Proteogenomics connects somatic mutations to signalling in breast cancer.

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here 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 provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins 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. In addition to 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 the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

Regulated Phosphosignaling Associated with Breast Cancer Subtypes and Druggability.

Aberrant phospho-signaling is a hallmark of cancer. We investigated kinase-substrate regulation of 33,239 phosphorylation sites (phosphosites) in 77 breast tumors and 24 breast cancer xenografts. Our search discovered 2134 quantitatively correlated kinase-phosphosite pairs, enriching for and extending experimental or binding-motif predictions. Among the 91 kinases with auto-phosphorylation, elevated EGFR, ERBB2, PRKG1, and WNK1 phosphosignaling were enriched in basal, HER2-E, Luminal A, and Luminal B breast cancers, respectively, revealing subtype-specific regulation. CDKs, MAPKs, and ataxia-telangiectasia proteins were dominant, master regulators of substrate-phosphorylation, whose activities are not captured by genomic evidence. We unveiled phospho-signaling and druggable targets from 113 kinase-substrate pairs and cascades downstream of kinases, including AKT1, BRAF and EGFR. We further identified kinase-substrate-pairs associated with clinical or immune signatures and experimentally validated activated phosphosites of ERBB2, EIF4EBP1, and EGFR. Overall, kinase-substrate regulation revealed by the largest unbiased global phosphorylation data to date connects driver events to their signaling effects.

Research-based PAM50 signature and long-term breast cancer survival.

PURPOSE: Multi-gene signatures provide biological insight and risk stratification in breast cancer. Intrinsic molecular subtypes defined by mRNA expression of 50 genes (PAM50) are prognostic in hormone-receptor positive postmenopausal breast cancer. Yet, for 25-40% in the PAM50 intermediate risk group, long-term risk remains uncertain. Our study aimed to (i) test the long-term prognostic value of the PAM50 signature in pre- and post-menopausal breast cancer; (ii) investigate if the PAM50 model could be improved by addition of other mRNAs implicated in oncogenesis. METHODS: We used archived FFPE samples from 1723 breast cancer survivors; high quality reads were obtained on 1253 samples. Transcript expression was quantified using a custom codeset with probes for > 100 targets. Cox models assessed gene signatures for breast cancer relapse and survival. RESULTS: Over 15 + years of follow-up, PAM50 subtypes were (P < 0.01) associated with breast cancer outcomes after accounting for tumor stage, grade and age at diagnosis. Results did not differ by menopausal status at diagnosis. Women with Luminal B (versus Luminal A) subtype had a > 60% higher hazard. Addition of a 13-gene hypoxia signature improved prognostication with > 40% higher hazard in the highest vs lowest hypoxia tertiles. CONCLUSIONS: PAM50 intrinsic subtypes were independently prognostic for long-term breast cancer survival, irrespective of menopausal status. Addition of hypoxia signatures improved risk prediction. If replicated, incorporating the 13-gene hypoxia signature into the existing PAM50 risk assessment tool, may refine risk stratification and further clarify treatment for breast cancer.

Proteogenomic characterization of human colon and rectal cancer.

Extensive genomic characterization of human cancers presents the problem of inference from genomic abnormalities to cancer phenotypes. To address this problem, we analysed proteomes of colon and rectal tumours characterized previously by The Cancer Genome Atlas (TCGA) and perform integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. Messenger RNA transcript abundance did not reliably predict protein abundance differences between tumours. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA 'microsatellite instability/CpG island methylation phenotype' transcriptomic subtype, but had distinct mutation, methylation and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates, including HNF4A (hepatocyte nuclear factor 4, alpha), TOMM34 (translocase of outer mitochondrial membrane 34) and SRC (SRC proto-oncogene, non-receptor tyrosine kinase). Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology.

QuantFusion: Novel Unified Methodology for Enhanced Coverage and Precision in Quantifying Global Proteomic Changes in Whole Tissues.

Single quantitative platforms such as label-based or label-free quantitation (LFQ) present compromises in accuracy, precision, protein sequence coverage, and speed of quantifiable proteomic measurements. To maximize the quantitative precision and the number of quantifiable proteins or the quantifiable coverage of tissue proteomes, we have developed a unified approach, termed QuantFusion, that combines the quantitative ratios of all peptides measured by both LFQ and label-based methodologies. Here, we demonstrate the use of QuantFusion in determining the proteins differentially expressed in a pair of patient-derived tumor xenografts (PDXs) representing two major breast cancer (BC) subtypes, basal and luminal. Label-based in-spectra quantitative peptides derived from amino acid-coded tagging (AACT, also known as SILAC) of a non-malignant mammary cell line were uniformly added to each xenograft with a constant predefined ratio, from which Ratio-of-Ratio estimates were obtained for the label-free peptides paired with AACT peptides in each PDX tumor. A mixed model statistical analysis was used to determine global differential protein expression by combining complementary quantifiable peptide ratios measured by LFQ and Ratio-of-Ratios, respectively. With minimum number of replicates required for obtaining the statistically significant ratios, QuantFusion uses the distinct mechanisms to "rescue" the missing data inherent to both LFQ and label-based quantitation. Combined quantifiable peptide data from both quantitative schemes increased the overall number of peptide level measurements and protein level estimates. In our analysis of the PDX tumor proteomes, QuantFusion increased the number of distinct peptide ratios by 65%, representing differentially expressed proteins between the BC subtypes. This quantifiable coverage improvement, in turn, not only increased the number of measurable protein fold-changes by 8% but also increased the average precision of quantitative estimates by 181% so that some BC subtypically expressed proteins were rescued by QuantFusion. Thus, incorporating data from multiple quantitative approaches while accounting for measurement variability at both the peptide and global protein levels make QuantFusion unique for obtaining increased coverage and quantitative precision for tissue proteomes.

Integrated Bottom-Up and Top-Down Proteomics of Patient-Derived Breast Tumor Xenografts.

Bottom-up proteomics relies on the use of proteases and is the method of choice for identifying thousands of protein groups in complex samples. Top-down proteomics has been shown to be robust for direct analysis of small proteins and offers a solution to the "peptide-to-protein" inference problem inherent with bottom-up approaches. Here, we describe the first large-scale integration of genomic, bottom-up and top-down proteomic data for the comparative analysis of patient-derived mouse xenograft models of basal and luminal B human breast cancer, WHIM2 and WHIM16, respectively. Using these well-characterized xenograft models established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium, we compared and contrasted the performance of bottom-up and top-down proteomics to detect cancer-specific aberrations at the peptide and proteoform levels and to measure differential expression of proteins and proteoforms. Bottom-up proteomic analysis of the tumor xenografts detected almost 10 times as many coding nucleotide polymorphisms and peptides resulting from novel splice junctions than top-down. For proteins in the range of 0-30 kDa, where quantitation was performed using both approaches, bottom-up proteomics quantified 3,519 protein groups from 49,185 peptides, while top-down proteomics quantified 982 proteoforms mapping to 358 proteins. Examples of both concordant and discordant quantitation were found in a ∼60:40 ratio, providing a unique opportunity for top-down to fill in missing information. The two techniques showed complementary performance, with bottom-up yielding eight times more identifications of 0-30 kDa proteins in xenograft proteomes, but failing to detect differences in certain posttranslational modifications (PTMs), such as phosphorylation pattern changes of alpha-endosulfine. This work illustrates the potency of a combined bottom-up and top-down proteomics approach to deepen our knowledge of cancer biology, especially when genomic data are available.

An Analysis of the Sensitivity of Proteogenomic Mapping of Somatic Mutations and Novel Splicing Events in Cancer.

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (∼80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor, raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identify gaps in sequence coverage, thereby benchmarking current technology and progress toward whole cancer proteome and transcriptome analysis.

Quality Assessments of Long-Term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues.

Clinical proteomics requires large-scale analysis of human specimens to achieve statistical significance. We evaluated the long-term reproducibility of an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomics strategy using one channel for reference across all samples in different iTRAQ sets. A total of 148 liquid chromatography tandem mass spectrometric (LC-MS/MS) analyses were completed, generating six 2D LC-MS/MS data sets for human-in-mouse breast cancer xenograft tissues representative of basal and luminal subtypes. Such large-scale studies require the implementation of robust metrics to assess the contributions of technical and biological variability in the qualitative and quantitative data. Accordingly, we derived a quantification confidence score based on the quality of each peptide-spectrum match to remove quantification outliers from each analysis. After combining confidence score filtering and statistical analysis, reproducible protein identification and quantitative results were achieved from LC-MS/MS data sets collected over a 7-month period. This study provides the first quality assessment on long-term stability and technical considerations for study design of a large-scale clinical proteomics project.

Reproducibility of Differential Proteomic Technologies in CPTAC Fractionated Xenografts.

The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC-MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation (source code is available from http://homepages.uc.edu/~wang2x7/Research.htm ). From these assessments, we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61 to 93% of the time. When comparing across different instruments and quantitative technologies, using multiple replicates, differential genes were reproduced by other data sets from 67 to 99% of the time. Projecting gene differences to biological pathways and networks increased the degree of similarity. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation.

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.

Genome remodelling in a basal-like breast cancer metastasis and xenograft.

Massively parallel DNA sequencing technologies provide an unprecedented ability to screen entire genomes for genetic changes associated with tumour progression. Here we describe the genomic analyses of four DNA samples from an African-American patient with basal-like breast cancer: peripheral blood, the primary tumour, a brain metastasis and a xenograft derived from the primary tumour. The metastasis contained two de novo mutations and a large deletion not present in the primary tumour, and was significantly enriched for 20 shared mutations. The xenograft retained all primary tumour mutations and displayed a mutation enrichment pattern that resembled the metastasis. Two overlapping large deletions, encompassing CTNNA1, were present in all three tumour samples. The differential mutation frequencies and structural variation patterns in metastasis and xenograft compared with the primary tumour indicate that secondary tumours may arise from a minority of cells within the primary tumour.

Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels.

Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. Although the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 min, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation, and cell death. Both pan-tumor and tissue-specific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples and motivates reexamination of collection protocols for phosphoprotein analysis.

Comprehensive quantitative analysis of ovarian and breast cancer tumor peptidomes.

Aberrant degradation of proteins is associated with many pathological states, including cancers. Mass spectrometric analysis of tumor peptidomes, the intracellular and intercellular products of protein degradation, has the potential to provide biological insights on proteolytic processing in cancer. However, attempts to use the information on these smaller protein degradation products from tumors for biomarker discovery and cancer biology studies have been fairly limited to date, largely due to the lack of effective approaches for robust peptidomics identification and quantification and the prevalence of confounding factors and biases associated with sample handling and processing. Herein, we have developed an effective and robust analytical platform for comprehensive analyses of tissue peptidomes, which is suitable for high-throughput quantitative studies. The reproducibility and coverage of the platform, as well as the suitability of clinical ovarian tumor and patient-derived breast tumor xenograft samples with postexcision delay of up to 60 min before freezing for peptidomics analysis, have been demonstrated. Moreover, our data also show that the peptidomics profiles can effectively separate breast cancer subtypes, reflecting tumor-associated protease activities. Peptidomics complements results obtainable from conventional bottom-up proteomics and provides insights not readily obtainable from such approaches.

Evaluation of Copanlisib in Combination with Eribulin in Triple-negative Breast Cancer Patient-derived Xenograft Models.

The PI3K pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple-negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the antitumor effect in TNBC. A panel of eight TNBC patient-derived xenograft (PDX) models was tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment-induced signaling changes were examined by reverse phase protein array, immunohistochemistry (IHC) and 18F-fluorodeoxyglucose PET (18F-FDG PET). Compared with each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin-induced mitotic arrest. The combination enhanced induction of apoptosis compared with each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan and AKT phosphorylation by IHC. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin and led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC. SIGNIFICANCE: In this research, we demonstrated that the pan-PI3K inhibitor copanlisib enhanced the cytotoxicity of eribulin in a panel of TNBC PDX models. The improved tumor growth inhibition was irrespective of PI3K pathway alteration and was corroborated by the enhanced mitotic arrest and apoptotic induction observed in PDX tumors after combination therapy compared with each drug alone. These data provide the preclinical rationale for the clinical testing in TNBC.

Combined KRAS-MAPK pathway inhibitors and HER2-directed drug conjugate is efficacious in pancreatic cancer.

Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients.

Supervised Risk Predictor of Breast Cancer Based on Intrinsic Subtypes.

PURPOSE: To improve on current standards for breast cancer prognosis and prediction of chemotherapy benefit by developing a risk model that incorporates the gene expression-based "intrinsic" subtypes luminal A, luminal B, HER2-enriched, and basal-like. METHODS: A 50-gene subtype predictor was developed using microarray and quantitative reverse transcriptase polymerase chain reaction data from 189 prototype samples. Test sets from 761 patients (no systemic therapy) were evaluated for prognosis, and 133 patients were evaluated for prediction of pathologic complete response (pCR) to a taxane and anthracycline regimen. RESULTS: The intrinsic subtypes as discrete entities showed prognostic significance (P = 2.26E-12) and remained significant in multivariable analyses that incorporated standard parameters (estrogen receptor status, histologic grade, tumor size, and node status). A prognostic model for node-negative breast cancer was built using intrinsic subtype and clinical information. The C-index estimate for the combined model (subtype and tumor size) was a significant improvement on either the clinicopathologic model or subtype model alone. The intrinsic subtype model predicted neoadjuvant chemotherapy efficacy with a negative predictive value for pCR of 97%. CONCLUSION: Diagnosis by intrinsic subtype adds significant prognostic and predictive information to standard parameters for patients with breast cancer. The prognostic properties of the continuous risk score will be of value for the management of node-negative breast cancers. The subtypes and risk score can also be used to assess the likelihood of efficacy from neoadjuvant chemotherapy.

Differential chromatin accessibility and transcriptional dynamics define breast cancer subtypes and their lineages.

Breast cancer is a heterogeneous disease, and treatment is guided by biomarker profiles representing distinct molecular subtypes. Breast cancer arises from the breast ductal epithelium, and experimental data suggests breast cancer subtypes have different cells of origin within that lineage. The precise cells of origin for each subtype and the transcriptional networks that characterize these tumor-normal lineages are not established. In this work, we applied bulk, single-cell (sc), and single-nucleus (sn) multi-omic techniques as well as spatial transcriptomics and multiplex imaging on 61 samples from 37 breast cancer patients to show characteristic links in gene expression and chromatin accessibility between breast cancer subtypes and their putative cells of origin. We applied the PAM50 subtyping algorithm in tandem with bulk RNA-seq and snRNA-seq to reliably subtype even low-purity tumor samples and confirm promoter accessibility using snATAC. Trajectory analysis of chromatin accessibility and differentially accessible motifs clearly connected progenitor populations with breast cancer subtypes supporting the cell of origin for basal-like and luminal A and B tumors. Regulatory network analysis of transcription factors underscored the importance of BHLHE40 in luminal breast cancer and luminal mature cells, and KLF5 in basal-like tumors and luminal progenitor cells. Furthermore, we identify key genes defining the basal-like ( PRKCA , SOX6 , RGS6 , KCNQ3 ) and luminal A/B ( FAM155A , LRP1B ) lineages, with expression in both precursor and cancer cells and further upregulation in tumors. Exhausted CTLA4-expressing CD8+ T cells were enriched in basal-like breast cancer, suggesting altered means of immune dysfunction among breast cancer subtypes. We used spatial transcriptomics and multiplex imaging to provide spatial detail for key markers of benign and malignant cell types and immune cell colocation. These findings demonstrate analysis of paired transcription and chromatin accessibility at the single cell level is a powerful tool for investigating breast cancer lineage development and highlight transcriptional networks that define basal and luminal breast cancer lineages.

Combining the Tyrosine Kinase Inhibitor Cabozantinib and the mTORC1/2 Inhibitor Sapanisertib Blocks ERK Pathway Activity and Suppresses Tumor Growth in Renal Cell Carcinoma.

Current treatment approaches for renal cell carcinoma (RCC) face challenges in achieving durable tumor responses due to tumor heterogeneity and drug resistance. Combination therapies that leverage tumor molecular profiles could offer an avenue for enhancing treatment efficacy and addressing the limitations of current therapies. To identify effective strategies for treating RCC, we selected ten drugs guided by tumor biology to test in six RCC patient-derived xenograft (PDX) models. The multitargeted tyrosine kinase inhibitor (TKI) cabozantinib and mTORC1/2 inhibitor sapanisertib emerged as the most effective drugs, particularly when combined. The combination demonstrated favorable tolerability and inhibited tumor growth or induced tumor regression in all models, including two from patients who experienced treatment failure with FDA-approved TKI and immunotherapy combinations. In cabozantinib-treated samples, imaging analysis revealed a significant reduction in vascular density, and single-nucleus RNA sequencing (snRNA-seq) analysis indicated a decreased proportion of endothelial cells in the tumors. SnRNA-seq data further identified a tumor subpopulation enriched with cell-cycle activity that exhibited heightened sensitivity to the cabozantinib and sapanisertib combination. Conversely, activation of the epithelial-mesenchymal transition pathway, detected at the protein level, was associated with drug resistance in residual tumors following combination treatment. The combination effectively restrained ERK phosphorylation and reduced expression of ERK downstream transcription factors and their target genes implicated in cell-cycle control and apoptosis. This study highlights the potential of the cabozantinib plus sapanisertib combination as a promising treatment approach for patients with RCC, particularly those whose tumors progressed on immune checkpoint inhibitors and other TKIs. SIGNIFICANCE: The molecular-guided therapeutic strategy of combining cabozantinib and sapanisertib restrains ERK activity to effectively suppress growth of renal cell carcinomas, including those unresponsive to immune checkpoint inhibitors.

Met induces diverse mammary carcinomas in mice and is associated with human basal breast cancer.

Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. The oncogene MET is associated with decreased survival in breast cancer, yet the role that MET plays in the various breast cancer subtypes is unclear. We describe a knockin mouse with mutationally activated Met (Met(mut)) that develops a high incidence of diverse mammary tumors with basal characteristics, including metaplasia, absence of progesterone receptor and ERBB2 expression, and expression of cytokeratin 5. With gene expression and tissue microarray analysis, we show that high MET expression in human breast cancers significantly correlated with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers. Few treatment options exist for breast cancers of the basal or trastuzumab-resistant ERBB2 subtypes. We conclude from these studies that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target.

A randomized phase 2 study of neoadjuvant carboplatin and paclitaxel with or without atezolizumab in triple negative breast cancer (TNBC) - NCI 10013.

Atezolizumab with chemotherapy has shown improved progression-free and overall survival in patients with metastatic PD-L1 positive triple negative breast cancer (TNBC). Atezolizumab with anthracycline- and taxane-based neoadjuvant chemotherapy has also shown increased pathological complete response (pCR) rates in early TNBC. This trial evaluated neoadjuvant carboplatin and paclitaxel with or without atezolizumab in patients with clinical stages II-III TNBC. The co-primary objectives were to evaluate if chemotherapy and atezolizumab increase pCR rate and tumor infiltrating lymphocyte (TIL) percentage compared to chemotherapy alone in the mITT population. Sixty-seven patients (ages 25-78 years; median, 52 years) were randomly assigned - 22 patients to Arm A, and 45 to Arm B. Median follow up was 6.6 months. In the modified intent to treat population (all patients evaluable for the primary endpoints who received at least one dose of combination therapy), the pCR rate was 18.8% (95% CI 4.0-45.6%) in Arm A, and 55.6% (95% CI 40.0-70.4%) in Arm B (estimated treatment difference: 36.8%, 95% CI 8.5-56.6%; p = 0.018). Grade 3 or higher treatment-related adverse events occurred in 62.5% of patients in Arm A, and 57.8% of patients in Arm B. One patient in Arm B died from recurrent disease during the follow-up period. TIL percentage increased slightly from baseline to cycle 1 in both Arm A (mean ± SD: 0.6% ± 21.0%) and Arm B (5.7% ± 15.8%) (p = 0.36). Patients with pCR had higher median TIL percentages (24.8%) than those with non-pCR (14.2%) (p = 0.02). Although subgroup analyses were limited by the small sample size, PD-L1-positive patients treated with chemotherapy and atezolizumab had a pCR rate of 75% (12/16). The addition of atezolizumab to neoadjuvant carboplatin and paclitaxel resulted in a statistically significant and clinically relevant increased pCR rate in patients with clinical stages II and III TNBC. (Funded by National Cancer Institute).