Examining a Unique Cysteine Residue in Regulating Differences Between ERK1 and ERK2 Signaling and Potential as Novel Therapeutic Targets

Abstract ID 99884 Poster Board 089 The extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is a well-studied kinase signaling pathway involved in cell growth and proliferation. Dysregulation of this pathway is responsible for uncontrolled growth and proliferation involved in cancer and other diseases. Several inhibitors of upstream kinases within the ERK1/2 pathway, namely targeting Raf and MEK1/2 at their ATP-binding or catalytic sites have been developed for clinical use. However, acquired drug resistance usually develops, often resulting in lack of durable patient response to kinase inhibitor therapies. Reactivation of ERK1/2 may contribute to the development of drug resistance. Thus, targeted inhibition of ERK1/2 may be an effective approach to overcome or prevent resistance to the current kinase inhibitors. Several new compounds have been identified to inhibit ERK1/2 by targeting the ATP-binding site and blocking all activity. As an alternative, our lab is developing kinase modulators which target substrate binding regions distinct from the ATP-binding site to inhibit select ERK1/2 functions associated with disease. We identified a compound, SF-3-030, that binds to cysteine residue (C254) of ERK2, near a substrate docking site that regulates the expression of oncogenic immediate early gene transcription factors, such as c-Fos and c-Myc. A375 melanoma cells, a cell line containing the constitutively active V600E mutation in BRAF, were used as a model to evaluate the role of this cysteine in regulating ERK1/2 signaling. CRISPR-Cas9 technology was used to generate isogenic A375 cell lines containing cysteine point mutations to compare phenotypes and ERK1/2 signaling with parent cells. Three distinct A375 mutant cell lines were generated and included homozygous mutations converting cysteine to alanine at positions 271 or 254 of ERK1 or ERK2, respectively, or in both isoforms. These cysteines are unique in that they are conserved in the ERK1/2 isoforms but are absent in all other major mitogen-activated protein kinases—namely ERK5, p38, and JNK. Examination of CRISPR-Cas9 off-target effects showed no mutations were introduced in other chromosomes. Cell Titer Blue cell viability assay was used to compare cell growth and revealed reduced cell viability in the ERK1 (C271A) mutant cell lines compared to the parent A375 cells. Interestingly, the ERK2 (C254A) mutation had cell viability as the parent cells. Immunoblotting examination of Fos family proteins showed increased expression of FosB in the ERK1 mutants but not the ERK2 mutants. These data are consistent with previous studies showing that increased Fos B expression is linked to inhibition of cell cycle progression and cell proliferation. Current studies are examining the cell viability of the ERK1/2 (C271A, C254A) mutant cell line and effects on signaling and Fos family protein regulation. These findings reveal potentially novel differences between the ERK1 and ERK2 functions and may challenge the prevailing data suggesting functional redundancy between ERK1/2 isoforms. These cysteines near a substrate docking site may represent a novel therapeutic target for modulating ERK1/2 activity in disease.