scholarly journals NKL Homeobox Genes NKX2-3 and NKX2-4 Deregulate Megakaryocytic-Erythroid Cell Differentiation in AML

2021 ◽  
Vol 22 (21) ◽  
pp. 11434
Author(s):  
Stefan Nagel ◽  
Claudia Pommerenke ◽  
Corinna Meyer ◽  
Roderick A. F. MacLeod
Keyword(s):  
Cell Line ◽  
Expression Profiling ◽  
Target Gene ◽  
Target Genes ◽  
Homeobox Genes ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Aberrant Expression ◽  
Erythroid Progenitor ◽  
Developmental Disturbance

NKL homeobox genes encode transcription factors that impact normal development and hematopoietic malignancies if deregulated. Recently, we established an NKL-code that describes the physiological expression pattern of eleven NKL homeobox genes in the course of hematopoiesis, allowing evaluation of aberrantly activated NKL genes in leukemia/lymphoma. Here, we identify ectopic expression of NKL homeobox gene NKX2-4 in an erythroblastic acute myeloid leukemia (AML) cell line OCI-M2 and describe investigation of its activating factors and target genes. Comparative expression profiling data of AML cell lines revealed in OCI-M2 an aberrantly activated program for endothelial development including master factor ETV2 and the additional endothelial signature genes HEY1, IRF6, and SOX7. Corresponding siRNA-mediated knockdown experiments showed their role in activating NKX2-4 expression. Furthermore, the ETV2 locus at 19p13 was genomically amplified, possibly underlying its aberrant expression. Target gene analyses of NKX2-4 revealed activated ETV2, HEY1, and SIX5 and suppressed FLI1. Comparative expression profiling analysis of public datasets for AML patients and primary megakaryocyte–erythroid progenitor cells showed conspicuous similarities to NKX2-4 activating factors and the target genes we identified, supporting the clinical relevance of our findings and developmental disturbance by NKX2-4. Finally, identification and target gene analysis of aberrantly expressed NKX2-3 in AML patients and a megakaryoblastic AML cell line ELF-153 showed activation of FLI1, contrasting with OCI-M2. FLI1 encodes a master factor for myelopoiesis, driving megakaryocytic differentiation and suppressing erythroid differentiation, thus representing a basic developmental target of these homeo-oncogenes. Taken together, we have identified aberrantly activated NKL homeobox genes NKX2-3 and NKX2-4 in AML, deregulating genes involved in megakaryocytic and erythroid differentiation processes, and thereby contributing to the formation of specific AML subtypes.

Dynamic Change in the Stochiometry of ETO2 and SCL Governs the Transition to Terminal Differentiation in Erythroid Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1169-1169
Author(s):  
Julie A. Lambert ◽  
Nicolas Goardon ◽  
Patrick Rodriguez ◽  
Sabine Herblot ◽  
Pierre Thibault ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
The Core ◽  
Undifferentiated Cells

Abstract As highly proliferative erythroid progenitors commit to terminal differentiation, they also progressively undergo growth arrest. To determine the mechanisms underlying the appropriate timing of erythroid gene expression and those associated with growth cessation, we analyzed the dynamical composition of the multiprotein complex nucleated by the bHLH transcription factor SCL, a crucial regulator of erythropoiesis that absolutely requires interaction with other factors to activate transcription. In progenitor cells, the SCL complex marks a subset of erythroid specific genes (alpha-globin, P4.2, glycophorin A) that are transcribed later in differentiating cells, conducting cells toward terminal maturation. To unravel the regulation of transcription by SCL, we used tagging/proteomics approaches in two differentiation-inducible erythroid cell lines, coupled with binding assays to immobilized DNA templates and chromatin immunoprecipitation. Our analyses reveal that the core complex comprised of known proteins (SCL, GATA-1, LMO2, Ldb1 and E2A) and two additional E protein family members, HEB and E2-2, did not change with differentiation. Strikingly, this complex recruits HDAC1-2 in undifferentiated cells which were exchanged with TRRAP, a chromatin remodelling factor, upon differentiation, suggesting an epigenetic regulation of erythroid differentiation mediated by the core SCL complex. Finally, we identified the corepressor ETO2 targeted via this complex through direct interaction with E2A/HEB. In vivo, ETO2 represses the transcription of SCL target genes both in transient assays and in chromatin. During erythroid differentiation, ETO2 remains associated with the SCL complex bound to erythroid promoters. However, the stoichiometry of ETO2 and SCL/HEB changes as SCL and HEB levels increase with erythroid differentiation, both in nuclear extracts and on DNA. To determine the functional consequence of this imbalance in activator to co-repressor ratio, we delivered ETO2 siRNA in primary hematopoietic cells and found an accelerated onset of SCL target genes on induction of erythroid differentiation, and conversely, these genes were decreased following ectopic ETO2 expression. Strikingly, inhibition of ETO2 expression in erythroid progenitors arrests cell proliferation, indicating that ETO2 is required for their expansion. We therefore analyzed gene expression in purified erythroid progenitors and differentiating erythroid cells (E1-E5) and found an inverse correlation between the mRNA levels of ETO2 and cyclin-dependent kinase inhibitors. Moreover, ETO2 siRNA treatment of primary erythroid progenitors results in increased p21 CDKI and Gfi1b expression, as assessed by real-time PCR. Finally, we show by chromatin immunoprecipitation that Gfi-1b, p21 and p27, are direct targets of the SCL- ETO2 complex. We therefore conclude that ETO2 regulates the erythroid lineage fate by repressing SCL marked erythroid genes in undifferentiated cells, and by controlling the expansion of erythroid progenitors. Our study elucidates the dual function of ETO2 in the erythroid lineage and sheds light on epigenetic mechanisms coordinating red blood cell proliferation and differentiation.

Regulation of Erythropoiesis by Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin A (DZNep)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 982-982
Author(s):  
Tohru Fujiwara ◽  
Haruka Saitoh ◽  
Yoko Okitsu ◽  
Noriko Fukuhara ◽  
Yasushi Onishi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Wide Range ◽  
The Impact ◽  
Chip Analysis

Abstract Abstract 982 Background. EZH2, a core component of Polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through mediating trimethylation of histone H3 at lysine 27 (H3K27), and is involved in various biological processes, including hematopoiesis. Overexpression of EZH2 has been identified in a wide range of solid tumors as well as hematological malignancies. Recent studies indicated that 3-deazaneplanocin A (DZNep), an inhibitor of EZH2, preferentially induces apoptosis in cancer cells, including acute myeloid leukemia and myelodysplastic syndromes, implying that EZH2 may be a potential new target for epigenetic treatment. On the other hand, whereas PRC2 complex has been reported to participate in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation (Yu et al. Mol Cell 2009; Ross et al. MCB 2012), the impact of DZNep on erythropoiesis has not been evaluated. Method. The K562 erythroid cell line was used for the analysis. The cells were treated with DZNep at doses of 0.2 and 1 microM for 72 h. Quantitative ChIP analysis was performed using antibodies to acetylated H3K9 and GATA-1 (Abcam). siRNA-mediated knockdown of EZH2 was conducted using Amaxa nucleofection technology™ (Amaxa Inc.). For transcription profiling, SurePrint G3 Human GE 8 × 60K (Agilent) and Human Oligo chip 25K (Toray) were used for DZNep-treated and EZH2 knockdown K562 cells, respectively. Gene Ontology was analyzed using the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/). Results. We first confirmed that DZNep treatment decreased EZH2 protein expression without significantly affecting EZH2 mRNA levels, suggesting that EZH2 was inhibited at the posttranscriptional level. We also confirmed that DZNep treatment significantly inhibited cell growth. Interestingly, the treatment significantly induced erythroid differentiation of K562 cells, as determined by benzidine staining. Transcriptional profiling with untreated and DZNep-treated K562 cells (1 microM) revealed that 789 and 698 genes were upregulated and downregulated (> 2-fold), respectively. The DZNep-induced gene ensemble included prototypical GATA-1 targets, such as SLC4A1, EPB42, ALAS2, HBA, HBG, and HBB. Concomitantly, DZNep treatment at both 0.2 and 1 microM upregulated GATA-1 protein level as determined by Western blotting, whereas the effect on its mRNA levels was weak (1.02- and 1.43-fold induction with 0.2 and 1 microM DZNep treatment, P = 0.73 and 0.026, respectively). Furthermore, analysis using cycloheximide treatment, which blocks protein synthesis, indicated that DZNep treatment could prolong the half-life of GATA-1 protein, suggesting that DZNep may stabilize GATA-1 protein, possibly by affecting proteolytic pathways. Quantitative ChIP analysis confirmed significantly increased GATA-1 occupancy as well as increased acetylated H3K9 levels at the regulatory regions of these target genes. Next, to examine whether the observed results of DZNep treatment were due to the direct inhibition of EZH2 or hitherto unrecognized effects of the compound, we conducted siRNA-mediated transient knockdown of EZH2 in K562 cells. Quantitative RT-PCR analysis demonstrated that siRNA-mediated EZH2 knockdown had no significant effect on the expression of GATA-1 as well as erythroid-lineage related genes. Furthermore, transcription profiles of the genes in the quantitative range of the array were quite similar between control and EZH2 siRNA-treated K562 cells, with a correlation efficient of 0.977. Based on our profiling results, we are currently exploring the molecular mechanisms by which DZNep promotes erythroid differentiation of K562 cells. Conclusion. DZNep promotes erythroid differentiation of K562 cells, presumably through a mechanism not directly related to EZH2 inhibition. Our microarray analysis of DZNep-treated K562 cells may provide a better understanding of the mechanism of action of DZNep. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Establishment of an erythroid cell line from primary CD36+ erythroid progenitor cells

2010 ◽  
Vol 38 (11) ◽  
pp. 994-1005.e2 ◽  
Author(s):  
Susan Wong ◽  
Keyvan Keyvanfar ◽  
Zhihong Wan ◽  
Sachiko Kajigaya ◽  
Neal S. Young ◽  
...  
Keyword(s):  
Cell Line ◽  
Progenitor Cells ◽  
Erythroid Cell ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

Role of Atypical p50:p50:Bcl-3 NFκlB Complex in Differentiation of Erythroid Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 568-568 ◽  
Author(s):  
Stephen Sawyer ◽  
Jingchun Chen
Keyword(s):  
Dna Binding ◽  
Transcriptional Control ◽  
Biological Significance ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Necrosis Factor Alpha ◽  
Erythroid Progenitor ◽  
Tnf Α

Abstract We recently reported that mouse and human primary erythroid progenitor cells and erythroid cell lines synthesize and respond to Tumor Necrosis Factor-alpha (TNF-α). The nuclear transcriptional control complex, NFκlB is central in signaling downstream from TNF-α; so we began to study the function of NFκlB in erythroid cells. We made three very interesting initial findings: 1) first we found that NFκlB binding to DNA increased very slowly in HCD57 erythroid cells treated with erythropoietin (EPO, the hormone required for red blood cell development). An inhibitory effect of adding a neutralizing antibody to TNF-α on EPO-stimulated NFκlB DNA suggested this increase in NFκlB was due to TNF-α rather than direct EPO signaling. 2) We also found that NFκlB binding to DNA increased 10-fold or greater during erythroid differentiation. We found greatly increased NFκlB DNA binding in HCD57 cells that differentiated due to over-expression of JunB, F-MEL induced by DMSO, or human UT7-EPO or murine HCD57 cells induced to differentiate with hemin. 3) Surprisingly, we found that the NFκlB DNA binding complex in mouse primary erythroid cells and the erythroid cells lines tested was almost exclusively composed of the atypical p50/p50/Bcl3 NFκlB rather than the canonical p65/p50 or the non-canonical p65/p52 NFκlB. When we begin to study the biological significance of this atypical NFκlB in EPO-mediated erythroid differentiation in vivo using genetic tools, we found marked deficiencies in the development of erythroid cells in either the nfkb1−/− mice (p50−/−) or the bcl3 −/− mice. The nfkb1−/− mice were mildly anemic. The number of red blood cells in the circulation of these mice was statistically lower than in control mice. The number of CFU-e was also reduced in nfkb1−/− mice. Using the Ter-119 and CD71 staining method, we noted that proerythroblasts and immature erythroid cells increased and mature erythroblasts decreased in either non-anemic bone marrow or anemic spleens of nfkb1−/− mice. Forward scatter of Ter-119+ cells also showed an increased size of the average immature erythroid cell in the bone a marrow of nfkb1 −/− mice, suggesting a block in differentiation and continued cell cycling of the immature erythroblasts. Similar erythroid defects were observed in the spleens of anemic bcl-3−/− mice. nfkb1−/− mice and bcl-3−/− mice are also apparently unable to produce new reticulocytes as effectively as wild type mice after induction of anemia. Our working hypothesis is without expression of either p50 or Bcl-3 NFκlB proteins, immature erythroid cells continue to proliferate and ineffectively differentiate. In summary, the atypical p50/p50/Bcl-3 NFκlB complex appears necessary for maximal differentiation of immature erythroid cells.

scholarly journals Graded Levels of GATA-1 Expression Modulate Survival, Proliferation, and Differentiation of Erythroid Progenitors

2005 ◽  
Vol 280 (23) ◽  
pp. 22385-22394 ◽  
Author(s):  
Xiaoqing Pan ◽  
Osamu Ohneda ◽  
Kinuko Ohneda ◽  
Fokke Lindeboom ◽  
Fumiko Iwata ◽  
...  
Keyword(s):  
Culture System ◽  
Target Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Critical Threshold ◽  
Erythroid Cells ◽  
Es Cell ◽  
Erythroid Progenitor ◽  
Proliferation And Differentiation

Transcription factor GATA-1 plays an important role in gene regulation during the development of erythroid cells. Several reports suggest that GATA-1 plays multiple roles in survival, proliferation, and differentiation of erythroid cells. However, little is known about the relationship between the level of GATA-1 expression and its nature of multifunction to affect erythroid cell fate. To address this issue, we developed in vitro embryonic stem (ES) culture system by using OP9 stromal cells (OP9/ES cell co-culture system), and cultured the mutant (GATA-1.05 and GATA-1-null) and wild type (WT)ES cells, respectively. By using this OP9/ES cell co-culture system, primitive and definitive erythroid cells were developed individually, and we examined how expression level of GATA-1 affects the development of erythroid cells. GATA-1.05 ES-derived definitive erythroid cells were immature with the appearance of proerythroblasts, and highly proliferated, compared with WT and GATA-1-null ES-derived erythroid cells. Extensive studies of cell cycle kinetics revealed that the GATA-1.05 proerythroblasts accumulated in S phase and expressed lower levels of p16INK4A than WT ES cell-derived proerythroblasts. We concluded that GATA-1 must achieve a critical threshold activity to achieve selective activation of specific target genes, thereby influencing the developmental decision of an erythroid progenitor cell to undergo apoptosis, proliferation, or terminal differentiation.

scholarly journals Differential Contribution of the Gata1 Gene Hematopoietic Enhancer to Erythroid Differentiation

2008 ◽  
Vol 29 (5) ◽  
pp. 1163-1175 ◽  
Author(s):  
Mikiko Suzuki ◽  
Takashi Moriguchi ◽  
Kinuko Ohneda ◽  
Masayuki Yamamoto
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Erythroid Differentiation ◽  
Artificial Chromosome ◽  
Erythroid Cell ◽  
Erythroid Progenitor ◽  
Dynamic Regulation ◽  
Gfp Reporter ◽  
Specific Manner ◽  
Gata Box

ABSTRACT GATA1 is a key regulator of erythroid cell differentiation. To examine how Gata1 gene expression is regulated in a stage-specific manner, transgenic mouse lines expressing green fluorescent protein (GFP) reporter from the Gata1 locus in a bacterial artificial chromosome (G1BAC-GFP) were prepared. We found that the GFP reporter expression faithfully recapitulated Gata1 gene expression. Using GFP fluorescence in combination with hematopoietic surface markers, we established a purification protocol for two erythroid progenitor fractions, referred to as burst-forming units-erythroid cell-related erythroid progenitor (BREP) and CFU-erythroid cell-related erythroid progenitor (CREP) fractions. We examined the functions of the Gata1 gene hematopoietic enhancer (G1HE) and the highly conserved GATA box in the enhancer core. Both deletion of the G1HE and substitution mutation of the GATA box caused almost complete loss of GFP expression in the BREP fraction, but the CREP stage expression was suppressed only partially, indicating the critical contribution of the GATA box to the BREP stage expression of Gata1. Consistently, targeted deletion of G1HE from the chromosomal Gata1 locus provoked suppressed expression of the Gata1 gene in the BREP fraction, which led to aberrant accumulation of BREP stage hematopoietic progenitor cells. These results demonstrate the physiological significance of the dynamic regulation of Gata1 gene expression in a differentiation stage-specific manner.

Vegf regulates embryonic erythroid development through Gata1 modulation

Blood ◽  
2010 ◽  
Vol 116 (12) ◽  
pp. 2141-2151 ◽  
Author(s):  
Benjamin Drogat ◽  
Joanna Kalucka ◽  
Laura Gutiérrez ◽  
Hamida Hammad ◽  
Steven Goossens ◽  
...  
Keyword(s):  
Target Genes ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Erythroid Progenitor ◽  
Expression Levels ◽  
Conditional Deletion ◽  
Differentiation Block ◽  
Erythroid Development

Abstract To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf164 isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our Vegf gain of function model. These results demonstrate that Vegf modulates Gata1 expression levels in vivo and provides new molecular insight into Vegf's ability to modulate erythropoiesis.

scholarly journals Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3626
Author(s):  
Panayiota L. Papasavva ◽  
Nikoletta Y. Papaioannou ◽  
Petros Patsali ◽  
Ryo Kurita ◽  
Yukio Nakamura ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Regulatory Networks ◽  
Target Genes ◽  
Human Peripheral Blood ◽  
Expression Patterns ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Small Rna Sequencing ◽  
Erythroid Cells ◽  
Mirna Regulation

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.

scholarly journals WT1 and DNMT3A Play an Essential Function and Represent Therapeutic Vulnerabilities in Certain AML Samples, As Shown By CRISPR/Cas9 Mediated Knockout in PDX Models In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 377-377
Author(s):  
Maryam Ghalandary ◽  
Yuqiao Gao ◽  
Martin Becker ◽  
Diana Amend ◽  
Klaus H. Metzeler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Cell Lines ◽  
Target Gene ◽  
Target Genes ◽  
Nsg Mice ◽  
Essential Function ◽  
Pdx Models

Abstract Background: The prognosis of patients with acute myeloid leukemia (AML) remains poor and novel therapeutic options are intensively needed. Targeted therapies specifically address molecules with essential function for AML and deciphering novel essential target genes is of utmost importance. Functional genomics via CRISPR\Cas9 technology paves the way for the systematic discovery of novel essential genes, but was so far mostly restricted to studying cell lines in vitro, lacking features of, e.g., primary tumor cells and the in vivo tumor microenvironment. To move closer to the clinical situation in patients, we used the CRISPR\Cas9 technology in patient-derived xenograft (PDX) models of AML in vivo. Methods: Primary tumor cells from seven patients with AML were transplanted into immunocompromised NSG mice and serially transplantable PDX models derived thereof. PDX models were selected which carry the AML specific mutations of interest at variant allele frequencies close to 0.5. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA; successfully transduced PDX cells were enriched by flow cytometry gating on a recombinant fluorochrome or by puromycin. The customized sgRNA library was designed using the CLUE (www.crispr-clue.de) platform and cloned into a lentiviral vector with five different sgRNAs per target gene, plus positive and negative controls (Becker et al., Nucleic Acids Res. 2020). PDX cells were lentivirally transduced with the CRISPR/Cas9 sgRNA library, transplanted into NSG mice, grown in vivo and cells re-isolated at advanced AML disease. sgRNA distribution was measured by next generation sequencing and compared to input control using the MAGeCK pipeline. Interesting dropout hits from PDX in vivo screens were validated by fluorochrome-guided competitive in vivo experiments in the PDX models, comparing growth of PDX AML cells with knockout of the gene of interest versus control knockout in the same mouse. PDX cells were transduced with lentiviral vectors expressing a single sgRNA, using in parallel three different sgRNAs per target gene. Targeting and control sgRNAs were marked by different fluorochromes; PDX cells expressing targeting or control sgRNA were mixed at a 1:1 ratio, injected into NSG mice and PDX models competitively grown until advanced disease stage, when cell distributions was determined by flow cytometry. Human AML cell lines were studied in vitro for comparison. Results: In search for genes with essential function in AML, we cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo. From the dropouts, we validated most interesting target genes using fluorochrome-guided competitive in vivo assays. Knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1. KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS, although with a stronger effect upon KRAS mutation, suggesting that patients with tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS. Surprising results were obtained for WT1 and DNMT3A. Both genes are frequently mutated in AML, but most AML cell lines tested in vitro do not show an essential function of any of the two genes, in published knockdown or knockout data, including from the Cancer Dependency Map database. On the contrary, knockout of either WT1 or DNMT3A was shown to enhance growth of AML cell lines and increase leukemogenesis in certain models. In PDX models in vivo, we found a clearly essential function for DNMT3A in all AML samples and WT1 in most samples tested and PDX in vivo results were discordant to cell line in vitro data, suggesting that cell line inherent features and/or the in vivo environment influence the function of WT1 and DNMT3A. Conclusion: We conclude that functional genomics in PDX models in vivo allows discovering essentialities hidden for cell line in vitro approaches. WT1 and DNMT3A harbor the potential to represent attractive therapeutic targets in AML under in vivo conditions, warranting further evaluation. Disclosures No relevant conflicts of interest to declare.

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