Metabolic plasticity in CLL: adaptation to the hypoxic niche.

Metabolic transformation in cancer is increasingly well understood. However, little is known about the metabolic responses of cancer cells that permit their survival in different microenvironments. We have used a nuclear magnetic resonance based approach to monitor metabolism in living primary chronic lymphoid leukemia (CLL) cells and to interrogate their real-time metabolic responses to hypoxia. Our studies demonstrate considerable metabolic plasticity in CLL cells. Despite being in oxygenated blood, circulating CLL cells are primed for hypoxia as measured by constitutively low level hypoxia-inducible factor (HIF-1α) activity and modest lactate production from glycolysis. Upon entry to hypoxia we observed rapid upregulation of metabolic rates. CLL cells that had adapted to hypoxia returned to the 'primed' state when re-oxygenated and again showed the same adaptive response upon secondary exposure to hypoxia. We also observed HIF-1α independent differential utilization of pyruvate in oxygenated and hypoxic conditions. When oxygenated, CLL cells released pyruvate, but in hypoxia imported pyruvate to protect against hypoxia-associated oxidative stress. Finally, we identified a marked association of slower resting glucose and glutamine consumption, and lower alanine and lactate production with Binet A0 stage samples indicating that CLL may be divided into tumors with higher and lower metabolic states that reflect disease stage.

Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production.

Despite recent therapeutic advancements, multiple myeloma (MM) remains incurable and new therapies are needed, especially for the treatment of elderly and relapsed/refractory patients. We have screened a panel of 100 off-patent licensed oral drugs for anti-myeloma activity and identified niclosamide, an anti-helminthic. Niclosamide, at clinically achievable non-toxic concentrations, killed MM cell lines and primary MM cells as efficiently as or better than standard chemotherapy and existing anti-myeloma drugs individually or in combinations, with little impact on normal donor cells. Cell death was associated with markers of both apoptosis and autophagy. Importantly, niclosamide rapidly reduced free light chain (FLC) production by MM cell lines and primary MM. FLCs are a major cause of renal impairment in MM patients and light chain amyloid and FLC reduction is associated with reversal of tissue damage. Our data indicate that niclosamides anti-MM activity was mediated through the mitochondria with rapid loss of mitochondrial membrane potential, uncoupling of oxidative phosphorylation and production of mitochondrial superoxide. Niclosamide also modulated the nuclear factor-κB and STAT3 pathways in MM cells. In conclusion, our data indicate that MM cells can be selectively targeted using niclosamide while also reducing FLC secretion. Importantly, niclosamide is widely used at these concentrations with minimal toxicity.

Treatment of primary CLL cells with bezafibrate and medroxyprogesterone acetate induces apoptosis and represses the pro-proliferative signal of CD40-ligand, in part through increased 15dDelta12,14,PGJ2.

B-cell chronic lymphocytic leukemia (CLL), the most common leukemia in older adults, remains largely incurable and novel treatments are urgently required. We previously reported powerful pro-apoptotic actions of bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) against Burkitts lymphoma cells. Here, we demonstrate that BEZ and MPA individually, and more potently when combined (BEZ+MPA), induce apoptosis of unsorted and CD19(+ve)-selected CLL cells and abrogate the pro-proliferative activity of CD40(L). This action was tumor cell specific, as the drugs had little impact on normal donor cells. The antiproliferative actions of BEZ+MPA were associated with the generation of reactive oxygen species (ROS), and the proapoptotic actions were associated with the generation of both ROS and mitochondrial superoxide (MSO). BEZ increased prostaglandin D(2) (PGD(2)) synthesis by CLL cells, and treatment with PGD(2) and its antineoplastic derivative 15dDelta(12,14,)PGJ(2) recapitulated BEZ-induced antiproliferative and proapoptotic actions. The PGD(2) receptor antagonist, BW868C, did not block BEZ or PGD(2) activity against CLL cells. The potency of BEZ+MPA against CLL cells mirrored that of chlorambucil, and BEZ+MPA combined with chlorambucil was more potent than either treatment alone. Given the known safety profiles of BEZ and MPA, our data warrant further investigation of their potential as novel therapy for CLL.

Histone deacetylases in acute myeloid leukaemia show a distinctive pattern of expression that changes selectively in response to deacetylase inhibitors.

Histone deacetylase inhibitors (HDIs) are a new class of drugs with significant antileukemic activity. To explore mechanisms of disease-specific HDI activity in acute myeloid leukaemia (AML), we have characterised expression of all 18 members of the histone deacetylase family in primary AML blasts and in four control cell types, namely CD34+ progenitors from umbilical cord, either quiescent or cycling (post-culture), cycling CD34+ progenitors from GCSF-stimulated adult donors and peripheral blood mononuclear cells. Only SIRT1 was consistently overexpressed (>2 fold) in AML samples compared with all controls, while HDAC6 was overexpressed relative to adult, but not neo-natal cells. HDAC5 and SIRT4 were consistently underexpressed. AML blasts and cell lines, exposed to HDIs in culture, showed both histone hyperacetylation and, unexpectedly, specific hypermethylation of H3 lysine 4. Such treatment also modulated the pattern of HDAC expression, with strong induction of HDAC11 in all myeloid cells tested and with all inhibitors (valproate, butyrate, TSA, SAHA), and lesser, more selective, induction of HDAC9 and SIRT4. The distinct pattern of HDAC expression in AML and its response to HDIs is of relevance to the development of HDI-based therapeutic strategies and may contribute to observed patterns of clinical response and development of drug resistance.

The in vitro effects of triiodothyronine on epidermal growth factor-induced trophoblast function.

The development of the human placenta involves a complex process of tightly regulated proliferation and invasion by extravillous trophoblast into the uterine decidua. Inadequate placentation is a feature of intrauterine growth restriction and other gestational pathology. There is some evidence that T(3) plays a role in the regulation of these processes and that T(3) may act synergistically with epidermal growth factor (EGF). The aim of this study was to define the expression of thyroid hormone receptors in extravillous trophoblast, elucidate the effects of T(3) on both proliferation and differentiation of human trophoblast cells of varying origins, and define the potential interaction between EGF and T(3) on these processes. Using immunohistochemistry, specific thyroid hormone receptor isoforms were localized in extravillous trophoblast in first- and second-trimester placental bed biopsies, indicating potential sensitivity to T(3). In studies of human trophoblast-derived cell lines and primary cultures of cytotrophoblast cells in vitro, T(3) and EGF exerted an antiproliferative effect on an extravillous-like cell line (SGHPL-4) but stimulated proliferation in JEG-3 choriocarcinoma cells. EGF enhanced survival of nonproliferative term primary cytotrophoblast cells and significantly enhanced invasion of fibrin gels by SGHPL-4 cells, an effect attenuated by T(3). Both T(3) and EGF also significantly enhanced SGHPL-4 motility. These results suggest that EGF and T(3) may act synergistically to regulate both proliferation and differentiated function of human trophoblast.

PTTG's C-terminal PXXP motifs modulate critical cellular processes in vitro.

Human pituitary tumor-transforming gene (PTTG), known also as securin, is a multifunctional protein implicated in the control of mitosis and the pathogenesis of thyroid, colon, oesophageal and other tumour types. Critical to PTTG function is a C-terminal double PXXP motif, forming a putative SH3-interacting domain and housing the gene's sole reported phosphorylation site. The exact role of phosphorylation and PXXP structure in the modulation of PTTG action in vitro remains poorly understood. We therefore examined the mitotic, transformation, proliferation and transactivation function of the C-terminal PXXP motifs of human PTTG. Live-cell imaging studies using an EGFP-PTTG construct indicated that PTTG's regulation of mitosis is retained regardless of phosphorylation status. Colony-formation assays demonstrated that phosphorylation of PTTG may act as a potent inhibitor of cell transformation. In proliferation assays, NIH-3T3 cells stable transfected and overexpressing mutations preventing PTTG phosphorylation (Phos-) showed significantly increased [3H]thymidine incorporation compared with WT, whereas mutants mimicking constitutive phosphorylation of PTTG (Phos+) exhibited reduced cell proliferation. We demonstrated that PTTG transactivation of FGF-2 in primary thyroid and PTTG-null cell lines was not affected by PTTG phosphorylation but was prevented by a mutant disrupting the PXXP motifs (SH3-). Taken together, our data suggest that PXXP structure and phosphorylation are likely to exert independent and critical influences upon PTTG's diverse actions in vitro.