Abstract Pancreatic cancer is the third leading cause of cancer-related deaths with a 5-year survival rate of less than 10%. Approximately 90% of reported cases are driven by KRAS mutations. The ...mutant KRAS acquires its functional localization through interactions with chaperone proteins via its polybasic polyisoprenylated tail. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) mimic this region possibly disrupting the polyisoprenylation-dependent interactions with chaperone proteins. Here, we determine the effect of newly developed PCAIs that were designed to be more water-soluble on cell viability, 3D spheroid invasion, spheroid degeneration and F-actin filaments on MIA PaCa-2 and PANC-1 cells. Of the seven new PCAIs analogs tested, NSL-AB-51 and NSL-AB-45 were the most potent, with EC50 values of 2.5 and 5.6 µM against MIA PaCa-2, and 1.7 and 4.5 µM against PANC-1 cells, respectively. At 5 and 10 µM, NSL-AB-51 decreased the invasion area of 3D spheroids by 98 and 99%, respectively. MIA PaCa-2 spheroids degenerated by 61% after 72 h treatment with 10 µM NSL-AB-51. Treatment with 1.0 μM NSL-AB-51 caused cell rounding and reduction of mean cell areas by 69% and F-actin filaments by 96%. Moreover, to identify the pharmacological target of the PCAIs, NSL-AB-45 was coupled to an epoxy-activated Sepharose resin for affinity trapping of PCAIs-interacting proteins. Affinity-analysis of supernatants and detergent membrane extracts from MIA PaCa-2 and PANC-1 cells revealed single Coomassie band of about 17 kDa that was identified as calmodulin by western blotting analysis. The calmodulin-PCAIs affinity interaction was found to be strongest in the presence of calcium and weakest in the presence of EDTA. These findings reveal that calmodulin is the likely target of PCAIs. Since calmodulin is the chaperone protein responsible for KRAS trafficking and localization to the inner surface of the plasma membrane, this finding strongly suggests, at least in part, an anticancer role for the PCAIs that involve disruption of KRAS functions. Citation Format: Kweku Ofosu-Asante, Jassy Mary Lazarte, Amarender Burra, Nazarius S. Lamango. The anticancer effects of polyisoprenylated cysteinyl amide inhibitors involves interaction with the KRAS chaperone calmodulin 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 566.
Abstract Mutated KRAS is the most common cancer driver gene in lung adenocarcinomas in all racial and ethnic groups, including Blacks/African Americans (B/AAs). Drugs developed to combat KRAS-driven ...cancers become less effective due to intrinsic changes in the cells resulting in resistance and relapse. Due to the enormous challenges encountered in developing direct KRAS inhibitors, agents targeting alternative downstream RAS mediator kinase enzymes of the MAPK and PI3K/AKT signaling pathways have been developed. However, other avenues that directly target KRAS and similar cancer drivers must be explored to obtain alternative effective therapies. PCAIs constitute a group of potential anticancer agents that we developed to specifically disrupt and suppress signaling by hyperactive G-proteins such as mutated KRAS. In previous studies, PCAIs stimulated MEK1/2, ERK1/2, p90RSK and AKT phosphorylation resulting in cell death. The present study aims to determine whether specific inhibitors of the kinases can reverse PCAIs-stimulated phosphorylation and cell death. PCAIs were thus tested against the KRAS mutant NCI- cell line H23 (from a B/AA patient) and compared to KRASG12C inhibitor Sotorasib and other kinase inhibitors drugs. The EC50 values for the PCAIs NSL-YHJ-2-27 and NSL-AB-45 were 4.1 ± 0.3 and 5.5 ± 0.1 μM, respectively, compared to Gefitinib (EGFR), Sotorasib (KRASG12C), Dabrafenib (BRAF), Selumetinib (MEK1/2), Ulixertinib (ERK1/2) and Ipatasertib (AKT) whose EC50 values were 83 ± 12.9 μM, 38 ± 5.5 μM, 33 ± 1.2 μM, 71 ± 5.3 μM, 11 ± 0.2 μM, 12 ± 1.0 μM, respectively. The results indicate that the PCAIs are more effective against NCI-H23 than the other kinase inhibitor drugs. Future work will determine the effects of co-treating cells with the PCAIs and the respective kinase inhibitors on cell viability and PCAIs-induced phosphorylation of the kinases. Supported by grants U54CA233396, U54CA233444, and U54CA233465 from the National Institutes of Health (NIH)/National Cancer Institute (NCI) and the Norris Comprehensive Cancer Center core grant, award number P30CA014089 from the NIH/NCI Citation Format: Justin K. Mensah-Mamfo, Kweku Ofosu-Asante, Jassy Mary Lazarte, Amarender Goud Burra, Sofia A. Lugo, Yong Huang, Ite A. Offringa, Nazarius S. Lamango. Polyisoprenylated cysteinyl amide inhibitors: Effects on RAS signaling pathway intermediates and viability of mutant KRAS African American lung cancer cell line 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 3395.
Abstract
KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) constitute a group of cancer therapy ...agents designed to specifically disrupt and suppress hyperactive G protein signaling, such as that triggered by RAS mutations. Here we determine the effects of PCAIs on the viability, G-proteins levels, downstream mediators, and apoptosis-related proteins on the KRAS-mutant, Black American-derived lung adenocarcinoma cell line, NCI-H23. Of the 17 PCAIs tested, compounds NSL-YHJ-2-27 and NSL-YHJ-2-46 showed the most potency with EC50 values of 2.7 and 3.3 µM, respectively. To determine the effect of the PCAIs on the levels of Mitogen-Activated Pathway Kinase (MAPK)-related enzymes, western blot analysis was performed. After 48 hours exposure to 5 μM of the compounds, 57 to 150% increases of BRAF, ERK1/2, MEK1/2, and p90RSK phosphorylation along with a 60 to 78% decrease of pCRAF were observed, indicating significant disruptions in RAS signaling. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 and 76%, respectively. The depletion of these key cytoskeletal proteins may account for the observed 52 and 42% reduction in cell migration after 24 h exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46, respectively. Western blot analysis was used to determine the effect PCAIs on caspase activation necessary for the initiation of apoptosis. After treatment with 5 μM of NSL-YHJ-2-27, full-length inactive caspase 3 and 7 levels dropped by 72 and 60%, while cleaved active caspase 3 and 7 levels increased by 274 and 130%, respectively. Treatment with 5 μM of NSL-YHJ-2-46 depleted full-length caspase 7 by 53% and increased cleaved active caspase 3 levels by 83%. These findings clearly show the direct effects of the PCAIs on the RAS signaling pathway, perhaps through the activation of proapoptotic isoforms of p90RSK. These results support the potential use of the PCAIs to serve as targeted therapies in cancers harboring RAS mutations.
Citation Format: Matthew D. Gregory, Pablo E. Puente, Nadine L. Belony, Jassy Mary S. Lazarte, Nada Tawfeeq, Yong Huang, Ite A. Offringa, Nazarius S. Lamango. Treatment of a Black American lung cancer cell line harboring KRAS mutations with polyisoprenylated cysteinyl amide inhibitors reveals effects on the MAP kinase signaling pathway, cell migration and apoptosis abstract. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C022.
The causative agent of acute hepatopancreatic necrosis disease (AHPND) is the bacterium,
, which secretes toxins into the gastrointestinal tract of its host.
toxins A and B (PirA
/PirB
) have been ...implicated in the pathogenesis of this disease, and are, therefore, the focus of studies developing treatments for AHPND. We previously produced recombinant antibodies based on the hagfish variable lymphocyte receptor B (VLRB) capable of neutralizing some viruses, suggesting that this type of antibody may have a potential application for treatment of AHPND. Here, recombinant PirA
/PirB
, produced using a bacterial expression system, were used as antigens to screen a hagfish VLRB cDNA library to obtain PirA
/PirB
-specific antibodies. A cell line secreting these antibodies was established by screening and cloning the DNA extracted from hagfish B cells. Supernatants collected from cells secreting the PirA
/PirB
antibodies were collected and concentrated, and used to passively immunize shrimp to neutralize the toxins PirA
or PirB
associated with AHPND. Briefly, 10 μg of PirA
and PirB
antibodies, 7C12 and 9G10, respectively, were mixed with the shrimp feed, and fed to shrimp for three days consecutive days prior to experimentally infecting the shrimp with
(containing toxins A and B), and resulting mortalities recorded for six days. Results showed significantly higher level of survival in shrimp fed with the PirB
-9G10 antibody (60%) compared to the group fed the PirA
-7C12 antibody (3%) and the control group (0%). This suggests that VLRB antibodies may be a suitable alternative to immunoglobulin-based antibodies, as passive immunization treatments for effective management of AHPND outbreaks within shrimp farms.
PDF
