Combinatorial drug screening identifies compensatory pathway interactions and adaptive resistance mechanisms

Abstract

Constitutively activated signaling molecules are often the primary drivers of malignancy, and are favored targets for therapeutic intervention. However, the effectiveness of targeted inhibition of cell signaling can be blunted by compensatory signaling which generates adaptive resistance mechanisms and reduces therapeutic responses. Therefore, it is important to identify and target these compensatory pathways with combinations of targeted agents to achieve durable clinical benefit. In this report, we demonstrate the use of high-throughput combinatorial drug screening as a discovery tool to identify compensatory pathways that generate resistance to the cytotoxic effects of targeted therapy. We screened 420 drug combinations in 14 different cell lines representing three cancer lineages, and assessed the ability of each combination to cause synergistic cytotoxicity. Drug substitution studies were used to validate the functionally important drug targets. Of the 84 combinations that caused robust synergy in multiple cell lines, none were synergistic in more than half of the lines tested, and we observed no pattern of lineage specificity in the observed synergies. This reflects the plasticity of cell signaling networks, even among cell lines of the same tissue of origin. Mechanistic analysis of one novel synergistic combination identified in the screen, the multi-kinase inhibitor Ro31-8220 and lapatinib, demonstrated compensatory crosstalk between the p70S6 kinase and EGF receptor pathways. In addition, we identified BAD as a node of convergence between these two pathways that may be playing a role in the enhanced apoptosis observed upon combination treatment.

Harnessing Connectivity in a Large-Scale Small-Molecule Sensitivity Dataset

“…Unbiased combination screening has been used to successfully identify acquired mechanisms of resistance to targeted therapies (43), though the scale of experiments required to assess combinations prohibits the routine use of this approach. Here, we developed a high-throughput method to rapidly assess interactions between small-molecule treatments.…”

Combining lapatinib and pertuzumab to overcome lapatinib resistance due to NRG1-mediated signalling in HER2-amplified breast cancer

“…Therefore, it could be interesting to further assess the cross-talk between neurotensinergic and the HER pathways in relation to lapatinib sensitivity and resistance. It may also be useful to use a high-throughput combinatorial drug screening approach to identify compensatory pathways that mediate lapatinib resistance and to target them with other targeted agents in combination with lapatinib to overcome its resistance and to achieve durable clinical in order benefit [44]. …”

p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling networks

“…This combination caused synergistic cytotoxicity in multiple cell lines and cancer lineages such as bladder (UMUC-6) and head and neck squamous cell carcinoma (HNSCC) (Cal27 and SCC61) (Figure 1 A-C). To be certain that the biological effects of these drugs were due to inhibition of the expected target enzyme and not a consequence of off-target effects, we determined that multiple pharmacophores with the same putative target elicited the same biological effects, as described previously[14]. BMS599626, lapatinib, and AG1478 were functionally equivalent (Figure 1 and data not shown), thus validating the HER family as the functional target.…”

“…We tested over 500 drug combinations for synergistic cytotoxic effects in 21 epithelial cancer cell lines representing three distinct cancer lineages 2 [14]. One combination that we identified as synergistic using the Bliss model of additivity is the combination of a HER-family kinase inhibitor and a dual PI3K/mTOR inhibitor.…”