Laure C. Bouchez

Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells

Stem Cell Expansion The ability to expand hematopoietic stem cells (HSCs) during ex-vivo culture has been an important goal for over 20 years. Using a high-throughput chemical screen, Boitano et al. (p. 1345, published online 5 August; see the Perspective by Sauvageau and Humphries) found that a purine derivative, StemRegenin1 (SR1), promoted the expansion of human HSCs. Treatment of HSCs with SR1 (which blocked the activity of the aryl hydrocarbon receptor) led to the expansion of CD34+ cells and a 12 to 17-fold increase in the number of HSCs that engraft immune deficient mice. The identification of a mechanism for ex vivo amplification may facilitate clinical application of hematopoietic stem cell therapies. Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.

Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4.

Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such compounds may provide mechanistic insight into the reprogramming process.

A Stem Cell–Based Approach to Cartilage Repair

Osteoarthritis and Kartogenin Osteoarthritis is characterized by progressive breakdown of articular cartilage and affects over 25 million people in the United States. Mesenchymal stem cells (MSCs), which reside in healthy and diseased joints, are multipotent adult stem cells that are able to differentiate into a variety of cell types. Johnson et al. (p. 717, published online 5 April) identified a small molecule, kartogenin, which was able to induce MSCs to differentiate into chondrocytes in vitro. When administered locally, kartogenin was efficacious in two animal models of osteoarthritis. A chemical screen using mesenchymal stem cells identifies a small molecule, kartogenin, that can promote chondrogenesis. Osteoarthritis (OA) is a degenerative joint disease that involves the destruction of articular cartilage and eventually leads to disability. Molecules that promote the selective differentiation of multipotent mesenchymal stem cells (MSCs) into chondrocytes may stimulate the repair of damaged cartilage. Using an image-based high-throughput screen, we identified the small molecule kartogenin, which promotes chondrocyte differentiation (median effective concentration = 100 nM), shows chondroprotective effects in vitro, and is efficacious in two OA animal models. Kartogenin binds filamin A, disrupts its interaction with the transcription factor core-binding factor β subunit (CBFβ), and induces chondrogenesis by regulating the CBFβ-RUNX1 transcriptional program. This work provides new insights into the control of chondrogenesis that may ultimately lead to a stem cell—based therapy for osteoarthritis.

R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5.

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4−/− crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.

CXCR4 expression in prostate cancer progenitor cells.

Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44+/CD133+ prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy.

CXCR4 activation maintains a stem cell population in tamoxifen-resistant breast cancer cells through AhR signalling

Background:Tamoxifen is commonly used for breast cancer therapy. However, tamoxifen resistance is an important clinical problem. Continuous treatment with conventional therapy may contribute to cancer progression in recurring cancers through the accumulation of drug-resistant cancer progenitors.Methods:To investigate signalling mechanisms important for the maintenance and viability of drug-resistant cancer progenitors, we used microarray analysis, PCR array for genes involved in cancer drug resistance and metabolism, flow cytometry, soft agar colony formation assay, in vivo tumourigenicity assay and immunohistochemical analysis using tamoxifen-sensitive and tamoxifen-resistant breast cancer MCF7 cells.Results:Downregulation of CXCR4 signalling by small molecule antagonist AMD3100 specifically inhibits growth of progenitor cell population in MCF7(TAM-R) cells both in vitro and in vivo. Microarray analysis revealed aryl hydrocarbon receptor (AhR) signalling as one of the top networks that is differentially regulated in MCF7(TAM-R) and MCF7 xenograft tumours treated with AMD3100. Further, small molecule antagonists of AhR signalling specifically inhibit the progenitor population in MCF7(TAM-R) cells and growth of MCF7(TAM-R) xenografts in vivo.Conclusion:The chemokine receptor CXCR4 maintains a cancer progenitor population in tamoxifen-resistant MCF7 cells through AhR signalling and could be a putative target for the treatment of tamoxifen-resistant breast cancers.

Pan-Src Family Kinase Inhibitors Replace Sox2 during the Direct Reprogramming of Somatic Cells **

Ectopic expression of the four transcription factors Oct4, Klf4, Sox2 and c-Myc reprograms adult somatic cells to induced pluripotent stem (iPS) cells.[1] Although iPS cells hold considerable promise as tools in research and drug discovery, the clinical application of iPS cells is hindered by the use of viruses that deliver the exogenous factors and modify the host genome. It is therefore of great interest to replace virally transduced factors with either proteins or small molecules. To date a number of compounds have been identified that facilitate reprogramming of somatic cells. Among these are kenpaullone[2], valproic acid[3] and inhibitors of TGFβ-signaling.[4] Here we have exploited a reporter based screen[2] to identify a new class of compounds that functionally replace Sox2: inhibitors of the Src family of kinases. These molecules provide novel tools to study the molecular mechanism of Sox2 in reprogramming. To screen for small molecule replacements of Sox2, mouse embryonic fibroblasts (MEFs) harboring the firefly luciferase (Fluc) gene in the Nanog locus[2] (NL-MEFs) were transduced with Oct4, Klf4 and c-Myc (OKM), seeded into 1536-well plates in standard growth media and assayed against a large chemical library[5] (750,000 compounds; 2.2 μM). Compounds that reproducibly and dose-dependently activated the NL reporter >2.5-fold over vehicle-treated controls (Figure 1a) were then counter-screened in a cell based SV40-driven Fluc assay to rule out false positives that directly and non-specifically induce luciferase signal.[2, 6] Figure 1 Chemical complementation of Sox2 To confirm that filtered hit compounds which activate Nanog gene expression also replace Sox2, iPS cell colony formation was used as a secondary assay. Specifically, Klf4 and c-Myc were delivered retrovirally to O4NR-MEFs[1b] (cells harboring a Doxycycline (Dox)-inducible Oct4 cDNA in the collagen locus and the neomycin-resistance gene in the Oct4 locus), and Oct4 expression was induced by addition of Dox to the culture media (day 0). Two days later, positive screen hits (1-10 μM) were added to OKM-expressing MEFs in place of Sox2. After 10 days of compound treatment, growth media was supplemented with neomycin to select for colonies that reactivated the endogenous Oct4 locus. The reactivation of epigenetically silenced pluripotency-associated genes is required for somatic cells to transition to the iPS cell state.[7] Dox-independent, neomycin resistant colonies were not observed in DMSO-treated (0.1%, v/v) controls, indicating that vehicle-treated cells had not removed the epigenetic silencing marks from the Oct4 promoter (which drives NeoR) and were thus not pluripotent. Among the compounds tested, one compound, iPYrazine (iPY; 10 μM), promoted the formation of neomycin-resistant iPS cell colonies (Figure 1b, blue bars) that survived and could be cultured in the absence of Dox. Transgenic Oct4 independent (minus Dox) growth of the iPY-treated iPS cells demonstrated that they had reactivated and relied on endogenous Oct4 to maintain the pluripotent state. In addition, OKM transduction combined with iPY treatment of MEFs carrying a GFP reporter under control of the endogenous Oct4 locus[8] also gave rise to stable, GFP-positive iPS cell lines (Figure S1, Supporting Information). iPS cells derived from O4NR-MEFs with iPY, Dox and KM-transduction grew as pluripotent stem cell colonies in the absence of Dox and iPY. Moreover, these cells were indistinguishable from ES cells by morphological criteria and expressed the pluripotency-associated markers Oct4 and SSEA1 (Figure 1c). We next tested the differentiation potential of the iPY-derived iPS cells in a teratoma assay by injecting 106 cells subcutaneously into NOD-SCID mice. Tumors were isolated 3 weeks later and histological analyses demonstrated that cell types of all three germ layers were present; these included neural tissues, bone, cartilage and ciliated epithelium (Figure 1d). Furthermore, iPY-derived iPS cells contributed to live chimeras, as shown in Figure 1d. The results from this series of analyses indicate that the iPY-derived, Sox2-free iPS cells are pluripotent. In order to identify the biological target of iPY, we profiled the compound against a biochemical panel of tyrosine kinases (51 kinases; Table S1). From this analysis, we found that iPY potently inhibited a number of tyrosine kinases at 5 μM. Commercially available inhibitors (Figures 2a-b and Table S2) of these candidate kinase targets were then assayed for their ability to replace Sox2 in the iPS cell colony formation assay. As shown in Figure 2b, the pan-Src family kinase (SFK) inhibitors Dasatinib[9] and PP1[10] (Figure 2b) were able to recapitulate the activity of iPY. Interestingly, both Dasatinib and PP1 were >2-fold more active than iPY and efficiently replaced Sox2 (Figure 2b). Moreover, the pan-SFK inhibitors gave rise to colonies with a similar efficiency to TGFβ inhibitors (SB-431542 and LY-364947). The latter have been reported to replace Sox2 and served as a positive control in this study.[4] In addition to TGFβ inhibitors, Ichida et al. have also reported that the SFK inhibitor PP1 is able to replace Sox2.[4a] Together with our work, these results indicate that iPY is likely playing a role in reprogramming by inhibiting Src kinases, although additional mechanisms cannot be excluded. Figure 2 Src family kinase and TGFβ-inhibitors recapitulate the Sox2 replacement activity of iPY SFKs are a class of proto-oncogene tyrosine kinases that include nine mammalian members (i.e., c-Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn and Frk).[11] Several members of the SFK family have been reported to influence embryonic stem (ES) cell self-renewal and differentiation.[12] For example, activation of c-Src signaling promotes ES cell differentiation.[13] Consistent with this observation we find that the activation of Src signaling in MEFs with JK239[14] potently inhibits 4-factor reprogramming (Figure 2c). Together, our results suggest that SFK signaling is an important mediator of somatic cell reprogramming, where activation of the SFK pathway prevents reprogramming and inhibition allows for reprogramming in the absence of exogenous Sox2. Previously, Ichida et al. demonstrated that small molecule mediated inhibition of TGFβ-signaling with LY-364947 or E-616452 can replace Sox2 through the activation of Nanog expression.[4a] The results from our screen, which rely on Nanog activation as a surrogate for the replacement of Sox2, suggest that the inhibition of SFK- and TGFβ-signaling may converge on a similar mechanism; that is, the function of Sox2 can be replaced during direct reprogramming by activating Nanog expression. Another potential scenario comes from the observation that both Nanog[15] and SFK inhibition[13] are capable of maintaining the self-renewing pluripotent state in ES cells. Thus, TGFβ inhibitor-mediated Nanog activation and pan-SFK inhibition may instead converge on a common mechanism in which the differentiation of newly formed iPS cells is prevented, thereby assisting in the transition to an undifferentiated state. In either case, it is interesting to note that inhibition of distinct signaling responses converge on a common end point. In summary, we applied a cell-based, high-throughput chemical screen to identify small molecules that replace Sox2 during somatic cell reprogramming. The identification of novel SFK inhibitors provides new chemical tools to study the mechanisms underlying direct reprogramming and may ultimately help to bring iPS cell technology one step closer to clinical application.

CXCR4 as biomarker for radioresistant cancer stem cells

Abstract Purpose: Radioresistance of cancer cells remains a fundamental barrier for maximum efficient radiotherapy. Tumor heterogeneity and the existence of distinct cell subpopulations exhibiting different genotypes and biological behaviors raise difficulties to eradicate all tumorigenic cells. Recent evidence indicates that a distinct population of tumor cells, called cancer stem cells (CSC), is involved in tumor initiation and recurrence and is a putative cause of tumor radioresistance. There is an urgent need to identify the intrinsic molecular mechanisms regulating the generation and maintenance of resistance to radiotherapy, especially within the CSC subset. The chemokine C-X-C motif receptor 4 (CXCR4) has been found to be a prognostic marker in various types of cancer, being involved in chemotaxis, stemness and drug resistance. The interaction of CXCR4 with its ligand, the chemokine C-X-C motif ligand 12 (CXCL12), plays an important role in modulating the tumor microenvironment, angiogenesis and CSC niche. Moreover, the therapeutic inhibition of the CXCR4/CXCL12 signaling pathway is sensitizing the malignant cells to conventional anti-cancer therapy. Content: Within this review we are summarizing the role of the CXCR4/CXCL12 axis in the modulation of CSC properties, the regulation of the tumor microenvironment in response to irradiation, therapy resistance and tumor relapse. Conclusion: In light of recent findings, the inhibition of the CXCR4/CXCL12 signaling pathway is a promising therapeutic option to refine radiotherapy.

Small-Molecule Regulators of Human Stem Cell Self-Renewal

Agents that selectively control the self-renewal and differentiation of human hematopoietic stem cells (hHSCs) can serve as useful tools for understanding the basic developmental biology of hematopoiesis. 2] The identification of molecules that regulate HSC fate might also lead to new therapies for the treatment of cancer and genetic blood diseases. For example, the ability to use cord-blood (CB)-derived HSCs would greatly facilitate the finding of matched donors for allogenic bone marrow transplants. 4] However, because engraftment depends on HSC number, and the number of human HSCs available from individual cord blood units is small, the widespread application of cord-blood HSCs is currently limited to pediatric transplants. 6] Thus, the ability to expand HSCs, especially CBderived HSCs, during ex vivo culture would have a major impact on the field of HSC transplantation. We recently identified the purine derivative stem regenin1 (SR1), which expands CB-derived HSCs ex vivo by selectively antagonizing the aryl hydrocarbon receptor (AhR). CB-derived HSCs expanded with SR1 maintain full multilineage potential and engraft efficiently in the NOD Scid Gamma (NSG) mouse transplant model. To identify alternative chemical scaffolds that induce HSC self-renewal through similar or distinct mechanisms, we screened additional chemical libraries (>100 000 discrete small heterocyclic chemical compounds) in a cell-based phenotypic screen, and now report a series of benzimidazoles, diarylamides, and flavonoids that induce HSC self-renewal (Scheme 1). This assay takes advantage of advances in screening technology developed in our group that permit lowvolume (~40 mL) screens to be conducted in a massively parallel fashion with the use of advanced automation. The primary screen was a seven-day assay using mobilized blood-derived CD34 cells in which the loss of CD34 expression (phenotypic hematopoietic stem and progenitor marker) was monitored by high-throughput flow cytometry. 8] A variety of heterocyclic compounds such as flavonoid, benzimidazole, phenylbenzamide, quinoxaline, benzoxazole, and benzothiazole derivatives were identified as potential hits. Secondary assays were then conducted to confirm the phenotypic changes observed in the primary screen. First, compounds were tested for their ability to significantly increase (up to twofold) the number of CD34-expressing cells in a dose-dependent fashion. Next, we determined whether compounds expand CD34 cells isolated from human cord blood. One thousand CB-derived CD34 cells were cultured with each compound, and the cell number and phenotype were measured weekly. After three weeks of culture, three compounds (SR2, SR3, and SR4) were able to expand the number of CD34 cells. Nucleated cell numbers in the cultures increased on average sevento tenfold in the compound-treated culture (SR2, SR3, and SR4) compared to the vehicle-treated culture (DMSO, 0.01 %; see the Supporting Information). Most importantly, 28– 48 % of compound-treated cells (SR2, SR3, and SR4) were CD34 compared to 13 % in vehicle control cultures (see Figure 1). These results suggest that SR2, SR3, and SR4 are expanding phenotypic hematopoietic stem and progenitor cells. Notably, when cultured in the absence of cytokines, these compounds did not induce proliferation of CD34 cells, neither did they have an effect on murine HSCs (LSK: Lin Sca-1 Kit). We next carried out a preliminary structure–activity study of SR2, SR3 and SR4, beginning with the benzimidazole (SR2) scaffold. A collection of benzimidazole derivatives was prepared in an expedient manner from 3-nitro-4-fluorobenzoic acid. The fluoro-group was displaced by nucleophilic aromatic substitution, thus allowing facile incorporation of the amine function at C4. The acid moiety was readily transformed into Scheme 1. Structures of SR1 and compounds identified in the primary screen that expand phenotypic HSCs (SR2–5).

Identification of Small Molecules Which Induce Skeletal Muscle Differentiation in Embryonic Stem Cells via Activation of the Wnt and Inhibition of Smad2/3 and Sonic Hedgehog Pathways

The multilineage differentiation capacity of mouse and human embryonic stem (ES) cells offers a testing platform for small molecules that mediate mammalian lineage determination and cellular specialization. Here we report the identification of two small molecules which drives mouse 129 ES cell differentiation to skeletal muscle with high efficiency without any genetic modification. Mouse embryoid bodies (EBs) were used to screen a library of 1,000 small molecules to identify compounds capable of inducing high levels of Pax3 mRNA. Stimulation of EBs with SMIs (skeletal muscle inducer, SMI1 and SMI2) from the screen resulted in a high percentage of intensively twitching skeletal muscle fibers 3 weeks after induction. Gene expression profiling studies that were carried out for mode of actions analysis showed that SMIs activated genes regulated by the Wnt pathway and inhibited expression of Smad2/3 and Sonic Hedgehog (Shh) target genes. A combination of three small molecules known to modulate these three pathways acted similarly to the SMIs found here, driving ES cells from 129 as well as Balb/c and C57Bl/6 to skeletal muscle. Taken together, these data demonstrate that the SMI drives ES cells to skeletal muscle via concerted activation of the Wnt pathway, and inhibition of Smad2/3 signaling and Shh pathways. This provides important developmental biological information about skeletal muscle differentiation from embryonic stem cells and may lead to the development of new therapeutics for muscle disease. Stem Cells 2016;34:299–310