Detecting drug resistance in TNBC

Triple-negative breast cancer (TNBC) represents roughly 15 percent of breast cancer diagnoses for women, and is one of the most aggressive types of cancer. This cancer is difficult to treat as it is, given that it is negative for estrogen, progesterone and HER2/neu receptors and therefore doesn't respond to therapies targeted to those receptors, even without the tendency towards metastasis. The primary treatment for TNBC is chemotherapy, but therapeutic resistance is a common problem. PI3K inhibitors—which target the phosphoinositide 3-kinase pathway, which sees increased signaling in TNBC—are a common cancer treatment, but they too offer only limited efficacy in TNBC.

To explore this, Dr. Steven Carr of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) and his team at the Broad Institute of MIT and Harvard used preclinical models to explore cellular events linked to sensitivity or resistance to PI3K treatment. Their work was published in Cancer Research in an article titled “Mass spectrometry-based proteomics reveals potential roles of NEK9 and MAP2K4 in resistance to PI3K inhibitors in triple negative breast cancers.”

As the authors note in their paper, buparlisib targets “all of the class I PI3-kinase isoforms,” but at present no clinically validated biomarkers exist for predicting responses to PI3K inhibitors. Though previous studies have examined genomic alterations with little success, the team hypothesized that “investigations at the levels of the proteome and the phosphorylation status of proteins, a key post-translational modification central to cellular signal transduction, could potentially identify aberrant signaling pathways or proteins associated with treatment response or resistance.”

The scientists started with a panel of patient-derived xenograft (PDX) models of TNBC that presented with different levels of responsiveness to buparlisib, a pan-PI3K inhibitor. They combined whole proteome data generated via liquid chromatography-high resolution tandem mass spectrometry (LC-MS/MS) and data from kinase and transcriptome profiling, and were able to find phosphoproteomic changes in PI3K, PLK1 (polo like kinase 1) and MAPK/MEK (mitogen-activated protein kinase) family members. Further analysis with kinomes revealed that NIMA related kinase 9 (NEK9) and mitogen-activated protein kinase kinase (MAP2K4) play a role in buparlisib resistance and might be regulators of TNBC. As noted in the paper, “Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro. A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4-dependent mechanisms.”

Within the six PDX models Carr and colleagues used, they found that “On a pathway level, buparlisib-induced changes in the transcriptome related to PIP3, PTEN, and mTOR signaling and several phases of the cell cycle, as well as to gene expression/transcription. Significant proteins in pathways related to nucleic acid, sugar, fatty acid, and amino acid metabolism as well as to steroid biosynthesis were upregulated by buparlisib treatment across all of the PDX models. In the phosphoproteome, buparlisib induced changes related to PI3K, AKT and mTOR signaling as well as the crosstalking PLK1 (Polo-Like Kinase 1) pathway.”

The team then took the two PDX models that were most sensitive to buparlisib treatment and the two that were most resistant and compared the changes in each following buparlisib treatment.

According to the Cancer Research paper, “With an adjusted p-value ≤0.05, 108 protein markers and 340 phosphosites were differentially expressed between the resistant and the sensitive tumors. These proteins are involved in pathways related to the Complement System, the Endosomal Vacuolar Pathway and Coagulation, as well as the MTA3 Pathway that has previously been shown to be down-regulated in ER-negative breast tumors. Furthermore, a Connectivity Map (CMAP) analysis showed that proteins that are significantly and relatively higher in the sensitive tumors after buparlisib treatment are very similar to the transcriptional changes that are observed in multiple cell lines (from various tissue origins, including breast) after treatment with many PI3K and mTOR inhibitors. In contrast, the same CMAP analysis showed that proteins that are relatively high in the resistant tumors show similar traits to a diverse set of treatments.”

The authors caution that their results are not wholly conclusive, noting that "Tumor heterogeneity, together with the low number of samples in this study, impeded our ability to identify a unified mechanism of buparlisib resistance with high confidence. However, markers shared between omic platforms showed a moderate positive correlation, with correlations between mRNA and protein levels very similar to two previous human cancer studies and a recent large PDX baseline study that included our six models."

In addition, their results show that NEK9 and MAP2K4 also impact survivin, which is “highly expressed in most cancers and is associated with chemotherapy resistance,” and the authors report that “a clear correlation between NEK9/MAP2K4 levels and survivin expression was observed in this study.” As such, the team argues that both targets should be further explored for their potential in TNBC and PI3K inhibitor treatment specifically, and point to their results in support of the importance of multi-omic investigations.