C-terminal Transactivation Domain Research Articles

β-catenin mediates endodermal commitment of human ES cells via distinct transactivation functions

Backgroundβ-catenin, acting as the core effector of canonical Wnt signaling pathway, plays a pivotal role in controlling lineage commitment and the formation of definitive endoderm (DE) during early embryonic development. Despite extensive studies using various animal and cell models, the β-catenin-centered regulatory mechanisms underlying DE formation remain incompletely understood, partly due to the rapid and complex cell fate transitions during early differentiation.ResultsIn this study, we generated new CTNNB1-/- human ES cells (hESCs) using CRISPR-based insertional gene disruption approach and systematically rescued the DE defect in these cells by introducing various truncated or mutant forms of β-catenin. Our analysis showed that a truncated β-catenin lacking both N- and C-terminal domains (ΔN148C) could robustly rescue the DE formation, whereas hyperactive β-catenin mutants with S33Y mutation or N-terminal deletion (ΔN90) had limited ability to induce DE lineage. Notably, the ΔN148C mutant exhibited significant nuclear translocation that was positively correlated with successful DE rescue. Transcriptomic analysis further uncovered that two weak β-catenin mutants lacking the C-terminal transactivation domain (CTD) activated primitive streak (PS) genes, whereas the hyperactive β-catenin mutants activated mesoderm genes.ConclusionOur study uncovered an unconventional regulatory function of β-catenin through weak transactivation, indicating that the levels of β-catenin activity determine the lineage bifurcation from mesendoderm into endoderm and mesoderm.

The structure of B-ARR reveals the molecular basis of transcriptional activation by cytokinin.

The phytohormone cytokinin has various roles in plant development, including meristem maintenance, vascular differentiation, leaf senescence, and regeneration. Prior investigations have revealed that cytokinin acts via a phosphorelay similar to the two-component system by which bacteria sense and respond to external stimuli. The eventual targets of this phosphorelay are type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs), containing the conserved N-terminal receiver domain (RD), middle DNA binding domain (DBD), and C-terminal transactivation domain. While it has been established for two decades that the phosphoryl transfer from a specific histidyl residue in ARABIDOPSIS HIS PHOSPHOTRANSFER PROTEINS (AHPs) to an aspartyl residue in the RD of B-ARRs results in a rapid transcriptional response to cytokinin, the underlying molecular basis remains unclear. In this work, we determine the crystal structures of the RD-DBD of ARR1 (ARR1RD-DBD) as well as the ARR1DBD-DNA complex from Arabidopsis. Analyses of the ARR1DBD-DNA complex have revealed the structural basis for sequence-specific recognition of the GAT trinucleotide by ARR1. In particular, comparing the ARR1RD-DBD and ARR1DBD-DNA structures reveals that unphosphorylated ARR1RD-DBD exists in a closed conformation with extensive contacts between the RD and DBD. In vitro and vivo functional assays have further suggested that phosphorylation of the RD weakens its interaction with DBD, subsequently permits the DNA binding capacity of DBD, and promotes the transcriptional activity of ARR1. Our findings thus provide mechanistic insights into phosphorelay activation of gene transcription in response to cytokinin.

Engineered coiled-coil HIF1α protein domain mimic.

The development of targeted anti-cancer therapeutics offers the potential for increased efficacy of drugs and diagnostics. Utilizing modalities agnostic to tumor type, such as the hypoxic tumor microenvironment (TME), may assist in the development of universal tumor targeting agents. The hypoxia-inducible factor (HIF), in particular HIF1, plays a key role in tumor adaptation to hypoxia, and inhibiting its interaction with p300 has been shown to provide therapeutic potential. Using a multivalent assembled protein (MAP) approach based on the self-assembly of the cartilage oligomeric matrix protein coiled-coil (COMPcc) domain fused to the critical residues of the C-terminal transactivation domain (C-TAD) of the α subunit of HIF1 (HIF1α), we generate HIF1α-MAP (H-MAP). The resulting H-MAP demonstrates picomolar binding affinity to p300, the ability to downregulate hypoxia-inducible genes, and in vivo tumor targeting capability.

Hypoxia and HIF-1α promote lytic de novo KSHV infection.

The current view is that the default pathway of Kaposi's sarcoma-associated herpesvirus (KSHV) infection is the establishment of latency, which is a prerequisite for lifelong infection and viral oncogenesis. This view about KSHV infection is supported by the observations that KSHV latently infects most of the cell lines cultured in vitro in the absence of any environmental stresses that may occur in vivo. The goal of this study was to determine the effect of hypoxia, a natural stress stimulus, on primary KSHV infection. Our data indicate that hypoxia promotes euchromatin formation on the KSHV genome following infection and supports lytic de novo KSHV infection. We also discovered that hypoxia-inducible factor-1α is required and sufficient for allowing lytic KSHV infection. Based on our results, we propose that hypoxia promotes lytic de novo infection in cells that otherwise support latent infection under normoxia; that is, the environmental conditions can determine the outcome of KSHV primary infection.

The C-terminal transactivation domain of MITF interacts promiscuously with co-activator CBP/p300

The microphthalmia-associated transcription factor (MITF) is one of four closely related members of the MiT/TFE family (TFEB, TFE3, TFEC) that regulate a wide range of cellular processes. MITF is a key regulator of melanocyte-associated genes, and essential to proper development of the melanocyte cell lineage. Abnormal MITF activity can contribute to the onset of several diseases including melanoma, where MITF is an amplified oncogene. To enhance transcription, MITF recruits the co-activator CREB-binding protein (CBP) and its homolog p300 to gene promoters, however the molecular determinants of their interaction are not yet fully understood. Here, we characterize the interactions between the C-terminal MITF transactivation domain and CBP/p300. Using NMR spectroscopy, protein pulldown assays, and isothermal titration calorimetry we determine the C-terminal region of MITF is intrinsically disordered and binds with high-affinity to both TAZ1 and TAZ2 of CBP/p300. Mutagenesis studies revealed two conserved motifs within MITF that are necessary for TAZ2 binding and critical for MITF-dependent transcription of a reporter gene. Finally, we observe the transactivation potential of the MITF C-terminal region is reliant on the N-terminal transactivation domain for function. Taken together, our study helps elucidate the molecular details of how MITF interacts with CBP/p300 through multiple redundant interactions that lend insight into MITF function in melanocytes and melanoma.

A novel HIF2A mutation causes dyslipidemia and promotes hepatic lipid accumulation

Hypoxia-inducible factor-2α (HIF-2α) is a transcription factor responsible for regulating genes related to angiogenesis and metabolism. This study aims to explore the effect of a previously unreported mutation c.C2473T (p.R825S) in the C-terminal transactivation domain (CTAD) of HIF-2α that we detected in tissue of patients with liver disease. We sequenced available liver and matched blood samples obtained during partial liver resection or liver transplantation performed for clinical indications including hepatocellular carcinoma and liver failure. In tandem, we constructed cell lines and a transgenic mouse model bearing the corresponding identified mutation in HIF-2α from which we extracted primary hepatocytes. Lipid accumulation was evaluated in these cells and liver tissue from the mouse model using Oil Red O staining and biochemical measurements. We identified a mutation in the CTAD of HIF-2α (c.C2473T; p.R825S) in 5 of 356 liver samples obtained from patients with hepatopathy and dyslipidemia. We found that introduction of this mutation into the mouse model led to an elevated triglyceride level, lipid droplet accumulation in liver of the mutant mice and in their extracted primary hepatocytes, and increased transcription of genes related to hepatic fatty acid transport and synthesis in the mutant compared to the control groups. In mutant mice and cells, the protein levels of nuclear HIF-2α and its target perilipin-2 (PLIN2), a lipid droplet-related gene, were also elevated. Decreased lipophagy was observed in mutant groups. Our study defines a subpopulation of dyslipidemia that is caused by this HIF-2α mutation. This may have implications for personalized treatment.

Transcriptional Regulation of the Acer truncatum B. Response to Drought and the Contribution of AtruNAC36 to Drought Tolerance.

Drought stress is one of the major environmental factors severely restricting plant development and productivity. Acer truncatum B, which is an economically important tree species, is highly tolerant to drought conditions, but the underlying molecular regulatory mechanisms remain relatively unknown. In this study, A. truncatum seedlings underwent a drought treatment (water withheld for 0, 3, 7, and 12 days), after which they were re-watered for 5 days. Physiological indices were measured and a transcriptome sequencing analysis was performed to reveal drought response-related regulatory mechanisms. In comparison to the control, the drought treatment caused a significant increase in antioxidant enzyme activities, with levels rising up to seven times, and relative electrical conductivity from 14.5% to 78.4%, but the relative water content decreased from 88.3% to 23.4%; these indices recovered somewhat after the 5-day re-watering period. The RNA sequencing analysis identified 9126 differentially expressed genes (DEGs), which were primarily involved with abscisic acid responses, and mitogen-activated protein kinase signaling. These DEGs included 483 (5.29%) transcription factor genes from 53 families, including ERF, MYB, and NAC. A co-expression network analysis was conducted and three important modules were analyzed to identify hub genes, one of which (AtruNAC36) was examined to clarify its function. The AtruNAC36 protein was localized to the nucleus and had a C-terminal transactivation domain. Moreover, it bounded specifically to the NACRS element. The overexpression of AtruNAC36 in Arabidopsis thaliana resulted in increased drought tolerance by enhancing antioxidant enzyme activities. These findings provide important insights into the transcriptional regulation mediating the A. truncatum response to drought. Furthermore, AtruNAC36 may be relevant for breeding forest trees resistant to drought stress.

Activation of HIF-1α C-terminal transactivation domain protects against hypoxia-induced kidney injury through hexokinase 2-mediated mitophagy

The transcription factor hypoxia-inducible factor-1α (HIF-1α), as a master regulator of adaptive responses to hypoxia, possesses two transcriptional activation domains [TAD, N-terminal (NTAD), and C-terminal (CTAD)]. Although the roles of HIF-1α NTAD in kidney diseases have been recognized, the exact effects of HIF-1α CTAD in kidney diseases are poorly understood. Here, two independent mouse models of hypoxia-induced kidney injury were established using HIF-1α CTAD knockout (HIF-1α CTAD−/−) mice. Furthermore, hexokinase 2 (HK2) and mitophagy pathway are modulated using genetic and pharmacological methods, respectively. We demonstrated that HIF-1α CTAD−/− aggravated kidney injury in two independent mouse models of hypoxia-induced kidney injury, including ischemia/reperfusion-induced kidney injury and unilateral ureteral obstruction-induced nephropathy. Mechanistically, we found that HIF-1α CTAD could transcriptionally regulate HK2 and subsequently ameliorate hypoxia-induced tubule injury. Furthermore, it was found that HK2 deficiency contributed to severe renal injury through mitophagy inhibition, while mitophagy activation using urolithin A could significantly protect against hypoxia-induced kidney injury in HIF-1α C-TAD−/− mice. Our findings suggested that the HIF-1α CTAD-HK2 pathway represents a novel mechanism of kidney response to hypoxia, which provides a promising therapeutic strategy for hypoxia-induced kidney injury.

β-Catenin interacts with the TAZ1 and TAZ2 domains of CBP/p300 to activate gene transcription

The transcriptional co-regulator β-catenin is a critical member of the canonical Wnt signaling pathway, which plays an important role in regulating cell fate. Deregulation of the Wnt/β-catenin pathway is characteristic in the development of major types of cancer, where accumulation of β-catenin promotes cancer cell proliferation and renewal. β-catenin gene expression is facilitated through recruitment of co-activators such as histone acetyltransferases CBP/p300; however, the mechanism of their interaction is not fully understood. Here we investigate the interaction between the C-terminal transactivation domain of β-catenin and CBP/p300. Using a combination of pulldown assays, isothermal titration calorimetry, and nuclear resonance spectroscopy we determine the disordered C-terminal region of β-catenin binds promiscuously to the TAZ1 and TAZ2 domains of CBP/p300. We then map the interaction site of the C-terminal β-catenin transactivation domain onto TAZ1 and TAZ2 using chemical-shift perturbation studies. Luciferase-based gene reporter assays indicate Asp750-Leu781 is critical to β-catenin gene activation, and mutagenesis revealed that acidic and hydrophobic residues within this region are necessary to maintain TAZ1 binding. These results outline a mechanism of Wnt/β-catenin gene regulation that underlies cell development and provides a framework to develop methods to block β-catenin dependent signaling.

Hypomorphic and dominant-negative impact of truncated SOX9 dysregulates Hedgehog–Wnt signaling, causing campomelia

Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygous Sox9 null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, invitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases invivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygous Sox9 null mutation (Sox9+/-) with the Sox9+/Y440X CD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While some Sox9+/Y440X mice survived, all Sox9+/- mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced in Sox9+/Y440X compared with Sox9+/-. Activating Sox9Y440X specifically in the chondrocyte-osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developing Sox9+/Y440X limbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9Y440X failed to interact with β-catenin and was unable to suppress transactivation of Ihh in cell-based assays. We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9Y440X, cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.

Chilo suppressalis heat shock proteins are regulated by heat shock factor 1 during heat stress.

Heat shock factor 1 (HSF1) functions to maintain cellular and organismal homeostasis by regulating the expression of target genes, including those encoding heat shock proteins (HSPs). In the present study, the gene encoding HSF1 was cloned from the rice pest Chilo suppressalis, and designated Cshsf1. The deduced protein product, CsHSF1, contained conserved domains typical of the HSF1 family, including a DNA-binding domain, two hydrophobic heptad repeat domains, and a C-terminal transactivation domain. Real-time quantitative PCR showed that Cshsf1 was highly expressed in hemocytes. Expression analysis in different developmental stages of C. suppressalis revealed that Cshsf1 was most highly expressed in male adults. RNAi-mediated silencing of Cshsf1 expression reduced C. suppressalis survival at high temperatures. To investigate the regulatory interactions between Cshsf1 and Cshsps, the promoters and expression patterns of 18 identified Cshsps in C. suppressalis were analysed; four types of heat shock elements (HSEs) were identified in promoter regions including canonical, tail-tail, head-head, and step/gap. The expression of Cshsp19.0, Cshsp21.7B, Cshsp60, Cshsp70 and Cshsp90 was positively regulated by Cshsf1; however, Cshsp22.8, Cshsp702, Cshsp705 and Cshsp706 gene expression was not altered. This study provides a foundation for future studies of HSF1 in insects during thermal stress.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Molecular cloning, expression and functional analysis of STAT2 in orange-spotted grouper, Epinephelus coioides

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signaling pathway. To better understand the functions of STAT2 in fish immune response, a STAT2 gene from orange-spotted grouper (Epinephelus coioides) (EcSTAT2) was cloned and characterized in this study. EcSTAT2 encoded a 802-amino acid peptide which shared 99.5% and 91.5% identity with giant grouper (Epinephelus lanceolatus) and leopard coral grouper (Plectropomus leopardus), respectively. Amino acid alignment analysis showed that EcSTAT2 contained five conserved domains, including N-terminal protein interaction domain, coiled coil domain (CCD), DNA binding domain (DBD), Src-homology 2 (SH2) domain, and C-terminal transactivation domain (TAD). Phylogenetic analysis indicated that EcSTAT2 clustered into fish STAT2 group and showed the nearest relationship to giant grouper STAT2. In healthy grouper, EcSTAT2 was distributed in all tissues tested, and the expression of EcSTAT2 was predominantly detected in spleen, kidney and gill. In vitro, EcSTAT2 expression was significantly increased in response to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization showed that EcSTAT2 was located in the cytoplasm in a punctate manner. EcSTAT2 overexpression significantly inhibited RGNNV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral genes and protein. Consistently, knockdown of EcSTAT2 using small interfering RNA (siRNA) promoted RGNNV replication. Furthermore, EcSTAT2 overexpression increased both interferon (IFN) and interferon stimulated genes (ISGs) expression. In addition, EcSTAT2 knockdown decreased the transcription levels of IFN and ISGs. Together, our data demonstrated that EcSTAT2 exerted antiviral activity against RGNNV through up-regulation of host interferon response.

Non-canonical function of an Hif-1α splice variant contributes to the sustained flight of locusts.

The hypoxia inducible factor (Hif) pathway is functionally conserved across metazoans in modulating cellular adaptations to hypoxia. However, the functions of this pathway under aerobic physiological conditions are rarely investigated. Here, we show that Hif-1α2, a locust Hif-1α isoform, does not induce canonical hypoxic responses but functions as a specific regulator of locust flight, which is a completely aerobic physiological process. Two Hif-1α splice variants were identified in locusts, a ubiquitously expressed Hif-1α1 and a muscle-predominantly expressed Hif-1α2. Hif-1α1 that induces typical hypoxic responses upon hypoxia exposure remains inactive during flight. By contrast, the expression of Hif-1α2, which lacks C-terminal transactivation domain, is less sensitive to oxygen tension but induced extensively by flying. Hif-1α2 regulates physiological processes involved in glucose metabolism and antioxidation during flight and sustains flight endurance by maintaining redox homeostasis through upregulating the production of a reactive oxygen species (ROS) quencher, DJ-1. Overall, this study reveals a novel Hif-mediated mechanism underlying prolonged aerobic physiological activity.

Molecular cloning of crtc2 and its expression in response to different feeding status in largemouth bass (Micropterus salmoides)

Crtc2, a member of the cAMP response element-binding protein (CREB)-regulated transcription coactivators family, plays crucial roles in variety of cellular functions and biological processes, including energy homeostasis, immunity, and endoplasmic reticulum (ER) stress in mammals. Thus far, its roles in teleosts are still rarely known. In the present study, the crtc2 gene was identified and characterized from largemouth bass (Micropterus salmoides), and then its transcription pattern in response to different feeding status was determined. The coding sequence of crtc2 gene is 2565 bp in length, encoding a protein with 854 amino acids. The putative Ms-crtc2 contains a typical N-terminal CREB-binding domain (CBD), a central regulatory domain (REG), a C-terminal transactivation domain (TAD), as well as conserved 14–3–3 binding sites, SIK2 and AMPK phosphorylation sites. Multiple alignments revealed that Ms-crtc2 shares high identity with its homologs in teleosts. Phylogenetic, syntenic, and gene structure analysis further demonstrated that crtc2 genes are highly conserved among vertebrates. Meanwhile, Ms-crtc2 was detectable in all tested tissues with the highest expression level in spleen. In a long-term fasting and refeeding experiment, we found that the transcriptional level of crtc2 was significantly increased during starvation, and then returned to normal level after refeeding in all tested tissues. Taken together, our findings reveal that the crtc2 gene may play important roles in regulating energy homeostasis and immune response in largemouth bass, which should be beneficial for further studies on the function of crtc2 in teleosts.

Retraction: Mapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3

Retraction: Mapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3

EP459: Rare clinical gene variant of SOX9: Acampomelic campomelic dysplasia

eP459: Rare clinical gene variant of SOX9: Acampomelic campomelic dysplasia

Biophysical investigation of the dual binding surfaces of human transcription factors FOXO4 and p53.

Cellular senescence is protective against external oncogenic stress, but its accumulation causes aging-related diseases. Forkhead box O4 (FOXO4) and p53 are human transcription factors known to promote senescence by interacting with each other and activating p21 transcription. Inhibition of the interaction is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the FOXO4-p53 interaction remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by NMR spectroscopy. NMR chemical shift perturbation analysis showed that the binding between FOXO4's forkhead domain (FHD) and p53's transactivation domain (TAD), and between FOXO4's C-terminal transactivation domain (CR3) and p53's DNA-binding domain (DBD), mediate the FOXO4-p53 interaction. Isothermal titration calorimetry data showed that both interactions have micromolar Kd values, and FOXO4 FHD-p53 TAD interaction has a higher binding affinity. We also showed that the intramolecular CR3-binding surface of FOXO4 FHD interacts with p53 TAD2, and FOXO4 CR3 interacts with the DNA/p53 TAD-binding surface of p53 DBD, suggesting a network of potentially competitive and/or coordinated interactions. Based on these results, we propose that a network of intramolecular and intermolecular interactions contributes to the two transcription factors' proper localisation on the p21 promoter and consequently promotes p21 transcription and cell senescence. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence.

FOXO transcription factors differ in their dynamics and intra/intermolecular interactions.

Transcription factors play key roles in orchestrating a plethora of cellular mechanisms and controlling cellular homeostasis. Transcription factors share distinct DNA binding domains, which allows to group them into protein families. Among them, the Forkhead box O (FOXO) family contains transcription factors crucial for cellular homeostasis, longevity and response to stress. The dysregulation of FOXO signaling is linked to drug resistance in cancer therapy or cellular senescence, however, selective drugs targeting FOXOs are limited, thus knowledge about structure and dynamics of FOXO proteins is essential. Here, we provide an extensive study of structure and dynamics of all FOXO family members. We identify residues accounting for different dynamic and structural features. Furthermore, we show that the auto-inhibition of FOXO proteins by their C-terminal trans-activation domain is conserved throughout the family and that these interactions are not only possible intra-, but also inter-molecularly. This indicates a model in which FOXO transcription factors would modulate their activities by interacting mutually.

FOXO1 Dependent Transcription Network Is a Targetable Vulnerability of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) often has an adverse prognosis and despite aggressive multimodal treatment with conventional and targeted therapies, the median survival of MCL patients remains approximately 4 years. Thus, there is a significant unmet need to find novel targets and rational combination treatments.Targeting lineage vulnerabilities driven by specific transcription factors has been broadly confirmed as an effective intervention in many human cancers. To identify the transcription program dependency of MCL cells, we conducted an unbiased domain-focused CRISPR-Cas9 screening against a library of 8,750 sgRNAs targeting 1,434 transcription factors in MCL cell lines (JEKO1, MAVER1, UPN1, and CCMCL1). We identified Forkhead Box O1 (FOXO1), EBF1, PAX5, and IRF4 as 4 transcription factors that are specifically required for MCL survival and growth. Chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis further revealed that the four transcription factors act together to orchestrate B cell lineage transcriptional program and MCL cell survival.Despite its well-recognized role as a tumor suppressor, FOXO1 has been implicated as a lineage specific transcription factor involved in mature B cell development. Genetic studies revealed a critical role of FOXO1 in germinal center dark zone formation and lymphomagenesis, raising the possibility that FOXO1 acts as a master transcription factor for lineage survival transcription program of MCL. Indeed, hierarchical interaction analysis revealed that FOXO1 functions as a pioneer factor that facilitate the chromatin access of other B cell lineage transcription factors. We demonstrated that interaction of FOXO1 to its cognate motif stabilizes B cell transcription factor complex and supports MCL progression. Along this line, we show that enforced expression of FOXO1 in myeloid leukemia cells induces transdifferentiation and B-cell specific gene expression. Mechanistically, we demonstrate through tiling CRISPR scanning screen that forkhead DNA binding and c-terminal transactivation domains of FOXO1 are specifically required for the viability of MCL cells.Given our finding of FOXO1 as a lineage-specific oncogene in MCL, we next explored the possibility of developing FOXO1-tageted inhibitors. We screened a library of potential small molecule inhibitors of FOXO1 (Forkhead BioTherapeutics) and identified cpd10 as one of the most potent and selective FOXO1 inhibitors (IC 50=76 ×/÷ 1.7. nM) Through the CCMCL1 MCL-NSG preclinical model, we found that cpd10 (100 mg/kg/daily) was well tolerated without overt toxicities. Importantly, prolonged treatment induced a robust cytotoxic response of MCL cells and suppressed MCL progression in vivo. Altogether, our findings identified FOXO1 as a MCL lineage-survival oncogene that can be exploited as a therapeutic target of future drug development. DisclosuresCantley: Petra Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Elemento: Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Janssen: Research Funding; Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy. Belvedere: Forkhead BioTherapeutics: Current Employment. Paik: Forkhead BioTherapeutics: Research Funding.