Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant cDNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS– MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)–mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially-passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAFV600E melanoma cells in culture, and the abundance of Notch1 pathway markers were increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.

Purpose Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains first-line therapy for the management of estrogen receptor (ESR1) positive breast cancer. However, ESR1 mutations or other ligand-independent ESR1 activation mechanisms limit the duration of response. The clinical efficacy of fulvestrant, a Selective Estrogen Receptor Downregulator (SERD) that competitively inhibits agonist binding to ESR1 and triggers receptor downregulation, has confirmed that ESR1 frequently remains engaged in endocrine therapy resistant cancers. We evaluated the activity of a new class of Selective Estrogen Receptor Modulators (SERM)/SERD hybrids (SSHs) that downregulate ESR1 in relevant models of endocrine-resistant breast cancer. Building on the observation that concurrent inhibition of ESR1 and the cyclin dependent kinases 4 and 6 (CDK4/6) significantly increased progression free survival in advanced patients, we explored the activity of different SERD- or SSH-CDK4/6 inhibitor combinations in models of endocrine therapy resistant ESR1+ breast cancer. Experimental Design SERDs, SSHs, and the CDK4/6 inhibitor palbociclib were evaluated as single agents or in combination in established cellular and animal models of endocrine therapy resistant ESR1+ breast cancer. Results The combination of palbociclib with a SERDs or an SSH was shown to effectively inhibit the growth of MCF-7 cell or ESR-1 mutant patient derived tumor xenografts. In tamoxifen-resistant MCF7 xenografts the palbociclib/SERDor SSH combination resulted in an increased duration of response as compared to either drug alone. Conclusion A SERD- or SSH-palbociclib combination has therapeutic potential in breast tumors resistant to endocrine therapies or those expressing ESR1 mutations.

Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains first-line treatment for estrogen receptor (ESR1) positive breast cancer. However, tumor resistance limits the duration of response. The clinical efficacy of fulvestrant, a Selective Estrogen Receptor Degrader (SERD) that triggers receptor degradation, has confirmed that ESR1 often remains engaged in endocrine therapy resistant cancers. Recently developed Selective Estrogen Receptor Modulators (SERM)/SERD hybrids (SSHs) that facilitate ESR1 degradation in breast cancer cells and reproductive tissues have been advanced as an alternative treatment for advanced breast cancer, particularly in the metastatic setting. RAD1901 is one SSH currently being evaluated clinically that is unique among ESR1 modulators in that it readily enters the brain, a common site of breast cancer metastasis. In this study, RAD1901 inhibited estrogen activation of ESR1 in vitro and in vivo, inhibited estrogen-dependent breast cancer cell proliferation and xenograft tumor growth, and mediated dose-dependent downregulation of ESR1 protein. However, doses of RAD1901 insufficient to induce ESR1 degradation were shown to result in activation of ESR1 target genes and in stimulation of xenograft tumor growth. RAD1901 is an SSH that exhibits complex pharmacology in breast cancer models, having dose-dependent agonist/antagonist activity displayed in a tissue-selective manner. It remains unclear how this unique pharmacology will impact the utility of RAD1901 for breast cancer treatment. However, being the only SERD currently known to access the brain, RAD1901 merits evaluation as a targeted therapy for the treatment of breast cancer brain metastases.

Therapies that efficiently induce apoptosis are likely to be required for durable clinical responses in patients with solid tumors. Using a pharmacological screening approach, we discovered that the combined inhibition of BCL-XL and the mTOR/4E-BP axis results in selective and synergistic induction of apoptosis in cellular and animal models of PIK3CA mutant breast cancers, including triple negative tumors. Mechanistically, inhibition of mTOR/4E-BP suppresses MCL-1 protein translation only in PIK3CA mutant tumors, creating a synthetic dependence on BCL-XL. This dual dependence on BCL-XL and MCL-1, but not on BCL-2, appears to be a fundamental property of diverse breast cancer cell lines, xenografts, and patient-derived tumors that is independent of molecular subtype or PIK3CA mutational status. Further, this dependence distinguishes breast cancers from normal breast epithelial cells, which are neither primed for apoptosis nor dependent on BCL-XL/MCL-1, suggesting a potential therapeutic window. By tilting the balance of pro- to anti-apoptotic signals in the mitochondria, dual inhibition of MCL-1 and BCL-XL also sensitizes breast cancer cells to standard of care cytotoxic and targeted chemotherapies. Together, these results suggest that patients with PIK3CA mutant breast cancers may benefit from combined treatment with inhibitors of BCL-XL and the mTOR/4E-BP axis, whereas alternative methods of inhibiting MCL-1 and BCL-XL may be effective in tumors lacking PIK3CA mutations.

No abstract