Mutagenesis Screen Research Articles

FLOWERING REPRESSOR AAA+ ATPase 1 is a novel regulator of perennial flowering in Arabis alpina.

Arabis alpina is a polycarpic perennial, in which PERPETUAL FLOWERING1 (PEP1) regulates flowering and perennial traits in a vernalization-dependent manner. Mutagenesis screens of the pep1 mutant established the role of other flowering time regulators in PEP1-parallel pathways. Here we characterized three allelic enhancers of pep1 (eop002, 085 and 091) which flower early. We mapped the causal mutations and complemented mutants with the identified gene. Using quantitative reverse transcriptase PCR and reporter lines, we determined the protein spatiotemporal expression patterns and localization within the cell. We also characterized its role in Arabidopsis thaliana using CRISPR and in A. alpina by introgressing mutant alleles into a wild-type background. These mutants carried lesions in an AAA+ ATPase of unknown function, FLOWERING REPRESSOR AAA+ ATPase 1 (AaFRAT1). AaFRAT1 was detected in the vasculature of young leaf primordia and the rib zone of flowering shoot apical meristems. At the subcellular level, AaFRAT1 was localized at the interphase between the endoplasmic reticulum and peroxisomes. Introgression lines carrying Aafrat1 alleles required less vernalization to flower and reduced number of vegetative axillary branches. By contrast, A. thaliana CRISPR lines showed weak flowering phenotypes. AaFRAT1 contributes to flowering time regulation and the perennial growth habit of A. alpina.

A missense mutation in Kcnc3 causes hippocampal learning deficits in mice

Although a wide variety of genetic tools has been developed to study learning and memory, the molecular basis of memory encoding remains incompletely understood. Here, we undertook an unbiased approach to identify novel genes critical for memory encoding. From a large-scale, invivo mutagenesis screen using contextual fear conditioning, we isolated in mice a mutant, named Clueless, with spatial learning deficits. A causative missense mutation (G434V) was found in the voltage-gated potassium channel, subfamily C member 3 (Kcnc3) gene in a region that encodes a transmembrane voltage sensor. Generation of a Kcnc3G434V CRISPR mutant mouse confirmed this mutation as the cause of the learning defects. While G434V had no effect on transcription, translation, or trafficking of the channel, electrophysiological analysis of the G434V mutant channel revealed a complete loss of voltage-gated conductance, a broadening of the action potential, and decreased neuronal firing. Together, our findings have revealed a role for Kcnc3 in learning and memory.

Abstract LB516A: The irreversible FLT3 inhibitor FF-10101 is active against a diversity of FLT3 inhibitor resistance mechanisms

Abstract Background: FLT3 tyrosine kinase inhibitors (TKIs) such as midostaurin and gilteritinib have improved clinical outcomes for patients with FLT3-mutant acute myeloid leukemia (AML), but resistance remains a problem. FF-10101 is the first irreversible, covalent inhibitor of FLT3 and has shown activity against FLT3 F691L and D835 mutations. We evaluated FF-10101 against an expanded panel of FLT3 resistance mutations and activating mechanisms of MAPK signaling associated with FLT3 TKI resistance in AML cell lines and patient samples. Results: FF-10101 was uniformly active against a series of previously unevaluated FLT3 TKI resistant D835 mutations (sensitivity = IC50 <10X of FLT3-ITD alone in Ba/F3 cells). FF-10101 was more active against F691L than gilteritinib (8-fold vs 14-fold increase in IC50 vs. FLT3-ITD, respectively). K429E and N676K mutations associated with clinical resistance to crenolanib and midostaurin, respectively, were also both FF-10101-sensitive. G697S and Y693N mutations, previously identified by random saturation mutagenesis screening to confer resistance to gilteritinib, also conferred moderate resistance to FF-10101 while Y693C conferred a high degree of resistance to both (IC50 >50X higher than FLT3-ITD). D698N, however, was FF-10101-sensitive and gilteritinib-resistant. Using a cell-based model, we confirmed C695 as the lone residue covalently bound by FF-10101. Moreover, a mutagenesis screen for FF-10101 resistance identified only C695Y/R mutations. Structural modeling showed an aromatic π-π interaction between FF-10101 and Y693 that orients an electrophilic acrylamide portion for covalent bonding of FLT3 at C695, likely explaining resistance conferred by Y693C. Confirmatory mutagenesis experiments showed that Y693F, by recapitulating the native aromatic interaction, retained FF-10101 sensitivity while the non-aromatic Y693S mutation conferred a high degree of resistance. Finally, FF-10101 exerted generally greater cytotoxicity than gilteritinib in FLT3-ITD+ AML cell lines (MOLM-14 and MV4-11) and three FLT3-mutant AML patient samples cultured in HS5 stromal cell conditioned media that models bone marrow microenvironment (BME) induced TKI resistance. In MOLM-14s, FF-10101 more effectively suppressed rebound ERK re-activation after 24h drug exposure than gilteritinib, potentially explaining this enhanced activity. Nevertheless, MOLM-14 and MV4-11s with NRAS G12C and Q61K co-mutations exhibited significant resistance to FF-10101 and gilteritinib. Conclusions: FF-10101 is active against nearly all mutations associated with FLT3 TKI resistance except for C695 mutations (site of FF-10101 binding) and the gilteritinib/crenolanib-resistant Y693C mutation (which has yet to be identified clinically). FF-10101 potentially overcomes resistance associated with the BME but likely remains vulnerable to RAS activating mutants. In report of phase 1 study of FF-10101 in relapsed/refractory AML (NCT03194685), 8/30 evaluable patients exhibited response (27%) with five responding patients previously receiving a FLT3 TKI, including one CRi after progression on gilteritinib. These results and our findings show that FF-10101 has potential to benefit AML patients that acquire certain FLT3 TKD resistance mutations or exhibit progression with other FLT3 TKIs, including gilteritinib. Citation Format: Timothy T. Ferng, Daisuke Terada, Makoto Ando, Theodore C. Tarver, Fihr Chaudhary, Kimberly C. Lin, Aaron C. Logan, Catherine C. Smith. The irreversible FLT3 inhibitor FF-10101 is active against a diversity of FLT3 inhibitor resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB516A.

Mutation of rpoS is Beneficial for Suppressing Organic Acid Secretion During 1,3-Propandiol Biosynthesis in Klebsiella pneumoniae

In this study, to reduce the formation of organic acid during 1,3-propanediol biosynthesis in Klebsiella pneumoniae, a method combining UV mutagenesis and high-throughput screening with pH color plates was employed to obtain K. pneumoniae mutants. When compared with the parent strain, the total organic acid formation by the mutant decreased, whereas 1,3-propanediol biosynthesis increased after 24h anaerobic shake flask culture. Subsequently, genetic changes in the mutant were analyzed by whole-genome sequencing and verified by signal gene deletion. Mutation of the rpoS gene was confirmed to contribute to the regulation of organic acid synthesis in K. pneumoniae. Besides, rpoS deletion eliminated the formation of 2,3-butanediol, the main byproduct produced during 1,3-propanediol fermentation, indicating the role of rpoS in metabolic regulation in K. pneumoniae. Thus, a K. pneumoniae mutant was developed, which could produce lower organic acid during 1,3-propanediol fermentation due to an rpoS mutation in this study.

Abstract 2967: ACTR5 maintains hepatocellular carcinoma via H3K9 epigenetic silencing of CDKN2A

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers (>800,000 new cases each year worldwide) and a leading cause of cancer mortality (~700,000 deaths per year globally), with an average 5-year survival rate of 20~30%. The prevalence of HCC and the unmet clinical needs emphasize the urge for novel regimens for these malignancies. In this study, we performed an epigenetics-focused CRISPR interference (CRISPRi) library screen in the HepG2-dCas9-Krab cells (a total of 3,669 sgRNAs targeting 728 chromatin regulator genes at their transcription start sites) and identified ACTR5 as a novel essential gene in HCC. CRISPR depletion of ACTR5 led to arrested cell cycle and attenuated HCC tumor progression in vivo. Transcriptomic analysis revealed a significantly increased CDKN2A expression in the sgACTR5 transduced HCC cells, which led to the activation of Rb and repression of CDK6 and E2F signaling. Meanwhile, histone modification profiling identified loss of H3K9me2 and Hek9me3 at the CDKN2A promoters upon ACTR5 depletion, indicating the involvement of the epigenetic silencing of tumor suppressors (including CDKN2A) underlying the ACTR5-driven HCC progression. Mechanistically, we found that CRISPR targeting the ACTR5’s interacting protein IES6 (also known as INO80C) recapitulated the sgACTR5 phenotypes, highlighting the collaborative roles between these two partners. We then conducted a high-density CRISPR gene tiling scan using a sgRNA library that targets every NGG protospacer adjacent motif (PAM) within the ACTR5 (284 sgRNAs) and IES6 (90 sgRNAs) coding exons. This saturating mutagenesis screen revealed the requirement of 2 regions (G502-S519 and I552-C569) within ACTR5’s 2nd Actin-fold domain for IES6 interaction. Reciprocally, our unbiased screen identified a previously uncharacterized domain (T163-V187) in IES6’s C-terminal region to mediate the ACTR5-IES6 complex formation. To investigate the clinical impact of ACTR5, we examined the response of HCC cells to various inhibitors upon ACTR5 depletion. While sgACTR5 did not affect the cellular response to sorafenib (an FDA-approved RAF and VEGF inhibitor for HCC patients), we found that suppression of ACTR5 sensitized the HCC cells to ribociclib (a CDK6 inhibitor) and UNC0642 (an H3K9 methyltransferase G9a and GLP inhibitor). Based on these observations, we rationalized a combination treatment of ribociclib/UNC0642 that synergistically suppresses the proliferation of the HCC cells. In summary, our study revealed a pivotal role of ACTR5 (and its complex member IES6) in supporting the CDK6-driven cell cycle progression via an H3K9me2 H3K9me3-mediated silencing of CDKN2A. These notions can be exploited to develop novel combinational therapies against the difficult to cure liver cancers. Citation Format: Xiaobao Xu, Lu Yang, Anthony Chan, Sheela Pangeni Pokharel, Mingli Li, Qiao Liu, Nicole Mattson, Chun-wei Chen. ACTR5 maintains hepatocellular carcinoma via H3K9 epigenetic silencing of CDKN2A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2967.

A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment.

Sphingosine-1-phosphate lyase is encoded by the Sgpl1 gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer from adrenal insufficiency, severe lymphopenia, and skin disorders. In this study, we used random mutagenesis screening to identify a mouse line carrying a missense mutation of Sgpl1 (M467K). This mutation caused similar pathologies as Sgpl1 knock-out mice in multiple organs, but greatly preserved its lifespan, which M467K mutation mice look normal under SPF conditions for over 40 weeks, in contrast, the knock-out mice live no more than 6 weeks. When treated with Imiquimod, Sgpl1M467K mice experienced exacerbated skin inflammation, as revealed by aggravated acanthosis and orthokeratotic hyperkeratosis. We also demonstrated that the IL17a producing Vγ6+ cell was enriched in Sgpl1M467K skin and caused severe pathology after imiquimod treatment. Interestingly, hyperchromic plaque occurred in the mutant mice one month after Imiquimod treatment but not in the controls, which resembled the skin disorder found in Sgpl1 deficient patients. Therefore, our results demonstrate that Sgpl1M467K point mutation mice successfully modeled a human disease after being treated with Imiquimod. We also revealed a major subset of γδT cells in the skin, IL17 secreting Vγ6 T cells were augmented by Sgpl1 deficiency and led to skin pathology. Therefore, we have, for the first time, linked the IL17a and γδT cells to SPL insufficiency.

High-level production of L-valine in Escherichia coli using multi-modular engineering

L-valine is a valuable amino acid in mammals that is used as the main component of feed additives. The low efficiency of the fermentation titer limits the industrial application of L-valine. Here, an L-valine-producing strain of Escherichia coli was obtained using a multi-modular strategy. Initially, a chassis strain was generated by mutagenesis and high-throughput screening. The L-valine biosynthetic pathway and transport module were modified to improve the L-valine titer. Subsequently, the transcription factors associated with L-valine biosynthesis were investigated. Overexpression of PdhR and inhibition of the expression of RpoS promoted L-valine synthesis. Finally, the NADPH supply was enhanced after the introduction of the heterologous Entner-Doudoroff (ED) pathway from Zymomonas mobilis. The strain VAL38 produced 92 g/L L-valine in a 5-L bioreactor with a yield of 0.34 g/g glucose. This strategy is provided as a reference for improving the production performance of cell factories for L-valine and its derivatives.

Genome-Scale Mutational Analysis of Cathode-Oxidizing Thioclava electrotropha ElOx9T.

Extracellular electron transfer (EET) – the process by which microorganisms transfer electrons across their membrane(s) to/from solid-phase materials – has implications for a wide range of biogeochemically important processes in marine environments. Though EET is thought to play an important role in the oxidation of inorganic minerals by lithotrophic organisms, the mechanisms involved in the oxidation of solid particles are poorly understood. To explore the genetic basis of oxidative EET, we utilized genomic analyses and transposon insertion mutagenesis screens (Tn-seq) in the metabolically flexible, lithotrophic Alphaproteobacterium Thioclava electrotropha ElOx9T. The finished genome of this strain is 4.3 MB, and consists of 4,139 predicted ORFs, 54 contain heme binding motifs, and 33 of those 54 are predicted to localize to the cell envelope or have unknown localizations. To begin to understand the genetic basis of oxidative EET in ElOx9T, we constructed a transposon mutant library in semi-rich media which was comprised of >91,000 individual mutants encompassing >69,000 unique TA dinucleotide insertion sites. The library was subjected to heterotrophic growth on minimal media with acetate and autotrophic oxidative EET conditions on indium tin oxide coated glass electrodes poised at –278 mV vs. SHE or un-poised in an open circuit condition. We identified 528 genes classified as essential under these growth conditions. With respect to electrochemical conditions, 25 genes were essential under oxidative EET conditions, and 29 genes were essential in both the open circuit control and oxidative EET conditions. Though many of the genes identified under electrochemical conditions are predicted to be localized in the cytoplasm and lack heme binding motifs and/or homology to known EET proteins, we identified several hypothetical proteins and poorly characterized oxidoreductases that implicate a novel mechanism(s) for EET that warrants further study. Our results provide a starting point to explore the genetic basis of novel oxidative EET in this marine sediment microbe.

Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1rd8 mouse model.

Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.

Ring Finger Protein 125 Is an Anti-Proliferative Tumor Suppressor in Hepatocellular Carcinoma.

Simple SummaryHCC is a leading cause of cancer-related deaths worldwide. However, its tremendous inter- and intra-tumor heterogeneity has made it difficult to identify driver genes for HCC. Transposon mutagenesis is a versatile in vivo tool to identify cancer genes in tissues of interest. Herein, we analyzed transposon mutagenesis screening data in the liver and discovered a novel tumor suppressor gene, RNF125. This gene functions as a negative regulator of cell proliferation through transcriptional suppression of multiple genes important for cell proliferation and liver regeneration. This gene is frequently inactivated in human HCC and has a significant impact on patient prognosis. Our findings identify a new regulatory network of cell proliferation mediated by RNF125 and its contribution to HCC development and progression.Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide and the only cancer with an increasing incidence in the United States. Recent advances in sequencing technology have enabled detailed profiling of liver cancer genomes and revealed extensive inter- and intra-tumor heterogeneity, making it difficult to identify driver genes for HCC. To identify HCC driver genes, we performed transposon mutagenesis screens in a mouse HBV model of HCC and discovered many candidate cancer genes (SB/HBV-CCGs). Here, we show that one of these genes, RNF125 is a potent anti-proliferative tumor suppressor gene in HCC. RNF125 is one of nine CCGs whose expression was >3-fold downregulated in human HCC. Depletion of RNF125 in immortalized mouse liver cells led to tumor formation in transplanted mice and accelerated growth of human liver cancer cell lines, while its overexpression inhibited their growth, demonstrating the tumor-suppressive function of RNF125 in mouse and human liver. Whole-transcriptome analysis revealed that RNF125 transcriptionally suppresses multiple genes involved in cell proliferation and/or liver regeneration, including Egfr, Met, and Il6r. Blocking Egfr or Met pathway expression inhibited the increased cell proliferation observed in RNF125 knockdown cells. In HCC patients, low expression levels of RNF125 were correlated with poor prognosis demonstrating an important role for RNF125 in HCC. Collectively, our results identify RNF125 as a novel anti-proliferative tumor suppressor in HCC.

Enhancing protease activity of Bacillus subtilis using UV-laser random mutagenesis and high-throughput screening

Increasing the peptide level of fermented soybean meal helps improve its nutritional value, so it is necessary to screen strains with high protease activity. In this study, UV-laser random mutagenesis was employed to treat Bacillus subtilis for the selection of high protease producers. A high-throughput screening (HTS) processed aided by a microtiter plate (MTP) was established to enhance the efficiency of screening. Five mutagenesis methods were employed to investigate the mutagenesis effect, among which the positive mutation rate was the highest in laser UV sequential mutation (LA-UV). Two mutants, UV-1–84 and LA-UV-1–11, were re-screened from 32 positive mutants, exhibiting excellent genetic stability and protease activity. Besides, fermented soybean meal test showed that peptide content of SBM fermented by mutants UV-1–84 and LA-UV-1–11 increased by 13.66% and 12.91% respectively, compared with the wild-type strain. The HTS process established in this study could be easily transferred to other similar high-yield strains.

Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris

BackgroundPichia pastoris is a widely used host organism for heterologous production of industrial proteins, such as cellulases. Although great progress has been achieved in improving protein expression in P. pastoris, the potential of the P. pastoris expression system has not been fully explored due to unknown genomic impact factors. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high-throughput screening (HTS), has been considered to be a promising strategy in strain improvement at the genome level.ResultsIn this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic HTS, was developed to improve heterogenous cellulase production. The droplet-based microfluidic platform based on a cellulase-catalyzed reaction of releasing fluorescence was established to be suitable for methanol-grown P. pastoris. The validation experiment showed a positive sorting efficiency of 94.4% at a sorting rate of 300 droplets per second. After five rounds of iterative ARTP mutagenesis and microfluidic screening, the best mutant strain was obtained and exhibited the cellulase activity of 11,110 ± 523 U/mL, an approximately twofold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region. The effects of point mutations and mutant genes on cellulase production were verified using reconstruction of point mutations and gene deletions. Intriguingly, the point mutation Rsc1G22V was observed in all the top-performing producers selected from each round, and gene deletion analysis confirmed that Rsc1, a component of the RSC chromatin remodeling complex, might play an important role in cellulase production.ConclusionsWe established a droplet-based microfluidic HTS system, thereby facilitating whole-cell directed evolution of P. pastoris for enhancing cellulase production, and meanwhile identified genomic alterations by whole-genome resequencing and genetic validation. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.

Forward Genetics in Apicomplexa Biology: The Host Side of the Story.

Forward genetic approaches have been widely used in parasitology and have proven their power to reveal the complexities of host-parasite interactions in an unbiased fashion. Many aspects of the parasite’s biology, including the identification of virulence factors, replication determinants, antibiotic resistance genes, and other factors required for parasitic life, have been discovered using such strategies. Forward genetic approaches have also been employed to understand host resistance mechanisms to parasitic infection. Here, we will introduce and review all forward genetic approaches that have been used to identify host factors involved with Apicomplexa infections, which include classical genetic screens and QTL mapping, GWAS, ENU mutagenesis, overexpression, RNAi and CRISPR-Cas9 library screens. Collectively, these screens have improved our understanding of host resistance mechanisms, immune regulation, vaccine and drug designs for Apicomplexa parasites. We will also discuss how recent advances in molecular genetics give present opportunities to further explore host-parasite relationships.

Characterizing BfpU, an Essential Protein in the Type IV Pilus of Enteropathogenic Escherichia coli

Type IV pili (T4Ps) are a family of surface appendages that are important for adhesion, colonization, biofilm formation, virulence, twitching motility and many other functions. BfpU is an essential periplasmic protein of the T4P of Enteropathogenic Escherichia coli. Previous crosslinking and localization studies have shown that BfpU might interact with the outer membrane secretin BfpB, which is also essential for T4P biogenesis. To date, BfpU has no known homologs and its function is unknown. To purify BfpU, we performed affinity and size exclusion chromatography. Preliminary characterization of BfpU via thermal stability assay and circular dichroism suggests a melting temperature of 80 °C, and its secondary structure is comprised of ~40% anti‐parallel β‐sheet and ~45% random coil. The secondary structure profile is reminiscent of PilP, an essential periplasmic protein anchored in the membrane that interacts with secretins in other T4P systems. Additionally, random mutagenesis is in progress to assess essential residues of BfpU that may be involved in protein interactions within this T4P system. Single residue mutations from this mutagenesis screening will be used to confirm interaction sites within BfpU. The results of these studies will help clarify the role of BfpU in T4P biogenesis and its interactions with BfpB.

Discovery of Novel Pain Therapies Without Side‐effects by Combining Targeted Mutagenesis and Phenotype‐based Drug Screening in Larval Zebrafish

IntroductionOpioid drugs provide effective pain relief but come with considerable side‐effects including addiction and respiratory depression which can be lethal with overdose. During the COVID19 pandemic, the opioid epidemic reached almost 100,000 deaths in the United States, highlighting the urgent need to identify pain killers with reduced liability so they can be safely prescribed. One approach to develop safe pain killers is to combine existing opioid analgesics with drugs reducing respiratory depression. Using our novel biotechnology platforms in larval zebrafish, we propose to identify new molecular targets and drugs with potent analgesic properties, reduced respiratory liability, and without addictive properties.MethodsCombining targeted mutagenesis and phenotype‐based drug screening approaches in larval zebrafish in vivo,we can quickly and effectively identify new molecular targets and screen new drug compounds to either target respiratory depression, analgesia (Zaig and Montandon, 2021, eLife), as well as addiction. We first screened customized drug libraries targeting voltage‐gated calcium channels and the adenylyl cyclase pathway, two molecular pathways involved in pain. We are also creating mutants using CRISPR‐cas9 in first generation zebrafish embryos to identify new molecular targets. Using our new models, we quantified respiratory depression by fentanyl in day post‐fertilization 7 larvae, as well as the nociceptive response to formalin.ResultsTo determine the role of calcium channels opioid‐mediated effects, we combined fentanyl and the calcium channel activators nefiracetam (1µM) or FLP‐64176 (1µM). Both nefiractem and FLP‐64176 prevented respiratory depression by fentanyl (1µM). To target the adenylyl cyclase pathway, we combined fentanyl and the phosphodiesterase inhibitors pentoxifylline (50µM), roflumilast (1µM), or dipyridamole (100µM). Pentaxifylline prevented respiratory depression by fentanyl but not roflumilast and dipyridamole. To perform targeted mutagenesis, we injected 4 single guide RNAs targeting the orpm1 gene, i.e. the gene coding for µ‐opioid receptors. Six days later, we measured respiratory depression by fentanyl in knockout larvae or controls. In oprm1‐/‐ knockout larvae, respiratory depression by fentanyl was substantially reduced compared to controls, therefore confirming the validity of our targeted mutagenesis approach. We are currently targeting the genes coding for calcium channels and phosphodiesterases to determine whether they can be molecular targets for respiratory depression and/or analgesia.DiscussionThe successful identification of new drug combinations to prevent respiratory depression while preserving analgesia suggests that our drug discovery platform in larval zebrafish will substantially accelerate the identification of new opioid drugs with reduced morbidity and mortality. Our platform is also being leveraged to discover non‐opioid pain killers with reduced side‐effects.

Discovering the Developmental Basis of Trachea‐Esophageal Birth Defects: Evidence for Endosome‐opathies

The trachea and esophagus (TE) arise from a common foregut tube during embryonic development. Disruptions in TE morphogenesis cause congenital trachea‐esophageal defects (TEDs) such as esophageal atresia, tracheoesophageal fistula and tracheoesophageal clefts. TEDs occur in approximately 1 in 3500 births, but their etiology is poorly understood. We have established the www.CLEARconsortium.org; a multidisciplinary team of clinicians, geneticists, bioinformaticians, stem cell and developmental biologists using patient genome sequencing, animal models and iPSC‐derived human organoids to discover the genetic and developmental basis of trachea‐esophageal birth defects. Using the complementary advantages of Xenopus and mouse models we have defined the conserved molecular and cellular mechanisms that regulate normal TE morphogenesis. We show that downstream of Hedgehog/Gli signaling endosome‐mediated epithelial remodeling regulates TE morphogenesis which when disrupted results in tracheoesophageal clefts similar to human Pallister Hall syndrome patients. Proband‐parent trio genome sequencing identified an enrichment of potential damaging de novo variants in genes encoding membrane/vesicular‐trafficking proteins, suggesting a common “endosome‐opathy” pathway. Ongoing CRISPR mutagenesis screens in Xenopus tropicalis assessing candidate causative variants from patients confirms that the endosome protein Itsn1 is essential for TE morphogenesis, suggesting that the ITSN1 variant is likely pathogenic in the patient. Finally, leveraging results from animal models we have generated multi‐lineage human esophageal organoids from iPSCs with patient mutations to identify how mutations impact human esophageal differentiation. Together these results significantly advance our understanding of TEDs with the goal of revealing phenotype‐genotype associations that will inform prognosis and clinical treatment.

Fluoropyrimidine Bioactivation and Metabolism by the Gut Microbiome

Despite decades of research into why populations of cancer patients differentially respond to the same treatment, variation in response to therapy regimens remains a significant challenge in the oncology field. However, our understanding of the causal mechanisms of interpatient variability remains lacking; up to 35% of patients require dose adjustments and 10% of patients are removed from the fluoropyrimidine capecitabine altogether. This research seeks to further our understanding of the microbiome’s role in fluoropyrimidine treatment by identifying gut bacterial strains responsible for capecitabine activation as well as biochemically characterizing drug inactivating enzymes from the human gut microbiome. Growth curve data show that knockouts of bacterial enzymes homologous to mammalian enzymes known to be involved in capecitabine metabolism increase bacterial resistance to capecitabine. Additionally, an unbiased transposon mutagenesis screen identifies enzymes known to increase resistance as well as enzymes both within and outside of nucleotide metabolism. These two pieces of data suggest that bacteria have the capacity to metabolize capecitabine, upending the paradigm that activation of this prodrug happens exclusively in the liver and tumor. Confirmation of these metabolites with high resolution triple quadrupole linear ion trap mass spectrometry is ongoing. Separately, previous studies indicate that gut bacteria are capable of degrading 5‐fluorouracil, however it is not clear how efficient these pathways are or to what extent they occur in the host. Enzyme kinetics of homologues of this enzyme within the microbiome show that degradative reaction for uracil is essentially irreversible. Additionally, this enzymatic characterization has identified the non‐enzymatic enantiomer of the degraded fluoropyrimidine as a potential inhibitor for this enzyme, which might allow for co‐dosing and inhibition of bacterial metabolism. Metagenomic sequencing of the degradative enzymes within patient samples is ongoing and will potentially reveal the main drivers of this activity in patients. These avenues of research will uncover new bacterial metabolism pathways of xenobiotics, compare these metabolic pathways between microbiota and host, and have the potential to lead to new advances in disrupting microbial metabolism of chemotherapeutics.

A saturation mutagenesis screen uncovers resistant and sensitizing secondary KRAS mutations to clinical KRASG12C inhibitors.

Mutant-specific inhibitors of KRASG12C, such as AMG510 (sotorasib) and MRTX849 (adagrasib), offer the unprecedented opportunity to inhibit KRAS, the most frequently mutated and heretofore undruggable oncoprotein. While clinical data are still limited, on-target mutations in KRASG12C at position 12 and other sites are emerging as major drivers of clinical relapse. We identified additional mutations in KRASG12C that impact inhibitor sensitivity through a saturation mutagenesis screen in the KRASG12C NCI-H358 non–small-cell lung cancer (NSCLC) cell line. We also identified individuals in population genetic databases harboring these resistance mutations in their germline and in tumors, including a subset that co-occur with KRASG12C, indicating that these mutations may preexist in patients treated with KRASG12C inhibitors. Notably, through structural modeling, we found that one such mutation (R68L) interferes with the critical protein–drug interface, conferring resistance to both inhibitors. Finally, we uncovered a mutant (S17E) that demonstrated a strong sensitizing phenotype to both inhibitors. Functional studies suggest that S17E sensitizes KRASG12C cells to KRASG12C inhibition by impacting signaling through PI3K/AKT/mTOR but not the MAPK signaling pathway. Our studies highlight the utility of unbiased mutation profiling to understand the functional consequences of all variants of a disease-causing genetic mutant and predict acquired resistant mutations in the targeted therapeutics.

Novel Protein-Protein Interactions Highlighting the Crosstalk between Hypoplastic Left Heart Syndrome, Ciliopathies and Neurodevelopmental Delays.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) affecting 1 in 5000 newborns. We constructed the interactome of 74 HLHS-associated genes identified from a large-scale mouse mutagenesis screen, augmenting it with 408 novel protein–protein interactions (PPIs) using our High-Precision Protein–Protein Interaction Prediction (HiPPIP) model. The interactome is available on a webserver with advanced search capabilities. A total of 364 genes including 73 novel interactors were differentially regulated in tissue/iPSC-derived cardiomyocytes of HLHS patients. Novel PPIs facilitated the identification of TOR signaling and endoplasmic reticulum stress modules. We found that 60.5% of the interactome consisted of housekeeping genes that may harbor large-effect mutations and drive HLHS etiology but show limited transmission. Network proximity of diabetes, Alzheimer’s disease, and liver carcinoma-associated genes to HLHS genes suggested a mechanistic basis for their comorbidity with HLHS. Interactome genes showed tissue-specificity for sites of extracardiac anomalies (placenta, liver and brain). The HLHS interactome shared significant overlaps with the interactomes of ciliopathy- and microcephaly-associated genes, with the shared genes enriched for genes involved in intellectual disability and/or developmental delay, and neuronal death pathways, respectively. This supported the increased burden of ciliopathy variants and prevalence of neurological abnormalities observed among HLHS patients with developmental delay and microcephaly, respectively.

A bacterial three-hybrid assay for forward and reverse genetic analysis of RNA-protein interactions.

This protocol describes a bacterial three-hybrid (B3H) assay, an in vivo system that reports on RNA-protein interactions and can be implemented in both forward and reverse genetic experiments. The B3H assay connects the strength of an RNA-protein interaction inside of living Escherichia coli cells to the transcription of a reporter gene (here, lacZ). We present protocols to (1) insert RNA and protein sequences into appropriate vectors for B3H experiments, (2) detect putative RNA-protein interactions with both qualitative and quantitative readouts and (3) carry out forward genetic mutagenesis screens. The B3H assay builds on a well-established bacterial two-hybrid system for genetic analyses. As a result, protein-protein interactions can be assessed in tandem with RNA interactions with a bacterial two-hybrid assay to ensure that protein variants maintain their functionality. The B3H system is a powerful complement to traditional biochemical methods for dissecting RNA-protein interaction mechanisms: RNAs and proteins of interest do not need to be purified, and their interactions can be assessed under native conditions inside of a living bacterial cell. Once cloning has been completed, an assay can be completed in under a week and a screen in 1-2 weeks.