RNA Expression Data from IL-15-/- and IL-15+/+ TAX-LUC Mouse Tumors
ABSTRACT: IL-15 is recognized as a promising candidate for tumor immunotherapy and has been described as both a promoter of cancer and a promoter of anti-cancer immunity. IL-15 was discovered in cells transformed by HTLV-1, the etiologic agent of adult T cell leukemia / lymphoma (ATL) and the human retrovirus that carries the Tax oncogene. We have developed the TAX-LUC mouse model of ATL in which Tax expression drives both malignant transformation and luciferase expression, enabling non-invasive imaging of tumorigenesis in real time. To identify the role of IL-15 in spontaneous development of lymphoma in vivo, an IL-15-/- TAX-LUC strain was developed and examined. The absence of IL-15 resulted in aggressive tumor growth and accelerated mortality and demonstrated that IL-15 was not required for Tax-mediated lymphoma but was essential for anti-tumor immunity. Further analysis revealed a unique transcriptional profile in tumor cells that arise in the absence of IL-15 that included a significant increase in the expression of IL-1α and IL-1α-regulated cytokines. Moreover, anti-IL-1α antibodies and an IL-1 receptor antagonist (Anakinra) were used to interrogate the potential of IL-1α targeted therapies in this model. Taken together, these findings identify IL-15 and IL-1α as therapeutic targets in lymphoma. We used microarrays to compare the gene expression profile of tumors in IL-15-/- TAX-LUC mice to IL-15+/+ TAX-LUC mice RNA was obtained from CD16/32HI and CD16/32LO cells harvested from n=2 IL-15+/+ (control)and n=2 IL-15-/- Tax tumors and was compared to look for alterations in gene expression in malignant and tumor infiltrating cells resulting from loss of IL-15 in vivo
Project description:IL-15 is recognized as a promising candidate for tumor immunotherapy and has been described as both a promoter of cancer and a promoter of anti-cancer immunity. IL-15 was discovered in cells transformed by HTLV-1, the etiologic agent of adult T cell leukemia/lymphoma (ATL) and the human retrovirus that carries the Tax oncogene. We have developed the TAX-LUC mouse model of ATL in which Tax expression drives both malignant transformation and luciferase expression, enabling non-invasive imaging of tumorigenesis in real time. To identify the role of IL-15 in spontaneous development of lymphoma in vivo, an IL-15(-/-) TAX-LUC strain was developed and examined. The absence of IL-15 resulted in aggressive tumor growth and accelerated mortality and demonstrated that IL-15 was not required for Tax-mediated lymphoma but was essential for anti-tumor immunity. Further analysis revealed a unique transcriptional profile in tumor cells that arise in the absence of IL-15 that included a significant increase in the expression of IL-1α and IL-1α-regulated cytokines. Moreover, anti-IL-1α antibodies and an IL-1 receptor antagonist (Anakinra) were used to interrogate the potential of IL-1α targeted therapies in this model. Taken together, these findings identify IL-15 and IL-1α as therapeutic targets in lymphoma.
Project description: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.
Project description:The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein induces growth transformation and is critical for the pathogenesis of the HTLV-1-induced adult T-cell leukemia (ATL). It stimulates the cell cycle and transactivates cellular genes. Here we show that the expression of interleukin-13 (IL-13) is upregulated as a consequence of Tax in HTLV-1-transformed T cells and ATL-derived cultures. IL-13 exerts proliferative and antiapoptotic functions and is linked to leukemogenesis, since it stimulates Hodgkin lymphoma cells by an autocrine mechanism. Overexpression of IL-13 RNA and protein was confirmed in HTLV-1-positive and Tax-transformed cells. Induction of endogenous IL-13 levels in tax-transfected Jurkat cells and in conditional Tax-expressing transformed T lymphocytes suggested that Tax can replace signals required for IL-13 synthesis. For functional analysis, the IL-13 promoter and deletion variants were cloned into luciferase reporter plasmids. Experiments with transfected human T lymphocytes revealed a 16-fold stimulation of the IL-13 promoter by Tax. Experiments with Tax mutants indicated that none of the classical transactivation pathways (SRF, CREB, and NF-kappaB) is sufficient for the transactivation; at least two different Tax functions are required for full transactivation. The IL-13 promoter is stimulated via two elements; one is a NF-AT binding P element, and the other is a putative AP-1 site. The following observations suggest that IL-13 may stimulate HTLV-1-transformed cells by an autocrine mechanism: (i) the HTLV-1-transformed cells express the IL-13 receptor on their surface, and (ii) STAT6, a downstream effector of IL-13 signaling, is constitutively activated. Thus, in summary, Tax, by transactivating the promoter, induces IL-13 overexpression that possibly leads to an autocrine stimulation of HTLV-1-infected cells.
Project description:The retrovirus, human T-cell-lymphotrophic virus-1 (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-I-encoded protein tax constitutively activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems that in turn activate the Jak3 (Janus kinase 3)/STAT5 (signal transducers and activators of transcription 5) pathway, suggesting a therapeutic strategy that involves targeting Jak3. We evaluated the action of the Jak3 inhibitor CP-690,550 on cytokine dependent ex vivo proliferation that is characteristic of peripheral blood mononuclear cells (PBMCs) from select patients with smoldering or chronic subtypes of ATL, or from those with HAM/TSP whose PBMCs are associated with autocrine/paracrine pathways that involve the production of IL-2, IL-9, IL-15, and their receptors. CP-690,550 at 50 nM inhibited the 6-day ex vivo spontaneous proliferation of PBMCs from ATL and HAM/TSP patients by 67.1% and 86.4%, respectively. Furthermore, CP-690,550 inhibited STAT5 phosphorylation in isolated ATL T cells ex vivo. Finally, in an in vivo test of biological activity, CP-690,550 treatment of mice with a CD8 T-cell IL-15-transgenic leukemia that manifests an autocrine IL-15/IL-15R? pathway prolonged the survival duration of these tumor-bearing mice. These studies support further evaluation of the Jak3 inhibitor CP-690,550 in the treatment of select patients with HTLV-I-associated ATL and HAM/TSP.
Project description:Infection with the human T-cell leukemia virus-1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia/lymphoma (ATL). Although the pathogenesis of these disorders is poorly understood, it involves complex interactions with the host immune system. Activation of infected T cells may play an important role in disease pathogenesis through induction of the oncogenic HTLV-1 Tax transactivator protein. To test this hypothesis, we employed transgenic mice in which Tax is regulated by the HTLV-1 LTR. T-cell receptor stimulation of LTR-Tax CD4(+) T cells induced Tax expression, hyper-proliferation, and immortalization in culture. The transition to cellular immortalization was accompanied by markedly increased expression of the antiapoptotic gene, mcl-1, previously implicated as important in T-cell survival. Immortalized cells exhibited a CD4(+)CD25(+)CD3(-) phenotype commonly observed in ATL. Engraftment of activated LTR-Tax CD4(+) T cells into NOD/Shi-scid/IL-2R? null mice resulted in a leukemia-like phenotype with expansion and tissue infiltration of Tax(+), CD4(+) lymphocytes. We suggest that immune activation of infected CD4(+) T cells plays an important role in the induction of Tax expression, T-cell proliferation, and pathogenesis of ATL in HTLV-1-infected individuals.
Project description:T cells infected with human T-cell leukemia virus type 1 (HTLV-1) transform into malignant/leukemic cells and develop adult T-cell leukemia (ATL) after a long latency period. The tax (transactivator from the X-gene region) and HBZ (HTLV-1 bZIP factor) genes of HTLV-1 play crucial roles in the development of ATL. The process and mechanism by which HTLV-1-infected T cells acquire malignancy and develop ATL remain to be elucidated. Constitutive expression of interleukin-2 (IL-2) receptor ?-chain (IL-2R?/CD25), induced by the tax and HBZ genes of HTLV-1, on ATL cells implicates the involvement of IL-2/IL-2R pathway in the growth and development of ATL cells in vivo. However, the leukemic cells in the majority of ATL patients appeared unresponsive to IL-2, raising controversies on the role of this pathway for the growth of ATL cells in vivo. Here, we report the establishment of 32 IL-2-dependent T-cell lines infected with HTLV-1 from 26 ATL patients, including eight leukemic cell lines derived from five ATL patients, while no T-cell lines were established without IL-2. We have shown that the IL-2-dependent ATL cell lines evolved into IL-2-independent/-unresponsive growth phase, resembling ATL cells in vivo. Moreover, the IL-2-dependent non-leukemic T-cell lines infected with HTLV-1 acquired IL-2-independency and turned into tumor-producing cancer cells as with the ATL cell lines. HTLV-1-infected T cells in vivo could survive and proliferate depending on IL-2 that was produced in vivo by the HTLV-1-infected T cells of ATL patients and patients with HTLV-1-associated diseases and, acts as a physiological molecule to regulate T-cell growth. These results suggest that ATL cells develop among the HTLV-1-infected T cells growing dependently on IL-2 and that most of the circulating ATL cells progressed to become less responsive to IL-2, acquiring the ability to proliferate without IL-2.
Project description:We completed clinical trials of rhIL-15 by bolus, subcutaneous, and continuous intravenous infusions (CIV). IL-15 administered by CIV at 2 mcg/kg/day yielded a 38-fold increase in 10- day number of circulating NK cells, a 358-fold increase in CD56bright NK cells and a 5.8-fold increase in CD8 T cells. However, IL-15 preparations administered as monotherapy were ineffective, due to actions of immunological checkpoints and due to the lack of tumor specific targeting by NK cells. To circumvent checkpoints, trials of IL-15 in combination with other anticancer agents were initiated. Tumor-bearing mice receiving IL-15 with antibodies to CTLA-4 and PD-L1 manifested marked prolongation of survival compared to mice receiving IL-15 with either agent alone. In translation, a phase I trial was initiated involving IL-15 (rhIL-15), nivolumab and ipilimumab in patients with malignancy (NCT03388632). In rhesus macaques CIV IL-15 at 20 ?g/kg/day for 10 days led to an 80-fold increase in number of circulating effector memory CD8 T cells. However, administration of ?c cytokines such as IL-15 led to paralysis/depression of CD4 T-cells that was mediated through transient expression of SOCS3 that inhibited the STAT5 signaling pathway. This lost CD4 helper role could be restored alternatively by CD40 agonists. In the TRAMP-C2 prostate tumor model the combination of IL-15 with agonistic anti-CD40 produced additive effects in terms of numbers of TRAMP-C2 tumor specific Spas/SCNC/9H tetramer positive CD8 T cells expressed and tumor responses. A clinical trial is being initiated for patients with cancer using an intralesional anti-CD40 in combination with CIV rhIL-15. To translate IL-15-mediated increases in NK cells, we investigated combination therapy of IL-15 with anticancer monoclonal antibodies including rituximab in mouse models of EL-4 lymphoma transfected with human CD20 and with alemtuzumab (CAMPATH-1H) in a xenograft model of adult T cell leukemia (ATL). IL-15 enhanced the ADCC and therapeutic efficacy of both antibodies. These results provided the scientific basis for trials of IL-15 combined with alemtuzumab (anti-CD52) for patients with ATL (NCT02689453), with obinutuzumab (anti-CD20) for patients with CLL (NCT03759184), and with avelumab (anti-PD-L1) in patients with T-cell lymphoma (NCT03905135) and renal cancer (NCT04150562). In the first trial, there was elimination of circulating ATL and CLL leukemic cells in select patients.
Project description:Transcriptional profiling of cytokines and its receptors in primary murine lymphoblastoid cells (pML cells), which are lymphomatous cells from HTLV-1 TAX transgenic mice. ATL is a T-cell malignancy caused by HTLV-I, and presents as an aggressive leukemia with characteristic widespread leukemic cell infiltration into visceral organs and skin. The molecular mechanisms associated with leukemic cell infiltration are poorly understood. We have employed mouse models of ATL to investigate the role of chemokines in this process. Transfer of splenic lymphomatous cells from transgenic to SCID mice rapidly reproduces a leukemia and lymphoma which is histologically identical to human disease. It could be shown that lymphomatous cells exhibit specific chemotactic activity in response to SDF-1α. Lymphomatous cells exhibited surface expression of CXCR4, the specific receptor of SDF-1α and chemotaxis was associated with down regulation of CXCR4 expression and phosphorylation of intracellular ERK1/2. AMD3100, a CXCR4 antagonist, was found to inhibit both SDF-1α - induced migration and phosphorylation of ERK1/2. Investigation of cultured cells from human ATL patients revealed identical findings. Employing the SCID mouse model it could be demonstrated that AMD3100 inhibited infiltration of lymphomatous cells into liver and lung tissues in vivo. These results demonstrate the involvement of the SDF-1α /CXCR4 interaction as one mechanism of leukemic cell migration and this may provide a novel target as part of combination therapy for ATL. Overall design: pML cells vs. pan T cells from C57BL6 mice.
Project description:Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.
Project description:Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia (ATL) and the neuroinflammatory disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein functions as a potent viral oncogene that constitutively activates the NF-?B transcription factor to transform T cells; however, the underlying mechanisms remain obscure. Here, using next-generation RNA sequencing we identified the IL-25 receptor subunit IL-17RB as an aberrantly overexpressed gene in HTLV-1 immortalized T cells. Tax induced the expression of IL-17RB in an I?B kinase (IKK) and NF-?B-dependent manner. Remarkably, Tax activation of the canonical NF-?B pathway in T cells was critically dependent on IL-17RB expression. IL-17RB and IL-25 were required for HTLV-1-induced immortalization of primary T cells, and the constitutive NF-?B activation and survival of HTLV-1 transformed T cells. IL-9 was identified as an important downstream target gene of the IL-17RB pathway that drives the proliferation of HTLV-1 transformed cells. Furthermore, IL-17RB was overexpressed in leukemic cells from a subset of ATL patients and also regulated NF-?B activation in some, but not all, Tax-negative ATL cell lines. Together, our results support a model whereby Tax instigates an IL-17RB-NF-?B feed-forward autocrine loop that is obligatory for HTLV-1 leukemogenesis.