Novel Therapeutic Strategy Targeting NHE1 and Its Upstream Activators in Acute Myeloid Leukemia

Increase in Tescalcin (TESC) gene expression and intracellular pH (pHi) have been associated with drug resistance in acute myeloid leukemia (AML). Tescalcin was shown to stabilize the membrane sodium/hydrogen exchanger (NHE1) that maintains a high pHi by H⁺ efflux in exchange for Na⁺. NHE1 has also been shown to be activated by PDGFR, PKC, calmodulin, p90-RSK and ROCK-RhoA, but their relevance to leukemogenesis and drug resistance in AML was unknown. We hypothesized that targeting NHE1 and its upstream activators might offer a novel and effective therapeutic strategy in AML.

AML cell lines and mononuclear cell fraction from peripheral blood (PB) or bone marrow (BM) of AML patients (comprising primarily myeloblasts as shown by microscopic review of cytospin preparations) were treated with inhibitors for 3 days (concentrations: 0.1nM to 10mM) that target potential activators of NHE1. The anti-leukemia effects of these inhibitors were evaluated by PrestoBlue® Cell Viability Reagent as a measure of viable cell number. Their effects on pHi and apoptosis were evaluated by SNARF-1 and Annexin V/7-AAD staining respectively by flow cytometry.

AML cell lines ML2, Kasumi-1, MOLM-13 and MV4-11 (IC₅₀ in mM: 12.2, 13.1, 11.6 and 9.2 respectively) were more sensitive than KG1, NB4, THP-1 and OCI-AML3 (IC₅₀ in mM: 30.7, 24.8, 119.2 and 49.4 respectively) to the growth inhibitory effects of NHE1 inhibitor, 5-(N,N-hexamethylene) amiloride (HMA), accompanied with a larger extent of cellular acidification and apoptosis induction in those 4 HMA-sensitive lines. To look for the upstream activators of NHE1 relevant to AML, the cell lines were treated with specific inhibitors targeting potential NHE1 activators. Both HMA-sensitive and insensitive cell lines were susceptible to the intracellular acidification and growth inhibition by PDGFR and p90-RSK inhibitors. Furthermore, FLT3 inhibitors, sorafenib and quizartinib, also reduced pHi of FLT3-ITD⁺ (Fms-Like Tyrosine Kinase 3 - Internal Tandem Duplication) AML cell lines, MOLM-13 and MV4-11, suggesting that FLT3-ITD might also activate NHE1, resulting in high pHi of FLT3-ITD⁺ AML. Different primary AML samples were treated with inhibitors to NHE1 (n=50), PDGFR (n=50) and p90-RSK (n=36) (Concentration: 100nM to 10mM) in vitro. Their response to the growth inhibitory effect of HMA, accompanied by effective pHi reduction (n=10), correlated with that of PDGFR and p90-RSK inhibitors (Pearson r=0.74, p<0.001 and r=0.73, p<0.001 respectively), supporting the proposition that these signaling pathways might be the critical and common activators of NHE1. Synergism of anti-leukemia effects could also be demonstrated between HMA and PDGFR inhibitors, calculated by Excess over Bliss Additivism (EOBA). To evaluate the clinical relevance of the study, serum was obtained from medical patients treated with high dose amiloride (20 mg daily), an NHE1 inhibitor, for underlying congestive heart failure. Compared with the serum of healthy volunteers, the amiloride-containing serum significantly reduced the pHi (n=10, p=0.001), induced apoptosis (n=4, p=0.04) and potentiated the inhibitory effects of PDGFR inhibitors (n=4, p=0.04) in primary AML samples.

NHE1 might be a potential target in drug-resistant AML and activated by PDGFR, PKC, p90-RSK or both in a patient-specific fashion. Therefore, employing specific inhibitors to target NHE1 and its upstream activators should be explored as novel therapeutic strategy in this group of patients.