Medicinal Chemistry and NMR Driven Discovery of Novel UDP-glucuronosyltransferase 1A Inhibitors That Overcome Therapeutic Resistance in Cells.

The UDP glucuronosyltransferases (UGT) deactivate many therapeutics via glucuronidation while being required for clearance of normal metabolites and xenobiotics. There are 19 UGT enzymes categorized into UGT1A and UGT2B families based on sequence conservation. This presents a challenge in terms of targeting specific UGTs to overcome drug resistance without eliciting overt toxicity. Here, we identified for the first time that UGT1A4 is highly elevated in acute myeloid leukemia (AML) patients and its reduction corresponded to objective clinical responses. To develop inhibitors to UGT1A4, we leveraged previous NMR-based fragment screening data against the C-terminal domain of UGT1A (UGT1A-C). NMR and medicinal chemistry strategies identified novel chemical matter based on fragment compounds with the capacity to bind ∼20 fold more tightly to UGT1A-C (Kd âˆ¼ 600 µM vs ∼30 µM). Some compounds differentially inhibited UGT1A4 versus UGT1A1 enzyme activity and restored drug sensitivity in resistant human cancer cells. NMR-based NOE experiments revealed these novel compounds recognised a region distal to the catalytic site suggestive of allosteric regulation. This binding region is poorly conserved between UGT1A and UGT2B C-terminal sequences, which otherwise exhibit high similarity. Consistently, these compounds did not bind to the C-terminal domain of UGT2B7 nor a triple mutant of UGT1A-C replaced with UGT2B7 residues in this region. Overall, we discovered a site on UGTs that can be leveraged to differentially target UGT1As and UGT2Bs, identified UGT1A4 as a therapeutic target, and found new chemical matter that binds the UGT1A C-terminus, inhibits glucuronidation and restores drug sensitivity.

XPO1 Enables Adaptive Regulation of mRNA Export Required for Genotoxic Stress Tolerance in Cancer Cells.

Exportin-1 (XPO1), the main soluble nuclear export receptor in eukaryotic cells, is frequently overexpressed in diffuse large B-cell lymphoma (DLBCL). A selective XPO1 inhibitor, selinexor, received approval as single agent for relapsed or refractory (R/R) DLBCL. Elucidating the mechanisms by which XPO1 overexpression supports cancer cells could facilitate further clinical development of XPO1 inhibitors. We uncovered here that XPO1 overexpression increases tolerance to genotoxic stress, leading to a poor response to chemoimmunotherapy. Upon DNA damage induced by MYC expression or exogenous compounds, XPO1 bound and exported EIF4E and THOC4 carrying DNA damage repair mRNAs, thereby increasing synthesis of DNA damage repair proteins under conditions of increased turnover. Consequently, XPO1 inhibition decreased the capacity of lymphoma cells to repair DNA damage and ultimately resulted in increased cytotoxicity. In a phase I clinical trial conducted in R/R DLBCL, the combination of selinexor with second-line chemoimmunotherapy was tolerated with early indication of efficacy. Overall, this study reveals that XPO1 overexpression plays a critical role in the increased tolerance of cancer cells to DNA damage while providing new insights to optimize the clinical development of XPO1 inhibitors. SIGNIFICANCE: XPO1 regulates the dynamic ribonucleoprotein nuclear export in response to genotoxic stress to support tolerance and can be targeted to enhance the sensitivity of cancer cells to endogenous and exogenous DNA damage. See related commentary by Knittel and Reinhardt, p. 3.

Molecular targeting of the UDP-glucuronosyltransferase enzymes in high-eukaryotic translation initiation factor 4E refractory/relapsed acute myeloid leukemia patients: a randomized phase II trial of vismodegib, ribavirin with or without decitabine.

Drug resistance underpins poor outcomes in many malignancies including refractory and relapsed acute myeloid leukemia (R/R AML). Glucuronidation is a common mechanism of drug inactivation impacting many AML therapies, e.g., cytarabine, decitabine, azacytidine and venetoclax. In AML cells, the capacity for glucuronidation arises from increased production of the UDP-glucuronosyltransferase 1A (UGT1A) enzymes. UGT1A elevation was first observed in AML patients who relapsed after response to ribavirin, a drug used to target the eukaryotic translation initiation factor eIF4E, and subsequently in patients who relapsed on cytarabine. UGT1A elevation resulted from increased expression of the sonic-hedgehog transcription factor GLI1. Vismodegib inhibited GLI1, decreased UGT1A levels, reduced glucuronidation of ribavirin and cytarabine, and re-sensitized cells to these drugs. Here, we examined if UGT1A protein levels, and thus glucuronidation activity, were targetable in humans and if this corresponded to clinical response. We conducted a phase II trial using vismodegib with ribavirin, with or without decitabine, in largely heavily pre-treated patients with high-eIF4E AML. Pre-therapy molecular assessment of patients' blasts indicated highly elevated UGT1A levels relative to healthy volunteers. Among patients with partial response, blast response or prolonged stable disease, vismodegib reduced UGT1A levels, which corresponded to effective targeting of eIF4E by ribavirin. In all, our studies are the first to demonstrate that UGT1A protein, and thus glucuronidation, are targetable in humans. These studies pave the way for the development of therapies that impair glucuronidation, one of the most common drug deactivation modalities. Clinicaltrials.gov: NCT02073838.

The eukaryotic translation initiation factor eIF4E reprograms alternative splicing.

Aberrant splicing is typically attributed to splice-factor (SF) mutation and contributes to malignancies including acute myeloid leukemia (AML). Here, we discovered a mutation-independent means to extensively reprogram alternative splicing (AS). We showed that the dysregulated expression of eukaryotic translation initiation factor eIF4E elevated selective splice-factor production, thereby impacting multiple spliceosome complexes, including factors mutated in AML such as SF3B1 and U2AF1. These changes generated a splicing landscape that predominantly supported altered splice-site selection for ~800 transcripts in cell lines and ~4,600 transcripts in specimens from high-eIF4E AML patients otherwise harboring no known SF mutations. Nuclear RNA immunoprecipitations, export assays, polysome analyses, and mutational studies together revealed that eIF4E primarily increased SF production via its nuclear RNA export activity. By contrast, eIF4E dysregulation did not induce known SF mutations or alter spliceosome number. eIF4E interacted with the spliceosome and some pre-mRNAs, suggesting its direct involvement in specific splicing events. eIF4E induced simultaneous effects on numerous SF proteins, resulting in a much larger range of splicing alterations than in the case of mutation or dysregulation of individual SFs and providing a novel paradigm for splicing control and dysregulation.

Subcellular Fractionation Suitable for Studies of RNA and Protein Trafficking.

The nuclear pore complex is the major conduit for trafficking between the nucleus and cytoplasm. Nuclear import and export of both proteins and RNAs represent important functional steps for many biological processes. One of the major means to study NPC activity and the nuclear and cytoplasmic distribution of proteins and RNAs is through biochemical fractionation. Here, we describe detailed methods to generate high quality nuclear and cytoplasmic fractions simultaneously capturing RNA and proteins which can be used subsequently for a wide array of biochemical characterizations including proteomics and next generation sequencings.

Identification and Characterization of the Interaction Between the Methyl-7-Guanosine Cap Maturation Enzyme RNMT and the Cap-Binding Protein eIF4E.

The control of RNA metabolism is an important aspect of molecular biology with wide-ranging impacts on cells. Central to processing of coding RNAs is the addition of the methyl-7 guanosine (m7G) "cap" on their 5' end. The eukaryotic translation initiation factor eIF4E directly binds the m7G cap and through this interaction plays key roles in many steps of RNA metabolism including nuclear RNA export and translation. eIF4E also stimulates capping of many transcripts through its ability to drive the production of the enzyme RNMT which methylates the G-cap to form the mature m7G cap. Here, we found that eIF4E also physically associated with RNMT in human cells. Moreover, eIF4E directly interacted with RNMT in vitro. eIF4E is only the second protein reported to directly bind the methyltransferase domain of RNMT, the first being its co-factor RAM. We combined high-resolution NMR methods with biochemical studies to define the binding interfaces for the RNMT-eIF4E complex. Further, we found that eIF4E competes for RAM binding to RNMT and conversely, RNMT competes for binding of well-established eIF4E-binding partners such as the 4E-BPs. RNMT uses novel structural means to engage eIF4E. Finally, we observed that m7G cap-eIF4E-RNMT trimeric complexes form, and thus RNMT-eIF4E complexes may be employed so that eIF4E captures newly capped RNA. In all, we show for the first time that the cap-binding protein eIF4E directly binds to the cap-maturation enzyme RNMT.

The Cap-Binding Complex CBC and the Eukaryotic Translation Factor eIF4E: Co-Conspirators in Cap-Dependent RNA Maturation and Translation.

The translation of RNA into protein is a dynamic process which is heavily regulated during normal cell physiology and can be dysregulated in human malignancies. Its dysregulation can impact selected groups of RNAs, modifying protein levels independently of transcription. Integral to their suitability for translation, RNAs undergo a series of maturation steps including the addition of the m7G cap on the 5' end of RNAs, splicing, as well as cleavage and polyadenylation (CPA). Importantly, each of these steps can be coopted to modify the transcript signal. Factors that bind the m7G cap escort these RNAs through different steps of maturation and thus govern the physical nature of the final transcript product presented to the translation machinery. Here, we describe these steps and how the major m7G cap-binding factors in mammalian cells, the cap binding complex (CBC) and the eukaryotic translation initiation factor eIF4E, are positioned to chaperone transcripts through RNA maturation, nuclear export, and translation in a transcript-specific manner. To conceptualize a framework for the flow and integration of this genetic information, we discuss RNA maturation models and how these integrate with translation. Finally, we discuss how these processes can be coopted by cancer cells and means to target these in malignancy.

The eukaryotic translation initiation factor eIF4E elevates steady-state m7G capping of coding and noncoding transcripts.

Methyl-7-guanosine (m7G) "capping" of coding and some noncoding RNAs is critical for their maturation and subsequent activity. Here, we discovered that eukaryotic translation initiation factor 4E (eIF4E), itself a cap-binding protein, drives the expression of the capping machinery and increased capping efficiency of ∼100 coding and noncoding RNAs. To quantify this, we developed enzymatic (cap quantification; CapQ) and quantitative cap immunoprecipitation (CapIP) methods. The CapQ method has the further advantage that it captures information about capping status independent of the type of 5' cap, i.e., it is not restricted to informing on m7G caps. These methodological advances led to unanticipated revelations: 1) Many RNA populations are inefficiently capped at steady state (∼30 to 50%), and eIF4E overexpression increased this to ∼60 to 100%, depending on the RNA; 2) eIF4E physically associates with noncoding RNAs in the nucleus; and 3) approximately half of eIF4E-capping targets identified are noncoding RNAs. eIF4E's association with noncoding RNAs strongly positions it to act beyond translation. Coding and noncoding capping targets have activities that influence survival, cell morphology, and cell-to-cell interaction. Given that RNA export and translation machineries typically utilize capped RNA substrates, capping regulation provides means to titrate the protein-coding capacity of the transcriptome and, for noncoding RNAs, to regulate their activities. We also discovered a cap sensitivity element (CapSE) which conferred eIF4E-dependent capping sensitivity. Finally, we observed elevated capping for specific RNAs in high-eIF4E leukemia specimens, supporting a role for cap dysregulation in malignancy. In all, levels of capping RNAs can be regulated by eIF4E.

Structural studies of the eIF4E-VPg complex reveal a direct competition for capped RNA: Implications for translation.

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5' end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5' end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg-eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E-eIF4G, eIF4E bound VPg-luciferase RNA conjugates, and these VPg-RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg-RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.

GLI1-Inducible Glucuronidation Targets a Broad Spectrum of Drugs.

Cancer therapies are plagued by resistance. Previously, we discovered a novel form of cancer drug resistance where the Glioma-associated protein 1 (GLI1) elevates UGT1A glucuronidation enzymes, thereby glucuronidating cytarabine and ribavirin, leading to resistance in leukemia patients. Here, we demonstrate that GLI1 imparts resistance to ∼40 compounds, including FDA-approved drugs with disparate chemotypes ( e.g., methotrexate and venetoclax). GLI1 indirectly elevates UGT1As via the chaperone calreticulin, which is required for resistance. Further, we demonstrate that resistant cells are more sensitive to ATP inhibitors, suggesting an Achilles' heel, which could be exploited in the future. In all, we identify GLI1-inducible glucuronidation as a broad-spectrum multidrug resistance pathway.

Targeting EIF4E signaling with ribavirin in infant acute lymphoblastic leukemia.

The poor outcomes in infant acute lymphoblastic leukemia (ALL) necessitate new treatments. Here we discover that EIF4E protein is elevated in most cases of infant ALL and test EIF4E targeting by the repurposed antiviral agent ribavirin, which has anticancer properties through EIF4E inhibition, as a potential treatment. We find that ribavirin treatment of actively dividing infant ALL cells on bone marrow stromal cells (BMSCs) at clinically achievable concentrations causes robust proliferation inhibition in proportion with EIF4E expression. Further, we find that ribavirin treatment of KMT2A-rearranged (KMT2A-R) infant ALL cells and the KMT2A-AFF1 cell line RS4:11 inhibits EIF4E, leading to decreases in oncogenic EIF4E-regulated cell growth and survival proteins. In ribavirin-sensitive KMT2A-R infant ALL cells and RS4:11 cells, EIF4E-regulated proteins with reduced levels of expression following ribavirin treatment include MYC, MCL1, NBN, BCL2 and BIRC5. Ribavirin-treated RS4:11 cells exhibit impaired EIF4E-dependent nuclear to cytoplasmic export and/or translation of the corresponding mRNAs, as well as reduced phosphorylation of the p-AKT1, p-EIF4EBP1, p-RPS6 and p-EIF4E signaling proteins. This leads to an S-phase cell cycle arrest in RS4:11 cells corresponding to the decreased proliferation. Ribavirin causes nuclear EIF4E to re-localize to the cytoplasm in KMT2A-AFF1 infant ALL and RS4:11 cells, providing further evidence for EIF4E inhibition. Ribavirin slows increases in peripheral blasts in KMT2A-R infant ALL xenograft-bearing mice. Ribavirin cooperates with chemotherapy, particularly L-asparaginase, in reducing live KMT2A-AFF1 infant ALL cells in BMSC co-cultures. This work establishes that EIF4E is broadly elevated across infant ALL and that clinically relevant ribavirin exposures have preclinical activity and effectively inhibit EIF4E in KMT2A-R cases, suggesting promise in EIF4E targeting using ribavirin as a means of treatment.

The Impact of Post-transcriptional Control: Better Living Through RNA Regulons.

Traditionally, cancer is viewed as a disease driven by genetic mutations and/or epigenetic and transcriptional dysregulation. While these are undoubtedly important drivers, many recent studies highlight the disconnect between the proteome and the genome or transcriptome. At least in part, this disconnect arises as a result of dysregulated RNA metabolism which underpins the altered proteomic landscape observed. Thus, it is important to understand the basic mechanisms governing post-transcriptional control and how these processes can be co-opted to drive cancer cell phenotypes. In some cases, groups of mRNAs that encode protein involved in specific oncogenic processes can be co-regulated at multiple processing levels in order to turn on entire biochemical pathways. Indeed, the RNA regulon model was postulated as a means to understand how cells coordinately regulate transcripts encoding proteins in the same biochemical pathways. In this review, we describe some of the basic mRNA processes that are dysregulated in cancer and the biological impact this has on the cell. This dysregulation can affect networks of RNAs simultaneously thereby underpinning the oncogenic phenotypes observed.

The eukaryotic translation initiation factor eIF4E harnesses hyaluronan production to drive its malignant activity.

The microenvironment provides a functional substratum supporting tumour growth. Hyaluronan (HA) is a major component of this structure. While the role of HA in malignancy is well-defined, the mechanisms driving its biosynthesis in cancer are poorly understood. We show that the eukaryotic translation initiation factor eIF4E, an oncoprotein, drives HA biosynthesis. eIF4E stimulates production of enzymes that synthesize the building blocks of HA, UDP-Glucuronic acid and UDP-N-Acetyl-Glucosamine, as well as hyaluronic acid synthase which forms the disaccharide chain. Strikingly, eIF4E inhibition alone repressed HA levels as effectively as directly targeting HA with hyaluronidase. Unusually, HA was retained on the surface of high-eIF4E cells, rather than being extruded into the extracellular space. Surface-associated HA was required for eIF4E's oncogenic activities suggesting that eIF4E potentiates an oncogenic HA program. These studies provide unique insights into the mechanisms driving HA production and demonstrate that an oncoprotein can co-opt HA biosynthesis to drive malignancy.

A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery.

The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway.

Importin 8 mediates m7G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E.

Regulation of nuclear-cytoplasmic trafficking of oncoproteins is critical for growth homeostasis. Dysregulated trafficking contributes to malignancy, whereas understanding the process can reveal unique therapeutic opportunities. Here, we focus on eukaryotic translation initiation factor 4E (eIF4E), a prooncogenic protein highly elevated in many cancers, including acute myeloid leukemia (AML). Typically, eIF4E is localized to both the nucleus and cytoplasm, where it acts in export and translation of specific methyl 7-guanosine (m(7)G)-capped mRNAs, respectively. Nuclear accumulation of eIF4E in patients who have AML is correlated with increased eIF4E-dependent export of transcripts encoding oncoproteins. The subcellular localization of eIF4E closely correlates with patients' responses. During clinical responses to the m(7)G-cap competitor ribavirin, eIF4E is mainly cytoplasmic. At relapse, eIF4E reaccumulates in the nucleus, leading to elevated eIF4E-dependent mRNA export. We have identified importin 8 as a factor that directly imports eIF4E into the nucleus. We found that importin 8 is highly elevated in untreated patients with AML, leading to eIF4E nuclear accumulation. Importin 8 only imports cap-free eIF4E. Cap-dependent changes to the structure of eIF4E underpin this selectivity. Indeed, m(7)G cap analogs or ribavirin prevents nuclear entry of eIF4E, which mirrors the trafficking phenotypes observed in patients with AML. Our studies also suggest that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context. These findings position nuclear trafficking of eIF4E as a critical step in its regulation and position the importin 8-eIF4E complex as a novel therapeutic target.

Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphomas.

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.

Sonic Hedgehog factor Gli1: As good as resistant.

Chemoresistance remains a major impediment in cancer therapy. Although major progress has been made in understanding the mechanisms underlying resistance in cancer, there is still more to learn. Our studies provide evidence that Gli1 drives a novel form of drug resistance involving Phase II drug metabolism enzymes, specifically the UGT1A family.

The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation.

Drug resistance is a major hurdle in oncology. Responses of acute myeloid leukaemia (AML) patients to cytarabine (Ara-C)-based therapies are often short lived with a median overall survival of months. Therapies are under development to improve outcomes and include targeting the eukaryotic translation initiation factor (eIF4E) with its inhibitor ribavirin. In a Phase II clinical trial in poor prognosis AML, ribavirin monotherapy yielded promising responses including remissions; however, all patients relapsed. Here we identify a novel form of drug resistance to ribavirin and Ara-C. We observe that the sonic hedgehog transcription factor glioma-associated protein 1 (GLI1) and the UDP glucuronosyltransferase (UGT1A) family of enzymes are elevated in resistant cells. UGT1As add glucuronic acid to many drugs, modifying their activity in diverse tissues. GLI1 alone is sufficient to drive UGT1A-dependent glucuronidation of ribavirin and Ara-C, and thus drug resistance. Resistance is overcome by genetic or pharmacological inhibition of GLI1, revealing a potential strategy to overcome drug resistance in some patients.