The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes.

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.

FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors.

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs.

Critical role of Lama4 for hematopoiesis regeneration and acute myeloid leukemia progression.

Impairment of normal hematopoiesis and leukemia progression are 2 well-linked processes during leukemia development and are controlled by the bone marrow (BM) niche. Extracellular matrix proteins, including laminin, are important BM niche components. However, their role in hematopoiesis regeneration and leukemia is unknown. Laminin α4 (Lama4), a major receptor-binding chain of several laminins, is altered in BM niches in mice with acute myeloid leukemia (AML). So far, the impact of Lama4 on leukemia progression remains unknown. We here report that Lama4 deletion in mice resulted in impaired hematopoiesis regeneration following irradiation-induced stress, which is accompanied by altered BM niche composition and inflammation. Importantly, in a transplantation-induced MLL-AF9 AML mouse model, we demonstrate accelerated AML progression and relapse in Lama4-/- mice. Upon AML exposure, Lama4-/- mesenchymal stem cells (MSCs) exhibited dramatic molecular alterations, including upregulation of inflammatory cytokines that favor AML growth. Lama4-/- MSCs displayed increased antioxidant activities and promoted AML stem cell proliferation and chemoresistance to cytarabine, which was accompanied by increased mitochondrial transfer from the MSCs to AML cells and reduced reactive oxygen species in AML cells in vitro. Similarly, we detected lower levels of reactive oxygen species in AML cells from Lama4-/- mice post-cytarabine treatment. Notably, LAMA4 inhibition or knockdown in human MSCs promoted human AML cell proliferation and chemoprotection. Together, our study for the first time demonstrates the critical role of Lama4 in impeding AML progression and chemoresistance. Targeting Lama4 signaling pathways may offer potential new therapeutic options for AML.

MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.

HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection During Cancer Treatment.

Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition. IMPLICATIONS: Our finding that NSCs can be selectively rescued while cancer cells remain sensitive to the treatment, provide a foundation for reduction of cognitive impairment in children with neurologic cancers.

Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing cytarabine efficacy.

The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.

The histone chaperone NAP1L3 is required for haematopoietic stem cell maintenance and differentiation.

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.

Distinct roles of mesenchymal stem and progenitor cells during the development of acute myeloid leukemia in mice.

Despite increasing evidence for the involvement of bone marrow (BM) hematopoietic stem cell niche in leukemogenesis, how BM mesenchymal stem and progenitor cells (MSPCs) contribute to leukemia niche formation and progression remains unclear. Using an MLL-AF9 acute myeloid leukemia (AML) mouse model, we demonstrate dynamic alterations of BM cellular niche components, including MSPCs and endothelial cells during AML development and its association with AML engraftment. Primary patient AML cells also induced similar niche alterations in xenografted mice. AML cell infiltration in BM causes an expansion of early B-cell factor 2+ (Ebf2+) MSPCs with reduced Cxcl12 expression and enhanced generation of more differentiated mesenchymal progenitor cells. Importantly, in vivo fate-mapping indicates that Ebf2+ MSPCs participated in AML niche formation. Ebf2+ cell deletion accelerated the AML development. These data suggest that native BM MSPCs may suppress AML. However, they can be remodeled by AML cells to form leukemic niche that might contribute to AML progression. AML induced dysregulation of hematopoietic niche factors like Angptl1, Cxcl12, Kitl, Il6, Nov, and Spp1 in AML BM MSPCs, which was associated with AML engraftment and partially appeared before the massive expansion of AML cells, indicating the possible involvement of the niche factors in AML progression. Our study demonstrates distinct dynamic features and roles of BM MSPCs during AML development.

Sipa1 deficiency-induced bone marrow niche alterations lead to the initiation of myeloproliferative neoplasm.

Mutations of signal-induced proliferation-associated gene 1 (SIPA1), a RAP1 GTPase-activating protein, were reported in patients with juvenile myelomonocytic leukemia, a childhood myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Sipa1 deficiency in mice leads to the development of age-dependent MPN. However, Sipa1 expression in bone marrow (BM) microenvironment and its effect on the pathogenesis of MPN remain unclear. We here report that Sipa1 is expressed in human and mouse BM stromal cells and downregulated in these cells from patients with MPN or MDS/MPN at diagnosis. By using the Sipa1-/- MPN mouse model, we find that Sipa1 deletion causes phenotypic and functional alterations of BM mesenchymal stem and progenitor cells prior to the initiation of the MPN. Importantly, the altered Sipa1-/- BM niche is required for the development of MDS/MPN following transplantation of normal hematopoietic cells. RNA sequencing reveals an enhanced inflammatory cytokine signaling and dysregulated Dicer1, Kitl, Angptl1, Cxcl12, and Thpo in the Sipa1-/- BM cellular niches. Our data suggest that Sipa1 expression in the BM niche is critical for maintaining BM niche homeostasis. Moreover, Sipa1 loss-induced BM niche alterations likely enable evolution of clonal hematopoiesis to the hematological malignancies. Therefore, restoring Sipa1 expression or modulating the altered signaling pathways involved might offer therapeutic potential for MPN.

The chromatin-remodeling factor CHD4 is required for maintenance of childhood acute myeloid leukemia.

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.

Selenite and methylseleninic acid epigenetically affects distinct gene sets in myeloid leukemia: A genome wide epigenetic analysis.

Selenium compounds have emerged as promising chemotherapeutic agents with proposed epigenetic effects, however the mechanisms and downstream effects are yet to be studied. Here we assessed the effects of the inorganic selenium compound selenite and the organic form methylseleninic acid (MSA) in a leukemic cell line K562, on active (histone H3 lysine 9 acetylation, H3K9ac and histone H3 lysine 4 tri-methylation, H3K4me3) and repressive (histone H3 lysine 9 tri-methylation, H3K9me3) histone marks by Chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq). Both selenite and MSA had major effects on histone marks but the effects of MSA were more pronounced. Gene ontology analysis revealed that selenite affected genes involved in response to oxygen and hypoxia, whereas MSA affected distinct gene sets associated with cell adhesion and glucocorticoid receptors, also apparent by global gene expression analysis using RNA sequencing. The correlation to adhesion was functionally confirmed by a significantly weakened ability of MSA treated cells to attach to fibronectin and linked to decreased expression of integrin beta 1. A striking loss of cellular adhesion was also confirmed in primary patient AML cells. Recent strategies to enhance the cytotoxicity of chemotherapeutic drugs by disrupting the interaction between leukemic and stromal cells in the bone marrow are of increasing interest; and organic selenium compounds like MSA might be promising candidates. In conclusion, these results provide new insight on the mechanism of action of selenium compounds, and will be of value for the understanding, usage, and development of new selenium compounds as anticancer agents.

Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies.

The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.

Understanding cytoskeleton regulators in glioblastoma multiforme for therapy design.

The cellular cytoskeleton forms the primary basis through which a cell governs the changes in size, shape, migration, proliferation, and forms the primary means through which the cells respond to their environment. Indeed, cell and tissue morphologies are used routinely not only to grade tumors but also in various high-content screening methods with an aim to identify new small molecules with therapeutic potential. This study examines the expression of various cytoskeleton regulators in glioblastoma multiforme (GBM). GBM is a very aggressive disease with a low life expectancy even after chemo- and radiotherapy. Cancer cells of GBM are notorious for their invasiveness, ability to develop resistance to chemo- and radiotherapy, and to form secondary site tumors. This study aims to gain insight into cytoskeleton regulators in GBM cells and to understand the effect of various oncology drugs, including temozolomide, on cytoskeleton regulators. We compare the expression of various cytoskeleton regulators in GBM-derived tumor and normal tissue, CD133-postive and -negative cells from GBM and neural cells, and GBM stem-like and differentiated cells. In addition, the correlation between the expression of cytoskeleton regulators with the clinical outcome was examined to identify genes associated with longer patient survival. This was followed by a small molecule screening with US Food and Drug Administration (FDA)-approved oncology drugs, and its effect on cellular cytoskeleton was compared to treatment with temozolomide. This study identifies various groups of cytoskeletal regulators that have an important effect on patient survival and tumor development. Importantly, this work highlights the advantage of using cytoskeleton regulators as biomarkers for assessing prognosis and treatment design for GBM.

Xeno-immunosuppressive properties of human decidual stromal cells in mouse models of alloreactivity in vitro and in vivo.

BACKGROUND AIMS: Human decidual stromal cells (hDSCs) may cure acute graft-versus-host disease (GVHD) in humans. We evaluated immunosuppression by xenogenic hDSCs in mice, both in vitro and in vivo. METHODS: hDSCs inhibited mouse lymphocyte proliferation in allo- and xeno-stimulation assays in mixed lymphocyte culture (MLC) and after mitogenic stimulation. The immunosuppressive effect of hDSCs was dose-dependent and strain-independent. Trans-well experiments showed that hDSCs needed cell-to-cell contact to be immunosuppressive. In a GVHD model, Balb/c mice were transplanted with bone marrow and splenocytes from C57BL/6 a donor. Varying doses of hDSCs (10(5)-10(6) per mouse) were infused at different time points. Recipient mice showed lower GVHD scores than untreated mice, but they did not have consistently improved survival. Histopathological investigation of liver, gastrointestinal tract and skin of animals with GVHD did not show any significant improvement from hDSC infusion. RESULTS: hDSCs were transduced with immunosuppressive genes including those encoding interleukin-10, prostaglandin-E2 receptor, indoleamine dioxygenase, interferon-γ and programmed death ligand-1. Transduced and untransduced hDSCs showed similar effects in vitro and in vivo. At a dose of 10(6)hDSCs per mouse, the majority of recipients died of embolism. CONCLUSIONS: hDSCs inhibit allo-reactivity, xeno-reactivity and mitogen-induced stimulation in mouse lymphocytes. Although the GVHD score was reduced by hDSC infusion, survival and GVHD histopathology were not improved. One reason for failure was fatal embolism.

Aberrant splicing of genes involved in haemoglobin synthesis and impaired terminal erythroid maturation in SF3B1 mutated refractory anaemia with ring sideroblasts.

Refractory anaemia with ring sideroblasts (RARS) is distinguished by hyperplastic inefficient erythropoiesis, aberrant mitochondrial ferritin accumulation and anaemia. Heterozygous mutations in the spliceosome gene SF3B1 are found in a majority of RARS cases. To explore the link between SF3B1 mutations and anaemia, we studied mutated RARS CD34(+) marrow cells with regard to transcriptome sequencing, splice patterns and mutational allele burden during erythroid differentiation. Transcriptome profiling during early erythroid differentiation revealed a marked up-regulation of genes involved in haemoglobin synthesis and in the oxidative phosphorylation process, and down-regulation of mitochondrial ABC transporters compared to normal bone marrow. Moreover, mis-splicing of genes involved in transcription regulation, particularly haemoglobin synthesis, was confirmed, indicating a compromised haemoglobinization during RARS erythropoiesis. In order to define the phase during which erythroid maturation of SF3B1 mutated cells is most affected, we assessed allele burden during erythroid differentiation in vitro and in vivo and found that SF3B1 mutated erythroblasts showed stable expansion until late erythroblast stage but that terminal maturation to reticulocytes was significantly reduced. In conclusion, SF3B1 mutated RARS progenitors display impaired splicing with potential downstream consequences for genes of key importance for haemoglobin synthesis and terminal erythroid differentiation.

Neural stem cell differentiation is dictated by distinct actions of nuclear receptor corepressors and histone deacetylases.

Signaling factors including retinoic acid (RA) and thyroid hormone (T3) promote neuronal, oligodendrocyte, and astrocyte differentiation of cortical neural stem cells (NSCs). However, the functional specificity of transcriptional repressor checkpoints controlling these differentiation programs remains unclear. Here, we show by genome-wide analysis that histone deacetylase (HDAC)2 and HDAC3 show overlapping and distinct promoter occupancy at neuronal and oligodendrocyte-related genes in NSCs. The absence of HDAC3, but not HDAC2, initiated a neuronal differentiation pathway in NSCs. The ablation of the corepressor NCOR or HDAC2, in conjunction with T3 treatment, resulted in increased expression of oligodendrocyte genes, revealing a direct HDAC2-mediated repression of Sox8 and Sox10 expression. Interestingly, Sox10 was required also for maintaining the more differentiated state by repression of stem cell programming factors such as Sox2 and Sox9. Distinct and nonredundant actions of NCORs and HDACs are thus critical for control of lineage progression and differentiation programs in neural progenitors.

RETRACTED: Vulnerability of glioblastoma cells to catastrophic vacuolization and death induced by a small molecule.

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer with marginal life expectancy. Based on the assumption that GBM cells gain functions not necessarily involved in the cancerous process, patient-derived glioblastoma cells (GCs) were screened to identify cellular processes amenable for development of targeted treatments. The quinine-derivative NSC13316 reliably and selectively compromised viability. Synthetic chemical expansion reveals delicate structure-activity relationship and analogs with increased potency, termed Vacquinols. Vacquinols stimulate death by membrane ruffling, cell rounding, massive macropinocytic vacuole accumulation, ATP depletion, and cytoplasmic membrane rupture of GCs. The MAP kinase MKK4, identified by a shRNA screen, represents a critical signaling node. Vacquinol-1 displays excellent in vivo pharmacokinetics and brain exposure, attenuates disease progression, and prolongs survival in a GBM animal model. These results identify a vulnerability to massive vacuolization that can be targeted by small molecules and point to the possible exploitation of this process in the design of anticancer therapies.

shRNA screening identifies JMJD1C as being required for leukemia maintenance.

Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Recent progress suggests that proteins involved in epigenetic control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic regulators for cell survival and proliferation. We used a mouse model of human AML induced by the MLL-AF9 fusion oncogene and an epigenetic short hairpin RNA (shRNA) library to screen for novel potential drug targets. As a counter-screen for general toxicity of shRNAs, we used normal mouse bone marrow cells. One of the best candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines, with the strongest effect observed in the MLL-rearranged ALL cell line SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia.