• T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells.

      Hombach, Andreas A; Schildgen, Verena; Heuser, Claudia; Finnern, Ricarda; Gilham, David E; Abken, Hinrich; Klinik I für Innere Medizin, Tumorgenetik, Kliniken der Universität zu Köln and Zentrum für Molekulare Medizin Köln, Josef-Stelzmann Strasse 9, Köln, Germany. (2007-04-01)
      Recombinant TCRs confer specificity to T cells and trigger their activation. Receptors with Ab-derived binding domains have the advantages of MHC-independent Ag recognition and of targeting a variety of chemically different molecules. We explored the impact of the position of a defined epitope within the target molecule on the efficacy of receptor-mediated T cell activation. T cells were grafted with recombinant immunoreceptors that recognize either the membrane distal N or the proximal A3 domain of carcinoembryonic Ag (CEA). Upon binding to isolated, solid-phase immobilized CEA, receptor-mediated T cell activation correlates with the binding efficiency, irrespectively, of the epitope position. Upon binding to CEA expressed on the cell membrane, in contrast, the A3 epitope mediates more efficiently T cell activation than the N epitope, although the N epitope is bound with higher affinity. The CEA N epitope when expressed in a more membrane proximal position, however, activated receptor grafted T cells with higher efficiency than in the distal position. The position of the targeted epitope within the molecule obviously has major impact on the efficacy of T cell activation independently of the binding efficiency of the immunoreceptor.
    • T cell receptor gamma gene status of human alpha/beta+ and gamma/delta+ T cell clones: absence of V9JP rearrangements in alpha/beta+ clones is not a result of a lack of rearrangements involving more 5' J gamma segments.

      Christmas, Stephen E; Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital & Holt Radium Institute, Manchester, United Kingdom. (1989-11)
      T cell receptor (TCR) gamma gene rearrangements were examined in panels of human T cell clones expressing TCR alpha/beta or gamma/delta heterodimers. Over half of the alpha/beta+ clones had both chromosomes rearranged to C gamma 2 but this was the case for only 20% of the gamma/delta+ clones. While more than half of the gamma/delta+ clones showed a V9JP rearrangement, this configuration was absent from all 49 alpha/beta+ clones analysed. However, this was not a result of all rearrangements being to the more 3' J gamma genes as 11 alpha/beta+ clones had rearrangement(s) to JP1, the most 5' J gamma gene segment. Both alpha/beta+ and gamma/delta+ clones showed a similar pattern of V gamma gene usage in rearrangements to J gamma 1 or J gamma 2 with a lower proportion of the more 3' genes being rearranged to J gamma 2 than for the more 5' genes. Several alpha/beta+ and several gamma/delta+ clones had noncoordinate patterns of rearrangement involving both C gamma 1 and C gamma 2. Eleven out of fourteen CD8+ clones tested had both chromosomes rearranged to C gamma 2 whereas all clones derived from CD4-8- cells and having unconventional phenotypes (CD4-8- or CD4+8+) had at least one C gamma 1 rearrangement. Twelve out of twenty-seven CD4+ clones also had this pattern, suggesting that CD4-8+ clones had a tendency to utilize more 3' J gamma gene segments than CD4+ clones. There was some evidence for interdonor variation in the proportions of TCR gamma rearrangements to C gamma 1 or C gamma 2 in alpha/beta+ clones as well as gamma/delta+ clones. The results illustrate the unique nature of the V9JP rearrangement in gamma/delta+ clones and the possible use of a sequential mechanism of TCR gamma gene rearrangements during T cell differentiation is discussed.
    • T cell-based immunotherapy of metastatic renal cell carcinoma: modest success and future perspective.

      Shablak, Alaaeldin; Hawkins, Robert E; Rothwell, Dominic G; Elkord, Eyad; Department of Medical Oncology, School of Cancer, Enabling Sciences and Technology, The University of Manchester, Manchester Academic Health Science Centre, The Christie NHS Foundation Trust, Manchester, United Kingdom. (2009-11-01)
      Metastatic renal cell carcinoma (MRCC) remains a challenging malignancy to treat. Cancer immunotherapies have been extensively explored in melanoma and RCC as they poorly respond to conventional cytotoxic agents but show responses to a variety of immunologic agents. The recent considerable success of T cell-based immunotherapy in melanoma warrants further efforts to apply this treatment to other cancers including MRCC. Although RCC is an immunosensitive cancer, similar attempts in MRCC have shown a very limited success. In this review, we summarize the clinical data on T cell-based immunotherapies for MRCC showing the modest success that has been achieved to date. More importantly, we discuss potential strategies for improving its efficacy for the treatment of MRCC in light of the important achievements for treating metastatic melanoma. In particular, the growing evidence of success by combining expanded tumor-infiltrating lymphocytes with lymphodepletion merits investigation in MRCC. Identifying new RCC-associated antigens, optimized methods, and conditions for detection, isolation, and/or modification and expansion of tumor-specific T cells are all important strategies to be pursued for improving T cell-based immunotherapy of MRCC.
    • T lymphocytes isolated from patients with advanced colorectal cancer are suitable for gene immunotherapy approaches.

      Sheen, Aali J; Sherlock, David J; Irlam, Joely J; Hawkins, Robert E; Gilham, David E; Cancer Research UK Department of Medical Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, University of Manchester, UK. (2003-04-07)
      Despite improvements in treatment, the 5-year survival for metastatic colorectal cancer remains poor. Novel approaches such as gene immunotherapy are being investigated to improve treatment. Retroviral gene transfer methods have been shown to transduce primary human T lymphocytes effectively resulting in the expression of therapeutic genes. However, a number of defects have been identified in T lymphocytes isolated from patients bearing tumour, which may have critical implications for the development of gene-targeted T cells as an anticancer therapy. To address this issue, primary T lymphocytes were isolated from patients with advanced colorectal cancer and tested for their ability to be transduced and to express subsequently a chimeric immune receptor consisting of a single-chain antibody fragment antigen-binding moiety specific for carcinoembryonic antigen (CEA) fused to the T cell receptor (TCR) CD3zeta chain. In 10 out of 10 patients, T lymphocytes were transduced, expanded in the absence of selection and tested for functional activity against CEA-expressing tumour cells. In each case, functional-specific cytotoxic activity was observed. Negligible activity was found in control cultures. This study highlights the feasibility of patient-derived T lymphocytes as a source of immune cells for autologous gene immunotherapy approaches.
    • T regulatory cells in cancer: recent advances and therapeutic potential.

      Elkord, Eyad; Alcantar-Orozco, E M; Dovedi, S J; Tran, D Q; Hawkins, Robert E; Gilham, David E; University of Salford, School of Environment and Life Sciences, Centre for Biochemistry, Drug Design and Cancer Research, Salford, UK. e.elkord@salford.ac.uk (2010-11)
      IMPORTANCE OF THE FIELD: The active suppression of immune responses against tumor is a major barrier to the likely success of cancer immunotherapy. There is now compelling evidence implicating T regulatory cells (Tregs) as being key players driving immune suppression. Elevated frequencies of Tregs within the peripheral circulation and tumor microenvironment of cancer patients correlate with poor prognosis and reduced survival. Understanding the mechanism of Treg elevation is critical for the development of new approaches aiming to modulate the frequency and function of Tregs to enhance the efficacy of cancer immune-based therapies. AREAS COVERED IN THIS REVIEW: This review focuses on current knowledge concerning Tregs in cancer and discusses putative mechanisms which underlie the expansion of Tregs in cancer patients. Additionally, we review current strategies to deplete/suppress Treg activity, the limitations of these strategies and future perspective for improving their efficacy. WHAT THE READER WILL GAIN: An insight of the current aspects concerning Treg subsets in cancer and an overview of recent advances in the identification of Treg-specific markers, in addition to the potential strategies to target Tregs for enhancing antitumor immunity. TAKE HOME MESSAGE: Mechanisms by which Treg functions modulate the immune response to tumors are becoming further understood. However, specific markers to tumor-specific/induced Tregs are yet to be clearly identified, which is a major limitation in optimizing strategies to specifically target Tregs in cancer. Despite this, strategies aimed at modulating Tregs in patients are providing some early encouraging results supporting the overall concept and indicating that further studies are clearly warranted.
    • T-cell receptor beta gene rearrangements in clones derived from human CD4-8- cells expressing natural killer cell activity.

      Christmas, Stephen E; Moore, M; Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, U.K. (1988-12)
      Clones derived from highly purified human peripheral blood Leu 19+ cells in the presence of phytohaemagglutinin (PHA) and interleukin-2 (IL-2) expressed cytotoxic activity against natural killer (NK)-resistant as well as NK-sensitive targets. All 66 clones analysed had a germ line configuration of T-cell receptor (TCR) beta genes and 38/40 also had unrearranged TCR gamma genes. The two exceptions were both CD3+ clones, but these did not have a cytotoxic repertoire noticeably different from CD3- clones without TCR gamma gene rearrangements. Clones were also obtained from highly purified CD4-8- cells, most of which were also cytotoxic for NK-resistant and NK-sensitive targets. About 90% of these clones were CD3+ but only around 50% remained negative for CD4 and CD8 while a significant number (12.7%) were positive for both CD4 and CD8. All clones analysed had rearranged TCR gamma genes and most had also rearranged TCR beta genes, including 20/25 of the clones which were CD3+4-8-. Many of the clones showed two rearrangements of TCR beta genes, and 3/4 CD3- clones had rearranged TCR beta as well as TCR gamma genes. There was no correlation between cytotoxic activity and TCR gene status or phenotype of these CD4-8- derived clones, except that clones which were Leu 19+ tended to have higher cytotoxic activity against NK-sensitive and NK-resistant targets than Leu 19-clones. The results strongly indicate that TCR beta and gamma gene products are not involved in the cytotoxicity mediated by these clones. They also suggest that some CD4-8- cells may be capable of limited differentiation in vitro.
    • T-cells from HLA-B*57:01+ human subjects are activated with abacavir through two independent pathways and induce cell death by multiple mechanisms.

      Bell, C; Faulkner, L; Martinsson, K; Farrell, J; Alfirevic, A; Tugwood, Jonathan D; Pirmohamed, M; Naisbitt, D; Park, K; MRC Centre for Drug Safety Science, Department of Pharmacology, University of Liverpool , Sherrington Building, Ashton Street, Liverpool L69 3GE, England. (2013-05-20)
      Susceptibility to abacavir hypersensitivity has been attributed to possession of the specific human leukocyte antigen allele HLA-B*57:01. HLA-B*57:01-restricted activation of CD8+ T-cells provides a link between the genetic association and the iatrogenic disease. The objectives of this study were to characterize the functionality of drug-responsive CD8+ T-cell clones generated from HLA-B*57:01+ drug-naive subjects and to explore the relationship between abacavir accumulation in antigen presenting cells and the T-cell response. Seventy-four CD8+ clones expressing different Vβ receptors were shown to proliferate and kill target cells via different mechanisms when exposed to abacavir. Certain clones were activated with abacavir in the absence of antigen presenting cells. Analysis of the remaining clones revealed two pathways of drug-dependent T-cell activation. Overnight incubation of antigen presenting cells with abacavir, followed by repeated washing to remove soluble drug, activated approximately 50% of the clones, and the response was blocked by glutaraldehyde fixation. In contrast, a 1 h antigen presenting cell pulse did not activate any of the clones. Accumulation of abacavir in antigen presenting cells was rapid (less than 1 h), and the intracellular concentrations were maintained for 16 h. However, intracellular abacavir was not detectable by mass spectrometry after pulsing. These data suggest that T-cells can be activated by abacavir through a direct interaction with surface and intracellular major histocompatibility complex (MHC) molecules. With the former, abacavir seemingly participates in the MHC T-cell receptor binding interaction. In contrast, the latter pathway likely involves MHC binding peptides displayed as a consequence of abacavir exposure, but not abacavir itself.
    • Tanning in human skin types II and III offers modest photoprotection against erythema.

      Sheehan, J M; Potten, Christopher S; Young, A R; Department of Photobiology, St John's Institute of Dermatology, St Thomas's Hospital, London, UK. j.sheehan@umds.ac.uk (1998-10)
      We have investigated the photoprotective properties of tanning using erythema as an endpoint. Previously unexposed buttock skin sites of 16 young, healthy adults (8 skin type II, and 8 skin type III) were exposed daily (Mon-Fri) for 2 weeks to 0.5 and 0.75 minimal erythema doses (MED) of solar-simulated radiation (SSR). Erythema and melanin levels were assessed daily both visually and quantitatively using a reflectance device. One week after the last tanning treatment, MED reassessments were made on pretreated sites and on adjacent nontreated sites, including sites from which stratum corneum was removed by tape stripping. Compared to skin type II, similar daily SSR treatments produced less erythema and more evident tanning in skin types III. Independent of skin type, all volunteers showed an increased MED value when assessed on the 0.75 MED- and 0.5 MED-treated sites compared to the MED value assessed on adjacent untreated sites. We express any increase in MED as an induced protection factor (IPF), i.e. (MED post-tan/MED pre-tan). Our data show mean IPF of 1.4 and of 2.1 in the 0.5 and 0.75 MED-treated sites respectively, in skin types II. Similar values were obtained in skin types III with IPF of 1.5 and 2.3 for the 0.5 and 0.75 MED-treated sites, respectively. In all cases, removal of the stratum corneum lowered the IPF by about 20%. Our results show that SSR-induced melanogenesis, whether in skin type II or III, offers only moderate protection against erythema and suggest that SSR-induced stratum corneum thickening affords less photoprotection than tanning.
    • Tanning salon exposure and molecular alterations.

      Whitmore, S Elizabeth; Morison, Warwick L; Potten, Christopher S; Chadwick, Caroline A; Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. (2001-05)
      BACKGROUND: Human studies of the short-term cellular effects of tanning salon exposures are lacking. Findings of such studies may prove extremely helpful in educating consumers considering or currently attending tanning salons. OBJECTIVE: Our purpose was to determine whether tanning salon exposure causes DNA alterations and p53 protein expression in epidermal keratinocytes and/or circulating peripheral lymphocytes. METHODS: Eleven subjects received 10 full-body tanning salon exposures over a 2-week period. UV-induced DNA cyclobutane pyrimidine dimers and p53 protein expression were examined, comparing pretreatment peripheral blood lymphocytes and epidermal biopsy specimens with analogous specimens obtained after the 10 tanning salon exposures. RESULTS: Cyclobutane pyrimidine dimers in DNA and p53 protein expression were detected in epidermal keratinocytes, but were absent in lymphocytes. CONCLUSION: Similar to outdoor sun exposure, short-term recreational tanning salon exposure causes molecular alterations believed essential in the development of skin cancer.
    • Target cells for the cytotoxic effects of carcinogens in the murine small bowel.

      Li, Y Q; Fan, Chun-Yang; O'Connor, Peter J; Winton, D J; Potten, Christopher S; CRC Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1992-03)
      Two direct-acting mutagens, N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU), and two agents requiring metabolic activation, 1-2-dimethylhydrazine (DMH) and N-nitrosodimethylamine (NDMA), were administered i.p. to mice. Sections of crypts of the small intestine were assayed for acute histological cell death at various times up to 12 h after treatment. Dead or dying cells exhibited the typical light microscopic morphological features of apoptosis. The incidence of apoptosis at each cell position along the side of longitudinal crypt sections was recorded and frequency plots of the incidence against cell position were determined. NEU (50 mg/kg) produced the highest incidence of cell death but this was closely followed by NDMA (50 mg/kg) and NMU (200 mg/kg). DMH (40 or 80 mg/kg) was the least cytotoxic but even here significantly elevated levels of cell death were observed. The highest incidence of cell death occurred 4-5 h after treatment with NEU, NMU and DMH and at 6 h after NDMA. The data obtained at 4 h after NEU suggest that approximately 22 cells out of a total crypt population of 250 cells are killed, but that for some cell positions near the crypt base (stem cell regions) up to 24% of the cells may be killed. Analysis of the changing shape of the frequency plots with time after treatment enabled the target cell position in the crypts for cytotoxicity to be estimated. This was at cell position 4 for NEU, NMU and DMH and at cell position 5 for NDMA. The stem cells in the crypts are believed to be located at the fourth cell position and hence at least NEU, NMU and DMH are targeting the stem cells with some specificity.
    • Targeted cytokine delivery to neuroblastoma.

      Dehal, P K; Embleton, Jim; Kemshead, John T; Hawkins, Robert E; Medical Oncology, Paterson Institute for Cancer Research, Wilmslow Road, Withington, Manchester M20 4BX, UK. (2002-08)
      The aim of this study was to construct a fusion protein from the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) and a single-chain Fv fragment (scFv D29) and to investigate its potential to activate cells of the immune system against neuroblastoma cells expressing neural cell adhesion molecule (NCAM). Mammalian cell expression of the scFv D29-GM-CSF fusion protein was compared using a number of vectors, including retroviral and adenoviral vectors. The resultant fusion protein, expressed by HeLa cells, was found by ELISA to bind immobilized recombinant NCAM. Moreover, FACS analysis confirmed binding to the human neuroblastoma cell line SKNBE and a murine neuroblastoma cell line engineered to express the glycosylphosphatidylinositol form of human NCAM (N2A-rKNIE). The fusion protein was also found to stimulate the proliferation of the FDC-P1 haemopoietic cell line, which is dependent on GM-CSF (or interleukin 3) for continued growth. In vitro clonogenic assays indicated that scFv-GM-CSF could selectively induce growth inhibition of SKNBE cells by murine lymphoid cells.
    • Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,N6-ethenoadenine and hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine.

      Hang, B; Singer, B; Margison, Geoffrey P; Elder, Rhoderick H; Donner Laboratory, Lawrence Berkeley National Laboratory, University of California, 94720, USA. (1997-11-25)
      It has previously been reported that 1,N6-ethenoadenine (epsilonA), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N4-ethenocytosine (epsilonC), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing epsilonA and Hx, but retained wild-type levels of activity for epsilonC and 8-oxoG. These findings indicate that (i) the previously identified epsilonA-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; (ii) the two structurally closely related mutagenic adducts epsilonA and epsilonC are repaired by separate gene products; and (iii) APNG does not contribute detectably to the repair of 8-oxoG.
    • [Targeted drugs a chance for a better cure]

      Bergh, Jonas; Jonsson, Bjorn; Manchester University, Christie Hospital, Manchester, England. jonas.bergh@ki.se (2010)
    • Targeted genetic dependency screen facilitates identification of actionable mutations in FGFR4, MAP3K9, and PAK5 in lung cancer.

      Fawdar, Shameem; Trotter, Eleanor W; Li, Yaoyong; Stephenson, Natalie L; Hanke, Franziska; Marusiak, Anna A; Edwards, Zoe C; Lentile, Sara; Waszkowycz, Bohdan; Miller, Crispin J; et al. (2013-07-23)
      Approximately 70% of patients with non-small-cell lung cancer present with late-stage disease and have limited treatment options, so there is a pressing need to develop efficacious targeted therapies for these patients. This remains a major challenge as the underlying genetic causes of ∼50% of non-small-cell lung cancers remain unknown. Here we demonstrate that a targeted genetic dependency screen is an efficient approach to identify somatic cancer alterations that are functionally important. By using this approach, we have identified three kinases with gain-of-function mutations in lung cancer, namely FGFR4, MAP3K9, and PAK5. Mutations in these kinases are activating toward the ERK pathway, and targeted depletion of the mutated kinases inhibits proliferation, suppresses constitutive activation of downstream signaling pathways, and results in specific killing of the lung cancer cells. Genomic profiling of patients with lung cancer is ushering in an era of personalized medicine; however, lack of actionable mutations presents a significant hurdle. Our study indicates that targeted genetic dependency screens will be an effective strategy to elucidate somatic variants that are essential for lung cancer cell viability.
    • Targeted immunotherapy of cancer with CAR T cells: achievements and challenges.

      Lipowska-Bhalla, Grazyna; Gilham, David E; Hawkins, Robert E; Rothwell, Dominic G; Clinical and Experimental Immunotherapy Group, School of Cancer and Enabling Sciences, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK. (2012-07)
      The adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a relatively new but promising approach in the field of cancer immunotherapy. This therapeutic strategy is based on the genetic reprogramming of T cells with an artificial immune receptor that redirects them against targets on malignant cells and enables their destruction by exerting T cell effector functions. There has been an explosion of interest in the use of CAR T cells as an immunotherapy for cancer. In the pre-clinical setting, there has been a considerable focus upon optimizing the structural and signaling potency of the CAR while advances in bio-processing technology now mean that the clinical testing of these gene-modified T cells has become a reality. This review will summarize the concept of CAR-based immunotherapy and recent clinical trial activity and will further discuss some of the likely future challenges facing CAR-modified T cell therapies.
    • Targeted in vivo infection with a retroviral vector carrying the interleukin-3 (multi-CSF) gene leads to immortalization and leukemic transformation of primitive hematopoietic progenitor cells.

      Just, Ursula; Katsuno, M; Stocking, C; Spooncer, Elaine; Dexter, T Michael; Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1993)
      To measure the effect of endogenous IL-3 (Multi-CSF) expression on hematopoietic cells in vivo, we have infected several kinds of hematopoietic cell populations with retroviral vectors carrying the IL-3 gene (M3MuV) in vitro and injected the virus-producing cells into mice to "target" the virus to sites of hematopoiesis. Mast cell lines (Elut cells) or multipotent cell lines (FDC-Pmix) were infected with MPSV-based replication defective retroviral vectors carrying either the neomycin resistance gene alone (M3neoV) or the neomycin gene plus the IL-3 gene (M3MuV). These cell lines produced infective retroviral particles consisting of the replication defective vectors and helper virus constitutively produced by the target cell populations. Irradiated and non-irradiated virus-producing Elut cells and the virus-producing FDC-Pmix cells were transplanted into syngeneic mice to "target" virus infection to the sites of hemopoiesis. Control mice injected with M3neoV-producing cells did not develop a disease up to 6 months following transplantation, whereas mice injected with M3MuV-producing cells developed a myeloproliferative disease within 3 months. Hematopoietic cell lines were rescued from diseased and control mice. In all cases these cell lines were of host origin. Cell lines derived from control mice were of basophil/mast cell morphology only, and required IL-3 for their continued proliferation (similar to cell lines derived from uninfected animals), whereas the cell lines generated from spleen and bone marrow cells of host mice with myeloproliferative disease carried the M3MuV vector, were G418 resistant and IL-3 independent. The biologic properties of M3MuV infected host derived cell lines varied considerably. Some were multipotential and could be induced to differentiate in response to stromal cells and serum factors, others were more restricted to the granulocyte/macrophage lineage but were also differentiation inducible, and some were blocked in differentiation at the myeloblast/promyelocyte stage. We conclude that the injected donor cells acted as "infectious centers" to facilitate the infection of host hematopoietic cells with the M3MuV vector. Our results indicate that the "targeted" in vivo infection of primitive hematopoietic cells with M3MuV can initiate the immortalization and leukaemogenesis of multipotential and lineage restricted progenitor cells.
    • Targeted molecular mechanisms of epoetin alfa.

      Langer, Corey J; Hirsch, Fred R; Cortés-Funes, Hernan; Sawyer, Stephen T; Thatcher, Nick; Department of Medical Oncology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA. cj_langer@fccc.edu (2003-08)
      Despite therapeutic improvements and ongoing efforts to develop more efficacious therapies, the majority of lung cancer patients face a poor prognosis. Therefore, the primary goal of current treatment is palliation, improvement and maintenance of quality of life (QOL), and (modest) prolongation of survival. Anemia frequently occurs in lung cancer patients and has been associated with decreased QOL, impaired treatment outcomes, and shortened survival time. Furthermore, anemia is a causative factor of tumor hypoxia, which compromises the efficacy of chemotherapy and radiotherapy. Thus, correction of even mild anemia seems to have a beneficial effect on QOL and cancer treatment outcomes. The current article describes the basis and mechanism for the use of recombinant human erythropoietin (rHuEPO, epoetin alfa), a molecular targeted therapy, for the treatment of cancer-related anemia, with a focus on lung cancer. Epoetin alfa has proven efficacy and safety in correcting anemia and improving QOL based on numerous clinical studies and over a decade of clinical practice. In addition, emerging data show that epoetin alfa may offer potential benefits beyond treating anemia, specifically in terms of treatment outcomes and cognitive function. Future research needs to be conducted to explore the potential for epoetin alfa to improve survival time in lung cancer patients.
    • Targeting breast cancer vaccines to dendritic cells: improved immunological responses with less protein?

      Armstrong, Anne C; Gilham, David E; Clinical and Experimental Immunotherapy Group, School of Cancer and Enabling Sciences, University of Manchester, Manchester Academic Healthcare Science Centre, Paterson Institute for Cancer Research, Wilmslow Road, Withington, Manchester, M20 4BX, UK. dgilham@picr.man.ac.uk. (2012)
      The central goal of cancer immunotherapy is to control tumors through the mobilization of the patient's immune system. Vaccines targeting the Her2/neu proto-oncogene have been tested with some early encouraging responses in breast cancer. However, a more effective set of vaccines targeting specific immune cell subtypes may provide a more potent means to stimulate anti-tumor immunity. Dendritic cell-specific antibodies fused with the Her2/neu protein proved effective at generating immune responses in preclinical models. Importantly, only low amounts of protein vaccine were required to generate this response, which has potentially significant implications for the future clinical development of Her2/neu-targeted vaccines and other vaccine targets.
    • Targeting cancer stem cell propagation with palbociclib, a CDK4/6 inhibitor: Telomerase drives tumor cell heterogeneity.

      Bonuccelli, Gloria; Peiris-Pages, Maria; Ozsvari, Bela; Martinez-Outschoorn, U; Sotgia, F; Lisanti, M; Paterson Building, University of Manchester, Manchester M20 4BX (2016-12-25)
      In this report, we systematically examined the role of telomerase activity in lung and ovarian cancer stem cell (CSC) propagation. For this purpose, we indirectly gauged telomerase activity, by linking the hTERT-promoter to eGFP. Using lung (A549) and ovarian (SKOV3) cancer cells, transduced with the hTERT-GFP reporter, we then employed GFP-expression levels to fractionate these cell lines into GFP-high and GFP-low populations. We functionally compared the phenotype of these GFP-high and GFP-low populations. More specifically, we now show that the cancer cells with higher telomerase activity (GFP-high) are more energetically activated, with increased mitochondrial mass and function, as well as increased glycolytic activity. This was further validated and confirmed by unbiased proteomics analysis. Cells with high telomerase activity also showed an increased capacity for stem cell activity (as measured using the 3D-spheroid assay) and cell migration (as measured using a Boyden chamber approach). These enhanced biological phenotypes were effectively inhibited by classical modulators of energy metabolism, which target either i) mitochondrial metabolism (i.e., oligomycin) or ii) glycolysis (i.e., 2-deoxy-glucose), or iii) by using the FDA-approved antibiotic doxycycline, which inhibits mitochondrial biogenesis. Finally, the level of telomerase activity also determined the ability of hTERT-high cells to proliferate, as assessed by measuring DNA synthesis via EdU incorporation. Consistent with these observations, treatment with an FDA-approved CDK4/6 inhibitor (PD-0332991/palbociclib) specifically blocked the propagation of both lung and ovarian CSCs. Virtually identical results were obtained with breast CSCs, which were also highly sensitive to palbociclib at concentrations in the nanomolar range. In summary, CSCs with high telomerase activity are among the most energetically activated, migratory and proliferative cell sub-populations. These observations may provide a mechanistic explanation for tumor metabolic heterogeneity, based on telomerase activity. FDA-approved drugs, such as doxycycline and palbociclib, were both effective at curtailing CSC propagation. Thus, these FDA-approved drugs could be used to target telomerase-high proliferative CSCs, in multiple cancer types. Finally, our experiments also allowed us to distinguish two different cellular populations of hTERT-high cells, one that was proliferative (i.e., replicative immortality) and the other that was non-proliferative (i.e., quiescent). We speculate that the non-proliferative population of hTERT-high cells that we identified could be mechanistically involved in tumor dormancy.
    • Targeting double-strand breaks to replicating DNA identifies a subpathway of DSB repair that is defective in ataxia-telangiectasia cells.

      Johnson, Robert T; Gotoh, Eisuke; Mullinger, Ann M; Ryan, Anderson J; Shiloh, Yosef; Ziv, Yael; Squires, Shoshana; Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, United Kingdom. johnsort@umdnj.edu (1999-08-02)
      The critical cellular defect(s) and basis for cell killing by ionizing radiation in ataxia-telangiectasia (A-T) are unknown. We use the topoisomerase I inhibitor camptothecin (CPT), which kills mainly S-phase cells and induces DSBs predominantly in replication forks, to show that A-T cells are defective in the repair of this particular subclass of DSBs. CPT-treated A-T cells reaching G2 have abnormally high levels of chromatid exchanges (viewed as prematurely condensed G2 chromosomes); aberrations in normal cells are mostly chromatid breaks. Transfectants of A-T cells with the wild-type ATM cDNA are corrected for CPT sensitivity, chromatid aberrations, and the DSB repair defect. These data suggest that in normal cells ATM, the A-T protein, probably recognizes DSBs in active replicons and targets the repair machinery to the breaks; in addition, the ATM protein is involved in the suppression of low-fidelity, adventitious rejoining between replication-associated DSBs. The loss of ATM functions therefore leads to genome destabilization, sensitivity to DSB-inducing agents and to the cancer-promoting illegitimate exchange events that follow.