Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML.

ABSTRACT: The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

IDH-mutant myeloid neoplasms are associated with seronegative rheumatoid arthritis and innate immune activation.

ABSTRACT: High prevalence of IDH mutations in seronegative rheumatoid arthritis (RA) with myeloid neoplasm, elevated 2-hydroxyglutarate, dysregulated innate immunity, and proinflammatory microenvironment suggests causative association between IDH mutations and seronegative RA. Our findings merit investigation of IDH inhibitors as therapeutics for seronegative IDH-mutated RA.

Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Targeting sphingosine kinase 1 induces MCL1-dependent cell death in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy where despite improvements in conventional chemotherapy and bone marrow transplantation, overall survival remains poor. Sphingosine kinase 1 (SPHK1) generates the bioactive lipid sphingosine 1-phosphate (S1P) and has established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers. The role and targeting of SPHK1 in primary AML, however, has not been previously investigated. Here we show that SPHK1 is overexpressed and constitutively activated in primary AML patient blasts but not in normal mononuclear cells. Subsequent targeting of SPHK1 induced caspase-dependent cell death in AML cell lines, primary AML patient blasts, and isolated AML patient leukemic progenitor/stem cells, with negligible effects on normal bone marrow CD34+ progenitors from healthy donors. Furthermore, administration of SPHK1 inhibitors to orthotopic AML patient-derived xenografts reduced tumor burden and prolonged overall survival without affecting murine hematopoiesis. SPHK1 inhibition was associated with reduced survival signaling from S1P receptor 2, resulting in selective downregulation of the prosurvival protein MCL1. Subsequent analysis showed that the combination of BH3 mimetics with either SPHK1 inhibition or S1P receptor 2 antagonism triggered synergistic AML cell death. These results support the notion that SPHK1 is a bona fide therapeutic target for the treatment of AML.

Hypoxia Induced ER Stress Response as an Adaptive Mechanism in Cancer.

It is evident that regions within tumors are deprived of oxygen, which makes the microenvironment hypoxic. Cancer cells experiencing hypoxia undergo metabolic alterations and cytoprotective adaptive mechanisms to survive such stringent conditions. While such mechanisms provide potential therapeutic targets, the mechanisms by which hypoxia regulates adaptive responses-such as ER stress response, unfolded protein response (UPR), anti-oxidative responses, and autophagy-remain elusive. In this review, we summarize the complex interplay between hypoxia and the ER stress signaling pathways that are activated in the hypoxic microenvironment of the tumors.

Conditional knockout mice demonstrate function of Klf5 as a myeloid transcription factor.

Krüppel-like factor 5 (Klf5) encodes a zinc-finger transcription factor and has been reported to be a direct target of C/EBPα, a master transcription factor critical for formation of granulocyte-macrophage progenitors (GMP) and leukemic GMP. Using an in vivo hematopoietic-specific gene ablation model, we demonstrate that loss of Klf5 function leads to a progressive increase in peripheral white blood cells, associated with increasing splenomegaly. Long-term hematopoietic stem cells (HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs) were all significantly reduced in Klf5(Δ/Δ) mice, and knockdown of KLF5 in human CD34(+) cells suppressed colony-forming potential. ST-HSCs, MPPs, and total numbers of committed progenitors were increased in the spleen of Klf5(Δ/Δ) mice, and reduced ß1- and ß2-integrin expression on hematopoietic progenitors suggests that increased splenic hematopoiesis results from increased stem and progenitor mobilization. Klf5(Δ/Δ) mice show a significant reduction in the fraction of Gr1(+)Mac1(+) cells (neutrophils) in peripheral blood and bone marrow and increased frequency of eosinophils in the peripheral blood, bone marrow, and lung. Thus, these studies demonstrate dual functions of Klf5 in regulating hematopoietic stem and progenitor proliferation and localization in the bone marrow, as well as lineage choice after GMP, promoting increased neutrophil output at the expense of eosinophil production.

Novel germ line DDX41 mutations define families with a lower age of MDS/AML onset and lymphoid malignancies.

Recently our group and others have identified DDX41 mutations both as germ line and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), suggesting that DDX41 acts as a tumor suppressor. To determine whether novel DDX41 mutations could be identified in families with additional types of hematologic malignancies, our group screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine (3%) with DDX41 mutations. As previously observed, MDS and AML were the most common malignancies, often of the erythroblastic subtype, and 1 family displayed early-onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germ line DDX41 mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical.

Ecotopic viral integration site 1 (EVI1) regulates multiple cellular processes important for cancer and is a synergistic partner for FOS protein in invasive tumors.

Ecotropic viral integration site 1 (EVI1) is an oncogenic dual domain zinc finger transcription factor that plays an essential role in the regulation of hematopoietic stem cell renewal, and its overexpression in myeloid leukemia and epithelial cancers is associated with poor patient survival. Despite the discovery of EVI1 in 1988 and its emerging role as a dominant oncogene in various types of cancer, few EVI1 target genes are known. This lack of knowledge has precluded a clear understanding of exactly how EVI1 contributes to cancer. Using a combination of ChIP-Seq and microarray studies in human ovarian carcinoma cells, we show that the two zinc finger domains of EVI1 bind to DNA independently and regulate different sets of target genes. Strikingly, an enriched fraction of EVI1 target genes are cancer genes or genes associated with cancer. We also show that more than 25% of EVI1-occupied genes contain linked EVI1 and activator protein (AP)1 DNA binding sites, and this finding provides evidence for a synergistic cooperative interaction between EVI1 and the AP1 family member FOS in the regulation of cell adhesion, proliferation, and colony formation. An increased number of dual EVI1/AP1 target genes are also differentially regulated in late-stage ovarian carcinomas, further confirming the importance of the functional cooperation between EVI1 and FOS. Collectively, our data indicate that EVI1 is a multipurpose transcription factor that synergizes with FOS in invasive tumors.

An optimized algorithm for detecting and annotating regional differential methylation.

BACKGROUND: DNA methylation profiling reveals important differentially methylated regions (DMRs) of the genome that are altered during development or that are perturbed by disease. To date, few programs exist for regional analysis of enriched or whole-genome bisulfate conversion sequencing data, even though such data are increasingly common. Here, we describe an open-source, optimized method for determining empirically based DMRs (eDMR) from high-throughput sequence data that is applicable to enriched whole-genome methylation profiling datasets, as well as other globally enriched epigenetic modification data. RESULTS: Here we show that our bimodal distribution model and weighted cost function for optimized regional methylation analysis provides accurate boundaries of regions harboring significant epigenetic modifications. Our algorithm takes the spatial distribution of CpGs into account for the enrichment assay, allowing for optimization of the definition of empirical regions for differential methylation. Combined with the dependent adjustment for regional p-value combination and DMR annotation, we provide a method that may be applied to a variety of datasets for rapid DMR analysis. Our method classifies both the directionality of DMRs and their genome-wide distribution, and we have observed that shows clinical relevance through correct stratification of two Acute Myeloid Leukemia (AML) tumor sub-types. CONCLUSIONS: Our weighted optimization algorithm eDMR for calling DMRs extends an established DMR R pipeline (methylKit) and provides a needed resource in epigenomics. Our method enables an accurate and scalable way of finding DMRs in high-throughput methylation sequencing experiments. eDMR is available for download at http://code.google.com/p/edmr/.

The double life of KLF5: Opposing roles in regulation of gene-expression, cellular function, and transformation.

The mechanisms by which cells control their growth and behavioral identities are complex and require adaptability to environmental changes. Transcription factors act as master controllers of many of these pivotal points through their ability to influence the expression of many thousands of downstream genes, and increasingly research is showing that transcription factor regulation of target genes can change in response to environmental stimuli and cell type such that their function is not prescribed but rather context-dependent. Krüppel like factor 5 (KLF5) is an example of such a transcription factor, where evidence of disparate effects on cell growth and differentiation in normal and transformed tissue are clear. Here we present and discuss the literature covering the differential roles of KLF5 in particular tissues and cancer states, and the mechanisms by which these differences are effected through the regulation of KLF5 protein function in response to different cellular states and the direct effect on target gene expression.

The vent-like homeobox gene VENTX promotes human myeloid differentiation and is highly expressed in acute myeloid leukemia.

Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus xvent2 gene, is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34(+) leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT-PCR documented expression of the gene in lineage positive hematopoietic subpopulations, with the highest expression in CD33(+) myeloid cells. Notably, expression levels of VENTX were negligible in normal CD34(+)/CD38(-) or CD34(+) human progenitor cells. In contrast to this, leukemic CD34(+)/CD38(-) cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34(+) cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34(+) human progenitor cells perturbs normal hematopoietic development, promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together, these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis.

The GM-CSF receptor utilizes ß-catenin and Tcf4 to specify macrophage lineage differentiation.

Granulocyte-macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor ß subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation and accumulation of ß-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised ß-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate ß-catenin stability, most likely via GSK3ß. Consistent with this pathway being active in primary cells we show that inhibition of GSK3ß activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signaling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.

Kinetic Effects of Transferrin-Conjugated Gold Nanoparticles on the Antioxidant Glutathione-Thioredoxin Pathway.

Nanoparticle-based therapeutics are being clinically translated for treating cancer. Even when thought to be biocompatible, nanoparticles are being increasingly identified as altering cell regulation and homeostasis. Antioxidant pathways are important for maintaining cell redox homeostasis and play important roles by maintaining ROS levels within tolerable ranges. Here, we sought to understand how a model of a relatively inert nanoparticle without any therapeutic agent itself could antagonize a cancer cell lines' antioxidant mechanism. A label-free protein expression approach was used to assess the glutathione-thioredoxin antioxidative pathway in a prostate cancer cell line (PC-3) after exposure to gold nanoparticles conjugated with a targeting moiety (transferrin). The impact of the nanoparticles was also corroborated through morphological analysis with TEM and classification of pro-apoptotic cells by way of the sub-G0/G1 population via the cell cycle and annexin V apoptosis assay. After a two-hour exposure to nanoparticles, major proteins associated with the glutathione-thioredoxin antioxidant pathway were downregulated. However, this response was acute, and in terms of protein expression, cells quickly recovered within 24 h once nanoparticle exposure ceased. The impact on PRDX-family proteins appears as the most influential factor in how these nanoparticles induced an oxidative stress response in the PC-3 cells. An apparent adaptive response was observed if exposure to nanoparticles continued. Acute exposure was observed to have a detrimental effect on cell viability compared to continuously exposed cells. Nanoparticle effects on cell regulation likely provide a compounding therapeutic advantage under some circumstances, in addition to the action of any cytotoxic agents; however, any therapeutic advantage offered by nanoparticles themselves with regard to vulnerabilities specific to the glutathione-thioredoxin antioxidative pathway is highly temporal.

Novel modes of MPL activation in triple-negative myeloproliferative neoplasms.

The identification of a somatic mutation associated with myeloid malignancy is of diagnostic importance in myeloproliferative neoplasms (MPNs). Individuals with no mutation detected in common screening tests for variants in JAK2, CALR, and MPL are described as 'triple-negative' and pose a diagnostic challenge if there is no other evidence of a clonal disorder. To identify potential drivers that might explain the clinical phenotype, we used an extended sequencing panel to characterise a cohort of 44 previously diagnosed triple-negative MPN patients for canonical mutations in JAK2, MPL and CALR at low variant allele frequency (found in 4/44 patients), less common variants in the JAK-STAT signalling pathway (12 patients), or other variants in recurrently mutated genes from myeloid malignancies (18 patients), including hotspot variants of potential clinical relevance in eight patients. In one patient with thrombocytosis we identified biallelic germline MPL variants. Neither MPL variant was activating in cell proliferation assays, and one of the variants was not expressed on the cell surface, yet co-expression of both variants led to thrombopoietin hypersensitivity. Our results highlight the clinical value of extended sequencing including germline variant analysis and illustrate the need for detailed functional assays to determine whether rare variants in JAK2 or MPL are pathogenic.

Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia.

The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia.

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure, and recurrence in acute myeloid leukemia (AML). Despite AML displaying extensive biological and clinical heterogeneity, LSC with high interleukin-3 receptor (IL3R) levels are a constant yet puzzling feature, as this receptor lacks tyrosine kinase activity. Here, we show that the heterodimeric IL3Rα/ßc receptor assembles into hexamers and dodecamers through a unique interface in the 3D structure, where high IL3Rα/ßc ratios bias hexamer formation. Importantly, receptor stoichiometry is clinically relevant as it varies across the individual cells in the AML hierarchy, in which high IL3Rα/ßc ratios in LSCs drive hexamer-mediated stemness programs and poor patient survival, while low ratios mediate differentiation. Our study establishes a new paradigm in which alternative cytokine receptor stoichiometries differentially regulate cell fate, a signaling mechanism that may be generalizable to other transformed cellular hierarchies and of potential therapeutic significance. SIGNIFICANCE: Stemness is a hallmark of many cancers and is largely responsible for disease emergence, progression, and relapse. Our finding that clinically significant stemness programs in AML are directly regulated by different stoichiometries of cytokine receptors represents a hitherto unexplained mechanism underlying cell-fate decisions in cancer stem cell hierarchies. This article is highlighted in the In This Issue feature, p. 1749.

The GM-CSF receptor family: mechanism of activation and implications for disease.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pluripotent cytokine produced by many cells in the body, which regulates normal and malignant hemopoiesis as well as innate and adaptive immunity. GM-CSF assembles and activates its heterodimeric receptor complex on the surface of myeloid cells, initiating multiple signaling pathways that control key functions such as cell survival, cell proliferation, and functional activation. Understanding the molecular composition of these pathways, the interaction of the various components as well as the kinetics and dose-dependent mechanics of receptor activation provides valuable insights into the function of GM-CSF as well as the related cytokines, interleukin-3 and interleukin-5. This knowledge provides opportunities for the development of new therapies to block the action of these cytokines in hematological malignancy and chronic inflammation.

Alternative modes of GM-CSF receptor activation revealed using activated mutants of the common beta-subunit.

Granulocyte/macrophage colony-stimulating factor promotes growth, survival, differentiation, and activation of normal myeloid cells and plays an important role in myeloid leukemias. The GM-CSF receptor (GMR) shares a signaling subunit, beta(c), with interleukin-3 and interleukin-5 receptors and has recently been shown to induce activation of Janus kinase 2 (JAK2) and downstream signaling via formation of a unique dodecameric receptor complex. In this study we use 2 activated beta(c) mutants that display distinct signaling capacity and have differential requirements for the GMR alpha-subunit (GMR-alpha) to dissect the signaling pathways associated with the GM-CSF response. The V449E transmembrane mutant selectively activates JAK2/signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) pathways, resulting in a high level of sensitivity to JAK and ERK inhibitors, whereas the extracellular mutant (FIDelta) selectively activates the phosphoinositide 3-kinase/Akt and IkappaKbeta/nuclear factorkappaB pathways. We also demonstrate a novel and direct interaction between the SH3 domains of Lyn and Src with a conserved proline-rich motif in GMR-alpha and show a selective requirement for Src family kinases by the FIDelta mutant. We relate the nonoverlapping nature of signaling by the activated mutants to the structure of the unique GMR complex and propose alternative modes of receptor activation acting synergistically in the mature liganded receptor complex.