cIAPs control RIPK1 kinase activity-dependent and -independent cell death and tissue inflammation.

Cellular inhibitor of apoptosis proteins (cIAPs) are RING-containing E3 ubiquitin ligases that ubiquitylate receptor-interacting protein kinase 1 (RIPK1) to regulate TNF signalling. Here, we established mice simultaneously expressing enzymatically inactive cIAP1/2 variants, bearing mutations in the RING domains of cIAP1/2 (cIAP1/2 mutant RING, cIAP1/2MutR ). cIap1/2MutR/MutR mice died during embryonic development due to RIPK1-mediated apoptosis. While expression of kinase-inactive RIPK1D138N rescued embryonic development, Ripk1D138N/D138N /cIap1/2MutR/MutR mice developed systemic inflammation and died postweaning. Cells expressing cIAP1/2MutR and RIPK1D138N were still susceptible to TNF-induced apoptosis and necroptosis, implying additional kinase-independent RIPK1 activities in regulating TNF signalling. Although further ablation of Ripk3 did not lead to any phenotypic improvement, Tnfr1 gene knock-out prevented early onset of systemic inflammation and premature mortality, indicating that cIAPs control TNFR1-mediated toxicity independent of RIPK1 and RIPK3. Beyond providing novel molecular insights into TNF-signalling, the mouse model established in this study can serve as a useful tool to further evaluate ongoing therapeutic protocols using inhibitors of TNF, cIAPs and RIPK1.

The miR-141/200c-STAT4 Axis Contributes to Leukemogenesis by Enhancing Cell Proliferation in T-PLL.

T-prolymphocytic leukemia (T-PLL) is a rare and mature T-cell malignancy with characteristic chemotherapy-refractory behavior and a poor prognosis. Molecular concepts of disease development have been restricted to protein-coding genes. Recent global microRNA (miR) expression profiles revealed miR-141-3p and miR-200c-3p (miR-141/200c) as two of the highest differentially expressed miRs in T-PLL cells versus healthy donor-derived T cells. Furthermore, miR-141/200c expression separates T-PLL cases into two subgroups with high and low expression, respectively. Evaluating the potential pro-oncogenic function of miR-141/200c deregulation, we discovered accelerated proliferation and reduced stress-induced cell death induction upon stable miR-141/200c overexpression in mature T-cell leukemia/lymphoma lines. We further characterized a miR-141/200c-specific transcriptome involving the altered expression of genes associated with enhanced cell cycle transition, impaired DNA damage responses, and augmented survival signaling pathways. Among those genes, we identified STAT4 as a potential miR-141/200c target. Low STAT4 expression (in the absence of miR-141/200c upregulation) was associated with an immature phenotype of primary T-PLL cells as well as with a shortened overall survival of T-PLL patients. Overall, we demonstrate an aberrant miR-141/200c-STAT4 axis, showing for the first time the potential pathogenetic implications of a miR cluster, as well as of STAT4, in the leukemogenesis of this orphan disease.

Oncogenic role and target properties of the lysine-specific demethylase KDM1A in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eµ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eµ-TCL1A vs iKdm1aKD;Eµ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti-B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.

The proto-oncogene TCL1A deregulates cell cycle and genomic stability in CLL.

Upregulation of the proto-oncogene T-cell leukemia/lymphoma 1A (TCL1A) is causally implicated in various B-cell and T-cell malignancies. High-level TCL1A correlates with aggressive disease features and inferior clinical outcomes. However, the molecular and cell biological consequences of, particularly nuclear, TCL1A are not fully elucidated. We observed here in mouse models of subcellular site-specific TCL1A-induced lymphomagenesis that TCL1A exerts a strong transforming impact via nuclear topography. In proteomic screens of TCL1A-bound molecules in chronic lymphocytic leukemia (CLL) cells and B-cell lymphoma lines, we identified regulators of cell cycle and DNA repair pathways as novel TCL1A interactors, particularly enriched under induced DNA damage and mitosis. By functional mapping and in silico modeling, we specifically identified the mitotic checkpoint protein, cell division cycle 20 (CDC20), as a direct TCL1A interactor. According to the regulatory impact of TCL1A on the activity of the CDC20-containing mitotic checkpoint and anaphase-promoting complexes during mitotic progression, TCL1A overexpression accelerated cell cycle transition in B-cell lymphoma lines, impaired apoptotic damage responses in association with pronounced chromosome missegregation, and caused cellular aneuploidy in Eµ-TCL1A mice. Among hematopoietic cancers, CDC20 levels seem particularly low in CLL. CDC20 expression negatively correlated with TCL1A and lower expression marked more aggressive and genomically instable disease and cellular phenotypes. Knockdown of Cdc20 in TCL1A-initiated murine CLL promoted aneuploidy and leukemic acceleration. Taken together, we discovered a novel cell cycle-associated effect of TCL1A abrogating controlled cell cycle transition. This adds to our concept of oncogenic TCL1A by targeting genome stability. Overall, we propose that TCL1A acts as a pleiotropic adapter molecule with a synergistic net effect of multiple hijacked pathways.

Noncanonical Function of AGO2 Augments T-cell Receptor Signaling in T-cell Prolymphocytic Leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a chemotherapy-refractory T-cell malignancy with limited therapeutic options and a poor prognosis. Current disease concepts implicate TCL1A oncogene-mediated enhanced T-cell receptor (TCR) signaling and aberrant DNA repair as central perturbed pathways. We discovered that recurrent gains on chromosome 8q more frequently involve the argonaute RISC catalytic component 2 (AGO2) gene than the adjacent MYC locus as the affected minimally amplified genomic region. AGO2 has been understood as a protumorigenic key regulator of miRNA (miR) processing. Here, in primary tumor material and cell line models, AGO2 overrepresentation associated (i) with higher disease burden, (ii) with enhanced in vitro viability and growth of leukemic T cells, and (iii) with miR-omes and transcriptomes that highlight altered survival signaling, abrogated cell-cycle control, and defective DNA damage responses. However, AGO2 elicited also immediate, rather non-RNA-mediated, effects in leukemic T cells. Systems of genetically modulated AGO2 revealed that it enhances TCR signaling, particularly at the level of ZAP70, PLCγ1, and LAT kinase phosphoactivation. In global mass spectrometric analyses, AGO2 interacted with a unique set of partners in a TCR-stimulated context, including the TCR kinases LCK and ZAP70, forming membranous protein complexes. Models of their three-dimensional structure also suggested that AGO2 undergoes posttranscriptional modifications by ZAP70. This novel TCR-associated noncanonical function of AGO2 represents, in addition to TCL1A-mediated TCR signal augmentation, another enhancer mechanism of this important deregulated growth pathway in T-PLL. These findings further emphasize TCR signaling intermediates as candidates for therapeutic targeting. SIGNIFICANCE: The identification of AGO2-mediated activation of oncogenic T cells through signal amplifying protein-protein interactions advances the understanding of leukemogenic AGO2 functions and underlines the role of aberrant TCR signaling in T-PLL.

Advanced Pathogenetic Concepts in T-Cell Prolymphocytic Leukemia and Their Translational Impact.

T-cell prolymphocytic leukemia (T-PLL) is the most common mature T-cell leukemia. It is a typically aggressively growing and chemotherapy-resistant malignancy with a poor prognosis. T-PLL cells resemble activated, post-thymic T-lymphocytes with memory-type effector functions. Constitutive transcriptional activation of genes of the T-cell leukemia 1 (TCL1) family based on genomic inversions/translocations is recognized as a key event in T-PLL's pathogenesis. TCL1's multiple effector pathways include the enhancement of T-cell receptor (TCR) signals. New molecular dependencies around responses to DNA damage, including repair and apoptosis regulation, as well as alterations of cytokine and non-TCR activation signaling were identified as perturbed hallmark pathways within the past years. We currently witness these vulnerabilities to be interrogated in first pre-clinical concepts and initial clinical testing in relapsed/refractory T-PLL patients. We summarize here the current knowledge on the molecular understanding of T-PLL's pathobiology and critically assess the true translational progress around this to help appraisal by caregivers and patients. Overall, the contemporary concepts on T-PLL's pathobiology are condensed in a comprehensive mechanistic disease model and promising interventional strategies derived from it are highlighted.

The Modes of Dysregulation of the Proto-Oncogene T-Cell Leukemia/Lymphoma 1A.

Incomplete biological concepts in lymphoid neoplasms still dictate to a large extent the limited availability of efficient targeted treatments, which entertains the mostly unsatisfactory clinical outcomes. Aberrant expression of the embryonal and lymphatic TCL1 family of oncogenes, i.e., the paradigmatic TCL1A, but also TML1 or MTCP1, is causally implicated in T- and B-lymphocyte transformation. TCL1A also carries prognostic information in these particular T-cell and B-cell tumors. More recently, the TCL1A oncogene has been observed also in epithelial tumors as part of oncofetal stemness signatures. Although the concepts on the modes of TCL1A dysregulation in lymphatic neoplasms and solid tumors are still incomplete, there are recent advances in defining the mechanisms of its (de)regulation. This review presents a comprehensive overview of TCL1A expression in tumors and the current understanding of its (dys)regulation via genomic aberrations, epigenetic modifications, or deregulation of TCL1A-targeting micro RNAs. We also summarize triggers that act through such transcriptional and translational regulation, i.e., altered signals by the tumor microenvironment. A refined mechanistic understanding of these modes of dysregulations together with improved concepts of TCL1A-associated malignant transformation can benefit future approaches to specifically interfere in TCL1A-initiated or -driven tumorigenesis.