Jonathan T. Lei

Proteogenomics connects somatic mutations to signalling in breast cancer

Summary Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. We describe quantitative mass spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers of which 77 provided high-quality data. Integrated analyses allowed insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. The 5q trans effects were interrogated against the Library of Integrated Network-based Cellular Signatures, thereby connecting CETN3 and SKP1 loss to elevated expression of EGFR, and SKP1 loss also to increased SRC. Global proteomic data confirmed a stromal-enriched group in addition to basal and luminal clusters and pathway analysis of the phosphoproteome identified a G Protein-coupled receptor cluster that was not readily identified at the mRNA level. Besides ERBB2, other amplicon-associated, highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

JAK kinases control IL-5 receptor ubiquitination, degradation, and internalization

IL‐5, IL‐3, and GM‐CSF are related hematopoietic cytokines, which regulate the function of myeloid cells and are mediators of the allergic inflammatory response. These cytokines signal through heteromeric receptors containing a specific α chain and a shared signaling chain, βc. Previous studies demonstrated that the ubiquitin (Ub) proteasome degradation pathway was involved in signal termination of the βc‐sharing receptors. In this study, the upstream molecular events leading to proteasome degradation of the IL‐5 receptor (IL‐5R) were examined. By using biochemical and flow cytometric methods, we show that JAK kinase activity is required for βc ubiquitination and proteasome degradation but only partially required for IL‐5R internalization. Furthermore, we demonstrate the direct ubiquitination of the βc cytoplasmic domain and identify lysine residues 566 and 603 as sites of βc ubiquitination. Lastly, we show that ubiquitination of the βc cytoplasmic domain begins at the plasma membrane, increases after receptor internalization, and is degraded by the proteasome after IL‐5R internalization. We propose an updated working model of IL‐5R down‐regulation, whereby IL‐5 ligation of its receptor activates JAK2/1 kinases, resulting in βc tyrosine phosphorylation, ubiquitination, and IL‐5R internalization. Once inside the cell, proteasomes degrade the βc cytoplasmic domain, and the truncated receptor complex is terminally degraded in the lysosomes. These data establish a critical role for JAK kinases and the Ub/proteasome degradation pathway in IL‐5R down‐regulation.

Separate endocytic pathways regulate IL-5 receptor internalization and signaling

Eosinophils are critically dependent on IL‐5 for their activation, differentiation, survival, and augmentation of cytotoxic activity. We previously showed that the cytoplasmic domain of the hematopoietic receptor, βc, which is shared by IL‐5, IL‐3, and GM‐CSF, is directly ubiquitinated and degraded by the proteasomes in a JAK2‐dependent manner. However, studies describing the spatial distribution, endocytic regulation, and trafficking of βc‐sharing receptors in human eosinophils are currently lacking. Using deconvolution microscopy and biochemical methods, we clearly demonstrate that IL‐5Rs reside in and are internalized by clathrin‐ and lipid raft‐dependent endocytic pathways. Microscopy analyses in TF1 cells and human eosinophils revealed significant colocalization of βc, IL‐5Rα, and Cy3‐labeled IL‐5 with transferrin‐ (clathrin) and cholera toxin‐B‐ (lipid raft) positive vesicles. Moreover, whereas internalized IL‐5Rs were detected in both clathrin‐ and lipid raft‐positive vesicles, biochemical data revealed that tyrosine phosphorylated, ubiquitinated, and proteasome‐degraded IL‐5Rs partitioned to the soluble, nonraft fractions (clathrin‐containing). Lastly, we show that optimal IL‐5‐induced signaling requires entry of activated IL‐5Rs into the intracellular compartment, as coimmunoprecipitation of key signaling molecules with the IL‐5R was completely blocked when either endocytic pathway was inhibited. These data provide the first evidence that IL‐5Rs segregate and traffic into two distinct plasma membrane compartments, and they further establish that IL‐5R endocytosis regulates signaling both positively and negatively.

Three Lysine Residues in the Common β Chain of the Interleukin-5 Receptor Are Required for Janus Kinase (JAK)-dependent Receptor Ubiquitination, Endocytosis, and Signaling

Background: A complete understanding of the role of βc ubiquitination in IL-5R biology is currently lacking. Results: We identified three βc lysine residues that are required for JAK kinase binding to the receptor. Conclusion: JAK kinase binding to βc via 3 lysines is essential for receptor ubiquitination. Significance: We provide new mechanistic details regarding IL-5R biology, which is important for understanding eosinophil physiology. Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory diseases including allergic asthma and hypereosinophilic syndrome. Eosinophil physiology is critically dependent on IL-5 and the IL-5 receptor (IL-5R), composed of a ligand binding α chain (IL-5Rα), and a common β chain, βc. Previously, we demonstrated that the βc cytoplasmic tail is ubiquitinated and degraded by proteasomes following IL-5 stimulation. However, a complete understanding of the role of βc ubiquitination in IL-5R biology is currently lacking. By using a well established, stably transduced HEK293 cell model system, we show here that in the absence of ubiquitination, βc subcellular localization, IL-5-induced endocytosis, turnover, and IL-5R signaling were significantly impaired. Whereas ubiquitinated IL-5Rs internalized into trafficking endosomes for their degradation, ubiquitination-deficient IL-5Rs accumulated on the cell surface and displayed blunted signaling even after IL-5 stimulation. Importantly, we identified a cluster of three membrane-proximal βc lysine residues (Lys457, Lys461, and Lys467) whose presence was required for both JAK1/2 binding to βc and receptor ubiquitination. These findings establish that JAK kinase binding to βc requires the presence of three critical βc lysine residues, and this binding event is essential for receptor ubiquitination, endocytosis, and signaling.

Loss of MutL Disrupts CHK2-Dependent Cell-Cycle Control through CDK4/6 to Promote Intrinsic Endocrine Therapy Resistance in Primary Breast Cancer

Significant endocrine therapy-resistant tumor proliferation is present in ≥20% of estrogen receptor-positive (ER+) primary breast cancers and is associated with disease recurrence and death. Here, we uncover a link between intrinsic endocrine therapy resistance and dysregulation of the MutL mismatch repair (MMR) complex (MLH1/3, PMS1/2), and demonstrate a direct role for MutL complex loss in resistance to all classes of endocrine therapy. We find that MutL deficiency in ER+ breast cancer abrogates CHK2-mediated inhibition of CDK4, a prerequisite for endocrine therapy responsiveness. Consequently, CDK4/6 inhibitors (CDK4/6i) remain effective in MutL-defective ER+ breast cancer cells. These observations are supported by data from a clinical trial where a CDK4/6i was found to strongly inhibit aromatase inhibitor-resistant proliferation of MutL-defective tumors. These data suggest that diagnostic markers of MutL deficiency could be used to direct adjuvant CDK4/6i to a population of patients with breast cancer who exhibit marked resistance to the current standard of care.Significance: MutL deficiency in a subset of ER+ primary tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy-resistant tumors. Therefore, markers of MutL dysregulation could guide CDK4/6 inhibitor use in the adjuvant setting, where the risk benefit ratio for untargeted therapeutic intervention is narrow. Cancer Discov; 7(10); 1168-83. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Functional Annotation of ESR1 Gene Fusions in Estrogen Receptor-Positive Breast Cancer.

SUMMARY RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER+) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1–6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression, explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective.

Mammary Ductal Environment Is Necessary for Faithful Maintenance of Estrogen Signaling in ER+ Breast Cancer

In this issue of Cancer Cell, Sflomos et al. (2016) describe a robust preclinical animal model of ER⁺ breast cancer. The authors identify the critical role of the breast microenvironment in determining hormone response of ER⁺ breast cancer cells and in driving the luminal phenotype of breast cancer.

Proteomic profiling identifies key coactivators utilized by mutant ERα proteins as potential new therapeutic targets

Approximately 75% of breast cancers are estrogen receptor alpha (ERα)-positive and are treatable with endocrine therapies, but often patients develop lethal resistant disease. Frequent mutations (10–40%) in the ligand-binding domain (LBD) codons in the gene encoding ERα (ESR1) have been identified, resulting in ligand-independent, constitutively active receptors. In addition, ESR1 chromosomal translocations can occur, resulting in fusion proteins that lack the LBD and are entirely unresponsive to all endocrine treatments. Thus, identifying coactivators that bind to these mutant ERα proteins may offer new therapeutic targets for endocrine-resistant cancer. To define coactivator candidate targets, a proteomics approach was performed profiling proteins recruited to the two most common ERα LBD mutants, Y537S and D538G, and an ESR1-YAP1 fusion protein. These mutants displayed enhanced coactivator interactions as compared to unliganded wild-type ERα. Inhibition of these coactivators decreased the ability of ESR1 mutants to activate transcription and promote breast cancer growth in vitro and in vivo. Thus, we have identified specific coactivators that may be useful as targets for endocrine-resistant breast cancers.

ESR1 fusions drive endocrine therapy resistance and metastasis in breast cancer

ABSTRACT Estrogen receptor alpha gene (ESR1) fusion transcripts have been identified in breast cancer but their role in breast cancer is not completely understood. Here, we report a causal role for ESR1 fusions in driving both endocrine therapy resistance and metastasis, and describe a therapeutic strategy to target ESR1 fusion-induced growth.

Abstract 1814: NF1 as an estrogen receptor-α co-repressor in breast cancer

NF1 has been best known as a GAP (GTPase Activating Protein) that inactivates Ras. However, we are now finding evidence that it also functions as an ER co-repressor, whose loss leads to endocrine therapy resistance. Sequencing tumor DNA from >600 ER+ breast cancers treated by tamoxifen adjuvant monotherapy, we found that frameshift (FS) and nonsense (NS) NF1 mutations, which can create an NF1-null state, strongly correlate with relapse risk (HR=2.6, submitted). Surprisingly, no recurrent missense NF1 mutations inactivating GAP activity were found in our cohort, and such mutations are rare in primary cancers in general. We thus posulated that complete loss of NF1 protein (e.g., caused by NS/FS mutations), but not GAP inactivation alone, is required to drive endocrine therapy resistance. Here we demonstrate that NF1 loss (by gene silencing) in ER+ breast cancer cells greatly enhances ligand-dependent ER transcriptional activity in vitro and in vivo, causing estradiol (E2) hypersensitivity and tamoxifen agonism. Mechanistically we show that NF1 can bind directly to ER, an interaction enhanced by tamoxifen but not by E2. Binding is mediated by leucine/isoleucine-rich motifs in NF1, analogous to other ER co-repressors. Mutations in these motifs (some of which are targeted by somatic mutation in cancer) inhibit ER binding and transcriptional activity without impacting GAP activity; conversely, inactivating GAP activity does not impact ER binding and repression. To validate NF1 as an ER co-repressor, we examined proteomic data from >100 breast cancer patients in the CPTAC data base and found that proteins whose levels are positively correlated with NF1 are highly enriched with factors known to bind nuclear receptors; by contrast, levels of another GAP, p120, which lacks ER binding sites, are negatively correlated with these molecules. Importantly, preclinical treatment studies indicate that while NF1-deficient ER+ breast cancer should not be treated by tamoxifen or aromatase inhibitors, fulvestrant, which degrades ER, remains effective. However, fulvestrant monotherapy can activate the Ras-MAP pathway, which may promote cell survival and acquired fulvestrant resistance unless combined with dabrafinib and trametinib to inhibit Raf and MEK —a clinical trial for this combination is in development. Our data suggest that NF1 is a dual negative regulator at the intersection of two potent oncogenic signaling pathways, Ras and ER. Combination therapy targeting both the ER and the Ras-Raf pathways should be investigated for NF1-deficient cancers driven by ER. Citation Format: Eric C. Chang, zeyi Zheng, Meenakshi Anurag, Jin Gao, Burcu Cakar, Xinhui Du, Jing Li, Philip Lavere, Jonathan T. Lei, Purba Singh, Sinem Seker, Wei Song, Jianheng Peng, Tiffany Nguyen, Doug Chan, Xi Chen, Kimberly C. Banks, Richarad B. Lanman, Maryam Shafaee, Susan Hilsenbeck, Charles Foulds, Matthew J. Ellis. NF1 as an estrogen receptor-α co-repressor in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1814.