S100A4 Expression Protects AML Cells from V-Atpase Mediated Apoptosis Via the Nuclear Receptor Coactivator, NCOA7

Despite advances in targeted therapies, the 5-year survival rate for Acute myeloid leukemia (AML) remains poor, particularly for patients over 60 years of age, underscoring the urgent need for novel therapeutic approaches. Our group's previous comparison of the nuclear proteome and transcriptome of AML primary blasts with normal CD34⁺ identified several differentially expressed proteins, including S100A4. This calcium-binding protein was significantly overexpressed (5.5-fold) in the nuclei of 11 out of 15 AML patients compared to cord blood derived CD34⁺ cells (Alanazi et al, Leukemia 2020). S100A4 is known to regulate various biological processes including cell cycle progression, proliferation, and differentiation. It has been studied in the context of solid tumor metastasis. However, its potential involvement in hematological malignancies is an emerging area of research, and the underlying mechanisms of its contribution to AML development and progression are not well understood. We demonstrate that S100A4 is essential for AML cell proliferation and survival through a series of in vitro experiments.

Knockdown (KD) of S100A4 using targeted shRNA significantly impaired the growth of AML cell lines, reducing growth by 7-fold (p<0.0001). Flow cytometry analysis revealed that S100A4 KD induced cell cycle arrest, with a 28.9% ± 2.1% increase in G1 phase cells and decrease in S and G2 phases cells compared to control cells (p<0.0001). Additionally, Annexin V/PI staining showed a 2.7-fold increase in early apoptotic cells and a 2.1-fold increase in late apoptotic/necrotic cells in S100A4-depleted cells (p<0.0001). These findings were validated in 5 primary AML samples, where S100A4 KD reduced growth and survival of AML patient blasts by an average of 3.6-fold (p<0.0001).

RNA-sequencing revealed substantial alterations in gene expression profiles following S100A4 KD in AML cell lines (THP-1, TF-1, NOMO-1, OCI-AML2). We identified 3,850 upregulated and 3,205 downregulated genes (fold change>1.5, adjusted p-value <0.05), with enrichment in processes related to inflammation, immune response, cell cycle regulation, apoptosis, and autophagy. Downregulation of S100A4 was associated with a 2.3-fold upregulation of NCOA7 across various AML cells (p<0.001). The mRNA expression data from an AML patient cohort (Ley et al. 2013) revealed a significant negative correlation between S100A4 and NCOA7 expression levels (-0.42, p=2.59×10⁻⁸). This is consistent with our observation that S100A4 KD leads to increased NCOA7 expression in AML cells.

Our study revealed a novel S100A4/NCOA7/V-ATPase axis in AML, where S100A4 KD led to 5-fold upregulation of NCOA7 protein expression (p<0.001). Further investigation into the functional relationship between NCOA7 and S100A4 revealed that NCOA7 CRISPR-cas9 rescued the survival defect in AML cells with S100A4 KD. V-ATPase activity is essential for maintaining normal intracellular pH homeostasis. Acidification of the cytoplasm is a characteristic feature of cells undergoing apoptosis. Intracellular pH measurements verified that V-ATPase activity was increased in S100A4 KD cells, leading to a decrease in intracellular pH. Pharmacological inhibition of V-ATPase in the S100A4-depleted cells using bafilomycin A and concanamycin A, lead to an increase in intracellular pH mirroring the rescue effect and suggesting a mechanism by which S100A4 promotes AML cell survival through downregulation of NCOA7 and activation of V-ATPase. Western blot analysis showed increased expression of V-ATPase subunits ATP6V1B2 (4.5-fold) and ATP6V0A1 (3.4-fold) in S100A4 KD cells (p < 0.001).

Furthermore, we investigated the role of S100A4 in modulating the response of AML cells to chemotherapeutic agents. Overexpression of S100A4 in MV4-11 cells resulted in resistance to Cytarabine and Daunorubicin (p<0.001). In contrast, S100A4 overexpression in KG-1 cells led to increased sensitivity to Cytarabine and Daunorubicin (p<0.0001), highlighting the cell line-specific effects of S100A4 on chemotherapy response. These findings suggest that the impact of S100A4 expression on chemotherapy efficacy may depend on the molecular and cytogenetic background of the AML cells.

Our study reveals a novel S100A4/NCOA7/V-ATPase axis in AML and suggests that targeting S100A4 may improve treatment outcomes by addressing drug resistance and enhancing therapeutic efficacy.

Knapper:Jazz Pharmacueticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria.