A dual role for PSIP1/LEDGF in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy. Current intensified therapeutic protocols coincide with severe side effects, and no salvage therapy is available for primary therapy-resistant or relapsed patients. This highlights the need to identify new therapeutic targets in T-ALL. PSIP1, dispensable for normal hematopoiesis, is a dependency factor in KMT2A-rearranged myeloid leukemia. Nonetheless, loss-of-function mutations suggest a tumor suppressor role for PSIP1 in T-ALL. Here, we demonstrate that the loss of Psip1 accelerates T-ALL initiation in mice which we correlated with reduced H3K27me3 binding. Contrastingly, loss of PSIP1 impaired cell proliferation in several T-ALL cell lines. In cell lines, PSIP1 down-regulation leads to a reduction of COX20, an assembly factor of the cytochrome c oxidase in the mitochondria, and to a reduction in mitochondrial respiration. This indicates that PSIP1 can exert a dual role in the context of T-ALL, either as a tumor suppressor gene during tumor initiation or as a dependency factor in tumor maintenance.

Focal deletions of a promoter tether activate the IRX3 oncogene in T-cell acute lymphoblastic leukemia.

Oncogenes can be activated in cis through multiple mechanisms including enhancer hijacking events and noncoding mutations that create enhancers or promoters de novo. These paradigms have helped parse somatic variation of noncoding cancer genomes, thereby providing a rationale to identify noncanonical mechanisms of gene activation. Here we describe a novel mechanism of oncogene activation whereby focal copy number loss of an intronic element within the FTO gene leads to aberrant expression of IRX3, an oncogene in T cell acute lymphoblastic leukemia (T-ALL). Loss of this CTCF bound element downstream to IRX3 (+224 kb) leads to enhancer hijack of an upstream developmentally active super-enhancer of the CRNDE long noncoding RNA (-644 kb). Unexpectedly, the CRNDE super-enhancer interacts with the IRX3 promoter with no transcriptional output until it is untethered from the FTO intronic site. We propose that 'promoter tethering' of oncogenes to inert regions of the genome is a previously unappreciated biological mechanism preventing tumorigenesis.

BCAT1 is a NOTCH1 target and sustains the oncogenic function of NOTCH1.

High levels of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) have been associated with tumor aggressiveness and drug resistance in several cancer types. Nevertheless, the mechanistic role of BCAT1 in T-cell acute lymphoblastic leukemia (T-ALL) remains uncertain. We provide evidence that Bcat1 was over-expressed following NOTCH1-induced transformation of leukemic progenitors and that NOTCH1 directly controlled BCAT1 expression by binding to a BCAT1 promoter. Further, using a NOTCH1 gain-of-function retroviral model of T-ALL, mouse cells genetically deficient for Bcat1 showed defects in developing leukemia. In murine T-ALL cells, Bcat1 depletion or inhibition redirected leucine metabolism towards production of 3-hydroxy butyrate (3-HB), an endogenous histone deacetylase inhibitor. Consistently, BCAT1 depleted cells showed altered protein acetylation levels which correlated with a pronounced sensitivity to DNA damaging agents. In human NOTCH1-dependent leukemias, high expression levels of BCAT1 may predispose to worse prognosis. Therapeutically, BCAT1 inhibition specifically synergized with etoposide to eliminate tumors in patient-derived xenograft models suggesting that BCAT1 inhibitors may have a part to play in salvage protocols for refractory T-ALL.

Myb overexpression synergizes with the loss of Pten and is a dependency factor and therapeutic target in T-cell lymphoblastic leukemia.

T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Although the prognosis of T-ALL has improved over time, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains poor. Therefore, further progress in the treatment of T-ALL requires a better understanding of its biology and the development of more effective precision oncologic therapies. The proto-oncogene MYB is highly expressed in diverse hematologic malignancies, including T-ALLs with genomic aberrations that further potentiate its expression and activity. Previous studies have associated MYB with a malignant role in the pathogenesis of several cancers. However, its role in the induction and maintenance of T-ALL remains relatively poorly understood. In this study, we found that an increased copy number of MYB is associated with higher MYB expression levels, and might be associated with inferior event-free survival of pediatric T-ALL patients. Using our previously described conditional Myb overexpression mice, we generated two distinct MYB-driven T-ALL mouse models. We demonstrated that the overexpression of Myb synergizes with Pten deletion but not with the overexpression of Lmo2 to accelerate the development of T-cell lymphoblastic leukemias. We also showed that MYB is a dependency factor in T-ALL since RNA interference of Myb blocked cell cycle progression and induced apoptosis in both human and murine T-ALL cell lines. Finally, we provide preclinical evidence that targeting the transcriptional activity of MYB can be a useful therapeutic strategy for the treatment of T-ALL.

A helicase-independent role of DHX15 promotes MYC stability and acute leukemia cell survival.

DHX15 has been implicated in RNA splicing and ribosome biogenesis, primarily functioning as an RNA helicase. To systematically assess the cellular role of DHX15, we conducted proteomic analysis to investigate the landscape of DHX15 interactome, and identified MYC as a binding partner. DHX15 co-localizes with MYC in cells and directly interacts with MYC in vitro. Importantly, DHX15 contributes to MYC protein stability at the post-translational level and independent of its RNA binding capacity. Mechanistic investigation reveals that DHX15 interferes the interaction between MYC and FBXW7, thereby preventing MYC polyubiquitylation and proteasomal degradation. Consequently, the abrogation of DHX15 drastically inhibits MYC-mediated transcriptional output. While DHX15 depletion blocks T cell development and leukemia cell survival as we recently reported, overexpression of MYC significantly rescues the phenotypic defects. These findings shed light on the essential role of DHX15 in mammalian cells and suggest that maintaining sufficient MYC expression is a significant contributor to DHX15-mediated cellular functions.

Targeting hyperactive platelet-derived growth factor receptor-ß signaling in T-cell acute lymphoblastic leukemia and lymphoma.

T cell acute lymphoblastic leukemia (T-ALL) and T cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematological malignancies. Current treatment consists of intensive chemotherapy, leading to 80% overall survival but are associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL and that screening T-ALL/TLBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

Mutations in the histone methyltransferase Ezh2 drive context-dependent leukemia in Xenopus tropicalis.

CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations.

RNA helicase DHX15 exemplifies a unique dependency in acute leukemia.

RNA-binding proteins (RBP) have emerged as essential regulators that control gene expression and modulate multiple cancer traits. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy derived from transformation of T-cell progenitors that normally undergo discrete steps of differentiation in the thymus. The implications of essential RBP during T-cell neoplastic transformation remain largely unclear. Systematic evaluation of RBP identifies RNA helicase DHX15, which facilitates the disassembly of the spliceosome and release of lariat introns, as a T-ALL dependency factor. Functional analysis using multiple murine T-ALL models demonstrates the essential importance of DHX15 in tumor cell survival and leukemogenesis. Moreover, single-cell transcriptomics reveals that DHX15 depletion in T-cell progenitors hinders burst proliferation during the transition from doublenegative to double-positive cells (CD4-CD8- to CD4+CD8+). Mechanistically, abrogation of DHX15 perturbs RNA splicing and leads to diminished levels of SLC7A6 and SLC38A5 transcripts due to intron retention, thereby suppressing glutamine import and mTORC1 activity. We further propose a DHX15 signature modulator drug ciclopirox and demonstrate that it has prominent anti-T-ALL efficacy. Collectively, our data highlight the functional contribution of DHX15 to leukemogenesis through regulation of established oncogenic pathways. These findings also suggest a promising therapeutic approach, i.e., splicing perturbation by targeting spliceosome disassembly, may achieve considerable anti-tumor efficacy.

SOX11 regulates SWI/SNF complex components as member of the adrenergic neuroblastoma core regulatory circuitry.

The pediatric extra-cranial tumor neuroblastoma displays a low mutational burden while recurrent copy number alterations are present in most high-risk cases. Here, we identify SOX11 as a dependency transcription factor in adrenergic neuroblastoma based on recurrent chromosome 2p focal gains and amplifications, specific expression in the normal sympatho-adrenal lineage and adrenergic neuroblastoma, regulation by multiple adrenergic specific (super-)enhancers and strong dependency on high SOX11 expression in adrenergic neuroblastomas. SOX11 regulated direct targets include genes implicated in epigenetic control, cytoskeleton and neurodevelopment. Most notably, SOX11 controls chromatin regulatory complexes, including 10 SWI/SNF core components among which SMARCC1, SMARCA4/BRG1 and ARID1A. Additionally, the histone deacetylase HDAC2, PRC1 complex component CBX2, chromatin-modifying enzyme KDM1A/LSD1 and pioneer factor c-MYB are regulated by SOX11. Finally, SOX11 is identified as a core transcription factor of the core regulatory circuitry (CRC) in adrenergic high-risk neuroblastoma with a potential role as epigenetic master regulator upstream of the CRC.

Pre-Clinical Evaluation of the Hypomethylating Agent Decitabine for the Treatment of T-Cell Lymphoblastic Lymphoma.

T-cell lymphoblastic lymphoma (T-LBL) is a rare and aggressive lymphatic cancer, often diagnosed at a young age. Patients are treated with intensive chemotherapy, potentially followed by a hematopoietic stem cell transplantation. Although prognosis of T-LBL has improved with intensified treatment protocols, they are associated with side effects and 10-20% of patients still die from relapsed or refractory disease. Given this, the search toward less toxic anti-lymphoma therapies is ongoing. Here, we targeted the recently described DNA hypermethylated profile in T-LBL with the DNA hypomethylating agent decitabine. We evaluated the anti-lymphoma properties and downstream effects of decitabine, using patient derived xenograft (PDX) models. Decitabine treatment resulted in prolonged lymphoma-free survival in all T-LBL PDX models, which was associated with downregulation of the oncogenic MYC pathway. However, some PDX models showed more benefit of decitabine treatment compared to others. In more sensitive models, differentially methylated CpG regions resulted in more differentially expressed genes in open chromatin regions. This resulted in stronger downregulation of cell cycle genes and upregulation of immune response activating transcripts. Finally, we suggest a gene signature for high decitabine sensitivity in T-LBL. Altogether, we here delivered pre-clinical proof of the potential use of decitabine as a new therapeutic agent in T-LBL.

CircFBXW7 in patients with T-cell ALL: depletion sustains MYC and NOTCH activation and leukemia cell viability.

Circular RNAs (circRNAs) are emerging as new players in leukemogenic mechanisms. In patients with T-cell Acute Lymphoblastic Leukemia (T-ALL), the recent report of a remarkable dysregulation of circRNAs incited further functional investigation. Here we focus on circFBXW7, highly expressed in T-cells, with a notably high abundance of the circular compared to linear transcript of FBXW7. Two T-ALL patient cohorts profiled with RNA-seq were analyzed in comparison with five populations of developing thymocytes as normal counterpart, quantifying circRNA and gene expression. CircFBXW7 expression was very heterogeneous in T-ALL patients allowing their stratification in two groups with low and high expression of this circRNA, not correlated with FBXW7 mutation status and T-ALL molecular subgroups. With a loss-of-function study in T-ALL in vitro, we demonstrate that circFBXW7 depletion increases leukemic cell viability and proliferation. Microarray profiling highlighted the effect of the circFBXW7 silencing on gene expression, with activation of pro-proliferative pathways, supporting a tumor suppressor role of circFBXW7 in T-ALL. Further, MYC and intracellular NOTCH1 protein levels, as well as expression of MYC target and NOTCH signaling genes were elevated after circFBXW7 depletion, suggesting an inhibitory role of circFBXW7 in these oncogenic axes. Plus, low circFBXW7 levels were associated with a particular gene expression profile in T-ALL patients, which was remarkably mirrored by the effects of circFBXW7 loss-of-function in vitro. CircFBXW7 depletion notably emerges as a new factor enhancing a proliferative phenotype and the activation of the MYC signaling pathway, key players in this aggressive malignancy.

Intrathymic dendritic cell-biased precursors promote human T cell lineage specification through IRF8-driven transmembrane TNF.

The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.

In vivo stabilization of a less toxic asparaginase variant leads to a durable antitumor response in acute leukemia.

Asparagine is a non-essential amino acid since it can either be taken up via the diet or synthesized by asparagine synthetase. Acute lymphoblastic leukemia (ALL) cells do not express asparagine synthetase or express it only minimally, which makes them completely dependent on extracellular asparagine for their growth and survival. This dependency makes ALL cells vulnerable to treatment with L-asparaginase, an enzyme that hydrolyzes asparagine. To date, all clinically approved L-asparaginases have significant L-glutaminase co-activity, associated with non-immune related toxic side effects observed during therapy. Therefore, reduction of L-glutaminase co-activity with concomitant maintenance of its anticancer L-asparaginase effect may effectively improve the tolerability of this unique drug. Previously, we designed a new alternative variant of Erwinia chrysanthemi (ErA; Erwinaze) with decreased L-glutaminase co-activity, while maintaining its L-asparaginase activity, by the introduction of three key mutations around the active site (ErA-TM). However, Erwinaze and our ErA-TM variant have very short half-lives in vivo. Here, we show that the fusion of ErA-TM with an albumin binding domain (ABD)-tag significantly increases its in vivo persistence. In addition, we evaluated the in vivo therapeutic efficacy of ABD-ErA-TM in a B-ALL xenograft model of SUP-B15. Our results show a comparable long-lasting durable antileukemic effect between the standard-of-care pegylated-asparaginase and ABD-ErA-TM L-asparaginase, but with fewer co-glutaminase-related acute side effects. Since the toxic side effects of current L-asparaginases often result in treatment discontinuation in ALL patients, this novel ErA-TM variant with ultra-low L-glutaminase co-activity and long in vivo persistence may have great clinical potential.

Oncogenic deubiquitination controls tyrosine kinase signaling and therapy response in acute lymphoblastic leukemia.

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

IRF2 is required for development and functional maturation of human NK cells.

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.

An interactive mass spectrometry atlas of histone posttranslational modifications in T-cell acute leukemia.

The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide and protein data for downstream analyses, exemplified by the PeptidoformViz tool. This data resource sets the stage for generalizing MS-based histone analysis and provides the first reusable histone dataset for epidrug development.

Engraftment of Allotransplanted Tumor Cells in Adult rag2 Mutant Xenopus tropicalis.

Modeling human genetic diseases and cancer in lab animals has been greatly aided by the emergence of genetic engineering tools such as TALENs and CRISPR/Cas9. We have previously demonstrated the ease with which genetically engineered Xenopus models (GEXM) can be generated via injection of early embryos with Cas9 recombinant protein loaded with sgRNAs targeting single or multiple tumor suppressor genes. What has been lacking so far is the possibility to propagate and characterize the induced cancers via transplantation. Here, we describe the generation of a rag2 knockout line in Xenopus tropicalis that is deficient in functional T and B cells. This line was validated by means of allografting experiments with primary tp53-/- and apc+/-/tp53-/- donor tumors. In addition, we optimized available protocols for the sub-lethal irradiation of wild-type X. tropicalis froglets. Irradiated animals also allowed the stable, albeit transient, engraftment of transplanted X. tropicalis tumor cells. The novel rag2-/- line and the irradiated wild-type froglets will further expand the experimental toolbox in the diploid amphibian X. tropicalis and help to establish it as a versatile and relevant model for exploring human cancer.

TXNIP Promotes Human NK Cell Development but Is Dispensable for NK Cell Functionality.

The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation.

The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency.

Natural killer (NK) cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human haematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2Rß expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations.