Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase.

Among breast cancers, triple-negative breast cancer (TNBC) is the most poorly understood and is refractory to current targeted therapies. Using a genetic screen, we identify the PTPN12 tyrosine phosphatase as a tumor suppressor in TNBC. PTPN12 potently suppresses mammary epithelial cell proliferation and transformation. PTPN12 is frequently compromised in human TNBCs, and we identify an upstream tumor-suppressor network that posttranscriptionally controls PTPN12. PTPN12 suppresses transformation by interacting with and inhibiting multiple oncogenic tyrosine kinases, including HER2 and EGFR. The tumorigenic and metastatic potential of PTPN12-deficient TNBC cells is severely impaired upon restoration of PTPN12 function or combined inhibition of PTPN12-regulated tyrosine kinases, suggesting that TNBCs are dependent on the proto-oncogenic tyrosine kinases constrained by PTPN12. Collectively, these data identify PTPN12 as a commonly inactivated tumor suppressor and provide a rationale for combinatorially targeting proto-oncogenic tyrosine kinases in TNBC and other cancers based on their profile of tyrosine-phosphatase activity.

Epigenetic markers and therapeutic targets for metastasis.

The last few years have seen an increasing number of discoveries which collectively demonstrate that histone and DNA modifying enzyme modulate different stages of metastasis. Moreover, epigenomic alterations can now be measured at multiple scales of analysis and are detectable in human tumors or liquid biopsies. Malignant cell clones with a proclivity for relapse in certain organs may arise in the primary tumor as a consequence of epigenomic alterations which cause a loss in lineage integrity. These alterations may occur due to genetic aberrations acquired during tumor progression or concomitant to therapeutic response. Moreover, evolution of the stroma can also alter the epigenome of cancer cells. In this review, we highlight current knowledge with a particular emphasis on leveraging chromatin and DNA modifying mechanisms as biomarkers of disseminated disease and as therapeutic targets to treat metastatic cancers.

Tumor self-seeding by circulating cancer cells.

Cancer cells that leave the primary tumor can seed metastases in distant organs, and it is thought that this is a unidirectional process. Here we show that circulating tumor cells (CTCs) can also colonize their tumors of origin, in a process that we call "tumor self-seeding." Self-seeding of breast cancer, colon cancer, and melanoma tumors in mice is preferentially mediated by aggressive CTCs, including those with bone, lung, or brain-metastatic tropism. We find that the tumor-derived cytokines IL-6 and IL-8 act as CTC attractants whereas MMP1/collagenase-1 and the actin cytoskeleton component fascin-1 are mediators of CTC infiltration into mammary tumors. We show that self-seeding can accelerate tumor growth, angiogenesis, and stromal recruitment through seed-derived factors including the chemokine CXCL1. Tumor self-seeding could explain the relationships between anaplasia, tumor size, vascularity and prognosis, and local recurrence seeded by disseminated cells following ostensibly complete tumor excision.

WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis.

Metastasis from lung adenocarcinoma can occur swiftly to multiple organs within months of diagnosis. The mechanisms that confer this rapid metastatic capacity to lung tumors are unknown. Activation of the canonical WNT/TCF pathway is identified here as a determinant of metastasis to brain and bone during lung adenocarcinoma progression. Gene expression signatures denoting WNT/TCF activation are associated with relapse to multiple organs in primary lung adenocarcinoma. Metastatic subpopulations isolated from independent lymph node-derived lung adenocarcinoma cell lines harbor a hyperactive WNT/TCF pathway. Reduction of TCF activity in these cells attenuates their ability to form brain and bone metastases in mice, independently of effects on tumor growth in the lungs. The WNT/TCF target genes HOXB9 and LEF1 are identified as mediators of chemotactic invasion and colony outgrowth. Thus, a distinct WNT/TCF signaling program through LEF1 and HOXB9 enhances the competence of lung adenocarcinoma cells to colonize the bones and the brain. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

Genes that mediate breast cancer metastasis to the brain.

The molecular basis for breast cancer metastasis to the brain is largely unknown. Brain relapse typically occurs years after the removal of a breast tumour, suggesting that disseminated cancer cells must acquire specialized functions to take over this organ. Here we show that breast cancer metastasis to the brain involves mediators of extravasation through non-fenestrated capillaries, complemented by specific enhancers of blood-brain barrier crossing and brain colonization. We isolated cells that preferentially infiltrate the brain from patients with advanced disease. Gene expression analysis of these cells and of clinical samples, coupled with functional analysis, identified the cyclooxygenase COX2 (also known as PTGS2), the epidermal growth factor receptor (EGFR) ligand HBEGF, and the alpha2,6-sialyltransferase ST6GALNAC5 as mediators of cancer cell passage through the blood-brain barrier. EGFR ligands and COX2 were previously linked to breast cancer infiltration of the lungs, but not the bones or liver, suggesting a sharing of these mediators in cerebral and pulmonary metastases. In contrast, ST6GALNAC5 specifically mediates brain metastasis. Normally restricted to the brain, the expression of ST6GALNAC5 in breast cancer cells enhances their adhesion to brain endothelial cells and their passage through the blood-brain barrier. This co-option of a brain sialyltransferase highlights the role of cell-surface glycosylation in organ-specific metastatic interactions.

Significance of glioma-associated oncogene homolog 1 (GLI1) expression in claudin-low breast cancer and crosstalk with the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway.

INTRODUCTION: The recently identified claudin-low subtype of breast cancer is enriched for cells with stem-like and mesenchymal-like characteristics. This subtype is most often triple-negative (lacking the estrogen and progesterone receptors (ER, PR) as well as lacking epidermal growth factor 2 (HER2) amplification) and has a poor prognosis. There are few targeted treatment options available for patients with this highly aggressive type of cancer. METHODS: Using a high throughput inhibitor screen, we identified high expression of glioma-associated oncogene homolog 1 (GLI1), the effector molecule of the hedgehog (Hh) pathway, as a critical determinant of cell lines that have undergone an epithelial to mesenchymal transition (EMT). RESULTS: High GLI1 expression is a property of claudin-low cells and tumors and correlates with markers of EMT and breast cancer stem cells. Knockdown of GLI1 expression in claudin-low cell lines resulted in reduced cell viability, motility, clonogenicity, self-renewal, and reduced tumor growth of orthotopic xenografts. We observed non-canonical activation of GLI1 in claudin-low and EMT cell lines, and identified crosstalk with the NFκB pathway. CONCLUSIONS: This work highlights the importance of GLI1 in the maintenance of characteristics of metastatic breast cancer stem cells. Remarkably, treatment with an inhibitor of the NFκB pathway reproducibly reduces GLI1 expression and protein levels. We further provide direct evidence for the binding of the NFκB subunit p65 to the GLI1 promoter in both EMT and claudin-low cell lines. Our results uncover crosstalk between NFκB and GLI1 signals and suggest that targeting these pathways may be effective against the claudin-low breast cancer subtype.

ASCL1 Drives Tolerance to Osimertinib in EGFR Mutant Lung Cancer in Permissive Cellular Contexts.

The majority of EGFR mutant lung adenocarcinomas respond well to EGFR tyrosine kinase inhibitors (TKI). However, most of these responses are partial, with drug-tolerant residual disease remaining even at the time of maximal response. This residual disease can ultimately lead to relapses, which eventually develop in most patients. To investigate the cellular and molecular properties of residual tumor cells in vivo, we leveraged patient-derived xenograft (PDX) models of EGFR mutant lung cancer. Subcutaneous EGFR mutant PDXs were treated with the third-generation TKI osimertinib until maximal tumor regression. Residual tissue inevitably harbored tumor cells that were transcriptionally distinct from bulk pretreatment tumor. Single-cell transcriptional profiling provided evidence of cells matching the profiles of drug-tolerant cells present in the pretreatment tumor. In one of the PDXs analyzed, osimertinib treatment caused dramatic transcriptomic changes that featured upregulation of the neuroendocrine lineage transcription factor ASCL1. Mechanistically, ASCL1 conferred drug tolerance by initiating an epithelial-to-mesenchymal gene-expression program in permissive cellular contexts. This study reveals fundamental insights into the biology of drug tolerance, the plasticity of cells through TKI treatment, and why specific phenotypes are observed only in certain tumors. SIGNIFICANCE: Analysis of residual disease following tyrosine kinase inhibitor treatment identified heterogeneous and context-specific mechanisms of drug tolerance in lung cancer that could lead to the development of strategies to forestall drug resistance. See related commentary by Rumde and Burns, p. 1188.

Patient-Derived Models of Cancer in the NCI PDMC Consortium: Selection, Pitfalls, and Practical Recommendations.

For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.

Mediators of vascular remodelling co-opted for sequential steps in lung metastasis.

Metastasis entails numerous biological functions that collectively enable cancerous cells from a primary site to disseminate and overtake distant organs. Using genetic and pharmacological approaches, we show that the epidermal growth factor receptor ligand epiregulin, the cyclooxygenase COX2, and the matrix metalloproteinases 1 and 2, when expressed in human breast cancer cells, collectively facilitate the assembly of new tumour blood vessels, the release of tumour cells into the circulation, and the breaching of lung capillaries by circulating tumour cells to seed pulmonary metastasis. These findings reveal how aggressive primary tumorigenic functions can be mechanistically coupled to greater lung metastatic potential, and how such biological activities may be therapeutically targeted with specific drug combinations.

Mammalian SWI/SNF chromatin remodeling complexes promote tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer.

Acquired resistance to tyrosine kinase inhibitors (TKI), such as osimertinib used to treat EGFR-mutant lung adenocarcinomas, limits long-term efficacy and is frequently caused by non-genetic mechanisms. Here, we define the chromatin accessibility and gene regulatory signatures of osimertinib sensitive and resistant EGFR-mutant cell and patient-derived models and uncover a role for mammalian SWI/SNF chromatin remodeling complexes in TKI resistance. By profiling mSWI/SNF genome-wide localization, we identify both shared and cancer cell line-specific gene targets underlying the resistant state. Importantly, genetic and pharmacologic disruption of the SMARCA4/SMARCA2 mSWI/SNF ATPases re-sensitizes a subset of resistant models to osimertinib via inhibition of mSWI/SNF-mediated regulation of cellular programs governing cell proliferation, epithelial-to-mesenchymal transition, epithelial cell differentiation, and NRF2 signaling. These data highlight the role of mSWI/SNF complexes in supporting TKI resistance and suggest potential utility of mSWI/SNF inhibitors in TKI-resistant lung cancers.

Tracing the origins of metastasis.

Cancer metastasis is often considered an orderly sequence of events leading to the colonization of distal organs by malignant cells. In fact, the evolution of metastatic disease is a dynamic process that is influenced by unique cellular lineages, altered microenvironments, distinct anatomical restrictions and multiple genetic and epigenetic alterations. These factors all contribute to variable clinical courses, likely requiring tailored therapy. As we inch closer towards personalized medicine, there is a renewed conceptual and technological focus on characterizing the cellular and genetic heterogeneity within tumours, to ultimately trace the origins of metastatic cells in different cancers.

Human WDR5 promotes breast cancer growth and metastasis via KMT2-independent translation regulation.

Metastatic breast cancer remains a major cause of cancer-related deaths in women, and there are few effective therapies against this advanced disease. Emerging evidence suggests that key steps of tumor progression and metastasis are controlled by reversible epigenetic mechanisms. Using an in vivo genetic screen, we identified WDR5 as an actionable epigenetic regulator that is required for metastatic progression in models of triple-negative breast cancer. We found that knockdown of WDR5 in breast cancer cells independently impaired their tumorigenic as well as metastatic capabilities. Mechanistically, WDR5 promotes cell growth by increasing ribosomal gene expression and translation efficiency in a KMT2-independent manner. Consistently, pharmacological inhibition or degradation of WDR5 impedes cellular translation rate and the clonogenic ability of breast cancer cells. Furthermore, a combination of WDR5 targeting with mTOR inhibitors leads to potent suppression of translation and proliferation of breast cancer cells. These results reveal novel therapeutic strategies to treat metastatic breast cancer.

Brain metastatic outgrowth and osimertinib resistance are potentiated by RhoA in EGFR-mutant lung cancer.

The brain is a major sanctuary site for metastatic cancer cells that evade systemic therapies. Through pre-clinical pharmacological, biological, and molecular studies, we characterize the functional link between drug resistance and central nervous system (CNS) relapse in Epidermal Growth Factor Receptor- (EGFR-) mutant non-small cell lung cancer, which can progress in the brain when treated with the CNS-penetrant EGFR inhibitor osimertinib. Despite widespread osimertinib distribution in vivo, the brain microvascular tumor microenvironment (TME) is associated with the persistence of malignant cell sub-populations, which are poised to proliferate in the brain as osimertinib-resistant lesions over time. Cellular and molecular features of this poised state are regulated through a Ras homolog family member A (RhoA) and Serum Responsive Factor (SRF) gene expression program. RhoA potentiates the outgrowth of disseminated tumor cells on osimertinib treatment, preferentially in response to extracellular laminin and in the brain. Thus, we identify pre-existing and adaptive features of metastatic and drug-resistant cancer cells, which are enhanced by RhoA/SRF signaling and the brain TME during the evolution of osimertinib-resistant disease.

Melanoma central nervous system metastases: An update to approaches, challenges, and opportunities.

Brain metastases are the most common brain malignancy. This review discusses the studies presented at the third annual meeting of the Melanoma Research Foundation in the context of other recent reports on the biology and treatment of melanoma brain metastases (MBM). Although symptomatic MBM patients were historically excluded from immunotherapy trials, efforts from clinicians and patient advocates have resulted in more inclusive and even dedicated clinical trials for MBM patients. The results of checkpoint inhibitor trials were discussed in conversation with current standards of care for MBM patients, including steroids, radiotherapy, and targeted therapy. Advances in the basic scientific understanding of MBM, including the role of astrocytes and metabolic adaptations to the brain microenvironment, are exposing new vulnerabilities which could be exploited for therapeutic purposes. Technical advances including single-cell omics and multiplex imaging are expanding our understanding of the MBM ecosystem and its response to therapy. This unprecedented level of spatial and temporal resolution is expected to dramatically advance the field in the coming years and render novel treatment approaches that might improve MBM patient outcomes.

Preclinical Models for the Study of Lung Cancer Pathogenesis and Therapy Development.

Experimental preclinical models have been a cornerstone of lung cancer translational research. Work in these model systems has provided insights into the biology of lung cancer subtypes and their origins, contributed to our understanding of the mechanisms that underlie tumor progression, and revealed new therapeutic vulnerabilities. Initially patient-derived lung cancer cell lines were the main preclinical models available. The landscape is very different now with numerous preclinical models for research each with unique characteristics. These include genetically engineered mouse models (GEMMs), patient-derived xenografts (PDXs) and three-dimensional culture systems ("organoid" cultures). Here we review the development and applications of these models and describe their contributions to lung cancer research.

Measuring Faculty Effort: A Quantitative Approach That Aligns Personal and Institutional Goals in Pathology at Yale.

US medical schools increasingly seek ways to reduce costs and improve productivity. One aspect of this effort has been the development of performance-based incentives for individual faculty. A myriad of such plans exist. Typically, they incentivize clinical revenue generation but vary widely in how teaching, investigation, and administrative contributions are recognized. In Pathology at Yale, we have developed a transparent metrically driven approach that recognizes all missions and allows faculty significant control over their career path. Although some metrics derive from traditional measures such as workload relative value units and one's level of grant support, the key concept underpinning our approach is to define one's contributions not in terms of the revenue generated, but rather on the effort devoted to each of our missions, benchmarked against national or local standards. Full-time faculty are paid a competitive rank-based salary and are expected to contribute at least 100% effort in support of the school's missions: clinical, research, education, administration, and professional service. Metrics define the effort assigned to each activity. Faculty achieving greater than 100% effort receive bonus compensation in proportion to their excess effort. By codifying explicitly how such effort is recognized into a single metric (% effort), we achieve a process that better aligns the professional and personal goals of faculty with the aims of the school. To facilitate its implementation, we have developed a web-based software platform called SWAY (Standardized Workload Analysis at Yale) that enables faculty to monitor their progress and record their activities in real time.

Preclinical Comparison of the Blood-brain barrier Permeability of Osimertinib with Other EGFR TKIs.

PURPOSE: Osimertinib is a potent and selective EGFR tyrosine kinase inhibitor (EGFR-TKI) of both sensitizing and T790M resistance mutations. To treat metastatic brain disease, blood-brain barrier (BBB) permeability is considered desirable for increasing clinical efficacy. EXPERIMENTAL DESIGN: We examined the level of brain penetration for 16 irreversible and reversible EGFR-TKIs using multiple in vitro and in vivo BBB preclinical models. RESULTS: In vitro osimertinib was the weakest substrate for human BBB efflux transporters (efflux ratio 3.2). In vivo rat free brain to free plasma ratios (Kpuu) show osimertinib has the most BBB penetrance (0.21), compared with the other TKIs (Kpuu ≤ 0.12). PET imaging in Cynomolgus macaques demonstrated osimertinib was the only TKI among those tested to achieve significant brain penetrance (C max %ID 1.5, brain/blood Kp 2.6). Desorption electrospray ionization mass spectroscopy images of brains from mouse PC9 macrometastases models showed osimertinib readily distributes across both healthy brain and tumor tissue. Comparison of osimertinib with the poorly BBB penetrant afatinib in a mouse PC9 model of subclinical brain metastases showed only osimertinib has a significant effect on rate of brain tumor growth. CONCLUSIONS: These preclinical studies indicate that osimertinib can achieve significant exposure in the brain compared with the other EGFR-TKIs tested and supports the ongoing clinical evaluation of osimertinib for the treatment of EGFR-mutant brain metastasis. This work also demonstrates the link between low in vitro transporter efflux ratios and increased brain penetrance in vivo supporting the use of in vitro transporter assays as an early screen in drug discovery.

Specific chromatin landscapes and transcription factors couple breast cancer subtype with metastatic relapse to lung or brain.

BACKGROUND: Few somatic mutations have been linked to breast cancer metastasis, whereas transcriptomic differences among primary tumors correlate with incidence of metastasis, especially to the lungs and brain. However, the epigenomic alterations and transcription factors (TFs) which underlie these alterations remain unclear. METHODS: To identify these, we performed RNA-seq, Chromatin Immunoprecipitation and sequencing (ChIP-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) of the MDA-MB-231 cell line and its brain (BrM2) and lung (LM2) metastatic sub-populations. We incorporated ATAC-seq data from TCGA to assess metastatic open chromatin signatures, and gene expression data from human metastatic datasets to nominate transcription factor biomarkers. RESULTS: Our integrated epigenomic analyses found that lung and brain metastatic cells exhibit both shared and distinctive signatures of active chromatin. Notably, metastatic sub-populations exhibit increased activation of both promoters and enhancers. We also integrated these data with chromosome conformation capture coupled with ChIP-seq (HiChIP) derived enhancer-promoter interactions to predict enhancer-controlled pathway alterations. We found that enhancer changes are associated with endothelial cell migration in LM2, and negative regulation of epithelial cell proliferation in BrM2. Promoter changes are associated with vasculature development in LM2 and homophilic cell adhesion in BrM2. Using ATAC-seq, we identified a metastasis open-chromatin signature that is elevated in basal-like and HER2-enriched breast cancer subtypes and associates with worse prognosis in human samples. We further uncovered TFs associated with the open chromatin landscapes of metastatic cells and whose expression correlates with risk for metastasis. While some of these TFs are associated with primary breast tumor subtypes, others more specifically correlate with lung or brain metastasis. CONCLUSIONS: We identify distinctive epigenomic properties of breast cancer cells that metastasize to the lung and brain. We also demonstrate that signatures of active chromatin sites are partially linked to human breast cancer subtypes with poor prognosis, and that specific TFs can independently distinguish lung and brain relapse.

Tumor progression and chromatin landscape of lung cancer are regulated by the lineage factor GATA6.

Lineage selective transcription factors (TFs) are important regulators of tumorigenesis, but their biological functions are often context dependent with undefined epigenetic mechanisms of action. In this study, we uncover a conditional role for the endodermal and pulmonary specifying TF GATA6 in lung adenocarcinoma (LUAD) progression. Impairing Gata6 in genetically engineered mouse models reduces the proliferation and increases the differentiation of Kras mutant LUAD tumors. These effects are influenced by the epithelial cell type that is targeted for transformation and genetic context of Kras-mediated tumor initiation. In LUAD cells derived from surfactant protein C expressing progenitors, we identify multiple genomic loci that are bound by GATA6. Moreover, suppression of Gata6 in these cells significantly alters chromatin accessibility, particularly at distal enhancer elements. Analogous to its paradoxical activity in lung development, GATA6 expression fluctuates during different stages of LUAD progression and can epigenetically control diverse transcriptional programs associated with bone morphogenetic protein signaling, alveolar specification, and tumor suppression. These findings reveal how GATA6 can modulate the chromatin landscape of lung cancer cells to control their proliferation and divergent lineage dependencies during tumor progression.

Perilesional edema in brain metastases: potential causes and implications for treatment with immune therapy.

BACKGROUND: Little is known about tumor-associated vasogenic edema in brain metastasis, yet it causes significant morbidity and mortality. Our purpose was to characterize edema in patients treated with anti-PD-1 and to study potential causes of vessel leakage in humans and in pre-clinical models. METHODS: We analyzed tumor and edema volume in 18 non-small cell lung (NSCLC) and 18 melanoma patients with untreated brain metastases treated with pembrolizumab on a phase II clinical trial. Melanoma brain metastases were stained with anti-CD34 to assess vessel density and its association with edema. We employed an in vitro model of the blood-brain barrier using short-term cultures from melanoma brain and extracranial metastases to determine tight junction resistance as a measure of vessel leakiness. RESULTS: Edema volumes are similar in NSCLC and melanoma brain metastases. While larger tumors tended to have more edema, the correlation was weak (R2 = 0.30). Patients responding to pembrolizumab had concurrent shrinkage of edema volume and vice versa (R2 = 0.81). Vessel density was independent of the degree of edema (R2 = 0.037). Melanoma brain metastasis cells in culture caused loss of tight junction resistance in an in vitro blood-brain barrier model system in some cases, whereas extracerebral cell cultures did not. CONCLUSIONS: Edema itself should not preclude using anti-PD-1 with caution, as sensitive tumors have resultant decreases in edema, and anti-PD-1 itself does not exacerbate edema in sensitive tumors. Additional factors aside from tumor mass effect and vessel density cause perilesional edema. Melanoma cells themselves can cause decline in tight junction resistance in a system void of immune cells, suggesting they secrete factors that cause leakiness, which might be harnessed for pharmacologic targeting in patients with significant perilesional edema.