Hexamethylene Amiloride Sensitizes AML to Venetoclax through Inducing Lysosomal Membrane Permeabilization

Chemotherapy remains the standard therapeutic approach for acute myeloid leukemia (AML). Although several new drugs have received approval for clinical usage, including the selective BCL2 inhibitor venetoclax, refractory disease or resistance continues to be a recurring theme, and exploring more effective therapies for AML treatment remains necessary. Intracellular pH dynamics are vital determinants for cancer progression and chemoresistance. Tumors maintain a more alkaline intracellular pH (pHi) dependent on proton exporters, including sodium-hydrogen exchangers (NHEs). Elevated NHE1 expression has emerged as a biomarker for the tumorigenesis and prognosis of solid tumors. However, whether targeting NHE1 with inhibitors could inhibit AML cell growth and the underlying mechanisms remain unknown.

We found that NHE1 was highly expressed in AML and predicted a worse prognosis based on data analysis of public database. Moreover, NHE1 knockdown inhibited the growth of AML cells. In addition, NHE1 inhibitor Hexamethylene amiloride (HA) significantly inhibited proliferation and promoted apoptosis in a concentration-dependent manner in both AML cell lines and primary AML cells.

Next, we combined HA with several first-line or novel drugs and found that HA combined with venetoclax significantly inhibited viability of AML cells. AML patients with higher NHE1 expression were less sensitive to BCL-2 inhibitors. This suggested that HA and venetoclax might be an effective combination to treat AML. As expected, HA synergized with venetoclax to inhibit proliferation and promote apoptosis. In vivo potential of HA combined with venetoclax was evaluated in a MV4-11-derived xenograft mouse model (Fig.1). The combinational regimen prolonged survival and alleviated leukemia burden in bone marrow, spleen and peripheral blood. We further validated the effect of HA with venetoclax with primary AML cells. Consistent with results in cell lines, HA combined with venetoclax also exerted synergistic effect in inhibiting viability and promoting apoptosis. RNA-Seq analysis revealed that lysosome pathway was significantly enriched in the combination group. We further verified the increased quantity of lysosome, and the swelling and rupture of lysosome with TEM (Fig.2). Moreover, HA combined with venetoclax induced obvious LGALS3 puncta formation in this cell line, which represented the occurrence of lysosomal membrane permeabilization (LMP), leading to the release of proteases which eventually results in cell death.

In conclusion, our results for the first time provide evidence that the NHE1 inhibitor HA or its combination with venetoclax efficiently inhibits the growth of AML in vitro and in vivo. This study might shed light on the clinical treatment of AML involving proton exporter inhibitors with venetoclax as combinatory regime.

No relevant conflicts of interest to declare.