Synthetic lethal interactions of DEAD/H-box helicases as targets for cancer therapy

DEAD/H-box helicases are implicated in virtually every aspect of RNA metabolism, including transcription, pre-mRNA splicing, ribosomes biogenesis, nuclear export, translation initiation, RNA degradation, and mRNA editing. Most of these helicases are upregulated in various cancers and mutations in some of them are associated with several malignancies. Lately, synthetic lethality (SL) and synthetic dosage lethality (SDL) approaches, where genetic interactions of cancer-related genes are exploited as therapeutic targets, are emerging as a leading area of cancer research. Several DEAD/H-box helicases, including DDX3, DDX9 (Dbp9), DDX10 (Dbp4), DDX11 (ChlR1), and DDX41 (Sacy-1), have been subjected to SL analyses in humans and different model organisms. It remains to be explored whether SDL can be utilized to identity druggable targets in DEAD/H-box helicase overexpressing cancers. In this review, we analyze gene expression data of a subset of DEAD/H-box helicases in multiple cancer types and discuss how their SL/SDL interactions can be used for therapeutic purposes. We also summarize the latest developments in clinical applications, apart from discussing some of the challenges in drug discovery in the context of targeting DEAD/H-box helicases

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

The EphB6 receptor is overexpressed in pediatric T cell acute lymphoblastic leukemia and increases its sensitivity to doxorubicin treatment

While impressive improvements have been achieved in T-ALL therapy, current treatment approaches fail in approximately 25% of patients and these patients have limited treatment options. Another significant group of patients is being overtreated, which causes long-lasting side effects. Identification of molecules controlling drug resistance in T-ALL is crucial for treatment optimisation in both scenarios. We report here the EphB6 receptor is frequently overexpressed in T-ALL. Remarkably, our observations indicate that EphB6 acts in T-ALL cells to enhance sensitivity to a DNA-damaging drug, doxorubicin, as interruption of EphB6 activity interferes with the efficiency of doxorubicin-induced eradication of T-ALL cells in cell culture and in xenograft animals. This effect relies on the protection of Akt kinase signaling, while Akt inhibition combined with doxorubicin application produces synergistic effects on the elimination of EphB6-deficient T-ALL cells. These data imply that EphB6 suppresses T-ALL resistance by interfering with Akt activity. Our observations highlight a novel role for EphB6 in reducing drug resistance of T-ALL and suggest that doxorubicin treatment should produce better results if personalised based on EphB6 levels. If successfully verified in clinical studies, this approach should improve outcomes for T-ALL patients resistant to current therapies and for patients, who are being overtreated.This work was sponsored by the Canadian Institutes of Health Research (CIHR) grant # 132191 and by the Saskatchewan Health Research Foundation (SHRF) grant # 2891. A.E.Z. was supported by SHRF Postdoctoral Fellowship

The intrinsically kinase-inactive EPHB6 receptor predisposes cancer cells to DR5-induced apoptosis by promoting mitochondrial fragmentation

Death Receptor 5 (DR5) is a promising target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells. However, the therapeutic usefulness of DR5 agonists is currently limited by the frequent resistance of malignant tumours to its activation. The identification of molecular mechanisms that determine outcomes of DR5 action is therefore crucial for improving the efficiency of DR5-activating reagents in cancer treatment. Here, we provide evidence that an intrinsically kinase-inactive member of the Eph group of receptor tyrosine kinases, EPHB6, induces marked fragmentation of the mitochondrial network in breast cancer cells of triple-negative origin, lacking expression of the estrogen, progesterone and HER2 receptors. Remarkably, this response renders cancer cells more susceptible to DR5-mediated apoptosis. EPHB6 action in mitochondrial fragmentation proved to depend on its ability to activate the ERK-DRP1 pathway, which increases the frequency of organelle fission. Moreover, DRP1 activity is also essential to the EPHB6-mediated pro-apoptotic response that we observe in the context of DR5 activation. These findings provide the first description of a member of the receptor tyrosine kinase family capable of producing a pro-apoptotic effect through the activation of ERK-DRP1 signaling and subsequent mitochondrial fragmentation. Our observations are of potential practical importance, as they imply that DR5-activating therapeutic approaches should be applied in a more personalized manner to primarily treat EPHB6-expressing tumours. Finally, our findings also suggest that the EPHB6 receptor itself may represent a promising target for cancer therapy, since EPHB6 and DR5 co-activation should support more efficient elimination of cancer cells.This work was supported by operating grants from the Canadian Breast Cancer Foundation (CBCF) grant # C7003 to A.F. and S.C.L., Canadian Institutes of Health Research (CIHR) grant # RSN 132192 and SHRF grant # 2891 to A.F., and by fall-in funds from a CIHR New Investigator Award #244917 to S.C.L

Ligand stimulation induces clathrin- and Rab5- dependent downregulation of the kinase-dead EphB6 receptor preceded by the disruption of EphB6-Hsp90 interaction

Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) serve to determine biological outputs of their signalling. Intrinsically kinase-deficient RTKs control a variety of biological responses, however, the mechanism of their downregulation is not well understood and its analysis is focused exclusively on the ErbB3 receptor. The Eph group of RTKs is represented by the EphA and EphB subclasses. Each bears one kinase-inactive member, EphA10 and EphB6, respectively, suggesting an important role for these molecules in the Eph signalling network. While EphB6 effects on cell behaviour have been assessed, the mechanism of its downregulation remains elusive. Our work reveals that EphB6 and its kinase-active relative, and signaling partner, EphB4, are down regulated in a similar manner in response to their common ligand, ephrin-B2. Following stimulation, both receptors are internalised through clathrin-coated pits and are degraded in lysosomes. Their targeting for lysosomal degradation relies on the activity of an early endosome regulator, the Rab5 GTPase, as this process is inhibited in the presence of a Rab5 dominant-negative mutant. EphB6 also interacts with the Hsp90 chaperone and EphB6 downregulation is preceded by their rapid dissociation. Moreover, the inhibition of Hsp90 results in EphB6 degradation, mimicking its ligand-induced downregulation. These processes appear to rely on overlapping mechanisms, since Hsp90 inhibition does not significantly enhance ligand-induced EphB6 elimination. Taken together, our observations define a novel mechanism for intrinsically kinase-deficient RTK downregulation and support an intriguing model, where Hsp90 dissociation acts as a trigger for ligand-induced receptor removal.This work was funded by the Canadian Institutes of Health Research grants and SHRF. O.A. was funded by a Graduate Research Award from the University of Saskatchewan

Structure of the EphB6 receptor ectodomain.

Eph receptors are the largest group amongst the receptor tyrosine kinases and are divided into two subgroups, A and B, based on ligand binding specificities and sequence conservation. Through ligand-induced and ligand-independent activities, Ephs play central roles in diverse biological processes, including embryo development, regulation of neuronal signaling, immune responses, vasculogenesis, as well as tumor initiation, progression, and metastasis. The Eph extracellular regions (ECDs) are constituted of multiple domains, and previous structural studies of the A class receptors revealed how they interact with ephrin ligands and simultaneously mediate Eph-Eph clustering necessary for biological activity. Specifically, EphA structures highlighted a model, where clustering of ligand-bound receptors relies on two distinct receptor/receptor interfaces. Interestingly, most unliganded A class receptors also form an additional, third interface, between the ligand binding domain (LBD) and the fibronectin III domain (FN3) of neighboring molecules. Structures of B-class Eph ECDs, on the other hand, have never been reported. To further our understanding of Eph receptor function, we crystallized the EphB6-ECD and determined its three-dimensional structure using X-ray crystallography. EphB6 has important functions in both normal physiology and human malignancies and is especially interesting because this atypical receptor innately lacks kinase activity and our understanding of the mechanism of action is still incomplete. Our structural data reveals the overall EphB6-ECD architecture and shows EphB6-LBD/FN3 interactions similar to those observed for the unliganded A class receptors, suggesting that these unusual interactions are of general importance to the Eph group. We also observe unique structural features, which likely reflect the atypical signaling properties of EphB6, namely the need of co-receptor(s) for this kinase-inactive Eph. These findings provide new valuable information on the structural organization and mechanism of action of the B-class Ephs, and specifically EphB6, which in the future will assist in identifying clinically relevant targets for cancer therapy