PDP1 is a key metabolic gatekeeper and modulator of drug resistance in FLT3-ITD-positive acute myeloid leukemia.

High metabolic flexibility is pivotal for the persistence and therapy resistance of acute myeloid leukemia (AML). In 20-30% of AML patients, activating mutations of FLT3, specifically FLT3-ITD, are key therapeutic targets. Here, we investigated the influence of FLT3-ITD on AML metabolism. Nuclear Magnetic Resonance (NMR) profiling showed enhanced reshuffling of pyruvate towards the tricarboxylic acid (TCA) cycle, suggesting an increased activity of the pyruvate dehydrogenase complex (PDC). Consistently, FLT3-ITD-positive cells expressed high levels of PDP1, an activator of the PDC. Combining endogenous tagging of PDP1 with genome-wide CRISPR screens revealed that FLT3-ITD induces PDP1 expression through the RAS signaling axis. PDP1 knockdown resulted in reduced cellular respiration thereby impairing the proliferation of only FLT3-ITD cells. These cells continued to depend on PDP1, even in hypoxic conditions, and unlike FLT3-ITD-negative cells, they exhibited a rapid, PDP1-dependent revival of their respiratory capacity during reoxygenation. Moreover, we show that PDP1 modifies the response to FLT3 inhibition. Upon incubation with the FLT3 tyrosine kinase inhibitor quizartinib (AC220), PDP1 persisted or was upregulated, resulting in a further shift of glucose/pyruvate metabolism towards the TCA cycle. Overexpression of PDP1 enhanced, while PDP1 depletion diminished AC220 resistance in cell lines and peripheral blasts from an AC220-resistant AML patient in vivo. In conclusion, FLT3-ITD assures the expression of PDP1, a pivotal metabolic regulator that enhances oxidative glucose metabolism and drug resistance. Hence, PDP1 emerges as a potentially targetable vulnerability in the management of AML.

Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome.

Gene expression is in part controlled by cis-regulatory elements (CREs) such as enhancers and repressive elements. Anecdotal evidence has indicated that a CRE and a promoter need to be biochemically compatible for promoter regulation to occur, but this compatibility has remained poorly characterized in mammalian cells. We used high-throughput combinatorial reporter assays to test thousands of CRE-promoter pairs from three Mb-sized genomic regions in mouse cells. This revealed that CREs vary substantially in their promoter compatibility, ranging from striking specificity to broad promiscuity. More than half of the tested CREs exhibit significant promoter selectivity. Housekeeping promoters tend to have similar CRE preferences, but other promoters exhibit a wide diversity of compatibilities. Higher-order transcription factors (TF) motif combinations may account for compatibility. CRE-promoter selectivity does not correlate with looping interactions in the native genomic context, suggesting that chromatin folding and compatibility are two orthogonal mechanisms that confer specificity to gene regulation.

The proteogenomic subtypes of acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis. We report a comprehensive proteogenomic analysis of bone marrow biopsies from 252 uniformly treated AML patients to elucidate the molecular pathophysiology of AML in order to inform future diagnostic and therapeutic approaches. In addition to in-depth quantitative proteomics, our analysis includes cytogenetic profiling and DNA/RNA sequencing. We identify five proteomic AML subtypes, each reflecting specific biological features spanning genomic boundaries. Two of these proteomic subtypes correlate with patient outcome, but none is exclusively associated with specific genomic aberrations. Remarkably, one subtype (Mito-AML), which is captured only in the proteome, is characterized by high expression of mitochondrial proteins and confers poor outcome, with reduced remission rate and shorter overall survival on treatment with intensive induction chemotherapy. Functional analyses reveal that Mito-AML is metabolically wired toward stronger complex I-dependent respiration and is more responsive to treatment with the BCL2 inhibitor venetoclax.

SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

Rewiring of B cell receptor signaling by Epstein-Barr virus LMP2A.

Epstein-Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.

Dissection of acute stimulus-inducible nucleosome remodeling in mammalian cells.

Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). While stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis-regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data sets and used it to analyze nucleosome dynamics at stimulus-regulated cis-regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing, however, different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.

Selective inactivation of hypomethylating agents by SAMHD1 provides a rationale for therapeutic stratification in AML.

Hypomethylating agents decitabine and azacytidine are regarded as interchangeable in the treatment of acute myeloid leukemia (AML). However, their mechanisms of action remain incompletely understood, and predictive biomarkers for HMA efficacy are lacking. Here, we show that the bioactive metabolite decitabine triphosphate, but not azacytidine triphosphate, functions as activator and substrate of the triphosphohydrolase SAMHD1 and is subject to SAMHD1-mediated inactivation. Retrospective immunohistochemical analysis of bone marrow specimens from AML patients at diagnosis revealed that SAMHD1 expression in leukemic cells inversely correlates with clinical response to decitabine, but not to azacytidine. SAMHD1 ablation increases the antileukemic activity of decitabine in AML cell lines, primary leukemic blasts, and xenograft models. AML cells acquire resistance to decitabine partly by SAMHD1 up-regulation. Together, our data suggest that SAMHD1 is a biomarker for the stratified use of hypomethylating agents in AML patients and a potential target for the treatment of decitabine-resistant leukemia.

High-throughput identification of human SNPs affecting regulatory element activity.

Most of the millions of SNPs in the human genome are non-coding, and many overlap with putative regulatory elements. Genome-wide association studies (GWAS) have linked many of these SNPs to human traits or to gene expression levels, but rarely with sufficient resolution to identify the causal SNPs. Functional screens based on reporter assays have previously been of insufficient throughput to test the vast space of SNPs for possible effects on regulatory element activity. Here we leveraged the throughput and resolution of the survey of regulatory elements (SuRE) reporter technology to survey the effect of 5.9 million SNPs, including 57% of the known common SNPs, on enhancer and promoter activity. We identified more than 30,000 SNPs that alter the activity of putative regulatory elements, partially in a cell-type-specific manner. Integration of this dataset with GWAS results may help to pinpoint SNPs that underlie human traits.

Promoter-Intrinsic and Local Chromatin Features Determine Gene Repression in LADs.

It is largely unclear whether genes that are naturally embedded in lamina-associated domains (LADs) are inactive due to their chromatin environment or whether LADs are merely secondary to the lack of transcription. We show that hundreds of human promoters become active when moved from their native LAD position to a neutral context in the same cells, indicating that LADs form a repressive environment. Another set of promoters inside LADs is able to "escape" repression, although their transcription elongation is attenuated. By inserting reporters into thousands of genomic locations, we demonstrate that escaper promoters are intrinsically less sensitive to LAD repression. This is not simply explained by promoter strength but by the interplay between promoter sequence and local chromatin features that vary strongly across LADs. Enhancers also differ in their sensitivity to LAD chromatin. This work provides a general framework for the systematic understanding of gene regulation by repressive chromatin.

Comparative proteomics reveals a diagnostic signature for pulmonary head-and-neck cancer metastasis.

Patients with head-and-neck cancer can develop both lung metastasis and primary lung cancer during the course of their disease. Despite the clinical importance of discrimination, reliable diagnostic biomarkers are still lacking. Here, we have characterised a cohort of squamous cell lung (SQCLC) and head-and-neck (HNSCC) carcinomas by quantitative proteomics. In a training cohort, we quantified 4,957 proteins in 44 SQCLC and 30 HNSCC tumours. A total of 518 proteins were found to be differentially expressed between SQCLC and HNSCC, and some of these were identified as genetic dependencies in either of the two tumour types. Using supervised machine learning, we inferred a proteomic signature for the classification of squamous cell carcinomas as either SQCLC or HNSCC, with diagnostic accuracies of 90.5% and 86.8% in cross- and independent validations, respectively. Furthermore, application of this signature to a cohort of pulmonary squamous cell carcinomas of unknown origin leads to a significant prognostic separation. This study not only provides a diagnostic proteomic signature for classification of secondary lung tumours in HNSCC patients, but also represents a proteomic resource for HNSCC and SQCLC.

Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures.

Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relation between cis- regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 minutes of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra- and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.

Mbd3/NuRD controls lymphoid cell fate and inhibits tumorigenesis by repressing a B cell transcriptional program.

Differentiation of lineage-committed cells from multipotent progenitors requires the establishment of accessible chromatin at lineage-specific transcriptional enhancers and promoters, which is mediated by pioneer transcription factors that recruit activating chromatin remodeling complexes. Here we show that the Mbd3/nucleosome remodeling and deacetylation (NuRD) chromatin remodeling complex opposes this transcriptional pioneering during B cell programming of multipotent lymphoid progenitors by restricting chromatin accessibility at B cell enhancers and promoters. Mbd3/NuRD-deficient lymphoid progenitors therefore prematurely activate a B cell transcriptional program and are biased toward overproduction of pro-B cells at the expense of T cell progenitors. The striking reduction in early thymic T cell progenitors results in compensatory hyperproliferation of immature thymocytes and development of T cell lymphoma. Our results reveal that Mbd3/NuRD can regulate multilineage differentiation by constraining the activation of dormant lineage-specific enhancers and promoters. In this way, Mbd3/NuRD protects the multipotency of lymphoid progenitors, preventing B cell-programming transcription factors from prematurely enacting lineage commitment. Mbd3/NuRD therefore controls the fate of lymphoid progenitors, ensuring appropriate production of lineage-committed progeny and suppressing tumor formation.

Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia.

The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia.

Stable Polycomb-dependent transgenerational inheritance of chromatin states in Drosophila.

Transgenerational epigenetic inheritance (TEI) describes the transmission of alternative functional states through multiple generations in the presence of the same genomic DNA sequence. Very little is known about the principles and the molecular mechanisms governing this type of inheritance. Here, by transiently enhancing 3D chromatin interactions, we established stable and isogenic Drosophila epilines that carry alternative epialleles, as defined by differential levels of Polycomb-dependent trimethylation of histone H3 Lys27 (forming H3K27me3). After being established, epialleles can be dominantly transmitted to naive flies and can induce paramutation. Importantly, epilines can be reset to a naive state by disruption of chromatin interactions. Finally, we found that environmental changes modulate the expressivity of the epialleles, and we extended our paradigm to naturally occurring phenotypes. Our work sheds light on how nuclear organization and Polycomb group (PcG) proteins contribute to epigenetically inheritable phenotypic variability.

HSP90 promotes Burkitt lymphoma cell survival by maintaining tonic B-cell receptor signaling.

Burkitt lymphoma (BL) is an aggressive B-cell neoplasm that is currently treated by intensive chemotherapy in combination with anti-CD20 antibodies. Because of their toxicity, current treatment regimens are often not suitable for elderly patients or for patients in developing countries where BL is endemic. Targeted therapies for BL are therefore needed. In this study, we performed a compound screen in 17 BL cell lines to identify small molecule inhibitors affecting cell survival. We found that inhibitors of heat shock protein 90 (HSP90) induced apoptosis in BL cells in vitro at concentrations that did not affect normal B cells. By global proteomic and phosphoproteomic profiling, we show that, in BL, HSP90 inhibition compromises the activity of the pivotal B-cell antigen receptor (BCR)-proximal effector spleen tyrosine kinase (SYK), which we identified as an HSP90 client protein. Consistently, expression of constitutively active TEL-SYK counteracted the apoptotic effect of HSP90 inhibition. Together, our results demonstrate that HSP90 inhibition impairs BL cell survival by interfering with tonic BCR signaling, thus providing a molecular rationale for the use of HSP90 inhibitors in the treatment of BL.

SAMHD1 is a biomarker for cytarabine response and a therapeutic target in acute myeloid leukemia.

The nucleoside analog cytarabine (Ara-C) is an essential component of primary and salvage chemotherapy regimens for acute myeloid leukemia (AML). After cellular uptake, Ara-C is converted into its therapeutically active triphosphate metabolite, Ara-CTP, which exerts antileukemic effects, primarily by inhibiting DNA synthesis in proliferating cells. Currently, a substantial fraction of patients with AML fail to respond effectively to Ara-C therapy, and reliable biomarkers for predicting the therapeutic response to Ara-C are lacking. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs, we show here that SAMHD1 reduces Ara-C cytotoxicity in AML cells. Mechanistically, dGTP-activated SAMHD1 hydrolyzes Ara-CTP, which results in a drastic reduction of Ara-CTP in leukemic cells. Loss of SAMHD1 activity-through genetic depletion, mutational inactivation of its triphosphohydrolase activity or proteasomal degradation using specialized, virus-like particles-potentiates the cytotoxicity of Ara-C in AML cells. In mouse models of retroviral AML transplantation, as well as in retrospective analyses of adult patients with AML, the response to Ara-C-containing therapy was inversely correlated with SAMHD1 expression. These results identify SAMHD1 as a potential biomarker for the stratification of patients with AML who might best respond to Ara-C-based therapy and as a target for treating Ara-C-refractory AML.

A High-Density Map for Navigating the Human Polycomb Complexome.

Polycomb group (PcG) proteins are major determinants of gene silencing and epigenetic memory in higher eukaryotes. Here, we systematically mapped the human PcG complexome using a robust affinity purification mass spectrometry approach. Our high-density protein interaction network uncovered a diverse range of PcG complexes. Moreover, our analysis identified PcG interactors linking them to the PcG system, thus providing insight into the molecular function of PcG complexes and mechanisms of recruitment to target genes. We identified two human PRC2 complexes and two PR-DUB deubiquitination complexes, which contain the O-linked N-acetylglucosamine transferase OGT1 and several transcription factors. Finally, genome-wide profiling of PR-DUB components indicated that the human PR-DUB and PRC1 complexes bind distinct sets of target genes, suggesting differential impact on cellular processes in mammals.

Elucidation of tonic and activated B-cell receptor signaling in Burkitt's lymphoma provides insights into regulation of cell survival.

Burkitt's lymphoma (BL) is a highly proliferative B-cell neoplasm and is treated with intensive chemotherapy that, because of its toxicity, is often not suitable for the elderly or for patients with endemic BL in developing countries. BL cell survival relies on signals transduced by B-cell antigen receptors (BCRs). However, tonic as well as activated BCR signaling networks and their relevance for targeted therapies in BL remain elusive. We have systematically characterized and compared tonic and activated BCR signaling in BL by quantitative phosphoproteomics to identify novel BCR effectors and potential drug targets. We identified and quantified ∼16,000 phospho-sites in BL cells. Among these sites, 909 were related to tonic BCR signaling, whereas 984 phospho-sites were regulated upon BCR engagement. The majority of the identified BCR signaling effectors have not been described in the context of B cells or lymphomas yet. Most of these newly identified BCR effectors are predicted to be involved in the regulation of kinases, transcription, and cytoskeleton dynamics. Although tonic and activated BCR signaling shared a considerable number of effector proteins, we identified distinct phosphorylation events in tonic BCR signaling. We investigated the functional relevance of some newly identified BCR effectors and show that ACTN4 and ARFGEF2, which have been described as regulators of membrane-trafficking and cytoskeleton-related processes, respectively, are crucial for BL cell survival. Thus, this study provides a comprehensive dataset for tonic and activated BCR signaling and identifies effector proteins that may be relevant for BL cell survival and thus may help to develop new BL treatments.