Mutant KRAS Enhances Stress Granules and Resistance to Proteasome Inhibition Via 15-d-PGJ2 in Multiple Myeloma

Introduction: Mutant KRAS leads to activation of the MEK/ERK pathway in approximately 20% of multiple myeloma (MM) patients. Stress granules (SGs) are cytosolic non-membranous structures composed of translational mRNAs, ribosomal proteins, and RNA-binding proteins, which form in response to different stress stimuli and chemotherapeutic treatment. We have previously shown that mutant KRAS upregulates SG formation. The molecular mechanisms underlying this process are unclear. The purpose of this study was to elucidate a) the mechanism by which mutant KRAS upregulates SG formation and b) to provide a molecular rationale for the targeting the cascades for SG formation in MM patients. Methods. We determined whether mutant KRAS is necessary for SG upregulation. To this purpose a panel of MM cell lines including mutant KRAS (KRASM), mutant NRAS (NRASm) and wildtype KRAS (KRASWT) was used. The gain- and loss-of-function of mutant KRAS was generated to suppress (KRASM/KO) in KRASM or switch on by expression of mutant KRAS G12A (KRASG12A) in KRASWT cells by a lentiviral vector system. SG formation was assessed by measuring the cellular distribution of SG resident proteins by immunofluorescence using anti-G3BP and anti-eIF4G antibodies. The quantitative readout for SG formation was analyzed by ImageJ analyze-particle tool as the SG index. Immunoblotting was used to detect inactivation of eIF2a or activity of MAPK. qPCR analysis was employed to detect the mRNA of COX2. MTT assays were used to detect the effect of SGs on the survival of MM cells alone or in combination with diclofenac sodium (DS), or bortezomib (Bzb). Results: We compared the induction of SGs in response to oxidative stress across a panel of MM cells with different Ras mutation status (KRASM, NRASM and KRASWT)to investigate the role mutant KRAS in GS formation. The level of SGs were significantly higher (p We investigated whether 15-d-PGJ2-depedent inactivation of elF4A is involved in SG upregulation to determine the mechanism through which mutant KRAS promotes SG formation. To do this, we assessed the effect of 15-d-PGJ2 on SG formation because that elF4A inactivation is controlled by 15-d-PGJ2, which is one of COX2-derived products. Treatment of KRASM cells with 15-d-PGJ2 significantly induced accumulation of SGs compared with untreated cells (40-fold increase vs control cells, p Conclusion: Our results suggest that mutant KRAS mutant upregulates SG formation by 15-d-PGJ2 and inhibition of COX2 activity enhances KRAS sensitivity to proteasome inhibition and targeting SG formation might be represent an effective strategy to treatment of KRAS mutant myeloma. Disclosures Davies: Janssen, Celgene: Other: Research Grant, Research Funding; Amgen, Celgene, Janssen, Oncopeptides, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Consultant/Advisor. Morgan: Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding. Walker: Celgene: Research Funding. van Rhee: Karyopharm Therapeutics: Consultancy; Kite Pharma: Consultancy; Adicet Bio: Consultancy; EUSA: Consultancy; Castleman Disease Collaborative Network: Consultancy; Sanofi Genzyme: Consultancy; Takeda: Consultancy.