The selective prolyl hydroxylase inhibitor IOX5 stabilizes HIF-1α and compromises development and progression of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.

Hematopoietic stem cell quiescence and DNA replication dynamics maintained by the resilient ß-catenin/Hoxa9/Prmt1 axis.

ABSTRACT: Maintenance of quiescence and DNA replication dynamics are 2 paradoxical requirements for the distinct states of dormant and active hematopoietic stem cells (HSCs), which are required to preserve the stem cell reservoir and replenish the blood cell system in response to hematopoietic stress, respectively. Here, we show that key self-renewal factors, ß-catenin or Hoxa9, largely dispensable for HSC integrity, in fact, have dual functions in maintaining quiescence and enabling efficient DNA replication fork dynamics to preserve the functionality of hematopoietic stem and progenitor cells (HSPCs). Although ß-catenin or Hoxa9 single knockout (KO) exhibited mostly normal hematopoiesis, their coinactivation led to severe hematopoietic defects stemmed from aberrant cell cycle, DNA replication, and damage in HSPCs. Mechanistically, ß-catenin and Hoxa9 function in a compensatory manner to sustain key transcriptional programs that converge on the pivotal downstream target and epigenetic modifying enzyme, Prmt1, which protects the quiescent state and ensures an adequate supply of DNA replication and repair factors to maintain robust replication fork dynamics. Inactivation of Prmt1 phenocopied both cellular and molecular phenotypes of ß-catenin/Hoxa9 combined KO, which at the same time could also be partially rescued by Prmt1 expression. The discovery of the highly resilient ß-catenin/Hoxa9/Prmt1 axis in protecting both quiescence and DNA replication dynamics essential for HSCs at different key states provides not only novel mechanistic insights into their intricate regulation but also a potential tractable target for therapeutic intervention.

Inhibition of PLK4 remodels histone methylation and activates the immune response via the cGAS-STING pathway in TP53-mutated AML.

Acute myeloid leukemia (AML) with TP53 mutation is one of the most lethal cancers and portends an extremely poor prognosis. Based on in silico analyses of druggable genes and differential gene expression in TP53-mutated AML, we identified pololike kinase 4 (PLK4) as a novel therapeutic target and examined its expression, regulation, pathogenetic mechanisms, and therapeutic potential in TP53-mutated AML. PLK4 expression was suppressed by activated p53 signaling in TP53 wild-type AML and was increased in TP53-mutated AML cell lines and primary samples. Short-term PLK4 inhibition induced DNA damage and apoptosis in TP53 wild-type AML. Prolonged PLK4 inhibition suppressed the growth of TP53-mutated AML and was associated with DNA damage, apoptosis, senescence, polyploidy, and defective cytokinesis. A hitherto undescribed PLK4/PRMT5/EZH2/H3K27me3 axis was demonstrated in both TP53 wild-type and mutated AML, resulting in histone modification through PLK4-induced PRMT5 phosphorylation. In TP53-mutated AML, combined effects of histone modification and polyploidy activated the cGAS-STING pathway, leading to secretion of cytokines and chemokines and activation of macrophages and T cells upon coculture with AML cells. In vivo, PLK4 inhibition also induced cytokine and chemokine expression in mouse recipients, and its combination with anti-CD47 antibody, which inhibited the "don't-eat-me" signal in macrophages, synergistically reduced leukemic burden and prolonged animal survival. The study shed important light on the pathogenetic role of PLK4 and might lead to novel therapeutic strategies in TP53-mutated AML.

A novel network pharmacology approach for leukaemia differentiation therapy using Mogrify®.

Acute myeloid leukaemia (AML) is a rapidly fatal blood cancer that is characterised by the accumulation of immature myeloid cells in the blood and bone marrow as a result of blocked differentiation. Methods which identify master transcriptional regulators of AML subtype-specific leukaemia cell states and their combinations could be critical for discovering novel differentiation-inducing therapies. In this proof-of-concept study, we demonstrate a novel utility of the Mogrify® algorithm in identifying combinations of transcription factors (TFs) and drugs, which recapitulate granulocytic differentiation of the NB4 acute promyelocytic leukaemia (APL) cell line, using two different approaches. In the first approach, Connectivity Map (CMAP) analysis of these TFs and their target networks outperformed standard approaches, retrieving ATRA as the top hit. We identify dimaprit and mebendazole as a drug combination which induces myeloid differentiation. In the second approach, we show that genetic manipulation of specific Mogrify®-identified TFs (MYC and IRF1) leads to co-operative induction of APL differentiation, as does pharmacological targeting of these TFs using currently available compounds. We also show that loss of IRF1 blunts ATRA-mediated differentiation, and that MYC represses IRF1 expression through recruitment of PML-RARα, the driver fusion oncoprotein in APL, to the IRF1 promoter. Finally, we demonstrate that these drug combinations can also induce differentiation of primary patient-derived APL cells, and highlight the potential of targeting MYC and IRF1 in high-risk APL. Thus, these results suggest that Mogrify® could be used for drug discovery or repositioning in leukaemia differentiation therapy for other subtypes of leukaemia or cancers.

HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia.

HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of ß-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced ß-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

KDM5B protein expressed in viable and fertile ΔARID mice exhibit no demethylase activity.

Post­translational modification of histones serve a crucial role in the control of gene transcription. Trimethylation of lysine 4 on histone 3 is associated with transcription activation. There are currently six known methylases and six known demethylases that can control the methylation status of this site. Lysine demethylase 5B (KDM5B) is one such demethylase, which can repress gene expression. In particular KDM5B has been found to be overexpressed in a number of cancer types, and small­molecular weight inhibitors of its demethylase activity have been identified. Previous characterisation of Kdm5b knock­out mice has revealed that this genotype leads to either embryonic or neonatal lethality. However, the ΔA­T rich interaction domain (ΔARID)­KDM5B strain of mice, which have the ARID domain and five amino acids within the Jumonji (Jmj)N domain spliced out from KDM5B, remain viable and fertile. In the present study, ΔARID­KDM5B was found to have no demethylase activity as determined by in vitro demethylase assays and by immunofluorescence in transfected Cos­1 cells. Furthermore, molecular dynamic simulations revealed conformational changes within the ΔARID­KDM5B structure compared with that in WT­KDM5B, particularly in the JmjC domain, which is responsible for the catalytic activity of WT­KDM5B. This supports the experimental data that shows the loss of demethylase activity. Since Kdm5b knock­out mice show varying degrees of lethality, these data suggest that KDM5B serves a crucial function in development in a manner that is independent of its demethylase activity.

Therapeutic Opportunities of Targeting Canonical and Noncanonical PcG/TrxG Functions in Acute Myeloid Leukemia.

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.

Functional reconstruction of human AML reveals stem cell origin and vulnerability of treatment-resistant MLL-rearranged leukemia.

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.

Reconstruction of Human AML Using Functionally and Immunophenotypically Defined Human Haematopoietic Stem and Progenitor Cells as Targeted Populations.

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.

Novel therapeutic strategies for MLL-rearranged leukemias.

MLL rearrangement is one of the key drivers and generally regarded as an independent poor prognostic marker in acute leukemias. The standard of care for MLL-rearranged (MLL-r) leukemias has remained largely unchanged for the past 50 years despite unsatisfying clinical outcomes, so there is an urgent need for novel therapeutic strategies. An increasing body of evidence demonstrates that a vast number of epigenetic regulators are directly or indirectly involved in MLL-r leukemia, and they are responsible for supporting the aberrant gene expression program mediated by MLL-fusions. Unlike genetic mutations, epigenetic modifications can be reversed by pharmacologic targeting of the responsible epigenetic regulators. This leads to significant interest in developing epigenetic therapies for MLL-r leukemia. Intriguingly, many of the epigenetic enzymes also involve in DNA damage response (DDR), which can be potential targets for synthetic lethality-induced therapies. In this review, we will summarize some of the recent advances in the development of epigenetic and DDR therapeutics by targeting epigenetic regulators or protein complexes that mediate MLL-r leukemia gene expression program and key players in DDR that safeguard essential genome integrity. The rationale and molecular mechanisms underpinning the therapeutic effects will also be discussed with a focus on how these treatments can disrupt MLL-fusion mediated transcriptional programs and impair DDR, which may help overcome treatment resistance.

Discovery of Reversible Inhibitors of KDM1A Efficacious in Acute Myeloid Leukemia Models.

Lysine-specific demethylase 1 (LSD1 or KDM1A) is a FAD-dependent enzyme that acts as a transcription corepressor or coactivator by regulating the methylation status of histone H3 lysines K4 and K9, respectively. KDM1A represents an attractive target for cancer therapy. While, in the past, the main medicinal chemistry strategy toward KDM1A inhibition was based on the optimization of ligands that irreversibly bind the FAD cofactor within the enzyme catalytic site, we and others have also identified reversible inhibitors. Herein we reported the discovery of 5-imidazolylthieno[3,2-b]pyrroles, a new series of KDM1A inhibitors endowed with picomolar inhibitory potency, active in cells and efficacious after oral administration in murine leukemia models.

Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia.

Internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3/ITD) occurs in about 30% of acute myeloid leukemia (AML) and is associated with poor response to conventional treatment and adverse outcome. Here, we reported that human FLT3/ITD expression led to axis duplication and dorsalization in about 50% of zebrafish embryos. The morphologic phenotype was accompanied by ectopic expression of a morphogen follistatin (fst) during early embryonic development. Increase in fst expression also occurred in adult FLT3/ITD-transgenic zebrafish, Flt3/ITD knock-in mice, and human FLT3/ITD AML cells. Overexpression of human FST317 and FST344 isoforms enhanced clonogenicity and leukemia engraftment in xenotransplantation model via RET, IL2RA, and CCL5 upregulation. Specific targeting of FST by shRNA, CRISPR/Cas9, or antisense oligo inhibited leukemic growth in vitro and in vivo. Importantly, serum FST positively correlated with leukemia engraftment in FLT3/ITD AML patient-derived xenograft mice and leukemia blast percentage in primary AML patients. In FLT3/ITD AML patients treated with FLT3 inhibitor quizartinib, serum FST levels correlated with clinical response. These observations supported FST as a novel therapeutic target and biomarker in FLT3/ITD AML.

HOTTIP lncRNA Promotes Hematopoietic Stem Cell Self-Renewal Leading to AML-like Disease in Mice.

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.

Transcriptional memory of cells of origin overrides ß-catenin requirement of MLL cancer stem cells.

While ß-catenin has been demonstrated as an essential molecule and therapeutic target for various cancer stem cells (CSCs) including those driven by MLL fusions, here we show that transcriptional memory from cells of origin predicts AML patient survival and allows ß-catenin-independent transformation in MLL-CSCs derived from hematopoietic stem cell (HSC)-enriched LSK population but not myeloid-granulocyte progenitors. Mechanistically, ß-catenin regulates expression of downstream targets of a key transcriptional memory gene, Hoxa9 that is highly enriched in LSK-derived MLL-CSCs and helps sustain leukemic self-renewal. Suppression of Hoxa9 sensitizes LSK-derived MLL-CSCs to ß-catenin inhibition resulting in abolishment of CSC transcriptional program and transformation ability. In addition, further molecular and functional analyses identified Prmt1 as a key common downstream mediator for ß-catenin/Hoxa9 functions in LSK-derived MLL-CSCs. Together, these findings not only uncover an unexpectedly important role of cells of origin transcriptional memory in regulating CSC self-renewal, but also reveal a novel molecular network mediated by ß-catenin/Hoxa9/Prmt1 in governing leukemic self-renewal.

PARPi potentiates with current conventional therapy in MLL leukemia.

Acute myeloid leukemias driven by MLL fusion proteins are commonly associated with poor prognosis and refractory treatment. Here, we provide evidence that olaparib can potentiate sensitivity of MLL leukemia cells to conventional chemotherapy and DNMT inhibitors offering new potential therapeutic strategies for MLL rearranged leukemias Using the primary mouse leukemia cells and human MLL-AF9 leukemic cell line, we demonstrate that treatment of MLL-AF9 leukemic cells with DNMT inhibitors or chemotherapies in combination with olaparib results in significant reduction in colony formation or cell growth while the single agent treatments had little impacts. Combining olaparib with DNMT inhibitor induce cell cycle block and apoptosis. Furthermore, we observe a significant increase in proportion of cells with >10 γH2AX DNA damage foci and double stranded breaks when treated with DNMT inhibitors or chemotherapy agents in combination with olaparib, thus providing possible mechanistic insights for the synergism.

Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions.

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1/Hoxa9-driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation.

Homoharringtonine (omacetaxine mepesuccinate) as an adjunct for FLT3-ITD acute myeloid leukemia.

An in vitro drug-screening platform on patient samples was developed and validated to design personalized treatment for relapsed/refractory acute myeloid leukemia (AML). Unbiased clustering and correlation showed that homoharringtonine (HHT), also known as omacetaxine mepesuccinate, exhibited preferential antileukemia effect against AML carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD). It worked synergistically with FLT3 inhibitors to suppress leukemia growth in vitro and in xenograft mouse models. Mechanistically, the effect was mediated by protein synthesis inhibition and reduction of short-lived proteins, including total and phosphorylated forms of FLT3 and its downstream signaling proteins. A phase 2 clinical trial of sorafenib and HHT combination treatment in FLT3-ITD AML patients resulted in complete remission (true or with insufficient hematological recovery) in 20 of 24 patients (83.3%), reduction of ITD allelic burden, and median leukemia-free and overall survivals of 12 and 33 weeks. The regimen has successfully bridged five patients to allogeneic hematopoietic stem cell transplantation and was well tolerated in patients unfit for conventional chemotherapy, including elderly and heavily pretreated patients. This study validated the principle and clinical relevance of in vitro drug testing and identified an improved treatment for FLT3-ITD AML. The results provided the foundation for phase 2/3 clinical trials to ascertain the clinical efficacy of FLT3 inhibitors and HHT in combination.