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 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.

Regulation and function of the NFE2 transcription factor in hematopoietic and non-hematopoietic cells.

The NFE2 transcription factor was identified over 25 years ago. The NFE2 protein forms heterodimers with small MAF proteins, and the resulting complex binds to regulatory elements in a large number of target genes. In contrast to other CNC transcription family members including NFE2L1 (NRF1), NFE2L2 (NRF2) and NFE2L3 (NRF3), which are widely expressed, earlier studies had suggested that the major sites of NFE2 expression are hematopoietic cells. Based on cell culture studies it was proposed that this protein acts as a critical regulator of globin gene expression. However, the knockout mouse model displayed only mild erythroid abnormalities, while the major phenotype was a defect in megakaryocyte biogenesis. Indeed, absence of NFE2 led to severely impaired platelet production. A series of recent data, also summarized here, shed new light on the various functional roles of NFE2 and the regulation of its activity. NFE2 is part of a complex regulatory network, including transcription factors such as GATA1 and RUNX1, controlling megakaryocytic and/or erythroid cell function. Surprisingly, it was recently found that NFE2 also has a role in non-hematopoietic tissues, such as the trophoblast, in which it is also expressed, as well as the bone, opening the door to new research areas for this transcription factor. Additional data showed that NFE2 function is controlled by a series of posttranslational modifications. Important strides have been made with respect to the clinical significance of NFE2, linking this transcription factor to hematological disorders such as polycythemias.

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.

1H, 13C, 15N Backbone and sidechain chemical shift assignments of the C-terminal domain of human UDP-glucuronosyltransferase 2B17 (UGT2B17-C).

UDP-glucuronosyltransferases are the principal enzymes involved in the glucuronidation of metabolites and xenobiotics for physiological clearance in humans. Though glucuronidation is an indispensable process in the phase II metabolic pathway, UGT-mediated glucuronidation of most prescribed drugs (> 55%) and clinical evidence of UGT-associated drug resistance are major concerns for therapeutic development. While UGTs are highly conserved enzymes, they manifest unique substrate and inhibitor specificity which is poorly understood given the dearth of experimentally determined full-length structures. Such information is important not only to conceptualize their specificity but is central to the design of inhibitors specific to a given UGT in order to avoid toxicity associated with pan-UGT inhibitors. Here, we provide the 1H, 13C and 15N backbone (~ 90%) and sidechain (~ 62%) assignments for the C-terminal domain of UGT2B17, which can be used to determine the molecular binding sites of inhibitor and substrate, and to understand the atomic basis for inhibitor selectivity between UGT2B17 and other members of the UGT2B subfamily. Given the physiological relevance of UGT2B17 in the elimination of hormone-based cancer drugs, these assignments will contribute towards dissecting the structural basis for substrate specificity, selective inhibitor recognition and other aspects of enzyme activity with the goal of selectively overcoming glucuronidation-based drug resistance.

Antagonistic roles of the ERK and p38 MAPK signalling pathways in globin expression, haem biosynthesis and iron uptake.

Late-stage erythroid cells synthesize large quantities of haemoglobin, a process requiring the co-ordinated regulation of globin and haem synthesis as well as iron uptake. In the present study, we investigated the role of the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in MEL (mouse erythroleukaemia) cell differentiation. We found that treatment of HMBA (hexamethylene bisacetamide)-induced MEL cells with the ERK pathway inhibitor UO126 results in an increase in intracellular haem and haemoglobin levels. The transcript levels of the genes coding for ß(major)-globin, the haem biosynthesis enzyme 5-aminolevulinate synthase 2 and the mitochondrial iron transporter mitoferrin 1 are up-regulated. We also showed enhanced expression of globin and transferrin receptor 1 proteins upon UO126 treatment. With respect to iron uptake, we found that ERK inhibitor treatment led to an increase in both haem-bound and total iron. In contrast, treatment of MEL cells with the p38 MAPK pathway inhibitor SB202190 had the opposite effect, resulting in decreased globin expression, haem synthesis and iron uptake. Reporter assays showed that globin promoter and HS2 enhancer-mediated transcription was under the control of MAPKs, as inhibition of the ERK and p38 MAPK pathways led to increased and decreased gene activity respectively. Our present results suggest that the ERK1/2 and p38α/ß MAPKs play antagonistic roles in HMBA-induced globin gene expression and erythroid differentiation. These results provide a novel link between MAPK signalling and the regulation of haem biosynthesis and iron uptake in erythroid cells.

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.

Thioredoxin-interacting protein regulates the differentiation of murine erythroid precursors.

Thioredoxin-interacting protein (TXNIP) is involved in various cellular processes including redox control, metabolism, differentiation, growth, and apoptosis. With respect to hematopoiesis, TXNIP has been shown to play roles in natural killer cells, dendritic cells, and hematopoietic stem cells. Our study investigates the role of TXNIP in erythropoiesis. We observed a rapid and significant increase of TXNIP transcript and protein levels in mouse erythroleukemia cells treated with dimethyl sulfoxide or hexamethylene bisacetamide, inducers of erythroid differentiation. The upregulation of TXNIP was not abrogated by addition of the antioxidant N-acetylcysteine. The increase of TXNIP expression was confirmed in another model of erythroid differentiation, G1E-ER cells, which undergo differentiation upon activation of the GATA1 transcription factor. In addition, we showed that TXNIP levels are induced following inhibition of p38 or c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. We also observed an increase in iron uptake and a decrease in transferrin receptor protein upon TXNIP overexpression, suggesting a role in iron homeostasis. In vivo, flow cytometry analysis of cells from Txnip(-/-) mice revealed a new phenotype of impaired terminal erythropoiesis in the spleen, characterized by a partial block between basophilic and late basophilic/polychromatic erythroblasts. Based on our data, TXNIP emerges as a novel regulator of terminal erythroid differentiation.

Abnormal differentiation of erythroid precursors in p45 NF-E2(-/-) mice.

The transcription factor p45 nuclear factor-erythroid-derived 2 (NF-E2) plays major roles in erythroid and megakaryocytic lineages. Here, we investigated the role of p45 NF-E2 in erythroid differentiation in vivo. Absence of p45 NF-E2 in mice leads to a twofold increase in serum erythropoietin levels. In the bone marrow of these animals, we found a different distribution of precursor populations compared to wild-type mice, suggesting abnormal differentiation. Loss of p45 NF-E2 was also associated with an increase in splenic erythropoiesis, as evidenced by an accumulation of early precursors, namely, late basophilic and polychromatic erythroblasts. These observations are consistent with a stress erythropoiesis phenotype and indicate that the spleen is likely compensating for ineffective erythropoiesis in the bone marrow. Analysis of bone marrow samples revealed increased GATA1 levels, as well as an increased proportion of erythroid cells arrested at the G(1) stage of cell cycle in p45 NF-E2-deficient mice. These results suggest that p45 NF-E2 is required for the differentiation of erythroid precursors.