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A. Chaturvedi et al.
Figure 4. Model of the combined activity of BAY1436032 and azaci- tidine on leukemia stem cells. BAY1436032 and azacitidine (AZA) as sin- gle agents induce the expression of genes involved in myeloid differ- entiation (PU.1, CEBPA, and GABPA) and show additive effects in combi- nation. MAP kinase sig- naling is synergistically inhibited by the combina- tion treatment mediated by inhibition of ERK1/2 phosphorylation and suppression of its down- stream targets (ELK1, ETS, CCND1). Suppression of the RB phosphorylation by the combined treatment of BAY1436032 and azaci- tidine suggests synergis- tic inhibition of the cyclinD1/CDK4 complex. Unphosphorylated retinoblastoma binds to E2F transcription factors and prevents the G1 to S transition of the cell cycle, thereby inhibiting cell proliferation.
Azacitidine also induces differentiation and reduces self- renewal15,16 letting us hypothesize that the combination of a mIDH1 inhibitor and azacitidine may act synergistically. Our data suggest that the combination of BAY1436032 and azacitidine has additive effects on differentiation, while it synergistically reduces LSC by 33,150-fold.
It was shown previously that mutant IDH1 activates MAPK/ERK signaling in leukemia and glioma cells.5,17 Therefore, we expected that a mIDH1 inhibitor would be able to reduce MAPK/ERK signaling. Data from pancreatic cancer cells suggested that treatment with azacitidine also inhibits MAPK/ERK signaling by promoter hypomethyla- tion and induction of expression of DUSP6, which dephos- phorylates members of the MAPK superfamily.18,19 However, the effect of azacitidine on MAPK signaling in leukemia patients is not known. We found that inhibition of the MAP kinase pathway is not a direct consequence of DNA hypomethylation of MAP kinase genes, but rather a transcriptional or posttranscriptional effect on genes that regulate the MAP kinase pathway.
We evaluated the consequences of MAPK/ERK inhibi- tion and found that reduced phosphorylation of ERK1/2 leads to suppression of cyclin D1, which is then unavail- able to bind CDK4. CDK4 in turn cannot phosphorylate Rb and consequently phosphorylation of Rb is strongly inhibited by the combined treatment of BAY1436032 and azacitidine, providing a mechanistic basis for reduced pro- liferation and self-renewal of AML cells (Figure 4).
Phosphorylation of Serine 795 of Rb is required for the dis- sociation of E2F1 from Rb, so that it can bind to DNA and induce transcription of genes involved in the G1 to S tran- sition.20 Similarly, the phosphorylation of Serine 807 and 811 is required to prime phosphorylation at other sites on Rb.21 E2F1 then remains bound to Rb and is unavailable for cell cycle progression, leading to the observed strong antileukemic effects.
NRAS mutations activate MAPK/ERK signaling and have been associated with resistance to mIDH2 inhibitor treat- ment.22,23 Combination treatment with a mIDH1/2 inhibitor with azacitidine and inhibition of MAPK/ERK signaling may indeed target a specific dependency in IDH mutated leukemias and may reduce the risk of relapse.
Although IDH1/2 mutations increase DNA methylation through the oncometabolite R-2HG,24 IDH1/2 mutated leukemias were not associated with better response to azacitidine monotherapy in myelodysplastic syndrome and AML patients.25,26 We conclude that the synergistic effect of BAY1436032 and azacitidine derives from specific inhibition of MAPK/ERK and RB/E2F signaling. This is sup- ported by the striking difference between sequential and simultaneous treatment with BAY1436032 and azacitidine. Simultaneous treatment during 5 days was 70-fold more efficient in eliminating LSC than sequential treatment, sug- gesting improved efficacy of the combination when both drugs are present in the leukemic cell at the same time. This strongly argues for the concurrent application of both
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haematologica | 2021; 106(2)