Single-Cell Profiling of Acute Myeloid Leukemia Identified ARMH1, a Novel Protein Associated with Proliferation, Migration, and Drug Resistance

Despite advancements in treatments, half of pediatric acute myeloid leukemia (AML) patients relapse (Chen, J. & Glasser, C.L., 2020), making it pertinent to identify novel biomarkers that can be used for better stratification of patients post end of induction (EOI) therapy as well as to develop targeted therapies that could lead to higher clinical remission (CR) rates.

Single-cell RNA sequencing (scRNAseq) analysis has revolutionized cancer research by identifying novel cell types, genes, and pathways. Our group used scRNAseq analysis of pediatric AML bone marrow (BM) samples collected at diagnosis (Dx) and EOI to identify the upregulation of multiple genes including a novel gene, Armadillo like helical domain containing 1 (ARMH1)/C1orf228, in cancerous blast cells (Thomas, B.E. et al., 2020). ARMH1 has not been previously associated with AML. Also, ARMH1 expression was observed to be significantly higher at Dx in samples from patients with relapse compared to samples from patients with CR (P<2.2e-16). Independent validation for ARMH1 blast-association using single cell and bulk transcriptome profiling revealed that ARMH1 expression was significantly higher in scRNAseq data of AML Dx blast cells compared to EOI non-blast cells (fold change, FC=1.8, P<2.2e-31) as well as in bulk RNAseq data of Dx samples with high blast% (>90%) compared to EOI samples (FC=1.8, P=.003) (Ulukaya, G. et al., 2021). Further analysis on other leukemias like T cell-acute lymphocytic leukemia (T-ALL) and mixed phenotypic acute leukemia (MPAL) also revealed higher expression of ARMH1 in the blast cells as compared to non-blast cells. Survival analysis on bulk RNAseq data using the survival genie platform (Dwivedi, B. et al., 2022) revealed that higher expression of ARMH1 was significantly associated with poor overall survival (OS) in both AML (P=.003) (Ulukaya, G. et al., 2021) and T-ALL (P=.014) datasets. Comparative gene expression analysis using the the Pediatric Single Cell Cancer Atlas - PedSCAtlas - tool (Mumme, H.L. et al., 2021), revealed negligible expression of ARMH1 in normal lymphoid, myeloid, and erythroid cell lineages, further confirming the leukemic blasts-specific over-expression of ARMH1.

ARMH1 encodes a cytosolic protein of yet unknown function. The malignant blast specificity of ARMH1 suggested a role in leukemic cells' survival/maintenance. To ascertain its functional involvement in cancer cells, ARMH1 was silenced using siRNAs in both normal and AML cell lines. Through in vitro assays, we demonstrate that ARMH1 silencing significantly reduced leukemic cells' capacity to proliferate (P=.0041) and migrate (P=.0001). We also observed slightly increased sensitivity to chemotherapy drug, Cytarabine in the ARMH1 silenced cancer cells compared to control cancer cells in which the gene is fully functional. However, suppression of ARMH1 expression had no effect on cell growth in the normal HEK293t cell line (P=0.913). On the other hand, overexpression of ARMH1 via transient transfection of ARMH1 gene into the AML cell line, made the cells more resistant to Cytarabine and significantly increased both migration (P=.041) and cell proliferation (P=.032).

In conclusion, we demonstrate for the first time that ARMH1, a still poorly understood protein, contributes to the increased proliferation and migration of malignant AML cells. With specific overexpression in blast cells of several pediatric leukemias and the newly discovered roles in proliferation, migration, and chemotherapy resistance, ARMH1 may be a good candidate for targeted treatment of pediatric leukemias.

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