RAG Expression Research Articles

ETV6-RUNX1 and RUNX1 directly regulate RAG1 expression: one more step in the understanding of childhood B-cell\xa0acute lymphoblastic leukemia leukemogenesis

HighlightsETV6-RUNX1 and RUNX1 directly promote RAG1 expression.ETV6-RUNX1 and RUNX1 preferentially bind to the −1200 bp enhancer of RAG1 and the −80 bp promoter of RAG1 gene respectively, and compete for these bindings.ETV6-RUNX1 and RUNX1 induce an excessive RAG recombinase activity.ETV6-RUNX1 participates directly in two events of the multi-hit ALL leukemogenesis: as an initiating event and as an activator of RAG1 expression.

Genome-Wide Analysis Identifies Rag1 and Rag2 as Novel Notch1 Transcriptional Targets in Thymocytes.

Recombination activating genes 1 (Rag1) and Rag2 are expressed in immature lymphocytes and essential for generating the vast repertoire of antigen receptors. Yet, the mechanisms governing the transcription of Rag1 and Rag2 remain to be fully determined, particularly in thymocytes. Combining cDNA microarray and ChIP-seq analysis, we identify Rag1 and Rag2 as novel Notch1 transcriptional targets in acute T-cell lymphoblastic leukemia (T-ALL) cells. We further demonstrate that Notch1 transcriptional complexes directly bind the Rag1 and Rag2 locus in not only T-ALL but also primary double negative (DN) T-cell progenitors. Specifically, dimeric Notch1 transcriptional complexes activate Rag1 and Rag2 through a novel cis-element bearing a sequence-paired site (SPS). In T-ALL and DN cells, dimerization-defective Notch1 causes compromised Rag1 and Rag2 expression; conversely, dimerization-competent Notch1 achieves optimal upregulation of both. Collectively, these results reveal Notch1 dimerization-mediated transcription as one of the mechanisms for activating Rag1 and Rag2 expression in both primary and transformed thymocytes. Our data suggest a new role of Notch1 dimerization in compelling efficient TCRβ rearrangements in DN progenitors during T-cell development.

MicroRNA miR-29c regulates RAG1 expression and modulates V(D)J recombination during B cell development.

Recombination activating genes (RAGs), consisting of RAG1 and RAG2, are stringently regulated lymphoid-specific genes, which initiate V(D)J recombination in developing lymphocytes. We report the regulation of RAG1 through a microRNA (miRNA), miR-29c, in a B cell stage-specific manner in mice and humans. Various lines of experimentation, including CRISPR-Cas9 genome editing, demonstrate the target specificity and direct interaction of miR-29c to RAG1. Modulation of miR-29c levels leads to change in V(D)J recombination efficiency in pre-B cells. The miR-29c expression is inversely proportional to RAG1 in a B cell developmental stage-specific manner, and miR-29c null mice exhibit a reduction in mature B cells. A negative correlation of miR-29c and RAG1 levels is also observed in leukemia patients, suggesting the potential use of miR-29c as a biomarker and a therapeutic target. Thus, our results reveal the role of miRNA in the regulation of RAG1 and its relevance in cancer.

Targeting of Glutamine Pathways Restores Sensitivity in Venetoclax Resistant Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

Targeting of Glutamine Pathways Restores Sensitivity in Venetoclax Resistant Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

IL6 Signaling Regulates Embryonic Hematopoietic Stem Cell Emergence

Recent studies have illustrated the significance of some proinflammatory factors in regulating embryonic hemtopoietic stem cells (HSCs) production ,such as TNFα、IFNγ and TLR4.Il6 is a powerful proinflammatory cytokine that plays a pivotal role in the regulation of immunity,hematopoiesis and other physiological processes.Il6-deficient mice leads to impaired survival and self-renewal of HSCs and early progenitors.Whereas Il6 signaling regulates aspects of adult hematopoiesis,little is known about its role in the developmental specification of HSCs.This has led us to explore the impact of loss of Il6 signaling on embryonic HSC emergence by injecting specific antisense morpholinos (MOs) into zebrafish embryos at 1-2 cell stage.Here we report that MO knockdown of Il6r and Il6 strongly reduced the number of cmyb+HSCs in or near the floor of the dorsal aorta (DA) at 36 hr post fertilization (36hpf) by wholemount in situ hybridization (WISH)compared with their standard MO(Std MO)siblings(Fig.1a).Consistent with the results above,the number of double-positive cmyb+kdrl+ HSCs was reduced about 50% in the floor of the DA in cmyb:GFP;kdrl:mCherry double transgenic embryos at 48 hpf by confocal microscopy when compared to control embryos(Fig. 1b).To distinguish these reductions were due to a defect in the initial specification of HSCs or in their subsequent maintenance,we performed WISH for runx1, both Il6r- and Il6- deficient embryos showed significant reduction in the number of runx1+ HSCs in the aortic floor at 24 and 28hpf.Meanwhile,Il6r and Il6 morphants had sustained reductions in CD41:eGFP+ HSCs colonizing the caudal hematopoietic tissue (CHT) at 72 hpf using transgenic CD41:eGFP animals. Additionally ,expression of rag1 in thymus was completely or nearly absent in Il6r- and Il6-deficient embryos at 96 hpf(Fig. 1c), further surpporting that T-cell defects in these embryos resulted from early HSPC defects. Together, these results demonstrate that Il6 signaling pathway is important both for early specification and subsequent maintenance of HSC fate.To begin to determine the mechanism by which Il6 signaling regulates embryonic HSCs emergence in vivo,we first tested the impact of loss of Il6 signaling on vasculogenesis.No vascular abnormalities or impaired arterial specification were observed in Il6r- or Il6-deficient embryos at 28hpf when assayed by WISH for kdrl,efnb2a and dlc at the MO doses used in this study,suggesting that the function of Il6 signaling is required specifically during HSC development independently of their role in developing vasculature.Besides,we found that loss of Il6 signaling caused no increased apoptotic endothelial cells within the DA.Primitive hematopoiesis was also unaffected in our study.Lastly,to investigate the connection between Notch and Il6 signaling in HSC specification,we utilized tp1:eGFP;kdrl:mCherry double transgenic embryos and found that the double-positive tp1+kdrl+ Notch signaling-activated HSCs in the ventral floor of the DA had no significant change at 32-36hpf when blocking the IL6 signaling;embryos treated with notch signaling inhibitor DAPT developed fewer cmyb+ HSCs in the ventral floor of DA at 48hpf by WISH than DMSO-treated siblings;however,when the embryos were injected 200pg Il6r mRNA simutaneously, the HSC defect caused by DAPT could be partially rescued (Fig. 1d).In summary,our studies unravel an unexpected developmental role for Il6 signaling and place its action downstream of Notch signaling to control HSC emergence.Figure legendFigure 1. a)Representative cmyb WISH images and qualitative phenotype distribution (normal/down,n≥30 embryos/condition)of Il6r,Il6 and std MO-injected embryos at 36 hpf. b) Maximum projections of 48 hpf cmyb:GFP;kdrl:mCherry double-transgenic embryos injected with Std, Il6r, and Il6 MOs. Arrowheads denote cmyb+kdrl+ HSCs along the DA (left figure).Enumeration of cmyb+kdrl+ HSCs shown in(right figure).Error bars represent mean ± SEM of Std (n = 24), Il6r(n = 22), and Il6(n = 24) morphants. c)phenotype distribution(normal/down/absent,n≥30 embryos/condition) of rag1 expression in Il6r,Il6 and std MO-injected embryos at 96 hpf.d)WISH samples and phenotype distribution(high/medium/low,n≥30 embryos/condition)of cmyb expression in DMSO-treated,DAPT-treated,DAPT-treated and Il6r mRNA-injected simutaneously embryos at 48 hpf . [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity is required for V(D)J recombination.

A whole-genome CRISPR/Cas9 screen identified ATP2A2, the gene encoding the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 protein, as being important for V(D)J recombination. SERCAs are ER transmembrane proteins that pump Ca2+ from the cytosol into the ER lumen to maintain the ER Ca2+ reservoir and regulate cytosolic Ca2+-dependent processes. In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage. SERCA2 deficiency in B cells leads to increased expression of SERCA3, and combined loss of SERCA2 and SERCA3 results in decreased ER Ca2+ levels, increased cytosolic Ca2+ levels, reduction in RAG1 and RAG2 gene expression, and a profound block in V(D)J recombination. Mice with B cells deficient in SERCA2 and humans with Darier disease, caused by heterozygous ATP2A2 mutations, have reduced numbers of mature B cells. We conclude that SERCA proteins modulate intracellular Ca2+ levels to regulate RAG1 and RAG2 gene expression and V(D)J recombination and that defects in SERCA functions cause lymphopenia.

Polystyrene microplastics reduce abundance of developing B cells in rainbow trout (Oncorhynchus mykiss) primary cultures

Environmental microplastic pollution (including polystyrene, PS) may have detrimental effects on the health of aquatic organisms. Accumulation of PS microplastics has been reported to affect innate immune cells and inflammatory responses in fish. To date, knowledge on effects of microplastics on the antibody response is still very limited. Here, we investigated effects of small (0.8–20 μm) PS microplastics on the abundance of B lineage cells in primary cultures of developing immune cells from the anterior kidney of rainbow trout. Both purchased PS microbeads and PS microparticles generated from consumer products were used as microplastic sources. We first show that rainbow trout phagocytic B cells efficiently took up small (0.83–3.1 μm) PS microbeads within hours of exposure. In addition, our data revealed that PS microplastic exposure most significantly decreased the abundance of a population of non-phagocytic developing B cells, using both flow cytometry and RT-qPCR. PS microplastics-induced loss of developing B cells further correlated with reduced gene expression of RAG1 and the membrane form of immunoglobulin heavy chains mu and tau. Based on the induced loss of developing B cells observed in our in vitro studies, we speculate that in vivo, chronic PS microplastic-exposure may lead to suboptimal IgM/IgT levels in response to pathogens in teleost species. Considering the highly conserved nature of vertebrate B lymphopoiesis it is likely that PS microplastics will similarly reduce antibody responses in higher vertebrate species, including humans. Further, RAG1 provides an effective biomarker to determine effects of PS microplastics on B cell development in teleost species.

Full length RAG2 expression enhances the DNA damage response in pre-B cells

V(D)J recombination by the RAG1 and RAG2 protein complex in developing lymphocytes includes DNA double strand break (DSB) intermediates. RAG2 undergoes export from the nucleus and enrichment at the centrosome minutes following production of DSBs by genotoxic stress, suggesting that RAG2 participates in cellular responses to DSBs such as those generated during V(D)J recombination. To determine the effect of RAG2 expression on cell viability following DSB generation, we measured pre-B cells that expressed either full length (FL) wild-type RAG2, or a T490A mutant of RAG2 that has increased stability and fails to undergo nuclear export following generation of DSBs. Each RAG2 construct was labeled with GFP at the N-terminus. Compared to the T490A mutant, cells expressing FL RAG2 exhibited elevated apoptosis by 24 h following irradiation, and this coincided with a greater amount of Caspase 3 cleavage measured in cell lysates. Pre-B cells expressing either RAG2 protein exhibited similar increases in phospho-p53 levels following irradiation. Interestingly, FL RAG2-expressing cells exhibited elevated division relative to the T490A clone beginning ~24 h following irradiation, as well as an increased percentage of cells proceeding through mitosis, suggesting an improved rate of recovery following the initial burst in apoptosis. Altogether, these data show that FL RAG2, but not its stable nuclear export-defective T490A mutant, participates in pre-B cell decisions between apoptosis versus DNA repair and cell cycle progression following DNA damage.

RAG deficiencies: Recent advances in disease pathogenesis and novel therapeutic approaches.

The RAG1 and RAG2 proteins initiate the process of V(D)J recombination and therefore play an essential role in adaptive immunity. While null mutations in the RAG genes cause severe combined immune deficiency with lack of T and B cells (T- B- SCID) and susceptibility to life-threatening, early-onset infections, studies in humans and mice have demonstrated that hypomorphic RAG mutations are associated with defects of central and peripheral tolerance resulting in immune dysregulation. In this review, we provide an overview of the extended spectrum of RAG deficiencies and their associated clinical and immunological phenotypes in humans. We discuss recent advances in the mechanisms that control RAG expression and function, the effects of perturbed RAG activity on lymphoid development and immune homeostasis, and propose novel approaches to correct this group of disorders.

Ethanol modulation of cerebellar neuroinflammation in a postnatal mouse model of fetal alcohol spectrum disorders

Fetal alcohol spectrum disorders (FASD) are alarmingly common, result in significant personal and societal loss, and there is no effective treatment for these disorders. Cerebellar neuropathology is common in FASD and causes aberrant cognitive and motor function. Ethanol-induced neuroinflammation is believed to contribute to neuropathological sequelae of FASD, and was previously demonstrated in the cerebellum in animal models of FASD. We now demonstrate neuroinflammation persists in the cerebellum several days following cessation of ethanol treatment in an early postnatal mouse model, with meaningful implications for timing of therapeutic intervention in FASD. We also demonstrate by Sholl analysis that ethanol decreases ramification of microglia cell processes in cells located near the Purkinje cell layer but not those near the external granule cell layer. Ethanol did not alter the expression of anti-inflammatory molecules or molecules that constitute NLRP1 and NLRP3 inflammasomes. Interestingly, ethanol decreased the expression of IL-23a (P19) and IL-12Rβ1 suggesting that ethanol may suppress IL-12 and IL-23 signaling. Fractalkine-fractalkine receptor (CX3CL1-CX3CR1) signaling is believed to suppress microglial activation and our demonstration that ethanol decreases CX3CL1 expression suggests that ethanol modulation of CX3CL1-CX3CR1 signaling may contribute to cerebellar neuroinflammation and neuropathology. We demonstrate ethanol alters the expression of specific molecules in the cerebellum understudied in FASD, but crucial for immune responses. Ethanol increases the expression of NOX-2 and NGP and decreases the expression of RAG1, NOS1, CD59a, S1PR5, PTPN22, GPR37, and Serpinb1b. These molecules represent a new horizon as potential targets for development of FASD therapy.

B7-H6 is a new potential biomarker and therapeutic target of T-lymphoblastic lymphoma.

BackgroundB7-H6 is a novel co-stimulatory protein exclusively expressed on a variety of cancer cells and associated with poor prognosis. T-cell lymphoblastic lymphoma (T-LBL) is a highly aggressive hematological malignancy whose treatment requires reliable prognostic biomarkers and therapeutic targets. However, the rare nature and delayed progression of T-LBL have limited its clinical management.MethodsThe expression of B7-H6 was analyzed by immunohistochemistry (IHC) in 65 T-LBL samples; the association with the clinicopathological characteristics and prognosis was also investigated. B7-H6-depleted Jurkat cells were also generated to investigate the effect of B7-H6 on cell proliferation, migration, and invasion. RNA sequencing was used to explore differentially expressed genes.ResultsB7-H6 was expressed in 61.5% (40/65) of T-LBL patients; of note, 38.5% (25/65) of patients showed membrane/cytoplasmic expression of B7-H6. Although the expression of B7-H6 varied across samples and did not correlate with patient survival, it was significantly associated with B symptoms, high ECOG scores (3 to 4), elevated serum lactate dehydrogenase level, and reduced complete remission at interim evaluation. B7-H6 underwent translocation into the nucleus of T-LBL cells, showing a specific nuclear localization sequence in the C-terminus. Moreover, the depletion of B7-H6 in Jurkat cells impaired cell proliferation, migration, and invasion. RNAseq showed the differential expression of RAG-1, which may be involved in the tumorigenesis of T-LBL.ConclusionsB7-H6 may serve as a novel prognostic biomarker and therapeutic target of T-LBL.

The Mechanisms of Peripheral Nerve Preconditioning Injury on Promoting Axonal Regeneration.

Two major factors contribute to the failure of axonal regrowth in the central nervous system (CNS), namely, the neuronal intrinsic regenerative capacity and the extrinsic local inhibitory microenvironments. However, a preconditioning peripheral nerve lesion could substantially enhance the regeneration of central axons following a subsequent spinal cord injury. In the present review, we summarize the molecular mechanisms of the preconditioning injury effect on promoting axonal regeneration. The injury signal transduction resulting from preconditioning peripheral nerve injury regulates the RAG expression to enhance axonal regeneration. Importantly, preconditioning peripheral nerve injury triggers interactions between neurons and nonneuronal cells to amplify and maintain their effects. Additionally, the preconditioning injury impacts mitochondria, protein, and lipid synthesis. All these coordinated changes endow axonal regeneration.

B7-H6 Is Associated with Poor Prognosis and a Potential Therapeutic Target of T-Lymphoblastic Lymphoma

Background:B7-H6 is a novel co-stimulatory protein that is exclusively expressed on a variety of cancer cells and associated with poor prognosis. T cell lymphoblastic lymphoma (T-LBL) is a highly aggressive hematological malignancy whose treatment requires reliable prognostic biomarkers and therapeutic targets. The rare nature and delayed progression of T-LBL has limited its clinical management. Methods:B7-H6 expression was analyzed by immunohistochemistry (IHC) in 65 T-LBL samples, and its association with clinicopathological characteristics and prognosis was analyzed. Jurkat cell with B7-H6 depletion was constructed to investigate the impact of B7-H6 on cell proliferation, migration, and invasion ability. RNA sequencing was used to explore aberrantly expressing genes. Results:In this study, we used immunohistochemistry to show the expression of B7-H6 in 61.5% (40/65) of the cohort, including 38.5% (25/65) patients with membrane/cytoplasmic expression of B7-H6. Although B7-H6 expression varied across samples and did not correlate with patient survival, it was significantly associated with B symptoms, higher ECOG score (3-4), elevated serum lactate dehydrogenase, and reduced complete remission at interim evaluation. B7-H6 underwent translocation into the nucleus of T-LBL cells and had a specific nuclear localization sequence in the C-terminus. Depleting Jurkat cells of B7-H6 impaired cell proliferation, migration, and invasion. RNAseq showed the differential expression of RAG-1 that could be involved in the tumorigenesis of T-LBL. Conclusions:B7-H6 may serve as a novel prognostic biomarker and therapeutic target of T-LBL. Disclosures Ke: Peking University Third Hospital:Current Employment.

Abstract 6265: Combining arsenic and topoisomerase inhibitors synergistically inhibit gastric cancer

Abstract Objectives: Arsenic compounds have cytotoxic activities in gastric cancer cells, however, the underlying mechanism is unclear and the clinical potential need to be explored. This study was aimed to investigate the mechanism of arsenic on gastric cancer and find a novel regimen for the treatment of gastric cancer. Methods: Gastric cancer cell lines were treated with arsenics and/or chemotherapeutic drugs. MTT assay, FACS, Western blotting, q-RT-PCR and CHIP were used to detect proliferation, modulation of proteins and genes. Results: We previously showed that NFATc3 was a target of Arsenic sulfide and its inhibition blocks gastric cancer growth. Here we show that the NFATc3 expression is critically important for the growth and survival of gastric cancer cells because knockdown of NFATc3 resulted in DNA damage and cell cycle arrest. Arsenic sulfide can target NFATc3 in gastric cancer cells and cause cell double-strand DNA breaks (DSB). However, overexpression of NFATc3 can block the DNA damage. Abolishing NFATc3 expression, whether by shRNA-induced silencing or Arsenic sulfide treatment, directly upregulated recombination activating gene 1 (RAG1, an endonuclease which is essential for immunoglobulin V(D)J recombination), leading to fatal DSB. NFATc3 targets the promoter of RAG1 for transcriptional inhibition. Moreover, we find that Arsenic sulfide and topoisomerase inhibitors (Irinotecan and Epirubicin, Doxorubicin, Etoposide and SN38) synergistically inhibit gastric cancer. Arsenic sulfide, as well as Arsenic Trioxide, enhanced the cytotoxic effect of Irinotecan and they synergistically induced NFATc3 degradation, resulted in disability of DNA repair system, as reflected by the decrease in the phosphorylated ATM/CHK2/CHK1 protein level and the dramatic increase in the phosphorylated H2AX protein level. More inspiring, a patient with advanced gastric cancer with liver and lymph-node metastasis who failed in first-line treatment achieved PR with 2 months treatment of Irinotecan combined with Realgar-Indigo Naturalis Formula, the main component of which is Arsenic sulfide. The progression-free survival was 4 months with no additional adverse event. Conclusions: Our research links arsenic compounds to the regulation of DNA damage repair and RAG1 expression as a mechanism for the cytotoxic effect. The combination of arsenic with topoisomerase inhibitors may represent a novel regimen for the treatment of gastric cancer and warrants further studies. Citation Format: Ting Kang, Maolin Ge, Shumin Lu, Qing Wang, Shouhai Zhu, Chuanying Zhu, Leizhen Zheng, Han Liu, Siyu Chen. Combining arsenic and topoisomerase inhibitors synergistically inhibit gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6265.

Clinical value of RAG1 expression and IKZF1 deletions in Philadelphia negative pediatric B cell precursor acute lymphoblastic leukemia

This study aimed to address the clinical impact of recombination-activating gene (RAG1) expression and tumor suppressor IKZF1 gene deletions in Philadelphia negative B-cell precursor acute lymphoblastic leukemia (B-ALL) patients. Fifty newly diagnosed pediatric Philadelphia negative B-ALL patients were included in this study. Using Bone Marrow samples, RAG1 expression was assessed by real time PCR and IKZF1 deletions were determined by multiplex real-time quantitative PCR. The expression of RAG1 was significantly higher in B-ALL patients as compared to the controls (p < .001). The B-ALL patients with RAG1 high expression (≥median) had lower response to induction of remission, shorter DFS, shorter overall survival, higher blast cells, and white cell counts in the peripheral blood as compared to those with low RAG1 expression levels (p < .01 for all). Likewise, there was significant association between IKZF1 deletion and high RAG1 expression. Based on our findings RAG1 high expression and IKZF1 deletions were associated with adverse prognosis in Philadelphia negative B-ALL. RAG1 could be used as therapeutic target in the treatment of B-ALL.

Aberrant RAG-mediated recombination contributes to multiple structural rearrangements in lymphoid blast crisis of chronic myeloid leukemia.

Blast crisis of chronic myeloid leukemia is associated with poor survival and the accumulation of genomic lesions. Using whole-exome and/or RNA sequencing of patients at chronic phase (CP, n = 49), myeloid blast crisis (MBC, n = 19), and lymphoid blast crisis (LBC, n = 20), we found 25 focal gene deletions and 14 fusions in 24 patients in BC. Deletions predominated in LBC (83% of structural variants). Transcriptional analysis identified the upregulation of genes involved in V(D)J recombination, including RAG1/2 and DNTT in LBC. RAG recombination is a reported mediator of IKZF1 deletion. We investigated the extent of RAG-mediated genomic lesions in BC. Molecular hallmarks of RAG activity; DNTT-mediated nucleotide insertions and a RAG-binding motif at structural variants were exclusively found in patients with high RAG expression. Structural variants in 65% of patients in LBC displayed these hallmarks compared with only 5% in MBC. RAG-mediated events included focal deletion and novel fusion of genes associated with hematologic cancer: IKZF1, RUNX1, CDKN2A/B, and RB1. Importantly, 8/8 patients with elevated DNTT at CP diagnosis progressed to LBC by 12 months, potentially enabling early prediction of LBC. This work confirms the central mutagenic role of RAG in LBC and describes potential clinical utility in CML management.

Reconstruction of rearranged T-cell receptor loci by whole genome and transcriptome sequencing gives insights into the initial steps of T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive tumor with leukemic presentation of mature T-lymphocytes. Here, we aimed at characterizing the initial events in the molecular pathogenesis of T-PLL and particularly, at determining the point in T-cell differentiation when the hallmark oncogenic events, that is, inv(14)(q11q32)/t(14;14)(q11;q32) and t(X;14)(q28;q11) occur. To this end, we mined whole genome and transcriptome sequencing data of 17 and 11 T-PLL cases, respectively. Mapping of the 14q32.1 locus breakpoints identified only TCL1A, which was moreover significantly overexpressed in T-PLL as compared to benign CD4+ and CD8+ T-cells, as the only common oncogenic target of aberrations. In cases with t(14;14), the breakpoints mapped telomeric and in cases with inv(14) centromeric or in the 3'-untranslated region of TCL1A. Regarding the T-cell receptor alpha (TRA) locus-TCL1A breakpoint junctions, all 17 breakpoints involved recombination signal sequences and 15 junctions contained nontemplated (N-) nucleotides. All T-PLL cases studied carried in-frame TRA rearrangements on the intact allele, which skewed significantly toward usage of distal/central TRAV/TRAJ gene segments as compared to the illegitimate TRA rearrangements. Our findings suggest that the oncogenic TRA-TCL1A/MTCP1 rearrangements in T-PLL occur during opening of the TRA locus, that is, during the progression from CD4+ immature single positive to early double positive thymocyte stage, just before physiologic TCL1A expression is silenced. The cell carrying such an oncogenic event continues maturation and rearranges the second TRA allele to achieve a functional T-cell receptor. Thereafter, it switches off RAG and DNTT expression in line with the mature T-cell phenotype at presentation of T-PLL.

Ezh1 Inhibits Commitment to Hemogenic Fate and HSPC Formation during Vertebrate Development

Understanding how hematopoietic stem cells (HSCs) are specified from mesodermal precursors is essential to the goal of generating patient-specific HSCs capable of multi-potent long-term function. HSCs are born from hemogenic endothelium in select arterial niches during embryonic development through a transdifferentiation process turned endothelial-to-hematopoietic transistion (EHT). Despite increasing efforts to recapitulate this process in vitro, current differentiation protocols largely fail to produce long-lived multi-lineage progenitors from human induced pluripotent stem cell (iPSC) sources. Recently, an in vitro loss-of-function screen in human hematopoietic progenitors identified the Polycomb group protein, Enhancer of Zeste Homolog 1 (EZH1), as a regulator of definitive hematopoietic commitment, as assayed by acquisition of lymphoid competence. To determine the mechanism by which Ezh1 regulates HSPC fate in vivo we employed functional knockdown and epistasis investigations using the zebrafish model. Morpholino-mediated knockdown of ezh1 promoted expression of the conserved HSC markers runx1 and c-myb in the ventral wall of the dorsal aorta (VDA) at 36 hours post fertilization (hpf), as assessed by whole mount in situ hybridization (WISH); additionally, expression of the lymphoid marker rag1 was found to be enhanced at 120 hpf, as assayed by WISH and fluorescent activated cell sorting (FACS), in line with our in vitro observations. An impact on HSPCs was confirmed and quantified by qPCR for runx1 (**p &lt; 0.01) and FACS using the CD41:GFP reporter line (**p &lt; 0.01), indicating significantly increased HSPC number. Importantly, this enhancement in HSPC production had no effect on gross vascular morphology of the niche as determined by confocal microscopy for flk1. Assessment of arterial versus venous fate indicated that while the latter was unchanged in morphant embryos, expression of the arterial markers, epbrinb2a, dll4, dlc and tbx20, was strongly reduced by WISH and qPCR (**p &lt; 0.01, *p &lt; 0.05, **p &lt; 0.01, and **** p &lt; 0.001, respectively). In contrast, markers of hemogenic commitment, gata2b, and scl/flk1, were significantly increased, suggesting that loss of ezh1 enhanced hematopoietic potential at the expense of maintaining arterial fate. Profiling of single-cell RNA-sequencing data obtained from sorted populations of E10.5 mouse embryos revealed EZH1 to be more highly expressed in cells undergoing the endothelial-to-hematopoietic transition, consistent with a role of EZH1 in regulating arterial verses hematopoietic fate. Gene set enrichment analysis (GSEA) from our prior in vitro studies revealed the Notch pathway to be significantly altered following EZH1 knockdown. As Notch signaling has been implicated in both arterial specification and HSC emergence, we next examined the potential role of Notch signaling in ezh1 knockdown-mediated HSPC expansion. Consistent with a hypothesized interaction, differential regulation of Notch ligands and receptors was observed in ezh1 morphants compared to wild-type siblings; specifically, expression of arterial ligands, dll4 and dlc were decreased, while hematopoietic ligands and receptors, jag1a and notch1a were enhanced. Notably, the effect on Notch signaling was specific to ezh1 knockdown, as ezh2 loss shows a distinct pattern and temporal impact, reducing HSC production rather than enhancing it, consistent with recent reports. The strong conservation of ezh1-mediated regulation of HSC number, and our identification of its mechanistic role at the level of Notch receptor/ligand interactions, position zebrafish as a platform to identify chemical mediators that can be used to regulate ezh1 function during in vitro differentiation to unlock multi-lineage HSC commitment of human iPSC for therapeutic application. Disclosures Daley: Epizyme, Inc: Other: Equity &amp; Consulting Fees; 28/7 Therapeutics: Other: Equity &amp; Consulting Fees.

De Novo Point Mutation of RPL31 in Diamond-Blackfan Anaemia: A Story from Patient to Zebrafish

Diamond Blackfan anemia (DBA) is a rare bone marrow failure disorder that usually manifests as severe macrocytic anemia and erythroid hypoplasia ,often accompanied by various physical malformations and increased frequency of cancer. The disease are associated with known ribosomal protein gene and rarely GATA1 mutations in most of DBA cases, with the genetic etiology unclear in the remaining patients. Here, we present one patient with Diamond-Blackfan anaemia who lacked documented mutations involving known DBA genes by screening.Whole-exome sequencing (WES) was performed and we identified a single missense mutation in RPL31.The missense variant c.254G&gt;T is predicted to give rise to a gene product with substitution p. Arg85Leu ,which is regarded as causative because the unaffected parents and sister of the patient do not possess the mutation,implying that this mutation was de novo. Previous studies showed that nearly all of the causative variants of RP genes observed in DBA are loss-of function mutations and RP haploinsufficiency accounts for most cases of DBA. Particularly, The coding region of RPL31 shares 89.6% amino acid identities with its zebrafish ortholog, which is highly conserved between these two species.All of above led us to explore the effects of RPL31 deficiency on morphology and erythropoietic status during embryonic development. Therefore, morpholino antisense oligonucleotides (MOs) targeting zebrafish rpl31, orthologs of human RPL31,was injected at a proper concentration into one-cell-stage zebrafish embryos to mimic RPL31 haploinsufficiency in DBA. The specificity of the rpl31-MO was valitated by western blot detection.Compared with wild-type embryos, rpl31 deficiency embryos showed developmental abnormalities such as pericardial edema, microcephaly ,delayed pigmentation and bent tail. We also performed haemoglobin staining at 48 hr post fertilization (48hpf) and found a significant decrease of erythrocyte production in the cardial vein of the morphants. Meanwhile,the number of lcr+red blood cells was reduced about 50% in lcr:GFP transgenic embryos at 48 hpf by fluorescence microscopy and 3dpf by flow cytometry when compared to control embryos.However,the myeloid and lymphoid lineages were preserved as analyzed by using lyz:dsRed transgenic line and by observing expression of rag1,respectively. Lots of evidence showed that many other ribosomal protein genes deficiency can up-regulate p53 network,resulting in consequent cell cycle arrest or apoptosis.so we want to investigate the connection between RPL31 and p53 signaling in erythropoiesis.However,p53 null can not completely rescue the erythroid defect phenotype when we injected rpl31-MO into p53-/- embryos,suggesting alternative pathways may be involved in this process. In summary, we reported a brand new point mutation in RPL31 gene and we phonecopied loss of function of RPL31 observed in the DBA patient in vivo by constructing a RPL31 deficiency zebrafish model,indicating preliminarily that the loss of function mutation in RPL31 is a candidate responsible for DBA.However,underlying molecular mechanisms and reasons of the specific erythroid impairments in RPL31 deficiency are still needed to be clarified.we will focused on these in the future. Disclosures No relevant conflicts of interest to declare.

Single Cell Transcriptome Mapping of Human Thymopoiesis Reveals a Continuum of Cell States during T-Lineage Specification and Commitment

The initial stages of thymopoiesis are characterized by the induction of T-lineage genes (specification) and the repression of alternative lineage genes (commitment). Multilineage (Thy1 and Thy2) and committed (Thy3) populations representing successive differentiation stages have been defined within CD34+ progenitor cells in the human thymus by the expression of CD7 and CD1a. However, due to intra-population heterogeneity and unstudied transitional states between populations, our understanding of the transcriptional programs that launch human thymopoiesis remains incomplete. Also, the interpretation of species specific gene expression profiles seen in these populations is confounded by species related differences in progenitor immunophenotypes. To our knowledge, whole transcriptome profiles of human thymic CD34+ progenitors at single cell resolution have not been reported. To resolve the transcriptional landscape of the initial stages of thymopoiesis, we performed single cell RNA-Seq (sRNA-Seq) of FACS sorted unfractionated human thymic CD34+CD4-CD8- cells from 3 donors using 10X (1 thymus, 5000 cells), Indrop (1 thymus, 2000 cells), or BD genomics (1 thymus, 192 index sorted cells) sequencing Averaged sRNA-Seq expression profiles recapitulated previous bulk RNASeq data validating our sRNA-Seq pipeline. Transcriptomes of index sorted cells mapped appropriately to those predicted to be Thy1,Thy2, or Thy3 cells from CD34, CD7, and CD1a RNA levels in the 10x and indrops data indicating the validity of identifying Thy1-3 cells by RNA levels. Seurat clustering showed that CD34+ thymocytes consist of a continuum of cell states rather than discrete populations. Nine cell states marked by gradual changes in CD2, CD44, CD7, CD1A, and CD34 RNA levels were seen: state 1 (Thy1), states 2-6 (Thy2), and states 7-9 (Thy3). Thy2 and Thy3 cells spanned several previously undescribed states with distinct expression profiles. Single cells co-expressing stem and T-lineage genes were seen in state 1 indicating an early onset of T-lineage priming that occurs prior to downregulation of stem cell genes. Within Thy2 cells, high expression of stem and alternative lineage genes was mostly restricted to states 2 and 3, which represent novel Thy2 subpopulations. State 2 cells co-expressed T-cell and high levels of innate immune genes, particularly IRF8. State 3 cells showed low CD2 expression and co-expressed T-cell and stem, myeloid, B-cell, and/ or NK genes. These results indicate concomitant priming of multiple lineage transcriptional programs rather than just the presence of unilineage gene program expressing subpopulations in the earliest thymic progenitor cells. In preliminary experiments, FACS sorted CD2 low Thy2 cells generated T-cells as well as a higher output of alternative lineage cells than CD2 high Thy2 cells. States 7-9 showed committed profiles with upregulated T-cell genes and silenced stem and alternative lineage genes. While state 9 (late Thy3) showed high RAG1 expression and cell cycle arrest, most Thy3 cells (states 7 and 8) were cycling indicating that commitment, which precedes T-cell receptor rearrangement, is not associated with proliferation arrest. Monocle pseudotime analysis finely resolved expression profiles along the differentiation trajectory within Thy1-3 populations: e.g. genes repressed earlier (e.g. MEIS1) vs later (e.g. DTX1). BCL11B, a key T-cell commitment gene was one of the earliest transcription factors (TF) to be induced during human thymopoiesis, and upregulation of TCF7 and BCL11B occurred concurrently. In contrast, in mice induction of Bcl11b occurs later after Tcf7 is fully upregulated. Thus, the initial stages of thymopoiesis are characterized by a continuum of multilineage transcriptional priming followed by a gradual transition to a T-lineage restricted gene program, a process similar to that recently described for lineage commitment of bone marrow hematopoietic progenitors but not previously reported in the thymus. SRNA-Seq yielded expression profiles along numerous data-points on the differentiation trajectory within heterogenous populations, providing high transcriptional resolution not possible with bulk data. We found differences in key TF expression profiles that could imply species specific regulatory mechanisms. Our data are a resource for elucidating transcriptional networks in T-cell development and T-cell leukemias. Disclosures No relevant conflicts of interest to declare.