A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.

MS4A3 promotes differentiation in chronic myeloid leukemia by enhancing common ß-chain cytokine receptor endocytosis.

The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common ß-chain (ßc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade ßc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo.

Genetic demultiplexing of pooled single-cell RNA-sequencing samples in cancer facilitates effective experimental design.

BACKGROUND: Pooling cells from multiple biological samples prior to library preparation within the same single-cell RNA sequencing experiment provides several advantages, including lower library preparation costs and reduced unwanted technological variation, such as batch effects. Computational demultiplexing tools based on natural genetic variation between individuals provide a simple approach to demultiplex samples, which does not require complex additional experimental procedures. However, to our knowledge these tools have not been evaluated in cancer, where somatic variants, which could differ between cells from the same sample, may obscure the signal in natural genetic variation. RESULTS: Here, we performed in silico benchmark evaluations by combining raw sequencing reads from multiple single-cell samples in high-grade serous ovarian cancer, which has a high copy number burden, and lung adenocarcinoma, which has a high tumor mutational burden. Our results confirm that genetic demultiplexing tools can be effectively deployed on cancer tissue using a pooled experimental design, although high proportions of ambient RNA from cell debris reduce performance. CONCLUSIONS: This strategy provides significant cost savings through pooled library preparation. To facilitate similar analyses at the experimental design phase, we provide freely accessible code and a reproducible Snakemake workflow built around the best-performing tools found in our in silico benchmark evaluations, available at https://github.com/lmweber/snp-dmx-cancer.

Reciprocal regulation of pancreatic ductal adenocarcinoma growth and molecular subtype by HNF4α and SIX1/4.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a 5-year survival of less than 5%. Transcriptomic analysis has identified two clinically relevant molecular subtypes of PDAC: classical and basal-like. The classical subtype is characterised by a more favourable prognosis and better response to chemotherapy than the basal-like subtype. The classical subtype also expresses higher levels of lineage specifiers that regulate endodermal differentiation, including the nuclear receptor hepatocyte nuclear factor 4 α (HNF4α). The objective of this study is to evaluate the role of HNF4α, SIX4 and SIX1 in regulating the growth and molecular subtype of PDAC. DESIGN: We manipulate the expression of HNF4α, SIX4 and SIX1 in multiple in vitro and in vivo PDAC models. We determine the consequences of manipulating these genes on PDAC growth, differentiation and molecular subtype using functional assays, gene expression analysis and cross-species comparisons with human datasets. RESULTS: We show that HNF4α restrains tumour growth and drives tumour cells toward an epithelial identity. Gene expression analysis of murine models and human tumours shows that HNF4α activates expression of genes associated with the classical subtype. HNF4α also directly represses SIX4 and SIX1, two mesodermal/neuronal lineage specifiers expressed in the basal-like subtype. Finally, SIX4 and SIX1 drive proliferation and regulate differentiation in HNF4α-negative PDAC. CONCLUSION: Our data show that HNF4α regulates the growth and molecular subtype of PDAC by multiple mechanisms, including activation of the classical gene expression programme and repression of SIX4 and SIX1, which may represent novel dependencies of the basal-like subtype.

Estrogen Receptor Alpha Mutations in Breast Cancer Cells Cause Gene Expression Changes through Constant Activity and Secondary Effects.

While breast cancer patients with tumors that express estrogen receptor α (ER) generally respond well to hormone therapies that block ER activity, a significant number of patients relapse. Approximately 30% of these recurrences harbor activating mutations in the ligand binding domain (LBD) of ER, which have been shown to confer ligand-independent function. However, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate the molecular effects of mutant ER, we developed multiple isogenic ER-mutant cell lines for the most common LBD mutations, Y537S and D538G. These mutations induced differential expression of thousands of genes, the majority of which were mutant allele specific and were not observed upon estrogen treatment of wild-type (WT) cells. These mutant-specific genes showed consistent differential expression across ER-mutant lines developed in other laboratories. WT cells with long-term estrogen exposure only exhibited some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations exhibited minor effects on ER genomic binding, with the exception of ligand independence, ER mutations conferred substantial differences in chromatin accessibility. Mutant ER was bound to approximately a quarter of mutant-enriched accessible regions that were enriched for other DNA binding factors, including FOXA1, CTCF, and OCT1. Overall, our findings indicate that mutant ER causes several consistent effects on gene expression, both indirectly and through constant activity. SIGNIFICANCE: This study demonstrates the multiple roles of mutant ER in breast cancer progression, including constant ER activity and secondary regulatory effects on gene expression and chromatin accessibility. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/539/F1.large.jpg.See related commentary by Hermida-Prado and Jeselsohn, p. 537 See related article by Williams and colleagues, p. 732.

Regulatory sharing between estrogen receptor α bound enhancers.

The human genome encodes an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by the combined action of multiple enhancers. We have previously shown that neighboring estrogen-responsive enhancers exhibit complex synergistic contributions to the production of an estrogenic transcriptional response. Here we sought to determine the molecular underpinnings of this enhancer cooperativity. We generated genetic deletions of four estrogen receptor α (ER) bound enhancers that regulate two genes and found that enhancers containing full estrogen response element (ERE) motifs control ER binding at neighboring sites, while enhancers with pre-existing histone acetylation/accessibility confer a permissible chromatin environment to the neighboring enhancers. Genome engineering revealed that two enhancers with half EREs could not compensate for the lack of a full ERE site within the cluster. In contrast, two enhancers with full EREs produced a transcriptional response greater than the wild-type locus. By swapping genomic sequences, we found that the genomic location of a full ERE strongly influences enhancer activity. Our results lead to a model in which a full ERE is required for ER recruitment, but the presence of a pre-existing permissible chromatin environment can also be needed for estrogen-driven gene regulation to occur.

ETV4 Is Necessary for Estrogen Signaling and Growth in Endometrial Cancer Cells.

Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and in vivo in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells. SIGNIFICANCE: Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1234/F1.large.jpg.

Sufficiency analysis of estrogen responsive enhancers using synthetic activators.

Multiple regulatory regions bound by the same transcription factor have been shown to simultaneously control a single gene's expression. However, it remains unclear how these regulatory regions combine to regulate transcription. Here, we test the sufficiency of promoter-distal estrogen receptor α-binding sites (ERBSs) for activating gene expression by recruiting synthetic activators in the absence of estrogens. Targeting either dCas9-VP16(10x) or dCas9-p300(core) to ERBS induces H3K27ac and activates nearby expression in a manner similar to an estrogen induction, with dCas9-VP16(10x) acting as a stronger activator. The sufficiency of individual ERBSs is highly correlated with their necessity, indicating an inherent activation potential that is associated with the binding of RNA polymerase II and several transcription factors. By targeting ERBS combinations, we found that ERBSs work independently to control gene expression when bound by synthetic activators. The sufficiency results contrast necessity assays that show synergy between these ERBSs, suggesting that synergy occurs between ERBSs in terms of activator recruitment, whereas directly recruiting activators leads to independent effects on gene expression.

Estrogen-independent molecular actions of mutant estrogen receptor 1 in endometrial cancer.

Estrogen receptor 1 (ESR1) mutations have been identified in hormone therapy-resistant breast cancer and primary endometrial cancer. Analyses in breast cancer suggest that mutant ESR1 exhibits estrogen-independent activity. In endometrial cancer, ESR1 mutations are associated with worse outcomes and less obesity, however, experimental investigation of these mutations has not been performed. Using a unique CRISPR/Cas9 strategy, we introduced the D538G mutation, a common endometrial cancer mutation that alters the ligand binding domain of ESR1, while epitope tagging the endogenous locus. We discovered estrogen-independent mutant ESR1 genomic binding that is significantly altered from wild-type ESR1. The D538G mutation impacted expression, including a large set of nonestrogen-regulated genes, and chromatin accessibility, with most affected loci bound by mutant ESR1. Mutant ESR1 is distinct from constitutive ESR1 activity because mutant-specific changes are not recapitulated with prolonged estrogen exposure. Overall, the D538G mutant ESR1 confers estrogen-independent activity while causing additional regulatory changes in endometrial cancer cells that are distinct from breast cancer cells.

MYC-Driven Small-Cell Lung Cancer is Metabolically Distinct and Vulnerable to Arginine Depletion.

PURPOSE: Small-cell lung cancer (SCLC) has been treated clinically as a homogeneous disease, but recent discoveries suggest that SCLC is heterogeneous. Whether metabolic differences exist among SCLC subtypes is largely unexplored. In this study, we aimed to determine whether metabolic vulnerabilities exist between SCLC subtypes that can be therapeutically exploited. EXPERIMENTAL DESIGN: We performed steady state metabolomics on tumors isolated from distinct genetically engineered mouse models (GEMM) representing the MYC- and MYCL-driven subtypes of SCLC. Using genetic and pharmacologic approaches, we validated our findings in chemo-naïve and -resistant human SCLC cell lines, multiple GEMMs, four human cell line xenografts, and four newly derived PDX models. RESULTS: We discover that SCLC subtypes driven by different MYC family members have distinct metabolic profiles. MYC-driven SCLC preferentially depends on arginine-regulated pathways including polyamine biosynthesis and mTOR pathway activation. Chemo-resistant SCLC cells exhibit increased MYC expression and similar metabolic liabilities as chemo-naïve MYC-driven cells. Arginine depletion with pegylated arginine deiminase (ADI-PEG 20) dramatically suppresses tumor growth and promotes survival of mice specifically with MYC-driven tumors, including in GEMMs, human cell line xenografts, and a patient-derived xenograft from a relapsed patient. Finally, ADI-PEG 20 is significantly more effective than the standard-of-care chemotherapy. CONCLUSIONS: These data identify metabolic heterogeneity within SCLC and suggest arginine deprivation as a subtype-specific therapeutic vulnerability for MYC-driven SCLC.

Lentiviral CRISPR Epigenome Editing of Inflammatory Receptors as a Gene Therapy Strategy for Disc Degeneration.

Degenerative disc disease (DDD) is a primary contributor to low-back pain, a leading cause of disability. Progression of DDD is aided by inflammatory cytokines in the intervertebral disc (IVD), particularly TNF-α and IL-1ß, but current treatments fail to effectively target this mechanism. The objective of this study was to explore the feasibility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) epigenome editing-based therapy for DDD, by modulation of TNFR1/IL1R1 signaling in pathological human IVD cells. Human IVD cells from the nucleus pulposus of patients receiving surgery for back pain were obtained and the regulation of TNFR1/IL1R1 signaling by a lentiviral CRISPR epigenome editing system was tested. These cells were tested for successful lentiviral transduction/expression of deactivated Cas9 fused to Krüppel Associated Box system and regulation of TNFR1/IL1R1 expression. TNFR1/IL1R1 signaling disruption was investigated through measurement of NF-κB activity, apoptosis, and anabolic/catabolic changes in gene expression postinflammatory challenge. CRISPR epigenome editing systems were effectively introduced into pathological human IVD cells and significantly downregulated TNFR1 and IL1R1. This downregulation significantly attenuated deleterious TNFR1 signaling but not IL1R1 signaling. This is attributed to less robust IL1R1 expression downregulation, and IL-1ß-driven reversal of IL1R1 expression downregulation in a portion of patient IVD cells. In addition, RNAseq data indicated novel transcription factor targets, IRF1 and TFAP2C, as being primary regulators of inflammatory signaling in IVD cells. These results demonstrate the feasibility of CRISPR epigenome editing of inflammatory receptors in pathological IVD cells, but highlight a limitation in epigenome targeting of IL1R1. This method has potential application as a novel gene therapy for DDD, to attenuate the deleterious effect of inflammatory cytokines present in the degenerative IVD.

The Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment.

The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor.

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines.

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers.

Clinical and Genomic Crosstalk between Glucocorticoid Receptor and Estrogen Receptor α In Endometrial Cancer.

Steroid hormone receptors are simultaneously active in many tissues and are capable of altering each other's function. Estrogen receptor α (ER) and glucocorticoid receptor (GR) are expressed in the uterus, and their ligands have opposing effects on uterine growth. In endometrial tumors with high ER expression, we surprisingly found that expression of GR is associated with poor prognosis. Dexamethasone reduced normal uterine growth in vivo; however, this growth inhibition was abolished in estrogen-induced endometrial hyperplasia. We observed low genomic-binding site overlap when ER and GR are induced with their respective ligands; however, upon simultaneous induction they co-occupy more sites. GR binding is altered significantly by estradiol with GR recruited to ER-bound loci that become more accessible upon estradiol induction. Gene expression responses to co-treatment were more similar to estradiol but with additional regulated genes. Our results suggest phenotypic and molecular interplay between ER and GR in endometrial cancer.

Multiplex Enhancer Interference Reveals Collaborative Control of Gene Regulation by Estrogen Receptor α-Bound Enhancers.

Multiple regulatory regions have the potential to regulate a single gene, yet how these elements combine to affect gene expression remains unclear. To uncover the combinatorial relationships between enhancers, we developed Enhancer-interference (Enhancer-i), a CRISPR interference-based approach that uses 2 different repressive domains, KRAB and SID, to prevent enhancer activation simultaneously at multiple regulatory regions. We applied Enhancer-i to promoter-distal estrogen receptor α binding sites (ERBS), which cluster around estradiol-responsive genes and therefore may collaborate to regulate gene expression. Targeting individual sites revealed predominant ERBS that are completely required for the transcriptional response, indicating a lack of redundancy. Simultaneous interference of different ERBS combinations identified supportive ERBS that contribute only when predominant sites are active. Using mathematical modeling, we find strong evidence for collaboration between predominant and supportive ERBS. Overall, our findings expose a complex functional hierarchy of enhancers, where multiple loci bound by the same transcription factor combine to fine-tune the expression of target genes.

CRISPR Epigenome Editing of AKAP150 in DRG Neurons Abolishes Degenerative IVD-Induced Neuronal Activation.

Back pain is a major contributor to disability and has significant socioeconomic impacts worldwide. The degenerative intervertebral disc (IVD) has been hypothesized to contribute to back pain, but a better understanding of the interactions between the degenerative IVD and nociceptive neurons innervating the disc and treatment strategies that directly target these interactions is needed to improve our understanding and treatment of back pain. We investigated degenerative IVD-induced changes to dorsal root ganglion (DRG) neuron activity and utilized CRISPR epigenome editing as a neuromodulation strategy. By exposing DRG neurons to degenerative IVD-conditioned media under both normal and pathological IVD pH levels, we demonstrate that degenerative IVDs trigger interleukin (IL)-6-induced increases in neuron activity to thermal stimuli, which is directly mediated by AKAP and enhanced by acidic pH. Utilizing this novel information on AKAP-mediated increases in nociceptive neuron activity, we developed lentiviral CRISPR epigenome editing vectors that modulate endogenous expression of AKAP150 by targeted promoter histone methylation. When delivered to DRG neurons, these epigenome-modifying vectors abolished degenerative IVD-induced DRG-elevated neuron activity while preserving non-pathologic neuron activity. This work elucidates the potential for CRISPR epigenome editing as a targeted gene-based pain neuromodulation strategy.

MYC Drives Progression of Small Cell Lung Cancer to a Variant Neuroendocrine Subtype with Vulnerability to Aurora Kinase Inhibition.

Loss of the tumor suppressors RB1 and TP53 and MYC amplification are frequent oncogenic events in small cell lung cancer (SCLC). We show that Myc expression cooperates with Rb1 and Trp53 loss in the mouse lung to promote aggressive, highly metastatic tumors, that are initially sensitive to chemotherapy followed by relapse, similar to human SCLC. Importantly, MYC drives a neuroendocrine-low "variant" subset of SCLC with high NEUROD1 expression corresponding to transcriptional profiles of human SCLC. Targeted drug screening reveals that SCLC with high MYC expression is vulnerable to Aurora kinase inhibition, which, combined with chemotherapy, strongly suppresses tumor progression and increases survival. These data identify molecular features for patient stratification and uncover a potential targeted treatment approach for MYC-driven SCLC.

Sox2 cooperates with Lkb1 loss in a mouse model of squamous cell lung cancer.

Squamous cell carcinoma (SCC) of the lung is the second most common subtype of lung cancer. With limited treatment options, the 5-year survival rate of SCC is only 15%. Although genomic alterations in SCC have been characterized, identifying the alterations that drive SCC is critical for improving treatment strategies. Mouse models of SCC are currently limited. Using lentiviral delivery of Sox2 specifically to the mouse lung, we tested the ability of Sox2 to promote tumorigenesis in multiple tumor suppressor backgrounds. Expression of Sox2, frequently amplified in human SCC, specifically cooperates with loss of Lkb1 to promote squamous lung tumors. Mouse tumors exhibit characteristic histopathology and biomarker expression similar to human SCC. They also mimic human SCCs by activation of therapeutically relevant pathways including STAT and mTOR. This model may be utilized to test the contribution of additional driver alterations in SCC, as well as for preclinical drug discovery.