Eat, Sleep, Console Approach or Usual Care for Neonatal Opioid Withdrawal.

BACKGROUND: Although clinicians have traditionally used the Finnegan Neonatal Abstinence Scoring Tool to assess the severity of neonatal opioid withdrawal, a newer function-based approach - the Eat, Sleep, Console care approach - is increasing in use. Whether the new approach can safely reduce the time until infants are medically ready for discharge when it is applied broadly across diverse sites is unknown. METHODS: In this cluster-randomized, controlled trial at 26 U.S. hospitals, we enrolled infants with neonatal opioid withdrawal syndrome who had been born at 36 weeks' gestation or more. At a randomly assigned time, hospitals transitioned from usual care that used the Finnegan tool to the Eat, Sleep, Console approach. During a 3-month transition period, staff members at each hospital were trained to use the new approach. The primary outcome was the time from birth until medical readiness for discharge as defined by the trial. Composite safety outcomes that were assessed during the first 3 months of postnatal age included in-hospital safety, unscheduled health care visits, and nonaccidental trauma or death. RESULTS: A total of 1305 infants were enrolled. In an intention-to-treat analysis that included 837 infants who met the trial definition for medical readiness for discharge, the number of days from birth until readiness for hospital discharge was 8.2 in the Eat, Sleep, Console group and 14.9 in the usual-care group (adjusted mean difference, 6.7 days; 95% confidence interval [CI], 4.7 to 8.8), for a rate ratio of 0.55 (95% CI, 0.46 to 0.65; P<0.001). The incidence of adverse outcomes was similar in the two groups. CONCLUSIONS: As compared with usual care, use of the Eat, Sleep, Console care approach significantly decreased the number of days until infants with neonatal opioid withdrawal syndrome were medically ready for discharge, without increasing specified adverse outcomes. (Funded by the Helping End Addiction Long-term (HEAL) Initiative of the National Institutes of Health; ESC-NOW ClinicalTrials.gov number, NCT04057820.).

PELP1/SRC-3-dependent regulation of metabolic PFKFB kinases drives therapy resistant ER+ breast cancer.

Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs frequently exist as a minority population in therapy resistant tumors. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in mammary intraductal (MIND) models. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer including tamoxifen (TamR) and paclitaxel (TaxR) resistant models, murine tumor cells, and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cell populations that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance.

A quality improvement project improving the value of iNO utilization in preterm and term infants.

OBJECTIVE: Inhaled NO (iNO) is used in the NICU for management of hypoxemic respiratory failure. The cost of iNO is significant and does not consistently improve outcomes in infants <34 weeks. PROJECT DESIGN: Our team used The Model for Improvement to design a quality improvement project to utilize iNO for appropriate indications, ensure response to therapy and initiate timely weaning. The project was carried out at a Level IV NICU and successful interventions spread to a smaller Level III NICU. RESULTS: This project demonstrated significant improvement in all measures; total iNO hours per month, average iNO hours per patient, and the percentage of prolonged iNO courses. With an estimated cost of $115/h, the cost per patient for iNO use declined by half from $21,620 to $10,580. CONCLUSIONS: Our team improved the value of iNO utilization at our institution and spread successful interventions to another NICU in our network.

Increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 activity in response to EGFR signaling contributes to non-small cell lung cancer cell survival.

Constitutive activation of the epidermal growth factor receptor (EGFR) because of somatic mutations of the EGFR gene is commonly observed in tumors of non-small cell lung cancer (NSCLC) patients. Consequently, tyrosine kinase inhibitors (TKI) targeting the EGFR are among the most effective therapies for patients with sensitizing EGFR mutations. Clinical responses to the EGFR-targeting TKIs are evaluated through 2-[18F]fluoro-2-deoxy-glucose (18FDG)-PET uptake, which is decreased in patients responding favorably to therapy and is positively correlated with survival. Recent studies have reported that EGFR signaling drives glucose metabolism in NSCLC cells; however, the precise downstream effectors required for this EGFR-driven metabolic effect are largely unknown. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is an essential glycolytic regulator that is consistently overexpressed in lung cancer. Here, we found that PFKFB3 is an essential target of EGFR signaling and that PFKFB3 activation is required for glycolysis stimulation upon EGFR activation. We demonstrate that exposing NSCLC cells harboring either WT or mutated EGFR to EGF rapidly increases PFKFB3 phosphorylation, expression, and activity and that PFKFB3 inhibition markedly reduces the EGF-mediated increase in glycolysis. Furthermore, we found that prolonged NSCLC cell exposure to the TKI erlotinib drives PFKFB3 expression and that chemical PFKFB3 inhibition synergizes with erlotinib in increasing erlotinib's anti-proliferative activity in NSCLC cells. We conclude that PFKFB3 has a key role in mediating glucose metabolism and survival of NSCLC cells in response to EGFR signaling. These results support the potential clinical utility of using PFKFB3 inhibitors in combination with EGFR-TKIs to manage NSCLC.

Vitamin D controls the capacity of human dendritic cells to induce functional regulatory T cells by regulation of glucose metabolism.

Tolerogenic dendritic cells (tolDCs) instruct regulatory T cells (Tregs) to dampen autoimmunity. Active vitamin D3 (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) imprints human monocyte-derived DCs with tolerogenic properties by reprogramming their glucose metabolism. Here we identify the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) as a critical checkpoint and direct transcriptional target of 1α,25(OH)2D3 in determining the tolDC profile. Using tracer metabolomics, we show that PFKFB4 activity is essential for glucose metabolism, especially for glucose oxidation, which is elevated upon 1α,25(OH)2D3 exposure. Pharmacological inhibition of PFKFB4 reversed the 1α,25(OH)2D3-mediated shift in metabolism, DC profile and function, as determined by expression of inhibitory surface markers and secretion of regulatory cytokines and factors. Moreover, PFKFB4 inhibition in 1α,25(OH)2D3-treated DCs blocked their hallmark capacity to induce suppressive Tregs. This work demonstrates that alterations in the bioenergetic metabolism of immune cells are central to the immunomodulatory effects induced by 1α,25(OH)2D3.

Therapeutic targeting of PFKFB3 with a novel glycolytic inhibitor PFK158 promotes lipophagy and chemosensitivity in gynecologic cancers.

Metabolic alterations are increasingly recognized as important novel anti-cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158-induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co-immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158-mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX-resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.

Lactoferrin: A Critical Player in Neonatal Host Defense.

Newborn infants are at a high risk for infection due to an under-developed immune system, and human milk has been shown to exhibit substantial anti-infective properties that serve to bolster neonatal defenses against multiple infections. Lactoferrin is the dominant whey protein in human milk and has been demonstrated to perform a wide array of antimicrobial and immunomodulatory functions and play a critical role in protecting the newborn infant from infection. This review summarizes data describing the structure and important functions performed by lactoferrin in protecting the neonate from infection and contributing to the maturation of the newborn innate and adaptive immune systems. We also briefly discuss clinical trials examining the utility of lactoferrin supplementation in the prevention of sepsis and necrotizing enterocolitis in newborn infants. The data reviewed provide rationale for the continuation of studies to examine the effects of lactoferrin administration on the prevention of sepsis in the neonate.

Potential clinical and immunotherapeutic utility of talimogene laherparepvec for patients with melanoma after disease progression on immune checkpoint inhibitors and BRAF inhibitors.

Talimogene laherparepvec is a genetically modified herpes simplex virus type 1-based oncolytic immunotherapy for the local treatment of unresectable subcutaneous and nodal tumors in patients with melanoma recurrent after initial surgery. We report on two patients with melanoma who, after progression on numerous systemic therapies, derived clinical benefit from talimogene laherparepvec in an expanded-access protocol (ClinicalTrials.gov, NCT02147951). Intralesional talimogene laherparepvec (day 1, ≤4 ml 10 PFU/ml; after 3 weeks, ≤4 ml 10 PFU/ml every 2 weeks) was administered until complete response, no injectable tumors, progressive disease, or intolerance occurred. Patient 1 was 71 years old, had stage IIIB disease, and had previously received granulocyte-macrophage colony-stimulating factor, vemurafenib, metformin, ipilimumab, dabrafenib, trametinib, and pembrolizumab. Patient 2 was 45 years old, had stage IIIC disease, and had previously received nivolumab/ipilimumab combination therapy. There were marked reductions in the number and size of melanoma lesions during treatment with talimogene laherparepvec. Both patients experienced mild-to-moderate nausea and vomiting, which were managed using ondansetron, metoclopramide, and pantoprazole. Both patients completed treatment with talimogene laherparepvec in the expanded-access protocol on 24 November 2015, but received talimogene laherparepvec in clinical practice. Patient 1 continues to receive therapy (>60 weeks); patient 2 experienced a complete response at 23 weeks. Immunohistochemistry of a biopsied dermal metastasis from patient 1 showed a marked infiltration of CD4 and CD8 T cells after 1 year of treatment. Talimogene laherparepvec was active in patients with advanced melanoma with disease progression following multiple previous systemic therapies; no new safety signals were identified.

Inhibition of 6-phosphofructo-2-kinase (PFKFB3) suppresses glucose metabolism and the growth of HER2+ breast cancer.

PURPOSE: Human epidermal growth factor receptor-2 (HER2) has been implicated in the progression of multiple tumor types, including breast cancer, and many downstream effectors of HER2 signaling are primary regulators of cellular metabolism, including Ras and Akt. A key downstream metabolic target of Ras and Akt is the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 isozyme (PFKFB3), whose product, fructose-2,6-bisphosphate (F26BP), is a potent allosteric activator of a rate-limiting enzyme in glycolysis, 6-phosphofructo-1-kinase (PFK-1). We postulate that PFKFB3 may be regulated by HER2 and contribute to HER2-driven tumorigenicity. METHODS: Immunohistochemistry and Kaplan-Meier analysis of HER2+ patient samples investigated the relevance of PFKFB3 in HER2+ breast cancer. In vitro genetic and pharmacological inhibition of PFKFB3 was utilized to determine effects on HER2+ breast cancer cells, while HER2 antagonist treatment assessed the mechanistic regulation on PFKFB3 expression and glucose metabolism. Administration of a PFKFB3 inhibitor in a HER2-driven transgenic breast cancer model evaluated this potential therapeutic approach in vivo. RESULTS: PFKFB3 is elevated in human HER2+ breast cancer and high PFKFB3 transcript correlated with poorer progression-free (PFS) and distant metastatic-free (DFMS) survival. Constitutive HER2 expression led to elevated PFKFB3 expression and increased glucose metabolism, while inhibition of PFKFB3 suppressed glucose uptake, F26BP, glycolysis, and selectively decreased the growth of HER2-expressing breast cancer cells. In addition, treatment with lapatinib, an FDA-approved HER2 inhibitor, decreased PFKFB3 expression and glucose metabolism in HER2+ cells. In vivo administration of a PFKFB3 antagonist significantly suppressed the growth of HER2-driven breast tumors and decreased 18F-2-deoxy-glucose uptake. CONCLUSIONS: Taken together, these data support the potential clinical utility of PFKFB3 inhibitors as chemotherapeutic agents against HER2+ breast cancer.

Targeting the sugar metabolism of tumors with a first-in-class 6-phosphofructo-2-kinase (PFKFB4) inhibitor.

Human tumors exhibit increased glucose uptake and metabolism as a result of high demand for ATP and anabolic substrates and this metabolotype is a negative prognostic indicator for survival. Recent studies have demonstrated that cancer cells from several tissue origins and genetic backgrounds require the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4), a regulatory enzyme that synthesizes an allosteric activator of glycolysis, fructose-2,6-bisphosphate. We report the discovery of a first-in-class PFKFB4 inhibitor, 5-(n-(8-methoxy-4-quinolyl)amino)pentyl nitrate (5MPN), using structure-based virtual computational screening. We find that 5MPN is a selective inhibitor of PFKFB4 that suppresses the glycolysis and proliferation of multiple human cancer cell lines but not non-transformed epithelial cells in vitro. Importantly, 5MPN has high oral bioavailability and per os administration of a non-toxic dose of 5MPN suppresses the glucose metabolism and growth of tumors in mice.

Different thresholds of ZEB1 are required for Ras-mediated tumour initiation and metastasis.

Ras pathway mutation is frequent in carcinomas where it induces expression of the transcriptional repressor ZEB1. Although ZEB1 is classically linked to epithelial-mesenchymal transition and tumour metastasis, it has an emerging second role in generation of cancer-initiating cells. Here we show that Ras induction of ZEB1 is required for tumour initiation in a lung cancer model, and we link this function to repression Pten, whose loss is critical for emergence of cancer-initiating cells. These two roles for ZEB1 in tumour progression can be distinguished by their requirement for different levels of ZEB1. A lower threshold of ZEB1 is sufficient for cancer initiation, whereas further induction is necessary for tumour metastasis.

Fructose-2,6-bisphosphate synthesis by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is required for the glycolytic response to hypoxia and tumor growth.

Fructose-2,6-bisphosphate (F2,6BP) is a shunt product of glycolysis that allosterically activates 6-phosphofructo-1-kinase (PFK-1) resulting in increased glucose uptake and glycolytic flux to lactate. The F2,6BP concentration is dictated by four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) with distinct kinase:phosphatase activities. PFKFB4 is over-expressed in human cancers, induced by hypoxia and required for survival and growth of several cancer cell lines. Although PFKFB4 appears to be a rational target for anti-neoplastic drug development, it is not clear whether its kinase or phosphatase activity is required for cancer cell survival. In this study, we demonstrate that recombinant human PFKFB4 kinase activity is 4.3-fold greater than its phosphatase activity, siRNA and genomic deletion of PFKFB4 decrease F2,6BP, PFKFB4 over-expression increases F2,6BP and selective PFKFB4 inhibition in vivo markedly reduces F2,6BP, glucose uptake and ATP. Last, we find that PFKFB4 is required for cancer cell survival during the metabolic response to hypoxia, presumably to enable glycolytic production of ATP when the electron transport chain is not fully operational. Taken together, our data indicate that the PFKFB4 expressed in multiple transformed cells and tumors functions to synthesize F2,6BP. We predict that pharmacological disruption of the PFKFB4 kinase domain may have clinical utility for the treatment of human cancers.

Estradiol stimulates glucose metabolism via 6-phosphofructo-2-kinase (PFKFB3).

Estradiol (E2) administered to estrogen receptor-positive (ER(+)) breast cancer patients stimulates glucose uptake by tumors. Importantly, this E2-induced metabolic flare is predictive of the clinical effectiveness of anti-estrogens and, as a result, downstream metabolic regulators of E2 are expected to have utility as targets for the development of anti-breast cancer agents. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) control glycolytic flux via their product, fructose-2,6-bisphosphate (F26BP), which activates 6-phosphofructo-1-kinase (PFK-1). We postulated that E2 might promote PFKFB3 expression, resulting in increased F26BP and glucose uptake. We demonstrate that PFKFB3 expression is highest in stage III lymph node metastases relative to normal breast tissues and that exposure of human MCF-7 breast cancer cells to E2 causes a rapid increase in [(14)C]glucose uptake and glycolysis that is coincident with an induction of PFKFB3 mRNA (via ER binding to its promoter), protein expression and the intracellular concentration of its product, F26BP. Importantly, selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor markedly reduces the E2-mediated increase in F26BP, [(14)C]glucose uptake, and glycolysis. Furthermore, co-treatment of MCF-7 cells with the PFKFB3 inhibitor and the anti-estrogen ICI 182,780 synergistically induces apoptotic cell death. These findings demonstrate for the first time that the estrogen receptor directly promotes PFKFB3 mRNA transcription which, in turn, is required for the glucose metabolism and survival of breast cancer cells. Importantly, these results provide essential preclinical information that may allow for the ultimate design of combinatorial trials of PFKFB3 antagonists with anti-estrogen therapies in ER(+) stage IV breast cancer patients.

Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism.

BACKGROUND: Unlike glycolytic enzymes that directly catabolize glucose to pyruvate, the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFBs) control the conversion of fructose-6-phosphate to and from fructose-2,6-bisphosphate, a key regulator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). One family member, PFKFB3, has been shown to be highly expressed and activated in human cancer cells, and derivatives of a PFKFB3 inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), are currently being developed in clinical trials. However, the effectiveness of drugs such as 3PO that target energetic pathways is limited by survival pathways that can be activated by reduced ATP and nutrient uptake. One such pathway is the process of cellular self-catabolism termed autophagy. We hypothesized that the functional glucose starvation induced by inhibition of PFKFB3 in tumor cells would induce autophagy as a pro-survival mechanism and that inhibitors of autophagy could increase the anti-tumor effects of PFKFB3 inhibitors. RESULTS: We found that selective inhibition of PFKFB3 with either siRNA transfection or 3PO in HCT-116 colon adenocarcinoma cells caused a marked decrease in glucose uptake simultaneously with an increase in autophagy based on LC3-II and p62 protein expression, acridine orange fluorescence of acidic vacuoles and electron microscopic detection of autophagosomes. The induction of autophagy caused by PFKFB3 inhibition required an increase in reactive oxygen species since N-acetyl-cysteine blocked both the conversion of LC3-I to LC3-II and the increase in acridine orange fluorescence in acidic vesicles after exposure of HCT-116 cells to 3PO. We speculated that the induction of autophagy might protect cells from the pro-apoptotic effects of 3PO and found that agents that disrupt autophagy, including chloroquine, increased 3PO-induced apoptosis as measured by double staining with Annexin V and propidium iodide in both HCT-116 cells and Lewis lung carcinoma (LLC) cells. Chloroquine also increased the anti-growth effect of 3PO against LLCs in vivo and resulted in an increase in apoptotic cells within the tumors. CONCLUSIONS: We conclude that PFKFB3 inhibitors suppress glucose uptake, which in turn causes an increase in autophagy. The addition of selective inhibitors of autophagy to 3PO and its more potent derivatives may prove useful as rational combinations for the treatment of cancer.

The ZEB1 transcription factor acts in a negative feedback loop with miR200 downstream of Ras and Rb1 to regulate Bmi1 expression.

Ras mutations are frequent in cancer cells where they drive proliferation and resistance to apoptosis. However in primary cells, mutant Ras instead can cause oncogene-induced senescence, a tumor suppressor function linked to repression of the polycomb factor Bmi1, which normally regulates cell cycle inhibitory cyclin-dependent kinase inhibitors (cdki). It is unclear how Ras causes repression of Bmi1 in primary cells to suppress tumor formation while inducing the gene in cancer cells to drive tumor progression. Ras also induces the EMT transcription factor ZEB1 to trigger tumor invasion and metastasis. Beyond its well-documented role in EMT, ZEB1 is important for maintaining repression of cdki. Indeed, heterozygous mutation of ZEB1 is sufficient for elevated cdki expression, leading to premature senescence of primary cells. A similar phenotype is evident with Bmi1 mutation. We show that activation of Rb1 in response to mutant Ras causes dominant repression of ZEB1 in primary cells, but loss of the Rb1 pathway is a hallmark of cancer cells and in the absence of such Rb1 repression Ras induces ZEB1 in cancer cells. ZEB1 represses miR-200 in the context of a mutual repression loop. Because miR-200 represses Bmi1, induction of ZEB1 leads to induction of Bmi1. Rb1 pathway status then dictates the opposing effects of mutant Ras on the ZEB1-miR-200 loop in primary versus cancer cells. This loop not only triggers EMT, surprisingly we show it acts downstream of Ras to regulate Bmi1 expression and thus the critical decision between oncogene-induced senescence and tumor initiation.

Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis.

Strategies targeting pathological angiogenesis have focused primarily on blocking vascular endothelial growth factor (VEGF), but resistance and insufficient efficacy limit their success, mandating alternative antiangiogenic strategies. We recently provided genetic evidence that the glycolytic activator phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) promotes vessel formation but did not explore the antiangiogenic therapeutic potential of PFKFB3 blockade. Here, we show that blockade of PFKFB3 by the small molecule 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) reduced vessel sprouting in endothelial cell (EC) spheroids, zebrafish embryos, and the postnatal mouse retina by inhibiting EC proliferation and migration. 3PO also suppressed vascular hyperbranching induced by inhibition of Notch or VEGF receptor 1 (VEGFR1) and amplified the antiangiogenic effect of VEGF blockade. Although 3PO reduced glycolysis only partially and transiently in vivo, this sufficed to decrease pathological neovascularization in ocular and inflammatory models. These insights may offer therapeutic antiangiogenic opportunities.

Rb1 family mutation is sufficient for sarcoma initiation.

It is thought that genomic instability precipitated by Rb1 pathway loss rapidly triggers additional cancer gene mutations, accounting for rapid tumour onset following Rb1 mutation. However, recent whole-genome sequencing of retinoblastomas demonstrated little genomic instability, but instead suggested rapid epigenetic activation of cancer genes. These results raise the possibility that loss of the Rb1 pathway, which is a hallmark of cancers, might be sufficient for cancer initiation. Yet, mutation of the Rb1 family or inactivation of the Rb1 pathway in primary cells has proven insufficient for tumour initiation. Here we demonstrate that traditional nude mouse assays impose an artificial anoikis and proliferation barrier that prevents Rb1 family mutant fibroblasts from initiating tumours. By circumventing this barrier, we show that primary fibroblasts with only an Rb1 family mutation efficiently form sarcomas in nude mice, and a Ras-ZEB1-Akt pathway then causes transition of these tumours to an invasive phenotype.

Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

Targeting 6-phosphofructo-2-kinase (PFKFB3) as a therapeutic strategy against cancer.

In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a key regulator of glycolysis, 6-phosphofructo-2-kinase (PFKFB3). This enzyme synthesizes fructose 2,6-bisphosphate (F26BP), which is an activator of 6-phosphofructo-1-kinase, a key step of glycolysis. Previously, a weak competitive inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), was found to reduce the glucose metabolism and proliferation of cancer cells. We have synthesized 73 derivatives of 3PO and screened each compound for activity against recombinant PFKFB3. One small molecule, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15), was selected for further preclinical evaluation of its pharmacokinetic, antimetabolic, and antineoplastic properties in vitro and in vivo. We found that PFK15 causes a rapid induction of apoptosis in transformed cells, has adequate pharmacokinetic properties, suppresses the glucose uptake and growth of Lewis lung carcinomas in syngeneic mice, and yields antitumor effects in three human xenograft models of cancer in athymic mice that are comparable to U.S. Food and Drug Administration-approved chemotherapeutic agents. As a result of this study, a synthetic derivative and formulation of PFK15 has undergone investigational new drug (IND)-enabling toxicology and safety studies. A phase I clinical trial of its efficacy in advanced cancer patients will initiate in 2013 and we anticipate that this new class of antimetabolic agents will yield acceptable therapeutic indices and prove to be synergistic with agents that disrupt neoplastic signaling.