Activation Of Integrated Stress Response Research Articles

Inability to rescue stalled ribosomes results in overactivation of the integrated stress response

Endogenous and exogenous chemical agents are known to compromise the integrity of RNA and cause ribosome stalling and collisions. Recent studies have shown that collided ribosomes serve as sensors for multiple processes, including ribosome quality control (RQC) and the integrated stress response (ISR). Since RQC and the ISR have distinct downstream consequences, it is of great importance that organisms activate the appropriate process. We previously showed that RQC is robustly activated in response to collisions and suppresses the ISR activation. However, the molecular mechanics behind this apparent competition were not immediately clear. Here we show that Hel2 does not physically compete with factors of the ISR, but instead its ribosomal-protein ubiquitination activity, and downstream resolution of collided ribosomes, is responsible for suppressing the ISR. Introducing a mutation in the RING domain of Hel2 – which inhibits its ubiquitination activity and downstream RQC but imparts higher affinity of the factor for collided ribosomes – resulted in increased activation of the ISR upon MMS-induced alkylation stress. Similarly, mutating Hel2’s lysine targets in uS10, which is responsible for RQC activation, resulted in increased Gcn4 target induction. Remarkably, the entire process of RQC appears to be limited by the action of Hel2, as the overexpression of this one factor dramatically suppressed activation of the ISR. Collectively, our data suggest that cells evolved Hel2 to bind collided ribosomes with a relatively high affinity, but kept its concentration relatively low, ensuring that it gets exhausted under stress conditions that cannot be resolved by quality control processes.

Abstract 3334: Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response

Abstract Integrated stress response (ISR) activation has been linked to many human diseases, including cancer and neurological diseases. The ISR controls protein synthesis and proteostasis. ISR main effector ATF4 transcriptional factor regulates the balance between survival and cell death. Under acute ISR, ATF4 promotes cell adaptation and survival. Sustained ISR leads to ATF4-mediated apoptosis. In many cancers, mutations or overexpression of oncogenes, results in enhanced cell proliferation and increased protein synthesis, which activate the ISR. Therefore, protein translation is reduced to maintain proteostasis. In other words, many cancers are primed by the ISR. Therefore, additional push by small molecule ISR-inducers will disrupt the balance and force cancer cells to enter apoptosis. Nelfinavir activates ATF4 through inhibition of eIF2α specific phosphatases CReP and GADD34. PG3 upregulates ATF4 through the HRI/eIF2α/ATF4 pathway. We hypothesize that combined treatment of PG3 and Nelfinavir will sustain upregulation of ATF4, and lead to ATF4-mediated cell apoptosis. Cell viability assays indicated that the combined treatment potently and synergistically inhibited cell viability in colorectal cancer cell lines HT29, SW480 and HCT116 p53−/−, osteosarcoma cell line U2OS and breast cancer cell lines MCF7 and T47D. The combined treatment sustained the induction of ATF4 and enhanced the expression of its proapoptotic target genes, CHOP and PUMA, and cell apoptosis. We confirmed that the enhanced induction of ATF4, CHOP and PUMA is through the ISR because ISRIB (ISR inhibitor) blocked the combined treatment-induced upregulation of ATF4, CHOP and PUMA. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response [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 3334.

Abstract 6565: Improving survival of atypical teratoid/rhabdoid tumor orthotopic xenografts through the combination of PI3K inhibitor paxalisib and nucleoside analog gemcitabine

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are an aggressive infantile brain tumor with a dismal four-year event-free survival of 37%. We have previously identified high activation of mTORC1 and mTORC2 within AT/RT and shown that the highly brain penetrant PI3K inhibitor paxalisib is capable of simultaneously inhibiting mTORC1/2 by acting upstream of both complexes. In two orthotopic xenograft models, paxalisib slowed tumor growth and significantly extended survival (CHLA-06: 40 to 54 days, p=0.001; BT-12: 21 to 35 days, p=0.02). RNAseq analysis following mTOR inhibition in AT/RT cell lines showed an upregulation of genes involved in the activation of the integrated stress response (ISR) (ATF4, CHOP, PPP1R15A t-test p<0.05). Analysis of primary pediatric brain tumors through the Children’s Brain Tumor Network showed increased levels of ATF4 mRNA and eIF2α protein and mRNA in AT/RT compared to other indolent pediatric brain tumors. Increased expression of ISR components suggests this pathway may be a dependency in AT/RT. Activation of this pathway is known to be cytoprotective, however persistent over-activation drives cell death through induction of pro-apoptotic factors. Gemcitabine is a pyrimidine analog functioning to inhibit DNA synthesis and induce DNA damage; notably, recent literature has correlated DNA damage to activation of the ISR with downstream phosphorylation of eIF2α. We hypothesize that due to high baseline activation of ISR for AT/RT, paxalisib combined with gemcitabine will result in an over-activation of the ISR leading to AT/RT cell death. Western blot analysis of combination therapy compared to DMSO control showed induction of phospho-eIF2α, ATF4, and CHOP, denoting an activation of the ISR. Combination therapy in AT/RT cells induces cell death (Western blot analysis cPARP, MUSE Annexin V assay, SynergyFinder BLISS score [viability] CHLA-06: 21.50, CHLA-266: 22.60) and synergizes to decrease cell growth (SynergyFinder BLISS score [inhibition] CHLA-06: 16.80, BT-12: 14.30, CHLA-266: 13.90, BT-37: 11.40, CHLA-05: 10.40). Paxalisib/gemcitabine combination therapy in murine orthotopic xenograft models of AT/RT has slowed tumor growth (bioluminescent imaging) and led to significant extension of survival. Mice bearing BT-37 orthotopic xenografts and treated with vehicle control had a median survival of 56 days, while all of the paxalisib/gemcitabine treated mice were alive at 100 days of treatment (p<0.0001). In CHLA-06 orthotopic tumors combination therapy extended median survival from 22 days for control to 82.5 days (p<0.0001). Based on these findings, the Pacific Pediatric Neuro-Oncology Consortium (PNOC) is planning to include paxalisib/gemcitabine combination therapy in the next international clinical trial for patients with relapsed/recurrent AT/RT. Citation Format: Tyler Findlay, Kristen Malebranche, Anupa Geethadevi, Charles Eberhart, Jeffrey Rubens, Eric Raabe. Improving survival of atypical teratoid/rhabdoid tumor orthotopic xenografts through the combination of PI3K inhibitor paxalisib and nucleoside analog gemcitabine [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 6565.

Abstract 350: Targeting prohibitins by fluorizoline leads to the activation of ISR and apoptosis through DELE1 and HRI by an impairment of the mitochondrial protein import machinery

Abstract Prohibitins are ubiquitously expressed within the cell, but they are mainly located at the inner mitochondrial membrane. These proteins are closely associated with tumour progression, invasion, and apoptotic resistance, being overexpressed in various primary tumours such as lymphoma and endometrial cancer. Notably, we have developed a novel synthetic molecule, fluorizoline, which induces apoptosis by selectively targeting PHBs in different cancer cell lines and primary samples from various haematological neoplasias. We have previously described that fluorizoline-induced apoptosis is mediated through the activation of the pro-apoptotic branch of the integrated stress response (ISR) pathway in HeLa and HAP1 cells, concretely, by the ATF4-CHOP-NOXA axis. In a recent study, we identified compensatory mechanisms for four ISR-related kinases. Importantly, HRI emerged as the primary kinase responsible for activating this pathway and promoting apoptosis, suggesting that mitochondrial stress could lead to this activation of the ISR.Here, we investigate the OMA1-DELE1-HRI molecular pathway responsible for sensing mitochondrial stress and activating the ISR cancer cell lines. Firstly, we demonstrate that fluorizoline treatment triggers the cleavage of large DELE1 (L-DELE1) to its cleaved form (S-DELE1) through western blot analysis, and its subsequent sorting to the cytosol as confirmed by confocal analysis. Using DELE1 KO lines generated by CRISPR/Cas9 technology, we demonstrate that DELE1 is involved in the activation of the ISR and apoptosis induced by fluorizoline. However, although we cannot rule out OMA1 involvement, fluorizoline-induced ISR activation does not strongly depend on OMA1. Interestingly, our data suggests that targeting PHBs, either by fluorizoline or by their downregulation, could result in mitochondrial protein import machinery impairment. This impairment may potentially be the cause of DELE1 accumulation in the cytosol, leading to the subsequent activation of the ISR by HRI upon fluorizoline treatment. Citation Format: Ismael Sánchez-Vera, Nekane Maritorena-Hualde, Max-Hinderk Schuler, Ana M- Cosialls, Rodolfo Lavilla, Gabriel Pons, Lucas T Jae, Daniel Iglesias-Serret, Joan Gil. Targeting prohibitins by fluorizoline leads to the activation of ISR and apoptosis through DELE1 and HRI by an impairment of the mitochondrial protein import machinery [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 350.

Abstract 7528: Coordinated regulation of critical immune checkpoint proteins by the integrated stress response (ISR) pathway in lung cancer

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Despite recent advances, high rates of recurrence highlight the need for novel treatment options. Groundbreaking discoveries in identifying and exploiting the PD-1/PD-L1 (Programmed Death 1/Programmed Death Ligand 1) immune checkpoint have resulted in the approval of monoclonal antibodies that disrupt this interaction as a first-line therapy for lung cancer patients. However, only ~20% of NSCLC patients benefit from checkpoint blockade. A critical question remains as to what mechanisms facilitate resistance to PD-1/PD-L1 therapy and whether other immune checkpoints or pathways can be pursued clinically in combination with this therapy. The integrated stress response (ISR) pathway represents an emerging therapeutic vulnerability. The ISR is a generalized response to maintain cellular homeostasis, and is known to be activated by hypoxia, heme deprivation, amino acid starvation, and oncogenic activation. We previously demonstrated that ISR activation potently induces PD-L1 in NSCLC and suppression of anti-tumor immunity (Suresh et al, Nature Cancer, 2020). We discovered that ISR pathway activation enhances PD-L1 translation through the bypass of inhibitory upstream open reading frames (uORFs) in the PD-L1 5' UTR and a mechanistic link to the translation initiation factor eIF5B. Our latest studies demonstrate that the immune checkpoint protein, CD155 (Cluster of Differentiation 155), is induced by ISR activation. We find that both PD-L1 and CD155 are induced by multiple arms of the ISR pathway, and CD155 harbors inhibitory uORFs in its 5' UTR. Importantly, we observed a significant correlation between PD-L1 and CD155 expression in a panel of primary human lung adenocarcinomas. We further demonstrated that ISR activation inhibits T cell function in vitro and promotes tumor growth in the CMT167 syngeneic mouse model. Analysis of immune cell infiltration using mass cytometry in this in vivo model is ongoing. Currently, we are determining the extent to which ISR inhibition suppresses tumorigenesis by promoting anti-tumor immunity and whether this may synergize with existing immune checkpoint therapies. Overall, these studies will set the stage for determining whether inhibition of the ISR pathway alone or in combination with immune checkpoint blockade will benefit lung cancer patients. Our studies may also lead to new therapeutic approaches to trigger anti-cancer immune responses and improve current strategies. Citation Format: Shayna Elizabeth Thomas-Jardin, Shruthy Suresh, Cheryl Lewis, Chul Ahn, Bret M. Evers, John D. Minna, Kathryn A. O'Donnell. Coordinated regulation of critical immune checkpoint proteins by the integrated stress response (ISR) pathway in lung 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 7528.

Abstract 4615: HC-7366, a potent GCN2 activator, complements belzutifan, a HIF-2⍺ antagonist, by providing combination benefit in belzutifan-sensitive models and monotherapy activity in belzutifan-resistant models

Abstract Recent approval of the HIF-2α antagonist belzutifan (Welireg) for patients with VHL disease has enabled the clinical opportunity to target the HIF-2 axis in cancer. This is particularly important for patients with clear cell renal cell carcinoma (ccRCC), in which 90% of tumors are VHL deficient, and approximately one in four primary tumors express HIF-2α exclusively. Clinical studies to date suggest an overall response rate (ORR) of 20-25% for belzutifan monotherapy, highlighting the need for a rational combination strategy to improve patient outcomes. This has led to multiple clinical trials testing belzutifan in combination with diverse therapeutic agents, including immune-checkpoint inhibitors, VEGFR-TKIs, and CDK4/6 inhibitors. HiberCell is developing a first-in-class GCN2 activator currently undergoing Ph1 clinical evaluation in patients with solid tumors (NCT05121948). GCN2 activation by HC-7366 drives the Integrated Stress Response (ISR), resulting in translation inhibition and delayed cell cycle progression in cancer cells. In preclinical studies, HC-7366 demonstrated significant antitumor activity associated with HIF suppression across diverse tumor models, including ccRCC (70% TGI), head and neck (33% regression), sarcoma (84% TGI) and prostate (100% TGI). Analysis of tumor samples revealed that HC-7366 inhibited both HIF-1α and HIF-2α, as well as multiple cell cycle regulators. Given the mechanistic link between HIF-2 and cell cycle regulation, and the noted combination benefit reported when CDK inhibitors are combined with belzutifan, we hypothesized that HC-7366 would enhance the antitumor effects of belzutifan in RCC. Combination studies in HIF-2 dependent 786-O and A-498 RCC xenografts demonstrated that HC-7366 improved the antitumor effects of belzutifan at clinically relevant doses. The combination of HC-7366 and belzutifan in A-498 drove robust tumor inhibition (~90% TGI). In 786-O, HC-7366 increased the number of complete responses from 2 of 8 tumors in the belzutifan-treated animals to 6 of 8 in the combination group. When tested in two belzutifan-resistant PDX models, HC-7366 resulted in significant monotherapy activity, achieving 17% regression as a maximal response, and inhibiting both HIF and cell cycle markers. Belzutifan is currently being tested in indications beyond RCC, so we evaluated the combination in VHLwt endometrial cancer model, MFE-280. Consistent with RCC models, the combination of HC-7366 and belzutifan resulted in tumor stasis with activation of ISR, enhanced inhibition of HIF and cell cycle markers in the combination vs belzutifan monotherapy. Together, these findings suggest that HC-7366 enhances belzutifan activity and serves as a rationale supporting clinical evaluation of this therapeutic combination. Citation Format: Michael E. Stokes, Feven Tameire, Paulina Wojnarowicz, Sho Fujisawa, Crissy Dudgeon, Sharon Huang, Nick Collette, Ben Harrison, Ashley LaCayo, Xiaohong Qiu, Takashi Kangas, Weiyu Zhang, Jeremy Drees, David Surguladze, Eric S. Lightcap, Nandita Bose. HC-7366, a potent GCN2 activator, complements belzutifan, a HIF-2⍺ antagonist, by providing combination benefit in belzutifan-sensitive models and monotherapy activity in belzutifan-resistant models [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 4615.

Abstract 7591: Tumor Treating Fields (TTFields) show efficacy in Triple-Negative breast cancer (TNBC) cells alone and in combination with PARP inhibitor Talazoparib

Abstract Breast cancer is the most common cancer in women worldwide, and the fifth leading cause of cancer-related deaths globally. Triple-negative breast cancer (TNBC), the most aggressive form of breast cancer, is usually treated with surgery, chemotherapy, and radiation. Tumor Treating Fields therapy are FDA-approved for the treatment of glioblastoma (GBM) and malignant pleural mesothelioma (MPM).TT Fields use alternating electric fields to inhibit tumor growth through mitotic disruption, cellular damage, integrated stress response activation, and immune mechanisms and inhibit cell proliferation, induce cell death, and cause cell cycle arrest.TT Fields can induce BRCAness and when combined with PARP inhibition, this combination may result in synthetic lethality. We explored the effect of TTFields on two TNBC cell lines MDA-MB436 (BRCA1-mutant, p53-null) and MDA-MB468 (BRCA1 wild-type, p53-mutant) treated with TTFields (150 kHz) for 24 hours. Western blot analysis showed elevation of p21 independent of p53 mutational status. We examined the combination of PARP inhibitor (Talazoparib) with TTFields in MDA-MB468 cells to determine the effect of the combination in BRCA wild-type cancer cells. Western blot analysis showed p21 upregulation after 24 hours in combination without an increase in p53. p21 acts as a G1-checkpoint protein and loss of p21 expression has been observed in a high percentage of human breast cancer cells. Our results suggest a rational and potentially effective therapy that could be further developed and used for the treatment of breast cancer. Our ongoing studies are exploring potential mechanisms of synergy between PARP inhibitor Talazoparib and TTFields using various TNBC models, and the effect of the combination on cell cycle arrest in cell culture and in vivo. Citation Format: Maryam Ghandali, Praveen Srinivasan, Yutong Xia, Eric T. Wong, Robert W. Sobol, Lanlan zhou, Benedito A. Carneiro, Stephanie L. Graff, Wafik S. El-Deiry. Tumor Treating Fields (TTFields) show efficacy in Triple-Negative breast cancer (TNBC) cells alone and in combination with PARP inhibitor Talazoparib [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 7591.

Abstract 4740: Acid ceramidase inhibition enhances venetoclax sensitivity in preclinical models of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic malignancy in urgent need of improved therapeutic strategies. AML blasts frequently rely on the anti-apoptotic protein, Bcl-2, for survival. The specific Bcl-2 inhibitor, venetoclax (VEN), is FDA-approved in combination with low-dose cytarabine (LDAC) or azacitidine for patients unfit for intensive induction chemotherapy. Unfortunately, responses are transient due to upregulation of compensatory survival proteins (e.g., Bcl-xL or Mcl-1) or resistance characterized by reprogrammed mitochondrial bioenergetics. Our research aims to improve current AML treatments and understanding of AML pathogenesis by targeting dysregulated sphingolipid metabolism. Ceramides are tumor-suppressor sphingolipids that mediate therapy-induced cell death. Sphingolipid dysregulation that results in decreased ceramide content and/or enhanced ceramide catabolism is an emerging AML hallmark. We’ve previously shown that acid ceramidase (AC), a ceramide catabolizing lysosomal enzyme, is overexpressed in AML and contributes to drug resistance. Here, we demonstrated that AC inhibition enhanced VEN cytotoxicity. Combining the AC inhibitor/ceramide analog, SACLAC, with VEN resulted in synergistic lethality in multiple AML cell lines in cell viability and apoptosis assays. Genetic inhibition of AC also improved VEN cytotoxicity. The SACLAC+VEN combination achieved Bliss scores (SynergyFinder 2.0) between 22 and 32 in cell lines, which indicate a highly synergistic combination. SACLAC+VEN efficacy was comparable to the FDA-approved combination of VEN+LDAC when tested in 67 primary AML patient samples. Pharmacological inhibition of AC also increased the efficacy of the VEN+LDAC combination. Mechanistically, the observed synergistic lethality was independent of changes to Bcl-2, Mcl-1, and Bcl-xL protein levels. Combined AC and Bcl-2 inhibition resulted in synergistic ceramide accumulation, integrated stress response (ISR) activation, and caspase-dependent apoptosis characterized by impaired mitochondrial function. Pretreatment with the pan-caspase inhibitor, zVAD-FMK, or ISR inhibitor, ISRIB, significantly rescued cell death. Ongoing studies aim to further characterize mitochondrial impairment and ISR activation induced by inhibiting AC and Bcl-2. Taken together, these results detail the first report of ISR activation and mitochondrial defects elicited by combined AC and Bcl-2 inhibition as contributory to cytotoxic impact (mechanism) and warrant additional studies of AC inhibitors with frontline AML therapeutics. Citation Format: Johnson Ung, Su-Fern Tan, Jeremy J. Shaw, Todd E. Fox, Maansi Taori, David F. Claxton, Kelsey H. Fisher-Wellman, Myles C. Cabot, David J. Feith, Thomas P. Loughran. Acid ceramidase inhibition enhances venetoclax sensitivity in preclinical models of 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 4740.

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.

Integrated Stress Response Potentiates Ponatinib-Induced Cardiotoxicity.

Mitochondrial dysfunction is a primary driver of cardiac contractile failure; yet, the cross talk between mitochondrial energetics and signaling regulation remains obscure. Ponatinib, a tyrosine kinase inhibitor used to treat chronic myeloid leukemia, is among the most cardiotoxic tyrosine kinase inhibitors and causes mitochondrial dysfunction. Whether ponatinib-induced mitochondrial dysfunction triggers the integrated stress response (ISR) to induce ponatinib-induced cardiotoxicity remains to be determined. Using human induced pluripotent stem cells-derived cardiomyocytes and a recently developed mouse model of ponatinib-induced cardiotoxicity, we performed proteomic analysis, molecular and biochemical assays to investigate the relationship between ponatinib-induced mitochondrial stress and ISR and their role in promoting ponatinib-induced cardiotoxicity. Proteomic analysis revealed that ponatinib activated the ISR in cardiac cells. We identified GCN2 (general control nonderepressible 2) as the eIF2α (eukaryotic translation initiation factor 2α) kinase responsible for relaying mitochondrial stress signals to trigger the primary ISR effector-ATF4 (activating transcription factor 4), upon ponatinib exposure. Mechanistically, ponatinib treatment exerted inhibitory effects on ATP synthase activity and reduced its expression levels resulting in ATP deficits. Perturbed mitochondrial function resulting in ATP deficits then acts as a trigger of GCN2-mediated ISR activation, effects that were negated by nicotinamide mononucleotide, an NAD+ precursor, supplementation. Genetic inhibition of ATP synthase also activated GCN2. Interestingly, we showed that the decreased abundance of ATP also facilitated direct binding of ponatinib to GCN2, unexpectedly causing its activation most likely because of a conformational change in its structure. Importantly, administering an ISR inhibitor protected human induced pluripotent stem cell-derived cardiomyocytes against ponatinib. Ponatinib-treated mice also exhibited reduced cardiac function, effects that were attenuated upon systemic ISRIB administration. Importantly, ISRIB does not affect the antitumor effects of ponatinib in vitro. Neutralizing ISR hyperactivation could prevent or reverse ponatinib-induced cardiotoxicity. The findings that compromised ATP production potentiates GCN2-mediated ISR activation have broad implications across various cardiac diseases. Our results also highlight an unanticipated role of ponatinib in causing direct activation of a kinase target despite its role as an ATP-competitive kinase inhibitor.

Nucleus pulposus cells regulate macrophages in degenerated intervertebral discs via the integrated stress response-mediated CCL2/7-CCR2 signaling pathway

Lower back pain (LBP), which is a primary cause of disability, is largely attributed to intervertebral disc degeneration (IDD). Macrophages (MΦs) in degenerated intervertebral discs (IVDs) form a chronic inflammatory microenvironment, but how MΦs are recruited to degenerative segments and transform into a proinflammatory phenotype remains unclear. We evaluated chemokine expression in degenerated nucleus pulposus cells (NPCs) to clarify the role of NPCs in the establishment of an inflammatory microenvironment in IDD and explored the mechanisms. We found that the production of C-C motif chemokine ligand 2 (CCL2) and C-C motif chemokine ligand 7 (CCL7) was significantly increased in NPCs under inflammatory conditions, and blocking CCL2/7 and their receptor, C-C chemokine receptor type 2(CCR2), inhibited the inductive effects of NPCs on MΦ infiltration and proinflammatory polarization. Moreover, activation of the integrated stress response (ISR) was obvious in IDD, and ISR inhibition reduced the production of CCL2/7 in NPCs. Further investigation revealed that activating Transcription Factor 3 (ATF3) responded to ISR activation, and ChIP-qPCR verified the DNA-binding activity of ATF3 on CCL2/7 promoters. In addition, we found that Toll-like receptor 4 (TLR4) inhibition modulated ISR activation, and TLR4 regulated the accumulation of mitochondrial reactive oxygen species (mtROS) and double-stranded RNA (dsRNA). Downregulating the level of mtROS reduced the amount of dsRNA and ISR activation. Deactivating the ISR or blocking CCL2/7 release alleviated inflammation and the progression of IDD in vivo. Moreover, MΦ infiltration and IDD were inhibited in CCR2-knockout mice. In conclusion, this study highlights the critical role of TLR4/mtROS/dsRNA axis-mediated ISR activation in the production of CCL2/7 and the progression of IDD, which provides promising therapeutic strategies for discogenic LBP.

GDF15 is a dynamic biomarker of the integrated stress response in the central nervous system.

Characterize Growth Differentiation Factor 15 (GDF15) as a secreted biomarker of the integrated stress response (ISR) within the central nervous system (CNS). We determined GDF15 levels utilizing invitro and invivo neuronal systems wherein the ISR was activated. Primarily, we used the murine model of vanishing white matter disease (VWMD), a neurological disease driven by persistent ISR in the CNS, to establish a link between levels of GDF15 in the cerebrospinal fluid (CSF) and ISR gene expression signature in the CNS. GDF15 was also determined in the CSF of VWM patients. GDF15 expression was increased concomitant to ISR activation in stress-induced primary astrocytes as well as in retinal ganglion cells following optic nerve crush, while treatment with 2Bact, a specific eIF2B activator, suppressed both the ISR and GDF15. In the VWMD model, CSF GDF15 levels corresponded with the magnitude of the ISR and were reduced by 2BAct. In VWM patients, mean CSF GDF15 was elevated >20-fold as compared to healthy controls, whereas plasma GDF15 was undifferentiated. These data suggest that CSF GDF15 is a dynamic marker of ISR activation in the CNS and may serve as a pharmacodynamic biomarker for ISR-modulating therapies.

Pharmacological inhibition of the integrated stress response accelerates disease progression in an amyotrophic lateral sclerosis mouse model.

The integrated stress response (ISR) regulates translation in response to diverse stresses. ISR activation has been documented in amyotrophic lateral sclerosis (ALS) patients and ALS experimental models. In experimental models, both ISR stimulation and inhibition prevented ALS neurodegeneration; however, which mode of ISR regulation would work in patients is still debated. We previously demonstrated that the ISR modulator ISRIB (Integrated Stress Response InhiBitor, an eIF2B activator) enhances survival of neurons expressing the ALS neurotoxic allele SOD1 G93A. Here, we tested the effect of two ISRIB-like eIF2B activators (2BAct and PRXS571) in the disease progression of transgenic SOD1G93A mice. After biochemical characterization in primary neurons, SOD1G93A mice were treated with 2BAct and PRXS571. Muscle denervation of vulnerable motor units was monitored with a longitudinal electromyographic test. We used a clinical score to document disease onset and progression; force loss was determined with the hanging wire motor test. Motor neuronal survival was assessed by immunohistochemistry. In primary neurons, 2BAct and PRXS571 relieve the ISR-imposed translational inhibition while maintaining high ATF4 levels. Electromyographic recordings evidenced an earlier and more dramatic muscle denervation in treated SOD1G93A mice that correlated with a decrease in motor neuron survival. Both compounds anticipated disease onset and shortened survival time. 2BAct and PRXS571 anticipate disease onset, aggravating muscle denervation and motor neuronal death of SOD1G93A mice. This study reveals that the ISR works as a neuroprotective pathway in ALS motor neurons and reveals the toxicity that eIF2B activators may display in ALS patients.

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

Abstract B044: Results of a phase 1 dose escalation clinical trial of NXP800, a novel GCN2 activator, in patients with advanced or metastatic solid tumors

Abstract Background: NXP800 is an antineoplastic, oral, small molecule discovered in a phenotypic screen for inhibitors of the heat shock factor 1 stress response. In a panel of human carcinoma cell lines NXP800 induced the expression of genes associated with activation of the integrated stress response (ISR). Orthogonal experimental approaches revealed that activation of the kinase GCN2 was required for ISR activation by NXP800. In tumor samples from ARID1a-mutated ovarian carcinoma xenografts, treatment with NXP800 resulted in substantial tumor growth inhibition and tumor regressions. Induction of the ISR effector ATF4 was observed. Here we present data on the safety, pharmacokinetics and pharmacodynamics of NXP800 from the dose escalation phase 1 trial in patients with advanced or metastatic solid tumors. Methods: This is a first-in-human, open label, dose escalation and expansion Phase 1 study in patients with advanced solid tumors (Part A, all comers) and ovarian cancer (Part B).Dose escalation was guided by a Bayesian modified continual reassessment method that targeted a dose-limiting toxicity DLT probability (DLTp) closest to 30% but <33%.The primary objective of the dose escalation was to identify a dose and dosing schedule for Part B, in which the clinical activity of NXP800 is being evaluated in patients with platinum resistant,ARID1a-mutated ovarian carcinoma. Results: Eighteen patients were treated in Part A: at baseline, the median age was 65 years (range 42-77), 9 (50%) were female and 14/18 (78%) had an ECOG score of 1. Two schedules (once per day [QD] and twice per day [BID] and a dose range of 50 to 150 mg/day were evaluated. The most common (incidence >20%) non-laboratory, treatment emergent adverse events (TEAEs) were vomiting, nausea, diarrhea, fatigue, and decreased appetite, all reported as Grade 1-2 except for nausea and diarrhea that were reported as Grade 3 in one patient each. The most common laboratory TEAEs were thrombocytopenia and aspartate aminotransferase increase, all reported as Grade 1-2 except in two patients in whom thrombocytopenia was reported as Grade 3. Two Grade 4 TEAEs that were considered unrelated to study drug and no Grade 5 TEAEs were reported. The DLT for the QD schedule was 150 mg/day, with an estimated Dose Limiting Toxicity Probability (DLTp) of 34% (no doses in the BID schedule had estimated DLTp of <33%). The maximum tolerated dose was 100 mg/day (DLTp=26%). Model-based simulations demonstrated that NXP800 exposures obtained following 50, 75 and 100 mg QD were comparable to the drug exposure achieved in xenograft model of ARID1a-mutated ovarian carcinoma. ATF4 protein and ISR genes were induced in peripheral blood mononuclear cell samples obtained from patients treated with NXP800. Conclusion: The NXP800 doses and dosing schedules selected for Part B are 50 and 75 mg/day in a QD schedule. At these dose levels, NXP800 was tolerable, achieved relevant systemic exposure levels and induced ISR PD biomarkers. Part B is open for enrollment of patients with platinum resistant, ARID1a-mutated ovarian carcinoma. Citation Format: Udai Banerji, Simon Pacey, Karen Finkelstein, Simon Rodney, Ana Filipa Palma DosReis, Giovanni Codaccipisanelli, Mark Sloan, Florence I Raynaud, Ruth Ruddle, Karen Swales, Matthew Tall, Shay Shemesh, Diane Marsolini, Megan Sardone, Justin Hay, Robin Bliss, Enrique Poradosu, Paul Workman. Results of a phase 1 dose escalation clinical trial of NXP800, a novel GCN2 activator, in patients with advanced or metastatic solid tumors [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 B044.

Repositioning of mifepristone as an integrated stress response activator to potentiate cisplatin efficacy in non-small cell lung cancer

Lung cancer, primarily non-small-cell lung cancer (NSCLC), is a significant cause of cancer-related mortality worldwide. Cisplatin-based chemotherapy is a standard treatment for NSCLC; however, its effectiveness is often limited due to the development of resistance, leading to NSCLC recurrence. Thus, the identification of effective chemosensitizers for cisplatin is of paramount importance. The integrated stress response (ISR), activated by various cellular stresses and mediated by eIF2α kinases, has been implicated in drug sensitivity. ISR activation globally suppresses protein synthesis while selectively promoting the translation of ATF4 mRNA, which can induce pro-apoptotic proteins such as CHOP, ATF3, and TRIB3. To expedite and economize the development of chemosensitizers for cisplatin treatment in NSCLC, we employed a strategy to screen an FDA-approved drug library for ISR activators. In this study, we identified mifepristone as a potent ISR activator. Mifepristone activated the HRI/eIF2α/ATF4 axis, leading to the induction of pro-apoptotic factors, independent of its known role as a synthetic steroid. Our in vitro and in vivo models demonstrated mifepristone's potential to inhibit NSCLC re-proliferation following cisplatin treatment and tumor growth, respectively, via the ISR-mediated cell death pathway. These findings suggest that mifepristone, as an ISR activator, could enhance the efficacy of cisplatin-based therapy for NSCLC, highlighting the potential of drug repositioning in the search for effective chemosensitizers.

SARS-CoV-2 variant-specific differences in inhibiting the effects of the PKR-activated integrated stress response

The integrated stress response (ISR) is a eukaryotic cell pathway that triggers translational arrest and the formation of stress granules (SGs) in response to various stress signals, including those caused by viral infections. The SARS-CoV-2 nucleocapsid protein has been shown to disrupt SGs, but SARS-CoV-2 interactions with other components of the pathway remains poorly characterized. Here, we show that SARS-CoV-2 infection triggers the ISR through activation of the eIF2α-kinase PKR while inhibiting a variety of downstream effects. In line with previous studies, SG formation was efficiently inhibited and the induced eIF2α phosphorylation only minimally contributed to the translational arrest observed in infected cells. Despite ISR activation and translational arrest, expression of the stress-responsive transcription factors ATF4 and CHOP was not induced in SARS-CoV-2 infected cells. Finally, we found variant-specific differences in the activation of the ISR between ancestral SARS-CoV-2 and the Delta and Omicron BA.1 variants in that Delta infection induced weaker PKR activation while Omicron infection induced higher levels of p-eIF2α, and greatly increased SG formation compared to the other variants. Our results suggest that different SARS-CoV-2 variants can affect normal cell functions differently, which can have an impact on pathogenesis and treatment strategies.

Extracellular Vesicle Trafficking Regulates Hematopoietic Stem and Progenitor Cell Fitness

Among regulatory pathways critical to hematopoietic stem and progenitor cell (HSPC) function, sphingolipid metabolism has recently been recognized for its role in safeguarding essential HSPC properties of self-renewal and lineage fate. In particular, de novo ceramide (Cer) synthesis appears to regulate a coordinated stress response that results in enhanced self-renewal of human HSPCs. However, the fundamental molecular mechanisms underlying these regenerative stress responses have yet to be uncovered and broadly validated. Moreover, alternative synthetic pathways regulating Cer and sphingomyelin (SM) levels may serve additional regulatory roles in HSPCs. Here we target neutral sphingomyelinase-2 (nSMase2/SMPD3)-dependent SM hydrolysis through pharmacologic (GW4869) and genetic (Cas9-mediated) interruption, and similarly confirm an adaptive ATF4-mediated integrated stress response (ISR). Functionally, transient inhibition led to durable improvements in HSPC reconstitution after primary (>8 fold at steady-state, p<0.001) and secondary (>20 fold at steady-state, p<0.001) transplantation. Surprisingly, lipidomic profiling of GW4869-treated HSPCs demonstrated neither global Cer depletion nor SM accumulation, suggesting an alternative mechanism underlying the observed gains in HSPC fitness. Sphingolipid metabolism also regulates the biogenesis of select subclasses of secreted extracellular vesicles (EVs). To segregate metabolic regulation of HSPCs by sphingolipids from their role in EV formation and secretion, we began to systematically modulate and compare several core EV biogenesis pathways by Cas9/ribonucleoprotein-mediated knockout (KO) of SMPD3, CD63 and TSG101 in HSPC ex vivo expansion cultures . Similar to pharmacologic nSMase2 inhibition, loss of all 3 genes suppressed EV secretion, whereas only depletion of SMPD3 or ESCRT protein TSG101, but not the tetraspanin protein CD63, led to ISR activation in HSPCs (p<0.05). As a functional corollary, clonogenicity and long-term transplantation outcomes were enhanced by KO of SMPD3 (1.5-fold; p<0.01)and TSG101 (2-fold; p<0.001), but reduced after CD63-KO (0.6-fold; p<0.05). One function of EVs is the export of protein and nucleic acid cargo from the cell. We reasoned the observed differences in stress response and HSPC fitness may reflect uniquely altered secretomes. Quantitative proteomic analysis of the vesicle secretome across KO cells revealed impaired secretion of mitochondrial (n=160, FDR=8.3E-22) and oxidative stress-related (n=82, FDR=2.8E-12) proteins from SMPD3- and TSG101- KO, but not CD63-KO HSPCs, highlighting the heterogeneity and specificity of EV cargo trafficking. We validated this observation by pharmacologic blockade of SMPD3 with GW4869, which similarly led to transient decreases in mitochondrial DNA (0.36-fold, p<0.01) and protein (0.6-fold, p<0.05) export. Finally, we confirmed that impaired mitochondrial cargo secretion into EVs (mtEV) after SMPD3 or TSG101, but not CD63, depletion led to transient accumulation of mitochondrial-sourced reactive oxygen species (p<0.01) that specifically triggered the observed ISR activation and downstream mTOR inhibition. Our study reveals a novel mechanism by which secretory cargo trafficking into EVs guards HSPC homeostasis and fitness. We further demonstrate that transient modulation of this pathway, wherein EV cargo retention activates a molecularly-defined stress response pathway, can durably improve long-term transplantation outcomes. Sphingolipid biology inextricably links cellular metabolism and EV biogenesis, making it likely that prior descriptions of Cer-dependent adaptive stress response involve modulation of EV secretion. Finally, our work underscores the high degree of specificity that governs EV secretion in regulating intrinsic cell function. In aggregate, we show that differential EV biogenesis pathways regulate HSPC fate and fitness, with important translational implications in safeguarding stemness and regenerative capacity during ex vivo expansion and therapeutic transplantation.

Pharmacologic activation of a compensatory integrated stress response kinase promotes mitochondrial remodeling in PERK-deficient cells

The integrated stress response (ISR) comprises the eIF2α kinases PERK, GCN2, HRI, and PKR, which induce translational and transcriptional signaling in response to diverse insults. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activation of compensatory eIF2α kinases to rescue ISR signaling and promote mitochondrial adaptation in PERK-deficient cells. We show that the HRI activator BtdCPU and GCN2 activator halofuginone promote ISR signaling and rescue ER stress sensitivity in PERK-deficient cells. However, BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and activation of the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and adaptive mitochondrial respiration, mimicking regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK signaling and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly selective ISR activators.

The Antihypertensive Guanabenz Exacerbates Integrated Stress Response and Disrupts the Brain Circadian Clock.

The circadian clock regulates a variety of biological processes that are normally synchronized with the solar day. Disruption of circadian rhythms is associated with health problems. Understanding the signaling mechanisms that couple cell physiology and metabolism to circadian timekeeping will help to develop novel therapeutic strategies. The integrated stress response (ISR) is activated by the cellular stressors to maintain physiological homeostasis by orchestrating mRNA translation. Aberrant ISR has been found in a number of neurological diseases that exhibit disrupted circadian rhythms and sleep. Recent work has started to uncover a critical role for the ISR in regulating the physiology of the circadian clock. Guanabenz (2,6-dichlorobenzylidene aminoguanidine acetate) is an orally bioavailable α2-adrenergic receptor agonist that has been used as an antihypertensive for decades. Recent studies demonstrated that guanabenz can regulate the ISR. Here, we assessed the effects of guanabenz on cellular and behavioral circadian rhythms using a multidisciplinary approach. We found that guanabenz can induce the ISR by increasing eIF2α phosphorylation in cultured fibroblasts as well as in the mouse brain. The hyperphosphorylation of eIF2α by guanabenz is associated with the shortened circadian period in cells and animals and the disruption of behavioral circadian rhythms in mice. Guanabenz administration disrupted circadian oscillations of the clock protein Per1 and Per2 in the mouse suprachiasmatic nucleus, the master pacemaker. These results uncover a significant yet previously unidentified role of guanabenz in regulating circadian rhythms and indicate that exacerbated ISR activation can impair the functions of the brain's circadian clock by disrupting clock gene expression.