TCF-1 and LEF-1 act upstream of Th-POK to promote the CD4(+) T cell fate and interact with Runx3 to silence Cd4 in CD8(+) T cells.

The transcription factors TCF-1 and LEF-1 are essential for early T cell development, but their roles beyond the CD4(+)CD8(+) double-positive (DP) stage are unknown. By specific ablation of these factors in DP thymocytes, we demonstrated that deficiency in TCF-1 and LEF-1 diminished the output of CD4(+) T cells and redirected CD4(+) T cells to a CD8(+) T cell fate. The role of TCF-1 and LEF-1 in the CD4-versus-CD8 lineage 'choice' was mediated in part by direct positive regulation of the transcription factor Th-POK. Furthermore, loss of TCF-1 and LEF-1 unexpectedly caused derepression of CD4 expression in T cells committed to the CD8(+) lineage without affecting the expression of Runx transcription factors. Instead, TCF-1 physically interacted with Runx3 to cooperatively silence Cd4. Thus, TCF-1 and LEF-1 adopted distinct genetic 'wiring' to promote the CD4(+) T cell fate and establish CD8(+) T cell identity.

Regulation of lymphoid-myeloid lineage bias through regnase-1/3-mediated control of Nfkbiz.

ABSTRACT: Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.

The E-Id Protein Axis Specifies Adaptive Lymphoid Cell Identity and Suppresses Thymic Innate Lymphoid Cell Development.

Innate and adaptive lymphoid development is orchestrated by the activities of E proteins and their antagonist Id proteins, but how these factors regulate early T cell progenitor (ETP) and innate lymphoid cell (ILC) development remains unclear. Using multiple genetic strategies, we demonstrated that E proteins E2A and HEB acted in synergy in the thymus to establish T cell identity and to suppress the aberrant development of ILCs, including ILC2s and lymphoid-tissue-inducer-like cells. E2A and HEB orchestrated T cell fate and suppressed the ILC transcription signature by activating the expression of genes associated with Notch receptors, T cell receptor (TCR) assembly, and TCR-mediated signaling. E2A and HEB acted in ETPs to establish and maintain a T-cell-lineage-specific enhancer repertoire, including regulatory elements associated with the Notch1, Rag1, and Rag2 loci. On the basis of these and previous observations, we propose that the E-Id protein axis specifies innate and adaptive lymphoid cell fate.

Three-step transcriptional priming that drives the commitment of multipotent progenitors toward B cells.

Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the "time-lapse" TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that ∼10%-30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.

Trends in cell medicine: from autologous cells to allogeneic universal-use cells for adoptive T-cell therapies.

In currently ongoing adoptive T-cell therapies, T cells collected from patients are given back to them after ex vivo activation and expansion. In some cases, T cells are transduced with chimeric antigen receptor (CAR) or T-cell receptor (TCR) genes during the ex vivo culture period in order to endow T cells with the desired antigen specificity. Although such strategies are effective in some types of cancer, there remain issues to be solved: (i) the limited number of cells, (ii) it is time-consuming, (iii) it is costly, and (iv) the quality can be unstable. Points (ii) and (iv) can be solved by preparing allogeneic T cells and cryopreserving them in advance and methods are being developed using healthy donor-derived T cells or pluripotent stem cells as materials. Whereas it is difficult to solve (i) and (iii) in the former case, all the issues can be cleared in the latter case. However, in either case, a new problem arises: rejection by the patient's immune system. Deletion of human leukocyte antigen (HLA) avoids rejection by recipient T cells, but causes rejection by NK cells, which can recognize loss of HLA class I. Various countermeasures have been developed, but no definitive solution is yet available. Therefore, further research and development are necessary.

Tracing the evolutionary history of blood cells to the unicellular ancestor of animals.

Blood cells are thought to have emerged as phagocytes in the common ancestor of animals followed by the appearance of novel blood cell lineages such as thrombocytes, erythrocytes, and lymphocytes, during evolution. However, this speculation is not based on genetic evidence and it is still possible to argue that phagocytes in different species have different origins. It also remains to be clarified how the initial blood cells evolved; whether ancient animals have solely developed de novo programs for phagocytes or they have inherited a key program from ancestral unicellular organisms. Here, we traced the evolutionary history of blood cells, and cross-species comparison of gene expression profiles revealed that phagocytes in various animal species and Capsaspora (C.) owczarzaki, a unicellular organism, are transcriptionally similar to each other. We also found that both phagocytes and C. owczarzaki share a common phagocytic program, and that CEBPα is the sole transcription factor highly expressed in both phagocytes and C. owczarzaki. We further showed that the function of CEBPα to drive phagocyte program in nonphagocytic blood cells has been conserved in tunicate, sponge, and C. owczarzaki. We finally showed that, in murine hematopoiesis, repression of CEBPα to maintain nonphagocytic lineages is commonly achieved by polycomb complexes. These findings indicate that the initial blood cells emerged inheriting a unicellular organism program driven by CEBPα and that the program has also been seamlessly inherited in phagocytes of various animal species throughout evolution.

Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Development of Immune Cell Therapy Using T Cells Generated from Pluripotent Stem Cells.

In the field of cancer immunotherapy, the effectiveness of a method in which patient-derived T cells are genetically modified ex vivo and administered to patients has been demonstrated. However, problems remain with this method, such as (1) time-consuming, (2) costly, and (3) difficult to guarantee the quality. To overcome these barriers, strategies to regenerate T cells using iPSC technology are being pursued by several groups in the last decade. The authors have been developing a method by which specific TCR genes are introduced into iPSCs and T cells are generated from those iPSCs (TCR-iPSC method). At present, our group is preparing this approach for clinical trial, where iPSCs provided from the iPSC project are transduced with WT1 antigen-specific TCR that had been already clinically tested, and killer T cells are generated from such TCR-iPSCs, to be administered to acute myeloid leukemia patients. While the adoptive T cell therapies have been mainly directed to be used in cancer immunotherapy, it is possible to apply these approaches to viral infections. Strategies by other groups to regenerate various types of T cells from iPSCs will also be introduced.

Conversion of T cells to B cells by inactivation of polycomb-mediated epigenetic suppression of the B-lineage program.

In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5 These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program.

Characteristic differences in the abundance of tumor-infiltrating lymphocytes and intratumoral developing T cells in thymoma, with special reference to PD-1 expression.

PURPOSE: Though programmed cell death-1 (PD-1) inhibitors mainly target tumor-infiltrating lymphocytes (TILs) expressing PD-1, developing T cells in thymus also express PD-1 in their process of maturation. To predict the therapeutic effect of PD-1 inhibitors for thymoma, it is necessary to clarify the proportions of TILs and intratumoral developing T cells. METHODS: The expressions of CD4, CD8, and PD-1 on T cells were analyzed by flow cytometry in 31 thymomas. The amount of T cell receptor excision circles (TRECs), which can be detected in newly formed naïve T cells in the thymus, was evaluated using sorted lymphocytes from thymomas by quantitative PCR. The expressions of granzyme B (GZMB) and lymphocyte activation gene-3 (LAG-3) in PD-1 + CD8 T cells were analyzed by image cytometry using multiplex immunohistochemistry. RESULTS: The PD-1 + rate in both CD4 and CD8 T cells was significantly higher in type AB/B1/B2 than in type A/B3 thymomas. The amounts of TRECs in CD4 and CD8 T cells were significantly higher in type AB/B1/B2 than in type A/B3 thymomas and comparable to normal thymus. PD-1 expression at each stage of T cell development of type AB/B1/B2 thymomas was comparable to that of normal thymus. Both the percentages and cell densities of PD-1 + CD8 T cells expressing GZMB or LAG-3, which are known to contain tumor-reactive T cells, were significantly lower in type AB/B1/B2 thymomas. CONCLUSION: Most PD-1 + T cells in type AB/B1/B2 thymomas are intratumoral developing T cells and are not TILs.

The E-Id protein axis modulates the activities of the PI3K-AKT-mTORC1-Hif1a and c-myc/p19Arf pathways to suppress innate variant TFH cell development, thymocyte expansion, and lymphomagenesis.

It is now well established that the E and Id protein axis regulates multiple steps in lymphocyte development. However, it remains unknown how E and Id proteins mechanistically enforce and maintain the naïve T-cell fate. Here we show that Id2 and Id3 suppressed the development and expansion of innate variant follicular helper T (TFH) cells. Innate variant TFH cells required major histocompatibility complex (MHC) class I-like signaling and were associated with germinal center B cells. We found that Id2 and Id3 induced Foxo1 and Foxp1 expression to antagonize the activation of a TFH transcription signature. We show that Id2 and Id3 acted upstream of the Hif1a/Foxo/AKT/mTORC1 pathway as well as the c-myc/p19Arf module to control cellular expansion. We found that mice depleted for Id2 and Id3 expression developed colitis and αß T-cell lymphomas. Lymphomas depleted for Id2 and Id3 expression displayed elevated levels of c-myc, whereas p19Arf abundance declined. Transcription signatures of Id2- and Id3-depleted lymphomas revealed similarities to genetic deficiencies associated with Burkitt lymphoma. We propose that, in response to antigen receptor and/or cytokine signaling, the E-Id protein axis modulates the activities of the PI3K-AKT-mTORC1-Hif1a and c-myc/p19Arf pathways to control cellular expansion and homeostatic proliferation.

[Development of "Off the Shelf" T Cells for Cancer Immunotherapy].

In the area of cancer immunotherapy, the efficacy of strategies in which patient derived T cells are genetically modified ex vivo and administered to patients has been demonstrated. However, some issues have remained to be addressed; the method using autologous T cells is costly and time consuming, and their quality is unstable. The time consuming problem can be solved by preparing allogeneic T cells in advance. Peripheral blood is being considered as a source of allogeneic T cells, and methods are being explored to avoid the risk of rejection or GVHD, but even so the issues of cost and quality stability still remain. On the other hand, use of pluripotent stem cells such as iPS cells or ES cells as material of T cells may solve the cost issue and achieve homogeneity of products. The authors group has been developing a method to generate T cells from iPS cells transduced with a certain T cell receptor gene, and is currently preparing for clinical trials. We believe that, when this strategy is realized, it becomes possible to deliver a universal and homogeneous T cell preparation immediately when needed.

Regeneration of antigen-specific T cells by using induced pluripotent stem cell (iPSC) technology.

In currently ongoing adoptive T-cell therapies, T cells collected from the patient are given back to the patient after ex vivo cell activation and expansion. In some cases, T cells are transduced with chimeric antigen receptor (CAR) or T-cell receptor (TCR) genes during the ex vivo culture period. Although such strategies have been shown to be effective in some types of cancer, there remain issues to be solved; these methods (i) are time-consuming, (ii) are costly and (iii) it is difficult to guarantee the quality because the products depend on patient-derived T cells. To address these issues, several groups including ours have developed methods in which cytotoxic cells are mass-produced by using induced pluripotent stem cell (iPSC) technology. For the regeneration of T cells, the basic idea is as follows: iPSCs produced from T cells inherit rearranged TCR genes, and thus all regenerated T cells should express the same TCR. Based on this idea, various types of T cells have been regenerated, including conventional cytotoxic T lymphocytes (CTLs), γδT cells, NKT cells and mucosal-associated invariant T (MAIT) cells. On the other hand, any cytotoxic cells can be used as the base cells into which CAR is introduced, and thus iPSC-derived NK cells have been developed. To apply the iPSC-based cell therapy in an allogeneic setting, the authors' group developed a method in which non-T-cell-derived iPSCs are transduced with exogenous TCR genes (TCR-iPSC method). This approach is being prepared for a clinical trial to be realized in Kyoto University Hospital, in which acute myeloid leukemia patients will be treated by the regenerated WT1 antigen-specific CTLs.

Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers.

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3' region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3'-untranslated region (UTR). Disruption of the Pd-l1 3'-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3'-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

A regulatory element in the 3'-untranslated region of CEBPA is associated with myeloid/NK/T-cell leukemia.

OBJECTIVES: CCAAT/enhancer-binding protein α (CEBPA) is an essential transcription factor for myeloid differentiation. Not only mutation of the CEBPA gene, but also promoter methylation, which results in silencing of CEBPA, contributes to the pathogenesis of acute myeloid leukemia (AML). We sought for another differentially methylated region (DMR) that associates with the CEBPA silencing and disease phenotype. METHODS: Using databases, we identified a conserved DMR in the CEBPA 3'-untranslated region (UTR). RESULTS: Methylation-specific PCR analysis of 231 AML cases showed that hypermethylation of the 3'-UTR was associated with AML that had a myeloid/NK/T-cell phenotype and downregulated CEBPA. Most of these cases were of an immature phenotype with CD7/CD56 positivity. These cases were significantly associated with lower hemoglobin levels than the others. Furthermore, we discovered that the CEBPA 3'-UTR DMR can enhance transcription from the CEBPA native promoter. In vitro experiments identified IKZF1-binding sites in the 3'-UTR that are responsible for this increased transcription of CEBPA. CONCLUSIONS: These results indicate that the CEBPA 3'-UTR DMR is a novel regulatory element of CEBPA related to myeloid/NK/T-cell lineage leukemogenesis. Transcriptional regulation of CEBPA by IKZF1 may provide a clue for understanding the fate determination of myeloid vs. NK/T-lymphoid progenitors.

A Phase I/IIa Study of Antidisialoganglioside Antibody Dinutuximab in Japanese Patients With Neuroblastoma.

Japanese patients with neuroblastoma completing induction therapy and high-dose chemotherapy received antidisialoganglioside antibody dinutuximab 17.5 mg/m2 for 4 days during each of 5 consecutive 28-day cycles. Patients also received macrophage colony-stimulating factor (M-CSF) or granulocyte colony-stimulating factor (G-CSF) during cycles 1, 3, and 5 combined with interleukin-2 teceleukin during cycles 2 and 4. A total of 25 patients (11 in the M-CSF group and 14 in the G-CSF group) were enrolled, and dose-limiting toxicity was assessed in the first 12 patients (6 in each group). The recommended doses of dinutuximab, M-CSF, and G-CSF were determined to be 17.5 mg/m2, 6.0×106 U/m2, and 5 µg/kg/d, respectively, whereas that of teceleukin was 0.75×106 IU/m2 during week 1 and 1×106 IU/m2 during week 2. The most common grade 3 or 4 adverse events in both groups were neutrophil count decreased, platelet count decreased, pyrexia, and alanine aminotransferase increased. Four patients (2 in each group) discontinued the treatment because of adverse events. At the end of the study, survival was confirmed in 22 patients (9 in the M-CSF group and 13 in the G-CSF group). From these results, we concluded that this combination regimen is a feasible treatment for Japanese patients with neuroblastoma.

[The origin of blood cells and myeloid cells].

In human hematopoiesis, cells of various lineages exist, such as neutrophils, lymphocytes, and erythrocytes. Unveiling the pathway from stem cells to the various lineages helps us understand the blood disorders and develop therapies for them. We have studied the developmental pathway of hematopoiesis for decades and found that myeloid potential is retained just before the differentiation into each lineage of the various lineage progenitors. This uniqueness of myeloid cells might reflect the character of mixed-phenotype leukemia and provide a very important clue in determining the evolutional history of blood cells. Recent studies concerning the differentiation pathways of megakaryocytes and granulocytes as well as the findings on the hemocytes of invertebrates have strongly supported the concept of the uniqueness of myeloid cells and enabled us to propose insights into the evolutional history of blood. In this paper, we discuss the origin of blood cells in the context of developmental pathways during ontogeny and phylogeny.

Long non-coding RNA MANCR is a target of BET bromodomain protein BRD4 and plays a critical role in cellular migration and invasion abilities of prostate cancer.

Androgen receptor (AR)-negative castration-resistant prostate cancer (CRPC) is highly aggressive and is resistant to most of the current therapies. Bromodomain and extra terminal domain (BET) protein BRD4 binds to super-enhancers (SEs) that drive high expression of oncogenes in many cancers. A BET inhibitor, JQ1, has been found to suppress the malignant phenotypes of prostate cancer cells, however, the target genes of JQ1 remain largely unknown. Here we show that SE-associated genes specific for AR-negative CRPC PC3 cells include genes involved in migration and invasion, and that JQ1 impairs migration and invasion of PC3 cells. We identified a long non-coding RNA, MANCR, which was markedly down-regulated by JQ1, and found that BRD4 binds to the MANCR locus. MANCR knockdown led to a significant decrease in migration and invasion of PC3 cells. Furthermore, RNA sequencing analysis revealed that expression of the genes involved in migration and invasion was altered by MANCR knockdown. In summary, our data demonstrate that MANCR plays a critical role in migration and invasion of PC3 cells.

Cascading suppression of transcriptional silencers by ThPOK seals helper T cell fate.

CD4 and the transcription factor ThPOK are essential for the differentiation of major histocompatibility complex class II-restricted thymocytes into the helper T cell lineage; their genes (Cd4 and Zbtb7b (called 'ThPOK' here)) are repressed by transcriptional silencer elements in cytotoxic T cells. The molecular mechanisms regulating expression of these genes during helper T cell lineage differentiation remain unknown. Here we showed that inefficient upregulation of ThPOK, induced by removal of the proximal enhancer from the ThPOK locus, resulted in the transdifferentiation of helper lineage-specified cells into the cytotoxic T cell lineage. Furthermore, direct antagonism by ThPOK of the Cd4 and ThPOK silencers generated two regulatory loops that initially inhibited Cd4 downregulation and later stabilized ThPOK expression. Our results show how an initial lineage-specification signal can be amplified and stabilized during the lineage-commitment process.

Discovery of ORN0829, a potent dual orexin 1/2 receptor antagonist for the treatment of insomnia.

Here, we present the design, synthesis, and SAR of dual orexin 1 and 2 receptor antagonists, which were optimized by balancing the antagonistic activity for orexin receptors and lipophilicity. Based on the prototype compound 1, ring construction and the insertion of an additional heteroatom into the resulting ring led to the discovery of orexin 1 and 2 receptor antagonists, which were 3-benzoyl-1,3-oxazinane derivatives. Within these derivatives, (-)-3h enabled a high dual orexin receptor antagonistic activity and a low lipophilicity. Compound (-)-3h exhibited potent sleep-promoting effects at a po dose of 1 mg/kg in a rat polysomnogram study, and optimal PK properties with a rapid Tmax and short half-lives in rats and dogs were observed, indicating a predicted human half-life of 0.9-2.0 h. Thus, (-)-3h (ORN0829; investigation code name, TS-142) was selected as a viable candidate and is currently in clinical development for the treatment of insomnia.