The role of NPM1 alternative splicing in patients with chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with heterogeneous clinical course. Recent studies revealed a link between NOTCH1 mutation and the overexpression of MYC and MYC-related genes involved in ribosome biogenesis and protein biosynthesis, such as nucleophosmin-1 (NPM1), in CLL cells. In the present study, we aim to evaluate the impact of the NOTCH1 mutation on the MYC and MYC induced NPM1 expression in CLL cells via quantification of their transcripts. METHODS: Using qRT-PCR, we analyzed the levels of MYC and three main NPM1 splice variants in 214 samples collected from CLL patients. We assessed the impact of each splice variant on CLL prognostic markers, including the IGHV, TP53, NOTCH1, SF3B1, and MYD88 mutational status, cytogenetic aberrations, and laboratory features. RESULTS: Significantly higher levels of NPM1.R1 transcripts in patients with unmutated compared to mutated IGHV status were found. The median time to first treatment (TTFT) in patients with a high level of NPM1.R1 was significantly shorter compared to the group with low NPM1.R1 levels (1.5 vs 33 months, p = 0.0002). Moreover, in Multivariate Cox Proportional Hazard Regression Model NPM1.R1 splice variant provided an independent prognostic value for TTFT. CONCLUSION: In conclusion, our study indicates the prognostic significance of the level of NPM1.R1 expression and suggests the importance of splicing alterations in the pathogenesis of CLL.

Optogenetics in cancer drug discovery.

INTRODUCTION: The discovery and domestication of biomolecules that respond to light has taken a light of its own, providing new molecular tools with incredible spatio-temporal resolution to manipulate cellular behavior. Areas covered: The authors herein analyze the current optogenetic tools in light of their current, and potential, uses in cancer drug discovery, biosafety and cancer biology. Expert opinion: The pipeline from drug discovery to the clinic is plagued with drawbacks, where most drugs fail in either efficacy or safety. These issues require the redesign of the pipeline and the development of more controllable/personalized therapies. Light is, aside from inexpensive, almost harmless if used appropriately, can be directed to single cells or organs with controllable penetration, and comes in a variety of wavelengths. Light-responsive systems can activate, inhibit or compensate cell signaling pathways or specific cellular events, allowing the specific control of the genome and epigenome, and modulate cell fate and transformation. These synthetic molecular tools have the potential to revolutionize drug discovery and cancer research.

Analysis of NPM1 splice variants reveals differential expression patterns of prognostic value in acute myeloid leukemia.

Mutations of the nucleophosmin-1 (NPM1) gene in cytogenetically normal (CN) acute myeloid leukemia (AML) identify a group of patients with more favorable prognosis. NPM1 encodes three main alternatively spliced isoforms R1(B23.1), R2(B23.2), and R3(B23.3). The expression of splice variants R1, R2 and R3 were higher in AML patients compared to normal cells of healthy volunteers (HVs), although RNA-seq analysis revealed enhanced R2 expression also in less differentiated cells of HVs as well as in AML cells. The variant R2, which lacks exons 11 and 12 coding for the nucleolar localization domain, might behave similar to the mutant form of NPM1 (NPM1mut). In accordance, in CN-AML high R2 expression was associated with favorable impact on outcome. Moreover, functional studies showed nucleolar localization of the eGFP-NPM1 wildtype and cytoplasmic localization of the eGFP-NPM1 mut protein. While the eGFP-NPM1 R2 splice variant localized predominantly in the nucleoplasm, we also could detect cytoplasmic expression for the R2 variant. These results support a unique biological consequence of R2 overexpression and in part explain our clinical observation, where that high R2 variant expression was associated with a better prognosis in CN-AML patients.

Functional relationships between plasmids and their significance for metabolism and symbiotic performance of Rhizobium leguminosarum bv. trifolii.

Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) is a soil bacterium establishing a highly specific symbiotic relationship with clover, which is based on the exchange of molecular signals between the host plant and the microsymbiont. The RtTA1 genome is large and multipartite, composed of a chromosome and four plasmids, which comprise approximately 65 % and 35 % of the total genome, respectively. Extrachromosomal replicons were previously shown to confer significant metabolic versatility to bacteria, which is important for their adaptation in the soil and nodulation competitiveness. To investigate the contribution of individual RtTA1 plasmids to the overall cell phenotype, metabolic properties and symbiotic performance, a transposon-based elimination strategy was employed. RtTA1 derivatives cured of pRleTA1b or pRleTA1d and deleted in pRleTA1a were obtained. In contrast to the in silico predictions of pRleTA1b and pRleTA1d, which were described as chromid-like replicons, both appeared to be completely curable. On the other hand, for pRleTA1a (symbiotic plasmid) and pRleTA1c, which were proposed to be unessential for RtTA1 viability, it was not possible to eliminate them at all (pRleTA1c) or entirely (pRleTA1a). Analyses of the phenotypic traits of the RtTA1 derivatives obtained revealed the functional significance of individual plasmids and their indispensability for growth, certain metabolic pathways, production of surface polysaccharides, autoaggregation, biofilm formation, motility and symbiotic performance. Moreover, the results allow us to suggest broad functional cooperation among the plasmids in shaping the phenotypic properties and symbiotic capabilities of rhizobia.

Phenotype profiling of Rhizobium leguminosarum bv. trifolii clover nodule isolates reveal their both versatile and specialized metabolic capabilities.

Rhizobium leguminosarum bv. trifolii (Rlt) are soil bacteria inducing nodules on clover, where they fix nitrogen. Genome organization analyses of 22 Rlt clover nodule isolates showed that they contained 3-6 plasmids and majority of them possessed large (>1 Mb), chromid-like replicon with exception of four Rlt strains. The Biolog phenotypic profiling comprising utilization of C, N, P, and S sources and tolerance to osmolytes and pH revealed metabolic versatility of the Rlt strains. Statistical analyses of our results showed a clear bias toward specific metabolic preferences, tolerance to unfavorable osmotic conditions, and increased nodulation activity of the strains having smaller amount of extrachromosomal DNA. The K5.4 and K4.15 lacking a large megaplasmid possessed substantially diverse metabolism and belonged to effective clover inoculants. In conclusion, besides overall metabolic versatility, some metabolic specialization may enable rhizobia to persist in variable environments and to compete successfully with other bacteria.

Intragenomic diversity of Rhizobium leguminosarum bv. trifolii clover nodule isolates.

BACKGROUND: Soil bacteria from the genus Rhizobium are characterized by a complex genomic architecture comprising chromosome and large plasmids. Genes responsible for symbiotic interactions with legumes are usually located on one of the plasmids, named the symbiotic plasmid (pSym). The plasmids have a great impact not only on the metabolic potential of rhizobia but also underlie genome rearrangements and plasticity. RESULTS: Here, we analyzed the distribution and sequence variability of markers located on chromosomes and extrachromosomal replicons of Rhizobium leguminosarum bv. trifolii strains originating from nodules of clover grown in the same site in cultivated soil. First, on the basis of sequence similarity of repA and repC replication genes to the respective counterparts of chromids reported in R. leguminosarum bv. viciae 3841 and R. etli CFN42, chromid-like replicons were distinguished from the pool of plasmids of the nodule isolates studied. Next, variability of the gene content was analyzed in the different genome compartments, i.e., the chromosome, chromid-like and 'other plasmids'. The stable and unstable chromosomal and plasmid genes were detected on the basis of hybridization data. Displacement of a few unstable genes between the chromosome, chromid-like and 'other plasmids', as well as loss of some markers was observed in the sampled strains. Analyses of chosen gene sequences allowed estimation of the degree of their adaptation to the three genome compartments as well as to the host. CONCLUSIONS: Our results showed that differences in distribution and sequence divergence of plasmid and chromosomal genes can be detected even within a small group of clover nodule isolates recovered from clovers grown at the same site. Substantial divergence of genome organization could be detected especially taking into account the content of extrachromosomal DNA. Despite the high variability concerning the number and size of plasmids among the studied strains, conservation of the location as well as dynamic distribution of the individual genes (especially replication genes) of a particular genome compartment were demonstrated. The sequence divergence of particular genes may be affected by their location in the given genome compartment. The 'other plasmid' genes are less adapted to the host genome than the chromosome and chromid-like genes.

repABC-based replication systems of Rhizobium leguminosarum bv. trifolii TA1 plasmids: incompatibility and evolutionary analyses.

Soil bacteria of the genus Rhizobium possess complex genomes consisting of a chromosome and in addition, often, multiple extrachromosomal replicons, which are usually equipped with repABC genes that control their replication and partition. The replication regions of four plasmids of Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) were identified and characterized. They all contained a complete set of repABC genes. The structural diversity of the rep regions of RtTA1 plasmids was demonstrated for parS and incα elements, and this was especially apparent in the case of symbiotic plasmid (pSym). Incompatibility assays with recombinant constructs containing parS or incα demonstrated that RtTA1 plasmids belong to different incompatibility groups. Horizontal acquisition was plausibly the main contributor to the origin of RtTA1 plasmids and pSym is probably the newest plasmid of this strain. Phylogenetic and incompatibility analyses of repABC regions of three closely related strains: RtTA1, R. leguminosarum bv. viciae 3841 and Rhizobium etli CFN42, provided data on coexistence of their replicons in a common genomic framework.