Next-generation hypomethylating agent SGI-110 primes acute myeloid leukemia cells to IAP antagonist by activating extrinsic and intrinsic apoptosis pathways

Abstract

Therapeutic efficacy of first-generation hypomethylating agents (HMAs) is limited in elderly acute myeloid leukemia (AML) patients. Therefore, combination strategies with targeted therapies are urgently needed. Here, we discover that priming with SGI-110 (guadecitabine), a next-generation HMA, sensitizes AML cells to ASTX660, a novel antagonist of cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2) and X-linked IAP (XIAP). Importantly, SGI-110 and ASTX660 synergistically induced cell death in a panel of AML cell lines as well as in primary AML samples while largely sparing normal CD34+ human progenitor cells, underlining the translational relevance of this combination. Unbiased transcriptome analysis revealed that SGI-110 alone or in combination with ASTX660 upregulated the expression of key regulators of both extrinsic and intrinsic apoptosis signaling pathways such as TNFRSF10B (DR5), FAS, and BAX. Individual knockdown of the death receptors TNFR1, DR5, and FAS significantly reduced SGI-110/ASTX660-mediated cell death, whereas blocking antibodies for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or FAS ligand (FASLG) failed to provide protection. Also, TNFα-blocking antibody Enbrel had little protective effect on SGI-110/ASTX660-induced cell death. Further, SGI-110 and ASTX660 acted in concert to promote cleavage of caspase-8 and BID, thereby providing a link between extrinsic and intrinsic apoptotic pathways. Consistently, sequential treatment with SGI-110 and ASTX660-triggered loss of mitochondrial membrane potential (MMP) and BAX activation which contributes to cell death, as BAX silencing significantly protected from SGI-110/ASTX660-mediated apoptosis. Together, these events culminated in the activation of caspases-3/-7, nuclear fragmentation, and cell death. In conclusion, SGI-110 and ASTX660 cooperatively induced apoptosis in AML cells by engaging extrinsic and intrinsic apoptosis pathways, highlighting the therapeutic potential of this combination for AML.

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Fig. 1: SGI-110 synergizes with ASTX660 to induce cell death and to suppress clonogenic growth of AML cell lines.
Fig. 2: SGI-110 cooperates with ASTX660 to induce cell death in a large proportion of primary AML blasts while showing minimal toxicity against normal CD34+ HPCs.
Fig. 3: SGI-110 and ASTX660 demonstrate on-target activity in AML cell lines.
Fig. 4: Sequential SGI-110/ASTX660 treatment upregulates extrinsic and intrinsic apoptosis signaling genes.
Fig. 5: Sequential SGI-110/ASTX660 treatment initiates death receptor-dependent apoptosis.
Fig. 6: Sequential SGI-110/ASTX660 treatment engages intrinsic apoptosis as well as a crosstalk between extrinsic and intrinsic apoptotic pathways.
Fig. 7: SGI-110 and ASTX660 cooperate to induce biochemical and morphological hallmarks of apoptosis.

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Acknowledgements

We thank S. Jordan (UCT Biobank, Frankfurt) for providing primary AML samples, H. Bönig (Institute for Transfusion Medicine and Immunohematology, University Hospital Frankfurt) for providing CD34+ HPCs, Astex Pharmaceuticals (Cambridge, UK) for providing SGI-110 and ASTX660, C. Hugenberg for expert secretarial assistance and Dr M. Bewerunge-Hudler and her team (Genomics and Proteomics Core Facility, German Cancer Research Center/DKFZ, Heidelberg, Germany) for their microarray service. We thank V. Ehrenfeld for assistance with image preparation. This work was supported by grants from the BMBF (to SF) and German Cancer Aid (to SF), and by Astex Pharmaceuticals, Cambridge, UK (to SF). MB is supported by grants from the DFG (CRC850), BMBF (CoNfirm; FKZ 01ZX1708F), and MIRACUM within the Medical Informatics Funding Scheme (FKZ 01ZZ1606A-H). PM is funded by MIRACUM.

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Correspondence to Simone Fulda.

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This work was supported in part by Astex Pharmaceuticals, Cambridge, UK (to SF). GAW is employee of Astex Pharmaceuticals. JD, TH, PM, MB, and MV declare no conflict of interest.

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Dittmann, J., Haydn, T., Metzger, P. et al. Next-generation hypomethylating agent SGI-110 primes acute myeloid leukemia cells to IAP antagonist by activating extrinsic and intrinsic apoptosis pathways. Cell Death Differ (2019). https://doi.org/10.1038/s41418-019-0465-8

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