RUNX1 Inhibition Using Lipid Nanoparticle-Mediated Silencing RNA Delivery as an Effective Treatment for Acute Leukemias.

Transcription factor RUNX1 plays key roles in the establishment and maintenance of the hematopoietic system. Although RUNX1 has been considered a beneficial tumor suppressor, several recent reports have described the tumor-promoting role of RUNX1 in a variety of hematopoietic neoplasms. In this study, we assessed the effect of RUNX1 depletion in multiple human leukemia cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, and confirmed that RUNX1 is in fact required for sustaining their leukemic proliferation. To achieve efficient RUNX1 inhibition in leukemia cells, we then examined the effect of lipid nanoparticle (LNP)-mediated delivery of RUNX1-targeting small interfering (si)RNA using two tumor-tropic LNPs. The LNPs containing RUNX1-targeting siRNA were efficiently incorporated into myeloid and T-cell leukemia cell lines and patient-derived primary human acute myeloid leukemia (AML) cells, downregulated RUNX1 expression, induced cell cycle arrest and apoptosis, and exhibited the growth-inhibitory effect in them. In contrast, the LNPs were not efficiently incorporated into normal cord blood CD34+ cells, indicating their minimum cytotoxicity. Thus, our study highlights RUNX1 as a potential therapeutic target to inhibit leukemogenesis, and provides the LNP-based siRNA delivery as a promising approach to deplete RUNX1 specifically in leukemia cells.

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line.

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.

Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin transactivates aryl hydrocarbon receptor-responsive element III in the tyrosine hydroxylase immunoreactive neurons of the mouse midbrain.

Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR-responsive element III (AHRE-III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could regulate AHRE-III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE-III enhancers, TH gene promoter and the c-myc-tagged luciferase gene. Single oral administrations of TCDD (0-2000 ng kg⁻¹ body weight) to the transgenic dams markedly enhanced TH-immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3 days and 8 weeks of age. The offspring of dams treated with 200 ng kg⁻¹ TCDD exhibited significant increases in the numbers of TH- and double (TH and c-myc)-ir neurons in area A9 compared with controls at 8 weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH-ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR-AHRE-III-mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR-AHRE-III signaling pathway and overgrowth of TH-ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD).

Combined inhibition of MET and EGFR suppresses proliferation of malignant mesothelioma cells.

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm associated with asbestos exposure. Although expression and activation of receptor tyrosine kinases (RTKs), including MET, have been reported in most MPM, specific RTK inhibitors showed less than the expected response in MPM cells. To determine whether the lack of response of MET inhibitors was due to cooperation with other RTKs, we determined activation status of MET and other RTKs, including epidermal growth factor receptor (EGFR) family of 20 MPM cell lines, and tested whether dual RTK inhibition is an effective therapeutic strategy. We detected MET upregulation and phosphorylation (thus indicating activation) in 14 (70%) and 13 (65%) cell lines, but treatment with MET-specific inhibitors showed weak or modest effect of suppression in most of the cell lines. Phospho-RTK array analysis revealed that MET was simultaneously activated with other RTKs, including EGFR, ErbB2, ErbB3 and platelet-derived growth factor receptor-beta. Combination of MET and EGFR inhibitors triggered stronger inhibition on cell proliferation and invasion of MPM cells than that of each in vitro. These results indicated that coactivation of RTKs was essential in mesothelioma cell proliferation and/or survival, thus suggesting that simultaneous inhibition of RTKs may be a more effective strategy for the development of molecular target therapy for MPM.

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells.

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.

A rapid protein expression and purification system using Chinese hamster ovary cells expressing retrovirus receptor.

Conventional stable protein expression systems using mammalian cells include a time-consuming step of antibiotic resistance-based cell cloning. Here, we report a rapid flow cytometry-based method for the collection of retrovirus vector-infected Chinese hamster ovary (CHO) cells that express desired proteins. The vector carries the genes for green fluorescent protein (GFP), as a marker, and glutathione-S-transferase (GST), to express the desired protein as a GST-fusion construct. To render CHO cells susceptible to retrovirus infection, they were forced to express EcoR, the receptor for retroviruses. After infection, cells expressing desired proteins were collected by flow cytometry as a GFP-positive population, and the desired proteins were purified by glutathione affinity chromatography. This method reduces the time required between infection of cells and purification of a desired protein from several months to approximately 2 weeks.

Identification of B cell adaptor for PI3-kinase (BCAP) as an Abl interactor 1-regulated substrate of Abl kinases.

In previous work we showed that Abl interactor 1 (Abi-1), by linking enzyme and substrate, promotes the phosphorylation of Mammalian Enabled (Mena) by c-Abl. To determine whether this mechanism extends to other c-Abl substrates, we used the yeast two-hybrid system to search for proteins that interact with Abi-1. By screening a human leukocyte cDNA library, we identified BCAP (B-cell adaptor for phosphoinositide 3-kinase) as another Abi-1-interacting protein. Binding experiments revealed that the SH3 domain of Abi-1 and the C-terminal polyproline structure of BCAP are involved in interactions between the two. In cultured cells, Abi-1 promoted phosphorylation of BCAP not only by c-Abl but also by v-Abl. The phosphorylation sites of BCAP by c-Abl were mapped to five tyrosine residues in the C-terminal region that are well conserved in mammals. These results show that Abi-1 promotes Abl-mediated BCAP phosphorylation and suggest that Abi-1 in general coordinates kinase-substrate interactions.