An in vitro drug-screening platform on patient samples was developed and validated to design personalized treatment for relapsed/refractory acute myeloid leukemia (AML). Unbiased clustering and correlation showed that homoharringtonine (HHT), also known as omacetaxine mepesuccinate, exhibited preferential antileukemia effect against AML carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD). It worked synergistically with FLT3 inhibitors to suppress leukemia growth in vitro and in xenograft mouse models. Mechanistically, the effect was mediated by protein synthesis inhibition and reduction of short-lived proteins, including total and phosphorylated forms of FLT3 and its downstream signaling proteins. A phase 2 clinical trial of sorafenib and HHT combination treatment in FLT3-ITD AML patients resulted in complete remission (true or with insufficient hematological recovery) in 20 of 24 patients (83.3%), reduction of ITD allelic burden, and median leukemia-free and overall survivals of 12 and 33 weeks. The regimen has successfully bridged five patients to allogeneic hematopoietic stem cell transplantation and was well tolerated in patients unfit for conventional chemotherapy, including elderly and heavily pretreated patients. This study validated the principle and clinical relevance of in vitro drug testing and identified an improved treatment for FLT3-ITD AML. The results provided the foundation for phase 2/3 clinical trials to ascertain the clinical efficacy of FLT3 inhibitors and HHT in combination.
Clinical outcome and mutations of 96 core-binding factor acute myeloid leukemia (AML) patients 18–60 years old were examined. Complete remission (CR) after induction was 94.6%. There was no significant difference in CR, leukemia-free-survival (LFS) and overall survival (OS) between t(8;21) (N=67) and inv(16) patients (N=29). Univariate analysis showed hematopoietic stem cell transplantation at CR1 as the only clinical parameter associated with superior LFS. Next-generation sequencing based on a myeloid gene panel was performed in 72 patients. Mutations in genes involved in cell signaling were associated with inferior LFS and OS, whereas those in genes involved in DNA methylation were associated with inferior LFS. KIT activation loop (AL) mutations occurred in 25 patients, and were associated with inferior LFS (P=0.003) and OS (P=0.001). TET2 mutations occurred in 8 patients, and were associated with significantly shorter LFS (P=0.015) but not OS. Patients negative for KIT-AL and TET2 mutations (N=41) had significantly better LFS (P<0.001) and OS (P=0.012) than those positive for both or either mutation. Multivariate analysis showed that KIT-AL and TET2 mutations were associated with inferior LFS, whereas age ⩾40 years and marrow blast ⩾70% were associated with inferior OS. These observations provide new insights that may guide better treatment for this AML subtype.
Gain-of-function kinase mutations are common in AML and usually portend an inferior prognosis. We reported a novel mechanism whereby kinase mutants induced intracellular alkalization characteristic in oncogenesis. Thirteen kinases were found to activate sodium/hydrogen exchanger (NHE1) in normal hematopoietic progenitors, of which FLT3-ITD, KRASG12D, and BTK phosphorylated NHE1 maintained alkaline intracellular pH (pHi) and supported survival of AML cells. Primary AML samples with kinase mutations also showed increased NHE1 phosphorylation and evidence of NHE1 addiction. Amiloride enhanced anti-leukemic effects and intracellular distribution of kinase inhibitors and chemotherapy. Co-inhibition of NHE1 and kinase synergistically acidified pHi in leukemia and inhibited its growth in vivo. Plasma from patients taking amiloride for diuresis reduced pHi of leukemia and enhanced cytotoxic effects of kinase inhibitors and chemotherapy in vitro. NHE1-mediated intracellular alkalization played a key pathogenetic role in transmitting the proliferative signal from mutated-kinase and could be exploited for therapeutic intervention in AML.
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. Disclosures No relevant conflicts of interest to declare.
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