Statins Kill Acute Myeloid Leukemia Cells through Low-Density Lipoprotein Receptor-Mediated Unfolded Protein Response Activation

Statins, widely known for their cholesterol-lowering properties, have also shown anti-tumor effects, including against acute myeloid leukemia (AML) cells. Previously, most evidence for these effects came from in vitro studies. However, the SWOG S0919 trial, which evaluated a combination of idarubicin, cytarabine, and pravastatin in relapsed/refractory AML cases, reported a high complete response (CR/CRi) rate of 75%. Moreover, a recent retrospective analysis of a large national cohort revealed that statin use in patients with myelodysplastic syndromes was associated with a reduced risk of AML progression and prolonged overall survival. These studies have rekindled interest in the anti-leukemic effects of statins. Nevertheless, the precise mechanisms underlying these effects remain unclear.

To elucidate these mechanisms, we conducted a genome-wide CRISPR-Cas9 knockout screening using THP-1 cells, a representative AML cell line, with or without statin treatment. The cells were cultured in the presence of either 2µM atorvastatin, 2µM fluvastatin, or dimethyl sulfoxide (DMSO) as a control for 20 days.

The screening analysis identified several key genes that are associated with statin-induced proliferation suppression. Notably, LDLR (low-density lipoprotein receptor) and LDLRAP1 (LDLR adaptor protein 1) were among the top hits, with false discovery rate (FDR) < 0.01 and log2-fold change (LFC) > 4. Conversely, MYLIP (myosin regulatory light chain interacting protein), a ubiquitin ligase targeting LDLR, was among the negative hits (FDR < 0.25), implicating that LDLR is a key factor in statin-mediated AML cell death.

Subsequent pathway analysis using the Hallmark gene sets from the Molecular Signatures Database (MSigDB) revealed significant enrichment of genes involved in the Unfolded Protein Response (UPR) pathway among the positive hits (FDR < 0.01) under either statin. The UPR pathway is known to induce cell death under excess endoplasmic reticulum (ER) stress, mainly caused by an increase in improperly folded proteins.

Based on these results, we hypothesized that UPR induced by LDLR kills AML cells under statin treatment. We confirmed that the expressions of LDLR and ER stress markers (BiP, ATF4, CHOP) were significantly upregulated after statin treatment in THP-1 cells. Western blotting demonstrated the activation of downstream signals of ER stress, including phospho-SAPK/JNK and cleaved caspase-3. To further investigate the biological role of LDLR, we created THP-1 cell lines with LDLR knocked out by CRISPR-Cas9. Strikingly, these knockout cells showed no significant increase in ER stress markers.

LDLR knockout cells exhibited significantly decreased apoptosis under either statin treatment (p < 0.01), as measured by Annexin V assay using flow cytometry. While 72h exposure to 2µM atorvastatin suppressed proliferation of control THP-1 cells by more than 50%, as determined by ATP assay, LDLR knockout cells showed over 50% survival even at 16µM atorvastatin. Conversely, THP-1 cells constitutively overexpressing LDLR were significantly more sensitive to statin treatments.

These findings indicate that AML cells are killed via a UPR mechanism induced by statin-mediated increased LDLR expression. While the mechanism by which excess LDLR induces UPR requires further investigation, our CRISPR screening has specified a pathway related to AML cell death under statin treatment. As high LDLR expression is an independent poor prognostic factor and cause of chemoresistance, statins may potentially improve outcomes for this challenging subpopulation. Furthermore, the identification of UPR stress as a major cause of AML cell death opens possibilities for developing novel molecular targeted therapies.

In conclusion, our study revealed that statin-induced AML cell death is mediated by LDLR-induced UPR activation, providing new insights into potential therapeutic strategies for AML treatment.