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Abstract Acute promyelocytic leukemia (APL) is characterised by the t(15;17)(q22;q21) leading to fusion of PML to the gene encoding the myeloid transcription factor Retinoic Acid Receptor α (RARα). Chromosomal translocations such as the t(15;17) are considered to be initiating events in leukemogenesis; however, sequencing of APL genomes has provided further evidence that the PML-RARα fusion is insufficient to induce leukemia, which depends upon the acquisition of cooperating mutations. The PML-RARα oncoprotein exerts a profound effect on nuclear architecture, disrupting multiprotein structures known as PML nuclear bodies (NBs). The function of these structures remains an enigma; however, their disruption in PML-RARα+ APL and acute lymphoblastic leukemia with the t(9;15)(p13;q24)/PAX5-PML fusion is associated with delocalisation of a number of component proteins including PML, which have been implicated in growth control and neoplastic transformation. It is now established that the PML moiety contributes to APL pathogenesis by conferring via the translocation a novel dimerisation capacity to RARα, but it has been unclear whether deregulation of PML and other NB components cooperates in leukemic transformation or impacts the response to differentiating agents. To address these questions, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues in the amino-terminal RING domain of Pml. Homozygous Pml RING mutant mice are viable, with no overt developmental defect; however, analysis of the bone marrow revealed significant expansion of the Lin(-)Sca-1(+)c-Kit(+) (LSK) population compared to wild type (WT) controls (p

Transcriptional deregulation is a key driver of acute myeloid leukemia (AML), a heterogeneous blood cancer with poor survival rates. Polycomb group (PcG) and Trithorax group (TrxG) genes, originally identified in Drosophila melanogaster several decades ago as master regulators of cellular identity and epigenetic memory, not only are important in mammalian development but also play a key role in AML disease biology. In addition to their classical canonical antagonistic transcriptional functions, noncanonical synergistic and nontranscriptional functions of PcG and TrxG are emerging. Here, we review the biochemical properties of major mammalian PcG and TrxG complexes and their roles in AML disease biology, including disease maintenance as well as drug resistance. We summarize current efforts on targeting PcG and TrxG for treatment of AML and propose rational synthetic lethality and drug-induced antagonistic pleiotropy options involving PcG and TrxG as potential new therapeutic avenues for treatment of AML. [ABSTRACT FROM AUTHOR]

An original leukemia treatment strategy: The heterogeneity of acute myeloid leukemia (AML) has problematized understanding this cancer's cell of origin and the role that this origin might play in the onset of treatment resistance. Zeisig et al. used cord blood and cells from patients with primary MLL-rearranged AML to determine that most MLL-AML cells are derived from either hematopoietic stem cells (HSCs) or common myeloid progenitor cells. They further showed that the ABCC3 inhibitor fidaxomicin together with doxorubicin extended survival in mouse models of treatment resistant MLL-AML derived from specifically HSCs. This work could have implications for the clinical treatment of AML with MLL rearrangement. Chemoresistance remains the major challenge for successful treatment of acute myeloid leukemia (AML). Although recent mouse studies suggest that treatment response of genetically and immunophenotypically indistinguishable AML can be influenced by their different cells of origin, corresponding evidence in human disease is still largely lacking. By combining prospective disease modeling using highly purified human hematopoietic stem or progenitor cells with retrospective deconvolution study of leukemia stem cells (LSCs) from primary patient samples, we identified human hematopoietic stem cells (HSCs) and common myeloid progenitors (CMPs) as two distinctive origins of human AML driven by Mixed Lineage Leukemia (MLL) gene fusions (MLL-AML). Despite LSCs from either MLL-rearranged HSCs or MLL-rearranged CMPs having a mature CD34−/lo/CD38+ immunophenotype in both a humanized mouse model and primary patient samples, the resulting AML cells exhibited contrasting responses to chemotherapy. HSC-derived MLL-AML was highly resistant to chemotherapy and expressed elevated amounts of the multispecific anion transporter ABCC3. Inhibition of ABCC3 by shRNA-mediated knockdown or with small-molecule inhibitor fidaxomicin, currently used for diarrhea associated with Clostridium difficile infection, effectively resensitized HSC-derived MLL-AML toward standard chemotherapeutic drugs. This study not only functionally established two distinctive origins of human LSCs for MLL-AML and their role in mediating chemoresistance but also identified a potential therapeutic avenue for stem cell–associated treatment resistance by repurposing a well-tolerated antidiarrhea drug already used in the clinic. [ABSTRACT FROM AUTHOR]

The INK4/ARF locus regulates senescence and is frequently altered in cancer. In normal cells, the INK4/ARF locus is found silenced by Polycomb repressive complexes (PRCs). Which are the mechanisms responsible for the recruitment of PRCs to INK4/ARF and their other target genes remains unclear. In a genetic screen for transcription factors regulating senescence, we identified the homeodomain-containing protein HLX1 (H2.0-like homeobox 1). Expression of HLX1 extends cellular lifespan and blunts oncogene-induced senescence. Using quantitative proteomics, we identified p16INK4a as the key target mediating the effects of HLX1 in senescence. HLX1 represses p16INK4a transcription by recruiting PRCs and HDAC1. This mechanism has broader implications, as HLX1 also regulates a subset of PRC targets besides p16INK4a. Finally, sampling members of the Homeobox family, we identified multiple genes with ability to repress p16INK4a. Among them, we found HOXA9 (Homeobox A9), a putative oncogene in leukaemia, which also recruits PRCs and HDAC1 to regulate p16INK4a. Our results reveal an unexpected and conserved interplay between homeodomain-containing proteins and PRCs with implications in senescence, development and cancer. [ABSTRACT FROM AUTHOR]

MLL fusion proteins are oncogenic transcription factors that are associated with aggressive lymphoid and myeloid leukemias. We constructed an inducible MLL fusion, MLL-ENL-ERtm, that rendered the transcriptional and transforming properties of MLL-ENL strictly dependent on the presence of 4-hydroxy-tamoxifen. MLL-ENL-ERtm-immortalized hematopoietic cells required 4-hydroxy-tamoxifen for continuous growth and differentiated terminally upon tamoxifen withdrawal. Microarray analysis performed on these conditionally transformed cells revealed Hoxa9 and Hoxa7 as well as the Hox coregulators Meis1 and Pbx3 among the targets upregulated by MLL-ENL-ERtm. Overexpression of the Hox repressor Bmi-1 inhibited the growth-transforming activity of MLL-ENL. Moreover, the enforced expression of Hoxa9 in combination with Meis1 was sufficient to substitute for MLL-ENL-ERtm function and to maintain a state of continuous proliferation and differentiation arrest. These results suggest that MLL fusion proteins impose a reversible block on myeloid differentiation through aberrant activation of a limited set of homeobox genes and Hox coregulators that are consistently expressed in MLL-associated leukemias. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) are critical for regulating HOX genes, aberration of which is a dominant mechanism for leukemic transformation. How HOX gene-associated lncRNAs regulate hematopoietic stem cell (HSC) function and contribute to leukemogenesis remains elusive. We found that HOTTIP is aberrantly activated in acute myeloid leukemia (AML) to alter HOXA-driven topologically associated domain (TAD) and gene expression. HOTTIP loss attenuates leukemogenesis of transplanted mice, while reactivation of HOTTIP restores leukemic TADs, transcription, and leukemogenesis in the CTCF-boundary-attenuated AML cells. Hottip aberration in mice abnormally promotes HSC self-renewal leading to AML-like disease by altering the homeotic/hematopoietic gene-associated chromatin signature and transcription program. Hottip aberration acts as an oncogenic event to perturb HSC function by reprogramming leukemic-associated chromatin and gene transcription. • HOTTIP reprograms 3D AML genome and drives leukemic transcription profile in AML • HOTTIP binds and regulates genes important for hematopoiesis and leukemogenesis • HOTTIP KO attenuates AML progression by impairing leukemic transcription program • Hottip aberration perturbs HSC self-renewal leading to AML-like disease in mice Luo et al. find that the lncRNA HOTTIP is overexpressed in acute myeloid leukemia (AML). They show that HOTTIP coordinates topologically associated domain organization in the AML genome, including the posterior HOXA genes and various key hematopoietic regulator loci, and is important for AML growth. [ABSTRACT FROM AUTHOR]

Acute myeloid leukaemia (AML) is a highly heterogenous blood cancer, in which the expansion of aberrant myeloid blood cells interferes with the generation and function of normal blood cells. Although key driver mutations and their associated inhibitors have been identified in the last decade, they have not been fully translated into better survival rates for AML patients, which remain dismal. In addition to DNA mutation, studies in mouse models strongly suggest that the cell of origin, where the driver mutation (such as MLL fusions) occurs, emerges as an additional factor that determines the treatment outcome in AML. To investigate its functional relevance in human disease, we have recently reported that AML driven by MLL fusions can transform immunophenotypically and functionally distinctive human hematopoietic stem cells (HSCs) or myeloid progenitors resulting in immunophenotypically indistinguishable human AML. Intriguingly, these cells display differential treatment sensitivities to current treatments, attesting the cell of origin as an important determinant governing treatment outcome for AML. To further facilitate this line of investigation, here we describe a comprehensive disease modelling protocol using human primary haematopoietic cells, which covers all the key steps, from the isolation of immunophenotypically defined human primary haematopoietic stem and progenitor populations, to oncogene transfer via viral transduction, the in vitro liquid culture assay, and finally the xenotransplantation into immunocompromised mice., Competing Interests: Competing interestsThe authors declare no conflict of interest., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

Non-random chromosomal translocations can be found in about half of acute leukaemia patients and mostly lead to either over-expression of proto-oncogenes or creation of novel fusion genes. To assess the oncogenic potential and characterize the underlying mechanisms mediated by these candidate oncoproteins, a retroviral transduction/transformation assay (RTTA) has been successfully employed to study the biological impacts of a number of proto-oncoproteins and novel fusion proteins in primary hematopoietic cells both in vitro and in vivo. To further widen the application of the RTTA, a lentiviral transduction/transformation assay (LTTA) has also been developed to target the most quiescent hematopoietic stem cells (HSCs). This chapter will cover both the RTTA and LTTA for studying candidate oncogenes involved in human leukaemia.

Saccharomyces cerevisiae Yaf9p and the mammalian leukemia-associated protein ENL share a high degree of similarity. To investigate the biological function of Yaf9p, this protein was used to search for interacting proteins in a two-hybrid system. Here, we demonstrate that Yaf9p binds directly to Swc4p, the yeast homolog of the mammalian DNA-methyltransferase-associated protein 1. Yaf9p and Swc4p associate through C-terminal domains, and both proteins coprecipitate in vitro in pull-down experiments and in vivo by immunoprecipitation. In living cells, Swc4p is present in a megadalton protein complex that shows a fractionation behavior in gel filtration similar to that of Esa1p, the histone acetyltransferase of the NuA4 complex. Recruitment of Yaf9p to DNA leads to promoter-specific transcriptional activation that can be inhibited by dominant negative Swc4p lacking the Yaf9p binding domain. Interference with Swc4p function also increases sensitivity to the microtubule toxin benomyl, a trait that corresponds to the known phenotype of a yaf9(-) knockout strain. In summary, the results suggest that Yaf9p and Swc4p form a protein pair that has a role in chromatin modification with possible implications also for the function of their mammalian counterparts.