Mass Spectrometry–Based Proteomics Reveals Potential Roles of NEK9 and MAP2K4 in Resistance to PI3K Inhibition in Triple-Negative Breast Cancers

Abstract

Activation of phosphoinositide 3-kinase (PI3K) signaling is frequently observed in triplenegative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity.To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4.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. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy. STATEMENT OF SIGNIFICANCEIntegrative phospho-proteogenomic analyses is used to determine intrinsic resistance mechanisms of triple negative breast tumors to PI3K inhibition.

Kinobead Profiling Reveals Reprogramming of B-cell Receptor Signaling in Response to Therapy Within Primary Chronic Lymphocytic Leukemia Cells

“…These latter two possibilities explain why we were only able to isolate and detect one member (RIOK2) of the atypical kinase subfamily by kinobeads. Nevertheless, consistent with studies on other cell types, [12][13][14][15] our results show that kinobeads were able to detect a substantial proportion of the kinome in malignant B-cells.…”

“…Kinobeads provide a powerful new tool to probe kinase function. [12][13][14][15] In this approach cell lysates are incubated with beads coated with broad-specificity type 1 KI, allowing binding of a large proportion of the expressed kinome. Bound kinases can then be profiled using mass-spectrometry (MS) or analyzed directly using immunoblotting.…”

BlackSheep: A Bioconductor and Bioconda package for differential extreme value analysis

“…Proteogenomic studies characterizing cancer have been completed by many groups, several of which also conducted phosphoproteome analysis (1)(2)(3)(4)(5)(6)(7). Outlier identification was used in a number of these studies to identify samples with aberrantly high levels of each phosphosite and phosphoprotein (1,8). In these studies, outlier identification and subsequent subtype enrichment was used to interpret phosphopeptide data at the protein-level, and highlight novel putative clinically relevant targets (1) or to nominate targets in a kinase inhibitor screen for sensitizers in drug-resistant cell lines (8).…”

“…Outlier identification was used in a number of these studies to identify samples with aberrantly high levels of each phosphosite and phosphoprotein (1,8). In these studies, outlier identification and subsequent subtype enrichment was used to interpret phosphopeptide data at the protein-level, and highlight novel putative clinically relevant targets (1) or to nominate targets in a kinase inhibitor screen for sensitizers in drug-resistant cell lines (8). This nonparametric method is of particular use for multi-omics studies, as non-parametric approaches are more robust to the various sources of technical noise present in these data sets, which violate assumptions in parametric tests.…”

Microscaled Proteogenomic Methods for Precision Oncology

“…Both by improving the functional annotation of genomic aberrations and by providing insights into pathway activation, this multi-dimensional approach to the characterization of human tumors has shown promise for the delineation of cancer biology and treatment options 1-5 . In addition, proteogenomics applied to patient-derived xenograft (PDX) samples has exposed potential predictive markers and mechanisms of tumor response and resistance 3,6,7 .…”