Fig. 6. Chemotherapy-induced necroptosis is impeded by A20 in AML.
A Immunoblot of necroptosis effectors in A20+/+ MLL-AF9 treated with vehicle, Doxorubicin (5 nM), or Cytarabine (500 nM) for 24 h (n = 2 independent experiments). B Immunoblot of CD34 + MLL-AF9;FLT3-ITD cells treated with Doxorubicin (0, 1, 2.5 nM) or Cytarabine (0, 250, 500 nM) for 24 h (n = 2 independent experiments). C Transmission electron microscopy of A20+/+ MLL-AF9 treated with vehicle or Doxorubicin (5 nM) for 24 h (n = 3 technical replicates). Scale bar = 6 μm. D LDH release assay of an A20High (JM62) and A20Low (JM18) PDX AML samples treated with doxorubicin (+, 250 nM; ++, 500 nM) and/or Necrostatin-1 (Nec-1, 30 μM) (n = 3 independent technical replicates). Student’s t test (unpaired, two-tailed) was used to determine significance. E Immunoblot of CD34 + MLL-AF9;FLT3-ITD cells expressing empty vector or wild-type A20 treated with Doxorubicin (0, 1, 2.5 nM) or Cytarabine (0, 250, 500 nM) for 24 h. F Colony numbers of CD34 + MLL-AF9;FLT3-ITD cells expressing empty vector, wild-type A20, or A20ZF4/7 following treatment with Doxorubicin (0, 1, 2.5 nM) (n = 3 independent experiments). Student’s t test (unpaired, two-tailed) was used to determine significance. G Timeline of AML patient sample collection pre- and post-chemotherapy. H Percent of AML cells with A20 overexpression (Z score >0.25) pre- and post-chemotherapy. I UMAP plots of single-cell RNA sequencing data (GSE116256) showing AML cell fate mapping (left), AML cells prior to chemotherapy in patient AML329 and AML420, and AML cells post-chemotherapy (right). J A20 expression in the indicated malignant cells pre- and post-chemotherapy. K Overview of A20-dependent suppression of necroptosis in AML. Error bars represent the standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001. For each immunoblot (A, B, E), all samples were derived from the same experiment, but different gels for each antibody were processed in parallel. Source data are provided as a source data file.