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Immune checkpoint inhibitors are a powerful new tool in the treatment of cancer, with prolonged responses in multiple diseases, including hematologic malignancies, such as Hodgkin lymphoma. However, in a recent report, we demonstrated that the PD-1 inhibitor nivolumab led to rapid progression in patients with adult T-cell leukemia/lymphoma (ATLL) (NCT02631746). We obtained primary cells from these patients to determine the cause of this hyperprogression. Analyses of clonality, somatic mutations, and gene expression in the malignant cells confirmed the report of rapid clonal expansion after PD-1 blockade in these patients, revealed a previously unappreciated origin of these malignant cells, identified a novel connection between ATLL cells and tumor-resident regulatory T cells (Tregs), and exposed a tumor-suppressive role for PD-1 in ATLL. Identifying the mechanisms driving this alarming outcome in nivolumab-treated ATLL may be broadly informative for the growing problem of rapid progression with immune checkpoint therapies.

Background Adult T-cell leukemia lymphoma (ATLL) is a chemotherapy-resistant malignancy with a median survival of less than one year that will afflict between one hundred thousand and one million individuals worldwide who are currently infected with human T-cell leukemia virus type 1. Recurrent somatic mutations in host genes have exposed the T-cell receptor pathway through nuclear factor κB to interferon regulatory factor 4 (IRF4) as an essential driver for this malignancy. We sought to determine if IRF4 represents a therapeutic target for ATLL and to identify downstream effectors and biomarkers of IRF4 signaling in vivo. Results ATLL cell lines, particularly Tax viral oncoprotein-negative cell lines, that most closely resemble ATLL in humans, were sensitive to dose- and time-dependent inhibition by a next-generation class of IRF4 antisense oligonucleotides (ASOs) that employ constrained ethyl residues that mediate RNase H-dependent RNA degradation. ATLL cell lines were also sensitive to lenalidomide, which repressed IRF4 expression. Both ASOs and lenalidomide inhibited ATLL proliferation in vitro and in vivo. To identify biomarkers of IRF4-mediated CD4 + T-cell expansion in vivo, transcriptomic analysis identified several genes that encode key regulators of ATLL, including interleukin 2 receptor subunits α and β, KIT ligand, cytotoxic T-lymphocyte-associated protein 4, and thymocyte selection-associated high mobility group protein TOX 2. Conclusions These data support the pursuit of IRF4 as a therapeutic target in ATLL with the use of either ASOs or lenalidomide.

Additional file 1: Figure S1. Sensitivity of cell lines to combination of lenalidomide and IRF4 ASO. Cells were treated for 2 days with either 10 uM IRF4 ASO, 2.5 uM lenalidomide, Combination, or Control ASO + DMSO Treatment. Resazurin proliferation assay were conducted and values are presented as normalized to control treatment for each cell line. Figure S2. IRF4 gene targets up-regulated in primary T-cells. Total RNA was obtained from flow sorted GFP+ CD45.2+ CD4+ CD8- T-cells harvested from the thymus of three mice in each group and submitted to the Genome Technology Access Center at Washington University for RNA sequencing (RNAseq) analysis. Normalized gene counts for each of the 9 samples were divided by the average number of normalized counts for the 3 MIG samples. Colors indicate fold increase for each gene in each sample over average control (MIG) value for each gene. Genes listed are those that were expressed (>10 reads) and elevated (>2 fold) in cells expressing WT IRF4 and IRF4 K59R. Figure S3. IRF4 gene targets confirmed in primary ATL. Data from an ATLL gene expression microarray study was downloaded from the Gene Expression Omnibus at the NCBI (GEO accession: GSE33615). In the original study, RNA was extracted from PBMCs isolated from patients with acute (n=26), chronic (n=20), lymphomatous (n=1), and smoldering (n=4) ATLL, and compared to RNA obtained from CD4+ cells from 21 normal subjects. In this study, values for CTLA4 and TOX2 were normalized to actin (ACTB) then represented as fold-Patient 10 (a smoldering ATLL sample with the lowest proviral load in the study). A) Correlation of TOX2 and CTLA4 to IRF4 expression in ATLL. B) Expression of TOX2, CTLA4, and IRF4 in IRF4 HI ATLL samples compared to normal T-cells. C) Expression of TOX2, CTLA4, and IRF4 in acute, chronic, lymphoma, and smoldering subtypes of ATLL.

The human T-cell leukemia virus-1 (HTLV-1) oncoprotein Tax drives cell proliferation and resistance to apoptosis early in the pathogenesis of adult T-cell leukemia (ATL). Subsequently, probably as a result of specific immunoediting, Tax expression is down-regulated and functionally replaced by somatic driver mutations of the host genome. Both amplification and point mutations of interferon regulatory factor 4 (IRF4) have been previously detected in ATL., K59R is the most common single-nucleotide variation of IRF4 and is found exclusively in ATL. High-throughput whole-exome sequencing revealed recurrent activating genetic alterations in the T-cell receptor, CD28, and NF-κB pathways. We found that IRF4, which is transcriptionally activated downstream of these pathways, is frequently mutated in ATL. IRF4 RNA, protein, and IRF4 transcriptional targets are uniformly elevated in HTLV-1–transformed cells and ATL cell lines, and IRF4 was bound to genomic regulatory DNA of many of these transcriptional targets in HTLV-1–transformed cell lines. We further noted that the K59R IRF4 mutant is expressed at higher levels in the nucleus than WT IRF4 and is transcriptionally more active. Expression of both WT and the K59R mutant of IRF4 from a constitutive promoter in retrovirally transduced murine bone marrow cells increased the abundance of T lymphocytes but not myeloid cells or B lymphocytes in mice. IRF4 may represent a therapeutic target in ATL because ATL cells select for a mutant of IRF4 with higher nuclear expression and transcriptional activity, and overexpression of IRF4 induces the expansion of T lymphocytes in vivo.

Intraflagellar transport (IFT) proteins are vital for the genesis and maintenance of cilia. Our identification of ift122 transcripts in zebrafish thrombocytes that lack primary cilia was unexpected. IFT proteins serve transport in cilia, whose narrow dimensions may have necessitated the evolution of IFT from vesicular transport in ancestral eukaryotes. We hypothesized that IFTs might also facilitate transport within the filopodia that form when thrombocytes are activated. To test this possibility, we knocked down ift122 expression by injecting antisense Morpholino oligonucleotides (MOs) into zebrafish embryos. Laser-induced arterial thrombosis showed prolonged time to occlusion (TTO) of the vessel, as would be expected with defective thrombocyte function. Acute effects in adult zebrafish were evaluated by Vivo-Morpholino (Vivo-MO) knockdown of ift122. Vivo-MO morphants showed a prolonged time to thrombocyte aggregation (TTA) in the plate tilt assay after thrombocyte activation by the following agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in thrombocyte function.

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.

Zebrafish has become an excellent model system to study mammalian hemostasis. Despite our extensive efforts to develop technologies to measure zebrafish hemostasis and even with previously established thrombocyte qualitative and quantitative functional assays, quantifying thrombocyte function for high throughput applications has been a challenge. In this paper, we have developed two quantitative methods to estimate thrombocyte aggregation: one by whole blood aggregometry and the other by flow cytometry. We found that it is possible to conduct whole blood aggregometry using only 2 µl of blood and the currently available aggregometer. Each of three agonists, arachidonic acid, ADP, and collagen yielded impedance curves similar to those obtained with human blood. We were also able to use flow cytometry to indirectly quantify the extent of thrombocyte aggregation by labeling whole blood with mepacrine, aggregating in the presence of each of the above agonists, separating the aggregates from the white blood cells by centrifugation, and then sorting the resulting white cell fraction for thrombocyte numbers. These methods have high throughput capabilities and have the potential to be used in large scale screens to detect and characterize mutants with thrombocyte functional defects or to identify genes involved in thrombocyte function by large scale knockdowns.

Morpholino and vivo-morpholino gene knockdown methods have been used to study thrombocyte function in zebrafish. However, a large-scale knockdown of the entire zebrafish genome using these technologies to study thrombocyte function is prohibitively expensive. We have developed an inexpensive gene knockdown method, which uses a hybrid of a control vivo-morpholino and a standard antisense oligonucleotide specific for a gene. This hybrid molecule is able to deliver antisense deoxyoligonucleotides into zebrafish thrombocytes because it piggybacks on a control vivo-morpholino. To validate use of this hybrid molecule in gene knockdowns, we targeted the thrombocyte specific αIIb gene with a hybrid of a control vivo-morpholino and an oligonucleotide antisense to αIIb mRNA. The use of this piggyback technology resulted in degradation of αIIb mRNA and led to thrombocyte functional defect. This piggyback method to knockdown genes is inexpensive since one control vivo-morpholino can be used to target many different genes by making many independent gene-specific oligonucleotide hybrids. Thus, this novel piggyback technology can be utilized for cost-effective large-scale knockdowns of genes to study thrombocyte function in zebrafish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a canonical member of a group of dioxins which are byproducts of industrial combustion and are dangerous environmental pollutants. TCDD has been shown to cause several abnormalities in humans and wildlife, and recently, some dioxins have been found to activate platelets. However, TCDD-mediated platelet activation pathways are elusive and virtually nothing is known about TCDD activation of fish thrombocytes. To investigate TCDD effect on thrombocyte function, we tested zebrafish blood in presence of TCDD using a thrombocyte functional assay. We found that TCDD activated thrombocytes. Further experiments showed that thrombocytes of fish treated with TCDD formed both aggregates and filopodia. To investigate the mechanism of TCDD-mediated activation of thrombocytes we used inhibitors for Gq, cyclooxygenase-1, aryl hydrocarbon receptor (AHR), c-src, Akt, and ERK1/2. We found that TCDD induces AHR which activates c-src and signals the activation of Akt and ERK1/2 which are ultimately involved in generation of thromboxane A2. Furthermore, we found that ADP potentiates TCDD action, which led to the discovery that ADP itself activates AHR in the absence of TCDD. Taken together, these results resolved the pathway of TCDD activation of thrombocytes and led to the finding that ADP is an activator of AHR.