Induction Of Integrated Stress Response Research Articles

Abstract 524: Using acquired resistance to explore the mechanism of action of the integrated stress response/GCN2 activator NXP800 - A new developmental agent for platinum-resistant ARID1A mutant ovarian cancer

Abstract Background: NXP800 is a potent, oral activator of the Integrated Stress response (ISR), inhibitor of heat shock factor 1 (HSF1) activation and tumor cell proliferation, which is in early clinical studies in ARID1A-mutated, platinum resistant, clear cell ovarian cancer (NCT05226507). We discovered NXP800 using multiparameter medicinal chemistry optimization of a hit identified from a cell-based phenotypic screen. Owing to the unbiased nature of phenotypic screening, target identification is crucial to understand the biological and therapeutic activity of hit compounds. Methods and Results:: By RNAseq profiling human cancer cells treated with NXP800 we identified changes in expression of genes regulated by HSF1 or ATF4 - effects accompanied by eIF2alpha (eIF2a) phosphorylation and resulting activation of the ISR. To further understand this response, we explored whether NXP800 resistance models could inform on its mechanism of action (MoA). We used ARID1A mutant SK-OV-3 human ovarian carcinoma cells that are 1) highly sensitive to NXP800, 2) model the target patient population and 3) MSI-high so likely to have an elevated mutation rate contributing to acquisition of resistance. We generated two independent NXP800-resistant SK-OV-3 cell lines in which NXP800-mediated ATF4 induction and concomitant inhibition of global translation were abolished. By whole exome sequencing, we identified a heterozygous L99P mutation in the alpha subunit of eIF2B (eIF2Ba), the nucleotide exchange factor for eIF2a. Expression of eIF2BaL99P, but not wild-type, in parental SK-OV-3 cells reduced sensitivity to NXP800 to the same level as cells with NXP800-induced resistance. Using fluorescence recovery after photobleaching to monitor the dynamic association of the eIF2B:eIF2a complex, we elucidated that the eIF2BaL99P mutation reduces NXP800-mediated inhibition of eIF2a-GFP recycling through eIF2B bodies. Phosphorylation of eIF2a is regulated by four stress-controlled kinases GCN2, HRI, PKR and PERK. Using systematic siRNA knockdown or small-molecule inhibitors, we showed that GCN2 alone is required for ISR activation by NXP800 and that ISR induction inhibited HSF1 activation. Furthermore, inactivation of GCN2 reduced the antiproliferative activity of NXP800 to the same extent observed in NXP800-resistant SK-OV-3 cells. In contrast, exposure of resistant or parental SK-OV-3 cells expressing eIF2BaL99P to GCN2 inhibitors did not cause further reduction in NXP800 sensitivity - confirming the importance of the eIF2BaL99P mutation in the resistance mechanism. Conclusions: We have used acquired resistance to understand the MoA of NXP800 as a potent activator of GCN2 and the ISR pathway. Further studies are underway to determine the exact proximal molecular target of NXP800 and the mechanism of GCN2/ISR activation. Citation Format: Marissa V. Powers, Rachel Hodgson, Swee Y. Sharp, Toby Roe, K. Elizabeth Allen, Susan Campbell, Robert te Poele, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Using acquired resistance to explore the mechanism of action of the integrated stress response/GCN2 activator NXP800 - A new developmental agent for platinum-resistant ARID1A mutant ovarian cancer [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 524.

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Mitochondrial integrated stress response controls lung epithelial cell fate.

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment.

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.

Abstract LB234: Activation of the integrated stress response by the developmental HSF1 pathway inhibitor NXP800

Abstract HSF1 is a stress-inducible transcription factor that regulates the eukaryotic heat shock response (HSR). HSF1 activation induces the expression of multiple proteins needed for cellular recovery from stress. HSF1 also plays a key role in tumorigenesis and regulates the expression of a cancer-specific gene signature which is unique to malignant cells and distinct from that activated during the HSR. We discovered NXP800, the first-in-class orally active HSF1 pathway inhibitor which is now undergoing Phase 1 clinical trial, based on a phenotypic pathway screen. Here we employed various techniques to investigate the mechanism of action of NXP800. Using RNAseq in a panel of human carcinoma cell lines, we identified overlapping gene expression changes in response to NXP800. These included genes regulated by HSF1 and interestingly also genes associated with activation of the integrated stress response (ISR). In contrast, we found no evidence for activation of the unfolded protein response. Consistent with the observed NXP800-induced phosphorylation of eIF2α which is a critical regulator of the ISR, NXP800 increased the protein expression of downstream ISR markers ATF4, CHOP and CHAC1, both in human tumor cells in vitro and in human tumor xenograft models in vivo. Induction of the ISR is controlled by four stress-activated protein kinases (PKs) that phosphorylate eIF2α. To further explore these kinases in the mechanism of action of NXP800 we used genetic knockdown by siRNA and inhibition by small-molecule tool compounds. Silencing each of the ISR-regulatory PKs revealed that GCN2 was required for ISR activation by NXP800. This was confirmed using two GCN2 inhibitors from different chemical series. Global phospho-proteome analysis showed that altered protein phosphorylation following NXP800 exposure was reversed upon co-treatment with a GCN2 inhibitor. We also demonstrated that activation of the ISR caused inhibition of HSF1 activation when stimulated with an HSF1 activator, thus confirming the link between ISR induction and inhibition of HSF1 activation. Activation of GCN2 and the ISR can occur in response to a variety of stimuli including amino acid deprivation. However, we did not detect a difference in the uptake of amino acids following exposure to NXP800, indicating that NXP800 does not directly impair amino acid uptake. Using an siRNA approach to determine if activation of the ISR components was contributing to growth inhibition following NXP800 exposure, we found that blocking the induction of ATF4 reduced the response of NXP800-sensitive SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. In summary, NXP800 acts on cancer cells to induce activation of the ISR pathway via GCN2, which then leads to inhibition of HSF1 activation. Further studies are underway to determine the precise molecular target of NXP800 and the mechanism of HSF1 pathway inhibition. Citation Format: Marissa V. Powers, Swee Y. Sharp, Eirini-Maria Lampraki, Toby Roe, Loredana Pellegrino, Maria Taskinen, Robert te Poele, Florence Raynaud, Suzanne Eccles, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Activation of the integrated stress response by the developmental HSF1 pathway inhibitor NXP800 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB234.

Contributions of Ccr4 and Gcn2 to the Translational Response of C. neoformans to Host-Relevant Stressors and Integrated Stress Response Induction.

In response to the host environment, the human pathogen Cryptococcus neoformans must rapidly reprogram its translatome from one which promotes growth to one which is responsive to host stress. In this study, we investigate the two events which comprise translatome reprogramming: the removal of abundant, pro-growth mRNAs from the translating pool, and the regulated entry of stress-responsive mRNAs into the translating pool. Removal of pro-growth mRNAs from the translating pool is controlled primarily by two regulatory mechanisms, repression of translation initiation via Gcn2, and decay mediated by Ccr4. We determined that translatome reprogramming in response to oxidative stress requires both Gcn2 and Ccr4, whereas the response to temperature requires only Ccr4. Additionally, we assessed ribosome collision in response to host-relevant stress and found that collided ribosomes accumulated during temperature stress but not during oxidative stress. The phosphorylation of eIF2α that occurred as a result of translational stress led us to investigate the induction of the integrated stress response (ISR). We found that eIF2α phosphorylation varied in response to the type and magnitude of stress, yet all tested conditions induced translation of the ISR transcription factor Gcn4. However, Gcn4 translation did not necessarily result in canonical Gcn4-dependent transcription. Finally, we define the ISR regulon in response to oxidative stress. In conclusion, this study begins to reveal the translational regulation in response to host-relevant stressors in an environmental fungus which is capable of adapting to the environment inside the human host. IMPORTANCE Cryptococcus neoformans is a human pathogen capable of causing devastating infections. It must rapidly adapt to changing environments as it leaves its niche in the soil and enters the human lung. Previous work has demonstrated a need to reprogram gene expression at the level of translation to promote stress adaptation. In this work, we investigate the contributions and interplay of the major mechanisms that regulate entry of new mRNAs into the pool (translation initiation) and the clearance of unneeded mRNAs from the pool (mRNA decay). One result of this reprogramming is the induction of the integrated stress response (ISR) regulon. Surprisingly, all stresses tested led to the production of the ISR transcription factor Gcn4, but not necessarily to transcription of ISR target genes. Furthermore, stresses result in differential levels of ribosome collisions, but these are not necessarily predictive of initiation repression as has been suggested in the model yeast.

Diverse injury responses of human oligodendrocyte to mediators implicated in multiple sclerosis.

Early multiple sclerosis lesions feature relative preservation of oligodendrocyte cell bodies with dying back retraction of their myelinating processes. Cell loss occurs with disease progression. Putative injury mediators include metabolic stress (low glucose/nutrient), pro-inflammatory mediators (interferon γ and tumour necrosis factor α), and excitotoxins (glutamate). Our objective was to compare the impact of these disease relevant mediators on the injury responses of human mature oligodendrocytes. In the current study, we determined the effects of these mediators on process extension and survival of human brain derived mature oligodendrocytes in vitro and used bulk RNA sequencing to identify distinct effector mechanisms that underlie the responses. All mediators induced significant process retraction of the oligodendrocytes in dissociated cell culture. Only metabolic stress (low glucose/nutrient) conditions resulted in delayed (4-6 days) non-apoptotic cell death. Metabolic effects were associated with induction of the integrated stress response, which can be protective or contribute to cell injury dependent on its level and duration of activation. Addition of Sephin1, an agonist of the integrated stress response induced process retraction under control conditions and further enhanced retraction under metabolic stress conditions. The antagonist ISRIB restored process outgrowth under stress conditions, and if added to already stressed cells, reduced delayed cell death and prolonged the period in which recovery could occur. Inflammatory cytokine functional effects were associated with activation of multiple signalling pathways (including Jak/Stat-1) that regulate process outgrowth, without integrated stress response induction. Glutamate application produced limited transcriptional changes suggesting a contribution of effects directly on cell processes. Our comparative studies indicate the need to consider both the specific injury mediators and the distinct cellular mechanisms of responses to them by human oligodendrocytes to identify effective neuroprotective therapies for multiple sclerosis.

Halofuginone triggers a transcriptional program centered on ribosome biogenesis and function in honey bees

We previously found that pharmacological inhibition of prolyl-tRNA synthetase by halofuginone has potent activity against Nosema ceranae, an important pathogen of honey bees. However, we also observed that prolyl-tRNA synthetase inhibition is toxic to bees, suggesting further work is necessary to make this a feasible therapeutic strategy. As expected, we found that pharmacological inhibition of prolyl-tRNA synthetase activity resulted in robust induction of select canonical ATF4 target genes in honey bees. However, our understanding of this and other cellular stress responses in general in honey bees is incomplete. Thus, we used RNAseq to identify novel changes in gene expression after halofuginone treatment and observed induction of genes involved in ribosome biogenesis, translation, tRNA synthesis, and ribosome-associated quality control (RQC). These results suggest that halofuginone, potentially acting through the Integrated Stress Response (ISR), promotes a transcriptional response to ribosome functional impairment in honey bees rather than the response designed to oppose amino acid limitation, which has been observed in other organisms after ISR induction. In support of this idea, we found that cycloheximide (CHX) administration also induced all tested target genes, indicating that this gene expression program could be induced by ribosome stalling in addition to tRNA synthetase inhibition. Only a subset of halofuginone-induced genes was upregulated by Unfolded Protein Response (UPR) induction, suggesting that mode of activation and cross-talk with other cellular signaling pathways significantly influence ISR function and cellular response to its activation. Future work will focus on understanding how the apparently divergent transcriptional output of the ISR in honey bees impacts the health and disease of this important pollinator species.

Abstract 2888: Reversible and dose-dependent suppression of motility of human breast cancer cells with ONC201 and/or TR57 in culture

Abstract The cytotoxicity and cytostatic effects of imipridone family drugs ONC201 and its analog TR57 (Madera, USA) in variety of cultured breast cancer cell line (estrogen dependent BT474) was manifested through induction of ISR (Integrated Stress Response), UPRER and UPRMT. The major consequences of drug treatment are inhibition of proliferation and mitochondrial structural and functional damage(s). Our preliminary data demonstrated that 24 h exposure of BT474 leads to dysregulation and suppression in mitochondrial Ca2+ uptake and Ca2+ capacity (Odinokova et al., 2021). In the present work, we extend our study to include the effect of these drugs on the rate of migration intact and treated cells in culture. Treatment of BT474 cells in culture for 24h resulted in decreased motility of cells from 17 ± 2 µm/h in the absence of the drugs to 5 ± 1 µm/h and to 3±1 µm/h in cells treated with 10µM of ONC201 or 50nM of TR57 (n=5). This suppression was fully reversible and removal of drugs for 120h resulted in time dependent restoration of the initial motility of BT474 cells, and TR57 caused larger inhibitory effect on motility as compared with ONC201. Buffering of extracellular Ca2+ with 5 mM EGTA or intracellular Ca2+ with 10 µM BAPTA, 30 min suppressed motility of cells (19 ± 2 µm/h vs 6 ± 1 µm/h). Gd3+, selective blocker of store operated Ca2+ entrance (SOCE) pathway at concentration of 10 µM suppressed the motility of BT474 cells, and rate of migration changed for from 16 ± 2 µm/h in control to 7± 2 µm/h, in Gd3+ treated cells. In line with our preliminary data, taken together these data indicate that ONC201 and/or TR57 induced dysfunction in mitochondrial Ca2+ homeostasis, which translates into the suppression of the migration of human breast cancer cells in culture. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Yana Evstratova, Margarita Kobyakova, Irina Odinokova, Alexander Stolyarov, Artyom Mishukov, Lee M. Graves, Ekhson Holmuhamedov. Reversible and dose-dependent suppression of motility of human breast cancer cells with ONC201 and/or TR57 in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2888.

HGG-42. PEDIATRIC H3K27M MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) SHOWS ROBUST RESPONSE TO IMIPRIDONE BASED COMBINATION THERAPY

ONC201 is a first-in-class small molecule imipridone therapy, which is known to selectively induce apoptosis of cancer cells independent of p53. This novel chemotherapeutic, as well as its analogs ONC206 and ONC212, has been shown to have potent preclinical efficacy against H3K27M mutant diffuse intrinsic pontine glioma (DIPG). We sought to identify synergy between imipridones and other FDA-approved chemotherapeutics. Seven patient-derived DIPG cell lines, six H3.3K27M mutant (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SUDIPG-27, SU-DIPG-29, SF8628), and one H3.1K27M mutant (SU-DIPG-36) were grown in culture and exposed to first and second generation imipridones, both as monotherapies and in combination with histone de-acetylase inhibitors [HDACi], Marizomib, Etoposide, and Temozolomide. A dose dependent response was demonstrated across all cell lines, with increased potency of ONC206 and ONC212 as compared to ONC201, with half maximal inhibitory concentration (IC50) of 0.11 µM, 0.03 µM, and 1.46 µM respectively. Strong synergy is demonstrated between ONC201 and Panobinostat with best combination index (CI) of 0.01. ONC201 similarly shows strong synergy with Romidepsin with best CI of 0.02, and Marizomib with best CI of 0.18. Combination of ONC201 and Etoposide or Entinostat shows some synergy, with best CI of 0.53 and 0.71 respectively. When combined with Temozolomide, some synergy is evident, however, there is overall poor efficacy, with lack of cell death even at the highest doses of Temozolomide. Second generation imipridones show a similar pattern of strong synergy with Panobinostat, Romidepsin, and Marizomib. Immunoblotting showed evidence of apoptosis, as measured by the induction of PARP cleavage, with a combination of imipridones and Panobinostat, as well as induction of integrated stress response with a combination of imipridones and Romidepsin. These results are indicative of promising synergy between imipridones and Panobinostat, Romidepsin, or Marizomib against H3K27M mutant DIPG, combinations which should be considered for future clinical trials.

HIV-induced neuroinflammation inhibits oligodendrocyte maturation via glutamate-dependent activation of the PERK arm of the integrated stress response.

Despite combined antiretroviral therapy (cART), HIV-associated neurocognitive disorder (HAND) affects 30-50% of HIV-positive patients. Importantly, persistent white matter pathologies, specifically corpus callosum thinning and disruption of white matter microstructures observed in patients with HAND despite viral control through cART, raise the possibility that HIV infection in the setting of suboptimal cART may perturb oligodendrocyte (OL) maturation, function and/or survival, influencing HAND persistence in the cART era. To examine the effect of HIV infection on OL maturation, we used supernatants of primary human monocyte-derived macrophages infected with HIV (HIV/MDMs) to treat primary cultures of rat oligodendrocyte precursor cells (OPCs) during their differentiation to mature OLs. Using immunostaining for lineage-specific markers, we found that HIV/MDMs significantly inhibited OPC maturation. Based on our previous studies, we examined the potential role of several signaling pathways, including ionotropic glutamate receptors and the integrated stress response (ISR), and found that AMPA receptors (AMPAR)/kainic acid (KA) receptors (KARs) mediated the HIV/MDMs-induced defect in OL maturation. We also found that the treatment of OPC cultures with glutamate or AMPAR/KAR agonists phenocopied this effect. Blocking ISR activation, specifically the PERK arm of the ISR, protected OPCs from HIV/MDMs-mediated inhibition of OL maturation. Further, while glutamate, AMPA, and KA activated the ISR, inhibition of AMPAR/KAR activation prevented ISR induction in OPCs and rescued OL maturation. Collectively, these data identify glutamate signaling via ISR activation as a potential therapeutic pathway to ameliorate white matter pathologies in HAND and highlight the need for further investigation of their contribution to cognitive impairment.

The Role of the Integrated Stress Response and the Actin Cytoskeleton During Wound Healing

Background and Hypothesis: 
 Chronic cutaneous wounds are a serious health concern afflicting millions of people. One of the primary factors preventing the closure of chronic wounds is the inability of keratinocytes to migrate across the wound bed. Epidermal keratinocytes migrate in a cohesive manner known as the keratinocyte collective cell migration (KCCM). Our lab has demonstrated that the integrated stress response (ISR) plays a key role in the KCCM. The ISR is initiated by stress-sensitive kinases, such as GCN2, and results in decreased global protein synthesis while preferentially increasing the translation of mRNAs encoding cytoprotective proteins. Wound repair also relies on the actin cytoskeleton, but the crosstalk between actin and the ISR is not well established. We hypothesize that the interaction between the ISR and the actin cytoskeleton is critical for KCCM during wound healing. 
 
 Methods: 
 Cutaneous wound healing was approximated in vitro using the KCCM-dependent scratch assay. Wild-type (WT) and GCN2-deleted (KO) keratinocytes were grown on coverslips, differentiated, scratched, and harvested at different times post-wounding. F-actin and vimentin (VIM) expression was monitored over time using fluorescent phalloidin-488 and immunofluorescence. In addition, WT keratinocytes were treated with actin-depolymerizing drugs and induction of ISR was measured using immunoblots. 
 
 Results: 
 Depolymerization of F-actin was observed along the leading edge of both wounded WT and GCN2-KO keratinocytes immediately following wounding. WT keratinocytes upregulated VIM expression at the leading edge whereas VIM expression remained unchanged in the wounded GCN2-KO keratinocytes. Treatment with latrunculin B and cytochalasin D, which both result in actin depolymerization, induced GCN2 phosphorylation in the differentiated WT keratinocytes. 
 
 Conclusion and Potential Impact: 
 F-actin depolymerization elicits a GCN2-mediated induction of the ISR. GCN2 and the ISR are critical components of the cutaneous wound repair process and their crosstalk with the actin cytoskeleton may serve as a novel therapeutic target in the treatment of chronic wounds.

Pharmacodynamic Responses to CC-90009, a Novel Cereblon E3 Ligase Modulator, in a Phase I Dose-Escalation Study in Relapsed or Refractory Acute Myeloid Leukemia (R/R AML)

Pharmacodynamic Responses to CC-90009, a Novel Cereblon E3 Ligase Modulator, in a Phase I Dose-Escalation Study in Relapsed or Refractory Acute Myeloid Leukemia (R/R AML)

EIF2B activator prevents neurological defects caused by a chronic integrated stress response.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.

Adult mouse eIF2B\u03b5 Arg191His astrocytes display a normal integrated stress response in vitro

Vanishing white matter (VWM) is a genetic childhood white matter disorder, characterized by chronic as well as episodic, stress provoked, neurological deterioration. Treatment is unavailable and patients often die within a few years after onset. VWM is caused by recessive mutations in the eukaryotic initiation factor 2B (eIF2B). eIF2B regulates protein synthesis rates in every cell of the body. In normal cells, various types of cellular stress inhibit eIF2B activity and induce the integrated stress response (ISR). We have developed a VWM mouse model homozygous for the pathogenic Arg191His mutation in eIF2Bε (2b5ho), representative of the human disease. Neuropathological examination of VWM patient and mouse brain tissue suggests that astrocytes are primarily affected. We hypothesized that VWM astrocytes are selectively hypersensitive to ISR induction, resulting in a heightened response. We cultured astrocytes from wildtype and VWM mice and investigated the ISR in assays that measure transcriptional induction of stress genes, protein synthesis rates and cell viability. We investigated the effects of short- and long-term stress as well as stress recovery. We detected congruent results amongst the various assays and did not detect a hyperactive ISR in VWM mouse astrocytes.

Abstract P3-06-08: Imipridone compounds inhibit breast cancer mTORC1 signaling through integrated stress response-mediated upregulation of endogenous mTORC1 inhibitor sestrin2

Abstract Breast cancer is a major cause of cancer-related death and there is a need for novel therapies with increased efficacy and decreased toxicity. The small molecule ONC201 was initially identified as a TRAIL pathway inducer. The compound has entered early phase clinical trials and is being tested in a range of solid tumors and hematological malignancies. Our previously published data demonstrate that ONC201 has potent anti-proliferative and pro-apoptotic effects in a broad range of breast cancer subtypes through TRAIL-dependent and TRAIL-independent mechanisms. Analogs of ONC201 with a shared pharmacophore have been developed and form the novel “imipridone” class. When compared with ONC201, imipridones ONC206, ONC212, and ONC213 showed increased potency of anti-proliferative or pro-apoptotic effects in breast cancer cells. We were interested in further defining the previously unstudied anti-proliferative effects of the imipridone compounds. Single agent efficacy of potent imipridone ONC212 in a xenograft model of TNBC was observed in the absence of apoptosis induction. This indicates that the anti-proliferative actions of the compound are sufficient for an in vivo anti-tumor effect. Our lab has previously shown that ONC201 activates an ATF4-dependent integrated stress response (ISR), essential for apoptosis induction in colon cancer cells. In contrast, in breast cancer cells, although ATF4 knockdown did not block cell death induced by ONC201 it did partially abrogate the anti-proliferative effects of the compound. The mammalian target of rapamycin complex 1 (mTORC1) is a well-known regulator of cellular growth and proliferation. mTORC1 signaling is inactivated in breast cancer cells following treatment with ONC201 and its analogs ONC212 and ONC213, regardless of whether the cells undergo apoptosis. Knockdown of ATF4 abrogated ONC201-mediated inhibition of p70 S6 kinase and ribosomal protein S6 phosphorylation, linking ISR induction to mTORC1 inhibition. We hypothesized that sestrin2, an endogenous mTORC1 inhibitor known to be upregulated following cellular stress, might represent a link between induction of ATF4 and inhibition of mTORC1. Treatment with ONC201 and its analogs ONC212 and ONC213 in multiple breast cancer cell lines resulted in sestrin2 upregulation. This was blocked by ATF4 knockdown. Furthermore, knockdown of sestrin2 abrogated the effects of ONC201 on mTORC1 signaling in breast cancers from multiple molecular subtypes. Previous mechanistic studies have focused exclusively on the relevance of ATF4 in the pro-apoptotic effects of ONC201. The novel findings described here help to elucidate the mechanism behind the potent and understudied anti-proliferative effects of the imipridones. Our findings also strengthen the preclinical rationale for testing of imipridone compounds against breast cancers regardless of molecular subtype. Citation Format: Ralff MD, Kline CLB, El-Deiry WS. Imipridone compounds inhibit breast cancer mTORC1 signaling through integrated stress response-mediated upregulation of endogenous mTORC1 inhibitor sestrin2 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-08.

Pharmacological eEF 2K activation promotes cell death and inhibits cancer progression

Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV-PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti-viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir-resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir-mediated anti-tumoral activity is severely compromised in eEF2K-deficient engrafted tumors in vivo Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti-tumoral properties of HIV-PIs.

Abstract 674: Early integrated stress response induction of ATF4 is required for the anticancer effects of the dual Akt/ERK inhibitor and TRAIL pathway inducer ONC201/TIC10

Abstract ONC201/TIC10 is a first-in-class small molecule inducer of the tumor necrosis apoptosis-inducing ligand (TRAIL) pathway, inhibiting both Akt and ERK and consequently activating FOXO3a. Here we present a hitherto unrecognized and early mechanism that contributes to ONC201/TIC10's cytotoxic effects. ONC201/TIC10 activates the integrated stress response, thereby engaging the ATF4 pathway and consequently inducing expression of the pro-apoptotic proteins CHOP and DR5. Cells that are resistant to ONC201/TIC10 do not exhibit ATF4 pathway activation. Knocking down ATF4 and CHOP partially abrogates ONC201/TIC10's cytotoxicity and ONC201/TIC10-induced DR5 upregulation. We also report here that ONC201/TIC10 does not only cause apoptosis but also inhibits cell cycle progression, potentially explaining its broad-spectrum cytotoxicity against 22 out of the 23 tumor cell lines tested. ONC201/TIC10-induced cell cycle arrest was associated with downregulation of Cyclin D1 expression. ONC201/TIC10 induces CHOP and DR5 in cells regardless of whether cells are susceptible to ONC201/TIC10-induced apoptosis. Levels of the X-linked inhibitor of apoptosis (XIAP) influence whether a cell succumbs to proliferation inhibition or apoptosis in response to ONC201/TIC10. Our findings show that ONC201/TIC10 upregulates the TRAIL pathway not only by inhibiting Akt and ERK, and activating FOXO3a but also by engaging the ATF4/CHOP/DR5 pathway that leads to complementary and potent tumor cell killing. Our findings also suggest that CHOP expression may serve as a pharmacodynamic marker for ONC201/TIC10 activity. Citation Format: Christina Leah Kline, Pieter van den Heuvel, Joshua E. Allen, David T. Dicker, Wafik S. El-Deiry. Early integrated stress response induction of ATF4 is required for the anticancer effects of the dual Akt/ERK inhibitor and TRAIL pathway inducer ONC201/TIC10. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 674. doi:10.1158/1538-7445.AM2015-674