An orthogonal IL-2 and IL-2Rß system drives persistence and activation of CAR T cells and clearance of bulky lymphoma.

Chimeric antigen receptor (CAR) T cells induce durable responses in patients with refractory hematological tumors. However, low CAR T cell activity, poor engraftment, or short in-patient persistence can lead to tumor progression or relapse. Furthermore, excessive CAR T cell expansion and activation can result in life-threatening cytokine release syndrome (CRS). Thus, in-patient control of the CAR T cell population is essential. Interleukin-2 (IL-2) is a critical cytokine for T cell proliferation and effector function, but its clinical use is limited by immune-mediated toxicity. Here, we report on an orthogonal IL-2 receptor and ligand system that enables specific in vivo control of CAR T cell expansion and activation, wherein an orthogonal human IL-2 (STK-009) selectively pairs with an orthogonal human IL-2Rß (hoRb) expressed on CAR T cells. STK-009 expands hoRb-expressing CAR T cells in the presence and absence of tumor antigen and maintains the presence of stem cell memory T cells (TSCM) and effector T cells. In preclinical models of human CAR-refractory lymphoma, STK-009 treatment resulted in systemic and intratumoral expansion and activation of hoRb-expressing anti­CD19-CD28ζ CAR T cells (SYNCAR). The orthogonal IL-2 receptor/ligand system delivers complete responses in large subcutaneous lymphomas, even with substantially reduced CAR T cell doses, by selectively expanding and activating CAR T cells in vivo. STK-009 withdrawal allowed normal CAR T cell contraction, thereby limiting CRS induced by tumor antigen­specific T cell activation. These data suggest that the orthogonal IL-2 receptor/ligand system provides the in vivo control necessary to maximize efficacy of CAR T therapies.

Single-cell transcriptomics identifies gene expression networks driving differentiation and tumorigenesis in the human fallopian tube.

The human fallopian tube harbors the cell of origin for the majority of high-grade serous "ovarian" cancers (HGSCs), but its cellular composition, particularly the epithelial component, is poorly characterized. We perform single-cell transcriptomic profiling of around 53,000 individual cells from 12 primary fallopian specimens to map their major cell types. We identify 10 epithelial subpopulations with diverse transcriptional programs. Based on transcriptional signatures, we reconstruct a trajectory whereby secretory cells differentiate into ciliated cells via a RUNX3high intermediate. Computational deconvolution of advanced HGSCs identifies the "early secretory" population as a likely precursor state for the majority of HGSCs. Its signature comprises both epithelial and mesenchymal features and is enriched in mesenchymal-type HGSCs (p = 6.7 × 10-27), a group known to have particularly poor prognoses. This cellular and molecular compendium of the human fallopian tube in cancer-free women is expected to advance our understanding of the earliest stages of fallopian epithelial neoplasia.

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.

Metformin induces distinct bioenergetic and metabolic profiles in sensitive versus resistant high grade serous ovarian cancer and normal fallopian tube secretory epithelial cells.

Metformin is a widely used agent for the treatment of diabetes and infertility, however, it has been found to have anti-cancer effects in a variety of malignancies including high grade serous ovarian cancer (HGSC). Studies describing the mechanisms by which metformin affects HGSC are ongoing, but detailed analysis of its effect on the cellular metabolism of both HGSC cells and their precursor, normal fallopian tube secretory epithelial cells (FTSECs), is lacking. We addressed the effects of metformin and the more potent biguanide, phenformin, on HGSC cell lines and normal immortalized FTSECs. Cell proliferation assays identified that FTSECs and a subset of HGSC cell lines are relatively resistant to the anti-proliferative effects of metformin. Bioenergetic and metabolomic analyses were used to metabolically differentiate the metformin-sensitive and metformin-resistant cell lines. Bioenergetically, biguanides elicited a significant decrease in mitochondrial respiration in all HGSC cells and FTSECs. However, biguanides had a greater effect on mitochondrial respiration in metformin sensitive cells. Metabolomic analysis revealed that metformin and phenformin generally induce similar changes in metabolic profiles. Biguanide treatment led to a significant increase in NADH in FTSECs and HGSC cells. Interestingly, biguanide treatment induced changes in the levels of mitochondrial shuttle metabolites, glycerol-3-phopshate (G3P) and aspartate, specifically in HGSC cell lines and not in FTSECs. Greater alterations in G3P or aspartate levels were also found in metformin sensitive cells relative to metformin resistant cells. These data identify bioenergetic and HGSC-specific metabolic effects that correlate with metformin sensitivity and novel metabolic avenues for possible therapeutic intervention.

Glucose deprivation elicits phenotypic plasticity via ZEB1-mediated expression of NNMT.

Glucose is considered the primary energy source for all cells, and some cancers are addicted to glucose. Here, we investigated the functional consequences of chronic glucose deprivation in serous ovarian cancer cells. We found that cells resistant to glucose starvation (glucose-restricted cells) demonstrated increased metabolic plasticity that was dependent on NNMT (Nicotinamide N-methyltransferase) expression. We further show that ZEB1 induced NNMT, rendered cells resistant to glucose deprivation and recapitulated metabolic adaptations and mesenchymal gene expression observed in glucose-restricted cells. NNMT depletion reversed metabolic plasticity in glucose-restricted cells and prevented de novo formation of glucose-restricted colonies. In addition to its role in glucose independence, we found that NNMT was required for other ZEB1-induced phenotypes, such as increased migration. NNMT protein levels were also elevated in metastatic and recurrent tumors compared to matched primary carcinomas, while normal ovary and fallopian tube tissue had no detectable NNMT expression. Our studies define a novel ZEB1/NNMT signaling axis, which elicits mesenchymal gene expression, as well as phenotypic and metabolic plasticity in ovarian cancer cells upon chronic glucose starvation. Understanding the causes of cancer cell plasticity is crucial for the development of therapeutic strategies to counter intratumoral heterogeneity, acquired drug resistance and recurrence in high-grade serous ovarian cancer (HGSC).

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.

Sphingosine kinase 1 is required for TGF-ß mediated fibroblastto- myofibroblast differentiation in ovarian cancer.

Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-ß-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-ß signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.

Rethinking ovarian cancer II: reducing mortality from high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSOC) accounts for 70-80% of ovarian cancer deaths, and overall survival has not changed significantly for several decades. In this Opinion article, we outline a set of research priorities that we believe will reduce incidence and improve outcomes for women with this disease. This 'roadmap' for HGSOC was determined after extensive discussions at an Ovarian Cancer Action meeting in January 2015.

Suboptimal cytoreduction in ovarian carcinoma is associated with molecular pathways characteristic of increased stromal activation.

OBJECTIVE: Suboptimal cytoreductive surgery in advanced epithelial ovarian cancer (EOC) is associated with poor survival but it is unknown if poor outcome is due to the intrinsic biology of unresectable tumors or insufficient surgical effort resulting in residual tumor-sustaining clones. Our objective was to identify the potential molecular pathway(s) and cell type(s) that may be responsible for suboptimal surgical resection. METHODS: By comparing gene expression in optimally and suboptimally cytoreduced patients, we identified a gene network associated with suboptimal cytoreduction and explored the biological processes and cell types associated with this gene network. RESULTS: We show that primary tumors from suboptimally cytoreduced patients express molecular signatures that are typically present in a distinct molecular subtype of EOC characterized by increased stromal activation and lymphovascular invasion. Similar molecular pathways are present in EOC metastases, suggesting that primary tumors in suboptimally cytoreduced patients are biologically similar to metastatic tumors. We demonstrate that the suboptimal cytoreduction network genes are enriched in reactive tumor stroma cells rather than malignant tumor cells. CONCLUSION: Our data suggest that the success of cytoreductive surgery is dictated by tumor biology, such as extensive stromal reaction and increased invasiveness, which may hinder surgical resection and ultimately lead to poor survival.

Large oncosomes contain distinct protein cargo and represent a separate functional class of tumor-derived extracellular vesicles.

Large oncosomes (LO) are atypically large (1-10 µm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep(TM)) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.

Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS.

High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC.

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.

A role for BRCA1 in uterine leiomyosarcoma.

Uterine leiomyosarcoma (ULMS) is a rare gynecologic malignancy with a low survival rate. Currently, there is no effective treatment for ULMS. Infrequent occurrences of human ULMS hamper the understanding of the initiation and progression of the disease, thereby limiting the ability to develop efficient therapies. To elucidate the roles of the p53 and BRCA1 tumor suppressor genes in gynecologic malignancies, we generated mice in which p53 and/or BRCA1 can be conditionally deleted using anti-Müllerian hormone type II receptor (Amhr2)-driven Cre recombinase. We showed that conditional deletion of p53 in mice results in the development of uterine tumors that resemble human ULMS and that concurrent deletion of p53 and BRCA1 significantly accelerates the progression of these tumors. This finding led to our hypothesis that BRCA1 may play a role in human ULMS development. Consistent with this hypothesis, we showed that the BRCA1 protein is absent in 29% of human ULMS and that BRCA1 promoter methylation is the likely mechanism of BRCA1 downregulation. These data indicate that the loss of BRCA1 function may be an important step in the progression of ULMS. Our findings provide a rationale for investigating therapies that target BRCA1 deficiency in ULMS.

The Tsc/Rheb signaling pathway controls basic amino acid uptake via the Cat1 permease in fission yeast.

The Tsc/Rheb signaling pathway plays critical roles in the control of growth and cell cycle. Studies in fission yeast have also implicated its importance in the regulation of amino acid uptake. Disruption of tsc2+, one of the tsc+ genes, has been shown to result in decreased arginine uptake and resistance to canavanine. A similar effect is also seen with other basic amino acids. We have identified a permease responsible for the uptake of basic amino acids by genetic complementation and disruption. SPAC869.11 (termed Cat1 for cationic amino acid transporter) contains 12 predicted transmembrane domains and its overexpression in wild type fission yeast leads to the increased uptake of basic amino acids and sensitivity to canavanine. Disruption of cat1+ in the deltatsc2 background interfered with the suppression of the canavanine-resistant phenotype of Atsc2 mutants by a dominant negative Rheb. In deltatsc2 mutant strains, the amount of Cat1 was not altered, but instead was mislocalized. This mislocalization was suppressed by the expression of dominant negative Rheb. In addition, we found that the loss of the E3 ubiquitin ligase, Pub1, also restores proper localization. These results provide a crucial link between Tsc/Rheb signaling and the regulation of the basic amino acid permease in fission yeast.

The TSC/Rheb/TOR signaling pathway in fission yeast and mammalian cells: temperature sensitive and constitutive active mutants of TOR.

The TSC/Rheb/TOR signaling pathway plays important roles in growth and cell cycle regulation. The main player TOR belongs to the PI3K-related protein kinase family. Recent studies utilizing fission yeast Tor2 have led to the identification of a number of amino acid changes that lead to inactivation as well as activation of TOR kinase. Also, constitutive active mutations in its upstream regulator, Rheb, have been identified. Isolation and characterization of temperature sensitive Tor2 mutants have established that this kinase functions as a key switch that determines cell fate between growth and sexual development. Introduction of Tor2 activating mutations into mTOR conferred nutrient independent activation of mTOR. Interestingly, these studies point to regions of TOR kinase important for its function.

Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast.

Rheb GTPase is a key player in the control of growth, cell cycle and nutrient uptake that is conserved from yeast to humans. To further our understanding of the Rheb pathway, we sought to identify hyperactivating mutations in the Schizosaccharomyces pombe Rheb, Rhb1. Hyperactive forms of Rhb1 were found to result from single amino acid changes at valine-17, serine-21, lysine-120 or asparagine-153. Expression of these mutants confers resistance to canavanine and thialysine, phenotypes which are similar to phenotypes exhibited by cells lacking the Tsc1/Tsc2 complex that negatively regulates Rhb1. The thialysine-resistant phenotype of the hyperactive Rhb1 mutants is suppressed by a second mutation in the effector domain. Purified mutant proteins exhibit dramatically decreased binding of GDP, while their GTP binding is not drastically affected. In addition, some of the mutant proteins show significantly decreased GTPase activities. Thus the hyperactivating mutations are expected to result in an increase in the GTP-bound/GDP-bound ratio of Rhb1. By using the hyperactive mutant, Rhb1(K120R), we have been able to demonstrate that Rhb1 interacts with Tor2, one of the two S. pombe TOR (Target of Rapamycin) proteins. These fission yeast results provide the first evidence for a GTP-dependent association of Rheb with Tor.

The Rheb family of GTP-binding proteins.

Rheb proteins represent a novel and unique family of the Ras superfamily GTP-binding proteins that is conserved from yeast to human. Biochemical studies establish that they bind and hydrolyze GTP. Molecular modeling studies reveal a few structural differences between Rheb and Ras, which may suggest that residues involved in biochemical activities differ between the two G-proteins. The function of Rheb has been studied in a number of organisms that point to the involvement of Rheb in cell growth and cell cycle progression. In addition, studies in fungi suggest that Rheb is involved in arginine uptake. Further studies in Drosophila and mammalian cells have shown that the effects of Rheb on growth and cell cycle progression are mediated by the effect on the insulin/TOR/S6K signaling pathway. These studies have also shown that a complex consisting of the tuberous sclerosis gene products, Tsc1/Tsc2, serves as a GTPase activating protein (GAP) for Rheb, implying Rheb's role in this genetic disorder. Finally, Rheb proteins have been shown to be farnesylated and small molecule inhibitors of protein farnesyltransferase can block the ability of Rheb to activate the TOR/S6K signaling.