Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

TEAD Proteins Associate With DNA Repair Proteins to Facilitate Cellular Recovery From DNA Damage.

Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage-induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.

A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer.

Cancer stem cells (CSCs) have been hypothesized to represent the driving force behind tumour progression and metastasis, making them attractive cancer targets. However, conclusive experimental evidence for their functional relevance is still lacking for most malignancies. Here we show that the leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) identifies intestinal CSCs in mouse tumours engineered to recapitulate the clinical progression of human colorectal cancer. We demonstrate that selective Lgr5+ cell ablation restricts primary tumour growth, but does not result in tumour regression. Instead, tumours are maintained by proliferative Lgr5- cells that continuously attempt to replenish the Lgr5+ CSC pool, leading to rapid re-initiation of tumour growth upon treatment cessation. Notably, CSCs are critical for the formation and maintenance of liver metastasis derived from colorectal cancers. Together, our data highlight distinct CSC dependencies for primary versus metastasic tumour growth, and suggest that targeting CSCs may represent a therapeutic opportunity for managing metastatic disease.

Targeting PTPRK-RSPO3 colon tumours promotes differentiation and loss of stem-cell function.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

S100a4 upregulation in Pik3caH1047R;Trp53R270H;MMTV-Cre-driven mammary tumors promotes metastasis.

BACKGROUND: PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers. We previously generated a conditionally activatable Pik3caH1047R;MMTV-Cre mouse model and found a few malignant sarcomatoid (spindle cell) carcinomas that had acquired spontaneous dominant-negative Trp53 mutations. METHODS: A Pik3caH1047R;Trp53R270H;MMTV-Cre double mutant mouse breast cancer model was generated. Tumors were characterized by histology, marker analysis, transcriptional profiling, single-cell RNA-seq, and bioinformatics. Cell lines were developed from mutant tumors and used to identify and confirm genes involved in metastasis. RESULTS: We found Pik3caH1047R and Trp53R270H cooperate in driving oncogenesis in mammary glands leading to a shorter latency than either alone. Double mutant mice develop multiple histologically distinct mammary tumors, including adenocarcinoma and sarcomatoid (spindle cell) carcinoma. We found some tumors to be invasive and a few metastasized to the lung and/or the lymph node. Single-cell RNA-seq analysis of the tumors identified epithelial, stromal, myeloid, and T cell groups. Expression analysis of the metastatic tumors identified S100a4 as a top candidate gene associated with metastasis. Metastatic tumors contained a much higher percentage of epithelial-mesenchymal transition (EMT)-signature positive and S100a4-expressing cells. CRISPR/CAS9-mediated knockout of S100a4 in a metastatic tumor-derived cell line disrupted its metastatic potential indicating a role for S100a4 in metastasis. CONCLUSIONS: Pik3caH1047R;Trp53R270H;MMTV-Cre mouse provides a preclinical model to mimic a subtype of human breast cancers that carry both PIK3CA and TP53 mutations. It also allows for understanding the cooperation between the two mutant genes in tumorigenesis. Our model also provides a system to study metastasis and develop therapeutic strategies for PIK3CA/TP53 double-positive cancers. S100a4 found involved in metastasis in this model can be a potential diagnostic and therapeutic target.

Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21.

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.

Homeostatic IL-23 receptor signaling limits Th17 response through IL-22-mediated containment of commensal microbiota.

Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. In mice, the presence of segmented filamentous bacteria (SFB) promotes Th17 differentiation and the development of autoimmune disease. Here, we demonstrate that the IL-23 pathway dynamically regulates the abundance of SFB as well as mucosal barrier function in the adult animal. Genetic or pharmacological inactivation of the pathway selectively perturbs the abundance of a small group of commensals, including SFB, and results in an impaired mucosal barrier. Defective barrier function leads to systemic dissemination of microbial products, provoking induction of the IL-23 pathway with dual consequences: IL-23 drives IL-22 production to reinforce mucosal barrier function and elicit antimicrobial activities, and it also drives the differentiation of Th17 cells in an attempt to combat escaped microbes in the lamina propria and in distal tissues. Thus, barrier defects generate a systemic environment that facilitates Th17 development.

Disruption of PH-kinase domain interactions leads to oncogenic activation of AKT in human cancers.

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.

A requirement for wild-type Ras isoforms in mutant KRas-driven signalling and transformation.

The mutant forms of KRas, NRas and HRas drive the initiation and progression of a number of human cancers, but less is known about the role of WT (wild-type) Ras alleles and isoforms in cancer. We used zinc-finger nucleases targeting HRas and NRas to modify both alleles of these genes in the mutant KRas-driven Hec1A endometrial cancer cell line, which normally expresses WT copies of these genes. The disruption of either WT isoform of Ras compromised growth-factor-dependent signalling through the ERK (extracellular-signal-regulated kinase) pathway. In addition, the disruption of HRas hindered the activation of Akt and subsequent downstream signalling. This was associated with decreased proliferation, increased apoptosis and decreased anchorage-independent growth in the HRas-disrupted cells. However, xenograft tumour growth was not significantly affected by the disruption of either NRas or HRas. As expected, deleting the mutant allele of KRas abolished tumour growth, whereas deletion of the remaining WT copy of KRas increased the tumorigenic properties of these cells; deleting a single copy of either HRas or NRas did not mimic this effect. The present study demonstrates that the WT copies of HRas, NRas and KRas play unique roles in the context of mutant KRas-driven tumours.

Adrenergic nerves regulate intestinal regeneration through IL-22 signaling from type 3 innate lymphoid cells.

The intestinal epithelium has high intrinsic turnover rate, and the precise renewal of the epithelium is dependent on the microenvironment. The intestine is innervated by a dense network of peripheral nerves that controls various aspects of intestinal physiology. However, the role of neurons in regulating epithelial cell regeneration remains largely unknown. Here, we investigated the effects of gut-innervating adrenergic nerves on epithelial cell repair following irradiation (IR)-induced injury. We observed that adrenergic nerve density in the small intestine increased post IR, while chemical adrenergic denervation impaired epithelial regeneration. Single-cell RNA sequencing experiments revealed a decrease in IL-22 signaling post IR in denervated animals. Combining pharmacologic and genetic tools, we demonstrate that ß-adrenergic receptor signaling drives IL-22 production from type 3 innate lymphoid cells (ILC3s) post IR, which in turn promotes epithelial regeneration. These results define an adrenergic-ILC3 axis important for intestinal regeneration.

Disruption of stem cell niche-confined R-spondin 3 expression leads to impaired hematopoiesis.

Self-renewal and differentiation of stem and progenitor cells are tightly regulated to ensure tissue homeostasis. This regulation is enabled both remotely by systemic circulating cues, such as cytokines and hormones, and locally by various niche-confined factors. R-spondin 3 (RSPO3) is one of the most potent enhancers of Wnt signaling, and its expression is usually restricted to the stem cell niche where it provides localized enhancement of Wnt signaling to regulate stem cell expansion and differentiation. Disruption of this niche-confined expression can disturb proper tissue organization and lead to cancers. Here, we investigate the consequences of disrupting the niche-restricted expression of RSPO3 in various tissues, including the hematopoietic system. We show that normal Rspo3 expression is confined to the perivascular niche in the bone marrow. Induction of increased systemic levels of circulating RSPO3 outside of the niche results in prominent loss of early B-cell progenitors and anemia but surprisingly has no effect on hematopoietic stem cells. Using molecular, pharmacologic, and genetic approaches, we show that these RSPO3-induced hematopoietic phenotypes are Wnt and RSPO3 dependent and mediated through noncanonical Wnt signaling. Our study highlights a distinct role for a Wnt/RSPO3 signaling axis in the regulation of hematopoiesis, as well as possible challenges related to therapeutic use of RSPOs for regenerative medicine.

Tumor-intrinsic expression of the autophagy gene Atg16l1 suppresses anti-tumor immunity in colorectal cancer.

Microsatellite-stable colorectal cancer (MSS-CRC) is highly refractory to immunotherapy. Understanding tumor-intrinsic determinants of immunotherapy resistance is critical to improve MSS-CRC patient outcomes. Here, we demonstrate that high tumor expression of the core autophagy gene ATG16L1 is associated with poor clinical response to anti-PD-L1 therapy in KRAS-mutant tumors from IMblaze370 (NCT02788279), a large phase III clinical trial of atezolizumab (anti-PD-L1) in advanced metastatic MSS-CRC. Deletion of Atg16l1 in engineered murine colon cancer organoids inhibits tumor growth in primary (colon) and metastatic (liver and lung) niches in syngeneic female hosts, primarily due to increased sensitivity to IFN-γ-mediated immune pressure. ATG16L1 deficiency enhances programmed cell death of colon cancer organoids induced by IFN-γ and TNF, thus increasing their sensitivity to host immunity. In parallel, ATG16L1 deficiency reduces tumor stem-like populations in vivo independently of adaptive immune pressure. This work reveals autophagy as a clinically relevant mechanism of immune evasion and tumor fitness in MSS-CRC and provides a rationale for autophagy inhibition to boost immunotherapy responses in the clinic.

IL-1R1-dependent signaling coordinates epithelial regeneration in response to intestinal damage.

Repair of the intestinal epithelium is tightly regulated to maintain homeostasis. The response after epithelial damage needs to be local and proportional to the insult. How different types of damage are coupled to repair remains incompletely understood. We report that after distinct types of intestinal epithelial damage, IL-1R1 signaling in GREM1+ mesenchymal cells increases production of R-spondin 3 (RSPO3), a Wnt agonist required for intestinal stem cell self-renewal. In parallel, IL-1R1 signaling regulates IL-22 production by innate lymphoid cells and promotes epithelial hyperplasia and regeneration. Although the regulation of both RSPO3 and IL-22 is critical for epithelial recovery from Citrobacter rodentium infection, IL-1R1-dependent RSPO3 production by GREM1+ mesenchymal cells alone is sufficient and required for recovery after dextran sulfate sodium-induced colitis. These data demonstrate how IL-1R1-dependent signaling orchestrates distinct repair programs tailored to the type of injury sustained that are required to restore intestinal epithelial barrier function.

Modeling Colorectal Cancer Progression Through Orthotopic Implantation of Organoids.

Colorectal cancer (CRC) related death has often been attributed to the presence of metastatic disseminated disease. A concise understanding of the molecular mechanism(s) that drive metastatic progression is therefore needed but has thus far been hampered by the limited number of CRC mouse models that progress toward this disease stage. In addition, preclinical evaluation of therapeutic modalities aimed at managing metastatic disease also rests on the availability of relevant in vivo models that faithfully recapitulate the key molecular features of metastatic human CRC. To overcome these limitations, we have recently developed methodologies that enable the study of CRC progression at relevant orthotopic sites. Here, we provide a detailed methodology that describes the injection of CRC derived cell lines and organoids directly into the colorectal mucosa. This results in the growth of a single tumor mass within the colon, that can spontaneously metastasize to the liver. Furthermore, we also present a surgical procedure to directly inject cells into the portal venous circulation to induce CRC tumor growth in the liver without the requirement of a primary tumor.

Elevated Serum Amino Acids Induce a Subpopulation of Alpha Cells to Initiate Pancreatic Neuroendocrine Tumor Formation.

The cellular origin of sporadic pancreatic neuroendocrine tumors (PNETs) is obscure. Hormone expression suggests that these tumors arise from glucagon-producing alpha cells or insulin-producing ß cells, but instability in hormone expression prevents linage determination. We utilize loss of hepatic glucagon receptor (GCGR) signaling to drive alpha cell hyperproliferation and tumor formation to identify a cell of origin and dissect mechanisms that drive progression. Using a combination of genetically engineered Gcgr knockout mice and GCGR-inhibiting antibodies, we show that elevated plasma amino acids drive the appearance of a proliferative population of SLC38A5+ embryonic progenitor-like alpha cells in mice. Further, we characterize tumors from patients with rare bi-allelic germline GCGR loss-of-function variants and find prominent tumor-cell-associated expression of the SLC38A5 paralog SLC7A8 as well as markers of active mTOR signaling. Thus, progenitor cells arise from adult alpha cells in response to metabolic signals and, when inductive signals are chronically present, drive tumor initiation.

A Clinically Applicable Gene-Expression Classifier Reveals Intrinsic and Extrinsic Contributions to Consensus Molecular Subtypes in Primary and Metastatic Colon Cancer.

PURPOSE: Four consensus molecular subtypes (CMS1-4) of colorectal cancer were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for colorectal cancer-related mortality, we examined the molecular and clinical attributes of CMS in metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: We developed a colorectal cancer-focused NanoString-based CMS classifier that is ideally suited to interrogate archival tissues. We successfully used this panel in the CMS classification of formalin-fixed paraffin-embedded (FFPE) tissues from mCRC cohorts, one of which is composed of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modeling of colorectal cancer in vivo at relevant sites. RESULTS: Using our classifier, we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in nonmetastatic early-stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared with other CMS. Assigning CMS to primary tumors and their matched metastases reveals mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs reveals differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic colorectal cancer. CONCLUSIONS: We describe the utility of a NanoString panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on colorectal cancer heterogeneity during disease progression.

The Hippo pathway effector TAZ induces TEAD-dependent liver inflammation and tumors.

The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.

Actionable Activating Oncogenic ERBB2/HER2 Transmembrane and Juxtamembrane Domain Mutations.

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) that include G660D, R678Q, E693K, and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small-molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.

pHUSH: a single vector system for conditional gene expression.

BACKGROUND: Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector. RESULTS: Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer. CONCLUSION: We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.

Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38 is a promising therapeutic target for multiple cancers.

To identify genes that could serve as targets for novel cancer therapeutics, we used a bioinformatic analysis of microarray data comparing gene expression between normal and tumor-derived primary human tissues. From this approach, we have found that maternal embryonic leucine zipper kinase (Melk), a member of the AMP serine/threonine kinase family, exhibits multiple features consistent with the potential utility of this gene as an anticancer target. An oligonucleotide microarray analysis of multiple human tumor samples and cell lines suggests that Melk expression is frequently elevated in cancer relative to normal tissues, a pattern confirmed by quantitative reverse transcription-PCR and Western blotting of selected primary tumor samples. In situ hybridization localized Melk expression to malignant epithelial cells in 96%, 23%, and 13% of colorectal, lung, and ovarian tissue tumor samples, respectively. Expression of this gene is also elevated in spontaneous tumors derived from the ApcMin and Apc1638N murine models of intestinal tumorigenesis. To begin addressing whether Melk is relevant for tumorigenesis, RNA interference-mediated silencing within human and murine tumor cell lines was done. We show that Melk knockdown decreases proliferation and anchorage-independent growth in vitro as well as tumor growth in a xenograft model. Together, these results suggest that Melk may provide a growth advantage for neoplastic cells and, therefore, inactivation may be therapeutically beneficial.