(2005) 24, 5931–5937 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc

Discovery of HTLV-1, the first human , its unique regulatory mechanisms, and insights into pathogenesis

Mitsuaki Yoshida*,1

1Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., 3 Ohkubo, Tsukuba, Ibaraki 300-2611, Japan

I briefly review the discovery and characterization of the In Japan something was there, but it was not clear first human retrovirus, human T- leukemia type 1, focusing on contributions from Japanese researchers. Discovery of ATL The unique regulatory mechanisms for the viral regulation The first key event for human retrovirus was the with Tax and Rex, etiology of ATL and possible discovery of adult T-cell leukemia (ATL) by Kiyoshi leukemogenic mechanism with Tax are also discussed Takatsuki and his colleagues in Japan in 1976 (Uchi- briefly. et al Oncogene (2005) 24, 5931–5937. doi:10.1038/sj.onc.1208981 yama ., 1977). The reports from his laboratory described unique characteristics of the leukemia such as Keywords: HTLV-1; ; tumor-suppressor unusual morphology of leukemic cells with lobulated ; cell cycle; epidemiology nuclei and surface phenotypes. The most striking feature of the disease was the Kyushu (West part of Japan) origin of most of the patients who were identified in Kyoto where Takatsuki’s group performed their research. Takatsuki’s findings were later confirmed by extensive epidemiology and a clear clustering of Introduction ATL cases in the Kyushu area strongly suggested a unique etiology (Hinuma et al., 1981a). At the time, When virologists and oncologists are challenged to this novel discovery elicited enormous attention from identify human tumor , they dream of the physicians, virologists, epidemiologists and oncologists, contribution of such viruses towards understanding but still there was no clue as to how to search for an and answering questions such as how and why etiology. cancers develop and how to prevent viral infection in order to prevent cancers. There was a time when were a particular focus because retroviruses Establishment of cell lines had been isolated as tumor viruses from many Isao Miyoshi who was interested in karyotype of tumor species in the 1970s. Such studies provided the cells tried to establish cell lines from peripheral conceptual revolution in understanding cancers, ulti- lymphoid cells of ATL patients (Miyoshi et al., 1980). mately contributing to the proposition of a ‘viral Prior to the availability of purified lymphokines, a cell oncogene’. Indeed, over the course of research, it line MT-1 was established from the sample of an ATL became apparent that viral originated from patient using cocultivation with cord blood lympho- their cell ‘proto-oncogenes’, which are widely cytes. Similar attempts then led to the establishing conserved in many animal species. Thus, ‘the oncogene’ of MT-2 and MT-4 cells, and these established lines became the ‘key player’ in research linking exhibited extremely high chromosomal abnormalities. findings between and humans and also the Otherwise, at that time, not much additional informa- different origins of cancers. However, the daunting tion was captured from these cell lines. Nevertheless, worldwide challenge remained to identify and isolate a these cell lines later became essential tools for diagnosis bona fide retrovirus in a human cancer. of ATL and identification of the retrovirus, providing The discovery of the first human retrovirus proceeded reproducible and consistent biological materials that rather independently in Japan and the USA. Below, I could be propagated in large quantity. Surprisingly, first summarize the history of discovery from Japanese some of these cell lines turned out to be of cord- researchers and then discuss human T-cell leukemia blood origin and not of ATL origin according to virus type 1 (HTLV-1) regulatory and pathogenic analysis (Miyoshi et al., 1981). These mechanisms. unexpected results were later explained by the transmis- sion of retrovirus from ATL cells into cord-blood cells and subsequent immortalization of the de novo *Correspondence: M Yoshida; E-mail: [email protected] infected cell. Discovery of HTLV-1, the first human retrovirus M Yoshida 5932 Identification of a unique antibody in ATL patients competent genomic structure is frequently deleted by acquisition of a cellular proto-oncogene resulting in the Identification of unique serum antibodies in ATL formation of a transforming, but replication defective, patients was another key criterion for a new retrovirus. virus. The newly isolated HTLV-1 was unusual in that it Postulating a viral infection in ATL patients, Yorio has a complete undeleted replication competent , Hinuma et al. (1981b) applied an indirect immunofluor- but it contained an additional pX sequence which could escence assay to some ATL sera employing a large panel encode multiple using alternative open reading of cells of human origin. After laborious screening, a frames. Thus, HTLV-1 has an ‘LTR-gag---pX- single cell line was identified to give a robustly positive LTR’ genomic structure (Figure 1; Seiki et al., 1982, signal. That was the MT-1 cell, and the putative antigens 1983), with constitution similar to that of Rous sarcoma in the cell line were preliminary termed ATL antigens virus. This unique genome structure distinguished (ATLA). Later, MT-2 and -4 were also identified to give HTLV-1 from any other animal retroviruses, thus positive signals in this assay. Without knowing what founding a new retroviral group. Other members of this ATLA were, screening for anti-ATLA was carried out viral group, HTLV-2, STLV or PTLV and BLV, are with sera from various types of leukemias, and the well characterized today. results were importantly informative: anti-ATLA were detected in all ATL patients, also in a minor fraction of healthy individuals restricted to the Kyushu area in Association with ATL and other diseases Japan (Hinuma et al., 1981a, b). Therefore, it was evident that something was there, but it was not clear As described above, unique antibodies that crossreact what was there. with MT-1 cells were detected in all ATL patients, but not in other leukemic or healthy individuals, except for a rare minor population (Shimoyama et al., 1986). The HTLV-1 proteins, p24 and p19, were identified at least The daybreak of HTLV-1 in Japan as a part of the antigens for the unique antibodies in ATL patients; therefore, close association of HTLV-1 Identification of retrovirus infection with ATL was immediately concluded (Yoshida et al., 1982). Furthermore, the epidemiology also During the corresponding period of these years, Robert revealed that the cumulative risk of ATL among virus C Gallo and his group at NIH, USA (Poeisz et al., carriers in Japan was about 3–5%, indicating that most 1980), identified and first reported a retrovirus in a of the HTLV-1 carriers are asymptomatic throughout T-cell line, HUT102, established from a patient with their (Tajima, 1990). Mycosis fungoides , which was later diagnosed as a case Subsequently, HTLV-1 infection was also discovered of ATL lymphoma. to associate with tropical spastic paraparesis (TSP) in Independently, molecular approaches were being Jamaica by Guy de The and his colleagues (Gessain applied to identify a retrovirus in the MT-2 cell line in et al., 1985) and with HTLV-1-associated myelopathy Japan by Yoshida et al. (1982). MT2 reacted positively (HAM) in Japan by Osame M et al. (1986). TSP and to sera from ATL patients. Particle fraction with similar HAM are now identified as identical myelopathies, density to animal retrovirus was purified from MT-2 which have lesions in the spinal cord. Interestingly, ATL culture fluid, and cDNA was prepared with the reverse and HAM/TSP are mutually exclusive. Onset of either transcriptase activity endogenous to those particles. This disease may depend on HLA types which determine the cDNA preparation was demonstrated to hybridize with magnitude of immune response to HTLV-1, or may cellular DNA of MT-2 but not of other cells. Thus, the depend on modes of infection, mucosal exposure to virus-like particles released from MT-2 cells were shown to contain both RNA templates and reverse transcrip- tase, and MT-2 cells had DNA sequence which hybridized with RNA from the particles, that is, the LTR gag pol env pX LTR integrated DNA. p24 and p19 were also Gag, Pol identified as components of the particles, which are 3 known today as viral core proteins. Thus, a retrovirus Env was convincingly demonstrated in MT-2 cells, and this retrovirus was reported as adult T-cell leukemia virus (ATLV). The US HTLV and Japanese ATLV were later mRNA Repression demonstrated to be identical at the sequence level and Tax 2 Activation named as HTLV-1 (Watanabe et al., 1984). 1 Rex Figure 1 Feed back regulation of HTLV-1 expression by Tax Evidences for new retrovirus and Rex. Spontaneous viral expression of Tax from doubly spliced viral transcript. (1) Tax further activates subsequent viral Generally, replication competent retroviruses have their transcription; (2) Rex encoded by the same mRNA as Tax suppresses splicing of viral RNA, (3) thus accumulated unspliced proviral genome organized as LTR-gag-pol-env-LTR, mRNAs express Gag, Pol and Env proteins, and then followed which respectively encode the viral core proteins, reverse by down-regulation of Tax/Rex expression and by shut-off of transcriptase and envelope proteins. The replication transcription

Oncogene Discovery of HTLV-1, the first human retrovirus M Yoshida 5933 HTLV-1 or primary viral infection in the peripheral Accordingly, the viral is efficient at blood (Barmak et al., 2003). More studies are appar- the initial stage of infection/gene expression and then is ently required. moderated quickly before host immunologic surveil- Other diseases including uveitis, arthropathy and lance could be effectively triggered against infected cells. Sjogren syndrome were also discussed in possible Such feedback regulation through the viral products was association with HTLV-1 infection, but further study uniquely described first for HTLV-1. is needed for the conclusion. Additional new accessory proteins were identified including p12, p35 and some others, which were encoded by of the viral message (Koralnik Unique regulation in the et al., 1992). These genes seemed not essential for the HTLV-1 has an extra sequence pX, in addition to gag, viral replication in vitro, but might be significant in pol and env, required for viral replication. However, pX pathogenesis. Physiologies of these proteins are still shows no sequence homology to host cell DNA, and under investigation. thus is not a typical oncogene. Molecular studies on pX ultimately revealed a unique regulatory system essential Etiology for ATL for HTLV-1 replication. In brief, pX encodes three proteins, Tax, Rex and p21, in overlapping reading The most important aspect of the new retroviral frames. Tax is a transcriptional activator of the viral isolation was not just novelty but an etiology for a genome (Kiyokawa et al., 1984; Sodroski et al., 1984; human leukemia. However, etiological proof for a Fujisawa et al., 1985) and Rex is a splicing suppressor of human disease is generally not easy unless an animal the viral transcripts (Kiyokawa et al., 1985; Seiki et al., model is available. Here, molecular biology provided 1985). The combination of these two proteins regulates effective tools. Association of HTLV-1 with ATL was sequential and transitory viral expression (Hidaka et al., quickly demonstrated after viral identification by pre- 1988; Seiki et al., 1988). ceding findings on unique antibodies in ATL as As summarized in Figure 2, an initial HTLV-1 introduced above. transcript is fully spliced into pX mRNA which encodes The most critical question thereafter was whether a termed p40-Tax and p27-Rex. Tax trans- ‘close association of HTLV-1 with ATL’ reflects its activates the transcription of the viral genome; thus viral causative role or whether the virus was just a passenger. expression is potently enhanced. Next, p27-Rex, which The nature of provirus integration of the retroviruses is encoded by the same pX mRNA in an alternate provided a critical tool for the discrimination. The reading frame, accumulates and suppresses splicing of retroviral are generally reverse transcribed into the viral transcripts. As a consequence, unspliced gag- provirus DNA, and the proviral genomes are integrated pol-env and env mRNAs are expressed, and the viral into host cell DNA at random sites. Since a tumor structural proteins are produced. The suppression of the originates from unlimited expansion of a single malig- viral RNA splicing reduces the level of fully spliced pX nant cell, the site for the proviral integration into tumor mRNA that codes for the trans-activator Tax, and thus cells would be uniform in individuals if the retroviral results in downregulation of viral gene expression. infection plays a causative role; but if the virus fortuitously infects leukemic cells, then the integration sites would be random. Southern blot analysis of Targets for Targeted process Phenotypes patients’ leukemic cell DNA clearly indicated clonal Tax binding HTLV Cell integration in each patient revealing two distinct bands Transcription Proliferation, Apoptosis, with cellular franking sequences. This finding clearly CBP-CREB LTR supported the virus playing a causative role in ATL p300-NF-kB c-fos, IL-6, IL-2Ra (Yoshida et al., 1984). Virtually all ATL cases were Bcl-x CBP-SRF clonally infected leukemic cells; therefore, the conclu- Transcription sion for a ‘causative role’ became generally accepted. As CBP-E47 P18ink4 controls, the sites for the integration in viral carriers are p300-p53 Bax p53-dep, DNA pol random except only in a few cases which show clonal p16-Cdk4 Cell cycle Signaling integration with higher vial burden. p15-Cdk4 Rb/E2F, Apc, PCNA hDlg Molecular pathogenesis of ATL hsMad-1 Cell cycle Check point DNA Topo-1 Animal retroviruses induce tumors basically through one of two mechanisms: either activation through a Malignant Transformation ‘viral oncogene’ or ‘insertional activation’ of a cellular Figure 2 Pleiotropic functions of Tax co-operate for malignant gene such as a proto-oncogene (Teich et al., 1985). transformation targeting many molecules and affecting many Neither mechanism was supported by early studies on signaling pathways. Most of the target genes promote cells towards HTLV-1 and ATL cells (Seiki et al., 1984). Naturally more proliferation and mutation, and reduce cellular apoptosis allowing for enhancement to fix into the cellular most attention has been focused on the Tax protein genome. These genome changes occur at a low and transitory because it was implicated as a potent transcriptional level in a limited in vivo cell population activator for the viral gene expression. In fact, Tax was

Oncogene Discovery of HTLV-1, the first human retrovirus M Yoshida 5934 reported to transform Rat-1 cells, a rat fibroblastic cell p16INK4 and p15INK4, which are otherwise frequently line (Pozzatti et al., 1990; Tanaka et al., 1990), and to deleted or hypermethylated in many human tumors. induce tumors in transgenic mice (Nerenberg et al., Tax was also found to bind to another tumor-suppressor 1987). protein, hDlg, and interfere with its interaction with another tumor-suppressor protein APC (Suzuki et al., Pleiotropic functions of Tax 1999). Through this binding, Tax affects the which controls cell proliferation. Transcriptional activation and repression Tax was a potent transcriptional activator for the viral gene Suppression of apoptosis, DNA repair and cell cycle expression. So, the natural expectation was that it checkpoint T-cell lines infected with HTLV-1 are would activate cellular gene(s). The first indication for resistant to apoptotic signals (Copeland et al., 1994). the activation of cellular gene was very intriguing (Inoue The mechanisms for this resistance were proposed to be et al., 1986). It was already known that ATL cells activation of NF-kB, transcriptional activation of Bcl-X express a high level of IL-2 receptor (Uchiyama et al., gene and repression of Bax gene. Most likely all or some 1985; a and b subunits for the receptor was not yet of these pathways cooperate together in infected Tcells. known), which is essential for T-cell growth; this Accumulation of mutations was enhanced by Tax suggested that the constitutive expression of IL-2R expression in an indicator gene exogenously inserted in might support abnormal cell growth of leukemic cells. rat cell line (Miyake et al., 1999). This phenotype may Tax was found to robustly activate a involve reduction of DNA repair activity and/or reporter construct consisting of IL-2R promoter and suppression of checkpoint activity in infected cells. reporter gene, and also induce expression of cellular IL- In this respect, Tax represses transcription of DNA 2R gene in a certain T-cell line through activation of polymerase b (Jeang et al., 1990) and inhibits NF-kB. This was exciting evidence which demonstrated DNA topoisomerase I (Suzuki et al., 2000), reducing how viral infection induces and supports abnormal the DNA repair activity. Furthermore, Tax binds to growth of ATL cells, although it is realized today that hsMad1 and suppresses G2/M cell cycle checkpoint (Jin the activation of IL-2Ra may not be sufficient to explain et al., 1998). The latter function of Tax accelerates cells the tumor states. This finding consequently triggered a into and through the mitotic phase without checking for huge numbers of investigators to uncover cellular gene chromosomal abnormalities. This property of Tax targets, which might explain abnormal cellular pheno- prevents the cell from either repairing chromosomal types. Today, many target genes have been identified abnormalities or sending unrepaired cells into apoptosis. including those involved in cell cycle, lymphokine/ receptor systems, apoptosis and differentiation. Thus, Pleiotropic functions of Tax cooperate as tumor promo- a mechanistic summary of Tax action is that it binds ter After uncovering so many divergent functions of enhancer-binding proteins such as NF-kB and CREB Tax, one can see a common directional theme on cellular and transcriptional cofactors such as CBP, and that it proliferation. That is, each of the different categories of bridges several of these factors to accelerate complex affected function in infected cells is directed towards formation on enhancer–promoter DNA, and thereby promotion of accelerated cell cycle and proliferation. enhances transcriptional initiation. When Tax cannot Therefore, it can be proposed that these multiple bind either partner, Tax will repress the transcription. functions of Tax cooperate together for malignant The latter case seems to be operating in Tax-induced transformation of HTLV-1-infected cells. Taken repression of transcription of DNA polymerase b (Jeang together, Tax induces abnormal cell growth by activa- et al., 1990). This unexpected latter effect raises the ting growth-promoting genes, by repressing growth- hypothesis that Tax-expressing cells would suffer from suppressing genes and also by inhibiting tumor insufficient DNA repair, and thus accumulate mutations suppressor proteins. During induced abnormal cell easily. proliferation, Tax also suppresses DNA repair capacity and bypasses cell cycle checkpoint by inactivation of Inhibition of tumor-suppressor proteins Study on pro- checkpoint function and thus enhances accumulation of tein–protein interaction focusing on a specific domain mutations. Furthermore, Tax inhibits apoptotic cell for understanding transcriptional activation (Hirai death even in cells with abnormally damaged . et al., 1994) opened up a new category of Tax function Over repeated cell cycles, some cells will fortuitously that targets tumor-suppressor proteins. Tax binds to accumulate the right combination of mutated DNA, and inactivates p16INK4 and p15INK4, which were which would trigger transformation and progress into known as tumor-suppressor proteins that keep the Rb malignant conversion. It can also be said that pleiotrop- signaling pathway active (Suzuki et al., 1996). As a ism of Tax might correspond to the multiple steps result, Cdk4 is activated and phosphorylates and required for tumorigenesis in general cancers, which inactivates the Rb protein. Inactivated Rb releases take place sequentially and at a low rate of incidence. E2F, and released E2F activates transcription of various genes required for DNA replication, thus compelling Is the laboratory bench far from the clinical bed? cells into a G1–S transition. The observation holds significant implication because it indicates that Tax Molecular biology of Tax is mainly based on studies in abrogates cell cycle arrest in G1 phase by inactivating vitro, which indicate that Tax targets various cellular

Oncogene Discovery of HTLV-1, the first human retrovirus M Yoshida 5935 genes frequently altered in spontaneous tumor cells. through promoter insertion to enhance cellular proto- This, therefore, strongly suggests that Tax would play a oncogene. Consequently, we postulated a new mechanism central role in induction of ATL. However, it is for tumorigenesis in ATL. However, what subsequent generally known that expression of many genes is HTLV-1 study discovered after extensive molecular differentially controlled in cultured cells and in vivo, investigation were mechanisms similar to those used by although the significance of this finding for transforma- DNA tumor viruses. Molecules and signaling pathways tion is not well understood. As a result of the limitations targeted by Tax were and include tumor-suppressor of extrapolating from ex vivo studies, the pathogenic proteins such as p16ink4/Rb, p53, APC, and were similar roles of Tax protein for in vivo leukemia remain to be to those targeted by transforming proteins of DNA carefully established. tumor viruses, Tantigen of SV40, E6/E7 of HPV and E1a of adenovirus. Furthermore, as generally accepted, the Leukemic cells without Tax expression It is reported actions of viral transforming genes are also shared with that a vast majority (over 95%) of ATL cells in vivo are the activation mechanisms identified in spontaneously absolutely negative for HTLV-1 expression (Kinoshita arising or genetically induced tumors in humans. These et al., 1989). However, most of them express some cellular similarities between cellular alterations induced HTLV-1, genes targeted by Tax such as IL-2Ra. This indicates that DNA tumor viruses and in spontaneous tumors strongly in vivo expression of IL-2Ra does not continuously require suggest that general transforming mechanisms are Tax. The molecular mechanisms to explain such phenom- operative in ATL induction. ena are not well understood. Chromosomal translocation at the T-cell receptor region was identified in some cases of ATL (Isobe et al., 1990), but such isolated data are not Prevention of HTLV-1 infection sufficient to establish a general mechanism. We need to Upon the identification of a new retrovirus, virologists identify one or more abnormalities fixed in leukemic cells and oncologists excitedly harbored the expectation that that functionally mimic the role of Tax in activating the we would be able to prevent virus infection and expression of a multitude of genes. replication and thus ultimately prevent the development of specific leukemia. This goal has been partly achieved Tax is not sufficient to dictate gene expression in vivo by elucidating and preventing the infection pathways. The viral expression in vivo is also mysterious in another HTLV-1 is mainly transmitted from (1) a mother to her aspect. T-cell lines infected with HTLV-1 were estab- child (Sugiyama et al., 1986), (2) husband to wife lished, and many of them express the viral genome (Tajima et al., 1986) and (3) donor to recipient of blood continuously at high levels. On the other hand, transfusion (Okochi et al., 1984). Viral transmission is regardless of whether the cell is leukemic or nonleuke- thought to require the transfer of live infected T mic, HTLV-1 is expressed in vivo at extremely low levels lymphocytes, that is, Tcells in breast milk, Tcells in and in many cases can be detected only by PCR. semen and fresh Tcells in blood carriers of HTLV-1 However, once the cells are harvested ex vivo, viral . Therefore, cessation of breast milk is now expression is frequently and quickly switched on. The applied to prevent HTLV-1 infection from an infected mechanism as to how viral expression is maintained mother to her child (Hino et al., 1994). In Japanese in vivo in a mostly latent form is not understood. The endemic areas, pregnant mothers are mostly examined elucidation of such mechanism may provide us with a for HTLV-1 antibodies, and once the mother is possible clue as to how to limit viral expression, identified to be infected, bottlefeeding is strongly ultimately enabling prevention of pathogenesis. suggested. The trial is now working quite well and is rather successful in preventing HTLV-1 infection in the Targeted genes are oriented for proliferation next generation. However, there is a concern whether this strategy can be the best choice in developing Most of the genes so far identified as the targets of Tax regions, where breast milk is valuable in transferring a are those for promoting cell cycle and proliferation. mother’s immunity to her children to protect against Abnormality of these genes might explain abnormal various infectious diseases. growth of infected cells. However, clonal expansion of To combat the transmission through blood trans- leukemic cells is not explained by these genes. No one fusion, sero-positive bloods are excluded from the blood knows what kind of mechanism is involved additionally. bank and infection through this pathway is now almost Also, we can ask whether unlimited proliferation is completely gone in Japan. development for equal to ‘malignancy’. This is a very basic question on preventing new infection has been challenging at the the nature of tumors and can also be asked about basic study level, and has achieved some success in general tumorigenesis. animal studies, but no vaccine has yet come to fruition for protecting humans. Expected in 1981 and answered in 2005 Prevention of pathogenesis Understanding the leukemogenesis A total of 12 million individuals are estimated to be HTLV-1 was characterized as a causative agent of ATL, HTLV-1 carriers throughout the world. Therefore, but the virus has no oncogene and does not function development of any strategy to halt viral replication

Oncogene Discovery of HTLV-1, the first human retrovirus M Yoshida 5936 and/or viral gene expression would save a half million HTLV-1 offers valuable clues for in vivo study of human carriers from death by ATL. Although molecular cancers has progressed rather extensively, no possible strategy for this has been effective. HTLV-1 provirus During the quarter century since the discovery of ATL, replicates very poorly through free virions in vivo, but the HTLV/ATL biological model has provided great the provirus is efficiently amplified through abnormal opportunities for scientists to study tumorigenesis replication of infected Tcells whose DNA carries the directly in humans. Points of particular note are (1) it proviral genomes. Therefore, the task of preventing is efficient and reliable to identify high-risk individuals pathogenesis remains extremely difficult. for ATL, (2) it is possible to follow malignant steps of smoldering, chronic and acute phase of ATL, (3) it is Treatment of ATL possible to obtain samples repeatedly from specific individuals, and (4) it is easy to isolate a single Acute ATL is aggressive T-cell leukemia and no effective malignant cell intact. In view of these advantages, it treatment is available. Unfortunately, even after ex- should be possible for scientists to answer key questions tensive advances in the molecular biology of HTLV-1 in human tumorigenesis. Even though the ATL patient and ATL, no effective treatment for ATL is available. population is small, from this small group valuable Although some therapies such as monoclonal antibody understanding of information applicable to general and combination therapies with available anticancer tumorigenesis of other cancers can be accrued. The drugs were reported to be transiently effective, no recent years have seen the total genome sequencing convincing therapeutic benefits have been established. for humans and the advent of molecular and genomic Long-term strategic challenges, such as genome-wide technologies such as genome-wide molecular profiling. molecular profiling for understanding the landscape of Unanswered questions and prematurely derived con- ATL cells in vivo and molecular targeting strategy for clusions may merit revisiting with new modern identifying new drugs, are significant future objectives. technologies.

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Oncogene