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Conjugated polymer nanoparticles (CPNs) have emerged as a new promising class of cancer theranostic agents due to their desirable optical features, such as high absorption coefficient and photoluminescence quantum yields, spanning from the ultraviolet to the near infrared, along with their photothermal and photodynamic properties. However, limited studies have been demonstrated up to now on the rational design of CPNs for specific biological purposes. In particular, it is not well understood how exactly the chemical structure of the conjugated polymer and the nanoparticle formulation approach (encapsulation versus nanoprecipitation) associates with the cytotoxicity, the intracellular uptake in vitro and the therapeutic behavior. For this reason, nanoprecipitated and encapsulated aqueous CPNs were formulated consisting of thiophene–quinoxaline type conjugated polymers varying as regards the number of the fluorine atoms (three versus four) on the repeat unit. The obtained CPNs were systematically examined in terms of cytotoxicity and intracellular uptake in vitro in three epithelial breast cell lines represented by one normal cell line, and two breast cancer cell lines composed of one luminal and one triple negative line. From the obtained results, it is presented that only the nanoprecipitated CPNs with the three fluorine atoms exhibited efficient intracellular uptake to all the epithelial cell lines tested, in addition to the visible fluorescence on the triple negative breast cancer cells. Moreover, evaluation of the comparison of the effect of the CPNs on cell proliferation and apoptosis of all cell lines with the corresponding antibiotic staurosporine was performed, indicating a putative therapeutic potential of nanoparticles under study.

Simple Summary Different types of cells with tumor-initiating cell (TIC) activity contribute to colorectal cancer (CRC) progression and resistance to anti-cancer treatment. In this study, we aimed to understand whether different cell types exist within a patient-derived tumor culture, distinguishable by different patterns of their gene expression. By mRNA sequencing of patient-derived CRC cultures at the single-cell level, we defined expression programs that closely resemble differentiated cell populations of the normal intestine. Here, cell type-associated subpopulations showed differences in functional properties such as cell growth and energy metabolism. Subsequent functional analyses in vitro and in vivo demonstrated that metabolic states are linked to TIC activity in primary CRC cultures. We also show that TIC activity is dependent on oxidative phosphorylation, which may therefore represent a target for novel therapies. Abstract Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC.

Summary Novel treatment options for metastatic colorectal cancer (CRC) are urgently needed to improve patient outcome. Here, we screen a library of non-characterized small molecules against a heterogeneous collection of patient-derived CRC spheroids. By prioritizing compounds with inhibitory activity in a subset of—but not all—spheroid cultures, NCT02 is identified as a candidate with minimal risk of non-specific toxicity. Mechanistically, we show that NCT02 acts as molecular glue that induces ubiquitination of cyclin K (CCNK) and proteasomal degradation of CCNK and its complex partner CDK12. Knockout of CCNK or CDK12 decreases proliferation of CRC cells in vitro and tumor growth in vivo. Interestingly, sensitivity to pharmacological CCNK/CDK12 degradation is associated with TP53 deficiency and consensus molecular subtype 4 in vitro and in patient-derived xenografts. We thus demonstrate the efficacy of targeted CCNK/CDK12 degradation for a CRC subset, highlighting the potential of drug-induced proteolysis for difficult-to-treat types of cancer.

Disseminated tumor cells (dTCs) can frequently be detected in the bone marrow (BM) of colorectal cancer (CRC) patients, raising the possibility that the BM serves as a reservoir for metastatic tumor cells. Identification of dTCs in BM aspirates harbors the potential of assessing therapeutic outcome and directing therapy intensity with limited risk and effort. Still, the functional and prognostic relevance of dTCs is not fully established. We have previously shown that CRC cell clones can be traced to the BM of mice carrying patient-derived xenografts. However, cellular interactions, proliferative state and tumorigenicity of dTCs remain largely unknown. Here, we applied a coculture system modeling the microvascular niche and used immunofluorescence imaging of the murine BM to show that primary CRC cells migrate toward endothelial tubes. dTCs in the BM were rare, but detectable in mice with xenografts from most patient samples (8/10) predominantly at perivascular sites. Comparable to primary tumors, a substantial fraction of proliferating dTCs was detected in the BM. However, most dTCs were found as isolated cells, indicating that dividing dTCs rather separate than aggregate to metastatic clones-a phenomenon frequently observed in the microvascular niche model. Clonal tracking identified subsets of self-renewing tumor-initiating cells in the BM that formed tumors out of BM transplants, including one subset that did not drive primary tumor growth. Our results indicate an important role of the perivascular BM niche for CRC cell dissemination and show that dTCs can be a potential source for tumor relapse and tumor heterogeneity.

As colorectal cancer (CRC) mortality remains high due to metastatic spread in patients with advanced disease stages, we here aimed at identifying novel metastasis regulators in CRC and at understanding their mechanism of action. In CRC, tumor progression and metastasis formation are driven by cells that possess tumor-initiating cell (TIC) activity. We therefore employed a collection of primary patient-derived CRC tumor spheroid cultures (TSC) that are enriched for cells with TIC activity to perform a gene activating in vivo screen, intending to identify genes that enhance metastasis formation. To this end, TSC (n=4) were transduced with a lentiviral trapping vector that integrates into the genome and thereby drives the overexpression of genes located in the vicinity of the integration site (IS). Transduced TSC were then serially transplanted under the kidney capsule of immunodeficient mice (NSG) and monitored for the induction of metastasis formation. Analysis of lentiviral IS by linear amplification-mediated (LAM)-PCR revealed an enrichment of an IS within the gene locus of Estrogen Receptor Binding Site Associated Antigen 9 (EBAG9) in metastases. RNA sequencing confirmed the trapping vector mediated upregulation of a truncated version of the EBAG9 gene (exon 5-7), which strongly suggested a contribution of EBAG9 to metastatic spread. To validate these findings, both the truncated (TR) as well as the physiologically occurring full length (FL) EBAG9 gene were overexpressed in TSC (n=4) and serially transplanted into NSG mice. In addition to an augmentation in metastasis upon EBAG9 overexpression compared to controls (FL: 1.4-fold, TR: 2.4-fold), we also detected the occurrence of lymphogenous metastases when EBAG9 levels were increased, indicating that EBAG9 not only contributes to metastatic spread but also modulates tropism. To further characterize the underlying processes, global gene and protein expression analyses were performed in primary TSC as well as in the CRC cell line DLD1. Compared to control transduced cells, EBAG9 overexpression induced expression changes in cell adhesion and microtubule molecules, which led us to investigate cell migration and adhesion as potential contributing mechanisms to metastasis formation. We here observed an increase both in cell migration as well as in adhesion to the extracellular matrix protein collagen I upon EBAG9 overexpression in DLD1. Interestingly, DLD1 adhesion to lymphatic endothelial cells was significantly higher in EBAG9 FL (p=0.008) and EBAG9 TR (p=0.016) overexpressing cells, underscoring the role of EBAG9 in tropism modulation. In summary, we here identified EBAG9 as novel metastasis regulator in CRC which will not only contribute to a better mechanistic understanding of metastatic spread but also to the development of novel future strategies for preventing and targeting advanced disease stages. Citation Format: Karin Laaber, Taronish D. Dubash, Anna Maria Melzer, Tania Christiansen, Nati Ha, Benedikt Brors, Martin Schneider, Friederike Herbst, Hanno Glimm, Claudia R. Ball. A gene activating in vivo screen identifies EBAG9 as novel metastasis regulator in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2852.

Tumor progression in colorectal cancer (CRC) is driven by a subpopulation of cells with tumor-initiating cell (TIC) activity. In the past, bulk experiments provided strong evidence for functional heterogeneity of the CRC TIC compartment with distinct types of TICs driving CRC progression. However, in these retrospective experiments direct assignment of functional states to individual functionally relevant cell types in CRC has not been possible. We here asked whether functional states of individual cells can be assigned to specific CRC cell subpopulations. Therefore, 4,663 single-cell profiles were generated by single-cell RNA-sequencing (scRNA-seq) of patient-derived CRC spheroid cultures (n=12). Non-negative matrix factorization identified 13 gene expression modules linked to cell states or cell types that resemble differentiation states of normal intestinal epithelial cells. To evaluate their presence in CRC patient tumors, the identified transcriptional programs were applied to publicly available expression data (TCGA cohort; n=459). Clustering of samples based on the contribution of individual expression signatures revealed six patient clusters with significantly different progression-free survival (p=0.043) and a trend towards different overall survival (p=0.12), indicating a relevance for patient outcome. Comparing cell state meta-signature expression in cell type-associated subpopulations revealed differences in functional states, i.e. proliferative and metabolic activity. Interestingly, stem-like cells showed significantly increased oxidative phosphorylation (OXPHOS) and decreased glycolysis scores, suggesting a link between metabolic states and cell differentiation. scRNA-seq of patient-derived organoid and xenograft models as well as primary tumors revealed similar trends. To assess whether metabolic states are linked to TIC activity, CRC spheroid cells were sorted according to mitochondrial membrane potential (MMP), and sphere forming cell (SFC) as well as TIC frequency were assessed by limiting dilutions. Strikingly, MMPhigh cells exhibited a pronounced increase of SFC frequencies in vitro compared to MMPlow cells in 80% of patients (n=5). In addition, TIC frequencies were strongly increased in the MMPhigh compared to the MMPlow subpopulation in vivo (n=2; 1/46,535 vs. 1/211,305 and 1/249 vs. 1/2,089), demonstrating that TIC activity relies on OXPHOS. In line with this, pretreatment with the OXPHOS inhibitor CCCP decreased SFC frequencies (n=3), indicating targetable metabolic vulnerability of CRC spheroid cells. In summary, we here show that transcriptional heterogeneity identifies functional states during CRC TIC differentiation. Targeting context-dependent regulation of tumor cell differentiation might unravel novel vulnerabilities for therapeutic intervention in human CRC. Citation Format: Martina K. Zowada, Stephan M. Tirier, Sebastian M. Dieter, Teresa Krieger, Ava Oberlack, Robert L. Chua, Mario Huerta, Foo Wei Ten, Karin Laaber, Jeongbin Park, Katharina Jechow, Torsten Müller, Mathias Kalxdorf, Mark Kriegsmann, Katharina Kriegsmann, Friederike Herbst, Jeroen Krijgsveld, Martin Schneider, Roland Eils, Hanno Glimm, Christian Conrad, Claudia R. Ball. Transcriptional heterogeneity identifies functional states of tumor-initiating cell differentiation in human colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1491.

The de novo DNA methyltransferases Dnmt3a and Dnmt3b are of crucial importance in hematopoietic stem cells. Dnmt3b has recently been shown to play a role in genic methylation. To investigate how Dnmt3b-mediated DNA methylation affects leukemogenesis, we analyzed leukemia development under conditions of high and physiological methylation levels in a tetracycline-inducible knock-in mouse model. High expression of Dnmt3b slowed leukemia development in serial transplantations and impaired leukemia stem cell (LSC) function. Forced Dnmt3b expression induced widespread DNA hypermethylation inMyc-Bcl2-induced leukemias, preferentially at gene bodies.MLL-AF9-induced leukemogenesis showed much less pronounced DNA hypermethylation upon Dnmt3b expression. Nonetheless, leukemogenesis was delayed in both models with a shared core set of DNA hypermethylated regions and suppression of stem cell-related genes. Acute myeloid leukemia patients with high expression of Dnmt3b target genes showed inferior survival. Together, these findings indicate a critical role for Dnmt3b-mediated DNA methylation in leukemia development and maintenance of LSC function.

Within primary colorectal cancer (CRC) a subfraction of all tumor-initiating cells (TIC) drives long-term progression in serial xenotransplantation. It has been postulated that efficient maintenance of TIC activity in vitro requires serum-free spheroid culture conditions that support a stem-like state of CRC cells. To address whether tumorigenicity is indeed tightly linked to such a stem-like state in spheroids, we transferred TIC-enriched spheroid cultures to serum-containing adherent conditions that should favor their differentiation. Under these conditions, primary CRC cells did no longer grow as spheroids but formed an adherent cell layer, up-regulated colon epithelial differentiation markers, and down-regulated TIC-associated markers. Strikingly, upon xenotransplantation cells cultured under either condition equally efficient formed serially transplantable tumors. Clonal analyses of individual lentivirally marked TIC clones cultured under either culture condition revealed no systematic differences in contributing clone numbers, indicating that phenotypic differentiation does not select for few individual clones adapted to unfavorable culture conditions. Our results reveal that CRC TIC can be propagated under conditions previously thought to induce their elimination. This phenotypic plasticity allows addressing primary human CRC TIC properties in experimental settings based on adherent cell growth.

Genes that regulate hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation are tightly controlled by regulatory regions. However, mapping such regions relies on surface markers and immunophenotypic definition of HSCs. Here, we use γ-retroviral integration sites (γRV ISs) from a gene therapy trial for 10 patients with Wiskott-Aldrich syndrome to mark active enhancers and promoters in functionally defined long-term repopulating HSCs. Integration site clusters showed the highest ATAC-seq signals at HSC-specific peaks and strongly correlated with hematopoietic risk variants. Tagged genes were significantly enriched for HSC gene sets. We were able to map over 3,000 HSC regulatory regions in late-contributing HSCs, and we used these data to identify miR-10a and miR-335 as two miRNAs regulating early hematopoiesis. In this study, we show that viral insertion sites can be used as molecular tags to assess chromatin conformation on functionally defined cell populations, thereby providing a genome-wide resource for regulatory regions in human repopulating long-term HSCs.

Combining high-coverage whole-genome sequencing with functional analyses, Giessler et al. demonstrate that tumor initiation and long-term tumor formation in human colorectal cancer are driven by multiple genomic subclones and that the functional heterogeneity of colorectal cancer tumor clone–initiating cells is not based on genomic architecture., A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.

Ectopic viral integration site 1 (EVI1), a transcription factor frequently overexpressed in myeloid neoplasias, has been implicated in the generation of malignancy-associated centrosomal aberrations and chromosomal instability. Here, we sought to investigate the underlying cause of centrosome amplification in EVI1-overexpressing cells. We found that overexpression of EVI1-HA in U2OS cells induced supernumerary centrosomes, which were consistently associated with enlarged nuclei or binuclear cells. Live cell imaging experiments identified cytokinesis failure as the underlying cause of this phenotype. In accordance with previous reports, EVI1 overexpression induced a partial cell cycle arrest in G0/1 phase, accompanied by elevated cyclin D1 and p21 levels, reduced Cdk2 activity and activation of the p53 pathway. Supernumerary centrosomes predominantly occurred in resting cells, as identified by low levels of the proliferation marker Ki-67, leading to the conclusion that they result from tetraploidization after cytokinesis failure and are confined to G0/1-arrested tetraploid cells. Depletion of p53 using siRNA revealed that further polyploidization of these cells was inhibited by the p53-dependent tetraploidy checkpoint.

Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in γ-retroviral (γ-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy.

SummaryHuman colon cancer harbors a small subfraction of tumor-initiating cells (TICs) that is assumed to be a functionally homogeneous stem-cell-like population driving tumor maintenance and metastasis formation. We found unexpected cellular heterogeneity within the TIC compartment, which contains three types of TICs. Extensively self-renewing long-term TICs (LT-TICs) maintained tumor formation in serial xenotransplants. Tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributed to tumor formation only in primary mice. Rare delayed contributing TICs (DC-TICs) were exclusively active in secondary or tertiary mice. Bone marrow was identified as an important reservoir of LT-TICs. Metastasis formation was almost exclusively driven by self-renewing LT-TICs. Our results demonstrate that tumor initiation, self-renewal, and metastasis formation are limited to particular subpopulations of TICs in primary human colon cancer. We identify LT-TICs as a quantifiable target for therapies aimed toward eradication of self-renewing tumorigenic and metastatic colon cancer cells.

The Dnmt2 enzyme utilizes the catalytic mechanism of eukaryotic DNA methyltransferases to methylate several tRNAs at cytosine 38. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile, and the biological function of Dnmt2 has remained elusive. Here, we show that endochondral ossification is delayed in newborn Dnmt2-deficient mice, which is accompanied by a reduction of the haematopoietic stem and progenitor cell population and a cell-autonomous defect in their differentiation. RNA bisulfite sequencing revealed that Dnmt2 methylates C38 of tRNA Asp(GTC), Gly(GCC), and Val(AAC), thus preventing tRNA fragmentation. Proteomic analyses from primary bone marrow cells uncovered systematic differences in protein expression that are due to specific codon mistranslation by tRNAs lacking Dnmt2-dependent methylation. Our observations demonstrate that Dnmt2 plays an important role in haematopoiesis and define a novel function of C38 tRNA methylation in the discrimination of near-cognate codons, thereby ensuring accurate polypeptide synthesis.

Even though a large proportion of patients with acute myeloid leukemia (AML) achieve a complete remission upon initial therapy, the majority of them eventually relapse with resistant disease. Overexpression of the gene coding for the transcription factor Ecotropic Virus Integration site 1 (EVI1) is associated with rapid disease recurrence and shortened survival. We therefore sought to identify EVI1 target genes that may play a role in chemotherapy resistance using a previously established in vitro model system for EVI1 positive myeloid malignancies. Gene expression microarray analyses uncovered the Cell Adhesion Molecule 1 (CADM1) gene as a candidate whose deregulation by EVI1 may contribute to drug refractoriness. CADM1 is an apoptosis inducing tumor suppressor gene that is inactivated by methylation in a variety of tumor types. In the present study we provide evidence that it may play a role in chemotherapy induced cell death in AML: CADM1 was induced by drugs used in the treatment of AML in a human myeloid cell line and in primary diagnostic AML samples, and its experimental expression in a cell line model increased the proportion of apoptotic cells. CADM1 up-regulation was abolished by ectopic expression of EVI1, and EVI1 expression correlated with increased CADM1 promoter methylation both in a cell line model and in primary AML cells. Finally, CADM1 induction was repressed in primary samples from AML patients at relapse. In summary, these data suggest that failure to up-regulate CADM1 in response to chemotherapeutic drugs may contribute to therapy resistance in AML.

More than 10 years ago, we developed an efficient protocol for serum-free retroviral transduction of human hematopoietic stem cells derived from mobilized peripheral blood. After upscaling of the methodology, serum-free retroviral gibbon-ape leukemia virus (GALV) pseudotype PG13/LN vector supernatant produced under strict good manufacturing practice (GMP) conditions was used in the first clinical gene-marking trial in Germany. In this study, we analyzed the titer and transduction efficiency of this serum-free clinical-grade retroviral supernatant 10 years after production to evaluate the long-term stability. Long-term storage and transport on dry ice resulted in modestly decreased titers and levels of transduction efficiency in CD34+ cells ranging from 38.4 to 49.1%. We conclude that the stability of retroviral vectors in serum-free medium allows extended storage and distribution of approved clinical-grade retroviral vector stocks to distant sites in multicenter clinical trials.

Within this study we have developed a novel approach to identify hematopoietic regulatory genes by re-analyzing genome-wide viral integration site (IS) data from a clinical gene therapy trial of 10 Wiskott-Aldrich syndrome patients. We observed a statistically enriched contribution of individual cell clones marked by IS to benign human long-term hematopoiesis within a dataset comprising 130,699 unique IS in the proximity of 12,437 genes. Furthermore, when we assessed the genomic distribution of the IS, we found that clusters of IS (CIS) are significantly over-represented at certain coding and non-coding genetic loci, indicating a viral marking of actively transcribed genes or a proliferation advantage of specific clones due to viral induced deregulation of adjacent genes. Notably, 42 % of the 50 most enriched regions encode for human HSC regulators such as MECOM, LMO2 and HMGA2. Based on a stepwise and stringent selection process evaluating IS number, location and CIS size, we chose the top 12 ranked miRNA genes for further analysis. Of those, three miRNAs (miR-132, miR-142, miR-146a) have described roles in hematopoiesis, strongly validating our selection strategy. In order to functionally investigate the influence of selected candidate genes on hematopoiesis we developed a lentiviral (LV) based pooled overexpression (OE) approach using uniquely barcoded (BC) vectors encoding for the top 8 candidate miRNA HSC regulators in lin- sca1+ ckit+ (LSK) cells. The LV based library consists of two vectors per gene including corresponding GFP control vectors, and harbor a unique BC cassette serving as a molecular tag to allow its unbiased amplification and high-throughput sequencing of up to 96 indexed samples simultaneously. To assess the influence of the selected miRNA candidates on hematopoietic differentiation in vitro, we performed serial CFU assays using BC library transduced LSK-SLAM cells as a starting population. Low infection rates were used to ensure one vector copy per cell. We detected a 1.58±0.03 [SEM] fold increase (p=0.003, n=5) in myeloid colony forming potential of miRNA library OE cells compared to GFP control cells without a shift in colony phenotypes. After three rounds of serial replating, colonies expressing 2/8 candidates were significantly overrepresented (1.6±0.23 to 2.0±0.20 fold; p=0.049 and p=0.005; n =5) while BC vectors encoding for two other candidate miRNAs were significantly reduced (0.4±0.11 to 0.5±0.14 fold, p=0.006 and p=0.027) compared to GFP control (fc=1.0±0.12). To address the influence of candidate miRNA OE on multi-lineage engraftment and self-renewal in vivo, we serially transplanted miRNA or GFP control library transduced LSK cells in a cohort of 18 recipient mice. We revealed efficient candidate BC representation in all mice with a median read count of 7.1E4 (min=2451, max=8.4E5) 20 weeks post-transplant. In line with our results in vitro, we observed a 1.23±0.23 (p=0.044) fold enrichment of the same two miRNA candidates and a depletion of the two other miRNA genes (0.63±0.09 to 0.68±0.13fold; p=0.002 and p=0.033) in PB samples already eight weeks after transplantation. Interestingly, BC sequencing of lineage sorted fractions suggests that one of the overrepresented candidates has a strong influence on T-cell differentiation (fc=1.5±0.2, p=0.029, 20 weeks post TP) whereas the other candidate impacts on myelopoiesis (fc=1.6±0.35, p=0.114). In summary, our systematic stepwise approach combining the comprehensive analysis of the complete integration site repertoire in a clinical gene therapy study with subsequent functional validation using a pooled lentiviral overexpression approach is a useful strategy to identify and characterize novel HSC regulators. Disclosures No relevant conflicts of interest to declare.

Kinetics of hematopoietic recovery driven by different types of human stem and progenitor cells after transplantation are not fully understood. Short-term repopulating cells (STRCs) dominate early hematopoiesis after transplantation. STRCs are highly enriched in adult mobilized peripheral blood compared with cord blood, but the length of their contribution to hematopoiesis remains unclear. To understand posttransplantation durability and lineage contribution of STRCs, we compared repopulation kinetics of mobilized peripheral blood (high STRC content) with cord blood transplants (low STRC content) in long-lived NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (IL2RG−/−) mice. This comparison demonstrates that quantitative contribution of human STRCs to hematopoiesis is restricted to the first 5 months after transplantation. The ratio of STRCs to long-term repopulating cells dramatically changes during ontogeny. This model enables to precisely determine early and late engraftment kinetics of defined human repopulating cell types and to preclinically assess the engraftment kinetics of engineered stem cell transplants.

Hematopoietic stem cell (HSC) regulation is controlled by extrinsic and intrinsic factors adapting blood cell production to the need of the organism. To search for novel HSC regulatory genes, our group has established a unique screening approach. By systematically analyzing the entire integration site (IS) repertoire of ten Wiskott-Aldrich syndrome (WAS) patients enrolled in clinical gene therapy trials, we hypothesize to identify novel key regulatory genes in hematopoiesis. By applying our screening pipeline based on the number and distance of IS to the transcription start sites (TSS) of genes, we observed a statistically significant number of therapeutic vector insertions close to 32 single genes in nine out of ten WAS patients including the Evl/miR-342 gene locus which has not been linked to hematopoiesis so far. Common insertion sites close to Evl/miR-342 accounted for up to 1.2% of relative sequencing reads within the peripheral blood (PB) of patients and clones harboring such integrations contributed to hematopoiesis for up to six years. We therefore hypothesized that the protein-coding gene Evl and/or its intronic miR-342 - which share a common genomic locus - may regulate hematopoiesis. Evl has been shown to play a pivotal role in actin cytoskeleton remodeling, and to interact with RAD51 complexes within homologous recombination. MiR-342 is a direct target of the transcription factor PU.1, which drives myeloid differentiation, and accelerates all-trans retinoic acid (ATRA)-induced differentiation of APL blasts. First of all, we investigated the candidate gene RNA expression in purified murine hematopoietic cell populations. Interestingly, we observed that Evl and miR-342 are not highly expressed in murine Lineage- Sca1+ ckit+ (LSK) cells but their expression increases profoundly with blood cell differentiation. While Evl expression was highest in lymphocytes (20-30 fold higher as compared to LSK cells), miR-342 was expressed at the highest level in macrophages (300 fold higher compared to LSK cells). To study the role of the candidates in hematopoiesis, we overexpressed Evl and miR-342 by using lentiviral vectors in murine primary LSK cells. Gene expression profiling of LSK cells overexpressing Evl revealed that 32% (62 out of 190) of the deregulated transcripts were involved in hematopoietic system development and function. Moreover, the top deregulated canonical pathways detected are essential for the development of B-cells (p=2.59*10-11). However, pathways important for myeloid cells such as immune cell trafficking and, more specifically, granulocytic adhesion and diapedesis (p=2.59*10-3) were significantly upregulated within miR-342- positive LSK cells. Functional analysis showed that Evl overexpression leads to a three- to fourfold increase of preB-cell colonies compared to control vector-transduced LSK cells. By contrast, miR-342 overexpressing cells formed a twofold higher number of myeloid colonies in semisolid medium. Next, the influence of Evl and its intronic miRNA on self-renewal and multilineage differentiation in vivo was investigated in serial bone marrow (BM) transplantation experiments. Within the PB of primary recipient mice, we detected a decrease of Evl-positive cells over time (week 4: 20.6 ± 9.6%; week 20: 4.7 ± 2.4%). Within the spleens a significantly higher donor-derived B-cell frequency was detectable (Evl: 63.6 ± 17.1%; Mock: 39.9 ± 18.3%). In line with our in vitro results, we detected a 4.3 fold higher frequency of Evl positive B-cells four weeks after secondary transplantation (Evl: 68.1 ± 9.8 %; Mock: 15.9 ± 7.5%). In summary, our data show that different hematopoietic differentiation programs are driven by one common gene locus depending on the expressed gene product. While the protein-coding gene Evl drives B-cell lymphopoiesis, its intronic miR-342 promotes myeloid differentiation. Disclosures No relevant conflicts of interest to declare.

Abstract 2398 Deregulated expression of the zinc finger transcription factor ecotropic viral integration site 1 (EVI1) is an independent poor prognostic marker for patients with acute myeloid leukemia. Moreover, we have recently shown that in clinical gene therapy of chronic granulomatous disease (cGD) the activation of this gene locus by strong promoter and enhancer elements within the gamma-retroviral vector LTR has led to clonal dominance and malignant transformation. Physiologically, EVI1 is essential for embryonic development and in regulating hematopoietic stem cell self-renewal. As very little is known about its molecular mechanism driving hematopoietic transformation towards leukemia, we aim to systematically analyze the role of deregulated EVI1 expression and its larger splice form MDS1/EVI1 in hematopoiesis. Lentiviral vector particles encoding for EVI1 (E) or MDS1/EVI1 (ME) and eGFP as marker protein were produced to stably overexpress the transgenes. Transgene expression was verified in myeloid HL60 cells by western blotting and immunofluorescence staining. Analysis of growth kinetics of human HL60 cells in suspension cultures revealed 1.5 – 3 folds lower cell counts of ME and E expressing cells as compared to eGFP control vector transduced cells. We further analyzed the expression of transferrin receptor CD71 which is mainly expressed on proliferating cells to promote iron uptake. Both, E and ME transduced cells, down-regulated CD71 during seven days in culture as compared to eGFP-transduced control cells. 64.2 ± 0.4% of ME cells and 31.1 ± 1.6% of E cells expressed CD71 compared to untransduced (97.4 ± 0.0%) and control vector cells (94.8 ± 0.8%) (p We then asked if the cell cycle arrest in G0/G1 is associated with genetic instability, as patients with insertional activation of EVI1 developed a myelodysplastic syndrome with monosomy 7. Analyzing global gene expression comparing mock and eGFP control vector transduced cells with EVI1 expressing hematopoietic cells revealed more than 2000 differentially regulated genes. Genes involved in cell cycle regulation, recombination, replication and DNA repair were significantly downregulated upon EVI1 expression compared to control cells. For further studying DNA damage repair capacity, we irradiated EVI1- and eGFP-control vector transduced cells. Staining of γ-H2AX, an indirect marker for DNA double strand breaks (DSB), revealed that EVI1+ γ-H2AX+ cells were enriched almost 2-fold in G0/G1 phase of the cell cycle as compared to control vector transduced cells. The number of DSB positive cells decreased within 6 hours without apoptosis indicating that most of the double-strand breaks were repaired by non-homologous end-joining. In summary, our data show that EVI1 overexpression causes G0 cell cycle arrest of hematopoietic cells possibly associated with genetic instability. DSB repair in EVI1+ cells may subsequently lead to the accumulation of additional mutations. Systematic investigation of EVI1 and MDS1/EVI1 overexpression in human hematopoietic cells will gain insights into mechanisms leading to clonal dominance and malignant transformation. Disclosures: No relevant conflicts of interest to declare.

Abstract 2431 Insertional activation of the zinc finger transcription factor ecotropic viral integration site 1 (EVI1) by promoter and enhancer elements within the gamma-retroviral vector LTR has led to clonal dominance and malignant transformation in clinical gene therapy of chronic granulomatous disease (cGD). EVI1 has been shown to inhibit stress induced cell death or TGFβ signaling and is essential for embryonic development. Cytogenetic rearrangements leading to the activation of the gene locus are a marker for poor prognosis in myeloid malignancies. Very little is known about its larger splice variant MDS1/EVI1 in normal or malignant hematopoiesis. We aim to systematically analyze the role of deregulated EVI1 and MDS1/EVI1 expression in hematopoiesis to further elucidate the stepwise progression of clonal selection up to malignant transformation. Lentiviral vector particles encoding for EVI1 (E) or MDS1/EVI1 (ME) and eGFP as marker protein were produced to stably overexpress the transgenes. Transgene expression was verified in myeloid HL60 cells by western blotting and immunofluorescence. Analysis of growth kinetics revealed a 1.5 – 3 fold lower proliferation of ME and E expressing cells as compared to eGFP control vector transduced cells. Additional analysis on cell cycle distribution revealed that in both, E and ME transduced cells, a higher percentage of cells could be detected in the G1/G0 phase of the cell cycle (52.5 ± 1.6% in ME cells and 55.2 ± 1.8% in E cells) compared to untransduced (48.9 ± 1.6%) and control vector cells (47.7 ± 0.3%) (p In summary, our data show that EVI1 overexpression causes G1 cell cycle arrest of hematopoietic cells that may be caused by genetic instability or inefficient DSB repair. No sign for clonal selection could be detected, neither in vitro nor in vivo, but EVI1 transplanted mice showed a delay in thrombocyte recovery. Systematic investigation of EVI1 and MDS1/EVI1 overexpression in human hematopoietic cells will help us to gain insights into regulatory processes of hematopoietic stem cells and mechanisms leading to dominant clones in gene modified hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 3760 Lentiviral vectors (LV) assure stable transgene expression in vivo, allowing to investigate genes and their functions. In recent years, lentiviral gene transfer was considered to facilitate the generation of transgenic mice with a higher yield of transgenic offspring as compared to commonly used DNA microinjection. We applied LV to generate a mouse model transgenic for SETBP1 and eGFP. Murine zygotes were infected at dE0.5 with lentiviral particles directly injected into the perivitelline space. Specific PCRs for either the SETBP1 transgene or for the WPRE element of the lentiviral construct verified complete lentiviral integration in newborn pups (F0). Lentiviral integration sites were detected using highly sensitive LAM-PCR in 65% of 31 analyzed F0 mice. Germline transmission was shown in a total of 33% vector positive offspring from 5 out of 9 F0 mice. However, no ectopic transcription and overexpression of neither SETBP1 nor eGFP could be detected in transgenic mice. We therefore analyzed the methylation status of the internal SFFV promoter (SFFVp) by bisulfite sequencing. Extensive methylation (around 90%) could be assessed in 18 of 18 analyzed CpGs within the promoter region in F0 animals and in all progeny determined (n=12). We transduced mES cells with LV.SFFV.Setbp1.IRES.eGFP or the corresponding eGFP-expressing control vector to exclude transgene effects on epigenetic silencing of SFFVp sequences in self-inactivating LVs. Differentiation of ES cells infected with the transgene vector and SFFV driven control vector led to a 1.8 – 3.5 fold decrease of eGFP expression. To analyze whether methylation of SFFVp sequences is a common event even in adult tissues, we analyzed the methylation status of peripheral blood in mice transplanted with bone marrow cells transduced with either gammaretroviral vectors (RV) or LV 3 months after transplantation (n=7). Interestingly, SFFVp sequences in peripheral blood of mice transplanted with LV transduced bone marrow were stronger methylated than CpGs of SFFVp in RV transplants. Our data demonstrate that the commonly used SFFV promotor is highly methylated with remarkable strength and frequency during development in vivo and differentiation in vitro. We conclude that lentiviral vectors using an internal SFFV promoter are not suitable for the generation of transgenic mice or constitutive expression studies in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.