Epigenetic alterations contribute to leukemogenesis in childhood acute myeloid leukemia and therefore are of interest for potential therapeutic strategies. Herein, we performed large-scale ribonucleic acid interference screens using small hairpin ribonucleic acids in acute myeloid leukemia cells and non-transformed bone marrow cells to identify leukemia-specific dependencies. One of the target genes displaying the strongest effects on acute myeloid leukemia cell growth and less pronounced effects on nontransformed bone marrow cells, was the chromatin remodeling factor CHD4. Using ribonucleic acid interference and CRISPR-Cas9 approaches, we showed that CHD4 was essential for cell growth of leukemic cells in vitro and in vivo. Loss of function of CHD4 in acute myeloid leukemia cells caused an arrest in the G0 phase of the cell cycle as well as downregulation of MYC and its target genes involved in cell cycle progression. Importantly, we found that inhibition of CHD4 conferred anti-leukemic effects on primary childhood acute myeloid leukemia cells and prevented disease progression in a patient-derived xenograft model. Conversely, CHD4 was not required for growth of normal hematopoietic cells. Taken together, our results identified CHD4 as a potential therapeutic target in childhood acute myeloid leukemia.
Nucleosome assembly proteins (NAPs) are histone chaperones with an important role in chromatin structure and epigenetic regulation of gene expression. We find that high gene expression levels of mouse Nap1l3 are restricted to haematopoietic stem cells (HSCs) in mice. Importantly, with shRNA or CRISPR-Cas9 mediated loss of function of mouse Nap1l3 and with overexpression of the gene, the number of colony-forming cells and myeloid progenitor cells in vitro are reduced. This manifests as a striking decrease in the number of HSCs, which reduces their reconstituting activities in vivo. Downregulation of human NAP1L3 in umbilical cord blood (UCB) HSCs impairs the maintenance and proliferation of HSCs both in vitro and in vivo. NAP1L3 downregulation in UCB HSCs causes an arrest in the G0 phase of cell cycle progression and induces gene expression signatures that significantly correlate with downregulation of gene sets involved in cell cycle regulation, including E2F and MYC target genes. Moreover, we demonstrate that HOXA3 and HOXA5 genes are markedly upregulated when NAP1L3 is suppressed in UCB HSCs. Taken together, our findings establish an important role for NAP1L3 in HSC homeostasis and haematopoietic differentiation.
Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate. The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML. In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs. The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells. To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells. Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo. Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation. Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. Disclosures No relevant conflicts of interest to declare.
Acute myeloid leukemia (AML) is an aggressive blood malignancy characterized by clonal accumulating of immature myeloid progenitors in the bone marrow and peripheral blood. Transcription factors are the most frequently mutated and dysregulated genes in AML and they have critical roles in AML pathogenesis and progression. In this study, we performed large-scale RNA interference screens in MLL-AF9 transformed AML cells and identified GTF2IRD1 as a novel transcription factor essential for the survival of various types of myeloid leukemic cells in vitro and in vivo, but not for primary normal hematopoietic cells. Inhibition of GTF2IRD1 reduced the frequency of primary childhood and adult AML cells, including cell populations enriched for leukemia-initiating cells. In animal models for AML, inhibition of GTF2IRD1 significantly delayed the disease progression. Inhibition of GTF2IRD1 caused an accumulation of quiescent AML cells in the G0 phase of the cell cycle but caused minor effects in apoptosis. In line with this, RNA sequencing analysis revealed a significant downregulation of E2F targets as a consequence of inhibition of GTF2IRD1. Taken together, we identified GTF2IRD1 as a transcription factor with a selective importance in AML and our findings may contribute to new therapeutic inventions for the disease.
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