Knock-in Mutation Research Articles

TMEM106B coding variant is protective and deletion detrimental in a mouse model of tauopathy.

TMEM106B is a risk modifier of multiple neurological conditions, where a single coding variant and multiple non-coding SNPs influence the balance between susceptibility and resilience. Two key questions that emerge from past work are whether the lone T185S coding variant contributes to protection, and if the presence of TMEM106B is helpful or harmful in the context of disease. Here, we address both questions while expanding the scope of TMEM106B study from TDP-43 to models of tauopathy. We generated knockout mice with constitutive deletion of TMEM106B, alongside knock-in mice encoding the T186S knock-in mutation (equivalent to the human T185S variant), and crossed both with a P301S transgenic tau model to study how these manipulations impacted disease phenotypes. We found that TMEM106B deletion accelerated cognitive decline, hind limb paralysis, tau pathology, and neurodegeneration. TMEM106B deletion also increased transcriptional correlation with human AD and the functional pathways enriched in KO:tau mice aligned with those of AD. In contrast, the coding variant protected against tau-associated cognitive decline, synaptic impairment, neurodegeneration, and paralysis without affecting tau pathology. Our findings reveal that TMEM106B is a critical safeguard against tau aggregation, and that loss of this protein has a profound effect on sequelae of tauopathy. Our study further demonstrates that the coding variant is functionally relevant and contributes to neuroprotection downstream of tau pathology to preserve cognitive function.

Abstract 4669: Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations

Abstract Background: Differences in profiles of acquired mutations of resistance to BTKis might have implications for treatment sequencing. We evaluated preclinical activity of BTKis (ibrutinib [Ibr], acalabrutinib [Acala], zanubrutinib [Zanu], and pirtobrutinib [Pirto]) against multiple clinically relevant BTK mutations and report on the clinical impact of BTK C481S in patients (pts) treated with Ibr. Methods: Inhibition of kinase activity by BTKis was evaluated in a cell-free kinase activity assay (Nanosyn). Cell-killing activity on CRISPR-generated BTK mutant knock-in TMD8 cells was assessed after 3 days of BTKi treatment. Time from first detection of C481S mutation to progressive disease (PD) was evaluated in pts treated with Ibr with previously untreated (n=247) or relapsed/refractory (R/R; n=172) CLL using pooled data from 5 clinical trials. Results: In vitro kinase activity was highest for Ibr compared with Acala or Zanu against C481S (Ibr IC50: 26.3 nM; Zanu: 84.8 nM; Acala: 338 nM) and T474I (Ibr: 1.2 nM; Zanu: 4.4 nM; Acala: 43.6 nM). Ibr maintained cell-killing activity against all tested BTK mutants, whereas limited activity was seen with Acala against C481S and A428D; with Zanu against C481S, L528W, and A428D; and with Pirto against T474I, L528W, V416L, and A428D (Table). In pts treated with Ibr, C481S was detected prior to clinical PD in 3 previously untreated pts and in 26 pts with R/R CLL; median (range) time from first detection of C481S to PD in these pts was 17.0 mo (11.2-34.3) and 10.0 mo (2.6-35.7), respectively. Conclusion: This study demonstrates activity of Ibr against all evaluated BTK variants, which are associated with resistance to covalent and noncovalent BTKis. Clinical data shows that pts who develop C481S mutations continue to derive benefit from Ibr for several months before PD. Together, these data suggest a role for Ibr in the treatment of pts with a broad spectrum of BTK variants. Table. Cell-killing activity of BTKis against wild type and mutant BTK EC50 (nM) BTK Wild type C481S T474I L528W V416L A428D Ibr 0.6 414.0 1.9 181.4 0.5 205.3 Acala 3.9 2973.8 43.3 3.5 106.3 3000.0 Zanu 0.7 2322.0 11.7 3000.0 0.6 1726.0 Pirto 8.2 12.6 2727.5 3000.0 1898.0 3000.0 Citation Format: Matthew S. Davids, Inhye E. Ahn, Jacob J. Riehm, Melih Acar, Vincent Girardi, Sima Patel, Tarikere Gururaja, Jennifer Woyach, Lloyd T. Lam, Edith Szafer-Glusman. Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4669.

Abstract 1942: Characterization of a panel of CRISPR/Cas9 engineered KRAS G12C inhibitor-resistant tumor models

Abstract Introduction: KRAS is one of the most frequent mutated oncogenes and has been recognized as undruggable for many years. AMG510, the first therapy to directly target KRAS, was approved by the FDA to treat non-small cell lung cancer (NSCLC) bearing KRAS G12C mutation. However, the emergence of resistance in patients remains a challenge and limits its clinical benefits, which calls for next-generation targeted therapy or combination strategies to overcome the resistance to KRAS G12C inhibitors. A variety of secondary mutations in KRAS attributing to the resistance have been identified, which requires robust in vitro and in vivo preclinical models to validate potential therapeutics targeting these mutations. Therefore, we generated a panel of KRAS G12C inhibitor-resistant tumor models to facilitate the development of possible strategies to overcome such resistance. Methods: First, a secondary KRAS mutation, including H95D, H95Q, H95R, Q61H and R68S, was introduced by using CRISPR/Cas9 technology in MIA PaCa cell line, which harbors a homozygous KRAS G12C mutation in addition to Y96D, Y96C and Y96S previously published by us. Point mutation knock-in was validated by sanger sequencing, and cell identity was confirmed by SNP assay. In vitro, the parental and mutated cells were treated with either AMG510 and MRTX849 and cell viability was measured by CellTiter-Glo. In vivo efficacy of KRAS G12C inhibitors, AMG510 and MRTX849, SOS1 inhibitor, BI-3406 and MEK inhibitor, Trametinib were evaluated in the Y96D mutated MIA PaCa subcutaneous xenograft. Results: Homozygous secondary point mutation knock-in in MIA PaCa cells was confirmed by sanger sequencing. Similar growth rate and morphology was observed in selected clones compared to the parental line. Similar to Y96D mutation previously published, R68S mutation was highly resistant to both KRAS G12C inhibitors, with IC50 increased more than 100 fold for both MRTX849 and AMG510, whereas H95D, H95Q and H95R mutation was more resistant to MRTX849, while Q61H had a minimal effect on either one of the inhibitors. In addition, cells expressing KRAS G12C/Y96D were also resistant to AMG510 and MRTX849 in in vivo study, whereas combination of BI-3406 (single treatment: TGI of 27%) and Trametinib (single treatment: TGI of 76%) suppressed tumor growth significantly with TGI of 93% on day 17 after randomization compared to control group (p<0.001). Also, the combination showed significant improvement compared to BI-3406 single treatment (p<0.001) whereas no significant improvement was observed compared to Trametinib single treatment (p>0.05). Conclusion: CRISPR/Cas9 engineered second site KRAS mutations in cells harboring KRAS G12C mutation displayed a differentially resistant profile to KRAS G12C inhibitors. Thus, H95D/Q/R, R68S and Y96D can be used as preclinical inhibitor-resistant models to evaluate clinical strategies to overcome resistance to KRAS-targeted therapies. Citation Format: Jun Zhou, Li Hua, Jian Feng, Ning Bao, Dan Zhang, Jingjing Wang, Marrit Putker, Ludovic Bourre. Characterization of a panel of CRISPR/Cas9 engineered KRAS G12C inhibitor-resistant tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1942.

Key role of the ryanodine receptor I4790K mutation in mediating diamide resistance in Plutella xylostella

The diamondback moth Plutella xylostella, a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017–0.917) of the 4790K allele, but not with either 4790M (0–0.183) or 4946E (0.017–0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella, and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest.

NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.

Targeted inhibition of SCFSKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma

Osteosarcoma(OS) is a highly aggressive bone cancer for which treatment has remained essentially unchanged for decades. Although OS is characterized by extensive genomic heterogeneity and instability, RB1 and TP53 have been shown to be the most commonly inactivated tumor suppressors in OS. We previously generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which largely recapitulates human OS with nearly complete penetrance. SKP2 is a repression target of pRb and serves as a substrate recruiting subunit of the SCFSKP2 complex. In addition, SKP2 plays a central role in regulating the cell cycle by ubiquitinating and promoting the degradation of p27. We previously reported the DKOAA transgenic model, which harbored a knock-in mutation in p27 that impaired its binding to SKP2. Here, we generated a novel p53-Rb1-SKP2 triple-knockout model (TKO) to examine SKP2 function and its potential as a therapeutic target in OS. First, we observed that OS tumorigenesis was significantly delayed in TKO mice and their overall survival was markedly improved. In addition, the loss of SKP2 also promoted an apoptotic microenvironment and reduced the stemness of DKO tumors. Furthermore, we found that small-molecule inhibitors of SKP2 exhibited anti-tumor activities in vivo and in OS organoids as well as synergistic effects when combined with a standard chemotherapeutic agent. Taken together, our results suggest that SKP2 inhibitors may reduce the stemness plasticity of OS and should be leveraged as next-generation adjuvants in this cancer.

Recovering a Targeted Mutation in Mice from Embryonic Stem Cell Chimeras or CRISPR-Cas Founders.

Following the production of chimeras from targeted embryonic stem (ES) cells or obtaining founders from CRISPR-Cas gene editing in preimplantation embryos, the desired targeted mutation must be recovered and established in the heterozygous state in a strain or stock of mice for further study. The breeding schemes for ES chimeras and CRISPR-Cas founders differ. For ES cell chimeras, we discuss the relative benefits of breeding from male or female chimeras. We discuss the importance of genetic background and provide practical advice for putting the mutation on inbred or outbred backgrounds or producing a coisogenic strain. For CRISPR-Cas founders, which will most likely be mosaic for different mutations, initial breeding strategies are discussed to maintain a desired genetic background at the same time as producing progeny to segregate different alleles. Strategies for testing the progeny to recognize indels, missense mutations, and knock-in mutations are discussed. In the event that ES cell chimeras or CRISPR-Cas founders produce no offspring or fail to transmit the mutant allele(s), there is a troubleshooting guide to pinpoint the problem. If heterozygous offspring from the chimeras or founders are normal, fertile, and of both sexes, the analysis of homozygous effects of the mutation can now begin; if not, possible dominant effects are considered.

Parkinson's LRRK2-G2019S risk gene mutation drives sex-specific behavioral and cellular adaptations to chronic variable stress.

Anxiety is a psychiatric non-motor symptom of Parkinson's that can appear in the prodromal period, prior to significant loss of midbrain dopamine neurons and motor symptoms. Parkinson's-related anxiety affects females more than males, despite the greater prevalence of Parkinson's in males. How stress, anxiety and Parkinson's are related and the basis for a sex-specific impact of stress in Parkinson's are not clear. We addressed this using young adult male and female mice carrying a G2019S knockin mutation of leucine-rich repeat kinase 2 (Lrrk2 G2019S) and Lrrk2 WT control mice. In humans, LRRK2 G2019S significantly elevates the risk of late-onset Parkinson's. To assess within-sex differences between Lrrk2 G2019S and control mice in stress-induced anxiety-like behaviors in young adulthood, we used a within-subject design whereby Lrrk2 G2019S and Lrrk2 WT control mice underwent tests of anxiety-like behaviors before (baseline) and following a 28 day (d) variable stress paradigm. There were no differences in behavioral measures between genotypes in males or females at baseline, indicating that the mutation alone does not produce anxiety-like responses. Following chronic stress, male Lrrk2 G2019S mice were affected similarly to male wildtypes except for novelty-suppressed feeding, where stress had no impact on Lrrk2 G2019S mice while significantly increasing latency to feed in Lrrk2 WT control mice. Female Lrrk2 G2019S mice were impacted by chronic stress similarly to wildtype females across all behavioral measures. Subsequent post-stress analyses compared cFos immunolabeling-based cellular activity patterns across several stress-relevant brain regions. The density of cFos-activated neurons across brain regions in both male and female Lrrk2 G2019S mice was generally lower compared to stressed Lrrk2 WT mice, except for the nucleus accumbens of male Lrrk2 G2019S mice, where cFos-labeled cell density was significantly higher than all other groups. Together, these data suggest that the Lrrk2 G2019S mutation differentially impacts anxiety-like behavioral responses to chronic stress in males and females that may reflect sex-specific adaptations observed in circuit activation patterns in some, but not all stress-related brain regions.

SYNCAS: Efficient CRISPR/Cas9 gene-editing in difficult to transform arthropods

The genome editing technique CRISPR/Cas9 has led to major advancements in many research fields and this state-of-the-art tool has proven its use in genetic studies for various arthropods. However, most transformation protocols rely on microinjection of CRISPR/Cas9 components into embryos, a method which is challenging for many species. Alternatively, injections can be performed on adult females, but transformation efficiencies can be very low as was shown for the two-spotted spider mite, Tetranychus urticae, a minute but important chelicerate pest on many crops. In this study, we explored different CRISPR/Cas9 formulations to optimize a maternal injection protocol for T. urticae. We observed a strong synergy between branched amphipathic peptide capsules and saponins, resulting in a significant increase of CRISPR/Cas9 knock-out efficiency, exceeding 20%. This CRISPR/Cas9 formulation, termed SYNCAS, was used to knock-out different T. urticae genes – phytoene desaturase, CYP384A1 and Antennapedia – but also allowed to develop a co-CRISPR strategy and facilitated the generation of T. urticae knock-in mutants. In addition, SYNCAS was successfully applied to knock-out white and white-like genes in the western flower thrips, Frankliniella occidentalis. The SYNCAS method allows routine genome editing in these species and can be a game changer for genetic research in other hard to transform arthropods.

SERCA2 dysfunction triggers hypertension by interrupting mitochondrial homeostasis and provoking oxidative stress

Background and aimSarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is critical in maintaining Ca2+ homeostasis. The cysteine 674 (C674) is the key redox regulatory cysteine in regulating SERCA2 activity, which is irreversibly oxidized in the renal cortex of hypertensive mice. We have reported that the substitution of C674 by serine causes SERCA2 dysfunction and increases blood pressure by induction of endoplasmic reticulum stress (ERS). This study is to explore whether the dysfunction of SERCA2 causes hypertension by interrupting mitochondrial homeostasis and inducing oxidative stress. Methods & resultsWe used heterozygous SERCA2 C674S gene mutation knock-in (SKI) mice, where one copy of C674 was substituted by serine to represent partial C674 oxidation. In renal proximal tubule (RPT) cells, the substitution of C674 by serine decreased mitochondrial Ca2+ content, increased mitochondrial membrane potential, ATP content, and reactive oxygen species (ROS), which could be reversed by ERS inhibitor 4-phenylbutyric acid or SERCA2 agonist CDN1163. In SKI RPT cells, the redox modulator Tempol alleviated oxidative stress, downregulated the protein expression of ERS markers and soluble epoxide hydrolase, upregulated the protein expression of dopamine D1 receptor, and reduced Na+/K+- ATPase activity. In SKI mice, SERCA2 agonists CDN1163 and [6]-Gingerol, or the redox modulator Tempol increased urine output and lowered blood pressure. ConclusionThe irreversible oxidation of C674 is not only an indicator of increased ROS, but also further inducing oxidative stress to cause hypertension. Activation of SERCA2 or inhibition of oxidative stress is beneficial to alleviate hypertension caused by SERCA2 dysfunction.

Abstract B168: Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations

Abstract Background: EGFR mutations are well validated clinical targets in non-small cell lung cancer (NSCLC). Osimertinib, a highly-selective EGFR mutation-targeting covalent drug, is increasingly used in the first line setting for patients with NSCLC bearing EGFR L858R mutation or exon 19 deletions (ex19del). In a subset of these patients, co-occurring L858R/C797x or ex19del/C797x mutations (“double mutants”) are emerging as an on-target resistance mechanism, necessitating the need for new therapies, particularly in patients with CNS metastases. Materials and methods: STX-241 was tested across a panel of in vitro biochemical, cell signaling, and proliferation assays for potency against EGFR L858R and ex19del single mutants and the corresponding C797S double mutants. Additionally, STX-241 was tested for in vivo activity in mice bearing human NSCLC cell line xenografts NCI-H3255 (L858R) and PC-9 (ex19del), and in a PC-9-derived ex19del/C797S double mutant knock-in xenograft. Free CNS penetration (Kp,uu) was determined in non-tumor bearing mice. Osimertinib and gefitinib (an approved reversible EGFR inhibitor) were used as benchmark molecules across assays. Results: STX-241, an ATP-competitive reversible EGFR inhibitor representing novel chemical matter, demonstrated potent and selective inhibition of recombinant EGFR L858R/C797S mutant protein relative to wild-type, with increased mutant residence time relative to gefitinib. Potent (high picomolar to low nanomolar) inhibition of L858R, ex19del, L858R/C797S, and ex19del/C797S mutants was observed in proliferation assays using engineered Ba/F3 and human NSCLC cell lines. STX-241 demonstrated >150x selectivity for all tested EGFR mutants relative to wild-type EGFR in engineered Ba/F3 cells and human cancer cells. In these assays, STX-241 selectivity exceeded that of the benchmark reversible EGFR inhibitor gefitinib. Strong potency and double mutant selectivity was also observed for STX-241 in pharmacodynamic assays measuring EGFR pathway activation (pEGFR). STX-241 was well tolerated at doses of 15 mg/kg BID or 50 mg/kg QD in mice, where regression of EGFR exon 19 or 21 mutant NSCLC xenografts was observed, concomitant with EGFR pathway suppression. Notably, using an isogenic pair of PC-9 (EGFR ex19del) NSCLC xenografts that differ only by the presence of a C797S mutation, STX-241 demonstrates no drop off in antitumor activity in the presence of C797S. In mouse pharmacokinetic studies measuring free CNS penetration, Kp,uu measurements were comparable to Osimertinib, run in parallel as a benchmark. Conclusions: STX-241 demonstrates strong potency and selectivity against EGFR L858R/C797S and ex19del/C797S double mutants, as well as robust CNS penetrance. These data warrant further exploration of this potential best-in-class inhibitor in the clinic for patients progressing on Osimertinib via C797x mutation. Citation Format: Raymond A Pagliarini, Benjamin C Milgram, Deanna R Borrelli, Erin O'Hearn, Michael R. Huff, Brendon Ladd, Natasja Brooijmans, Weixue Wang, Petr Kuzmic, Angel Guzman-Perez, Darrin D Stuart. Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations [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 B168.

Genetic Knockin Approaches to Reconstructing DLBCL-Subtypes from Human Germinal Center B-Cells

Background and Significance: The clinical and genetic heterogeneity of DLBCL has led to the recent classification of the disease into five genetically distinct subtypes. The MCD/C5 subtype of DLBCL is defined by genetic alterations of CD79B at the first tyrosine residue of its ITAM motif (CD79B Y196H), crucial for signaling downstream of the BCR, and MYD88 (MYD88 L265P), a signaling adaptor protein essential for Toll Like receptor signaling. CD79B Y196H and MYD88 L265P mutations together define the MCD/C5 subtype and were found to be co-dependent for proliferation and oncogenic BCR-signaling in vitro. However, the mechanism of epistatic interaction between CD79B Y196H and MYD88 L265P is unknown. The MCD/C5 DLBCL subtype is associated with particularly poor clinical outcomes, however only a few human cell lines and a genetic mouse model are available for preclinical testing. Here, we developed a novel genetic approach to engineer the MCD/C5 subtype of DLBCL in primary human tonsillar germinal center B cells. To this end, we introduced the CD79B Y196H and MYD88 L265P mutations as genetic knockin alleles utilizing CRISPR-Cas9 homology directed repair templates (HDRT). In addition, we developed humanized NBSGW mice carrying a mouse Tslp-transgene to enable development of lymph nodes and secondary lymphoid organs to propagate genetically engineered GC B-cells carrying CD79B Y196H and MYD88 L265P knockin mutations for lymphoma development ( Figure 1). Results: Primary human germinal center B-cells were isolated from tonsils and grown on follicular dendritic feeder cells expressing CD40L and IL21. Single strand DNA templates were designed for CD79B, with and without Y196H mutation. Constructs were linked to fluorescent reporters by T2A sites and flanked by homology arms of ~280 bp targeting the guide RNA cut site. The target site was selected to disrupt the endogenous gene, while placing the construct under transcriptional control of endogenous promoter/enhancer elements. We achieved a knock-in efficiency of 5-8%, prior to single-cell cloning and genotyping to confirm the presence of heterozygous CD79B Y196H mutant CD79B Y196 wild-type mutations ( Figure 1). Preliminary biochemical studies show a delayed response and reduced intensity of signaling downstream of the BCR upon stimulation in CD79B Y196H mutant, compared to CD79B Y196 wild-type knockin germinal center B-cells. A similar approach was taken to design temples encoding Exon 5 of MYD88, with and without the L265P mutation, utilizing two guide RNAs to excise and replace Exon 5, linked to a mCherry reporter by a T2A site. Upon successfully introducing the MYD88 HDR templates into our CD79B wild-type and mutant clones to generate four distinct genotypes based on wildtype and CD79B Y196H and MYD88 L265P mutations alone or in combination. In addition, traditional NSG mice lack lymph nodes and secondary lymphoid organs. Lack of lymphoid follicles in NSG mice, owing to defective IL7Rα-signaling, contributed to lack of lymphoma formation observed in the previous model. In our model, we substituted the missing IL7Rα-signal with transgenic expression of murine Tslp, which enables development of embryonic lymphoid tissue inducer (LTi) cells that are required for the development of secondary lymphoid organs and germinal center follicles supporting initiation of human DLBCL development. Conclusion: Previous approaches to model DLBCL starting from germinal center B-cells were based on lentiviral overexpression of mutant oncogenes. Thereby, tonsillar germinal center B-cells with lentiviral BCL2 and BCL6 overexpression did not form lymphomas in NSG mice, however, were permissive to malignant transformation by MYC. Building on this previous work, we propose an improved model for preclinical testing for genetically defined DLBCL subtypes. Our two main improvements involve (i) genetic modeling based on knockin-strategies targeting endogenous loci and transcriptional control elements to phenocopy physiological gene expression levels and transcriptional regulation, and (ii) substitution of defective IL7Rα-signaling by transgenic expression of murine Tslp to rescue development of LTi cells. Transgenic expression of mTslp enables LTi-dependent formation of lymph nodes and secondary lymphoid organs supporting lymphoma development.

Dynamic Recruitment of Inhibitory Complexes Controls Oncogenic Signaling in B-Cell Malignancies

Background and Significance: Initiation of B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. High expression levels on the surface of B-ALL cells are associated with poor clinical outcomes in pediatric (COG P9906) and adult (ECOG E2993) B-ALL cohorts. In DLBCL, aggressive disease is associated with CD25-inactivation by shedding and high serum levels of soluble CD25. Results: Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ, immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors and inhibitory phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ and phosphatases, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early stages of B-cell development (e.g. B-ALL) CD25-loss induced hyperactivation of oncogenic signaling followed by cell death. In contrast, genetic deletion of CD25 in models for mature B-cell lymphoma accelerated proliferation of malignant cells. Biochemical and interactome studies revealed a critical role of CD25 in feedback regulation of oncogenic signaling in B-cell malignancies: Signaling from BCR-ABL1 and RAS-oncogenes in B-ALL as well as oncogenic BCR-signaling in DLBCL induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268). Importantly, CD25-phosphorylation stabilized its expression at the cell membrane and opposed both inactivation by CD25-shedding and internalization. Moreover CD25-S 268 tail-phosphorylation enabled CD25 to recruit inhibitory receptors, including CD22, that bear ITIM-motifs for the activation of inhibitory phosphatases (e.g. SHP1, Figure 1). Thereby, the positively charged cytoplasmic tail of CD25 engages negatively charged residues in the cytoplasmic tails of the BCR signaling chain CD79B and the inhibitory CD22-ITIM. Molecular dynamics simulations revealed that the ternary complex between CD25, CD79B and CD22 enables recruitment and activation of the phosphatase SHP1 within reach of its substrate, the BCR signaling chain CD79B. Thereby, the SH2-domain of SHP1 engages the CD22-ITIM motif, while its phosphatase domain makes contact with the CD79B-ITAM to dephosphorylate and terminate BCR-signaling. This complex, while engaging and activating SHP1, is held together by CD25 ( Figure 1). To test functional consequences of destabilization of this complex, we generated a knockin mouse model carrying a single point mutation (S268A) in the cytoplasmic tail of CD25. The CD25 S268A knockin mutation abolished recruitment and activation of SHP1. In addition, the S268A-mutant failed to assemble CD22 and CD79B in complex with SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function and failure to terminate oncogenic signaling resulted in constitutive activation of ERK and NF-κB, autonomous Ca 2+-oscillations. Hyperactivation of oncogenic signaling resulted in exhaustion and cell death in B-ALL cells, as opposed to excessive proliferation and acceleration of disease in mature B-cell lymphoma. Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, these findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and provides an explanation for the common feature of CD25 shedding and high serum levels of functionally inactive soluble CD25 in aggressive B-cell lymphomas.

Type 3 Innate Lymphoid Cells Promote Acute Myeloid Leukemia Immune Evasion and Expansion

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy originated in the bone marrow with poor overall survival rates of less than 30%. The immune system has been shown to play an important role in detecting and eliminating leukemic cells. Recent studies have described a population of immune cells termed Innate lymphoid cells (ILCs). ILCs are known to regulate both innate and adaptive immunity in an antigen-independent manner and are believed to be among the first immune cells to interact with malignantly transformed cells. Natural killer (NK) cells are a subset of ILCs and are known to inhibit AML progression, however the impact of other ILCs is not yet fully understood. Type 3 ILCs (ILC3s) have key roles in maintaining homeostasis in mucosal immunity through their ability to secrete cytokines including IL-17, IL-22, TNFα and GM-CSF (pro-myeloid). An expansion of ILC3s has recently been observed in solid tumors, however the overall functional impact of ILC3s in the setting of AML remains poorly understood and warrants further investigation. We first utilized an MLL-partial tandem duplication and FLT3-internal tandem duplication Flt3-ITD double knock-in mutation murine model of AML that recapitulates two clinically relevant mutations in human AML. Using this AML model, we discovered an expansion of ILC3s in the bone marrow of leukemic mice compared to wild-type control mice (2.364% +/- 0.40% AML vs 0.045% +/- 0.40% WT, n=5, p<0.0001). We also observed an increase in ILC3s in the bone marrow of untreated AML patients relative to age matched donor controls (7.013% +/- 2.6% vs. 2.328% +/- 2.6%). Our published data have previously shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. AHR is an important transcription factor in ILC3 development. Therefore, we hypothesized that the ILC3 increase was due at least in part to AHR-mediated increased ILC3 differentiation. To address this hypothesis, we isolated the ILC precursor (ILCP) from normal human donors and co-cultured with AML cells. By regulating AHR activity in this system, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (28.02% +/- 3.89% without AML, 60% +/- 5.5% with AML and 27% +/- 5% AML+AHR inhibitor, n=15, p<0.001). Next, we sought to determine the direct impact of ILC3s on AML proliferation and differentiation that could contribute to AML relapse. We utilized a colony forming assay (CFU) assay in the presence or absence of ILC3s. Bone marrow from primary untreated AML patients was cultured alone or with ILC3s (Lin-CD94-NKp44+). AML samples co-cultured with ILC3s had significantly increased colony formation relative to AML alone (69.4 CFU AML+ILC3 vs 42.7 CFU AML alone, n=8, p<0.05). Post-culture, we observed significant increases in the transcript of GM-CSF and TNFα in ILC3s co-cultured with AML marrow as compared to ILC3s alone. Furthermore, in our immunocompetent murine AML model, we observed significantly higher production of GM-CSF and TNFα by ILC3s in the bone marrow of leukemic mice relative to nonleukemic controls (GM-CSF: 11.08% +/- 1.6 AML vs 1.128% +/- 1.6 WT, n=10, p<0.0001; TNFα: 5.456% +/- 1.3 AML vs 1.307% +/- 1.3 WT, n=10, p<0.05), suggesting a potential mechanism of ILC3-mediated increased colony formation. Lastly, to determine if ILC3 expansion impacted NK cell immune surveillance, we co-cultured normal donor human ILC3s with AML cells, separated by transwell, for 48 hours before subjecting the AML cells to an NK cell cytotoxicity assay. We observed significantly reduced NK cell killing of AML cells when co-cultured with ILC3s compared to controls, suggesting ILC3s protect AML cells from NK cell-mediated killing (24.3% killing +/- 3.81% AML alone vs 14.57% +/- 1.35% AML with ILC3, n=18, p<0.01). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion to both support AML proliferation and protect AML blasts from immune surveillance. Future studies will focus in exploring how to target these interactions to design effective therapeutic approaches and improve patient clinical outcomes.

Synthetic Introns Identify the Novel RNA Splicing Factor GPATCH8 As Required for Mis-Splicing Induced By SF3B1 Mutations

Disclosures Mutations in the core RNA splicing factor SF3B1 are common across MDS, CLL, and clonal hematopoiesis. Prior studies have elucidated that mutations in SF3B1 result in neomorphic widespread changes in splicing due to usage of aberrant intronic branchpoint nucleotides. However, the molecular basis by which mutant SF3B1 induces mis-splicing is not established and therapeutic means to correct mis-splicing due to mutant SF3B1 do not exist. Recently, the aberrant gain-of-function splicing activity of mutant SF3B1 was harnessed to regulate expression of proteins in a mutant selective manner using synthetic version of endogenous mRNA sequences uniquely recognized by mutant SF3B1. Here, we utilized this technology to engineer a synthetic intron derived from MAP3K7 that interrupts the coding sequence of the fluorescent protein mEmerald such that mutant SF3B1 produces lower mEmerald expression than SF3B1 wild-type (WT) cells (Fig.A). This fluorescent splicing reporter was then used to perform positive enrichment whole genome CRISPR screens to identify genes whose deletion corrects SF3B1 mutant aberrant splicing. GPATCH8 was the single most robust hit. GPATCH8 knockout strongly corrected mis-splicing of both synthetic and endogenous MAP3K7 introns (Fig.A). A previously unexplored protein, GPATCH8 has domains characteristic of RNA splicing factors including a G-patch motif, thought to be important in activating RNA helicases. Interestingly, recent work suggests that mutations in SF3B1 result in mis-splicing by disrupting physical interaction of SF3B1 to the RNA helicase DHX15 via the G-patch domain containing protein SUGP1. We therefore compared SUGP1 to GPATCH8 activity, evaluating first their transcriptome-wide binding sites using TRIBE-seq. We found that both proteins predominantly target intronic sequences over other genomic regions suggesting roles in splicing. RNA-seq of SF3B1 mutant and WT cells with or without GPATCH8/SUGP1 deletion revealed that ~33% of SF3B1 mutant splicing alterations are corrected by GPATCH8 deletion, while ~60% of SF3B1 mutant splicing alterations are recapitulated upon SUGP1 deletion. GPATCH8 regulated splicing events have stronger branchpoints indicating a potential role for GPATCH8 in 3' splice site recognition. To further understand the connection between GPATCH8 and mutant SF3B1, we performed mass spectrometry studies of immunoprecipitated endogenous GPATCH8 in SF3B1 WT and mutant knockin cells. This revealed strong interaction between GPATCH8 and DHX15. GPATCH8 interacts with DHX15 at the same sites previously shown to be occupied by SUGP1 in crystal structures of the SUGP1/DHX15 interaction. These data and further biochemical studies elucidated that GPATCH8 competes with SUGP1 for interaction to DHX15. As such, deletion of GPATCH8 corrects SF3B1 mutant mis-splicing by enhancing the interaction of SUGP1 and DHX15 to the mutant SF3b complex. Given that GPATCH8 is required for a large proportion of the splicing alterations induced by mutant SF3B1, we next evaluated the phenotypic effect of correcting SF3B1 mutant mis-splicing. We first tested the impact of anti-Gpatch8 shRNAs in bone marrow from WT mice as well as animals with conditional knockin of Sf3b1 K700E, K666N, or R625H mutations. While each Sf3b1 mutation results in impaired colony formation of hematopoietic precursors in methylcellulose assays, silencing of Gpatch8 rescued colony formation from hematopoietic precursors of each of the different Sf3b1 mutant mice and was tolerated by normal hematopoietic precursors (Fig.B). Moreover, while CRISPR base-edited knockin of the SF3B1 K700E mutation in adult CD34 + cells impaired erythroid development, this erythroid differentiation defect was rescued by GPATCH8 deletion in the same cells (Fig.B). These data identify GPATCH8 as a novel RNA splicing factor involved in quality control of RNA branchpoint selection whose expression is required for mis-splicing by the different mutant forms of SF3B1. GPATCH8 antagonizes the activity of SUGP1 by competing for interaction with DHX15. These findings suggest that disrupting GPATCH8/DHX15 interaction could have important therapeutic benefit for the multitude of SF3B1 mutant hematopoietic diseases. This study also demonstrates the power of synthetic intronic splicing assays for discovery of trans factors and druggable proteins required by leukemia-associated mutant RNA splicing factors. Abdel-Wahab: AbbVie, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Loxo/Lilly: Consultancy; Nurix Therapeutics: Research Funding; Minovia Therapeutics: Research Funding; Amphista Therapeutics: Consultancy; AstraZeneca: Consultancy; Harmonic Discovery: Current holder of stock options in a privately-held company. Figure 1

Conserved and Unique Regulatory Mechanisms of RUNX1 in Hematopoietic Stem/Progenitor Cell Subpopulations in Both Normal Hematopoiesis and FPD/AML Development

RUNX1 is a RUNT domain core-binding factor indispensable for the establishment of definitive hematopoiesis in vertebrate. In humans, heterozygous RUNX1 germline mutations are closely associated with Familial Platelet Disorder with propensity to develop AML (FPD/AML), a rare autosomal dominant disorder characterized by thrombocytopenia, platelet dysfunction, and markedly elevated risk for myelodysplastic syndrome and leukemia. RUNX1 regulates hematopoietic genes directly or indirectly, acting as a context-dependent activator or repressor of gene transcription. Here we have examined how RUNX1 regulates various subpopulations of hematopoietic stem/progenitor cells (HSPCs) to provide important insights into its physiologic and pathogenic roles and mechanisms.We used two complementary mouse models: a conditional RUNX1 knockout mouse, i,e, Mx-Cre;Runx1flox/flox, and a tetracycline-inducible FPD/AML patient derived RUNX1 S291fsX300 mutation knock-in crossed to a MLL-PTD knock-in mouse, i.e. MLL-PTD;RUNX1 S291fsX300. We monitored hematopoiesis and/or disease progression in both models by tracking CBCs in peripheral blood and analyzing bone marrow cells by flow cytometry. The bone marrow samples were sorted by flow cytometry to yield HSPC subpopulations: HSC (LSK CD34-Flt3-), GMP (LK FCRII/III+CD34+), and MEP (LK FCRII/III-CD34-). Cells were analyzed for transcriptome by deep RNA-seq, epigenetics by ATAC-seq, and gene targets by anti-RUNX1 CHIP-seq. Focusing on the HSC, GMP, and MEP subpopulations, we found that RUNX1 deletion in the hematopoietic system causes thrombocytopenia and HSC engraftment defects, and dramatic changes in HSPC subpopulations: RUNX1 deficiency resulted in an increase MegaK-primed HSCs via blocking their direct differentiation to megakaryocytes leading to thrombocytopenia, and caused a marked increase of GMPs and a significant decrease of MEPs. Induction of RUNX1 S291fsX300 mutant in MLL-PTD;RUNX1 S291fsX300 mice mimicked that of RUNX1 deletion, resulting similarly in thrombocytopenia, an expansion of Mega-HSCs, increased GMPs, and decreased MEPs. “Correction” of the RUNX1S291fsX300 mutation by doxycycline withdraw reversed the FPD phenotype as well as the cellular distribution changes in HSCs, GMPs and MEPs. RNA-seq analyses of DEGs found that RUNX1 acts both as an activator and a suppressor in gene transcription in HSPCs. In HSCs, among 1879 DEGs, 608 are upregulated and 1271 are downregulated in Runx1 -/- compared with WT cells. In GMPs, 2042 DEGs were seen, among which 908 are upregulated and 1134 are downregulated whereas in MEPs, 316 transcripts were upregulated and 1214 were downregulated amongst 1530 DEGs in Runx1 -/- cells. Combining the DEGs from RNA-seq with annotated ATAC-seq peaks, we discovered through gene ontology analyses that a set of systemic development genes are commonly regulated by RUNX1 via chromosome accessibility in all HSPC subpopulations, with potentially shared targets such as Jam2, Bcar1, Irf4 Insrr , Cxcl12, Tnfsf4, Rap1gap, Aldh1a1. In contrast, in HSCs, a unique set of hemostasis/platelet regulatory genes such as Itga2b, Gp5, Pdgfb and Vwf were seen as potential targets, consistent with our recent findings ( Blood Adv 7:2590). In GMPs, RUNX1 appears to specifically regulate inflammatory and innate immunity genes with potential targets such as Gfi1b, Ets1, Tnfrsf11a while in MEP, RUNX1 specifically regulates cell motility and migration gene sets affecting targets such as Notch1, Ifitm1, Hoxa7, Cd300a Cxcl10 Fut7, suggesting a role of RUNX1 in modulating MEP movement/location in the bone marrow. RUNX1 CHIP-seq analyses are under way to further clarify the potential targets in each HSPC subpopulation. Our studies indicate that RUNX1 regulates common as well as distinct transcriptional events in various HSPC subpopulations by context-dependent activation and suppression mechanisms. RUNX1 plays a universal role in the systemic developmental program of various HSPC subpopulations but appears to engage in unique lineage specific factors to deliver subpopulation-specific functions such as mega-K cell fate-priming of HSCs, inflammatory program in GMPs, and bone marrow location signals in MEPs. Ongoing analyses of the FPD/AML mouse genomic data in this context are expected to reveal contributions of patient RUNX1 mutations to the disease development.

A homozygous variant in INTS11 links mitosis and neurogenesis defects to a severe neurodevelopmental disorder

The INTS11 endonuclease is crucial in modulating gene expression and has only recently been linked to human neurodevelopmental disorders (NDDs). However, how INTS11 participates in human development and disease remains unclear. Here, we identify a homozygous INTS11 variant in two siblings with a severe NDD. The variant impairs INTS11 catalytic activity, supported by its substrate's accumulation, and causes G2/M arrest in patient cells with length-dependent dysregulation of genes involved in mitosis and neural development, including the NDD gene CDKL5. The mutant knockin (KI) in induced pluripotent stem cells (iPSCs) disturbs their mitotic spindle organization and thus leads to slow proliferation and increased apoptosis, possibly through the decreased neurally functional CDKL5-induced extracellular signal-regulated kinase (ERK) pathway inhibition. The generation of neural progenitor cells (NPCs) from the mutant iPSCs is also delayed, with long transcript loss concerning neurogenesis. Our work reveals a mechanism underlying INTS11 dysfunction-caused human NDD and provides an iPSC model for this disease.

Context-dependent ciliary regulation of hedgehog pathway repression in tissue morphogenesis.

A fundamental problem in tissue morphogenesis is identifying how subcellular signaling regulates mesoscale organization of tissues. The primary cilium is a paradigmatic organelle for compartmentalized subcellular signaling. How signaling emanating from cilia orchestrates tissue organization-especially, the role of cilia-generated effectors in mediating diverse morpho-phenotypic outcomes-is not well understood. In the hedgehog pathway, bifunctional GLI transcription factors generate both GLI-activators (GLI-A) and GLI-repressors (GLI-R). The formation of GLI-A/GLI-R requires cilia. However, how these counterregulatory effectors coordinate cilia-regulated morphogenetic pathways is unclear. Here we determined GLI-A/GLI-R requirements in phenotypes arising from lack of hedgehog pathway repression (derepression) during mouse neural tube and skeletal development. We studied hedgehog pathway repression by the GPCR GPR161, and the ankyrin repeat protein ANKMY2 that direct cAMP/protein kinase-A signaling by cilia in GLI-R generation. We performed genetic epistasis between Gpr161 or Ankmy2 mutants, and Gli2/Gli3 knockouts, Gli3R knock-in and knockout of Smoothened, the hedgehog pathway transducer. We also tested the role of cilia-generated signaling using a Gpr161 ciliary localization knock-in mutant that is cAMP signaling competent. We found that the cilia-dependent derepression phenotypes arose in three modes: lack of GLI-R only, excess GLI-A formation only, or dual regulation of either lack of GLI-R or excess GLI-A formation. These modes were mostly independent of Smoothened. The cAMP signaling-competent non-ciliary Gpr161 knock-in recapitulated Gpr161 loss-of-function tissue phenotypes solely from lack of GLI-R only. Our results show complex tissue-specific GLI-effector requirements in morphogenesis and point to tissue-specific GLI-R thresholds generated by cilia in hedgehog pathway repression. Broadly, our study sets up a conceptual framework for rationalization of different modes of signaling generated by the primary cilium in mediating morphogenesis in diverse tissues.

Abstract 15747: AMPK Cysteine Oxidation by SOD3-Derived H 2 O 2 Mediates Exercise-Induced Restoration of Skeletal Muscle Angiogenesis in Type2 Diabetes

Background: Exercise restores decreased capillary density in skeletal muscle (SKM) (capillary rarefaction) in type2 diabetes (T2D) that has eNOS uncoupling/excess O 2 - , which is required for treatment of diabetic peripheral vascular disease (PAD) via unknown mechanisms. AMP-activated protein kinase (AMPK) is a key angiogenic/metabolic kinase mainly regulated by phosphorylation and is required for baseline, but not exercise-induced capillary density in SKM under healthy conditions. AMPK senses H 2 O 2 signal to increase its activity via oxidative modification of Cys299/304 in vitro. In addition, extracellular SOD (SOD3) that catalyzes the dismutation of O 2 - to H 2 O 2 is increased in response to exercise. However, the role of AMPK and SOD3 in exercise-induced SKM angiogenesis in T2D has not been reported. Oxidation of Cysteine residues to generate cysteine sulfenic acid (Cys-OH) is a key event in H 2 O 2 -mediated signaling. Results: Here we show that reduced CD31+ capillary density in SKM of high fat diet-induced T2D mice was restored (2.4-fold) by voluntary wheel exercise training (2 weeks) with enhanced SOD3 activity (224%) and extracellular H 2 O 2 levels (3.2-fold). Of note, exercise in T2D mice increased AMPK activity (by p-ACC) via inducing CysOH formation of AMPK (3.6-fold), but not p-AMPK induction, which was inhibited by SOD3 -/- /T2D mice that reduced exercise-induced H 2 O 2 . To demonstrate functional significance of AMPK Cys oxidation, we generated CRISPR/Cas9-genome editing “redox-dead” AMPK-Cys299/304Ala (AMPK-C/A) knock-in (KI) mutant mice and found that AMPK-C/A KI mice showed significant inhibition of exercise-induced angiogenesis (44%) via reducing angiogenic PGC1α and VEGF protein expression in SKM of T2D mice. In vitro, C2C12 skeletal muscles overexpressing SOD3 treated with palmitate (mimic T2D with exercise) induced AMPK-CysOH formation and VEGF secretion. Its conditioned media promoted angiogenic responses such as EC migration (1.8-fold) and tube formation (1.7-fold), which was inhibited by C2C12 cells overexpressing AMPK C/A mutant. Conclusion: Novel exercise-induced AMPK CysOH formation at Cys299/304 restores angiogenesis in SKM of T2D via SOD3-mediated H 2 O 2 , which may contribute to revascularization in diabetic PAD.

Mild weight loss and activation of RAAS in gene-edited mice with a E958A knock-in mutation in the AF2-LBD domain of the mineralocorticoid receptor gene

Mild weight loss and activation of RAAS in gene-edited mice with a E958A knock-in mutation in the AF2-LBD domain of the mineralocorticoid receptor gene