Targeting Cholesterol Homeostasis Enhances Phagocytosis of Acute Myeloid Leukemia Cells
Authors: Ghosh Somadri , Reinbach Katrin , Peña-Martínez Pablo , Ramakrishnan Ramprasad , André Oscar , Rodriguez Zabala Maria , Nordenfelt Pontus , Järås Marcus 
Published: December 2024

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and is associated with poor outcome. While allogeneic stem cell transplantation can be curative, it is often accompanied by severe side-effects. Recently, there has been emerging recognition that the innate immune system is suppressed in AML and new therapies that restore it could have therapeutic potential. In particular macrophages, which belong to innate immunity, are suppressed by AML cells that protect them from phagocytosis.

To identify cell surface proteins that protect AML cells from phagocytosis, we here performed a CRISPR-Cas9 dropout screen targeting about 1,000 genes encoding cell surface proteins expressed by AML cells. In the screen, we co-cultured THP-1 cells (an AML cell line) with primary macrophages for three hours. The MHC class-I molecule HLA-A, a known inhibitor of phagocytosis, scored as the top hit confirming that the screen was robust. MFSD2A, SCARB2 and LRP4, all associated with cholesterol homeostasis, were also among the top hits. While validations confirmed that disruption of any of these genes increased phagocytosis of THP-1 cells, the most pronounced effect was observed following MFSD2A knockdown (KD). MFSD2A disruption in THP-1 cells resulted in a 2.0-fold (p&lt;0.001) increase in phagocytosis by macrophages. This was further validated in the AML cell lines Monomac6 (MM6) and MOLM-13, each showing a corresponding 1.5-fold (p&lt;0.01) increase in phagocytosis.

To identify the mechanism behind the enhanced phagocytosis upon MFSD2A KD, RNA sequencing was performed in THP-1 cells. Gene set enrichment analysis suggested an altered cholesterol homeostasis in the MFSD2A KD cells. This finding was verified by a colorimetric assay showing that MFSD2A KD resulted in significantly (p&lt;0.05) reduced cholesterol levels. Additionally, the MFSD2A KD cells exhibited downregulation of several genes (DLG1, RFTN1, and CAV2) related to assembly of lipid rafts. Immunofluorescence staining of the lipid raft marker ganglioside GM1, using the cholera toxin B-subunit, confirmed a significant reduction of lipid rafts (p&lt;0.0001). As HLA-A that inhibits macrophages is localized to lipid rafts, we next evaluated the cell surface expression of HLA-A. In line with the reduced phagocytic activity and decrease in lipid rafts, MFSD2A KD led to a 2.2-fold (p&lt;0.0001) reduction in HLA-A expression. Overexpressing a MFSD2A single guide RNA-resistant cDNA in the MFSD2A KD cells significantly restored the phagocytosis to basal levels as well as increased the HLA-A expression, confirming the specificity of the MFSD2A KD.

To further explore if suppression of cholesterol synthesis affects phagocytosis of target cells, AML cells were treated with Lovastatin, an HMG CoA Reductase (HMGCR) inhibitor. Similar to MFSD2A disruption, Lovastatin treatment of THP-1 cells decreased lipid rafts, reduced HLA-A expression, and enhanced phagocytosis. Supplementing the media with mevalonate, the downstream product of HMGCR enzymatic activity, significantly reversed the effects of Lovastatin, revealing a previously unknown dependency between cholesterol biosynthesis and phagocytosis in AML. Similar effects were observed in the AML cell lines MM6, MOLM-13, MV4-11, HNT-34 and OCI-AML3. To assess the clinical relevance of these findings, we tested the effect of Lovastatin on 26 primary AML patient samples of pediatric and adult origin. Following overnight treatment, an increased phagocytosis was observed in 65% of the samples (p&lt;0.0001). In contrast, no effect of Lovastatin was observed in CD34+ normal bone marrow cells. Moreover, HLA-A expression was also attenuated upon treatment across the responders (p=0.009).

Taken together, we here identified MFSD2A on AML cells as a novel regulator of phagocytosis by controlling cholesterol homeostasis. Mechanistically, MFSD2A disruption lowers cholesterol levels, reduces lipid rafts and HLA-A expression. Pharmacological inhibition of cholesterol synthesis by Lovastatin results in enhanced phagocytosis of AML cells. These findings reveal cholesterol homeostasis as a novel dependency of AML cells, findings that may translate into new therapeutic opportunities.