FOXC2 Promotes Vasculogenic Mimicry in Ovarian Cancer.

FOXC2 is a forkhead family transcription factor that plays a critical role in specifying mesenchymal cell fate during embryogenesis. FOXC2 expression is associated with increased metastasis and poor survival in various solid malignancies. Using in vitro and in vivo assays in mouse ovarian cancer cell lines, we confirmed the previously reported mechanisms by which FOXC2 could promote cancer growth, metastasis, and drug resistance, including epithelial-mesenchymal transition, stem cell-like differentiation, and resistance to anoikis. In addition, we showed that FOXC2 expression is associated with vasculogenic mimicry in mouse and human ovarian cancers. FOXC2 overexpression increased the ability of human ovarian cancer cells to form vascular-like structures in vitro, while inhibition of FOXC2 had the opposite effect. Thus, we present a novel mechanism by which FOXC2 might contribute to cancer aggressiveness and poor patient survival.

HOXB13 controls cell state through super-enhancers.

Expression of the homeodomain transcription factor HOXB13 has been demonstrated in several malignancies but its role in tumorigenesis remains elusive. We observed high levels of HOXB13 in poorly differentiated pediatric tumors including a highly aggressive childhood neoplasm - malignant rhabdoid tumor. In a xenograft model of rhabdoid tumor, knockout of HOXB13 diminished tumor growth while partial knockdown of HOXB13 promoted differentiation of tumor cells into bone. These results suggest that HOXB13 enhances rhabdoid malignancy by interfering with mesenchymal stem cell differentiation. Consistent with this hypothesis, overexpression of HOXB13 in mesenchymal progenitor cells inhibited adipogenic, myogenic, and osteogenic differentiation. Mechanistically, we demonstrated that HOXB13 binds to super-enhancer regions regulating genes involved in differentiation and tumorigenesis.

A Pilot Feasibility Study of Yttrium-90 Liver Radioembolization Followed by Durvalumab and Tremelimumab in Patients with Microsatellite Stable Colorectal Cancer Liver Metastases.

LESSONS LEARNED: Radioembolization with yttrium-90 resin microspheres can be combined safely with full doses of durvalumab and tremelimumab in patients with metastatic colorectal cancer. Regional radioembolization with yttrium-90 resin microspheres did not result in any hepatic or extrahepatic responses to a combination of durvalumab and tremelimumab. The lack of immunomodulatory responses to yttrium-90 on biopsies before and after treatment rules out a potential role for this strategy in converting a "cold tumor" into an "inflamed," immune responsive tumor. BACKGROUND: PD-1 inhibitors have been ineffective in microsatellite stable (MSS) metastatic colorectal cancer (CRC). Preclinical models suggest that radiation therapy may sensitize MSS CRC to PD-1 blockade. METHODS: Patients with MSS metastatic CRC with liver-predominant disease who progressed following at least one prior line of treatment were treated with yttrium-90 (Y90) radioembolization to the liver (SIR-Spheres; Sirtex, Woburn, MA) followed 2-3 weeks later by the combination of durvalumab and tremelimumab. A Simon two-stage design was implemented, with a planned expansion to 18 patients if at least one response was noted in the first nine patients. RESULTS: Nine patients enrolled in the first stage of the study, all with progressive disease (PD) during or after their first two cycles of treatment. Per preplanned design, the study was closed because of futility. No treatment-related grade 3 or greater toxicities were recorded. Correlative studies with tumor biopsies showed low levels of tumor-infiltrating lymphocyte (TIL) infiltration in tumor cancer islands before and after Y90 radioembolization. CONCLUSION: Y90 radioembolization can be added safely to durvalumab and tremelimumab but did not promote tumor-directed immune responses against liver-metastasized MSS CRC.

Immune overdrive signature in colorectal tumor subset predicts poor clinical outcome.

The prognostic value of immune cell infiltration within the tumor microenvironment (TME) has been extensively investigated via histological and genomic approaches. Based on the positive prognostic value of T cell infiltration, Immunoscore has been developed and validated for predicting risk of recurrence for colorectal cancer (CRC). Also, association between a consensus T helper 1 (Th-1) immune response and favorable clinical outcomes has been observed across multiple cancer types. Here, we reanalyzed public genomic data sets from The Cancer Genome Atlas (TCGA) and NCBI Gene Expression Omnibus (NCBI-GEO) and performed multispectral immunohistochemistry (IHC) on a cohort of colorectal tumors. We identified and characterized a risk group, representing approximately 10% of CRC patients, with high intratumoral CD8+ T cell infiltration, but poor prognosis. These tumors included both microsatellite instable (MSI) and stable (MSS) phenotypes and had a high density of tumor-associated macrophages (TAMs) that expressed CD274 (programmed death-ligand 1 [PD-L1]), TGF-ß activation, and an immune overdrive signature characterized by the overexpression of immune response and checkpoint genes. Our findings illustrate that CRC patients may have poor prognosis despite high CD8+ T cell infiltration and provide CD274 as a simple biomarker for identifying these patients.

FOXC2 augments tumor propagation and metastasis in osteosarcoma.

Osteosarcoma is a highly malignant tumor that contains a small subpopulation of tumor-propagating cells (also known as tumor-initiating cells) characterized by drug resistance and high metastatic potential. The molecular mechanism by which tumor-propagating cells promote tumor growth is poorly understood. Here, we report that the transcription factor forkhead box C2 (FOXC2) is frequently expressed in human osteosarcomas and is important in maintaining osteosarcoma cells in a stem-like state. In osteosarcoma cell lines, we show that anoikis conditions stimulate FOXC2 expression. Downregulation of FOXC2 decreases anchorage-independent growth and invasion in vitro and lung metastasis in vivo, while overexpression of FOXC2 increases tumor propagation in vivo. In osteosarcoma cell lines, we demonstrate that high levels of FOXC2 are associated with and required for the expression of osteosarcoma tumor-propagating cell markers. In FOXC2 knockdown cell lines, we show that CXCR4, a downstream target of FOXC2, can restore osteosarcoma cell invasiveness and metastasis to the lung.

Integrin αvß3 drives slug activation and stemness in the pregnant and neoplastic mammary gland.

Although integrin αvß3 is linked to cancer progression, its role in epithelial development is unclear. Here, we show that αvß3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. Whereas αvß3 is a luminal progenitor marker in the virgin gland, we noted increased αvß3 expression in MaSCs at midpregnancy. Accordingly, mice lacking αvß3 or expressing a signaling-deficient receptor showed defective mammary gland morphogenesis during pregnancy. This was associated with decreased MaSC expansion, clonogenicity, and expression of Slug, a master regulator of MaSCs. Surprisingly, αvß3-deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. Transforming growth factor ß2 (TGF-ß2) induced αvß3 expression, enhancing Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells, αvß3 was necessary and sufficient for Slug activation, tumorsphere formation, and tumor initiation. Thus, pregnancy-associated MaSCs require a TGF-ß2/αvß3/Slug pathway, which may contribute to breast cancer progression and stemness.

Succinate dehydrogenase inhibition leads to epithelial-mesenchymal transition and reprogrammed carbon metabolism.

BACKGROUND: Succinate dehydrogenase (SDH) is a mitochondrial metabolic enzyme complex involved in both the electron transport chain and the citric acid cycle. SDH mutations resulting in enzymatic dysfunction have been found to be a predisposing factor in various hereditary cancers. Therefore, SDH has been implicated as a tumor suppressor. RESULTS: We identified that dysregulation of SDH components also occurs in serous ovarian cancer, particularly the SDH subunit SDHB. Targeted knockdown of Sdhb in mouse ovarian cancer cells resulted in enhanced proliferation and an epithelial-to-mesenchymal transition (EMT). Bioinformatics analysis revealed that decreased SDHB expression leads to a transcriptional upregulation of genes involved in metabolic networks affecting histone methylation. We confirmed that Sdhb knockdown leads to a hypermethylated epigenome that is sufficient to promote EMT. Metabolically, the loss of Sdhb resulted in reprogrammed carbon source utilization and mitochondrial dysfunction. This altered metabolic state of Sdhb knockdown cells rendered them hypersensitive to energy stress. CONCLUSIONS: These data illustrate how SDH dysfunction alters the epigenetic and metabolic landscape in ovarian cancer. By analyzing the involvement of this enzyme in transcriptional and metabolic networks, we find a metabolic Achilles' heel that can be exploited therapeutically. Analyses of this type provide an understanding how specific perturbations in cancer metabolism may lead to novel anticancer strategies.

Musashi-2 regulates normal hematopoiesis and promotes aggressive myeloid leukemia.

RNA-binding proteins of the Musashi (Msi) family are expressed in stem cell compartments and in aggressive tumors, but they have not yet been widely explored in the blood. Here we demonstrate that Msi2 is the predominant form expressed in hematopoietic stem cells (HSCs), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of human MSI2 in a mouse model increases HSC cell cycle progression and cooperates with the chronic myeloid leukemia-associated BCR-ABL1 oncoprotein to induce an aggressive leukemia. MSI2 is overexpressed in human myeloid leukemia cell lines, and its depletion leads to decreased proliferation and increased apoptosis. Expression levels in human myeloid leukemia directly correlate with decreased survival in patients with the disease, thereby defining MSI2 expression as a new prognostic marker and as a new target for therapy in acute myeloid leukemia (AML).

Constitutively active AKT depletes hematopoietic stem cells and induces leukemia in mice.

Human cancers, including acute myeloid leukemia (AML), commonly display constitutive phosphoinositide 3-kinase (PI3K) AKT signaling. However, the exact role of AKT activation in leukemia and its effects on hematopoietic stem cells (HSCs) are poorly understood. Several members of the PI3K pathway, phosphatase and tensin homolog (Pten), the forkhead box, subgroup O (FOXO) transcription factors, and TSC1, have demonstrated functions in normal and leukemic stem cells but are rarely mutated in leukemia. We developed an activated allele of AKT1 that models increased signaling in normal and leukemic stem cells. In our murine bone marrow transplantation model using a myristoylated AKT1 (myr-AKT), recipients develop myeloproliferative disease, T-cell lymphoma, or AML. Analysis of the HSCs in myr-AKT mice reveals transient expansion and increased cycling, associated with impaired engraftment. myr-AKT-expressing bone marrow cells are unable to form cobblestones in long-term cocultures. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) rescues cobblestone formation in myr-AKT-expressing bone marrow cells and increases the survival of myr-AKT mice. This study demonstrates that enhanced AKT activation is an important mechanism of transformation in AML and that HSCs are highly sensitive to excess AKT/mTOR signaling.

Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera.

We report that TG101348, a selective small-molecule inhibitor of JAK2 with an in vitro IC50 of approximately 3 nM, shows therapeutic efficacy in a murine model of myeloproliferative disease induced by the JAK2V617F mutation. In treated animals, there was a statistically significant reduction in hematocrit and leukocyte count, a dose-dependent reduction/elimination of extramedullary hematopoiesis, and, at least in some instances, evidence for attenuation of myelofibrosis. There were no apparent toxicities and no effect on T cell number. In vivo responses were correlated with surrogate endpoints, including reduction/elimination of JAK2V617F disease burden assessed by quantitative genomic PCR, suppression of endogenous erythroid colony formation, and in vivo inhibition of JAK-STAT signal transduction as assessed by flow cytometric measurement of phosphorylated Stat5.

MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.

BACKGROUND: The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR). METHODS AND FINDINGS: DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 x 10(12)/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. CONCLUSIONS: Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.