Metastatic BRAFV600E CRC carries an extremely poor prognosis and is in urgent need of better treatments. In this regard, a recent breakthrough was the approval of encorafenib plus cetuximab for treatment of chemorefractory BRAFV600E metastatic CRC patients [13]. However, objective responses only occur in ~20% of patients and overall survival is only increased by ~4 months, warranting the search for strategies to enhance the efficacy of this treatment regime. Herein, we demonstrate that a limitation of BRAF inhibitors when used either alone or in combination with EGFR inhibitors, is that they fail to effectively induce apoptosis in BRAFV600E CRC cell lines. Notably, despite their failure to induce apoptosis, we found that BRAF inhibitors induced expression of the pro-apoptotic protein BIM and repressed expression of the pro-survival protein MCL-1, suggesting these agents may prime BRAFV600E cells to undergo apoptosis.
An important element of this study was the investigation of the mechanism underpinning apoptotic resistance of BRAFV600E CRC cells to BRAF inhibitors, which was investigated by transcriptomic profiling of basal expression levels of pro-survival genes. This analysis revealed high basal expression of both MCL-1 and BCL-XL, and lower expression of BCL-2, BCL-w and BFL1 in all CRCs including the BRAFV600E subset. Notably, despite the high basal expression of both BCL-XL and MCL-1, a greater enhancement of apoptosis was observed when encorafenib was combined with a BCL-XL inhibitor compared to an MCL-1 inhibitor. While this finding may reflect differences in target inhibition between current BCL-XL and MCL-1 inhibitors, a further explanation may be the capacity of the encorafenib+BCL-XL combination to inhibit both BCL-XL as well as MCL-1, the latter through encorafenib-mediated transcriptional and/or post-translational effects (Fig. 1). Comparatively, when encorafenib is combined with an MCL-1 inhibitor, the apoptotic threshold may remain elevated due to the sustained presence of high BCL-XL. In addition, we also demonstrate a key role for BIM induction in encorafenib+BCL-XL inhibitor-induced apoptosis. The encorafenib-induced transcriptional and/or post-translational stabilization of BIM [34,35,36,37], and suppression of MCL-1 expression, combined with a BCL-XL inhibitor, may result in sufficient levels of BAX and BAK being released from pro-survival proteins allowing BIM to activate BAX and BAK, and initiate apoptosis [38].
While these findings reveal that combining BRAF and BCL-XL inhibitors represents a promising therapeutic approach for BRAFV600E CRC, the clinical use of BCL-XL inhibitors is currently limited by their on-target toxicity of thrombocytopenia [30, 31]. We found that combining encorafenib with the BCL-XL degrading PROTAC DT2216 and AZD0466, a novel BCL-2/BCL-XL inhibitor-dendrimer conjugate [21, 39], significantly enhanced apoptosis. Furthermore, combination of encorafenib with AZD0466 significantly reduced tumour growth in vivo and was generally well tolerated in mice. AZD0466 is currently being tested in phase I/II clinical trials for the treatment of haematological malignancies (ClinicalTrials.gov identifier: NCT05205161 and NCT04865419), and our findings suggest that combining encorafenib with AZD0466 may also be a promising approach for treating BRAFV600E tumours.
The concept of combining MAPK pathway inhibitors with BH3 mimetics has been explored in colorectal cancer and other tumour types. Specifically, a pre-clinical study in BRAFV600E melanoma demonstrated synergistic induction of apoptosis when BRAF or MEK inhibitors were combined with a MCL-1 inhibitor, which aligned with MCL-1 being the predominant pro-survival protein expressed in melanoma cells [17]. A further study in KRAS-mutant NSCLC and other KRAS-mutant solid tumour cell lines also reported synergistic cell killing when MAPK pathway inhibitors were combined with BH3 mimetics [40]. Excitingly, these studies have prompted the initiation of a clinical trial of the MEK inhibitor trametinib in combination with the BCL-2/BCL-XL inhibitor navitoclax in patients with advanced or metastatic solid tumours harbouring KRAS or NRAS mutations (Clinicaltrials.gov identifier: NCT02079740).
The high expression of multiple pro-survival proteins in CRC cells suggests alternative strategies for inducing apoptosis could be through the combined inhibition of BCL-XL and MCL-1. However, pre-clinical studies of these combinations have demonstrated acute liver toxicity, which is consistent with the cooperative role of BCL-XL with MCL-1 in hepatocyte survival [41, 42]. The current approach of using a targeted therapy (BRAF inhibitor) in combination with a BCL-XL inhibitor therefore has the potential to minimize these toxicities, while providing the added benefit of inducing pro-apoptotic proteins such as BIM.
In summary, our findings demonstrate that BRAF inhibitors alone and in combination with the EGFR inhibitor cetuximab fail to induce extensive levels of apoptosis in BRAFV600E CRC cells. Importantly, we reveal that this can be overcome by combining a BRAF inhibitor with a BCL-XL inhibitor, in both in vitro and in vivo models of BRAFV600E CRC, suggesting this combination regimen is worthy of clinical validation.
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