ahctf1 and kras mutations combine to amplify oncogenic stress and restrict liver overgrowth in a zebrafish model of hepatocellular carcinoma.

The nucleoporin (NUP) ELYS, encoded by AHCTF1, is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using both larval and adult zebrafish models of hepatocellular carcinoma (HCC), in which the expression of an inducible mutant kras transgene (krasG12V) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly decreases liver volume, while non-hyperplastic tissues are unaffected. We demonstrate that in the context of cancer, ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly, and chromosome segregation, leading to DNA damage and activation of a Tp53-dependent transcriptional programme that induces cell death and cell cycle arrest. Heterozygous expression of both ahctf1 and ranbp2 (encoding a second nucleoporin), or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocks krasG12V-driven hepatocyte hyperplasia. Gene expression analysis of patient samples in the liver hepatocellular carcinoma (LIHC) dataset in The Cancer Genome Atlas shows that high expression of one or more of the transcripts encoding the 10 components of the NUP107-160 subcomplex, which includes AHCTF1, is positively correlated with worse overall survival. These results provide a strong and feasible rationale for the development of novel cancer therapeutics that target ELYS function and suggest potential avenues for effective combinatorial treatments.

PHLPP1 regulates CFTR activity and lumen expansion through AMPK.

Complex organ development depends on single lumen formation and its expansion during tubulogenesis. This can be achieved by correct mitotic spindle orientation during cell division, combined with luminal fluid filling that generates hydrostatic pressure. Using a human 3D cell culture model, we have identified two regulators of these processes. We find that pleckstrin homology leucine-rich repeat protein phosphatase (PHLPP) 2 regulates mitotic spindle orientation, and thereby midbody positioning and maintenance of a single lumen. Silencing the sole PHLPP family phosphatase in Drosophila melanogaster, phlpp, resulted in defective spindle orientation in Drosophila neuroblasts. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) is the main channel regulating fluid transport in this system, stimulated by phosphorylation by protein kinase A and inhibited by the AMP-activated protein kinase AMPK. During lumen expansion, CFTR remains open through the action of PHLPP1, which stops activated AMPK from inhibiting ion transport through CFTR. In the absence of PHLPP1, the restraint on AMPK activity is lost and this tips the balance in the favour of channel closing, resulting in the lack of lumen expansion and accumulation of mucus.

Development of NanoLuc-targeting protein degraders and a universal reporter system to benchmark tag-targeted degradation platforms.

Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12F36V (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic NanoLuc-targeting PROTACs (NanoTACs) to hijack the CRL4CRBN complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate.

The RNA helicase Ddx21 controls Vegfc-driven developmental lymphangiogenesis by balancing endothelial cell ribosome biogenesis and p53 function.

The development of a functional vasculature requires the coordinated control of cell fate, lineage differentiation and network growth. Cellular proliferation is spatiotemporally regulated in developing vessels, but how this is orchestrated in different lineages is unknown. Here, using a zebrafish genetic screen for lymphatic-deficient mutants, we uncover a mutant for the RNA helicase Ddx21. Ddx21 cell-autonomously regulates lymphatic vessel development. An established regulator of ribosomal RNA synthesis and ribosome biogenesis, Ddx21 is enriched in sprouting venous endothelial cells in response to Vegfc-Flt4 signalling. Ddx21 function is essential for Vegfc-Flt4-driven endothelial cell proliferation. In the absence of Ddx21, endothelial cells show reduced ribosome biogenesis, p53 and p21 upregulation and cell cycle arrest that blocks lymphangiogenesis. Thus, Ddx21 coordinates the lymphatic endothelial cell response to Vegfc-Flt4 signalling by balancing ribosome biogenesis and p53 function. This mechanism may be targetable in diseases of excessive lymphangiogenesis such as cancer metastasis or lymphatic malformation.

Disruption of exon-bridging interactions between the minor and major spliceosomes results in alternative splicing around minor introns.

Vertebrate genomes contain major (>99.5%) and minor (<0.5%) introns that are spliced by the major and minor spliceosomes, respectively. Major intron splicing follows the exon-definition model, whereby major spliceosome components first assemble across exons. However, since most genes with minor introns predominately consist of major introns, formation of exon-definition complexes in these genes would require interaction between the major and minor spliceosomes. Here, we report that minor spliceosome protein U11-59K binds to the major spliceosome U2AF complex, thereby supporting a model in which the minor spliceosome interacts with the major spliceosome across an exon to regulate the splicing of minor introns. Inhibition of minor spliceosome snRNAs and U11-59K disrupted exon-bridging interactions, leading to exon skipping by the major spliceosome. The resulting aberrant isoforms contained a premature stop codon, yet were not subjected to nonsense-mediated decay, but rather bound to polysomes. Importantly, we detected elevated levels of these alternatively spliced transcripts in individuals with minor spliceosome-related diseases such as Roifman syndrome, Lowry-Wood syndrome and early-onset cerebellar ataxia. In all, we report that the minor spliceosome informs splicing by the major spliceosome through exon-definition interactions and show that minor spliceosome inhibition results in aberrant alternative splicing in disease.

Early developmental arrest and impaired gastrointestinal homeostasis in U12-dependent splicing-defective Rnpc3-deficient mice.

Splicing is an essential step in eukaryotic gene expression. While the majority of introns is excised by the U2-dependent, or major class, spliceosome, the appropriate expression of a very small subset of genes depends on U12-dependent, or minor class, splicing. The U11/U12 65K protein (hereafter 65K), encoded by RNPC3, is one of seven proteins that are unique to the U12-dependent spliceosome, and previous studies including our own have established that it plays a role in plant and vertebrate development. To pinpoint the impact of 65K loss during mammalian development and in adulthood, we generated germline and conditional Rnpc3-deficient mice. Homozygous Rnpc3-/- embryos died prior to blastocyst implantation, whereas Rnpc3+/- mice were born at the expected frequency, achieved sexual maturity, and exhibited a completely normal lifespan. Systemic recombination of conditional Rnpc3 alleles in adult (Rnpc3lox/lox ) mice caused rapid weight loss, leukopenia, and degeneration of the epithelial lining of the entire gastrointestinal tract, the latter due to increased cell death and a reduction in cell proliferation. Accompanying this, we observed a loss of both 65K and the pro-proliferative phospho-ERK1/2 proteins from the stem/progenitor cells at the base of intestinal crypts. RT-PCR analysis of RNA extracted from purified preparations of intestinal epithelial cells with recombined Rnpc3lox alleles revealed increased frequency of U12-type intron retention in all transcripts tested. Our study, using a novel conditional mouse model of Rnpc3 deficiency, establishes that U12-dependent splicing is not only important during development but is indispensable throughout life.

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

BAK/BAX-Mediated Apoptosis Is a Myc-Induced Roadblock to Reprogramming.

Despite intensive efforts to optimize the process, reprogramming differentiated cells to induced pluripotent stem cells (iPSCs) remains inefficient. The most common combination of transcription factors employed comprises OCT4, KLF4, SOX2, and MYC (OKSM). If MYC is omitted (OKS), reprogramming efficiency is reduced further. Cells must overcome several obstacles to reach the pluripotent state, one of which is apoptosis. To directly determine how extensively apoptosis limits reprogramming, we exploited mouse embryonic fibroblasts (MEFs) lacking the two essential mediators of apoptosis, BAK and BAX. Our results show that reprogramming is enhanced in MEFs deficient in BAK and BAX, but only when MYC is part of the reprogramming cocktail. Thus, the propensity for Myc overexpression to elicit apoptosis creates a significant roadblock to reprogramming under OKSM conditions. Our results suggest that blocking apoptosis during reprogramming may enhance the derivation of iPSCs for research and therapeutic purposes.

Mis-expression of grainyhead-like transcription factors in zebrafish leads to defects in enveloping layer (EVL) integrity, cellular morphogenesis and axial extension.

The grainyhead-like (grhl) transcription factors play crucial roles in craniofacial development, epithelial morphogenesis, neural tube closure, and dorso-ventral patterning. By utilising the zebrafish to differentially regulate expression of family members grhl2b and grhl3, we show that both genes regulate epithelial migration, particularly convergence-extension (CE) type movements, during embryogenesis. Genetic deletion of grhl3 via CRISPR/Cas9 results in failure to complete epiboly and pre-gastrulation embryonic rupture, whereas morpholino (MO)-mediated knockdown of grhl3 signalling leads to aberrant neural tube morphogenesis at the midbrain-hindbrain boundary (MHB), a phenotype likely due to a compromised overlying enveloping layer (EVL). Further disruptions of grhl3-dependent pathways (through co-knockdown of grhl3 with target genes spec1 and arhgef19) confirm significant MHB morphogenesis and neural tube closure defects. Concomitant MO-mediated disruption of both grhl2b and grhl3 results in further extensive CE-like defects in body patterning, notochord and somite morphogenesis. Interestingly, over-expression of either grhl2b or grhl3 also leads to numerous phenotypes consistent with disrupted cellular migration during gastrulation, including embryo dorsalisation, axial duplication and impaired neural tube migration leading to cyclopia. Taken together, our study ascribes novel roles to the Grhl family in the context of embryonic development and morphogenesis.

The Pu.1 target gene Zbtb11 regulates neutrophil development through its integrase-like HHCC zinc finger.

In response to infection and injury, the neutrophil population rapidly expands and then quickly re-establishes the basal state when inflammation resolves. The exact pathways governing neutrophil/macrophage lineage outputs from a common granulocyte-macrophage progenitor are still not completely understood. From a forward genetic screen in zebrafish, we identify the transcriptional repressor, ZBTB11, as critical for basal and emergency granulopoiesis. ZBTB11 sits in a pathway directly downstream of master myeloid regulators including PU.1, and TP53 is one direct ZBTB11 transcriptional target. TP53 repression is dependent on ZBTB11 cys116, which is a functionally critical, metal ion-coordinating residue within a novel viral integrase-like zinc finger domain. To our knowledge, this is the first description of a function for this domain in a cellular protein. We demonstrate that the PU.1-ZBTB11-TP53 pathway is conserved from fish to mammals. Finally, Zbtb11 mutant rescue experiments point to a ZBTB11-regulated TP53 requirement in development of other organs.

The A2b adenosine receptor antagonist PSB-603 promotes oxidative phosphorylation and ROS production in colorectal cancer cells via adenosine receptor-independent mechanism.

PURPOSE: Adenosine is a multifaceted regulator of tumor progression. It modulates immune cell activity as well as acting directly on tumor cells. The A2b adenosine receptor (A2b-AR) is thought to be an important mediator of these effects. In this study we sought to analyze the contribution of the A2b-AR to the behavior of colorectal cancer cells. PRINCIPAL RESULTS: The A2b-AR antagonist PSB-603 changed cellular redox state without affecting cellular viability. Quantification of cellular bioenergetics demonstrated that PSB-603 increased basal oxygen consumption rates, indicative of enhanced mitochondrial oxidative phosphorylation. Unexpectedly, pharmacological and genetic approaches to antagonize AR-related signalling of PSB-603 did not abolish the response, suggesting that it was AR-independent. PSB-603 also induced acute increases in reactive oxygen species, and PSB-603 synergized with chemotherapy treatment to increase colorectal cancer cell death, consistent with the known link between cellular metabolism and chemotherapy response. MAJOR CONCLUSIONS: PSB-603 alters cellular metabolism in colorectal cancer cells and increases their sensitivity to chemotherapy. Although requiring more mechanistic insight into its A2b-AR-independent activity, our results show that PSB-603 may have clinical value as an anti-colorectal cancer therapeutic.

Focusing the Spotlight on the Zebrafish Intestine to Illuminate Mechanisms of Colorectal Cancer.

Colorectal cancer, encompassing colon and rectal cancer, arises from the epithelial lining of the large bowel. It is most prevalent in Westernised societies and is increasing in frequency as the world becomes more industrialised. Unfortunately, metastatic colorectal cancer is not cured by chemotherapy and the annual number of deaths caused by colorectal cancer, currently 700,000, is expected to rise. Our understanding of the contribution that genetic mutations make to colorectal cancer, although incomplete, is reasonably well advanced. However, it has only recently become widely appreciated that in addition to the ongoing accumulation of genetic mutations, chronic inflammation also plays a critical role in the initiation and progression of this disease. While a robust and tractable genetic model of colorectal cancer in zebrafish, suitable for pre-clinical studies, is not yet available, the identification of genes required for the rapid proliferation of zebrafish intestinal epithelial cells during development has highlighted a number of essential genes that could be targeted to disable colorectal cancer cells. Moreover, appreciation of the utility of zebrafish to study intestinal inflammation is on the rise. In particular, zebrafish provide unique opportunities to investigate the impact of genetic and environmental factors on the integrity of intestinal epithelial barrier function. With currently available tools, the interplay between epigenetic regulators, intestinal injury, microbiota composition and innate immune cell mobilisation can be analysed in exquisite detail. This provides excellent opportunities to define critical events that could potentially be targeted therapeutically. Further into the future, the use of zebrafish larvae as hosts for xenografts of human colorectal cancer tissue, while still in its infancy, holds great promise that zebrafish could one day provide a practical, preclinical personalized medicine platform for the rapid assessment of the metastatic potential and drug-sensitivity of patient-derived cancers.

Genetic basis of hindlimb loss in a naturally occurring vertebrate model.

Here we genetically characterise pelvic finless, a naturally occurring model of hindlimb loss in zebrafish that lacks pelvic fin structures, which are homologous to tetrapod hindlimbs, but displays no other abnormalities. Using a hybrid positional cloning and next generation sequencing approach, we identified mutations in the nuclear localisation signal (NLS) of T-box transcription factor 4 (Tbx4) that impair nuclear localisation of the protein, resulting in altered gene expression patterns during pelvic fin development and the failure of pelvic fin development. Using a TALEN-induced tbx4 knockout allele we confirm that mutations within the Tbx4 NLS (A78V; G79A) are sufficient to disrupt pelvic fin development. By combining histological, genetic, and cellular approaches we show that the hindlimb initiation gene tbx4 has an evolutionarily conserved, essential role in pelvic fin development. In addition, our novel viable model of hindlimb deficiency is likely to facilitate the elucidation of the detailed molecular mechanisms through which Tbx4 functions during pelvic fin and hindlimb development.

A hypermorphic epithelial ß-catenin mutation facilitates intestinal tumorigenesis in mice in response to compounding WNT-pathway mutations.

Activation of the Wnt/ß-catenin pathway occurs in the vast majority of colorectal cancers. However, the outcome of the disease varies markedly from individual to individual, even within the same tumor stage. This heterogeneity is governed to a great extent by the genetic make-up of individual tumors and the combination of oncogenic mutations. In order to express throughout the intestinal epithelium a degradation-resistant ß-catenin (Ctnnb1), which lacks the first 131 amino acids, we inserted an epitope-tagged ΔN(1-131)-ß-catenin-encoding cDNA as a knock-in transgene into the endogenous gpA33 gene locus in mice. The resulting gpA33(ΔN-Bcat) mice showed an increase in the constitutive Wnt/ß-catenin pathway activation that shifts the cell fate towards the Paneth cell lineage in pre-malignant intestinal epithelium. Furthermore, 19% of all heterozygous and 37% of all homozygous gpA33(ΔN-Bcat) mice spontaneously developed aberrant crypt foci and adenomatous polyps, at frequencies and latencies akin to those observed in sporadic colon cancer in humans. Consistent with this, the Wnt target genes, MMP7 and Tenascin-C, which are most highly expressed in benign human adenomas and early tumor stages, were upregulated in pre-malignant tissue of gpA33(ΔN-Bcat) mice, but those Wnt target genes associated with excessive proliferation (i.e. Cdnn1, myc) were not. We also detected diminished expression of membrane-associated α-catenin and increased intestinal permeability in gpA33(ΔN-Bcat) mice in challenge conditions, providing a potential explanation for the observed mild chronic intestinal inflammation and increased susceptibility to azoxymethane and mutant Apc-dependent tumorigenesis. Collectively, our data indicate that epithelial expression of ΔN(1-131)-ß-catenin in the intestine creates an inflammatory microenvironment and co-operates with other mutations in the Wnt/ß-catenin pathway to facilitate and promote tumorigenesis.

Glycoprotein A33 deficiency: a new mouse model of impaired intestinal epithelial barrier function and inflammatory disease.

The cells of the intestinal epithelium provide a selectively permeable barrier between the external environment and internal tissues. The integrity of this barrier is maintained by tight junctions, specialised cell-cell contacts that permit the absorption of water and nutrients while excluding microbes, toxins and dietary antigens. Impairment of intestinal barrier function contributes to multiple gastrointestinal disorders, including food hypersensitivity, inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Glycoprotein A33 (GPA33) is an intestinal epithelium-specific cell surface marker and member of the CTX group of transmembrane proteins. Roles in cell-cell adhesion have been demonstrated for multiple CTX family members, suggesting a similar function for GPA33 within the gastrointestinal tract. To test a potential requirement for GPA33 in intestinal barrier function, we generated Gpa33(-/-) mice and subjected them to experimental regimens designed to produce food hypersensitivity, colitis and CAC. Gpa33(-/-) mice exhibited impaired intestinal barrier function. This was shown by elevated steady-state immunosurveillance in the colonic mucosa and leakiness to oral TRITC-labelled dextran after short-term exposure to dextran sodium sulphate (DSS) to injure the intestinal epithelium. Gpa33(-/-) mice also exhibited rapid onset and reduced resolution of DSS-induced colitis, and a striking increase in the number of colitis-associated tumours produced by treatment with the colon-specific mutagen azoxymethane (AOM) followed by two cycles of DSS. In contrast, Gpa33(-/-) mice treated with AOM alone showed no increase in sporadic tumour formation, indicating that their increased tumour susceptibility is dependent on inflammatory stimuli. Finally, Gpa33(-/-) mice displayed hypersensitivity to food allergens, a common co-morbidity in humans with IBD. We propose that Gpa33(-/-) mice provide a valuable model to study the mechanisms linking intestinal permeability and multiple inflammatory pathologies. Moreover, this model could facilitate preclinical studies aimed at identifying drugs that restore barrier function.

Quantitative intratumoural microdistribution and kinetics of (131)I-huA33 antibody in patients with colorectal carcinoma.

BACKGROUND: The ability of recombinant antibodies to adequately penetrate into tumours is a key factor in achieving therapeutic effect; however, the behaviour of antibodies at a cellular level in tumours is poorly understood. The purpose of this study was to investigate those factors that influence the macroscopic and microscopic intratumoural distribution of an IgG1-humanized antibody, huA33, in colorectal tumours. METHODS: Twelve patients were infused with radiolabelled huA33 at 7 days prior to elective surgery for colorectal carcinoma. Macroscopic huA33 uptake was determined by both gamma well counter and autoradiography measurements of the resected tumour specimens. Microscopic uptake was then quantitated at a cellular level and compared to vascular penetrance. The impact of variation in tumour antigen (GPA33) expression, tumour size, specimen type (primary vs metastatic), presence of macroscopic necrosis, and tumour vasculature on huA33 uptake were examined. RESULTS: The I-huA33 uptake in whole tumour sections was (mean ± SD) 5.13 ± 2.71 × 10(-3)% injected dose per gram (ID/g). GPA33 was expressed in all viable tumour cells, and huA33 uptake was excellent regardless of tumour size and specimen type. In tumours with macroscopically evident central necrosis (n = 5), huA33 uptake in tumour necrotic centres was lower than in viable peripheries (0.606 ± 0.493 vs 2.98 ± 2.17 × 10(-3)%ID, p = 0.06). However, when corrected for low cell viability in necrotic centres, uptake of huA33 at the cellular level was highly comparable to that in the more viable tumour periphery (7.10 ± 5.10 × 10(-9) vs 3.82 ± 3.67 × 10(-9)%ID/cell, p = 0.4). In the five patients who exhibited macroscopic necrosis in their tumours, huA33 showed excellent tissue penetration, with a maximum penetration distance of 26 µm in peripheral tumour regions and 118 µm in central regions. No correlation was observed between (131)I-huA33 uptake in tumour on a cellular basis and tumour vascularity. CONCLUSIONS: In patients with colorectal carcinoma, monoclonal antibody huA33 effectively targets viable tumour cells in all cellular milieus examined, including effective penetration into necrotic tumour centres, a novel and therapeutically important finding.

Grainyhead-like 3 regulation of endothelin-1 in the pharyngeal endoderm is critical for growth and development of the craniofacial skeleton.

Craniofacial development is a highly conserved process that requires complex interactions between neural crest cells (NCCs) and pharyngeal tissues derived from all three germ layers. Signals emanating from the pharyngeal endoderm drive differentiation of NCCs into craniofacial cartilage, and disruption of this process underpins several human craniofacial defects (CFD). Here, we demonstrate that morpholino (MO)-mediated knockdown in zebrafish of the highly conserved transcription factor grainyhead-like 3 (grhl3), which is selectively expressed in the pharyngeal endoderm, leads to severe hypoplasia of the lower jaw cartilages. Phylogenetic analysis of conserved grhl-binding sites in gene regulatory regions identified endothelin-1 (edn1) as a putative direct grhl3 target gene, and this was confirmed by chromatin precipitation (ChIP) assays in zebrafish embryos. Injection of sub-phenotypic concentrations of MOs targeting both grhl3 and edn1 induced jaw abnormalities, and injection of edn1 mRNA into grhl3-morphants rescued both pharyngeal expression of the downstream effectors of edn1, and jaw cartilage formation. This study sheds new light on the role of endodermal endothelin-1 in vertebrate jaw development, and highlights potential new genetic defects that could underpin human CFD.

The drug vehicle and solvent N-methylpyrrolidone is an immunomodulator and antimyeloma compound.

N-methyl-2-pyrrolidone (NMP) is a common solvent and drug vehicle. We discovered unexpected antineoplastic and immunomodulatory activity of NMP in a cMYC-driven myeloma model. Coincident to this, NMP was identified as an acetyllysine mimetic and candidate bromodomain ligand. Accordingly, NMP-treated cells demonstrated transcriptional overlap with BET-bromodomain inhibition, including downregulation of cMYC and IRF4. NMP's immunomodulatory activity occurred at sub-BET inhibitory concentrations, and, despite phenotypic similarities to lenalidomide, its antimyeloma activity was independent of the IMiD targets cereblon and Ikaros-1/3. Thus, low-affinity yet broad-spectrum bromodomain inhibition by NMP mediates biologically potent, cereblon-independent immunomodulation and at higher doses targets malignant cells directly via BET antagonism. These data reveal that NMP is a functional acetyllysine mimetic with pleotropic antimyeloma and immunomodulatory activities. Our studies highlight the potential therapeutic benefits of NMP, the consequences of current human NMP exposures, and the need for reassessment of scientific literature where NMP was used as an "inert" drug-delivery vehicle.

Minor class splicing shapes the zebrafish transcriptome during development.

Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.

Physiological expression of the PI3K-activating mutation Pik3ca(H1047R) combines with Apc loss to promote development of invasive intestinal adenocarcinomas in mice.

PIK3CA, the gene encoding the p110α catalytic subunit of PI3K (phosphoinositide 3-kinase), is mutated in approximately 20% of sporadic CRCs (colorectal cancers), but the role of these mutations in the pathogenesis of CRC remains unclear. In the present study we used a novel mouse model to investigate the role of the Pik3caH1047R mutation, the most common PIK3CA mutation in CRC, during the development and progression of intestinal cancer. Our results demonstrate that Pik3caH1047R, when expressed at physiological levels, is insufficient to initiate intestinal tumorigenesis; however, in the context of Apc (adenomatous polyposis coli) loss, which is observed in 80% of CRCs and by itself results in benign intestinal adenomas, the Pik3caH1047R mutation promotes the development of highly aggressive and invasive adenocarcinomas in both the small and large intestines. The results of the present study show that an activating Pik3ca mutation can act in tandem with Apc loss to drive the progression of gastrointestinal cancer and thus this disease may be susceptible to therapeutic targeting using PI3K pathway inhibitors.