Conditional Allele Research Articles

Trp53-Loss in Hematopoietic Stem Cells Drives the Evolutionary Process of Leukemic Transformation in a Jak2V617F-Driven Myeloproliferative Neoplasms Mouse Model

Myeloproliferative neoplasms (MPNs) are clonal disorders resulting from genetic lesions in hematopoietic stem cells (HSCs). The development of post-MPN AML is treatment resistant and associated with very poor outcomes. This leukemic transformation is due to TP53 inactivation in approximately 50% of cases. Existing mouse models with double mutant Jak2V617F and Trp53 have demonstrated that Trp53-lossalone is sufficient to induce leukemic transformation in Jak2V617F-driven MPN. These models were generated either by crossing Jak2V617F transgenic mice with Trp53R172H knock-in or Trp53 knock-out mice, or by retroviral expression of Jak2V617F in Trp53 knock-out bone morrow (BM) cells. These germline mutated models do not accurately model the sequential somatic Trp53 mutations that arise in Jak2V617F MPN, or track their clonal expansion in vivo. Here, we used CRISPR-Cas9 to induce Trp53-loss in HSCs of Jak2V617F mice to generate a novel mouse model of high molecular risk MPN that transforms to AML. We isolated lineage -/low Sca1 +c-Kit + (LSK) cells from BM of donor mice expressing a conditional allele of mutant Jak2 from its endogenous locus ( Jak2fl-V617F) in combination with an inducible Cas9-IRES-GFP allele and CreER-fusion allele, both from the Rosa26 locus ( Rosa26CreER/lsl-Cas9-IRES-GFP). LSKs were transduced with lentivirus expressing a sgRNA targeting exon 4 of Trp53 (sgTrp53), located in the DNA-binding domain of Trp53. These transfected sgTrp53 LSKs, along with control groups transduced with either empty vector (EV) or a sgRNA targeting Catsper1 (sgCatsper1, off target cutting control), were transplanted into lethally irradiated recipients. Successful engraftment and CreER activity, induced by a 2-week period of Tamoxifen chow, were confirmed by flow cytometry. Validation of Trp53-editing was performed using amplicon sequencing, revealing the most common mutations were frameshift mutations. A subset of these frameshift mutations exhibited an extremely high frequency, conferring a selective advantage within the cell populations in sgTrp53 mice. We further examined the levels of p53 induced by nutlin-3a (an MDM2 inhibitor) in edited BM cells from three groups of mice and found that Trp53 editing leads to a reduction in p53 levels and prevents the loss of viability in response to MDM2 inhibition. This indicates that CRISPR-Cas9 mediated Trp53 mutations result in a loss of function. SgTrp53 mice recapitulated the evolution of leukemic transformation of MPN, showing a PV phenotype for 8 weeks post CreER activation, characterized by elevated hematocrit but normal leukocytes and platelet counts, consistent with the phenotype exhibited by the EV and sgCatsper1 recipients. Subsequently, after 12 weeks, this MPN phenotype transformed to acute leukemia. This disease progression was associated with significantly shortened survival of sgTrp53 recipients, compared to the sgCatsper1 or EV control groups. Histopathologic analysis further supported these findings, showing leukocytosis and the presence of immature blast cells in the PB, spleen and BM of sgTrp53 mice, whereas such cells were absent in the control groups. Notably, the frequency of blast cells was correlated with the frequency of Trp53 frameshift mutations, consistent with what has been reported in MPN patients. Additionally, CRISPR-Cas9 mediated Trp53-loss primarily affected the erythroid lineage, driving expansion of erythroblasts, megakaryocyte-erythroid progenitors (MEP), and Lineage -/low Sca1 -c-Kit + in sgTrp53 mice. The erythroblasts showed aberrant co-expression of CD41 and CD71. Through secondary transplants with unfractionated spleen cells, whole BM cells, or sorted MEP cell populations, we confirmed that the leukemia driven by CRISPR-cas9 mediated Trp53-loss in the Jak2V617F-driven PV is transplantable, and that the MEP compartment in leukemic sgTrp53 mice contains the leukemia initiation population. In total, we used CRISPR-Cas9 to induce Trp53-loss in HSCs of Jak2V617F mice, faithfully replicating the process of clonal expansion and evolution observed in the leukemic transformation of MPN. These mouse models of secondary AML are valuable for testing new therapies targeting disease initiating stem cells. Moreover, our models represent the distinct stages of MPN progression, offering a platform to evaluate preventive and therapeutic interventions at various stages of this disease.

Abstract 084: Smooth Muscle Cullin-3 Regulates The Renal Baroreceptor Mechanism Via Lamin AC

Cullin-3 (CUL3) is a critical component of the CUL3-Ring-Ligase (CRL) ubiquitin ligase complex. Patients carrying mutations in CUL3 exhibit early onset of hypertension (HTN). We demonstrated that conditional ablation of CUL3 in vascular smooth muscle cells (VSMCs) results in vascular dysfunction, arterial stiffness, and severe HTN in mice. We hypothesized that 1) conditional deletion of CUL3 in VSMCs (S-CUL3KO) results in severe HTN via the renin-angiotensin system (RAS)-dependent mechanism, and 2) S-CUL3KO mice exhibit an impaired baroreceptor mechanism regulating renin. Mice carrying a conditional allele of CUL3 were bred with mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter (ISM-CRE, control). S-CUL3KO mice exhibited elevated systolic blood pressure (SBP) 3 weeks after tamoxifen administration (S-CUL3KO: 164±1 vs. ISM-CRE: 124±4 mmHg, p<0.05). To test the first hypothesis captopril or candesartan (10 mg/kg/day and 100 mg/kg/day in drinking water, respectively) were administered to S-CUL3KO once severe HTN was already established. Captopril or candesartan treatment for 1 week reversed elevated SBP to 117±4 mmHg (p<0.05) and 127±2 mmHg (p<0.05), respectively, in S-CUL3KO. Despite the severity of HTN in S-CUL3KO, the level of renin mRNA in the kidney was surprisingly unaltered (p>0.05) when compared to the normotensive control mice. The protein expression of lamin AC, a crucial component of the nuclear mechanotransducer controlling renin expression, was significantly increased in S-CUL3KO kidneys (0.11±0.02 vs. 0.05±0.01 normalized RFU, p<0.05). This suggests lamin AC may be a target protein of CUL3-ubiquitin ligase complex. Supporting this concept, treatment of A10 smooth muscle cells with MLN4914, an inhibitor of the Cullin pathway, results in a 2-fold increase in lamin AC expression (0.007±0.001 vs. 0.0154±0.0004, normalized RFU, p<0.05). Furthermore, co-immunoprecipitation of CUL3 in HEK293 cells immunoprecipitated lamin AC. Our findings support the hypothesis that 1) CUL3 regulates RAS via the renal baroreceptor mechanism and 2) lamin AC is a potential target of the CRL3-mediated regulation. Understanding CUL3 target proteins might help the development of new strategies to mitigate HTN.

NOTCH and AKT Signalling Interact to Drive Mammary Tumour Heterogeneity.

A better understanding of the mechanisms generating tumour heterogeneity will allow better targeting of current therapies, identify potential resistance mechanisms and highlight new approaches for therapy. We have previously shown that in genetically modified mouse models carrying conditional oncogenic alleles, mammary tumour histotype varies depending on the combination of alleles, the cell type to which they are targeted and, in some cases, reproductive history. This suggests that tumour heterogeneity is not a purely stochastic process; rather, differential activation of signalling pathways leads to reproducible differences in tumour histotype. We propose the NOTCH signalling pathway as one such pathway. Here, we have crossed conditional knockout Notch1 or Notch2 alleles into an established mouse mammary tumour model. Notch1/2 deletion had no effect on tumour-specific survival; however, loss of Notch alleles resulted in a dose-dependent increase in metaplastic adenosquamous carcinomas (ASQCs). ASQCs and adenomyoepitheliomas (AMEs) also demonstrated a significant increase in AKT signalling independent of Notch status. Therefore, the NOTCH pathway is a suppressor of the ASQC phenotype, while increased PI3K/AKT signalling is associated with ASQC and AME tumours. We propose a model in which PI3K/AKT and NOTCH signalling act interact to determine mouse mammary tumour histotype.

The longevity and reversibility of quiescence in Schizosaccharomyces pombe are dependent upon the HIRA histone chaperone

ABSTRACT Quiescence (G0) is a reversible non-dividing state that facilitates cellular survival in adverse conditions. Here, we demonstrate that the HIRA histone chaperone complex is required for the reversibility and longevity of nitrogen starvation-induced quiescence in Schizosaccharomyces pombe. The HIRA protein, Hip1 is not required for entry into G0 or the induction of autophagy. Although hip1Δ cells retain metabolic activity in G0, they rapidly lose the ability to resume proliferation. After a short period in G0 (1 day), hip1Δ mutants can resume cell growth in response to the restoration of a nitrogen source but do not efficiently reenter the vegetative cell cycle. This correlates with a failure to induce the expression of MBF transcription factor-dependent genes that are critical for S phase. In addition, hip1Δ G0 cells rapidly progress to a senescent state in which they can no longer re-initiate growth following nitrogen source restoration. Analysis of a conditional hip1 allele is consistent with these findings and indicates that HIRA is required for efficient exit from quiescence and prevents an irreversible cell cycle arrest.

Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

Ezh2 promotes mammary tumor initiation through epigenetic regulation of the Wnt and mTORC1 signaling pathways

The regulation of gene expression through histone posttranslational modifications plays a crucial role in breast cancer progression. However, the molecular mechanisms underlying the contribution of histone modification to tumor initiation remain unclear. To gain a deeper understanding of the role of the histone modifier Enhancer of Zeste homology 2 (Ezh2) in the early stages of mammary tumor progression, we employed an inducible mammary organoid system bearing conditional Ezh2 alleles that faithfully recapitulates key events of luminal B breast cancer initiation. We showed that the loss of Ezh2 severely impairs oncogene-induced organoid growth, with Ezh2-deficient organoids maintaining a polarized epithelial phenotype. Transcriptomic profiling showed that Ezh2-deficient mammary epithelial cells up-regulated the expression of negative regulators of Wnt signaling and down-regulated genes involved in mTORC1 (mechanistic target of rapamycin complex 1) signaling. We identified Sfrp1, a Wnt signaling suppressor, as an Ezh2 target gene that is derepressed and expressed in Ezh2-deficient epithelium. Furthermore, an analysis of breast cancer data revealed that Sfrp1 expression was associated with favorable clinical outcomes in luminal B breast cancer patients. Finally, we confirmed that targeting Ezh2 impairs mTORC1 activity through an indirect mechanism that up-regulates the expression of the tumor suppressor Pten. These findings indicate that Ezh2 integrates the mTORC1 and Wnt signaling pathways during early mammary tumor progression, arguing that inhibiting Ezh2 or therapeutically targeting Ezh2-dependent programs could be beneficial for the treatment of early-stage luminal B breast cancer.

Acetaldehyde and defective mismatch repair increase colonic tumours in a Lynch syndrome model with Aldh1b1 inactivation.

ALDH1B1 expressed in the intestinal epithelium metabolises acetaldehyde to acetate, protecting against acetaldehyde-induced DNA damage. MSH2 is a key component of the DNA mismatch repair (MMR) pathway involved in Lynch syndrome (LS)-associated colorectal cancers. Here, we show that defective MMR (dMMR) interacts with acetaldehyde, in a gene/environment interaction, enhancing dMMR-driven colonic tumour formation in a LS murine model of Msh2 conditional inactivation (Lgr5-CreER; Msh2flox/-, or Msh2-LS) combined with Aldh1b1 inactivation. Conditional (Aldh1b1flox/flox) or constitutive (Aldh1b1-/-) Aldh1b1 knockout alleles combined with the conditional Msh2flox/- intestinal knockout mouse model of LS (Msh2-LS) received either ethanol, which is metabolised to acetaldehyde, or water. We demonstrated that 41.7% of ethanol-treated Aldh1b1flox/flox Msh2-LS mice and 66.7% of Aldh1b1-/- Msh2-LS mice developed colonic epithelial hyperproliferation and adenoma formation, in 4.5 and 6 months, respectively, significantly greater than 0% in water-treated control mice. Significantly higher numbers of dMMR colonic crypt foci precursors and increased plasma acetaldehyde levels were observed in ethanol-treated Aldh1b1flox/flox Msh2-LS and Aldh1b1-/- Msh2-LS mice compared with those in water-treated controls. Hence, ALDH1B1 loss increases acetaldehyde levels and DNA damage that interacts with dMMR to accelerate colonic, but not small intestinal, tumour formation.

Long-read sequence analysis of MMEJ-mediated CRISPR genome editing reveals complex on-target vector insertions that may escape standard PCR-based quality control

CRISPR genome editing is a powerful tool for elucidating biological functions. To modify the genome as intended, it is essential to understand the various modes of recombination that can occur. In this study, we report complex vector insertions that were identified during the generation of conditional alleles by CRISPR editing using microhomology-mediated end joining (MMEJ). The targeting vector contained two loxP sequences and flanking 40-bp microhomologies. The genomic regions corresponding to the loxP sequences were cleaved with Cas9 in mouse embryonic stem cells. PCR screening for targeted recombination revealed a high frequency of bands of a larger size than expected. Nanopore sequencing of these bands revealed complex vector insertions mediated not only by MMEJ but also by non-homologous end joining and homologous recombination in at least 17% of the clones. A new band appeared upon improving the PCR conditions, suggesting the presence of unintentionally modified alleles that escape standard PCR screening. This prompted us to characterize the recombination of each allele of the genome-edited clones using heterozygous single nucleotide polymorphisms, leading to confirmation of the presence of homozygous alleles. Our study indicates that careful quality control of genome-edited clones is needed to exclude complex, unintended, on-target vector insertion.

Cover Image, Volume 61, Issue 3–4

Cover illustration: The cover image is based on the Technical Note A CRISPR/Cas9‐engineered mouse carrying a conditional knockout allele for the early growth response‐1 transcription factor by Vineet K. Maurya et al., https://doi.org/10.1002/dvg.23515.image

Spatiotemporal control of genome engineering in cone photoreceptors

BackgroundCones are essential for color recognition, high resolution, and central vision; therefore cone death causes blindness. Understanding the pathophysiology of each cell type in the retina is key to developing therapies for retinal diseases. However, studying the biology of cone cells in the rod-dominant mammalian retina is particularly challenging. In this study, we used a bacterial artificial chromosome (BAC) recombineering method to knock in the “CreERT2” sequence into the Gnat2 and Arr3 genes, respectively and generated three novel inducible CreERT2 mice with different cone cell specificities.ResultsThese models (Gnat2CreERT2, Arr3T2ACreERT2, and Arr3P2ACreERT2) express temporally controllable Cre recombinase that achieves conditional alleles in cone photoreceptors. Cre-LoxP recombination can be induced as early as postnatal day (PD) two upon tamoxifen injection at varying efficiencies, ranging from 10 to 15% in Gnat2CreERT2, 40% in Arr3T2ACreERT2, and 100% in Arr3P2ACreERT2. Notably, knocking in the P2A-CreERT2 cassette does not affect cone cell morphology and functionality. Most cone-phototransduction enzymes, including Opsins, CNGA3, etc. are not altered except for a reduction in the Arr3 transcript.ConclusionsThe Arr3P2ACreERT2 mouse, an inducible cone-specific Cre driver, is a valuable line in studying cone cell biology, function, as well as its relationship with rod and other retinal cells. Moreover, the Cre activity can be induced by delivering tamoxifen intragastrically as early as PD2, which will be useful for studying retinal development or in rapid degenerative mouse models.

Dnajb11-Kidney Disease Develops from Reduced Polycystin-1 Dosage but not Unfolded Protein Response in Mice.

Heterozygous DNAJB11 mutation carriers manifest with small cystic kidneys and renal failure in adulthood. Recessive cases with prenatal cystic kidney dysplasia were recently described. Our in vitro and mouse model studies investigate the proposed disease mechanism as an overlap of autosomal-dominant polycystic kidney disease and autosomal-dominant tubulointerstitial kidney disease pathogenesis. We find that DNAJB11 loss impairs cleavage and maturation of the autosomal-dominant polycystic kidney disease protein polycystin-1 (PC1) and results in dosage-dependent cyst formation in mice. We find that Dnajb11 loss does not activate the unfolded protein response, drawing a fundamental contrast with the pathogenesis of autosomal-dominant tubulointerstitial kidney disease. We instead propose that fibrosis in DNAJB11 -kidney disease may represent an exaggerated response to polycystin-dependent cysts. Patients with heterozygous inactivating mutations in DNAJB11 manifest with cystic but not enlarged kidneys and renal failure in adulthood. Pathogenesis is proposed to resemble an overlap of autosomal-dominant polycystic kidney disease (ADPKD) and autosomal-dominant tubulointerstitial kidney disease (ADTKD), but this phenotype has never been modeled in vivo . DNAJB11 encodes an Hsp40 cochaperone in the endoplasmic reticulum: the site of maturation of the ADPKD polycystin-1 (PC1) protein and of unfolded protein response (UPR) activation in ADTKD. We hypothesized that investigation of DNAJB11 would shed light on mechanisms for both diseases. We used germline and conditional alleles to model Dnajb11 -kidney disease in mice. In complementary experiments, we generated two novel Dnajb11-/- cell lines that allow assessment of PC1 C-terminal fragment and its ratio to the immature full-length protein. Dnajb11 loss results in a profound defect in PC1 cleavage but with no effect on other cystoproteins assayed. Dnajb11-/- mice are live-born at below the expected Mendelian ratio and die at a weaning age with cystic kidneys. Conditional loss of Dnajb11 in renal tubular epithelium results in PC1 dosage-dependent kidney cysts, thus defining a shared mechanism with ADPKD. Dnajb11 mouse models show no evidence of UPR activation or cyst-independent fibrosis, which is a fundamental distinction from typical ADTKD pathogenesis. DNAJB11 -kidney disease is on the spectrum of ADPKD phenotypes with a PC1-dependent pathomechanism. The absence of UPR across multiple models suggests that alternative mechanisms, which may be cyst-dependent, explain the renal failure in the absence of kidney enlargement.

CLIC4 Regulates Endothelial Barrier Control by Mediating PAR1 Signaling via RhoA.

Endothelial CLICs (chloride intracellular channel proteins) CLIC1 and CLIC4 are required for the GPCRs (G-protein-coupled receptors) S1PR1 (sphingosine-1-phosphate receptor 1) and S1PR3 to activate the small GTPases Rac1 (Ras-related C3 botulinum toxin substrate 1) and RhoA (Ras homolog family member A). To determine whether CLIC1 and CLIC4 function in additional endothelial GPCR pathways, we evaluated CLIC function in thrombin signaling via the thrombin-regulated PAR1 (protease-activated receptor 1) and downstream effector RhoA. We assessed the ability of CLIC1 and CLIC4 to relocalize to cell membranes in response to thrombin in human umbilical vein endothelial cells (HUVEC). We examined CLIC1 and CLIC4 function in HUVEC by knocking down expression of each CLIC protein and compared thrombin-mediated RhoA or Rac1 activation, ERM (ezrin/radixin/moesin) phosphorylation, and endothelial barrier modulation in control and CLIC knockdown HUVEC. We generated a conditional murine allele of Clic4 and examined PAR1-mediated lung microvascular permeability and retinal angiogenesis in mice with endothelial-specific loss of Clic4. Thrombin promoted relocalization of CLIC4, but not CLIC1, to HUVEC membranes. Knockdown of CLIC4 in HUVEC reduced thrombin-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Knockdown of CLIC1 did not reduce thrombin-mediated RhoA activity but prolonged the RhoA and endothelial barrier response to thrombin. Endothelial-specific deletion of Clic4 in mice reduced lung edema and microvascular permeability induced by PAR1 activating peptide. CLIC4 is a critical effector of endothelial PAR1 signaling and is required to regulate RhoA-mediated endothelial barrier disruption in cultured endothelial cells and murine lung endothelium. CLIC1 was not critical for thrombin-mediated barrier disruption but contributed to the barrier recovery phase after thrombin treatment.

BIOL-23. LOSS OF MICRORNAS COOPERATES WITH TUMOR SUPPRESSORS TO INITIATE MYC-DEPENDENT METASTATIC MEDULLOBLASTOMA

Abstract To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma (MB) instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and the cerebellar tumors were histologically indistinguishable from human MB. The minimum genetic mutations required for MB formation were Dicer1 and Trp53, while additional loss of Pten produced more invasive tumors with leptomeningeal metastasis. Analysis of tumor transcriptome and MB subtype-specific markers show that Dicer tumors most resemble human Sonic Hedgehog (SHH) MB. The presence of leptomeningeal metastasis, Trp53 loss and upregulation of Mycn and Gli2 in the setting of activated Shh signaling suggests that this model phenocopies extremely high risk p53-mutated SHH MB, which carries poor prognosis and is associated with the majority of treatment failures in SHH MB. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the external granule cell layer (EGL), suggesting that tumors may arise from cerebellar granule neuron precursors (CGNPs). However, targeting CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Dicer tumors exhibit reduced microRNAs and RNA-induced silencing complexes consistent with loss of Dicer. One of these downregulated microRNAs, miR-101, is a regulator of Mycn and its overexpression leads to Mycn downregulation and inhibition of MB growth. Likewise, genetic and pharmacologic inhibition of Myc leads to inhibition of MB growth. These studies demonstrate a role for microRNAs in Myc-dependent MB development and show an alternative pathway for SHH MB tumorigenesis.

Abstract A06: Murine models of mutant p53 clonal hematopoiesis elucidate mechanisms of expansion of p53 mutant subclones by MDM2 inhibition

Abstract Unleashing the pro-apoptotic effects of p53 by pharmacologic inhibition of MDM2 has been an attractive therapeutic strategy in hematologic malignancies with intact p53. However, concerns were expressed regarding the selection of TP53-mutant subclones with potential GOF activities. It is not clear whether pre-existing sub-clones with TP53 mutation(s) undergo malignant transformation or clonal expansion after exposure to MDM2i therapy. We generated traceable conditional somatic TP53R172H mutant mouse models to monitor and track the expansion of p53 mutant myeloid cells over the course of therapy with MDM2i. We combined a myeloid-specific Cre line, LysM-CreERT2, or Mx1-cre with a conditional knock-in mutant Trp53 allele that constitutively expresses WT Trp53 and converts to mutant (Mut) Trp53R172H upon Cre-mediated recombination. To trace the recombined hematopoietic cells, we used the Rosa26mTmG reporter allele as previously described and performed a pulse-chase experiment. LysM-CreERT2;mTmG;Trp53R172H/fx mice did not develop leukemia after 1 year upon multiple tamoxifen inductions, suggesting that Trp53 mutation in mature myeloid cells is not leukemogenic. Having a GFP marker in myeloid cells allowed us to monitor the population of GFP+ myeloid cells in peripheral blood after each tamoxifen pulse. The GFP+ cells slowly disappeared from peripheral blood over time and were completely undetectable after 1 month following induction, suggesting that p53 mutation in myeloid cells does not extend the half-life of myeloid cells. Next, to assess if the Mdm2i therapy may cooperate with Trp53 mutation and transform the myeloid cells to AML, we induced the p53 mutation by tamoxifen injection and treated LysM-CreERT2;mTmG;Trp53fx/fx and LysM-CreERT2;mTmG;Trp53R172H/fx mice with the MDM2i, idasanutlin, for 10 days. There were no significant differences in the number of GFP+ myeloid cells between the two groups at baseline, however, in the p53-mutant group, the population of GFP+ cells was significantly decreased and in some mice was fully eradicated after MDM2i treatment. We transplanted LysM-CreERT2;mTmG;Trp53R172H/fx and control mice with syngeneic MLL-AF9 transformed leukemia cells and treated them with idasanutlin 1 week after transplant. Strikingly, the population of GFP+ cells was highly expanded in p53 mutant mice transplanted with AML after MDM2i treatment, suggesting that MDM2 inhibition promotes the proliferation of p53-mutant myeloid subclones in the context of AML. We sorted the GFP+ cells and transplanted them into syngeneic mice. No leukemia arose after transplantation. We here established the first in vivo model of p53-mutant clonal hematopoiesis using a new p53 mutant conditional allele. The expanded population of p53 mutant subclone did not transform into AML. However, in the presence of fully transformed AML cells, the p53 mutant clonal hematopoiesis expanded. This finding is consistent with reports of transitory expansion of p53 mutant cells in patients with myelofibrosis treated with MDM2 inhibitor. Citation Format: Russell Pourebrahim, Rafel Heinz Montoya, Lauren Ostermann, Michael Andreeff. Murine models of mutant p53 clonal hematopoiesis elucidate mechanisms of expansion of p53 mutant subclones by MDM2 inhibition [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A06.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells

Abstract Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγR) that engage the Fc region of IgG. Studies investigating the role of FcγR on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγR. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also express the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knock-out alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity (ADCC) of antibody-coated B cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B cell lymphomas. This project was funded by fellowships from Burroughs Wellcome Fund (BWF) and the Cancer Research Institute to O.A.A., and grants from the Parker Institute for Cancer Immunotherapy (PICI), and the NIH (AI068129 and AI146581) to L.L.L.

Regulation of Renin-Angiotensin System Activity and Blood Pressure by Vascular Smooth Muscle Cullin-3

Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) in smooth muscle cells (SMCs) cause vascular dysfunction, arterial stiffness, and severe hypertension. We hypothesized that conditional deletion of CUL3 in SMCs causes severe hypertension through a renin-angiotensin system (RAS)-dependent mechanism. Mice carrying a conditional allele of CUL3 were bred to mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter (ISM-CRE, control). Tamoxifen was administered to generate smooth muscle CUL3 knockout (S-CUL3KO). Primary aortic SMCs were used to elucidate the contribution of CUL3 to intracellular angiotensin II-mediated calcium flux. Calcium response in SMCs was analyzed via ratiometric confocal fluorescent microscopyS-CUL3KO mice demonstrated hypertension following three weeks of tamoxifen, mean arterial pressure (MAP) was 108±1 vs. 140±1 mmHg (ISM-CRE vs. S-CUL3KO). Aorta from S-CUL3KO mice exhibited impaired vasorelaxation to acetylcholine (ACh, max relaxation: 84±4% vs. 16±9%, p<0.05) and sodium nitroprusside (SNP, max relaxation: 95±3% vs. 56±9%, p<0.05). Captopril administration (7 days) markedly reduced MAP to 84±0.3 mmHg and improved vasorelaxation in response to both ACh (72±6%) and SNP (91±4%) in S-CUL3KO mice, suggesting RAS-dependency of the hypertension. Additionally, candesartan treatment (7 days) substantially decreased MAP from 138±1 to 96±3 mmHg suggesting that hypertension in S-CUL3KO group is angiotensin type-1 receptor mediated. Q-PCR and RNAScope analyses demonstrated no difference in renin expression in the kidney between the S-CUL3KO and control mice suggesting a blunting of the baroreceptor mechanism regulating renin mRNA expression. Equilibrium angiotensin II plasma levels in both groups were not different (2517±352 and 2379±549 pmol/L, p>0.05). Preliminary data demonstrated that acute application of 10 μM of angiotensin II to S-CUL3KO SMCs resulted in a sustained increase in intracellular calcium which was abolished by a preincubation with 2 μM of candesartan.Our data suggest that CUL3 may play a vital role in baroreceptor mechanism in the kidney and regulate angiotensin II response in SMCs which are essential for responding to changes in perfusion pressure. Ultimately, understanding CUL3 target proteins may benefit the development of novel treatments for hypertension. R35 HL144807 (to CDS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Syngap1 Disruption Induced by Recombination between Inverted loxP Sites Is Associated with Hippocampal Interneuron Dysfunction.

SYNGAP1 haploinsufficiency in humans causes intellectual disability (ID). SYNGAP1 is highly expressed in cortical excitatory neurons and, reducing its expression in mice accelerates the maturation of excitatory synapses during sensitive developmental periods, restricts the critical period window for plasticity, and impairs cognition. However, its specific role in interneurons remains largely undetermined. In this study, we investigated the effects of conditional Syngap1 disruption in medial ganglionic eminence (MGE)-derived interneurons on hippocampal interneuron firing properties and excitatory synaptic inputs, as well as on pyramidal cell synaptic inhibition and synaptic integration. We show that conditional Syngap1 disruption in MGE-derived interneurons results in cell-specific impairment of firing properties of hippocampal Nkx2.1 fast-spiking interneurons, with enhancement of their AMPA receptor (AMPAR)-mediated excitatory synaptic inputs but compromised short-term plasticity. In contrast, regular-spiking Nkx2.1 interneurons are largely unaffected. These changes are associated with impaired pyramidal cell synaptic inhibition and enhanced summation of excitatory responses. Unexpectedly, we found that the Syngap1flox allele used in this study contains inverted loxP sites and that its targeted recombination in MGE-derived interneurons induces some cell loss during embryonic development and the reversible inversion of the sequence flanked by the loxP sites in postmitotic cells. Together, these results suggest that Syngap1 plays a role in cell-specific regulation of hippocampal interneuron function and inhibition of pyramidal cells in mice. However, because of our finding that the Syngap1flox allele used in this study contains inverted loxP sites, it will be important to further investigate interneuron function using a different Syngap1 conditional allele.

Generation of floxed Spag6l mice and disruption of the gene by crossing to a Hprt-Cre line.

Mouse sperm-associated antigen 6 like (SPAG6L) is an axoneme central apparatus protein, essential for the normal function of the ependymal cell and lung cilia, and sperm flagella. Accumulated evidence has disclosed multiple biological functions of SPAG6L, including ciliary/flagellar biogenesis and polarization, neurogenesis, and neuronal migration. Conventional Spag6l knockout mice died of hydrocephalus, which impedes further investigation of the function of the gene in vivo. To overcome the limitation of the short lifespan of conventional knockout mice, we developed a conditional allele by inserting two loxP sites in the genome flanking exon 3 of the Spag6l gene. By crossing the floxed Spag6l mice to a Hrpt-Cre line which expresses Cre recombinase ubiquitously in vivo, mutant mice that are missing SPAG6L globally were obtained. Homozygous mutant Spag6l mice showed normal appearance within the first week after birth, but reduced body size was observed after 1week, and all developed hydrocephalus and died within 4 weeks of age. The phenotype mirrored that of the conventional Spag6l knockout mice. The newly established floxed Spag6l model provides a powerful tool to further investigate the role of the Spag6l gene in individual cell types and tissues.

Collagen XIX is required for pheromone recognition and glutamatergic synapse formation in mouse accessory olfactory bulb.

In mammals, the accessory olfactory bulb (AOB) receives input from vomeronasal sensory neurons (VSN) which detect pheromones, chemical cues released by animals to regulate the physiology or behaviors of other animals of the same species. Cytoarchitecturally, cells within the AOB are segregated into a glomerular layer (GL), mitral cell layer (MCL), and granule cell layer (GCL). While the cells and circuitry of these layers has been well studied, the molecular mechanism underlying the assembly of such circuitry in the mouse AOB remains unclear. With the goal of identifying synaptogenic mechanisms in AOB, our attention was drawn to Collagen XIX, a non-fibrillar collagen generated by neurons in the mammalian telencephalon that has previously been shown to regulate the assembly of synapses. Here, we used both a targeted mouse mutant that lacks Collagen XIX globally and a conditional allele allowing for cell-specific deletion of this collagen to test if the loss of Collagen XIX causes impaired synaptogenesis in the mouse AOB. These analyses not only revealed defects in excitatory synapse distribution in these Collagen XIX-deficient mutants, but also showed that these mutant mice exhibit altered behavioral responses to pheromones. Although this collagen has been demonstrated to play synaptogenic roles in the telencephalon, those roles are at perisomatic inhibitory synapses, results here are the first to demonstrate the function of this unconventional collagen in glutamatergic synapse formation.

Abstract 2733: TYMS as a novel target for AAV-based therapy

Abstract The purpose of this study is to develop a strategy for treating pancreatic neuroendocrine tumor (PanNET) through blocking Thymidylate Synthase (TYMS or TS), a DNA metabolic enzyme. Elevated TYMS levels are frequently observed in human PanNET and are associated with worse prognosis and resistance to cytotoxic agents such as 5FU. Elevated TYMS levels directly promote tumorigenesis in vitro and ectopic expression of human TS (hTS) in transgenic mice promotes adenoma development in the endocrine pancreas in vivo. Pancreatic islet tumor formation in hTS transgenic mice occurred with a long latency period, suggesting that additional somatic events are required for PanNET formation and progression. Men1 was shown to be a frequent target for somatic mutation in PanNETs (44% have MEN1 mutations). Therefore, we established a new mouse model designated hTS/Men1-/-, where hTS is overexpressed in pancreatic islet cells carrying conditional Men1 null alleles. We observed that ectopic hTS cooperates with Men1 inactivation to significantly accelerate PanNET progression and shorten survival. Since traditional TYMS inhibitors, such as 5-FU, consistently induce tumor resistance and cannot cure advanced disease, we have now tested the ability of TS-shRNA delivered using adeno-associated viral (AAV) vectors as a pharmacological safe and effective anti-tumor strategy. We packaged TS shRNA under the mouse insulin promoter (mIP) for islet-cell specific expression using capsid optimized Y to F double mutant AAV (AAV8-2M) to generate viral particles of scAAV8-2M-mIP-GFP-hTS-shRNA. We treated hTS/Men1-/- mice with intraperitoneal (IP) injection (1x1011 viral genomes/mice). Treated and control mice were analyzed for PanNET progression, TYMS expression and survival. In addition, we tested a human PanNET tumor cell line (BON) that was isogenic with or without hTS expression for tumor formation in xenograft model. We confirmed sustained reduction of TYMS levels 3 weeks after IP delivery of AA8-2M-mIP-GFP-hTS-shRNA into PanNET tissues of hTS/Men1-/- mice. In addition, AA8-2M-mIP-GFP-hTS-shRNA delivery blocked PanNET tumor progression and enhanced the survival of treated mice as compared to vector controls. We also confirmed that TYMS knock-down in BON cells by hTS-shRNA reduced xenograft tumor growth as compared to non-specific shRNA controls. In conclusion, the ability of AAV-mediated TS shRNA to block PanNET tumor progression directly demonstrates the importance of elevated TYMS in tumorigenesis and offers a novel anti-cancer strategy. Citation Format: Vinod Vijayakurup, Benjamin Meyer, Akbar Nawab, Frederic J. Kaye, Maria V. Guijarro, Maria Zajac-Kaye. TYMS as a novel target for AAV-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2733.