Triple combination of BET plus PI3K and NF-κB inhibitors exhibit synergistic activity in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell lymphoproliferative malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). ATL is an orphan disease with no curative drug treatment regimens urgently needing new combination therapy. HTLV-1-infected cells rely on viral proteins, Tax and HBZ (HTLV-1-b-ZIP factor), to activate the transcription of various host genes that are critical for promoting leukemic transformation. Inhibition of bromodomain and extraterminal motif (BET) protein was previously shown to collapse the transcriptional network directed by BATF3 super-enhancer and thereby induced ATL cell apoptosis. In the current work, by using xenograft, ex vivo, and in vitro models, we demonstrated that I-BET762 (BETi) synergized with copanlisib (PI3Ki) and bardoxolone methyl (NF-κBi) to dramatically decrease the growth of ATL cells. Mechanistically, the triple combination exhibited synergistic activity by down-regulating the expression of c-MYC while upregulating the level of the glucocorticoid-induced leucine zipper (GILZ). The triple combination also enhanced apoptosis induction by elevating the expression of active caspase-3 and cleaved PARP. Importantly, the triple combination prolonged the survival of ATL-bearing xenograft mice and inhibited the proliferation of ATL cells from peripheral blood mononuclear cells (PBMCs) of both acute and smoldering/chronic ATL patients. Therefore, our data provide the rationale for a clinical trial exploring the multiagent combination of BET, PI3K/AKT, and NF-κB inhibitors for ATL patients and expands the potential treatments for this recalcitrant malignancy.

Genome-wide CRISPR screen identifies CDK6 as a therapeutic target in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild-type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.

Germinal epimutation of Fragile Histidine Triad (FHIT) gene is associated with progression to acute and chronic adult T-cell leukemia diseases.

BACKGROUND: Human T cell Leukemia virus type 1 (HTLV-I) is etiologically linked to adult T cell leukemia/lymphoma (ATL) and an inflammatory neurodegenerative disease called HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The exact genetic or epigenetic events and/or environmental factors that influence the development of ATL, or HAM/TSP diseases are largely unknown. The tumor suppressor gene, Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), is frequently lost in cancer through epigenetic modifications and/or deletion. FHIT is a tumor suppressor acting as genome caretaker by regulating cellular DNA repair. Indeed, FHIT loss leads to replicative stress and accumulation of double DNA strand breaks. Therefore, loss of FHIT expression plays a key role in cellular transformation. METHODS: Here, we studied over 400 samples from HTLV-I-infected individuals with ATL, TSP/HAM, or asymptomatic carriers (AC) for FHIT loss and expression. We examined the epigenetic status of FHIT through methylation specific PCR and bisulfite sequencing; and correlated these results to FHIT expression in patient samples. RESULTS: We found that epigenetic alteration of FHIT is specifically found in chronic and acute ATL but is absent in asymptomatic HTLV-I carriers and TSP/HAM patients' samples. Furthermore, the extent of FHIT methylation in ATL patients was quantitatively comparable in virus-infected and virus non-infected cells. We also found that longitudinal HTLV-I carriers that progressed to smoldering ATL and descendants of ATL patients harbor FHIT methylation. CONCLUSIONS: These results suggest that germinal epigenetic mutation of FHIT represents a preexisting mark predisposing to the development of ATL diseases. These findings have important clinical implications as patients with acute ATL are rarely cured. Our study suggests an alternative strategy to the current "wait and see approach" in that early screening of HTLV-I-infected individuals for germinal epimutation of FHIT and early treatment may offer significant clinical benefits.

Short-course IL-15 given as a continuous infusion led to a massive expansion of effective NK cells: implications for combination therapy with antitumor antibodies.

BACKGROUND: Full application of cytokines as oncoimmunotherapeutics requires identification of optimal regimens. Our initial effort with intravenous bolus recombinant human interleukin-15 (rhIL-15) was limited by postinfusional reactions. Subcutaneous injection and continuous intravenous infusion for 10 days (CIV-10) provided rhIL-15 with less toxicity with CIV-10 giving the best increases in CD8+ lymphocytes and natural killer (NK) cells. To ease rhIL-15 administration, we shortened time of infusion. Treatment with rhIL-15 at a dose of 3-5 µg/kg as a 5-day continuous intravenous infusion (CIV-5) had no dose-limiting toxicities while effector cell stimulation was comparable to the CIV-10 regimen. METHODS: Eleven patients with metastatic cancers were treated with rhIL-15 CIV-5, 3 µg (n=4), 4 µg (n=3), and 5 µg/kg/day (n=4) in a phase I dose-escalation study (April 6, 2012). RESULTS: Impressive expansions of NK cells were seen at all dose levels (mean 34-fold), including CD56bright NK cells (mean 144-fold for 4 µg/kg), as well as an increase in CD8+ T cells (mean 3.38-fold). At 5 µg/kg/day, there were no dose-limiting toxicities but pulmonary capillary leak and slower patient recovery. This led to our choice of the 4 µg/kg as CIV-5 dose for further testing. Cytolytic capacity of CD56bright and CD56dim NK cells was increased by interleukin-15 assayed by antibody-dependent cellular cytotoxicity (ADCC), natural cytotoxicity and natural killer group 2D-mediated cytotoxicity. The best response was stable disease. CONCLUSIONS: IL-15 administered as CIV-5 substantially expanded NK cells with increased cytotoxic functions. Tumor-targeting monoclonal antibodies dependent on ADCC as their mechanism of action including alemtuzumab, obinutuzumab, avelumab, and mogamulizumab could benefit from those NK cell expansions and provide a promising therapeutic strategy. TRIAL REGISTRATION NUMBERS: NCT01572493, NCT03759184, NCT03905135, NCT04185220 and NCT02689453.

Enhanced efficacy of JAK1 inhibitor with mTORC1/C2 targeting in smoldering/chronic adult T cell leukemia.

Adult T-cell leukemia (ATL) is an aggressive T-cell lymphoproliferative malignancy of regulatory T lymphocytes (Tregs), caused by human T-cell lymphotropic virus 1 (HTLV-1). Interleukin 2 receptor alpha (IL-2Rα) is expressed in the leukemic cells of smoldering/chronic ATL patients, leading to constitutive activation of the JAK/STAT pathway and spontaneous proliferation. The PI3K/AKT/mTOR pathway also plays a critical role in ATL cell survival and proliferation. We previously performed a high-throughput screen that demonstrated additive/synergistic activity of Ruxolitinib, a JAK1/2 inhibitor, with AZD8055, an mTORC1/C2 inhibitor. However, effects of unintended JAK2 inhibition with Ruxolitinib limits it therapeutic potential for ATL patients, which lead us to evaluate a JAK1-specific inhibitor. Here, we demonstrated that Upadacitinib, a JAK-1 inhibitor, inhibited the proliferation of cytokine-dependent ATL cell lines and the expression of p-STAT5. Combinations of Upadacitinib with either AZD8055 or Sapanisertib, mTORC1/C2 inhibitors, showed anti-proliferative effects against cytokine-dependent ATL cell lines and synergistic effect with reducing tumor growth in NSG mice bearing IL-2 transgenic tumors. Importantly, the combination of these two agents inhibited ex vivo spontaneous proliferation of ATL cells from patients with smoldering/chronic ATL. Combined targeting of JAK/STAT and PI3K/AKT/mTOR pathways represents a promising therapeutic intervention for patients with smoldering/chronic ATL.

A novel model of alternative NF-κB pathway activation in anaplastic large cell lymphoma.

Aberrant activation of NF-κB is the most striking oncogenic mechanism in B-cell lymphoma; however, its role in anaplastic large cell lymphomas (ALCL) has not been fully established and its activation mechanism(s) remain unclear. Using ALCL cell line models, we revealed the supporting roles for NFKB2 and the NIK pathway in some ALCL lines. To investigate the detailed activation mechanisms for this oncogenic pathway, we performed specifically designed alternative NF-κB reporter CRISPR screens followed by the RNA-seq analysis, which led us to identify STAT3 as the major mediator for NIK-dependent NF-κB activation in ALCL. Consistently, p-STAT3 level was correlated with NFKB2 nuclear accumulation in primary clinical samples. Mechanistically, we found that in NIK-positive ALK- ALCL cells, common JAK/STAT3 mutations promote transcriptional activity of STAT3 which directly regulates NFKB2 and CD30 expression. Endogenous expression of CD30 induces constitutive NF-κB activation through binding and degrading of TRAF3. In ALK+ ALCL, the CD30 pathway is blocked by the NPM-ALK oncoprotein, but STAT3 activity and resultant NFKB2 expression can still be induced by NPM-ALK, leading to minimal alternative NF-κB activation. Our data suggest combined NIK and JAK inhibitor therapy could benefit patients with NIK-positive ALK- ALCL carrying JAK/STAT3 somatic mutations.

Essential role of the linear ubiquitin chain assembly complex and TAK1 kinase in A20 mutant Hodgkin lymphoma.

More than 70% of Epstein-Barr virus (EBV)-negative Hodgkin lymphoma (HL) cases display inactivation of TNFAIP3 (A20), a ubiquitin-editing protein that regulates nonproteolytic protein ubiquitination, indicating the significance of protein ubiquitination in HL pathogenesis. However, the precise mechanistic roles of A20 and the ubiquitination system remain largely unknown in this disease. Here, we performed high-throughput CRISPR screening using a ubiquitin regulator-focused single-guide RNA library in HL lines carrying either wild-type or mutant A20. Our CRISPR screening highlights the essential oncogenic role of the linear ubiquitin chain assembly complex (LUBAC) in HL lines, which overlaps with A20 inactivation status. Mechanistically, LUBAC promotes IKK/NF-κB activity and NEMO linear ubiquitination in A20 mutant HL cells, which is required for prosurvival genes and immunosuppressive molecule expression. As a tumor suppressor, A20 directly inhibits IKK activation and HL cell survival via its C-terminal linear-ubiquitin binding ZF7. Clinically, LUBAC activity is consistently elevated in most primary HL cases, and this is correlated with high NF-κB activity and low A20 expression. To further understand the complete mechanism of NF-κB activation in A20 mutant HL, we performed a specifically designed CD83-based NF-κB CRISPR screen which led us to identify TAK1 kinase as a major mediator for NF-κB activation in cells dependent on LUBAC, where the LUBAC-A20 axis regulates TAK1 and IKK complex formation. Finally, TAK1 inhibitor Takinib shows promising activity against HL in vitro and in a xenograft mouse model. Altogether, these findings provide strong support that targeting LUBAC or TAK1 could be attractive therapeutic strategies in A20 mutant HL.

IL-2 receptors preassemble and signal in the ER/Golgi causing resistance to antiproliferative anti-IL-2Rα therapies.

Interleukin-2 (IL-2) and IL-15 play pivotal roles in T cell activation, apoptosis, and survival, and are implicated in leukemias and autoimmune diseases. Their heterotrimeric receptors share their ß- and γc-chains, but have distinct α-chains. Anti-IL-2Rα (daclizumab) therapy targeting cell surface-expressed receptor subunits to inhibit T cell proliferation has only brought limited success in adult T cell leukemia/lymphoma (ATL) and in multiple sclerosis. We asked whether IL-2R subunits could already preassemble and signal efficiently in the endoplasmic reticulum (ER) and the Golgi. A combination of daclizumab and anti-IL-2 efficiently blocked IL-2-induced proliferation of IL-2-dependent wild-type (WT) ATL cells but not cells transfected with IL-2, suggesting that in IL-2-producing cells signaling may already take place before receptors reach the cell surface. In the Golgi fraction isolated from IL-2-producing ATL cells, we detected by Western blot phosphorylated Jak1, Jak3, and a phosphotyrosine signal attributed to the γc-chain, which occurred at much lower levels in the Golgi of WT ATL cells. We expressed EGFP- and mCherry-tagged receptor chains in HeLa cells to study their assembly along the secretory pathway. Confocal microscopy, Förster resonance energy transfer, and imaging fluorescence cross-correlation spectroscopy analysis revealed partial colocalization and molecular association of IL-2 (and IL-15) receptor chains in the ER/Golgi, which became more complete in the plasma membrane, further confirming our hypothesis. Our results define a paradigm of intracellular autocrine signaling and may explain resistance to antagonistic antibody therapies targeting receptors at the cell surface.

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

Adult T cell leukemia/lymphoma (ATLL) is a frequently incurable disease associated with the human lymphotropic virus type I (HTLV-I). RNAi screening of ATLL lines revealed that their proliferation depends on BATF3 and IRF4, which cooperatively drive ATLL-specific gene expression. HBZ, the only HTLV-I encoded transcription factor that is expressed in all ATLL cases, binds to an ATLL-specific BATF3 super-enhancer and thereby regulates the expression of BATF3 and its downstream targets, including MYC. Inhibitors of bromodomain-and-extra-terminal-domain (BET) chromatin proteins collapsed the transcriptional network directed by HBZ and BATF3, and were consequently toxic for ATLL cell lines, patient samples, and xenografts. Our study demonstrates that the HTLV-I oncogenic retrovirus exploits a regulatory module that can be attacked therapeutically with BET inhibitors.

Chimeric antigen receptor modified T cells that target chemokine receptor CCR4 as a therapeutic modality for T-cell malignancies.

With the emerging success of treating CD19 expressing B cell malignancies with ex vivo modified, autologous T cells that express CD19-directed chimeric antigen receptors (CAR), there is intense interest in expanding this evolving technology to develop effective modalities to treat other malignancies including solid tumors. Exploiting this approach to develop a therapeutic modality for T cell malignancies for which the available regimens are neither curative, nor confer long term survival we generated a lentivirus-based CAR gene transfer system to target the chemokine receptor CCR4 that is over-expressed in a spectrum of T cell malignancies as well as in CD4+ CD25+ Foxp3+ T regulatory cells that accumulate in the tumor microenvironment constituting a barrier against anti-tumor immunity. Ex vivo modified, donor-derived T cells that expressed CCR4 directed CAR displayed antigen-dependent potent cytotoxicity against patient-derived cell lines representing ATL, CTCL, ALCL and a subset of HDL. Furthermore, these CAR T cells also eradicated leukemia in a mouse xenograft model of ATL illustrating the potential utility of this modality in the treatment of a wide spectrum of T cell malignancies.

Cytokine receptor signaling is required for the survival of ALK- anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations.

Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)- anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK- ALCL, we treated ALK- cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK- ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK- ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK- ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK- ALCL who are phosphorylated STAT3^.

Augmented efficacy of brentuximab vedotin combined with ruxolitinib and/or Navitoclax in a murine model of human Hodgkin's lymphoma.

Despite relative success of therapy for Hodgkin's lymphoma (HL), novel therapeutic agents are needed for patients with refractory or relapsed disease. Recently, anti-PD1 immunotherapy or treatment with the anti-CD30 toxin conjugate brentuximab vedotin (BV) have been associated with remissions; however, the median responses of complete responses (CRs) with the latter were only 6.7 mo. To obtain curative therapy, other effective agents, based on HL biology, would have to be given in combination with BV. Hodgkin's Reed-Sternberg (HRS) cells secrete cytokines including IL-6 and -13, leading to constitutive activation of JAK/STAT signaling. In the present study the JAK1/2 inhibitor ruxolitinib reduced phosphorylation of STAT3 and STAT6 and expression of c-Myc in the HL cell line HDLM-2. These changes were enhanced when, on the basis of a matrix screen of drug combinations, ruxolitinib was combined with the Bcl-2/Bcl-xL inhibitor Navitoclax. The combination augmented expression of Bik, Puma, and Bax, and attenuated Bcl-xL expression and the phosphorylation of Bad. The use of the two-agent combination of either ruxolitinib or Navitoclax with BV or the three-agent combination strongly activated Bax and increased activities of cytochrome c and caspase-9 and -3 that, in turn, led to cleavage of poly(ADP ribose) polymerase and Mcl-1. Either ruxolitinib combined with Navitoclax or BV alone prolonged survival but did not cure HDLM-2 tumor-bearing mice, whereas BV combined with ruxolitinib and/or with Navitoclax resulted in a sustained, complete elimination of the HDLM-2 HL. These studies provide scientific support for a clinical trial to evaluate BV combined with ruxolitinib in select patients with HL.

Selective targeting of JAK/STAT signaling is potentiated by Bcl-xL blockade in IL-2-dependent adult T-cell leukemia.

Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. HTLV-1-encoded protein Tax (transactivator from the X-gene region) up-regulates Bcl-xL (B-cell lymphoma-extra large) expression and activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems, resulting in amplified JAK/STAT signaling. Inhibition of JAK signaling reduces cytokine-dependent ex vivo proliferation of peripheral blood mononuclear cells (PBMCs) from ATL patients in smoldering/chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in ATL cell lines. The selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen combined with >450 potential therapeutic agents, and Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. The combination was noted to strongly activate BAX (Bcl-2-associated X protein), effect mitochondrial depolarization, and increase caspase 3/7 activities that lead to cleavage of PARP (poly ADP ribose polymerase) and Mcl-1 (myeloid cell leukemia 1). Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, whereas the combination dramatically lowered tumor burden and prolonged survival in an ATL murine model. This combination strongly blocked ex vivo proliferation of five ATL patients' PBMCs. These studies provide support for a therapeutic trial in patients with smoldering/chronic ATL using a drug combination that inhibits JAK signaling and antiapoptotic protein Bcl-xL.

A new domain in the Toll/IL-1R domain-containing adaptor inducing interferon-ß factor protein amino terminus is important for tumor necrosis factor-α receptor-associated factor 3 association, protein stabilization and interferon signaling.

Toll/IL-1R domain-containing adaptor inducing interferon-ß (IFN-ß) factor (TRIF) is a key adaptor for Toll-like receptor (TLR) 3 and TLR4 signaling. Using a novel cDNA isolate encoding a TRIF protein with a 21-residue deletion (Δ160-181) from its amino-terminal half, we investigated the impact of this deletion on TRIF functions. Transfection studies consistently showed higher expression levels of the (Δ160-181) TRIF compared to wild-type (wt) TRIF, an effect unrelated to apoptosis, cell lines or plasmid amplification. Colocalization of wt and (Δ160-181) TRIF proteins led to a dramatic reduction of their respective expressions, suggesting that wt/(Δ160-181) TRIF heterocomplexes are targeted for degradation. We demonstrated that wt TRIF associates with tumor necrosis factor-α receptor-associated factor 3 (TRAF3) better than (Δ160-181) TRIF, culminating in its greater ubiquitination and proteolysis. This explains, in part, the differential expression levels of the two TRIF proteins. Despite higher expression levels in transfected cells, (Δ160-181) TRIF inefficiently transactivated the IFN pathway, whereas the nuclear factor-κB (NF-κB) pathway activation remained similar to that by wt TRIF. In coexpression studies, (Δ160-181) TRIF marginally contributed to the IFN pathway activation, but still enhanced NF-κB signaling with wt TRIF. Therefore, this 21 amino acid sequence is crucial for TRAF3 association, modulation of TRIF stability and activation of the IFN pathway.

Phase 1 trial of IL-15 trans presentation blockade using humanized Mikß1 mAb in patients with T-cell large granular lymphocytic leukemia.

In the present study, Hu-Mikß1, a humanized mAb directed at the shared IL-2/IL-15Rß subunit (CD122) was evaluated in patients with T-cell large granular lymphocytic (T-LGL) leukemia. Hu-Mikß1 blocked the trans presentation of IL-15 to T cells expressing IL-2/IL-15Rß and the common γ-chain (CD132), but did not block IL-15 action in cells that expressed the heterotrimeric IL-15 receptor in cis. There was no significant toxicity associated with Hu-Mikß1 administration in patients with T-LGL leukemia, but no major clinical responses were observed. One patient who had previously received murine Mikß1 developed a measurable Ab response to the infused Ab. Nevertheless, the safety profile of this first in-human study of the humanized mAb to IL-2/IL-15Rß (CD122) supports its evaluation in disorders such as refractory celiac disease, in which IL-15 and its receptor have been proposed to play a critical role in the pathogenesis and maintenance of disease activity.

Development of an IL-15-autocrine CD8 T-cell leukemia in IL-15-transgenic mice requires the cis expression of IL-15Rα.

IL-15 has growth-promoting effects on select lymphoid subsets, including natural killer (NK) cells, NK T cells, intraepithelial lymphocytes (IELs), CD8 T cells, and γδ-T cells. Constitutive expression of murine IL-15 in IL-15-transgenic mice was reported to cause T-NK leukemia. We investigated whether IL-15 expression is sufficient for leukemic transformation using a human IL-15-transgenic (IL-15Tg) mouse model. We noted that 100% of the mice observed over a 2-year period (n > 150) developed fatal expansions of CD8 T cells with NK markers, and determined that these cells expressed IL-15 receptor alpha (IL-15Rα). The expression of IL-15Rα on CD8 T cells appears to be required for uncontrolled aggressive lymphoproliferation, because none of the IL-15Rα(-/-)-IL-15Tg mice that we followed for more than 2 years developed the fatal disease despite controlled expansion of CD8 T cells. In addition, in contrast to IL-15Tg mice, in which leukemia-like CD8 T cells expressed IL-15Rα persistently, acutely activated normal CD8 T cells only transiently expressed IL-15Rα. Inhibition of DNA methylation enabled sustained IL-15Rα expression induced by activation. We present a scenario for IL-15Tg mice in which CD8 T cells that acquire constitutive persistent IL-15Rα expression are at a selective advantage and become founder cells, outgrow other lymphocytes, and lead to the establishment of a leukemia-like condition.