Smaller Isoform Research Articles

SF3B1 Mutations Induce Oncogenic IRAK4 Isoforms and Activate Targetable Innate Immune Pathways in MDS and AML

SF3B1 mutations are the most frequently occurring splicing factor mutations in MDS and AML, however the misspliced genes that contribute the malignant state in SF3B mutant MDS or AML remains unclear. We determined that SF3B1 mutant cases of MDS express a longer, active isoform of interleukin 1 receptor associated kinase (IRAK4). IRAK4 is a serine/threonine kinase that is downstream of toll-like receptor (TLR) signaling and leads to activation of oncogenic signaling states, including NF-kB and MAPK. Examination of IRAK4 by RNA sequencing showed that normal cells predominantly express small IRAK4 isoforms resulting from exclusion of the part of exon 6. These isoforms are targeted for proteosomal degradation leading to diminished IRAK4 expression and activation in normal cells. In contrast, a large proportion of MDS/AML samples with SF3B1 mutation show increased expression of an IRAK4 isoform that retains full exon 6, encoding the full-length protein (IRAK4-Long). Consequently, we show that expression of mutant SF3B1-K700E in leukemic cells is associated with increased NF-kB activity, suggesting that mutations in SF3B1 instruct expression of IRAK4 RNA isoforms with maximal functional potential. Furthermore, SF3B1 mutant MDS and AML cells exhibited a block in hematopoietic differentiation in clonogenic assays. This differentiation block was ameliorated with pharmacologic inhibition of IRAK4 with CA-4948, a potent oral clinically useful small-molecule inhibitor of IRAK4. CA-4948 blocked TLR-stimulated cytokine release in various cell models and also led to decreased leukemic burden in mice xenografted with SF3B1 mutant MDS/AML cells. Finally, we determined that SF3B1 mutation induced IRAK4 activation led to TRAF6 mediated K63 ubiquitination of critical cell cycle and regulatory proteins directly implicated in oncogenesis. We had recently shown that U2AF1 mutations can lead to IRAK4 activation via retention of exon 4 (Smith et al, Nat Cell Bio, 2019). Our data now demonstrate that SF3B1 leads to overactivation of IRAK4 via retention of a different exon (exon 6), thus reinforcing that IRAK/TRAF6 activation is a common downstream oncogenic pathway in splicing factor mutated MDS/AML. Taken together, in this study, we find that mutations in SF3B1 induce expression of therapeutically targetable "active" IRAK4 isoforms and provide a genetic link between a spliceosome mutation and oncogenic innate immune signaling in MDS and AML. Disclosures Booher: Curis: Employment. Ramachandra:Aurigene: Employment. Samson:Curis: Employment. Will:Novartis Pharmaceuticals: Research Funding. Steidl:BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; Aileron Therapeutics: Consultancy, Research Funding. Starczynowski:Kurome Therapeutics: Consultancy. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria.

Unexpected Compensatory Increase in Shank3 Transcripts in Shank3 Knock-Out Mice Having Partial Deletions of Exons.

Genetic variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, which encodes excitatory postsynaptic core scaffolds cause numerous brain disorders. Several lines of Shank3 knock-out (KO) mice with deletions of different Shank3 exons have previously been generated and characterized. The different Shank3 KO mouse lines have both common and line-specific phenotypes. Shank3 isoform diversity is considered a mechanism underlying phenotypic heterogeneity, and compensatory changes through regulation of Shank3 expression may contribute to this heterogeneity. However, whether such compensatory changes occur in Shank3 KO mouse lines has not been investigated in detail. Using previously reported RNA-sequencing analyses, we identified an unexpected increase in Shank3 transcripts in two different Shank3 mutant mouse lines (Shank3B and Shank3ΔC) having partial deletions of Shank3 exons. We validated an increase in Shank3 transcripts in the hippocampus, cortex, and striatum, but not in the cerebellum, of Shank3B heterozygous (HET) and KO mice, using qRT-PCR analyses. In particular, expression of the N-terminal exons 1–12, but not the more C-terminal exons 19–22, was observed to increase in Shank3B mice with deletion of exons 13–16. This suggests a selective compensatory activation of upstream Shank3 promoters. Furthermore, using domain-specific Shank3 antibodies, we confirmed that the increased Shank3 transcripts in Shank3B KO mice produced a small Shank3 isoform that was not detected in wild-type mice. Taken together, our results illustrate another layer of complexity in the regulation of Shank3 expression in the brain, which may also contribute to the phenotypic heterogeneity of different Shank3 KO mouse lines.

Assembling the jigsaw puzzle: CBX2 isoform 2 and its targets in disorders/differences of sex development

Background One of the defining moments of human life occurs early during embryonic development, when individuals sexually differentiate into either male or female. Perturbation of this process can lead to disorders/differences of sex development (DSD). Chromobox protein homolog 2 (CBX2) has two distinct isoforms, CBX2.1 and CBX2.2: the role of CBX2.1 in DSD has been previously established, yet to date the function of the smaller isoform CBX2.2 remains unknown. Methods The genomic DNA of two 46,XY DSD patients was analysed using whole exome sequencing. Furthermore, protein/DNA interaction studies were performed using DNA adenine methyltransferase identification (DamID) to identify putative binding partners of CBX2. Finally, in vitro functional studies were used to elucidate the effect of wild-type and variant CBX2.2 on selected downstream targets. Results Here, we describe two patients with features of DSD i.e. atypical external genitalia, perineal hypospadias and no palpable gonads, each patient carrying a distinct CBX2.2 variant, p.Cys132Arg (c.394T>C) and p.Cys154fs (c.460delT). We show that both CBX2.2 variants fail to regulate the expression of genes essential for sexual development, leading to a severe 46,XY DSD defect, likely because of a defective expression of EMX2 in the developing gonad. Conclusion Our study indicates a distinct function of the shorter form of CBX2 and by identifying several of its unique targets, can advance our understanding of DSD pathogenesis and ultimately DSD diagnosis and management.

Pannexin-1 limits the production of proinflammatory cytokines during necroptosis.

The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) controls the execution of necroptosis, a regulated form of necrosis that occurs in apoptosis-deficient conditions. Active oligomerized MLKL triggers the exposure of phosphatidylserine residues on the cell surface and disrupts the plasma membrane integrity by forming lytic pores. MLKL also governs endosomal trafficking and biogenesis of small extracellular vesicles as well as the production of proinflammatory cytokines during the early steps of necroptosis; however, the molecular basis continues to be elucidated. Here, we find that MLKL oligomers activate Pannexin-1 (PANX1) channels, concomitantly to the loss of phosphatidylserine asymmetry. This plasma membrane "leakiness" requires the small GTPase RAB27A and RAB27B isoforms, which regulate intracellular vesicle trafficking, docking, and fusion with the plasma membrane. Although cells in which PANX1 is silenced or inhibited normally undergo necroptotic death, they display enhanced production of cytokines such as interleukin-8, indicating that PANX1 may tamper with inflammation. These data identify a novel signaling nexus between MLKL, RAB27, and PANX1 and propose ways to interfere with inflammation associated with necroptosis.

Abstract 5159: Isoform specific expression of cadherin10 is associated with progression and drug resistance in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer related death worldwide accounting for 1.69 million of total 8.8 million cancer deaths in 2015 alone. Nearly 85% of all lung cancer cases are classified as non-small cell lung cancer (NSCLC) with a 5-year survival rate of 16%, despite of all advanced targeted treatment modalities. Like any other cancer, cadherin proteins play a critical role in lung cancer progression, metastasis and drug resistance. Classical type I and type II cadherin proteins are calcium-dependent transmembrane glycoproteins with a cytoplasmic tail that mediates downstream signaling and a large extracellular N-terminal head that engages in homophilic and heterophilic adhesion. While alterations of type I cadherins (N-cadherin and E-cadherin) are well studied, functions as well as alterations of type II cadherins, such as cadherin-8, -9, -10, -12, and -18, remain largely unknown. Recent whole genome sequencing of patient samples towards Precision Medicine initiatives revealed 2,687 donors affected by 9,151 cadherin10 (CDH10) mutations alone across the 63 cancer projects worldwide. The majority of mutations in CDH10 are missense substitutions largely distributed throughout the entire gene length, without any specific hotspot. CDH10 copy number amplification is also reported in a significantly large number of lung cancer patients. Alternative splicing of this gene potentially results in multiple transcript variants. To resolve the conundrum of Loss-of-heterozygosity (LOH) and copy number amplification, we decided to look further in to expression and function of CDH10 using array of lung cancer cell lines and human lung cancer patient samples. Here we report, for the first time, that full-length CDH10 expression as well as the tumor suppressive function are compromised in the course of NSCLC development, while aberrant smaller isoforms appear in the cell lines and tumors that drive the oncogenic progression. Expression of such smaller isoforms were further altered when the cells were treated with commonly used chemotherapy drugs, such as Cisplatin, Doxorubicin and Paclitaxel indicating a direct relationship between drug resistance and appearance of these smaller isoforms. We found that the downregulation of full-length CDH10 using siRNA results in an increase in proliferation and invasion, indicating a potential anti-oncogenic function of full-length CDH10 protein. Therefore, we found that mutations and alternative splicing potentially downregulates full-length CDH10 expression along with its tumor suppressive function. However, appearance of smaller aberrant isoforms accounts for tumorigenic progression and drug resistance. Citation Format: Rajib Ghosh, Phattrakorn Powan, Liying W. Rojanasakul, Yon Rojanasakul. Isoform specific expression of cadherin10 is associated with progression and drug resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5159.

Gene Regulatory Networks Governing the Generation and Regeneration of Blood

Blood is an example of a highly regenerative tissue and its regeneration depends on the presence of stem cells residing in the bone marrow in humans. A better understanding of how these stem cells are programmed would benefit their use in clinical practice and shed light on the mechanisms by which the unique properties of stem cells are established. Our approach is to delineate the gene regulatory networks (GRNs) that specify these cells during their development in the embryo, and we use the amphibian experimental model because a wealth of evidence shows that the mechanisms used are conserved in mammals including humans. Blood stem cells are made during the intraembryonic wave of hematopoiesis during embryonic development where they emerge from endothelial precursors in the floor of the dorsal aorta (DA). These cells are derived from lateral plate mesoderm and so we have focused on the subset of cells in the lateral plate mesoderm fated to become blood and endothelium known as definitive hemangioblasts. We have found that their programming results from the activities of vascular endothelial growth factor A (VEGFA) and bone morphogenetic protein (BMP) signaling and the inhibition by miRNA of transforming growth factor beta signaling. VEGFA is first generated in the somites adjacent to the lateral plate mesoderm, and one of the responses of the lateral plate mesoderm is to activate endogenous VEGFA expression. BMP has multiple inputs into the programming of these cells via the activation of the transcription factor (TF), Gata2, and of the VEGFA receptor. These actions culminate in the expression of the leukemia-associated TF, Scl/Tal1, which is essential for blood fate specification. The activity of VEGFA in driving endothelial development resides in the small isoform, but the medium and large isoforms are required to initiate the blood stem cell program in the floor of the DA. The expression of the small isoform is dependent on the blood TF with leukemia connections, Tel1/Etv6, whereas the larger isoforms depend on another transcription-associated factor with leukemia connections, Eto2, raising the possibility that the regulation of VEGFA expression may be the mode of action of these leukemic factors. The action of Tel1/Etv6 in directly activating VEGFA expression in the somites was unexpected because this TF had only been reported to repress transcription. Using chromatin immunoprecipitation technology, we were able to show that Tel1/Etv6 does indeed work by repressing the expression of a VEGFA repressor, FoxC3, but it also acts directly to activate VEGFA expression, working together with Klf4. Finally, we have also looked at the mesodermal population that gives rise to the earlier waves of hematopoiesis, which do not generate a stem cell. We find significant differences including differential use of TFs of the E-Twenty-Six (ETS) family. In conclusion, we have elucidated the GRN responsible for preparing the lateral mesoderm for blood stem cell production.

T128. Regulation of Dendritic Spine Morphology by Small Isoform of Ankyrin-G and Homer1

T128. Regulation of Dendritic Spine Morphology by Small Isoform of Ankyrin-G and Homer1

Rab39a and Rab39b Display Different Intracellular Distribution and Function in Sphingolipids and Phospholipids Transport.

Rab GTPases define the identity and destiny of vesicles. Some of these small GTPases present isoforms that are expressed differentially along developmental stages or in a tissue-specific manner, hence comparative analysis is difficult to achieve. Here, we describe the intracellular distribution and function in lipid transport of the poorly characterized Rab39 isoforms using typical cell biology experimental tools and new ones developed in our laboratory. We show that, despite their amino acid sequence similarity, Rab39a and Rab39b display non-overlapping intracellular distribution. Rab39a localizes in the late endocytic pathway, mainly at multivesicular bodies. In contrast, Rab39b distributes in the secretory network, at the endoplasmic reticulum/cis-Golgi interface. Therefore, Rab39a controls trafficking of lipids (sphingomyelin and phospholipids) segregated at multivesicular bodies, whereas Rab39b transports sphingolipids biosynthesized at the endoplasmic reticulum-Golgi factory. Interestingly, lyso bis-phosphatidic acid is exclusively transported by Rab39a, indicating that both isoforms do not exert identical functions in lipid transport. Conveniently, the requirement of eukaryotic lipids by the intracellular pathogen Chlamydia trachomatis rendered useful for dissecting and distinguishing Rab39a- and Rab39b-controlled trafficking pathways. Our findings provide comparative insights about the different subcellular distribution and function in lipid transport of the two Rab39 isoforms.

Sumoylation of a small isoform of NFATc1 is promoted by PIAS proteins and inhibits transactivation activity

The NFAT family of transcription factors plays an important role in immune system development and function. NFATc1 and NFATc2 are highly expressed in peripheral T cells, and several isoforms are produced via the use of different promoters and polyadenylation sites. The specific isoforms with relatively long C-termini, NFATc1/C and NFATc2/A, have been shown to be modified by SUMO within their specific C-terminal regions, which regulates NFAT protein localization and transactivation activity. Here, we demonstrate that an isoform NFATc1/A, which has a short C-terminus and does not contain the sumoylation sites found in the long isoforms, is also modified by SUMO. NFATc1/A sumoylation increased with low level expression of SUMO E3 ligases, specifically PIAS1, PIAS3, and PIASy, in co-transfected cells. PIAS3 interacted with NFATc1/A and an active site mutant failed to promote NFATc1/A sumoylation, indicating a role for PIAS3 as a SUMO E3 ligase. A lysine residue at 351 within the central regulatory domain was identified as the major SUMO attachment site in both co-transfection and in vitro assays. Sumoylation of NFATc1/A did not affect nuclear translocation upon ionomycin and phorbol 12-myristate 13-acetate treatment. However, although sumoylation of NFATc1/A slightly increased protein stability, it inhibited transactivation activity for reporter genes driven by promoters containing NFAT sites. Our results indicate that the transactivation activity of NFATc1/A is negatively regulated by PIAS protein-mediated sumoylation, and that SUMO is a general regulator of NFAT family members with either long or short C-termini.

Distribution of sperm antigen 6 (SPAG6) and 16 (SPAG16) in mouse ciliated and non-ciliated tissues.

The cilia and flagella of eukaryotic cells serve many functions, exhibiting remarkable conservation of both structure and molecular composition in widely divergent eukaryotic organisms. SPAG6 and SPAG16 are the homologous in the mice to Chlamydomonas reinhardtii PF16 and PF20. Both proteins are associated with the axonemal central apparatus and are essential for ciliary and flagellar motility in mammals. Recent data derived from high-throughput studies revealed expression of these genes in tissues that do not contain motile cilia. However, the distribution of SPAG6 and SPAG16 in ciliated and non-ciliated tissues is not completely understood. In this work, we performed a quantitative analysis of the expression of Spag6 and Spag16 genes in parallel with the immune-localization of the proteins in several tissues of adult mice. Expression of mRNA was higher in the testis and tissues bearing motile cilia than in the other analyzed tissues. Both proteins were present in ciliated and non-ciliated tissues. In the testis, SPAG6 was detected in spermatogonia, spermatocytes, and in the sperm flagella whereas SPAG16 was found in spermatocytes and in the sperm flagella. In addition, both proteins were detected in the cytoplasm of cells from the brain, spinal cord, and ovary. A small isoform of SPAG16 was localized in the nucleus of germ cells and some neurons. In a parallel set of experiments, we overexpressed EGFP-SPAG6 in cultured cells and observed that the protein co-localized with a subset of acetylated cytoplasmic microtubules. A role of these proteins stabilizing the cytoplasmic microtubules of eukaryotic cells is discussed.

Interaction between the BAG1S isoform and HSP70 mediates the stability of anti-apoptotic proteins and the survival of osteosarcoma cells expressing oncogenic MYC

BackgroundThe oncoprotein MYC has the dual capacity to drive cell cycle progression or induce apoptosis, depending on the cellular context. BAG1 was previously identified as a transcriptional target of MYC that functions as a critical determinant of this cell fate decision. The BAG1 protein is expressed as multiple isoforms, each having an array of distinct biochemical functions; however, the specific effector function of BAG1 that directs MYC-dependent cell survival has not been defined.MethodsIn our studies the human osteosarcoma line U2OS expressing a conditional MYC-ER allele was used to induce oncogenic levels of MYC. We interrogated MYC-driven survival processes by modifying BAG1 protein expression. The function of the separate BAG1 isoforms was investigated by depleting cells of endogenous BAG1 and reintroducing the distinct isoforms. Flow cytometry and immunoblot assays were performed to analyze the effect of specific BAG1 isoforms on MYC-dependent apoptosis. These experiments were repeated to determine the role of the HSP70 chaperone complex in BAG1 survival processes. Finally, a proteomic approach was used to identify a set of specific pro-survival proteins controlled by the HSP70/BAG1 complex.ResultsLoss of BAG1 resulted in robust MYC-induced apoptosis. Expression of the larger isoforms of BAG1, BAG1L and BAG1M, were insufficient to rescue survival in cells with oncogenic levels of MYC. Alternatively, reintroduction of BAG1S significantly reduced the level of apoptosis. Manipulation of the BAG1S interaction with HSP70 revealed that BAG1S provides its pro-survival function by serving as a cofactor for the HSP70 chaperone complex. Via a proteomic approach we identified and classified a set of pro-survival proteins controlled by this HSP70/BAG1 chaperone complex that contribute to the BAG1 anti-apoptotic phenotype.ConclusionsThe small isoform of BAG1, BAG1S, in cooperation with the HSP70 chaperone complex, selectively mediates cell survival in MYC overexpressing tumor cells. We identified a set of specific pro-survival clients controlled by the HSP70/BAG1S chaperone complex. These clients define new nodes that could be therapeutically targeted to disrupt the survival of tumor cells driven by MYC activation. With MYC overexpression occurring in most human cancers, this introduces new strategies for cancer treatment.

Role of CD97 small isoform in human cervical carcinoma.

Various studies revealed that elevated expression of CD97 in carcinomas is associated with the dedifferentiation, aggressiveness and metastasis of tumour. CD97 is a protein member of the epidermal growth factor seven-transmembrane (EGF-TM7) family of class II TM7 receptors. Our previous study suggested that the overexpression of CD97 in cervical cancer was correlated with the aggressiveness of the tumour and that CD97 might be an independent poor prognostic factor for cervical cancer patients. Based on these data, we have investigated the role of CD97 small isoform in cervical cancer proliferation, migration and invasion in vitro. Three cervical cancer cell lines were tested and the CD97 small isoform was found to be expressed predominantly in the SiHa cells. The mobile and invasive ability of different cervical cancer cell lines was not correlated positively with total CD97 protein expression but was with the level of the CD97 small isoform. Functional significance was assessed 48hours after transient knockdown using siRNA targeting CD97 small isoform (CD97/EGF1,2,5) or a scrambled control sequence in cervical cancer cell lines. As a result, decreased ability of migration and invasion was found in CD97 small isoform RNAi cells, which showed, however, no change in cell proliferation. This study shed light on the role of the CD97 small isoform in tumour progression and provides a basis for further studies to determine its function in vivo.

Adenosine Deaminase in Synovial Fluid at Rheumatoid Arthritis is Citrullinated

AIM: The objective of this article is to compare accumulation and citrullination of small (SADA) and large (LADA) isoforms of adenosine deaminase (ADA) in the synovial fluids (SFs) of patients with rheumatoid arthritis (RA), ankylosing spondylitis, gout, juvenile idiopathic and reactive arthritis. MATERIALS AND METHODS: The SFs of patients, diagnosed in the Medical Center “Muratsan”, were taken from knee joints. The methods of ion-exchange and gel-chromatography were applied for separation and purification of ADA isoforms from the SFs of above mentioned five arthritis types. The activities and citrullination state of ADA isoforms were studied using appropriate colorimetric assays. RESULTS: This work revealed accumulation of SADA in RA SFs and of LADA in the SFs of four other arthritis types. The average protein citrullination, measured as A 530 /A 280 ratio, was of 0.07±0.04 (n=5) in SADA purified from RA SFs with the initial ADA activity between 35-60 U/L. In SADA samples from RA SFs with the initial ADA activity of 70-190 U/L, the average value of protein citrullination was higher by an order (0.83±0.28, n=8). The citrullination of LADA was not registered in any of all five studied arthritis types. CONCLUSION: In SFs of RA, the citrullinated SADA is accumulated. In four other studied arthritis types, LADA is accumulated. One can suggest: in RA the citrullination of SADA prevents its binding to dipeptidyl peptidase IV and transformation to LADA. The obtained data permit the assumption of being SADA as a new citrulline containing auto-antigen applicable in RA diagnosis.

Anion-Mediated Effects on the Size and Mechanical Properties of Enzymatically Crosslinked Suckerin Hydrogels.

The suckerin family of proteins, identified from the squid sucker ring teeth assembly, offers unique mechanical properties and potential advantages over other natural biomaterials. In this study, a small suckerin isoform, suckerin-12, is used to create enzymatically crosslinked, macro-scale hydrogels. Upon exposure to specific salt conditions, suckerin-12 hydrogels contracted into a condensed state where mechanical properties are found to be modulated by the salt anion present. The rate of contraction is found to correlate well with the kosmotropic arm of the Hofmeister anion series. However, the observed changes in hydrogel mechanical properties are better explained by the ability of the salt to neutralize charges in suckerin-12 by deprotonization or charge screening. Thus, by altering the anions in the condensing salt solution, it is possible to tune the mechanical properties of suckerin-12 hydrogels. The potential for suckerins to add new properties to materials based on naturally-derived proteins is highlighted.

Antifreeze Glycoproteins Bind Irreversibly to Ice.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) inhibit ice growth via an adsorption-inhibition mechanism that assumes irreversible binding of AF(G)Ps to embryonic ice crystals and the inhibition of further growth. The irreversible binding of antifreeze glycoproteins (AFGPs) to ice has been questioned and remains poorly understood. Here, we used microfluidics and fluorescence microscopy to investigate the nature of the binding of small and large AFGP isoforms. We found that both AFGP isoforms bind irreversibly to ice, as evidenced by microfluidic solution exchange experiments. We measured the adsorption rate of the large AFGP isoform and found it to be 50% faster than that of AFP type III. We also found that the AFGP adsorption rate decreased by 65% in the presence of borate, a well-known inhibitor of AFGP activity. Our results demonstrate that the adsorption rate of AFGPs to ice is crucial for their ice growth inhibition capability.

Genome-Wide Association Analyses Reveal the Importance of Alternative Splicing in Diversifying Gene Function and Regulating Phenotypic Variation in Maize.

Alternative splicing (AS) enhances transcriptome diversity and plays important roles in regulating plant processes. Although widespread natural variation in AS has been observed in plants, how AS is regulated and contribute to phenotypic variation is poorly understood. Here, we report a population-level transcriptome assembly and genome-wide association study to identify splicing quantitative trait loci (sQTLs) in developing maize (Zea mays) kernels from 368 inbred lines. We detected 19,554 unique sQTLs for 6570 genes. Most sQTLs showed small isoform usage changes without involving major isoform switching between genotypes. The sQTL-affected isoforms tend to display distinct protein functions. We demonstrate that nonsense-mediated mRNA decay, microRNA-mediated regulation, and small interfering peptide-mediated peptide interference are frequently involved in sQTL regulation. The natural variation in AS and overall mRNA level appears to be independently regulated with different cis-sequences preferentially used. We identified 214 putative trans-acting splicing regulators, among which ZmGRP1, encoding an hnRNP-like glycine-rich RNA binding protein, regulates the largest trans-cluster. Knockout of ZmGRP1 by CRISPR/Cas9 altered splicing of numerous downstream genes. We found that 739 sQTLs colocalized with previous marker-trait associations, most of which occurred without changes in overall mRNA level. Our findings uncover the importance of AS in diversifying gene function and regulating phenotypic variation.

Abstract 3341: Inhibiting the destruction of the oncogene ZNF217 promotes breast cancer metastasis to lung

Abstract The oncogene and transcription factor ZNF217 is overexpressed in 20-30% of breast cancers. Its overexpression correlates strongly with poor prognosis in patients and causes accelerated tumor progression, metastasis, and chemoresistance in vivo. While several studies have examined the regulation of ZNF217 at the gene and mRNA levels, little is known about how ZNF217 is regulated as a protein. In this study we identify the regions of ZNF217 that are required for protein turnover and other cancer phenotypes, including increased invasion and metastasis. Due to the high expression and aberrant localization of ZNF217 in some human breast tumors, both ZNF217 protein expression levels and localization of ZNF217 may be critical determinants of ZNF217's function in vivo. To investigate the role of ZNF217 protein on cancer progression, we first examined ZNF217 protein expression in human breast tumors by Western analysis. In addition to full-length ZNF217 protein, smaller ZNF217 proteins were detected and were even more prominent than full-length ZNF217 in both human breast tumors and cell lines, but the importance of these smaller isoforms remains unknown. To investigate the function of the smaller ZNF217 proteins and to determine the requirements of the regions of ZNF217 protein to promote breast cancer, we examined the consequences of removing regions of ZNF217 using truncation mutants overexpressed in human breast cancer cells. As a structure-function analysis, we tested both truncation mutants and full-length ZNF217 for their ability to promote phenotypes usually associated with ZNF217 overexpression, including protein turnover, increased branching of organoids, tumor burden, and metastasis. For protein turnover, these mutants were expressed in breast cancer cells and treated with cycloheximide to inhibit protein synthesis. This assay identified the regions of ZNF217 required for ZNF217 protein turnover. Further analysis of the sequence in this region identified a putative destruction box required for the protein turnover. We next determined which regions of ZNF217 are required for organoid branching and metastatic tumor burden. Both the N-terminus and C-terminus of ZNF217 were required for organoid branching. Interestingly, the same region identified to be required for ZNF217 protein turnover was not required for metastatic tumor burden. In fact, overexpression of breast cancer cells expressing stabilized ZNF217 truncation mutants significantly increased the metastatic tumor burden in vivo when assayed by tail vein injection. These results suggest that regulation of ZNF217 protein expression may be clinically valuable in generating a clinical assay used to generate personalized treatment strategies for patients with advanced metastatic breast cancer and high ZNF217 expression. Citation Format: Beth Facchine, Junmin Wu, Megan Fabry, Matt Messana, William Kaliney, Laurie Littlepage. Inhibiting the destruction of the oncogene ZNF217 promotes breast cancer metastasis to lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3341.

CD45RB Status Defines TCR Priming Affinity and CD8+ T Cell Memory Persistence

Abstract T cell receptor (TCR) affinity plays a critical role in shaping T cell differentiation, but few studies have investigated the role of TCR affinity in the context of the endogenous CD8+ T cell memory pool. While naïve CD8+ T cells express primarily large CD45 isoforms (denoted as CD45RBhi), CD8+ T cell memory cells are traditionally described as expressing small isoforms (CD45RBlo). Interestingly, although CD8+ T cell memory to acute infection with LCMV included CD45RBlo cells, we found that over half of the CD8+ T cell memory pool remained CD45RBhi. The 2D micropipette affinity assay revealed that CD45RBhi memory cells had a 29-fold lower affinity for antigen relative to CD45RBlo cells. CD45RBloand CD45RBhi memory cells expressed similar levels of granzyme B and IFN-γ, but RNASeq analysis revealed that these populations differentially expressed over 150 genes. Phenotypically, the CD45RBhi population contained a higher frequency of the CD62LhiCD27hiCD127hiKLRG1lo persistent memory phenotype compared to CD45RBlo cells. We found that from early to late memory time-points, portions of each of the CD45RBhi and CD45RBlo populations underwent conversion to CD62Lhi, demonstrating that CD45RB status is distinct from the TCM phenotype. Adoptive transfer of purified CD45RBhi and CD45RBlo populations into naïve mice revealed that CD45RBlo memory cells underwent significant cell loss relative to CD45RBhi memory cells. Over time, the majority of CD45RBlo cells homeostatically converted to the CD45RBhipersistent memory phenotype. Together, these data demonstrate a novel role for CD45RB expression as a marker of TCR priming affinity, and describe a novel way in which CD45RB status can functionally categorize the CD8+ T cell memory pool.

GAD65 autoantibody characteristics in patients with co-occurring type 1 diabetes and epilepsy may help identify underlying epilepsy etiologies

BackgroundAutoantibodies against the smaller isoform of glutamate decarboxylase (GAD65Ab) reflect autoimmune etiologies in Type 1 diabetes (T1D) and several neurological disorders, including Stiff Person Syndrome (SPS). GAD65Ab are also reported in cases of epilepsy, indicating an autoimmune component. GAD65Ab in patients with co-occurring T1D, epilepsy or SPS may be part of either autoimmune pathogenesis. To dissect the etiologies associated with GAD65Ab, we analyzed GAD65Ab titer, epitope specificity and enzyme inhibition in GAD65Ab-positive patients diagnosed with epilepsy (n = 28), patients with epilepsy and T1D (n = 10), patients with SPS (n = 20), and patients with T1D (n = 42).ResultsGAD65Ab epitope pattern in epilepsy differed from T1D and SPS patients. Four of 10 patients with co-occurring T1D and epilepsy showed GAD65Ab profiles similar to T1D patients, while lacking GAD65Ab characteristics found in GAD65Ab-positive epilepsy patients. One of these patients responded well to anti-epileptic drugs (AEDs), while another patient did not require medication for seizure control. The third patient was refractory due to a diagnosis of meningioma. The response of the remaining patient to AEDs was unknown. GAD65Ab in the remaining six patients with T1D and epilepsy showed profiles similar to those in epilepsy patients.ConclusionsDifferent autoimmune responses associated with T1D, epilepsy and SPS are reflected by disease-specific GAD65Ab patterns. Moreover, the epileptic etiology in patients diagnosed with both T1D and epilepsy may present two different etiologies regarding their epileptic condition. In one group T1D co-occurs with non-autoimmune epilepsy. In the other group GAD65Ab are part of an autoimmune epileptic condition.

Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin-2 Clearance Receptor.

The Stabilin receptors are systemic clearance receptors for some classes of chemically modified nucleic acid therapeutics. In this study, the recombinant human secreted ecto-domain of the small isoform of Stabilin-2 (s190) was purified from cell culture and evaluated for direct binding with a multitude of antisense oligonucleotides (ASOs) using a fluorescence polarization-based assay. The tested ASOs varied in their backbone composition, modification of the ribose 2′ position, overall length of the oligo, and sequence of the nucleotide bases. A fully phosphorothioate (PS) ASO with a 5–10–5 pattern of flanking 2′-O-methoxyethyl modifications was then used to test the effects of pH and salt concentration on receptor binding. These tests concluded that the PS backbone was the primary determinant for ASO binding and that decreasing pH and increasing salt generally increased the rate of ligand dissociation and fit within the biological parameters expected of a constitutive recycling receptor. These results will be useful in the rational design of therapeutic oligonucleotides for enhancing their affinity or avoidance of the Stabilin receptors.