Integrated Stress Response Kinases Research Articles

Cytotoxic stress caused by azalamellarin D (AzaD) interferes with cellular protein translation by targeting the nutrient-sensing kinase mTOR.

Analogs of pyrrole alkaloid lamellarins exhibit anticancer activity by modulating multiple cellular events. Lethal doses of several lamellarins were found to enhance autophagy flux in HeLa cells, suggesting that lamellarins may modulate protein homeostasis through the interference of proteins or kinases controlling energy and nutrient metabolism. To further delineate molecular mechanisms and their targets, our results herein show that azalamellarin D (AzaD) cytotoxicity could cause translational attenuation, as indicated by a change in eIF2α phosphorylation. Intriguingly, acute AzaD treatment promoted the phosphorylation of GCN2, a kinase that transduces the integrated stress response (ISR), and prolonged exposure to AzaD could increase the levels of the phosphorylated forms of eIF2α and the other ISR kinase PKR. However, the effects of AzaD on ISR signaling were marginally abrogated in cells with genetic deletion of GCN2 and PKR, and evaluation of protein target engagement by CETSA revealed no significant interaction between AzaD and ISR kinases. Further investigation revealed that acute AzaD treatment negatively affected mTOR phosphorylation and signaling. The analyses by CETSA and computational modeling indicated that mTOR may be a possible protein target for AzaD. These findings indicate the potential for developing lamellarins as novel agents for cancer treatment.

Abstract 1233: Novel inhibitor targeting triple integrated stress response kinase HRI, PERK, and GCN2 provides new insights into overcoming resistance to proteasome inhibitors in multiple myeloma

Abstract The introduction of novel treatments for Multiple myeloma (MM), particularly proteasome inhibitors (PI) such as bortezomib (BTZ), has greatly improved outcomes. However, many patients eventually experience relapse. Therefore, new approaches for managing patients with resistance to PI should be explored. The integrated stress response (ISR) is an evolutionarily conserved intracellular signaling network that is activated in response to both intrinsic and extrinsic stresses. Stresses are sensed by four specialized kinases: HRI, PERK, PKR, and GCN2, which converge on the phosphorylation of eIF2α. This phosphorylation triggers the translation of ATF4, thus promoting cell survival and homeostasis. Recent studies investigating the contribution of the four ISR kinases in overcoming resistance to PI treatment, have revealed the potential of a selective GCN2 inhibitor to overcome this resistance. Here, we observed that PI simultaneously induced mitochondrial stress, ER stress, and amino acid starvation, leading to the activation of HRI, PERK, and GCN2, respectively. However, the contribution of crosstalk among ISR kinases has not been considered thus far. To gain a comprehensive understanding of ISR kinases in BTZ resistance, we generated single or combination patterns of double and triple KO of the four ISR kinases and reevaluated their contribution to BTZ treatment. Sensitivity screening using the HAP1 single, double, and triple KO cell library showed that HRI primarily, and to a lesser extent, PERK and GCN2, contribute to BTZ resistance. Consistent with the HAP1 KO screening, MM cell line-derived KO cells revealed a similar dependency on these three kinases. Building upon our observation of the significance of the HRI/PERK/GCN2 triple kinase, we developed a novel and orally available small molecule, CRD-1367799, which inhibits these triple kinases in cells. CRD-1367799 effectively dampened the activity of these ISR kinases, leading to a complete inhibition of eIF2α phosphorylation and ATF4 activation. Additionally, we observed a distinct anti-growth effect when combined with BTZ in vitro and in vivo, which was not replicated by the GCN2 or PERK-selective inhibitor. Furthermore, we uncovered the mechanism of cell death induction in combination with proteasome inhibitors. Notably, the apoptosis inducers NOXA and CHOP were upregulated, but the mechanism did not seem to rely on previously known processes such as ATF4-mediated transcriptional induction. In summary, our novel findings on the contribution of the triple ISR kinase provides us with new insights for overcoming resistance to proteasome inhibitors with HRI/PERK/GCN2 inhibitor. To the best of our knowledge, the use of a triple kinase inhibitor in this context has not been reported thus far, making CRD-1367799 a new therapeutic strategy for MM with PI. Citation Format: Yasumichi Inoue, Koki Hikami, Shunsuke Ebara, Midori Sugiyama, Robert Oda, Akio Mizutani, Misaki Yoshida, Hiroko Yamakawa, Shigehiro Yagishita, Akinobu Hamada, Hidetoshi Hayashi, Masaki Ri, Shinsuke Iida, Daisuke Morishita. Novel inhibitor targeting triple integrated stress response kinase HRI, PERK, and GCN2 provides new insights into overcoming resistance to proteasome inhibitors in multiple myeloma [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 1233.

Abstract 1873: AST-05X: A novel GCN2 inhibitor as a promising anti-cancer approach for sarcoma

Abstract Background Integrated stress response (ISR) is an adaptive signaling pathway that responds to stress and plays an important role in cell fate determination. This pathway enables cellular adaptation and survival under stress conditions by activating specific genes that reduce protein synthesis. Tumors exist in a state characterized by heightened intrinsic and extrinsic stress, heavily relying on a balanced ISR to manage the metabolic demands for rapid growth. ISR (Integrated Stress Response) consists of four major kinases: GCN2, PERK, HRI, and PKR. GCN2 is activated by stress due to amino acid deprivation. Amino acids are crucial for cell survival, and cancer cells, require higher amounts than normal cells, particularly depending on conditionally essential amino acids like glutamine, asparagine, and arginine. GCN2 activation acts as a key mechanism for cancer cells to overcome amino acid deficiencies, making GCN2 a potential target, especially in conjunction with anticancer drugs like asparaginase. In this study, we aimed to identify a potential GCN2 inhibitor to explore a promising anti-cancer approach in sarcoma, a rare cancer with no new therapeutic alternatives. Method The activity verification of ISR kinases was characterized using in-vitro enzyme assays. In-vitro panel assays were employed to determine the selectivity profiling for oncology kinases. The modulation of the ISR pathway was validated using a sarcoma cell line (HT-1080) and a cell line derived from sarcoma patients. Western blot or ELISA assays were employed to measure phospho-GCN2, ATF4, phospho-eIF2a, and CHOP under both basal conditions and amino acid starvation conditions. Gene expression profiling (GEP) of sarcoma patient tissue samples(n=17) was conducted through RNA sequencing (RNAseq) analysis. Results AST-05X is a selective and potent GCN2 inhibitor. AST-05X inhibits the expression of GCN2 and ISR downstream factors (p-eIF2a, ATF, CHOP) in vitro cell-based experiments under amino acid deprivation. In addition, based on the results of ISR-related gene expression profiling GEP analysis, we identified potential CDx biomarkers for evaluating the diagnosis and treatment response of sarcoma. Conclusion This study is currently investigating the efficacy of the drug in sarcoma patient-derived xenograft models. AST-05X is a GCN2 inhibitor that has the potential to validate the inhibition of GCN2 as a promising anti-cancer approach for sarcoma, a rare cancer with limited therapeutic alternatives. Citation Format: Youngki Choi, Jinback Lim, Hyo-Hyun Park, Jinho Kang, Hyosong Kim, Kumhee Yun, Hun Jung. AST-05X: A novel GCN2 inhibitor as a promising anti-cancer approach for sarcoma [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 1873.

Activation of GCN2 By HC-7366 Results in Significant Anti-Tumor Efficacy As Monotherapy and Overcomes Resistance Mechanisms When Combined with Venetoclax in AML

Venetoclax (anti-BCL2) is approved for elderly patients with acute myeloid leukemia (AML) in combination with hypomethylating agents such as azacitidine or decitabine. However, several resistance mechanisms arise, impeding responses in patients. These resistance mechanisms include TP53 or FLT3-ITD mutations, suppression of pro-apoptotic proteins NOXA or PUMA, increases in oxidative phosphorylation and energy metabolism, or elevated expression of S100A8/A9. Recent reports have highlighted that hyperactivation of the integrated stress response (ISR) pathway overcomes venetoclax resistance through NOXA activation and glycolytic activity inhibition. However, these studies used drugs that indirectly activate the ISR with other off-target effects. General Control Nonderepressible 2 (GCN2) is an ISR kinase that senses and responds to nutrient stress conditions, and activation of GCN2 results in antitumor activity. We are developing HC-7366, a first-in-class, first-in-human direct GCN2 activator, and are currently evaluating this agent in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in AML, and how it impacts venetoclax resistance. AML patients with TP53 mutations are known to have lower response rates to venetoclax.The in vivo efficacy of HC-7366 in both CDX and PDX models was independent of TP53 mutation, resulting in 100% complete response and 100% tumor growth inhibition in TP53-mutated MOLM-16 and KG-1 tumor models, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of the ISR as evidenced by increased expression of the ATF4 targets ASNS and PSAT1. A genome-wide CRISPR screen in KG-1 cells revealed that GCN2 sgRNAs were significantly enriched in HC-7366-treated cells, highlighting the importance of GCN2 activation in mediating the mechanism of action of HC-7366. Interestingly, BCL2 sgRNAs were significantly depleted in HC-7366 treated cells, suggesting a synthetic lethal relationship with BCL2 inhibitors, such as venetoclax, when combined with HC-7366. Indeed, in the MV4-11 FLT3-ITD mutant model (a differentiated subtype of AML that shows limited response to venetoclax), the combination of HC-7366 and venetoclax produced substantial benefit resulting in 26% tumor regression and enhanced activation of the ISR pathway. HC-7366 treatment also increased mRNA and protein levels of NOXA and PUMA while reducing mitochondrial respiration and glycolysis in a GCN2-dependent manner, leading to a low energetic state. Consistent with its impact on cellular energetics, global metabolomic analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with glycolysis and the TCA cycle. Additionally, HC-7366 reduced protein levels of S100A8/A9, also known to correlate with venetoclax resistance. Our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax. The potential of HC-7366 to counteract multiple known resistance mechanisms to venetoclax could provide a viable treatment option for patients with relapsed/refractory AML when used in combination with venetoclax.

Abstract A30: Activation of GCN2 by HC-7366 results in significant anti-tumor efficacy as monotherapy and in combination with Venetoclax in AML models

Abstract The integrated stress response (ISR) is an adaptive signaling pathway that cells utilize to respond to a wide range of extrinsic and intrinsic stresses, many of which are important for tumorigenesis. Activation of ISR plays a dual role in cell fate decisions; while the ISR promotes survival, prolonged activation of ISR induces apoptosis. Activation of General Control Nonderepressible 2 (GCN2), an ISR kinase that senses and responds to nutrient stress conditions results in anti-tumor activity. We are developing HC-7366, a first-in-class, first-in-human GCN2 activator, and are currently evaluating it in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in acute myeloid leukemia (AML). Higher expression of GCN2 and ISR markers such as ATF4 has been observed in primitive or minimally differentiated AML cells, suggesting that AML may be particularly sensitive to HC-7366. Encouragingly, in vivo efficacy studies in MOLM-16 and KG-1 tumor models showed 100% complete response and 100% tumor growth inhibition, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of ISR as evidenced by increased expression of the ATF4 targets, ASNS and PSAT1, confirming that HC-7366 is functioning as a GCN2 activator in vivo. In the MV4-11 model, a differentiated subtype of AML that shows limited response to venetoclax, the combination of HC-7366 and venetoclax produced strong benefit resulting in 26% tumor regression. Enhanced activation of ISR pathway was again observed when HC-7366 was combined with venetoclax. HC-7366 treatment also impacted possible venetoclax resistance mechanisms by increasing the levels of PUMA while reducing levels of S100A8/A9 proteins. To investigate the effects of the compound on primary AML, we performed an ex vivo screen using cells from AML patients and treatment with HC-7366 showed a remarkable decrease in cell proliferation. Furthermore, in a xenotransplantable model of patient-derived AML, we found that HC-7366 significantly reduced mature myeloid (CD33+) AML cells in the bone marrow as compared to standard of care (SOC) agents, including venetoclax. We confirmed that activation of ISR, reduction in cell viability, and inhibition of protein synthesis following treatment with HC-7366 was dependent on GCN2 using CRISPR knockout cells. In addition, HC-7366 reduced mitochondrial respiration in MOLM-16 cells, suggesting effects on cellular bioenergetics. Metabolomics analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with amino acid metabolism, urea cycle, and oxidative stress. Together, our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax, supporting its investigation in clinical trials in patients with AML. Citation Format: Feven Tameire, Paulina M. Wojnarowicz, Sho Fujisawa, Sharon Huang, Owen Reilly, Crissy Dudgeon, Nicholas Collette, Jeremy Drees, Kathryn Bieging-Rolett, Takashi O Kangas, Weiyu Zhang, Maria Fumagalli, Iman Dewji, Yunfang Li, Anissa SH Chan, Xiaohong Qiu, Ben Harrison, Ashley LaCayo, Kirk A. Staschke, Alan C. Rigby, Savithri Ramurthy, Eric S. Lightcap, David Surguladze, Nandita Bose. Activation of GCN2 by HC-7366 results in significant anti-tumor efficacy as monotherapy and in combination with Venetoclax in AML models [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A30.

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 1640: DP-9024, an investigational small molecule modulator of the Integrated Stress Response kinase PERK, causes B-cell cancer growth inhibition as single agent and in combination with standard-of-care agents

Abstract Background: The Integrated Stress Response (ISR) is a major adaptive stress response pathway in cancer cell maintenance. The ISR kinase family member PERK controls one of the three arms of the Unfolded Protein Response (UPR). The UPR is considered an Achilles’ heel in B-cell cancers. Multiple myeloma (MM) and B-cell lymphomas are dependent on a well-balanced UPR pathway to cope with the high demand for protein folding and their secretory nature. Given the double-edge sword nature of the UPR, the activation of PERK and downstream pathway can have cytoprotective or cytotoxic effects. In B-cell cancers the UPR is at close to maximum cytoprotective capacity, such that further pharmacological stimulation of PERK can potentially be leveraged to cause a cancer cell cytotoxic response and induce antitumoral effects. Methods: Modulation of ISR kinases was characterized using enzymatic assays. Kinome selectivity profiling was determined using enzymatic and cellular assays. Cellular assays of PERK activation assessed ATF4 by ELISA. Cellular assays of GCN2 modulation assessed phospho-GCN2 and ATF4 by Western blot or ELISA (under basal or low amino acid conditions). DP-9024-induced upregulation of components of the ISR/UPR pathway (ATF4, CHOP) or the apoptosis pathway (PARP and Caspase 3/7) was measured by Western blot or ELISA assays. Compound-mediated PERK activation was investigated mechanistically using a cellular nanoBRET dimerization assay. In vivo upregulation of tumoral ATF4 was determined in a MM PK/PD xenograft model. In vivo inhibition of tumor growth was determined in MM and B-cell lymphoma xenografts. Results: DP-9024 was designed as a selective and potent modulator of PERK and GCN2. DP-9024 was found to upregulate the ISR/UPR pathway (ATF4, CHOP). The mechanism by which DP-9024 treatment induced the UPR pathway was found to be through the dimerization and activation of PERK. Upregulation of the UPR pathway downstream of PERK led to induction of apoptosis (PARP and Caspase 3/7) in MM and B-cell lymphoma lines in vitro. DP-9024 mediated activation of the UPR pathway in cell lines with high basal level of endoplasmic reticulum (ER) stress led to growth arrest in combination with FDA approved therapies. Oral dosing of DP-9024 in MM xenograft models induced ATF4, and combination efficacy was observed in MM and B-cell lymphoma xenografts in combination with FDA approved agents in vivo. Conclusions: The ISR/UPR is a targetable vulnerability in cancers with high basal levels of ER stress. DP-9024 increases UPR signaling via activating PERK dimerization. This novel mechanism leads to antitumoral effects in B-cell cancers in vitro and in vivo likely through the induction of unresolved ER stress, which may potentially provide an alternative mechanism to current UPR targeting therapies. Citation Format: Gada Al-Ani, Qi Groer, Aaron J. Rudeen, Kristin M. Elliott, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javed, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DP-9024, an investigational small molecule modulator of the Integrated Stress Response kinase PERK, causes B-cell cancer growth inhibition as single agent and in combination with standard-of-care agents [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 1640.

Abstract 1639: DP-9149, an investigational small molecule modulator of the Integrated Stress Response kinase GCN2, pre-clinically causes solid tumor growth inhibition as a single agent and regression in combination with standard of care agents

Abstract Background: The Integrated Stress Response (ISR) is one of the major adaptive stress response pathways in cancer and plays an important role in cell fate determination. Oncogene addicted solid tumors are under high stress levels, both extrinsic as well as intrinsic, and are dependent on a well-balanced ISR pathway activity to cope with the high demand for accelerated growth. The ISR is well known to be a double edge sword of survival and cell death and depending on context, the activation of the ISR kinase, GCN2, and downstream pathway can have either cytoprotective or cytotoxic effects. Given the context-dependent nature of the ISR pathway, the inhibition or stimulation of GCN2 in solid tumors can be pharmacologically leveraged to induce anti-tumoral effects. Methods: Modulation of ISR kinases was characterized using enzymatic assays. Kinome selectivity profiling was determined using enzymatic and cellular assays. Cellular modulation of the ISR pathway (phospho-GCN2, ATF4, CHOP) or the apoptosis pathway (PARP and Caspase3/7) was assessed via Western blot or ELISA. In vivo upregulation of tumoral ATF4 was determined in a fibrosarcoma PK/PD xenograft model. In vivo inhibition of tumor growth was determined in solid tumor xenografts. Results: Selective and potent modulators of GCN2 kinase with favorable drug-like properties were designed. These compounds were found to upregulate components of the ISR pathway (phospho-GCN2, ATF4, CHOP). The mechanism by which GCN2 modulator DP-9149 treatment induced the ISR pathway was found to be through the direct binding and activation of GCN2. Upregulation of the ISR pathway downstream of GCN2 led to induction of a programmed cell death pathway in oncogene-driven solid tumor cell lines in vitro. DP-9149-mediated activation of the ISR pathway led to cell growth arrest both as a single agent and in combination with standard-of-care (SOC) agents. Furthermore, oral dosing of DP-9149 in RAS mutant and other oncogene-driven xenograft models in vivo induced ATF4, and significantly inhibited tumor growth as a single agent and in combination with SOC agents. Additionally, therapeutic agents targeting the tumor microenvironment, including anti-angiogenic agents, synergized with DP-9149 to induce tumor regressions in vivo. Conclusions: The ISR is a targetable vulnerability in oncogene addicted solid tumors. Upregulating the ISR by paradoxical activation of the ISR family member kinase, GCN2, by DP-9149 can be leveraged as a novel mechanism to cause anti-tumoral effects in solid tumors in vitro and in vivo, likely through the induction of an unresolved stress response. In particular, DP-9149 exhibited robust activity in RAS mutant cancers and in VHL-mutant renal cancers as a single agent and in combination with SOC agents in vivo. Citation Format: Gada Al-Ani, Qi Groer, Kristin M. Elliott, Aaron J. Rudeen, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javid, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DP-9149, an investigational small molecule modulator of the Integrated Stress Response kinase GCN2, pre-clinically causes solid tumor growth inhibition as a single agent and regression in combination with standard of care agents [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 1639.

The Role of the Integrated Stress Response in the Biologic Response of Keratinocytes to UVB Exposure

Background and Hypothesis:Ultraviolet B (UVB) irradiation from the sun can disrupt normal epidermal keratinocyte function, leading to the development of skin cancer. The integrated stress response (ISR) is a conserved cellular mechanism against environmental stressors, such as UV irradiation, through the phosphorylation of the alpha subunit of the initiation factor eIF2 (eIF2a-P). Previously, we demonstrated that the ISR kinase GCN2 plays a role in the response of keratinocytes exposed to UV irradiation. However, the role of other components of the ISR in UV-induced stress has not been well elucidated. We hypothesize that in addition to GCN2, other components of ISR such as PERK, GCN1, and ZAK1 are involved in apoptotic pathways induced by prolonged UVB exposure. Methods:Gene-specific knockouts (KO) were generated using CRISPR/Cas9 and validated through immunoblotting. Wild-type (WT) and KO NTERT keratinocytes were irradiated with UVB at 0, 100, 200, and 400J/m2. Six hours post-UVB, cell photomicrographs were taken at 0 and 400J/m2. Measurements for apoptosis were then performed by measuring enzymatic levels of caspase-3. Results:Measurements of caspase-3 following UVB exposure on WT NTERT, PERK-KO, and GCN2-KO showed no difference between the groups. GCN1-KO had significantly greater caspase-3 activity at 400J/m2, whereas ZAK1-KO had significantly reduced caspase-3 activity at the same concentration. GCN2/PERK double-KO had significantly increased caspase-3 activity at 400 J/m2 compared to the individual KO cells. Conclusion and Potential Impact:Collectively, our data suggests that the ISR may be involved in the apoptotic responses that ensue in keratinocytes exposed to UVB. How GCN2 and PERK could co-interact with GCN1 or ZAK1 to facilitate apoptotic fate in keratinocytes still warrants further research. Nevertheless, elucidating the role of the ISR in normal keratinocyte response to UV irradiation will allow us to define novel therapeutic targets for the treatment or prevention of UV-related skin malignancies.

Abstract 3040: The eIF2 kinase GCN2 controls expression of key amino acid transporters and is critical for prostate cancer growth and progression

Abstract The Integrated Stress Response (ISR) is central for adaptation and survival of tumor cells in response to exogenous and endogenous stresses. The ISR features a family of protein kinases that phosphorylate the eukaryotic translation initiation factor eIF2 in response to distinct stresses. While phosphoryation of eIF2 reduces global protein synthesis, there is paradoxical induced translation of select stress adapative gene transcripts, including ATF4, which is central for ISR-directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer adaptation to stress. Recently, eIF2 phosphorylation and ATF4 were suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinase(s), its mode of activation, and mechanisms by which the ISR facilitate PCa progression are not known.We discovered that the eIF2 kinase GCN2 (EIF2AK4) is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduced growth in androgen-sensitive and castration-resistant PCa cell lines in culture and cell line-derived and patient-derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2. A transcriptome analysis of PCa cells treated with a specific small molecular inhibitor of GCN2 indicates that this eIF2 kinase was critical for expression of multiple SLC genes, including amino acid transporters previously implicated in different cancers. We determined that GCN2 inhibition decreases intracellular amino acid levels, and reduced aminoacylation of select tRNAs, accounting for reduced growth in PCa cells. Using CRISPR-based phenotypic screens and genome-wide gene expression analyses of control and GCN2-depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is a key SLC gene induced by GCN2 and is essential for PCa proliferation. SLC3A2 has been reported to associate with many SLC7 family transporters, allowing for the localization of these transporters to the plasma membrane for amino acid uptake. Of importance, expression of SLC3A2 partially restored amino acid levels and growth due to loss of GCN2. Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including SLC3A2, which provides for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising new therapeutic targets for the treatment of PCa. Citation Format: Ricardo Cordova, Jagannath Misra, Nur P. Damayanti, Parth H. Amin, Kenneth R. Carlson, Angela J. Klunk, Emily T. Mirek, Marcus J. Miller, Tracy G. Anthony, Roberto Pili, Ronald C. Wek, Kirk A. Staschke. The eIF2 kinase GCN2 controls expression of key amino acid transporters and is critical for prostate cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3040.

Targeting GCN2 Regulation of Amino Acid Homeostasis in Prostate Cancer

The Integrated Stress Response (ISR) plays a critical role in the adaptation and survival of tumor cells to exogenous and endogenous stresses. The ISR features four protein kinases (PERK, GCN2, PKR, and HRI), each activated by different stresses, that phosphorylate the eukaryotic translation initiation factor eIF2, resulting in repression of global protein synthesis. Paradoxically, eIF2 phosphorylation also enhances translation of select gene transcripts, including the transcription factor ATF4, which is central for ISR‐directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer stress adaptation. Moreover, eIF2 phosphorylation and ATF4 have recently been suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinases, their mode of activation, and the mechanisms by which the ISR facilitate PCa progression are unknown.We discovered that GCN2 is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduces growth in androgen‐sensitive and castration‐resistant PCa cell lines in culture and cell line‐derived and patient‐derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2.A transcriptome analysis of PCa cells treated with a specific GCN2 small molecular inhibitor indicates that GCN2 is critical for expression of SLCgenes involved in metabolite transport. We found that GCN2 inhibition decreases intracellular amino acid levels accounting for reduced growth in PCa cells. Using CRISPR‐based phenotypic screens and genome‐wide gene expression analyses of wild‐type and GCN2‐depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is induced by GCN2 and is essential for PCa proliferation. SLC3A2 engages with many nutrient transporters, allowing for their localization to the plasma membrane. Importantly, expression of SLC3A2 reduced GCN2 activation and rescued decreased amino acid levels and growth inhibition due to loss of GCN2.Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including 4F2 (SLC3A2), which provide for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising therapeutic targets for both androgen‐sensitive and castration‐resistant prostate cancer.

The Integrated Stress Response and Phosphorylated Eukaryotic Initiation Factor 2α in Neurodegeneration.

The proposed molecular mechanisms underlying neurodegenerative pathogenesis are varied, precluding the development of effective therapies for these increasingly prevalent disorders. One of the most consistent observations across neurodegenerative diseases is the phosphorylation of eukaryotic initiation factor 2α (eIF2α). eIF2α is a translation initiation factor, involved in cap-dependent protein translation, which when phosphorylated causes global translation attenuation. eIF2α phosphorylation is mediated by 4 kinases, which, together with their downstream signaling cascades, constitute the integrated stress response (ISR). While the ISR is activated by stresses commonly observed in neurodegeneration, such as oxidative stress, endoplasmic reticulum stress, and inflammation, it is a canonically adaptive signaling cascade. However, chronic activation of the ISR can contribute to neurodegenerative phenotypes such as neuronal death, memory impairments, and protein aggregation via apoptotic induction and other maladaptive outcomes downstream of phospho-eIF2α-mediated translation inhibition, including neuroinflammation and altered amyloidogenic processing, plausibly in a feed-forward manner. This review examines evidence that dysregulated eIF2a phosphorylation acts as a driver of neurodegeneration, including a survey of observations of ISR signaling in human disease, inspection of the overlap between ISR signaling and neurodegenerative phenomenon, and assessment of recent encouraging findings ameliorating neurodegeneration using developing pharmacological agents which target the ISR. In doing so, gaps in the field, including crosstalk of the ISR kinases and consideration of ISR signaling in nonneuronal central nervous system cell types, are highlighted.

Abstract 2385: Evaluation of GCN2 chemical probes: Activity in leukemia

Abstract Cellular stress signals activate adaptive signaling pathways of the mammalian integrated stress response (ISR), which converge at the phosporylation of eIF2α. Drug-like chemical inhibitors targeting major ISR kinases have been identified, with the exception of GCN2. We synthesized and evaluated a series of GCN2 inhibitors based on a triazolo[4,5-d]pyrimidine scaffold. Several compounds potently inhibited GCN2 in vitro in orthogonal assays and displayed good selectivity over the related kinases PERK, HRI, and IRE1. The compounds had low solubility at pH 7.4, but sufficient permeability to inhibit phosporylation of eIF2α in HEK293T cells with an IC50 < 150 nM. In a screen of the NCI-60 cancer cell line panel, the leukemia cells were the only group of cancer cell lines to uniformly exhibit growth inhibition. Given the previously reported genetic association of Gcn2 with resistance to asparaginase, which is a first line clinical leukemia therapy, pharmacologic inhibition of Gcn2 may overcome resistance to asparaginase treatment in leukemia. Citation Format: Lea Lough, Dan Sherman, Eric Ni, Palaniraja Thandapani, Ioannis Aifantis, Timothy Cardozo. Evaluation of GCN2 chemical probes: Activity in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2385.

Triazolo[4,5-d]pyrimidines as Validated General Control Nonderepressible 2 (GCN2) Protein Kinase Inhibitors Reduce Growth of Leukemia Cells

Cellular stress signals activate adaptive signaling pathways of the mammalian integrated stress response (ISR), of which the unfolded protein response (UPR) is a subset. These pathways converge at the phosporylation of eIF2α. Drug-like, potent and selective chemical inhibitors (valid chemical probes) targeting major ISR kinases have been previously identified, with the exception of GCN2. We synthesized and evaluated a series of GCN2 inhibitors based on a triazolo[4,5-d]pyrimidine scaffold. Several compounds potently inhibited GCN2 in vitro and displayed good selectivity over the related kinases PERK, HRI, and IRE1. The compounds inhibited phosporylation of eIF2α in HEK293T cells with an IC50 < 150 nM, validating them as chemical probes for cellular studies. These probes were screened against the National Cancer Institute NCI-60 human cancer cell line panel. Uniform growth inhibition was observed in the leukemia group of cell lines. Growth inhibition in the most sensitive cell lines coincided with high GCN2 mRNA expression levels. Oncomine analysis revealed high GCN2 expression accompanied by lower asparagine synthetase (ASNS) expression in patient-derived acute lymphoblastic leukemias with B-Cell origins (B-ALL) as well. Notably, asparaginase, which depletes amino acids and triggers GCN2 activity, is a licensed, first-line B-ALL treatment. Thus, we hypothesize that leukemias exhibiting high GCN2 expression and low ASNS expression may be susceptible to pharmacologic GCN2 inhibition.

Abstract 230: The role of integrated stress response (ISR) transcription factor ATF4 in metabolic adaptation of tumors to c-Myc activation

Abstract The ability of cancer cells to adapt to non-cell autonomous (extrinsic) and cell autonomous (intrinsic) stresses is critical for maintaining cell viability and growth. The Integrated Stress Response (ISR) pathway is essential in cancer cell adaptation to extrinsic stresses such as hypoxia and nutrient deprivation both in vitro and in vivo. The ISR kinases, PERK and GCN2, phosphorylate eIF2α, transiently inhibiting protein translation during times of stress. Phosphorylation of eIF2α promotes the preferential translation of the transcription factor ATF4, which activates a gene expression program that enhances uptake and synthesis of amino acids, antioxidant defense and chaperone expression to promote recovery from nutrient deprivation and ER stress. Here, we demonstrate the importance of ISR signaling in MYC oncogene induced intrinsic stress both in vitro and in vivo. We previously reported that activation of PERK, during c-Myc induction is vital for activating cytoprotective autophagy and suppressing c-Myc induced apoptosis. Here, we demonstrate GCN2 is also robustly activated during c-Myc induction. GCN2 activation after c-Myc induction regulates ATF4 transcription and translation. ATF4 was necessary for survival and suppression of oxidative stress during c-Myc activation. We have identified ATF4 targets that regulate amino acid uptake and involved in amelioration of oxidative stress. Furthermore, siRNA mediated knockdown or genetic knockout of GCN2 was synthetic lethal with c-Myc activation. Interestingly, PERK is still able to phosphorylate eIF2α in the absence of GCN2. However, eIF2α phosphorylation was completely inhibited in the absence of both kinases suggesting that these kinases compensate for each other and both are required for optimal phosphorylation of eIF2α after c-Myc activation. Consistent with our in vitro data, we observe higher levels of p-eIF2α and ATF4 in lymphoma cells compared to WT B cells in vivo in a mouse model of Myc driven lymphoma. These findings highlight a novel mechanism of stress response pathway activation to support c-Myc driven tumorigenesis involving an amino acid sensor kinase GCN2. Citation Format: Feven Tameire, Maria A. Monroy, Constantinos Koumenis. The role of integrated stress response (ISR) transcription factor ATF4 in metabolic adaptation of tumors to c-Myc activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 230.

Novel dengue virus inhibitor 4-HPR activates ATF4 independent of protein kinase R–like Endoplasmic Reticulum Kinase and elevates levels of eIF2α phosphorylation in virus infected cells

Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N-(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection (Fraser et al., 2014b). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R–like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2α in DENV infection using guanabenz, a specific inhibitor of eIF2α dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic.

Signaling through alternative Integrated Stress Response pathways compensates for GCN2 loss in a mouse model of soft tissue sarcoma.

The tumor microenvironment is characterized by deficiencies in oxygen and nutrients, such as glucose and amino acids. Activation of the GCN2 arm of the Integrated Stress Response (ISR) in response to amino acid deprivation is one mechanism by which tumor cells cope with nutrient stress. GCN2 phosphorylates the alpha subunit of the eukaryotic translation initiation factor eIF2, leading to global downregulation of translation to conserve amino acids and initiation of a transcriptional program through ATF4 to promote recovery from nutrient deprivation. Loss of GCN2 results in decreased tumor cell survival in vitro under amino acid deprivation and attenuated tumor growth in xenograft tumor models. However, it is not known what effects GCN2 loss has on the growth of autochthonous tumors that arise in their native microenvironment. Here, we demonstrate in a genetically engineered mouse model of soft tissue sarcoma that loss of GCN2 has no effect on tumor growth or animal survival. The sarcomas displayed compensatory activation of PERK or phospho-eIF2α independent upregulation of ATF4 in order to maintain ISR signaling, indicating that this pathway is critical for tumorigenesis. These results have important implications for the development and testing of small molecule inhibitors of ISR kinases as cancer therapeutics.

Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. We have developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2alpha phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest.