Exon Clusters Research Articles

Memory consolidation in honey bees is enhanced by down-regulation of Down syndrome cell adhesion molecule and changes its alternative splicing.

Down syndrome cell adhesion molecule (Dscam) gene encodes a cell adhesion molecule required for neuronal wiring. A remarkable feature of arthropod Dscam is massive alternative splicing generating thousands of different isoforms from three variable clusters of alternative exons. Dscam expression and diversity arising from alternative splicing have been studied during development, but whether they exert functions in adult brains has not been determined. Here, using honey bees, we find that Dscam expression is critically linked to memory retention as reducing expression by RNAi enhances memory after reward learning in adult worker honey bees. Moreover, alternative splicing of Dscam is altered in all three variable clusters after learning. Since identical Dscam isoforms engage in homophilic interactions, these results suggest a mechanism to alter inclusion of variable exons during memory consolidation to modify neuronal connections for memory retention.

Abstract C140: BI 1810631 is a novel EGFR wild-type sparing, HER2-selective small molecule inhibitor that efficiently blocks HER2 mutant-driven lung cancer

Abstract Here we report pre-clinical characterization of the clinical compound BI 1810631, a HER2 specific, EGFR wild-type-sparing tyrosine kinase inhibitor (TKI). BI 1810631 has shown early signs of clinical activity in patients carrying tumors with HER2 aberrations [NCT04886804], mainly comprising HER2 mutations (Heymach et al. 2023). In non-small cell lung cancer, activating mutations in HER2 are found in 2-4% of patients and predominantly cluster in exon 20 within the tyrosine kinase domain (TKD), of which the most frequent variant is A775_G776insYVMA. Studies across tumor cell lines and xenograft mouse models show that BI 1810631 is a potent and selective inhibitor of HER2-driven oncogenic signaling. Compound-mediated reduction of cell growth and survival was observed in mutant HER2 driven Ba/F3 cell systems, in human tumor cell lines in vitro and translated into tumor regressions in a genome-engineered HER2-YVMA mutant xenograft model. The in vivo efficacy of BI 1810631 was further confirmed in HER2 exon 20 mutant patient-derived tumor models. Corresponding biomarker studies in vitro and in vivo showed modulation of pharmacodynamic markers, corroborating the on-target mechanism of action of the compound. These findings demonstrate that HER2 mutations can be effectively addressed by BI 1810631 and support the ongoing and future clinical trials. Citation Format: Ralph A. Neumüller, Birgit Wilding, Dirk Scharn, Anke Baum, Martin Augsten, Irene Waizenegger, Shinji Kohsaka, Valeria Santoro, Paolo Chetta, Lydia Woelflingseder, Johannes Popow, Daniel Gerlach, Peter Ettmayer, Thomas Gerstberger, Julian Fuchs, Matthias Treu, Stephan Zahn, Mark Pearson, Mark Petronczki, Darryl B. McConnell, Norbert Kraut, Flavio Solca. BI 1810631 is a novel EGFR wild-type sparing, HER2-selective small molecule inhibitor that efficiently blocks HER2 mutant-driven lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C140.

Mutations in the B30.2 and the central helical scaffold domains of pyrin differentially affect inflammasome activation

Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory disorder. FMF is caused by mutations in the MEFV gene, encoding pyrin, an inflammasome sensor. The best characterized pathogenic mutations associated with FMF cluster in exon 10. Yet, mutations have been described along the whole MEFV coding sequence. Exon 10 encodes the B30.2 domain of the pyrin protein, but the function of this human-specific domain remains unclear. Pyrin is an inflammasome sensor detecting RhoA GTPase inhibition following exposure to bacterial toxins such as TcdA. Here, we demonstrate that the B30.2 domain is dispensable for pyrin inflammasome activation in response to this toxin. Deletion of the B30.2 domain mimics the most typical FMF-associated mutation and confers spontaneous inflammasome activation in response to pyrin dephosphorylation. Our results indicate that the B30.2 domain is a negative regulator of the pyrin inflammasome that acts independently from and downstream of pyrin dephosphorylation. In addition, we identify the central helical scaffold (CHS) domain of pyrin, which lies immediately upstream of the B30.2 domain as a second regulatory domain. Mutations affecting the CHS domain mimic pathogenic mutations in the B30.2 domain and render the pyrin inflammasome activation under the sole control of the dephosphorylation. In addition, specific mutations in the CHS domain strongly increase the cell susceptibility to steroid catabolites, recently described to activate pyrin, in both a cell line model and in monocytes from genotype-selected FMF patients. Taken together, our work reveals the existence of two distinct regulatory regions at the C-terminus of the pyrin protein, that act in a distinct manner to regulate positively or negatively inflammasome activation. Furthermore, our results indicate that different mutations in pyrin regulatory domains have different functional impacts on the pyrin inflammasome which could contribute to the diversity of pyrin-associated autoinflammatory diseases.

Discovery of potent and selective HER2 inhibitors with efficacy against HER2 exon 20 insertion-driven tumors, which preserve wild-type EGFR signaling.

Oncogenic alterations in human epidermal growth factor receptor 2 (HER2) occur in approximately 2% of patients with non-small cell lung cancer and predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. Most clinical-grade tyrosine kinase inhibitors are limited by either insufficient selectivity against wild-type (WT) epidermal growth factor receptor (EGFR), which is a major cause of dose-limiting toxicity or by potency against HER2 exon 20 mutant variants. Here we report the discovery of covalent tyrosine kinase inhibitors that potently inhibit HER2 exon 20 mutants while sparing WT EGFR, which reduce tumor cell survival and proliferation in vitro and result in regressions in preclinical xenograft models of HER2 exon 20 mutant non-small cell lung cancer, concomitant with inhibition of downstream HER2 signaling. Our results suggest that HER2 exon 20 insertion-driven tumors can be effectively treated by a potent and highly selective HER2 inhibitor while sparing WT EGFR, paving the way for clinical translation.

NOTCH3 mutations in a cohort of Portuguese patients within CADASIL spectrum phenotype

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common inherited cerebral small vessel disease. It is caused by mutations in the NOTCH3 gene, which encodes a membranebound receptor protein with three main distinct functional domains. Thus far, several different NOTCH3 mutations, most of them cysteine altering variants, have been described and although they tend to cluster in certain exons, their distribution varies in different geographically populations. Therefore, in this study, we describe the mutation analysis of NOTCH3 gene in 24 Portuguese families with small vessel disease suspected to have CADASIL from the central region of Portugal. The genetic analysis revealed 15 different heterozygous variants, eight pathogenic cysteine altering variants, six cysteine sparing variants and one nonsense variant, located mainly in the exons 4, 8 and 11. Thus, in our population, the genetic testing should initially be focused on these exons. In addition, the genetic findings broaden the mutational and clinical spectrum of CADASIL related phenotype and provide additional evidences for genetic counseling and clinical management.

Abstract 1472: Novel EGFR WT sparing, HER2 selective inhibitors for the treatment of HER2 exon 20 insertion driven tumors address a clear unmet medical need

Abstract Activating mutations in ERBB2 receptors are tractable oncogenes in 2-3% of NSCLC patients for whom no approved targeted therapies are available. In this indication, oncogenic mutations in HER2 predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. We initiated a drug discovery program aiming at discovering novel HER2 selective inhibitors sparing EGFR WT activity. Focus was set on the most frequent HER2 mutation (ERBB2 A775 insYVMA), which is least sensitive to current compounds tested in clinical trials. Here, we report the identification and pharmacological characterization of novel selective HER2 exon 20 mutation TKIs that differ from currently tested TKIs such as poziotinib, TAK-788 or BDTX-189. We could demonstrate that selective inhibition of oncogenic HER2 signaling abrogates oncogenic signaling in in vitro models. Cell survival and proliferation was reduced, which translated into tumor regressions in preclinical CRISPR engineered xenotransplantation models of HER2 exon 20 mutants. The in vivo efficacy was confirmed in patient-derived tumor models. Our results suggest that HER2 exon 20 insertions can be effectively treated by a potent and highly selective HER2 inhibitor that spares EGFR wild type. These findings warrant the upcoming clinical testing in HER2 mutant NSCLC patients in order to effectively treat this aggressive type of cancer. Citation Format: Ralph A. Neumüller, Birgit Wilding, Dirk Scharn, Dietrich Böse, Valeria Santoro, Daniel Gerlach, Peter Ettmayer, Thomas Gerstberger, Julian Fuchs, Matthias Treu, Stephan Zahn, Anke Baum, Paolo Chetta, Mark Pearson, Darryl B. McConnell, Norbert Kraut, Flavio Solca. Novel EGFR WT sparing, HER2 selective inhibitors for the treatment of HER2 exon 20 insertion driven tumors address a clear unmet medical need [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1472.

Complex RNA Secondary Structures Mediate Mutually Exclusive Splicing of Coleoptera Dscam1.

Mutually exclusive splicing is an important mechanism for expanding protein diversity. An extreme example is the Down syndrome cell adhesion molecular (Dscam1) gene of insects, containing four clusters of variable exons (exons 4, 6, 9, and 17), which potentially generates tens of thousands of protein isoforms through mutually exclusive splicing, of which regulatory mechanisms are still elusive. Here, we systematically analyzed the variable exon 4, 6, and 9 clusters of Dscam1 in Coleoptera species. Through comparative genomics and RNA secondary structure prediction, we found apparent evidence that the evolutionarily conserved RNA base pairing mediates mutually exclusive splicing in the Dscam1 exon 4 cluster. In contrast to the fly exon 6, most exon 6 selector sequences in Coleoptera species are partially located in the variable exon region. Besides, bidirectional RNA–RNA interactions are predicted to regulate the mutually exclusive splicing of variable exon 9 of Dscam1. Although the docking sites in exon 4 and 9 clusters are clade specific, the docking sites-selector base pairing is conserved in secondary structure level. In short, our result provided a mechanistic framework for the application of long-range RNA base pairings in regulating the mutually exclusive splicing of Coleoptera Dscam1.

Intronic enhancer region governs transcript-specific Bdnf expression in rodent neurons.

Brain-derived neurotrophic factor (BDNF) controls the survival, growth, and function of neurons both during the development and in the adult nervous system. Bdnf is transcribed from several distinct promoters generating transcripts with alternative 5' exons. Bdnf transcripts initiated at the first cluster of exons have been associated with the regulation of body weight and various aspects of social behavior, but the mechanisms driving the expression of these transcripts have remained poorly understood. Here, we identify an evolutionarily conserved intronic enhancer region inside the Bdnf gene that regulates both basal and stimulus-dependent expression of the Bdnf transcripts starting from the first cluster of 5' exons in mouse and rat neurons. We further uncover a functional E-box element in the enhancer region, linking the expression of Bdnf and various pro-neural basic helix-loop-helix transcription factors. Collectively, our results shed new light on the cell-type- and stimulus-specific regulation of the important neurotrophic factor BDNF.

RNA secondary structures in Dscam1 mutually exclusive splicing: unique evolutionary signature from the midge.

The Drosophila melanogaster gene Dscam1 potentially generates 38,016 distinct isoforms via mutually exclusive splicing, which are required for both nervous and immune functions. However, the mechanism underlying splicing regulation remains obscure. Here we show apparent evolutionary signatures characteristic of competing RNA secondary structures in exon clusters 6 and 9 of Dscam1 in the two midge species (Belgica antarctica and Clunio marinus). Surprisingly, midge Dscam1 encodes only ∼6000 different isoforms through mutually exclusive splicing. Strikingly, the docking site of the exon 6 cluster is conserved in almost all insects and crustaceans but is specific in the midge; however, the docking site-selector base-pairings are conserved. Moreover, the docking site is complementary to all predicted selector sequences downstream from every variable exon 9 of the midge Dscam1, which is in accordance with the broad spectrum of their isoform expression. This suggests that these cis-elements mainly function through the formation of long-range base-pairings. This study provides a vital insight into the evolution and mechanism of Dscam1 alternative splicing.

The recurrent SETBP1 c.2608G\u2009>\u2009A, p.(Gly870Ser) variant in a patient with Schinzel-Giedion syndrome: an illustrative case of the utility of whole exome sequencing in a critically ill neonate

BackgroundSchinzel-Giedion syndrome (SGS) is a multiple malformation syndrome mainly characterized by severe intellectual disability, distinctive facial features, and multiple congenital anomalies, including skeletal abnormalities, genitourinary and renal malformations, cardiac defects, as well as an increased pediatric cancer risk. Recently, SGS has been associated with de novo heterozygous deleterious variants in the SETBP1 gene; to date, nine different variants, clustering in exon 4 of SETBP1, have been identified in 25 patients.Case presentationIn this study, by using Whole Exome Sequencing (WES), we identified a patient with a recurrent missense mutation in SETBP1, the c.2608G > A, p.(Gly870Ser) variant, previously reported as likely pathogenic. This finding allowed us to confirm the suspected clinical diagnosis of SGS. Clinical features of patients carrying the same variant, including our patient, were evaluated by a review of medical records.ConclusionsOur study confirms SGS as a severe disorder potentially presenting at birth as a critically ill neonate and demonstrates the causal role of the c.2608G > A, p.(Gly870Ser) variant in the etiology of the syndrome. Moreover, although the cohort of SETBP1-patients reported in the literature is still small, our study reports for the first time the prevalence of the variant (about 27%, 7/26). Finally, given the heterogeneity of clinical presentations of affected patients hospitalized in Neonatal Intensive Care Units (NICU) and/or Pediatric Intensive Care Units (PICU), in agreement with emerging data from the literature, we suggest that WES should be used in the diagnosis of unexplained syndromic conditions, and even as part of a standard first-line diagnostic approach, as it would allow a better diagnosis, counseling and management of affected patients and their families.

Srrm234, but not canonical SR and hnRNP proteins, drive inclusion of Dscam exon 9 variable exons.

Alternative splicing of pre-mRNA is a major mechanism to diversify protein functionality in metazoans from a limited number of genes. The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene, which is important for neuronal wiring and phagocytosis of bacteria, can generate up to 38,016 isoforms by mutually exclusive alternative splicing in four clusters of variable exons. However, it is not understood how a specific exon is chosen from the many variables and how variable exons are prevented from being spliced together. A main role in the regulation of Dscam alternative splicing has been attributed to RNA binding proteins (RBPs), but how they impact on exon selection is not well understood. Serine-arginine rich (SR) proteins and hnRNP proteins are the two main types of RBPs with major roles in exon definition and splice site selection. Here, we analyzed the role of SR and hnRNP proteins in Dscam exon 9 alternative splicing in mutant Drosophila melanogaster embryos because of their essential function for development. Strikingly, loss or overexpression of canonical SR and hnRNP proteins even when multiple proteins are depleted together, does not affect Dscam alternative exon selection very dramatically. Conversely, noncanonical SR protein Serine–arginine repetitive matrix 2/3/4 (Srrm234) is a main determinant of exon inclusion in the Dscam exon 9 cluster. Since long-range base-pairings are absent in the exon 9 cluster, our data argue for a small complement of regulatory factors as main determinants of exon inclusion in the Dscam exon 9 cluster.

Non-Methylation-Linked Mechanism of REST-Induced Neuroglobin Expression Impacts Mitochondrial Phenotypes in a Mouse Model of Amyotrophic Lateral Sclerosis

Neuroglobin (Ngb) is a REST/NRSF-regulated protein, active in reactive oxygen species detoxification and cytochrome c inhibition, which provides a beneficial outcome in pathologies as Alzheimer's disease and strokes. Considering that oxidative stress and cell death are typical hallmarks of amyotrophic lateral sclerosis (ALS), we sought to explore Ngb's involvement along this disease progression. Ngb transcription was detected to be two-fold down-regulated in late-stage SODG93A mice, similarly as previously described for Alzheimer disease. Interestingly, in accordance with REST/NRSF transcription, Ngb expression is higher in spinal cords than in cortices. Hence, downstream REST/NRSF mechanisms were studied. A methylation cluster in Ngb's exon 1 (Chr12:87101763–87102586) was selected to assess methylation alterations, based on significantly altered positions in GEO DataSets of human c9orf72 and sporadic ALS cases. However, only the methylation percentage on position Chr12.87102586 was significantly increased in SODG93A mice. A larger impact can therefore be expected from the detected altered REST splicing; with levels of alternatively spliced, gene-activating REST4 to be lower than those of the gene-inhibitory full variant. To look further into the link between Ngb and ALS, we generated a double mutant Ngb-/-SODG93A mouse model, which shows an earlier onset and severity of hind limb deficits. Mitochondria derived thereof showed an altered mean volume, granularity and Ca2+-induced swelling as compared to NgbWt/WtSODG93A mice. These results indicate Ngb to be involved in and affected by the SOD1G93A pathology, which could in part be attributed to its role in halting destabilizing events of mitochondrial swelling and phenotypes.

Role and convergent evolution of competing RNA secondary structures in mutually exclusive splicing

ABSTRACTExon or cassette duplication is an important means of expanding protein and functional diversity through mutually exclusive splicing. However, the mechanistic basis of this process in non-arthropod species remains poorly understood. Here, we demonstrate that MRP1 genes underwent tandem exon duplication in Nematoda, Platyhelminthes, Annelida, Mollusca, Arthropoda, Echinodermata, and early-diverging Chordata but not in late-diverging vertebrates. Interestingly, these events were of independent origin in different phyla, suggesting convergent evolution of alternative splicing. Furthermore, we showed that multiple sets of clade-conserved RNA pairings evolved to guide species-specific mutually exclusive splicing in Arthropoda. Importantly, we also identified a similar structural code in MRP exon clusters of the annelid, Capitella teleta, and chordate, Branchiostoma belcheri, suggesting an evolutionarily conserved competing pairing-guided mechanism in bilaterians. Taken together, these data reveal the molecular determinants and RNA pairing-guided evolution of species-specific mutually exclusive splicing spanning more than 600 million years of bilaterian evolution. These findings have a significant impact on our understanding of the evolution of and mechanism underpinning isoform diversity and complex gene structure.

Profile of Calreticulin (CALR) Somatic Mutations in a Cohort of Pakistani Patients with Myeloproliferative Neoplasms

Introduction:Calreticulin (CALR) is a calcium binding multifunctional protein encoded by the CALR gene, whose job is to bind to mis-folded proteins and prevent them from being exported from the endoplasmic reticulum to the golgi apparatus. Outside the endoplasmic reticulum it is found both intra- and extracellularly on the cell surface and has been implicated in diverse processes including proliferation, apoptosis, phagocytosis and immunogenic cell death.In December 2013, somatic mutations in exon 9 of CALR were identified as the second most prevalent acquired nucleotide changes in JAK2 mutation negative essential thrombocythosis (ET) and primary myelofibrosis (PMF) patients but not in polycythaemia vera (PV) patients. At that time CALR mutations were found mutually exclusive with JAK2 and MPL. Recently there have been some reports on co-occurence of JAK2 and CALR mutations, not only in ET and PMF but also in some PV cases. Indels in CALR have also been reported in a JAK2 mutation negative PV patient. All CALR mutations are insertions or deletions resulting in a frameshift and cluster in exon 9 which is the last exon of the gene. Thus far, more than 50 different types of mutations in CALR have been reported. Two specific mutations, Type I (52bp deletion) and Type II (5bp insertion), are most prevalent. Overall, these two mutation types are found in more than 80% of patients with mutant CALR. One report found absence of Type II mutations and scatter point mutations in PMF patients of India. In the light of currently available literature, reported from North America (USA), Europe (Cyprus/Italy/Spain), Eastern Europe (Poland), and East Asia (Mainland China/Taiwan China), indel mutations in CALR exon 9 occur with a similar frequency in both ET and PMF patients regardless of ethnic diversity. CALR mutations have been shown to have important diagnostic and prognostic significance in ET and PMF patients and will likely be incorporated into the World Health Organization diagnostic criteria for MPN.Methods:Clinical and hematological features were obtained from 51 MPN patients. We studied 10 patients with PV, 17 patients with ET, 12 patients with MF and 9 patients with Undifferentiated MPN. JAK2 V617F mutation was analyzed by DNA tetra-primer amplification refractory mutation system (ARMS-PCR). CALR mutations were identified by bi-directional Sanger sequencing.Results:In our cohort, CALR mutations were detected in 7 patients (13.7%; 7/51) of which one patients was positive for JAK2 V617F as well. All those who showed positive results for CALR mutations were ET patients (41.17%; 7/17). Type I mutation was found in 3 patients (42.8%; 3/7) and among them 2 had two new scattered point mutations (c.1081C>G and c.1086C>G) as well, Type II in 2 patients (28.5%; 2/7), and others in 2 patients (28.5%; 2/7). Among those 2 patients with CALR mutations other than Type I or Type II, one had novel CALR mutation pattern of 10bp deletion (c.1130_1139del) whereas other had reported CALR 12bp deletion (c.1214_1225del) profile.When CALR positive ET patients were statistically compared with JAK2 V617F positive patient, insignificant differences were found in platelet counts (p-value 0.198) and hemoglobin level (p-value 0.380) which might be due to the fact that there was only one JAK2 V617F positive patient in the cohort. No differences were also observed in hemoglobin, white blood cell, platelet counts, hepatomegaly, and splenomegaly between CALR positive and CALR negative patients (p-value >0.05).Conclusion:In our cohort of 51 MPN patients CALR mutations were found associated with ET only that is different from other studies worldwide. Prevalence of both scatter point and Type I and Type II CALR mutations are in agreement with worldwide studies but differ from the study conducted on Indian population which shows the need to conduct studies with large sample size in this region to explore the CALR mutation profile in the patients of MPN here. DisclosuresNo relevant conflicts of interest to declare.

Long-range RNA pairings contribute to mutually exclusive splicing.

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA–RNA interactions in gene regulatory networks.

Somatic and Germline Diversification of a Putative Immunoreceptor within One Phylum: Dscam in Arthropods.

Arthropod Dscam, the homologue of the human Down Syndrome cell adhesion molecule, is a receptor used by the nervous and immune systems. Unlike in vertebrates, evolutionary pressure has selected and maintained a vast Dscam diversity of isoforms, known to specifying neuronal identity during the nervous system differentiation. This chapter examines the different modes of Dscam diversification in the context of arthropods' evolution and that of their immune system, where its role is controversial. In the single Dscam gene of insects and crustaceans, mutually exclusive alternative splicing affects three clusters of duplicated exons encoding the variable parts of the receptor. The Dscam gene produces over 10,000 isoforms. In the more basal arthropods such as centipedes, Dscam diversity results from a combination of many germline genes (over 80) with, in about half of those, the possibility of alternative splicing affecting only one exon cluster. In the even more basal arthropods, such as chelicerates, no splicing possibility is detected, but there exist dozens of germline Dscam genes. Compared to controlling the expression of multiple germline genes, the somatic mutually alternative splicing within a single gene may offer a simplified way of expressing a large Dscam repertoire. Expressed by hemocytes, Dscam is considered a phagocytic receptor but is also encountered in solution. More information is necessary about its binding to pathogens, its role in phagocytosis, its possible role in specifying hemocyte identity, its kinetics of expression, and the regulation of its RNA splicing to understand how its diversity is linked to immunity.

Genetic Variation of EPAS1 Gene in Tibetan Pigs and Three Low-Altitude Pig Breeds in China

Endothelial PAS domain protein 1 (EPAS1), also called hypoxia-inducible factor-2, is a key regulatory factor of hypoxic responses and plays an essential role in high-altitude adaptation in mammalian species. In this study, polymorphisms of EPAS1 were detected in 217 individuals from 2 Tibetan pig populations and 3 low-altitude pig breeds by DNA pooling, PCR-SSCP, PCR-RFLP and DNA sequencing methods. A total of 14 synonymous polymorphisms were identified in the coding region. The analysis suggested that SNP1 (G963A), SNP7 (C1632T), SNP10 (G1929A) and SNP11 (G1947A) showed potential association with high-altitude environment because of their particular variation patterns in Tibetan pigs. Linkage disequilibrium (LD) of these SNPs was analyzed. One common LD block including 5 SNPs clustering in exon 12 was identified in all studied pig populations. Haplotype H1 (AGGTC) in LD block was dominant in Tibetan pigs (76.6 and 74.2% in Linzhi (LZ) and Chayu (CY) pigs, respectively) and segregated at higher frequency than that in low-altitude pig breeds (52.3, 58.7 and 56.2% in Wuzhishan (WZS), Min (M) and Laiwu (LW) pigs, respectively), indicating that H1 may relate to adaptation to high altitude in Tibetan pigs. These findings raise hope that EPAS1 gene can be a candidate gene that involved in adaptation of high altitude in Tibetan pigs.

Neuroimaging of Movement Disorders

1389 Objectives Cancer cell lines, established from either primary or metastatic sites of cancer patients, have contributed to understanding cancer biology and the selection/validation of imaging radiotracers. Cell lines with a shorter doubling time (DT) are expected to have more aggressive behavior. In the present study, we assessed correlations between the DTs of hematological malignant cell lines and the abundance of messages for Ki67, PCNA, TK1, thymidylate synthase (TYMS), HK1, or transporters (SLC29A1-2) Methods A NCI 60 exon dataset (interrogating over 1 million exon clusters within the known and predicted transcribed regions of the entire genome) was selected from NIH Gene Expression Omnibus repository, which contains 178 chips including 6 hematological malignant cell lines with triplicates for each: HL-60, RPMI-8226, K-562, SR, CCRF-CEM, and MOLT-4. Using Partek, commercial software, we obtained an average abundance of the transcripts of interest above. Correlations between the abundance of genes and NCI documented doubling times were determined by Pearson correlation testing (α=0.05 for a one-tailed test). Results The correlation coefficients r of HK1 message (phosphorylating FDG) vs. DTs was -0.87 (p= 0.01 Conclusions In these cell lines, HK1 message abundance appeared to have a stronger correlation to DTs of hematological malignant cancer cell lines than message levels for TK1, TYMS, and conventional proliferation markers Ki67 and PCNA. The correlation is negative, supporting higher HK1 abundance and shorter DT. Since message levels and protein levels can sometimes be disparate, our findings should be validated using protein quantitation and in vivo. Research Support NIH T32 training grant

Comprehensive analysis of mutually exclusive alternative splicing in C. elegans.

Mutually exclusive selection of one exon in a cluster of exons is a rare form of alternative pre-mRNA splicing, yet suggests strict regulation. However, the repertoires of regulation mechanisms for the mutually exclusive (ME) splicing in vivo are still unknown. Here, we experimentally explore putative ME exons in C. elegans to demonstrate that 29 ME exon clusters in 27 genes are actually selected in a mutually exclusive manner. Twenty-two of the clusters consist of homologous ME exons. Five clusters have too short intervening introns to be excised between the ME exons. Fidelity of ME splicing relies at least in part on nonsense-mediated mRNA decay for 14 clusters. These results thus characterize all the repertoires of ME splicing in this organism.

Abstract B266: Ponatinib, a potent KIT inhibitor, suppresses the emergence of secondary resistance mutations in a gastrointestinal stromal tumor (GIST) model system.

Abstract Background: Approximately 80% of GISTs contain primary activating mutations in KIT, the majority of which cluster in exon 11. Imatinib is approved for treatment of metastatic and/or unresectable GIST; however patients often relapse due to the acquisition of secondary resistance mutations in KIT, typically located in the ATP-binding pocket or activation loop (A-loop). Sunitinib is approved for 2nd line treatment, but does not effectively inhibit A-loop mutants. Ponatinib is a multi-targeted tyrosine kinase inhibitor (TKI) with potent activity against BCR-ABL that has been approved for treatment of CML and Ph+ ALL patients resistant or intolerant to prior TKIs. We have previously shown that ponatinib also potently inhibits both activated KIT and KIT containing imatinib- or sunitinib-resistance mutations, including those at the gatekeeper residue and within the A-loop. V654A was identified as the most recalcitrant mutant, although ponatinib was still capable of significantly inhibiting tumor growth in a V654A mouse model. To survey more broadly for mutations that might confer substantial resistance to ponatinib, an accelerated KIT mutagenesis screen was performed. Results: Ba/F3 cells dependent on exon 11 mutant KIT (Δ557-8) were chemically mutagenized to introduce random mutations and grown in the presence of TKIs to identify secondary resistance mutations. Importantly, resistant clones that grew in the presence of imatinib or sunitinib recapitulated the spectrum of mutations observed clinically. In the presence of imatinib, mutations in the ATP pocket and A-loop were observed, with the T670I gatekeeper mutation conferring the highest level of resistance. In the presence of sunitinib, mutations were observed primarily in the A-loop (D816, D820, N822 and Y823), but not the ATP pocket. In contrast, in the presence of 40 nM ponatinib, only the V654A mutation persisted, in a small number of clones. In the presence of 80 nM ponatinib the development of resistance mutations was completely suppressed. Importantly, levels of ponatinib achieved in patients dosed once daily with 45 mg ponatinib (145 nM peak; 64 nM trough) exceed these target levels. The results of mutagenesis studies using other primary mutant backgrounds, and the newly approved TKI regorafenib, will also be described. Conclusion: Preclinical analysis suggests that, in an exon 11 mutant KIT background, ponatinib can inhibit development of a broad spectrum of potential resistance mutations, including multiple problematic mutations within the A-loop. Thus far, the only secondary mutation identified that is not completely suppressed at clinically achievable trough concentrations is V654A. These studies provide further support for an ongoing phase 2 study of ponatinib in patients with TKI-resistant GIST (NCT01874665). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B266. Citation Format: Alexa B. Schrock, Tzu-Hsiu Chen, Victor M. Rivera, Joseph M. Gozgit. Ponatinib, a potent KIT inhibitor, suppresses the emergence of secondary resistance mutations in a gastrointestinal stromal tumor (GIST) model system. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B266.