Francine Rezzoug

Cells Expressing Early Cardiac Markers Reside in the Bone Marrow and Are Mobilized Into the Peripheral Blood After Myocardial Infarction

The concept that bone marrow (BM)–derived cells participate in cardiac regeneration remains highly controversial and the identity of the specific cell type(s) involved remains unknown. In this study, we report that the postnatal BM contains a mobile pool of cells that express early cardiac lineage markers (Nkx2.5/Csx, GATA-4, and MEF2C). These cells are present in significant amounts in BM harvested from young mice but their abundance decreases with age; in addition, the responsiveness of these cells to gradients of motomorphogens SDF-1, HGF, and LIF changes with age. FACS analysis, combined with analysis of early cardiac markers at the mRNA and protein levels, revealed that cells expressing these markers reside in the nonadherent, nonhematopoietic CXCR4+/Sca-1+/lin−/CD45− mononuclear cell (MNC) fraction in mice and in the CXCR4+/CD34+/AC133+/CD45− BMMNC fraction in humans. These cells are mobilized into the peripheral blood after myocardial infarction and chemoattracted to the infarcted myocardium in an SDF-1-CXCR4–, HGF-c-Met–, and LIF-LIF-R–dependent manner. To our knowledge, this is the first demonstration that the postnatal BM harbors a nonhematopoietic population of cells that express markers for cardiac differentiation. We propose that these potential cardiac progenitors may account for the myocardial regenerative effects of BM. The present findings provide a novel paradigm that could reconcile current controversies and a rationale for investigating the use of BM-derived cardiac progenitors for myocardial regeneration.

Plasmacytoid precursor dendritic cells facilitate allogeneic hematopoietic stem cell engraftment

Bone marrow transplantation offers great promise for treating a number of disease states. However, the widespread application of this approach is dependent upon the development of less toxic methods to establish chimerism and avoid graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FCs) have been shown to enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. In the present studies, we have identified the main subpopulation of FCs as plasmacytoid precursor dendritic cells (p-preDCs). FCs and p-preDCs share many phenotypic, morphological, and functional features: both produce IFN-α and TNF-α, both are activated by toll-like receptor (TLR)-9 ligand (CpG ODN) stimulation, and both expand and mature after Flt3 ligand (FL) treatment. FL-mobilized FCs, most of which express a preDC phenotype, significantly enhance engraftment of HSCs and induce donor-specific tolerance to skin allografts. However, p-preDCs alone or p-preDCs from the FC population facilitate HSC engraftment less efficiently than total FCs. Moreover, FCs depleted of preDCs completely fail to facilitate HSC engraftment. These results are the first to define a direct functional role for p-preDCs in HSC engraftment, and also suggest that p-preDCs need to be in a certain state of maturation/activation to be fully functional.

TNF-α Is Critical to Facilitate Hemopoietic Stem Cell Engraftment and Function

The use of tolerogenic cells as an approach to induce tolerance to solid organ allografts is being aggressively pursued. A major limitation to the clinical application of cell-based therapies has been the ability to obtain sufficient numbers and also preserve their tolerogenic state. We previously reported that small numbers of bone marrow-derived CD8+/TCR− graft facilitating cells (FC) significantly enhance hemopoietic stem cell (HSC) engraftment in allogeneic and syngeneic recipients. Although the majority of FC resemble precursor plasmacytoid dendritic cells (p-preDC), p-preDC do not replace FC in facilitating function. In the present studies, we investigated the mechanism of FC function. We show for the first time that FC significantly enhance HSC clonogenicity, increase the proportion of multipotent progenitors, and prevent apoptosis of HSC. These effects require direct cell:cell contact between FC and HSC. Separation of FC from HSC by transwell membranes completely abrogates the FC effect on HSC. p-preDC FC do not replace FC total in these effects on HSC function. FC produce TNF-α, and FC from TNF-α-deficient mice exhibit impaired facilitation in vivo and loss of the in vitro effects on HSC. Neutralizing TNF-α in FC similarly blocks the FC effect. The antiapoptotic effect of FC is associated with up-regulation of Bcl-3 transcripts in HSC and blocking of TNF-α is associated with abrogation of up-regulation of Bcl-3 transcripts. These data demonstrate a critical role for TNF-α in mediating FC function. FC may have a significant impact upon the safe use of chimerism to establish tolerance to transplanted organs and tissue.

Alcohol modulates expression of DNA methyltranferases and methyl CpG-/CpG domain-binding proteins in murine embryonic fibroblasts.

Fetal alcohol syndrome (FAS), presenting with a constellation of neuro-/psychological, craniofacial and cardiac abnormalities, occurs frequently in offspring of women who consume alcohol during pregnancy, with a prevalence of 1-3 per 1000 livebirths. The present study was designed to test the hypothesis that alcohol alters global DNA methylation, and modulates expression of the DNA methyltransferases (DNMTs) and various methyl CpG-binding proteins. Murine embryonic fibroblasts (MEFs), utilized as an in vitro embryonic model system, demonstrated ∼5% reduction in global DNA methylation following exposure to 200mM ethanol. In addition, ethanol induced degradation of DNA methyltransferases (DNMT-1, DNMT-3a, and DNMT-3b), as well as the methyl CpG-binding proteins (MeCP-2, MBD-2 and MBD-3), in MEF cells by the proteasomal pathway. Such degradation could be completely rescued by pretreatment of MEF cells with the proteasomal inhibitor, MG-132. These data support a potential epigenetic molecular mechanism underlying the pathogenesis of FAS during mammalian development.

NK cells play a critical role in the regulation of class I-deficient hemopoietic stem cell engraftment: evidence for NK tolerance correlates with receptor editing.

The role that NK cells play in the rejection of hemopoietic stem cell (HSC) and tolerance induction has remained controversial. In this study, we examined whether NK cells play a direct role in the rejection of HSC. Purified HSC from MHC class II-deficient mice engrafted readily in congenic mice, while HSC from class I-deficient donors (β2-microglobulin−/− (β2m−/−)) failed to engraft. Recipient mice lacking CD8+, CD4+, or T cells also rejected HSC from class I-deficient donors, pointing directly to NK cells as the effector in rejection of HSC. Recipients, deficient in or depleted of NK cells, engrafted readily with β2m−/− HSC. Expression of the activating Ly-49D and inhibitory Ly-49G2 receptors on recipient NK cells was significantly decreased in these β2m−/−→B6 chimeras, and the proportion of donor NK cells expressing Ly-49D was also significantly decreased. Notably, β2m−/− chimeras accepted β2m−/− HSC in second transplants, demonstrating that NK cells in the chimeras had been tolerized to β2m−/−. Taken together, our data demonstrate that NK cells play a direct role in the regulation of HSC engraftment, and down-regulation and/or deletion of specific NK subsets in mixed chimeras can contribute to the induction of NK cell tolerance in vivo. Moreover, our data show that bone marrow-derived elements significantly contribute to NK cell development and tolerance.

Flt3-ligand-mobilized peripheral blood, but not Flt3-ligand-expanded bone marrow, facilitating cells promote establishment of chimerism and tolerance.

Facilitating cells (CD8+/TCR−) (FCs) enhance engraftment of limiting numbers of hematopoietic stem cells (HSCs). The primary component of FCs is precursor‐plasmacytoid dendritic cells (p‐preDCs), a tolerogenic cell expanded by Flt3‐ligand (FL). In this study, we evaluated the function and composition of FL‐expanded FCs. FL treatment resulted in a significant increase of FCs in bone marrow (BM) and peripheral blood (PB). When FL‐expanded FCs were transplanted with c‐Kit+/Sca‐1+/Lin− (KSL) cells into allogeneic recipients, BM‐FCs exhibited significantly impaired function whereas PB‐FCs were potently functional. A significant upregulation of P‐selectin expression and downregulation of VCAM‐1 (vascular cell adhesion molecule 1) were present on FL‐expanded PB‐FCs compared with FL BM‐FCs. Stromal cell–derived factor‐1 (SDF‐1), and CXCR4 transcripts were significantly increased in FL PB‐FCs and decreased in FL BM‐FCs. Supernatant from FL PB‐FCs primed HSC migration to SDF‐1, confirming production of the protein product. The FL PB‐FCs contained a predominance of p‐preDCs and natural killer (NK)–FCs, and NK‐FCs were lacking in FL BM‐FCs. The impaired function for BM‐FCs was restored within 5 days after cessation of treatment. Taken together, these data suggest that FCs may enhance HSC homing and migration via the SDF‐1/CXCR4 axis and adhesion molecule modulation. These findings may have implications in development of strategies for retaining function of ex vivo manipulated FCs and HSCs.

Suppression of Chondrogenesis by Id Helix-Loop-Helix Proteins in Murine Embryonic Orofacial Tissue

Inhibitors of differentiation (Id) proteins are helix-loop-helix (HLH) transcription factors lacking a DNA-binding domain. Id proteins modulate cell proliferation, apoptosis and differentiation in embryonic/fetal tissue. Perturbation of any of these processes in cells of the developing orofacial region results in orofacial anomalies. Chondrogenesis, a process integral to normal orofacial ontogenesis, is known to be modulated, in part, by Id proteins. In the present study, the mRNA and protein expression patterns of Id1, Id2, Id3 and Id4 were examined in developing murine orofacial tissue in vivo, as well as in murine embryonic maxillary mesenchymal cells in vitro. The functional role of Ids during chondrogenesis was also explored in vitro. Results reveal that cells derived from developing murine orofacial tissue (1) express Id1, Id2, Id3 and Id4 mRNAs and proteins on each of gestational days 12-14, (2) express all four Id proteins in a developmentally regulated manner, (3) undergo chondrogenesis and express genes encoding various chondrogenic marker proteins (e.g. Runx2, Type X collagen, Sox9) when cultured under micromass conditions and (4) can have their chondrogenic potential regulated via alteration of Id protein function through overexpression of a basic HLH factor. In summary, results from the current report reveal for the first time the expression of all four Id proteins in cells derived from developing murine orofacial tissue, and demonstrate a functional role for the Ids in regulating the ability of these cells to undergo chondrogenesis.

Fms-Related Tyrosine Kinase 3 Expression Discriminates Hematopoietic Stem Cells Subpopulations With Differing Engraftment-Potential : Identifying the Most Potent Combination

Background. Fms-related tyrosine kinase 3 (Flt3)-ligand (FL) promotes the proliferation, differentiation, development, and mobilization of hematopoietic cells. We previously found that FL-mobilized hematopoietic stem cells (HSC) engraft efficiently, whereas FL-expanded bone marrow HSC do not. The function of FL-mobilized c-Kit+ Sca-1+Lin− (KSL) subpopulations has not been systematically evaluated. A precise definition of the repopulating ability is needed to define which HSC subpopulations are critical for long-term chimerism and tolerance induction. FL significantly mobilized c-Kithi and c-Kitlo Sca-1+Lin− cells into peripheral blood (PB). Here, we evaluated the influence of Flt3 expression on long-term repopulating ability of HSC subpopulations. Methods. c-Kithi or c-Kitlo KSL cells were sorted from PB of FL-treated green fluorescent protein-positive donors. The function of these cells was evaluated using competitive reconstitution assays, colony-forming units spleen, and colony forming cell assays. The function of c-Kithi CD34−Flt3− KSL, c-Kithi CD34+Flt3− KSL, c-Kithi CD34+Flt3+ KSL were investigated in an in vivo transplantation model. Results. Only FL-mobilized PB c-Kithi KSL cells exhibited high spleen colony-forming unit activity, generated high numbers of both lymphoid and myeloid colonies in vitro, and rescued ablated recipients. FL-mobilization expanded both c-Kithi CD34+Flt3− cells (short-term HSC) and c-Kithi CD34−Flt3− KSL cells (long-term HSC). There was a significant decrease in c-Kithi CD34+Flt3+ KSL late multipotent progenitors in PB. A combination of c-Kithi CD34+Flt3− and c-Kithi CD34−Flt3− KSL cells offered the most effective rescue of ablated recipients. Conclusions. These data suggest that engraftment of purified HSC is influenced by both short- and long-term repopulating populations and that Flt3 expression may be useful for selecting the most critical HSC subpopulations for transplantation.

Discovery of a Family of Genomic Sequences Which Interact Specifically with the c-MYC Promoter to Regulate c-MYC Expression.

G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.

Abstract 1521: hTERT G-quadruplex-targeted oligonucleotides inhibit glioblastoma cell growth

Glioblastoma is one of the most common and deadly forms of brain cancer, representing roughly 75% of all brain malignancies. These tumors generally have poor prognoses and are resistant to conventional therapy. It has recently been shown that as many as 80% of all glioblastomas contain mutations in a G-rich 68 base pair region of the hTERT promoter. hTERT is the catalytic subunit of telomerase, a holoenzyme responsible for lengthening the ends of chromosomes, thereby conferring immortality to cells. Normally, hTERT is not expressed in somatic cells and its expression is tightly controlled in stem cells. However, hTERT is upregulated in up-to 95% of human tumors and is considered a key activator of cancer progression and a sign of poor clinical outcome. Therefore, hTERT has been under investigation for the past decade as a potential therapeutic target. We have shown that the mutations in hTERT promoter occur in a G-rich region that is part of a silencer element which forms a secondary G-quadruplex structure required for function, and that these mutations destabilize the G-quadruplex structure, allowing hTERT expression. We have also demonstrated that oligonucleotides encoding the G-quadruplex forming sequence in the c-MYC promoter can stabilize the G-quadruplex structure and downregulate c-MYC expression. Therefore, we hypothesized that oligonucleotides targeted to the G-quadruplex of the hTERT promoter could downregulate this gene expression and inhibit glioblastoma cell proliferation in a similar manner. We designed several G-quadruplex-forming oligonucleotides covering the mutated sites in the hTERT promoter either in totality (68 nucleotides) or separately (25 nucleotides) to stabilize the G-quadruplex region. Two glioblastoma cell lines (A172 and U87) and one neuroblastoma cell line (CHP134) were exposed to these oligonucleotides and evaluated for growth inhibition using the MTT assay and for gene expression by QRT-PCR. All oligonucleotides tested were found to induce between 40 to 90% growth inhibition in the 3 cell lines. The cell growth inhibition was both time and dose dependent and showed effectiveness as early as 3 days suggesting that this effect is not solely due to telomere shortening. Four oligonucleotides with the most consistent efficacy in growth inhibition were evaluated for their effect on hTERT gene expression in the 3 cell lines at 4 day exposure and revealed that two of the G-quadruplex forming oligonucleotides significantly decreased hTERT expression compared to untreated cells. In conclusion, we have defined G-quadruplex oligonucleotides targeted to the hTERT promoter that downregulate hTERT gene expression and are effective growth inhibitors in glioblastoma cells. Our findings indicate that downregulation of hTERT with targeted oligonucleotides affects non-canonical functions of hTERT conferring an advantage to this therapeutic approach. Note: This abstract was not presented at the meeting. Citation Format: Alex West, Francine Rezzoug, Shelia D. Thomas, Donald M. Miller. hTERT G-quadruplex-targeted oligonucleotides inhibit glioblastoma cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1521. doi:10.1158/1538-7445.AM2017-1521