NOVEL ERK INHIBITOR SENSITIZES MAPK INHIBITOR RESISTANT CELLS

In recent years, “targeted” cancer therapies have shown promising results. With the discovery and development of “personalized” novel cancer treatments encouraging clinical responses in a subset of patients with advanced systemic disease have been observed.

This has been particularly evident with several of the recently developed kinase inhibitors that target oncogenic forms of EGFR, HER2, BCR-ABL, ALK, JAK2, and BRAF. In addition, several MEK inhibitors are developed to target oncogenic RAS. Although these drugs initially exhibit responses, they fail to sustain results due to the emergence of acquired resistance. The median duration of response with the EGFR inhibitor gefitinib (Iressa®, FDA approved) in non-small cell lung cancer patients is 7 months. Additionally, response duration for the BRAF inhibitor vemurafenib is approximately 6-7 months.  In phase II clinical trials, median progression free survival time in patients bearing K-RAS mutant tumors treated with selumetinib (MEK inhibitor) was 5.3 months. Multiple mechanisms are associated to limiting the efficacy of these targeted agents. Therefore, further studies are needed to develop effective strategies to overcome the aquisition of resistance to targeted agents. Formulation of effective drug combinations as well as identification of novel compounds with anti-neoplastic properties could be useful in this context.

 

MEK signal transduction

A recent study by Morris and colleagues published in Cancer Discovery (April 29th, 2013) reported the identification and characterization of a novel and selective ERK inhibitor. This inhibitor was found to induce tumor regression in BRAF, NRAS, and KRAS xenograft models and also inhibited MAPK signaling and cell proliferation in BRAF or MEK inhibitor resistant models. In addition, this ERK inhibitor was found to be effective in tumor cells that are resistant to concurrent treatment with BRAF and MEK inhibitors.

In this study, Morris et al. screened nearly 5 million compounds’ ability to bind the unphosphorylated form of ERK2 and observed ATP competitive compound, SCH772984, selectively bound and inhibited ERK1/2 activity at a nanomolar range. SCH772984 was effective in blocking ERK activation in a dose-dependent manner in V600EBRAF mutant and KRAS mutant cell lines. A known mechanism of resistance to MAPK inhibitors is via negative feedback activation of the MAPK pathway through ERK activation (for details please refer to my previous post titled “resistance to BRAF inhibitors in melanoma”). In this study, negative feedback activation via increased ERK phosphorylation was also observed following treatment with SCH772984. However, SCH772984 was found to be effective in blocking further downstream signaling of ERK as it maintained complete inhibition of ERK substrate p90 ribosomal S6 kinase.

Next, to test the efficacy of SCH772984 in the context of clinically observed resistance mechanisms to the MAPK inhibitors (such as BRAF amplification, MEK1 mutation, RAS mutation), researchers generated resistant cell lines and tested the efficacy of SCH772984. In all scenarios tested, sensitivity to SCH772984 treatment was observed as compared to the MAPK inhibitors. A recent clinical study concurrently treated patients with BRAF and MEK inhibitors and found to double the progression free survival period in patients whose tumors harbored MAPK activation especially in V600EBRAF mutant melanoma.

In the near future this combination could potentially become the standard-of-care for V600EBRAF mutant melanoma. However, due to heterogeneous nature of tumor cells, it is possible that acquired resistance may emerge to this combination treatment. To that end, Morris et al. tested the efficacy of SCH772984 in BRAF and MEK inhibitor combination resistant models of melanoma and colorectal cancer. Reduced activation of ERK and inhibition of cellular proliferation was noted in this model following SCH772984 treatment.

Taken together this study implicates a new ERK inhibitor SCH772984 that may exhibit encouraging therapeutic responses for the treatment of patients with BRAF, NRAS, and KRAS mutations including patients who relapse on current BRAF or MEK inhibitor therapy.



References:

1. Akinleye A, Furqan M, Mukhi N, Ravella P, Liu D. MEK and the inhibitors: from bench to bedside. J Hematol Oncol. 2013;6:27.

2. Cohen MH, Williams GA, Sridhara R, Chen G, Pazdur R. FDA drug approval summary: gefitinib (ZD1839) (Iressa) tablets. Oncologist. 2003;8:303-6.

3. Morris EJ, Jha S, Restaino CR, Dayananth P, Zhu H, Cooper A, et al. Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors. Cancer Discov. 2013.

When can B cells make IL-17?

IL-17 cytokines are best known for their roles in describe the imageimmune defense against bacterial and fungal infections and in the pathogenesis of inflammatory autoimmune diseases including rheumatoid arthritis(RA), multiple sclerosis(MS), and psoriasis.   Cells that are known to be producers of IL-17 include CD4+ TH17 T cells, CD8+ TC17 T cells, type 3 innate lymphoid cells (ILC3/lymphoid tissue-inducer cells), gd T cells, and NKT cells.  However, in a recent article in Nature Immunology, Bermejo et. al demonstrate that B cells are primary producers of IL-17 during infection with the protozoan parasite,Trypanosoma cruzi.  Furthermore, B cells activated IL-17 production through an entirely novel pathway.

IL-17 comprises a family of six related homo- or hetero-dimeric functioning cytokines (IL-17A – IL-17F) that signal through a complex family of multimeric IL-17 receptors (IL-17RA – IL-17RE).  IL-17 cytokines activate a unique signaling pathway through the adaptor protein Act1 which leads to induction of several transcription factors including NF-kB, IκBζ, C/EBPδ, C/EBPβ, MAPK, and PI3K.

In the study by Bermejo et. al, the authors sought to identify all of the cell types producing IL-17 during in vivo infection of mice with T. Cruzi.  Interestingly, the majority of splenic cells producing IL-17 at 10 and 19 days post-infection were CD3 and instead expressed CD19 and B220, markers of B cells.  Further characterization of these cells revealed expression of the plasmablast marker CD138, but not germinal center B cell markers.  IL-17+B220+ cells were absent in T. Cruzi infected B cell-deficient μMT mice and these mice were less able to control the infection and had higher levels of IFN-gamma and TNF.  Thus, B cells were not only the major IL-17 producers during T. Cruzi infection, but played a major role in protective immune responses and limiting immune pathology.

In mice, the generation of IL-17 producing TH17 cells is driven by IL-6, IL-23, and the transcription factors RORgammaT, ROR-alpha, and Ahr.  However, IL-17 producing B cells were still generated in T. Cruzi infected mice or B cells lacking IL-6, IL-23R, RORgammaT, Ahr, or treated with inhibitors of ROR-alpha.  In vitro exposure of purified B cells to T. Cruzi induced production of IL-17; however this was not mimicked by incubation of B cells with TLR2, TLR4, or TLR9 ligands, further indicating the lack of a role for inflammatory cytokines upregulated by TLRs in inducing IL-17 in B cells.  T cells did not produce IL-17 in response to T. Cruzi.  Thus the known regulators of IL-17 producing cells were not involved in the unique induction of IL-17 in B cells by T. Cruzi.

The authors focused on T. Cruzi trans-sialidase, a surface GPI-anchored enzyme, as being the potential IL-17 inducing signal, due to its previously described activity as a B cell mitogen.  Treatment of B cells with recombinant enzymatically active trans-sialidase recapitulated IL-17 production and a blocking antibody against the enzymatic site of trans-sialidase completely inhibited the induction of IL-17 during T. Cruzi exposure.  Through a series of experiments, the authors determined that trans-sialidase-mediated sialylation of the surface marker CD45 was required for B cell IL-17 production.  The signaling pathway downstream of CD45 leading to IL-17 induction was found to involve Src kinases, Btk, and Tec.

Finally, the authors examined if this phenomenon occurs in human B cells as well.  CD19+ B cells were isolated from tonsils and the production of IL-17 was also seen in response to T. Cruzi exposure in a CD45 and Btk dependant fashion.

In conclusion, this was an exciting study that demonstrated not only that B cells are major IL-17 producers during parasitic infections, but also identified a unique signaling pathway that mediates this effect in both mice and humans.  Many questions remain about how this unique signal transduction pathway operates only in plasmablast B cells but not other cell types, despite the widespread expression of CD45 by hematopoietic cells.  Furthermore, the role of IL-17 production by B cells in immune responses to other pathogens that express trans-sialidases as well as the role for these B cells in IL-17-driven immune pathologies remains to be explored.

Further Reading:

Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells.  Bermejo DA, Jackson SW, Gorosito-Serran M, Acosta-Rodriguez EV, Amezcua-Vesely MC, Sather BD, Singh AK, Khim S, Mucci J, Liggitt D, Campetella O, Oukka M, Gruppi A, Rawlings DJ. Nat Immunol. 2013 May;14(5):514-22. doi: 10.1038/ni.2569.

IL-17-producing B cells combat parasites.  León B, Lund FE. Nat Immunol. 2013 May;14(5):419-21. doi: 10.1038/ni.2593.

Recent advances in the IL-17 cytokine family.  Gaffen SL. Curr Opin Immunol. 2011 Oct;23(5):613-9. doi: 10.1016/j.coi.2011.07.006.

Development and evolution of RORγt+ cells in a microbe’s world.  Eberl G. Immunol Rev. 2012 Jan;245(1):177-88. doi: 10.1111/j.1600-065X.2011.01071.x.


New in MS Research: Interplay Between IFN-beta, B-cells and Monocytes

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS), characterized by the presence of scar tissues (plaques) localized within the brain’s white matter and spinal cord. These plaques are results of myelin-degeneration (demyelination) and axonal death. Although MS has been classically considered to be a T-cell-mediated disease, the high efficacy of B cell-depleting therapies have demonstrated the critical role of B-lymphocytes and the humoral immune response in MS pathogenesis, albeit the underlying mechanisms remain unclear. In approximately 90% of MS patients, there is increased levels of intrathecally synthesized IgG in the MS-plaques as well as Cerebral Spinal Fluid (CSF), which manifests B-cell clonal expansions within the CNS.

B-cell-lineage cells differentiate into antibody-secreting plasma cells that are the source of persistent IgG, in the presence of key factors such as interleukin-6 (IL-6), B-cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL). IL-6 promotes terminal differentiation of B cells to plasma cells and is essential for the survival and Ig secretion. In conjunction with APRIL, BAFF regulates, B-cell survival, differentiation and is essential for initiation of T-cell independent B-cell responses. 

B cell resized 600

Type I IFNs (IFN-α, IFN-β, IFN-κ, and IFN-ω) are cytokines expressed by many cell types in response to viral or microbial infections, which bind to- and trigger specific Toll-like receptors (TLRs) that induce a large number of genes modulating and linking the innate and the adaptive immune responses.

Despite the development of other new treatments, IFN-β has been the first-line disease-modifying drug treatment for patients with relapsing-remitting multiple sclerosis (RRMS). Thus, understanding the molecular mechanisms of the anti-inflammatory effect of IFN-β in RRMS may provide insight into MS pathogenesis.  

TLRs are a family of non-catalytic pattern recognition receptors that recognize and bind to specific molecular patterns of pathogen-derived and endogenous damage-associated components. In addition to their key role in mediating innate immunity, TLRs have also been shown to play an important part in the activation of the adaptive immune system by inducing proinflammatory cytokines such as TNF-α, IL-1, IL-6, IL-12, and IFN.

Several studies have shown that of the 11 TLRs identified in humans, endosomal TLRs 7, 8 and 9 which recognize pathogen-derived and synthetic nucleic acids, also recognize endogenous immune complexes containing self-nucleic acids in certain autoimmune disorders such as MS. Interestingly, B-cells express both TLR7 and TLR9. TLR7 recognizes guanosine- and uridine-rich single-stranded RNAs (ssRNAs), whereas TLR9 recognizes hypomethylated CpG-rich double-stranded DNAs.  Upon activation by their specific ligands, these TLRs induce B cell proliferation and differentiation into Ig-secreting cells.

describe the image

In a recent study published in the European journal of Immunology, Coccia’s group has demonstrated the essential interactions between monocytes and B cells for the release of effective humoral immune response that elicits TLR7-mediated -induced B-cell differentiation into Ig secreting cells. Furthermore, they have shown a clear deficiency in this cross-talk interaction in MS patients; the peripheral blood mononuclear cell (PBMC) of MS patients exhibit substantially lowered TLR7-induced Ig production (compared to Healthy donors). However, results obtained after one-month long IFN-β therapy showed that lower humoral immune response in MS subjects was replenished, through IFN-β–induced secretion of TLR7- triggering cytokines, which mediated the selective increase in IgM and IgG to levels comparable to Healthy donors’. This data revealed that the IFN-β enhancement of TLR7-induced B-cell responses in MS patients occurs in at least two steps: 1) Regulation of TLR7 gene expression, and 2) Secretion of B-cell differentiation factors, in particular IL-6 and BAFF.

Finally, the last and perhaps the most significant finding of Coccia’s new study, is reporting, for the first time, the presence of a defect in TLR7 gene expression and signaling in monocytes of MS patients. Lack of TLR7-driven IgM and IgG production, absence of IL-6 and a significant reduction in BAFF expression in samples of MS patient-IFN-β treated PBMCs that were depleted of monocytes, evince IFN-β therapeutic mechanism by fine-tuning monocyte functions, through stimulation of TLR7 which subsequently effects B cell differentiation.

The discovery of the tight regulation of both TLR expression and TLR-induced responses in maintenance of immune environment’s homeostasis, as well as IFN-β-mediated- TLR7 function recovery are indicative of the critical changes in PBMC microenvironment induced by IFN-β therapy; within this microenvironment, leukocyte subsets establish critical immune regulatory interactions which determine the fate of the host’s immune tolerance processes.

Coccia’s new study has revealed new insights, which are not only crucial for the better understanding of the MS immunopathology, but also significant for development of new MS therapeutic strategies which target TLR expression and/or TLR-induced responses.


Further Reading:

IFN-β therapy modulates B-cell and monocyte crosstalk via TLR7 in multiple sclerosis patients.

GENETIC AND EPIGENTIC CHANGES IN ACUTE MYELOID LEUKEMIA

Acute myeloid leukemia (AML), a cancer of hematopoietic cells, is a molecularly heterogeneous disease. AML is associated with several genetic changes that alter normal hematopoietic growth and differentiation, resulting in the accumulation of large numbers of abnormal immature myeloid cells in the bone marrow and peripheral blood. These cells are capable of dividing and proliferating, but cannot differentiate into mature hematopoietic cells. Recurrent structural alterations of chromosomes are the established diagnostic and prognostic markers in AML. Several studies using targeted sequencing (determination of DNA sequence of specific areas of interest within the genome) identified recurrent gene mutations that contained diagnostic and prognostic information, including mutations in FLT3, NPM1, KIT, CEBPA, and TET2 genes. In addition, massively parallel sequencing (high-throughput approaches of DNA sequencing, also called next-generation sequencing) has discovered recurrent mutations in DNMT3A and IDH1/2 genes that may also provide prognostic information for some patients. Even though these genetic abnormalities may play an essential role in the pathogenesis of AML, nearly 50% of AML patients have normal karyotype (an organized profile of a person’s chromosomes).No. of mutations in AML resized 600 Based on the cytogenetic analysis, AML patients are classified into three major risk categories: favorable, intermediate and unfavorable. AML patients with PML-RARA, RUNX1-RUNX1T1, or MYTH11-CBFB gene fusions (as a result of chromosomal rearrangements) profile belong to favorable- risk category and have shown relatively good response to chemotherapy. Patients with complex genetic alterations (e.g. monosomy karyotype) are categorized into unfavorable-risk profile. Majority of the AML patients exhibit normal karyotype and belong to intermediate-risk category. Some of these patients respond well to chemotherapy while others don’t. Since nearly 50% of AML patients have normal chromosomal profile, better molecular characterization of pathogenesis of AML is required for better approaches to therapy. In addition, next-generation sequencing (NGS) studies have revealed that even though AML usually harbor hundreds of mutated genes, only a limited number of mutated  genes serve as driver mutations (i.e. causing the tumor). Among the different adult cancer types sequenced extensively so far, AML has had the fewest mutations discovered. Therefore, identification of a significant number of novel driver mutations present at low frequency in AML will help to better understand the leukemogenesis.

A study recently published in the New England Journal of Medicine (May 1st, 2013) by researchers at The Cancer Genome Atlas (TCGA) group led by Timothy J. Ley broadly classified the genomic alterations that frequently underlie the development of AML. This study also suggested potential new drug targets and treatment strategies for AML. In this study the genome of 200 newly diagnosed adult cases of AML patients, representing all of the known subtypes, was analyzed by performing whole-genome sequencing (50 cases) and whole-exome sequencing (150 cases). In addition, RNA and micro-RNA sequencing and DNA-methylation analysis was also performed. 

Each AML genome was compared to the describe the imagenormal genome derived from a skin sample of the same patient. The recurrently mutated genes discovered in this study were grouped into nine categories that were defined according to biologic function and that are considered to play a role in AML pathogenesis. Some of these groups include: tumor suppressor genes, transcription-factor fusions, activated signaling genes and epigenetic modifiers (DNA-methylation related genes and chromatin-modifying genes) with the latter being the most frequently mutated class of genes found in this study. At least one potential driver mutation was identified in nearly all AML samples including genes that are well established as being associated with AML pathogenesis (eg. FLT3, NPM1, DNMT3A, IDH1, IDH2, and CEBPA).

This study was the first to observe recurrent mutations in cohesin genes, which are important in cell division, in 13% of cases of AML samples. In addition, this study also observed a mutation in microRNA 142 (miR-142). Overall this study provided a detailed understanding of the genetic and epigenetic changes associated with adult de novo AML. Future studies are warranted to understand the relationship between these alterations and treatment results.

 

References:

1. Bacher U, Schnittger S, Haferlach T. Molecular genetics in acute myeloid leukemia. Curr Opin Oncol. 2010;22:646-55.

2. Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer. 2003;3:650-65.

3. Yamashita Y, Yuan J, Suetake I, Suzuki H, Ishikawa Y, Choi YL, et al. Array-based genomic resequencing of human leukemia. Oncogene. 2010;29:3723-31.

4. Network TCGAR. Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia. N Engl J Med. 2013.

The nuances of using CFSE to monitor lymphocyte proliferation

Measuring proliferation of lymphocytes such as T cells isolated from peripheral blood monuclear cells (PBMC) using carboxyfluorescein diacetate succinimidyl ester (CFSE) is not a foolproof protocol.  CFSE can be toxic to cells and non-optimal CFSE labeling conditions can thus hamper proliferation of cells and obscure interpretation of results.  An article in Nature Protocols by Quah et al., details CFSE labeling conditions and how to achieve optimal results.

CFSE is a fluorescent cell membrane permeable dye with similar excitation and emission properties as fluorescein isothiocyanate (FITC).  Thus CFSE can be assayed in flow cytometry by the same channels that detect the fluorescence intensity of FITC.  The CFSE precursor, carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) that is used to label cells is non-fluorescent, but once inside cells, acetate groups are removed by intracellular esterases, causing the resulting CFSE molecule to become fluorescent and also less membrane permeable.  Furthermore, the succinimidyl ester group of CFSE covalently couples to primary amine groups, thus remaining bound to proteins inside cells for long time periods.  As a cell divides, the intensity of CFSE staining in the resultant daughter cells will be half that of the parent, allowing easy flow cytometric assessment of the number of cell divisions that have occurred since labeling.

While CFSE is commonly used to assess lymphocyte proliferation, CFSE can be toxic and impair cell division.  According to Quah et al., four parameters of the labeling conditions must be considered to minimize this toxicity:

1. The concentration of the cells.

2. The concentration of CFSE.

3. The duration of cell labeling.

4. The presence of amino acids in the labeling media.

CFSE will bind to free amines in aqueous conditions and thus reduce the remaining CFSE concentration. To avoid the loss of CFSE to amino acids in the labeling media, PBS is the recommended diluent for CFSE prior to adding to cells.  Cells are uniformly suspended in PBS with serum, and the CFSE/PBS stock is immediately mixed rapidly with the cells and allowed to incubate for the optimal amount of time.

Regarding cell and CFSE concentration, these two parameters must be considered in the context of the other.  Cells at higher concentrations can be labeled with higher concentrations of CFSE with a minimal effect of CFSE toxicity on cell division.  For instance, cells at a concentration of 50 x 106/ml can be labeled with 5uM CFSE, but cells at a concentration of 1 x 106/ml will experience significant toxicity if labeled with 5uM CFSE but will do well with 1uM CFSE.  The time of labeling is also important, and longer incubation times will increase toxicity.  Quah et al. recommended 5 minutes of incubation with CFSE before washing the cells.

To assess proliferation, after CFSE labeling, cells are washed and then stimulated with a mitogenic signal.  For instance, T cells can be stimulated with anti-CD3 + anti-CD28, PHA, SEB, PMA + ionomycin or other stimuli.  Then the cells will be allowed to divide for a number of days which must also be optimized depending on the stimulus used.

T cells will die if left unstimulated in vitro and as they proliferate, they can undergo activation induced cell death (AICD).  Thus, some amount of cell loss must be anticipated.  In an accompanying protocol in Nature Methods, Hawkins et al. detail the incorporation of cell count beads during flow cytometry to more accurately measure the degree of cell proliferation.

Thus, there are many nuances to consider when using CFSE to label cells for assays such as proliferation.  I recommend reading both of these protocols to achieve robust assay performance.

Further Reading:

Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.  Quah BJ, Warren HS, Parish CR. Nat Protoc. 2007;2(9):2049-56.

Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data.  Hawkins ED, Hommel M, Turner ML, Battye FL, Markham JF, Hodgkin PD. Nat Protoc. 2007;2(9):2057-67.

 


Tissue Resident CD8+ Memory T cells: A front line defense?

Memory CD8+ T cells continually Tcell cytolysiscirculate in the blood, lymph, and secondary lymphatic organs as they patrol for the presence of secondary infections.  Recently, a class of effector memory CD8+ T cells has also been shown to migrate to and reside long-term in non-lymphoid tissues. These tissue resident memory T cells can quickly respond to tissue infections with their cognate pathogen.  CD8+ T cells mainly function in killing of infected cells though cytolysis and by producing effector cytokines, including IFN-gamma to activate other immune cells.  In a recent article in Nature Immunology, Schenkel et. al demonstrate an additional function for these tissue resident memory CD8+ T cells: as major producers of chemokines that recruit circulating memory T cell forces to the site of infection.

In this study, Lymphocytic choriomeningitis virus (LCMV) was injected intraperitoneally into female mice that had been adoptively transferred with naïve TCR transgenic CD8+ T cells specific for the gp33 epitope expressed by LCMV.  Two months later, mice were transcervically infected with a Vaccinia virus strain engineered to express gp33 (VV-gp33) and the kinetics of the CD8 T cell recall response in the mucosal tissues of the female reproductive tract were assessed.  Gp33-specific tissue resident memory CD8+ T cells were required for the rapid (within 2 days) recruitment of additional circulating gp33-specific T cells into the tissues.  However, if the secondary infection was with a Vaccinia virus strain expressing a different antigen (OVA), then relatively few memory T cells were seen accumulating in the reproductive tract tissue.  This specificity was also seen when gp33 versus OVA peptides were transcervically injected into the tissues instead of infection with VV.

To derive the mechanisms underlying the recruitment of T cells to infected tissue sites, chemokine expression was assessed.  CXCL9 as well as multiple other chemokines were found to be rapidly induced in various cells residing in the reproductive tissues including endothelial cells, tissue dendritic cells, and memory CD8+ T cells.  Production of IFN-gamma by the tissue resident memory CD8+ T cells was required for both recruitment of additional memory CD8+ T cells to the site as well as CXCL9 production by endothelial cells.

Antigen presenting cells as well as activated T cells produce copious amounts of IFN-gamma and it is expected that IFN-gamma production would elicit CXCL9 expression as the alternate name of CXCL9 is monokine induced by IFN-gamma (MIG).  In this study however, it was production of IFN-gamma by the antigen-specific (gp33) tissue resident memory CD8+ T cells that was critical in the expression of chemokines such as CXCL9 and further recruitment of additional memory CD8+ T cells into the infected tissues.  Antigen presenting cells alone were not as productive in this process because when the secondary infection was instead performed with an OVA-antigen expressing vaccinia virus, significant numbers of OVA-specific T cells were not recruited to the infected tissues.

Thus, this study demonstrated that antigen-specific CD8+ memory T cells that reside in tissues function to significantly amplify innate immune alarms to secondary infections by recruiting additional circulating CD8+ memory T cells to the infected site.

Further Reading:

Sensing and alarm function of resident memory CD8(+) T cells.  Schenkel JM, Fraser KA, Vezys V, Masopust D. Nat Immunol. 2013 May;14(5):509-13. doi: 10.1038/ni.2568. Epub 2013 Mar 31.

Hidden memories: frontline memory T cells and early pathogen interception.  Masopust D, Picker LJ. J Immunol. 2012 Jun 15;188(12):5811-7. doi: 10.4049/jimmunol.1102695.

Chemokine monokine induced by IFN-gamma/CXC chemokine ligand 9 stimulates T lymphocyte proliferation and effector cytokine production.  Whiting D, Hsieh G, Yun JJ, Banerji A, Yao W, Fishbein MC, Belperio J, Strieter RM, Bonavida B, Ardehali A. J Immunol. 2004 Jun 15;172(12):7417-24.

The Crucial Connection Between Metabolism and the Immune System.

Over the past couple of decades, the field of immunology has been growing at an exponential pace. Today, immunologists that continue to study the intracellular and extracellular components are also creating new therapies that suppress the immune system, increase the immune system, and even fix the dysregulation of the immune system. Likewise, there has been an increase in the study of organism metabolism and intracellular metabolism in various pathologies; such as diabetes and cancer. Interestingly, there is also an increase in studying how immune cells function in terms of their intracellular metabolism, how these metabolic pathways affect the phenotype and activation of immune cells, and how the immune system affects the metabolic functions of its host organism also known as immunometabolism1.

There are multiple metabolic pathways that cells use to make ATP. It has been discovered that some cells may preferentially use the glycolytic pathway to make ATP, even when the components are available for aerobic respiration; an event called the Warburg effect1. On the other hand, some cells may use the glycolytic pathway along with the Krebs cycle and electron transport chain (ETC), known as oxidative phosphorylation (OxPhos), to consume materials to make energy1. However, not all immune cells act alike. For instance, activated neutrophils preferentially use the Warburg effect1.  Interestingly, this metabolic pathway makes the most hydrogen peroxide, which is part of the substance neutrophils use for granulocytic release against pathogens1. Likewise, dendritic cells that have been activated via a toll-like receptor agonist and express inducible nitric oxide synthase (iNOS) are also found to use the Warburg effect and, like neutrophils, the metabolite of iNOS plays a functional role in activated dendritic cells1. Finally, the

macrophage

M1 pro-inflammatory macrophages also use the Warburg pathways to make ATP1. Glycolysis and oxidative phosphorylation are linked when the pyruvate from the glycolytic pathway is used to make acetyl-CoA using the Krebs cycle. Immune cells that use this pathway are activated T-cells, immunosuppressive M2 macrophages, and the pro-inflammatory Th17 T-cells1. Finally, fatty acid oxidation, the process of using lipids to make ATP, is utilized by memory T-cells, regulatory T-cells, and alternatively-activated macrophages1.

Metabolism in the immune system is more than just connected to the cell’s activation. Metabolism is involved in the homeostasis between immune cells as well as between immune cells and their stromal host cells1. Dysregulation of metabolites has been found in many pathologies and the role of metabolite fluctuation caused by host immune cells versus pathogens is an active area of study2. Likewise, metabolism has been shown to be involved in class switching effector T-cells into memory T-cells and is involved in the act of bringing immune cells into quiescence3.

An interesting example of a link between immunity and metabolism can be found in an article published in Nature April 11, 2013 by Tannahill et al. This paper demonstrates that the metabolite succinate, a key component in the Krebs cycle, is integral in lipopolysaccharide-induced macrophage activation4. Furthermore, they demonstrated that the activation of toll-like receptor 4, through LPS stimulation, leads to an increase of intracellular glutamate uptake and upregulation of succinate production under the “gamma-Amminobutyric acid (GABA) shunt” metabolic pathway4. The metabolite succinate then stabilizes hypoxia-inducible factor-1α; a protein that is involved in IL-1β production4. 

The study of metabolism is more than just an academic exercise. Many therapies have been proposed that look at changing the metabolic system for alteration of the dysregulated immune response. One of these is metformin, a drug that is used to help regulate type 2 diabetes2. However, metformin is now actively investigated as an anti-cancer therapeutic, having an effect on changing the tumor immune microenvironment from a pro-tumor phenotype to an anti-tumor phenotype2. As we continue to learn more about the effects of host metabolism on the immune system, the role of the immune system in organism metabolism, and the intracellular metabolic pathways involved in various immune cells during different states of function, we will hopefully be able to develop more therapies that repair immune dysregulation as well as develop immunotherapies for treating various metabolic diseases.

Further Reading

1. Pearce, E. L. & Pearce, E. J. Metabolic pathways in immune cell activation and quiescence. Immunity 38, 633-643, doi:10.1016/j.immuni.2013.04.005 (2013).

2. Mathis, D. & Shoelson, S. E. Immunometabolism: an emerging frontier. Nature reviews. Immunology 11, 81, doi:10.1038/nri2922 (2011).

3. Finlay, D. & Cantrell, D. A. Metabolism, migration and memory in cytotoxic T cells. Nature reviews. Immunology 11, 109-117, doi:10.1038/nri2888 (2011).

4. Tannahill, G. M. et al. Succinate is an inflammatory signal that induces IL-1beta through HIF-1alpha. Nature 496, 238-242, doi:10.1038/nature11986 (2013).

Proof-of-principle study of the first-ever autologous iPSC-derived cell transplant in non-human primates

Shinya Yamanaka was awarded the Noble Prize for Medicine last year for his work on cellular reprogramming and creating induced pluripotent stem cells (iPSC).  Shinya Yamanaka found four transcription factors (Oct-3/4, Sox2, c-Myc, and Klf4) that determine pluripotency and he was able to reprogram differentiated adult cells into pluripotent cells that can then be re-differentiated into fully specialized tissue.  His findings raised great expectations, especially in the field of cellular therapy and regenerative medicine.  However, the path to the therapeutic use of iPSC is long and not without complications.  It was believed that iPSCs would avoid any immunogenic response because these cells can be developed from a patient’s own somatic cells. However, Zhao et al. challenged this notion when they discovered that iPSCs derived from C57BL/6 (B6) mice by the standard retroviral approach formed teratomas once transplanted into syngenic host mice and induced a rapid T cell dependent immune response1.

Recently, a group of researchers led by Dr. Su-Chun Zhang at the Waisman Center on the University of Wisconsin-Madison campus have shown that utilizing iPSCs as an autologous cell therapy is feasible and without any immunological reaction or rejection.  Dr. Zhang was the first to derive neural cells from embryonic stem cells (ESCs), as well as from iPSCs, and now has shown the first proof-of-principle that autologous iPSC-derived cells can engraft and survive in the primate brain.

In a new study published in Cell Reports, Zhang’s group reports on the successful generation of iPSCs from fibroblasts obtained from 8-10 year old rhesus monkeys (Macaca mulatta) using retroviruses containing the four Yamanaka factors, subsequent neuronal differentiation and cell transplantation back into the donor monkeys2.  The rhesus iPSCs were differentiated into neuroepithelia with the characteristic neural tube-like rosettes and expression of neuroectoderm transcription factors Pax6 and Sox1.  The neuronal rosettes were then expanded and further differentiated into neurons so that by the time for cell transplantation (day 42), 37% of the cells were bIII-tubulin+ neurons, 16% were S100b+ immature astrocytes, and 47% were Nestin+ progenitors.

primate ipsc cell pic
To examine the feasibility of transplanting autologous iPSC-derived neural progenitors in Parkinson’s disease, Zhang created parkinsonism in the rhesus monkeys by unilateral intracarotid artery injection of a neurotoxin 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP).  A year to 18 months after the MPTP infusion, all the monkeys developed a stable hemiparkinsonian condition distinguished by the characteristic tremors, bradykinesia, imbalance and impairment in motor skills.  At day 30 of cell culture the iPSC-derived neural progenitors were labeled with a GFP (green fluorescent protein) lentivirus and cells were transplanted at day 42 into the striatum and substantia nigra of the same monkey from which the iPSC were derived.

The monkey did not receive any immune suppression and engraftment of the GFP-labeled iPSC-derived neural progenitors was assessed at 6 months post-transplantation using stereological analysis on serial coronal sections.  Distinct grafts were present in the injected regions where 63% of the cells were microtubule-associated protein 2 (MAP2)+ neurons, 22% were glial fibrillary acidic protein (GFAP)+ astrocytes, and 10% were myelin basic protein (MBP)+ oligodendrocytes.  The GFP+ neurons showed long fibers extending into the surrounding host tissue and some neurons were present outside of the graft region expressing markers of mature neuronal differentiation.  Additionally, there was an absence of markers for pluripotent stem cells (OCT4, NANOG, SOX17, and Brachyury) and no positive staining for Ki67 (labels mitotic cells) indicating that the grafted progenitors had terminally differentiated.

Zhao et al. showed that the teratomas formed from transplanted iPSCs had an immunological rejection by syngenic mice1.  However, Zhang demonstrates that the autologous iPSC-derived neural cell transplants in the primate brain do not undergo immunological rejections suggested by the lack of CD3 and CD8 (lymphocyte markers) staining.  There was minimal response of endogenous glia (astrocytes and microglia) since staining for human leukocyte antigen D-related (HLA-DR; microglia and macrophage marker) was seen throughout the brain including the grafted regions.

Despite the positive engraftment and differentiation of the iPSC-derived neural progenitors, they did not see any behavioral improvement in the parkinsonian monkeys.  A potential explanation is that the GFP+ neurons were mostly g-Aminobutyric acid (GABA+) and few were positive for tyrosine hydroxylase (TH+).  TH catalyzes the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine.  Deficiency in TH has been implicated in giving rise to parkinsonian characteristics3.  Also, the number of transplanted cells may not have been enough to replace the dopamine-making cells in the primate brain.

However, this study provides hope for cell therapy using autologous iPSC-derived cells.  The iPSC-derived neural progenitors survived and differentiated into mature neurons, astrocytes, and oligodendrocytes in the primate brain with no evidence of immune rejection or teratoma formation.  The transplanted cells structurally integrated into the host brain and with characteristic features of neurons indicating by extending long processes and features of oligodendrocytes indicated by staining for myelin basic protein, suggestive of myelination.  This proof-of-principle study of the first-ever transplant of iPSC-derived cells back into the same non-human primate presents hope for personalized regenerative medicine and the neurodegenerative patient population.

Further reading:

1. Zhao, Tongbiao; Zhang, Zhen-Ning; Rong, Zhili; and Xu, Yang.  Immunogenicity of induced pluripotent stem cells.  Nature. 474, 212–215, 9 June 2011

2. Emborg ME, Liu Y, Xi J, Chang X, Yin Y, Lu J, Joers V, Swanson C, Holden JE, and Zhang Su.  Induced pluripotent stem cell-derived neural stem cells survive and mature in the nonhuman primate brain.  Cell Reports 3, 1-5, March 28, 2013.

3. Goodwill KE, Sabatier C, Marks C, Raag R, Fitzpatrick PF, Stevens RC. Crystal Structure of tyrosine hydroxylase at 2.3a and its implications for inherited neurodegenerative diseases. Nature Structural Biology 4 (7): 578–585, 1997

ATM INHIBITOR SENSITIZES GLIOBLASTOMA TO IONIZING RADIATION THERAPY

A brain tumor is an abnormal growth of tissue in the brain. Brain tumors may develop from neural elements within the brain (primary brain tumor), or they may represent spread of distant cancers (secondary brain tumor). According to the National Cancer Institute, in 2013 the estimated new cases and deaths from brain tumor in the United States would be 23,130 and 14,080 respectively. There are many types of primary brain tumors. Primary brain tumors are named according to the type of cells or the part of the brain in which they originate. For example, most primary brain tumors begin in glial cells. This type of tumor is called a glioma. Among adults, the most common types are:

  • Meningioma: This tumor arises in the meninges. It’s usually benign (grade I) and grows slowly.
  • Oligodendroglioma: This tumor arises from cells that make the fatty substance that covers and protects nerves. It usually occurs in the cerebrum. It’s most common in middle-aged adults.
  • Astrocytoma: This tumor arises from star-shaped glial cells called astrocytes.

Among these, glioblastoma multiforme (GBM) is the most common and deadliest of malignant primary brain tumors in adults. Classified as a Grade IV (most serious) astrocytoma, GBM develops from the lineage of star-shaped glial cells, called astrocytes that support nerve cells. Even though GBM is one of the most aggressive forms of human cancers, the incident of this disease is fairly less.  Every year, in the United States approximately 7 out of 100,000 people are diagnosed with high-grade glioblastoma. Uncontrolled cellular proliferation, diffuse infiltration, and resistance to apoptosis are considered as hallmarks of GBM. The current standard of treatment in glioblastoma involves surgery followed by radiation therapy, and chemotherapy with the DNA alkylating agent temozolomide (TMZ).  The efficacy of this current treatment strategy is limited as the median survival is just over one year because of development of resistance. Therefore, more effective treatment plans need to be developed and explored. In a recent study published in the peer review journal Clinical Cancer Research (2013 Apr 25) Thorpe and colleagues reported that combined treatment of ataxia telangiectasia mutated (ATM) inhibitor and radiation significantly increased survival of mice bearing glioblastoma with p53 mutation compared to treatment with ATM inhibitor or radiation alone.

The nuclear protein kinase ataxia-telangiectasia mutated (ATM) plays a crucial role in DNA-damage response by transducing double-strand breaks. This kinase phosphorylates several effectors that play key roles in cell cycle progression and DNA repair pathways.

gLIOBLASYTOMA -resized-600 (1)

Ionizing radiation is the most effective therapy for glioblastoma. Ionizing radiation works by damaging the DNA of exposed tissue leading to cellular death. However, tumor cells evade death and contribute to radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. Therefore, combined treatment of ionizing radiation with inhibitors of DNA damage checkpoint may overcome the problem of resistance in glioblastoma. Since ATM is associated with DNA damage repair there are several small molecule ATM inhibitors that disrupt ATM function and enhances sensitivity of tumor cells to radiation as well as chemotherapeutic agents. ATM inhibitor KU-60019 is a potent inhibitor effectively blocking activation of key ATM targets and radiosensitizing human glioma cells in vitro. In their study Thorpe et al. (2013) showed that mice bearing glioblastoma survived longer periods of time (145 days) when treated with KU-60019 and radiation than mice treated with radiation alone (58 days) or KU-60019 alone (66 days). In addition, this study also noted that mice bearing glioma with mutant p53 are more sensitive to KU-60019 dependent radiosensitization than mice bearing glioma with p53 wild-type. Altogether this study suggests that ATM kinase inhibition may be an effective strategy as adjuvant therapy for patients with mutant p53 brain cancers.

References:

1.Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006, 444(7120):756-760.

2.Biddlestone-Thorpe L, Sajjad M, Rosenberg E, Beckta JM, Valerie NC, Tokarz M, Adams BR, Wagner AF, Khalil A, Gilfor D et al: ATM kinase inhibition preferentially sensitizes p53 mutant glioma to ionizing radiation. Clin Cancer Res 2013.

3. http://www.cancer.gov/cancertopics/wyntk/brain/page2

 

Cell-based therapy for Parkinson’s disease: past, present and future.

Parkinson’s disease (PD) is a chronic neurodegenerative condition effecting dopaminergic neurons of the midbrain. PD manifests itself around age 50 with mainly motor symptoms, such as tremor (shaking), slowness of movement, rigidity and postural instability. Number of pharmaceutical agents (e.g., L-Dopa and MAO-B inhibitors) has been used for symptomatic relief in PD patients, but the ultimate therapy target is the replacement of degenerating dopaminergic neurons with new, healthy neurons.

describe the imageCell replacement therapy for PD dates back to mid 80s with the transplantation of adrenal medullary tissue into patients’ striatum [1-3], which resulted only moderate improvements. At the same time, researchers in Sweden performed transplantation of fetal ventral mesencephalic tissue from aborted fetuses [4, 5]. These early studies observed important and persistent improvement based on numerous clinical outcomes. Moreover, postmortem examination of the brains of PD patients, who received ventral mesencephalic tissue transplantation, showed sustained survival of the graft and re-innervation of the striatum [6]. With the lift of federal funding ban on using fetal tissue for research and therapy by President Clinton in 1993, United States also began clinical trials utilizing fetal ventral mesencephalic tissue [7, 8]. Unfortunately, not only the patients didn’t display any significant improvements following transplantation in these trials, they developed additional abnormal, involuntary movements (i.e., graft-induced dyskinesia), due to surgery, which was also observed in other trials.

Close examination of the transplantation studies using fetal ventral mesencephalic tissue revealed few noteworthy outcomes:

1. Younger patients with newly developed pathology showed significant improvements over older patients with severe PD pathology.

2. Some patients showed continued improvements 3-4 years after surgery, while they did not display any benefits during the first year, indicating that the improvement in clinical parameters may take a while to appear over time. Regardless, it is clear that patients respond differently to the transplants of dopaminergic neurons, making the clinical outcomes fluctuate considerably.

3. Preparation of the fetal tissues, as well as selection of patients for transplantation, varied significantly from center to center carrying out the clinical trials, further indicating the need for standardizing tissue preparation, patient selection and implantation site.

Compared to the aforementioned points, the use of fetal ventral mesencephalic tissue for grafting constitutes one of the biggest problems in cell based therapy for PD. It has been challenging to standardize the number and the quality of the fetal dopaminergic cells in graft preparations. Furthermore, the purity of the preparations also varies from batch to batch. Lastly, many ethical -and sometimes legal- issues surround fetal tissues/cells significantly limiting their clinical applicability. Do we have an alternative source that is free of these concerns/problems? The answer is yes, but not at the moment. With the isolation of human embryonic stem cells (hESCs) in 1998 and the introduction of human induced pluripotent stem cells (iPSCs) in 2007, stem cell derived dopaminergic neurons are at the top of everyone’s list when it comes to replacing degenerating neurons in PD. hESCs have been the primary source to produce dopaminergic neurons so far [9-11], but with the popularity and the advantages of iPSCs, the focus is more likely to shift to iPSC-derived dopaminergic neurons in future transplantation efforts.

Number of studies utilizing stem cell derived dopaminergic neurons in animal models of PD reported promising results over the years. However, we are far from using these cells in clinical trials. Many issues, such as long-term stability of the transplanted cells, sustained functional recovery, ability to re-innervate the host striatum, generation of GMP grade cells and long-terms safety especially with regards to tumor formation, remain to be determined. To be able to answer these concerns are critical for successful clinical translation of stem cell derived dopaminergic neurons. Nevertheless, the target is in front of everyone, and the field of regenerative medicine is moving at an incredible speed to reach it.  It should also be noted that an increasing number of novel therapeutic approaches (e.g., gene therapy and growth factor infusions) have been under development -in addition to cell transplantations- with the aim of restoring dopaminergic function in PD patients.

While we are looking ahead with the promise of stem cell derived dopaminergic neurons for future of cell-based therapy in PD, there are many lessons to be learnt from the early clinical trials using fetal ventral mesencephalic tissue. There is no question that fetal dopamine neurons will serve as a reference and a standard against stem cell derived neurons for future clinical trials, since we know that the transplants survived, re-innervated the striatum, and generated adequate symptomatic relief in some patients for more than a decade following surgery. For PD patients, who are interested in cell-based therapy now, the decision of whether to wait for clinical trials utilizing stem cell derived neurons or to proceed with currently available fetal tissue grafts remains a somewhat difficult question and should take into consideration the aforementioned strengths and weaknesses of each approach.

 

References:

[1] Backlund EO, Granberg PO, Hamberger B, et al. Transplantation of adrenal medullary tissue to striatum in parkinsonism. First clini- cal trials. J Neurosurg 1985;62:169–173.

[2] Herrera-Marschitz M, Stromberg I, Olsson D, Ungerstedt U, Olson L. Adrenal medullary implants in the dopamine-denervated rat striatum. II. Acute behavior as a function of graft amount and location and its modulation by neuroleptics. Brain Res 1984;297:53–61.

[3] Madrazo I, Drucker-Colin R, Diaz V, Martinez-Mata J, Torres C, Becerril JJ. Open microsurgical autograft of adrenal medulla to the right caudate nucleus in two patients with intractable Parkinson’s disease. N Engl J Med 1987;316:831–834.

[4] Lindvall O, Brundin P, Widner H, et al. Grafts of fetal dopamine neurons survive and improve motor function in Parkinson’s dis- ease. Science 1990;247:574–577.

[5] Widner H, Tetrud J, Rehncrona S, et al. Bilateral fetal mesence- phalic grafting in two patients with parkinsonism induced by 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). N Engl J Med 1992;327:1556–1563.

[6] Kordower JH, Rosenstein JM, Collier TJ, et al. Functional fetal nigral grafts in a patient with Parkinson’s disease: chemoanatomic, ultrastructural, and metabolic studies. J Comp Neurol 1996;370:203–230.

[7] Freed CR, Greene PE, Breeze RE, et al. Transplantation of embry- onic dopamine neurons for severe Parkinson’s disease. N Engl J Med 2001;344:710–719.

[8] Olanow CW, Goetz CG, Kordower JH, et al. A double-blind con- trolled trial of bilateral fetal nigral transplantation in Parkinson’s disease. Ann Neurol 2003;54:403–414.

[9] Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD. Effi- cient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000;18:675–679.

[10] Cho MS, Lee YE, Kim JY, et al. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 2008;105:3392–3397.

[11] Kawasaki H, Suemori H, Mizuseki K, et al. Generation of dopami- nergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A 2002;99:1580–1585.