HQP8361 is effective in suppressing the growth of AZD9291-resistant EGFRm NSCLC cell lines with hyper-activated c-Met. To determine the single agent activity of HQP8361 against the growth of human NSCLC cells, we first analyzed its effects on the growth of a panel of human NSCLC cell lines including those with acquired resistance to AZD9291. Among 18 cell lines tested, only the AZD9291-resistant cell line, HCC827/AR, responded to HQP8361 with an IC50 of around 1 µM (Figs. 1A). By examining MET and p-MET levels in these cell lines, we found that HCC827/AR cells possessed the highest levels of both MET and p-MET, whereas other cell lines expressed low levels of MET and undetectable levels of p-MET (Figs. 1B and 1C). We established two additional AZD9291-resistant cell lines from HCC827 named HCC829/AR0.5 and HCC827/AR2 through repeated treatment of HCC827 cells with fixed high concentrations of AZD9291 at 0.5 µM and 2 µM, respectively (Fig. S1). Like HCC827/AR cells, both HCC829/AR0.5 and HCC827/AR2 exhibited high levels of MET and p-MET (Fig. 1D) and responded better than HCC827 cells to HQP8361 (Fig. 1E). These data clearly suggest that HQP8361 has single agent activity only in cell lines with hyper-activated MET.
HQP8361, when combined with AZD9291, synergistically decreases the survival of AZD9291-resistant cell lines with hyper-activated MET and enhances induction of apoptosis. Given that MET amplification is an important mechanism accounting for the emergence of acquired resistance to EGFR-TKIs including AZD9291 and that targeting MET can overcome acquired AZD9291 resistance caused by this mechanism (1, 9), we then examined the effects of the HQP8361 and AZD9291 combination on the survival and apoptosis induction of different AZD9291-resistant cell lines. Treatment with each agent alone minimally decreased the survival of HCC827/AR cells, while the combination very effectively decreased survival with CIs < 1 (Fig. 2A), indicating synergistic effect. However, the combination was ineffective in both PC-9/AR and PC-9/3M cell lines (Fig. 2A). Consistently, the combination enhanced induction apoptosis in HCC827/AR cells, but not in PC-9/AR cells as measured by both annexin V-positive cells (Fig. 2B) and PARP cleavage (Fig. 2C). Colony formation assay, which allows repeated treatment of cells, also demonstrated that the combination of HQP8361 and AZD9291 was much more effective than either single agent alone in inhibiting the formation and growth of HCC827/AR cells (Fig. 2D). Similarly, the HQP8361 and AZD9291 combination synergistically decreased the survival (Fig. 2E) and enhanced the induction of apoptosis (Figs. 2F and 2G) of both HCC827/AR0.5 and HCC827/AR2 cells. These findings suggest that the combination of HQP8361 and AZD9291 is very effective against AZD9291-resistant cells with MET amplification or MET hyper-activation.
The combination of HQP8361 and AZD9291 enhances Bim elevation and Mcl-1 reduction accompanied with augmented suppression of mTOR signaling. To understand the mechanisms by which the combination of HQP8361 and AZD9291 enhances induction of apoptosis, we conducted time-course analyses of Bim and Mcl-1 alterations in HCC827/AR cells in response to the combination treatment. As presented in Fig. 3A, Mcl-1 levels were reduced the most and Bim levels were increased the most in cells exposed to the combination, in comparison with levels in cells exposed to either HQP8361 or AZD9291 alone. These effects occurred early at 8 h and extended to 36 h after treatment in parallel with increased PARP cleavage. Consistently, enhanced Bim elevation and Mcl-1 reduction by the HQP8361 and AZD9291 combination was also observed in HCC827/AR0.5 cells (Fig. S2). Similar effects on Mcl-1 and Bim were caused by the combination of crizotinib and AZD9291, although the reduction of Mcl-1 was greater in HCC827/AR cells treated with HQP8361 and AZD9291 than with crizotinib and AZD9291 given that crizotinib alone apparently decreased Mcl-1 levels (Fig. 3B).
Moreover, we assessed the impact of the HQP8361 and AZD9291 combination on signaling pathways downstream of EGFR and c-Met that may modulate Bim and Mcl-1 levels in this cell line. Interestingly, the combination did not enhance suppression of ERK1/2 phosphorylation until a very late time point (e.g., 36 h; Fig. 3A). This differed from the effect of the crizotinib and AZD9291 combination, which substantially enhanced reduction of p-ERK1/2 levels (Fig. 3B). Augmented inhibition of S6 phosphorylation (S235/236) was clearly detected early at 8 h, whereas enhanced suppression of Akt (both S473 and T450) and 4EBP1 (T37/46) phosphorylation was observed a little later, at or after 16 h post treatment. We noted that the combination exerted much more substantial suppression of S6 phosphorylation than of Akt and 4EBP1 phosphorylation (Fig. 3A). Strong suppression of S6 phosphorylation by the combination was also observed in HCC827/AR0.5 cells (Fig. S2). Similar effects were seen with the combination of crizotinib and AZD9291 (Fig. 3B). Nonetheless, the combination of HQP8361 and AZD9291 enhanced suppression of mTOR signaling in HCC827/AR cells.
We noted that treatment with HQP8361 alone increased the levels of p-MET in both HCC827/AR and HCC827/AR0.5 cells; this effect could be suppressed when combined with AZD9291. Further, the combination of HQP8361 and AZD9291 clearly decreased total MET levels (Figs. 3A and S2). In contrast, crizotinib alone abolished MET phosphorylation (Fig. 3B). However, both HQP8361 and particularly crizotinib increased the levels of both ErbB3 and p-ErbB3 in HCC827/AR cells. When combined with AZD9291, increased ErbB3 phosphorylation was abolished despite further increase in ErbB3 levels (Fig. 3B).
The combination of HQP8361 and AZD9291 regulates Bim and Mcl-1 levels through modulation of their protein stability. Considering that AZD9291 combined with crizotinib, a MET and ALK dual inhibitor, enhances the elevation of Bim levels by delaying its degradation (9), we next analyzed whether the combination of HQP8361 and AZD9291 affects the degradation of Bim and Mcl-1, resulting in enhanced Bim elevation and Mcl-1 reduction in HCC827/AR cells. By conducting a CHX chase assay, we observed that Bim was degraded much more slowly and Mcl-1 was degraded much faster in cells exposed to the combination of HQP8361 and AZD9291 than in those exposed to DMSO, HQP8361 or AZD9291 alone (Fig. 4). These results demonstrate that the combination of HQP8361 and AZD9291 slows Bim degradation while facilitating Mcl-1 degradation in HCC827/AR cells.
Enhanced Bim elevation and Mcl-1 contribute to augmented induction of apoptosis in HCC827/AR cells. To demonstrate the role of Bim elevation in enhancing apoptosis by the HQP8361 and AZD9291 combination, we generated Bim knockout cell lines from HCC827/AR cells and then examined the impact of Bim loss on the induction of apoptosis by the HQP8361 and AZD9291 combination. As presented in Figs. 5A and 5B, the combination of HQP8361 and AZD9291 enhanced the induction of apoptosis as measured by PARP cleavage and annexin V-positive cells in the control HCC827/AR cells as demonstrated above, but these effects were significantly attenuated in the two tested Bim KO cell lines. In HCC827/AR cells expressing ectopic Mcl-1, the combination of HQP8361 and AZD9291 induced much less cleavage of PARP and caspase-3 and significantly fewer annexin V-positive cells than in vector-control cells (Figs. 5C and 5D), indicating that enforced Mcl-1 expression in HCC827/AR cells compromises the ability of the HQP8361 and AZD9291 combination to enhance the induction of apoptosis.
The combination of HQP8361 and AZD9291 significantly inhibits the growth of HCC827/AR tumors in vivo with modulation of the levels of Bim, Mcl-1 and other protein markers. We lastly determined the effect of the HQP8361 and AZD9291 combination on the growth of HCC827/AR xenografts in nude mice. While treatment with HQP8361 or AZD9291 alone had limited inhibitory effect on the growth of HCC827/AR tumors, the combination of HQP8361 and AZD9291 potently and significantly retarded the growth of HCC827/AR tumors in comparison with either single agent treatment in terms of tumor sizes (Figs. 6A and 6B) and weights (Fig. 6C). Mice receiving the combination treatment had comparable body weights with those in other groups (Fig. 6D), indicating that the combination does not accordingly increase toxicity while enhancing anti-tumor activity.
We also analyzed alterations of several protein markers in these tumor tissues and found that the combination of AZD9291 and HQP8361 effectively decreased Mcl-1 levels and increased the levels of Bim and cleaved PARP, whereas each single agent had limited or no modulatory effects on the levels of these proteins (Fig. 6E). These results indicate that the HQP8361 and AZD9291 combination enhances the modulation of Bim and Mcl-1 levels accompanied with augmented induction of apoptosis in vivo. In agreement with the data generated in cell cultures, the combination of HQP8361 and AZD9291 was more effective than each single agent in decreasing the levels of p-S6, p-4EBP1 and p-Akt, but did not affect p-ERK levels (Fig. 6E), indicating that the combination also effectively enhances the suppression of mTOR signaling in vivo.