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Combination drug treatment decreases tumor growth and increases plasma cytokine levels in humanized mice. A. Schematic representation of generation of humanized mice with a functioning immune system. B. Flow cytometry showing CD45, CD3, CD56, CD14, and CD16 expression in mice peripheral blood. C. Mice imaging showing tumor burden fifteen days after post treatment with DAC (0.01 mg/kg), C92 (30 mg/kg) and combination. D. Photon intensity on the whole mouse body fifteen days after post treatment with DAC (0.01 mg/kg), C92 (30 mg/kg) and combination. E. Mice body weight fifteen days after post treatment with DAC (0.01 mg/kg), C92 (30 mg/kg) and combination. F-H . Cytokine expression of F. TNFα, G. IFNγ, and H. CXCL10 respectively assessed by ELISA assay in plasma from retroorbital bleeds of euthanized mice 15 days after DAC (0.01 mg/kg), C92 (30 mg/kg), and combination treatment. I-J. Immunohistochemistry performed on spleen tissue showing I. CXCL10 cytokines and J. CD8 T cell infiltration respectively 15 days after DAC, C92, and combination treatment. K. Graphical model showing Necroptosis pathway activation by DAC and C92 STING agonist combination therapy in AML cells depleted of TP53. DAC and C92 combination therapy demonstrate increased TP53 independent STING pathway that leads to interferon transcription and triggers a ZNFX1-ZBP1 PANoptosis cell death pathway. Activated ZBP1 along with RIPK3 and MLKL phosphorylation are key features of necroptosis, with membrane rupture occurring as a part of this cell death pathway. In cell lines where ZNFX1 is depleted, ZBP1 is completely abrogated and necroptosis does not occur. All data are presented as mean +/- SEM with p-values derived from two-tailed unpaired Student’s t test or ANOVA as appropriate. * p<0.05, p<0.01, p<0.001, p<0.0001. All experiments were performed at least 3 times.

Journal: bioRxiv

Article Title: STING agonists in combination with epigenetic drugs potentiate ZNFX1-driven inflammatory necroptosis in TP53 -mutated AML

doi: 10.64898/2026.07.27.741029

Figure Lengend Snippet: Combination drug treatment decreases tumor growth and increases plasma cytokine levels in humanized mice. A. Schematic representation of generation of humanized mice with a functioning immune system. B. Flow cytometry showing CD45, CD3, CD56, CD14, and CD16 expression in mice peripheral blood. C. Mice imaging showing tumor burden fifteen days after post treatment with DAC (0.01 mg/kg), C92 (30 mg/kg) and combination. D. Photon intensity on the whole mouse body fifteen days after post treatment with DAC (0.01 mg/kg), C92 (30 mg/kg) and combination. E. Mice body weight fifteen days after post treatment with DAC (0.01 mg/kg), C92 (30 mg/kg) and combination. F-H . Cytokine expression of F. TNFα, G. IFNγ, and H. CXCL10 respectively assessed by ELISA assay in plasma from retroorbital bleeds of euthanized mice 15 days after DAC (0.01 mg/kg), C92 (30 mg/kg), and combination treatment. I-J. Immunohistochemistry performed on spleen tissue showing I. CXCL10 cytokines and J. CD8 T cell infiltration respectively 15 days after DAC, C92, and combination treatment. K. Graphical model showing Necroptosis pathway activation by DAC and C92 STING agonist combination therapy in AML cells depleted of TP53. DAC and C92 combination therapy demonstrate increased TP53 independent STING pathway that leads to interferon transcription and triggers a ZNFX1-ZBP1 PANoptosis cell death pathway. Activated ZBP1 along with RIPK3 and MLKL phosphorylation are key features of necroptosis, with membrane rupture occurring as a part of this cell death pathway. In cell lines where ZNFX1 is depleted, ZBP1 is completely abrogated and necroptosis does not occur. All data are presented as mean +/- SEM with p-values derived from two-tailed unpaired Student’s t test or ANOVA as appropriate. * p<0.05, p<0.01, p<0.001, p<0.0001. All experiments were performed at least 3 times.

Article Snippet: Briefly, sections (5 μm) were deparaffinized, endogenous peroxidase was inactivated in 3% peroxide for 10 min, and antigen retrieval in 0.1 M sodium citrate was performed in a pressure cooker before the sections were blocked with 5% BSA and incubated overnight at 4 °C with polyclonal antibodies against CXCL10/IP-10 (Cat# 10937-1-AP, RRID:AB_2088002) and CD8 (Mouse anti-Human CD8 Antibody, Cat# 188-10258-IHC, RayBiotech, RRID:AB_2857849).

Techniques: Clinical Proteomics, Flow Cytometry, Expressing, Imaging, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Activation Assay, Phospho-proteomics, Membrane, Derivative Assay, Two Tailed Test