Kinase inhibitor pulldown assay (KiP) for clinical proteomics.

Protein kinases are frequently dysregulated and/or mutated in cancer and represent essential targets for therapy. Accurate quantification is essential. For breast cancer treatment, the identification and quantification of the protein kinase ERBB2 is critical for therapeutic decisions. While immunohistochemistry (IHC) is the current clinical diagnostic approach, it is only semiquantitative. Mass spectrometry-based proteomics offers quantitative assays that, unlike IHC, can be used to accurately evaluate hundreds of kinases simultaneously. The enrichment of less abundant kinase targets for quantification, along with depletion of interfering proteins, improves sensitivity and thus promotes more effective downstream analyses. Multiple kinase inhibitors were therefore deployed as a capture matrix for kinase inhibitor pulldown (KiP) assays designed to profile the human protein kinome as broadly as possible. Optimized assays were initially evaluated in 16 patient derived xenograft models (PDX) where KiP identified multiple differentially expressed and biologically relevant kinases. From these analyses, an optimized single-shot parallel reaction monitoring (PRM) method was developed to improve quantitative fidelity. The PRM KiP approach was then reapplied to low quantities of proteins typical of yields from core needle biopsies of human cancers. The initial prototype targeting 100 kinases recapitulated intrinsic subtyping of PDX models obtained from comprehensive proteomic and transcriptomic profiling. Luminal and HER2 enriched OCT-frozen patient biopsies subsequently analyzed through KiP-PRM also clustered by subtype. Finally, stable isotope labeled peptide standards were developed to define a prototype clinical method. Data are available via ProteomeXchange with identifiers PXD044655 and PXD046169.

TGIF1 functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

A prominent function of TGIF1 is suppression of transforming growth factor beta (TGF-ß) signaling, whose inactivation is deemed instrumental to the progression of pancreatic ductal adenocarcinoma (PDAC), as exemplified by the frequent loss of the tumor suppressor gene SMAD4 in this malignancy. Surprisingly, we found that genetic inactivation of Tgif1 in the context of oncogenic Kras, KrasG12D , culminated in the development of highly aggressive and metastatic PDAC despite de-repressing TGF-ß signaling. Mechanistic experiments show that TGIF1 associates with Twist1 and inhibits Twist1 expression and activity, and this function is suppressed in the vast majority of human PDACs by KrasG12D /MAPK-mediated TGIF1 phosphorylation. Ablating Twist1 in KrasG12D ;Tgif1KO mice completely blunted PDAC formation, providing the proof-of-principle that TGIF1 restrains KrasG12D -driven PDAC through its ability to antagonize Twist1. Collectively, these findings pinpoint TGIF1 as a potential tumor suppressor in PDAC and further suggest that sustained activation of TGF-ß signaling might act to accelerate PDAC progression rather than to suppress its initiation.

DPYSL3 modulates mitosis, migration, and epithelial-to-mesenchymal transition in claudin-low breast cancer.

A Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomic analysis prioritized dihydropyrimidinase-like-3 (DPYSL3) as a multilevel (RNA/protein/phosphoprotein) expression outlier specific to the claudin-low (CLOW) subset of triple-negative breast cancers. A PubMed informatics tool indicated a paucity of data in the context of breast cancer, which further prioritized DPYSL3 for study. DPYSL3 knockdown in DPYSL3-positive ([Formula: see text]) CLOW cell lines demonstrated reduced proliferation, yet enhanced motility and increased expression of epithelial-to-mesenchymal transition (EMT) markers, suggesting that DPYSL3 is a multifunctional signaling modulator. Slower proliferation in DPYSL3-negative ([Formula: see text]) CLOW cells was associated with accumulation of multinucleated cells, indicating a mitotic defect that was associated with a collapse of the vimentin microfilament network and increased vimentin phosphorylation. DPYSL3 also suppressed the expression of EMT regulators SNAIL and TWIST and opposed p21 activated kinase 2 (PAK2)-dependent migration. However, these EMT regulators in turn induce DPYSL3 expression, suggesting that DPYSL3 participates in negative feedback on EMT. In conclusion, DPYSL3 expression identifies CLOW tumors that will be sensitive to approaches that promote vimentin phosphorylation during mitosis and inhibitors of PAK signaling during migration and EMT.

gpGrouper: A Peptide Grouping Algorithm for Gene-Centric Inference and Quantitation of Bottom-Up Proteomics Data.

In quantitative mass spectrometry, the method by which peptides are grouped into proteins can have dramatic effects on downstream analyses. Here we describe gpGrouper, an inference and quantitation algorithm that offers an alternative method for assignment of protein groups by gene locus and improves pseudo-absolute iBAQ quantitation by weighted distribution of shared peptide areas. We experimentally show that distributing shared peptide quantities based on unique peptide peak ratios improves quantitation accuracy compared with conventional winner-take-all scenarios. Furthermore, gpGrouper seamlessly handles two-species samples such as patient-derived xenografts (PDXs) without ignoring the host species or species-shared peptides. This is a critical capability for proper evaluation of proteomics data from PDX samples, where stromal infiltration varies across individual tumors. Finally, gpGrouper calculates peptide peak area (MS1) based expression estimates from multiplexed isobaric data, producing iBAQ results that are directly comparable across label-free, isotopic, and isobaric proteomics approaches.

Kinome Reprogramming Is a Targetable Vulnerability in ESR1 Fusion-Driven Breast Cancer.

Transcriptionally active ESR1 fusions (ESR1-TAF) are a potent cause of breast cancer endocrine therapy (ET) resistance. ESR1-TAFs are not directly druggable because the C-terminal estrogen/anti-estrogen-binding domain is replaced with translocated in-frame partner gene sequences that confer constitutive transactivation. To discover alternative treatments, a mass spectrometry (MS)-based kinase inhibitor pulldown assay (KIPA) was deployed to identify druggable kinases that are upregulated by diverse ESR1-TAFs. Subsequent explorations of drug sensitivity validated RET kinase as a common therapeutic vulnerability despite remarkable ESR1-TAF C-terminal sequence and structural diversity. Organoids and xenografts from a pan-ET-resistant patient-derived xenograft model that harbors the ESR1-e6>YAP1 TAF were concordantly inhibited by the selective RET inhibitor pralsetinib to a similar extent as the CDK4/6 inhibitor palbociclib. Together, these findings provide preclinical rationale for clinical evaluation of RET inhibition for the treatment of ESR1-TAF-driven ET-resistant breast cancer. SIGNIFICANCE: Kinome analysis of ESR1 translocated and mutated breast tumors using drug bead-based mass spectrometry followed by drug-sensitivity studies nominates RET as a therapeutic target. See related commentary by Wu and Subbiah, p. 3159.

Proteogenomic Approaches for the Identification of NF1/Neurofibromin-depleted Estrogen Receptor-positive Breast Cancers for Targeted Treatment.

NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader, together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ patient-derived xenograft (PDX) models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pulldown. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An IHC assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Significance: A major challenge for targeting the consequence of tumor suppressor disruption is the accurate assessment of protein functional inactivation. NF1 can repress both RAS and ER signaling, and a ComboMATCH trial is underway to treat the patients with binimetinib and fulvestrant. Herein we report a MS-verified NF1 IHC assay that can determine a threshold for NF1 loss to predict treatment response. These approaches may be used to identify and expand the eligible patient population.

Transcriptional Reprogramming Differentiates Active from Inactive ESR1 Fusions in Endocrine Therapy-Refractory Metastatic Breast Cancer.

Genomic analysis has recently identified multiple ESR1 gene translocations in estrogen receptor alpha-positive (ERα+) metastatic breast cancer (MBC) that encode chimeric proteins whereby the ESR1 ligand binding domain (LBD) is replaced by C-terminal sequences from many different gene partners. Here we functionally screened 15 ESR1 fusions and identified 10 that promoted estradiol-independent cell growth, motility, invasion, epithelial-to-mesenchymal transition, and resistance to fulvestrant. RNA sequencing identified a gene expression pattern specific to functionally active ESR1 gene fusions that was subsequently reduced to a diagnostic 24-gene signature. This signature was further examined in 20 ERα+ patient-derived xenografts and in 55 ERα+ MBC samples. The 24-gene signature successfully identified cases harboring ESR1 gene fusions and also accurately diagnosed the presence of activating ESR1 LBD point mutations. Therefore, the 24-gene signature represents an efficient approach to screening samples for the presence of diverse somatic ESR1 mutations and translocations that drive endocrine treatment failure in MBC. SIGNIFICANCE: This study identifies a gene signature diagnostic for functional ESR1 fusions that drive poor outcome in advanced breast cancer, which could also help guide precision medicine approaches in patients harboring ESR1 mutations.

The bone microenvironment increases phenotypic plasticity of ER+ breast cancer cells.

Estrogen receptor-positive (ER+) breast cancer exhibits a strong bone tropism in metastasis. How the bone microenvironment (BME) impacts ER signaling and endocrine therapy remains poorly understood. Here, we discover that the osteogenic niche transiently and reversibly reduces ER expression and activities specifically in bone micrometastases (BMMs), leading to endocrine resistance. As BMMs progress, the ER reduction and endocrine resistance may partially recover in cancer cells away from the osteogenic niche, creating phenotypic heterogeneity in macrometastases. Using multiple approaches, including an evolving barcoding strategy, we demonstrated that this process is independent of clonal selection, and represents an EZH2-mediated epigenomic reprogramming. EZH2 drives ER+ BMMs toward a basal and stem-like state. EZH2 inhibition reverses endocrine resistance. These data exemplify how epigenomic adaptation to BME promotes phenotypic plasticity of metastatic seeds, fosters intra-metastatic heterogeneity, and alters therapeutic responses. Our study provides insights into the clinical enigma of ER+ metastatic recurrences despite endocrine therapies.

Neurofibromin Is an Estrogen Receptor-α Transcriptional Co-repressor in Breast Cancer.

We report that neurofibromin, a tumor suppressor and Ras-GAP (GTPase-activating protein), is also an estrogen receptor-α (ER) transcriptional co-repressor through leucine/isoleucine-rich motifs that are functionally independent of GAP activity. GAP activity, in turn, does not affect ER binding. Consequently, neurofibromin depletion causes estradiol hypersensitivity and tamoxifen agonism, explaining the poor prognosis associated with neurofibromin loss in endocrine therapy-treated ER+ breast cancer. Neurofibromin-deficient ER+ breast cancer cells initially retain sensitivity to selective ER degraders (SERDs). However, Ras activation does play a role in acquired SERD resistance, which can be reversed upon MEK inhibitor addition, and SERD/MEK inhibitor combinations induce tumor regression. Thus, neurofibromin is a dual repressor for both Ras and ER signaling, and co-targeting may treat neurofibromin-deficient ER+ breast tumors.

Microscaled proteogenomic methods for precision oncology.

Cancer proteogenomics promises new insights into cancer biology and treatment efficacy by integrating genomics, transcriptomics and protein profiling including modifications by mass spectrometry (MS). A critical limitation is sample input requirements that exceed many sources of clinically important material. Here we report a proteogenomics approach for core biopsies using tissue-sparing specimen processing and microscaled proteomics. As a demonstration, we analyze core needle biopsies from ERBB2 positive breast cancers before and 48-72 h after initiating neoadjuvant trastuzumab-based chemotherapy. We show greater suppression of ERBB2 protein and both ERBB2 and mTOR target phosphosite levels in cases associated with pathological complete response, and identify potential causes of treatment resistance including the absence of ERBB2 amplification, insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification, and candidate resistance mechanisms including androgen receptor signaling, mucin overexpression and an inactive immune microenvironment. The clinical utility and discovery potential of proteogenomics at biopsy-scale warrants further investigation.

Twist1 Activation in Muscle Progenitor Cells Causes Muscle Loss Akin to Cancer Cachexia.

Cancer cachexia is characterized by extreme skeletal muscle loss that results in high morbidity and mortality. The incidence of cachexia varies among tumor types, being lowest in sarcomas, whereas 90% of pancreatic ductal adenocarcinoma (PDAC) patients experience severe weight loss. How these tumors trigger muscle depletion is still unfolding. Serendipitously, we found that overexpression of Twist1 in mouse muscle progenitor cells, either constitutively during development or inducibly in adult animals, caused severe muscle atrophy with features reminiscent of cachexia. Using several genetic mouse models of PDAC, we detected a marked increase in Twist1 expression in muscle undergoing cachexia. In cancer patients, elevated levels of Twist1 are associated with greater degrees of muscle wasting. Finally, both genetic and pharmacological inactivation of Twist1 in muscle progenitor cells afforded substantial protection against cancer-mediated cachexia, which translated into meaningful survival benefits, implicating Twist1 as a possible target for attenuating muscle cachexia in cancer patients.

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

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.

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.

Proteogenomic integration reveals therapeutic targets in breast cancer xenografts.

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities.