BCL6 inhibition ameliorates resistance to ruxolitinib in <i>CRLF2</i>-rearranged acute lymphoblastic leukemia

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an intractable disease and most cases harbor genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits a CRLF2 gene rearrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient as a treatment, so combinatorial therapies targeting multiple signals are needed. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, we explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and found that elevated BCL6 expression was one such mechanism. Upregulated BCL6 suppressed the expression of TP53 along with its downstream cell cycle inhibitor p21 (CDKN2A) and pro-apoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells some degree of resistance to therapy. BCL6 inhibition (with FX1) alone was able to upregulate TP53 and restore the TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis- sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged the survival of xenografted mice. These findings provide one mechanism for the insufficiency of JAK inhibition for the treatment of CRLF2-rearranged ALL and indicate BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition

Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene.

Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines

Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.

CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies

The plant alkaloid conophylline inhibits matrix formation of fibroblasts

Conophylline is a vinca alkaloid from leaves of the tropical plant Ervatamia microphylla and has been shown to mimic the effect of the growth and differentiation factor activin A on pancreatic progenitor cells. However, whereas activin A stimulates fibrosis of pancreatic stellate cells, conophylline inhibits it, suggesting that this compound may serve as an antifibrotic drug. Here, we investigated the effects of conophylline on human foreskin fibroblasts, especially focusing on extracellular matrix (ECM) proteins. A gene microarray analysis revealed that conophylline remarkably suppressed expression of the gene for hyaluronan synthase 2 (HAS2) and of its antisense RNA, whereas the expression of collagen genes was unaffected. Of note, immunostaining experiments revealed that conophylline substantially inhibits incorporation of versican and collagens into the ECM in cells treated with transforming growth factor \u3b2 (TGF-\u3b2), which promotes collagen synthesis, but not in cells not treated with TGF-\u3b2. Moreover, a protein biosynthesis\ua0 assay disclosed that conophylline decreases collagen biosynthesis, concomitant with a decrease in total protein biosynthesis, indicating that conophylline-mediated inhibition of fibrosis is not specific to collagen synthesis. Conophylline neither affected TGF-\u3b2-induced nuclear translocation of SMAD family member 2/3 (SMAD2/3) nor phosphorylation of SMAD2. However, conophylline substantially inhibited phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting that conophylline inhibits HAS2 expression via TGF-\u3b2-mediated activation of the ERK1/2 pathway. Taken together, our results indicate that conophylline may be a useful inhibitor of ECM formation in fibrosis

Confirmatory assays examining the <i>PIGA</i> gene locus in FLAER-negative and -positive cell clones.

<p>(A and B) Representative results of PCR examining FLAER-negative and -positive single cell clones derived from HCT116 (A) and DLD-1 (B) infected with the promoter-trap <i>PIGA</i> targeting vector. PCR was conducted with primer pairs F1-R1, F2-R2 and F3-R3 that yielded PCR products of 1,140 bp, 1,184 bp, and 1,031 bp in size, respectively. For the location of primers, see Fig. 1A. Control: parental cell lines. (C) Southern blot analysis of FLAER-negative and -positive single cell clones. Genomic DNA was digested with XbaI, separated on a gel, and hybridized with a probe shown in Fig. 1A. The positions of the DNA size standards are shown to the right in kilobase. P: parental cell lines.</p

A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

<div><p>The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human <i>CDKN2A</i> gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human <i>PIGA</i> gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest <i>PIGA</i> gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.</p></div