Haploinsufficient Cells Research Articles

Cul-4 inhibition rescues spastin levels and reduces defects in hereditary spastic paraplegia models.

Hereditary spastic paraplegias (HSPs) are degenerative motor neuron diseases characterized by progressive spasticity and weakness in the lower limbs. The most common form of HSP is due to SPG4 gene haploinsufficiency. SPG4 encodes the microtubule severing enzyme spastin. Although, there is no cure for SPG4-HSP, strategies to induce a spastin recovery are emerging as promising therapeutic approaches. Spastin protein levels are regulated by poly-ubiquitination and proteasomal-mediated degradation, in a neddylation-dependent manner. However, the molecular players involved in this regulation are unknown. Here, we show that the Cullin-4-RING E3 ubiquitin ligase complex (CRL4) regulates spastin stability. Inhibition of CRL4 increases spastin levels by preventing its poly-ubiquitination and subsequent degradation in spastin-proficient and in patient derived SPG4 haploinsufficient cells. To evaluate the role of CRL4 complex in spastin regulation in vivo, we developed a Drosophila melanogaster model of SPG4 haploinsufficiency which show alterations of synapse morphology and locomotor activity, recapitulating phenotypical defects observed in patients. Downregulation of the CRL4 complex, highly conserved in Drosophila, rescues spastin levels and the phenotypical defects observed in flies. As a proof of concept of possible pharmacological treatments, we demonstrate a recovery of spastin levels and amelioration of the SPG4-HSP-associated defects both in the fly model and in patient-derived cells by chemical inactivation of the CRL4 complex with NSC1892. Taken together, these findings show that CRL4 contributes to spastin stability regulation and that it is possible to induce spastin recovery and rescue of SPG4-HSP defects by blocking the CRL4-mediated spastin degradation.

Collaborative effect of Csnk1a1 haploinsufficiency and mutant p53 in Myc induction can promote leukemic transformation

AbstractIt is still not fully understood how genetic haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) contributes to malignant transformation of hematopoietic stem cells. We asked how compound haploinsufficiency for Csnk1a1 and Egr1 in the common deleted region on chromosome 5 affects hematopoietic stem cells. Additionally, Trp53 was disrupted as the most frequently comutated gene in del(5q) MDS using CRISPR/Cas9 editing in hematopoietic progenitors of wild-type (WT), Csnk1a1–/+, Egr1–/+, Csnk1a1/Egr1–/+ mice. A transplantable acute leukemia only developed in the Csnk1a1–/+Trp53–edited recipient. Isolated blasts were indefinitely cultured ex vivo and gave rise to leukemia after transplantation, providing a tool to study disease mechanisms or perform drug screenings. In a small-scale drug screening, the collaborative effect of Csnk1a1 haploinsufficiency and Trp53 sensitized blasts to the CSNK1 inhibitor A51 relative to WT or Csnk1a1 haploinsufficient cells. In vivo, A51 treatment significantly reduced blast counts in Csnk1a1 haploinsufficient/Trp53 acute leukemias and restored hematopoiesis in the bone marrow. Transcriptomics on blasts and their normal counterparts showed that the derived leukemia was driven by MAPK and Myc upregulation downstream of Csnk1a1 haploinsufficiency cooperating with a downregulated p53 axis. A collaborative effect of Csnk1a1 haploinsufficiency and p53 loss on MAPK and Myc upregulation was confirmed on the protein level. Downregulation of Myc protein expression correlated with efficient elimination of blasts in A51 treatment. The “Myc signature” closely resembled the transcriptional profile of patients with del(5q) MDS with TP53 mutation.

CTCF Is a Haploinsufficient Tumor Suppressor in B-Cell Lymphoma

Alteration of epigenetic regulators is a hallmark of diffuse large B cell lymphoma (DLBCL), the most common type of non-Hodgkin B cell malignancy. It has been recently shown that haploinsufficiency of the cohesin complex ATPase subunit Smc3 cooperates with the oncogene IμBcl6 to induce lymphoma in mice, by a mechanism that involves decreased intra-topological associating domain (TAD) chromosomal interactions and downregulation of tumor suppressor genes. The transcription factor CTCF is an obligate partner of the cohesin complex in defining TADs and regulatory gene loops, acting as the boundaries of TADs and restricting the interaction of promoters and enhancers. Alterations in CTCF have been shown in several cancer types, including lymphomas. In this study, we aim to define the role of CTCF during B-cell lymphoma and lymphomagenesis. We generated germinal center (GC) B cell-specific conditional knockout mice for CTCF by crossing the CTCF floxed mice to the Ighg1-Cre (Cγ1-Cre) allele. In these experiments, we compared haploinsufficient CTCF conditional knockout mice-in which only one allele of CTCF was knocked out-versus wild-type mice expressing the Cγ1-Cre. Eight days after immunization with the T-cell-dependent antigen sheep red blood cells, we excised spleens and surveyed B cell populations by flow cytometry and immunohistochemistry. Remarkably, CTCF haploinsufficient mice contained a similar percentage of follicular GC B cells compared to wild-type mice (Live B220 +CD95 +GL7 + or live B220 +CD95 +CD38 DIM splenocytes). However, CTCF haploinsufficient GCs displayed a bias in the GC subpopulations, namely an increased percentage of light zone centrocytes (LZ: GC + markers CXCR4 loCD86 hi) and decreased dark zone centroblasts (DZ: GC + markers CXCR4 hiCD86 lo). Because biases in GCs were an early indication of malignant transformation in previous studies, we wondered whether CTCF haploinsufficiency would cooperate with lymphoma oncogenes to induce B-cell lymphoma. To address the lymphomagenic potential of CTCF haploinsufficiency, we crossed CTCF conditional knockout mice to the vavP-Bcl2 mice that develop GC-derived follicular-like lymphoma with long latency. We harvested bone marrow blood cell precursors from Cγ1-Cre (“WT”), Cγ1-Cre;CTCF +/- (“CTCF”), vavP-BCL2 +;Cγ1-Cre (“BCL2”), and vavP-BCL2 +;Cγ1-Cre;CTCF +/- (“BCL2/CTCF”) and transplanted them into lethally irradiated C57BL6/J (30 per group). Mice that survived more than two weeks after irradiation and bone marrow transplant were considered stably engrafted and monitored for any signs of disease. Lethargic mice were sacrificed and macroscopically inspected for the presence of tumors. Survival analysis by Kaplan-Meier showed that BCL2/CTCF mice had the shortest survival from all four groups, having a median survival of 230 days, while survival for the BCL2 alone group did not fall below 50% up to day 370. Immunohistochemistry against B220 + was performed in tissue sampling from dying mice to confirm the cause of death. Curiously, CTCF haploinsufficiency accelerated BCL2-induced lymphomagenesis but did not affect the survival of IμBCL6-induced lymphoma. To explore the mechanistic basis of CTCF haploinsufficiency-induced lymphoma in mice, we FACS sorted DZ and LZ GC B-cells from 8-day immunized mice and explored global transcriptomics by RNA-sequencing. While the transition from DZ to LZ involves the regulation of around ~1,500 genes, we failed to detect changes in gene expression between wild-type and CTCF haploinsufficiency cells. ATAC-seq assay on the same cells showed little to no change in chromatin accessibility between cells from CTCF haploinsufficient and wild-type mice. TOBIAS footprinting analysis, which identifies biases in Tn5 transposase activity due to protein binding, showed that CTCF haploinsufficient cells maintained a deep depletion in CTCF binding. Hi-C chromosomal conformation analysis identified an overlapping set of gene regulatory loops that lose interactions and showed decreased signal by gene set enrichment analysis. In this study, we characterized the role of CTCF haploinsufficiency phenotypically and are currently exploring the molecular mechanisms that contribute to the acceleration of the lymphomagenesis induced by the BCL2 oncogene.

Abstract 2601: Lrig1 is a haplo-insufficient tumor suppressor and Lrig1-expressing cells can function as cells-of-origin of tumor development

Abstract LRIG1, leucine-rich repeats and immunoglobulin-like domains protein 1, is deleted or downregulated in many cancer types and has been reported to function as a tumor suppressor. LRIG1 is preferentially expressed in adult stem/progenitor cells and regulates their biological properties and quiescence by repressing the ERBB signaling. We recently reported LRIG1 to be overexpressed in human prostate cancer and to function as a feedback tumor suppressor. Human LRIG1 transgenically expressed in the mouse prostate inhibited the development and growth of both Hi-Myc and TRAMP tumors. The relationship between Lrig1-expressing adult stem/progenitor cells and cells-of-origin for cancer remains poorly understood. To address this, we deleted the Pten tumor suppressor gene in Lrig1-expressing cells by crossing Lrig1-CreERT2 mouse with Ptenfl/fl animals followed by Cre activation via tamoxifen administration. This knockout of Pten in Lrig1-expressing cells allowed us to assess the tumor suppressive function of Lrig1 as Lrig1-CreERT2 is a knock-in model in which one allele of Lrig1 is inactivated. We also generated a Lrig1-lineage tracing mouse model to label endogenous Lrig1-expressing cells by crossing Lrig1-CreERT2 mouse with CAG-tdTomato animals. Cre recombinase in these animals was activated by tamoxifen and epithelial tissues were harvested and analyzed for tomato signal by fluorescent microscopy. Lrig1-lineage tracing model showed that Lrig1-expressing cells in mouse prostate and oral epithelia are of CK5+ and CK8- phenotype suggesting their basal lineage. Targeted deletion of Pten in the Lrig1-expressing cellular compartments resulted in hyperplasic lesions in oral mucosa as early as 4 weeks and prevalent papilloma by 8 weeks. Pten deletion in the mouse prostate led to hyperplasia and high-grade prostate intraepithelial neoplasia (HGPIN) in a time-dependent manner. Consistent with these hyperplastic and tumor phenotypes, we observed elevated Ki-67 and pAkt staining indicating increased cell proliferation. Since Lrig1-CreERT2 is a knock-in model in which one allele of Lrig1 is inactivated, our data suggest that even one allelic loss of Lrig1 promotes more aggressive tumor phenotypes compared to those reported earlier with Pten deletion in CK5+ basal cells (CK5-CreERT2:Ptenfl/fl) in which similar lesions in oral mucosa and prostate tissues developed only after 20-24 weeks of Pten deletion. These results suggested that LRIG1 is a haplo-insufficient tumor suppressor as mono-allelic deletion of Lrig1 is sufficient to promote the tumorigenic process in mouse epithelial tissues induced by Pten loss. Our study also suggests that the Lrig-1 expressing cells can function as the cells-of-origin of cancer. We propose that Lrig1 may be used as a prognostic marker in cancers of epithelial origin and higher levels may portend a better prognosis. Citation Format: Moyi Wang, Qiuhui Li, Amanda Tracz, Jason Kirk, Anmbreen Jamroze, Rahul Kumar, Dean G. Tang. Lrig1 is a haplo-insufficient tumor suppressor and Lrig1-expressing cells can function as cells-of-origin of tumor development [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 2601.

P53-Independent Pathways in Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare inherited bone marrow failure syndrome that manifests as a variably penetrant macrocytic anemia, which can spontaneously enter remission for unclear reasons. Rpl5 (uL18) is one of the commonly mutated genes in DBA and is associated with a severe phenotype including morphological defects and reduced likelihood of spontaneous remission. Our previous work established both cellular [mouse embryonic stem (mES) cell] and murine genetic models of Rpl5 haploinsufficiency (Rpl5Skax23-Jus/1/+). Rpl5Skax23-Jus/1/+ mutant mice exhibited increased mortality at all ages, reduced fetal liver cellularity, and a block in erythroid differentiation at the CFU-E/proerythroblast stage at E12.5 that started to resolve by E14.5. Newborn mutants are born with macrocytic anemia and the majority exhibited histological evidence of a ventricular septal defect (VSD). In contrast, mutants that survived until weaning lacked anemia and VSD, suggesting that the presence or absence of VSD at E12.5 may identify animals that are more or less likely to spontaneously remit from anemia. Our previous studies demonstrated that Rpl5+/- mES cells have a p53-independent cell cycle defect with a delay in the G2/M transition. Additional preliminary studies indicate that Rpl5 haploinsufficient mES cells also demonstrate no p53 induction and a reduced apoptotic response to oxidative stress. Currently, we are exploring the p53-independent pathways that trigger and/or lead to resolution of anemia in Rpl5Skax23-Jus/1/+ mutant mice. We performed RNA-seq to identify, in an unbiased fashion, dysregulated pathways in Rpl5 haploinsufficient E12.5 fetal liver erythroid progenitors. We plan to next validate novel genes and pathways that may drive or predict erythroid failure and spontaneous DBA remission in our model.

ZEB2 haploinsufficient Mowat-Wilson syndrome induced pluripotent stem cells show disrupted GABAergic transcriptional regulation and function.

Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.

Error-prone repair of stalled replication forks drives mutagenesis and loss of heterozygosity in haploinsufficient BRCA1 cells

Germline mutations in the BRCA genes are associated with a higher risk of carcinogenesis, which is linked to an increased mutation rate and loss of the second unaffected BRCA allele (loss of heterozygosity, LOH). However, the mechanisms triggering mutagenesis are not clearly understood. The BRCA genes contain high numbers of repetitive DNA sequences. We detected replication forks stalling, DNA breaks, and deletions at these sites in haploinsufficient BRCA cells, thus identifying the BRCA genes as fragile sites. Next, we found that stalled forks are repaired by error-prone pathways, such as microhomology-mediated break-induced replication (MMBIR) in haploinsufficient BRCA1 breast epithelial cells. We detected MMBIR mutations in BRCA1 tumor cells and noticed deletions-insertions (>50bp) at the BRCA1 genes in BRCA1 patients. Altogether, these results suggest that under stress, error-prone repair of stalled forks is upregulated and induces mutations, including complex genomic rearrangements at the BRCA genes (LOH), in haploinsufficient BRCA1 cells.

Effects of Recloning on the Telomere Lengths of Mouse Terc+/- Nuclear Transfer-Derived Embryonic Stem Cells.

Haploinsufficiency of genes that participate in telomere elongation and maintenance processes, such as telomerase RNA component (Terc) and telomere reverse transcriptase (Tert), often leads to premature aging-related diseases such as dyskeratosis congenita and aplastic anemia. Previously, we reported that when mouse Terc+/- tail tip fibroblasts (TTFs) were used as donor cells for somatic cell nuclear transfer (SCNT, also known as cloning), the derivative embryonic stem cells (ntESCs) had elongated telomeres. In the present work, we are interested to know if an additional round of SCNT, or recloning, could lead to further elongation of telomeres. Terc+/- TTFs were used to derive the first-generation (G1) ntESCs, followed by a second round of SCNT using G1-Terc+/- ntESCs as donor cells to derive G2-Terc+/- ntESCs. Multiple lines of G1- and G2-Terc+/- ntESCs were efficiently established, and all expressed major pluripotent markers and supported efficient chondrocyte differentiation in vitro. Compared with donor TTFs, telomere lengths of G1 ntESCs were elongated to the level comparable with that in wild-type ntESCs. Interestingly, recloning did not further elongate the telomere lengths of Terc+/- ntESCs. Together, our work demonstrates that while a single round of SCNT is a viable means to reprogram Terc haploinsufficient cells to the ESC state, and to elongate these cells' telomere lengths, a second round of SCNT does not necessarily further elongate the telomeres.

Npm1 haploinsufficiency in collaboration with MEIS1 is sufficient to induce AML in mice

AbstractNPM1 is among the most frequently mutated genes in acute myeloid leukemia (AML). Mutations in the NPM1 gene result in the increased export of NPM1 to the cytoplasm (NPM1c) and are associated with multiple transforming events including the aberrant upregulation of MEIS1 that maintains stem cell and cell cycle–associated pathways in NPM1c AML. However, another consequence of the NPM1c mutation is the inadequate levels of NPM1 wild-type in the nucleus and nucleolus, caused by the loss of one wild-type allele in addition to enforced NPM1 nuclear export. The contribution of NPM1 haploinsufficiency independently of the NPM1 mutation to AML development and its relationship with MEIS1 function is poorly understood. Using mouse models, our study shows that NPM1 haploinsufficiency paired with MEIS1 overexpression is sufficient to induce a fully penetrant AML in mice that transcriptionally resembles human NPM1c AML. NPM1 haploinsufficiency alters MEIS1-binding occupancies such that it binds the promoter of the oncogene structural maintenance of chromosome protein 4 (SMC4) in NPM1 haploinsufficient AML cells but not in NPM1 wild-type–harboring Hoxa9/Meis1-transformed cells. SMC4 is higher expressed in haploinsufficient and NPM1c+ AML cells, which are more vulnerable to the disruption of the MEIS1-SMC4 axis compared with AML cells with nonmutated NPM1. Taken together, our study underlines that NPM1 haploinsufficiency on its own is a key factor of myeloid leukemogenesis and characterizes the MEIS1-SMC4 axis as a potential therapeutic target in this AML subtype.

B Cell-Intrinsic IRF4 Haploinsufficiency Impairs Affinity Maturation.

The germinal center (GC) reaction is a coordinated and dynamic ensemble of cells and processes that mediate the maturation and selection of high-affinity GC B cells (GCBs) from lower-affinity precursors and ultimately results in plasma cell and memory cell fates that exit the GC. It is of great interest to identify intrinsic and extrinsic factors that control the selection process. The transcription factor IRF4, induced upon BCR and CD40 signaling, is essential for the acquisition of plasma cell and GCB cell fates. We hypothesized that beyond this early requirement, IRF4 continuously operates at later phases of the B cell response. We show that IRF4 is expressed in GCBs at levels greater than seen in resting cells and plays a role in efficient selection of high-affinity GCBs. Halving Irf4 gene copy number in an Ag-specific murine B cell model, we found that Ag presentation, isotype switching, GC formation and zonation, somatic hypermutation rates, and proliferation were comparable with cells with a full Irf4 allelic complement. In contrast, Irf4 haploinsufficient GCBs exhibited impaired generation of high-affinity cells. Mechanistically, we demonstrate suboptimal Blimp-1 regulation among high-affinity Irf4 haploinsufficient GCBs. Furthermore, in cotransfer settings, we observed a marked disadvantage of Irf4 haploinsufficient cells for GC entry, evidential of ineffective recruitment of T cell help. We propose that, analogous to its role in early GC entry, IRF4 continues to function in the late phase of the Ab response to promote productive T follicular helper cell interactions and to activate optimal Blimp-1 expression during GC selection and affinity maturation.

Necessity of Smc3 in Embryonic and Adult Hematopoiesis with an Emphasis on Myeloid Compartment

SMC3 encodes a subunit of cohesin associated with recurrent heterozygous mutations in acute myeloid leukemia (AML) and other myeloid malignancies. About one-third of these mutations are nonsense and splice-site mutations and the rest are missense. To understand whether these missense mutations might have dominant-negative effects or phenocopy loss-of-function effects, we compared the consequences of Smc3 deficient and haploinsufficient mouse models.We found that homozygous deletion of Smc3 during embryogenesis (Smc3fl/fl/Vav1-Cre+/-) is embryonic lethal. Grossly, the E13.5 Smc3fl/fl/Vav1-Cre+/- embryos were indistinguishable in size and appearance from other genotypes, except the lack of obvious fetal livers and near complete absence of CD45+Gr1+CD11b+ fetal liver cells, consistent with myeloid biased hematopoietic failure.Likewise, somatic deletion of homozygous Smc3 alleles in adult mice led to complete hematopoietic failure. In Smc3fl/fl/ERT2-Cre+/- mice, tamoxifen-induced Smc3 deletion led to rapid bone marrow (BM) failure and death. In competitive transplantation, the Smc3fl/fl/ERT2-Cre+/- BM cells were quickly outcompeted after tamoxifen treatment, indicating complete loss of hematopoietic stem/progenitor cell (HSPC) functions. Again, the effect was most pronounced within the myeloid compartment. Thus, Smc3 is necessary for both embryonic and adult hematopoiesis.We examined the consequences of Smc3 haploinsufficiency using three mouse models: Smc3fl/+/CMV-Cre+/-, Smc3fl/+/Vav1-Cre+/-, and Smc3fl/+/ERT2-Cre+/- . We found that Smc3 haploinsufficiency led to a normal number of female pups in X-linked CMV-Cre intercrosses, and the female pups had no obvious defects in growth or blood counts, implying tolerance of embryonic heterozygous deletion of Smc3 . In ex vivo methylcellulose assays, Smc3 haploinsufficiency did not result in increased number of colonies, and the Smc3 haploinsufficient BM cells did not replate beyond two weeks. In competitive transplantations, we observed a significant competitive disadvantage in the Smc3fl/+/ERT2-Cre+/- BM cells, most pronounced in the Gr1+ myeloid cells.The patterns of missense and nonsense mutations observed in AML patients suggested that SMC3 likely acts as a tumor suppressor, andthat haploinsufficiency would augment pre-leukemic self-renewal. However, we observed a competitive disadvantage across multiple experiments. This contradicts the competitive advantage observed when Smc3 haploinsufficiency was induced using Mx1-Cre (Viny et al . JEM 212 (11): 1819-1832). Therefore, we examined whether Smc3 haploinsufficiency might augment self-renewal under specific conditions of hematopoietic stress, including pIpC. We again observed a marked competitive disadvantage in the Smc3fl/+/ERT2-Cre+/- BM cells following tamoxifen induction, although the myeloid competitive disadvantage was partially ameliorated in the pIpC-treated group, whereas 5-FU treatment augmented the myeloid competitive disadvantage.In AML patients, SMC3 mutations frequently co-occurred with DNMT3A mutations. We, therefore, asked whether Smc3 haploinsufficiency might lead to a competitive advantage if it occurred in the background of Dnmt3a haploinsufficiency. With the addition of Dnmt3a haploinsufficiency, the severe myeloid competitive disadvantage was partially abrogated, but the significant competitive disadvantage in other lineages remained intact; even with constitutive Dnmt3a haploinsufficiency, Smc3 haploinsufficiency did not result in competitive growth of BM cells.In summary, our data demonstrate that homozygous Smc3 deletion was incompatible with embryonic or adult hematopoiesis, with the greatest defects noted within myeloid compartment, indicating that AML-associated SMC3 mutations are unlikely to have dominant-negative effects. Smc3 haploinsufficiency resulted in a myeloid competitive disadvantage, which was partially ameliorated by pIpC treatment, and accelerated by the stem cell stress of 5-FU. In the presence of concurrent Dnmt3a haploinsufficiency, the myeloid disadvantage was partially abrogated, while the disadvantage in other lineages remained unperturbed, suggesting interactions between the mutations. DisclosuresNo relevant conflicts of interest to declare.

BMP Receptor Inhibition Enhances Tissue Repair in Endoglin Heterozygous Mice.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFβ/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/− mice, a model for HHT1. Eng+/− mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6Chigh/CD206−) at the expense of reparative M2-like macrophages (Ly6Clow/CD206+). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFβ-induced differentiation of Eng+/− monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/− mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/− mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/− mice.

Molecular Dissection of Somatic Skeletal Disease in Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by heterozygous NF1 gene mutations. Patients with NF1 present with pleiotropic somatic secondary manifestations, including development of bone pseudarthrosis after fracture. Somatic NF1 gene mutations were reproducibly identified in patient-derived pseudarthrosis specimens, suggesting a local mosaic cell population including somatic pathologic cells. The somatic cellular pathogenesis of NF1 pseudarthroses remains unclear, though defects in osteogenesis have been posited. Here, we applied time-series single-cell RNA-sequencing (scRNA-seq) to patient-matched control and pseudarthrosis-derived primary bone stromal cells (BSCs). We show that osteogenic specification to an osteoblast progenitor cell population was evident for control bone-derived cells and haploinsufficient pseudarthrosis-derived cells. Similar results were observed for somatic patient fracture-derived NF1-/- cells; however, expression of genetic pathways associated with skeletal mineralization were significantly reduced in NF1-/- cells compared with fracture-derived NF1+/- cells. In mice, we show that Nf1 expressed in bone marrow osteoprogenitors is required for the maintenance of the adult skeleton. Results from our study implicate impaired Clec11a-Itga11-Wnt signaling in the pathogenesis of NF1-associated skeletal disease. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

CACNA1S haploinsufficiency confers resistance to New World arenavirus infection

Understanding the genetics of susceptibility to infectious agents is of great importance to our ability to combat disease. Here, we show that voltage-gated calcium channels (VGCCs) are critical for cellular binding and entry of the New World arenaviruses Junín and Tacaribe virus, suggesting that zoonosis via these receptors could occur. Moreover, we demonstrate that α1s haploinsufficiency renders cells and mice more resistant to infection by these viruses. In addition to being more resistant to infection, haploinsufficient cells and mice required a lower dosage of VGCC antagonists to block infection. These studies underscore the importance of genetic variation in susceptibility to both viruses and pharmaceutics.

CRISPR/Cas9-Mediated TERT Disruption in Cancer Cells.

Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated, such that in most adult somatic cells, TERT is inactivated and telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere attrition to achieve proliferative immortality. Therefore, inactivating TERT has been considered to be a promising means of cancer therapy. Here we applied the CRISPR/Cas9 gene editing system to target the TERT gene in cancer cells. We report that disruption of TERT severely compromises cancer cell survival in vitro and in vivo. Haploinsufficiency of TERT in tumor cells is sufficient to result in telomere attrition and growth retardation in vitro. In vivo, TERT haploinsufficient tumor cells failed to form xenograft after transplantation to nude mice. Our work demonstrates that gene editing-mediated TERT knockout is a potential therapeutic option for treating cancer.

Deconstructing the Clonal Advantage and Clonal Stability of 5q- Candidate Genes in Del(5q) MDS on a Single Cell Level

Hematopoietic Stem/Progenitor cells (HSPCs) with 5q haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) acquire a clonal advantage in the bone marrow and out-compete normal hematopoiesis. A critical, yet unsolved question remains: how does genetic haploinsufficiency in del(5q) cells contribute to the clonal advantage of HSPCs? We investigated the role of haploinsufficiency for three candidate genes in the common deleted region on chromosome 5 (Csnk1a1, Egr1 and Apc) in direct competition with each other and wild-type (wt) cells on a single cell level by employing a novel lentiviral genetic barcoding strategy. We introduced genotype and cell-specific barcodes into HSPCs from murine models haploinsufficient for Csnk1a1, Egr1 or Apc. Barcoded HSPCs were sort-purified, genotypes mixed and subsequently competitively transplanted into lethally irradiated mice and re-transplanted after 16 weeks in a secondary transplant. The barcoded progeny was reliably recovered from peripheral blood and relative contribution of the barcoded clones to differentiated blood lineages was followed over 32 weeks. Despite heterogeneity in clonal evolution among the mice, all haploinsufficient clones had the potential to outcompete wt clones (3 of 5 mice in the primary transplant, 3 out of 4 mice in the secondary transplant). Csnk1a1 haploinsufficient clones showed the largest clonal abundance and clonal persistence. Expansion of oligoclonal Csnk1a1 haploinsufficient HSPCs was further enhanced in the secondary transplant in all mice. Egr1 haploinsufficient clones showed potential for prominent oligoclonal expansion in one mouse, but decreased in abundance in all secondary transplants. Apc haploinsufficient clones showed persistence but not expansion in 3 out of 5 mice. These results were validated by conventional competitive transplants, which demonstrated that Csnk1a1 and Egr1 haploinsufficient cells achieved the highest advantage over wt hematopoiesis in the primary transplant and more enhanced in the secondary transplant. Since Csnk1a1 regulates β-catenin protein stability, we hypothesized that the clonal expansion of Csnk1a1 haploinsufficient HSPCs is dependent on β-catenin levels. We performed a second genetic barcoding competitive transplant, comparing Csnk1a1-/+ HSPC directly to double haploinsufficient Csnk1a1-/+/Ctnnb1-/+ (β-catenin encoding) HSPCs. We included additional double haploinsufficient mutants Csnk1a1-/+/Apc-/+ and Csnk1a1-/+/Egr1-/+. Results showed pronounced expansion of Csnk1a1-/+ clones, while Csnk1a1-/+/Ctnnb1-/+ clones were outcompeted over time, suggesting that the advantage of Csnk1a1-/+ clones is β-catenin dependent. Csnk1a1-/+/Egr1-/+ and Csnk1a1-/+/Apc-/+ clones were less advantageous than Csnk1a1-/+ clones. To further investigate the mechanism of clonal fitness in Csnk1a1-/+ haploinsufficient HSPCs, we performed droplet based single cell RNA sequencing of Csnk1a1-/+ and wt Lin-Sca1+cKit+ (LSK) HSPCs. Csnk1a1 -/+ LSK were characterized by a higher fraction of cells expressing cell cycle genes compared to wt cells. In line, transcriptional alterations in the most primitive HSCs suggest that the clonal advantage is conveyed by canonical Wnt signaling activating downstream targets such E2F proteins. Csnk1a1-/+ haploinsufficient multipotent progenitors and myeloid/lymphoid primed progenitors expressed marked upregulation of metabolic pathways, mitochondrial respiration, cell cycle and differentiation, ubiquitination/proteasome system and deregulation of ribosome biogenesis. In conclusion, we demonstrate using a novel genetic barcoding approach in a competitive transplant setting that Csnk1a1-/+ haploinsufficient HSPCs have the potential for oligoclonal expansion and clonal persistence. Wnt/β-catenin signaling plays a central role in the clonal expansion. Interestingly, in Csnk1a1 haploinsufficiency the HSC state is preserved and the increased proliferation and metabolic activation are hallmark features of differentiating progenitor cells at MPP stage, increasing with cell cycle activation, thus ensuring clonal stability and preventing HSC exhaustion over time. Disclosures Brümmendorf: University Hospital of the RWTH Aachen: Employment; Janssen: Consultancy; Pfizer: Consultancy, Research Funding; Merck: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Ebert:Celgene: Research Funding; Deerfield: Research Funding; Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute..

Transcriptomes of Dravet syndrome iPSC derived GABAergic cells reveal dysregulated pathways for chromatin remodeling and neurodevelopment

Dravet syndrome (DS) is an early onset refractory epilepsy typically caused by de novo heterozygous variants in SCN1A encoding the α-subunit of the neuronal sodium channel Nav1.1. The syndrome is characterized by age-related progression of seizures, cognitive decline and movement disorders. We hypothesized that the distinct neurodevelopmental features in DS are caused by the disruption of molecular pathways in Nav1.1 haploinsufficient cells resulting in perturbed neural differentiation and maturation. Here, we established DS-patient and control induced pluripotent stem cell derived neural progenitor cells (iPSC NPC) and GABAergic inter-neuronal (iPSC GABA) cells. The DS-patient iPSC GABA cells showed a shift in sodium current activation and a perturbed response to induced oxidative stress. Transcriptome analysis revealed specific dysregulations of genes for chromatin structure, mitotic progression, neural plasticity and excitability in DS-patient iPSC NPCs and DS-patient iPSC GABA cells versus controls. The transcription factors FOXM1 and E2F1, positive regulators of the disrupted pathways for histone modification and cell cycle regulation, were markedly up-regulated in DS-iPSC GABA lines. Our study highlights transcriptional changes and disrupted pathways of chromatin remodeling in Nav1.1 haploinsufficient GABAergic cells, providing a molecular framework that overlaps with that of neurodevelopmental disorders and other epilepsies.

MiRNA-376c-3p Mediates TWIST-1 Inhibition of Bone Marrow-Derived Stromal Cell Osteogenesis and Can Reduce Aberrant Bone Formation of TWIST-1 Haploinsufficient Calvarial Cells

Key transcription factors, which activate or repress master gene regulators and signaling pathways, tightly regulate self-renewal and cell lineage differentiation of bone marrow-derived stromal cells (BMSC). Among these factors is the basic helix-loop-helix transcription factor Twist-related protein 1 (TWIST-1), which is important in BMSC self-renewal, life span, and differentiation. Another layer of gene regulation comes from microRNAs (miRNAs). miRNAs are short noncoding RNAs that interfere with translation of specific target mRNAs and thereby regulate diverse biological processes, including BMSC lineage commitment. However, little is known of how TWIST-1-regulated miRNAs control osteogenic commitment, and influence the fate of bone precursor cells. In this study, we have discovered a novel TWIST-1-regulated miRNA, miR-376c-3p. Reduced miR-376c-3p expression by a miR-376c-3p inhibitor or due to TWIST-1 haploinsufficiency promotes alkaline phosphatase (ALP) activity, mineral deposition, and expression of osteoblast-associated genes in BMSC and calvarial cells. Conversely, overexpression of miR-376c-3p using a miR-376c-3p mimic inhibited BMSC proliferation and the osteogenic potential of BMSC and TWIST-1 haploinsufficient calvarial cells. This was demonstrated by a decrease in insulin growth factor 1 receptor (IGF1R) levels, Akt signaling, ALP activity, mineral deposition, and expression of osteoblast-associated genes. Thus, miR-376c-3p reduces IGF1R/Akt signaling in BMSC and is one mechanism by which osteogenesis may be inhibited. Overall, we have identified miR-376c-3p as a TWIST-1-regulated miRNA, which plays an important role in the osteogenesis of bone precursor cells and can mediate TWIST-1 inhibition of osteogenesis. Furthermore, overexpression of miRNA-376c-3p in TWIST-1 haploinsufficient calvarial cells can decrease the aberrant osteogenesis of these cells, which contributes to increased calvarial bone volume and premature fusion of the coronal sutures.

SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling

While multiple mechanisms of BRAFV600-mutant melanoma resistance to targeted MAPK signaling inhibitors (MAPKi) have been reported, the epigenetic regulation of this process remains undetermined. Here, using a CRISPR–Cas9 screen targeting chromatin regulators, we discover that haploinsufficiency of the histone deacetylase SIRT6 allows melanoma cell persistence in the presence of MAPKi. Haploinsufficiency, but not complete loss of SIRT6 promotes IGFBP2 expression via increased chromatin accessibility, H3K56 acetylation at the IGFBP2 locus, and consequent activation of the IGF-1 receptor (IGF-1R) and downstream AKT signaling. Combining a clinically applicable IGF-1Ri with BRAFi overcomes resistance of SIRT6 haploinsufficient melanoma cells in vitro and in vivo. Using matched melanoma samples derived from patients receiving dabrafenib + trametinib, we identify IGFBP2 as a potential biomarker for MAPKi resistance. Our study has not only identified an epigenetic mechanism of drug resistance, but also provides insights into a combinatorial therapy that may overcome resistance to standard-of-care therapy for BRAFV600-mutant melanoma patients.

PRMT1 mediates RANKL-induced osteoclastogenesis and contributes to bone loss in ovariectomized mice

Protein arginine methylation is a novel form of posttranslational modification mediated by protein arginine methyltransferase (PRMTs). PRMT1, a major isoform of the PRMT family, is responsible for various biological functions, including cellular differentiation. Although the important function that PRMT1 plays in various tissues is being increasingly recognized, its role in receptor activation of NF-κB ligand (RANKL)-induced osteoclastogenesis or osteoporosis has not yet been described. Here, we show that PRMT1 is essential for RANKL-induced osteoclastogenesis in vitro and for bone loss in vivo. RANKL treatment increased the expression of PRMT1 and its nuclear localization in bone marrow-derived macrophages (BMDMs) in a c-Jun N-terminal kinase (JNK)-dependent manner. Silencing PRMT1 attenuated RANKL-induced osteoclastogenesis by decreasing tartrate-resistant acid phosphatase (TRAP)-positive cells and inhibiting F-actin ring formation and bone resorption, which was confirmed in a separate experiment using haploinsufficient cells from PRMT1+/- mice. Our results also revealed that PRMT1 regulates the transcription activity of NF-κB by directly interacting with it in RANKL-treated BMDMs. An in vivo study showed that the haploinsufficiency of PRMT1 reduced the enzyme activity of TRAP and increased the bone mineral density in the metaphysis of ovariectomized (OVX) mice. Finally, treatment with estrogen (E2) downregulated the RANKL-induced expression of PRMT1, suggesting that estrogen may exert an inhibitory effect on osteoclastogenesis by suppressing PRMT1 expression. Our results suggest that PRMT1 plays an important role in the progression of osteoporosis and that it might be a good therapeutic target for postmenopausal osteoporosis.