scholarly journals Coagulation FXIII-A Protein Expression Defines Three Novel Sub-populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized by Distinct Gene Expression Signatures

2019 ◽  
Vol 9 ◽  
Author(s):  
Katalin Gyurina ◽  
Bettina Kárai ◽  
Anikó Ujfalusi ◽  
Zsuzsanna Hevessy ◽  
Gábor Barna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Distinct Gene ◽  
Gene Expression Signatures

scholarly journals Coagulation FXIII-a Protein Expression Defines Three Novel Sub-Populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized By Distinct Gene Expression Signatures

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2523-2523
Author(s):  
Katalin Gyurina ◽  
Bettina Kárai ◽  
Anikó Ujfalusi ◽  
Zsuzsanna Hevessy ◽  
Gábor Barna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Signature ◽  
Go Analysis ◽  
Expression Signature ◽  
Distinct Gene ◽  
Gene Expression Signatures

Background Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). FXIII-A expression determined by flow cytometry (FC) exhibited characteristically distinct expression patterns in subgroups of lymphoblasts. The FXIII-A negative subgroup was significantly associated with the 'B-other' genetic category and had an unfavorable disease outcome. Methods RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology (GO) analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed (DE) genes were validated by RT-Q-PCR. Results We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the expression of FXIII-A protein, as determined by FC. We observed three well-defined categories of FXIII-A protein expression: FXIII-A negative (<20% FXIII-A positive blasts), FXIII-A dim (20-80% FXIII-A positive blasts), and FXIII-A bright (>80% FXIII-A positive blasts). The intensity of the FXIII-A expression increased continuously, which excluded the existence of a distinct FXIII-A negative and a FXIII-A bright subpopulation within the FXIII-A dim subgroup (Image 1/A). These three subgroups defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. There were 26 DE genes found when comparing the FXIII-A negative with the FXIII-A bright subgroup. The FXIII-A dim vs. bright comparison resulted in 155 DE genes and there were 88 DE genes identified between the FXIII-A negative and dim subgroups (Image 1/B). Expression of F13A1 gene was detected and readily validated by RT-Q-PCR in every sample. Intensity of gene expression; however, was characteristically different among samples of the three different FXIII-A protein expression subgroups with an increasing intensity in terms of relative fold-changes measured by RT-Q-PCR from the FXIII-A negative, through dim to bright subgroups. We selected 13/45 genes based on fold-change results detected by microarray, whereas an additional 32/45 genes were selected according to enriched functional categories of potential interest as defined by the GO analysis. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2, validated by RT-Q-PCR according to the FXIII-A status, followed the pattern of intensity of the expression of the F13A1 gene. Of these genes, ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 appear to have a role in leukemia and other forms of cancer. NUP43 has not yet been shown to be associated with any forms of human cancer in contrast to other members of the NUP gene family. PLAC8 which is a trophoblast lineage marker was most intensively expressed in the FXIII-A dim subgroup. This gene has been shown to be aberrantly activated in various types of cancer arising in mammals and mammalian cancer cell lines, but not in any subtype of human ALL (Image 1/C). Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of 'B-other' samples. We identified 14 DE genes by microarray overlapping with DE genes of the 'B-other' subgroup defined by the COALL Group. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the 'B-other' subgroup. Conclusions We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL. Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Validated genes proved biologically and clinically relevant. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment. Figure Disclosures No relevant conflicts of interest to declare.

Gene Expression Profiling Data in Lymphoma and Leukemia: Review of the Literature and Extrapolation of Pertinent Clinical Applications

2006 ◽  
Vol 130 (4) ◽  
pp. 483-520 ◽  
Author(s):  
Cherie H. Dunphy
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Hodgkin Lymphoma ◽  
Expression Profiling ◽  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Clinical Applications

Abstract Context.—Gene expression (GE) analyses using microarrays have become an important part of biomedical and clinical research in hematolymphoid malignancies. However, the methods are time-consuming and costly for routine clinical practice. Objectives.—To review the literature regarding GE data that may provide important information regarding pathogenesis and that may be extrapolated for use in diagnosing and prognosticating lymphomas and leukemias; to present GE findings in Hodgkin and non-Hodgkin lymphomas, acute leukemias, and chronic myeloid leukemia in detail; and to summarize the practical clinical applications in tables that are referenced throughout the text. Data Source.—PubMed was searched for pertinent literature from 1993 to 2005. Conclusions.—Gene expression profiling of lymphomas and leukemias aids in the diagnosis and prognostication of these diseases. The extrapolation of these findings to more timely, efficient, and cost-effective methods, such as flow cytometry and immunohistochemistry, results in better diagnostic tools to manage the diseases. Flow cytometric and immunohistochemical applications of the information gained from GE profiling assist in the management of chronic lymphocytic leukemia, other low-grade B-cell non-Hodgkin lymphomas and leukemias, diffuse large B-cell lymphoma, nodular lymphocyte–predominant Hodgkin lymphoma, and classic Hodgkin lymphoma. For practical clinical use, GE profiling of precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, and acute myeloid leukemia has supported most of the information that has been obtained by cytogenetic and molecular studies (except for the identification of FLT3 mutations for molecular analysis), but extrapolation of the analyses leaves much to be gained based on the GE profiling data.

Pax5 Haploinsufficiency Cooperates with BCR-ABL1 to Induce Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 293-293 ◽  
Author(s):  
ChristoPher B Miller ◽  
Charles G Mullighan ◽  
Xiaoping Su ◽  
Jing Ma ◽  
Michael Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Point Mutation ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Lymphoid Development ◽  
B Lymphoid

Abstract Genes regulating B lymphoid development are somatically mutated in over 40% of B-progenitor acute lymphoblastic leukemia (ALL) cases, with the most common targets being the transcription factors PAX5, IKZF1 (encoding Ikaros), and EBF1. Notably, BCR-ABL1 ALL is characterized by a high frequency of mutations of IKZF1 (85%), PAX5 (55%), and CDKN2A/B (encoding INK4/ARF, 55%), suggesting that these lesions cooperate with BCR-ABL1 in lymphoid leukemogenesis. To examine cooperativity between Pax5 haploinsufficiency and BCR-ABL1, we transplanted Pax5+/+ and Pax5+/− bone marrow cells transduced with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 transduced Pax5+/− marrow developed B progenitor cell ALL with significantly higher penetrance and decreased latency when compared to animals transplanted with BCR-ABL1 transduced Pax5+/+ marrow (median survival 36 vs. 60 days, P=0.0003). The latency of tumor onset was further decreased in the presence of Arf haploinsufficiency (Pax5+/+Arf+/+ 60 days, Pax5+/−Arf+/+ 36 days, Pax5+/−Arf+/− 21 days, P&lt;0.0001). All leukemias were of B cell lineage and were transplantable to secondary recipients. In addition, Southern blot analysis revealed the Pax5+/−Arf+/+ leukemias to be monoclonal, where as the Pax5+/−Arf+/− leukemias were oligoclonal. Importantly, the Pax5+/− leukemias exhibited a more immature B cell immunophenotype than Pax5 wild type leukemias. Moreover, a proportion of the Pax5+/− leukemias (19%) exhibited a very immature early pro B cell immunophenotype (Cd19−, Bp1−), suggesting the possibility of acquired lesions in other key regulators of normal B cell differentiation. To explore this possibility and to identify the total complement of genetic lesions required to generate overt leukemia, we performed genome-wide copy number analysis on 30 murine leukemias (15 Pax5+/+, 15 Pax5+/−) using a custom CGH microarray (Agilent) that interrogated 477,000 autosomal loci, including 18,000 probes covering 20 genes encoding B lymphoid transcription factors and genes targeted by recurring copy number abnormalities (CNAs) in human BCR-ABL1 ALL (Bcl11a, Cdkn2a, Ebf1, Ikzf1, Ikzf2, Ikzf3, Il7r, Lef1, Mdm2, Mef2c, Myb, Pax5, Pten, Rb1, Sfpi1, Sox4, Stat5a, Tcf3, Tcf4, and Trp53). This analysis identified focal recurring CNAs in multiple genes including Cdkn2a/b, Ebf1, Ikzf1, Ikzf2, Ikzf3, and Pax5, each of which is a target of mutation in human B-ALL. Overall, there were on average 3.5 CNAs in Pax5+/+ leukemias versus 0.7 CNAs in Pax5+/− leukemias. Genomic resequencing was also performed on Pax5 and revealed three missense mutations in the DNA binding paired domain (R38H, P80R and G85R), one of which (P80R) is the most common PAX5 point mutation in human B-ALL. All three point mutations are predicted to impair DNA binding of Pax5. Interestingly, the majority of the pro-B cell leukemias that arose in the Pax5+/−Arf+/+ animals were found to harbor mutations (CNAs or point mutation) of the retained Pax5 allele, consistent with the immature immunophenotype. To further explore the relationship between our murine model and human BCR-ABL1 ALL, we performed gene expression profiling of Pax5+/+ and Pax5+/− leukemias and compared their signatures to those of human BCR-ABL1 ALL and stage-specific murine B lymphoid developmental signatures using gene set enrichment analysis (GSEA). This analysis identified significant similarity between murine and human BCR-ABL1 leukemias, thus providing further evidence that this model closely recapitulates human BCR-ABL1 ALL. Notably, Pax5+/− leukemias, or Pax5+/+ leukemias that acquired additional mutations of B-lymphoid regulators exhibited a less mature gene expression profile than leukemias lacking B-lymphoid regulatory mutations. These data indicate that loss of Pax5 contributes to leukemogenesis, that additional genomic alterations in genes regulating B lymphoid development and cell cycle regulators/tumor suppressors (Arf) are frequent events in BCR-ABL1 acute lymphoblastic leukemia, and that these lesions result in impaired B-lymphoid maturation in B-ALL. The genetic complexity of BCR-ABL1 ALL is likely to have important therapeutic implications for this poor prognosis subtype of leukemia.

High CD45 (PTPRC) Expression Is Associated with An Overall Poor Outcome in Childhood Acute Lymphoblastic Leukemia Treated on the ALL-BFM 2000 Protocol and Exerts An Especially Pronounced Effect in Intermediate Risk Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 742-742
Author(s):  
Gunnar Cario ◽  
Rita Mitlohner ◽  
Martin Zimmermann ◽  
Renja Romey ◽  
Peter Rhein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Protein Expression ◽  
Lymphoblastic Leukemia ◽  
Treatment Resistance ◽  
Intermediate Risk ◽  
Prognostic Relevance ◽  
Risk Patients ◽  
Low Expression

Abstract Abstract 742 Further improvement of outcome in childhood acute lymphoblastic leukemia (ALL) could be achieved by identifying additional high-risk (HR) patients who then may benefit from an intensified treatment. In trial ALL-BFM 2000, the HR group was defined by inadequate initial response to induction treatment [poor prednisone response on treatment day eight (PPR), non remission on treatment day 33, and/or a high load of minimal residual disease (MRD, ≥10E-3) after 12 weeks of treatment (TP2)] and/or by positive cytogenetics for a t(4;11) or t(9;22). No MRD already on treatment day 33 defined standard risk (SR) patients, a measurable MRD at a low level characterized the intermediate risk (IR) group. Of importance, the majority of relapses occurred within this heterogeneous group of patients. In order to identify potential new stratification markers we earlier compared gene expression profiles of MRD resistance (HR) and sensitive (SR) ALL in a case-control setting (Cario et al, Blood 2005). Subsequently, we aimed at confirming the potential prognostic relevance of genes identified and their respective proteins in representative study populations. CD45 (also PTPRC, protein-tyrosine phosphatase, receptor-type, C) was one of these candidate genes. In order to assess its prognostic relevance, CD45 gene expression was first analyzed by quantifiable RT-PCR in a set of 555 precursor B-ALL (pB-ALL); its protein expression subsequently in 422 pB-ALL patients by flow cytometry. About one third of patients were included in both study sets. Normalization of protein expression was done by assessing the density of surface expression relative to its density on normal lymphocytes. The 90th percentile was used as a cut-off to distinguish a CD45-high from a CD45-low expression group in both analyses. In gene expression analysis we observed a significant association of a high CD45 expression with a high white blood cell count at diagnosis (WBC) (P = 0.0004), NCI-HR (P = 0.03) as well as presence of the MLL-AF4 rearrangement (P < 0.0001). Moreover, a high CD45 expression was associated with in-vivo treatment resistance as defined by MRD (P = 0.0025). Analyzing CD45 protein expression confirmed the association of a high expression with a high WBC (P < 0.0001), NCI-HR (P = 0.0002) as well as presence of the MLL-AF4 rearrangement (P < 0.0001). Moreover, although the association to treatment resistance was lower (P = 0.055), patients with a high CD45 expression had a significantly worse 5-years EFS probability of 62±8% compared to 82±2% for those in the low-expression group (P=0.002). Focussing on the IR group, patients with a high CD45 expression had a very poor outcome (EFS 45±15%) as compared to those with a low expression (EFS 86±3%, P < 0.0001). This effect was mainly related to a higher cumulative relapse incidence (55±16% vs. 13±3%, P < 0.0001). Of interest, no significant differences in EFS were seen in HR patients. Based on our results, consideration of CD45 protein expression may serve as additional stratification tool in BFM-based protocols to further refine true non-high-risk patients with a low risk of relapse by identifying additional patients at high relapse risk. Of importance, in view of the fact that CD45 expression was not prognostic in the high-risk group, patients with a high CD45 expression currently treated on non high risk arms, may potentially benefit from an intensified treatment in the HR arm. Disclosures: No relevant conflicts of interest to declare.

C-myc protein expression in B-cell acute lymphoblastic leukemia, prognostic significance?

2014 ◽  
Vol 38 (9) ◽  
pp. 1061-1066 ◽  
Author(s):  
Ashleigh Allen ◽  
Kamraan Gill ◽  
Daniela Hoehn ◽  
Maria Sulis ◽  
Govind Bhagat ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Prognostic Significance ◽  
Cell Acute Lymphoblastic Leukemia

scholarly journals Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group

Blood ◽  
2011 ◽  
Vol 118 (11) ◽  
pp. 3080-3087 ◽  
Author(s):  
Jinghui Zhang ◽  
Charles G. Mullighan ◽  
Richard C. Harvey ◽  
Gang Wu ◽  
Xiang Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
B Cell ◽  
High Frequency ◽  
Lymphoblastic Leukemia ◽  
Cell Development ◽  
B Cell Development ◽  
Ras Signaling ◽  
Somatic Alterations

Abstract We sequenced 120 candidate genes in 187 high-risk childhood B-precursor acute lymphoblastic leukemias, the largest pediatric cancer genome sequencing effort reported to date. Integrated analysis of 179 validated somatic sequence mutations with genome-wide copy number alterations and gene expression profiles revealed a high frequency of recurrent somatic alterations in key signaling pathways, including B-cell development/differentiation (68% of cases), the TP53/RB tumor suppressor pathway (54%), Ras signaling (50%), and Janus kinases (11%). Recurrent mutations were also found in ETV6 (6 cases), TBL1XR1 (3), CREBBP (3), MUC4 (2), ASMTL (2), and ADARB2 (2). The frequency of mutations within the 4 major pathways varied markedly across genetic subtypes. Among 23 leukemias expressing a BCR-ABL1-like gene expression profile, 96% had somatic alterations in B-cell development/differentiation, 57% in JAK, and 52% in both pathways, whereas only 9% had Ras pathway mutations. In contrast, 21 cases defined by a distinct gene expression profile coupled with focal ERG deletion rarely had B-cell development/differentiation or JAK kinase alterations but had a high frequency (62%) of Ras signaling pathway mutations. These data extend the range of genes that are recurrently mutated in high-risk childhood B-precursor acute lymphoblastic leukemia and highlight important new therapeutic targets for selected patient subsets.

scholarly journals Clinical application of whole transcriptome sequencing for the classification of patients with acute lymphoblastic leukemia

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wencke Walter ◽  
Rabia Shahswar ◽  
Anna Stengel ◽  
Manja Meggendorfer ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Copy Number Variations ◽  
Current Standard ◽  
Genetic Classification ◽  
Genetic Characteristics ◽  
Standard Methods

Abstract Background Considering the clinical and genetic characteristics, acute lymphoblastic leukemia (ALL) is a rather heterogeneous hematological neoplasm for which current standard diagnostics require various analyses encompassing morphology, immunophenotyping, cytogenetics, and molecular analysis of gene fusions and mutations. Hence, it would be desirable to rely on a technique and an analytical workflow that allows the simultaneous analysis and identification of all the genetic alterations in a single approach. Moreover, based on the results with standard methods, a significant amount of patients have no established abnormalities and hence, cannot further be stratified. Methods We performed WTS and WGS in 279 acute lymphoblastic leukemia (ALL) patients (B-cell: n = 211; T-cell: n = 68) to assess the accuracy of WTS, to detect relevant genetic markers, and to classify ALL patients. Results DNA and RNA-based genotyping was used to ensure correct WTS-WGS pairing. Gene expression analysis reliably assigned samples to the B Cell Precursor (BCP)-ALL or the T-ALL group. Subclassification of BCP-ALL samples was done progressively, assessing first the presence of chromosomal rearrangements by the means of fusion detection. Compared to the standard methods, 97% of the recurrent risk-stratifying fusions could be identified by WTS, assigning 76 samples to their respective entities. Additionally, read-through fusions (indicative of CDKN2A and RB1 gene deletions) were recurrently detected in the cohort along with 57 putative novel fusions, with yet untouched diagnostic potentials. Next, copy number variations were inferred from WTS data to identify relevant ploidy groups, classifying an additional of 31 samples. Lastly, gene expression profiling detected a BCR-ABL1-like signature in 27% of the remaining samples. Conclusion As a single assay, WTS allowed a precise genetic classification for the majority of BCP-ALL patients, and is superior to conventional methods in the cases which lack entity defining genetic abnormalities.

scholarly journals Precise Modeling of IKZF1 Alterations in Human B-Cell Acute Lymphoblastic Leukemia Cell Lines Reveals Distinct Chemosensitivity, Homing, and Engraftment Properties

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 549-549
Author(s):  
Jason H. Rogers ◽  
Rohit Gupta ◽  
Jaime M. Reyes ◽  
Lorenzo Brunetti ◽  
Michael C. Gundry ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Sanger Sequencing ◽  
Lymphoblastic Leukemia ◽  
Clonal Cell ◽  
Ribonucleoprotein Complexes ◽  
Clonal Cell Lines ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Around 20% of pediatric and the majority of adults with B-cell acute lymphoblastic leukemia (B-ALL) suffer relapse, and prognosis after relapse is very poor. Therefore, identifying those at risk for treatment failure and improving their outcome is imperative. In B-ALL, deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are associated with an increased risk of relapse. IKZF1 encodes the IKAROS protein, which is a master lymphoid regulatory transcription factor and chromatin remodeler. Somatic IKZF1 lesions are thought to be secondarily acquired, arising in lymphoblasts with existing driver genetic lesions, most commonly co-occurring with BCR-ABL1 fusion, activating kinase fusions of Ph-like disease and deregulated DUX4 and ERG. In B-ALL, mono- or bi-allelic deletions of the entire gene, as well as intragenic deletions occur. One of the most common perturbations of IKZF1 in B-ALL is an intragenic deletion of a 50-kilobase (kb) region containing exons 4-7, resulting in the expression of a dominant-negative isoform, IK6. Recently published clinical data show potentially conflicting results over the benefits of therapy intensification in IKZF1-mutant cases (Clappier et al., 2015; Hinze et al., 2017; & Yeoh, et al., 2018). Human cell models of these deletions are needed, as there may be unknown functional differences among mutation types, and the available body of data relies on clinical statistical associations, in vitro RNA interference, viral overexpression of IK6, and mouse models. We used the CRISPR/Cas9 system in the human B-ALL cell lines Nalm-6 and REH by electroporation with sgRNA-Cas9 ribonucleoprotein complexes (RNPs) to generate IKZF1-mutant clones. We identified single cell-derived clonal lines with IKZF1 frameshift mutations in one or both alleles by Sanger sequencing and TIDE decomposition. We confirmed ablation of protein expression by immunoblotting. We treated the IKZF1-mutant clonal cell lines with chemotherapeutic agents commonly used to treat B-ALL and calculated the IC50 by Annexin V/7-AAD double-negative population after 48-72 hour treatment. Compared to IKZF1-wild type Nalm-6 cells, Nalm-6 IKZF1-/- clones exhibited profound resistance to dexamethasone and modest but significant resistance to most other chemotherapeutics tested including vincristine, asparaginase, and daunorubicin. In contrast, these cell lines were more sensitive to the nucleoside analog, cytarabine (Panel A). We next analyzed gene expression profiles by RNA-seq and observed that IKZF1-/- clones are characterized by a stem cell-like gene expression signature and activation of the JAK/STAT pathway (Panel B). Transplantation into immunodeficient NOD scid gamma (NSG) mice demonstrated that IKZF1 deletion leads to enhanced engraftment, significantly increased bone marrow homing, and reduced survival time (Panel D). We also employed a novel CRISPR/Cas9 homology-directed repair (HDR) strategy to generate clonal cell lines expressing IK6 under control of the endogenous promoter, which represents a significant advantage to many previous studies utilizing viral overexpression. We electroporated the cells with sgRNA-Cas9 RNPs along with a 3kb commercially synthesized double-stranded DNA HDR template that knocks-in exon 8 with a GFP tag directly following exon 3. Using this strategy, we were able to isolate heterozygous clones (IKZF1IK6/+) from both Nalm-6 (Panel C) and REH cell lines using flow cytometry sorting for GFP-positive cells. We confirmed precise HDR by Sanger sequencing and immunoblotting. When transplanted into immunodeficient mice, IKZF1IK6/+cells showed delayed engraftment and disease onset, but profound splenic infiltration, consistent with a more indolent, infiltrative disease phenotype (Panels D & E). Ongoing drug treatment assays suggest the chemosensitivity profiles of IKZF1IK6/+ and IKZF1IK6/-clonal cell lines are distinct from their isogenic IKZF1-/-counterparts. Our data support clinical studies reporting that IKZF1-mutated B-ALL is an aggressive, infiltrative, and treatment-resistant disease. Notable differences in drug response and in vivo dynamics in xenografts exist between IKZF1-/-cells and IKZF1IK6/+cells. Detailed delineation of the exact IKZF1 status in ALL patients at diagnosis may be informative in more accurately determining risk stratification and the most effective therapeutic regimen. Disclosures No relevant conflicts of interest to declare.

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