Oncogenic Kinase Cascades Induce Molecular Mechanisms That Protect Leukemic Cell Models from Lethal Effects of De Novo dNTP Synthesis Inhibition

Abstract

The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.

Ferroptosis-related molecular patterns reveal immune escape, inflammatory development and lipid metabolism characteristics of the tumor microenvironment in acute myeloid leukemia

“…Finally, to explore the clinical treatment options for patients with different ferroptosis-related phenotypes, we used drug prediction to identify five anticancer drugs and nine small molecule drugs that may have therapeutic benefits for patients with low FSscores and 20 small molecule drugs that may have therapeutic benefits for patients with high FSscores. Among them, ABT.263 (navitoclax) has been confirmed to be effective for the treatment of AML in cell and mouse experiments (73)(74)(75)(76). The combination of hyperforin and Akt inhibitor AKT inhibitor VIII significantly promotes the apoptosis of AML U937 cells (77).…”

RNA interference protocol to silence oncogenic drivers in leukemia cell lines

“…We effectively decrease proteins by electroporation and RNAi in MOLM-13 acute myeloid leukemia cells (voltage: 1,600 V, width: 10 ms, 3 pulses), in RS4-11 B cell precursor leukemia cells (voltage: 1,750 V, width: 20 ms, 1 pulse), and in HEL erythroleukemia cells (voltage: 1,400 V, width: 20 ms, 2 pulses). For K562 chronic myeloid leukemia cells and NB4 promyelocytic leukemia cells, see our recent publications ( Pons et al., 2019 , 2021 ). Transfer the cells into the 6 well plate (as shown in Figure 5 ) with the prewarmed medium.…”

Inhibitors of class I HDACs and of FLT3 combine synergistically against leukemia cells with mutant FLT3

“…Moreover, cells were confirmed to be free of mycoplasma every 1–4 months. The p53 status of the cells is wild-type for MV4-11 cells (Yan et al 2020 ) and MOLM-13 cells (Thompson et al 2010 ), mutant for RS4-11 cells (Demir et al 2020 ), null for K562 cells (Pons et al 2021 ). Marbotinib has been patented (Mahboobi et al 2019 ; Sellmer et al 2020 ).…”