ERK Signaling Research Articles

Formoterol Acting via β2-Adrenoreceptor Restores Mitochondrial Dysfunction Caused by Parkinson's Disease-Related UQCRC1 Mutation and Improves Mitochondrial Homeostasis Including Dynamic and Transport.

Formoterol, a β2-adrenergic receptor (β2AR) agonist, shows promise in various diseases, but its effectiveness in Parkinson's disease (PD) is debated, with unclear regulation of mitochondrial homeostasis. This study employed a cell model featuring mitochondrial ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) variants associated with familial parkinsonism, demonstrating mitochondrial dysfunction and dynamic imbalance, exploring the therapeutic effects and underlying mechanisms of formoterol. Results revealed that 24-h formoterol treatment enhanced cell proliferation, viability, and neuroprotection against oxidative stress. Mitochondrial function, encompassing DNA copy number, repatriation, and complex III-linked respiration, was comprehensively restored, along with the dynamic rebalance of fusion/fission events. Formoterol reduced extensive hypertubulation, in contrast to mitophagy, by significantly upregulating protein Drp-1, in contrast to fusion protein Mfn2, mitophagy-related protein Parkin. The upstream mechanism involved the restoration of ERK signaling and the inhibition of Akt overactivity, contingent on the activation of β2-adrenergic receptors. Formoterol additionally aided in segregating healthy mitochondria for distribution and transport, therefore normalizing mitochondrial arrangement in mutant cells. This study provides preliminary evidence that formoterol offers neuroprotection, acting as a mitochondrial dynamic balance regulator, making it a promising therapeutic candidate for PD.

Immunomodulatory Effects of Fluoroquinolones in Community-Acquired Pneumonia-Associated Acute Respiratory Distress Syndrome.

Community-acquired pneumonia is reported as one of the infectious diseases that leads to the development of acute respiratory distress syndrome. The innate immune system is the first line of defence against microbial invasion; however, its dysregulation during infection, resulting in an increased pathogen load, stimulates the over-secretion of chemokines and pro-inflammatory cytokines. This phenomenon causes damage to the epithelial-endothelial barrier of the pulmonary alveoli and the leakage of the intravascular protein into the alveolar lumen. Fluoroquinolones are synthetic antimicrobial agents with immunomodulatory properties that can inhibit bacterial proliferation as well as exhibit anti-inflammatory activities. It has been demonstrated that the structure of fluoroquinolones, particularly those with a cyclopropyl group, exerts immunomodulatory effects. Its capability to inhibit phosphodiesterase activity leads to the accumulation of intracellular cAMP, which subsequently enhances PKA activity, resulting in the inhibition of transcriptional factor NF-κB and the activation of CREB. Another mechanism reported is the inhibition of TLR and ERK signalling pathways. Although the sequence of events has not been completely understood, significant progress has been made in comprehending the specific mechanisms underlying the immunomodulatory effects of fluoroquinolones. Here, we review the indirect immunomodulatory effects of FQs as an alternative to empirical therapy in patients diagnosed with community-acquired pneumonia.

A comprehensive review of the botany, phytochemistry, pharmacology, and toxicology of Murrayae Folium et Cacumen.

Ethnopharmacological relevance: Murrayae Folium et Cacumen (MFC) is a plant considered to be a traditional Chinese medicine with culinary value as well. The dry leaves and twigs of Murraya paniculata and M. exotica are used to treat stomach aches, rheumatism, toothaches, swelling, and insect and snake bites. They are also used to prepare spicy chicken dishes. Aim of the review: This review comprehensively summarizes the available information on the botanical characterization, phytochemistry, pharmacological activities, pharmacodynamics, pharmacokinetics, and toxicity of MFC. Methods: Relevant scientific literature up to August 2023 was included in the study. Chinese and English studies on MFC were collected from databases, including PubMed, Elsevier, Web of Science, Springer, Science Direct, Wiley, ACS, and CNKI (Chinese). Doctoral and Master's dissertations were also included. Results: In total, 720 compounds have been identified and reported in the literature, including flavonoids, coumarins, alkaloids, sterols, phenylpropenols, organic acids, spirocyclopentenones, and volatile oils. Flavonoids and coumarins are the two most important bioactive compounds responsible for these pharmacological activities. MFC has anti-inflammatory, anti-bacterial, anti-microbial, anti-diabetic, anti-tumor, anti-oxidant, anti-depressant, potential anti-Alzheimer's disease, chondroprotective, and analgesic properties. The pharmacological effects include interrupting the STAT3/NF-κB/COX-2 and EGFR signaling pathways, downregulating EpCAM expression, inhibiting NF-κB and ERK signals, inhibiting the EP/cAMP/PKA signaling pathway and miR-29a/Wnt/β-catenin signaling activity, and upregulating Foxo3a expression. Conclusion: This review demonstrates that the chemical constituents, pharmacological activities, pharmacodynamics, pharmacokinetics, and toxicity of MFC support its use in traditional Chinese botanical medicines. MFC contains a wide range of chemical compounds. Flavonoids and coumarins promote strong pharmacological activity and, are low-toxicity natural phytomedicines that are widely used in medicine, food, ornamentation, and cosmetics, making MFC a promising compound for development and use in the treatment of several medical conditions.

Inhibition of NF-κB and ERK signaling pathways in osteoclasts and M1 macrophage polarization: Mechanistic insights into the anti-osteoporotic effects of Pseudolaric acid B

Osteoporosis, characterized by bone metabolism disruption leading to gradual bone loss and increased fracture susceptibility, is linked to the excessive activation of osteoclasts. Pseudolaric acid B (PAB), identified as an NF-κB signaling inhibitor crucial for osteoclastogenesis, is explored here for its protective effects in osteoporosis. Noncytotoxic PAB's impact on osteoclast differentiation was assessed through cell viability and osteoclast formation assays, with subsequent testing of osteoclast function via bone resorption assays. Quantitative real-time polymerase chain reaction evaluated PAB's genetic-level impact on osteoclastogenesis. Network pharmacology, western blot, and luciferase reporter gene assays were employed to elucidate PAB's regulatory mechanism. In an in vivo model of osteoporosis induced by ovariectomy (OVX) in mice, micro-CT, H&E staining, and TRAP staining facilitated histomorphometry analysis, while flow cytometry verified macrophage polarization. PAB demonstrated inhibitory effects on osteoclast formation and bone resorption in BMM and RAW264.7 cells, suppressing osteoclast-specific genes. Bioinformatic analysis, western blot, and luciferase assay results indicated PAB's inhibition of IκBα phosphorylation in the NF-κB signaling pathway and ERK in MAPKs, elucidating its mechanism. In vivo experiments confirmed PAB's attenuation of osteoporosis by reducing osteoclast formation in OVX mice. PAB further facilitated macrophage conversion from M1 to M2 and suppressed IL-1β, TNF-α, and IL-6 synthesis. In conclusion, PAB prevents osteoporosis by inhibiting RANKL-induced osteoclastogenesis through NF-κB and ERK signaling pathway suppression, coupled with macrophage polarization. These findings indicate the potential therapeutic role of PAB in osteoporosis.

A Natural Peptide from A Traditional Chinese Medicine Has the Potential to Treat Chronic Atrophic Gastritis by Activating Gastric Stem Cells.

Chronic atrophic gastritis (AG) is initiated mainly by Helicobacter pylori infection, which may progress to stomach cancer following the Correa's cascade. The current treatment regimen is H. pylori eradication, yet evidence is lacking that this treatment is effective on later stages of AG especially gastric gland atrophy. Here, using AG mouse model, patient samples, gastric organoids, and lineage tracing, this study unraveled gastric stem cell (GSC) defect as a crucial pathogenic factor in AG in mouse and human. Moreover, a natural peptide is isolated from a traditional Chinese medicine that activated GSCs to regenerate gastric epithelia in experimental AG models and revitalized the atrophic gastric organoids derived from patients. It is further shown that the peptide exerts its functions by stabilizing the EGF-EGFR complex and specifically activating the downstream ERK and Stat1 signaling. Overall, these findings advance the understanding of AG pathogenesis and open a new avenue for AG treatment.

Discovery of TK-642 as a highly potent, selective, orally bioavailable pyrazolopyrazine-based allosteric SHP2 inhibitor

Src homology-2-containing protein tyrosine phosphatase 2 (SHP2) is a promising therapeutic target for cancer therapy. In this work, we presented the structure-guided design of 5,6-fused bicyclic allosteric SHP2 inhibitors, leading to the identification of pyrazolopyrazine-based TK-642 as a highly potent, selective, orally bioavailable allosteric SHP2 inhibitor (SHP2WT IC50 = 2.7 nmol/L) with favorable pharmacokinetic profiles (F = 42.5%; t1/2 = 2.47 h). Both dual inhibition biochemical assay and docking analysis indicated that TK-642 likely bound to the “tunnel” allosteric site of SHP2. TK-642 could effectively suppress cell proliferation (KYSE-520 cells IC50 = 5.73 μmol/L) and induce apoptosis in esophageal cancer cells by targeting the SHP2-mediated AKT and ERK signaling pathways. Additionally, oral administration of TK-642 also demonstrated effective anti-tumor effects in the KYSE-520 xenograft mouse model, with a T/C value of 83.69%. Collectively, TK-642 may warrant further investigation as a promising lead compound for the treatment of esophageal cancer.

Abstract 5527: Spatiotemporal analysis of growth factor regulation of osteosarcoma AKT and ERK signaling dynamics in an ex vivo lung metastasis model

Abstract In the present study, we investigated the relationships between spatiotemporal distribution of growth factors in the lung microenvironment, metastatic osteosarcoma cell signaling, and intratumoral gene expression heterogeneity. Osteosarcoma is an aggressive bone malignancy that commonly metastasizes to the lungs, resulting in significant patient mortality. During metastasis, disseminated cancer cells are exposed to microenvironment-derived growth factors that result in signaling pathway activation. Two such pathways are MAPK/ERK and PI3K/AKT, which regulate complex transcriptional programs underlying cell proliferation and survival decisions. In this study, we utilized precision cut lung slices (PCLS) obtained from mice with established osteosarcoma metastases to ex vivo model associations between the spatiotemporal distributions of growth factors in the lung, osteosarcoma signaling dynamics, and intratumoral gene expression heterogeneity, with single cell precision. To accomplish this, we engineered osteosarcoma cells that co-expressed genetically-encoded ERK and AKT sensors, engrafted them into PCLS tissues, then recorded sensor dynamics via live-cell microscopy. These tissues were subsequently fixed and immuno-stained for measurement of growth factor expression in resident lung cells and spatial correlation with observed tumor cell ERK-AKT signaling dynamics. Using this approach, we found that growth factor expression was diffusely increased within areas bordering osteosarcoma metastases. FGF2 and IGF1, which we have previously demonstrated to potently induce ERK signaling in osteosarcoma cells, were both significantly upregulated in these regions. However, despite relatively uniform overexpression of growth factors adjacent to tumors, individual osteosarcoma cells displayed stochastics patterns of ERK and AKT signaling, and downstream gene targets. These findings revealed that upregulation of lung growth factors, and the inherent systems-level properties of osteosarcoma signaling networks, can converge to promote intratumoral signaling and gene expression heterogeneity. Together, our results, using a novel PCLS and live-cell biosensor imaging approach, provide new insight into how spatial and temporal changes in microenvironmental signaling composition are integrated by metastatic osteosarcoma cells to promote heterogeneity. Citation Format: Rawan Makkawi, Ryan D. Roberts, Jeremy Copperman, Alexander E. Davies. Spatiotemporal analysis of growth factor regulation of osteosarcoma AKT and ERK signaling dynamics in an ex vivo lung metastasis model [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 5527.

Abstract 5535: Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors

Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that commonly metastasizes to distant organs, such as the lung, resulting in the poor clinical outcomes. TNBC cells commonly overexpress epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) which when bound by microenvironmental growth factors, results in the activation of downstream effector kinases ERK-AKT and initiates transcriptional programs that control cell fate. Tumor cells, however, simultaneously receive many growth factor inputs from their respective microenvironments, many of which stimulate RTKs other than EGFR, that converge at ERK and AKT resulting in different transcriptional and cell fate outputs. This raises questions as to how tumor cells perceive, decode, and respond to a complex set of microenvironmental signals and what the relevance of this process may be for disease pathogenesis and treatment. To this end, our study aimed to determine how disseminated breast cancer cells perceive and respond to a complex growth factor milieu upon seeding and engraftment in the lung. Using live-cell microscopy techniques and TNBC cells carrying biosensors for ERK and Akt signaling pathways, we monitored the dynamic signaling behaviors of single tumor cells, in real-time, as they adapted to the environment in living human lung tissue. Strikingly, we found that individual disseminated tumor cells are less responsive to growth factors once seeded in the lung, as compared to outside this tissue, despite the presence of RTK stimulating ligands. We showed that partial activation of EGFR is necessary for complete activation of ERK signaling by other growth factor-RTK pairs, and expression of downstream ERK target genes, whereas AKT signaling was unaffected. Using quantitative modeling and experimentation, we showed that reshaping of ERK signaling response dynamics occurred via dysregulation of negative regulators that are dominantly controlled by EGFR signaling. Together, these results provide novel insight into how tumor cells perceive and respond to complex microenvironmental signals, which has important implications for drug targeting strategies. Citation Format: Vaibhav Murthy, Cemal Erdem, Jeremy Copperman, Marc Birtwistle, Alexander Davies. Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors [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 5535.

Abstract 1672: PKC-ι and 14-3-3 are crucial regulators of c-Raf and the Erk pathway in human medulloblastoma

Abstract Medulloblastoma is the second most common childhood cancer, behind leukemia, and the most common pediatric brain tumor, comprising 20% of all childhood brain cancers. Current treatment options are limited to radiation therapy, surgical resection, and chemotherapy, which are extremely invasive. This underscores the need for more targeted and effective therapeutic treatments to improve patient outcomes. The following study will focus on exploring the PKC-ι specific inhibitor 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and its effects on medulloblastoma cell lines Daoy and ONS-76. Our in-vitro treatments showed that PKC-ι functions an oncogenic protein that is necessary for Daoy and ONS-76 cell proliferation and that ICA-1S was found to be cytostatic to both Daoy and ONS-76 cells. The inhibition of PKC-ι in both cell lines resulted in a decreased phosphorylation of PKC-ι as well as downregulation of 14-3-3 and crucial components of the c-Raf/Erk pathway including total and phosphorylated levels of c-Raf, Mek1/2, and Erk1/2. This comes as a result of ICA-1S blocking the association of PKC-ι with 14-3-3 while PKC-ι and c-Raf were not found to directly associate with each other, indicating indirect modulation of c-Raf by PKC-ι. Additionally, PKC-ι inhibition decreased its association with Stat3. Expression levels of Stat1, Stat3, c-Fos, and c-Myc transcription factors were significantly downregulated, which is likely the result of reduced transcriptional activation due to upstream inhibition of PKC-ι and Erk signaling. Treatment with ICA-1S was also shown to substantially decrease cellular invasion of Daoy and ONS-76 cells and induce apoptosis. Taken together, these data suggest that PKC-ι likely plays a critical role in the regulation of the c-Raf/Erk pathway, which controls the proliferation and invasion of human medulloblastoma. Citation Format: Luke Lajmi, Wishrawana S. Ratnayake, Joseph Palathinkal, Sloan Breedy, Aaron Todman, Mildred Acevedo-Duncan. PKC-ι and 14-3-3 are crucial regulators of c-Raf and the Erk pathway in human medulloblastoma [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 1672.

Abstract 7070: MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition

Abstract Recent studies have connected dysregulation of H2A.Z deposition to glioblastoma as well as lung, pancreatic, and metastatic breast cancers. The H2AZ1 and H2AZ2 isoforms, collectively known as H2A.Z, are histone variants that regulate gene expression, stem cell identity, and differentiation during development and in homeostatic tissue. Most tumors originate from epithelial tissues. We utilize skin epidermis, an accessible model of epithelial tissue, to understand the regulatory mechanisms controlling proliferation and differentiation. Primary human epidermal keratinocytes retain their abilities to proliferate, differentiate, and stratify in vitro. First, we observed that progenitor keratinocytes express high levels of H2A.Z mRNA and protein, and that H2A.Z levels significantly and progressively decrease during four days of calcium-induced differentiation. Using ChIP-sequencing, we find a significant, genome-wide decrease in H2A.Z chromatin occupancy in differentiated keratinocytes. Therefore, we hypothesized that H2A.Z is critical for progenitor function. Knock down of either H2AZ1 or H2AZ2 significantly impaired keratinocyte proliferation in holoclone assays. Furthermore, H2AZ1 or H2AZ2 knockdown inhibited epidermal stratification in organotypic culture. Canonically, two chromatin remodeling complexes, SRCAP and EP400, deposit H2A.Z into nucleosomes. While both SRCAP and EP400 knockdown diminish keratinocyte proliferation, only SRCAP knockdown decreases H2A.Z deposition in epidermal keratinocytes. Finally, we find that MEK and ERK signaling is required to sustain expression of SRCAP, H2AZ1, and H2AZ2. MEK or ERK inhibition is sufficient to significantly decrease H2A.Z occupancy genome-wide in progenitor keratinocytes. Citation Format: Stephenie Droll, Benny Zhang, Celia Xue, Maxwell Levine, Xiaomin Bao. MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition [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 7070.

Abstract 1966: The FLT3 tyrosine kinase receptor ITD mutation controls its expression and drug resistance in acute myeloid leukemia

Abstract In approximately 23% of AML patients, Fms-like tyrosine kinase 3 (FLT3) contains a gain-of-function, internal tandem duplication (ITD) mutation that is associated with an unfavourable prognosis. FLT3 is therefore seen as a promising therapeutic avenue for AML and as a result, a more complete understanding of its function and signalling may lead to additional targets and resistance mechanisms being identified. To date, several FLT3 inhibitors including Quizartinib (AC220), a second-generation ITD-selective tyrosine kinase inhibitor, have been developed, but single-agent clinical trials have not been overwhelmingly successful. In most preclinical studies, the inhibitory effects of FLT3 inhibitors are mainly evaluated using mutant-expressing models, however, most AML patients harbour both a wild-type and mutant FLT3 allele as well as presenting with high plasma levels of FLT3 ligand (FLT3L). We hypothesized that FLT3L could act through WT-FLT3 to influence the efficacy of FLT3 inhibitors in cells with heterozygous mutations. In this study, we have examined the role of FLT3 inhibition and FLT3L activation on the cellular localisation and downstream signalling in several AML cell lines, some of which express ITD mutations. We also looked at mechanisms by which FLT3L could impair the efficacy of FLT3 inhibitors. Our results revealed that the majority of FLT3 in the MV4-11 and MOLM-13 cells was intracellular. Inhibition of ITD-FLT3 with quizartinib led to a dramatic relocalisation of FLT3 to the cell surface. This effect was more pronounced in MV4-11 (FLT3ITD/ITD) than the heterozygous MOLM-13 (FLT3ITD/WT) cells that also express WT. This was accompanied by inhibition of ERK, AKT and STAT5 signalling pathways and resulted in cell death. Quizartinib induced cell death only in AML lines expressing FLT3-ITD mutations (MOLM-13 and MV4-11), and this could be antagonised by FLT3 ligand (FLT3L). The largest inhibitory effects were seen in heterozygous cells expressing a mutated and wild-type allele. FLT3 inhibition was associated with downregulation of the anti-apoptotic protein Mcl1 and upregulation of the pro-apoptotic BH3-only protein, Bim. Our experiments indicated that both of these proteins were regulated by both WT and ITD-FLT3 through the MAPK pathway. These results suggest that activation of FLT3 signalling by FLT3L confers resistance to quizartinib through upregulation of Mcl-1 and suppression of Bim expression. Taken together, our data suggest that FLT3 cell surface localisation and expression are controlled by ITD mutations and are key components of drug resistance. The data also suggests a novel therapeutic approach in patients with high plasma FLT3L levels, especially when using type II inhibitors such as quizartinib, is to co-target the MAPK/ERK pathway to abrogate the FLT3Lmediated resistance in FLT3-ITD AML. Citation Format: Taha Alqahtani, David MacEwan. The FLT3 tyrosine kinase receptor ITD mutation controls its expression and drug resistance in 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 1966.

Abstract 3576: CRISPR Cas9 mediated MADD deletion can induce autophagy via downregulation of MAPK and PI3K/AKT/mTOR signaling in anaplastic thyroid cancer

Abstract Thyroid cancer is a highly prevalent endocrine malignancy with one of the fastest growing incidences of all cancers in the United States. Anaplastic thyroid cancer (ATC) has an incidence of about 1.7% of all thyroid cancers, but it causes over 40% of thyroid cancer-related deaths with a median survival period of less than one year. This is why agents that inhibit the aberrant signaling pathways which drive ATC aggressiveness are of significant interest. MAPK-activating Death Domain-activating protein (MADD) plays a key role in cancer cell survival and proliferation. MADD is constitutively expressed at higher levels in most tumor tissues and cancer cells relative to normal tissues. Studies using shRNA suggest that MADD prevents apoptosis in ATC cells. For a more definitive and mechanistic analysis of MADD in ATC, we used conditional CRISPR-Cas9 gene editing to target MADD. We observed that MADD deletion in ATC cells induced spontaneous apoptosis and also activated the autophagy pathway in human ATC cells through downregulation of ERK and mTOR signals involving the MAPK and PI3K/Akt/mTOR signaling pathways. Transcriptomic analysis of publicly available TCGA data showed several PI3K/AKT/mTOR pathway genes were positively correlated with MADD levels. RNA sequencing data of ATC lines demonstrated that enhanced activity of the PI3K/AKT/mTOR signaling pathway was correlated with a worse prognosis in ATC. Targeted MADD deletion in ATC cells combined with treatment using ERK/PI3K/Akt/mTOR/autophagy inhibitors resulted in enhanced cell death compared with MADD deletion alone. Further, MADD deleted 8505C-ATC cells showed significantly delayed tumor formation in an orthotopic nude mouse model. These findings suggest that combining MADD deletion with inhibitors of PI3K/Akt/mTOR pathway could provide an attractive new therapeutic option for ATC. Citation Format: Velavan Bakthavachalam, Manikannan Madhayan, Mark Sanborn, Jalees Rehman, Prabhakar Bellur. CRISPR Cas9 mediated MADD deletion can induce autophagy via downregulation of MAPK and PI3K/AKT/mTOR signaling in anaplastic thyroid 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 3576.

Abstract 608: NEOS-223 is a small molecule kinase inhibitor which induces apoptosis in selected cancer models

Abstract Due to their critical role in cancer signaling pathways, members of the kinase family have emerged as one of the most comprehensively pursued targets in pharmacological cancer research. Deregulation of kinase activities leads to a variety of upstream and downstream signaling pathway changes in cancer cell. The Ras/Raf/MEK/ERK and PI3K/Akt/mTOR cascades are activated by genetic alterations in selected upstream signaling molecules such as receptor tyrosine kinases. Recently developed therapies which target such kinases (EGFR, BRAF, MEK) with kinase inhibitors have notably improved the prognosis within various cancer patient groups. However, patients with different mutations of EGFR, RAS, and BRAF do not respond completely. In this study the potential of NEOS-223 as a new kinase-targeted therapy in selected cancer models was investigated. Multiple cancer cell lines were assessed for cell growth as well as the direct inhibitory effects of NEOS-223 alone and in combination with other clinically approved kinase inhibitors. NEOS-223 treatment caused cell growth inhibition, blocked colony growth, and induced apoptosis in lung, pancreatic, melanoma, and colon cancer cell lines in vitro and demonstrated potent antitumor activity in in vivo xenograft models of colon and lung cancer. We were particularly interested in examining different components of the ERK and PI3K signaling pathways in selected sensitive cell lines, it was identified that NEOS-223 treatment led to downregulation of the expression of pERK and pAKT signaling. Blockage of the PI3K/mTOR pathways showed inhibitory synergy with NEOS-223 in lung cancer in vitro. Most importantly, in vivo data showed that while administration of NEOS-223 alone decreased tumor growth, the addition of PI3K/Akt/mTOR inhibitor onatasertib significantly enhanced anticancer effect in a lung cancer model. In lung and pancreatic cancer models, inhibition of EGFR by the small molecule drug gefitinib strongly showed synergistic effect with NEOS-223 both in vitro and in vivo. This may be because EGFR potentially activates both the mitogen activated protein kinase (MAPK/ERK) and the PI3K/mTOR signaling cascades in different mutant cancer models. These studies reveal that NEOS-223 inhibits cell proliferation and induces apoptosis via suppressing the PI3K/Akt/mTOR pathway and further suggest that the combination of PI3K/Akt/mTOR and EGFR inhibitors and that NEOS-223 would be a strong potential chemotherapeutic strategy against lung and pancreatic cancers. Citation Format: Harish Potu, Sara A. Little, Stephanie Fedorchak, Sumathi Chittamuru, Timothy M Murphy, Roseanne Wexler, Sarah Di Croce, Melanie George, Laxman S. Desai. NEOS-223 is a small molecule kinase inhibitor which induces apoptosis in selected cancer 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 608.

Abstract 6955: Activation of ERK pathway by mutant Ras oncoproteins derived from THP-1 cell exosomes

Abstract The study of exosomes has attracted worldwide research due to its ability to modulate cellular activities, especially in the progression of cancer. Exosomes are membrane vesicles that can deliver lipids, proteins, and nucleic acids from one cell to another through membrane fusion, providing a unique form of cell-to-cell communication. It has been shown that KRAS G12V oncoprotein has been detected inside various pancreatic cancer cell derived exosomes. Our goal for this study was to examine the effect of KRAS G12V oncoprotein, detected inside exosomes isolated from THP-1 cell line supernatant, to activate MAPK pathway in MCF-7 breast cancer and AsPC-1 pancreatic cancer cells. To this end, we developed MILLIPLEX® immunoassays to detect cell signaling proteins as well as the exosome markers (CD9, CD63 and CD81), using Luminex® xMAP® technology which allows multiplex detection of proteins in a small volume of sample. Exosomes were isolated from THP-1 derived cell supernatant, using Qiagen exoEasy Maxi kit according to the kit protocol. KRAS G12V oncoprotein was detected in isolated THP-1 exosomes. As a control, THP-1 exosomes were enriched with exosome markers such as CD9, CD63 and CD81. After 48-hour incubation of AsPC-1 cells with KRAS G12V containing THP-1 exosomes, significant increase in KRAS G12V levels were detected in AsPC-1 cells. More importantly, this increase in KRAS G12V levels corresponded with an increase in phosphorylated ERK signaling, but not JNK, p38, NFκB, Akt, STAT3 and STAT3 pathways. In contrast, 48-hour incubation of MCF-7 cells with KRAS G12V containing THP-1 exosomes had no effect on any signaling pathways. Using MILLIPLEX® multiplex immunoassays, our data supports research findings that exosomes contain mitogenic signaling components that may enhance progression of cancer. Citation Format: Joseph B. Hwang, Laura Marquardt, Osama Sait, Brooke Gilliam, Qiang Xiao. Activation of ERK pathway by mutant Ras oncoproteins derived from THP-1 cell exosomes [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 6955.

Abstract 4735: Targeting CDK1/2-driven mechanisms of resistance to BRAF and EGFR inhibition in BRAF(V600E) colorectal cancer restores therapeutic response

Abstract Purpose: Metastatic colorectal cancer (mCRC) is the second leading cause of cancer death worldwide. Roughly 8% of mCRC tumors harbor a BRAF(V600E) mutation, associated with the worst prognosis and limited treatment options. Most CRC research and most clinical trials of targeted therapies have narrowly focused on targeting vertical mechanisms of MAPK re-activation (i.e., EGFR-MAPK), with incremental advances for patients. New approaches are needed to address how therapeutic resistance is mediated by coordinated upregulation of multiple signaling pathways. Methods and Recent Advances: Using an innovative high-throughput kinase-activity mapping (HT-KAM) platform and kinase network resource (PhosphoAtlas), we discovered that SRC kinases drive resistance to BRAF ± anti-EGFR therapy independently of ERK signaling by inducing adaptive transcriptional changes via beta-catenin. This compensatory activation of SRC was mediated by an autocrine PGE2-loop that could be blocked with COX2 inhibitors (COX2i). Using patient-derived xenograft (PDX) models established from pre-treatment biopsies in clinical trial participants, we showed that targeting COX2 in combination with standard of care (SOC) BRAFi + EGFRi is an effective drug repurposing strategy to stabilize tumor growth in vivo. New, Unpublished Findings: Building on our drug discovery pipeline to find and target orthogonal modalities of therapeutic resistance, we leveraged our bank of residual PDX tumors collected after 21-day exposure to SOC therapy, and now report that CDK1 and CDK2 are at the center of a stress response program that enables BRAF(V600E) CRC tumor cells to survive under therapeutic pressure. We used HT-KAM to interrogate the activity of 165 kinases and kinase families, and integrated these profiles using computational methods to model signaling pathways. Out of the reprogrammed kinomes of BRAFi + EGFRi resistant tumors, the CDK1/2 kinases and mechanisms of cell cycle checkpoint emerged as critical dependencies. We validated these findings using multiplex immuno-fluorescence in residual tumors, protein/gene expression profile of tissues and cell lines (e.g., upregulation of Cyclins and CDC25s), and cell viability assay using dinaciclib, a CDK1/2 inhibitor. Considering that COX2i has minimal toxicity, yet improved tumor growth inhibition when added to SOC, we evaluated whether jointly blocking inflammatory pathway (COX2) and cell cycle checkpoints (CDK1/2) could overcome resistance. We found that co-targeting CDK1/2 along with BRAFi + EGFRi ± COX2i is well-tolerated in mice and promotes significant, durable suppression of tumor growth in multiple PDX models, including tumor regression, over 9 weeks of treatment. Conclusion: When tumors progress on SOC BRAFi + EGFRi, a therapeutic window remains open to switch to a CDK1/2i-containing regimen and restore cancer control. Citation Format: Jean-Philippe Coppe, Denise P. Muñoz, Chloe E. Atreya, Yeonjoo Hwang, Mehdi Bouhaddou, Louise E. Heinrich, Mohammad Naser, Ons Zakraoui, Roxana Pirker, Rupa Guha, Christina Moelders, Alessandro Scapozza, Cecilia Schwarzer, Layla Phillips, Changjun Wang, Laura van 't Veer, Veronica Steri, Scott R. VandenBerg. Targeting CDK1/2-driven mechanisms of resistance to BRAF and EGFR inhibition in BRAF(V600E) colorectal cancer restores therapeutic 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 4735.

Abstract 592: A novel pMEK inhibitor ABM-4095 for the treatment of pancreatic cancer

Abstract The MEK-ERK pathway is often activated in tumors by mutations of upstream BRAF- or RAS-proteins. The RAS is frequently mutated in several cancers, especially in pancreatic cancer with an about 95% rate. Pancreatic cancer has the highest mortality rate with a 5-year survival rate of 8% and a median survival of 6 months. Targeted therapies have been extensively evaluated in pancreatic cancer, however, survival improvement of this aggressive disease using a targeted strategy has been minimal. The only currently approved targeted therapy is erlotinib in combination with the chemotherapy drug gemcitabine. To date, four MEK inhibitors that block RAF-activation have been approved by the FDA, but their ability to inhibit ERK signal activated by RAS-mutations is limited due to the induction of MEK phosphorylation by relief of ERK-dependent feedback inhibition of RAF. Here, we report a novel small molecule, ATP-uncompetitive, pMEK inhibitor ABM-4095 that potently prevents phosphorylation of MEK by RAF with moderate inhibition of MEK kinase activity. Most of MEK inhibitors approved by FDA or in clinical trials increase pMEK levels instead. ABM-4095 has a good cell permeability. Its anti-cancer efficacy has been demonstrated both in the in vitro cancer cell line proliferation assay and in vivo xenograft models. ABM-4095 showed a synergy effect with erlotinib in pancreatic cancer cells. In a Miapaca-2 xenograft model, ABM-4095 showed good dose-dependent tumor inhibition as a single agent or combining with the KRAS G12C inhibitor AMG-510, and tumor regressions were observed. In summary, ABM-4095 is a potent pMEK inhibitor with a high activity against the RAS-mutant pancreatic cancer in vitro and in vivo. Detailed preclinical results will be presented. Citation Format: Min Xu, Yong Hu, Lanjiao Zhao, Xiuquan Chen, Youqin Chen, Chen Yang, Jizhi Li, Chen Chen, Xiaobing Lv. A novel pMEK inhibitor ABM-4095 for the treatment of pancreatic 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 592.

Abstract 1730: Unraveling the oncogenic potential of KDM2A with gastric organoids

Abstract Background: Gastric adenocarcinoma (GAC), prevalent and often lethal, imposes a serious global health challenge. Therefore, it is critical to identify novel oncogenes (drivers) that can be therapeutically targeted. Of epigenetic modifications, histone lysine methylation is a hallmark of epigenetic and transcriptional regulation of gene expression. Therefore, investigating the involvement of histone methylation modifiers in GAC is paramount to gaining a comprehensive understanding of the disease but also to discover novel targets. Our analysis of the GAC genome indicates that these epigenetic modifiers are amplified in GAC. Objective and Hypothesis: The overall objective is to define the oncogenic role of KDM2A in GAC pathogenesis and progression. The central hypothesis is that KDM2A promotes GAC by upregulating the expression of oncogenic proteins via the activation of epigenomic signatures/pathways.KDM2A (FBXL11) is a histone lysine demethylase that demethylates H3K36me2 and represses target genes involved in various biological processes, including cell proliferation, DNA damage repair, and stem cell maintenance. Previously, we reported that KDM2A was necessary for the tumorigenic and metastatic capabilities of non-small lung cancer cells through transcriptionally repressing the expression of DUSP3 and HDAC3, which led to activation of ERK signaling and upregulation of cell cycle-associated genes. Results: Our analysis of the GAC genome indicates that KDM2A is one of the top demethylases frequently amplified and overexpressed (14%). In addition, KDM2A expression was significantly higher in GAC samples than in normal tissues, and high expression of KDM2A was associated with poor survival in GAC patients. To examine the effect of KDM2A knockdown on characteristics of GAC, we used gastric organoids generated from KDM2Aflox/flox mouse stomach. We expressed wild-type (Flag-KDM2A) and catalytic mutant (Flag-KDM2A-mut) in these organoids. Ectopic expression of KDM2A wild-type, but not mutant, increased the proliferation of organoids. We showed that KDM2A knockdown decreased the proliferation and invasion of two GAC cells, GA0518 and AGS. In addition, we determine the effect of overexpression of KDM2A on cell proliferation by comparing the control organoids to KDM2A overexpressed using IHC staining of Ki-67 (a proliferation marker) and other activated oncogenic signaling PI3KCA/mTOR/AKT, EGFR. We showed that GA5018 cells were very sensitive to the KDM2A inhibitor (daminozide) alone and in combination with erlotinib (EGFRi inhibitor) and RAD001 (RAD1 mTOR inhibitor). Significance and Innovation: In contrast to advances in cellular signaling pathways in GAC pathogenesis, how GAC development is epigenetically regulated remains largely unknown. Our results from using organoids indicate that KDM2A is an oncoprotein. Citation Format: Shilpa S. Dhar, Calena Brown, Constantin Zod, Jaffer A. Ajani, Min Gyu Lee. Unraveling the oncogenic potential of KDM2A with gastric organoids [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 1730.

Noggin promotes osteogenesis in human adipose-derived mesenchymal stem cells via FGFR2/Src/Akt and ERK signaling pathway

The balance between Noggin and bone morphogenetic proteins (BMPs) is important during early development and skeletal regenerative therapies. Noggin binds BMPs in the extracellular space, thereby preventing BMP signaling. However, Noggin may affect cell response not necessarily through the modulation of BMP signaling, raising the possibility of direct Noggin signaling through yet unspecified receptors. Here we show that in osteogenic cultures of adipose-derived stem cells (ASCs), Noggin activates fibroblast growth factor receptors (FGFRs), Src/Akt and ERK kinases, and it stabilizes TAZ proteins in the presence of dexamethasone. Overall, this leads ASCs to increased expression of osteogenic markers and robust mineral deposition. Our results also indicate that Noggin can induce osteogenic genes expression in normal human bone marrow stem cells and alkaline phosphatase activity in normal human dental pulp stem cells. Besides, Noggin can specifically activate FGFR2 in osteosarcoma cells. We believe our findings open new research avenues to further explore the involvement of Noggin in cell fate modulation by FGFR2/Src/Akt/ERK signaling and potential applications of Noggin in bone regenerative therapies.

Dysregulated fibrinolysis and plasmin activation promote the pathogenesis of osteoarthritis.

Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3'-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.

Synthesis and biological evaluation of novel benzothiazole derivatives as potential anticancer and antiinflammatory agents.

Introduction: Cancer, a significant global health concern, necessitates innovative treatments. The pivotal role of chronic inflammation in cancer development underscores the urgency for novel therapeutic strategies. Benzothiazole derivatives exhibit promise due to their distinctive structures and broad spectrum of biological effects. This study aims to explore new anti-tumor small molecule drugs that simultaneously anti-inflammatory and anticancer based on the advantages of benzothiazole frameworks. Methods: The compounds were characterized by nuclear magnetic resonance (NMR), liquid chromatograph-mass spectrometer (LC-MS) and high performance liquid chromatography (HPLC) for structure as well as purity and other related physicochemical properties. The effects of the compounds on the proliferation of human epidermoid carcinoma cell line (A431) and human non-small cell lung cancer cell lines (A549, H1299) were evaluated by MTT method. The effect of compounds on the expression levels of inflammatory factors IL-6 and TNF-α in mouse monocyte macrophages (RAW264.7) was assessed using enzyme-linked immunosorbent assay (ELISA). The effect of compounds on apoptosis and cell cycle of A431 and A549 cells was evaluated by flow cytometry. The effect of compounds on A431 and A549 cell migration was evaluated by scratch wound healing assay. The effect of compounds on protein expression levels in A431 and A549 cells was assessed by Western Blot assay. The physicochemical parameters, pharmacokinetic properties, toxicity and drug similarity of the active compound were predicted using Swiss ADME and admetSAR web servers. Results: Twenty-five novel benzothiazole compounds were designed and synthesized, with their structures confirmed through spectrogram verification. The active compound 6-chloro-N-(4-nitrobenzyl) benzo[d] thiazol-2-amine (compound B7) was screened through a series of bioactivity assessments, which significantly inhibited the proliferation of A431, A549 and H1299 cancer cells, decreased the activity of IL-6 and TNF-α, and hindered cell migration. In addition, at concentrations of 1, 2, and 4μM, B7 exhibited apoptosis-promoting and cell cycle-arresting effects similar to those of the lead compound 7-chloro-N-(2, 6-dichlorophenyl) benzo[d] thiazole-2-amine (compound 4i). Western blot analysis confirmed that B7 inhibited both AKT and ERK signaling pathways in A431 and A549 cells. The prediction results of ADMET indicated that B7 had good drug properties. Discussion: This study has innovatively developed a series of benzothiazole derivatives, with a focus on compound B7 due to its notable dual anticancer and anti-inflammatory activities. B7 stands out for its ability to significantly reduce cancer cell proliferation in A431, A549, and H1299 cell lines and lower the levels of inflammatory cytokines IL-6 and TNF-α. These results position B7B7 as a promising candidate for dual-action cancer therapy. The study's mechanistic exploration, highlighting B7's simultaneous inhibition of the AKT and ERK pathways, offers a novel strategy for addressing both the survival mechanisms of tumor cells and the inflammatory milieu facilitating cancer progression.