Regina Cencic - Academia.edu (original) (raw)

Papers by Regina Cencic

Proceedings of the National Academy of Sciences of the United States of America, Mar 1, 2017

RNA

Inhibition of eukaryotic translation initiation through unscheduled RNA clamping of the DEAD-box ... more Inhibition of eukaryotic translation initiation through unscheduled RNA clamping of the DEAD-box (DDX) RNA helicases eIF4A1 and eIF4A2 has been documented for pateamine A (PatA) and rocaglates—two structurally different classes of compounds that share overlapping binding sites on eIF4A. Clamping of eIF4A to RNA causes steric blocks that interfere with ribosome binding and scanning, rationalizing the potency of these molecules since not all eIF4A molecules need to be engaged to elicit a biological effect. In addition to targeting translation, PatA and analogs have also been shown to target the eIF4A homolog, eIF4A3—a helicase necessary for exon junction complex (EJC) formation. EJCs are deposited on mRNAs upstream of exon-exon junctions and, when present downstream from premature termination codons (PTCs), participate in nonsense-mediated decay (NMD), a quality control mechanism aimed at preventing the production of dominant-negative or gain-of-function polypeptides from faulty mRNA ...

Cellular and Molecular Life Sciences, 2021

Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of c... more Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of cap-dependent translation. This heterotrimeric complex consists of a cap binding subunit (eIF4E), a DEAD-box RNA helicase (eIF4A), and a large bridging protein (eIF4G). In mammalian cells, there are two genes encoding eIF4A (eIF4A1 and eIF4A2) and eIF4G (eIF4G1 and eIF4G3) paralogs that can assemble into eIF4F complexes. To query the essential nature of the eIF4F subunits in normal development, we used CRISPR/Cas9 to generate mouse strains with targeted ablation of each gene encoding the different eIF4F subunits. We find that Eif4e, Eif4g1, and Eif4a1 are essential for viability in the mouse, whereas Eif4g3 and Eif4a2 are not. However, Eif4g3 and Eif4a2 do play essential roles in spermatogenesis. Crossing of these strains to the lymphomaprone Eμ-Myc mouse model revealed that heterozygosity at the Eif4e or Eif4a1 loci significantly delayed tumor onset. Lastly, tumors derived from Eif4e ∆38 fs/+ /Eμ-Myc or Eif4a1 ∆5 fs/+ /Eμ-Myc mice show increased sensitivity to the chemotherapeutic agent doxorubicin, in vivo. Our study reveals that eIF4A2 and eIF4G3 play non-essential roles in gene expression regulation during embryogenesis; whereas reductions in eIF4E or eIF4A1 levels are protective against tumor development in a murine Myc-driven lymphoma setting.

Scientific Reports, 2021

Rocaglates are a class of eukaryotic translation initiation inhibitors that are being explored as... more Rocaglates are a class of eukaryotic translation initiation inhibitors that are being explored as chemotherapeutic agents. They function by targeting eukaryotic initiation factor (eIF) 4A, an RNA helicase critical for recruitment of the 40S ribosome (and associated factors) to mRNA templates. Rocaglates perturb eIF4A activity by imparting a gain-of-function activity to eIF4A and mediating clamping to RNA. To appreciate how rocaglates could best be enabled in the clinic, an understanding of resistance mechanisms is important, as this could inform on strategies to bypass such events as well as identify responsive tumor types. Here, we report on the results of a positive selection, ORFeome screen aimed at identifying cDNAs capable of conferring resistance to rocaglates. Two of the most potent modifiers of rocaglate response identified were the transcription factors FOXP3 and NR1I3, both of which have been implicated in ABCB1 regulation—the gene encoding P-glycoprotein (Pgp). Pgp has pr...

Nucleic Acids Research, 2020

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targetin... more Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5′ leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation...

Nucleic Acids Research, 2020

We report on the synthesis of siRNAs containing both 2′-5′- and 3′-5′-internucleotide linkages an... more We report on the synthesis of siRNAs containing both 2′-5′- and 3′-5′-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2′-5′ linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5′-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5′p-rN1-(2′-5′)-N2 unit in the antisense strand does not alter the ‘clover leaf’ bend and sugar puckers that are critical for anchoring the 5′-phosphate to Ago 2 MID domain. Importantly, 2′-5′-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results dem...

Nucleic Acids Research, 2020

Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in tra... more Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4G...

Cell Reports, 2020

Highlights d Rocaglates produce distinct inhibitory effects on translation initiation d Rocaglate... more Highlights d Rocaglates produce distinct inhibitory effects on translation initiation d Rocaglates interfere with eIF4F release from the cap structure d Rocaglates exert a bystander effect on translation initiation by sequestering eIF4F

RNA, 2020

The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node u... more The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity—generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anticancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F, and several potent small molecules (rocaglates, hippuristanol, pateamine A) affecting its activity have been identified and shown to demonstrate anticancer activity in vitro and in vivo in preclinical models. Recently, a number of new small molecules have been reported as having the capacity to target and inhibit eIF4A. Here, we undertook a comparative analysis of their biological activity and...

Blood, 2010

2995 Background: Multiple myeloma (MM) cell survival is dependent on the ability to produce and s... more 2995 Background: Multiple myeloma (MM) cell survival is dependent on the ability to produce and secrete immunoglobulins, requiring precise coordination of protein synthesis and degradation. Inhibition of these processes may be responsible for the exquisite sensitivity of MM to drugs such as bortezomib and histone deacetylase inhibitors, which respectively block proteosomal and aggresomal-mediated protein degradation. We hypothesized that MM cells would also be sensitive to inhibition of mRNA translation and have tested silvestrol, a plant-derived natural product that has previously been shown to inhibit ribosome recruitment and to be cytotoxic to B lymphocytes. Mehods: Silvestrol was resuspended in DMSO and diluted for use in RPMI medium. A panel of human MM cell lines, representing a range of chromosomal abnormalities observed in this disease, were cultured in RPMI with 10% FCS and appropriate supplementation. Primary mouse embryonic fibroblasts (MEFs) were prepared and passaged 7–...

Cell Chemical Biology, 2019

Rocaglates share a common cyclopentabenzo[b]furan core that inhibit eukaryotic translation initia... more Rocaglates share a common cyclopentabenzo[b]furan core that inhibit eukaryotic translation initiation by modifying the behaviour of the RNA helicase, eIF4A. Working as interfacial inhibitors, rocaglates stabilize the association between eIF4A and RNA which can lead to the formation of steric barriers that block initiating ribosomes. There is significant interest in the development and expansion of rocaglate derivatives as several members of this family have been shown to possess potent anti-neoplastic activity in vitro and in vivo. To further our understanding of rocaglate diversity and drug design, herein we explore the RNA clamping activity of >200 unique rocaglate derivatives. Through this, we report on the identification and characterization of a potent class of synthetic rocaglates called amidino-rocaglates. These compounds are among the most potent rocaglates documented to date, and taken together, this work offers important information that will guide the future design of rocaglates with improved biological properties.

Molecular Cancer Therapeutics, 2019

CDK4/6 inhibitors are FDA-approved drugs for estrogen receptor–positive (ER+) breast cancer and a... more CDK4/6 inhibitors are FDA-approved drugs for estrogen receptor–positive (ER+) breast cancer and are being evaluated to treat other tumor types, including KRAS-mutant non–small cell lung cancer (NSCLC). However, their clinical utility is often limited by drug resistance. Here, we sought to better understand the resistant mechanisms and help devise potential strategies to overcome this challenge. We show that treatment with CDK4/6 inhibitors in both ER+ breast cancer and KRAS-mutant NSCLC cells induces feedback upregulation of cyclin D1, CDK4, and cyclin E1, mediating drug resistance. We demonstrate that rocaglates, which preferentially target translation of key cell-cycle regulators, effectively suppress this feedback upregulation induced by CDK4/6 inhibition. Consequently, combination treatment of CDK4/6 inhibitor palbociclib with the eukaryotic initiation factor (eIF) 4A inhibitor, CR-1-31-B, is synergistic in suppressing the growth of these cancer cells in vitro and in vivo. Furth...

Cell Chemical Biology, 2019

Highlights d CRISPR/Cas9 engineering harnessed to generate a targetagnostic phenotypic screen d G... more Highlights d CRISPR/Cas9 engineering harnessed to generate a targetagnostic phenotypic screen d GFP tracing of c-MYC expression in multiple myeloma cells d Cardiac glycosides and cytoskeletal disruptors are potent MYC expression inhibitors

Scientific Reports, 2019

Translation is a highly regulated process that is perturbed in human cancers, often through activ... more Translation is a highly regulated process that is perturbed in human cancers, often through activation of the PI3K/mTOR pathway which impacts directly on the ribosome recruitment phase of translation initiation. While significant research has focused on “drugging” components of the PI3K/mTOR network, efforts have also been directed towards inhibiting eukaryotic initiation factor (eIF) 4F-dependent translation. Small molecule inhibitors of this complex have been identified, characterized, and used to validate the rationale of targeting this step to curtail tumor cell growth and modulate chemotherapy response. One such class of compounds are the rocaglates, secondary metabolites from the plant genus Aglaia, which target the RNA helicase subunit of eIF4F, eIF4A. Here we explore the ability of synthetic derivatives of aglaiastatins and an aglaroxin derivative to target the translation process in vitro and in vivo and find the synthetic derivative oxo-aglaiastatin to possess such activit...

Nature Communications, 2019

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie smal... more Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor s...

Proceedings of the National Academy of Sciences of the United States of America, Mar 6, 2018

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by parasites ... more Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in parasites. Rocaglates are a class of natural products derived from plants of the genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with (CM) and (blood-stage malaria), and can also block replication of different clinical isolates of in human erythrocytes infected ex vivo including drug-resistant isolates. In vivo, a single dosing of CR-1-31B in -infected animals is suffici...

Nature Chemistry, 2017

The lissoclimides are a family of labdane diterpenoids bearing an unusual succinimide motif, many... more The lissoclimides are a family of labdane diterpenoids bearing an unusual succinimide motif, many of which were reported to have potent cytotoxic activity against representative mammalian cancer cell lines. Our short semi-synthesis and analogue-oriented synthesis approaches have provided a series of lissoclimide natural products and analogues that permitted expansion of the structure-activity relationships (SAR) in this family. The semi-synthesis approach yielded significant quantities of chlorolissoclimide that permitted evaluation against the NCI's 60 cancer cell line panel and, most critically, allowed us to obtain an X-ray co-crystal structure of the synthetic secondary metabolite with the eukaryotic 80S ribosome. While it shares a binding site with other imide-based natural product translation inhibitors, two particularly interesting attractive interactions surfaced from this study: first, a hydrogen bond to a protein component of the ribosome, and second, a face-on halogen-π interaction between the ligand's alkyl chloride and a guanine residue. Our analogue-oriented synthesis supplemented our array of lissoclimide compounds, many of which were tested against aggressive human cancer cell lines, and whose cytotoxicity was correlated to their protein synthesis inhibitory activity. Finally, computational modeling was used to explain the SAR of certain key compounds, setting the stage for structure-guided design of better translation inhibitors.

Endocrinology, 2017

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spe... more Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.

Antimicrobial agents and chemotherapy, Jun 3, 2017

The permeation of antibiotics through bacterial membranes to their target site is a crucial deter... more The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity, but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold improved potency against a range of laboratory and clinical bacterial strains, which we named P10. Whole genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that the restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resist...

Proceedings of the National Academy of Sciences, 2017

Significance IFIT1 is an antiviral effector of host innate-immunity that selectively recognizes t... more Significance IFIT1 is an antiviral effector of host innate-immunity that selectively recognizes the 5′-end of viral mRNAs, which are often capped to mimic host mRNA, and blocks their translation. Our X-ray structural analysis reveals that the cap and four additional nucleotides are encircled by IFIT1 through a central tunnel in an adaptable manner, which gives it the flexibility required to defend against many different viruses, and to deter their ability to rapidly evolve. Host mRNA, normally ribose methylated at the first and second nucleotides following the cap, avoids IFIT1 recognition through tight complementary interfaces at these positions. This study uncovers the molecular basis for how IFIT1 selectively recognizes viral mRNAs and will help guide development of viral vaccines and mRNA therapeutics.

Proceedings of the National Academy of Sciences of the United States of America, Mar 1, 2017

RNA

Inhibition of eukaryotic translation initiation through unscheduled RNA clamping of the DEAD-box ... more Inhibition of eukaryotic translation initiation through unscheduled RNA clamping of the DEAD-box (DDX) RNA helicases eIF4A1 and eIF4A2 has been documented for pateamine A (PatA) and rocaglates—two structurally different classes of compounds that share overlapping binding sites on eIF4A. Clamping of eIF4A to RNA causes steric blocks that interfere with ribosome binding and scanning, rationalizing the potency of these molecules since not all eIF4A molecules need to be engaged to elicit a biological effect. In addition to targeting translation, PatA and analogs have also been shown to target the eIF4A homolog, eIF4A3—a helicase necessary for exon junction complex (EJC) formation. EJCs are deposited on mRNAs upstream of exon-exon junctions and, when present downstream from premature termination codons (PTCs), participate in nonsense-mediated decay (NMD), a quality control mechanism aimed at preventing the production of dominant-negative or gain-of-function polypeptides from faulty mRNA ...

Cellular and Molecular Life Sciences, 2021

Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of c... more Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of cap-dependent translation. This heterotrimeric complex consists of a cap binding subunit (eIF4E), a DEAD-box RNA helicase (eIF4A), and a large bridging protein (eIF4G). In mammalian cells, there are two genes encoding eIF4A (eIF4A1 and eIF4A2) and eIF4G (eIF4G1 and eIF4G3) paralogs that can assemble into eIF4F complexes. To query the essential nature of the eIF4F subunits in normal development, we used CRISPR/Cas9 to generate mouse strains with targeted ablation of each gene encoding the different eIF4F subunits. We find that Eif4e, Eif4g1, and Eif4a1 are essential for viability in the mouse, whereas Eif4g3 and Eif4a2 are not. However, Eif4g3 and Eif4a2 do play essential roles in spermatogenesis. Crossing of these strains to the lymphomaprone Eμ-Myc mouse model revealed that heterozygosity at the Eif4e or Eif4a1 loci significantly delayed tumor onset. Lastly, tumors derived from Eif4e ∆38 fs/+ /Eμ-Myc or Eif4a1 ∆5 fs/+ /Eμ-Myc mice show increased sensitivity to the chemotherapeutic agent doxorubicin, in vivo. Our study reveals that eIF4A2 and eIF4G3 play non-essential roles in gene expression regulation during embryogenesis; whereas reductions in eIF4E or eIF4A1 levels are protective against tumor development in a murine Myc-driven lymphoma setting.

Scientific Reports, 2021

Rocaglates are a class of eukaryotic translation initiation inhibitors that are being explored as... more Rocaglates are a class of eukaryotic translation initiation inhibitors that are being explored as chemotherapeutic agents. They function by targeting eukaryotic initiation factor (eIF) 4A, an RNA helicase critical for recruitment of the 40S ribosome (and associated factors) to mRNA templates. Rocaglates perturb eIF4A activity by imparting a gain-of-function activity to eIF4A and mediating clamping to RNA. To appreciate how rocaglates could best be enabled in the clinic, an understanding of resistance mechanisms is important, as this could inform on strategies to bypass such events as well as identify responsive tumor types. Here, we report on the results of a positive selection, ORFeome screen aimed at identifying cDNAs capable of conferring resistance to rocaglates. Two of the most potent modifiers of rocaglate response identified were the transcription factors FOXP3 and NR1I3, both of which have been implicated in ABCB1 regulation—the gene encoding P-glycoprotein (Pgp). Pgp has pr...

Nucleic Acids Research, 2020

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targetin... more Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5′ leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation...

Nucleic Acids Research, 2020

We report on the synthesis of siRNAs containing both 2′-5′- and 3′-5′-internucleotide linkages an... more We report on the synthesis of siRNAs containing both 2′-5′- and 3′-5′-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2′-5′ linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5′-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5′p-rN1-(2′-5′)-N2 unit in the antisense strand does not alter the ‘clover leaf’ bend and sugar puckers that are critical for anchoring the 5′-phosphate to Ago 2 MID domain. Importantly, 2′-5′-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results dem...

Nucleic Acids Research, 2020

Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in tra... more Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4G...

Cell Reports, 2020

Highlights d Rocaglates produce distinct inhibitory effects on translation initiation d Rocaglate... more Highlights d Rocaglates produce distinct inhibitory effects on translation initiation d Rocaglates interfere with eIF4F release from the cap structure d Rocaglates exert a bystander effect on translation initiation by sequestering eIF4F

RNA, 2020

The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node u... more The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity—generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anticancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F, and several potent small molecules (rocaglates, hippuristanol, pateamine A) affecting its activity have been identified and shown to demonstrate anticancer activity in vitro and in vivo in preclinical models. Recently, a number of new small molecules have been reported as having the capacity to target and inhibit eIF4A. Here, we undertook a comparative analysis of their biological activity and...

Blood, 2010

2995 Background: Multiple myeloma (MM) cell survival is dependent on the ability to produce and s... more 2995 Background: Multiple myeloma (MM) cell survival is dependent on the ability to produce and secrete immunoglobulins, requiring precise coordination of protein synthesis and degradation. Inhibition of these processes may be responsible for the exquisite sensitivity of MM to drugs such as bortezomib and histone deacetylase inhibitors, which respectively block proteosomal and aggresomal-mediated protein degradation. We hypothesized that MM cells would also be sensitive to inhibition of mRNA translation and have tested silvestrol, a plant-derived natural product that has previously been shown to inhibit ribosome recruitment and to be cytotoxic to B lymphocytes. Mehods: Silvestrol was resuspended in DMSO and diluted for use in RPMI medium. A panel of human MM cell lines, representing a range of chromosomal abnormalities observed in this disease, were cultured in RPMI with 10% FCS and appropriate supplementation. Primary mouse embryonic fibroblasts (MEFs) were prepared and passaged 7–...

Cell Chemical Biology, 2019

Rocaglates share a common cyclopentabenzo[b]furan core that inhibit eukaryotic translation initia... more Rocaglates share a common cyclopentabenzo[b]furan core that inhibit eukaryotic translation initiation by modifying the behaviour of the RNA helicase, eIF4A. Working as interfacial inhibitors, rocaglates stabilize the association between eIF4A and RNA which can lead to the formation of steric barriers that block initiating ribosomes. There is significant interest in the development and expansion of rocaglate derivatives as several members of this family have been shown to possess potent anti-neoplastic activity in vitro and in vivo. To further our understanding of rocaglate diversity and drug design, herein we explore the RNA clamping activity of >200 unique rocaglate derivatives. Through this, we report on the identification and characterization of a potent class of synthetic rocaglates called amidino-rocaglates. These compounds are among the most potent rocaglates documented to date, and taken together, this work offers important information that will guide the future design of rocaglates with improved biological properties.

Molecular Cancer Therapeutics, 2019

CDK4/6 inhibitors are FDA-approved drugs for estrogen receptor–positive (ER+) breast cancer and a... more CDK4/6 inhibitors are FDA-approved drugs for estrogen receptor–positive (ER+) breast cancer and are being evaluated to treat other tumor types, including KRAS-mutant non–small cell lung cancer (NSCLC). However, their clinical utility is often limited by drug resistance. Here, we sought to better understand the resistant mechanisms and help devise potential strategies to overcome this challenge. We show that treatment with CDK4/6 inhibitors in both ER+ breast cancer and KRAS-mutant NSCLC cells induces feedback upregulation of cyclin D1, CDK4, and cyclin E1, mediating drug resistance. We demonstrate that rocaglates, which preferentially target translation of key cell-cycle regulators, effectively suppress this feedback upregulation induced by CDK4/6 inhibition. Consequently, combination treatment of CDK4/6 inhibitor palbociclib with the eukaryotic initiation factor (eIF) 4A inhibitor, CR-1-31-B, is synergistic in suppressing the growth of these cancer cells in vitro and in vivo. Furth...

Cell Chemical Biology, 2019

Highlights d CRISPR/Cas9 engineering harnessed to generate a targetagnostic phenotypic screen d G... more Highlights d CRISPR/Cas9 engineering harnessed to generate a targetagnostic phenotypic screen d GFP tracing of c-MYC expression in multiple myeloma cells d Cardiac glycosides and cytoskeletal disruptors are potent MYC expression inhibitors

Scientific Reports, 2019

Translation is a highly regulated process that is perturbed in human cancers, often through activ... more Translation is a highly regulated process that is perturbed in human cancers, often through activation of the PI3K/mTOR pathway which impacts directly on the ribosome recruitment phase of translation initiation. While significant research has focused on “drugging” components of the PI3K/mTOR network, efforts have also been directed towards inhibiting eukaryotic initiation factor (eIF) 4F-dependent translation. Small molecule inhibitors of this complex have been identified, characterized, and used to validate the rationale of targeting this step to curtail tumor cell growth and modulate chemotherapy response. One such class of compounds are the rocaglates, secondary metabolites from the plant genus Aglaia, which target the RNA helicase subunit of eIF4F, eIF4A. Here we explore the ability of synthetic derivatives of aglaiastatins and an aglaroxin derivative to target the translation process in vitro and in vivo and find the synthetic derivative oxo-aglaiastatin to possess such activit...

Nature Communications, 2019

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie smal... more Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor s...

Proceedings of the National Academy of Sciences of the United States of America, Mar 6, 2018

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by parasites ... more Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in parasites. Rocaglates are a class of natural products derived from plants of the genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with (CM) and (blood-stage malaria), and can also block replication of different clinical isolates of in human erythrocytes infected ex vivo including drug-resistant isolates. In vivo, a single dosing of CR-1-31B in -infected animals is suffici...

Nature Chemistry, 2017

The lissoclimides are a family of labdane diterpenoids bearing an unusual succinimide motif, many... more The lissoclimides are a family of labdane diterpenoids bearing an unusual succinimide motif, many of which were reported to have potent cytotoxic activity against representative mammalian cancer cell lines. Our short semi-synthesis and analogue-oriented synthesis approaches have provided a series of lissoclimide natural products and analogues that permitted expansion of the structure-activity relationships (SAR) in this family. The semi-synthesis approach yielded significant quantities of chlorolissoclimide that permitted evaluation against the NCI's 60 cancer cell line panel and, most critically, allowed us to obtain an X-ray co-crystal structure of the synthetic secondary metabolite with the eukaryotic 80S ribosome. While it shares a binding site with other imide-based natural product translation inhibitors, two particularly interesting attractive interactions surfaced from this study: first, a hydrogen bond to a protein component of the ribosome, and second, a face-on halogen-π interaction between the ligand's alkyl chloride and a guanine residue. Our analogue-oriented synthesis supplemented our array of lissoclimide compounds, many of which were tested against aggressive human cancer cell lines, and whose cytotoxicity was correlated to their protein synthesis inhibitory activity. Finally, computational modeling was used to explain the SAR of certain key compounds, setting the stage for structure-guided design of better translation inhibitors.

Endocrinology, 2017

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spe... more Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.

Antimicrobial agents and chemotherapy, Jun 3, 2017

The permeation of antibiotics through bacterial membranes to their target site is a crucial deter... more The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity, but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold improved potency against a range of laboratory and clinical bacterial strains, which we named P10. Whole genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that the restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resist...

Proceedings of the National Academy of Sciences, 2017

Significance IFIT1 is an antiviral effector of host innate-immunity that selectively recognizes t... more Significance IFIT1 is an antiviral effector of host innate-immunity that selectively recognizes the 5′-end of viral mRNAs, which are often capped to mimic host mRNA, and blocks their translation. Our X-ray structural analysis reveals that the cap and four additional nucleotides are encircled by IFIT1 through a central tunnel in an adaptable manner, which gives it the flexibility required to defend against many different viruses, and to deter their ability to rapidly evolve. Host mRNA, normally ribose methylated at the first and second nucleotides following the cap, avoids IFIT1 recognition through tight complementary interfaces at these positions. This study uncovers the molecular basis for how IFIT1 selectively recognizes viral mRNAs and will help guide development of viral vaccines and mRNA therapeutics.