The CCAAT enhancer-binding protein α (C/EBPα) plays an important role in myeloid cell differentiation and in the enhancement of C/EBPα expression/activity, which can lead to granulocytic differentiation in acute myeloid leukemia (AML) cells. We found that styryl quinazolinones induce upregulation of C/EBPα expression, and thereby induce myeloid differentiation in human myeloid leukemia cell lines. We screened a series of active styryl quinazolinones and evaluated the structure⁻activity relationship (SAR) of these small molecules in inducing C/EBPα expression-thereby prompting the leukemic cells to differentiate. We observed that compound 78 causes differentiation at 3 μM concentration, while 1 induces differentiation at 10 μM concentration. We also observed an increase in the expression of neutrophil differentiation marker CD11b upon treatment with 78. Both the C/EBPα and C/EBPε levels were found to be upregulated by treatment with 78. These SAR findings are inspiration to develop further modified styryl quinazolinones, in the path of this novel differentiation therapy, which can contribute to the care of patients with AML.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by Adenosine DeAminases acting on double-stranded RNA(dsRNA) (ADAR), occurs predominantly in the 3' untranslated regions (3'UTRs) of spliced mRNA. Here we uncover an unanticipated link between ADARs (ADAR1 and ADAR2) and the expression of target genes undergoing extensive 3'UTR editing. Using METTL7A (Methyltransferase Like 7A), a novel tumor suppressor gene with multiple editing sites at its 3'UTR, we demonstrate that its expression could be repressed by ADARs beyond their RNA editing and double-stranded RNA (dsRNA) binding functions. ADARs interact with Dicer to augment the processing of pre-miR-27a to mature miR-27a. Consequently, mature miR-27a targets the METTL7A 3'UTR to repress its expression level. In sum, our study unveils that the extensive 3'UTR editing of METTL7A is merely a footprint of ADAR binding, and there are a subset of target genes that are equivalently regulated by ADAR1 and ADAR2 through their non-canonical RNA editing and dsRNA binding-independent functions, albeit maybe less common. The functional significance of ADARs is much more diverse than previously appreciated and this gene regulatory function of ADARs is most likely to be of high biological importance beyond the best-studied editing function. This non-editing side of ADARs opens another door to target cancer.

C/EBPalpha is a transcription factor essential for terminal differentiation of several cell types. It has not known if C/EBPalpha protein is expressed and functions in the prostate gland.

Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.

Monocyte differentiation involves the participation of lineage-restricted transcription factors, although the mechanisms by which this process occurs are incompletely defined. Within the hematopoietic system, members of the Kruppel-like family of factors (KLFs) play essential roles in erythrocyte and T lymphocyte development. Here we show that KLF4/GKLF is expressed in a monocyte-restricted and stage-specific pattern during myelopoiesis and functions to promote monocyte differentiation. Overexpression of KLF4 in HL-60 cells confers the characteristics of mature monocytes. Conversely, KLF4 knockdown blocked phorbol ester-induced monocyte differentiation. Forced expression of KLF4 in primary common myeloid progenitors (CMPs) or hematopoietic stem cells (HSCs) induced exclusive monocyte differentiation in clonogenic assays, whereas KLF4 deficiency inhibited monocyte but increased granulocyte differentiation. Mechanistic studies demonstrate that KLF4 is a target gene of PU.1. Consistently, KLF4 can rescue PU.1-/- fetal liver cells along the monocytic lineage and can activate the monocytic-specific CD14 promoter. Thus, KLF4 is a critical regulator in the transcriptional network controlling monocyte differentiation.

Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.

Metabolic reprogramming is widely known as a hallmark of cancer cells to allow adaptation of cells to sustain survival signals. In this report, we describe a novel oncogenic signaling pathway exclusively acting in mutated epidermal growth factor receptor (EGFR) non-small cell lung cancer (NSCLC) with acquired tyrosine kinase inhibitor (TKI) resistance. Mutated EGFR mediates TKI resistance through regulation of the fatty acid synthase (FASN), which produces 16-C saturated fatty acid palmitate. Our work shows that the persistent signaling by mutated EGFR in TKI-resistant tumor cells relies on EGFR palmitoylation and can be targeted by Orlistat, an FDA-approved anti-obesity drug. Inhibition of FASN with Orlistat induces EGFR ubiquitination and abrogates EGFR mutant signaling, and reduces tumor growths both in culture systems and in vivo Together, our data provide compelling evidence on the functional interrelationship between mutated EGFR and FASN and that the fatty acid metabolism pathway is a candidate target for acquired TKI-resistant EGFR mutant NSCLC patients.

SALL4 is a zinc finger transcription factor that belongs to the spalt-like (SALL) gene family. It plays important roles in the maintenance of self-renewal and pluripotency of embryonic stem cells, and its expression is repressed in most adult organs. SALL4 re-expression has been observed in different types of human cancers, and dysregulation of SALL4 contributes to the pathogenesis, metastasis, and even drug resistance of multiple cancer types. Surprisingly, little is known regarding how SALL4 expression is controlled, but recently microRNAs (miRNAs) have emerged as important regulators of SALL4. Due to the ability of regulating targets differentially in specific tissues, and recent advances in systemic and organ specific miRNA delivery mechanisms, miRNAs have emerged as promising therapeutic targets for cancer treatment. In this review, we summarize current knowledge of the interaction between SALL4 and miRNAs in mammalian development and cancer, paying particular attention to the emerging roles of the Let-7/Lin28 axis. In addition, we discuss the therapeutic prospects of targeting SALL4 using miRNA-based strategies, with a focus on the Let-7/LIN28 axis.

Point mutations within the TERT promoter are the most recurrent somatic noncoding mutations identified across different cancer types, including glioblastoma, melanoma, hepatocellular carcinoma, and bladder cancer. They are most abundant at -146C > T and -124C > T, and rarer at -57A > C, with the latter originally described as a familial case, but subsequently shown also to occur somatically. All three mutations create de novo E26-specific (ETS) binding sites and result in activation of the TERT gene, allowing cancer cells to achieve replicative immortality. Here, we used a systematic proteomics screen to identify transcription factors preferentially binding to the -146C > T, -124C > T, and -57A > C mutations. Although we confirmed binding of multiple ETS factors to the mutant -146C > T and -124C > T sequences, we identified E4F1 as a -57A > C-specific binder and ZNF148 as a TERT wild-type (WT) promoter binder that showed reduced interaction with the -124C > T allele. Both proteins are activating transcription factors that bind specifically to the -57A > C and WT (at position 124) TERT promoter sequence in corresponding cell lines, and up-regulate TERT transcription and telomerase activity. Our work describes new regulators of TERT gene expression with possible roles in cancer.

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterized by the genetic translocation t(9;22)(q34;q11.2) encoding for the BCR-ABL fusion oncogene. However, many molecular mechanisms of the disease progression still remain poorly understood. A growing body of evidence suggests that the epigenetic abnormalities are involved in tyrosine kinase resistance in CML, leading to leukaemic clone escape and disease propagation. Here we show that, by applying cellular reprogramming to primary CML cells, aberrant DNA methylation contributes to the disease evolution. Importantly, using a BCR-ABL inducible murine model, we demonstrate that a single oncogenic lesion triggers DNA methylation changes, which in turn act as a precipitating event in leukaemia progression.

DNA methylation was first described almost a century ago; however, the rules governing its establishment and maintenance remain elusive. Here we present data demonstrating that active transcription regulates levels of genomic methylation. We identify a novel RNA arising from the CEBPA gene locus that is critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extend the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene-selective demethylation of therapeutic targets in human diseases.

Oncogenic transcription factors such as RUNX1/ETO, which is generated by the chromosomal translocation t(8;21), subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq) to identify the core RUNX1/ETO-responsive transcriptional network of t(8;21) cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.

Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML.

Adenosine-to-inosine (A-to-I) RNA editing entails the enzymatic deamination of adenosines to inosines by adenosine deaminases acting on RNA (ADARs). Dysregulated A-to-I editing has been implicated in various diseases, including cancers. However, the precise factors governing the A-to-I editing and their physiopathological implications remain as a long-standing question. Herein, we unravel that DEAH box helicase 9 (DHX9), at least partially dependent of its helicase activity, functions as a bidirectional regulator of A-to-I editing in cancer cells. Intriguingly, the ADAR substrate specificity determines the opposing effects of DHX9 on editing as DHX9 silencing preferentially represses editing of ADAR1-specific substrates, whereas augments ADAR2-specific substrate editing. Analysis of 11 cancer types from The Cancer Genome Atlas (TCGA) reveals a striking overexpression of DHX9 in tumors. Further, tumorigenicity studies demonstrate a helicase-dependent oncogenic role of DHX9 in cancer development. In sum, DHX9 constitutes a bidirectional regulatory mode in A-to-I editing, which is in part responsible for the dysregulated editome profile in cancer.

Chromatin-modifying enzymes, and specifically the protein arginine methyltransferases (PRMTs), have emerged as important targets in cancer. Here, we investigated the role of CARM1 in normal and malignant hematopoiesis. Using conditional knockout mice, we show that loss of CARM1 has little effect on normal hematopoiesis. Strikingly, knockout of Carm1 abrogates both the initiation and maintenance of acute myeloid leukemia (AML) driven by oncogenic transcription factors. We show that CARM1 knockdown impairs cell-cycle progression, promotes myeloid differentiation, and ultimately induces apoptosis. Finally, we utilize a selective, small-molecule inhibitor of CARM1 to validate the efficacy of CARM1 inhibition in leukemia cells in vitro and in vivo. Collectively, this work suggests that targeting CARM1 may be an effective therapeutic strategy for AML.

Hematopoietic stem cell transplantation (HSCT) is a frequent therapeutic approach to restore hematopoiesis in patients with hematologic diseases. Patients receive a hematopoietic stem cell (HSC)-enriched donor cell infusion also containing immune cells, which may have a beneficial effect by eliminating residual neoplastic cells. However, the effect that donor innate immune cells may have on the donor HSCs has not been deeply explored. Here, we evaluate the influence of donor natural killer (NK) cells on HSC fate, concluded that NK cells negatively affect HSC frequency and function, and identified interferon-gamma (IFNγ) as a potential mediator. Interestingly, improved HSC fitness was achieved by NK cell depletion from murine and human donor infusions or by blocking IFNγ activity. Thus, our data suggest that suppression of inflammatory signals generated by donor innate immune cells can enhance engraftment and hematopoietic reconstitution during HSCT, which is particularly critical when limited HSC numbers are available and the risk of engraftment failure is high.

Leukemic stem cells (LSCs) can acquire non-mutational resistance following drug treatment leading to therapeutic failure and relapse. However, oncogene-independent mechanisms of drug persistence in LSCs are incompletely understood, which is the primary focus of this study. We integrated proteomics, transcriptomics, and metabolomics to determine the contribution of STAT3 in promoting metabolic changes in tyrosine kinase inhibitor (TKI) persistent chronic myeloid leukemia (CML) cells. Proteomic and transcriptional differences in TKI persistent CML cells revealed BCR-ABL-independent STAT3 activation in these cells. While knockout of STAT3 inhibited the CML cells from developing drug-persistence, inhibition of STAT3 using a small molecule inhibitor sensitized the persistent CML cells to TKI treatment. Interestingly, given the role of phosphorylated STAT3 as a transcription factor, it localized uniquely to genes regulating metabolic pathways in the TKI-persistent CML stem and progenitor cells. Subsequently, we observed that STAT3 dysregulated mitochondrial metabolism forcing the TKI-persistent CML cells to depend on glycolysis, unlike TKI-sensitive CML cells, which are more reliant on oxidative phosphorylation. Finally, targeting pyruvate kinase M2, a rate-limiting glycolytic enzyme, specifically eradicated the TKI-persistent CML cells. By exploring the role of STAT3 in altering metabolism, we provide critical insight into identifying potential therapeutic targets for eliminating TKI-persistent LSCs.

The phosphoinositide 3-kinase (PI3K) pathway represents the most hyperactivated oncogenic pathway in triple-negative breast cancer (TNBC), a highly aggressive tumor subtype encompassing ∼15% of breast cancers and which possesses no targeted therapeutics. Despite critical contributions of its signaling arms to disease pathogenesis, PI3K pathway inhibitors have not achieved expected clinical responses in TNBC, owing largely to a still-incomplete understanding of the compensatory cascades that operate downstream of PI3K. Here, we investigated the contributions of long noncoding RNAs (lncRNAs) to PI3K activities in clinical and experimental TNBC and discovered a prominent role for LINC01133 as a PI3K-AKT signaling effector. We found that LINC01133 exerted protumorigenic roles in TNBC and that it governed a previously undescribed mTOR Complex 2 (mTORC2)-dependent pathway that activated AKT in a PI3K-independent manner. Mechanistically, LINC01133 induced the expression of the mTORC2 component PROTOR1/PRR5 by competitively coupling away its negative messenger RNA (mRNA) regulator, the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1). PROTOR1/PRR5 in turn was sufficient and necessary for LINC01133-triggered functions, casting previously unappreciated roles for this Rictor-binding protein in cellular signaling and growth. Notably, LINC01133 antagonism undermined cellular growth, and we show that the LINC01133-PROTOR1/PRR5 pathway was tightly associated with TNBC poor patient survival. Altogether, our findings uncovered a lncRNA-driven signaling shunt that acts as a critical determinant of malignancy downstream of the PI3K pathway and as a potential RNA therapeutic target in clinical TNBC management.

The oncogenic transcription factor TAL1/SCL induces an aberrant transcriptional program in T-cell acute lymphoblastic leukemia (T-ALL) cells. However, the critical factors that are directly activated by TAL1 and contribute to T-ALL pathogenesis are largely unknown. Here, we identified AT-rich interactive domain 5B (ARID5B) as a collaborating oncogenic factor involved in the transcriptional program in T-ALL. ARID5B expression is down-regulated at the double-negative 2-4 stages in normal thymocytes, while it is induced by the TAL1 complex in human T-ALL cells. The enhancer located 135 kb upstream of the ARID5B gene locus is activated under a superenhancer in T-ALL cells but not in normal T cells. Notably, ARID5B-bound regions are associated predominantly with active transcription. ARID5B and TAL1 frequently co-occupy target genes and coordinately control their expression. ARID5B positively regulates the expression of TAL1 and its regulatory partners. ARID5B also activates the expression of the oncogene MYC Importantly, ARID5B is required for the survival and growth of T-ALL cells, and forced expression of ARID5B in immature thymocytes results in thymus retention, differentiation arrest, radioresistance, and tumor formation in zebrafish. Our results indicate that ARID5B reinforces the oncogenic transcriptional program by positively regulating the TAL1-induced regulatory circuit and MYC in T-ALL, thereby contributing to T-cell leukemogenesis.

Acute Myeloid Leukemia (AML) with MLL gene rearrangements demonstrate unique gene expression profiles driven by MLL-fusion proteins. Here, we identify the circadian clock transcription factor SHARP1 as a novel oncogenic target in MLL-AF6 AML, which has the worst prognosis among all subtypes of MLL-rearranged AMLs. SHARP1 is expressed solely in MLL-AF6 AML, and its expression is regulated directly by MLL-AF6/DOT1L. Suppression of SHARP1 induces robust apoptosis of human MLL-AF6 AML cells. Genetic deletion in mice delays the development of leukemia and attenuated leukemia-initiating potential, while sparing normal hematopoiesis. Mechanistically, SHARP1 binds to transcriptionally active chromatin across the genome and activates genes critical for cell survival as well as key oncogenic targets of MLL-AF6. Our findings demonstrate the unique oncogenic role for SHARP1 in MLL-AF6 AML.