Targeted engagement of ß-catenin-Ikaros complexes in refractory B-cell malignancies.

In most cell types, nuclear ß-catenin functions as prominent oncogenic driver and pairs with TCF7-family factors for transcriptional activation of MYC. Surprisingly, B-lymphoid malignancies not only lacked expression and activating lesions of ß-catenin but critically depended on GSK3ß for effective ß-catenin degradation. Our interactome studies in B-lymphoid tumors revealed that ß-catenin formed repressive complexes with lymphoid-specific Ikaros factors at the expense of TCF7. Instead of MYC-activation, ß-catenin was essential to enable Ikaros-mediated recruitment of nucleosome remodeling and deacetylation (NuRD) complexes for transcriptional repression of MYC. To leverage this previously unrecognized vulnerability of B-cell-specific repressive ß-catenin-Ikaros-complexes in refractory B-cell malignancies, we examined GSK3ß small molecule inhibitors to subvert ß-catenin degradation. Clinically approved GSK3ß-inhibitors that achieved favorable safety prof les at micromolar concentrations in clinical trials for neurological disorders and solid tumors were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of ß-catenin, repression of MYC and acute cell death. Preclinical in vivo treatment experiments in patient-derived xenografts validated small molecule GSK3ß-inhibitors for targeted engagement of lymphoid-specific ß-catenin-Ikaros complexes as a novel strategy to overcome conventional mechanisms of drug-resistance in refractory malignancies. HIGHLIGHTS: Unlike other cell lineages, B-cells express nuclear ß-catenin protein at low baseline levels and depend on GSK3ß for its degradation.In B-cells, ß-catenin forms unique complexes with lymphoid-specific Ikaros factors and is required for Ikaros-mediated tumor suppression and assembly of repressive NuRD complexes. CRISPR-based knockin mutation of a single Ikaros-binding motif in a lymphoid MYC superenhancer region reversed ß-catenin-dependent Myc repression and induction of cell death. The discovery of GSK3ß-dependent degradation of ß-catenin as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3ß-inhibitors for the treatment of refractory B-cell malignancies. GRAPHICAL ABSTRACT: Abundant nuclear ß-cateninß-catenin pairs with TCF7 factors for transcriptional activation of MYCB-cells rely on efficient degradation of ß-catenin by GSK3ßB-cell-specific expression of Ikaros factors Unique vulnerability in B-cell tumors: GSK3ß-inhibitors induce nuclear accumulation of ß-catenin.ß-catenin pairs with B-cell-specific Ikaros factors for transcriptional repression of MYC.

Dual blockade of macropinocytosis and asparagine bioavailability shows synergistic anti-tumor effects on KRAS-mutant colorectal cancer.

Mutations of KRAS gene are found in various types of cancer, including colorectal cancer (CRC). Despite intense efforts, no pharmacological approaches are expected to be effective against KRAS-mutant cancers. Macropinocytosis is an evolutionarily conserved actin-dependent endocytic process that internalizes extracellular fluids into large vesicles called macropinosomes. Recent studies have revealed macropinocytosis's important role in metabolic adaptation to nutrient stress in cancer cells harboring KRAS mutations. Here we showed that KRAS-mutant CRC cells enhanced macropinocytosis for tumor growth under nutrient-depleted conditions. We also demonstrated that activation of Rac1 and phosphoinositide 3-kinase were involved in macropinocytosis of KRAS-mutant CRC cells. Furthermore, we found that macropinocytosis was closely correlated with asparagine metabolism. In KRAS-mutant CRC cells engineered with knockdown of asparagine synthetase, macropinocytosis was accelerated under glutamine-depleted condition, and albumin addition could restore the glutamine depletion-induced growth suppression by recovering the intracellular asparagine level. Finally, we discovered that the combination of macropinocytosis inhibition and asparagine depletion dramatically suppressed the tumor growth of KRAS-mutant CRC cells in vivo. These results indicate that dual blockade of macropinocytosis and asparagine bioavailability could be a novel therapeutic strategy for KRAS-mutant cancers.

The therapeutic potential of multiclonal tumoricidal T cells derived from tumor infiltrating lymphocyte-1derived iPS cells.

Tumor-infiltrating lymphocytes (TIL), which include tumor-specific T lymphocytes with frequency, are used for adoptive cell transfer therapy (ACT) in clinical practice. The optimization of TIL preparation has been investigated to reduce the senescence and increase the abundance of TIL, as both the quality and quantity of the transferred cells have great influence on the outcome of TIL-based ACT (TIL-ACT). Considering the effects of cell reprogramming on senescence, we expected that the anti-tumor effect could be enhanced by TIL regeneration. To confirm this hypothesis, we established tumor-specific TIL-derived iPS cells (TIL-iPSC) with human colorectal cancer specimens. T cells differentiated from TIL-iPSC (TIL-iPS-T) retained not only intrinsic T cell functions and tumor specificity, but also exhibited improved proliferation capacity and additional killing activity. Moreover, less differentiated profiles and prolonged persistency were seen in TIL-iPS-T compared with primary cells. Our findings imply that iPSC technology has great potential for TIL-ACT.

Uncoupling of macrophage inflammation from self-renewal modulates host recovery from respiratory viral infection.

Tissue macrophages self-renew during homeostasis and produce inflammatory mediators upon microbial infection. We examined the relationship between proliferative and inflammatory properties of tissue macrophages by defining the impact of the Wnt/ß-catenin pathway, a central regulator of self-renewal, in alveolar macrophages (AMs). Activation of ß-catenin by Wnt ligand inhibited AM proliferation and stemness, but promoted inflammatory activity. In a murine influenza viral pneumonia model, ß-catenin-mediated AM inflammatory activity promoted acute host morbidity; in contrast, AM proliferation enabled repopulation of reparative AMs and tissue recovery following viral clearance. Mechanistically, Wnt treatment promoted ß-catenin-HIF-1α interaction and glycolysis-dependent inflammation while suppressing mitochondrial metabolism and thereby, AM proliferation. Differential HIF-1α activities distinguished proliferative and inflammatory AMs in vivo. This ß-catenin-HIF-1α axis was conserved in human AMs and enhanced HIF-1α expression associated with macrophage inflammation in COVID-19 patients. Thus, inflammatory and reparative activities of lung macrophages are regulated by ß-catenin-HIF-1α signaling, with implications for the treatment of severe respiratory diseases.

Suppressing neutrophil-dependent angiogenesis abrogates resistance to anti-VEGF antibody in a genetic model of colorectal cancer.

We tested cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice, which recapitulate key genetic abnormalities accumulating during colorectal cancer (CRC) tumorigenesis in humans, for responsiveness to anti-VEGF therapy. We found that even tumors in cis-ApcΔ716/Smad4+/-KrasG12D mice, although highly aggressive, were suppressed by anti-VEGF treatment. We tested the hypothesis that inflammation, a major risk factor and trigger for CRC, may affect responsiveness to anti-VEGF. Chemically induced colitis (CIC) in cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice promoted development of colon tumors that were largely resistant to anti-VEGF treatment. The myeloid growth factor G-CSF was markedly increased in the serum after induction of colitis. Antibodies blocking G-CSF, or its target Bv8/PROK2, suppressed tumor progression and myeloid cell infiltration when combined with anti-VEGF in CIC-associated CRC and in anti-VEGF-resistant CRC liver metastasis models. In a series of CRC specimens, tumor-infiltrating neutrophils strongly expressed Bv8/PROK2. CRC patients had significantly higher plasma Bv8/PROK2 levels than healthy volunteers and high plasma Bv8/PROK2 levels were inversely correlated with overall survival. Our findings establish Bv8/PROK2 as a translational target in CRC, in combination with anti-VEGF agents.

Chemosensitivity of Patient-Derived Cancer Stem Cells Identifies Colorectal Cancer Patients with Potential Benefit from FGFR Inhibitor Therapy.

Some colorectal cancer patients harboring FGFR (fibroblast growth factor receptor) genetic alterations, such as copy number gain, mutation, and/or mRNA overexpression, were selected for enrollment in several recent clinical trials of FGFR inhibitor, because these genetic alterations were preclinically reported to be associated with FGFR inhibitor sensitivity as well as poor prognosis, invasiveness, and/or metastatic potential. However, few enrolled patients were responsive to FGFR inhibitors. Thus, practical strategies are eagerly awaited that can stratify patients for the subset that potentially responds to FGFR inhibitor chemotherapy. In the present study, we evaluated the sensitivity to FGFR inhibitor erdafitinib on 25 patient-derived tumor-initiating cell (TIC) spheroid lines carrying wild-type RAS and RAF genes, both in vitro and in vivo. Then, we assessed possible correlations between the sensitivity and the genetic/genomic data of the spheroid lines tested. Upon their exposure to erdafitinib, seven lines (7/25, 28%) responded significantly. Normal colonic epithelial stem cells were unaffected by the inhibitors. Moreover, the combination of erdafitinib with EGFR inhibitor erlotinib showed stronger growth inhibition than either drug alone, as efficacy was observed in 21 lines (84%) including 14 (56%) that were insensitive to erdafitinib alone. The in vitro erdafitinib response was accurately reflected on mouse xenografts of TIC spheroid lines. However, we found little correlation between their genetic/genomic alterations of TIC spheroids and the sensitivity to the FGFR inhibitor. Accordingly, we propose that direct testing of the patient-derived spheroids in vitro is one of the most reliable personalized methods in FGFR-inhibitor therapy of colorectal cancer patients.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

Tubular ß-catenin and FoxO3 interactions protect in chronic kidney disease.

The Wnt/ß-catenin signaling pathway plays an important role in renal development and is reexpressed in the injured kidney and other organs. ß-Catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/ß-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular ß-catenin signaling in CKD remains unclear, we genetically stabilized (i.e., activated) ß-catenin specifically in murine proximal tubules. Mice with increased tubular ß-catenin signaling were protected in 2 murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional T cell factor/lymphoid enhancer factor-dependent ß-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular ß-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine γ-lyase as a potentially novel transcriptional target of ß-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturned the conventional dogma about ß-catenin signaling and CKD by showing a protective effect of proximal tubule ß-catenin in CKD and identified a potentially new transcriptional target of ß-catenin/FoxO3 signaling that has therapeutic potential for CKD.

MicroRNA-9-5p-CDX2 Axis: A Useful Prognostic Biomarker for Patients with Stage II/III Colorectal Cancer.

A lack of caudal-type homeobox transcription factor 2 (CDX2) protein expression has been proposed as a prognostic biomarker for colorectal cancer (CRC). However, the relationship between CDX2 levels and the survival of patients with stage II/III CRC along with the relationship between microRNAs (miRs) and CDX2 expression are unclear. Tissue samples were collected from patients with stage II/III CRC surgically treated at Kyoto University Hospital. CDX2 expression was semi-quantitatively evaluated by immunohistochemistry (IHC). The prognostic impacts of CDX2 expression on overall survival (OS) and relapse-free survival (RFS) were evaluated by multivariable statistical analysis. The expression of miRs regulating CDX2 expression and their prognostic impacts were analyzed using The Cancer Genome Atlas Program for CRC (TCGA-CRC). Eleven of 174 CRC tissues lacked CDX2 expression. The five-year OS and RFS rates of patients with CDX2-negative CRC were significantly lower than those of CDX2-positive patients. Multivariate analysis of clinicopathological features revealed that CDX2-negative status is an independent marker of poor prognosis in stage II/III CRC. miR-9-5p was shown to regulate CDX2 expression. TCGA-CRC analysis showed that high miR-9-5p expression was significantly associated with poor patient prognosis in stage II/III CRC. In conclusion, CDX2, the post-transcriptional target of microRNA-9-5p, is a useful prognostic biomarker in patients with stage II/III CRC.

A novel mouse model demonstrates that oncogenic melanocyte stem cells engender melanoma resembling human disease.

Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.

Hedgehog stimulates hair follicle neogenesis by creating inductive dermis during murine skin wound healing.

Mammalian wounds typically heal by fibrotic repair without hair follicle (HF) regeneration. Fibrosis and regeneration are currently considered the opposite end of wound healing. This study sought to determine if scar could be remodeled to promote healing with HF regeneration. Here, we identify that activation of the Sonic hedgehog (Shh) pathway reinstalls a regenerative dermal niche, called dermal papilla, which is required and sufficient for HF neogenesis (HFN). Epidermal Shh overexpression or constitutive Smoothened dermal activation results in extensive HFN in wounds that otherwise end in scarring. While long-term Wnt activation is associated with fibrosis, Shh signal activation in Wnt active cells promotes the dermal papilla fate in scarring wounds. These studies demonstrate that mechanisms of scarring and regeneration are not distant from one another and that wound repair can be redirected to promote regeneration following injury by modifying a key dermal signal.

A Chemosensitivity Study of Colorectal Cancer Using Xenografts of Patient-Derived Tumor-Initiating Cells.

Current genomic and gene expression analyses provide versatile tools to improve cancer chemotherapy. However, it is still difficult to predict whether each patient responds to a particular regimen or not. To predict chemosensitivity in each patient with colorectal cancer, we developed an evaluation method using the primary tumor-initiating cells (TIC, aka cancer stem cells) xenografted in nude mice subcutaneously (patient-derived spheroid xenografts; PDSX). Simultaneously, we also prepared the conventional patient-derived xenografts (PDX) from the same patients' tumors and compared the dosing results with those of PDSXs. We further compared the chemosensitivities of PDSXs with those of 7 patients who had been given regimens such as FOLFOX and FOLFIRI to treat their metastatic lesions. As per the results, the PDSX method provided much more precise and predictable tumor growth with less variance than conventional PDX, although both retained the epithelial characteristics of the primary tumors. Likewise, drug-dosing tests showed essentially the same results in PDXs and PDSXs, with stronger statistical power in PDSXs. Notably, the cancer chemosensitivity in each patient was precisely reflected in that of the PDSX mice along the clinical course until the resistance emerged at the terminal stage. This "paraclinical" xenograft trials using PDSXs may help selection of chemotherapy regimens efficacious for each patient, and, more importantly, avoiding inefficient ones by which the patient can lose precious time and QOL. Furthermore, the PDSX method may be employed for evaluations of off-label uses of cancer chemotherapeutics and compassionate uses of yet-unapproved new drugs in personalized therapies. Mol Cancer Ther; 17(10); 2187-96. ©2018 AACR.

SMAD4 Suppresses WNT-Driven Dedifferentiation and Oncogenesis in the Differentiated Gut Epithelium.

The cell of origin of colon cancer is typically thought to be the resident somatic stem cells, which are immortal and escape the continual cellular turnover characteristic of the intestinal epithelium. However, recent studies have identified certain conditions in which differentiated cells can acquire stem-like properties and give rise to tumors. Defining the origins of tumors will inform cancer prevention efforts as well as cancer therapies, as cancers with distinct origins often respond differently to treatments. We report here a new condition in which tumors arise from the differentiated intestinal epithelium. Inactivation of the differentiation-promoting transcription factor SMAD4 in the intestinal epithelium was surprisingly well tolerated in the short term. However, after several months, adenomas developed with characteristics of activated WNT signaling. Simultaneous loss of SMAD4 and activation of the WNT pathway led to dedifferentiation and rapid adenoma formation in differentiated tissue. Transcriptional profiling revealed acquisition of stem cell characteristics, and colabeling indicated that cells expressing differentiated enterocyte markers entered the cell cycle and reexpressed stem cell genes upon simultaneous loss of SMAD4 and activation of the WNT pathway. These results indicate that SMAD4 functions to maintain differentiated enterocytes in the presence of oncogenic WNT signaling, thus preventing dedifferentiation and tumor formation in the differentiated intestinal epithelium.Significance: This work identifies a mechanism through which differentiated cells prevent tumor formation by suppressing oncogenic plasticity. Cancer Res; 78(17); 4878-90. ©2018 AACR.

Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine.

Acting downstream of many growth factors, extracellular signal-regulated kinase (ERK) plays a pivotal role in regulating cell proliferation and tumorigenesis, where its spatiotemporal dynamics, as well as its strength, determine cellular responses. Here, we uncover the ERK activity dynamics in intestinal epithelial cells (IECs) and their association with tumour characteristics. Intravital imaging identifies two distinct modes of ERK activity, sustained and pulse-like activity, in IECs. The sustained and pulse-like activities depend on ErbB2 and EGFR, respectively. Notably, activation of Wnt signalling, the earliest event in intestinal tumorigenesis, augments EGFR signalling and increases the frequency of ERK activity pulses through controlling the expression of EGFR and its regulators, rendering IECs sensitive to EGFR inhibition. Furthermore, the increased pulse frequency is correlated with increased cell proliferation. Thus, ERK activity dynamics are defined by composite inputs from EGFR and ErbB2 signalling in IECs and their alterations might underlie tumour-specific sensitivity to pharmacological EGFR inhibition.

Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis.

In colorectal cancer patients, a high density of cytotoxic CD8+ T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/ß-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8+ T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8+ T cells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.

An improved method for culturing patient-derived colorectal cancer spheroids.

Recent advances allowed culturing and examination of patient-derived colorectal cancer (PD-CRC) cells as organoids or spheroids. To be applied to practical personalized medicine, however, current methods still need to be strengthened for higher efficiency. Here we report an improved method to propagate PD-CRC tumor initiating cells (TICs) in spheroid culture. We established > 100 cancer spheroid lines derived from independent colorectal cancer patients employing a serum-containing medium with additional inhibitors, Y27632 and SB431542. Because colorectal cancer spheroids showed wide-range growth rates depending on the patient tumors, we searched for supplementary factors that accelerated proliferation of slow-growing CRC-TIC spheroids. To this end, we introduced a convenient growth-monitoring method using a luciferase reporter. We found that epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF) were critical for steady propagation of a subset of CRC-TIC spheroids carrying the wild-type RAS and RAF genes. We also identified 5'-(N-ethyl-carboxamido)-adenosine (NECA), an adenosine receptor agonist, as an essential supplement for another subset of spheroids. Based on these results, we propose to optimize culture conditions for CRC-TIC spheroids by adjusting to the respective tumor samples. Our method provides a versatile tool that can be applied to personalized chemotherapy evaluation in prospective clinical studies.

Accurate diagnosis of mismatch repair deficiency in colorectal cancer using high-quality DNA samples from cultured stem cells.

Mismatch repair (MMR)-deficient or microsatellite instability (MSI) colorectal cancer includes two subtypes; Lynch syndrome and sporadic MSI cancer, both of which generate multiple neoantigens due to unrepaired mutations. Although such patients respond very well to immune checkpoint therapy, their diagnosis can be confused by low quality DNA samples owing to formalin fixation and/or low cancer cell content. Here we prepared high-quality DNA samples from in vitro-cultured cancer spheroids that consisted of the pure cell population. We evaluated their diagnostic power by on-chip electrophoresis, mutational burden assessment, and direct sequencing. Because formalin-fixed paraffin-embedded (FFPE) tissues are widely used as the DNA source, we compared such samples with spheroid DNA. Additionally, we performed immunohistochemistry (IHC) for MMR proteins on spheroids as well as primary tumor sections. Of 111 cases of colorectal cancer patients, we found seven MSI-high cases in which all diagnostic results agreed on spheroid-based assays, whereas the results with the FFPE DNA were less reliable though analyzable. Importantly, there was an MSS case that appeared as MSI by IHC on primary tumor sections. Based on these results, we propose to employ cultured cancer spheroids as the source of both DNA and IHC specimens for more reliable clinical diagnosis.

A mouse model for embryonal tumors with multilayered rosettes uncovers the therapeutic potential of Sonic-hedgehog inhibitors.

Embryonal tumors with multilayered rosettes (ETMRs) have recently been described as a new entity of rare pediatric brain tumors with a fatal outcome. We show here that ETMRs are characterized by a parallel activation of Shh and Wnt signaling. Co-activation of these pathways in mouse neural precursors is sufficient to induce ETMR-like tumors in vivo that resemble their human counterparts on the basis of histology and global gene-expression analyses, and that point to apical radial glia cells as the possible tumor cell of origin. Overexpression of LIN28A, which is a hallmark of human ETMRs, augments Sonic-hedgehog (Shh) and Wnt signaling in these precursor cells through the downregulation of let7-miRNA, and LIN28A/let7a interaction with the Shh pathway was detected at the level of Gli mRNA. Finally, human ETMR cells that were transplanted into immunocompromised host mice were responsive to the SHH inhibitor arsenic trioxide (ATO). Our work provides a novel mouse model in which to study this tumor type, demonstrates the driving role of Wnt and Shh activation in the growth of ETMRs and proposes downstream inhibition of Shh signaling as a therapeutic option for patients with ETMRs.

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction.

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/ß-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/ß-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.