Combination of CDK4/6 inhibitory and anti-hypoxia miRNA-6883 lipid nanoparticles with ferroptosis inducers or MEK inhibitors in preclinical breast and colorectal cancer models

Introduction MicroRNA 6883-5p (miR-6883) inhibits cancer proliferation and hypoxia signaling by silencing the cyclin-dependent kinases CDK4/6, inducing downstream degradation of Hypoxia-Inducible Factor 1alpha (HIF1alpha). Prior work demonstrated that CDK4/6 inhibition (CDK4/6i) induces HIF1alpha degradation through a VHL-independent mechanism via the E3 ubiquitin ligase SMURF2. Notably, several preclinical studies have reported synergy between CDK4/6i and MEK inhibition (MEKi). Others have reported that CDK4/6i sensitizes cancer cells to ferroptosis inducing agents, though this observation is not unequivocally supported and may be context-dependent. Here, we investigate combinations of miR-6883 lipid nanoparticles (LNPs) with MEKi or ferroptosis inducers using in vitro assays and in vivo murine xenograft models. Methods A hypoxia response element (HRE)-driven reporter (Addgene #118706) was transduced into HCT116 colorectal carcinoma and SK-BR-3 HER2+ breast cancer (BC) cells. For in vivo experiments, 2 million HCT116-HRE cells were injected subcutaneously into nude mice and tumors ≥150mm3 were treated with 0, 1, or 10µg LNP-encapsulated miRNA. For viability assays, 4,000 cells/well were plated and allowed to adhere overnight. 48-72h after treatment, viability was assessed by CellTiter-Glo®. In vitro hypoxia experiments were conducted in a 0.5% O2 hypoxia workstation. Results As a single agent, miR-6883 LNPs significantly attenuated HIF signal in the HCT116-HRE xenograft model, with a 94.3% reduction in luminescence observed 3 days post-treatment with 1µg miR-6883 compared to baseline. Ki67 was significantly reduced with a 10µg dose, supporting the antiproliferative activity of miR-6883. In vitro, the SK-BR-3-HRE model demonstrated a significant reduction in HIF activity with miR-6883 treatment under 0.5% O 2 , suggesting that the anti-hypoxia effect of miR-6883 extends to BC. In vitro viability assays were conducted in HCT116 (colorectal), SK-BR-3 (HER2+), MCF-7 (ER+), MDA-MB-231 (triple-negative), and MDA-MB-468 (triple-negative). Combination of small-molecule CDK4/6i and ferroptosis inducers (RSL3 or erastin) or MEKi (trametinib) revealed largely positive synergy scores. Accordingly, miR-6883 LNPs sensitized cells to doses of RSL3, erastin, or trametinib that were ineffective as monotherapies. Conclusions Our data support the anti-hypoxia and antiproliferative activity of miR-6883, and provide a basis for combination therapies with ferroptosis inducers or MEKi. Future experiments will employ BC xenografts and assess long-term tumor growth inhibition of combination therapies.