Primary Adult T-cell Leukemia/lymphoma Cells Research Articles

Single-Cell RNA Sequencing Revealed the YY1/EZH2/MLH1 Axis As a Possible Therapeutic Target of Intractable Adult T-Cell Leukemia

Background: Due to clinical heterogeneity of adult T-cell leukemia/lymphoma (ATL) and its diverse genetic abnormality, common therapeutic targets of ATL remains unclear. Whereas the YY1/EZH2 axis regulates global epigenetic modification of genes in ATL, its downstream target genes have not been fully elucidated. Meanwhile, DNA mismatch repair proteins may be targeted in ATL. Here we explored underlying molecular axes for ATL progression through comprehensive single-cell RNA-sequencing (sc-RNA-seq). Methods: Peripheral blood of 42 ATL patients and asymptomatic HTLV-1 carriers in addition to two cell lines of ATL43T and ED+ were subjected to the following analyses. Molecular markers associated with their clinical phenotypes were extracted through sc-RNA-seq analysis. Expression levels of DNA mismatch repair proteins (MLH1, MSH2) and target molecules were validated by flow cytometry. Finally, their function was verified through lentiviral shRNA-dependent knockdown and molecular-targeting reagents. Results: While the frequencies of CADM1+ cells among CD4+ T cells were generally correlated with HTLV-1 proviral loads (R = 0.76, p < 0.001), substantial CADM1+ cells were detected but did not increase in some long-remitted cases; thus, CADM1+ cells are biologically heterogeneous. sc-RNA-seq of CADM1+ cells of four ATL cases identified a cluster of CD48-deficient cells. in silico promoter analysis of the cluster extracted a transcription factor YY1 as a candidate for common master regulators. EZH2 and the putative driver genes (CELF2, PTPRC, CBLB, ATXN1, IKZF2) were identified as differentially expressed genes (DEGs) in the cluster. The EZH2-overexpressed cluster was increased in acute-type ATL compared with remitted ATL and smoldering-type ATL. Interestingly, the cluster increased moderately in the other smoldering-type ATL, which required UVB irradiation due to intractable skin lesion. Given that YY1 and EZH2 were up-regulated in acute-type ATL, the cluster may represent tumor aggressiveness. Furthermore, biphasic expression of YY1/EZH2 was observed in 2 acute-type ATL, which indicates that the EZH2 high ATL cells confer malignant signature. In-house analysis on microarray dataset GSE55851 specified the MLH1 associated with YY1/EZH2. MLH1 within CADM1+ fraction was down-regulated in ATL patients with remission (n = 7) but maintained in progressive ATL (n = 6) (p < 0.05). Lentiviral shYY1 knockdown resulted in down-regulation of MLH1 in both ATL43T and ED+ (p < 0.01). The EZH1/2 inhibitor valemetostat down-regulated EZH2 (p = 0.018) and MLH1 (p = 0.014) in primary ATL cells. Finally, knockdown of either YY1 or MLH1 suppressed proliferation of ATL43T and ED+ (p < 0.0001). Conclusions: Our findings indicate that down-regulation of MLH1 through YY1/EZH2 inhibition plays a key role in the treatment of aggressive ATL.

North American Adult T-Cell Leukemia/Lymphoma Has Frequent Mutations in CCR4 and Responds in Vitro to a Small Molecule CCR4 Antagonist, CCR4-351

Adult T-cell leukemia/lymphoma (ATLL) is a rare and aggressive disease of malignant CD4+ T cells that develops in human T-lymphotropic virus-1 (HTLV-1) carriers. Patients diagnosed with ATLL have the worst survival among all peripheral T-cell lymphomas. There is an urgent need for in-depth understanding of its pathogenesis and development of new treatment strategies. We and others have shown that ATLLs diagnosed in the Japanese (J-ATLL) and North American (NA-ATLL) patients have differences in clinical characteristics and immune-genetic landscape. Among the most frequently mutated genes in J-ATLL cohorts is CCR4, a chemokine receptor that is targeted by C-terminal truncation mutations in one third of the cases. These mutations result in a more stabilized surface CCR4 and have been reported to cause elevated Akt activation following chemokine stimulation. Since our previous targeted exon sequencing study did not include CCR4, this work was initiated to assess CCR4 mutation frequency in NA-ATLL cases and to evaluate the preclinical activity of a small molecule CCR4 antagonist. This is because, although an anti-CCR4 mAb, mogamulizumab, has been approved in Japan to treat ATLL, this therapy failed in a Phase 2 trial among patients outside of Japan. Thus, mogamulizumab is not available as a routine treatment for NA-ATLL patients. We compiled CCR4 gene status data from bulk RNA-seq (n=10) and whole genome sequencing (WGS) analysis (n = 10). Out of a total of 17 NA-ATLL cases, 8 (47.1%) carried CCR4 gene alterations that included in-frame and frameshift indels, as well as nonsense mutations targeted to the C-terminus cytoplasmic tail and also the trans-membrane domain. In subtype analysis, 6 out 14 acute cases (42.9%) and 2 out of 3 (66.7%) of chronic cases carried at least one copy of mutated CCR4 gene, indicating that the mutation frequency in NA-ATLL is possibly even higher than that reported for the Japanese cohorts ( Figure 1). We then evaluated cell surface CCR4 expression using flow cytometry. The primary ATLL cells (CD4 +CD7 -CD8 -) from all 7 diagnostic samples uniformly express markedly increased levels of CCR4 relative to healthy control CD4 T cells. Interestingly, CCR4 expression among 8 ATLL cell lines is highly variable, implying that in vivo, tumor microenvironment factors may also contribute to sustained CCR4 upregulation in addition to the influence from the HTLV-1 oncoprotein, HBZ. FLX475 is an orally active small molecule CCR4 antagonist that is currently in a Phase 1/2 study (NCT03674567) in several types of advanced cancer including EBV+ NK/T cell lymphoma. So far, favorable clinical antitumor activity has been observed that includes complete responses with FLX475 monotherapy and encouraging combination activity (Lin C.-C. et al ESMO-IO 2022 Dec; #187P). Using several CCR4-positive cell lines, we examined in vitro anti-ATLL activity of CCR4-351, a CCR4 antagonist that is a highly related analog of FLX475. CCR4 is the exclusive homing receptor for two chemokine molecules, CCL17 and CCL22. In a transwell assay, both chemokines can elicit strong chemotaxis activity in a J-ATLL cell line ED40515(+). However, only CCL22 but not CCL17 can induce significant chemotaxis in two NA-ATLL cell lines, ATL#13 and ATL#29. The exact cause of this difference between these cell lines awaits future studies. In these transwell-based assays, CCR4-351 exhibited potent chemotaxis inhibitory activity with IC 50 values ranging from 7-60 nM ( Figure 2). In addition, when the cell lines were cultured under growth factor-deprivation conditions, CCR4-351 treatment caused a mild reduction in total cell viability, suggesting an additional anti-ATLL mechanism of action. In summary, in this first study of the CCR4 pathway status in NA-ATLL, we found that nearly half of the cases in our ATLL cohort carried a mutated CCR4 gene. Contrary to primary ATLL cells, ATLL cell lines exhibit a wide variation in CCR4 expression. CCL22 but not CCL17 induced strong chemotaxis behavior in NA-ATLL cell lines, which was potently inhibited by a small molecule CCR4 antagonist, CCR4-351. Since extramedullary presentation is frequently seen in NA-ATLL and central nervous system involvement is an adverse prognostic feature, inhibiting chemotaxis with a CCR4 antagonist such as FLX475 may be an effective therapeutic approach.

Primary cells from patients with adult T cell leukemia/lymphoma depend on HTLV-1 Tax expression for NF-κB activation and survival

Adult T cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic infection with human T cell leukemia virus type 1 (HTLV-1). The viral oncoprotein Tax initiates T cell transformation through activation of critical cellular pathways, including NF-κB. Unexpectedly, Tax protein is not detectable in most ATL cells, in contrast to the HTLV-1 HBZ protein which antagonizes Tax effects. Here, we demonstrate that primary ATL cells from patients with acute or chronic ATL express very low levels of Tax mRNA and protein. Critically, survival of these primary ATL cells is dependent on continued Tax expression. Mechanistically, Tax extinction results in reversal of NF-κB activation, P53/PML activation and apoptosis. Tax drives interleukin-10 (IL-10) expression and recombinant IL-10 rescues the survival of tax-depleted primary ATL cells. These results demonstrate the critical role of continued Tax and IL-10 expression for the survival of primary ATL cells, highlighting their relevance as therapeutic targets.

TAS‐116 (pimitespib), a heat shock protein 90 inhibitor, shows efficacy in preclinical models of adult T‐cell leukemia

Adult T‐cell leukemia/lymphoma (ATL) is a highly chemoresistant malignancy of peripheral T lymphocytes caused by human T‐cell leukemia virus type 1 infection, for which there is an urgent need for more effective therapeutic options. The molecular chaperone heat shock protein 90 (HSP90) plays a crucial role in nuclear factor‐κB (NF‐κB)‐mediated antiapoptosis in ATL cells, and HSP90 inhibitors are new candidate therapeutics for ATL. Accordingly, we investigated the anti‐ATL effects of a novel oral HSP90 inhibitor, TAS‐116 (pimitespib), and the mechanisms involved in ex vivo and in vivo preclinical models. TAS‐116 achieved IC50 values of less than 0.5 μmol/L in 10 ATL‐related cell lines and less than 1 μmol/L in primary peripheral blood cells of nine ATL patients; no toxicity was observed toward CD4+ lymphocytes from healthy donors, indicating the safety of this agent. Given orally, TAS‐116 also showed significant inhibitory effects against tumor cell growth in ATL cell‐xenografted mice. Furthermore, gene expression profiling of TAS‐116‐treated Tax‐positive or ‐negative cell lines and primary ATL cells using DNA microarray and multiple pathway analysis revealed the significant downregulation of the NF‐κB pathway in Tax‐positive cells and cell‐cycle arrest in Tax‐negative cells and primary ATL cells. TAS‐116 suppressed the activator protein‐1 and tumor necrosis factor pathways in all examined cells. These findings strongly indicate the efficacy of TAS‐116, regardless of the stage of ATL progression, and its potential application as a novel clinical anti‐ATL therapeutic agent.

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.

Development of a Unique T Cell Receptor Gene-Transferred Tax-Redirected T Cell Immunotherapy for Adult T Cell Leukemia.

Adult T cell leukemia/lymphoma (ATL) is an aggressive peripheral T cell neoplasm caused by infection with human T cell lymphotropic virus type-1 (HTLV-1). Its prognosis remains extremely poor. Tax, the most important regulatory protein for HTLV-1, is associated with the aggressive proliferation of host cells and is also a major target antigen for CD8+ cytotoxic T cells (CTLs). Based on our previous findings that Tax-specific CTLs with a T cell receptor (TCR) containing a unique amino-acid sequence motif exhibit strong HLA-A*24:02-restricted, Tax301-309-specific activity against HTLV-1, we aimed to develop a Tax-redirected T cell immunotherapy for ATL. TCR-ɑ/β genes were cloned from a previously established CTL clone and transduced into peripheral blood mononuclear cells (PBMCs) of healthy volunteers using a retroviral siTCR vector. Then the cytotoxic efficacy against HTLV-1-infected T cells or primary ATL cells was assessed both in vitro and in vivo. The redirected CTLs (Tax-siCTLs) produced a large amount of cytokines and showed strong killing activity against ATL/HTLV-1-infected T cells in vitro, although they did not have universal activity against ATL cells. Next, in a xenograft mouse model using an HTLV-1-infected T cell line (MT-2), in all mice treated with Tax-siCTLs, the tumor rapidly diminished and finally disappeared without normal tissue damage, although all mice that were untreated or treated with non-gene-modified PBMCs died because of tumor progression. Our findings confirm that Tax-siCTLs can exert strong anti-ATL/HTLV-1 effects without a significant reaction against normal cells and have the potential to be a novel immunotherapy for ATL patients.

Cell death induced by dorsomorphin in adult T-cell leukemia/lymphoma is AMPK-independent.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasm with poor prognosis that develops after chronic infection with human T-cell leukemia virus type 1 (HTLV-1). Although AMP-activated protein kinase (AMPK) is a critical cellular energy sensor, it has recently become clear that AMPK can act as a tumor regulator. Here, we assessed the expression of AMPK in primary ATL cells and the effects of dorsomorphin, an AMPK inhibitor, on primary ATL cells and HTLV-1-infected T-cell lines. AMPK expression in acute and chronic ATL patients was significantly higher than in asymptomatic HTLV-1 carriers and healthy donors. Dorsomorphin induced apoptosis in peripheral blood mononuclear cells from ATL patients. Dorsomorphin also induced dose- and time-dependent apoptosis in HTLV-1-infected T-cell lines. Dorsomorphin increased the production of intracellular reactive oxygen species (ROS) and induced ataxia telangiectasia-mutated Ser1981 phosphorylation and p53 accumulation. These results indicated that dorsomorphin induces apoptosis via ROS-mediated DNA damage in HTLV-1-infected T-cell lines. Furthermore, dorsomorphin suppressed the growth of human ATL tumor xenografts in NOD/SCID mice. Together, these data suggest that AMPK could be a candidate therapeutic target for ATL and that dorsomorphin could be a therapeutic agent for ATL.

High expression of NAMPT in adult T-cell leukemia/lymphoma and anti-tumor activity of a NAMPT inhibitor

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature T lymphocytes induced by human T-cell leukemia virus-1 and has a poor outcome. New molecular targets for the prevention and treatment of ATL are needed urgently. We previously reported high expression of Sirtuin 1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, in primary acute-type ATL cells. NAD+ biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) modulates Sirtuin 1 activity. Here, we examined the expression and effects of inhibiting NAMPT, a rate-limiting enzyme in NAD+ biosynthesis, in ATL cells. We found that peripheral blood mononuclear cells from patients with acute-type ATL expressed significantly higher levels of NAMPT protein than cells from healthy subjects. FK866, a NAMPT inhibitor, induced apoptosis of freshly isolated ATL cells ex vivo and HTLV-1-infected T-cell lines in vitro, which was accompanied by activation of caspases, DNA fragmentation, and disruption of mitochondrial transmembrane potential. However, a pan-caspase inhibitor failed to prevent this FK866-induced cell death, while FK866 increased the caspase-independent cell death mediator endonuclease G. Intriguingly, FK866 also activated autophagy, as demonstrated by increases in protein levels of autophagosome marker LC3-II. Thus, FK866 simultaneously activated apoptosis and autophagy. Finally, FK866 treatment markedly decreased the growth of human ATL tumor xenografts in immunodeficient mice. We showed that NAMPT is highly expressed in primary ATL cells ex vivo, and that FK866 induces autophagy and caspase-dependent and -independent cell death pathways in vitro and has an anti-tumor activity in vivo. These results suggest a novel therapeutic strategy for patients with this fatal disease.

HTLV-1 viral oncogene HBZ drives bone destruction in adult T cell leukemia.

Osteolytic bone lesions and hypercalcemia are common, serious complications in adult T cell leukemia/lymphoma (ATL), an aggressive T cell malignancy associated with human T cell leukemia virus type 1 (HTLV-1) infection. The HTLV-1 viral oncogene HBZ has been implicated in ATL tumorigenesis and bone loss. In this study, we evaluated the role of HBZ on ATL-associated bone destruction using HTLV-1 infection and disease progression mouse models. Humanized mice infected with HTLV-1 developed lymphoproliferative disease and continuous, progressive osteolytic bone lesions. HTLV-1 lacking HBZ displayed only modest delays to lymphoproliferative disease but significantly decreased disease-associated bone loss compared with HTLV-1-infected mice. Gene expression array of acute ATL patient samples demonstrated increased expression of RANKL, a critical regulator of osteoclasts. We found that HBZ regulated RANKL in a c-Fos-dependent manner. Treatment of HTLV-1-infected humanized mice with denosumab, a monoclonal antibody against human RANKL, alleviated bone loss. Using patient-derived xenografts from primary human ATL cells to induce lymphoproliferative disease, we also observed profound tumor-induced bone destruction and increased c-Fos and RANKL gene expression. Together, these data show the critical role of HBZ in driving ATL-associated bone loss through RANKL and identify denosumab as a potential treatment to prevent bone complications in ATL patients.

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. [Display omitted] DisclosuresEzoe: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.

In vitro effects of arsenic trioxide, interferon α and zidovudine in adult T cell leukemia/lymphoma cells.

Despite the efficacy of combination chemotherapy with arsenic trioxide (ATO), interferon α (IFN) and zidovudine (AZT) for adult T cell leukemia/lymphoma (ATL), the precise mechanism underlying this combination treatment effect is unknown. In the present study, ATO/IFN/AZT was examined in an ATL leukemic cell line (S1T, non-Tax expressing), a human T-lymphotropic virus 1 (HTLV-1)-infected cell line (MT2, Tax-expressing) and primary ATL cells from patients with acute and chronic ATL. IFN/AZT marginally inhibited MT2 cell proliferation, but substantially inhibited S1T cell proliferation. IFN/AZT increased the cleavage of numerous caspases and PARP in S1T cells, and regulated the signal transducer and activator of transcription 1 and nuclear factor-κB signaling pathway. These effects represent the potential anti-ATL mechanisms of INF/AZT in vitro. In addition, the combination of ATO and IFN/AZT demonstrated synergistic effects on S1T cells. Therefore, the Tax-independent mechanism underlying the anti-ATL effect of ATO must be further elucidated.

Cyclin-dependent kinase 9 is a novel specific molecular target in adult T-cell leukemia/lymphoma

AbstractCyclin-dependent kinase 9 (CDK9), a subunit of the positive transcription elongation factor b (P-TEFb) complex, regulates gene transcription elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). The deregulation of CDK9/P-TEFb has important implications for many cancer types. BAY 1143572 is a novel and highly selective CDK9/P-TEFb inhibitor currently being investigated in phase 1 studies. We evaluated the therapeutic potential of BAY 1143572 in adult T-cell leukemia/lymphoma (ATL). As a result of CDK9 inhibition and subsequent inhibition of phosphorylation at serine 2 of the RNAPII CTD, BAY 1143572 decreased c-Myc and Mcl-1 levels in ATL-derived or human T-cell lymphotropic virus type-1 (HTLV-1)–transformed lines and primary ATL cells tested, leading to their growth inhibition and apoptosis. Median inhibitory concentrations for BAY 1143572 in ATL-derived or HTLV-1–transformed lines (n = 8), primary ATL cells (n = 11), and CD4+ cells from healthy volunteers (n = 5) were 0.535, 0.30, and 0.36 μM, respectively. Next, NOG mice were used as recipients of tumor cells from an ATL patient. BAY 1143572–treated ATL-bearing mice (once daily 12.5 mg/kg oral application) demonstrated significantly decreased ATL cell infiltration of the liver and bone marrow, as well as decreased human soluble interleukin-2 receptor levels in serum (reflecting the ATL tumor burden), compared with untreated mice (n = 8 for both). BAY 1143572–treated ATL-bearing mice demonstrated significantly prolonged survival compared with untreated ATL-bearing mice (n = 7 for both). Collectively, this study indicates that BAY 1143572 showed strong potential as a novel treatment of ATL.

Development and Molecular Analysis of Synthetic Lethality By Targeting EZH1 and EZH2 in Non-Hodgkin Lymphomas

Epigenetic program is a molecular basis of cellular identity and functions. We have shown the transcriptome (Cancer Cell, 2012) and the underlying histone methylation patterns (Blood, 2016) of adult T-cell leukemia-lymphoma (ATL). The epigenetic landscape of ATL is defined as "genome-wide H3K27me3 accumulation". Similar accumulation has been detected in multiple cancers, particularly in hematological malignancies. Relief of the cumulative methylation may restore the aberrant transcriptome to ideal expression signature, leading to a promising treatment. Due to variation characteristics, EZH2 is recognized as a druggable target. However, extrinsic regulation of methylation pattern is still challenging without knowledge of molecule(s) and their combination that needs to be targeted to reprogram the epigenome in anticipation of synthetic lethality.To redefine the conventional concept, we first examined expression and global occupancy patterns of H3K27 methyltransferases EZH1 and EZH2. EZH2 expression was low in CD4+ T cells and significantly high in ATL. In contrast, basal EZH1 was very high in mature lymphocytes compared with undifferentiated lineages and also high in ATL. ChIP-on-chip analysis revealed significant changes in distributions of both EZH1 and EZH2 in primary ATL cells, which caused abnormal H3K27me3 accumulation. Importantly, over 80% of H3K27me3 are specifically induced by either EZH1 (28.9%) or EZH2 (14.4%), or by both (39.3%). Moreover, they have differential functions by regulating specific targets; EZH2 suppresses various transcription regulators, establishing complex gene regulatory network. EZH1 would rather directly induce H3K27me3 at genes involving functional processes such as lymphocyte activation. We detected EZH1 and EZH2 in functional polycomb repressive complex 2 (PRC2) in ATL and DLBCL cells. Unexpectedly, EZH1/2 were co-localized with H3K4me3 at a small population of the lymphoma genome. The "EZH1/2 + H3K4me3" non-canonical targets such as cell cycle activators were highly expressed in ATL.We next compared functions of EZH1/2. Single depletion of EZH1 or EZH2 suppressed T- and B-lymphoma cell proliferation, which supports their unique functions. The broad reduction of H3K27me3 was achieved by EZH2 inhibition; however, genome-wide ChIP demonstrated that EZH2 inhibition or depletion triggered strong compensatory action of EZH1, followed by rebound accumulation of H3K27me3 at many functional genes in EZH1+ lymphoma cells. We found that a transcription factor YY1 affects PRC2 recruitment and EZH1 compensation after EZH2 inhibition. Double depletion of EZH1+EZH2 or YY1+EZH2 surmounted the epigenetic homeostasis. In addition, depletion of EZH1 significantly enhanced effects of EZH2 inhibitors in ATL and DLBCL cells (5.47~205-fold). These data indicate requirements for the aberrant epigenome, namely, fluctuating EZH2 (upregulation or mutation) and underlying stable EZH1.We have succeeded in developing an unprecedented EZH1/2 dual-inhibitor (IC50 9.0 nM (EZH1); 10 nM (EZH2)). The new drug successfully inhibited genome-wide EZH1/2 occupancies without compensation, resulting in effective H3K27me3 reduction and gene reactivation compared with the single inhibitor. Of note, the dual-inhibitor suppressed expression of the non-canonical EZH1/2 + H3K4me3 target genes. Besides H3K27me3 reprogramming, EZH1/2 inhibition could modulate genes through H3K4me3 regulation and indirect influence on the secondary regulatory network and signaling pathways such as NF-kB.The EZH1/2 dual-inhibitor significantly blocked primary ATL cell survival and in vivo tumor growth of ATL and DLBCL models. Furthermore, we evaluated drug efficacy in various models of hematological malignancies including ATL, DLBCL (EZH2 WT and mutant), Burkitt lymphoma, and PTCL, which are all originated from EZH1+ differentiated lymphocytes. All of them showed higher sensitivity against the EZH1/2 inhibitor compared with the EZH2 solo inhibitor (5.2~200-fold). We did not detect strong adverse effects in normal T cells.These integrated molecular and phenotypic analyses provide a novel concept "EZH1+EZH2 dual-targeting" and demonstrate validity of the new drug in the treatment of malignant lymphomas. Taking these results, a Phase I clinical trial against T- and B-cell non-Hodgkin lymphomas including ATL is now under way in Japan. DisclosuresYamagishi:Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Ohsugi:Daiichi Sankyo Co., Ltd.: Research Funding. Utsunomiya:Daiichi Sankyo Co., Ltd.: Speakers Bureau. Tsukasaki:Daiichi Sankyo Co., Ltd.: Consultancy; Takeda: Research Funding. Tobinai:Zenyaku Kogyo: Honoraria; Takeda: Honoraria, Research Funding; Mundipharma KK: Honoraria, Research Funding; Kyowa Hakko Kirin: Research Funding; Celgene: Research Funding; Daiichi Sankyo Co., Ltd.: Consultancy; SERVIER: Research Funding; Ono Pharmaceutical: Research Funding; Eisai: Honoraria, Research Funding; Chugai Pharma: Research Funding; Abbvie: Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; HUYA Bioscience: Honoraria. Araki:Daiichi Sankyo Co., Ltd.: Employment.

Proof of Merit in New Diet Therapy Against Adult T-Cell Leukemia/Lymphoma: Applying Specific Amino Acid Dependency of Leukemia/Lymphoma Cells

Introduction:Adult T-cell leukemia/lymphoma (ATL) is highly aggressive malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). Because ATL cells are very aggressive, cytotoxic combination chemotherapy is used as first-line therapy. However, ATL cells are often resistant to chemotherapy. The intensive chemotherapy induces severe myelosuppression, which requires frequent transfusions, and organ damages. Unfortunately, accumulation of these adverse events disables continuation of treatment in some patients. Recently antibody therapy which targets CC chemokine receptor 4 (CCR4) expressed on ATL cells has been approved for the treatment of relapsed ATL, but lymphoma-like massive ATL cells in lymph nodes or spleen are known to be resistant to antibody therapy. Therefore, we sought to develop a new strategy, and finally focused on amino acids as asparaginase is used for treatment of acute lymphoblastic leukemia.Methods:Amino acid dependency of HTLV-1-infected cells, including ATL cells, was examined in vitro using HTLV-1-infected cell-lines and ATL cells which were serially passaged after purification by flow cytometry from the sample of an acute-type ATL patient. These cells were cultured with stromal cells in the medium which lacked one of twenty amino acids for 2 weeks. Proliferation of ATL cells and influence on stromal cells was examined, and we picked up amino acids which were crucial only for ATL cells. We next prepared diets which lacked specific amino acids based on in-vitro screening, and examined the effect in vivo using xeno-transplantation models. In a lymphoma model, massed ATL cells were transplanted intraperitoneally into adult NOG mice, which were fed complete diets. Diets were unchanged or changed to restricted ones after 6 weeks, and maximum tumor size was compared after 10 weeks. In a leukemia model, primary ATL cells, which were sorted from peripheral blood of an acute-type ATL patient, were transplanted intravenously into neonatal NOG mice within 24 hours from the birth. Recipient mice were brought up by the mother fed complete diets. After 4 weeks, recipient mice were weaned from breast, and fed complete diets or restricted ones. Peripheral blood was followed over time by flow cytometry, and infiltration of ATL cells into various organs was analyzed after 8 weeks.Results:In-vitro studies revealed that HTLV-1-infected cells have dependency on specific amino acids in common, and it was noteworthy that ATL cells could not proliferate in valine-deficient conditions while the influence on co-cultured stromal cells was limited. Therefore, we specially prepared valine-depleted diets, and confirmed that density of valine was very low in peripheral blood and bone marrow of mice fed valine-depleted diets. In-vivo lymphoma models showed that no masses were observed macroscopically in all of mice fed valine-depleted diets. In-vivo systemic leukemia models showed that valine-restricted diets preferentially reduced ATL cells but not hemoglobin or platelets in peripheral blood as shown in figure 1. Additionally, pathological examinations showed that valine-deficient diets strongly prevented ATL cells from proliferating and infiltrating into organs, such as spleen, liver, and skin. As far as we examined histologically, there were no significant organ damages.Discussion and Conclusions:Our study uncovered that adult T-cell leukemia/lymphoma cells failed to proliferate in valine-depleted conditions. The inhibition induced by dietary valine restriction was drastic, whereas ATL cells could proliferate to greater or lesser degrees in conditions lacking other amino acids, even if essential amino acids were depleted. Additionally, valine is indispensable for the proliferation and maintenance of hematopoietic stem cells (HSCs) as we previously reported. Therefore, valine-deficient diets could successfully reduce leukemic stem cells of ATL. Moreover, there were no severe complications such as anemia, thrombocytopenia, and organ damages which are often seen in chemotherapy recipients. Lymphoma-like massive ATL cells, which are often resistant to antibody therapies, were also vulnerable to valine-depleted conditions. The issues seen in currently available therapies against aggressive leukemia and lymphoma could be solved by this diet therapy itself or by adding this therapy to other treatments with reduced toxicity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Induction of apoptosis by HBI-8000 in adult T-cell leukemia/lymphoma is associated with activation of Bim and NLRP3.

Adult T‐cell leukemia/lymphoma (ATL) is an aggressive T‐cell malignancy caused by human T‐cell lymphotropic virus 1. Treatment options for acute ATL patients include chemotherapy, stem cell transplantation, and recently the anti‐chemokine (C‐C motif) receptor 4 antibody, although most patients still have a poor prognosis and there is a clear need for additional options. HBI‐8000 is a novel oral histone deacetylase inhibitor with proven efficacy for treatment of T‐cell lymphomas that recently received approval in China. In the present study, we evaluated the effects of HBI‐8000 on ATL‐derived cell lines and primary cells obtained from Japanese ATL patients. In most cases HBI‐8000 induced apoptosis in both primary ATL cells and cell lines. In addition, findings obtained with DNA microarray suggested Bim activation and, interestingly, the contribution of the NLR family, pyrin domain containing 3 (NLRP3) inflammasome pathway in HBI‐8000‐induced ATL cell death. Further investigations using siRNAs confirmed that Bim contributes to HBI‐8000‐induced apoptosis. Our results provide a rationale for a clinical investigation of the efficacy of HBI‐8000 in patients with ATL. Although the role of NLRP3 inflammasome activation in ATL cell death remains to be verified, HBI‐8000 may be part of a novel therapeutic strategy for cancer based on the NLRP3 pathway.

The Combination of Belinostat with Zidovudine for Treatment of HTLV-I Related Adult T-Cell Leukemia-Lymphoma

Adult T-cell leukemia-lymphoma (ATLL) is an aggressive malignancy with poor prognosis that is incurable by conventional drugs. Histone deacetylase (HDAC) inhibitors (HDIs) are broadly active anti-neoplastic agents that can be cautiously exploited in the treatment of ATLL. The HTLV-I provirus is clonally integrated in virtually all ATLL cells. The HTLV-I 5'LTR promoter is trans -activated by the viral protein Tax through binding of CREB and p300/CBP. Tax binding enhances p300/CBP histone acetyl transferase (HAT) activity resulting in histone acetylation, chromatin unwinding, and transcription of the viral genome. HAT activity is countered by HDACs, and also by the viral-encoded protein HTLV-I bZip factor (HBZ), which is transcribed from the negative proviral strand at the 3' end. The 5' LTR is often mutated or deleted in ATLL cells thus HBZ is the only viral gene consistently expressed in ATLL. We hypothesized that HDAC inhibitors, which can be potent anti-neoplastic drugs, would reactivate HTLV-I in ATLL tumors containing intact provirus, thus killing virus-infected cells and provoking an immune response in vivo. Supporting this notion, adding the old generation HDI valproic acid (VPA) to AZT/IFNa in one subject suffering from aggressive acute-type ATLL resulted in HTLV-I proviral load reduction, followed by a complete molecular and prolonged clinical remission that lasted several years. We recently tested other more potent HDIs on primary ATLL cells and low-passage cell lines, which are rare and difficult to establish. In general, HDIs induced dose-dependent Tax expression and apoptosis in ATLL cells. Belinostat, a pan HDI, caused H3 acetylation, blocked HBZ protein expression, and induced Tax protein expression in ATLL cells resulting in concomitant apoptosis. ATLL cells exhibit high constitutive expression of nuclear factor kappa B (NF-kB), which is known to be activated by Tax and to play a major role in HTLV-I mediated T-cell transformation and ATLL development. Paradoxically, while belinostat induced Tax, it also inhibited NF-kB nuclear activity resulting in apoptosis. Further, adding AZT to belinostat augmented ATLL cell death. By contrast, IFNα diminished apoptosis induced by belinostat in freshly isolated ATLL cells from some of our patients. Based on these preclinical data, we have developed a clinical trial using belinostat in combination with AZT as consolidation therapy for ATLL. Our primary goal using this approach is to eradicate residual ATLL cells and HTLV-I infected reservoirs that normally persist regardless of treatment. DisclosuresNo relevant conflicts of interest to declare.

Anti-CCR4 monoclonal antibody KW-0761 (mogamulizumab) or investigator's choice in subjects with relapsed or refractory adult T-cell leukemia-lymphoma (ATL)

The receptor for macrophage derived chemokine (MDC) and thymus- and activation-regulated chemokine (TARC) CC chemokine receptor 4 (CCR4) is over expressed in several T-cell malignancies including HTLV-1 related ATL where approximately 90% of malignant cells have been shown to over express this chemokine receptor. The HTLV-1 transactivator gene (Tax) does not directly induce CCR4 expression. Rather, expression of CCR4 is controlled by the constitutive activation of several transcription factors in HTLV-1 infected cells. Inhibiting the expression of these transcription factors with small-interfering RNAs has been shown to block CCR4 expression and also reduce proliferation of the affected cells. Patients with CCR4 positive ATL are more likely to have skin involvement and shorter overall survival (OS; median 9.5 months) compared with CCR4-negative (20.6 months) patients. Mogamulizumab is a defucosylated, humanized, monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity (Potelligent®) against primary ATL cells that bind to CCR4.

A new ATL xenograft model and evaluation of pyrrolidine dithiocarbamate as a potential ATL therapeutic agent

Adult T-cell leukemia/lymphoma (ATL) is caused by human T-lymphotrophic virus type 1 infection and is one of the most refractory malignant T-cell lymphomas. Improvement of ATL therapy options requires the establishment of appropriate ATL animal models. In this study, we successfully generated an ATL mouse model by xenotransplantation of primary peripheral blood mononuclear cells (PBMCs) isolated from ATL patients (ATL cells) into nonobese diabetic/severe combined immunodeficiency/Jak3-null mice (NOJ mice). To generate the model, the ATL S1T cell line was subcutaneously injected into mice. Primary ATL cells were then transplanted subcutaneously, intraperitoneally, or intravenously. ATL cells infiltrated multiple organs, and elevated human soluble interleukin 2 receptor (IL-2R) levels were detected in peripheral blood. Injection of one million primary ATL cells was needed for successful engraftment into host mice. Thawed cells, frozen long-term in liquid nitrogen, could also be transplanted; however, more cells were required to achieve similar results. The median mouse survival time was proportional to the number of cells injected. Successful secondary transplantation of ATL cells from one NOJ mouse into another was achieved and confirmed by T-cell receptor analysis. Finally, we examined the effects of the antioxide pyrrolidine dithiocarbamate (PDTC) as an antitumor agent in vivo. PDTC administration inhibited the increase of soluble IL-2R and improved mouse survival, suggesting that this compound has potential as an anti-ATL agent. We demonstrated that ATL cells could be stably xenotransplanted into NOJ mice using primary cells. This model will be useful in the establishment of novel therapies to treat ATL.

Using HDAC Inhibitors to Eradicate Adult T-Cell Leukemia-Lymphoma

Adult T-cell leukemia-lymphoma (ATLL) is an aggressive malignancy with a poor prognosis caused by HTLV-I, which is endemic in Japan, the Caribbean, and South America. ATLL cannot be cured with conventional chemotherapy thus urging the development of new therapeutic strategies. Histone deacetyalse (HDAC) inhibitors are broadly active anti-neoplastic agents, which have shown efficacy in T-cell lymphomas. At our institution we encounter a relatively high number of ATLL cases as compared to other regions in the U.S. We have conducted a pilot trial using AZT/interferon-α(IFNα) plus the HDAC inhibitor valproic acid (VPA) during maintenance therapy in acute (leukemia-type) ATLL. We hypothesized that VPA would reactivate HTLV-I leading to a cytotoxic T-cell immune response followed by reduction of residual blood circulating ATLL clones that normally persist during AZT/IFNα therapy despite its suppressive effects. Supporting this notion, the addition of VPA to AZT/IFNα resulted in reduction of HTLV-I proviral loads in treated patients and a sustained molecular response in one subject who is still alive >4.5 years later. More recently, we have tested newly available and more potent HDAC inhibitors using fresh primary leukemic ATLL cells and low-passage cell lines. Through HDAC inhibition, these agents reactivated HTLV-I Tax gene expression and induced apoptosis in a dose-dependent manner. Belinostat, which is currently FDA-approved for the treatment of relapsed or refractory peripheral T-cell lymphoma, augmented ATLL cell death when added to AZT/IFNα at nanomolar concentrations. Therefore, we have proposed a new pilot clinical trial using belinostat for the treatment of ATLL during post-induction therapy. The role of HDAC inhibitors in ATLL, pre-clinical laboratory studies involving these agents, and clinical trial design will be discussed at the meeting DisclosuresOff Label Use: The use of HDAC inhibitors in adult T-cell Leukemia-lymphoma.

Comprehensive Analysis of Surface Antigens on Adult T-Cell Leukemia/Lymphoma (ATL) Cells and Search for ATL-Initiating Cell Markers

Adult T-cell leukemia/lymphoma (ATL) is highly aggressive malignancy caused by human T-cell leukemia virus type 1 (HTLV1). Intensive combination chemotherapy can initially reduce ATL cells, but relapses are common. Today many kinds of tumors are considered to be organized in a hierarchy of heterogeneous cell populations, with only a small proportion of cancer initiating cells (CICs) capable of sustaining tumor formation and growth. Even if most of tumor cells are killed, remaining chemo-resistant CICs can be causes of relapses. However, CICs of ATL have not been identified yet. Our first goal is to identify ATL-initiating cells using in-vitro and in-vivo models.Because co-culture of HTLV1-infected ATL cells and normal lymphocytes results in extraordinarily high HTLV1 integration into normal lymphocytes, which is not seen in the bodies of patients, purification of ATL cells is necessary for evaluation of tumor-specific proliferation. We reported that HTLV1-infected ATL cells could be specifically enriched in the CD4+CADM1+ fraction, while the HTLV-1 basic leucine zipper (HBZ) gene was not detected in CD4+CADM1- cells by PCR analysis. To purify ATL cells, CD4+CADM1+ gating was used.First, we performed comprehensive analysis of surface antigens specifically on ATL cells. Peripheral blood mononuclear cells (PBMCs) were isolated from typical acute-type ATL patients and healthy volunteers. Using flow cytometry and gating, surface expression of 105 antigens, including activated T-cell markers, cell adhesive molecules, and lineage markers, was analyzed. The expression on ATL cells was compared with other CD4-positive non-ATL lymphocytes. Statistical hierarchical clustering analysis of 10 sample groups showed that ATL cells were all grouped into the same cluster, and markers specific for acute-type ATL cells were therefore picked up as candidate markers.Second, CD4+CADM1+ cells were classified by differences in expression of those candidate markers and sorted using 12-color flow cytometry. Although growing primary ATL cells in vitro is very difficult, it was reported that only a small proportion of ATL cells can survive and proliferate on the murine MS-5 stromal cell line, which also allows the proliferation of some ALL and hematopoietic progenitor cells. We repeated these 14-days in-vitro assays and tried to identify the cells capable of surviving and proliferating on MS-5, which we consider to be ATL-initiating cells. Although most of picked-up markers didn’t have any impacts on survival and proliferation of ATL cells, several surface antigens affected the results. After confirmation and prioritization, CD25+ and CD71- fractions were finally picked up. Combining these two fractions, we found that CD71-CD25+CD4+CADM1+ cells could survive and proliferate on MS-5, while other CD4+CADM1+ cells died off in many cases. Even in a case where CD71-CD25+ cells accounted only for 1.1% among the CD4+CADM1+ fraction, CD71-CD25+ cells exclusively survived and proliferated in vitro (figure 1). Moreover, CD71-CD25+ cells could generate CD71+ and CD25- cells on MS-5. In serial culture models, the CD71-CD25+ cells could serially generate other CD4+CADM1+ cells in vitro.Then, we focused on the clinical course of actual ATL patients, and followed up the CD71-CD25+ ATL cells before and after chemotherapy. After chemotherapy, the frequency of CD71-CD25+ cells among CD4+CADM1+ cells became obviously higher, and they therefore seemed to be resistant to chemotherapy. In a case where robust reduction of ATL cells was achieved, more than 90% of remaining ATL cells were in the CD71-CD25+ fraction.Lastly, we compared the in-vivo proliferation of CD71-CD25+ ATL cells and other CD4+CADM1+ cells. Both cells were transplanted intravenously into neonatal NOD/Shi-scid/IL-2Rγnull (NOG) mice within 48 hours from birth. CD71-CD25+CD4+CADM1+ cells could proliferate in vivo while others couldn’t as far as we tried. Moreover, flow cytometric analysis of murine peripheral blood in 70 days after transplantation showed that CD71-CD25+CD4+CADM1+ cells could generate ATL cells with other phenotype seen in primary ATL patients.In summary, we successfully found that CD71-CD25+CD4+CADM1+ cells could generate other ATL cells in vitro and in vivo. This research suggested ATL-initiating cells could be in the CD71-CD25+CD4+CADM1+ fraction. [Display omitted] DisclosuresTojo:Novartis: Research Funding, Speakers Bureau.