Mutant KRAS and Brafs Upregulate Stress Granules and Mediate Drug Resistance, Which Can be Modulated By Cox2 Inhibition in Multiple Myeloma

Introduction: Mutant RAS leads to activation of the RAS/RAF/MEK/ERK pathway in approximately 50% of multiple myeloma (MM). Stress granules (SGs) are non-membranous structures composed of translational mRNAs, ribosomal proteins, and RNA-binding proteins, which form in response to different stress stimuli and chemotherapeutic treatment. TheKRAS effector pathway mediates the upregulation of SGs conferring resistance to chemotherapeutic agents in solid tumors. However, it is unclear if SGs are upregulated in KRAS mutant MM and if this is associated with chemotherapeutic resistance. The purpose of this study was to characterize the role of RAS/RAF mutants in SG formation and to provide a molecular rationale for the clinical testing of combinations targeting RAS mutation with inhibition of SG formation.

Methods. To investigate the association of RAS mutation status with SG formation, we used two K-RAS mutant positive (KRAS^M), two BRAF mutant positive (BRAF^M),and 2 wildtype KRAS (KRAS^WT) and wild type BRAF (BRAF^WT) MM cell lines as models.We assessed the cellular distribution of SG proteins by immunofluorescence using anti-G3BP and anti-eIF4G antibodies. To establish a quantitative readout for SG formation (SG index), we applied the ImageJ analyze-particle tool to calculate the fraction of the SG area in the total cell area based on G3BP and eIF4G immunofluorescence (SG area/cell area). 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.

Results:Immunofluorescence analysis showed that exposure of KRAS^M MM cells lines to oxidative stress via treatment with sodium arsenite (SA))was associated with the induction of SGs as indicated by accumulation of cytoplasmic puncta of G3BP and elF4G at high level, compared with RAS^WTMM cells. Quantitative analysis of SG formation by Image J analyze-particle tool revealed that the SG index for G3BP (13.225 ±3.2) in KRAS^M was significantly higher compared with KRAS^WT(2.936 ±1.2,P =0.0004) in response to SA. The SG index for elF4G(32.89 ±6.2) in KRAS^M was significantly higher than that in KRAS^WT(4.96, ±2.3, p<5.03E-5). These results indicate that mutant KRAS may play a regulatory role in SG formation.

Given that BRAF mutation leads to activation of MAPK pathway, we next determined if BRAF mutants regulate SGs in response to SA. MM cells harboring K601N mutation, showed a high level of induction of SG formation following treatment with SA, compared with BRAF^WTcells. The SG index for G3BP (11.52 ±0.89) in BRAF^M was significant higher than that in BRAF^WTcells (4.02 ±1.92; P=1.03E-6). Similarly, the SG index for elF4G (19.2±1.3) in BRAF^Mwas significant upregulated compared with BRAF^WT(6.3 ±2.8, p=5.3E-5). MM cells harboring BRAF mutation at D4594N displayed intermediate positive of SG formation. These results suggest that the presence of the BRAF mutation promotes SG formation in response to oxidative stress.

Since SGs have been shown to contribute to mutant KRAS-mediated resistance to chemotherapeutic reagents in solid tumors and mutant KRAS enhances SG formation by up-regulating cox biosynthesis, we next determine if inhibition of COX2 activity by diclofenac sodium (DS), a pharmacological inhibitor of COX2, was able to overcome Bzb resistance in KRAS and BRAF mutant MM cells. MTT assay showed that the viability index (0.8±0.16) reduced to (0.49 ±0.015) in KRAS^MMM cells treated by 10 nM of Bortezomib (Bzb) alone or to (0.46 ±0.016) with 200 mM of DS alone. A significant decrease of cell viability (0.22±0.01, P<0.001) was observed when treatment with Bzb and DS were combined. Similar synergistic effects of Bzb and DS were observed in BRAF^M cells, compared with BRAF^WT. Taken together, these results indicate that inhibition of COX2 activity enhance KRAS and BRAF mutant MM cell sensitivity to proteasome inhibition.

Conclusion: Our results suggest that KRAS mutant and BRAF mutant MM cells significantly upregulate SG formation and inhibition of COX2 activity enhances KRAS and BRAF mutant MM cell sensitivity to proteasome inhibition. Further experiments are ongoing to determine the molecular mechanism by which KRAS and BRAF mutant MM cells upregulate SG formation and their role in MM cell resistance to chemotherapeutic reagents.