Fuhong He

Identification of functional cooperative mutations of SETD2 in human acute leukemia

Acute leukemia characterized by chromosomal rearrangements requires additional molecular disruptions to develop into full-blown malignancy, yet the cooperative mechanisms remain elusive. Using whole-genome sequencing of a pair of monozygotic twins discordant for MLL (also called KMT2A) gene–rearranged leukemia, we identified a transforming MLL-NRIP3 fusion gene and biallelic mutations in SETD2 (encoding a histone H3K36 methyltransferase). Moreover, loss-of-function point mutations in SETD2 were recurrent (6.2%) in 241 patients with acute leukemia and were associated with multiple major chromosomal aberrations. We observed a global loss of H3K36 trimethylation (H3K36me3) in leukemic blasts with mutations in SETD2. In the presence of a genetic lesion, downregulation of SETD2 contributed to both initiation and progression during leukemia development by promoting the self-renewal potential of leukemia stem cells. Therefore, our study provides compelling evidence for SETD2 as a new tumor suppressor. Disruption of the SETD2-H3K36me3 pathway is a distinct epigenetic mechanism for leukemia development.

MLL fusion proteins preferentially regulate a subset of wild-type MLL target genes in the leukemic genome

MLL encodes a histone methyltransferase that is critical in maintaining gene expression during embryonic development and hematopoiesis. 11q23 translocations result in the formation of chimeric MLL fusion proteins that act as potent drivers of acute leukemia. However, it remains unclear what portion of the leukemic genome is under the direct control of MLL fusions. By comparing patient-derived leukemic cell lines, we find that MLL fusion-bound genes are a small subset of that recognized by wild-type MLL. In an inducible MLL-ENL model, MLL fusion protein binding and changes in H3K79 methylation are limited to a specific portion of the genome, whereas wild-type MLL distributes to a much larger set of gene loci. Surprisingly, among 223 MLL-ENL-bound genes, only 12 demonstrate a significant increase in mRNA expression on induction of the fusion protein. In addition to Hoxa9 and Meis1, this includes Eya1 and Six1, which comprise a heterodimeric transcription factor important in several developmental pathways. We show that Eya1 has the capacity to immortalize hematopoietic progenitor cells in vitro and collaborates with Six1 in hematopoietic transformation assays. Altogether, our data suggest that MLL fusions contribute to the development of acute leukemia through direct activation of a small set of target genes.

PU.1 is essential for MLL leukemia partially via crosstalk with the MEIS/HOX pathway

Mixed lineage leukemia (MLL) fusion proteins directly activate the expression of key downstream genes such as MEIS1, HOXA9 to drive an aggressive form of human leukemia. However, it is still poorly understood what additional transcriptional regulators, independent of the MLL fusion pathway, contribute to the development of MLL leukemia. Here we show that the transcription factor PU.1 is essential for MLL leukemia and is required for the growth of MLL leukemic cells via the promotion of cell-cycle progression and inhibition of apoptosis. Importantly, PU.1 expression is not under the control of MLL fusion proteins. We further identified a PU.1-governed 15-gene signature, which contains key regulators in the MEIS-HOX program (MEIS1, PBX3, FLT3, and c-KIT). PU.1 directly binds to the genomic loci of its target genes in vivo, and is required to maintain active expression of those genes in both normal hematopoietic stem and progenitor cells and in MLL leukemia. Finally, the clinical significance of the identified PU.1 signature was indicated by its ability to predict survival in acute myelogenous leukemia patients. Together, our findings demonstrate that PU.1 contributes to the development of MLL leukemia, partially via crosstalk with the MEIS/HOX pathway.

Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells

The ‘Yamanaka factors’ (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

Downregulation of RUNX1/CBFβ by MLL fusion proteins enhances hematopoietic stem cell self-renewal

RUNX1/CBFβ (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFβ recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFβ. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfβ levels accelerates MLL-AF9-mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFβ by MLL fusion proteins and suggest that targeting RUNX1/CBFβ levels may be a potential therapy for MLLs.

Regulation of MEIS1 by distal enhancer elements in acute leukemia.

Aberrant activation of the three-amino-acid-loop extension homeobox gene MEIS1 shortens the latency and accelerates the onset and progression of acute leukemia, yet the molecular mechanism underlying persistent activation of the MEIS1 gene in leukemia remains poorly understood. Here we used a combined comparative genomics analysis and an in vivo transgenic zebrafish assay to identify six regulatory DNA elements that are able to direct green fluorescent protein expression in a spatiotemporal manner during zebrafish embryonic hematopoiesis. Analysis of chromatin characteristics and regulatory signatures suggests that many of these predicted elements are potential enhancers in mammalian hematopoiesis. Strikingly, one of the enhancer elements (E9) is a frequent integration site in retroviral-induced mouse acute leukemia. The genomic region corresponding to enhancer E9 is differentially marked by H3K4 monomethylation and H3K27 acetylation, hallmarks of active enhancers, in multiple leukemia cell lines. Decreased enrichment of these histone marks is associated with downregulation of MEIS1 expression during hematopoietic differentiation. Further, MEIS1/HOXA9 transactivate this enhancer via a conserved binding motif in vitro, and participate in an autoregulatory loop that modulates MEIS1 expression in vivo. Our results suggest that an intronic enhancer regulates the expression of MEIS1 in hematopoiesis and contributes to its aberrant expression in acute leukemia.

Loss of Asxl1 Alters Self-Renewal and Cell Fate of Bone Marrow Stromal Cell, Leading to Bohring-Opitz-like Syndrome in Mice

Summary De novo ASXL1 mutations are found in patients with Bohring-Opitz syndrome, a disease with severe developmental defects and early childhood mortality. The underlying pathologic mechanisms remain largely unknown. Using Asxl1-targeted murine models, we found that Asxl1 global loss as well as conditional deletion in osteoblasts and their progenitors led to significant bone loss and a markedly decreased number of bone marrow stromal cells (BMSCs) compared with wild-type littermates. Asxl1−/− BMSCs displayed impaired self-renewal and skewed differentiation, away from osteoblasts and favoring adipocytes. RNA-sequencing analysis revealed altered expression of genes involved in cell proliferation, skeletal development, and morphogenesis. Furthermore, gene set enrichment analysis showed decreased expression of stem cell self-renewal gene signature, suggesting a role of Asxl1 in regulating the stemness of BMSCs. Importantly, re-introduction of Asxl1 normalized NANOG and OCT4 expression and restored the self-renewal capacity of Asxl1−/− BMSCs. Our study unveils a pivotal role of ASXL1 in the maintenance of BMSC functions and skeletal development.

Rictor/mammalian target of rapamycin 2 regulates the development of Notch1 induced murine T-cell acute lymphoblastic leukemia via forkhead box O3.

Mammalian target of rapamycin (mTOR) is composed of two distinct biochemical complexes, mTORC1 and mTORC2. In response to nutrients and growth factors, mTORC1 is known to control cellular growth by regulating the translational regulators S6 kinase 1 and 4E binding protein 1, whereas mTORC2 mediates cell proliferation and survival by activating Akt through phosphorylation at Ser473. Studies have shown that the deregulation of mTORC2 leads to the development of myeloproliferative disorder and leukemia in the phosphatase and tensin homolog deleted on chromosome ten (PTEN)-deleted mouse model. However, the mechanism by which mTORC2 specifically affects leukemogenesis is still not fully understood. Here, we investigated the role of mTORC2 in NOTCH1-driven T-cell acute lymphoblastic leukemia (T-ALL) in a Rictor-deficient mouse model. We found that, by deleting Rictor, an essential component of mTORC2, leukemia progression was significantly suppressed by arresting a greater proportion of Rictor(△/△) leukemic cells at the G0 phase of the cell cycle. Furthermore, the absence of Rictor led to the overexpression of chemotaxis-related genes, such as CCR2, CCR4 and CXCR4, which contributed to the homing and migration of Rictor-deficient T-ALL cells to the spleen but not the bone marrow. In addition, we demonstrated that inactivation of mTORC2 caused the overexpression of forkhead box O3 and its downstream effectors and eased the progression of leukemia in T-ALL mice. Our study thus indicates that forkhead box O3 could be a potential drug target for the treatment of T-ALL leukemia.

Loss of Asxl2 leads to myeloid malignancies in mice.

ASXL2 is frequently mutated in acute myeloid leukaemia patients with t(8;21). However, the roles of ASXL2 in normal haematopoiesis and the pathogenesis of myeloid malignancies remain unknown. Here we show that deletion of Asxl2 in mice leads to the development of myelodysplastic syndrome (MDS)-like disease. Asxl2−/− mice have an increased bone marrow (BM) long-term haematopoietic stem cells (HSCs) and granulocyte–macrophage progenitors compared with wild-type controls. Recipients transplanted with Asxl2−/− and Asxl2+/− BM cells have shortened lifespan due to the development of MDS-like disease or myeloid leukaemia. Paired daughter cell assays demonstrate that Asxl2 loss enhances the self-renewal of HSCs. Deletion of Asxl2 alters the expression of genes critical for HSC self-renewal, differentiation and apoptosis in Lin−cKit+ cells. The altered gene expression is associated with dysregulated H3K27ac and H3K4me1/2. Our study demonstrates that ASXL2 functions as a tumour suppressor to maintain normal HSC function.

PDGFRBmutation and tyrosine kinase inhibitor resistance in Ph-like acute lymphoblastic leukemia

Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) comprises ∼10% to 15% of childhood ALL cases, many of which respond exquisitely to tyrosine kinase inhibitors (TKIs), for example, imatinib in PDGFRB-rearranged ALL. However, some cases developed drug resistance to TKIs and the mechanisms are poorly understood. In this study, we identified a novel PDGFRB fusion gene, namely AGGF1-PDGFRB, and functionally characterized its oncogenic potential in vitro. Further genomic profiling of longitudinally collected samples during treatment revealed the emergence of a mutation, PDGFRBC843G , which directly conferred resistance to all generations of ABL TKIs, including imatinib, dasatinib, nilotinib, and ponatinib. PDGFRB-mutant leukemia cells are highly sensitive to multitarget kinase inhibitor CHZ868, suggesting potential therapeutic options for some patients resistant to ABL TKIs. In summary, we describe a complex clonal evolution pattern in Ph-like ALL and identified a novel PDGFRB point mutation that drives leukemia relapse after ABL TKI treatment.