Enapotamab Vedotin, an AXL-Specific Antibody-Drug Conjugate, Demonstrates Antitumor Efficacy in Patient-Derived Xenograft Models of Soft Tissue Sarcoma.

Doxorubicin (doxo) remains the standard of care for patients with advanced soft tissue sarcoma (STS), even though response rates to doxo are only around 14% to 18%. We evaluated enapotamab vedotin (EnaV), an AXL-specific antibody-drug conjugate (ADC), in a panel of STS patient-derived xenografts (PDX). Eight models representing multiple STS subtypes were selected from our STS PDX platform (n = 45) by AXL immunostaining on archived passages. Models were expanded by unilateral transplantation of tumor tissue into the left flank of 20 NMRI nu/nu mice. Once tumors were established, mice were randomized into an EnaV treatment group, or a group treated with isotype control ADC. Treatment efficacy was assessed by tumor volume evaluation, survival analysis, and histological evaluation of tumors, and associated with AXL expression. EnaV demonstrated significant tumor growth delay, regression, and/or prolonged survival compared to isotype control ADC in 5/8 STS PDX models investigated. Experimental passages of responding models were all found positive for AXL at varying levels, but no linear relationship could be identified between the level of expression and level of response to EnaV. One model was found negative for AXL on experimental passage and did not respond to EnaV. This study provides a preclinical rationale for the evaluation of AXL-targeting ADCs in the treatment of AXL-expressing sarcomas.

Wnt signaling regulates the lineage differentiation potential of mouse embryonic stem cells through Tcf3 down-regulation.

Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.

Cancer stemness in Wnt-driven mammary tumorigenesis.

Wnt signaling plays a central role in mammary stem cell (MaSC) homeostasis and in breast cancer. In particular, epigenetic alterations at different members of the Wnt pathway have been identified among triple-negative, basal-like breast cancers. Previously, we developed a mouse model for metaplastic breast adenocarcinoma, a subtype of triple-negative breast cancer, by targeting a hypomorphic mutations in the endogenous Apc gene (Apc (1572T/+)). Here, by employing the CD24 and CD29 cell surface antigens, we have identified a subpopulation of mammary cancer stem cells (MaCSCs) from Apc (1572T/+) capable of self-renewal and differentiation both in vivo and in vitro. Moreover, immunohistochemical analysis of micro- and macrolung metastases and preliminary intravenous transplantation assays suggest that the MaCSCs underlie metastasis at distant organ sites. Expression profiling of the normal and tumor cell subpopulations encompassing MaSCs and CSCs revealed that the normal stem cell compartment is more similar to tumor cells than to their own differentiated progenies. Accordingly, Wnt signaling appears to be active in both the normal and cancer stem cell compartments, although at different levels. By comparing normal with cancer mouse mammary compartments, we identified a MaCSC gene signature able to predict outcome in breast cancer in man. Overall, our data indicate that constitutive Wnt signaling activation affects self-renewal and differentiation of MaSCs leading to metaplasia and basal-like adenocarcinomas.

Alterations in Wnt-ß-catenin and Pten signalling play distinct roles in endometrial cancer initiation and progression.

Endometrioid endometrial cancer arises through a gradual series of histological changes, each accompanied by specific alterations in gene expression and activity. Activation of the Wnt-ß-catenin pathway and loss of PTEN activity are frequently observed in endometrial cancers. However, the specific roles played by alterations in these pathways in the initiation and progression of endometrial cancer are currently unclear. Here, we investigated the effects of loss of Pten and Apc gene function in the mouse endometrium by employing tissue-specific and inducible mutant alleles, followed by immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of their corresponding cancerous lesions. Loss of the Apc function in the endometrium leads to cytoplasmic and nuclear ß-catenin accumulation in association with uterine hyperplasia and squamous cell metaplasia, but without malignant transformation. Loss of Pten function also resulted in squamous metaplasia but, in contrast to loss of Apc function, it initiates endometrial cancer. On the other hand, loss of Apc function in the endometrium accelerates Pten-driven endometrial tumourigenesis. Analysis of compound heterozygous mice confirmed that somatic loss of the wild-type Pten allele represents the rate-limiting initiation step in endometrial cancer. Simultaneous loss of Pten and Apc resulted in endometrial cancer characterized by earlier onset and a more aggressive malignant behaviour. These observations are indicative of the synergistic action between the Wnt-ß-catenin and Pten signalling pathways in endometrial cancer onset and progression.

Wnt5a promotes human colon cancer cell migration and invasion but does not augment intestinal tumorigenesis in Apc1638N mice.

Whereas aberrant activation of canonical Wnt/ß-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling data sets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed protumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression.

Induced Wnt5a expression perturbs embryonic outgrowth and intestinal elongation, but is well-tolerated in adult mice.

Wnt5a is essential during embryonic development, as indicated by mouse Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5 dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in Wnt5a knockout mice. However, Wnt5a induction from 13.5 dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5 dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages.

Generation and characterization of an inducible transgenic model for studying mouse esophageal biology.

BACKGROUND: To facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein. RESULTS: First, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetO-HIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we subsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNP-rtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 7-10 days. CONCLUSIONS: We show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. Moreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 7-10 days.

A targeted constitutive mutation in the APC tumor suppressor gene underlies mammary but not intestinal tumorigenesis.

Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/beta-catenin signaling. Notably, genotype-phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/beta-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/beta-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc(+/1572T) mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc(+/1572T) mice suggests that specific dosages of Wnt/beta-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.

ß-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis.

Objective Deregulation of the Wnt signalling pathway by mutations in the Apc or ß-catenin genes underlies colorectal carcinogenesis. As a result, ß-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear ß-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate ß-catenin at tyrosine residues, which is thought to increase ß-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of ß-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous ß-catenin gene was introduced. Results This study provided in vivo evidence that ß-catenin(E654) is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the ß-catenin(E654) targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of ßcatenin. Surprisingly, the expression of ß-catenin(E654) did not affect histological grade or induce tumour invasiveness. Conclusions A thus far unknown mechanism was uncovered in which Y654 phosphorylation of ß-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that ß-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling.

Barrett's oesophageal adenocarcinoma encompasses tumour-initiating cells that do not express common cancer stem cell markers.

Accumulating evidence has suggested that tumours have a hierarchical organization in which only the cancer stem cells (CSCs) have tumour-initiating properties. Several surface antigens have been employed to isolate CSCs from various malignancies, although not from oesophageal adenocarcinoma (EA). We tested whether Barrett's oesophagus (BE) and EA might serve as a model for the CSC concept. In vivo assays were performed by transplantation of serially diluted bulk EA cells into NOD-SCID mice to establish the presence and frequency of tumour-initiating cells. These were found to be present as ca. 1 in 64 000 cells. The transplanted tumours fully recapitulated the primary lesions. Subsequently, a panel of previously established CSC markers was employed for immunohistochemistry. CD24, CD29 and CD44 showed heterogeneous staining in EA. Nuclear beta-catenin accumulation increased during progression from metaplasia to dysplasia and was often observed in the basal compartment with CD24 and CD29 staining. However, the overall staining patterns were not such to clearly point out specific candidate markers. Accordingly, all markers were employed to sort the corresponding subpopulations of cancer cells and transplant them at low multiplicities in NOD-SCID mice. No increased tumour-initiating capacity of sorted EA cells was observed upon transplantation. These results indicate that tumour-initiating cells are present in EA, thus reflecting a hierarchical organization. However, antibodies directed against novel surface antigens are needed to detect subpopulations enriched for CSCs in EA by transplantation assays.