Julie O'Neal

An activated receptor tyrosine kinase, TEL/PDGFβR, cooperates with AML1/ETO to induce acute myeloid leukemia in mice

The t(8;21)(q22;q22) translocation, occurring in 40% of patients with acute myeloid leukemia (AML) of the FAB-M2 subtype (AML with maturation), results in expression of the RUNX1-CBF2T1 [AML1-ETO (AE)] fusion oncogene. AML/ETO may contribute to leukemogenesis by interacting with nuclear corepressor complexes that include histone deacetylases, which mediate the repression of target genes. However, expression of AE is not sufficient to transform primary hematopoietic cells or cause disease in animals, suggesting that additional mutations are required. Activating mutations in receptor tyrosine kinases (RTK) are present in at least 30% of patients with AML. To test the hypothesis that activating RTK mutations cooperate with AE to cause leukemia, we transplanted retrovirally transduced murine bone marrow coexpressing TEL-PDGFRB and AE into lethally irradiated syngeneic mice. These mice (19/19, 100%) developed AML resembling M2-AML that was transplantable in secondary recipients. In contrast, control mice coexpressing with TEL-PDGFRB and a DNA-binding-mutant of AE developed a nontransplantable myeloproliferative disease identical to that induced by TEL-PDGFRB alone. We used this unique model of AML to test the efficacy of pharmacological inhibition of histone deacetylase activity by using trichostatin A and suberoylanilide hydroxamic acid alone or in combination with the tyrosine kinase inhibitor, imatinib mesylate. We found that although imatinib prolonged the survival of treated mice, histone deacetylase inhibitors provided no additional survival benefit. These data demonstrate that an activated RTK can cooperate with AE to cause AML in mice, and that this system can be used to evaluate novel therapeutic strategies.

RelB is the NF-κB subunit downstream of NIK responsible for osteoclast differentiation

NF-κB inducing kinase (NIK) is required for osteoclastogenesis in response to pathologic stimuli, and its loss leads to functional blockade of both alternative and classical NF-κB caused by cytoplasmic retention by p100. We now show that deletion of p100 restores the capacity of NIK-deficient osteoclast (OC) precursors to differentiate and normalizes RelB and p65 signaling. Differentiation of NIK−/− precursors is also restored by overexpression of RelB, but not p65. Additionally, RelB−/− precursors fail to form OCs in culture, and this defect is rescued by re-expression of RelB, but not by overexpression of p65. To further support the role of RelB in OCs, we challenged RelB−/− mice with TNF-α in vivo and found a diminished osteoclastogenic response. We then examined tumor-induced osteolysis in both RelB−/− and NIK−/− mice by using the B16 melanoma model. Growth of tumor cells in the bone marrow was similar to WT controls, but the absence of either RelB or NIK completely blocked the tumor-induced loss of trabecular bone. Thus, the alternative NF-κB pathway, culminating in activation of RelB, has a key and specific role in the differentiation of OCs that cannot be compensated for by p65.

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. Mol Cancer Ther; 12(8); 1461–70. ©2013 AACR.

6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27

The control of glucose metabolism and the cell cycle must be coordinated in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are well established regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent allosteric activator of 6-phosphofructo-1-kinase (Pfk-1). PFKFB3 is overexpressed in human cancers, regulated by HIF-1α, Akt and PTEN, and required for the survival and growth of multiple cancer types. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, activates cyclin-dependent kinases (Cdks). In particular, F2,6BP stimulates the Cdk-mediated phosphorylation of the Cip/Kip protein p27 (threonine 187), which in turn results in p27’s ubiquitination and proteasomal degradation. As p27 is a potent suppressor of the G1/S transition and activator of apoptosis, we hypothesized that the known requirement of PFKFB3 for cell cycle progression and prevention of apoptosis may be partly due to the ability of F2,6BP to activate Cdks. In this study, we demonstrate that siRNA silencing of endogenous PFKFB3 inhibits Cdk1 activity, which in turn stabilizes p27 protein levels causing cell cycle arrest at G1/S and increased apoptosis in HeLa cells. Importantly, we demonstrate that the increase in apoptosis and suppression of the G1/S transition caused by siRNA silencing of PFKFB3 expression is reversed by co-siRNA silencing of p27. Taken together with prior publications, these observations support a model whereby PFKFB3 and F2,6BP function not only as regulators of Pfk-1 but also of Cdk1 activity, and therefore serve to couple glucose metabolism with cell proliferation and survival in transformed cells.

Neurobeachin (NBEA) is a target of recurrent interstitial deletions at 13q13 in patients with MGUS and multiple myeloma

OBJECTIVE Chromosome 13 deletions (del[13]), detected by metaphase cytogenetics, predict poor outcomes in multiple myeloma (MM), but the gene(s) responsible have not been conclusively identified. We sought to identify tumor-suppressor genes on chromosome 13 using a novel array comparative genomic hybridization (aCGH) strategy. MATERIALS AND METHODS We identified DNA copy number losses on chromosome 13 using genomic DNA isolated from CD138-enriched bone marrow cells (tumor) from 20 patients with MM, monoclonal gammopathy of undetermined significance, or amyloidosis. We used matched skin biopsy (germline) genomic DNA to control for copy number polymorphisms and a novel aCGH array dedicated to chromosome 13 to map somatic DNA gains and losses at ultra-high resolution (>385,000 probes; median probe spacing 60 bp). We analyzed microarray expression data from an additional 262 patient samples both with and without del[13]. RESULTS Two distinct minimally deleted regions at 13q14 and 13q13 were defined that affected the RB1 and NBEA genes, respectively. RB1 is a canonical tumor suppressor previously implicated in MM. NBEA is implicated in membrane trafficking in neurons, protein kinase A binding, and has no known role in cancer. Noncoding RNAs on chromosome 13 were not affected by interstitial deletions. Both the RB1 and NBEA genes were deleted in 40% of cases (8 of 20; 5 patients with del[13] detected by traditional methods and 3 patients with interstitial deletions detected by aCGH). Forty-one additional MM patient samples were used for complete exonic sequencing of RB1, but no somatic mutations were found. Along with RB1, NBEA gene expression was significantly reduced in cases with del[13]. CONCLUSIONS The NBEA gene at 13q13, and its expression are frequently disrupted in MM. Additional studies are warranted to evaluate the role of NBEA as a novel candidate tumor-suppressor gene.

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

Background: The regulation of glucose metabolism by estradiol is poorly defined. Results: We find that estradiol stimulates glucose metabolism in part by stimulating the production of fructose 2,6-bisphosphate by PFKFB3. Conclusion: PFKFB3 is a downstream target of estradiol required to stimulate glucose metabolism. Significance: Combined targeting of PFKFB3 and the estrogen receptor may prove beneficial to ER+ stage IV breast cancer patients. 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 [14C]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, [14C]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.

Clinical development of cancer therapeutics that target metabolism.

Glucose and glutamine metabolism in cancer cells are markedly elevated relative to non-transformed normal cells. This metabolic reprogramming enables the production of adenosine triphosphate and the anabolic precursors needed for survival, growth and motility. The recent observations that mutant oncogenic proteins and the loss of tumor suppressors activate key metabolic enzymes suggest that selective inhibition of these enzymes may yield effective cancer therapeutics with acceptable toxicities. In support of this concept, pre-clinical studies of small molecule antagonists of several metabolic enzymes in tumor-bearing mice have demonstrated reasonable therapeutic indices. We will review the rationale for targeting metabolic enzymes as a strategy to treat cancer and will detail the results of several recent clinical trials of metabolic inhibitors in advanced cancer patients.

Glucose availability and glycolytic metabolism dictate glycosphingolipid levels.

Cancer therapeutics has seen an emergence and re‐emergence of two metabolic fields in recent years, those of bioactive sphingolipids and glycolytic metabolism. Anaerobic glycolysis and its implications in cancer have been at the forefront of cancer research for over 90 years. More recently, the role of sphingolipids in cancer cell metabolism has gained recognition, notably ceramides essential role in programmed cell death and the role of the glucosylceramide synthase (GCS) in chemotherapeutic resistance. Despite this knowledge, a direct link between these two fields has yet to be definitively drawn. Herein, we show that in a model of highly glycolytic cells, generation of the glycosphingolipid (GSL) glucosylceramide (GlcCer) by GCS was elevated in response to increased glucose availability, while glucose deprivation diminished GSL levels. This effect was likely substrate dependent, independent of both GCS levels and activity. Conversely, leukemia cells with elevated GSLs showed a significant change in GCS activity, but no change in glucose uptake or GCS expression. In a leukemia cell line with elevated GlcCer, treatment with inhibitors of glycolysis or the pentose phosphate pathway (PPP) significantly decreased GlcCer levels. When combined with pre‐clinical inhibitor ABT‐263, this effect was augmented and production of pro‐apoptotic sphingolipid ceramide increased. Taken together, we have shown that there exists a definitive link between glucose metabolism and GSL production, laying the groundwork for connecting two distinct yet essential metabolic fields in cancer research. Furthermore, we have proposed a novel combination therapeutic option targeting two metabolic vulnerabilities for the treatment of leukemia. J. Cell. Biochem. 116: 67–80, 2015.

Collaborative control of cell cycle progression by the RNA exonuclease Dis3 and ras is conserved across species

Dis3 encodes a conserved RNase that degrades or processes all RNA species via an N-terminal PilT N terminus (PIN) domain and C-terminal RNB domain that harbor, respectively, endonuclease activity and 3′–5′ exonuclease activity. In Schizosaccharomyces pombe, dis3 mutations cause chromosome missegregation and failure in mitosis, suggesting dis3 promotes cell division. In humans, apparently hypomorphic dis3 mutations are found recurrently in multiple myeloma, suggesting dis3 opposes cell division. Except for the observation that RNAi-mediated depletion of dis3 function drives larval arrest and reduces tissue growth in Drosophila, the role of dis3 has not been rigorously explored in higher eukaryotic systems. Using the Drosophila system and newly generated dis3 null alleles, we find that absence of dis3 activity inhibits cell division. We uncover a conserved CDK1 phosphorylation site that when phosphorylated inhibits Dis3’s exonuclease, but not endonuclease, activity. Leveraging this information, we show that Dis3’s exonuclease function is required for mitotic cell division: in its absence, cells are delayed in mitosis and exhibit aneuploidy and overcondensed chromosomes. In contrast, we find that modest reduction of dis3 function enhances cell proliferation in the presence of elevated Ras activity, apparently by accelerating cells through G2/M even though each insult by itself delays G2/M. Additionally, we find that dis3 and ras genetically interact in worms and that dis3 can enhance cell proliferation under growth stimulatory conditions in murine B cells. Thus, reduction, but not absence, of dis3 activity can enhance cell proliferation in higher organisms.

Deletion of Rb1 induces both hyperproliferation and cell death in murine germinal center B cells.

The retinoblastoma gene (RB1) has been implicated as a tumor suppressor in multiple myeloma (MM), yet its role remains unclear because in the majority of cases with 13q14 deletions, un-mutated RB1 remains expressed from the retained allele. To explore the role of Rb1 in MM, we examined the functional consequences of single- and double-copy Rb1 loss in germinal center B cells, the cells of origin of MM. We generated mice without Rb1 function in germinal center B cells by crossing Rb1(Flox/Flox) with C-γ-1-Cre (Cγ1) mice expressing the Cre recombinase in class-switched B cells in a p107(-/-) background to prevent p107 from compensating for Rb1 loss (Cγ1-Rb1(F/F)-p107(-/-)). All mice developed normally, but B cells with two copies of Rb1 deleted (Cγ1-Rb1(F/F)-p107(-/-)) exhibited increased proliferation and cell death compared with Cγ1-Rb1(+/+)-p107(-/-) controls ex vivo. In vivo, Cγ1-Rb1(F/F)-p107(-/-) mice had a lower percentage of splenic B220+ cells and reduced numbers of bone marrow antigen-specific secreting cells compared with control mice. Our data indicate that Rb1 loss induces both cell proliferation and death in germinal center B cells. Because no B-cell malignancies developed after 1 year of observation, our data also suggest that Rb1 loss is not sufficient to transform post-germinal center B cells and that additional, specific mutations are likely required to cooperate with Rb1 loss to induce malignant transformation.