Mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.

Despite being considered druggable and attractive therapeutic targets, most of the solute carrier (SLC) membrane transporters remain pharmacologically underexploited. One of the reasons for this is a lack of reliable chemical screening assays, made difficult by functional redundancies among SLCs. In this study we leveraged synthetic lethality between the lactate transporters SLC16A1 and SLC16A3 in a screening strategy that we call paralog-dependent isogenic cell assay (PARADISO). The system involves five isogenic cell lines, each dependent on various paralog genes for survival/fitness, arranged in a screening cascade tuned for the identification of SLC16A3 inhibitors. We screened a diversity-oriented library of ∼90,000 compounds and further developed our hits into slCeMM1, a paralog-selective and potent SLC16A3 inhibitor. By implementing chemoproteomics, we showed that slCeMM1 is selective also at the proteome-wide level, thus fulfilling an important criterion for chemical probes. This study represents a framework for the development of specific cell-based drug discovery assays.

Pancreatic islets of Langerhans contain several specialized endocrine cell types, which are commonly identified by the expression of single marker genes. However, the established marker genes cannot capture the complete spectrum of cellular heterogeneity in human pancreatic islets, and existing bulk transcriptome datasets provide averages across several cell populations. To dissect the cellular composition of the human pancreatic islet and to establish transcriptomes for all major cell types, we performed single-cell RNA sequencing on 70 cells sorted from human primary tissue. We used this dataset to validate previously described marker genes at the single-cell level and to identify specifically expressed transcription factors for all islet cell subtypes. All data are available for browsing and download, thus establishing a useful resource of single-cell expression profiles for endocrine cells in human pancreatic islets.

Post-translational modifications of histones alter chromatin structure and play key roles in gene expression and specification of cell states. Small molecules that target chromatin-modifying enzymes selectively are useful as probes and have promise as therapeutics, although very few are currently available. G9a (also named euchromatin histone methyltransferase 2 (EHMT2)) catalyzes methylation of lysine 9 on histone H3 (H3K9), a modification linked to aberrant silencing of tumor-suppressor genes, among others. Here, we report the discovery of a novel histone methyltransferase inhibitor, BRD4770. This compound reduced cellular levels of di- and trimethylated H3K9 without inducing apoptosis, induced senescence, and inhibited both anchorage-dependent and -independent proliferation in the pancreatic cancer cell line PANC-1. ATM-pathway activation, caused by either genetic or small-molecule inhibition of G9a, may mediate BRD4770-induced cell senescence. BRD4770 may be a useful tool to study G9a and its role in senescence and cancer cell biology.

Type 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Insulin expression is primarily restricted to the pancreatic β cells, which are physically or functionally depleted in diabetes. Identifying targetable pathways repressing insulin in non-β cells, particularly in the developmentally related glucagon-secreting α cells, is an important aim of regenerative medicine. Here, we perform an RNA interference screen in a murine α cell line to identify silencers of insulin expression. We discover that knockdown of the splicing factor Smndc1 triggers a global repression of α cell gene-expression programs in favor of increased β cell markers. Mechanistically, Smndc1 knockdown upregulates the β cell transcription factor Pdx1 by modulating the activities of the BAF and Atrx chromatin remodeling complexes. SMNDC1's repressive role is conserved in human pancreatic islets, its loss triggering enhanced insulin secretion and PDX1 expression. Our study identifies Smndc1 as a key factor connecting splicing and chromatin remodeling to the control of insulin expression in human and mouse islet cells.

Metastatic melanoma is hallmarked by its ability of phenotype switching to more slowly proliferating, but highly invasive cells. Here, we tested the impact of signal transducer and activator of transcription 3 (STAT3) on melanoma progression in association with melanocyte inducing transcription factor (MITF) expression levels. We established a mouse melanoma model for deleting Stat3 in melanocytes with specific expression of human hyperactive NRASQ61K in an Ink4a-deficient background, two frequent driver mutations in human melanoma. Mice devoid of Stat3 showed early disease onset with higher proliferation in primary tumors, but displayed significantly diminished lung, brain, and liver metastases. Whole-genome expression profiling of tumor-derived cells also showed a reduced invasion phenotype, which was further corroborated by 3D melanoma model analysis. Notably, loss or knockdown of STAT3 in mouse or human cells resulted in the upregulation of MITF and induction of cell proliferation. Mechanistically we show that STAT3-induced CAAT Box Enhancer Binding Protein (CEBP) expression was sufficient to suppress MITF transcription. Epigenetic analysis by ATAC-seq confirmed that CEBPa/b binding to the MITF enhancer region silenced the MITF locus. Finally, by classification of patient-derived melanoma samples, we show that STAT3 and MITF act antagonistically and hence contribute differentially to melanoma progression. We conclude that STAT3 is a driver of the metastatic process in melanoma and able to antagonize MITF via direct induction of CEBP family member transcription.

The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein-protein interactions, condensate formation, and chemical degradation.

Aberrations in genes coding for subunits of the BRG1/BRM associated factor (BAF) chromatin remodeling complexes are highly abundant in human cancers. Currently, it is not understood how these mostly loss-of-function mutations contribute to cancer development and how they can be targeted therapeutically. The cancer-type-specific occurrence patterns of certain subunit mutations suggest subunit-specific effects on BAF complex function, possibly by the formation of aberrant residual complexes. Here, we systematically characterize the effects of individual subunit loss on complex composition, chromatin accessibility and gene expression in a panel of knockout cell lines deficient for 22 BAF subunits. We observe strong, specific and sometimes discordant alterations dependent on the targeted subunit and show that these explain intracomplex codependencies, including the synthetic lethal interactions SMARCA4-ARID2, SMARCA4-ACTB and SMARCC1-SMARCC2. These data provide insights into the role of different BAF subcomplexes in genome-wide chromatin organization and suggest approaches to therapeutically target BAF-mutant cancers.

Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.

We provide a catalog for the effects of the human kinome on cell survival in response to DNA-damaging agents, covering all major DNA repair pathways. By treating 313 kinase-deficient cell lines with ten diverse DNA-damaging agents, including seven commonly used chemotherapeutics, we identified examples of vulnerability and resistance that are kinase specific. To investigate synthetic lethal interactions, we tested the response to carmustine for 25 cell lines by establishing a phenotypic fluorescence-activated cell sorting (FACS) assay designed to validate gene-drug interactions. We show apoptosis, cell cycle changes, and DNA damage and proliferation after alkylation- or crosslink-induced damage. In addition, we reconstitute the cellular sensitivity of DYRK4, EPHB6, MARK3, and PNCK as a proof of principle for our study. Furthermore, using global phosphoproteomics on cells lacking MARK3, we provide evidence for its role in the DNA damage response. Our data suggest that cancers with inactivating mutations in kinases, including MARK3, are particularly vulnerable to alkylating chemotherapeutic agents.

Patients with refractory or relapsed haematological malignancies have few treatment options and short survival times. Identification of effective therapies with genomic-based precision medicine is hampered by intratumour heterogeneity and incomplete understanding of the contribution of various mutations within specific cancer phenotypes. Ex-vivo drug-response profiling in patient biopsies might aid effective treatment identification; however, proof of its clinical utility is limited.

STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B-expressing mice lacked a hematopoietic phenotype, the STAT5BN642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8+ lymphoma or leukemia, indicating that the STAT5BN642H mutation drives cancer development. Persistent and enhanced levels of STAT5BN642H tyrosine phosphorylation in transformed CD8+ T cells led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5BN642H-expressing CD8+ T cells, which were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5BN642H mutation who respond poorly to conventional chemotherapy.

Maintaining sufficient levels of Pdx1 activity is a prerequisite for proper regulation of blood glucose homeostasis and beta cell function. Mice that are haploinsufficient for Pdx1 display impaired glucose tolerance and lack the ability to increase beta cell mass in response to decreased insulin signaling. Several studies have shown that post-translational modifications are regulating Pdx1 activity through intracellular localization and binding to co-factors. Understanding the signaling cues converging on Pdx1 and modulating its activity is therefore an attractive approach in diabetes treatment. We employed a novel technique called Nanofluidic Proteomic Immunoassay to characterize the post-translational profile of Pdx1. Following isoelectric focusing in nano-capillaries, this technology relies on a pan specific antibody for detection and it therefore allows the relative abundance of differently charged protein species to be examined simultaneously. In all eukaryotic cells tested we find that the Pdx1 protein separates into four distinct peaks whereas Pdx1 protein from bacteria only produces one peak. Of the four peaks in eukaryotic cells we correlate one of them to a phosphorylation Using alanine scanning and mass spectrometry we map this phosphorylation to serine 61 in both Min6 cells and in exogenous Pdx1 over-expressed in HEK293 cells. A single phosphorylation is also present in cultured islets but it remains unaffected by changes in glucose levels. It is present during embryogenesis but is not required for pancreas development.

In chronic myeloid leukemia (CML) resistance against one or more BCR-ABL1 tyrosine kinase inhibitors (TKI) remains a clinical challenge. Preclinical data suggest that TKI combinations may overcome resistance. We report on a heavily pre-treated 78 year-old female patient with CML who developed multi-resistant blast crisis with bone marrow fibrosis and a Ph- clone. Treatment with ponatinib resulted in blast cell clearance, decrease in fibrosis, and disappearance of BCR-ABL1, but also in severe thrombocytopenia with bleedings requiring platelet transfusions. We therefore switched from ponatinib to bosutinib. During bosutinib, platelet counts recovered. However, after 6 months, BCR-ABL1 mRNA levels increased to > 1%. Therefore, we ´switched back´ to ponatinib, and this was again followed by disappearance of BCR-ABL1 and a decrease in platelets. During the next 2 years, we applied ponatinib and bosutinib in continuous rotation-cycles and added hydroxyurea in order to suppress all sub-clones and to balance between efficacy and potential side effects following the principle of personalized medicine. With this approach the patient remained in complete molecular response and reached normal blood counts and a normal quality of life without vascular or other side effects. In conclusion, TKI rotation is a novel potent approach to suppress multiple resistant sub-clones and to balance between clinical efficacy and side effects in patients with advanced CML. Clinical trials are now warranted to show that TKI-rotation is in general safe and effective in these patients.

SMNDC1 is a Tudor domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor. Here, we study the specific contributions of the SMNDC1 Tudor domain to protein-protein interactions, subcellular localization, and molecular function. To perturb the protein function in cells, we develop small molecule inhibitors targeting the dimethylarginine binding pocket of the SMNDC1 Tudor domain. We find that SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation behavior is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein's Tudor domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDC1-dependent genes. These compounds will enable further pharmacological studies on the role of SMNDC1 in the regulation of nuclear condensates, gene regulation and cell identity.

Transcription factor fusion proteins can transform cells by inducing global changes of the transcriptome, often creating a state of oncogene addiction. Here, we investigate the role of epigenetic mechanisms in this process, focusing on Ewing sarcoma cells that are dependent on the EWS-FLI1 fusion protein. We established reference epigenome maps comprising DNA methylation, seven histone marks, open chromatin states, and RNA levels, and we analyzed the epigenome dynamics upon downregulation of the driving oncogene. Reduced EWS-FLI1 expression led to widespread epigenetic changes in promoters, enhancers, and super-enhancers, and we identified histone H3K27 acetylation as the most strongly affected mark. Clustering of epigenetic promoter signatures defined classes of EWS-FLI1-regulated genes that responded differently to low-dose treatment with histone deacetylase inhibitors. Furthermore, we observed strong and opposing enrichment patterns for E2F and AP-1 among EWS-FLI1-correlated and anticorrelated genes. Our data describe extensive genome-wide rewiring of epigenetic cell states driven by an oncogenic fusion protein.

Antiapoptotic Bcl-2 family members have recently (re)emerged as key drug targets in cancer, with a tissue- and tumor-specific activity profile of available BH3 mimetics. In multiple myeloma, MCL-1 has been described as a major gatekeeper of apoptosis. This discovery has led to the rapid establishment of clinical trials evaluating the impact of various MCL-1 inhibitors. However, our understanding about the clinical impact and optimal use of MCL-1 inhibitors is still limited. We therefore explored mechanisms of acquired MCL-1 inhibitor resistance and optimization strategies in myeloma. Our findings indicated heterogeneous paths to resistance involving baseline Bcl-2 family alterations of proapoptotic (BAK, BAX, and BIM) and antiapoptotic (Bcl-2 and MCL-1) proteins. These manifestations depend on the BH3 profile of parental cells that guide the enhanced formation of Bcl-2:BIM and/or the dynamic (ie, treatment-induced) formation of Bcl-xL:BIM and Bcl-xL:BAK complexes. Accordingly, an unbiased high-throughput drug-screening approach (n = 528) indicated alternative BH3 mimetics as top combination partners for MCL-1 inhibitors in sensitive and resistant cells (Bcl-xL>Bcl-2 inhibition), whereas established drug classes were mainly antagonistic (eg, antimitotic agents). We also revealed reduced activity of MCL-1 inhibitors in the presence of stromal support as a drug-class effect that was overcome by concurrent Bcl-xL or Bcl-2 inhibition. Finally, we demonstrated heterogeneous Bcl-2 family deregulation and MCL-1 inhibitor cross-resistance in carfilzomib-resistant cells, a phenomenon linked to the MDR1-driven drug efflux of MCL-1 inhibitors. The implications of our findings for clinical practice emphasize the need for patient-adapted treatment protocols, with the tracking of tumor- and/or clone-specific adaptations in response to MCL-1 inhibition.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo However, Cdk6-/- erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeleton organization. Cdk6-/- erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We also found that Cdk6 regulates the transcription of a panel of genes involved in actin (de-)polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia - and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.