Relapsed follicular lymphoma (FL) can arise from common progenitor cells (CPCs). Conceptually, CPC-defining mutations are somatic alterations shared by the initial and relapsed tumours, mostly B-cell leukaemia/lymphoma 2 (BCL2)/immunoglobulin heavy locus (IGH) translocations and other recurrent gene mutations. Through complementary approaches for highly sensitive mutation detection, we do not find CPC-defining mutations in highly purified BCL2/IGH-negative haematopoietic progenitor cells in clinical remission samples from three patients with relapsed FL. Instead, we find cells harbouring the same BCL2/IGH translocation but lacking CREB binding protein (CREBBP), lysine methyltransferase 2D (KMT2D) and other recurrent gene mutations. Thus, (i) the BCL2/IGH translocation can precede CPC-defining mutations in human FL, and (ii) BCL2/IGH-translocated cells can persist in clinical remission.

Adoptive transfer of TCR transgenic T cells holds great promise for treating various cancers. So far, mainly semi-randomly integrating vectors have been used to genetically modify T cells. These carry the risk of insertional mutagenesis, and the sole addition of an exogenous TCR potentially results in the mispairing of TCR chains with endogenous ones. Established approaches using nonviral vectors, such as transposons, already reduce the risk of insertional mutagenesis but have not accomplished site-specific integration. Here, we used CRISPR-Cas9 RNPs and adeno-associated virus 6 for gene targeting to deliver an engineered TCR gene specifically to the TCR alpha constant locus, thus placing it under endogenous transcriptional control. Our data demonstrate that this approach replaces the endogenous TCR, functionally redirects the edited T cells' specificity in vitro, and facilitates potent tumor rejection in an in vivo xenograft model.

Acute erythroid leukemia (AEL) is a rare and aggressive form of acute leukemia, the biology of which remains poorly understood. Here we demonstrate that the ParaHox gene CDX4 is expressed in patients with acute erythroid leukemia, and that aberrant expression of Cdx4 induced homogenously a transplantable acute erythroid leukemia in mice. Gene expression analyses demonstrated upregulation of genes involved in stemness and leukemogenesis, with parallel downregulation of target genes of Gata1 and Gata2 responsible for erythroid differentiation. Cdx4 induced a proteomic profile that overlapped with a cluster of proteins previously defined to represent the most primitive human erythroid progenitors. Whole-exome sequencing of diseased mice identified recurrent mutations significantly enriched for transcription factors involved in erythroid lineage specification, as well as TP53 target genes partly identical to the ones reported in patients with AEL. In summary, our data indicate that Cdx4 is able to induce stemness and inhibit terminal erythroid differentiation, leading to the development of AEL in association with co-occurring mutations.

Acute myeloid leukemia (AML) is characterized by high mortality, underlining the necessity for identifying tumor suppressors that counteract the leukemogenic potential of bona fide oncogenes such as the homeobox genes HOXA9 and CDX2. Homeobox genes are aberrantly expressed in the majority of patients with cytogenetically normal (CN)-AML and expression of CDX2 positively correlates with HOXA9 expression. Aberrant expression of Cdx2 in murine hematopoietic cells rapidly induced aggressive AML in mice. Recently, it was shown that a microRNA, miR-196, is encoded in the mammalian paralogous HOX gene cluster and that it has extensive evolutionarily conserved complementarity to sites in the 3' prime untranslated regions (3' UTR) of HOX genes (e.g. HOXA9, A7 and B8), directly regulating their expression in MLL-rearranged leukemic cells. To understand the role of miR-196b in AML more precisely, we first tried to identify transcripts generating mature miR-196b in human hematopoietic stem and progenitor cells (HSPC). We identified two novel non-coding transcripts encoding the miR-196b hairpin precursor sequence expressed from the HOXA9-10 locus in bone marrow HSPC (Online Supplementary Figure S1A, B) [NCBI accession number: MF139050, 486 basepairs (bp) and MF139051, 396 bp]. MF139051, a splice variant of the MF139050 transcript, showed 99.47% homology to MF139050. Both transcripts displayed high homology to the transcripts of other vertebrates and mammalian species. Retrovirally engineered expression of both transcripts resulted in significant overexpression of the mature miR-196b in HEK293T cells compared to the vector control (Figure 1A; Online Supplementary Figure S1C, D). Endogenous expression levels of both transcripts in the human CD34 bone marrow compartment were higher than those in mononuclear cells (Figure 1B). Second, we identified a highly conserved 802 bp long miR-196b promoter region, validated by luciferase reporter assay, located 201 bp upstream of the miR-196b stem loop precursor sequence on human chromosome 7 (Figure 1C; Online Supplementary Figure S1E, F). Transcription factor binding site prediction tools and published chromatin immunoprecipitation sequencing data showed, respectively, potential binding of transcription factors including SP1 and enrichment for proteins such as EZH2 on this promoter (Online Supplementary Figure S1G, H; Online Supplementary Table SW1). Previously, the miR-196b promoter region was described in the context of murine development, demonstrating in vivo enrichment for Cdx2 and Hoxd13. Next, we asked whether expression of miR-196b itself as well as the ratio between miR-196b expression and the expression of its direct target HOXA9 is perturbed in CN-AML patients: miRNA-sequencing analysis of healthy bone marrow-derived hematopoietic subpopulations revealed highest expression of miR-196b in the lymphoid-primed multi-potent progenitor subpopulation, following the same expression pattern as known for its target HOXA9, whose expression is also highest in immature cells (Figure 1D; Online Supplementary Table SE1). This resulted in a significant correlation between the expression levels of mature miR-196b or MF139051 and HOXA9 in HSPC (Online Supplementary Figure S2A; Online Supplementary Table SW2). In contrast, expression levels of miR-196b and of HOXA9 remained at similar levels across all three functionally validated leukemic bone marrow subpopulations from CN-AML patients (Figure 1D; Online Supplementary Table SE1). In contrast to the expression levels of the miR-196b transcript MF139051 and miR-196b, HOXA9 expression was dramatically increased in leukemic CD34 bone marrow compared to CD34 normal bone marrow cells. This difference could be confirmed in functionally validated leukemic stem cells compared to normal HSPC and in published data (Online Supplementary Figures S2B and S3; Online Supplementary Tables SW3-4). Based on this, the correlation between the expression levels of the mature miR-196b and its transcripts versus the expression of HOXA9 was lost in leukemic cells (Supplementary Figures S4). The difference in expression patterns resulted in a 111-fold higher ratio between the expression values of HOXA9 and mature miR-196b in CN-AML compared to normal CD34 bone marrow and a 57-fold higher ratio between NPM1c AML and normal CD34 bone marrow, which also held true for the short transcript MF139051 calculated for the same AML groups (Figure 1E; Online Supplementary Table SW4). This implies that compared to normal CD34 hematopoietic cells there are many fewer miR-196b transcripts and mature miRNA per HOXA9 transcript in CN-AML, including NPM1-mutated cases, previously shown to have particularly high HOX gene expression. Overexpression of miR-196b in normal murine HSPC did not affect clonogenicity and cell growth in vitro (Figure 2A; Online Supplementary Figure S5A, B). miR196b increased the proportion of circulating myeloid cells and three of 14 mice developed AML after a long latency, indicating that cooperating events may contribute to disease induction. (Figure 2B, F; Online Supplementary Table SW5). To test the impact of miR-196b expression in the context of Cdx2-induced AML, we retrovirally coexpressed Cdx2/miR-196b or Cdx2 alone in murine HSPC (Online Supplementary Figure S5C). Co-expression of miR-196b considerably reduced the Cdx2-induced proliferation, clonogenicity and spleen colony formation compared to those of Cdx2-transduced cells (Figure 2C, D; Online Supplementary Figure S5D-F). Cdx2-transplanted mice developed AML with a median latency of 193 days. In contrast, only 40% of the Cdx2/miR-196b-transplanted mice developed AML with a significantly longer latency (Figure 2E; Online Supplementary Table SW5). Moreover, miR-196b impaired growth and colony formation in the CDX2 and HOX gene-positive human AML cell lines OCI-AML3 and NB4 in vitro, in contrast to the CDX2 and HOX gene-negative human AML cell line Kasumi-1. Furthermore, miR-196b reduced engraftment of OCI-AML3 in NSG mice (Online Supplementary Figure S6A-K; Online Supplementary Table SW6). Overexpression of miR-196b did not induce apoptosis, cell cycle arrest or senescence in OCI-AML3 and NB4 cells (data not shown), suggesting that miR-196b preferentially targets selfrenewal by reducing HOX gene expression, thereby reducing clonogenicity and engraftment potential. Overexpression of miR-196b in HSPC alone resulted in 155 differentially expressed genes compared to the control. Among these 155 genes, 44% of the downregulated ones were significantly enriched for known miR-196b targets, whereas the upregulated ones did not show any miR-196b target enrichment (Online Supplementary Tables SE2-5; Online Supplementary Figure S7A-C). In Cdx2-transduced cells, overexpression of miR-196b induced 524 differentially expressed genes compared to Cdx2 alone (Online Supplementary Figure S7D; Online Supplementary Tables SE6 and SE7). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) II1 Apr 2018PD14-03 PREDICTION OF TREATMENT RESISTANCE UNDER ABIRATERONE AND ENZALUTMIDE VIA DETECTION OF AR-V7 IN PERIPHERAL WHOLE BLOOD OF CASTRATION-RESISTANT PROSTATE CANCER PATIENTS Anna-Katharina Seitz, Silvia Thoene, Andreas Bietenbeck, Roman Nawroth, Robert Tauber, Mark Thalgott, Sebastian Schmid, Ramona Secci, Margitta Retz, Jürgen Gschwend, Jürgen Ruland, Christof Winter, and Matthias Heck Anna-Katharina SeitzAnna-Katharina Seitz More articles by this author , Silvia ThoeneSilvia Thoene More articles by this author , Andreas BietenbeckAndreas Bietenbeck More articles by this author , Roman NawrothRoman Nawroth More articles by this author , Robert TauberRobert Tauber More articles by this author , Mark ThalgottMark Thalgott More articles by this author , Sebastian SchmidSebastian Schmid More articles by this author , Ramona SecciRamona Secci More articles by this author , Margitta RetzMargitta Retz More articles by this author , Jürgen GschwendJürgen Gschwend More articles by this author , Jürgen RulandJürgen Ruland More articles by this author , Christof WinterChristof Winter More articles by this author , and Matthias HeckMatthias Heck More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.789AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen receptor splice variant 7 (AR-V7) expression in circulating tumor cells (CTCs) was demonstrated to predict poor treatment response in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone or enzalutamide. To develop a practical and robust liquid profiling approach for direct quantification of AR-V7 in peripheral whole blood without the need of CTC capturing and to determine its potential to predict treatment response in mCRPC patients. METHODS Whole blood samples from a prospective biorepository of 85 mCRPC patients before treatment initiation with abiraterone (n=56) or enzalutamide (n=29) were analyzed with droplet digital PCR. The association of AR-V7 status with prostate-specific antigen (PSA) response defined by PSA decline ≥50% as well as PSA-progression-free survival (PSA-PFS), clinical PFS, and overall survival (OS) was assessed. RESULTS High AR-V7 expression levels in whole blood were detectable in 18% (15/85) of patients. No patient with high AR-V7 expression achieved PSA response, and AR-V7 status was an independent predictor of PSA response in multivariable logistic regression analysis (p=0.03). High AR-V7 expression was associated with shorter PSA-PFS (median 2.4 vs. 3.7 months, p<0.001), shorter clinical PFS (median 2.7 vs. 5.5 months, p<0.001), and shorter OS (median 4.0 vs. 13.9 months, p<0.001). On multivariable Cox regression analysis, high AR-V7 expression remained an independent predictor of shorter PSA-PFS (Hazard ratio (HR) 7.0, 95% CI 2.3 - 20.7, p<0.001), shorter clinical PFS (HR 2.3, 95% CI 1.1 - 4.9, p=0.02), and shorter OS (HR 3.0, 95 %CI 1.4 - 6.3, p=0.005). CONCLUSIONS AR-V7 mRNA level testing in whole blood is a simple and promising approach to predict poor treatment outcome in mCRPC patients undergoing treatment with abiraterone or enzalutamide. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e305 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Anna-Katharina Seitz More articles by this author Silvia Thoene More articles by this author Andreas Bietenbeck More articles by this author Roman Nawroth More articles by this author Robert Tauber More articles by this author Mark Thalgott More articles by this author Sebastian Schmid More articles by this author Ramona Secci More articles by this author Margitta Retz More articles by this author Jürgen Gschwend More articles by this author Jürgen Ruland More articles by this author Christof Winter More articles by this author Matthias Heck More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The transcription factor Foxp1 is critical for early B cell development. Despite frequent deregulation of Foxp1 in B cell lymphoma, the physiological functions of Foxp1 in mature B cells remain unknown. Here, we used conditional gene targeting in the B cell lineage and report that Foxp1 disruption in developing and mature B cells results in reduced numbers and frequencies of follicular and B-1 B cells and in impaired antibody production upon T cell-independent immunization in vivo. Moreover, Foxp1-deficient B cells are impaired in survival even though they exhibit an increased capacity to proliferate. Transcriptional analysis identified defective expression of the prosur-vival Bcl-2 family gene Bcl2l1 encoding Bcl-xl in Foxp1-deficient B cells, and we identified Foxp1 binding in the regulatory region of Bcl2l1. Transgenic overexpression of Bcl2 rescued the survival defect in Foxp1-deficient mature B cells in vivo and restored peripheral B cell numbers. Thus, our results identify Foxp1 as a physiological regulator of mature B cell survival mediated in part via the control of Bcl-xl expression and imply that this pathway might contribute to the pathogenic function of aberrant Foxp1 expression in lymphoma.

In hyper-IgE syndromes (HIES), a group of primary immunodeficiencies clinically overlapping with atopic dermatitis, early diagnosis is crucial to initiate appropriate therapy and prevent irreversible complications. Identification of underlying gene defects such as in DOCK8 and STAT3 and corresponding molecular testing has improved diagnosis. Yet, in a child and her newborn sibling with HIES phenotype molecular diagnosis was misleading. Extensive analyses driven by the clinical phenotype identified an intronic homozygous DOCK8 variant c.4626 + 76 A > G creating a novel splice site as disease-causing. While the affected newborn carrying the homozygous variant had no expression of DOCK8 protein, in the index patient molecular diagnosis was compromised due to expression of altered and wildtype DOCK8 transcripts and DOCK8 protein as well as defective STAT3 signaling. Sanger sequencing of lymphocyte subsets revealed that somatic alterations and reversions revoked the predominance of the novel over the canonical splice site in the index patient explaining DOCK8 protein expression, whereas defective STAT3 responses in the index patient were explained by a T cell phenotype skewed towards central and effector memory T cells. Hence, somatic alterations and skewed immune cell phenotypes due to selective pressure may compromise molecular diagnosis and need to be considered with unexpected clinical and molecular findings.

Background: It has been demonstrated that androgen receptor splice variant 7 (AR-V7) expression in circulating tumor cells (CTCs) predicts poor treatment response in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone or enzalutamide. Objective: To develop a practical and robust liquid profiling approach for direct quantification of AR-V7 in peripheral whole blood without the need for CTC capture and to determine its potential for predicting treatment response in mCRPC patients. Design, setting, and participants: Whole blood samples from a prospective biorepository of 85 mCRPC patients before treatment initiation with abiraterone (n = 56) or enzalutamide (n = 29) were analyzed via droplet digital polymerase chain reaction. Outcome measurements and statistical analysis: The association of AR-V7 status with prostate-specific antigen (PSA) response defined by PSA decline >= 50% and with PSA-progression-free survival (PSA-PFS), clinical PFS, and overall survival (OS) was assessed. Results and limitations: High AR-V7 expression levels in whole blood were detectable in 18% (15/85) of patients. No patient with high AR-V7 expression achieved a PSA response, and AR-V7 statuswas an independent predictor of PSA response in multivariable logistic regression analysis (p = 0.03). High AR-V7 expression was associated with shorter PSAPFS (median 2.4 vs 3.7 mo; p < 0.001), shorter clinical PFS (median 2.7 vs 5.5 mo; p < 0.001), and shorter OS (median 4.0 vs. 13.9 mo; p < 0.001). On multivariable Cox regression analysis, high AR-V7 expression remained an independent predictor of shorter PSA-PFS (hazard ratio [HR] 7.0, 95% confidence interval [CI] 2.3-20.7; p < 0.001), shorter clinical PFS (HR 2.3, 95% CI 1.1-4.9; p = 0.02), and shorter OS (HR 3.0, 95% CI 1.4-6.3; p = 0.005). Conclusions: Testing of AR-V7 mRNA levels in whole blood is a simple and promising approach to predict poor treatment outcome in mCRPC patients receiving abiraterone or enzalutamide. Patient summary: We established a method for determining AR-V7 status in whole blood. This test predicted treatment resistance in patients with metastatic castration-resistant prostate cancer undergoing treatment with abiraterone or enzalutamide. Prospective validation is needed before application to clinical practice. (C) 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.

The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2(Y641F) is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2(Y641F) by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2(Y641F). The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2(Y641F) can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

The histone methyl-transferase EZH2 is frequently affected by gain-of-function mutations in germinal-center lymphoma. To further test if these EZH2 mutations can be driver mutations in lymphomagenesis, we have previously generated and characterized a mouse line transgenic for EZH2Y641F. This mouse model leads to an acceleration of lymphoma development in combination with Myc. We have now further investigated this model system and have used RNAseq and ChIPseq to characterize the effects of mutated EZH2 on gene expression and the epigenome.

Abstract Abstract 227 The histone methyl-transferase EZH2 is frequently mutated in germinal-center derived diffuse-large B cell lymphoma (DLBCL) and follicular lymphoma. We and others have demonstrated that these mutations in tyrosine 641 of EZH2 represent a dominant gain-of-function, enhancing the capability to trimethylate H3K27 in combination with wildtype activity (Yap et al, Blood 2011; Sneeringer et al, PNAS 2010). To further test if these EZH2 mutations can be driver mutations in lymphomagenesis we have now generated and characterized a mouse line transgenic for EZH2 Y641F (the most commonly observed mutant form). Transgenic mice were created on the C57/Bl6 background by pronuclear microinjection of a construct where EZH2 Y641F is expressed from a lymphoid specific Emu-lck promoter. Spleen cells isolated from the transgenic mice displayed a global increase in H3K27me3 when analyzed by Western Blot. However none of the mice showed signs of lymphoma even when observed for prolonged periods of time. As EZH2 mutations often coincide with other mutations in lymphoma, we also combined the expression of EZH2 Y641F with the over-expression of Myc by crossing one of the transgenic lines with Emu-myc transgenic mice. In this combination model, we observed a dramatic acceleration of lymphoma development with the combination of Myc and EZH2 Y641F as compared to Myc alone (Myc alone (n=26) 137.5 days, Myc+EZH2 Y641F (n=20) 51 days, p < 0.0001 Log-Rank (Mantel-Cox) Test). The resulting lymphomas are characterized by a generalized massive lymphadenopathy, splenomegaly (spleen weight 0.416 +/− 0.133 g), bone marrow infiltration (72 +/− 8 %) and lymphocytosis (WBC 138.5 +/− 77.1 ×1000/μl). They show histological features of high-grade disease, and initiate disease in secondary recipients. In contrast to lymphomas observed with Emu-myc alone, all of the lymphomas observed in mice transgenic for both, Myc and EZH2 Y641F, presented with a mature B cell phenotype (B220+IgM+). This shift in differentiation can also be observed in the bone marrow even before development of overt lymphomas. While Myc transgenics mainly show an increase in the B220+IgM−compartment, this is shifted towards B220+IgM+ in double transgenics. Cell cycle analysis by in vivo Brdu incorporation before disease onset shows a drastic increase in the fraction of cells in cycle in the splenic B cells of double transgenic mice (43.3 and 44.9 % cells in S phase) as compared to mice transgenic for Myc alone (10.6 % and 14.2 % cells in S phase). Southern Blot analysis using a probe specific for JH4 showed more than one clonal rearrangement in a majority of the lymphomas in the double-transgenic mice. In summary, EZH2 Y641F can collaborate with Emu-myc in high efficiency lymphoma induction. This new murine lymphoma model now provides a powerful tool to study global changes in the epigenome caused by this frequent mutation and may be a useful system for testing novel treatments. Disclosures: No relevant conflicts of interest to declare.

Karsten Spiekermann, Wolfgang Hiddemann, Michaela Feuring-Buske and Christian Buske Konstantin Petropoulos, Aniruddha Deshpande, Leticia Quintanilla-Martinez, Stefan K. Bohlander, Vijay P. S. Rawat, Silvia Thoene, Vegi M. Naidu, Natalia Arseni, Bernhard Heilmeier, Klaus Metzeler, myeloid leukemia gene expression in human acute HOX correlated with deregulated progenitors depending on its N-terminal domain and is closely gene expression in murine HOX Overexpression of CDX2 perturbs

Abstract Abstract 739 Recent studies suggest that a variety of regulatory molecules active in embryonic development such as clustered and non-clustered homeobox genes play an important role in normal and malignant hematopoiesis. Since it was shown that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, it is of particular interest to examine the expression profile and function of its only recently discovered human homologue VENTX in hematopoietic development. Expression of the VENTX gene was analyzed in normal human hematopoiesis and AML patients samples by microarray and qPCR. To test the impact of the constitutive expression of VENTX on human progenitor cells, CD34+ cord blood (CB) cells were retrovirally transduced with VENTX or the empty control vector and analyzed using in vitro and in vivo assays. So far we and others have not been able to identify a murine Xenopus xvent gene homologue. However, we were able to document the expression of this gene by qPCR in human lineage positive hematopoietic subpopulations. Amongst committed progenitors VENTX was significantly 13-fold higher expressed in CD33+ BM myeloid cells (4/4 positive) compared to CD19+ BM lymphoid cells (5/7 positive, p=0.01). Of note, expression of VENTX was negligible in normal CD34+/CD38− but detectable in CD34+ BM human progenitor cells. In contrast to this, leukemic CD34+/CD38− from AML patients (n=3) with translocation t(8,21) showed significantly elevated expression levels compared to normal CD34+ BM cells (n=5) (50-fold higher; p≤0.0001). Furthermore, patients with normal karyotype NPM1c+/FLT3-LM− (n=9), NPM1c−/FLT3-LM+ (n=8) or patients with t(8;21) (n=9) had an >100-fold higher expression of VENTX compared to normal CD34+ BM cells and a 5- to 7.8-fold higher expression compared to BM MNCs. Importantly, lentivirus-mediated long-term silencing of VENTX in human AML cell lines (mRNA knockdown between 58% and 75%) led to a significant, reduction in cell number compared to the non-silencing control construct (>79% after 120h). Suggesting that growth of human leukemic cell lines depends on VENTX expression in vitro. As we observed that VENTX is aberrantly expressed in leukemic CD34+ cells with negligible expression in normal counterparts, we assessed the impact of forced VENTX gene expression in normal CD34+ human progenitor cells on the transcription program. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development (CD11b, CD125, CD9,CD14 and M-CSF), and downregulates genes involved in early lymphoid development (IL-7, IL-9R, LEF1/TCF and C-JUN) and erythroid development such as EPOR, CD35 and CD36. We then tested whether enforced expression of VENTX in CD34+ cells is able to alter the hematopoietic development of early human progenitors as indicated by gene expression and pathway analyses. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells induced a significant increase in the number of myeloid colonies compared to the GFP control with 48 ± 6.5 compared to 28.9 ± 4.8 CFU-G per 1000 initially plated CD34+ cells (n=11; p=0.03) and complete block in erythroid colony formation with an 81% reduction of the number of BFU-E compared to the control (n=11; p<0.003). In a feeder dependent co-culture system, VENTX impaired the development of B-lymphoid cells. In the NOD/SCID xenograft model, VENTX expression in CD34+ CB cells promoted generation of myeloid cells with an over 5-fold and 2.5-fold increase in the proportion of human CD15+ and CD33+ primitive myeloid cells compared to the GFP control (n=5, p=0.01). Summary: Overexpression of VENTX perturbs normal hematopoietic development, promotes generation of myeloid cells and impairs generation of lymphoid cells in vitro and in vivo. Whereas VENTX depletion in human AML cell lines impaired their growth.Taken together, these data extend our insights into the function of human embryonic mesodermal factors in human hematopoiesis and indicate a role of VENTX in normal and malignant myelopoiesis. Disclosures: No relevant conflicts of interest to declare.

Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus xvent2 gene, is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34+ leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT–PCR documented expression of the gene in lineage positive hematopoietic subpopulations, with the highest expression in CD33+ myeloid cells. Notably, expression levels of VENTX were negligible in normal CD34+/CD38− or CD34+ human progenitor cells. In contrast to this, leukemic CD34+/CD38− cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells perturbs normal hematopoietic development, promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together, these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis.