SUV39H1 regulates the progression of MLL-AF9-induced acute myeloid leukemia.
ABSTRACT: Epigenetic regulations play crucial roles in leukemogenesis and leukemia progression. SUV39H1 is the dominant H3K9 methyltransferase in the hematopoietic system, and its expression declines with aging. However, the role of SUV39H1 via its-mediated repressive modification H3K9me3 in leukemogenesis/leukemia progression remains to be explored. We found that SUV39H1 was down-regulated in a variety of leukemias, including MLL-r AML, as compared with normal individuals. Decreased levels of Suv39h1 expression and genomic H3K9me3 occupancy were observed in LSCs from MLL-r-induced AML mouse models in comparison with that of hematopoietic stem/progenitor cells. Suv39h1 overexpression increased leukemia latency and decreased the frequency of LSCs in MLL-r AML mouse models, while Suv39h1 knockdown accelerated disease progression with increased number of LSCs. Increased Suv39h1 expression led to the inactivation of Hoxb13 and Six1, as well as reversion of Hoxa9/Meis1 downstream target genes, which in turn decelerated leukemia progression. Interestingly, Hoxb13 expression is up-regulated in MLL-AF9-induced AML cells, while knockdown of Hoxb13 in MLL-AF9 leukemic cells significantly prolonged the survival of leukemic mice with reduced LSC frequencies. Our data revealed that SUV39H1 functions as a tumor suppressor in MLL-AF9-induced AML progression. These findings provide the direct link of SUV39H1 to AML development and progression.
Project description:Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1- cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.
Project description:Molecular genetic changes in acute myeloid leukemia (AML) play crucial roles in leukemogenesis, including recurrent chromosome translocations, epigenetic/spliceosome mutations and transcription factor aberrations. Six1, a transcription factor of the Sine oculis homeobox (Six) family, has been shown to transform normal hematopoietic progenitors into leukemia in cooperation with Eya. However, the specific role and the underlying mechanism of Six1 in leukemia maintenance remain unexplored. Here, we showed increased expression of SIX1 in AML patients and murine leukemia stem cells (c-Kit+ cells, LSCs). Importantly, we also observed that a higher level of Six1 in human patients predicts a worse prognosis. Notably, knockdown of Six1 significantly prolonged the survival of MLL-AF9-induced AML mice with reduced peripheral infiltration and tumor burden. AML cells from Six1-knockdown (KD) mice displayed a significantly decreased number and function of LSC, as assessed by the immunophenotype, colony-forming ability and limiting dilution assay. Further analysis revealed the augmented apoptosis of LSC and decreased expression of glycolytic genes in Six1 KD mice. Overall, our data showed that Six1 is essential for the progression of MLL-AF9-induced AML via maintaining the pool of LSC.
Project description:FLT3 is one of the most frequently mutated genes in acute leukemias. However, the role in leukemogenesis of wild-type (wt) FLT3, which is highly expressed in many hematologic malignancies, is unclear. We show here that in mouse models established by retroviral transduction of leukemic fusion proteins, deletion of Flt3 strongly inhibits MLL-ENL and to lesser extent p210(BCR-ABL)-induced leukemogenesis, but has no effect in MLL-AF9 or AML1-ETO9a models. Flt3 acts at the level of leukemic stem cells (LSCs), as a fraction of LSCs in MLL-ENL, but not in MLL-AF9-induced leukemia, expressed Flt3 in vivo, and Flt3 expression on LSCs was associated with leukemia development in this model. Furthermore, efficiency of MLL-ENL, but not of MLL-AF9-induced leukemia induction was significantly enhanced after transduction of Flt3(+) compared to Flt3(-) wt myeloid progenitors. However, Flt3 is not required for immortalization of bone marrow cells in vitro by MLL-ENL and does not affect colony formation by MLL-ENL LSCs in vitro, suggesting that in vitro models do not reflect the in vivo biology of MLL-ENL leukemia with respect to Flt3 requirement. We conclude that wt Flt3 plays a role in leukemia initiation in vivo, which is, however, not universal.
Project description:Hematopoietic stem cells (HSCs) engage in complex bidirectional signals with the hematopoietic microenvironment (HM), and there is emerging evidence that leukemia stem cells (LSCs) may use similar interactions. Using a syngeneic retroviral model of MLL-AF9 induced acute myeloid leukemia (AML), we have identified 2 different stages of leukemia progression, propagated by "pre-LSCs" and established leukemia (LSCs) and compared the homing properties of these distinctive entities to that of normal HSCs. The homing and microlocalization of pre-LSCs was most similar to long-term HSCs and was dependent on cell-intrinsic Wnt signaling. In contrast, the homing of established LSCs was most similar to that of committed myeloid progenitors and distinct from HSCs. Although osteoblast-derived Dickkopf-1, a potent Wnt inhibitor known to impair HSC function, dramatically impaired normal HSC localization within the bone marrow, it did not affect pre-LSCs, LSC homing, or AML development. Mechanistically, cell-intrinsic Wnt activation was observed in human and murine AML samples, explaining the independence of MLL-AF9 LSCs from niche-derived Wnt signals. These data identify differential engagement of HM associated with leukemic progression and identify an LSC niche that is physically distinct and independent of the constraints of Wnt signaling that apply to normal HSCs.
Project description:BACKGROUND:Overexpression of Wilms' tumor-1 (WT1) transcription factor facilitates proliferation in acute myeloid leukemia (AML). However, whether WT1 is enriched in the leukemia-initiating cells (LICs) and leukemia stem cells (LSCs) and facilitates the self-renewal of LSCs remains poorly understood. METHODS:MLL-AF9-induced murine leukemia model was used to evaluate the effect of knockdown of wt1 on the self-renewal ability of LSC. RNA sequencing was performed on WT1-overexpressing cells to select WT1 targets. Apoptosis and colony formation assays were used to assess the anti-leukemic potential of a deubiquitinase inhibitor WP1130. Furthermore, NOD/SCID-IL2R? (NSG) AML xenotransplantation and MLL-AF9-induced murine leukemia models were used to evaluate the anti-leukemogenic potential of WP1130 in vivo. RESULTS:We found that wt1 is highly expressed in LICs and LSCs and facilitates the maintenance of leukemia in a murine MLL-AF9-induced model of AML. WT1 enhanced the self-renewal of LSC by increasing the expression of BCL2L2, a member of B cell lymphoma 2 (BCL2) family, by direct binding to its promoter region. Loss of WT1 impaired self-renewal ability in LSC and delayed the progression of leukemia. WP1130 was found to modify the WT1-BCL2L2 axis, and WP1130-induced anti-leukemic activity was mediated by ubiquitin proteasome-mediated destruction of WT1 protein. WP1130 induced apoptosis and decreased colony formation abilities of leukemia cells and prolonged the overall survival in the THP1-based xenograft NSG mouse model. WP1130 also decreased the frequency of LSC and prolonged the overall survival in MLL-AF9-induced murine leukemia model. Mechanistically, WP1130 induced the degradation of WT1 by positively affecting the ubiquitination of WT1 protein. CONCLUSIONS:Our results indicate that WT1 is required for the development of AML. WP1130 exhibits anti-leukemic activity by inhibiting the WT1-BCL2L2 axis, which may represent a new acute myeloid leukemia therapy target.
Project description:Patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML), and while in mice the expression of this fusion oncogene also results in the development of myeloid leukemia, it is with long latency. To identify mutations that cooperate with Mll-AF9, we infected neonatal wild-type (WT) or Mll-AF9 mice with a murine leukemia virus (MuLV). MuLV-infected Mll-AF9 mice succumbed to disease significantly faster than controls presenting predominantly with myeloid leukemia while infected WT animals developed predominantly lymphoid leukemia. We identified 88 candidate cancer genes near common sites of proviral insertion. Analysis of transcript levels revealed significantly elevated expression of Mn1, and a trend toward increased expression of Bcl11a and Fosb in Mll-AF9 murine leukemia samples with proviral insertions proximal to these genes. Accordingly, FOSB and BCL11A were also overexpressed in human AML harboring MLL gene translocations. FOSB was revealed to be essential for growth in mouse and human myeloid leukemia cells using shRNA lentiviral vectors in vitro. Importantly, MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo BM viral transduction and transplantation assay. Together, our data identified genes that define transcription factor networks and important genetic pathways acting during progression of leukemia induced by MLL fusion oncogenes.
Project description:BACKGROUND:Chromosomal translocation-induced expression of the chromatin modifying oncofusion protein MLL-AF9 promotes acute myelocytic leukemia (AML). Whereas WNT/?-catenin signaling has previously been shown to support MLL-AF9-driven leukemogenesis, the mechanism underlying this relationship remains unclear. METHODS:We used two novel small molecules targeting WNT signaling as well as a genetically modified mouse model that allow targeted deletion of the WNT protein chaperone Wntless (WLS) to evaluate the role of WNT signaling in AML progression. ATAC-seq and transcriptome profiling were deployed to understand the cellular consequences of disrupting a WNT signaling in leukemic initiating cells (LICs). FINDINGS:We identified Six1 to be a WNT-controlled target gene in MLL-AF9-transformed leukemic initiating cells (LICs). MLL-AF9 alters the accessibility of Six1 DNA to the transcriptional effector TCF7L2, a transducer of WNT/?-catenin gene expression changes. Disruption of WNT/SIX1 signaling using inhibitors of the Wnt signaling delays the development of AML. INTERPRETATION:By rendering TCF/LEF-binding elements controlling Six1 accessible to TCF7L2, MLL-AF9 promotes WNT/?-catenin-dependent growth of LICs. Small molecules disrupting WNT/?-catenin signaling block Six1 expression thereby disrupting leukemia driven by MLL fusion proteins.
Project description:Leukemia stem cells (LSCs) are thought to share several properties with hematopoietic stem cells (HSCs), including cell-cycle quiescence and a capacity for self-renewal. These features are hypothesized to underlie leukemic initiation, progression, and relapse, and they also complicate efforts to eradicate leukemia through therapeutic targeting of LSCs without adverse effects on HSCs. Here, we show that acute myeloid leukemias (AMLs) with genomic rearrangements of the MLL gene contain a non-quiescent LSC population. Although human CD34(+)CD38(-) LSCs are generally highly quiescent, the C-type lectin CD93 is expressed on a subset of actively cycling, non-quiescent AML cells enriched for LSC activity. CD93 expression is functionally required for engraftment of primary human AML LSCs and leukemogenesis, and it regulates LSC self-renewal predominantly by silencing CDKN2B, a major tumor suppressor in AML. Thus, CD93 expression identifies a predominantly cycling, non-quiescent leukemia-initiating cell population in MLL-rearranged AML, providing opportunities for selective targeting and eradication of LSCs.
Project description:Leukemias exhibit a dysregulated developmental program mediated through both genetic and epigenetic mechanisms. Although IKZF2 is expressed in hematopoietic stem cells (HSCs), we found that it is dispensable for mouse and human HSC function. In contrast to its role as a tumor suppressor in hypodiploid B-acute lymphoblastic leukemia, we found that IKZF2 is required for myeloid leukemia. IKZF2 is highly expressed in leukemic stem cells (LSCs), and its deficiency results in defective LSC function. IKZF2 depletion in acute myeloid leukemia (AML) cells reduced colony formation, increased differentiation and apoptosis, and delayed leukemogenesis. Gene expression, chromatin accessibility, and direct IKZF2 binding in MLL-AF9 LSCs demonstrate that IKZF2 regulates a HOXA9 self-renewal gene expression program and inhibits a C/EBP-driven differentiation program. Ectopic HOXA9 expression and CEBPE depletion rescued the effects of IKZF2 depletion. Thus, our study shows that IKZF2 regulates the AML LSC program and provides a rationale to therapeutically target IKZF2 in myeloid leukemia.
Project description:FOXM1, a known transcription factor, promotes cell proliferation in a variety of cancer cells. Here we show that Foxm1 is required for survival, quiescence and self-renewal of MLL-AF9 (MA9)-transformed leukemia stem cells (LSCs) in vivo. Mechanistically, Foxm1 upregulation activates the Wnt/?-catenin signaling pathways by directly binding to ?-catenin and stabilizing ?-catenin protein through inhibiting its degradation, thereby preserving LSC quiescence, and promoting LSC self-renewal in MLL-rearranged AML. More importantly, inhibition of FOXM1 markedly suppresses leukemogenic potential and induces apoptosis of primary LSCs from MLL-rearranged AML patients in vitro and in vivo in xenograft mice. Thus, our study shows a critical role and mechanisms of Foxm1 in MA9-LSCs, and indicates that FOXM1 is a potential therapeutic target for selectively eliminating LSCs in MLL-rearranged AML.