AML Cells Utilize TNF-Driven JNK Signaling As a Critical NF-κB-Independent Survival Signal

Leukemic Stem Cells (LSCs) isolated from Acute Myeloid Leukemia (AML) patients are highly sensitive to NF-κB inhibition-induced cell death in in vitro culture when compared to normal hematopoietic stem/progenitor cells (HSPCs). This suggests blocking NF-κB could be an effective strategy for treating AML. However, NF-κB inhibitor treatment alone is unable to clear AML tumors in vivo. We propose this is most likely due to elements within the niche microenvironment stimulating compensatory survival/proliferation signals in AML cells that can overcome NF-κB inhibition.

We utilize a multi-faceted AML model that includes a murine system created by MLL-AF9 transduced hematopoietic cells, several established human AML cell lines, and primary AML tumors isolated from patients. We observed elevated Tumor Necrosis Factor-α (TNF) levels in the peripheral blood of AML M3, 4, and 5 patients. Our studies suggest this TNF is produced directly by the AML cells, and that AML cells utilize TNF to stimulate JNK as a survival signal parallel to NF-κB in leukemic cells and a death signal in HSPCs.

In healthy HSPCs, stimulation with exogenous TNF induces RIP1/3-mediated necroptosis and caspase-mediated apoptosis, resulting in cell death and loss of normal hematopoietic function. Inactivation of JNK signaling can partially rescue TNF-mediated loss of function in HSPCs. Additionally, blocking NF-κB in HSPCs sensitizes them to TNF-mediated cell death, which can also be partially prevented by inactivation of JNK signaling. These data suggest that JNK acts as a TNF-mediated death signal in HSPCs.

In AML, we found TNF signaling promotes the in vitro growth of leukemic cells as well as enhancing the in vivo development of AML tumors by stimulation of both NF-κB and JNK signaling in parallel. We determined that TNF-mediated JNK signaling in leukemic cells results in the propagation of a critical survival signal. These cells convert TNF-JNK stimulation from a death to survival signal by limiting JNK activity duration as well as upregulating the expression of the AP1 family transcription factor c-Jun. Therefore, TNF-JNK signaling in leukemic cells does not drive apoptotic cell death, but instead drives c-Jun activity and the subsequent production of the anti-apoptotic genes c-Flip and Mcl-1.

We found inactivation of any part of the TNF-JNK-AP1 signaling axis can repress the growth of leukemic cells in vitro and delay leukemogenesis in vivo. We also show that blocking any portion of the TNF-JNK-AP1 signaling axis sensitizes TNF-expressing leukemic cells, including LSCs, to NF-κB inhibitor treatment while at the same time protecting HSPCs from such treatment.

In conclusion, our studies suggest that the inadequate ability of NF-κB inhibition to clear AML tumors in vivo is due to the TNF-mediated activation of the JNK-AP1 signaling axis that can compensate for NF-κB signal repression in LSCs. We found that many types of AMLs produce TNF, and this TNF acts both in an autocrine fashion to promote LSC survival and self-renewal through parallel activation of NF-κB and JNK-AP1, as well as in a paracrine fashion to repress normal hematopoiesis. Therefore, we propose that inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more thorough treatment for AML patients with elevated peripheral blood TNF.