Targeted therapies such as kinase inhibitors that inhibit tumor-driving mutations are expanding in importance in cancer therapy with the continued identification of mutations across tumor types and the development of many selective inhibitors of these mutant enzymes. But with this potential comes difficulties in identifying which patients to treat and how to predict and counteract the development of resistance. A recent study by Ahronian et al. evaluated resistance in colorectal cancer and highlighted many of the difficulties in combatting resistance to BRAFi (Ahronian et al., 2015). They found multiple resistance mechanisms across tumors and often more than one resistance mechanism in a particular patient. More importantly, they found that when cells became resistant to one combination of drugs, there was often cross-resistance to other potential combination therapies. This emphasizes the difficulty of targeting individual resistance mechanisms e.g. by adding an inhibitor of a downstream kinase such as MEK to circumvent resistance to BRAF inhibition. Tumor heterogeneity is also a significant concern with studies showing branched evolution in mutations and resistance mechanisms that can contribute to acquired resistance. Such heterogeneity can occur both temporally as well as geographically within the same patient (Shi et al., 2014).

The development of a therapeutic strategy that circumvents different molecularly distinct resistance mechanisms could potentially address these issues. Here we present evidence from cell lines with both experimentally acquired and different molecularly defined resistance mechanisms and from tumor samples from patients with clinically acquired and intrinsic BRAFi resistance, that pharmacological inhibition of autophagy with CQ can achieve this goal. In the cell lines where it was experimentally feasible, we could also demonstrate a similar effect with genetic inhibition of autophagy. Broad applicability is suggested because the various resistance mechanisms that could be overcome included examples that did and did not involve cross-resistance to MEK inhibition, mutational activation of both KRAS and NRAS, EGFR overexpression, paradoxical activation of the ERK pathway by vemurafenib, and a PTEN mutation. Taken together, our data suggest that regardless of the resistance mechanism to the BRAF inhibitor, autophagy inhibition was able to improve the response to BRAFi.

We have seen rapid clinical responses (in as little as six weeks) in patients with both high and low-grade tumors with acquired BRAFi resistance. These first clinical responses have also been sustained. Patient #1 demonstrated control and reduction of her metastatic tumors for greater than seven months, whereas she had previously shown MRI-measurable tumor growth in as little as two weeks. Moreover, patient #1 has experienced clinical benefit when the autophagy inhibitor was added to vemurafenib despite having demonstrated subsequent acquisition of clinical resistance to MEK inhibition after the acquisition of BRAF resistance. Also, the first patient we reported (Levy et al., 2014) maintained sustained tumor regression on the combination of vemurafenib and CQ for more than 2 ½ years without significant clinical complications. Although these early clinical results are encouraging, our findings are in a limited number of patients and further clinical investigation is required to verify if this strategy of combining autophagy inhibition with BRAF inhibition provides a durable and widely applicable response in BRAFV600E tumors.

In summary, pre-clinical and clinical experience invariably shows that tumor cells rapidly evolve ways around inhibition of mutated kinase pathways like the RAF pathway targeted here. However, based on our results, we hypothesize that by targeting an entirely different cellular process, i.e. autophagy, upon which these same tumor cells rely, it may be feasible to overcome such resistance and thus re-establish effective tumor control. Importantly, our data suggest that this strategy can work even when different resistance mechanisms apply. This can be done using CQ, which is an approved, safe, and inexpensive drug and, perhaps, other more potent autophagy inhibitors that are under development (Egan et al., 2015; Goodall et al., 2014; Ronan et al., 2014). Importantly, in the context of BRAF mutant pediatric brain cancers where BRAF inhibition is already being tested, it should be feasible to quickly test this hypothesis in clinical trials.