Translation Initiation Factor eIF2α Research Articles

Efficacy of GCN2 inhibition by a novel small molecule AP030 in acute leukemia.

6532 Background: GCN2 is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR) that is activated in response to amino acid scarcity. Active GCN2 phosphorylates translation initiation factor eIF2α resulting in the attenuation of global protein synthesis. ISR signaling primarily promotes cell survival but may trigger cell death, dependent on the cellular context. In hematological tumors GCN2 promotes tumor cell survival under conditions of nutrient scarcity. Several GCN2 inhibitors are in clinical development for treatment of both solid and hematological tumors. Here, we describe the preclinical results of a novel, selective ATP-competitive inhibitor of GCN2. Methods: GCN2 inhibition by AP030 was determined using a biochemical Lanthascreen assay and a cell based HTRF assay measuring eIF2a phosphorylation following stimulation with Borrelidin. Kinase selectivity was determined using KinomeScan, and bespoke biochemical and cell-based assays. Interaction of AP030 with GCN2 was determined using X-ray crystallography. AP030 activity in disease-relevant tumor cell lines was determined using cell viability, caspase activation, protein expression and qPCR gene expression endpoints. Inhibition of hematological tumor growth and induction of cell death was investigated in patient samples, and ALL and AML in vivo animal models. Results: Potent inhibition of GCN2 in the Lanthascreen assay was demonstrated with AP030 (Ki of 4.4nM). Following stimulation with Borrelidin, AP030 inhibited eIF2a phosphorylation with an IC50 of 50.8nM and inhibited downstream targets CHAC1 and DDIT3 measured by qPCR. X-ray crystallography confirmed that AP030 binds to the ATP-binding site of GCN2. The KinomeScan confirmed that AP030 was highly selective against the human kinome. AP030 led to partial or complete reduction of AML cell line viability as a single agent and acted synergistically with asparaginase in ALL cell lines. Caspase 3/7 induction was observed in the AML and ALL cell lines evaluated and AP030 increased the proportion of cells undergoing apoptosis in AML patient primary samples. In the CCRF-CEM ALL systemic in vivo tumor model AP030 inhibited tumor growth in combination with asparaginase (TGI 79.33%). Treatment with 0.5-5mg/kg (q.d.) of AP030 resulted in dose-dependent tumor growth inhibition and regression of the MOLM-16 AML in vivo model (TGI 97.37% 5mg/kg). RNA sequencing of residual tumor cells revealed disruption of amino acid and protein metabolism, consistent with GCN2 inhibition. Conclusions: Targeting hematological tumor reliance on the GCN2 arm of the ISR during nutrient scarcity is a novel approach to preventing tumor cell survival. AP030 a novel, potent, selective, ATP-competitive kinase inhibitor has been shown to lead to impressive efficacy in acute leukemia. Based on these preclinical results, a phase 1/2 study has been initiated in AML.

The impact of single-stranded RNAs on the dimerization of double-stranded RNA-dependent protein kinase PKR

The RNA-binding protein PKR serves as a crucial antiviral innate immune factor that globally suppresses translation by sensing viral double-stranded RNA (dsRNA) and by phosphorylating the translation initiation factor eIF2α. Recent findings have unveiled that single-stranded RNAs (ssRNAs), including in vitro transcribed (IVT) mRNA, can also bind to and activate PKR. However, the precise mechanism underlying PKR activation by ssRNAs, remains incompletely understood. Here, we developed a NanoLuc Binary Technology (NanoBiT)-based in vitro PKR dimerization assay to assess the impact of ssRNAs on PKR dimerization. Our findings demonstrate that, akin to double-stranded polyinosinic:polycytidylic acid (polyIC), an encephalomyocarditis virus (EMCV) RNA, as well as NanoLuc luciferase (Nluc) mRNA, can induce PKR dimerization. Conversely, homopolymeric RNA lacking secondary structure fails to promote PKR dimerization, underscoring the significance of secondary structure in this process. Furthermore, adenovirus VA RNA 1, another ssRNA, impedes PKR dimerization by competing with Nluc mRNA. Additionally, we observed structured ssRNAs capable of forming G-quadruplexes induce PKR dimerization. Collectively, our results indicate that ssRNAs have the ability to either induce or inhibit PKR dimerization, thus representing potential targets for the development of antiviral and anti-inflammatory agents.

Baseline unfolded protein response signaling adjusts the timing of the mammalian cell cycle.

The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G1-to-S/G2 transition. Treatment with the small molecule ISRIB to bypass the effects of PERK-dependent phosphorylation of the translation initiation factor eIF2α had no such effect, suggesting that cell cycle timing depends on PERK's kinase activity but is independent of eIF2α phosphorylation. Using complementary light and electron microscopy and flow cytometry-based analyses, we also demonstrate that the ER enlarges before mitosis. Together, our results suggest coordination between UPR signaling and the cell cycle to maintain ER physiology during cell division.

Mitochondria-Modulating Liposomes Reverse Radio-Resistance for Colorectal Cancer.

Complete remission of colorectal cancer (CRC) is still unachievable in the majority of patients by common fractionated radiotherapy, leaving risks of tumor metastasis and recurrence. Herein, clinical CRC samples demonstrated a difference in the phosphorylation of translation initiation factor eIF2α (p-eIF2α) and the activating transcription factor 4 (ATF4), whose increased expression by initial X-ray irradiation led to the resistance to subsequent radiotherapy. The underlying mechanism is studied in radio-resistant CT26 cells, revealing that the incomplete mitochondrial outer membrane permeabilization (iMOMP) triggered by X-ray irradiation is key for the elevated expression of p-eIF2α and ATF4, and therefore radio-resistance. This finding guided to discover that metformin and 2-DG are synergistic in reversing radio resistance by inhibiting p-eIF2α and ATF4. Liposomes loaded with metformin and 2-DG (M/D-Lipo) are thus prepared for enhancing fractionated radiotherapy of CRC, which achieved satisfactory therapeutic efficacy in both local and metastatic CRC tumors by reversing radio-resistance and preventing T lymphocyte exhaustion.

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 618.

Cryo-EM structure of Influenza A virus NS1 and antiviral protein kinase PKR complex

Influenza A virus is the cause of a widespread human disease with high morbidity and mortality rates. The influenza virus encodes non-structural protein 1 (NS1), an exceedingly multifunctional virulence component. NS1 plays essential roles in viral replication and evasion of the cellular innate immune system. Protein kinase RNA-activated also known as protein kinase R (PKR) phosphorylates translation initiation factor eIF-2α on serine 51 to inhibit protein synthesis in virus-infected mammalian cells. Consequently, PKR activation inhibits mRNA translation, which results in the assert of both viral protein synthesis and cellular and possibly apoptosis in response to virus infection. Host signaling pathways are important in the replication of influenza virus, but the mechanisms involved remain to be characterized. Herein, the structure of NS1 and PKR complex was determined using Cryo-EM. We found the N91, E94, and G95 residues of PKR bind directly with N188, D125, and K126, respectively, of NS1. Furthermore, the study shows that PKR peptide offers a potential treatment for Influenza A virus infections.

Translation initiation factor eIF1A rescues hygromycin B sensitivity caused by deleting the carboxy-terminal tail in the GPN-loop GTPase Npa3.

The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

Cell competition: emerging signaling and unsolved questions.

Multicellular communities have an intrinsic mechanism that optimizes their structure and function via cell-cell communication. One of the driving forces for such self-organization of the multicellular system is cell competition, the elimination of viable unfit or deleterious cells via cell-cell interaction. Studies in Drosophila and mammals have identified multiple mechanisms of cell competition caused by different types of mutations or cellular changes. Intriguingly, recent studies have found that different types of "losers" of cell competition commonly show reduced protein synthesis. In Drosophila, the reduction in protein synthesis levels in loser cells is caused by phosphorylation of the translation initiation factor eIF2α via a bZip transcription factor Xrp1. Given that a variety of cellular stresses converge on eIF2α phosphorylation and thus global inhibition of protein synthesis, cell competition may be a machinery that optimizes multicellular fitness by removing stressed cells. In this review, we summarize and discuss emerging signaling mechanisms and critical unsolved questions, as well as the role of protein synthesis in cell competition.

A second-generation eIF4A RNA helicase inhibitor exploits translational reprogramming as a vulnerability in triple-negative breast cancer

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.

PABP-driven secondary condensed phase within RSV inclusion bodies activates viral mRNAs for ribosomal recruitment

Inclusion bodies (IBs) of respiratory syncytial virus (RSV) are formed by liquid-liquid phase separation (LLPS) and contain internal structures termed “IB-associated granules” (IBAGs), where anti-termination factor M2-1 and viral mRNAs are concentrated. However, the mechanism of IBAG formation and the physiological function of IBAGs are unclear. Here, we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein (mRNP) condensates formed by secondary LLPS. Mechanistically, the RSV nucleoprotein (N) and M2-1 interact with and recruit PABP to IBs, promoting PABP to bind viral mRNAs transcribed in IBs by RNA-recognition motif and drive secondary phase separation. Furthermore, PABP-eIF4G1 interaction regulates viral mRNP condensate composition, thereby recruiting specific translation initiation factors (eIF4G1, eIF4E, eIF4A, eIF4B and eIF4H) into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment. Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase.

Abstract A019: eIF4A and PDCD4 expression in TNBC underlies sensitivity to natural product translation inhibitor

Abstract Despite recent advances in immunotherapy and adjuvant-therapy, resistance to cancer therapeutics is common and requires novel approaches. Moreover, highly aggressive triple-negative breast cancers lack effective targeted treatments. Deregulation of translation factors is associated with the increased proliferation of TNBC cells, heightened CSC activity, and poor patient outcomes. Thus targeting translation regulatory factors upregulated in TNBC is a promising approach to developing novel treatments. Pateamine A (PatA), a natural marine macrodiolide product, inhibits RNA cap-dependent translation by binding and sequestering the eukaryotic translation initiation factor eIF4A. eIF4A promotes the translation of polypurine-rich mRNA sequences, which are enriched within breast cancer oncogenes. Here we report the anticancer efficacy of novel PatA analogs we have synthesized against breast cancer cell lines. The PatA analogs exhibited selective cytotoxicity to breast and colorectal cancers compared to non-cancerous cells measured by Cell Titer-Blue viability studies. Novel analogs show greater antiproliferative potency against models of triple-negative than against human mammary epithelial cells (HMLE). To further elucidate eIF4A as a molecular target, we conducted analyses of patient tissue samples in the Cancer Genome Atlas (TCGA). Our analyses reveal the overexpression of eIF4A in tumor tissues compared to normal tissues and gene correlation studies reveal the upregulation of key co-expressed tumor drivers including beta-catenin, fibronectin, and epithelial-to-mesenchymal transition (EMT) markers such as SNAIL and SLUG. Further analyses show a downregulation of tumor suppressor protein PDCD4, an endogenous inhibitor of eIF4A, in cancer tissues. We have generated eIF4A shRNA knockdown and PDCD4 shRNA knockdown cell lines to uncover the eIF4A-PDCD4 regulatory axis across breast adenocarcinoma and colorectal adenocarcinoma cell types. We show that the loss of eIF4A attenuates the mesenchymal and stem-like phenotype of breast cancer stem cells revealed by phenotypic assays. Our results suggest that targeting translation initiator eIF4A is an effective approach against highly proliferative and metastatic breast cancer types. The presented findings underscore the potential of eIF4A as a promising molecular target and supports translational control as a viable approach against aggressive cancers. Citation Format: Alagu Subramanian, Haoran Xue, Kenneth G Hull, Daniel Romo, Joseph Taube. eIF4A and PDCD4 expression in TNBC underlies sensitivity to natural product translation inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A019.

Ascomylactam C Induces an Immunogenic Cell Death Signature via Mitochondria-Associated ER Stress in Lung Cancer and Melanoma.

Ascomylactam C (AsC) is a new 13-membered-ring macrocyclic alkaloid, which was first isolated and identified in 2019 from the secondary metabolites of the mangrove endophytic fungus Didymella sp. CYSK-4 in the South China Sea. AsC has been found to have a broad-spectrum cytotoxic activity. However, the antitumor effects in vivo and mechanisms of AsC remain unclear. The aim of this study was to describe the effects of AsC on lung cancer and melanoma cells and to explore the antitumor molecular mechanism of AsC. In vitro, we used plate colony formation experiments and demonstrated the ability of AsC to inhibit low-density tumor growth. An Annexin V/PI cell apoptosis detection experiment revealed that AsC induced tumor cell apoptosis. In vivo, AsC suppressed the tumor growth of LLC and B16F10 allograft significantly in mice, and promoted the infiltration of CD4+ T and CD8+ T cells in tumor tissues. Mechanistically, by analyses of Western blotting, immunofluorescence and ELISA analysis, we found that AsC increased ROS formation, induced endoplasmic reticulum (ER) stress, activated the protein kinase RNA-like ER kinase (PERK)/eukaryotic translation initiation factor (eIF2α)/activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) signaling pathway, and induced immunogenic cell death (ICD) of tumor cells. Our results suggest that AsC may be a potentially promising antitumor drug candidate.

Cerebral Spinal Fluid Attenuates the Efficacy of Methotrexate Against Acute Lymphoblastic Leukemia Cells

The anti-folate methotrexate is a critical component of acute lymphoblastic leukemia (ALL) therapy. In addition to systemic administration, methotrexate is given intrathecally, or directly into the cerebral spinal fluid (CSF) via lumbar puncture, for central nervous system (CNS) leukemia treatment and prophylaxis. While IT methotrexate played a critical role in improving outcomes for ALL, CNS ALL relapse remains a major cause of treatment failure and therapy related morbidity. However, attempts to develop more efficacious and less toxic CNS ALL therapies have been largely unsuccessful and IT methotrexate has remained the standard of care for 60+ years. Based on our prior work showing that ALL cells are more poised to undergo apoptosis in nutrient poor CSF, we initially hypothesized that ALL chemosensitivity will be increased in CSF (Basile et al., 2020). While this was confirmed for several ALL drugs including cytarabine and anthracyclines, we strikingly found that methotrexate was significantly less efficacious and potent against ALL cells in CSF relative to standard tissue culture media or the more physiologically relevant human plasma-like media (HPLM). CSF also attenuated the sensitivity of ALL cells to other anti-folates including raltitrexed, trimetrexate, and pralatrexate. To define the mechanism(s) underlying this methotrexate resistance induced by CSF, we explored previously well-described mechanisms of methotrexate resistance in leukemia and other cancers. However, to date this work suggests that the molecular etiology of this CSF-induced relative methotrexate resistance is not entirely explained by diminished leukemia proliferation in CSF, altered methotrexate cellular influx/efflux, methotrexate metabolism, folate analog rescue, dysregulated expression of methotrexate target proteins, or compensation by nucleotide salvage pathways. To complement our work examining known mechanisms of methotrexate resistance, we also used a more discovery-based approach and compared the gene expression patterns of leukemia cells in CSF versus tissue culture media. Preliminary bioinformatic analyses identified dysregulation of multiple genes involved in the integrated stress response (ISR) and unfolded protein response (UPR) pathways in leukemia cells in CSF. The ISR/UPR are mechanisms by which cells adapt to diverse stress stimuli in the microenvironment, including potentially nutrient poor CSF which is low in glucose, proteins, and lipids relative to plasma. The hallmark event in this pathway is the phosphorylation of the translation initiation factor eIF2α, which inhibits global protein synthesis and enables the translation of selected genes that together enable cell survival. Supporting our gene expression data, ALL cells in CSF showed increased phosphorylation of eIF2α and a decrease in global protein synthesis. Intriguingly, it has been shown that activation of the UPR can divert metabolites from glycolysis to mitochondrial one-carbon metabolism with resulting resistance to anti-folates such as methotrexate (Reich et al., 2020). Accordingly, we next tested the effect of UPR/ISR activation on leukemia cell sensitivity to methotrexate in tissue culture media. Activation of the UPR with thapsigargin, a sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor that triggers ER stress, increased methotrexate resistance in leukemia cells. Similarly, BtdCPU a small-molecule activator of heme regulated inhibitor (HRI) kinase that phosphorylates eIF2α and activates the ISR, also enhanced leukemia resistance to methotrexate. Building upon these results, current investigations are focused on testing the role of UPR/IST activation in methotrexate resistance in leukemia cells in CSF and modulating this pathway to overcome resistance. While methotrexate is clearly an efficacious and critical component of CNS leukemia treatment and prophylaxis, we have shown that CSF attenuates the overall efficacy of methotrexate in targeting leukemia cells. Moreover, we anticipate that our current work defining the mechanisms driving this resistance may identify novel approaches for maximizing methotrexate efficacy and more completely eradicating leukemia cells in CSF and the CNS. Finally, this work highlights the importance of critically evaluating even long-established standards of care.

A highly efficient human cell-free translation system.

Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

Activation of the integrated stress response by inhibitors of its kinases

Phosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases.

Nutrient-dependent regulation of β-cell proinsulin content

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic β-cells remains largely unknown. Here, we first examined β-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3days) and found that the proinsulin pool size responds to each feeding within 1 to 2h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2α, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2α rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that β-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles.

Abstract A017: Deciphering the role of integrated stress response (ISR) in the developmental stages of mutant KRAS lung cancer

Abstract Lung cancer is the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) constitutes 80% of all lung malignancies with 15-25% attributed to mutations in the KRAS gene. KRAS mutations expose cells to stress-inducing conditions, e.g., genotoxic, proteotoxic and metabolic stress, which disrupt normal proliferation and tissue homeostasis. Successful proliferation of tumors is dependent on stress-adaptive pathways, which promote growth and contribute to resistance to chemotherapeutic drugs. Thus, inhibition of the adaptation process is considered a suitable anti-tumor approach for the treatment of KRAS cancers including lung cancer. An important mechanism of adaptation to stress involves the phosphorylation of the translation initiation factor eIF2α at serine 52 (p-eIF2α). Increased p-eIF2α is the nodal point of the integrated stress response (ISR), a master regulator of translational and transcriptional reprogramming that determines survival and adaptation to stress. We demonstrated the tumorigenic function of p-eIF2α in a mouse model of mutant KRAS cancer (Nature Communications 2021). We delineate the role of the ISR in lineage diversity and tumor heterogeneity during KRAS LUAD progression by single-cell RNA sequencing (sc-RNA seq). As such, the ISR was found to drive high stemness and epithelial to mesenchymal transition (EMT) programs in KRAS LUAD cells. The ISR also employs a high-plasticity cell-state mechanism for successful lung tumor progression and evolution. The involvement of the ISR in the mentioned processes further emphasizes its role as master regulator of pro-tumorigenic pathways and highlights the therapeutic potential of ISR inhibitors as novel targets in KRAS lung cancer. Our recent findings demonstrate that increased p-eIF2α stimulates the activity of the gene trans-activator proteins YAP and TAZ in mutant KRAS lung tumors. Genetic approaches identify a stimulatory effect of p-eIF2 on the expression of YAP/TAZ-dependent genes with roles in proliferation and epithelial to mesenchymal transition (EMT) including genes of WNT signaling pathway. We provide strong evidence that stimulation of YAP/TAZ activity by p-eIF2α occurs, at least partially, through the upregulation of the transcription factor ATF4 in tumor cells. Our findings demonstrate that p-eIF2α is a focal point of different tumorigenic pathways, intimately implicated in mutant KRAS-mediated transformation and tumor resistance to mutant KRAS inactivation. Citation Format: Shiqi Diao, Nour Ghaddar, Jia Yi Zou, Shuo Wang, Antonis E. Koromilas. Deciphering the role of integrated stress response (ISR) in the developmental stages of mutant KRAS lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A017.

Abstract 3731: The polyamine-hypusine circuit controls an oncogenic translational program essential for malignant transformation in MYC-driven lymphoma

Abstract The MYC oncoprotein is activated in a broad spectrum of human malignancies and transcriptionally reprograms the genome to drive cancer cell growth. Given these functions, it is unclear if targeting a single effector of MYC will have therapeutic benefit. MYC activates the polyamine-hypusine circuit, which post-translationally modifies a single lysine residue (Lys-50) of eukaryotic translation initiation factor eIF5A in a process coined hypusination via two enzymes,deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). The roles of this circuit in cancer are unclear. Here we report essential intrinsic roles for hypusinated eIF5A in the development and maintenance of MYC-driven lymphoma, where loss of eIF5A hypusination completely abolishes malignant transformation of MYC-overexpressing B cells in transgenic mice predestined to develop lymphoma. Mechanistically, integrating RNA-seq, Ribo-seq and proteomic analyses revealed that efficient translation of select targets is dependent upon eIF5A hypusination, including regulators of G1-to-S phase cell cycle progression and DNA replication. Thus, this circuit controls MYC’s proliferative response at several levels and is activated in many tumor types. These findings suggest the hypusine circuit as a therapeutic target for a broad spectrum of malignancies. Citation Format: Shima Nakanishi, Jiannong Li, Anders E. Berglund, Youngchul Kim, Yonghong Zhang, Ling Zhang, Chunying Yang, Raghavendra G. Mirmira, John L. Cleveland. The polyamine-hypusine circuit controls an oncogenic translational program essential for malignant transformation in MYC-driven lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3731.

Luteolin protects DYT-PRKRA cells from apoptosis by suppressing PKR activation.

DYT-PRKRA is a movement disorder caused by mutations in the PRKRA gene, which encodes for PACT, the protein activator of interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR. PACT brings about PKR's catalytic activation by a direct binding in response to stress signals and activated PKR phosphorylates the translation initiation factor eIF2α. Phosphorylation of eIF2α is the central regulatory event that is part of the integrated stress response (ISR), an evolutionarily conserved intracellular signaling network essential for adapting to environmental stresses to maintain healthy cells. A dysregulation of either the level or the duration of eIF2α phosphorylation in response to stress signals causes the normally pro-survival ISR to become pro-apoptotic. Our research has established that the PRKRA mutations reported to cause DYT-PRKRA lead to enhanced PACT-PKR interactions causing a dysregulation of ISR and an increased sensitivity to apoptosis. We have previously identified luteolin, a plant flavonoid, as an inhibitor of the PACT-PKR interaction using high-throughput screening of chemical libraries. Our results presented in this study indicate that luteolin is markedly effective in disrupting the pathological PACT-PKR interactions to protect DYT-PRKRA cells against apoptosis, thus suggesting a therapeutic option for using luteolin to treat DYT-PRKRA and possibly other diseases resulting from enhanced PACT-PKR interactions.