Mef2C is a lineage-restricted target of Scl/Tal1 and regulates megakaryopoiesis and B-cell homeostasis.

The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. Here, we identified a novel Scl target gene, transcription factor myocyte enhancer factor 2 C (Mef2C) from Scl(fl/fl) fetal liver progenitor cell lines. Analysis of Mef2C(-/-) embryos showed that Mef2C, in contrast to Scl, is not essential for specification into primitive or definitive hematopoietic lineages. However, adult VavCre(+)Mef2C(fl/fl) mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced, whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore, megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.

The Prostate Cancer Foundation 16th Annual Scientific Retreat: meeting report.

The 16th Annual Prostate Cancer Foundation (PCF) Scientific Retreat was held September 23-25, 2009 at Incline Village, NV. This report highlights some of the game-changing research reports from this conference.

Reprogrammed mouse fibroblasts differentiate into cells of the cardiovascular and hematopoietic lineages.

Forced expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the murine fibroblast genome, generating induced pluripotent stem (iPS) cells. Although the differentiation potential of these cells is thought to be equivalent to that of embryonic stem (ES) cells, it has not been rigorously determined. In this study, we sought to identify the capacity of iPS cells to differentiate into Flk1-positive progenitors and their mesodermal progeny, including cells of the cardiovascular and hematopoietic lineages. Immunostaining of tissues from iPS cell-derived chimeric mice demonstrated that iPS cells could contribute in vivo to cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. To compare the in vitro differentiation potential of murine ES and iPS cells, we either induced embryoid body (EB) formation of each cell type or cultured the cells on collagen type IV (ColIV), an extracellular matrix protein that had been reported to direct murine ES cell differentiation to mesodermal lineages. EB formation and exposure to ColIV both induced iPS cell differentiation into cells that expressed cardiovascular and hematopoietic markers. To determine whether ColIV-differentiated iPS cells contained a progenitor cell with cardiovascular and hematopoietic differentiation potential, Flk1-positive cells were isolated by magnetic cell sorting and exposed to specific differentiation conditions, which induced differentiation into functional cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. Our data demonstrate that murine iPS cells, like ES cells, can differentiate into cells of the cardiovascular and hematopoietic lineages and therefore may represent a valuable cell source for applications in regenerative medicine. Disclosure of potential conflicts of interest is found at the end of this article.

Polymorphisms and clinical outcome in recurrent ovarian cancer treated with cyclophosphamide and bevacizumab.

PURPOSE: This study was designed to evaluate the associations between angiogenesis gene polymorphisms and clinical outcome in ovarian cancer patients treated with low-dose cyclophosphamide and bevacizumab. EXPERIMENTAL DESIGN: Seventy recurrent/metastatic epithelial ovarian cancer patients were enrolled in a phase II clinical trial. Genomic DNA was available from 53 blood samples. Polymorphisms were analyzed using the PCR-RFLP protocol. A 5' end 33P gammaATP-labeled PCR protocol was used to analyze dinucleotide repeats. RESULTS: Patients genotyped A/A or A/T for the IL-8 T-251A gene polymorphism had a statistically significant lower response rate (19%; 0%) than those homozygous T/T (50%; P = 0.006, Fisher's exact test). Patients carrying a minimum one C allele (C/C; C/T) of the CXCR2 C+785T polymorphism showed a median progression-free survival (PFS) of 7.4 months compared with the PFS of 3.7 months for those homozygous T/T (P = 0.026, log-rank test). Patients with the VEGF C+936T polymorphism C/T genotype had a longer median PFS of 11.8 months, compared with those with the C/C and T/T genotype, which had median PFS of 5.5 months and 3.2 months, respectively (P = 0.061, log-rank test). Patients carrying both AM 3'end alleles < 14 CA repeats had the shortest median PFS of 3.4 months; patients with at least one allele > 14 repeats or both alleles > 14 repeats showed a median PFS of 6.4 months and 7.2 months, respectively (P = 0.008, log-rank test). CONCLUSION: Our data suggest that the IL-8 A-251T polymorphism may be a molecular predictor of response to bevacizumab-based chemotherapy. The CXCR2 C+785T, VEGF C+936T single nucleotide polymorphisms and the AM 3' dinucleotide repeat polymorphisms may be molecular markers for PFS in ovarian cancer patients.

Molecular determinants of cetuximab efficacy.

PURPOSE: To investigate whether mRNA expression levels of cyclin D1 (CCND1), cyclooxygenase 2 (Cox-2), epidermal growth factor receptor (EGFR), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF), all members of the EGFR signaling pathway, are associated with clinical outcome in patients with EGFR-expressing metastatic colorectal cancer (CRC) treated with cetuximab. PATIENTS AND METHODS: Thirty-nine patients with metastatic CRC, refractory to both irinotecan and oxaliplatin, were enrolled on IMCL-0144 and treated with single-agent cetuximab. The intratumoral mRNA levels of CCND1, Cox-2, EGFR, IL-8, and VEGF were assessed from paraffin-embedded tissue samples using laser-capture microdissection and quantitative real-time polymerase chain reaction. RESULTS: There were 21 women and 18 men with a median age of 64 years (range, 35 to 83 years). Higher gene expression levels of VEGF were associated with resistance to cetuximab (P = .038; Kruskal-Wallis test). The combination of low gene expression levels of Cox-2, EGFR, and IL-8 was significantly associated with overall survival (13.5 v 2.3 months; P = .028; log-rank test). Both findings were independent of skin toxicity that was itself significantly correlated to survival. Patients with a lower mRNA amount of EGFR had a longer overall survival compared with patients that had a higher mRNA amount (7.3 v 2.2 months; P = .09; log-rank test). Patients with lower expression of Cox-2 had a significantly higher rate of grade 2 to 3 skin reactions under cetuximab treatment. CONCLUSION: This pilot study suggests that gene expression levels of Cox-2, EGFR, IL-8, and VEGF in patients with metastatic CRC may be useful markers of clinical outcome in single-agent cetuximab treatment.

Genomic profiling associated with recurrence in patients with rectal cancer treated with chemoradiation.

PURPOSE: Stage II and III adenocarcinoma of the rectum has an overall 5-year survival rate of approximately 50%, and tumor recurrence remains a major problem despite an improvement in local control through chemotherapy and radiation. The efficacy of chemoradiation therapy may be significantly compromised as a result of interindividual variations in clinical response and host toxicity. Therefore, it is imperative to identify those patients who will benefit from chemoradiation therapy and those who will develop recurrent disease. In this study, we tested whether a specific pattern of 21 polymorphisms in 18 genes involved in the critical pathways of cancer progression (i.e., drug metabolism, tumor microenvironment, cell cycle regulation, and DNA repair) will predict the risk of tumor recurrence in rectal cancer patients treated with chemoradiation. PATIENTS AND METHODS: A total of 90 patients with Stage II or III rectal cancer treated with chemoradiation were genotyped using polymerase chain reaction (PCR)-based techniques for 21 polymorphisms. RESULTS: A polymorphism in interleukin (IL)-8 was individually associated with risk of recurrence. Classification and regression tree analysis of all polymorphisms and clinical variables developed a risk tree including the following variables: node status, IL-8, intracellular adhesion molecule-1, transforming growth factor-beta, and fibroblast growth factor receptor 4. CONCLUSION: Genomic profiling may help to identify patients who are at high risk for developing tumor recurrence, and those who are more likely to benefit from chemoradiation therapy. A larger prospective study is needed to validate these preliminary data using germline polymorphisms on tumor recurrences in rectal cancer patients treated with chemoradiation.

Epidermal growth factor receptor gene polymorphisms predict pelvic recurrence in patients with rectal cancer treated with chemoradiation.

An association between epidermal growth factor receptor (EGFR) signaling pathway and response of cancer cells to ionizing radiation has been reported. Recently, a polymorphic variant in the EGFR gene that leads to an arginine-to-lysine substitution in the extracellular domain at codon 497 within subdomain IV of EGFR has been identified. The variant EGFR (HER-1 497K) may lead to attenuation in ligand binding, growth stimulation, tyrosine kinase activation, and induction of proto-oncogenes myc, fos, and jun. A (CA)(n) repeat polymorphism in intron 1 of the EGFR gene that alters EGFR expression in vitro and in vivo has also been described. In the current pilot study, we assessed both polymorphisms in 59 patients with locally advanced rectal cancer treated with adjuvant or neoadjuvant chemoradiation therapy using PCR-RFLP and a 5'-end [gamma-(33)P]ATP-labeled PCR protocol. We tested whether either polymorphism alone or in combination can be associated with local recurrence in the setting of chemoradiation treatment. We found that patients with HER-1 497 Arg/Arg genotype or lower number of CA repeats (both alleles <20) tended to have a higher risk of local recurrence (P = 0.24 and 0.31, respectively). Combined analysis showed the highest risk for local recurrence was seen in patients who possessed both a HER-1 497 Arg allele and <20 CA repeats (P = 0.05, log-rank test). Our data suggest that the HER-1 R497K and EGFR intron 1 (CA)(n) repeat polymorphisms may be potential indicators of radiosensitivity in patients with rectal cancer treated with chemoradiation.

Gene polymorphisms of epidermal growth factor receptor and its downstream effector, interleukin-8, predict oxaliplatin efficacy in patients with advanced colorectal cancer.

BACKGROUND: Researchers have recently reported an association between the epidermal growth factor receptor (EGFR) pathway and platinum-chemotherapy sensitivity in cancer patients. The (CA)(n) repeat polymorphism in intron 1 of the EGFR gene has been identified and found to alter EGFR expression in vitro as well as in vivo. A higher number of these CA repeats is associated with lower EGFR levels, whereas a low number of repeats is associated with higher EGFR levels. A second key polymorphism within the EGFR pathway (HER1 R497K) is a single nucleotide change (G-A) in codon 497 of the EGFR gene, which leads to an arginine-lysine substitution in the extracellular domain of subdomain IV. Furthermore, interleukin-8 (IL-8), recently identified as an EGFR downstream effector, plays a vital role in tumor angiogenesis and progression. Three other polymorphisms, each related to the IL-8 gene, have also been identified as playing a pivotal role in the EGFR pathway: T-251A in the promoter region of the IL-8 gene, G+2607C in exon 2 of the IL-8 receptor CXCR1 gene, and C+785T in exon 11 of the IL-8 receptor CXCR2 gene. PATIENTS AND METHODS: In this study, we employed a 5'-end 33P-gATP-labeled polymerase chain reaction (PCR) protocol as well as the PCR-restriction fragment length polymorphism method in order to determine the genotypes for the previously mentioned polymorphisms in 105 patients with metastatic colorectal cancer. Tests were conducted to establish whether these polymorphisms could predict clinical outcome to 5-flourouracil/oxaliplatin chemotherapy. RESULTS: Among all patients assessed, those possessing < 20 EGFR CA repeats were more likely to show disease progression than were patients with >or= 20 CA repeats (P = 0.019; log-rank test). Also, patients with the CXCR1 GC genotype were found to have an increased relative risk of time to tumor progression that was 1.55 (95% CI, 0.8-3.0) times that of patients with the homozygous GG genotype (P = 0.17; log-rank test). CONCLUSION: Overall, our data suggest that gene polymorphisms active in the EGFR pathway may be associated with the sensitivity of colorectal cancer patients to platinum-based chemotherapy.

Hematopoietic stem cell development in the placenta.

The placenta is a highly vascularized organ that mediates fetal-maternal exchange during pregnancy and is thereby vital for the survival and growth of the developing embryo. In addition to having this well-established role in supporting pregnancy, the placenta was recently shown to function as a hematopoietic organ. The placenta is unique among other fetal hematopoietic organs, as it is capable of both generating multipotential hematopoietic cells de novo and establishing a major hematopoietic stem cell (HSC) pool in the conceptus, while protecting HSCs from premature differentiation. The mouse placenta contains two distinct vascular regions that support hematopoiesis: the large vessels in the chorionic plate where HSCs/progenitors are thought to emerge and the labyrinth vasculature where nascent HSCs/progenitors may colonize for expansion and possible functional maturation. Defining how this cytokine- and growth factor rich organ supports HSC generation, maturation and expansion may ultimately help to establish culture protocols for HSC expansion or de novo generation from pluripotent cells.

Isolation and analysis of hematopoietic stem cells from the placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion. In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment. We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

Isolation and visualization of mouse placental hematopoietic stem cells.

This unit describes the isolation of hematopoietic stem cells (HSCs) from the mouse placenta. The placenta was recently identified as an important hematopoietic site that generates HSCs de novo and provides a transitory niche for a large pool of HSCs during midgestation. This protocol includes a dissection technique for murine placenta, the mechanical and enzymatic steps of placental tissue dissociation, and phenotypical identification and isolation of HSCs. It also contains a method for immunohistochemical analysis of placenta tissue sections to visualize developing HSCs in the placenta.

The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation.

The mouse placenta was unveiled as an important reservoir for hematopoietic stem cells (HSCs), yet the origin of placental HSCs was unknown. By tracking developing HSCs by expression of Runx1-lacZ and CD41, we have found that HSCs emerge in large vessels in the placenta. Analysis of Ncx1(-/-) embryos, which lack a heartbeat, verified that HSC development is initiated in the placental vasculature independent of blood flow. However, fewer CD41+ hematopoietic cells were found in Ncx1(-/-) placentas than in controls, implying that some HSCs/progenitors colonize the placenta via circulation and/or HSC emergence is compromised without blood flow. Importantly, placentas from Ncx1(-/-) embryos possessed equal potential to generate myelo-erythroid and B and T lymphoid cells upon explant culture, verifying intact multilineage hematopoietic potential, characteristic of developing HSCs. These data suggest that, in addition to providing a niche for a large pool of HSCs prior to liver colonization, the placenta is a true site of HSC generation.

Collagen IV induces trophoectoderm differentiation of mouse embryonic stem cells.

The earliest segregation of lineages in the developing embryo is the commitment of cells to the inner cell mass or the trophoectoderm in preimplantation blastocysts. The exogenous signals that control commitment to a particular cell lineage are poorly understood; however, it has been suggested that extracellular "niche" and extracellular matrix, in particular, play an important role in determining the developmental fate of stem cells. Collagen IV (ColIV) has been reported to direct embryonic stem (ES) cell differentiation to mesodermal lineages in both mouse and human ES cells. To define the effects of ColIV on ES cell differentiation and to identify the resulting heterogeneous cell types, we performed microarray analyses and determined global gene expression. We observed that ColIV induced the expression of mesodermal genes specific to hematopoietic, endothelial, and smooth muscle cells and, surprisingly, also a panel of trophoectoderm-restricted markers. This effect was specific to collagen IV, as no trophoblast differentiation was seen on collagen I, laminin, or fibronectin. Stimulation with basic fibroblast growth factor (FGF) or FGF4 increased the number of trophoectodermal cells. These cells were isolated under clonal conditions and successfully differentiated into a variety of trophoblast derivatives. Interestingly, differentiation of ES cells to trophoblastic lineages was only seen in ES cell lines maintained on embryonic feeder layers and was caudal-type homeobox protein 2 (Cdx2)-dependent, consistent with Cdx2's postulated role in trophoectoderm commitment. Our data suggest that, given the appropriate extracellular stimuli, mouse embryonic stem cells can differentiate into trophoectoderm. Disclosure of potential conflicts of interest is found at the end of this article.

ABCB1, SLCO1B1 and UGT1A1 gene polymorphisms are associated with toxicity in metastatic colorectal cancer patients treated with first-line irinotecan.

We tested specific gene polymorphisms known to be involved in the irinotecan (CPT-11) metabolic pathway. The combination of at least one SLCO1B1 521 T allele, one ABCB1 1236 C allele and one UGT1A1*28 variant 7 repeat demonstrated a statistically significant association with Grade 3/4 toxicities in metastatic colorectal cancer patients.

ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia.

In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3-internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC(50) approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC(50) = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G(0)/G(1) phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)-FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC(50) approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC(50) = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

Molecular determinants of irinotecan efficacy.

Molecular markers predicting the efficacy of CPT-11 based chemotherapies in patients with colorectal cancer (CRC) are unknown. Therefore, we investigated whether mRNA levels of drug targets (Topoisomerase I, TS), enzymes involved in 5-FU metabolism (DPD), in angiogenesis (EGFR, IL-8, VEGF) and in DNA-repair/drug detoxification (ERCC1, GST-P1) are associated with the clinical outcome of patients with CRC treated with first-line CPT-11 based chemotherapy. Thirty three patients with metastatic CRC were included in the study. Intratumoral gene expression levels were assessed from paraffin-embedded tissue samples, using laser capture microdissection and quantitative Real-Time PCR. Complete response was observed in 1 patient, partial response in 12 patients, stable disease in 13 patients and progressive disease in 6 patients. Response was inevaluable for 1 patient. Patients with complete response or partial response were classified as responders, while patients with stable disease or progressive disease were classified as nonresponders. High intratumoral mRNA levels of EGFR, ERCC1 and GSPT-P1 were each significantly associated with response to CPT-11 based chemotherapy. Recursive partitioning analysis showed that mRNA levels of EGFR and ERCC1 are primarily responsible for delineating responders from nonresponders. Also, the combination of high intratumoral gene expression levels of both EGFR and ERCC1 was significantly associated with progression-free survival. The mRNA levels of EGFR had a significant correlation with expression levels of ERCC1, GST-P1 and VEGF. This small retrospective study suggests that gene expression levels of EGFR, ERCC1 and GST-P1 may be useful in predicting the clinical outcome of patients with metastatic CRC treated with first-line CPT-11 based chemotherapy.

Cyclin D1 and epidermal growth factor polymorphisms associated with survival in patients with advanced colorectal cancer treated with Cetuximab.

The study aimed to investigate whether polymorphisms in genes of the EGFR signaling pathway are associated with clinical outcome in advanced colorectal cancer (CRC) patients treated with single-agent Cetuximab. Polymorphisms of interest in the EGFR pathway include: cyclin D1 (CCND1) A870G, cyclooxygenase 2 (Cox-2) G-765C, epidermal growth factor (EGF) A61G, epidermal growth factor receptor (EGFR) codon R497 K, EGFR CA dinucleotide repeat in intron 1, interleukin (IL)-8 T-251A and vascular endothelial growth factor (VEGF) C936 T gene polymorphisms. Thirty-nine metastatic CRC patients were enrolled in the IMCL-0144 trial and treated with single-agent Cetuximab. Using the polymerase chain reaction-restriction fragment length polymorphism method, gene polymorphisms of CCND1, COX-2, EGF, EGFR, IL-8 and VEGF were assessed from genomic DNA extracted from blood samples. A significant association was found between the CCND1 A870G polymorphism and overall survival in our 39 CRC subjects. Patients with the AA homozygous genotype survived for a median of 2.3 months [95% confidence interval (CI)=2.1-5.7], whereas those with any G allele (AG, GG genotype) survived for a median of 8.7 months (95% CI=4.4-13.5) (P=0.019, log-rank test). When we analysed the cyclin D1 and EGF polymorphisms together, patients with favourable genotypes (EGF any A allele and CCND1 any G allele) showed a median survival time of 12 months (95% CI=4.8-15.2), whereas patients with any two unfavourable genotypes (EGF GG or CCND1 AA) showed a median survived time of 4.4 months (95% CI=2.1-5.7) (P=0.004, log-rank test). The findings of this pilot study suggest that the cyclin D1 A870G and the EGF A61G polymorphisms may be useful molecular markers for predicting clinical outcome in CRC patients treated with single-agent Cetuximab.