CBFß-SMMHC Inhibition Triggers Apoptosis by Disrupting MYC Chromatin Dynamics in Acute Myeloid Leukemia.

The fusion oncoprotein CBFß-SMMHC, expressed in leukemia cases with chromosome 16 inversion, drives leukemia development and maintenance by altering the activity of the transcription factor RUNX1. Here, we demonstrate that CBFß-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFß-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers, where it represses MYC expression by mediating the replacement of the SWI/SNF complex component BRG1 with the polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFß-SMMHC inhibitor with the BET inhibitor JQ1 eliminates inv(16) leukemia in human cells and a mouse model. Enhancer-interaction analysis indicated that the three enhancers are physically connected with the MYC promoter, and genome-editing analysis demonstrated that they are functionally implicated in deregulation of MYC expression. This study reveals a mechanism whereby CBFß-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.

Disruption of sugar nucleotide clearance is a therapeutic vulnerability of cancer cells.

Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.

Diet-induced developmental acceleration independent of TOR and insulin in C. elegans.

Dietary composition has major effects on physiology. Here, we show that developmental rate, reproduction, and lifespan are altered in C. elegans fed Comamonas DA1877 relative to those fed a standard E. coli OP50 diet. We identify a set of genes that change in expression in response to this diet and use the promoter of one of these (acdh-1) as a dietary sensor. Remarkably, the effects on transcription and development occur even when Comamonas DA1877 is diluted with another diet, suggesting that Comamonas DA1877 generates a signal that is sensed by the nematode. Surprisingly, the developmental effect is independent from TOR and insulin signaling. Rather, Comamonas DA1877 affects cyclic gene expression during molting, likely through the nuclear hormone receptor NHR-23. Altogether, our findings indicate that different bacteria elicit various responses via distinct mechanisms, which has implications for diseases such as obesity and the interactions between the human microbiome and intestinal cells.

Integration of metabolic and gene regulatory networks modulates the C. elegans dietary response.

Expression profiles are tailored according to dietary input. However, the networks that control dietary responses remain largely uncharacterized. Here, we combine forward and reverse genetic screens to delineate a network of 184 genes that affect the C. elegans dietary response to Comamonas DA1877 bacteria. We find that perturbation of a mitochondrial network composed of enzymes involved in amino acid metabolism and the TCA cycle affects the dietary response. In humans, mutations in the corresponding genes cause inborn diseases of amino acid metabolism, most of which are treated by dietary intervention. We identify several transcription factors (TFs) that mediate the changes in gene expression upon metabolic network perturbations. Altogether, our findings unveil a transcriptional response system that is poised to sense dietary cues and metabolic imbalances, illustrating extensive communication between metabolic networks in the mitochondria and gene regulatory networks in the nucleus.

Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag.

Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety.

Repression of essential cell cycle genes increases cellular fitness.

A network of transcription factors (TFs) coordinates transcription with cell cycle events in eukaryotes. Most TFs in the network are phosphorylated by cyclin-dependent kinase (CDK), which limits their activities during the cell cycle. Here, we investigate the physiological consequences of disrupting CDK regulation of the paralogous repressors Yhp1 and Yox1 in yeast. Blocking Yhp1/Yox1 phosphorylation increases their levels and decreases expression of essential cell cycle regulatory genes which, unexpectedly, increases cellular fitness in optimal growth conditions. Using synthetic genetic interaction screens, we find that Yhp1/Yox1 mutations improve the fitness of mutants with mitotic defects, including condensin mutants. Blocking Yhp1/Yox1 phosphorylation simultaneously accelerates the G1/S transition and delays mitotic exit, without decreasing proliferation rate. This mitotic delay partially reverses the chromosome segregation defect of condensin mutants, potentially explaining their increased fitness when combined with Yhp1/Yox1 phosphomutants. These findings reveal how altering expression of cell cycle genes leads to a redistribution of cell cycle timing and confers a fitness advantage to cells.

U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3' End Formation.

Recurrent mutations in the splicing factor U2AF35 are found in several cancers and myelodysplastic syndrome (MDS). How oncogenic U2AF35 mutants promote transformation remains to be determined. Here we derive cell lines transformed by the oncogenic U2AF35(S34F) mutant and identify aberrantly processed pre-mRNAs by deep sequencing. We find that in U2AF35(S34F)-transformed cells the autophagy-related factor 7 (Atg7) pre-mRNA is abnormally processed, which unexpectedly is not due to altered splicing but rather selection of a distal cleavage and polyadenylation (CP) site. This longer Atg7 mRNA is translated inefficiently, leading to decreased ATG7 levels and an autophagy defect that predisposes cells to secondary mutations, resulting in transformation. MDS and acute myeloid leukemia patient samples harboring U2AF35(S34F) have a similar increased use of the ATG7 distal CP site, and previous studies have shown that mice with hematopoietic cells lacking Atg7 develop an MDS-like syndrome. Collectively, our results reveal a basis for U2AF35(S34F) oncogenic activity.

Consequences of aneuploidy in human fibroblasts with trisomy 21.

An extra copy of chromosome 21 causes Down syndrome, the most common genetic disease in humans. The mechanisms contributing to aneuploidy-related pathologies in this syndrome, independent of the identity of the triplicated genes, are not well defined. To characterize aneuploidy-driven phenotypes in trisomy 21 cells, we performed global transcriptome, proteome, and phenotypic analyses of primary human fibroblasts from individuals with Patau (trisomy 13), Edwards (trisomy 18), or Down syndromes. On average, mRNA and protein levels were increased by 1.5-fold in all trisomies, with a subset of proteins enriched for subunits of macromolecular complexes showing signs of posttranscriptional regulation. These results support the lack of evidence for widespread dosage compensation or dysregulation of chromosomal domains in human autosomes. Furthermore, we show that several aneuploidy-associated phenotypes are present in trisomy 21 cells, including lower viability and increased dependency on serine-driven lipid synthesis. Our studies establish a critical role of aneuploidy, independent of triplicated gene identity, in driving cellular defects associated with trisomy 21.

The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage.

Vertebrate mammals express a protein called Ki-67 which is most widely known as a clinically useful marker of highly proliferative cells. Previous studies of human cells indicated that acute depletion of Ki-67 can elicit a delay at the G1/S boundary of the cell cycle, dependent on induction of the checkpoint protein p21. Consistent with those observations, we show here that acute Ki-67 depletion causes hallmarks of DNA damage, and the damage occurs even in the absence of checkpoint signaling. This damage is not observed in cells traversing S phase but is instead robustly detected in mitotic cells. The C-terminal chromatin-binding domain of Ki-67 is necessary and sufficient to protect cells from this damage. We also observe synergistic effects when Ki-67 and p53 are simultaneously depleted, resulting in increased levels of chromosome bridges at anaphase, followed by the appearance of micronuclei. Therefore, these studies identify the C terminus of Ki-67 as an important module for genome stability.

Hinfp is a guardian of the somatic genome by repressing transposable elements.

Germ cells possess the Piwi-interacting RNA pathway to repress transposable elements and maintain genome stability across generations. Transposable element mobilization in somatic cells does not affect future generations, but nonetheless can lead to pathological outcomes in host tissues. We show here that loss of function of the conserved zinc-finger transcription factor Hinfp causes dysregulation of many host genes and derepression of most transposable elements. There is also substantial DNA damage in somatic tissues of Drosophila after loss of Hinfp. Interference of transposable element mobilization by reverse-transcriptase inhibitors can suppress some of the DNA damage phenotypes. The key cell-autonomous target of Hinfp in this process is Histone1, which encodes linker histones essential for higher-order chromatin assembly. Transgenic expression of Hinfp or Histone1, but not Histone4 of core nucleosome, is sufficient to rescue the defects in repressing transposable elements and host genes. Loss of Hinfp enhances Ras-induced tissue growth and aging-related phenotypes. Therefore, Hinfp is a physiological regulator of Histone1-dependent silencing of most transposable elements, as well as many host genes, and serves as a venue for studying genome instability, cancer progression, neurodegeneration, and aging.

Prostaglandin E2 stimulates cAMP signaling and resensitizes human leukemia cells to glucocorticoid-induced cell death.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.

KLF7 promotes pancreatic cancer growth and metastasis by up-regulating ISG expression and maintaining Golgi complex integrity.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Currently, there is no effective therapy for PDAC, and a detailed molecular and functional evaluation of PDACs is needed to identify and develop better therapeutic strategies. Here we show that the transcription factor Krüppel-like factor 7 (KLF7) is overexpressed in PDACs, and that inhibition of KLF7 blocks PDAC tumor growth and metastasis in cell culture and in mice. KLF7 expression in PDACs can be up-regulated due to activation of a MAP kinase pathway or inactivation of the tumor suppressor p53, two alterations that occur in a large majority of PDACs. ShRNA-mediated knockdown of KLF7 inhibits the expression of IFN-stimulated genes (ISGs), which are necessary for KLF7-mediated PDAC tumor growth and metastasis. KLF7 knockdown also results in the down-regulation of Discs Large MAGUK Scaffold Protein 3 (DLG3), resulting in Golgi complex fragmentation, and reduced protein glycosylation, leading to reduced secretion of cancer-promoting growth factors, such as chemokines. Genetic or pharmacologic activation of Golgi complex fragmentation blocks PDAC growth and metastasis similar to KLF7 inhibition. Our results demonstrate a therapeutically amenable, KLF7-driven pathway that promotes PDAC growth and metastasis by activating ISGs and maintaining Golgi complex integrity.

The coordinate actions of calcineurin and Hog1 mediate the stress response through multiple nodes of the cell cycle network.

Upon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the phosphatase calcineurin (CN) in the stress response and demonstrate that CN activates the Hog1/p38 pathway in both yeast and human cells. In yeast, the MAPK Hog1 is transiently activated in response to several well-studied osmostressors. We show that when a stressor simultaneously activates CN and Hog1, CN disrupts Hog1-stimulated negative feedback to prolong Hog1 activation and the period of cell cycle arrest. Regulation of Hog1 by CN also contributes to inactivation of multiple cell cycle-regulatory transcription factors (TFs) and the decreased expression of cell cycle-regulated genes. CN-dependent downregulation of G1/S genes is dependent upon Hog1 activation, whereas CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner to inhibit multiple nodes of the cell cycle-regulatory network. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors.

Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin.

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.

Genomic Characterization of Endothelial Enhancers Reveals a Multifunctional Role for NR2F2 in Regulation of Arteriovenous Gene Expression.

RATIONALE: Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap. OBJECTIVE: To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated. METHODS AND RESULTS: Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, HEY2 (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial HEY2 expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation. CONCLUSIONS: By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of HEY2 established a novel mechanism by which artery-specific expression can be achieved.

Mitochondrial UPR repression during Pseudomonas aeruginosa infection requires the bZIP protein ZIP-3.

Mitochondria generate most cellular energy and are targeted by multiple pathogens during infection. In turn, metazoans employ surveillance mechanisms such as the mitochondrial unfolded protein response (UPRmt) to detect and respond to mitochondrial dysfunction as an indicator of infection. The UPRmt is an adaptive transcriptional program regulated by the transcription factor ATFS-1, which induces genes that promote mitochondrial recovery and innate immunity. The bacterial pathogen Pseudomonas aeruginosa produces toxins that disrupt oxidative phosphorylation (OXPHOS), resulting in UPRmt activation. Here, we demonstrate that Pseudomonas aeruginosa exploits an intrinsic negative regulatory mechanism mediated by the Caenorhabditis elegans bZIP protein ZIP-3 to repress UPRmt activation. Strikingly, worms lacking zip-3 were impervious to Pseudomonas aeruginosa-mediated UPRmt repression and resistant to infection. Pathogen-secreted phenazines perturbed mitochondrial function and were the primary cause of UPRmt activation, consistent with these molecules being electron shuttles and virulence determinants. Surprisingly, Pseudomonas aeruginosa unable to produce phenazines and thus elicit UPRmt activation were hypertoxic in zip-3-deletion worms. These data emphasize the significance of virulence-mediated UPRmt repression and the potency of the UPRmt as an antibacterial response.

The VEGF receptor neuropilin 2 promotes homologous recombination by stimulating YAP/TAZ-mediated Rad51 expression.

Vascular endothelial growth factor (VEGF) signaling in tumor cells mediated by neuropilins (NRPs) contributes to the aggressive nature of several cancers, including triple-negative breast cancer (TNBC), independently of its role in angiogenesis. Understanding the mechanisms by which VEGF-NRP signaling contributes to the phenotype of such cancers is a significant and timely problem. We report that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double-strand break repair. Mechanistically, we provide evidence that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. We also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to cisplatin and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ. These findings reveal roles for VEGF-NRP2 and YAP/TAZ in DNA repair, and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ, and Rad51 that contributes to resistance to platinum chemotherapy.

Prosurvival kinase PIM2 is a therapeutic target for eradication of chronic myeloid leukemia stem cells.

A major obstacle to curing chronic myeloid leukemia (CML) is the intrinsic resistance of CML stem cells (CMLSCs) to the drug imatinib mesylate (IM). Prosurvival genes that are preferentially expressed in CMLSCs compared with normal hematopoietic stem cells (HSCs) represent potential therapeutic targets for selectively eradicating CMLSCs. However, the discovery of such preferentially expressed genes has been hampered by the inability to completely separate CMLSCs from HSCs, which display a very similar set of surface markers. To overcome this challenge, and to minimize confounding effects of individual differences in gene expression profiles, we performed single-cell RNA-seq on CMLSCs and HSCs that were isolated from the same patient and distinguished based on the presence or absence of BCR-ABL. Among genes preferentially expressed in CMLSCs is PIM2, which encodes a prosurvival serine-threonine kinase that phosphorylates and inhibits the proapoptotic protein BAD. We show that IM resistance of CMLSCs is due, at least in part, to maintenance of BAD phosphorylation by PIM2. We find that in CMLSCs, PIM2 expression is promoted by both a BCR-ABL-dependent (IM-sensitive) STAT5-mediated pathway and a BCR-ABL-independent (IM-resistant) STAT4-mediated pathway. Combined treatment with IM and a PIM inhibitor synergistically increases apoptosis of CMLSCs, suppresses colony formation, and significantly prolongs survival in a mouse CML model, with a negligible effect on HSCs. Our results reveal a therapeutically targetable mechanism of IM resistance in CMLSCs. The experimental approach that we describe can be generally applied to other malignancies that harbor oncogenic fusion proteins or other characteristic genetic markers.

Alveolar progenitor cells in the mammary gland are dependent on the ß4 integrin.

Understanding how progenitor cell function is regulated in the mammary gland is an important developmental problem that has significant implications for breast cancer. Although it had been assumed that the expression the α6ß4 integrin (ß4) is restricted to the basal lineage, we report that alveolar progenitor cells in the mouse mammary gland also express this integrin based on analysis of single cell RNA-Seq data. Subsequent experiments using a mouse mammary epithelial cell line (NMuMG) confirmed this finding and revealed that ß4 is essential for maintaining progenitor function as assessed by serial passage mammosphere assays. These data were substantiated by analyzing the alveolar progenitor population isolated from nulliparous mouse mammary glands. Based on the finding that the alveolar progenitor cells express Whey Acidic Protein (WAP), WAP-Cre mice were crossed with itgß4flox/flox mice to generate conditional knock-out of ß4 in alveolar progenitor cells. These itgß4flox/floxWAP-Cre+ mice exhibited significant defects in alveologenesis and milk production during pregnancy compared to itgß4flox/floxWAP-Cre- mice, establishing a novel role for the ß4 integrin in alveolar progenitor function and alveologenesis.