Construction of Human Neuromuscular Disease-Related Gene Site-Specific Mutant Cell Line by Cas9 Mutation System

Objective: to construct human neuromuscular disease-related gene site-specific mutant cell line by Cas9 mutation system. Methods: according to the principle of CRISPR/Cas9 target design, the exon region of CXCR4 gene sequence was found in the National Center for Biotechnology Information (NCBI) of the United States. Two sgRNAs were designed. Lenticrisprv2 was used as the vector to construct the lenticrisprv2-sgrna recombinant plasmid, which was transformed into the sensitive stbl3 strain. The monoclonal sequencing was selected to verify and expand the culture of the plasmid, then it was transferred to 293T cells for packaging to a slow virus. The virus was collected and infected with 4T1 cells. The monoclonal cells were isolated and cultured by puromycin screening and limited dilution method. The genomic DNA of the selected monoclonal cells was extracted and the DNA fragment near the knockout site was amplified by PCR and sequenced. Results: one cell line had 6 deletion mutations, including DYSF mutation site of neuromuscular disease gene and HEK293T cell model knocked out by DYSF mutation site of neuromuscular disease gene. Conclusion: the recombinant plasmid targeting CXCR4 gene was obtained by CRISPR/Cas9 system, and the human neuromuscular disease-related gene site-specific mutant cell line was successfully constructed.

SETBP1 Mutations Accelerate NRAS-Mutant Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive, rare form of early childhood leukemia driven by Ras pathway mutations. Mutations in SET binding protein 1 (SETBP1) are a strong predictor of relapse in JMML, and are associated with reduced five-year event-free survival. Although some mechanisms of oncogenesis have been established for SETBP1 mutations, it remains unclear why they are associated with poor prognosis and relapse. The goal of this study was to understand how SETBP1 modulates the biology of Ras-driven leukemias and to determine whether there are therapeutic vulnerabilities of SETBP1-JMML that can be exploited. Here, we present novel findings on the synergy of SETBP1 and NRAS, and provide pre-clinical evidence for therapeutic intervention. To address our central question of how SETBP1 mutations modulate Ras pathway-driven leukemia, we first set out to determine whether mutant SETBP1 promotes the growth of hematopoietic progenitors in the context of a Ras pathway mutation. To this end, we performed mouse hematopoietic colony forming unit assays in the absence of exogenous cytokines. Both NRAS-G12D and PTPN11-E76K alone formed a modest number of colonies, and the addition of SETBP1-D868N significantly augmented colony number with either Ras pathway mutation. The combination of NRAS-G12D and SETBP1-D868Nconfer robust serial replating, indicating that the SETBP1-D868N promotes oncogenic transformation and progenitor self-renewal in the NRAS-G12D mutant cells. To understand how SETBP1 modulates therapeutic response, a novel NRAS/SETBP1-mutant cell line was generated and analyzed with a chemical screen of essential cell growth and survival pathways. This screen revealed dependencies on the mTOR/AKT/PI3K and Raf/MEK/ERK pathways. An immunoblot analysis revealed that mutant SETBP1 enhanced NRAS-driven ERK and mTOR pathway activation. Inhibitors of these pathways, such as rapamycin and trametinib were highly efficacious against our cell line. The combination of trametinib and rapamycin had sub-nanomolar efficacy in our NRAS/SETBP1-hematopoietic cell line and exhibited greater than bliss additivity at several time points. To evaluate the efficacy in vivo, our SETBP1/NRAS-mutant cell line was injected into mice. At the onset of disease, mice were given once-daily treatment of trametinib, rapamycin, combination treatment, or DMSO control. The median survival of mice receiving DMSO was 19.5-days post-transplant, compared to 21 days for rapamycin, 35 days for the combination treatment, and 42 days for trametinib. Treatment with trametinib significantly increased the median survival to beyond rapamycin or DMSO, doubling the survival time of the mice. Our data demonstrates that SETBP1 mutations accelerate NRAS-driven oncogenesis and enhance MAPK pathway activation by NRAS-G12D. Despite enhanced transforming potential, SETBP1-mutant cells are still sensitive to inhibitors of the RAS/ERK/MAPK pathway. Trametinib, an inhibitor of this pathway, doubles overall survival in our murine model of NRAS/SETBP1-mutant leukemia, thus providing encouraging pre-clinical data for the use of trametinib in SETBP1-mutant disease. Disclosures No relevant conflicts of interest to declare.

TFIIIB Subunit Bdp1 Participates in RNA Polymerase III Transcription in the Protozoan Parasite Leishmania major

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.

Unique Primitive Erythropoiesis Defect In Rpl5-Deficient Murine Embryonic Stem Cell Model of Diamond Blackfan Anemia

Abstract 877 Background: Diamond Blackfan anemia (DBA), a rare inherited bone marrow failure syndrome, is characterized mainly by erythroid hypoplasia but is also associated with congenital anomalies, short stature and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS17, RPS19, RPS24, RPL5, RPL11, RPL35a), which renders erythroid precursors highly sensitive to death by apoptosis. The ontogeny and basis of the hematopoietic defect are unclear. The typical presentation of anemia occurs at 2–3 months of age, although there are rare cases of hydrops fetalis. Marked phenotypic variations exist among members of the same family and also between subsets of patients with different mutations. Methods: We studied in vitro hematopoietic differentiation of two murine embryonic stem (ES) cell lines: YHC074, Rps19 mutant with the pGT0Lxf gene trap vector inserted in intron 3 of Rps19, and D050B12, Rpl5 mutant with the FlipRosaβgeo gene trap vector inserted in intron 3 of Rpl5. Wild-type parental cell lines were used as controls. For primary differentiation and generation of embryoid bodies (EBs), ES cells were cultured in serum-supplemented methylcellulose medium containing stem cell factor (SCF). After 7 days, the cultures were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 12 for hematopoietic percentage. For secondary differentiation into definitive hematopoietic colonies, day 10 EBs were disrupted, and individual cells were suspended in serum-supplemented methylcellulose medium containing SCF, IL-3, Il-6 and epo. Definitive hematopoietic colonies were counted on day 10. Primitive erythropoiesis differentiation assays were performed by disruption of day 4 EBs, followed by suspension of cells in methylcellulose medium containing plasma-derived serum and epo. Primitive erythropoiesis colonies were counted on day 7. Results: We confirmed haploinsufficient expression (∼50% wild type) of Rps19 in YHC074 and Rpl5 protein in D050B12 by Western blot analysis. By polysome analysis, we found a selective reduction in the 40S subunit peak in the Rps19 mutant cell line and in the 60S subunit peak in the Rpl5 mutant cell line. Both types of mutants produced a significantly decreased number of EBs, particularly hematopoietic EBs, compared to parental cell lines. EB size was not compromised in the Rps19 mutant cell line, while Rpl5 mutant ES cells produced significantly smaller EBs, compared to its parental cells. Upon differentiation of cells to definitive hematopoietic colonies, both Rps19 and Rpl5 mutants showed a similar reduction in the erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation ratio. Primitive erythropoiesis was conserved in the Rps19 mutant (Figure 1. 1, top panel). By contrast, the Rpl5 mutant demonstrated a severe primitive erythropoiesis defect (Figure 1. 1, bottom panel). For confirmation of these results in an isogenic background, we stably transfected YHC074 ES cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones expressed Rps19 at the wild-type level. Upon differentiation of a chosen clone, we demonstrated correction of the EB defect and the definitive erythropoiesis defect, suggesting that the hematopoietic differentiation defects seen are directly related to levels of Rps19 protein. We are currently working on correction of the D050B12 ES cells in a similar manner. Conclusion: Murine ES cell lines with Rps19 and Rpl5 mutations exhibit ribosomal protein haploinsufficiency, demonstrate respective ribosome assembly defects, and recapitulate the major DBA hematopoietic differentiation defect. In addition, a unique defect in primitive erythropoiesis in the Rpl5 mutant ES cell line suggests that the Rpl5 mutation in this mouse strain affects early-stage embryogenesis, a finding which may offer insight into the ontogeny of DBA hematopoiesis and may offer an explanation for phenotypic variations seen in patients (such as hydrops fetalis). Disclosures: No relevant conflicts of interest to declare.

Variable Loop Glycan Dependency of the Broad and Potent HIV-1-Neutralizing Antibodies PG9 and PG16

ABSTRACT The HIV-1-specific antibodies PG9 and PG16 show marked cross-isolate neutralization breadth and potency. Antibody neutralization has been shown to be dependent on the presence of N-linked glycosylation at position 160 in gp120. We show here that (i) the loss of several key glycosylation sites in the V1, V2, and V3 loops; (ii) the generation of pseudoviruses in the presence of various glycosidase inhibitors; and (iii) the growth of pseudoviruses in a mutant cell line (GnT1−/−) that alters envelope glycosylation patterns all have significant effects on the sensitivity of virus to neutralization by PG9 and PG16. However, the interaction of antibody is not inhibited by sugar monosaccharides corresponding to those found in glycans on the HIV surface. We show that some of the glycosylation effects described are isolate dependent and others are universal and can be used as diagnostic for the presence of PG9 and PG16-like antibodies in the sera of HIV-1-infected patients. The results suggest that PG9 and PG16 recognize a conformational epitope that is dependent on glycosylation at specific variable loop N-linked sites. This information may be valuable for the design of immunogens to elicit PG9 and PG16-like antibodies, as well as constructs for cocrystallization studies.