Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells

Adult T-cell leukemia/lymphoma (ATLL) originates from human T-cell leukemia virus type 1 (HTLV-1) infection due to the activation of the nuclear factor-κB (NF-κB) signaling pathway to maintain proliferation and survival. An important mechanism of the activated NF-κB signaling pathway in ATLL is the activation of the macroautophagy (herafter referred to as autophagy in the remainder of this manuscript)-lysosomal degradation of p47 (NSFL1C), a negative regulator of the NF-κB pathway. Therefore, we considered the use of chloroquine (CQ) or hydroxychloroquine (HCQ) (CQ/HCQ) as an autophagy inhibitor to treat ATLL; these drugs were originally approved by the FDA as antimalarial drugs and have recently been used to treat autoimmune diseases, such as systemic lupus erythematosus (SLE). In this paper, we determined the therapeutic efficacy of CQ/HCQ, as NF-κB inhibitors, in ATLL mediated by blockade of p47 degradation. Administration of CQ/HCQ to ATLL cell lines and primary ATLL cells induced cell growth inhibition in a dose-dependent manner, and the majority of cells underwent apoptosis after CQ administration. As to the molecular mechanism, autophagy was inhibited in CQ-treated ATLL cells, and activation of the NF-κB pathway was suppressed with the restoration of the p47 level. When the antitumor effect of CQ/HCQ was examined using immunodeficient mice transplanted with ATLL cell lines, CQ/HCQ significantly suppressed tumor growth and improved the survival rate in the ATLL xenograft mouse model. Importantly, HCQ selectively induced ATLL cell death in the ATLL xenograft mouse model at the dose used to treat SLE. Taken together, our results suggest that the inhibition of autophagy by CQ/HCQ may become a novel and effective strategy for the treatment of ATLL.

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation.

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify novel Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation.Author SummaryInfection with the retrovirus HTLV-1 leads to the development of either CD4+CD25+ leukemia/lymphoma (ATLL) or a demyelinating neuroinflammatory disease (HAM/TSP) in a subset of infected individuals. The HTLV-1 Tax protein is a regulatory protein which regulates viral gene expression and persistently activates cellular signaling pathways such as NF-κB to drive the clonal expansion and longevity of HTLV-1 infected CD4+ T cells. Polyubiquitination of Tax is a key mechanism of NF-κB activation by assembling and activating IκB kinase (IKK) signaling complexes; however, the host factors regulating Tax ubiquitination have remained elusive. Here, we have identified the E3/E4 ubiquitin conjugation factor UBE4B as a novel Tax binding protein that promotes both K48- and K63-linked polyubiquitination of Tax. Knockdown or knockout of UBE4B impairs Tax-induced NF-κB activation and triggers apoptosis of HTLV-1 transformed cells. Therefore, UBE4B is an integral host factor that supports HTLV-1 Tax polyubiquitination, NF-κB activation and cell survival.

Genome-Wide CRISPR Library Screening Identifies CDK6 As Genetic Vulnerability in Adult T-Cell Leukemia/Lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive hematological malignancy associated with the retrovirus human T-cell lymphotropic virus type I. Clinical outcomes of currently available chemotherapies are generally inferior with extremely poor prognosis. Previous studies have recently utilized next generation sequencing technology for the identification of mutated genes that may be pivotal in the pathogenesis of ATLL. However, the identification of indispensable genes for the proliferation and/or survival of ATLL cells remains a formidable challenge due to the complexity of genomic/epigenetic alterations in the ATLL genome. To investigate previously undescribed therapeutic targets in ATLL, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening to identify genetic vulnerabilities in ATLL cells. Three ATLL cell lines were transduced with lentiviral construct for Cas9 nuclease, followed by lentiviral delivery of the human CRISPR Brunello pooled library (Addgene 73178) of 76,456 single-guide RNAs targeting 19,144 protein-coding genes to cause DNA double-stranded cleavage by the Cas9 nuclease and loss-of-function of the respective genes. Compared with the control cell lines, 23 essential genes, including BATF3 (which we previously discovered by shRNA library screening; Nakagawa et al., Cancer Cell. 34:286-297. 2018) and novel genes (CDK6, JUNB, STAT3, and CCND2) were identified to be involved in ATLL cell proliferation and/or survival. Among these, CDK6 (cyclin-dependent kinase 6), a critical regulatory serine/threonine kinase that forms heterodimers with D-type cyclins, had the best score. The CDK6/D-type cyclin complex regulates E2F transcription factors through the phosphorylation of Rb (retinoblastoma protein), resulting in G1-S transition of the cell cycle. Utilizing publicly available microarray data from peripheral T-cell lymphoma patients, we demonstrated the higher expression of CDK6 in ATLL than that of the other subtypes of T-cell lymphomas, which prompted us to focus on CDK6 as a therapeutic molecular target in ATLL. In confirmatory experiments, two sgRNAs targeting the coding sequences of CDK6 exhibited strong toxicity in five ATLL cell lines in a temporal fashion, which was mediated by G1 cell arrest and partially through apoptosis. We confirmed on-target effect of the sgCDK6 by successfully rescuing cells from toxicity using retroviruses expressing sgRNA-resistant CDK6 cDNA in two ATLL cell lines. The knockout of CDK6 and decrease in the level of phosphorylated Rb were confirmed by immunoblot of sgCDK6-transduced ATLL cell lines. Collectively, the data showed an essential role for CDK6 in cellular proliferation and survival in ATLL. Of the 19,144 genes examined, CDK6 was considered the best vulnerable target for ATLL; therefore, we extended our analysis to evaluate the pharmacological inhibition of CDK6 in ATLL cells. Palbociclib, FDA-approved CDK4/6 inhibitor for breast cancer, was toxic in 11 ATLL cell lines and in four primary ATLL cells but the range of IC50 values were relatively broad (9-6500 nM) among ATLL lines. Because aproximately 20% of ATLL patients carry genetic alteration in a cell cycle/apoptosis regulator TP53 gene, we hypothesized that TP53 alteration may affect the sensitivity of ATLL cells to palbociclib. First, we assessed TP53 status of ATLL cell lines by Sanger sequencing and immunoblotting and showed that six TP53-altered ATLL cell lines exhibited significantly higher IC50 for palbociclib compared with five TP53-intact ATLL cell lines (p<0.05). Notably, CRISPR-mediated TP53 knockout in a TP53-intact ATLL cell line, which exhibited increased resistance to palbociclib, supported the notion that TP53 is a potential molecular biomarker for palbociclib in ATLL. As a further confirmation of our in vitro findings, treatment of a TP53-intact ATLL cell line-xenograft mouse model with palbociclib showed significant inhibition of tumor growth without systemic toxicity. Through the findings of this study, we have provided evidence that CDK6 can serve as a novel therapeutic target for ATLL. We proposes that CDK6 inhibition with CDK4/6 inhibitor palbociclib, together with the biomarker anaylsis including TP53 status, is worth to be evaluated as a therapeutic strategy for ATLL in future clinical study. Disclosures Teshima: Novartis: Honoraria, Research Funding. Waldmann:Bioniz: Membership on an entity's Board of Directors or advisory committees. Staudt:Nanostring: Patents & Royalties. Nakagawa:akeda Pharmaceutical Company Limited: Research Funding.

S-Phase Progression of North American ATLL Cells Is Critically Regulated By the Proto-Oncogene BCL6 and Targetable By PARP Inhibition

Adult T-cell leukemia/lymphoma (ATLL) is a disease of malignant T cells that develops in human T-lymphotropic virus-1 (HTLV-1) carriers. ATLL is one of the most aggressive form of NHL with an abysmal survival and no promising targeted therapies. We and others have shown that ATLLs diagnosed in the Japanese (J-ATLL) and North American (NA-ATLL) patients have very different clinical behavior, with the NA-ATLL variant showing a more aggressive clinical course and carrying more frequent epigenetic mutations compared to J-ATLLs. The most frequently mutated epigenetic modifier gene in NA-ATLL is EP300 which is mutated in 20% of our patients compared to only 5.7% of J-ATLLs. EP300 encodes p300, a lysine acetyltransferase with a multitude of protein substrates including histone H3 and a number of important transcription factors, among which are p53 and BCL6. Previously, we have demonstrated that p300 mutations correlate with reduced p53 levels and compromised p53 transcriptional activity. In this study, we discovered for the first time that the B cell lymphoma oncogene BCL6 is expressed in primary ATLL samples and in vitro cultured ATLL cells. As expected, p300 mutation corresponds to reduced BCL6 acetylation. More importantly, notable differences in BCL6-regulated transcriptome were uncovered between EP300 WT and mutated samples based on RNA-seq analysis. Functional importance of BCL6 was then investigated using the siRNA-based knock-down approach in combination with a small compound BCL6 inhibitor, FX1. Unexpectedly, we discovered that nearly all cultured NA-ATLL samples are very sensitive to BCL6 inactivation (FX1 IC50 = 36+/-14 uM), while the 6 J-ATLL cell lines tested showed a wide range of FX1 sensitivity. This disparity is also seen in the pattern of BCL6-regulated genes. Specifically, based on clustering analysis, BCL6-regulated Reactome Pathways are more closely related among 4 NA-ATLL cell lines relative to those among 4 J-ATLL cell lines. Finally, we demonstrate that continued expression of BCL6 is critical for the survival of NA-ATLL cells going through the S-phase of cell cycle since the S-phase fraction was selectively depleted following BCL6 inactivation by either BCL6 siRNA transfection or FX1 treatment. Prompted by the recognition that the S-phase could be an "achilles heel" of NA-ATLL cells, we tested the sensitivity of a number of NA-ATLL samples (both primary and cell lines) and J-ATLL cell lines to 3 S-phase directed drugs: the PARP inhibitor, Olaparib, the Wee1 inhibitor, AZD-1775, and the ATR inhibitor, AZD-6738. While virtually all ATLL samples tested showed sensitivity to the Wee1 (IC50 = 0.2 to 1.1 uM) and ATR (IC50 = 1.0 to 14.5 uM) inhibitors, Olaparib sensitivity demonstrated ethnic and genotype specificity. Specifically, except the one and only EP300-mutated sample, 5/6 J-ATLL cell lines are resistant to Olaparib. In comparison, among the 10 NA-ATLL samples tested, 6/10 are Olaparib sensitive (IC50 =20-40 uM). The 4 Olaparib resistant samples carried inactivating mutations in either TP53 or RICTOR/PIK3CD. In summary, this study provides additional insights into the functional consequences of EP300 mutations in NA-ATLL. For the first time, we demonstrate that BCL6 is expressed in NA-ATLL and is critically required for survival of these cells when they transit through the S-phase of cell cycle. Furthermore, our discovery that NA-ATLLs are often sensitive to Olaparib treatment in vitro warrants follow up investigation that could lead to a novel targeted therapeutic strategy for this devastating malignancy. Disclosures Verma: Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding. Sica:Physician's Education Resources (PER): Honoraria.

Ectonucleosidase CD39 Is Highly Expressed on ATLL Cells and Suppresses the Immune Response through the Adenosine Pathway

Background: Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm, linked to the human T-cell lymphotropic virus, HTLV-1. Patients with ATLL are often at the risk of opportunistic infections. Some studies suggested that ATLL cells originate from HTLV-1-infected regulatory T cells (Tregs). It could be possible that this immunocompromised state is caused by the function of ATLL cells having similar phenotypes with Tregs. In this study, we examined the expression of immunosuppressive molecules associated with Tregs in ATLL cells, and analyzed their roles in the function of ATLL cells. Methods: The protocol of this study was approved by the Investigational Review Board of Osaka University Hospital. Peripheral blood mononuclear cells (PBMCs) were collected from 10 asymptomatic HTLV-1 carriers and 22 ATLL patients (1 with smoldering type, 5 with chronic type, 2 with lymphoma type, and 14 with acute type) after getting informed consent. PBMCs from 3 ATLL patients were separated into CD4+ CD7- CADM1+ATLL cells and adjacent CD4+CD7+ CADM1-normal T cells using Fluorescence-activated Cell Sorter (FACS), and cells in each fraction were subjected to total RNA sequencing experiments. Based on the results, we examined the expression patterns of CD39 and CD73 in HTLV-1 carriers or each type of ATLL patients, and also analyzed the immune functions of these molecules in ATLL tumor cells. Results: We compared whole transcriptome of ATLL cells and normal CD4+cells. Bioinformatic analyses showed that many genes associated with immunosuppressive functions were elevated or downregulated in ATLL cells. Among these genes we focused on CD39, CD73 and CD26, because they have recently been reported to be strongly associated with the functions of Tregs. CD39, expressed on normal Tregs, and extrinsic CD73 have immunosuppressive potential by catalyzing adenosine from extracellular ATP, and CD26 has opposite potential by resolving adenosine, which have a strong anti-inflammatory function and plays major role in Treg-mediated immunosuppression. We found that all of 4 ATLL cell lines (MJ, MT1, MT2, MT4) expressed CD39, but not CD73 just as human effector Tregs. Tumor cells from 12 acute ATLL patients (86%) and 2 chronic ATLL patients (40%) expressed CD39, but the expressions of CD73 were various. Also in asymptomatic carriers, we could detect CD39 and/or CD73 positive in CD7- CADM1+ abnormal fraction of CD4+cells. On the other hand, CD26, normally expressed on human CD4+Th cells other than effector Tregs, was negative in ATLL cell lines and primary ATLL cells except for cells in abnormal fraction of one asymptomatic carrier. CD39 negative cases in chronic/smoldering type tended to show slower disease progression after the blood collection. Next, the role of CD39 and/or CD73 in ATLL cells was assessed in vitro and in vivo. As expected, CD39+ ATLL cells converted significantly more extracellular ATP than CD39- ATLL cells, and mass spectrometry analysis of AMP/adenosine concentration identified the AMPase activity of CD73+ ATLL cells. Furthermore, we established CD39 knockout (KO) cells from ATL cell-line MJ using CRISPR/Cas9 system, and performed in vitro suppression assays for assessment of immunosuppressive function. Although wild type MJ suppressed the growth of normal CD4+ and CD8+ T cells, KO MJ did little. Next, we analyzed the role of CD39 in the progression of tumor cells in vivo. We transplanted mouse T-cell lymphoma cell-line EG7-OVA artificially expressing CD39 or mock into mice subcutaneously. The coinjection of immunoadjuvant poly(I:C) significantly suppressed the tumor growth of mock cells, but the tumor sizes of CD39 expressing cells were almost the same as those of mock cells without poly(I:C) injection (Figure). Conclusion: In this study, we reported that most of ATLL cells in acute type patients express CD39+ CD26- just as Tregs, and that CD39- KO of ATLL cell line cancelled its immunosuppressive effects, and forcibly expressed CD39 on tumor cells rejected the anti-tumor immunity in vivo. From these data, we clarified the pathological mechanism of immunosuppressive function in ATLL cells, and also showed that CD39 expression could be used as a prognostic clue and be a new therapeutic target of ATLL. Disclosures Ezoe: TAIHO Phamaceutical Co., Ltd.: Research Funding. Yokota:Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding. Ichii:Novartis Pharma K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Celgene K.K.: Speakers Bureau. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Targeting the HTLV-I-Regulated BATF3/IRF4 Transcriptional Network in Adult T-Cell Leukemia/Lymphoma

After neonatal HTLV-I infection through breast feeding, approximately 5% of HTLV-I carriers eventually develop Adult T-Cell Leukemia/Lymphoma (ATLL) with a latency of ~50 years, suggesting that acquired genetic and epigenetic changes in cellular genes act in concert with HTLV-I to initiate and maintain oncogenic transformation. We and others have recently utilized next generation sequencing technology to identify mutated genes that could be pivotal in the pathogenesis of ATLL. However, due to the complexity of genomic/epigenetic alteration in the ATLL genome, the identification of indispensable genes for proliferation and/or survival of ATLL cells remains a formidable challenge. To discover essential regulatory networks that are required for the proliferation and survival of ATLL cells, we performed a pooled shRNA screen in 8 ATLL cell lines using a library enriched for shRNAs targeting lymphoid regulatory factors and discovered that two BATF3 shRNAs and one IRF4 shRNA were highly toxic for all ATLL lines, but had little if any effect in other T cell and B cell lines. It is recently shown that a transcriptional complex of Irf4 and Batf binds to AP1-IRF composite (AICE) DNA motifs and plays key roles in the differentiation and function of certain mouse helper T cell subsets. A close paralogue of Batf, Batf3, is an indispensable transcription factor in a mouse dendritic cell subset, but also appears to play a redundant role with Batf in the differentiation of TH2 cells and can substitute for Batf in Batf knockout T cells. Our observations from shRNA screening suggested that IRF4 and BATF3 may cooperate to drive a transcriptional program that is essential for ATLL viability. We next used genome-wide chromatin precipitation (ChIP-seq) to identify the loci that are bound by BATF3 and IRF4. The set of binding peaks and the associated genes in IRF4 and BATF3 ChIP-seq intersected significantly. By integrating the ChIP-seq and gene expression profiling data of shBATF3- and shIRF4-ATLL cells, we defined a set of 68 BATF3-IRF4 direct target genes. Gene set enrichment analysis using gene expression profiling data from primary T cell lymphomas demonstrated that BATF3-IRF4 direct target genes were significantly enriched among genes that are more highly expressed in ATLL than in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), suggesting that the BATF3 and IRF4 cooperatively regulate transcription in primary ATLL cells. HBZ is unique among HTLV-I viral proteins in being maintained in expression in all ATLL cases, suggesting that it may help maintain the malignant phenotype. Given that BATF3 and IRF4 are essential regulators in ATLL, we hypothesized possible relationship between HBZ and BATF3-IRF4 complex. We defined HBZ direct target genes by integrating the ChIP-seq and gene expression profiling data of HBZ-knockout ATLL cell lines by CRISPR/Cas9. Notably we discovered that BATF3 was among these. BATF3 mRNA and protein expression decreased following HBZ inactivation. The above considerations suggested that pharmacologic inhibition of the BATF3-IRF4 regulatory network might be a means to attack the HBZ oncogenic program therapeutically. ChIP-seq analysis of two enhancer marks, H3K27ac and BRD4, identified super-enhancers at the BATF3 locus in two ATLL cell lines. The small molecule JQ1 prevents the BET-protein BRD4 from interacting with chromatin, which is required for the function of super-enhancers. JQ1 treatment reduced BATF3 mRNA and protein levels in all ATLL lines tested, correlating with the eviction of BRD4 from the BATF3 super-enhancer. MYC mRNA and protein expression was also broadly downmodulated by JQ1. JQ1 treatment was consistently toxic for all ATLL cell lines tested at dose ranges that killed cell line models of T-ALL and DLBCL, which are known to rely on BET-proteins. In a dose-dependent manner, JQ1 also reduced the viability of primary ATLL samples and downregulated their expression BATF3 and MYC mRNA. Finally, we treated mouse xenograft models of ATLL with the BET-protein inhibitor CPI-203, a JQ1 analog with superior bioavailability in mice. In two different xenograft models, we observed significant tumor regression or growth inhibition, without evidence of systemic toxicity. Our study demonstrates that the HTLV-I virus exploits a regulatory module that can potentially be attacked therapeutically with BET protein inhibitors. Disclosures Yu: Celgene Corporation: Employment.

Adult T-Cell Leukemia/Lymphoma Cells Were Eliminated By T Cells With Anti-CD38 Chimeric Antigen Receptor Through CD38 Expression Induced By All-Trans Retinoic Acid

Patients with adult T-cell leukemia and lymphoma (ATLL) often succumb to death even though multi-anti-cancer drugs are used. Thus, it is essential for establishing a novel therapeutic strategy for ATLL. We have previously developed a chimeric antigen receptor against CD38 (anti-CD38-CAR) and showed powerful cytotoxicity of anti-CD38-CAR to B-cell lymphoma cells as well as to myeloma cells expressing CD38. Unfortunately, as CD38 is poorly expressed on the cell surface of ATLL cells, it is required to induce CD38 to apply our anti-CD38-CAR. Here, we investigated cytotoxicity of T cells transduced with anti-CD38-CAR against ATLL cell lines and cells obtained from ATLL patients through CD38 induction by all-trans retinoic acid (ATRA), which is clinically available for acute promyelocytic leukemia. We evaluated an effect of ATRA on cytotoxicity of T cells bearing anti-CD38-CAR against ATLL cells through flow cytometry. We firstly confirmed the expression of anti-CD38-CAR on human T cells retrovirally transduced (10-70%). Then, secondly, we prepared ATLL cell lines (MT-2, MT-4, S1T, Hut102, and Su9T: >95%, <5%, <5%, 15%, and <5% at CD38 expression, respectively). We co-incubated CD38-specific T cells with ATLL cell line cells for 3 days. MT-2 cells were entirely abrogated by T cells harboring anti-CD38-CAR. However, others were restrictedly succumbed to death after 3-day co-culture with T cells carrying anti-CD38-CAR. Next, we investigated whether ATRA could enhance CD38 expression on the cell surface of ATLL cell lines and exert a cytotoxicity of T cells with anti-CD38-CAR. Intriguingly, even 10nM of ATRA augmented CD38 expression in MT-4, S1T, and Hut102 cells (>80%), but not in Su9T cells. Co-culture experiments in the presence of ATRA showed that MT-4, S1T, and Hut102 but Su9T cells were efficiently eliminated by T cells bearing anti-CD38-CAR, leading to a positive correlation of cytotoxicity with CD38 expression level. We tested whether ATLL cells obtained from 3 patients were disrupted by T cells bearing anti-CD38-CAR. CD38 was expressed in the cells from patients at a variety of expression ratio (0-30%). Intriguingly, CD38 expression was significantly enhanced in ATLL cells from 2 of 3 individual patients with ATRA (>50%). And resultantly, T cells bearing anti-CD38-CAR exerted more powerful cytotoxicity against ATLL cells with CD38 enhanced by ATRA (cytotoxicity of T cells with anti-CD38-CAR in CD38-positive ATLL fraction: >90%). ATRA exerted enhancing effect on the cytotoxicity of T cells bearing anti-CD38-CAR against ATLL cells through the augmentation of CD38 expression. These results may provide us a rationale for novel clinical settings of T cells carrying anti-CD38-CAR on patients with ATLL using ATRA. Disclosures: No relevant conflicts of interest to declare.

Downregulation of CDKN1A in Adult T-Cell Leukemia/Lymphoma despite Overexpression of CDKN1A in Human T-Lymphotropic Virus 1-Infected Cell Lines

ABSTRACT Human T-lymphotropic virus 1 (HTLV-1) causes an aggressive malignancy of T lymphocytes called adult T-cell leukemia/lymphoma (ATLL), and expression of HTLV-1 Tax influences cell survival, proliferation, and genomic stability in the infected T lymphocytes. Cyclin-dependent kinase inhibitor 1A (CDKN1A/p21waf1/Cip1) is upregulated by Tax, without perturbation of cell cycle control. During an analysis of the gene expression profiles of ATLL cells, we found very low expression of CDKN1A in ATLL-derived cell lines and ATLL cells from patient samples, and epigenetic abnormalities including promoter methylation are one of the mechanisms for the low CDKN1A expression in ATLL cells. Three HTLV-1-infected cell lines showed high levels of expression of both CDKN1A and Tax, but expression of CDKN1A was detected in only two of six ATLL-derived cell lines. In both the HTLV-1-infected and ATLL cell lines, we found that activated Akt phosphorylates CDKN1A at threonine 145 (T145), leading to cytoplasmic localization of CDKNIA. In HTLV-1-infected cell lines, cytoplasmic CDKN1A did not inhibit the cell cycle after UV irradiation; however, following treatment with LY294002, a PI3K inhibitor, CDKN1A was dephosphorylated and relocalized to the nucleus, resulting in suppression of the cell cycle. In the ATLL cell lines, treatment with LY294002 did not inhibit the cell cycle but induced apoptosis with the cytoplasmic localization. Therefore, the low CDKN1A expression in ATLL cells may be a key player in ATLL leukemogenesis, and the abnormal genomic methylation may influence the expression of not only HTLV-1 Tax but also CDKN1A during long-term development of ATLL from the HTLV-1-infected T lymphocytes.