Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

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.

Murine Oncostatin M Has Opposing Effects on the Proliferation of OP9 Bone Marrow Stromal Cells and NIH/3T3 Fibroblasts Signaling through the OSMR.

The IL-6 family cytokine Oncostatin M (OSM) is involved in cell development, growth, hematopoiesis, inflammation, and cancer. Intriguingly, OSM has proliferative and antiproliferative effects depending on the target cell. The molecular mechanisms underlying these opposing effects are not fully understood. Previously, we found OSM upregulation in different myeloproliferative syndromes. However, OSM receptor (OSMR) expression was detected on stromal cells but not the malignant cells themselves. In the present study, we, therefore, investigated the effect of murine OSM (mOSM) on proliferation in stromal and fibroblast cell lines. We found that mOSM impairs the proliferation of bone marrow (BM) stromal cells, whereas fibroblasts responded to mOSM with increased proliferation. When we set out to reveal the mechanisms underlying these opposing effects, we detected increased expression of the OSM receptors OSMR and LIFR in stromal cells. Interestingly, Osmr knockdown and Lifr overexpression attenuated the OSM-mediated effect on proliferation in both cell lines indicating that mOSM affected the proliferation signaling mainly through the OSMR. Furthermore, mOSM induced activation of the JAK-STAT, PI3K-AKT, and MAPK-ERK pathways in OP9 and NIH/3T3 cells with differences in total protein levels between the two cell lines. Our findings offer new insights into the regulation of proliferation by mOSM.

Phosphorylation of BECLIN-1 by BCR-ABL suppresses autophagy in chronic myeloid leukemia.

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL+ cells.

NPM1c alters FLT3-D835Y localization and signaling in acute myeloid leukemia.

Activating mutations in FMS-like tyrosine kinase receptor-3 (FLT3) and Nucleophosmin-1 (NPM1) are most frequent alterations in acute myeloid leukemia (AML), and are often coincidental. The mutational status of NPM1 has strong prognostic relevance to patients with point mutations of the FLT3 tyrosine kinase domain (TKD), but the biological mechanism underlying this effect remains unclear. In the present study, we investigated the effect of the coincidence of NPM1c and FLT3-TKD. Although expression of FLT3-TKD is not sufficient to induce a disease in mice, coexpression with NPM1c rapidly leads to an aggressive myeloproliferative disease in mice with a latency of 31.5 days. Mechanistically, we could show that FLT3-TKD is able to activate the downstream effector molecule signal transducer and activator of transcription 5 (STAT5) exclusively in the presence of mutated NPM1c. Moreover, NPM1c alters the cellular localization of FLT3-TKD from the cell surface to the endoplasmic reticulum, which might thereby lead to the aberrant STAT5 activation. Importantly, aberrant STAT5 activation occurs not only in primary murine cells but also in patients with AML with combined FLT3-TKD and NPM1c mutations. Thus, our data indicate a new mechanism, how NPM1c mislocalizes FLT3-TKD and changes its signal transduction ability.

Gaming the system: using transactional analysis to explore dysfunctional processes in clinical supervision.

OBJECTIVE: This paper discusses the ongoing relevance of concepts derived from transactional analysis to understanding the challenges that can emerge in clinical supervision under the Competency Based Fellowship Program. CONCLUSIONS: Defensive game playing has face validity as a framework for understanding dysfunctional processes in clinical supervision. Being aware of these concepts may aid trainees and supervisors in promoting effective clinical supervision practice.

Erratum: Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL-15 production in FLT3-ITD-mutant leukemia cells.

This corrects the article DOI: 10.1038/nm.4484.

Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL-15 production in FLT3-ITD-mutant leukemia cells.

Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD+ leukemia cells. This synergized with the allogeneic CD8+ T cell response, leading to long-term survival in six mouse models of FLT3-ITD+ AML. Sorafenib-related IL-15 production caused an increase in CD8+CD107a+IFN-γ+ T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD+ AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8+ T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.