Discovery of YAP1/TAZ pathway inhibitors through phenotypic screening with potent anti-tumor activity via blockade of Rho-GTPase signaling.

This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.

Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21.

USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.

Heteromeric HSFA2/HSFA3 complexes drive transcriptional memory after heat stress in Arabidopsis.

Adaptive plasticity in stress responses is a key element of plant survival strategies. For instance, moderate heat stress (HS) primes a plant to acquire thermotolerance, which allows subsequent survival of more severe HS conditions. Acquired thermotolerance is actively maintained over several days (HS memory) and involves the sustained induction of memory-related genes. Here we show that FORGETTER3/ HEAT SHOCK TRANSCRIPTION FACTOR A3 (FGT3/HSFA3) is specifically required for physiological HS memory and maintaining high memory-gene expression during the days following a HS exposure. HSFA3 mediates HS memory by direct transcriptional activation of memory-related genes after return to normal growth temperatures. HSFA3 binds HSFA2, and in vivo both proteins form heteromeric complexes with additional HSFs. Our results indicate that only complexes containing both HSFA2 and HSFA3 efficiently promote transcriptional memory by positively influencing histone H3 lysine 4 (H3K4) hyper-methylation. In summary, our work defines the major HSF complex controlling transcriptional memory and elucidates the in vivo dynamics of HSF complexes during somatic stress memory.

FORGETTER2 protein phosphatase and phospholipase D modulate heat stress memory in Arabidopsis.

Plants can mitigate environmental stress conditions through acclimation. In the case of fluctuating stress conditions such as high temperatures, maintaining a stress memory enables a more efficient response upon recurring stress. In a genetic screen for Arabidopsis thaliana mutants impaired in the memory of heat stress (HS) we have isolated the FORGETTER2 (FGT2) gene, which encodes a type 2C protein phosphatase (PP2C) of the D-clade. Fgt2 mutants acquire thermotolerance normally; however, they are defective in the memory of HS. FGT2 interacts with phospholipase D α2 (PLDα2), which is involved in the metabolism of membrane phospholipids and is also required for HS memory. In summary, we have uncovered a previously unknown component of HS memory and identified the FGT2 protein phosphatase and PLDα2 as crucial players, suggesting that phosphatidic acid-dependent signaling or membrane composition dynamics underlie HS memory.

Characterization of the Menin-MLL Interaction as Therapeutic Cancer Target.

Inhibiting the interaction of menin with the histone methyltransferase MLL1 (KMT2A) has recently emerged as a novel therapeutic strategy. Beneficial therapeutic effects have been postulated in leukemia, prostate, breast, liver and in synovial sarcoma models. In those indications, MLL1 recruitment by menin was described to critically regulate the expression of disease associated genes. However, most findings so far rely on single study reports. Here we independently evaluated the pathogenic functions of the menin-MLL interaction in a large set of different cancer models with a potent and selective probe inhibitor BAY-155. We characterized the inhibition of the menin-MLL interaction for anti-proliferation, gene transcription effects, and for efficacy in several in vivo xenografted tumor models. We found a specific therapeutic activity of BAY-155 primarily in AML/ALL models. In solid tumors, we observed anti-proliferative effects of BAY-155 in a surprisingly limited fraction of cell line models. These findings were further validated in vivo. Overall, our study using a novel, highly selective and potent inhibitor, shows that the menin-MLL interaction is not essential for the survival of most solid cancer models. We can confirm that disrupting the menin-MLL complex has a selective therapeutic benefit in MLL-fused leukemia. In solid cancers, effects are restricted to single models and more limited than previously claimed.

Functional diversity of inhibitors tackling the differentiation blockage of MLL-rearranged leukemia.

INTRODUCTION: The chromosomal rearrangements of the mixed-lineage leukemia gene MLL (KMT2A) have been extensively characterized as a potent oncogenic driver in leukemia. For its oncogenic function, most MLL-fusion proteins exploit the multienzyme super elongation complex leading to elevated expression of MLL target genes. High expression of MLL target genes overwrites the normal hematopoietic differentiation program, resulting in undifferentiated blasts characterized by the capacity to self-renew. Although extensive resources devoted to increased understanding of therapeutic targets to overcome de-differentiation in ALL/AML, the inter-dependencies of targets are still not well described. The majority of inhibitors potentially interfering with MLL-fusion protein driven transformation have been characterized in individual studies, which so far hindered their direct cross-comparison. METHODS: In our study, we characterized head-to-head clinical stage inhibitors for BET, DHODH, DOT1L as well as two novel inhibitors for CDK9 and the Menin-MLL interaction with a focus on differentiation induction. We profiled those inhibitors for global gene expression effects in a large cell line panel and examined cellular responses such as inhibition of proliferation, apoptosis induction, cell cycle arrest, surface marker expression, morphological phenotype changes, and phagocytosis as functional differentiation readout. We also verified the combination potential of those inhibitors on proliferation and differentiation level. RESULTS: Our analysis revealed significant differences in differentiation induction and in modulating MLL-fusion target gene expression. We observed Menin-MLL and DOT1L inhibitors act very specifically on MLL-fused leukemia cell lines, whereas inhibitors of BET, DHODH and P-TEFb have strong effects beyond MLL-fusions. Significant differentiation effects were detected for Menin-MLL, DOT1L, and DHODH inhibitors, whereas BET and CDK9 inhibitors primarily induced apoptosis in AML/ALL cancer models. For the first time, we explored combination potential of the abovementioned inhibitors with regards to overcoming the differentiation blockage. CONCLUSION: Our findings show substantial diversity in the molecular activities of those inhibitors and provide valuable insights into the further developmental potential as single agents or in combinations in MLL-fused leukemia.

The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies.

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.

BRUSHY1/TONSOKU/MGOUN3 is required for heat stress memory.

Plants encounter biotic and abiotic stresses many times during their life cycle and this limits their productivity. Moderate heat stress (HS) primes a plant to survive higher temperatures that are lethal in the naïve state. Once temperature stress subsides, the memory of the priming event is actively retained for several days preparing the plant to better cope with recurring HS. Recently, chromatin regulation at different levels has been implicated in HS memory. Here, we report that the chromatin protein BRUSHY1 (BRU1)/TONSOKU/MGOUN3 plays a role in the HS memory in Arabidopsis thaliana. BRU1 is also involved in transcriptional gene silencing and DNA damage repair. This corresponds with the functions of its mammalian orthologue TONSOKU-LIKE/NFΚBIL2. During HS memory, BRU1 is required to maintain sustained induction of HS memory-associated genes, whereas it is dispensable for the acquisition of thermotolerance. In summary, we report that BRU1 is required for HS memory in A. thaliana, and propose a model where BRU1 mediates the epigenetic inheritance of chromatin states across DNA replication and cell division.

Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling.

Plants as sessile organisms can adapt to environmental stress to mitigate its adverse effects. As part of such adaptation they maintain an active memory of heat stress for several days that promotes a more efficient response to recurring stress. We show that this heat stress memory requires the activity of the FORGETTER1 (FGT1) locus, with fgt1 mutants displaying reduced maintenance of heat-induced gene expression. FGT1 encodes the Arabidopsis thaliana orthologue of Strawberry notch (Sno), and the protein globally associates with the promoter regions of actively expressed genes in a heat-dependent fashion. FGT1 interacts with chromatin remodelers of the SWI/SNF and ISWI families, which also display reduced heat stress memory. Genomic targets of the BRM remodeler overlap significantly with FGT1 targets. Accordingly, nucleosome dynamics at loci with altered maintenance of heat-induced expression are affected in fgt1. Together, our results suggest that by modulating nucleosome occupancy, FGT1 mediates stress-induced chromatin memory.

HSFA2 orchestrates transcriptional dynamics after heat stress in Arabidopsis thaliana.

In nature, stress is typically chronic or recurring and stress exposure can prime modified responses to recurring stress. Such stress priming may occur at the level of transcription. Here, we discuss the connection between plant stress memory, transcription, and chromatin modifications using the example of recurring heat stress.

A JUMONJI Protein with E3 Ligase and Histone H3 Binding Activities Affects Transposon Silencing in Arabidopsis.

Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity.

A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory.

In nature, plants often encounter chronic or recurring stressful conditions. Recent results indicate that plants can remember a past exposure to stress to be better prepared for a future stress incident. However, the molecular basis of this is poorly understood. Here, we report the involvement of chromatin modifications in the maintenance of acquired thermotolerance (heat stress [HS] memory). HS memory is associated with the accumulation of histone H3 lysine 4 di- and trimethylation at memory-related loci. This accumulation outlasts their transcriptional activity and marks them as recently transcriptionally active. High accumulation of H3K4 methylation is associated with hyper-induction of gene expression upon a recurring HS. This transcriptional memory and the sustained accumulation of H3K4 methylation depend on HSFA2, a transcription factor that is required for HS memory, but not initial heat responses. Interestingly, HSFA2 associates with memory-related loci transiently during the early stages following HS. In summary, we show that transcriptional memory after HS is associated with sustained H3K4 hyper-methylation and depends on a hit-and-run transcription factor, thus providing a molecular framework for HS memory.

Epigenetic responses to heat stress at different time scales and the involvement of small RNAs.

The hypothesis that plants can benefit from a memory of past stress exposure has recently attracted a lot of attention. Here, we discuss two different examples of heat stress memory to elucidate the potential benefits that epigenetic responses may provide at both the level of acclimation of the individual plant and adaptation at a species-wide level. Specifically, we discuss how microRNAs regulate the heat stress memory and thereby increase survival upon a recurring heat stress. Secondly, we review how a prolonged heat stress in a small interfering RNA-deficient background induces retrotransposition that is transmitted to the next generation, thus creating genetic variation for natural selection to act on. Collectively, these studies reveal a crucial role of short RNAs in heat stress memory across different time scales.