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In: Leukemia, Springer Science and Business Media LLC, Vol. 33, No. 7 ( 2019-7), p. 1687-1699

Type of Medium: Online Resource

ISSN: 0887-6924 , 1476-5551

URL: Article

DOI: 10.1038/s41375-019-0380-5

Language: English

Publisher: Springer Science and Business Media LLC

Publication Date: 2019

detail.hit.zdb_id: 2008023-2

In: Blood, American Society of Hematology, Vol. 136, No. Supplement 1 ( 2020-11-5), p. 18-18

Abstract: Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell malignancy, caused by human T-cell leukemia virus type-1 (HTLV-1) infection. To elucidate immune microenvironment and heterogeneity of HTLV-1-infected normal and leukemic cells, we performed multi-omics single cell analysis, evaluating whole-transcriptome, 101 surface marker proteins, and T/B-cell receptor repertoires in the same single cells. We analyzed 236,192 peripheral blood mononuclear cells (PBMCs) from 31 ATL patients (35 samples including 4 sequential samples), 11 HTLV-1-infected carriers, and 4 healthy donors. In our analysis, expression of HTLV-1-related genes, such as HBZ, clearly identified a distinct cluster of HTLV-1-infected cells within non-malignant CD4+ T cells. These cells are characterized by a CD45RO+CD62L-CD7-CCR4+CD25+CD73+ memory/effector phenotype. By contrast, malignant ATL cells were segregated into different clusters across patients, suggestive of inter-tumor heterogeneity. Transcriptome analysis of CD4+ T cells revealed up-regulation of interferon (IFN) responses and down-regulation of TNFa signaling in malignant ATL cells compared with HTLV-1-infected normal CD4+ T cells. Likewise, sequential sample analysis showed that progression from indolent to aggressive disease enhanced IFN responses, suggesting a pivotal role of this pathway in the ATL pathogenesis. Surface marker protein analysis demonstrated that HTLV-1 infection up-regulated the expression of stimulatory and inhibitory immune checkpoint molecules (such as OX40 and TIGIT, respectively), which was further augmented by ATL progression. Within malignant cells, we identified a fraction of cycling cells present across most ATL samples. This fraction showed an enhanced T-cell activation markers, such as CD25 and HLA-DR, and their frequency was increased in aggressive subtypes. On the other hand, in HTLV-1-infected carriers, HTLV-1-infected CD4+ T cells contained a small population of malignant-like cells showing clonal expansion. The degree of clonal expansion was significantly correlated with HTLV-1 viral load in PBMCs. These results clarify the heterogeneity within HTLV-1-infected cells and ATL malignant cells, pointing to its relevance during ATL initiation and progression. We also observed dynamic changes of the immune microenvironment in ATL. Although the relative frequencies of other cell types remained almost the same or reduced, only myeloid cells were increased in ATL patients compared with in HTLV-1-infected carriers. Re-clustering of myeloid cells identified a novel cluster of monocytes expressing FCGR1A, encoding CD64, a biomarker of IFN-stimulated gene levels. Transcriptome analysis revealed increased IFN signaling and decreased TNFa in myeloid cells from ATL patients compared with HTLV-1-infected carriers. Similar expression signatures changes were also observed in various immune cell types, such as B, CD8+ T, and NK cells, in ATL patients. In addition, substantial changes of surface marker proteins were also found in ATL patients. Particularly, T-cell activation markers, such as HLA-DR, and inhibitory immune checkpoint molecules, such as PD-1 and TIM-3, were up-regulated in CD8+ T cells from ATL patients. A co-culture experiment of ATL cell lines with PBMCs from healthy volunteers demonstrated that ATL cells induced immune-phenotypic changes of myeloid and CD8+ T cells, similar to those observed in ATL patient by our single-cell analysis, confirming the role of ATL cells in the modulation of the immune system. Taken together, the composition and function of immune microenvironment is dramatically altered in ATL patients, which may contribute to immunosuppression and disease progression in ATL. In summary, our multi-omics single-cell analysis comprehensively dissects the cellular and molecular architecture in HTLV-1-infected carriers and ATL patients. In particular, our approach clearly defines HTLV-1-infected cells by the expression of HTLV-1-related genes, leading to the detailed characterization of HTLV-1-infected cells and elucidation of their difference from ATL malignant cells. These findings will help to devise novel diagnostic and therapeutic strategies for HTLV-1-related disorders. Disclosures Kogure: Takeda Pharmaceutical Company Limited.: Honoraria. Shimoda:Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company: Honoraria; Novartis: Honoraria, Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Shire plc: Honoraria; Celgene: Honoraria; Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding. Kataoka:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Genomics: Current equity holder in private company.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2020-141755

Language: English

Publisher: American Society of Hematology

Publication Date: 2020

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 140, No. Supplement 1 ( 2022-11-15), p. 1737-1739

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2022-159869

Language: English

Publisher: American Society of Hematology

Publication Date: 2022

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 138, No. Supplement 1 ( 2021-11-05), p. 714-714

Abstract: PD-L2 is a ligand for PD-1 immune checkpoint. In contrast to another ligand PD-L1, little is known about the biological relevance and regulatory mechanism of PD-L2 in cancer. Here we found by pan-cancer transcriptome analysis that PD-L2 was highly expressed in limited cancer types, especially in diffuse large B-cell lymphoma (DLBCL). In particular, PD-L2 expression was elevated in patients with PD-L2 genetic alterations, such as copy number amplifications and rearrangements causing promoter replacement or 3′-untranslated region (UTR) disruption. To evaluate the effect of PD-L1 and PD-L2 on the tumor microenvironment and clarify their similarities and differences at a similar expression level, we generated a A20-ovalbumin (OVA) murine B-cell lymphoma cell line lacking Pd-l1 and introduced exogenous Pd-l1 or Pd-l2 expression. Analysis of A20-OVA model showed that Pd-l2 expression accelerated tumor growth and attenuated CD8 + T cell infiltration in vivo, similar to Pd-l1 expression. Then, we performed multi-omics single-cell analysis in this model, constructing transcriptomic, surface phenotypic, and immune repertoire maps of & gt; 20,000 cells from mock-, Pd-l1-, and Pd-l2-expressing A20-OVA tumors. Importantly, Pd-l1- and Pd-l2-expressing tumors exhibited similar cellular dynamics as well as transcriptomic and surface phenotypic changes in the tumor microenvironment. Specifically, a significant decrease of CD8 + T cells, particularly effector/memory cells showing high clonality, and regulatory T cells as well as a significant increase of myeloid-derived cells, including monocytes/macrophages and plasmacytoid dendritic cells (DCs), were observed in Pd-l1- and Pd-l2-expressing tumors. Differentially expressed gene analysis demonstrated the downregulation of response to bacterial molecules, including lipopolysaccharide, and antigen processing and presentation pathways in monocytes/macrophages and conventional and plasmacytoid DCs, respectively, in Pd-l1- and Pd-l2-expressing tumors. In line with this, pro-inflammatory cytokine‒inducible markers, such as Ly6A/E and I-A/I-E, were down-regulated in various cell types in Pd-l1- and Pd-l2-expressing tumors. These results suggest that delineates pleiotropic effects shared by PD-L1 and PD-L2, mainly enhancing anti-inflammatory, pro-tumorigenic responses in the tumor microenvironment. Given similar functions of PD-L1 and PD-L2, we hypothesized that the expression level of PD-1 ligands determines their biological relevance. Therefore, we aimed to dissect PD-L2 regulatory landscape by performing CRISPR tiling screening targeting 51 candidate regulatory elements predicted from Hi-C and DNase-seq data of a human transformed B cell line (GM12878). In addition to known cis-regulatory elements including the canonical transcription start site (TSS) and 3′-UTR, we identified a novel TSS, which was validated by cap analysis of gene expression (CAGE) with sequencing (CAGE-seq). Pan-cancer and -tissue expression analyses revealed that this novel element was expressed in 13% of DLBCL, but not in normal tissues nor other cancer types, suggestive of a unique PD-L2 regulatory mechanism in DLBCL. In addition, we identified an element located in the PD-L1 promoter which function as a distal silencer, suggesting functional complexity of this regulatory element. CRISPR-mediated knockout of other PD-L1 exons did not affect PD-L2 expression, suggesting that a silencer function in the PD-L1 promoter is independent of PD-L1 expression. The identified PD-L2 regulatory elements can be occupied by an array of trans-regulatory factors. Indeed, ENCODE ChIP-seq of GM12878 revealed that many chromatin-associated proteins (CAPs) were bound within the PD-L1/PD-L2 topology associating domain. Therefore, to determine key regulators, we performed loss-of-function CRISPR screening for 103 CAPs. This CRISPR screening identified seven negative (such as IRF4 and BATF) and two positive regulators of PD-L2 expression. CRISPR/Cas9-based inhibition exhibited differential usage of canonical and novel TSSs among these factors. Taken together, our findings reveal lineage-specific complex network of cis-regulatory elements and CAPs in regulating PD-L2 expression. These data provide insights into the molecular mechanisms underlying immune evasion and help refining immune-based therapeutic strategy in DLBCL. Disclosures Koya: 10x Genomics: Honoraria. Kogure: Takeda Pharmaceutical: Honoraria. Kataoka: Bristol-Myers Squibb: Research Funding; Japan Blood Products Organization: Research Funding; Teijin Pharma: Research Funding; Shionogi: Research Funding; Asahi Genomics: Current holder of individual stocks in a privately-held company; Otsuka Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Janssen Pharmaceutical: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; AstraZeneca: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Celgene: Honoraria; Ono Pharmaceutical: Honoraria, Research Funding; Mochida Pharmaceutical: Research Funding; JCR Pharmaceuticals: Research Funding; MSD: Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2021-150101

Language: English

Publisher: American Society of Hematology

Publication Date: 2021

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Annals of Hematology, Springer Science and Business Media LLC, Vol. 94, No. 6 ( 2015-6), p. 989-994

Type of Medium: Online Resource

ISSN: 0939-5555 , 1432-0584

URL: Article

DOI: 10.1007/s00277-015-2309-z

Language: English

Publisher: Springer Science and Business Media LLC

Publication Date: 2015

detail.hit.zdb_id: 1458429-3

In: Bioscience, Biotechnology, and Biochemistry, Informa UK Limited, Vol. 62, No. 4 ( 1998-01), p. 727-734

Type of Medium: Online Resource

ISSN: 0916-8451 , 1347-6947

URL: Article

DOI: 10.1271/bbb.62.727

Language: English

Publisher: Informa UK Limited

Publication Date: 1998

detail.hit.zdb_id: 2110940-0

SSG: 12

In: Blood, American Society of Hematology, Vol. 136, No. 6 ( 2020-08-6), p. 684-697

Abstract: The linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-κB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B-cell–like (ABC) diffuse large B-cell lymphoma (DLBCL), and expression of HOIP, which parallels LUBAC activity, is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-κB activation, and analysis of the human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-κB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-κB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicate that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage–induced cell death and is a suitable therapeutic target for B-cell lymphomas.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.2019002654

Language: English

Publisher: American Society of Hematology

Publication Date: 2020

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 136, No. Supplement 1 ( 2020-11-5), p. 29-30

Abstract: Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy with a dismal prognosis, caused by HTLV-1. Although our previous study, mainly using whole-exome sequencing and SNP array karyotyping, discovered many driver mutations and copy number alterations (CNAs), the whole-genome landscape of ATL still remains elusive. To this end, we have performed high-depth whole-genome sequencing (WGS) of 155 ATL cases with a median sequencing depth of 96-fold for tumors. Among them, 75 cases were also analyzed by RNA sequencing (RNA-seq). In total, we detected 1,952,490 single nucleotide variants (SNVs) and 159,141 insertion-deletions (4.0 SNVs and 0.3 indels/Mb/case), 10,279 SVs (66.3 SVs/case), and 3,975 CN altered segments (25.7 segments/case). Using several driver discovery algorithms (dNdScv, MutSig2CV, and DriverPower), we identified 47 significantly mutated genes, 19 of which were mutated in more than 10% of cases. These included several novel mutations, such as those affecting XPO1 (7.1%), ZNF292 (6.5%), and ITGB1 (5.2%). Using GISTIC2.0, we identified 13 significant CNAs, such as IRF4 amplifications and CDKN2A deletions, consistent with previous SNP array data. To detect significantly recurrent SVs, we calculated SV breakpoint frequency and identified 13 genes affected by SVs, including the previously identified genes (such as CARD11, CD274, and TP73). In addition, we investigated recurrent mutations in non-coding elements by DriverPower and LARVA and discovered 12 recurrently mutated elements. Among them, the most frequent were splice site mutations, including those of HLA-A and HLA-B, most of which caused loss of function as revealed by RNA-seq. By contrast, we found recurrent mutations in TP73 splice site, which induced skipping of exons 2 and 3, generating a dominant-negative variant similar to their SVs. In addition, recurrent non-coding elements contained several novel regions, such as 3´-untranslated region (UTR) of NFKBIZ and 5´- UTR of TMSB4X. Altogether, a total of 56 genes were recurrently altered. The median number of driver alterations was eight per case, and at least one driver alteration was found in 149 cases (96.1%). Among 56 driver genes, 40 (71.4%) genes were affected by more than one alteration class. Some drivers, such as CDKN2A, IKZF2, and CD274, were affected almost exclusively by CNAs and/or SVs, while showing quite high alteration frequencies (11.6-29.0%). These observations suggest that WGS presented a substantially different overview of driver alterations from our previous study. The overall numbers of mutations and SVs were linked to these driver alterations, suggesting their etiology. In particular, inactivation of EP300 and immune-related molecules, such as HLA-A, HLA-B, and CD58, were associated with an increased number of mutations and SVs, especially deletions and tandem duplications. By contrast, cases with TP53-altered cases harbored more inversions and translocations. These results emphasize a pivotal role of immune evasion for acquiring genetic alterations to drive ATL progression. To define molecular subgroups in ATL, we integrated the 56 identified genetic drivers using non-negative matrix factorization clustering and identified two robust subgroups with discrete clinical and genetic characteristics. Group 1 was enriched with alterations affecting distal components of T-cell receptor (TCR)/NF-κB signaling (such as CARD11, PRKCB, and IRF4) and immune-related molecules (HLA-A, HLA-B, and CD58), whereas proximal regulators of TCR/NF-κB signaling (PLCG1, VAV1, and CD28) and a JAK/STAT signaling molecule (STAT3) were more frequently altered in group 2. In addition, group 1 cases had a larger number of mutations, SVs, and CNAs than group 2 cases. Clinically, most cases with lymphoma subtype were classified into group 1, whereas group 2 mainly consisted of cases with leukemic subtypes. Moreover, group1 cases showed a worse overall survival than group 2, independently of clinical subtype. These results suggest the biological and clinical relevance of the molecular classification of ATL. In summary, our WGS analysis not only identifies novel somatic alterations but also extends the overview of ATL genome. We also propose a new molecular classification of ATL, with its clinical relevance, which can lead to the future improvement of patient management. Disclosures Kogure: Takeda Pharmaceutical Company Limited.: Honoraria. Nosaka:Kyowa Kirin Co.Ltd: Honoraria; Chugai pharmaceutical Co. Ltd: Honoraria; Novartis international AG: Honoraria; Celgene K.K: Honoraria; Eisai Co., Ltd: Honoraria; Merck Sharp & Dohme K.K.: Honoraria; Bristol-Myer Squibb: Honoraria. Imaizumi:Kyowa Kirin Co. Ltd.: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Eisai: Honoraria. Utsunomiya:Kyowa Kirin: Honoraria; Celgene: Honoraria. Shah:Celgene: Research Funding; BMS: Research Funding; Physicians Education Resource: Honoraria. Janakiram:Takeda, Fate, Nektar: Research Funding. Ramos:NIH: Research Funding. Takaori-Kondo:Astellas Pharma: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; Eisai: Research Funding; Nippon Shinyaku: Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; MSD: Honoraria. Miyazaki:Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Celgene: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Otsuka Pharmaceutical: Honoraria; Novartis Pharma KK: Honoraria; Astellas Pharma Inc.: Honoraria. Matsuoka:Chugai Pharmaceutical Co. Ltd: Research Funding; Bristol-Myers Squibb: Research Funding; Kyowa Kirin Co. Ltd.: Research Funding. Ishitsuka:Takeda: Other: Personal fees, Research Funding; mundiharma: Other: Personal fees; Taiho Pharmaceuticals: Other: Personal fees, Research Funding; Janssen Pharmaceuticals: Other: Personal fees; Novartis: Other: Personal fees; Pfizer: Other: Personal fees; Astellas Pharma: Other, Research Funding; Genzyme: Other; Sumitomo Dainippon Pharma: Other, Research Funding; Eisai: Other, Research Funding; Mochida: Other, Research Funding; Shire: Other; Otsuka Pharmaceutical: Other; Ono Pharmaceutical: Other, Research Funding; Teijin Pharma: Research Funding; MSD: Research Funding; Asahi kasei: Research Funding; Eli Lilly: Research Funding; Daiichi Sankyo: Other; Huya Japan: Other; Celgene: Other: Personal Fees; Kyowa Hakko Kirin: Other: Personal fees, Research Funding; BMS: Other: Personal fees; Chugai Pharmaceutical: Other: Personal fees, Research Funding. Ogawa:Asahi Genomics Co., Ltd.: Current equity holder in private company; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding. Shimoda:Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria; Shire plc: Honoraria; Celgene: Honoraria; Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Novartis: Honoraria, Research Funding. Kataoka:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Genomics: Current equity holder in private company.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2020-134360

Language: English

Publisher: American Society of Hematology

Publication Date: 2020

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detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 140, No. Supplement 1 ( 2022-11-15), p. 3034-3035

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2022-163203

Language: English

Publisher: American Society of Hematology

Publication Date: 2022

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 130, No. Suppl_1 ( 2017-12-07), p. 730-730

Abstract: Programmed cell death ligand 1 (PD-L1) plays a key role in tumor immune escape by negatively regulating cytotoxic T-cells (CTLs) via PD-1 receptors. Thus, those tumors with high PD-L1 expression are thought to be sensitive to PD-1/PD-L1 blockade, reactivating CTL reactions to tumors. However, the mechanism that regulates PD-L1 expression in tumor cells has not been fully elucidated, understanding of which would help to develop effective anti-tumor immunotherapy. Recently, we reported that disruption of 3'-UTR of PD-L1 led to a remarkably enhanced expression of PD-L1 in a wide variety of human cancers, particularly adult T-cell leukemia/lymphoma (ATL) and diffuse large B-cell lymphoma. In these cancers, stability of PD-L1 transcripts is negatively regulated via their 3'-UTR sequence, whose disruption thus, results in markedly elevated PD-L1 expression and immune evasion (Kataoka et al., Nature, 2016). It has been well established that non-coding regions (i.e., 5'- and 3'-untranslated regions (UTRs)) play important roles in the regulation of mRNA expression, which is accomplished by several transacting factors that bind to cis-regulatory elements within the UTR. Based on this knowledge, we investigated the mechanism that controls PD-L1 expression through 3'-UTR sequence, primarily focusing on those transacting RNA-binding proteins (RNA-BPs). To determine the relevant regions within the 3'-UTR which are capable of repressing PD-L1, several cell lines were transfected with luciferase reporter vectors, in which a luciferase coding sequence was concatenated to various deletion mutants of the PD-L1 3'-UTR (Panel A). We identified two critical sequences, segment 5 and 2 within the PD-L1 3'-UTR, whose existence significantly decreased luciferase expression (Panel B; 293T data, mean ± SD, * denotes t-test p & lt; .05). Importantly, the deletion of these sequences showed a similar effect on gene expression in various cell lines derived from different tissues. To confirm this finding, we used a CRISPR-mediated tiled 3'-UTR editing in situ technique. We designed all possible single-guide (sg) RNAs in the PD-L1 3'-UTR. The library was virally infected into cells and those with high expression of PD-L1 were concentrated by FACS, and the enrichment of each sgRNA was evaluated by high-throughput sequencer. The enriched sgRNAs localized to positions compatible with the luciferase assay, confirming that the two regions are actually responsible for the regulation of PD-L1 expression. Next, we searched RNA-BPs that bind to these regions in mass spectrometry, utilizing flag-peptide-tagged RNA pull-down method (Panel C). In brief, we synthesized PD-L1 3'-UTR RNA segments along with β-actin mRNA as control in vitro, and conjugated a flag-peptide to their 3'-ends. Using these RNA-baits, an immunoprecipitation experiment was performed in 293T cells and the co-immunoprecipitated proteins were analyzed by mass spectrometry. The data obtained from different segments were compared to each other. In addition to those proteins binding to multiple regions within the PD-L1 3'-UTR, we found proteins that specifically interacted with the repressive segments (segment 5 and 2) identified through luciferase assays. Finally, to confirm that the proteins detected by mass spectrometry actually suppress PD-L1 expression, we performed knock-down experiments using siRNA designed for the RNA-BPs that are presumed to interact with the segment 5 and 2. Three siRNA constructs per gene were transfected to 293T cells and their effect on PD-L1 expression was evaluated by RQ-PCR. Panel D shows relative PD-L1 expressions for each siRNA targets with median line, which are grouped according to relevant PD-L1 3'-UTR segments. The negative regulatory effect of these RNA-PBs on PD-L1 expression was largely confirmed. PD-L1 protein level was also increased, when these genes were knocked out by CRISPR/Cas9 system. Expression of these genes in ATL and other lymphomas was also evaluated. In summary, we identified critical sequences within the PD-L1 3'-UTR and RNA-BPs that bind to these sequences and negatively regulate PD-L1 expression. Our findings should not only provide novel insight into the molecular mechanisms by which PD-L1 expression in tumor cell is regulated but also help to identify potential targets for immune therapy. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V130.Suppl_1.730.730

Language: English

Publisher: American Society of Hematology

Publication Date: 2017

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detail.hit.zdb_id: 80069-7