Hence, these kinases might represent new targets to increase radiosensitivity in HNSCC. To test this hypothesis, clonogenic survival as says were performed with inhibitors against these various kinases in combination with radiotherapy in 3 UT SCC the Tubacin alpha-tubulin phosphorylated kinases. As shown in Figure 2A, AKT inhibition significantly decreased survival after 4 Gy in UT SCC24A and UT SCC40. This effect was supra additive in UT SCC40. In all three cell lines AKT inhibition with or without radiotherapy clearly de creased pAKT levels. SFK inhibition Inhibitors,Modulators,Libraries only decreased survival after 4 Gy in UT SCC24A, and this was not a synergistic effect. Inhibitors,Modulators,Libraries Western blot analyses also showed only a clear decrease in pSFK Inhibitors,Modulators,Libraries levels in UT SCC24A cells. MEK inhibition significantly decreased survival after 4 Gy in all cell lines, which was supra additive in UT SCC24A.

MEK inhibition increased pMEK1 2 levels in all cell lines. In contrast, downstream pERK1 2 levels were decreased after MEK inhibition, indicating that the kinase activity of Inhibitors,Modulators,Libraries MEK1 2 was decreased despite a higher level of phosphorylated MEK1 2. However, this inhibition of ERK1 2 did only lead to reduced pMSK1 levels in UT SCC40. Inhibition of p38 in combination with radiotherapy also led to a reduction of survival in UT SCC24A, which was a supra additive effect. Similar to what was seen using the MEK inhibitor, p38 inhibition did not lead to reduced p p38 levels. rather p p38 levels were increased in UT SCC24A that showed a synergistic effect of p38 inhibition and radiotherapy.

However, no decrease in downstream pMSK1 levels were seen in any of the three cell lines after p38 inhibition indicating that the effect of p38 in hibition was not related to effects on MSK1 activity. As shown in Inhibitors,Modulators,Libraries Figures 2E and 2F, both STAT5 and STAT6 inhibition led to a significantly decreased survival after 4 Gy in all cell lines. For STAT6 inhibition this was only an additive effect, while STAT5 inhibition and 4 Gy had a supra additive ef fect on cell survival in UT SCC40. Both pSTAT5 and pSTAT6 levels were low and difficult to detect on western blot. Reduction of pSTAT5 was observed in UT SCC40 and of pSTAT6 in UT SCC5 and UT SCC40. Discussion In this study, an antibody based array was used to de termine which activated kinases involved in growth fac tor signaling were correlated with radiosensitivity in HNSCC.

This screen resulted in multiple kinases of dif ferent pathways, which could be potential targets to in crease radiosensitivity. normally Pathways known to be associated with radiosensitivity were found, including the RAS RAF ERK and the PI3 K AKT pathways, valida ting our approach. In addition, kinases not known to be involved in radiosensitivity were identified, including STAT5 and STAT6. Moreover, inhibitors of these kinases were able to decrease survival after radiotherapy, par ticularly inhibitors against MEK1 2, STAT5 and STAT6.