Targeting AXL kinase sensitizes leukemic stem and progenitor cells to venetoclax treatment in acute myeloid leukemia.

The abundance of genetic abnormalities and phenotypic heterogeneities in acute myeloid leukemia (AML) poses significant challenges to the development of improved treatments. Here, we demonstrated that a key growth arrest-specific gene 6/AXL axis is highly activated in cells from patients with AML, particularly in stem/progenitor cells. We developed a potent selective AXL inhibitor that has favorable pharmaceutical properties and efficacy against preclinical patient-derived xenotransplantation (PDX) models of AML. Importantly, inhibition of AXL sensitized AML stem/progenitor cells to venetoclax treatment, with strong synergistic effects in vitro and in PDX models. Mechanistically, single-cell RNA-sequencing and functional validation studies uncovered that AXL inhibition, alone or in combination with venetoclax, potentially targets intrinsic metabolic vulnerabilities of AML stem/progenitor cells and shows a distinct transcriptomic profile and inhibits mitochondrial oxidative phosphorylation. Inhibition of AXL or BCL-2 also differentially targets key signaling proteins to synergize in leukemic cell killing. These findings have a direct translational impact on the treatment of AML and other cancers with high AXL activity.

The retinoic acid receptor co-factor NRIP1 is uniquely upregulated and represents a therapeutic target in acute myeloid leukemia with chromosome 3q rearrangements.

Aberrant expression of Ecotropic Viral Integration Site 1 (EVI1) is a hallmark of acute myeloid leukemia (AML) with inv(3) or t(3;3), which is a disease subtype with especially poor outcome. In studying transcriptomes from AML patients with chromosome 3q rearrangements, we identified a significant upregulation of the Nuclear Receptor Interacting Protein 1 (NRIP1) as well as its adjacent non-coding RNA LOC101927745. Utilizing transcriptomic and epigenomic data from over 900 primary samples from patients as well as genetic and transcriptional engineering approaches, we have identified several mechanisms that can lead to upregulation of NRIP1 in AML. We hypothesize that the LOC101927745 transcription start site harbors a context-dependent enhancer that is bound by EVI1, causing upregulation of NRIP1 in AML with chromosome 3 abnormalities. Furthermore, we showed that NRIP1 knockdown negatively affects the proliferation and survival of 3qrearranged AML cells and increases their sensitivity to all-trans retinoic acid, suggesting that NRIP1 is relevant for the pathogenesis of inv(3)/t(3;3) AML and could serve as a novel therapeutic target in myeloid malignancies with 3q abnormalities.

Sildenafil triggers tumor lethality through altered expression of HSP90 and degradation of PKD2.

The repurposing of existing drugs has emerged as an attractive additional strategy to the development of novel compounds in the fight against cancerous diseases. Inhibition of phosphodiesterase 5 (PDE5) has been claimed as a potential approach to target various cancer subtypes in recent years. However, data on the treatment of tumors with PDE5 inhibitors as well as the underlying mechanisms are as yet very scarce. Here, we report that treatment of tumor cells with low concentrations of Sildenafil was associated with decreased cancer cell proliferation and augmented apoptosis in vitro and resulted in impaired tumor growth in vivo. Notably, incubation of cancer cells with Sildenafil was associated with altered expression of HSP90 chaperone followed by degradation of protein kinase D2, a client protein previously reported to be involved in tumor growth. Furthermore, the involvement of low doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, in combination with low concentrations of Sildenafil, synergistically and negatively impacted on the viability of cancer cells in vivo. Taken together, our study suggests that repurposing of already approved drugs, alone or in combination with oncology-dedicated compounds, may represent a novel cancer therapeutic strategy.

Stk33 is required for spermatid differentiation and male fertility in mice.

Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.

NFATC1 activation by DNA hypomethylation in chronic lymphocytic leukemia correlates with clinical staging and can be inhibited by ibrutinib.

B cell receptor (BCR) signaling is a key for survival of chronic lymphocytic leukemia (CLL) cells, and BCR signaling inhibitors are clinically active. However, relapse and resistance to treatment require novel treatment options. To detect novel candidate therapeutic targets, we performed a genome-wide DNA methylation screen with custom arrays and identified aberrant promoter DNA methylation in 2,192 genes. The transcription factor NFATC1 that is a downstream effector of BCR signaling was among the top hypomethylated genes and was concomitantly transcriptionally upregulated in CLL. Intriguingly, NFATC1 promoter DNA hypomethylation levels were significantly variant in clinical trial cohorts from different disease progression stages and furthermore correlated with Binet disease staging and thymidine kinase levels, strongly suggesting a central role of NFATC1 in CLL development. Functionally, DNA hypomethylation at NFATC1 promoter inversely correlated with RNA levels of NFATC1 and dysregulation correlated with expression of target genes BCL-2, CCND1 and CCR7. The inhibition of the NFAT regulator calcineurin with tacrolimus and cyclosporin A and the BCR signaling inhibitor ibrutinib significantly reduced NFAT activity in leukemic cell lines, and NFAT inhibition resulted in increased apoptosis of primary CLL cells. In summary, our results indicate that the aberrant activation of NFATC1 by DNA hypomethylation and BCR signaling plays a major role in the pathomechanism of CLL.

Micro-ribonucleic acid-155 is a direct target of Meis1, but not a driver in acute myeloid leukemia.

Micro-ribonucleic acid-155 (miR-155) is one of the first described oncogenic miRNAs. Although multiple direct targets of miR-155 have been identified, it is not clear how it contributes to the pathogenesis of acute myeloid leukemia. We found miR-155 to be a direct target of Meis1 in murine Hoxa9/Meis1 induced acute myeloid leukemia. The additional overexpression of miR-155 accelerated the formation of acute myeloid leukemia in Hoxa9 as well as in Hoxa9/Meis1 cells in vivo However, in the absence or following the removal of miR-155, leukemia onset and progression were unaffected. Although miR-155 accelerated growth and homing in addition to impairing differentiation, our data underscore the pathophysiological relevance of miR-155 as an accelerator rather than a driver of leukemogenesis. This further highlights the complexity of the oncogenic program of Meis1 to compensate for the loss of a potent oncogene such as miR-155. These findings are highly relevant to current and developing approaches for targeting miR-155 in acute myeloid leukemia.

Thrombotic Microangiopathy after Allogeneic Stem Cell Transplantation: A Comparison of Eculizumab Therapy and Conventional Therapy.

We report the results of a single-center analysis of a cohort of 39 patients treated between 1997 and 2016 for transplantion-associated thrombotic microangiopathy. We evaluated 2 subgroups of patients: 24 patients treated between 1997 and 2014 who received conventional therapy and 15 patients treated with the complement-inhibiting monoclonal antibody eculizumab between 2014 and 2016. The conventional therapy group was treated predominantly with defibrotide alone or in combination with plasmapheresis or rituximab. Despite an initial response rate of 61%, only 4 patients (16%) were long-term survivors, 2 of whom had a low-risk thrombotic microangiopathy without multiorgan damage. Progression of thrombotic micorangiopathy and bacterial/fungal infections contributed equally to treatment failure. The overall response rate in the eculizumab group was significantly higher, at 93%. In addition, we were able to stop eculizumab treatment in 5 patients (33%), all of whom had high-risk thrombotic microangiopathy, due to sustained recovery. Despite the very good response in the eculizumab-treated group, we did not observe a significant improved overall survival, due primarily to a high rate of infection-related mortality (70%). Therefore, further studies are needed to identify the optimal therapeutic management approach for transplantation-associated thrombotic microangiopathy to improve its dismal outcome.

Circular RNAs of the nucleophosmin (NPM1) gene in acute myeloid leukemia.

In acute myeloid leukemia, there is growing evidence for splicing pattern deregulation, including differential expression of linear splice isoforms of the commonly mutated gene nucleophosmin (NPM1). In this study, we detect circular RNAs of NPM1 and quantify circRNA hsa_circ_0075001 in a cohort of NPM1 wild-type and mutated acute myeloid leukemia (n=46). Hsa_circ_0075001 expression correlates positively with total NPM1 expression, but is independent of the NPM1 mutational status. High versus low hsa_circ_0075001 expression defines patient subgroups characterized by distinct gene expression patterns, such as lower expression of components of the Toll-like receptor signaling pathway in high hsa_circ_0075001 expression cases. Global evaluation of circRNA expression in sorted healthy hematopoietic controls (n=10) and acute myeloid leukemia (n=10) reveals circRNA transcripts for 47.9% of all highly expressed genes. While circRNA expression correlates globally with parental gene expression, we identify hematopoietic differentiation-associated as well as acute myeloid leukemia subgroup-specific circRNA signatures.

PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation.

BACKGROUND: Initially identified as a molecule that regulates the final step of glycolysis, the M2 isoform of pyruvate kinase (PKM2) was recently reported to have a central role in the metabolic reprogramming of cancer cells as well as participating in cell cycle progression and gene transcription. Despite intensive efforts, the intricate molecular mechanisms through which PKM2 regulates tumor progression remain elusive. METHODS: The proliferation and apoptosis of various pancreatic cancer cells using lentiviral-mediated PKM2 abrogation were assessed in vitro via Western blot and flow cytometric assay while the in vivo experiments involved tumor xenograft on chicken chorionallantoic membranes and immunohistochemistry on human tissue specimens. In order to decipher the molecular mechanism of HIF-1α and p65/RelA regulation by PKM2 in cancer cells cultivated in hypoxic atmosphere or normoxia we involved various biochemical assays such as Western blotting, immunoprecipitation, reporter gene assay and ELISA. RESULTS: Strong expression of PKM2 was observed in 68 % of human pancreatic adenocarcinoma specimens and almost all analyzed pancreatic cancer cell lines. Abrogation of PKM2 resulted in impaired proliferation and augmented apoptosis in vitro as well as impaired tumor growth and decreased blood vessel formation in vivo. Furthermore, deletion of PKM2 negatively impacted hypoxia-induced HIF-1α accumulation and promoter activity ultimately resulting in impaired secretion of VEGF. CONCLUSIONS: Our study suggests that in hypoxic pancreatic tumors PKM2 interferes both with NF-κB/p65 and HIF-1α activation that ultimately triggers VEGF-A secretion and subsequent blood vessel formation.

Comprehensive analysis of mammalian miRNA* species and their role in myeloid cells.

Processing of pre-miRNA through Dicer1 generates an miRNA duplex that consists of an miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep-sequencing libraries from mouse and human tissue. Comparisons of miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibited a highly dominant strand. Conversely, 10% of miRNA duplexes showed a comparable expression of both strands, whereas the remaining 40% exhibited variable ratios across the examined libraries, as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, which implies an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* targeted the insulin-like growth factor 1 receptor/phosphatidylinositol 3-kinase axis and that high miR-223* levels were associated with increased overall survival in patients with acute myeloid leukemia. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal and leukemic cell states. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA.

miR-148a-3p and DDX6 functional link promotes survival of myeloid leukemia cells.

Regulation of gene expression at the RNA level is an important regulatory mechanism in cancer. However, posttranscriptional molecular pathways underlying tumorigenesis remain largely unexplored. In this study, we uncovered a functional axis consisting of microRNA (miR)-148a-3p, RNA helicase DDX6, and its downstream target thioredoxin-interacting protein (TXNIP) in acute myeloid leukemia (AML). Using a DROSHA-knockout cell system to evaluate miR-mediated gene expression control, we comprehensively profiled putative transcripts regulated by miR-148a-3p and identified DDX6 as a direct target of miR-148a-3p in AML cells. DDX6 depletion induced cell cycle arrest, apoptosis, and differentiation, although delaying leukemia development in vivo. Genome-wide assessment of DDX6-binding transcripts and gene expression profiling of DDX6-depleted cells revealed TXNIP, a tumor suppressor, as the functional downstream target of DDX6. Overall, our study identified DDX6 as a posttranscriptional regulator that is required for AML survival. We proposed the regulatory link between miR-148a-3p and DDX6 as a potential therapeutic target in leukemia.

Elucidating the importance and regulation of key enhancers for human MEIS1 expression.

Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is a critical factor in the pathogenesis of a large subset of acute myeloid leukemia (AML). Despite the clinical relevance of MEIS1, its regulation is largely unknown. To understand the transcriptional regulatory mechanisms contributing to human MEIS1 expression, we created a knock-in green florescent protein (GFP) reporter system at the endogenous MEIS1 locus in a human AML cell line. Using this model, we have delineated and dissected a critical enhancer region of the MEIS1 locus for transcription factor (TF) binding through in silico prediction in combination with oligo pull-down, mass-spectrometry and knockout analysis leading to the identification of FLI1, an E-twenty-six (ETS) transcription factor, as an important regulator of MEIS1 transcription. We further show direct binding of FLI1 to the MEIS1 locus in human AML cell lines as well as enrichment of histone acetylation in MEIS1-high healthy and leukemic cells. We also observe a positive correlation between high FLI1 transcript levels and worse overall survival in AML patients. Our study expands the role of ETS factors in AML and our model constitutes a feasible tool for a more detailed understanding of transcriptional regulatory elements and their interactome.

Evidence for high bi-allelic expression of activating Ly49 receptors.

Stochastic expression is a hallmark of the Ly49 family that encode the main MHC class-I-recognizing receptors of mouse natural killer (NK) cells. This highly polygenic and polymorphic family includes both activating and inhibitory receptor genes and is one of genome's fastest evolving loci. The inhibitory Ly49 genes are expressed in a stochastic mono-allelic manner, possibly under the control of an upstream bi-directional early promoter and show mono-allelic DNA methylation patterns. To date, no studies have directly addressed the transcriptional regulation of the activating Ly49 receptors. Our study shows differences in DNA methylation pattern between activating and inhibitory genes in C57BL/6 and F1 hybrid mouse strains. We also show a bias towards bi-allelic expression of the activating receptors based on allele-specific single-cell RT-PCR in F1 hybrid NK cells for Ly49d and Ly49H expression in Ly49h(+/-) mice. Furthermore, we have identified a region of high sequence identity with possible transcriptional regulatory capacity for the activating Ly49 genes. Our results also point to a likely difference between NK and T-cells in their ability to transcribe the activating Ly49 genes. These studies highlight the complex regulation of this rapidly evolving gene family of central importance in mouse NK cell function.

Mitochondrial metabolism: powering new directions in acute myeloid leukemia.

There has been an explosion of knowledge about the role of metabolism and the mitochondria in acute myeloid leukemia (AML). We have also recently seen several waves of novel therapies change the treatment landscape for AML, such as the selective B-cell lymphoma 2 (BCL-2) inhibitor venetoclax. In this new context, we review the rapidly advancing literature on the role of metabolism and the mitochondria in AML pathogenesis, and how these are interwoven with the mechanisms of action for novel therapeutics in AML. We also review the role of oxidative phosphorylation (OxPhos) in maintaining leukemia stem cells (LSCs), how recurrent genomic alterations in AML alter downstream metabolism, and focus on how the BCL-2 pathway and the mitochondria are inextricably linked in AML. Thus, we provide an overview of the mitochondria and metabolism in the context of our new therapeutic world for AML and outline how targeting these vulnerabilities may produce novel therapeutic strategies.

Expression of the leukemic prognostic marker CD7 is linked to epigenetic modifications in chronic myeloid leukemia.

BACKGROUND: Expression levels of the cell surface glycoprotein, CD7, and the serine protease, elastase 2 (ELA2), in the leukemic cells of patients with chronic myeloid leukemia (CML) have been associated with clinical outcome. However, little is known about the mechanisms that underlie the variable expression of these genes in the leukemic cells. RESULTS: To address this question, we compared the level of their expression with the DNA methylation and histone acetylation status of 5' sequences of both genes in leukemic cell lines and primitive (lin-CD34+) leukemic cells from chronic phase CML patients. DNA methylation of the ELA2 gene promoter did not correlate with its expression pattern in lin-CD34+ cells from chronic phase CML patient samples even though there was clear differential DNA methylation of this locus in ELA2-expressing and non-expressing cell lines. In contrast, we found a strong relation between CD7 expression and transcription-permissive chromatin modifications, both at the level of DNA methylation and histone acetylation with evidence of hypomethylation of the CD7 promoter region in the lin-CD34+ cells from CML patients with high CD7 expression. CONCLUSION: These findings indicate a link between epigenetic modifications and CD7 expression in primitive CML cells.

MicroRNA-708 is a novel regulator of the Hoxa9 program in myeloid cells.

MicroRNAs (miRNAs) are commonly deregulated in acute myeloid leukemia (AML), affecting critical genes not only through direct targeting, but also through modulation of downstream effectors. Homeobox (Hox) genes balance self-renewal, proliferation, cell death, and differentiation in many tissues and aberrant Hox gene expression can create a predisposition to leukemogenesis in hematopoietic cells. However, possible linkages between the regulatory pathways of Hox genes and miRNAs are not yet fully resolved. We identified miR-708 to be upregulated in Hoxa9/Meis1 AML inducing cell lines as well as in AML patients. We further showed Meis1 directly targeting miR-708 and modulating its expression through epigenetic transcriptional regulation. CRISPR/Cas9 mediated knockout of miR-708 in Hoxa9/Meis1 cells delayed disease onset in vivo, demonstrating for the first time a pro-leukemic contribution of miR-708 in this context. Overexpression of miR-708 however strongly impeded Hoxa9 mediated transformation and homing capacity in vivo through modulation of adhesion factors and induction of myeloid differentiation. Taken together, we reveal miR-708, a putative tumor suppressor miRNA and direct target of Meis1, as a potent antagonist of the Hoxa9 phenotype but an effector of transformation in Hoxa9/Meis1. This unexpected finding highlights the yet unexplored role of miRNAs as indirect regulators of the Hox program during normal and aberrant hematopoiesis.