Jimmy Larsson

Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia

Mansouri et al. applied targeted deep sequencing to identify mutations within NF-κB core complex genes in CLL. NFKBIE, the gene encoding the inhibitory IκBε molecule, was most frequently mutated, especially in poor-prognostic subgroups of CLL. The authors show that NFKBIE mutations were associated with significantly reduced IkBε expression and p65 inhibition, ultimately leading to NF-κB activation and a more aggressive disease.

Inactivation of Pmel Alters Melanosome Shape But Has Only a Subtle Effect on Visible Pigmentation

PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel −/−). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel −/− melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation.

The gene expression profile of PDGF-treated neural stem cells corresponds to partially differentiated neurons and glia.

We have previously shown that platelet-derived growth factor AA (PDGF-AA) stimulates the expansion of neuronal progenitors from neural stem cells, but is unable to replace fibroblast-growth factor 2 (FGF-2) as a stem cell mitogen. In the present study, we compared gene expression in neural stem cells that were grown in the presence of FGF-2 and in cells cultured with PDGF-AA or in the absence of growth factor, which induces differentiation. The genetic program elicited by PDGF-AA (156 significantly regulated genes) was not unique, but an intermediate between the ones of FGF-2-cultured stem cells and differentiated cells. These observations are compatible with the hypothesis that PDGF-AA induces a partial differentiation of neural stem cells, which retain the ability to proliferate, rather than acting solely as an instructing agent for neuronal differentiation. Finally, the transcriptional signature of stem cells grown with FGF-2 included a large number of genes over-expressed in gliomas and a core set of conserved genes periodically expressed during the eukaryote cell cycle.

Functional genomics of monensin sensitivity in yeast: implications for post-Golgi traffic and vacuolar H+-ATPase function

We have screened a complete collection of yeast knockout mutants for sensitivity to monensin, an ionophore that interferes with intracellular transport. A total of 63 sensitive strains were found. Most of the strains were deleted for genes involved in post-Golgi traffic, with an emphasis on vacuolar biogenesis. A high correlation was thus seen with VPS and VAM genes, but there were also significant differences between the three sets of genes. A weaker correlation was seen with sensitivity to NaCl, in particular rate of growth effects. Interestingly, all 14 genes encoding subunits of the vacuolar H+-ATPase (V-ATPase) were absent in our screen, even though they appeared in the VPS or VAM screens. All monensin-sensitive mutants that could be tested interact synthetically with a deletion of the A subunit of the V-ATPase, Vma1. Synthetic lethality was limited to mutations affecting endocytosis or retrograde transport to Golgi. In addition, vma1 was epistatic over the monensin sensitivity of vacuolar transport mutants, but not endocytosis mutants. Deletions of the two isoforms of the V-ATPase a subunit, Vph1 and Stv1 had opposite effects on the monensin sensitivity of a ypt7 mutant. These findings are consistent with a model where monensin inhibits growth by interfering with the maintenance of an acidic pH in the late secretory pathway. The synthetic lethality of vma1 with mutations affecting retrograde transport to the Golgi further suggests that it is in the late Golgi that a low pH must be maintained.

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development.

Background: Angiogenesis is implicated in many pathological conditions. The role of the proteins involved remains largely unknown, and few vascular‐specific drug targets have been discovered. Previously, in a screen for angiogenesis regulators, we identified Paladin (mouse: X99384, human: KIAA1274), a protein containing predicted S/T/Y phosphatase domains. Results: We present a mouse knockout allele for Paladin with a β‐galactosidase reporter, which in combination with Paladin antibodies demonstrate that Paladin is expressed in the vasculature. During mouse embryogenesis, Paladin is primarily expressed in capillary and venous endothelial cells. In adult mice Paladin is predominantly expressed in arterial pericytes and vascular smooth muscle cells. Paladin also displays vascular‐restricted expression in human brain, astrocytomas, and glioblastomas. Conclusions: Paladin, a novel putative phosphatase, displays a dynamic expression pattern in the vasculature. During embryonic stages it is broadly expressed in endothelial cells, while in the adult it is selectively expressed in arterial smooth muscle cells. Developmental Dynamics 241:770–786, 2012.

Nuclear receptor binding protein 2 is induced during neural progenitor differentiation and affects cell survival.

We previously identified nuclear receptor binding protein 2 (NRBP2) in a screen for genes induced by differentiation of neural stem/progenitor cells. Here we show that during embryonic mouse brain development NRBP2 was expressed in the walls of the third and fourth ventricles, and in the hippocampus. In the adult brain, Purkinje cells of the cerebellum and neurons in the CA3 region of the hippocampus were main sites of NRBP2 expression. Analysis of a pediatric medulloblastoma showed that clusters of NRBP2 positive tumor cells co-expressed neurofilament, but not GFAP. Thus, NRBP2 was associated with neuronal differentiation both in normal and malignant brain tissue. We report that NRBP2 is a 55-60 kDa protein with mainly cytoplasmic location. In vitro, NRBP2 protein levels increased as neural stem/progenitor cells differentiated, and its down regulation by siRNA rendered neural progenitor cells more vulnerable to apoptosis. NRBP2 has no previously assigned function and our studies suggest a role for NRBP2 in neural progenitor cell survival.

Transposon mutagenesis reveals fludarabine-resistance mechanisms in chronic lymphocytic leukemia

Purpose: To identify resistance mechanisms for the chemotherapeutic drug fludarabine in chronic lymphocytic leukemia (CLL), as innate and acquired resistance to fludarabine-based chemotherapy represents a major challenge for long-term disease control. Experimental Design: We used piggyBac transposon-mediated mutagenesis, combined with next-generation sequencing, to identify genes that confer resistance to fludarabine in a human CLL cell line. Results: In total, this screen identified 782 genes with transposon integrations in fludarabine-resistant pools of cells. One of the identified genes is a known resistance mediator DCK (deoxycytidine kinase), which encodes an enzyme that is essential for the phosphorylation of the prodrug to the active metabolite. BMP2K, a gene not previously linked to CLL, was also identified as a modulator of response to fludarabine. In addition, 10 of 782 transposon-targeted genes had previously been implicated in treatment resistance based on somatic mutations seen in patients refractory to fludarabine-based therapy. Functional characterization of these genes supported a significant role for ARID5B and BRAF in fludarabine sensitivity. Finally, pathway analysis of transposon-targeted genes and RNA-seq profiling of fludarabine-resistant cells suggested deregulated MAPK signaling as involved in mediating drug resistance in CLL. Conclusions: To our knowledge, this is the first forward genetic screen for chemotherapy resistance in CLL. The screen pinpointed novel genes and pathways involved in fludarabine resistance along with previously known resistance mechanisms. Transposon screens can therefore aid interpretation of cancer genome sequencing data in the identification of genes modifying sensitivity to chemotherapy. Clin Cancer Res; 22(24); 6217–27. ©2016 AACR.

In situ genotyping of a pooled strain library after characterizing complex phenotypes

In this work, we present a proof‐of‐principle experiment that extends advanced live cell microscopy to the scale of pool‐generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single‐molecule fluorescence time‐lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E. coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter.

Somatic PRDM2 c.4467delA mutations in colorectal cancers control histone methylation and tumor growth

The chromatin modifier PRDM2/RIZ1 is inactivated by mutation in several forms of cancer and is a putative tumor suppressor gene. Frameshift mutations in the C-terminal region of PRDM2, affecting (A)8 or (A)9 repeats within exon 8, are found in one third of colorectal cancers with microsatellite instability, but the contribution of these mutations to colorectal tumorigenesis is unknown. To model somatic mutations in microsatellite unstable tumors, we devised a general approach to perform genome editing while stabilizing the mutated nucleotide repeat. We then engineered isogenic cell systems where the PRDM2 c.4467delA mutation in human HCT116 colorectal cancer cells was corrected to wild-type by genome editing. Restored PRDM2 increased global histone 3 lysine 9 dimethylation and reduced migration, anchorage-independent growth and tumor growth in vivo. Gene set enrichment analysis revealed regulation of several hallmark cancer pathways, particularly of epithelial-to-mesenchymal transition (EMT), with VIM being the most significantly regulated gene. These observations provide direct evidence that PRDM2 c.4467delA is a driver mutation in colorectal cancer and confirms PRDM2 as a cancer gene, pointing to regulation of EMT as a central aspect of its tumor suppressive action.

Linking FOXO3 , NCOA3 , and TCF7L2 to Ras pathway phenotypes through a genome-wide forward genetic screen in human colorectal cancer cells

BackgroundThe Ras pathway genes KRAS, BRAF, or ERBBs have somatic mutations in ~ 60% of human colorectal carcinomas. At present, it is unknown whether the remaining cases lack mutations activating the Ras pathway or whether they have acquired mutations in genes hitherto unknown to belong to the pathway.MethodsTo address the second possibility and extend the compendium of Ras pathway genes, we used genome-wide transposon mutagenesis of two human colorectal cancer cell systems deprived of their activating KRAS or BRAF allele to identify genes enabling growth in low glucose, a Ras pathway phenotype, when targeted.ResultsOf the 163 recurrently targeted genes in the two different genetic backgrounds, one-third were known cancer genes and one-fifth had links to the EGFR/Ras/MAPK pathway. When compared to cancer genome sequencing datasets, nine genes also mutated in human colorectal cancers were identified. Among these, stable knockdown of FOXO3, NCOA3, and TCF7L2 restored growth in low glucose but reduced MEK/MAPK phosphorylation, reduced anchorage-independent growth, and modulated expressions of GLUT1 and Ras pathway related proteins. Knockdown of NCOA3 and FOXO3 significantly decreased the sensitivity to cetuximab of KRAS mutant but not wild-type cells.ConclusionsThis work establishes a proof-of-concept that human cell-based genome-wide forward genetic screens can assign genes to pathways with clinical importance in human colorectal cancer.