Panoply™ Human FLT4 Over-expressing Stable Cell Line

Aberrant receptor tyrosine kinase (RTK) signaling enables cancer cells to regulate survival, proliferation, and death, leading to tumorigenesis and chemoresistance. In leukemia, the expression of the RTK FMS-related tyrosine kinase 4 (FLT4), also known as VEGFR-3 (vascular endothelial growth factor receptor-3), is dysregulated and associated with cancer progression. However, the underlying consequences of its dysregulation remain to be determined. Furthermore, chemotherapy requires cancer cells to retain wild-type p55 (wt) to respond to DNA damage through tumor suppressor activity (i.e., apoptosis). The activity of p53 is primarily limited by its two main negative regulators, MDM2 and MDMX, which inactivate it by promoting its degradation and/or cytoplasmic localization. In this study, researchers found that activation of FLT4 (either through overexpression or binding to its ligand VEGF-C) leads to increased MDM2/MDMX stability, p53 inactivation, and resistance to DNA damage therapies. Through immunoprecipitation and mass spectrometry analysis, the researchers observed that FLT4 induced phosphorylation of MDMX at Ser-314, a consensus site for CDK4/6. These data show that phosphorylation of MDMX at Ser-314 increases MDMX stability, which in turn affects the degradation of MDM2 and p53, and this effect can be reversed by the CDK4/6 inhibitor Palbociclib. More importantly, leukemia cells treated with Palbociclib were more sensitive to DNA damage-induced apoptosis and showed reduced cell proliferation.

After demonstrating that FLT4 can stabilize the MDM2/MDMX complex, the researchers aimed to investigate whether this effect of FLT4 on leukemia cells would have any impact on p53-mediated cell death and the response to DNA damaging agents. First, the researchers observed increased proliferation (Figure 1A) and colony formation (Figure 1B) in FLT4-overexpressing REH cells. To assess the role of FLT4 in the DNA damage response, they treated FLT4-overexpressing REH cells with doxorubicin and etoposide, two commonly used genotoxic drugs in leukemia treatment, for 5 hours and 3 hours, respectively (Figure 1C and 1F). The results showed that FLT4-overexpressing cells had higher levels of MDM2 compared to the control group. Furthermore, under treatment with both genotoxic drugs, FLT4-overexpressing cells exhibited lower p53 levels in the DNA damage response (Figure 1C and 1F). Since p53 regulates cell death, they investigated the effect of reduced p53 levels on DNA damage stress-induced apoptosis. After treatment with the genotoxic drugs, the researchers performed Annexin V and DAPI staining and measured the amount of apoptosis. Interestingly, compared to the control group, FLT4-overexpressing cells showed lower levels of apoptosis after treatment with both doxorubicin and etoposide (Figure 1D and 1G), and this effect was concentration-dependent (Figure 1E and 1H).

Figure 1. FLT4 overexpression decreases p53 and promotes cell survival and resistance to chemotherapy. (Dubuissez M, et al., 2023)