Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor‐risk FLT3‐ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8‐chloro‐adenosine (8‐Cl‐Ado) is a ribose‐containing, RNA‐directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8‐Cl‐Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8‐Cl‐Ado in AML. 8‐Cl‐Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8‐Cl‐Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3‐ITD mutation. FLT3‐ITD is associated with high expression of miR‐155. Furthermore, we demonstrate that 8‐Cl‐Ado inhibits miR‐155 expression levels accompanied by induction of DNA‐damage and suppression of cell proliferation, through regulation of miR‐155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3‐ITD + MV4−11 AML cells with 8‐Cl‐Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3‐ITD AML patients.

Background BCL‐2 inhibition through venetoclax (VEN) targets acute myeloid leukemia (AML) blast cells and leukemic stem cells (LSCs). Although VEN-containing regimens yield 60–70% clinical response rates, the vast majority of patients inevitably suffer disease relapse, likely because of the persistence of drug-resistant LSCs. We previously reported preclinical activity of the ribonucleoside analog 8-chloro-adenosine (8-Cl-Ado) against AML blast cells and LSCs. Moreover, our ongoing phase I clinical trial of 8-Cl-Ado in patients with refractory/relapsed AML demonstrates encouraging clinical benefit. Of note, LSCs uniquely depend on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. VEN inhibits OXPHOS in LSCs, which eventually may escape the antileukemic activity of this drug. FAO is activated in LSCs isolated from patients with relapsed AML. Methods Using AML cell lines and LSC-enriched blast cells from pre-treatment AML patients, we evaluated the effects of 8-Cl-Ado, VEN and the 8-Cl-Ado/VEN combination on fatty acid metabolism, glycolysis and OXPHOS using liquid scintillation counting, a Seahorse XF Analyzer and gene set enrichment analysis (GSEA). Western blotting was used to validate results from GSEA. HPLC was used to measure intracellular accumulation of 8-Cl-ATP, the cytotoxic metabolite of 8-Cl-Ado. To quantify drug synergy, we created combination index plots using CompuSyn software. The log-rank Kaplan–Meier survival test was used to compare the survival distributions of the different treatment groups in a xenograft mouse model of AML. Results We here report that VEN and 8-Cl-Ado synergistically inhibited in vitro growth of AML cells. Furthermore, immunodeficient mice engrafted with MV4-11-Luc AML cells and treated with the combination of VEN plus 8-Cl-Ado had a significantly longer survival than mice treated with either drugs alone (p ≤ 0.006). We show here that 8-Cl-Ado in the LSC-enriched population suppressed FAO by downregulating gene expression of proteins involved in this pathway and significantly inhibited the oxygen consumption rate (OCR), an indicator of OXPHOS. By combining 8-Cl-Ado with VEN, we observed complete inhibition of OCR, suggesting this drug combination cooperates in targeting OXPHOS and the metabolic homeostasis of AML cells. Conclusion Taken together, the results suggest that 8-Cl-Ado enhances the antileukemic activity of VEN and that this combination represents a promising therapeutic regimen for treatment of AML.

Key Points Teriflunomide, the active metabolite of leflunomide, downregulates c-Myc expression through inhibition of PIM kinases. Leflunomide together with lenalidomide significantly extended survival in an in vivo MM model.

The immunosuppressive agent leflunomide has been used in the treatment of over 300,000 patients with rheumatoid arthritis. Its active metabolite, teriflunomide (Ter), directly inhibits dihydroorotate dehydrogenase (DHODH), an enzyme involved in nucleoside synthesis. We report that Ter not only shows in vitro anti-proliferative activity in pancreatic cancer (PC) cells as a single agent but also synergizes with the chemotherapeutic gemcitabine (Gem) in growth inhibition of PC cells. The growth-inhibitory effects of Ter are not solely caused by inhibition of DHODH. Through a kinase screening approach, we identified the PIM-3 serine-threonine kinase as a novel direct target. Subsequent dose-response kinase assays showed that Ter directly inhibited all three PIM family members, with the highest activities against PIM-3 and -1. The PIM-3 kinase was the PIM family member most often associated with PC oncogenesis and was also the kinase inhibited the most by Ter among more than 600 kinases investigated. Ter in PC cells induced changes in phosphorylation and expression of PIM downstream targets, consistent with the effects achieved by overexpression or downregulation of PIM-3. Finally, pharmacological inhibition of PIM proteins not only diminished PC cell proliferation, but also small-molecule pan-PIM and PIM-3 inhibitors synergized with Gem in growth inhibition of PC cells.