly ~20% more expressed in MLL-AF10-ICs compared to HOXA9-ICs (Figure 3A). It is unclear why such a “not-so-drastic” difference would make big differences in oncogenesis as the reviewer pointed out. As shown in (Figure 4—figure supplement 2 and Figure 5—figure supplement 1A), endogenous Myc expression is decreased if the human MYC transgene is overexpressed, suggesting that endogenous Myc expression is downregulated by the excess amount of MYC or a population with low endogenous Myc expression preferentially survived upon selection of MYC-transduced cells. This suggests that there is an upper threshold of MYC levels that a cell can persevere as excess MYC proteins make hematopoietic progenitors prone to apoptosis. As a result, the most proliferative cells would express MYC as much as possible yet not at an exceeding level that induce apoptosis. We speculate that this 20% increase of MYC levels makes a big difference in leukemogenic capacity. HOXA9 alone likely cannot maintain Myc expression at such a high level, which may be the reason why it cannot induce leukemia in vivo efficiently. Hence additional MYC expression to HOXA9-transduced cells confers leukemogenic ability in vivo. We mentioned this in the discussion of the revised manuscript.

8. Moreover, Myc silencing at ~50% expression level of control completely abrogated colony formation in MLL-ENL-IC and HOXA9-IC (Figure 1E). Is there fine-tuning system that requires critical expression level of Myc? Or did the authors obtain much more silencing effect at the protein level? Please clarify this point.

As mentioned above, we speculate that there is a fine-tuning system that regulates Myc expression. However in this case, we think this is due to the nature of the experiment. When a gene critical for proliferation such as Myc is knocked down, cells with higher knockdown efficiency will be quickly removed from the population. Consequently, the viable cells remaining after selection tend to show relatively mild knockdown like ~50%. We provided western blot data of MYC expression in cells transduced with shRNA against Myc in Figure 1—figure supplement 2 to support the knockdown at protein levels. We saw similar phenomenon in the knockdown experiment of MENIN, an essential cofactor of MLL fusions (Yokoyama and Cleary, 2008). Hence, we think that the reason why we see only ~50% knock down of MYC is because MYC-depleted cells are quickly depleted and therefore the expression profile reflects on the cells with relatively mild knockdown.

9. In relation to the above questions, given that Myc expression is upregulated by HOXA9 like by MLL-AF10, there should be common genetic pathways regulated both HOXA9 and MYC. I would request the authors to provide the gene list and pathways commonly regulated by HOXA9 and MYC in Figure 2.

To identify the pathways upregulated by HOXA9 and MYC, we performed RNA-seq analysis of cKit+ cells and compared to those of HOXA9-ICs and MYC-ICs in Figure 2—figure supplement 1B 2A, B. Genes involved in lipid metabolism appeared to be the pathways commonly regulated by HOXA9 and MYC.

10. In discussion (page 21, lines 6-8) the authors claim that MLL fusion proteins promote oncogenesis by activating both MYC- and HOXA9-related pathways. However, the cooperative effect of MYC for HOXA9 is much weaker than that of MEIS1 as is shown in Figure 4E. Given that MEIS1 is the direct target of MLL fusions, I wonder whether MYC is dispensable in the presence of MEIS1. Since there have been several studies on MEIS1 function in leukemogenesis, the authors should show common and/or distinct downstream genes/pathways between MYC- and MEIS1-driven leukemogenesis.

MYC knockdown of HOXA9/MEIS1 cells resulted in a drastic attenuation of proliferation (Figure 4—figure supplement 1B), indicating that HOXA9/MEIS1 leukemia cells critically require MYC expression. Thus, HOXA9/MEIS1-mediated leukemia uses the MYC-dependent pathway as MYC/HOXA9-meidated leukemia. Because MEIS1 is known to associate with HOXA9, it likely enhances HOXA9-meidated functions to promote leukemogenesis. RT-qPCR analysis of 3-independent clones of HOXA9/vector- and HOXA9/MEIS1-transduced cells showed that HOXA9/MEIS1-transduced cells expressed Myc nearly twice as much as HOXA9/vector-transduced cells (Figure 4—figure supplement 1A). Thus, one possible mechanism by which MEIS1 contributes to leukemic transformation is that MEIS1 increases MYC expression cooperatively with HOXA9.

However, it should be noted that MEIS1 may confer additional oncogenic ability independently of HOXA9, as the MYC/MEIS1 combination also caused leukemia in vivo (Figure 4E). Thus, MEIS1-specific functions in oncogenesis is a very interesting topic as suggested by the reviewer.

We have performed RNA-seq analysis for HOXA9-MYC- and HOXA9-MEIS1-LCs and analyzed by gene set enrichment analysis (GSEA). In HOXA9-MEIS1-expressiong cells, genes involved in Tnfα signaling or Kras pathways were uniquely enriched. The gene sets for Tnfα signaling included leukemia associated genes such as Jun and Trib1 (Yoshino et al., 2021; Zhou et al., 2017). Although this is not the focus of the present study, this comparison suggests that MEIS1-specific downstream pathways can also be the causatives of MLL-leukemia. We have added these data in Figure 4—figure supplement 4A, B and mentioned in the revised manuscript.

11. BCL2 and SOX4 knockout showed delay in MLL-AF10- and HOXA9/MEIS1-induced leukemogenesis (Figure 7A, B). Please provide the data wheter these delays are caused by increased apoptosis by presenting the Annexin V staining as well as Caspase 3 cleavage and H2AX expression.

We have analyzed apoptotic state of leukemia cells harvested from the moribund recipient mice, the data of which were described in the Figure 7—figure supplement 2B, C. While leukemia with HOXA9/MEIS1 were slightly apoptotic in the absence of Bcl2/Sox4, we did not detect substantial differences in apoptotic signals for MLL-AF10-expressing leukemic cells. These results are partly consistent with our in vivo experiments showing both Bcl2 and Sox4 KO more significantly delayed the onset of HOXA9-MEIS1-induced leukemia compared to that of MLL-AF10-induced leukemia. Nonetheless, Bcl2 and Sox4 knockout did not cause marked differences of apoptotic properties in leukemia cells compared to the wildtype controls. We reason that these full-blown leukemia cells have acquired enough anti-apoptotic functions via multiple pathways at the time of disease presentation, therefore, the differences of anti-apoptotic properties of the leukemia cells between WT and KO are less obvious.

https://doi.org/10.7554/eLife.64148.sa2

Article and author information

Author details

  1. Ryo Miyamoto

    Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    Contribution
    Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Akinori Kanai

    Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
    Contribution
    Data curation, Formal analysis, Investigation, Visualization
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1555-6768
  3. Hiroshi Okuda

    Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  4. Yosuke Komata

    Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    Contribution
    Data curation, Formal analysis, Investigation, Visualization
    Competing interests
    No competing interests declared
  5. Satoshi Takahashi

    1. Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    2. Department of Hematology and Oncology, Kyoto University Graduate School of Medicine, Kyoto, Japan
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  6. Hirotaka Matsui

    Department of Molecular Laboratory Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
    Contribution
    Resources, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  7. Toshiya Inaba

    Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
    Contribution
    Resources, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  8. Akihiko Yokoyama

    1. Tsuruoka Metabolomics Laboratory, National Cancer Center, Tsuruoka, Japan
    2. Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    ayokoyam@ncc-tmc.jp
    Competing interests
    received a research grant from Dainippon Sumitomo Pharma Co. Ltd.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5639-8068

Funding

Japan Society for the Promotion of Science (16H05337)

  • Akihiko Yokoyama

Japan Society for the Promotion of Science (19H03694)

  • Akihiko Yokoyama

Japan Society for the Promotion of Science (19K16791)

  • Ryo Miyamoto

The Yamagata Prefectural Government (Research grant)

  • Akihiko Yokoyama

The City of Tsuruoka (Research grant)

  • Akihiko Yokoyama

Dainippon Sumitomo Pharma Co., Ltd. (Research grant)

  • Akihiko Yokoyama

The Friends of Leukemia Research Fund (Research grant)

  • Akihiko Yokoyama

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Yuzo Sato, Boban Stanojevic, Makiko Okuda, Megumi Nakamura, Etsuko Kanai, Aya Nakayama, Hagumu Sato, Ikuko Yokoyama, Kanae Ito, and Ayako Yokoyama for technical assistance. We thank Drs. Yoshihide Tsujimoto and Hans Clevers for providing us the knockout mouse lines of Bcl2 and Sox4, respectively. These mouse lines were provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan. We also thank all members of the Shonai Regional Industry Promotion Center for their administrative support. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants (16H05337 and 19H03694 to AY; 19K16791 to RM) and in part by research funds from the Yamagata prefectural government, the City of Tsuruoka, Dainippon Sumitomo Pharma Co. Ltd., and the Friends of Leukemia Research Fund.

Ethics

Animal experimentation: All animal experimental protocols were approved by the National Cancer Center (Tokyo Japan) Institutional Animal Care and Use Committee.

Senior and Reviewing Editor

  1. Erica A Golemis, Fox Chase Cancer Center, United States

Publication history

  1. Received: October 19, 2020
  2. Preprint posted: October 21, 2020 (view preprint)
  3. Accepted: July 4, 2021
  4. Version of Record published: July 26, 2021 (version 1)

Copyright

© 2021, Miyamoto et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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