p65/RelA NF‐κB fragments generated by RIPK3 activity regulate tumorigenicity, cell metabolism, and stemness characteristics

Receptor‐interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces caspase‐mediated p65/RelA cleavage, resulting in N‐terminal 1‐361 and C‐terminal 362‐549 fragments. We show here that a noncleavable p65/RelA D361E mutant expressed in DA1‐3b leukemia cells decreases mouse survival times and that coexpression of p65/RelA fragments increases the tumorigenicity of B16F1 melanoma cells. This aggressiveness in vivo did not correlate with NF‐κB activity measured in vitro. The fragments and p65/RelA D361E mutant induced different expression profiles in DA1‐3b and B16F1 cells. Stemness markers were affected: p65/RelA D361E increased ALDH activity in DA1‐3b cells, and fragment expression increased melanoma sphere formation in B16/F1 cells. p65/RelA fragments and the D361E noncleavable mutant decreased oxidative or glycolytic cell metabolism, with differences observed between models. Thus, p65/RelA cleavage initiated by kinase‐independent RIPK3 activity in cancer cells is not neutral and induces pleiotropic effects in vitro and in vivo that may vary across tumor types.