Regulation of proton partitioning in kinase-activating acute myeloid leukemia and its therapeutic implication.

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.

Saliva is more sensitive than nasopharyngeal or nasal swabs for diagnosis of asymptomatic and mild COVID-19 infection.

We aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples. We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.

Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia.

Internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3/ITD) occurs in about 30% of acute myeloid leukemia (AML) and is associated with poor response to conventional treatment and adverse outcome. Here, we reported that human FLT3/ITD expression led to axis duplication and dorsalization in about 50% of zebrafish embryos. The morphologic phenotype was accompanied by ectopic expression of a morphogen follistatin (fst) during early embryonic development. Increase in fst expression also occurred in adult FLT3/ITD-transgenic zebrafish, Flt3/ITD knock-in mice, and human FLT3/ITD AML cells. Overexpression of human FST317 and FST344 isoforms enhanced clonogenicity and leukemia engraftment in xenotransplantation model via RET, IL2RA, and CCL5 upregulation. Specific targeting of FST by shRNA, CRISPR/Cas9, or antisense oligo inhibited leukemic growth in vitro and in vivo. Importantly, serum FST positively correlated with leukemia engraftment in FLT3/ITD AML patient-derived xenograft mice and leukemia blast percentage in primary AML patients. In FLT3/ITD AML patients treated with FLT3 inhibitor quizartinib, serum FST levels correlated with clinical response. These observations supported FST as a novel therapeutic target and biomarker in FLT3/ITD AML.

Homoharringtonine (omacetaxine mepesuccinate) as an adjunct for FLT3-ITD acute myeloid leukemia.

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.

Suppression of SOX7 by DNA methylation and its tumor suppressor function in acute myeloid leukemia.

SOX7 belongs to the SOX (Sry-related high-mobility group [HMG] box) gene family, a group of transcription factors containing in common a HMG box domain. Its role in hematologic malignancies and, in particular, acute myeloid leukemia (AML) is completely unknown. Here, we showed that SOX7 expression was regulated by DNA hypermethylation in AML but not in acute lymphoblastic leukemia or normal bone marrow cells. In cell lines (KG1, ML2, and K562) and in primary CD34(+) AML samples, SOX7 expression could be induced by the DNA demethylating agent 5-aza-2'-deoxycytidine. Overexpression of SOX7 in K562 cells inhibited cell proliferation, with cell cycle delay in S/G2/M phases and reduced clonogenic activity. Apoptosis was unaffected. Ectopic expression of SOX7 in K562 and THP-1 cells, as well as primary CD33(+)CD34(+) AML cells, abrogated leukemia engraftment in xenogeneic transplantation. SOX7 expression inhibited the Wnt/ß-catenin pathway through direct protein binding to ß-catenin, and the antileukemia effects of SOX7 in THP-1 cells were significantly reduced by deletion of its ß-catenin binding site. The results provided unequivocal evidence for a novel tumor suppressor role of SOX7 in AML via a negative modulatory effect on the Wnt/ß-catenin pathway.