EGF enhances HSC regeneration following myelosuppressive injury

Maintenance of the hematopoietic system requires constant replenishment of mature blood cells from HSCs.  For patients with malignant and non-malignant disorders of the blood and immune system, myeloablation and subsequent HSC transplantation is often necessary. describe the imageHowever, exposure to ionizing radiation to induce myeloablation also causes DNA damage that can induce cell-cycle arrest or apoptosis of HSCs and their progenitor cells 1.  Studies have shown that treatment with cytokines can prevent cell-cycle arrest.  For example, administration of stem cell factor (SCF) before radiation exposure protected mice from radiation-induced lethality by inducing HSCs into late S phase 2, which is the most radioresistant phase of the cell cycle.  Further studies to identify additional cytokines that mediate HSC regeneration following radiation exposure are critical for the development of therapies to minimize myelosuppression in patients receiving chemotherapy.

Recently, in Nature Medicine, Doan et al discovered a new function of epidermal growth factor (EGF) signaling in regulation of HSC regeneration following myelosuppressive injury 3.  The authors previously generated a mouse model in which pro-apoptotic proteins, BAK and BAX, were deleted in Tie2+ bone marrow endothelial cells 4.  Mice lacking BAK and BAX expression demonstrated significantly increased numbers of HSCs and progenitor cells and increased survival following total body irradiation (TBI) compared to wild-type mice expressing the pro-apoptotic proteins.  This was the first indication that bone marrow endothelial cells might have therapeutic potential in enhancing hematopoietic reconstitution following myelosuppression.  However, the mechanism through which these cells regulate hematopoietic regeneration was unknown.

In their most recent study, Doan et al performed a cytokine array on bone marrow serum from mice lacking BAK and BAX expression and found a significant enrichment of EGF compared to wild-type mice 3.  Using multiparametric flow cytometry, they demonstrated that ~9% of c-Kit+Sca-1+LinSLAM+ HSCs express functional EGF receptor (EGFR), and expression increased by 6-fold following irradiation.  Systemic administration of EGF augmented HSC recovery in vivo and improved the survival of mice following TBI compared to saline-treated control mice.  In contrast, administration of erlotinib, an EGFR antagonist, suppressed HSC regeneration and significantly decreased the survival of mice following TBI, further suggesting that EGFR signaling is critical for radioprotection of bone marrow HSCs and progenitor cells.  They found that EGFR signaling promotes HSC proliferation by activation of the PI3K-AKT pathway.  In addition, EGF treatment inhibited expression of the p53 upregulated modulator of apoptosis (PUMA), an essential mediator of radiation-induced HSC apoptosis.

describe the imageIn summary, EGF promotes HSC cycling and survival following radiation-induced myelosuppression.  The study by Doan et al was the first demonstration that bone marrow HSCs express functional EGFR, and that EGFR signaling plays a role in HSC self-renewal.  The results of this study suggest that EGF may have therapeutic potential to enhance hematopoietic regeneration in patients receiving myelosuppressive chemotherapy or undergoing HSC transplantation.




1. Liu, Y. et al. p53 regulates hematopoietic stem cell quiescence. Cell Stem Cell 4, 37-48, doi:10.1016/j.stem.2008.11.006 (2009).

2. Zsebo, K. M. et al. Radioprotection of mice by recombinant rat stem cell factor. Proc Natl Acad Sci U S A 89, 9464-9468 (1992).

3. Doan, P. L. et al. Epidermal growth factor regulates hematopoietic regeneration after radiation injury. Nat Med, doi:10.1038/nm.3070 (2013).

4. Doan, P. L. et al. Tie2(+) Bone Marrow Endothelial Cells Regulate Hematopoietic Stem Cell Regeneration Following Radiation Injury. Stem Cells, doi:10.1002/stem.1275 (2012).

The Promise of Immortalized Neural Stem Cells in CNS Cell-Based Therapies

Cell replacement therapy (CRT) and cell-based therapy (CBT) have provided promising therapeutic strategies for treatment of several human neurological diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD) and malignant gliomas (GBM).  The four most-studied cell types considered viable candidates for development of CRT and CBT for these neurological diseases consist of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs). Although generation of different types of neurons and glial cells in vitro have been demonstrated by all these pluripotent cells, there are significant obstacles to the clinical utilization of stem cell-derived neurons or glial cells in CBT: First concern, aroused by previous studies, involves the long- term survival and phenotypic stability of stem cell-derived neurons or glial cells in vivo following transplantation. Second limitation is the high risk of any highly purified populations of neuronal cell type derived from ESCs, iPSCs, MSCs or NSCs, containing other neuronal/glial cell types, which may cause unfavorable interactions among grafted cells and/or with host central nervous system (CNS). Finally, the subpopulation (regardless of how small) of ESCs, iPSCs, MSCs or NSCs that did not completely differentiate, introduce a significant risk of tumorigenesis within the host CNS following transplantation. Furthermore, there are practical caveats, such as sustainable clinically approved, industrial quantity of these cells, which remain to be addressed.

In a recent review article published in the Journal of Neuropathology Seung U. Kim’s group have proposed utilization of immortalized human NSC lines as the cell-source for CBT in neurological diseases, as the best suited candidate. Kim’s group have previously generated clonally derived several immortalized human NSC lines, one of which has been particularly well characterized and currently used as a glioma therapy agent in phase II clinical trials. This particular line, named HB1.F3, was originally obtained from a fetal human telencephalon at 15 weeks gestation and immortalized by an amphotropic replication-incompetent retroviral vector, pLCN.v-myc, which encodes the v-myc oncogene.  This method of immortalization is not only safe, but also overcomes the issue of spontaneous differentiation, resulting in a non- tumorigenic, homogeneous NSC line.

Stem Cells,Cell-based therapies,PD,AD,ALS

HB1.F3’s exhibit normal human karyotype of 46XX, they are self-renewing and multipotent, capable of differentiating into neurons, astrocytes and oligodendrocytes, both in vivo and in vitro.  They express genes that encode for neurotrophic factors, such as for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF)-1, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), which can potentially make them a therapeutic agent rendering neuroprotection for neurons affected by injury or disease.

Kim’s group has reported functional improvement in a rat model of PD following HB1.F3 transplantation into the striatum. In yet another study, they show functional recovery in HD rat model, upon intravascular (iv) administration of HB1.F3s; their data suggests that the improvements observed here is due to the neuroprotection provided by HB1.F3s’ secretion of BDNF, since this factor has been previously shown to block neuronal injury under pathological conditions in animal models of HD. Another interesting outcome to this study is the integration of HB1.F3s in the striatum and homing to the site of neuronal injury, following their iv administration, indicative of their ability to freely cross the BBB. 

In AD patients, low levels of acetylcholine (ACh) is one contributing cause of cognitive impairment. The lack of sufficient ACh is due to the decreased activity of choline acetyltransferase (ChAT) that synthesizes ACh. Kim’s group transduced HB1.F3s, over-expressing the ChAT gene (F3.ChAT) and transplanted these NSCs into the brain of AD animal models. Their results show the functional recovery of presynaptic cholinergic system and fully restored learning and memory. Moreover, they generated motor neurons from HB1.F3s- encoding Olig2 basic helix loop helix (bHLH) transcription factor gene with sonic hedgehog (Shh) protein (F3.Olig2-Shh)- and transplanted them into L5 of the spinal cord of ALS animal model. This resulted in significantly delayed onset of the disease and prolonged average survival.

Neural Stem Cells,Huntington's,Parkinson's,Alzheimers

Utilization of HB1.F3s in human clinical trials was one of the first FDA permitted clinical trials in the United States, to use genetically modified human stem cells in maligant brain tumor CBT. Furthermore, the findings reported here do indicate that immortalized human NSCs are an effective source of cells for genetic manipulation and gene transfer into the CNS, for treatment of several neurological disorders. However, autologous iPSC-derived CNS cells seem to be a more promising strategy for CRT. This is mainly due to the risks associated with introducing immortalized cells, which may not survive long term post-transplantation. Nonetheless, all the mentioned stem cell sources have interesting characteristics that make each type suitable for treating different disorders.


Further Reading:

Neural Stem Cell-Based Treatment for Neurodegenerative Diseases

Contact and Encirclement of Glioma Cells in Vitro is an Intrinsic Behavior of a Clonal Human Neural Stem Cell Line.

Defining Human PBMC T cell activation markers. Part 2: CD71 and CD95

In a previous posting, I discussed the use of T cell activation markers as a strategy for assessing the function of T cells from human peripheral blood mononuclear cells (PBMC). Following T cell receptor (TCR) activation, T cells will express a series of activation markers that include chemokine and cytokine receptors, adhesion molecules, co-stimulatory molecules, and MHC-class II proteins. Understanding what these activation markers are, when they are expressed, and their role in T cell function during normal responses and disease states is important when selecting markers for assessing T cell biology for studies on human PBMC.

In the previous posting, I discussed two immediate early activation markers for assessing the activation status of human PBMC T cells: CD69 and CD40L.  In this article, the second in this series, I will discuss two additional mid-early T cell activation markers that can be assessed by flow cytometry: CD71 and CD95.

CD71 (TFRC, Transferrin Receptor, TfR) is a cell surface iron transport receptor that is upregulated in proliferating cells by 24-48 hours following T cell activation and expression continues to rise and is maintained for several days.  Thus CD71 can be considered a mid-early activation marker as compared with late activation markers that are not appreciably upregulated until even later time points.  CD71 has been shown to associate with the TCRz chain and ZAP70 and may participate in TCR signaling, and is an essential factor for proliferating T cells.

The inability of CD71 to be upregulated following TCR activation may be associated with T cell dysfunction.  As was similarly discussed for CD69, Critchley-Thorne et. al, 2007 showed that PBMC T cells from metastatic melanoma patients had reduced CD71 upregulation compared with healthy controls, and this corresponded with multiple other functional defects in T cells from these patients.  Thus CD71 may be aberrantly expressed by T cells in human disease.

fas signalingCD95 (Fas, APO-1, TNFRSF6) is a member of the TNF-receptor superfamily and is best known for its role in mediating activation-induced cell death in activated T cells following binding to its ligand, CD95L/FasL induced on antigen-presenting cells (APCs).  However, CD95 can also play additional, non-apoptotic roles in the modulation of T cell function.  CD95 ligation has been shown to inhibit TCR signaling and activation of naïve T cells.  However, this negative co-stimulatory effect appears to be dose-dependent, as low doses of CD95 agonists had the opposite effect and strongly promoted activation and proliferation of T cells.  Like CD71, CD95 expression can be detected by 24 hours following T cell activation and continues to increase over the course of several days.

Due to its differential roles in regulation of T cell apoptosis and activation, dysregulated expression of CD95 or its ligand CD95L could be avenues for T cell dysfunction in various human diseases.  Indeed, Strauss et. al, showed that regulation of CD95L expression may play a role in immune evasion during viral infections. CD95L was upregulated in HIV-infected APCs, and led to suppressed T cell activation.  Interferons are known to enhance CD95 expression, and our group (Critchley-Thorne et. al, 2009) has shown reduced upregulation of CD95 in PBMC T cells from breast cancer patients following T cell activation in the presence of interferons, indicating the lack of full T cell activation under these conditions.

Thus both CD71 and CD95 are upregulated in the mid-early phase of T cell activation and dysfunctional expression may be useful measures of T cell dysfunction in various disease states. Thus, these may be useful markers when assessing the phenotype and function of human PBMCs.


Additional Reading:

Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function.  Reddy M, Eirikis E, Davis C, Davis HM, Prabhakar U. J Immunol Methods. 2004 Oct;293(1-2):127-42.

Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Biselli R, Matricardi PM, D’Amelio R, Fattorossi A. Scand J Immunol. 1992 Apr;35(4):439-47.

Surface markers of lymphocyte activation and markers of cell proliferation.  Shipkova M, Wieland E.  Clin Chim Acta. 2012 Sep 8;413(17-18):1338-49.

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.  Caruso A, Licenziati S, Corulli M, Canaris AD, De Francesco MA, Fiorentini S, Peroni L, Fallacara F, Dima F, Balsari A, Turano A.   Cytometry. 1997 Jan 1;27(1):71-6.

Transferrin receptor induces tyrosine phosphorylation in T cells and is physically associated with the TCR zeta-chain.  Salmerón A, Borroto A, Fresno M, Crumpton MJ, Ley SC, Alarcón B. J Immunol. 1995 Feb 15;154(4):1675-83.

Transferrin synthesis by inducer T lymphocytes.  Lum JB, Infante AJ, Makker DM, Yang F, Bowman BH. J Clin Invest. 1986 Mar;77(3):841-9.

Down-regulation of the interferon signaling pathway in T lymphocytes from patients with metastatic melanoma.  Critchley-Thorne RJ, Yan N, Nacu S, Weber J, Holmes SP, Lee PP. PLoS Med. 2007 May;4(5):e176.

Pro- and anti-apoptotic CD95 signaling in T cells.  Paulsen M, Janssen O. Cell Commun Signal. 2011 Apr 8;9:7.

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling.  Strauss G, Lindquist JA, Arhel N, Felder E, Karl S, Haas TL, Fulda S, Walczak H, Kirchhoff F, Debatin KM.  J Exp Med 2009, 206:1379-1393.

Impaired interferon signaling is a common immune defect in human cancer.  Critchley-Thorne RJ, Simons DL, Yan N, Miyahira AK, Dirbas FM, Johnson DL, Swetter SM, Carlson RW, Fisher GA, Koong A, Holmes S, Lee PP. Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):9010-5.

*Image courtesy of*



Inhibition of cell death or apoptosis has been implicated in the chemotherapeutic resistance of tumor cells. TRAIL (tumor-necrosis-factor-related apoptosis inducing ligand; is involved with induction of apoptosis in tumor cells thereby represents an attractive target for the development of anticancer therapy. A type II transmembrane protein and a member of tumor necrosis factor family, TRAIL is expressed in wide range of tissues. Even though it is a membrane protein, TRAIL is also expressed in soluble form. Induction of TRAIL-mediated apoptosis involves binding of TRAIL with its receptor TR1 (also known as death receptor 4, DTRAIL signaling resized 600R4) and TR2 (DR5). TRAIL also binds with two other receptors TR3 and TR4. Unlike TR1 and TR2, these receptors have incomplete death domains. Elevated expression of TR3 and TR4 in normal cells is thereby suggested to protect normal cells from TRAIL-induced death signaling. Induction of apoptotic signaling involves binding of TRAIL to DR4 or DR5. This results in homotrimerization and activation of receptors, enabling the receptors’ death domain to recruit the adaptor protein Fas-associated death domain along with pro-caspase- 8 and pro-caspase- 10. These all together then form the multi-protein death-inducing signaling complex, (DISC). Inside the DISC procaspeses become autoactivated and become caspase-8/10. Activated caspase-8/10 then activates downstream effector caspase-3 or caspase-7. Finally, cleavage of downstream substrates by effector caspases results in DNA fragmentation leading to apoptosis.

Several studies assessed efficacy of TRAIL-based therapies in cancer as a single agent or in combination with other anti-cancer agents. In monotherapy, monoclonal antibodies conatumumab, AMG 655, CS-1008, dulanermin, exatumumab, and mapatumumab exhibited responses in follicular lymphoma, lung adenocarcinoma and in a rare form cancer synovial sarcoma with minimal toxicity. In addition, efficacy of TRAIL-monoclonal antibodies has also been evaluated in combination with anti-cancer agents such as paclitaxel, cisplatin, carboplatin, gemcitabine, histone deacetylase inhibitors, proteosome inhibitors, and kinase inhibitors. However, several cases of adverse effects and toxicities were reported in these studies. In addition, recent TRAIL-based therapies are expensive to produce for clinical applications and may also be limited by endogenous TRAIL level as well as dependency on tumor suppressor gene p53 as a regulator of TRAIL gene transcription. Therefore, studies are required to overcome the shortcoming of current TRAIL-based therapies.

A preclinical study published recently in Science Translational Medicine (Allen et al., 2013) reported that small-molecule drug TRAIL-inducing compound 10 (TIC10) can trigger tumor cell death in a variety of cancers including breast, colon, lung, and also gliobalstoma, a type of brain tumor that is very difficult to treat. The anti-tumor activity of TIC10 is mediated by the activation of TRAIL in Foxo3a dependent manner. In their study, Allen et al. observed significant tumor regression in human colon, triple-negative breast, non-small cell lung, and brain cancer xenograft-bearing mice following single-dose treatment of TIC10. Inactivation of both phosphoinositide 3-kinase/Akt (PI3-K/Akt) and mitoHuman glioblastoma bearing mouse recovered following TIC10 treatment compared to untreated.gen-activated protein kinase (MAPK) pathways was noted following TIC10 treatment in this in vivo study that resulted in translocation of the transcription factor Foxo3a into the nucleus, where Foxo3a induced expression of TRAIL gene to activate apoptosis.

In the pre-clinical study, small-molecule drug TIC10 showed potent anti-tumor activity with limited toxicity through sustained induction of TRAIL even in refractory brain malignancy in a p53-independent manner. This revives the hope of TRAIL-based therapies in cancer. However, clinical trials need to be conducted first in order to confirm safety and efficacy of TIC10 before it can ultimately be used within the clinical setting.



1.         Allen JE, Krigsfeld G, Mayes PA, et al. Dual Inactivation of Akt and ERK by TIC10 Signals Foxo3a Nuclear Translocation, TRAIL Gene Induction, and Potent Antitumor Effects. Sci Transl Med. 2013;5(171):171ra117.

2.         Hellwig CT, Rehm M. TRAIL signaling and synergy mechanisms used in TRAIL-based combination therapies. Mol Cancer Ther. 2012;11(1):3-13.

3.         Dimberg LY, Anderson CK, Camidge R, Behbakht K, Thorburn A, Ford HL. On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics. Oncogene. 2012.


Epithelial to Mesenchymal Transition: the Key to Cancer Metastasis

We have all seen science fiction movies where alien invaders, in search of a fertile planet like earth, start a full scale war against humanity. A common theme in many of these stories, but often overlooked, is the role of the scout. Before any invasion, a solitary member of the invaders, fortified for the harsh journey, is sent on a quest to scout out the new land. As soon as this scout finds a promised land, the colony is formed and the invasion begins. This concept of invasion is seen in nature with viral and bacterial infections, where fortified virus or sporulated bacteria are able to survive harsh conditions and then proliferate in their host system upon arrival. In an ironic display, cancer metastasis follows a similar system; cancer cells are able to leave the primary tumor, travel long distances in harsh conditions, and form colonies in other tissues within an organism. These metastases have grave consequences. In fact in certain cancers, such as melanoma, it is metastasis to vital areas like the brain that makes it life-threatening. One of the biggest areas in cancer biology research is elucidating the mechanisms involved in cancer metastasis, which includes the concept of the epithelial to mesenchymal transition (EMT). It is this process that gives the cancer “scouts” the means to invade vasculature, fortify themselves for a journey to the metastatic site, and resist most therapies at all locations in the tumor-bearing patient. In normal development, the epithelial to mesenchymal transition is a reversible system that is involved in embryonic gastrulation1 and cardiac development1,2. In adulthood, EMT is involved in wound healing3. Unfortunately, EMT is also involved in pathological events such as fibrosis of injured tissue1 and cancer development and progression1-4. Here, we focus on EMT’s role in tumorigenesis, cancer proliferation, treatment resistance, and metastasis.

The epithelial to mesenchymal transition occurs in many different solid tumors of epithelial origin. Many tumors are carcinoma based, which implies that they are usually confined from motility by the basement membrane1 but the process of converting toward a cancer cell that has mesenchymal characteristics allows cancer cells to infiltrate the circulatory and lymphatic systems, causing motility that will later lead to metastasis1,5. As a cancer progresses, epithelial features, such as cellular adhesion marker  E-cadherin3 and intracellular adhesion components such as tight junctions, cytokeratins, and desmosomes, are downregulated5. Coordinating with this downregulation is an upregulation of mesenchymal markers such as N-Cadherin, Vimentin, and Fibronectin3. This phenotypic shift is orchestrated by a coordination of many different mechanisms: epigenetic changes, post-translational modifications of proteins, transcriptional silencing by noncoding-RNAs (ncRNAs), and activation of epithelial to mesenchymal transcription factors (EMT-TFs)3. However, while all mechanisms of EMT development are important, this article will focus on the main orchestrators of EMT: the EMT-TFs.

EMT-TFs are many, but the main transcription factors fall into three families: SNAIL, ZEB, and TWIST. SNAIL has three main transcription factors: SNAIL (Snail1), SLUG (Snail2), and SMUC (Snail3)5. These proteins are zinc finger nucleases that are at the crux of EMT phenotype. In fact, it has been noted that SNAIL, and possibly SLUG, directly repress the expression of E-cadherin by binding to its promoter, CDH13. Furthermore, the SNAIL family has been shown to repress desmoplakin, adherens junctions, occulidins, and cytokeratin upon activation5. While not much is known about SMUC’s role in normal or pathological development, SNAIL, unlike SLUG, is so crucial to cancer metastasis that has been implicated in being an independent prognostic factor in the metastatic potential and severity of cancers5. Interestingly, not only does the SNAIL family propagate the characteristics of EMT transition, they also upregulate other EMT-TFs, such as ZEB1 and ZEB2, to further propagate the mechanism of EMT 5.

The Zinc-finger E-box-binding homeobox (ZEB) family are also a family of transcription factors with zinc-finger nuclease properties3. Like SNAIL, ZEB1 and ZEB2 bind to the E-cadherin promoter5. Moreover, ZEB1 and ZEB2 also downregulate tight/gap junctions, desmosomes and markers of polarity5. In addition, the ZEB family has been implicated in repressing P- and R-cadherins, other markers that inhibit the motility of epithelial cells5. ZEB1 is usually not found on non-cancerous cells but is found highly expressed on many cancer types5. In contrast, ZEB2 is expressed on normal epithelial cells, but is vastly upregulated in cancerous cells5.

describe the imageThe final group is the TWIST family, a basic helix-loop-helix transcription factor involved in different steps in embryonic development1. Interestingly, while TWISTs are vital to embryogenesis, they are absent in normal adult epithelium5. As a cancerous cell progresses , TWIST1 and TWIST2 appear and their reactivity increases in correlation with the tumorigenic progress5. Interestingly, while TWIST1 is involved  in regulating some of SLUG’s EMT effects, and directly drives expression of N-cadherin, it is not directly associated with the downregulation of E-cadherin5.

It is through these EMT-TFs that EMT is involved in tumorigenesis and tumor progression. EMT-TFs are involved in suppressing senescence and increasing cell cycle proliferation5. However, EMT-TFs are not enough to push tumorigenesis on their own, but require another event of tumorigenesis3. Therefore, EMT-TFs may act as a facilitator of tumorigenesis, but not be tumorigenic factors by themselves. EMT is also crucial for tumor invasiveness and metastasis. Besides the aforementioned changes in cellular adhesion molecules, activation of the EMT-TFs causes upregulation of matrix-metalloproteinases (MMP), enzymes involved in degradation of the extracellular matrix and invasion of cancer cells3. It has been demonstrated that the SNAIL family activates the expression of MMP1, MMP2, MMP7, and MT1-MP 5. Furthermore, the upregulation of MMPs additionally activates EMT-TFs, thus forming a feed-forward loop3. EMT events may also go beyond extracellular degradation; TWIST1 is known to be involved in the formation of invadopodia which has been correlated with invasiveness3.

The invasion of metastasis is not just due to intrinsic changes but also to changes in the host, and target microenvironment4. For instance, TGF-β, a cytokine that normally acts as a tumor suppressor, can enhance tumor invasion in later-stage tumors2. Furthermore, TGF-β activates the SNAIL and TWIST families of the EMT-TFs2,5. TGF-β may be produced by myeloid derived suppressor cells and CD11b+/F4/80+ tumor associated macrophages (TAMs) in the primary tumor microenvironment, thus perpetuating the EMT phenotype4. TAMs also express the cytokines fibroblast growth factor (FGF), epidermal growth factor (EGF), and macrophage colony stimulating factor (CSF-1) which are involved in EMT-based invasion as well as recruitment of immune cells toward a metastatic-favored microenvironment4. Moreover, TNF-α, usually correlated with  an anti-tumor phenotype, also stabilizes SNAIL expression, thus implicating it in facilitating EMT3. Besides immune cells, other stromal cells are involved in EMT initiating and metastasis. For instance, mesenchymal stem cells (MSCs) and cancer associated fibroblasts (CAFs) are both involved in EMT initiation and propagation4.

One of the more clinically relevant aspects of EMT in cancer progression is the ability of EMT to confer therapy resistance. Resistance to doxorubicin is breast cancer correlates with higher levels of ZEB15 and both ZEB1 and ZEB2 have been shown to guard against cisplatin therapy5. TWIST1 mediates resistance to paciltaxel and TWIST1 and TWIST2 block daunorubicin by inhibiting degradation of the anti-apoptotic protein, Bcl-2, in bladder, ovarian, and prostate cancer5. Along the same lines, the EMT has been shown to confer upon cells a cancer stem cell-like phenotype2,4, a cancer phenotype also known for its therapy resistance5. TGF-β-driven EMT activates protein involved in stem cell phenotype, such as Sox2, PDGFB, and LIF 2. Furthermore, ZEB1 has been shown to be vital in the formation and maintenance of stem cell phenotype in some cancers5. However, while activation of the EMT pathway may confer a cancer stem cell-like phenotype, it is not necessary to get this phenotype3 as EMT-TFs are not necessarily involved in dedifferentiation3. In fact, some reports shine controversy on EMT-TFs’ role in stem cell development: while colorectal cancer spheroids show higher levels of SNAIL, other studies have shown that overexpression of SNAIL and SLUG in ovarian cancer drives these cancers away from a stem cell phenotype5.

cancer patientBesides the difficulty in treatment that EMT poses, one of the main obstacles that is troubling the cancer metastasis field is difficulty in identification of EMT/MET in cancer in vivo4. Because EMT is an orchestration between tumor and it’s microenvironment, researchers have been unable to definitively demonstrate the role of EMT beyond stromal epithelium4. The field needs better phenotypic markers  to identify tumor epithelial cells from normal epithelial cells as well as a way to trace lineages of human cancers in vivo 4. Another is MET, the reverse of EMT and the end result of metastasis. To date, bona fide evidence for MET is only found in vitro studies and xenograft experiments4. MET explains why the phenotype of metastatic tumors mirror the primary site, but it is not an explanation for the system4. Several methods have been proposed to more appropriately study EMT, such as intravital 2-photon microscopy4. However, the sporadic nature of the EMT event makes observation, even in this system, very difficult4. In such a case, we are left to the spontaneous tumor-forming mouse models or studying xenograft models of immortalized cancer lines that are known to be highly metastatic. In addition, since there are no anatomically distinguishable between mesenchymal and epithelial cells, people have proposed the solutions of creating tumor lines the express reporter genes linked to promoters for epithelial/mesenchymal fates4. If one were able to combine an intravital two-photon system with a xenograft of a highly metastatic cancer line transduced with mesenchymal/epithelial reporter constructs, this would be the most feasible model to study EMT in real-time. As technology advances to detect individual cell populations in real-time, the expectation to solidify the mechanism of epithelial to mesenchymal transition in metastasis will increase the reliably of current EMT findings.



1          Lim, J. & Thiery, J. P. Epithelial-mesenchymal transitions: insights from development. Development 139, 3471-3486, doi:10.1242/dev.071209 (2012).

2          Massagué, J. TGFβ signalling in context. Nat Rev Mol Cell Biol 13, 616-630, doi:10.1038/nrm3434 (2012).

3          Craene, B. D. & Berx, G. Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer 13, 97-110, doi:10.1038/nrc3447 (2012).

4          Gao, D., Vahdat, L. T., Wong, S., Chang, J. C. & Mittal, V. Microenvironmental regulation of epithelial-mesenchymal transitions in cancer. Cancer Res 72, 4883-4889, doi:10.1158/0008-5472.can-12-1223 (2012).

5          Sánchez-Tilló, E. et al. EMT-activating transcription factors in cancer: beyond EMT and tumor invasiveness. Cell Mol Life Sci 69, 3429-3456, doi:10.1007/s00018-012-1122-2 (2012).


T cell dysfunction in cancer: Anergy, Exhaustion, or Senescence?

Immune responses against cancer have been shown to be effective in eliminating tumors.  However anti-tumor immunity is limited by dysfunctional T cells which have been described in cancer patients.  Understanding how dysfunctional T cells arise in cancer and the potential mechanisms for restoring functionality are critical for developing effective immunotherapeutics.  In the current issue of Current Opinion in Immunology, Dr. Weiping Zou’s group, (Crespo. et. al.,) review the various types of T cell dysfunction that occur in the tumor microenvironment.

Anergy, exhaustion, and senescence are three different mechanisms underlying T cell hyporesponsiveness, which share distinguishing phenotypic features but arise by different mechanisms and under different experimental settings.  However, what is the difference between these three types of T cell dysfunction and which contribute to impaired T cell responses in the setting of cancer?

Anergy: T cell anergy generally refers to a hyporesponsive state in T cells induced by triggering the TCR either without adequate concomitant co-stimulation through CD28 or in the presence of high co-inhibitory molecule signaling.  Without both TCR and CD28 signals, IL-2 is not effectively transcribed and instead, anergy-associated genes such as GRAIL are expressed which contribute to impaired TCR signaling via negative feedback.

In the tumor microenvironment cancer, altered expression of B7 family members by APCs leads to an enhanced expression of B7 family co-inhibitory molecules including PD-L1 and a reduction in the B7 family co-stimulatory molecules CD80 and CD86.  Thus, T cell activation in this environment could lead to induction of anergy.

Exhaustion: T cell exhaustion occurs as a result of chronic over-stimulation, such as occurs in the settings of chronic viral infections including hepatitis C virus (HCV) and HIV, autoimmunity, and cancer.  Exhausted T cells progressively lose the ability to express effector cytokines including IL-2, IFNg, and TNFα.  They also express multiple inhibitory receptors including PD-1 and LAG-3, lose cytotoxic and proliferative potential, and may ultimately be driven to apoptosis.

Because anti-tumor T cells are persistently exposed to antigen in the tumor microenvironment, exhaustion is a likely mechanism contributing to T cell dysfunction in cancer patients.  As such, exhausted T cells have been described in patients with melanoma, ovarian cancer and hepatocellular carcinoma.

Senescence:  Senescence is thought to occur due to the natural life span, or aging of cells.  However, senescent T cells have been observed in the settings of chronic inflammation and persistent infection in young individuals, indicating other factors beyond a person’s age, such as DNA damage, drive acquisition of this state.  Senescent T cells are marked by deficient CD28 expression, telomere shortening, expression of regulatory receptors such as TIM-3 and KLRG-1, and inability to progress through the cell cycle.

Cells with features of senescence have been described in patients with lung cancer, head and neck cancer, hepatocellular carcinoma, melanoma and lymphoma.  Thus, senescence may also contribute to T cell dysfunction in cancer patients.

In conclusion, there is evidence that anergy, exhaustion, and senescence may all be contributing toward T cell dysfunction in cancer.  In the review, Crespo. et. al., make the point that distinguishing cells in these states may be complicated as they overlap phenotypically and in expression of various markers.  Furthermore, the mechanisms mediating establishment of these three states is not well defined.  However, it is important to clarify the mechanisms by which T cells gain and maintain dysfunction in cancer in order to best develop effective immunotherapeutics.


Further Reading:

 T cell anergy, exhaustion, senescence, and stemness in the tumor microenvironment.  Crespo J, Sun H, Welling TH, Tian Z, Zou W.  Curr Opin Immunol. 2013 Jan 5.

The three main stumbling blocks for anticancer T cells.  Baitsch L, Fuertes-Marraco SA, Legat A, Meyer C, Speiser DE. Trends Immunol. 2012 Jul;33(7):364-72.

Induction of T cell anergy: integration of environmental cues and infectious tolerance. P. Chappert, R.H. Schwartz.  Curr. Opin. Immunol., 22 (2010), pp. 552–559.

T cell exhaustion.  E.J. Wherry.  Nat Immunol, 12 (2011), pp. 492–499.

T-cell senescence: a culprit of immune abnormalities in chronic inflammation and persistent infection.  A.N. Vallejo, C.M. Weyand, J.J. Goronzy.  Trends Mol Med, 10 (2004), pp. 119–124.

Recent Findings in Multiple Sclerosis Treatment

Multiple sclerosis (MS) is believed to be a neurodegenerative autoimmune disorder, in which the body’s immune system attacks its own healthy tissue, specifically the myelin sheath surrounding the axons of the central nervous system (CNS). The word sclerosis refers to the scar tissues or the plaques within brain’s white matter, observed through the Magnetic Resonance Imaging (MRI) of MS patients’ brains. These plaques are results of myelin-degeneration (demyelination) and axonal death. The progression of MS symptoms is directly proportional to the failure of remylination by oligodentrocytes, leading to neurodegeneration; this demyelination disrupts the proper conduction of action potentials from CNS to different target organs and will eventually results in permanent disability caused by chronically demyelinated plaques. Some of the common symptoms include changes in sensation, muscle weakness, abnormal muscle spasms, or difficulty moving and maintaining balance, problems in speech or swallowing, as well as visual problems.

Multiple Sclerosis,MS,MRI,Inflammatory

Therefore, in addition to inhibiting the autoimmunity, preventing permanent neurodegeneration as well as functional recovery of oligodentrocytes, are current therapeutic targets in MS clinical research. The focus of this article is to discuss the several recent studies that have reported promising results to this end.

Acid-Sensing Ion Channels (ASICs) are neuronal voltage-insensitive cationic channels activated by extracellular hydrogen ions (H+), and mediate entering and excessive accumulation of sodium (Na+) and calcium ions (Ca2+) inside the neuron’s cytoplasm. This intra-axonal accrual of Na+ and Ca2+ ions causes cellular injury and subsequent neurodegeneration in the CNS. Over-expression of ASIC1 has been observed in acute MS lesions (oligodendrocytes and axons with an injury co-express ASIC1 in chronic MS lesions) and believed to play a role in the development of irreversible tissue damage.

Moreover, amiloride, a potassium sparing diuretic (causes excretion of large amounts of potassium from the body), blocks ASICs by acting as “channel-blocker” and has been used for hypertension and congestive heart failure management. In a recent translational clinical study, effects of ASIC1-inhibition was tested in 14 patients with primary progressive MS, by comparing the rates of brain atrophy and tissue damage before and during amiloride treatment for 3 years. The results of this preliminary study show a significant decrease in the rate of whole-brain atrophy during the amiloride treatment period, which indicates reduced neurodegeneration (cell damage) through ASIC blocking. Although further studies with larger populations are needed to confirm the robustness of these observations, this is a safe, inexpensive promising potential neuroprotective MS treatment that may be utilized in conjunction with anti-inflammatory agents.

MS,Multiple sclerosis,Myelin,autoimmune,axon

As mentioned previously, failure of oligodendrocytes to remyelinate leads to the severe clinical impairments associated with MS, which makes myelin- regeneration a significant therapeutic goal. Even though oligodendrocyte precursor cells are present, they fail to mature and myelinate in MS brain. Some of the key factors stimulating migration, maturation and survival of myelinating oligodendrocytes are components of extracellular matrix (ECM).

The ECM component in areas with MS lesions have significant differences when compared with the healthy adult brain tissue: two of these ECM abnormalities are the increased expression of Laminin, as well as upregulation of Fibronectin molecule that is absent in the normal brain’s white matter. Therefore, it has been speculated that fibronectin expression in the injury environment may inhibit oligodendrocyte maturation, contributing to remyelination failure in MS plaques.

A recent study suggests that the MS inflammation in the CNS causes astrocytes to accumulate fibronectin, which impair remyelination within the chronic lesions. These findings offer new strategic clinical approaches to promote remyelination through inhibiting fibronectin aggregation and its clearance from the inflammatory sites within the parenchyma.

Interestingly, another group have recently demonstrated a strong remyelinating effect of testosterone mediated by its receptor. They propose promotion of remyelination in males with MS, through utilizing synthetic drug which specifically bine the brain androgen receptors, employing testosterone as remyelinating agents.

Although there is no approved method to efficiently treat MS up to date, there are increasing reports on not only the disease’s underlying mechanisms, but also promising clinical strategies that are rapidly moving to human clinical trials. To learn about the role of the Blood Brain Barrier and other 2013 findings in MS, visit


Multidrug-Resistance in Cancer: ABC-Transporters

Multidrug-resistance (MDR) is the chief limitation to the success of chemotherapy. According to the National Cancer Institute, multidrug-resistance is a phenomenon where cancer cells adopt to anti-tumor drugs in such a way that makes the drugs less effective. Studies have shown that 40% of all human cancers develop MDR. Deaths due to cancer occur in most of the cases when the tumor metastasizes. Chemotherapy is the only choice of treatment in metastatic cancer, and MDR limits that option.

Cancer one resized 600As shown in Figure 1, tumor cells adopt several mechanisms to evade death induced by anti-tumor agents. These include changes in apoptotic pathways and activation of cell-cycle check points to increase DNA repair. Alternatively, cancer cells develop resistance by increased expression of multidrug-resistant proteins and altered anti-tumor drug transport mechanisms. Members of the ABC transporters (ATP-binding cassette) are known to be associated with this phenomenon, as the human genome express over 48 genes in this transporter family alone. These proteins bind ATP in their ATP binding domain and use the energy to transport various molecules across the cell, thus they are known as ABC proteins. Among these proteins, P-glycoprotein (Pgp, ABCB1), multidrug resistance-associated protein (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2) are chiefly responsible for drug resistance in tumor cells. Studies are warranted to determine the role of other members of ABC transporters including MRP2, MRP3, MRP4, MRP5, ABCA2 and BSEP in drug-resistance.


cancerPlasma membrane glycoprotein (Pgp) was the first ABC-transporter detected in various cancers exerting resistance to a variety of chemically unrelated cytotoxic agents including anti-tumor drugs such as doxorubicin, vinblastine, ritonavir, indinavir and paclitaxel. It works as an energy-dependent efflux pump and can recognize a wide range of substrates. Even though this protein normally protects us from endogenous and exogenous toxins by transporting them out of the cells, the transporter causes a major problem in the bioavailability of anti-tumor drugs to tumor cells during chemotherapy.

Clinical Significance

Pgp plays an important role in altering the pharmacokinetics of a wide variety of drugs. This efflux pump creates a major physiological barrier in pharmacokinetics of drugs because of its localization at the site of drug absorption and elimination. Tumors with detectable levels of Pgp are 3-4 fold more susceptible to chemotherapeutic failure than Pgp negative tumors. Therefore the role of Pgp in the development of MDR is very significant: it has been used as a potential target for reversing clinical MDR. To overcome Pgp mediated drug resistance several inhibitors are developed and currently in clinical trials which include verapamil, cyclosporin A, quinine, and tamoxifen.

Multidrug Resistance Protein 1 (MRP1)

Like Pgp, MRP1 is also overexpressed in tumor cells and represents a major obstacle to drug delivery. MRP1 is ubiquitiously expressed in the lung, testis, kidney, and peripheral blood mononuclear cells in humans.

Clinical Significance

High levels of expression of MRP1 protein was observed in non-small-cell lung cancer. In breast cancer, there is also a significant expression of this protein which may increase the chance of treatment failure. Studies have also shown that over expression of MRP causes resistance to methotrexate (MTX) and antifoliates such as ZD1694 in colorectal cancer. Development of MRP1 inhibitors is in progress. In preclinical studies, effective inhibition of MRP1 was observed following treatment with MK571 and ethacrynicacid.

Breast Cancer Resistance Protein (BCRP)

BCRP belongs to a novel branch of the ABC-transporter family. The members of this subfamily are about half the size of the full-length ABC transporters, thus known as half-transporters. Overexpression of BCRP was reported in the plasma membrane of  drug-resistant ovary, breast, colon, gastric cancer, and fibrosarcoma cell lines. Even though the normal physiological function of BCRP has not been determined, it is possible that BCRP plays an important role in drug disposition. The overexpression of this protein causes reduced accumulation of chemotherapeutic agents such as mitoxantrone, irinotecan, SN-38, topotectan, and flavopiridol.

Clinical Significance

Since BCRP is expressed in the gastrointestinal tract, it is thought that this protein may affect the bioavailability of the drugs. Its overexpression in several types of cancer makes it a relevant target of strategies aimed at defeating multidrug-resistance. Some of the potent inhibitors of BCRP are Fumitremorgin C, reserpine and tryprostatin A.

Cancer defends itself against chemotherapeutic regimes by several mechanisms including MDR. Therefore, a detail understanding of ABC-transporters mediated drug resistance would help to formulate strategies to overcome this problem. Screening of novel inhibitors of ABC-transporters which are not effluxed by these transporters is currently in progress. One of these drugs, ixabepilone, has been approved in the United States for the treatment of breast cancer patients pretreated with an anti-tumor agent. Ongoing efforts to circumvent MDR also include development of potentially effective alternative strategies (e.g drug delivery using liposomes or nanoparticles, inhibition of expression of MDR associated ABC-transporters using monoclonal antibodies). Promising experimental data on these strategies suggest their potentialily to overcome important causes of MDR to significantly improve cancer treatment.



1.         Türk D, Hall MD, Chu BF, et al. Identification of compounds selectively killing multidrug-resistant cancer cells. Cancer Res. 2009;69(21):8293-8301.

2.         Litman T, Druley TE, Stein WD, Bates SE. From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance. Cell Mol Life Sci. 2001;58(7):931-959.

3.         Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer. 2002;2(1):48-58.

4.         Gottesman MM, Ludwig J, Xia D, Szakács G. Defeating drug resistance in cancer. Discov Med. 2006;6(31):18-23.

5.         Nobili S, Landini I, Mazzei T, Mini E. Overcoming tumor multidrug resistance using drugs able to evade P-glycoprotein or to exploit its expression. Med Res Rev. 2012;32(6):1220-1262.

Upcoming Immunology Conferences: March – June, 2013

antibodiesI previously posted about 2013 Conferences in Tumor Immunology and Cancer ImmunotherapyThis listing will include other upcoming Immunology-related conferences.


Keystone Symposium: Understanding Dendritic Cell Biology to Improve Human Disease

March 3 – 8, 2013.

Keystone, Colorado, USA.

Registration Deadline: March 3, 2013.


Gordon Research Conference: Cell Biology of Megakaryocytes & Platelets

March 10 – 15, 2013.

Galveston, Texas, USA

Application Deadline: February 10, 2013.


World Immune Regulation Meeting (WIRM) VII: Innate and Adaptive Immune Response and Role of Tissues in Immune Regulation

March 13 – 16, 2013.

Congress Center, Davos, Switzerland.

Registration is still open online.


Keystone Symposium: Host Response in Tuberculosis

This is a joint meeting with the Keystone meeting on Tuberculosis: Understanding the Enemy

March 13 – 18, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: March 3, 2013.


Keystone Symposium: Tuberculosis: Understanding the Enemy

This is a joint meeting with the Keystone meeting on Host Response in Tuberculosis.

March 13 – 18, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: March 3, 2013.


Keystone Symposium: Immune Activation in HIV Infection: Basic Mechanisms and Clinical Implications

April 3 – 8, 2013.

Breckenridge, Colorado, USA.

Registration Deadline: April 3, 2013.


Canadian Society for Immunology 26th Annual Spring Meeting.

April 5 – 8, 2013.

TELUS Whistler Conference Centre, Whistler, British Columbia, Canada.

Registration is open online.  Early registration ends March 1, 2013.

Abstract Submission Deadline: March 1, 2013.


Keystone Symposium: Immunopathology of Type 1 Diabetes

April 4 – 9, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: April 4, 2013.


Keystone Symposium: Advances in the Knowledge and Treatment of Autoimmunity

April l4 – 9, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: April 4, 2013.


Molecular Pattern Recognition Receptors

April 11 – 13, 2013.

Boston, Massachusetts, USA.

Early Registration Deadline: March 1, 2013.


Clinical Immunology Society Annual Meeting: Regulation and Dysregulation of Immunity

April 25 – 28, 2013.

Miami, Florida, USA.

Pre-Registration Deadline: April 3, 2013.


T cell Function and Modulation Meeting

April 28 – May 1, 2013.

Makena Beach & Golf Resort, Maui (Makena), HI.

Registration can be submitted online and is limited to the first 125 attendees.


IMMUNOLOGY 2013, AAI Annual Meeting and Centennial Celebration

May 3 – 7, 2013.

Hawaii Convention Center, Honolulu, Hawaii, USA.

Registration is open online.  Early registration ends March 18, 2013.

Abstract Submission Deadline: February 13, 2013.


Keystone Symposium: The Innate Immune Response in the Pathogenesis of Infectious Disease

May 5 – 10, 2013.

A Universidade Federal de Ouro Preto, Ouro Preto, Brazil.

Early Registration Deadline: March 5, 2013.


Cell Symposia: Microbiome and Host Health

May 12 – 14, 2013.

Lisbon, Portugal.

Abstract Submission Deadline: February 8, 2013.

Early Registration Deadline: March 9, 2013.


30 Years of HIV Science: Imagine the Future

May 21 – 23, 2013.

The International Research Centre in Paris, the Institut Pasteur Conference Centre, Paris, France.

Abstract Submission Deadline: March 1, 2013.

Early Registration Deadline: March 20, 2013.


Abcam: Allergy & Asthma 2013

May 23 – 24, 2013.

Bruges, Belgium.

Oral Abstract Submission Deadline: February 22, 2013.

Poster Abstract Submission Deadline: March 25, 2013.

Early Registration Deadline: March 25, 2013.


ISIR 2013 – Building Bridges in Reproductive Immunology.

May 28 – June 1, 2013.

Boston Park Plaza Hotel, Boston, Massachusetts, USA.

Registration is open online.

Abstract Submission Deadline: February 15, 2013.


78th Cold Spring Harbor Symposium on Quantitative Biology: Immunity & Tolerance

May 29 – June 3, 2013.

Cold Spring Harbor Laboratory, New York, USA

Abstract and Registration Deadline: March 15, 2013.


6th International Singapore Symposium of Immunology.

June 5 – 6, 2013.

Matrix Level 2 Auditorium, Biopolis, Singapore.

Registration is open online.

Abstract Submission Deadline: April 5, 2013.


Cell Symposium: Immunometabolism: From Mechanisms to Therapy

June 9 – 11, 2013.

The Sheraton Centre Toronto Hotel, Toronto, Canada.

Abstract Submission Deadline: February 22, 2013.

Early Registration Deadline: April 5, 2013.


Gordon Research Conference: Mucosal Health & Disease

June 9 – 14, 2013.

Stonehill College, Easton, Massachusetts, USA.

Application Deadline: May 12, 2013.


Gordon Research Conference: Phagocytes

June 9 – 14, 2013.

Waterville Valley, New Hampshire, USA.

Application Deadline: May 12, 2013.


European Academy of Allergy & Clinical Immunology and World Allergy Organization: World Allergy & Asthma Congress

June 22 – 26, 2013.

Milan, Italy.

Early Registration Deadline: February 20, 2013.


Aegean Conference: 10th International Conference on Innate Immunity

June 23 – 28, 2013.

Kos, Greece.

Early Registration and Abstract Submission Deadline: March 15, 2013.

Gordon Research Conference: Apoptotic Cell Recognition & Clearance

June 23 – 28, 2013.

University of New England, Biddeford, Maine, USA.

Application Deadline: May 26, 2013.


Abcam: Inflammasomes in Health and Disease

June 24 – 25, 2013.

Boston, Massachusetts, USA.

Oral Abstract Submission Deadline: April 26, 2013.

Poster Abstract Submission Deadline: May 17, 2013.

Early Registration Deadline: April 26, 2013.


FOCIS 2013

June 27 – 30, 2013.

Boston, Massachusetts, USA.

Late-breaking Abstract Submission Deadline: April, 2, 2013.

Registration Deadline for Poster Presenters:  March 28, 2013.

Websites that list upcoming Conferences & Events in Immunology, Tumor Immunology, and Cancer Immunotherapy:

The American Association of Immunologists (AAI) Meetings and Events Calendar

Nature Reviews Immunology’s list of conferences

Cancer Immunity Journal’s List of Conferences

A novel role of TPO in the regulation of HSC DNA repair

HSC transplantation is routinely used to treat patients with malignant and non-malignant disorders of the blood and immune system, but its therapeutic application is often restricted by difficulties in in vitro maintenance and expansion of HSCs.  Studies aimed at understanding the mechanisms governing self-renewal of HSCs within hematopoietic tissues identified a set of growth factors and cytokines that can augment in vitro expansion of HSCs, including stem cell factor (SCF), fms-like tryrosine kinase 3 ligand (FLT3l) and thrombopoietin (TPO) 1.  TPO and its receptor, Mpl, are primarily known for their role in megakaryopoiesis, but TPO has also been shown to support HSC quiescence during adult hematopoiesis, with the loss of signaling associated with bone marrow failure and thrombocytopenia 2.

Recently, in Cell Stem Cell, de Lavel et al. identified a novel role of TPO in the regulation of DNA repair in HSCs 3.  Exposure to genotoxic agents, such as ionizing radiation (IR), induces DNA damage comprised of double-strand breaks (DSBs).  DNA damage is repaired through two main pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ).  DNA repair is essential for cell survival, and studies have shown that NHEJ is necessary for HSC maintenance 4,5.  In their study, de Lavel et al. found that γH2AX foci, a marker of DSB formation, were significantly increased in Mpl-deficient HSCs and in their progenitors following IR exposure.  Moreover, a TPO injection into mice prior to IR reduced the number of γH2AX foci in HSCs in vivo, while HSCs exposed to IR in the absence of TPO demonstrated an increased number of γH2AX foci.  Other experiments showed that TPO modulates the efficiency of the NHEJ pathway by increasing the phosphorylation of the DNA-PK catalytic subunit, a major enzyme involved in NHEJ.  Pharmacological or genetic inhibition of DNA-PK abrogated TPO-mediated DNA repair.  Interestingly, the other cytokines involved in HSC maintenance and expansion, SCF and FLT3l, did not have the same effects as TPO, suggesting that DNA repair activity is a specific function of TPO.

clinical trialIn short, TPO regulates NHEJ-mediated DNA repair of DSBs by stimulating DNA-PK activity in HSCs.  This is the first demonstration that a cytokine involved in HSC maintenance may also regulate DSB repair machinery.  Since TPO treatment prior to IR exposure reduces DNA damage, TPO agonists could potentially be given to patients prior to receiving chemotherapy to reduce the risk of developing oncogenic mutations and defects in HSC function.  Romiplostim, a TPO peptide mimetic, and eltrombopag, a non-peptide TPO mimetic, have been successfully used for the treatment of immune thrombocytopenic purpura (ITP) and are approved by the FDA 6.  de Lavel et al showed that injection of romiplostim prior to IR exposure also completely abolished persistent DNA damage in HSCs, similar to TPO.  Thus, these TPO agonists might also be suited for clinical applications involving protection of normal HSC from DNA-damaging agents.



1          Ohmizono, Y. et al. Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand. Leukemia 11, 524-530 (1997).

2          Ballmaier, M., Germeshausen, M., Krukemeier, S. & Welte, K. Thrombopoietin is essential for the maintenance of normal hematopoiesis in humans: development of aplastic anemia in patients with congenital amegakaryocytic thrombocytopenia. Ann N Y Acad Sci 996, 17-25 (2003).

3          de Laval, B. et al. Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic Stem and Progenitor Cell Mutagenesis in Response to DNA Damage. Cell Stem Cell 12, 37-48, doi:10.1016/j.stem.2012.10.012 (2013).

4         Rossi, D. J. et al. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 447, 725-729, doi:10.1038/nature05862 (2007).

5          Nijnik, A. et al. DNA repair is limiting for haematopoietic stem cells during ageing. Nature 447, 686-690, doi:10.1038/nature05875 (2007).

6          Kuter, D. J. New thrombopoietic growth factors. Clin Lymphoma Myeloma 9 Suppl 3, S347-356, doi:10.3816/CLM.2009.s.034 (2009).