Pooled multicolour tagging for visualizing subcellular protein dynamics.

Imaging-based methods are widely used for studying the subcellular localization of proteins in living cells. While routine for individual proteins, global monitoring of protein dynamics following perturbation typically relies on arrayed panels of fluorescently tagged cell lines, limiting throughput and scalability. Here, we describe a strategy that combines high-throughput microscopy, computer vision and machine learning to detect perturbation-induced changes in multicolour tagged visual proteomics cell (vpCell) pools. We use genome-wide and cancer-focused intron-targeting sgRNA libraries to generate vpCell pools and a large, arrayed collection of clones each expressing two different endogenously tagged fluorescent proteins. Individual clones can be identified in vpCell pools by image analysis using the localization patterns and expression level of the tagged proteins as visual barcodes, enabling simultaneous live-cell monitoring of large sets of proteins. To demonstrate broad applicability and scale, we test the effects of antiproliferative compounds on a pool with cancer-related proteins, on which we identify widespread protein localization changes and new inhibitors of the nuclear import/export machinery. The time-resolved characterization of changes in subcellular localization and abundance of proteins upon perturbation in a pooled format highlights the power of the vpCell approach for drug discovery and mechanism-of-action studies.

Oncogenic TYK2 P760L kinase is effectively targeted by combinatorial TYK2, mTOR and CDK4/6 kinase blockade.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase/signal transducer and activator of transcription pathway, which is central in cytokine signaling. Previously, germline TYK2 mutations have been described in two patients developing de novo T-cell acute lymphoblastic leukemias (T-ALL) or precursor B-ALL. The mutations (P760L and G761V) are located within the regulatory pseudokinase domain and lead to constitutive activation of TYK2. We demonstrate the transformation capacity of TYK2 P760L in hematopoietic cell systems including primary bone marrow cells. In vivo engraftment of TYK2 P760L-expressing cell lines led to development of leukemia. A kinase inhibitor screen uncovered that oncogenic TYK2 acts synergistically with the PI3K/AKT/mTOR and CDK4/6 pathways. Accordingly, the TYK2-specific inhibitor deucravacitinib (BMS986165) reduces cell viability of TYK2 P760L-transformed cell models and ex vivo cultured TYK2 P760L-mutated patient- derived xenograft cells most efficiently when combined with mTOR or CDK4/6 inhibitors. Our study thereby pioneers novel treatment options for patients suffering from TYK2-driven acute leukemia.

Metabolic modulation of transcription: The role of one-carbon metabolism.

While it is well known that expression levels of metabolic enzymes regulate the metabolic state of the cell, there is mounting evidence that the converse is also true, that metabolite levels themselves can modulate gene expression via epigenetic modifications and transcriptional regulation. Here we focus on the one-carbon metabolic pathway, which provides the essential building blocks of many classes of biomolecules, including purine nucleotides, thymidylate, serine, and methionine. We review the epigenetic roles of one-carbon metabolic enzymes and their associated metabolites and introduce an interactive computational resource that places enzyme essentiality in the context of metabolic pathway topology. Therefore, we briefly discuss examples of metabolic condensates and higher-order complexes of metabolic enzymes downstream of one-carbon metabolism. We speculate that they may be required to the formation of transcriptional condensates and gene expression control. Finally, we discuss new ways to exploit metabolic pathway compartmentalization to selectively target these enzymes in cancer.

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

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.

Molecular and Pharmacological Bladder Cancer Therapy Screening: Discovery of Clofarabine as a Highly Active Compound.

BACKGROUND: The heterogeneity of bladder cancers (BCs) is a major challenge for the development of novel therapies. However, given the high rates of recurrence and/or treatment failure, the identification of effective therapeutic strategies is an urgent clinical need. OBJECTIVE: We aimed to establish a model system for drug identification/repurposing in order to identify novel therapies for the treatment of BC. DESIGN, SETTING, AND PARTICIPANTS: A collection of commercially available BC cell lines (n = 32) was comprehensively characterized. A panel of 23 cell lines, representing a broad spectrum of BC, was selected to perform a high-throughput drug screen. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Positive hits were defined as compounds giving >50% inhibition in at least one BC cell line. RESULTS AND LIMITATIONS: Amongst >1700 tested chemical compounds, a total of 471 substances exhibited antineoplastic effects. Clofarabine, an antimetabolite drug used as third-line treatment for childhood acute lymphoblastic leukaemia, was amongst the limited number of drugs with inhibitory effects on cell lines of all intrinsic subtypes. We, thus, reassessed the substance and confirmed its inhibitory effects on commercially available cell lines and patient-derived cell cultures representing various disease stages, intrinsic subtypes, and histologic variants. To verify these effects in vivo, a patient-derived cell xenograft model for urothelial carcinoma (UC) was used. Well-tolerated doses of clofarabine induced complete remission in all treated animals (n = 12) suffering from both early- and late-stage disease. We further took advantage of another patient-derived cell xenograft model originating from the rare disease entity sarcomatoid carcinoma (SaC). Similarly to UC xenograft mice, clofarabine induced subcomplete to complete tumour remissions in all treated animals (n = 8). CONCLUSIONS: The potent effects of clofarabine in vitro and in vivo suggest that our findings may be of high clinical relevance. Clinical trials are needed to assess the value of clofarabine in improving BC patient care. PATIENT SUMMARY: We used commercially available cell lines for the identification of novel drugs for the treatment of bladder cancer. We confirmed the effects of one of these drugs, clofarabine, in patient-derived cell lines and two different mouse models, thereby demonstrating a potential clinical relevance of this substance in bladder cancer treatment.

Functional Precision Medicine Provides Clinical Benefit in Advanced Aggressive Hematologic Cancers and Identifies Exceptional Responders.

Personalized medicine aims to match the right drug with the right patient by using specific features of the individual patient's tumor. However, current strategies of personalized therapy matching provide treatment opportunities for less than 10% of patients with cancer. A promising method may be drug profiling of patient biopsy specimens with single-cell resolution to directly quantify drug effects. We prospectively tested an image-based single-cell functional precision medicine (scFPM) approach to guide treatments in 143 patients with advanced aggressive hematologic cancers. Fifty-six patients (39%) were treated according to scFPM results. At a median follow-up of 23.9 months, 30 patients (54%) demonstrated a clinical benefit of more than 1.3-fold enhanced progression-free survival compared with their previous therapy. Twelve patients (40% of responders) experienced exceptional responses lasting three times longer than expected for their respective disease. We conclude that therapy matching by scFPM is clinically feasible and effective in advanced aggressive hematologic cancers. SIGNIFICANCE: This is the first precision medicine trial using a functional assay to instruct n-of-one therapies in oncology. It illustrates that for patients lacking standard therapies, high-content assay-based scFPM can have a significant value in clinical therapy guidance based on functional dependencies of each patient's cancer.See related commentary by Letai, p. 290.This article is highlighted in the In This Issue feature, p. 275.

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.

PURPOSE: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. EXPERIMENTAL DESIGN: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. RESULTS: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra-S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. CONCLUSIONS: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573.

Therapeutic targeting of chromatin: status and opportunities.

The molecular characterization of mechanisms underlying transcriptional control and epigenetic inheritance since the 1990s has paved the way for the development of targeted therapies that modulate these pathways. In the past two decades, cancer genome sequencing approaches have uncovered a plethora of mutations in chromatin modifying enzymes across tumor types, and systematic genetic screens have identified many of these proteins as specific vulnerabilities in certain cancers. Now is the time when many of these basic and translational efforts start to bear fruit and more and more chromatin-targeting drugs are entering the clinic. At the same time, novel pharmacological approaches harbor the potential to modulate chromatin in unprecedented fashion, thus generating entirely novel opportunities. Here, we review the current status of chromatin targets in oncology and describe a vision for the epigenome-modulating drugs of the future.

Heterogeneous modulation of Bcl-2 family members and drug efflux mediate MCL-1 inhibitor resistance in multiple myeloma.

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.

APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.

High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells.

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.

An inhibitor-mediated beta-cell dedifferentiation model reveals distinct roles for FoxO1 in glucagon repression and insulin maturation.

OBJECTIVE: The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. METHODS: The murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. RESULTS: We show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. CONCLUSIONS: Our study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation.

Cell-surface SLC nucleoside transporters and purine levels modulate BRD4-dependent chromatin states.

Metabolism negotiates cell-endogenous requirements of energy, nutrients and building blocks with the immediate environment to enable various processes, including growth and differentiation. While there is an increasing number of examples of crosstalk between metabolism and chromatin, few involve uptake of exogenous metabolites. Solute carriers (SLCs) represent the largest group of transporters in the human genome and are responsible for the transport of a wide variety of substrates, including nutrients and metabolites. We aimed to investigate the possible involvement of SLC-mediated solutes uptake and cellular metabolism in regulating cellular epigenetic states. Here, we perform a CRISPR-Cas9 transporter-focused genetic screen and a metabolic compound library screen for the regulation of BRD4-dependent chromatin states in human myeloid leukaemia cells. Intersection of the two orthogonal approaches reveal that loss of transporters involved with purine transport or inhibition of de novo purine synthesis lead to dysfunction of BRD4-dependent transcriptional regulation. Through mechanistic characterization of the metabolic circuitry, we elucidate the convergence of SLC-mediated purine uptake and de novo purine synthesis on BRD4-chromatin occupancy. Moreover, adenine-related metabolite supplementation effectively restores BRD4 functionality on purine impairment. Our study highlights the specific role of purine/adenine metabolism in modulating BRD4-dependent epigenetic states.

Acute BAF perturbation causes immediate changes in chromatin accessibility.

Cancer-associated, loss-of-function mutations in genes encoding subunits of the BRG1/BRM-associated factor (BAF) chromatin-remodeling complexes1-8 often cause drastic chromatin accessibility changes, especially in important regulatory regions9-19. However, it remains unknown how these changes are established over time (for example, immediate consequences or long-term adaptations), and whether they are causative for intracomplex synthetic lethalities, abrogating the formation or activity of BAF complexes9,20-24. In the present study, we use the dTAG system to induce acute degradation of BAF subunits and show that chromatin alterations are established faster than the duration of one cell cycle. Using a pharmacological inhibitor and a chemical degrader of the BAF complex ATPase subunits25,26, we show that maintaining genome accessibility requires constant ATP-dependent remodeling. Completely abolishing BAF complex function by acute degradation of a synthetic lethal subunit in a paralog-deficient background results in an almost complete loss of chromatin accessibility at BAF-controlled sites, especially also at superenhancers, providing a mechanism for intracomplex synthetic lethalities.

Precision Medicine in Hematology 2021: Definitions, Tools, Perspectives, and Open Questions.

During the past few years, our understanding of molecular mechanisms and cellular interactions relevant to malignant blood cell disorders has improved substantially. New insights include a detailed knowledge about disease-initiating exogenous factors, endogenous (genetic, somatic, epigenetic) elicitors or facilitators of disease evolution, and drug actions and interactions that underlie efficacy and adverse event profiles in defined cohorts of patients. As a result, precision medicine and personalized medicine are rapidly growing new disciplines that support the clinician in making the correct diagnosis, in predicting outcomes, and in optimally selecting patients for interventional therapies. In addition, precision medicine tools are greatly facilitating the development of new drugs, therapeutic approaches, and new multiparametric prognostic scoring models. However, although the emerging roles of precision medicine and personalized medicine in hematology and oncology are clearly visible, several questions remain. For example, it remains unknown how precision medicine tools can be implemented in healthcare systems and whether all possible approaches are also affordable. In addition, there is a need to define terminologies and to relate these to specific and context-related tools and strategies in basic and applied science. To discuss these issues, a working conference was organized in September 2019. The outcomes of this conference are summarized herein and include a proposal for definitions, terminologies, and applications of precision and personalized medicine concepts and tools in hematologic neoplasms. We also provide proposals aimed at reducing costs, thereby making these applications affordable in daily practice.

STAT3 promotes melanoma metastasis by CEBP-induced repression of the MITF pathway.

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.

5-Arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones with selective inhibitory activity against some leukemia cell lines.

The data on the pharmacology of 4-thiazolidinones showed that 5-ene-2-(imino)amino-4-thiazolidinones are likely to comprise one of the most promising groups of compounds possessing anticancer properties. A series of 5-arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones was designed, synthesized, and studied against 10 leukemia cell lines, including the HL-60, Jurkat, K-562, Dami, KBM-7, and some Ba/F3 cell lines. The structure-activity relationship analysis shows that almost all tested 5-arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones were characterized by ІС50 values lower or comparable to that of the control drug chlorambucil. Among the tested compounds, (5Z)-5-(2-methoxybenzylidene)- (12), (5Z)-(2-ethoxybenzylidene)- (21), (5Z)-5-(2-benzyloxybenzylidene)- (25), and (5Z)-5-(2-allyloxybenzylidene)-2-(4-hydroxyphenylamino)thiazol-4(5H)-ones (28) possessed the highest antileukemic activity at submicromolar concentrations (ІС50 = 0.10-0.95 µM).

Hematoxylin binds to mutant calreticulin and disrupts its abnormal interaction with thrombopoietin receptor.

Somatic mutations of calreticulin (CALR) have been identified as a main disease driver of myeloproliferative neoplasms, suggesting that development of drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD) abolishes the ability of mutant CALR to oncogenically activate the thrombopoietin receptor (MPL). We therefore hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing a selective growth inhibitory effect on cells that depend on oncogenic CALR for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR-mutated cells than in CALR wild-type cells. Additionally, knockout or C-terminal truncation of CALR eliminated drug hypersensitivity in CALR-mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, disruption of the mutant CALR-MPL interaction, inhibition of the JAK2-STAT5 pathway, and reduction at the intracellular level of mutant CALR upon drug treatment. Our data indicate that small molecules targeting the GBD of CALR can selectively kill CALR-mutated cells by disrupting the CALR-MPL interaction and inhibiting oncogenic signaling.

Rational discovery of molecular glue degraders via scalable chemical profiling.

Targeted protein degradation is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are molecular glues that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, we describe a scalable strategy toward glue degrader discovery that is based on chemical screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led us to identify compounds that induce ubiquitination and degradation of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, our data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.