Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML.
We determined that TNF stimulation drives the JNK-AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC.We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF-JNK-AP1 signaling pathway.Our data suggest that co-inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.
Affiliation: Molecular Biology Program, Department of Biology, Loyola University Chicago, Chicago, IL 60660.
- JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors/genetics/metabolism*
- Leukemia, Myeloid, Acute/genetics/metabolism*/pathology
- NF-kappa B/antagonists & inhibitors/genetics/metabolism*
- Tumor Necrosis Factor-alpha/genetics/metabolism*/pharmacology
- Blotting, Western
- Cell Line, Tumor
- Cell Survival/drug effects/genetics
- Cells, Cultured
- Gene Expression Regulation, Leukemic/drug effects
- HL-60 Cells
- K562 Cells
- Leukemia, Monocytic, Acute/genetics/metabolism
- Leukemia, Myelomonocytic, Acute/genetics/metabolism
- Leukemia, Promyelocytic, Acute/genetics/metabolism
- Mice, Knockout
- Receptors, Tumor Necrosis Factor/genetics/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Signal Transduction/drug effects/genetics
- Transcription Factor AP-1/genetics/metabolism
- U937 Cells
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fig1: Many types of AML cells produce TNF. (A) TNF concentrations in PB of AML patients as shown by ELISA from individual patient samples. (B) TNF expression in LC isolated from individual AML patient samples as shown by qRT-PCR assay. Horizontal bars in A and B indicate the mean. (C) Microarray analysis of TNF, TNFR1, and R2 in AML patient samples. t(8:21), t(15:17), and inv(16) are AML subtypes M2, M3, and M4, respectively. MLL leukemias include MLL-AF4 (B-ALL) and MLL-AF9 (AML-M5). CD34+ HSPCs, CD33+ myeloid progenitors, and/or MNC from healthy donors were used as controls. (D) Relative mRNA levels of TNF produced in established human AML cell lines. Values shown are mean ± 1 SD. (E) To generate WT and Tnfr−/− murine LCs, HSPCs isolated from mice were transduced with MSCV containing leukemia fusions creating PLC. PLCs were transplanted into recipient mice. Spleens were harvested for LCs after leukemia developed. (F) Murine MA9-LCs produce TNF as shown by Western blot assay. Each lane represents LC from an individual mouse. TNF protein levels were examined in murine MA9-LC with or without PMA (phorbol 12-myristate 13-acetate) stimulation by intracellular antibody staining and read by flow cytometry. Results shown are representative of three individual experiments. * indicates P < 0.05 compared with healthy donor or other control as determined by Student’s t test two-tailed analysis (A, B, and D).
We found that TNF levels are significantly higher in the PB of AML patients having subtypes M3, M4, and M5 as compared with healthy donors (Fig. 1 A). To study whether TNF is produced directly by LC, we first examined LC isolated from these patients for TNF transcription and found that TNF mRNA levels correlate with TNF protein levels in PB (Fig. 1 B). We then examined TNF expression by cDNA array in 106 AML patients with four different leukemic fusions. When compared with hematopoietic cells (CD34+ HSPC, CD33+ myeloid progenitors), many types of LC, especially AML subtypes t(15:17) M3, inv(16) M4, and MLL-AF9 (MA9) M5 cells, express higher levels of TNF (Fig. 1 C). We also found elevated TNF expression in many established human AML cell lines (Fig. 1 D). Furthermore, in our murine AML model, MA9-LC generated from transplanted HSPC expressing the MA9 fusion gene in an MSCV retrovirus (pre-LC [PLC]; Fig. 1 E) produces Tnf, as determined by Western blotting and intracellular staining assays (Fig. 1 F). These data suggest that a significant portion of circulating TNF in many AML patients may be produced directly by tumor cells.