Journal articles: 'CRISPR/Cas9, flow cytometry, order of event'

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Lanjewar, S. N., and K. R. Bondioli. "205 Optimization of Transfection Efficiency for CRISPR/Cas9-Induced Genomic Editing in Porcine Fibroblast Cells." Reproduction, Fertility and Development 30, no. 1 (2018): 243. http://dx.doi.org/10.1071/rdv30n1ab205.

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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system creates DNA double-stranded breaks (DSB) at specific sequences and allows efficient genomic modification, even in species previously resistant to gene editing. The DSB can be repaired using non-homologous end joining (creating insertions/deletions) or by homology directed repair (HDR) using a donor DNA with small changes at the cut site, giving rise to precise targeted modifications. Despite growing interest in genome editing using RNA-guided endonucleases, the efficiency of HDR is only 0.5 to 20%. The objective of this study was to optimize transfection conditions in order to increase efficiency of HDR for CRISPR/Cas9 targeted genomic editing of porcine cells. We utilised the Swedish mutation of the porcine APP gene causing early-onset Alzheimer’s disease. We first tested co-transfection of 2 plasmids, one containing our guide RNA (gRNA) and another containing the Cas9 nuclease, using square-wave electroporation. Upon analysis via T7 endonuclease assay I, this method failed to produce a DNA DSB at the target site. Next, we tested transfection of a single vector containing both the gRNA and Cas9 nuclease. Three gRNAs targeting exon 17 of the porcine APP gene were constructed and inserted into CRISPR/Cas9 pGuide-it plasmids expressing green fluorescent protein (GFP). Plasmid DNA was transfected into cultured porcine fibroblast cells by 3 methods: Lipofectamine 2000, square-wave electroporation, and exponential-wave electroporation. To determine which method yielded the highest transfection rates, cells were analysed using flow cytometry to detect GFP expression. The transfection efficiency, percentage of cells expressing GFP, was analysed by one-way ANOVA and individual pair wise comparisons. Twelve microliters of Lipofectamine 2000 per well of a 6-well plate with 200 ng of plasmid DNA per μL of Lipofectamine was used to optimize transfection rates, as suggested by the manufacturer. Removal of transfection media after 48 h yielded higher transfection rates than removal after 24 h (6.9% ± 0.7 v. 2.2% ± 0.1; P = 0.02). For electroporation, 12.5 and 25 μg of plasmid DNA per mL was added to 0.2- and 0.4-mm gap cuvettes, respectively, each containing cell suspensions of 1 × 106 cells mL−1. Square-wave electroporation was performed at 300 V for three 1-ms pulses in 0.2-mm cuvettes. Exponential-wave electroporation was performed at 350 V and 500 μFD in both 0.2-mm and 0.4-mm cuvettes. Exponential-wave electroporation containing 25 μg of plasmid DNA/mL of cell suspension yielded the highest average transfection efficiency, 22.8% (P < 0.00001), compared with square-wave electroporation and transfection using optimized Lipofectamine 2000 conditions (9.1 and 1%, respectively). All 3 gRNAs resulted in similar transfection rates. In conclusion, efficiency of transfection of the CRISPR/Cas9 system into porcine cells is optimized using exponential-wave electroporation of a single plasmid CRISPR system.

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Kaur, Surinder, Stephen C. Alley, Matt Szapacs, Amanda Wilson, Eugene Ciccimaro, Dian Su, Neil Henderson, et al. "2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A – Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine)." Bioanalysis 14, no. 9 (May 2022): 505–80. http://dx.doi.org/10.4155/bio-2022-0078.

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The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “Context of Use – COU”); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

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Corat, Marcus A. F., Jean-Yves Metais, and Cynthia E. Dunbar. "Progress Towards Creation of a Rhesus Macaque Animal Model for PNH Disease Via Crispr/Cas9 Technology to Knock out the PIG-a Gene." Blood 124, no. 21 (December 6, 2014): 4389. http://dx.doi.org/10.1182/blood.v124.21.4389.4389.

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Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem/progenitor cell (HSPC) disease characterized by severe intravascular hemolysis, bone marrow failure, and propensity to thrombosis, causing early death in untreated patients. PNH has been linked to acquired somatic loss-of-function mutations in the X-linked PIG-A gene in HSPC, with resultant disruption of the first step of the biosynthesis of GPI anchors and loss of cell-surface expression of GPI-linked proteins such as CD55 or CD59. PNH red cells are sensitive to complement-mediated lysis due to loss of these GPI-linked proteins. Even though the molecular mechanism of PNH has been well-characterized, the apparent clonal dominance of PIG-A mutant HSPC over residual unmutated HSPC is still poorly understood. Murine models for PNH generated via conditional knockout of PigA do not recapitulate the hemolysis, thrombotic phenotype, or clonal dominance. We took advantage of the newly-described CRISPR-Cas9 gene editing technology to disrupt the PIG-A gene via non-homologous end joining DNA repair and generate a relevant animal model for PNH, in order to investigate these important pathophysiologic questions in rhesus macaques, a species phylogenetically closely related to humans. We constructed a lentiviral vector expressing GFP, Cas9, and one of a series of five guide RNAs manually designed to target the rhesus macaque PIG-A gene at several sites within exon 2, where most human missense mutations are clustered. Two of these five gRNAs also 100% matched homologous sequences in the human PIG-A gene, while three of the gRNAs had 1-2 mismatches with the human sequence. We characterized the efficiency of each guide at the genotypic level using the SURVEYOR assay for locus disruption and direct sequencing, and at the phenotypic level by flow cytometry for GPI-linked proteins. We used human K562 or rhesus macaque FRhK-4 cell lines for proof of concept in vitro studies. The majority of human K562 cells transduced with vectors expressing guide RNAs targeting human PIG-A sequences in exon 2 gradually lost expression of cell surface CD55 and CD59, approximately 70% of double negative for CD55 and CD59 markers by 3 weeks following transduction, in contrast to K562 cells transduced with control vectors not expressing gRNA. It is notable that of the 5 guides tested in K562 cells, the 2 guides resulting in efficient knockdown of CD55 and CD59 were targeted to regions with 100% homology between the rhesus and the human sequence of PIG-A. The other 3 guides with only 1 or 2 mismatches to the human sequence were very inefficient at gene disruption in human cells, suggesting high fidelity and limited off-target effects The SURVEYOR assay confirmed disruption of the K562 PIG-A gene by these two gRNAs (see Figure). Upon sequencing, we demonstrated a variety of indels consisting of insertions or deletions of 1 to 40 nucleotides at the target site in the PIG-A gene. Analysis for off-target indels is ongoing. Similar experiments were carried out in the FRhK4 rhesus cell line, with the SURVEYOR assay demonstrating gene disruption with all five gRNA targeting sequences, all 100% homologous to the rhesus PIG-A gene targets (see Figure) . In FRhK-4 rhesus cells all the PIG-A gRNAs, knocked down CD55 and CD59 expression. gRNAs #4.1, #6.2 and #9.1 showed higher efficiency than the others, with approximately 78% of double negative cells for CD55 and CD59 markers 3 weeks following transduction. We have validated the lentiCRISPR-/Cas9 approach for use in rhesus cells for PIG-A knockout in vitro, and the guide-RNAs have shown effectiveness and specificity. We are in the process of implementing the technique in primary rhesus macaque CD34+ HSPC prior to proceeding to transplantation of these cells in an autologous rhesus model, allowing investigation of the mechanisms of clonal dominance and thrombosis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Pabst, Gabriel, Johannes Foßelteder, Angelika Schlacher, Lisa Auinger, Daniel Martinez-Krams, Asiri Ediriwickrema, Karl Kashofer, et al. "Modeling the Development of SRSF2 Mutated Myeloid Malignancies By CRISPR/Cas9 Mediated Genome Engineering of Primary Human Hematopoietic Stem and Progenitor Cells." Blood 138, Supplement 1 (November 5, 2021): 2160. http://dx.doi.org/10.1182/blood-2021-149591.

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Abstract Introduction: Acute Myeloid Leukemia (AML) is a malignant disease of the bone marrow that can arise from a premalignant condition called clonal hematopoiesis of indeterminate potential (CHIP). Mutations in Serine and Arginine-rich Splicing Factor 2 (SRSF2) are detected in CHIP and mediate a high risk for AML development. Here we used CRISPR/Cas9-mediated genome engineering to introduce a heterozygous SRSF2P95H mutation into primary human hematopoietic stem and progenitor cells (HSPCs) and investigated its functional consequences using both in vitro and in vivo assays. Methods: We used CRISPR/Cas9 technology to introduce a heterozygous mutant (mut) SRSF2P95H into the endogenous SRSF2 gene locus of healthy cord blood HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6) (Figure A). This allows for targeted in-frame integration of mut and/or wildtype (WT) SRSF2 cDNA under the control of the endogenous SRSF2 promoter. Notably, an integrated fluorescent reporter enables the isolation and tracking of heterozygously mutated HSPCs (Figure B). Methylcellulose colony and long-term competition assays of SRSF2 mut and WT HSPCs were performed in vitro. Cells were analyzed by flow cytometry and characterized cytomorphologically. In addition, bulk RNA-seq analyses were performed to characterize differential gene expression and abnormal splicing events. Xenotransplantation into NSG-SGM3 mice was performed in order to assess stem cell characteristics and the in vivo leukemogenic potential of SRSF2 mut HSPCs. Finally, we investigated the mutation-specific effect of the splicing inhibitor Indisulam to determine if SRSF2 mut cells are particularly vulnerable to splicing inhibition. Results: Colony assays (n=9) revealed impaired erythroid and increased monocytic differentiation of SRSF2 mut HSPCs. Quantification of colonies showed a lower frequency of erythroid BFU-E in SRSF2 mut compared to SRSF2 WT HSPCs (mean ± SD; 33.3 ± 12.5% vs. 17.4 ± 10.8%, p=0.00002). In contrast, the frequency of myeloid CFU-M colonies was higher in SRSF2 mut HSPCs compared to SRSF2 WT HSPCs (38.3 ± 7.3% vs. 22.6 ± 6.8%, p = 0.0003) (Figure C). Long-term in vitro competition assays revealed an outgrowth of SRSF2 mut over WT cells in 2 out of 7 donors. Strikingly, after three months of in vitro culture, in one donor, the SRSF2 mut cells developed a blast-like morphology with strong CD34 expression (Figure D). To assess stem cell characteristics and the leukemogenic potential in vivo, we transplanted SRSF2 mut HSPCs from 4 different donors into immunodeficient NSG-SGM3 mice (n=11). SRSF2 mut cells showed a myeloid-skewed engraftment. Cytomorphologic analysis of long-term engrafted SRSF2 mut myeloid cells revealed dysplastic changes such as nuclear abnormalities and extensive cytoplasmic vacuolization. In 4 out of 11 xenografts, human engraftment substantially increased over time with a parallel outgrowth of the SRSF2 mut clone and the appearance of blast-like cells resembling transformation into myeloid leukemia (Figure E). Comparative RNA-seq analysis identified 138 differentially spliced genes, with exon skipping being the dominant altered splicing type. Gene ontology (GO) analysis on differentially expressed genes revealed "Acute Myeloid Leukemia" among the most enriched terms (p-val = 8.2E-07, min FDR = 1.486E-04). When testing the SRSF2-mutation specific effect of the splicing inhibitor Indisulam, SRSF2 mut HSPCs show a significantly lower IC-50 than WT cells (977nM vs. 3574 nM). Strikingly, in competition- and CFU-assays, Indisulam preferentially eradicates SRSF2 mut hematopoietic cells, while sparing WT cells. Conclusion: Using our CRISPR/Cas9 approach, we can successfully introduce heterozygous SRSF2P95H mutants in primary human HSPCs. Mutant SRSF2P95H leads to increased monocytic differentiation, impaired erythroid differentiation, and phenocopy SRSF2P95H driven diseases in patients. Importantly, we show for the first time that the SRSF2 mutation alone is sufficient to induce dysplastic features and even transform healthy human HSPCs into AML-like blasts. Our model allows the identification and therapeutic investigation of specific cellular vulnerabilities caused by SRSF2 mutations and highlights Indisulam as a potential compound to specifically treat individuals carrying a SRSF2 mutation. Figure 1 Figure 1. Disclosures Ediriwickrema: Nanosive SAS: Patents & Royalties. Greinix: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Therakos: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Celgene: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc.. Reinisch: Pfizer: Consultancy; Celgene: Research Funding.

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Adriaanse, Fabienne R. S., Sadie M. Sakurada, Shondra M. Pruett-Miller, Ronald W. Stam, Michel C. Zwaan, and Tanja A. Gruber. "Non-Coding HOX Fusions in Pediatric Non-Down Syndrome Acute Megakaryoblastic Leukemia." Blood 134, Supplement_1 (November 13, 2019): 533. http://dx.doi.org/10.1182/blood-2019-127014.

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The homeobox (HOX) genes are a highly conserved family of transcription factors involved in embryonic patterning as well as adult hematopoiesis. Dysregulation of HOX genes, in particular upregulation of HOXA cluster genes, is a frequent event in Acute Myelogenous Leukemia (AML). Recently, we performed a detailed genomic analysis on pediatric non-Down Syndrome Acute Megakaryoblastic Leukemia (non-DS-AMKL) and identified novel fusions involving a HOX cluster gene in 14.9% of the cases. While most fusions were predicted to lead to an in-frame functional protein, several fusions included a non-coding HOX antisense gene (PLEK-HOXA11-AS, C8orf76-HOXA11-AS, HOXA10-AS-CD164) that were predicted to result in a loss of function of these regulatory transcripts. The functional consequence of these events, however, remain unknown. HOXA11-AS (human) and Hoxa11os (mouse) have been previously shown to have mutually exclusive expression with the Hoxa11 transcript throughout development. We therefore hypothesized that loss of function of non-coding HOX antisense genes as a result of these structural variations would cause upregulation of nearby coding HOXA genes that in turn promote leukemogenesis. To test this hypothesis, using CRISPR-Cas9 technology, we genome edited the human AMKL cell line CMK to carry the PLEK-HOXA11-AS translocation. qRT-PCR of HOXA11-AS and HOXA9-11 transcripts in this cell line recapitulated the pattern seen in patient specimens. Specifically, HOXA11-AS expression was significantly diminished while HOXA10 and HOXA11 transcripts were upregulated 1.8-2.5-fold when compared to parental CMK cells (p=0.0385 and p=0.006 respectively). To further investigate the loss of HOXA11-ASin vivo a CRISPR-Cas9 Hoxa11os knockout mouse model was established. qRT-PCR on bone marrow confirmed the loss of Hoxa11os transcripts in heterozygous (Hoxa11os1+/-) and homozygous (Hoxa11os-/-) mice of both genders (p=<0.0001-0.0012). Consistent with Hoxa11os knockdown, Hoxa11 transcripts were upregulated in male (1.8-fold p=0.0023 Hoxa11os+/-, and 2-fold p=0.0052 Hoxa11os-/-)and female (1.3-fold p=0.0074 Hoxa11os+/- and 2.2-fold p=0.0226 Hoxa11os-/-) bone marrow compared to wild type gender matched littermates. Interestingly, flow cytometry analysis of progenitor subsets revealed gender specific findings. We found a significant increase in the frequency of the lineage negative, Sca-1 and c-Kit positive (LSK) population in males (0.13% of total bone marrow Hoxa11os+/+, 0.19% p=0.0214 Hoxa11os+/-, and 0.25% p=0.0001 Hoxa11os-/-) compared to wild type male littermates but not in female mice at 8 weeks of age. In contrast an increase in the megakaryocyte-erythroid (MEP) population was seen only in the female setting (0.07% Hoxa11os+/+, 0.15% p=0.0055 Hoxa11os+/-, and 0.165% p<0.0001 Hoxa11os-/-). Limiting dilution colony forming assay confirmed the higher LSK frequency with a 2-fold increase in the number of colonies for male knockout marrow compared to wild type marrow in contrast to the female setting where no significant differences were seen. As hormonal signals have been shown to regulate expression of HOX genes and differences in clonogenicity of male and female stem cells has been previously demonstrated, we reasoned this phenomenon could be secondary to extrinsic stimuli in vivo. The relatively uniform Hoxa11 levels in male and female knockout mice, however, suggested that cell intrinsic factors may also play a role. We therefore overexpressed HOXA11 into male and female wild type bone marrow ex vivo for colony forming assays to determine if elevated levels of the HOXA11 protein led to functional differences. This assay demonstrated a clear enhancement of self-renewal in male but not female bone marrow in contrast to HOXA9 overexpression which serially replated in both genders. Combined these data demonstrate that loss of function alterations in Hoxa11os transcripts lead to upregulation of Hoxa11 and gender specific hematopoietic progenitor cell perturbations. Ongoing efforts include competitive transplant studies as well as RNA and ChIP sequencing to identify gender specific downstream targets of Hoxa11 in the hematopoietic compartment in order to understand the selective expansion of progenitor subsets and male specific self-renewal capacity of this protein. These data will contribute to our understanding on how HOXA11-AS translocations promote oncogenesis. Disclosures Zwaan: Daiichi Sankyo: Consultancy; Sanofi: Consultancy; Roche: Consultancy; Pfizer: Research Funding; BMS: Research Funding; Incyte: Consultancy; Celgene: Consultancy, Research Funding; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Janssen: Consultancy. Gruber:Bristol-Myers Squibb: Consultancy.

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Ivy, Kathryn S., Candace H. Cote, and Paul Brent Ferrell. "IDH2 Mutations Induce Altered STAT Signaling and Cytokine Responses Which Are Restored By Enasidenib." Blood 132, Supplement 1 (November 29, 2018): 1468. http://dx.doi.org/10.1182/blood-2018-99-117783.

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Abstract Acute myeloid leukemia (AML) is a heterogeneous myeloid malignancy characterized by mutational and clonal heterogeneity. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are common, occurring in approximately 15-20% of patients, and actionable, with recently approved inhibitors for both mutations. These inhibitors lead to leukemia cell differentiation in vitro, in vivo and in patients. Healthy myeloid differentiation is governed by precise regulation of intracellular signaling, but this regulation is disrupted in AML. Given that signal transducer and activator of transcription (STAT) proteins are involved in both leukemogenesis and myeloid differentiation, we sought to determine the role of phosphorylated STATs and other signaling proteins in mIDH AML and inhibitor-induced differentiation of mIDH2 AML. To simultaneously dissect single cell signaling, differentiation and epigenetic changes, we employed fluorescence flow cytometry and mass cytometry to study an in vitro model of mIDH2R140Q leukemia, consisting of a parental TF-1 leukemia cell line and a CRISPR/Cas9 gene-edited mIDH2R140Q-mutated cell line. In order to generate provoked signaling profiles, we stimulated both the wild-type IDH2 and mutant IDH2 cells with nine cytokines. Additionally, we treated both cell lines and primary mIDH2 AML samples ex vivo with enasidenib and measured changes in high-dimensional single cell phenotype and phospho-protein expression via mass cytometry. mIDH2 leukemia cells displayed elevated basal pSTAT1, pSTAT3 and pNFkB-s529 expression, and concomitant low basal IkBa levels when compared to parental cells. Further, mIDH2 cells had increased PMA induced pS6 and IL1β induced pNFkB-s529 than the parental cell line, while wtIDH2 cells had higher levels of cytokine-induced pSTAT1, pSTAT3 and pSTAT5. After prolonged enasidenib treatment (28 days), model mIDH2 cells expressed lower basal pSTAT1, pSTAT3 and pNFkB-s529 than vehicle-treated mIDH2 cells. Further, when stimulated with GM-CSF, enasidenib-treated mIDH2 leukemia cells showed increased response at pSTAT3 and pSTAT5 as compared to vehicle-treated leukemia cells. We dissected enasidenib-induced differentiation using 18 cell surface markers and visualized results using t-distributed stochastic neighbor embedding (tSNE). Presence of mIDH2 leads to baseline expression differences including higher CD90 and CD71. Following treatment with enasidenib, mIDH2 leukemia cells had increased CD45 and CD11b expression as compared to vehicle-treated controls. Additionally, prolonged treatment with enasidenib increased proliferation as shown by increased Ki67 and decreased histone hypermethylation at suppressive histone marks, H3K27 and H3K9, while 7 days of enasidenib did not result in changes to these marks. We further explored the impact of mIDH2 in primary leukemia samples. First, we developed an ex vivo stromal co-culture system that allowed for treatment and expansion of four primary mIDH2 AML samples for 16 days. While the basal levels of signaling markers were sample dependent, consistently across all samples enasidenib-treated primary AML was more sensitive to IL-6 and GM-CSF-induced pSTAT1, pSTAT3 and pSTAT5 signaling. The enasidenib-treated AML also showed increased expression of mature myeloid markers, including CD33 and CD11c. Here we have shown the presence of mIDH2 mutations leads to decreased STAT signaling in response to cytokine stimulation as compared to wtIDH2 AML cells. We have also demonstrated increased response of pNFkB-s529 and pS6 to cytokine stimulation in mIDH2 AML cells as compared to wtIDH2 AML cells. Moreover, in both mIDH2 AML cell line and primary samples, IDH2 inhibitor-induced differentiation restored sensitivity to cytokine responses and reduced histone hypermethylation. Future work exploring these aberrant signaling events may reveal precise connections between mutated IDH, associated epigenetic changes, and intracellular signaling, potentially uncovering synergistic therapeutic strategies for mIDH AML. Disclosures Ferrell: Incyte: Research Funding.

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Haase-Kohn, Cathleen, Markus Laube, Cornelius K. Donat, Birgit Belter, and Jens Pietzsch. "CRISPR/Cas9 Mediated Knockout of Cyclooxygenase-2 Gene Inhibits Invasiveness in A2058 Melanoma Cells." Cells 11, no. 4 (February 21, 2022): 749. http://dx.doi.org/10.3390/cells11040749.

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The inducible isoenzyme cyclooxygenase-2 (COX-2) is an important hub in cellular signaling, which contributes to tumor progression by modulating and enhancing a pro-inflammatory tumor microenvironment, tumor growth, apoptosis resistance, angiogenesis and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the functional knockout effect of COX-2 in A2058 human melanoma cells. COX-2 knockout was validated by Western blot and flow cytometry analysis. When comparing COX-2 knockout cells to controls, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor development in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic activity of cells under normoxia and experimental hypoxia as demonstrated by using the radiotracers [18F]FDG and [18F]FMISO. Finally, a pilot protein array analysis in COX-2 knockout cells verified significantly altered downstream signaling pathways that can be linked to cellular and molecular mechanisms of cancer metastasis closely related to the enzyme. Given the complexity of the signaling pathways and the multifaceted role of COX-2, targeted suppression of COX-2 in melanoma cells, in combination with modulation of related signaling pathways, appears to be a promising therapeutic approach.

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Beigl, Tobias B., Ine Kjosås, Emilie Seljeseth, Nina Glomnes, and Henriette Aksnes. "Efficient and crucial quality control of HAP1 cell ploidy status." Biology Open 9, no. 11 (November 12, 2020): bio057174. http://dx.doi.org/10.1242/bio.057174.

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ABSTRACTThe near-haploid human cell line HAP1 recently became a popular subject for CRISPR/Cas9 editing, since only one allele requires modification. Through the gene-editing service at Horizon Discovery, there are at present more than 7500 edited cell lines available and the number continuously increases. The haploid nature of HAP1 is unstable as cultures become diploid with time. Here, we demonstrated some fundamental differences between haploid and diploid HAP1 cells, hence underlining the need for taking control over ploidy status in HAP1 cultures prior to phenotyping. Consequently, we optimized a procedure to determine the ploidy of HAP1 by flow cytometry in order to obtain diploid cultures and avoid ploidy status as an interfering variable in experiments. Furthermore, in order to facilitate this quality control, we validated a size-based cell sorting procedure to obtain the diploid culture more rapidly. Hence, we provide here two streamlined protocols for quality controlling the ploidy of HAP1 cells and document their validity and necessity.This article has an associated First Person interview with the co-first authors of the paper.

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Mishima, Yuji, Jiantao Shi, Michele Moschetta, Yujia Shen, Salomon Manier, Aldo M. Roccaro, Francois Mercier, et al. "In Vivo Analysis of Clonal Evolution of Multiple Myeloma." Blood 128, no. 22 (December 2, 2016): 799. http://dx.doi.org/10.1182/blood.v128.22.799.799.

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Abstract Introduction According to the clonal evolution model, tumor progression proceeds in a branching rather than in a linear manner, leading to substantial clonal diversity and coexistence of genetically heterogeneous sets of subclones. Unlike many cancers, in which the evolutionary history can only be inferred from the established disease, Multiple Myeloma (MM) has well defined precursor states, which offer a unique opportunity to study the sequential evolution of the disease. In MM, multiple subclones can co-exist because they are of similar fitness, potentially interact with each other or with the surrounding microenvironment and further disseminate to spatially separate areas of the bone marrow (BM). Therefore, in order to accurately predict the course of the disease, we require methods to estimate clone-specific growth rates within the BM and define clones that have the propensity of dissemination. Methods We developed a MM metastatic xenograft model by performing tumor-bearing bone chip implantation to SCID-beige mice (SCID-murine model) and examining tumor clones that are present in the implanted bone chips (primary sites) compared to those present in the distant BM sites (metastatic sites). To obtain a perspective of clonal heterogeneity in vivo, we used the "rainbow" system by which fluorescent proteins were infected into cells using lentiviruses to label the cells with 15 distinctive fluorescence profiles (rainbow MM cells). Rainbow MM cells with equal proportion of all 15 colors were injected into donorfemurs and implanted into recipient mice. After paralysis, the mice were sacrificed and tumor cells were analyzed using flow cytometry and confocal microscopy. To further investigate the dynamics of heterogeneity at the single cell level, similar experiments were performed using a DNA-barcode library. For genomic and transcriptomic characterization, primary and metastatic tumor clones were purified by sorting and underwent whole exome and RNA sequencing. To identify key regulators of the metastatic process, we conducted in vivo CRISPR library screening of the most critical targets identified. Briefly, the MM cell library was prepared by transduction of sgRNAs targeted for 20 genes and control sgRNAs to MM.1S cells stably expressing Cas9. The cell library was used in SCID-murine model and the fractions of each sgRNA were calculated in the primary and metastatic sites to identify genes that facilitate tumor metastasis. Results We found that the 15 rainbow subpopulations were present with equal distribution in the primary sites but not at the metastatic sites. Specific subclones (winner clones) had a greater advantage of growing in the metastatic site. Interestingly, the winner clones were similar between the bilateral femurs of most of the mice, suggesting the existence of potential metastatic subclones. Experiments using DNA-barcoding further demonstrated that single clones could become disproportionately present in the metastatic sites, even though they account for a smaller fraction of the primary tumors. Confocal imaging showed the difference in cluster structures between primary and metastatic tumors. Most of the clusters in the metastatic sites consisted of cells of single colors. RNA sequencing analysis of two human MM cell lines derived from these mouse models demonstrated a distinct gene expression profile of the metastatic tumors compared to the primary sites. By intersecting differentially expressed genes, we identified 110 shared up-regulated genes and 238 shared down-regulated genes, which we designated as the "metastatic signature". Gene Set enrichment analysis of the metastatic signaturein publicly available MM patient datasets (GSE6477 and GSE2658) demonstrated that this signature significantly correlated with overall survival and with clinical progression from MGUS/smoldering MM to overt myeloma and relapsed disease. Finally, the CRISPR in vivo screening prioritized two transcription factors as the key regulatory molecules, namely EGR3 and ATF3. Conclusions Here, we demonstrate that in vivo clonal evolution can be characterized using an in vivo model of MM. The data defines specific subclones that have a higher metastatic potential and are likely driver clones for tumor metastasis in MM. On the molecular level, a metastatic gene signature was found and two genes were identified as potential regulator of MM metastasis. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Hatake:Chugai: Research Funding; Meiji-Seika: Consultancy; Kyowa Kirin: Honoraria, Research Funding; Otsuka: Consultancy. Scadden:Dr. Reddy's: Consultancy; Bone Therapeutics: Consultancy; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Teva: Consultancy; Apotex: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; GlaxoSmithKline: Research Funding. Ghobrial:Amgen: Honoraria; BMS: Honoraria, Research Funding; Noxxon: Honoraria; Takeda: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria.

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Foßelteder, Johannes, Angelika Schlacher, Gabriel Pabst, Bettina Amtmann, Wolfgang Schöll, Karl Kashofer, Christine Beham-Schmid, et al. "Introduction and Genetic Correction of Calreticulin Mutations in Human Hematopoietic Stem and Progenitor Cells Sheds Light on MPN Pathogenesis." Blood 138, Supplement 1 (November 5, 2021): 2541. http://dx.doi.org/10.1182/blood-2021-147919.

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Abstract Introduction: Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite recent advances in understanding mutant CALR, the detailed mechanisms are not fully elucidated, and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we first aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo. Second, we aimed to correct CALR mutations in patient-derived HSPCs to study their dependence on the initial driver event to exert an MPN phenotype. Methods: We used CRISPR/Cas9 to introduce heterozygous CALR mutations into the endogenous gene locus of healthy cord blood-derived HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6). Briefly, Cas9-sgRNA ribonucleoprotein (RNP) was used to cut the DNA. Simultaneously AAV6, carrying either a mutation-bearing or a wildtype control cDNA, was co-delivered to allow for targeted in-frame integration. This way, mutant CALR remains under the control of the endogenous promoter. Concurrent integration of a fluorescent reporter downstream of the mutated exon, enabled purification and tracking of modified cells via flow cytometry. Purified CRISPR-modified HSPCs were used for in vitro collagen-based colony-forming assays, proliferation and differentiation assays in liquid culture, and intrafemoral transplantation into immunodeficient NSG mice to assess their pathogenic potential. Results: Our CRISPR/Cas9 KI strategy enabled us to efficiently generate and enrich for heterozygous CALR mutant human HSPCs. Modified cells harbor the mutation at the endogenous CALR locus with intact gene regulatory regions. Correct integration and transcript expression were confirmed on DNA and RNA level by sanger sequencing. Additionally, CALR mutant protein expression was confirmed via immunohistochemistry using a diagnostically approved mutant-specific antibody. Type-1 and type-2 CALR mutations led to TPO-independent growth of CD34 + HSPC-derived cells and a two-fold (p<0.01) increase of megakaryocyte colonies in collagen-based media compared to wildtype control KI. These findings were corroborated by significantly enhanced CD41 + CD42b + megakaryocyte formation of CALR mutant HSPCs upon liquid culture differentiation. When transplanted into sublethally irradiated immunodeficient NSG mice, CALR mutant HSPCs showed robust engraftment in the bone marrow with a myeloid lineage skewing, outcompetition of wildtype cells and increased formation of CALR mutant CD41 + megakaryocyte progenitors. To investigate, if removal of type-1 and type-2 CALR mutations can ameliorate MPNs, we utilized our KI strategy to correct both CALR mutations in MPN patient-derived HSPCs by replacing them with wildtype sequences. A successful correction was confirmed on DNA and RNA level and by the absence of mutant CALR protein. Opposite to the results from introducing CALR mutations, correcting the mutations led to a two-fold decrease in megakaryocyte colony formation. Interestingly this was only seen in ET and post-ET MF samples, whereas primary MF samples were unaffected, underscoring the importance of other secondary genetic driver events in the pathogenesis of primary MF. Conclusion: Our system allows us to investigate human MPN pathogenesis prospectively and shed light on the transforming mechanisms of mutant CALR in primary HSPCs. We could show that CALR mutations prime HSPCs toward the formation of platelet-producing megakaryocytes. Genetic correction of CALR mutations in MPN patient-derived HSPCs revealed a dependence on the oncogenic mutant CALR driver event in ET and post-ET MF patients, opening the possibility of an ex vivo gene correction approach to remove mutant CALR in patient-derived HSPCs . Lastly, since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant model also provides a powerful new strategy to generate MPN xenotransplants with defined genotypes for the evaluation of novel therapies. Disclosures Greinix: Celgene: Consultancy; Therakos: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Novartis: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

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Castro, Jesus, Mariana Gaelzer, and Scott Welford. "Abstract 209: Elucidating the role of NMDA subunit NR2B in non-tumor and tumor-bearing mice." Cancer Research 82, no. 12_Supplement (June 15, 2022): 209. http://dx.doi.org/10.1158/1538-7445.am2022-209.

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Abstract Therapeutic options for glioblastoma multiforme (GBM), the most common and fatal brain tumor, have been limited over the course of recent decades, leaving radiation as one of the few effective therapies. While advancements in radiation therapy have improved life expectancy and patient outcome, exposure to radiotherapy causes normal tissue toxicity and damage to the central nervous system, resulting in cognitive impairment and negative patient side effects. In the hippocampus, the memory processing center of the brain, radiotherapy-induced elevated glutamate levels lead to over-excitation of N-methyl-D-aspartate receptors (NMDAR) and thus cell death. To investigate the NMDAR, we repurposed a NMDA subtype specific inhibitor (Ifenprodil) and treated non-tumor mice before radiation. We found that Ifenprodil rescues cell death and cognition by inhibiting the NR2B subunit of NMDA receptors and preventing over-excitement-induced excitotoxicity, despite presence of high glutamate levels. In order to relieve glioma patients of the negative side effects of radiotherapies, we aim to further investigate the critical role of NR2B inhibition in non-tumor and tumor-bearing mice to reduce excitotoxicity and prevent damage to the central nervous system, opening avenues for therapeutic targeting and drug development. To accomplish this, we designed promotor-specific plasmids, only activated when present in their designed neuronal cell types, to initiate CRISPR/Cas9 genome editing and knock out the NR2B subunit of NMDAR. With NR2B knocked out across 5 different cell types, we exposed hippocampal organotypic slice cultures, derived from non-tumor mice, to 2 Gy radiation and ran flow cytometry to reveal which neuronal NR2B subunits play critical roles in excitotoxic phenotypes. To further these findings, we utilized a recently developed in-utero-electroporation mouse model that produces glioblastomas in immunocompetent mice by inactivating select tumor-suppressor genes via CRISPR/Cas9 genome editing at embryonic day 14.5. By applying different CRISPR/Cas9 gRNAs into our tumor-bearing mice, we can not only explore the robust role of NR2B inhibition in tumor brains with varying expressions, but also shed light onto how NR2B inhibition protects normal brain while leaving tumors radiosensitive. In conclusion, this work seeks to further develop our understanding of radiotherapy-induced cytotoxicity in order to prevent cognitive impairment and negative side effects in patients, improving their quality of life. Ultimately, the results from our findings will lead to the advancement of targeted drug development and therapeutic options. Citation Format: Jesus Castro, Mariana Gaelzer, Scott Welford. Elucidating the role of NMDA subunit NR2B in non-tumor and tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 209.

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Dong, Mengmeng, Enfan Zhang, Haimeng Yan, Ruyi Xu, He Huang, Anyong Xie, and Zhen Cai. "Macrophages Promote DNA Repair of Double Strand Break in Multiple Myeloma Cells By Non-Homologous End Joining(NHEJ), Nevertheless Decrease Its Accuracy." Blood 134, Supplement_1 (November 13, 2019): 3087. http://dx.doi.org/10.1182/blood-2019-126753.

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Multiple myeloma (MM) is a hematological malignancy of B cells, characterized by clonal proliferation of malignant plasma cells. DNA damage and genomic instability play an important role in the pathogenesis of MM. Based on the characteristics of high heterogeneity and genomic instability of MM, and the protective effect of MΦs on MM cells (MMCs), our study intended to further clarify whether MΦs affect MMCs DNA damage response (DDR) and DNA repair, and the relationship between MΦs and genomic instability of MMCs. We found that the content of MΦs in bone marrow biopsy of MM patients was related to the results of cytogenetics (tested by FISH). The higher the content of MΦs, the more complicated the cytogenetic abnormalities of patients, especially in the IgH translocation, D13S319 locus deletion and RB1 deletion. In our study, MΦs were harvested from peripheral blood monocytes (PBMCs) , which were incubated for 7 days with M-CSF. Flow cytometry was used to detect M-CSF induced macrophages (MΦs) in vitro, and CD163 and CD206 were highly expressed in MΦs which implied that MΦs tended to be M2 type. The incubated MΦs were used in the following experiments. Our study showed that MΦs reduced the baseline γH2AX of MMCs, and contributed to MMCs surviving in the case of genomic instability detected by Western blot and immunofluorescence. We also confirmed that MΦs contribute to repairing the DNA damage in myeloma cells with the methods of comet assay. In the case of severe injury of MMCs' DNA, MΦs promoted the DDR and DNA damage repair. We examined the effects of macrophages on HR and NHEJ using U2OS cells. HR repair was measured in U2OS-HR cells loaded with SCR reporter (HR reporter) while NHEJ repair was measured in U2OS-NHEJ cells loaded with vGEJ reporter (NHEJ Reporter). We found that macrophages increased NHEJ but had no sense on total HR. In order to detect NHEJ level in endogenous genes, we adopted paired gRNA-CRISPR/Cas9 system. AAVS1 and HBB were used as detection genes, and the sequence of about 250 bp near the NHEJ interface was sequenced by NGS. The results proclaimed that the MΦs co-culture group significantly increased the efficiency of NHEJ, and decreased proportion of accurate NHEJ repair. In addition, analysis of the length of base sequence loss showed that the probability of base loss >3 bp in the MΦs co-culture group was higher than that MMCs in the group cultured alone. In the HBB site, MΦs also prolonged the average length of base loss in NHEJ. Furthermore, we used gRNA-CRISPR/Cas9 technology to cause fixed-point cleavage in AAVS1 and HBB respectively, and detected translocation by PCR and NGS with comparing the translocation reads between different groups. The results showed that MΦs promoted the probability of chromosomal translocation, which was of great importance in MM's occurrence and progression. Disclosures No relevant conflicts of interest to declare.

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Wakabayashi, Aoi, Maryanne Kihiu, Malini Sharma, Mathieu Quesnel-Vallieres, Osheiza Abdulmalik, Scott A. Peslak, Cheryl A. Keller, et al. "Interrogating Post-Transcriptional Mechanisms of Fetal Hemoglobin Regulation." Blood 138, Supplement 1 (November 5, 2021): 3079. http://dx.doi.org/10.1182/blood-2021-151488.

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Abstract Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta-hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD through inducing HbF is hydroxyurea, however the mechanism of action is unknown with variable effectiveness among patients. Thus, there remains a strong interest to identify more robust means of upregulating HbF, such as specific inhibition of HbF repressors. BCL11A and LRF are well-characterized transcription factors that independently repress the fetal type b-globin like genes HBG1 and HBG2 but their therapeutic potential is limited by challenging druggability and critical developmental function. However, upstream regulation of these factors, such as post-transcriptional mechanisms, are not well studied and may house novel therapeutic targets. To this end, we employed a CRISPR/Cas9 based screening approach to interrogate a library of RNA binding proteins (RBP) in the context of HbF regulation. Using HUDEP2 cells, a human adult-type erythroid progenitor cell line, we screened 341 human RBPs and identified four candidate RBPs, none of which have previously been implicated in HbF regulation. Of these candidates, RNA Binding Motif 12 (RBM12) showed the greatest level of HbF induction following in vitro depletion. Depletion of RBM12 protein in HUDEP2 cells and human CD34 + hematopoietic stem and progenitor cells (HSPC) via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by HbF flow cytometry, HBG1/2 mRNA, and protein (γ-globin). Cell viability and maturation of RBM12 perturbed cells were largely intact. Additionally, RBM12 depletion in CD34 + HSPCs derived from SCD patients resulted in reduced percentage of sickled cells under hypoxic conditions. Unexpectedly, reduction of RBM12 had minimal effect on BCL11A and LRF expression suggesting that RBM12 may regulate HbF through a pathway that is indirectly related or independent of these transcription factors. RBM12 is an RBP that is widely expressed across diverse cell types and contains multiple RNA recognition motifs (RRM). While it has been implicated in various cancers and neurological disorders, its functions are not well studied. As an RBP, RBM12 can carry out several roles of post-transcriptional regulation, such as pre-mRNA splicing, mRNA transport, stabilization, and translation. As these activities are executed in different cellular compartments, we set out to narrow down RBM12 function by assessing its subcellular localization. Immunofluorescence staining revealed strong nuclear presence of RBM12, suggesting that it functions via mRNA biogenesis and/or processing. RNASeq and LC-MS/MS analysis of RBM12 KO CD34 + HSPCs revealed modest changes in the transcriptome and proteome. In order to gain mechanistic insight into RBM12 in the context of HbF regulation, we performed cDNA rescue experiments in RBM12-deficient HUDEP2 clones. Overexpression of full length RBM12 restored HbF repression. Notably, four out of the five RRMs were dispensable for HbF silencing, but RRM1 was essential for this activity. Interestingly, an extended form of RRM1 was also sufficient for HbF silencing. Mechanistic studies of this RRM1 module are underway and will be discussed. In sum, the identification of RBM12 as a regulator of HbF production represents a previously undescribed post-transcriptional layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this understudied RBP in the context of HbF regulation which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. Disclosures Blobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy.

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Berrien-Elliott, Melissa M., Wong Pamela, Carly Neal, Julia A. Wagner, Michelle Becker-Hapak, Tim Schappe, Matthew L. Cooper, Emily M. Mace, and Todd A. Fehniger. "Primary Human NK Cell Gene-Editing Reveals a Critical Role for NKG2A in Cytokine-Induced Memory-like NK Cell Responses." Blood 134, Supplement_1 (November 13, 2019): 3237. http://dx.doi.org/10.1182/blood-2019-129162.

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Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell anti-leukemia potential is unclear. Paradigm-shifting reports have shown that NK cells exhibit "memory-like" properties following hapten exposure, virus infection, or combined cytokine pre-activation. Human cytokine-induced memory-like (ML) NK cells display enhanced re-stimulation responses to numerous activating stimuli, including tumor target cells. This has been translated in clinical trials as cellular therapy for rel/ref AML patients (NCT#), and the dose escalation of a phase 1/2 study has been completed (PMID). Donor memory-like NK cells expanded in patients' blood and bone marrow and retained enhanced functionality ex vivo, with 7 of 11 patients achieving CR/CRi. Since NK cell recognition depend on signals from multiple activating and inhibitory receptors, we developed mass cytometry panels to immunophenotype and track the diversity and effector functions of these human in vivo-differentiated memory-like NK cells. Previous work showed that that in vivo-differentiated memory-like (ML) NK cells were distinct from baseline (BL) NK cells from the same donor, as well as NK cells from normal donor PBMC. Multidimensional analyses revealed a memory-like phenotype: CD56hi CD11blo CD62L+ NKG2Ahi NKp30hi Ki-67+. Furthermore, Citrus analyses revealed that higher NKG2A expression was significantly correlated with treatment failure. NKG2A is a C-type lectin receptor with two immunoreceptor tyrosine-based inhibitory motifs. Signaling through NKG2A is achieved when it engages its ligand, HLA-E. HLA-E is a non-classical major histocompatibility complex class I molecule that is expressed abundantly on many normal tissue types as well as tumors, including AML. Based on these findings that NKG2A is upregulated on memory-like NK cells and the intensity of NKG2A on memory-like NK cells correlated with patient responses, we hypothesized that NKG2A/HLA-E interactions represent a major barrier to memory-like NK cell responses. CRISPR based gene editing of primary human NK cells has been technically challenging. In order to interrogate the role NKG2A may play in limiting ML NK cell responses, we optimized the MaxCyte GT electroporation system to introduce Cas9 and guide RNA into freshely isolated, purified human NK cells. As proof of principle, we introduced Cas9 and gRNA targeting CD56 into NK cells and assessed CD56 expression a week later. We observed a 96.5% ± 0.8% (SD) reduction in median CD56 expression as determined by flow cytometry, with little impact on cell viability (90.3% ± 2.7% live v 87.0% ± 3.1% live ΔCD56) after electroporation. Next we introduced Cas9 and gRNA against NKG2A into freshly isolated, purified human NK cells. After electroporation, cells were briefly incubated with IL-12/IL-15/IL-18, overnight. The cytokines are washed away and the cells incubated for 4 days in low-dose IL-15, which was required for their survival. NKG2A frequency was decreased 64.72% (ΔNKG2A v Control; 42.3-85.7% range, ± 13.18% SD) by 4 days post-electroporation. We compared the ability of these cells to respond to HLA-E+ K562 leukemia targets and observed a significantly enhanced ML NK cell response by ΔNKG2A ML NK cells compared to control ML NK cells (19.04 ± 5.9% IFN-γ+ v 34.9 ± 8.8% ΔNKG2A IFNγ+; Mean ± S.D.). Finally, we infused ΔNKG2A ML NK or control ML NK cells into NSG recipient mice and assessed the spleen at D7 and D14 for persistence and NKG2A expression. We were able to detect ΔNKG2A ML NK and control ML NK cells at both time points and the ΔNKG2A ML NK cells remain NKG2A-negative, post-transfer. Using gene-editing approaches, the data reveal an important inhibitory role for NKG2A on ML NK cell responses against HLA-E+ targets. Primary human NK cells are notoriously difficult to modify by virus or electroporation. Indeed, most reports utilize expanded NK cells or cord-blood differentiated NK cells which were edited in the stem cell stage. This report demonstrates that mature NK cells can be modified with little ex vivo manipulation with high efficiency and viability. This method has broad potential to expand our understanding of human NK cell biology using genetic loss or gain of function techniques, as exemplified by identification of NKG2A as a critical ML NK cell checkpoint. Figure Disclosures Cooper: Wugen: Consultancy, Equity Ownership, Patents & Royalties. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).

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Pilunov, Artem, Dmitrii Romaniuk, Savely Sheetikov, Alexandra Khmelevskaya, Anton Shmelev, Nadia Bykova, Alina Shomuradova, et al. "Development of T-Cell Therapy Targeting Hematopoietic Minor Histocompatibility Antigen HA-1." Blood 134, Supplement_1 (November 13, 2019): 5749. http://dx.doi.org/10.1182/blood-2019-130552.

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Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only curative therapy for hematological malignancies yet in nearly one-third of patients, it is followed by a relapse of the disease contributing to high mortality. In fully HLA-matched allo-HSCT graft versus leukemia reaction is driven by the recognition of the minor histocompatibility antigens (MiHAs) - endogenous polymorphic peptides presented by MHC. Particularly, HA-1 MiHA is a promising target for immunotherapy. HA-1 is presented by frequent among Caucasians HLA allele - A*02:01. The single nucleotide variation in ARGHAP45 gene which generates the MiHA has the optimal allelic distribution, thus immunogenic mismatch occurs in 30% of allo-HSCT. Also, ARGHAP45 is overexpressed in certain types of leukemia. Here we aim to develop HA-1-specific T-cells for post-transplant relapse therapy. To obtain the sequences of HA-1-specific T-cell receptors (TCRs), naive CD8+ T-cells from 3 HLA-A*02:01 positive and HA-1 negative donors were expanded in vitro using autologous dendritic cells pulsed with HA-1 peptide. Antigen-specific cells were enriched by CD137 marker expression or by HLA-tetramer staining, RNA from positive and negative fractions was isolated for cDNA library preparation. The α and β TCR-repertoires were sequenced using the Illumina MiSeq system. The representative enrichment plot is shown in Figure 1 (A - α chains, B - β chains). Each circle represents a unique TCR. The vertical axis shows the normalized frequency in enriched fraction, the horizontal axis shows the normalized frequency in tetramer or CD137 negative flowthrough. HA-1-specific TCRs are denoted by green filled circles.TCRs were considered to be HA-1-specific if they were significantly enriched in CD137+ or tetramer+ fraction (exact Fisher test, p=0.05). In total, 49 α and 80 β chains were described. To determine the degree of similarity between HA-1-specific TCRs Levenstein distance was calculated between amino acid sequences of complementarity-determining region 3 for both chains. Sequences of previously published HA-1-specific TCRs were also included in the analysis (Verdijk et.al., Haematologica, 2002; Bleakley et.al., 2017, WO2018058002A1). α chains demonstrated low degree of mutual similarity, the majority of sequences did not belong to any cluster (Figure 2A, sequences with the Levenstein distance <3 are connected). In contrast, a significant proportion of β chains were organized in a few clusters containing sequences from all 3 donors and previously published data (Figure 2B). We selected 14 α and 12 β HA-1-specific TCR chains (marked by the black dots in Figure 2). Clones were picked to represent separate clusters of similarity to Levenstein metrics, and unique sequences. Selected α and β-chains were cloned for subsequent functional screening in different combinations. Besides, we developed the modular lentiviral backbone for manufacturing HA-1 specific transgenic CD8+ T-cells. Our approach utilizes Golden Gate Cloning, which allows rapid assembly of lentiviral backbone carrying any combination of TCR α and β chains fused with the selective marker for sorting via p2A peptides. We used truncated CD34 as a transduced cell surface marker for the rapid separation of transduced cells by clinical-grade antibodies and subsequent expansion. In order to prevent the mispairing of transgenic TCR with endogenous one, CRISPR/Cas9 knockout strategy of endogenous TCR chains was developed. We used guide RNAs specific to TRAC,TRBC1 and TRBC2 genes and recombinant Cas9. The efficiency was demonstrated on Jurkat E6-1 cell line, the knockout was confirmed both by flow cytometry and genotyping of the modified cells using fragment analysis. Constant regions of the transgenic TCRs were modified to prevent cleavage by Cas9, the resistance was confirmed by in vitro Cas9 digestion assay. Moreover additional cysteines were introduced in the constant regions of transgenic TCRs for increased transgenic TCR stability. Cytotoxic activity of modified cells will be confirmed on lymphoblastoid cell lines and patient leukemia samples, cytokine secretion of modified cells will be detected using ELISPOT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. Disclosures No relevant conflicts of interest to declare.

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Shen, Yandong, Kyle R. Crassini, Michael E. O'Dwyer, Michael Francis O'Neill, Richard Christopherson, Stephen P. Mulligan, and Oliver Giles Best. "The Dual PI3/PIM-Kinase Inhibitor, Ibl-202, Is Highly Synergistic with Venetoclax Against CLL Cells, and TP53-Knock-out Cells, and Under Conditions That Mimic the Tumor Microenvironment." Blood 132, Supplement 1 (November 29, 2018): 1870. http://dx.doi.org/10.1182/blood-2018-99-115574.

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Abstract Background The B-cell receptor (BCR) signaling pathway and the pro-survival Bcl-2-family of proteins play crucial roles in the pathogenesis of chronic lymphocytic leukemia (CLL). Constitutive activity of the BCR signaling pathway and overexpression of Bcl-2 promote CLL-cell survival, proliferation and drug resistance. BCR-targeted therapies, most notably ibrutinib and idelalisib and the Bcl-2 inhibitor Venetoclax, demonstrate the potential of targeting these pathways. However, there is no evidence that these novel agents are curative in the event of relapse. Treatment options remain limited for patients treated with these agents, in particular those with TP53 lesions. In our recent study (Crassini et al., BJH 2018) we demonstrated efficacy of the PI3/PIM kinase inhibitor, IBL-202 (Inflection Biosciences, Ltd), against CLL cells. In the current study we investigated the effects of combining IBL-202 with Venetoclax on primary CLL cells and both wild-type and TP53 deficient OSU-CLL cells. Methods Primary CLL cells were co-cultured with CD40L-expressing fibroblasts. We established a TP53 knock-out OSU-CLL cell line (OSU-TP53ko) using the CRISPr-Cas9 system. Cell viability was assessed using the mitochondrial dye DilC1(5), propidium iodide and flow cytometry. Synergy between IBL-202 and Venetoclax was evaluated by determining combination indices (CI) using the Compusyn software. The effects of the drugs on cell cycle and proliferation of the OSU-CLL cell lines were assessed using propidium iodide or carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry. The effects of the drugs on the migratory capacity of CLL cells were assessed by determining changes in CXCR4 expression and CLL-cell migration along an SDF-1a gradient. The mechanisms of action of the drugs were investigated by immunoblotting. Results IBL-202 and Venetoclax were highly synergistic against primary CLL cells co-cultured with CD40L-fibroblasts, with a CI of 0.4 at a fractional effect of 0.9 (Figure A and B). Synergy between the drugs was consistent with a significant (P < 0.05) reduction in the IC50 for both drugs. Synergy was also observed against wild-type (WT) and TP53ko OSU-CLL cells, with CI values of < 0.5 at fractional effects of 0.5 and 0.9. Synergy was consistent with significantly greater cytotoxic effects of the drugs in combination (Figure C. WT : P = 0.002 and TP53ko : P = 0.002). IBL-202 and Venetoclax in combination induced cell cycle arrest and slowed the proliferation of both cell lines. Immunoblotting of primary CLL cells showed IBL-202, alone and in combination with Venetoclax, inhibited AKT phosphorylation and reduced the expression of Mcl-1 and Bcl-xL. A greater than additive effect of IBL-202 and Venetoclax was observed on the migratory capacity of CLL cells, reducing the number of cells migrating towards SDF1-a. The effects of the drugs on cell migration were consistent with reduced expression of CXCR4. Conclusions The synergy we observed between IBL-202 and venetoclax against primary CLL cells cultured under conditions that mimic the tumor microenvironment suggests this drug combination may be effective against CLL cells within the lymph nodes and bone marrow. Furthermore, the efficacy of the combination against the TP53ko OSU-CLL cell line suggests the combination may be a highly effective treatment strategy for poor risk CLL disease. Figure A and B - Synergy of IBL-202 and Venetoclax against primary CLL cells. Figure C - Cytotoxic effects of IBL-202 in combination with Venetoclax against OSU and OSU-TP53ko CLL cells Disclosures O'Dwyer: Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees.

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Gupta, Dipti, Yannis Hara, Samuel Lessard, Sarah Sturtevant, Yukio Nakamura, Sriram Krishnamoorthy, Melanie Demers, and Alexandra Hicks. "Catalytic Activity of Heme-Regulated eIF2 Alpha Kinase (HRI) Regulates Fetal Hemoglobin." Blood 136, Supplement 1 (November 5, 2020): 7. http://dx.doi.org/10.1182/blood-2020-139991.

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Sickle cell disease (SCD) is a hereditary disorder occurring due to a mutation in the β- globin gene resulting in hemoglobin polymerization and sickling of red blood cells that drives an array of severe pathophysiologies. SCD patients with hereditary persistence of fetal hemoglobin mutations show amelioration of disease symptoms. HRI is a heme sensing eIF2α kinase belonging to the integrated stress response pathway, primarily regulating the hemoglobin synthesis in RBCs. Under low levels of heme, HRI undergoes auto-phosphorylation and subsequently phosphorylates its substrate eIF2α. This impedes the global protein translation and selectively activates ATF4 translation thereby initiating a transcriptional stress response. HRI protein deletion has been shown to induce fetal hemoglobin (HbF) in vitro by reducing BCL11A levels (Grevet et al., 2018). It has also been shown that ATF4 binds to enhancer region of BCL11A promoting its expression, revealing a direct regulation of HRI from eIF2α to ATF4 to BCL11A to γ-globin (Huang P, 2020). As it is well-established that protein kinases can exert dual kinase and scaffold functions, we explored whether the regulation of HRI on HbF is driven by its catalytic or non-catalytic activity. We generated HRI kinase dead (KD) mutant HUDEP-2 cells, with a mutation (K196R) in HRI ATP binding site that is predicted to result in loss of its auto-phosphorylation (Bauer B. N., 2001). HRI KD mutant HUDEP-2 was created using CRISPR Cas9 and homologous recombination approach, followed by clonal selection that yielded two homozygous clones, referred as KD clones. To further compare the scaffold and kinase function of HRI, we created HRI knockout (KO) in HUDEP-2 cell line using CRISPR Cas9 approach. We isolated clonal populations with 3 clones each with heterozygous and homozygous editing. Western analysis of KD clones showed that HRI protein levels remain unchanged. About 50% and a 100% loss in HRI protein level was observed in heterozygous and homozygous KO clones respectively. To confirm the loss in kinase activity of HRI, we differentiated KD clones for 7 days in vitro and subjected them to iron depleted conditions. As expected, HRI WT cells under iron depleted conditions showed robust activation in the p-eIF2α levels indicative of increase in HRI activity. KD clones lacked increase in p-eIF2α levels under these conditions confirming the loss of kinase activity of HRI in KD clones. HRI KD and KO clones were characterized to evaluate the HbF regulation by differentiating in vitro for 7-8 days. KD clones showed 4-5-fold induction in HbF levels measured by flow cytometry. Heterozygous and homozygous KO clones showed an equally strong activation in HbF levels. KD and KO clones showed downregulation in BCL11A and ATF4 protein levels. These results indicate that HRI regulates HbF as a function of its catalytic activity. We further employed RNA-seq to study the global transcriptomics in HRI WT and KD mutant clones at day 0 and day 8, under steady state and iron depleted conditions. RNA-seq confirmed the strong activation of HBG1/2 levels and robust silencing of BCL11A levels in KD clones at day 8. A weaker induction in HBG1/2 levels and minor reduction on BCL11A levels were observed at day 0. These results indicate that HRI may play a role in HbF silencing in a stage specific manner. HRI WT cells under iron depleted condition showed activation of ATF4 pathway, indicative of activation of HRI stress pathway. As expected, HRI KD population showed no regulation of ATF4 pathway under iron depleted conditions. Interestingly, we did not observe any changes in the BCL11A gene expression in WT cells under iron depleted conditions. Further evaluation needs to be carried out in order to understand the regulation of BCL11A by ATF4 under low iron mediated stress. Fewer gene changes were observed in KD populations under iron depleted conditions indicative of HRI playing a vital role as a heme sensor. Overall, our results provide evidence to support that HRI regulates HbF though its catalytic activity and demonstrates its role as heme sensor in HUDEP-2 cells. Disclosures Gupta: Sanofi: Current Employment. Hara:Sanofi: Current Employment. Lessard:Sanofi: Current Employment. Sturtevant:Sanofi: Current Employment. Krishnamoorthy:Sanofi: Current Employment. Demers:Sanofi: Current Employment. Hicks:Sanofi: Current Employment.

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Wenthe, Sophia, Kelsie Becklin, Brett Napiwocki, Emma Kozurek, Branden Moriarity, and Jong Hyuk Kim. "Abstract 198: Unveiling chromatin accessibility landscape and convergent oncogenic pathway in angiosarcoma models using induced pluripotent stem cells." Cancer Research 82, no. 12_Supplement (June 15, 2022): 198. http://dx.doi.org/10.1158/1538-7445.am2022-198.

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Abstract Angiosarcoma is a rare soft tissue sarcoma that forms malignant vessels. Angiosarcomas are aggressive and highly metastatic, resulting in a poor prognosis. Recurrent somatic mutations in TP53 and genes involved in PI3K/AKT/mTOR pathway such as PIK3CA and PIK3R1 are identified in angiosarcomas. However, angiosarcomas are genomically complex, and the oncogenic mechanisms are virtually unknown. Due to its rarity, establishment of experimental tumor models is an unmet need for angiosarcoma research. In this study, we used human induced pluripotent stem cells (iPSCs) to develop a novel, reliable model for angiosarcoma recapitulating the genomic complexity and the tumor immune landscape. Specifically, we induced TP53 mutations in human iPSCs using CRISPR/Cas9 with validation of p53 deficiency by gene sequencing and Western blotting. We then established protocols to differentiate genetically engineered iPSCs to mesoderm and subsequently bi-potential hemangioblasts, which are considered the putative cell-of-origin of angiosarcoma. We found that isogenic wild-type (WT) and TP53 mutant iPSCs were capable of generating hemangioblasts, with no significant differences in morphology or growth patterns observed between WT and mutant cells. Putative iPSC-derived hemangioblasts were CD34+ by flow cytometry and contained primitive endothelial cells with the capacity to form tube-like structures in Matrigel. RNA-seq data libraries were generated to profile global gene expression in non-differentiated cells, mesodermal precursors, hemangioblasts, and endothelial cells derived from WT and p53 mutant iPSCs during differentiation over time (day 0, 2, 5, 8). Principal component analysis revealed that gene expression patterns were altered between WT and p53 mutant cells during differentiation, representing distinct gene signatures unique to each cell type. Intriguingly, transcriptomic alteration of p53 mutant iPSC-derived cells was more variable than that of WT. Our data also showed that p53 mutation induced dysregulation of genes associated with chromosome maintenance, extracellular matrix organization, hemostasis, and receptor tyrosine kinase signaling in iPSC-derived hemangioblasts when compared isogenic WT controls. Our data highlights the role that mutant p53 plays in the induction of genomic instability and transcriptional programs that regulate hemogenic and endothelial function during differentiation. Additionally, ATAC-seq data were generated from WT and p53 mutant iPSC-derived cells to determine chromatin accessibility dynamics and identify key transcription factors that activate convergent vascular tumorigenic pathways. We are currently generating iPSC harboring co-mutations in TP53 and PIK3CA in order to determine the phenotype and tumorigenic capacity of engineered iPSC-derived hemangioblasts in xenograft models. Citation Format: Sophia Wenthe, Kelsie Becklin, Brett Napiwocki, Emma Kozurek, Branden Moriarity, Jong Hyuk Kim. Unveiling chromatin accessibility landscape and convergent oncogenic pathway in angiosarcoma models using induced pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 198.

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Aruljothi, Charlesantony, Subin S. George, Patrick Somers, Justin Blum, Chelsea Thorsheim, Maxim Pimkin, and Vikram R. Paralkar. "Regulation of Ribosomal RNA Synthesis in Myeloid Progenitors By Cell Type Specific Transcription Factors." Blood 136, Supplement 1 (November 5, 2020): 11–12. http://dx.doi.org/10.1182/blood-2020-138776.

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Haematopoiesis relies on the ability of hematopoietic stem cells to progress through a systematic hierarchy to produce lineage-restricted progenitors that terminally differentiate into phenotypically distinct types of mature hematopoietic cells. This process is precisely coordinated by the combinatorial activity of lineage-specifying transcription factors (TFs). Indeed, the critical transcriptional program of every hematopoietic cell type, and indeed of all cell types throughout the body, requires a set of core TFs for its proper execution. A frequently-overlooked component of the cellular transcriptional program is the transcription of ribosomal RNA (rRNA), the major component of ribosomes. Ribosomal RNA comprises 90% of total cellular RNA, and its transcription from hundreds of rDNA genes by RNA polymerase I is one of the most intense transcriptional processes in the cell. Different progenitor and mature cell types in the hematopoietic tree have different sizes, ribosome abundances, and rates of protein synthesis. Tight control of ribosome abundance is essential for a normal cellular proteome, and different cell types within the hematopoietic tree have varied rRNA transcription rates. However, there has been limited study of the molecular basis of lineage-specific regulation of rDNA transcription in hematopoiesis. We explored the binding to rDNA of over twenty key hematopoietic TFs that were determined by the Broad Institute DepMap database to be crucial for survival of hematopoietic cell lines. Using a customized bioinformatics pipeline, we mapped over three hundred ChIP-Seq datasets for these factors (generated by us as well as publicly available from ENCODE and GEO) to mouse and human rDNA assemblies, and found that several essential hematopoietic TFs such as MYC, MYB, RUNX1, PU.1, CEBPA and others bind to rDNA at conserved sites and motifs (Fig A). MYC is well-known as a master regulator of rDNA, and RUNX1 was recently reported to bind rDNA, but most of the others have never been linked to rDNA, and their functional roles in regulating rDNA transcription have not been explored. We picked for further study CEBPA, a crucial TF required for specification of granulocyte-monocyte progenitors (GMPs). For our experiments, we used the mouse HOXA9-ER cell line, which mimics GMPs. We used CRISPR/Cas9 and homologous recombination to fuse FKBPV degron into bi-allelic endogenous loci of the Cebpa gene in HOXA9-ER cells, and, upon addition of dTAG-13 (the ligand for FKBPV), the CEBPA-FKBPV fusion protein could be rapidly degraded within 2 hours (Fig B, C), providing us an experimental system to study the immediate consequences of CEBPA loss. In order to quantify the rate of rRNA transcription, we devised an assay titled "47S FISH-Flow" that combined fluorescent in-situ hybridization (FISH) using probes against nascent 47S rRNA with flow cytometry (Fig D, E). This assay not only allows us to quantify the rate of rRNA transcription on a per-cell basis in millions of cells, but also allows us to separately gate and quantify rRNA transcription in different stages of the cell cycle, eliminating a major confounder in bulk cell studies - cell cycle distribution. Using 47S FISH-Flow, we observed that degradation of CEBPA in the HOXA9-ER mouse GMP cell line led to decrease in synthesis of 47S rRNA within hours (Fig F) before any change in cell cycle or growth kinetics, and was followed by growth arrest in 24 hours. In summary, we show that several critical hematopoietic TFs show abundant, conserved binding to rDNA, and the depletion of CEBPA rapidly reduces nascent rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by a wide range of lineage-specific hematopoietic TFs. Figure Disclosures No relevant conflicts of interest to declare.

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Meyer, Tatjana, Nikolaus Jahn, Anna Dolnik, Peter Paschka, Verena I. Gaidzik, Daniela Weber, Lars Bullinger, Hartmut Döhner, Konstanze Döhner, and Jan Kroenke. "BRCA1/2 Containing Complex 3 (BRCC36) Is Recurrently Mutated in AML with t(8;21) and Associated with Increased Sensitivity to Chemotherapy through Impairment of the DNA Damage Repair Pathway." Blood 132, Supplement 1 (November 29, 2018): 1487. http://dx.doi.org/10.1182/blood-2018-99-114143.

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Abstract Introduction BRCA1/BRCA2-containing complex 3 (BRCC36) is a Lys63-specific deubiquitinating enzyme (DUB) involved in DNA damage repair. Mutations in BRCC36 have been identified in 2-3% of patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). The role of BRCC36 mutations in de novo AML and their impact on DNA damage-inducing cytotoxic chemotherapy sensitivity is not clear. Aim We aimed to determine the incidence of BRCC36 mutations in AML and their impact on outcome and drug sensitivity in vitro. Methods We analyzed the entire coding region of BRCC36 for mutations in 191 AML cases with t(8;21) (q22;q22.1) and 95 cases with inv(16) (p13.1q22) using a customized targeted sequencing panel. Data for de novo AML was derived from The Cancer Genome Atlas Research Network (TCGA) data set (NEJM 2013). Lentiviral CRISPR/Cas9 was used to inactivate BRCC36 in t(8;21)-positive AML cell lines - Kasumi-1 and SKNO-1 - and murine hematopoietic stem and progenitor cells (LSKs). Knockout was confirmed by a cleavage assay as well as Western blot. AML1-ETO-9a was expressed by a retroviral vector. Cell lines and LSK cells were treated with different concentrations of doxorubicin or cytarabine and their viability was assessed seven days post treatment. DNA damage was assessed through phospho-γH2AX staining using flow-cytometry. Results BRCC36 mutations were identified in 7 out of 191 patients (3.7%) with t(8;21) AML and none of 95 patients with inv(16). In the TCGA data set one out of 200 patients (0.5%) with de novo AML had a BRCC36 mutation. This patient had a complex karyotype and would be considered as secondary AML with myelodysplastic-associated changes according to the 2016 WHO classification. Six of the 7 mutations were missense or nonsense mutations that were predicted to be deleterious to BRCC36 function. One mutation affected a splice site at exon 6, resulting in an impaired splicing capability. With intensive standard chemotherapy all patients with BRCC36 mutations achieved a complete remission and had an estimated relapse-free and overall survival of 100% after a median follow up of 4.2 years. Given its role in DNA damage repair, we hypothesized that BRCC36 inactivation sensitizes AML cells to DNA-damage inducing drugs. In order to test this, we generated BRCC36 knockout Kasumi-1 and SKNO-1 cell lines using CRISPR-Cas9. BRCC36 inactivation had no impact on cell growth on either of the cell lines. However, we found that BRCC36 knockout cells were significantly more sensitive to doxorubicin as compared to the parental cells with normal BRCC36. This was accompanied by a significant increase in DNA damage as assessed by phospho-γH2AX in BRCC36 knockout vs control cells after doxorubicin treatment. In contrast, BRCC36 inactivation had no impact on cytarabine sensitivity. We next assessed drug sensitivity in primary murine leukemic cells expressing AML1-ETO-9a. Again, inactivation of BRCC36 resulted in a significant higher sensitivity to doxorubicin but not cytarabine. Conclusion We found BRCC36 to be recurrently mutated in t(8;21)-positive AML Inactivation of BRCC36 was associated with impairment of the DNA damage repair pathway and thus higher sensitivity to DNA damage-inducing chemotherapy. This might be also reflected by the favorable clinical outcome of patients with BRCC36 mutated t(8;21)-positive AML, a finding which has to be confirmed in a large patient cohort. Disclosures Paschka: Pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Otsuka: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Other: Travel support; Janssen: Other: Travel support; Bristol-Meyers Squibb: Other: Travel support, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Travel support; Astex: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Bullinger:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding.

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Stopka, Tomas, Jarmila Vargova, Karina Vargova, Nina Dusilkova, Vojtech Kulvait, Vit Pospisil, Jiri Zavadil, Marek Trneny, and Pavel Klener. "Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Is a New Biomarker for Mantle Cell Lymphoma: Expression, Localization, and Phosphorylation Study." Blood 128, no. 22 (December 2, 2016): 1767. http://dx.doi.org/10.1182/blood.v128.22.1767.1767.

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Abstract Mantle cell lymphoma (MCL) is a relatively distinct B-cell non-Hodgkin lymphoma subtype with aggressive and often recurrent clinical course. At diagnosis, MCL often manifests with leukemization, a feature more common to chronic lymphocytic leukemia (CLL). Common features and differences between MCL and CLL were not yet explored by comprehensive global approaches, despite such understanding potentially being very neat for deciphering pathogenesis and tailoring therapies of these clinically distinct diseases. In our study, we have compared MCL(n=10), CLL(n=10) and normal control(n=8) B-cell samples using the Affymetrix Human Genome HG-U133 Plus 2.0 Array. We studied different mRNA levels of ~47.000 transcripts represented on the array. The comparative analyses identified a set of 892 differentially expressed genes between MCL and NBC; and 774 differentially expressed genes between CLL and NBC. In order to find MCL/CLL-specific biomarkers we focused on the intersection of differently expressed genes in both groups (CLL vs NBC and MCL vs NBC). There were 222 mRNAs in the intersection, 216 of them were deregulated in the same direction in both groups while 6 mRNAs were deregulated in the opposite direction. This set of 6 disease-specific mRNAs contained previously reported biomarkers (CD200, LEF1), and also the Myristoylated alanine-rich C-kinase substrate (MARCKS) that has not yet been studied in MCL. Thus we utilized the validation patient groups (NMCL=6, NCLL=8) and confirmed differential expression of MARCKS on protein levels by flow cytometry and immunofluorescence. As MARCKS was previously shown to either bound to the cell membrane, to reside in the cytosol, or alternatively become transmitted to nuclei, we investigated subcellular localization of MARCKS using immunofluorescence (IF). The cytoplasmic MARCKS signal in MCL was significantly higher than in CLL while the opposite was observed for the nuclear IF signal. The ratio between cytoplasmic and nuclear signal was 2.5 for MCL and 0.8 for CLL (p < 0,0001). The active forms of MARCKS were shown to become phosphorylated on serineresidues and this prompted us to study the phosphorylation forms of MARCKS in MCL. Indeed, one of the residues, Ser159/163, was hyperphosphorylated in the MCL cytoplasm and its level and distribution markedly differed from CLL or NBC. We next searched for regulatory mechanisms upstream of the MARCKS expression in MCL vs CLL. MARCKS is a predicted target of several microRNAs (according to DIANA-TarBase v7.0), among them also of miR-155 (that is differentially expressed between MCL and CLL). To further investigate the regulatory relationship between mir-155 and MARCKS we utilized a CLL cell line MEC-1 and using the CRISPR/Cas9 technology we prepared individual cell clones that were mutated within the mature miR-155 sequence that recognizes MARCKS mRNA. As expected, the miR-155-MEC-1 mutants expressed markedly higher level of MARCKS compared to the control MEC-1 cells. In conclusion, our work identified a set of six differentially expressed mRNAs when comparing MCL and CLL, among them, MARCKS. We further showed that MARCKS is differentially expressed, localized, and phosphorylated between MCL and CLL, and that MARCKS is partly controlled by oncogenic microRNA miR-155. MARCKS may play an important role in MCL pathogenesis and can serve as useful MCL biomarker. Grant support: GAČR 16-05649S & P305/12/1033, AZV: 16-27790A and 16-31586A. Institutional support: CZ.1.05/1.1.00/02.0109, UNCE 204021, LH15170, PRVOUK P24, LQ1604. Disclosures No relevant conflicts of interest to declare.

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Barrio, Santiago, Larissa Haertle, Umair Munawar, Lucia Martin, Isabel Cuenca, Cornelia Vogt, Andoni Garitano-Trojaola, et al. "Clonal Competition Models to Understand Progression and Resistance in Myeloma." Blood 134, Supplement_1 (November 13, 2019): 1807. http://dx.doi.org/10.1182/blood-2019-130812.

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Background: Progression and relapse in Multiple Myeloma (MM) is induced by changes in the clonal tumor composition. In order to better understand the mechanisms underlying these dynamics, we developed clonal competition models based on the co-culture of fluorescent labelled isogenic MM cells, with or without the alteration under study. Methods: To understand the effect of mono- and bi-allelic TP53 lesions, we use the AMO1 cell line, one of few myeloma cell lines harbouring wild type TP53 (WT). After modification with CRISPR / CAS9, we selected subclones with mono- and/or bi-allelic deletion of TP53. For the characterization of alterations in RAS, we selected OPM2 cells, one of the few lines with the RAS pathway intact. Furthermore, we generated the KRAS WT, G12A and A146T sublines by stable transfection with Sleeping Beauty vectors. To study mutations related to resistance to IMIDs and PIs, we introduced mutations in the target genes IKZF1 (WT, A152T, Q170D or R439H), CUL4B (KO), and PSMB5 (WT or A20T ) in AMO1 and L363, cell lines sensitive to IMiD or PI treatment. In addition, all WT and mutant sublines were also stably transformed with E-GFP or LSS-mkate2-RFP for flow cytometry analysis. Results: We recently demonstrated that lesions in TP53, both mono- and bi-allelic, induce a growth advantage to the affected cells. In the current study, we also observed an increased fitness in KRAS mutated cells (G12A or A146T vs WT) independent of treatment. We co-cultivated KRAS mutant with WT cells at a ratio 1:3 in two independent experiments, with the color labelling switched (red/green wt/mutant and vice-versa). KRAS G12A clone significantly expanded and reached 50% of the cells at day 40. Likewise, A146T clone outcompeted WT cells, but the time required to represent the majority of cells in the coculture was longer. We next explored the effects of resistance mutations and drug exposure. Both the IKZF1 A152T and CUL4B KO mutants outcompete WT cells in the presence of Lenalidomide (LEN). The same effect was observed for the PSMB5 A20T mutant exposed to Bortezomib (BOR). This selection ("Survival Fitness") did not occur without the presence of the drug. Thus, resistance related mutations seem only to provide a fitness advantage under drug exposition. In addition, both the CUL4B KO and PSMB5 A20T mutants were overcome by WT cells when the drug was removed from co-culture, suggesting that these lesions provide a survival disadvantage without the selective pressure of IMiD or PI. This may provide an explanation for the low mutation rate in this gene in recent sequencing publications, as usually samples are not obtained under selective pressure but in treatment free intervals. IKZF1 mutations outside the IMiDs / CRBN binding area (Q170D and R439H) provided no advantage to the cells. Conclusions: Our clonal competition assays provide novel insights on the impact of point mutations on the fitness of affected myeloma subclones, either with or without the selective pressure of therapy. Figure Disclosures Martinez-Lopez: Celgene: Honoraria, Other: Advisory boards and Non-Financial Support ; Amgen: Honoraria, Other: Non-Financial Support ; F. Hoffmann-La Roche Ltd: Honoraria; Janssen: Honoraria, Other: Advisory boards and Non-Financial Support ; BMS: Honoraria, Other: Advisory boards; Incyte: Honoraria, Other: Advisory boards; Novartis: Honoraria, Other: Advisory boards; VIVIA Biotech: Honoraria.

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Couch, Tyler A., Zachary C. Murphy, Michael Getman, Ryo Kurita, Yukio Nakamura, and Laurie A. Steiner. "Human Erythroblasts with c-Kit Activating Mutations Have Reduced Cell Culture Costs and Remain Capable of Terminal Maturation and Enucleation." Blood 132, Supplement 1 (November 29, 2018): 2315. http://dx.doi.org/10.1182/blood-2018-99-117157.

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Abstract There is a constant need for red blood cells for transfusion therapy in the treatment of anemias and acute injury. As all blood products for transfusion come from donors, there are concerns over shortages and safety. Furthermore, many patients with transfusion-dependent anemias risk alloiumminization. The in vitro production of red blood cells would address these problems, especially as they can be genetically engineered to prevent alloimmunization. Numerous erythroid culture systems now exist for the in vitro production of red blood cells. Hematopoietic stem and progenitor cells (HSPCs) obtained from umbilical cord or peripheral blood can be differentiated into erythrocytes, however, they are limited in expansion. While umbilical cord HSPCs have greater expandability than peripheral blood, the resulting erythrocytes contain fetal globins. Pluripotent stem cells can also be used as a starting source, however only a small percentage of the cells can be differentiated into erythroblasts which also suffer from low enucleation rates. Presently, the cost of in vitro production of a unit of red cells is greater than an order of magnitude higher than obtaining it from a donor largely due to the medium and cytokine costs (Timmins & Nielsen, Trends Biotechnol, 2009). A relatively new approach of immortalizing early erythroblasts allowing unlimited expansion as well as terminal maturation and enucleation shows great therapeutic promise (Kurita et al., PLoS One, 2013; Huang et al., Mol Ther, 2014; Trakarnsanga et al., Nat Commun, 2017). However, these immortalized erythroblasts are still reliant on two costly cytokines: stem cell factor (SCF) and erythropoietin (Epo). Mutations in exon 17 of the receptor tyrosine kinase gene KIT are frequently seen in acute myeloid leukemias, gastrointestinal stromal tumors, and mast cells leading to mastocytosis. These mutations cause the c-Kit protein to spontaneously activate and transduce signal in the absence of SCF (Kit-ligand). To generate an SCF-independent HUDEP-2 cell line (Kurita et al., PLoS One, 2013), we used CRISPR/Cas9 to introduce missense and frameshifting mutations within the vicinity of Asp816 in exon 17 of the KIT gene. The resulting monoclonal cell lines were selected for by removing SCF from the expansion medium and were subsequently named KIT-CAT (KIT with Constitutively Activating Transformation). To better understand what KIT mutations allowed or impaired terminal maturation, monoclonal cell lines were genotyped by Sanger sequencing. Three cell lines with unique genotypes were chosen for further analysis. All three KIT-CAT lines had a shorter doubling time compared to HUDEP-2 cells (16.7 vs 18.9 hrs, p=0.020) and were no longer dependent on SCF or Epo. However, two of the three KIT-CAT lines showed more robust proliferation with Epo in the expansion medium. The addition of SCF to the medium caused no increase in c-Kit activation by Western blotting for phosphorylation at Tyr703. Furthermore, the low molecular weight and immature form of c-Kit is also phosphorylated in KIT-CAT cells, but not HUDEP-2 cells, indicating c-Kit activation occurs before trafficking to the cell membrane where SCF would bind (Tabone-Eglinger et al., Clin Cancer Res, 2008). Key features of erythroblast maturation are the decrease in cell and nuclear size which can be measured using imaging flow cytometry (McGrath et al., Methods, 2017). While in expansion phase, all 3 cell lines were larger in cell and nuclear area compared to the parental HUDEP-2 line. By day 6 of maturation, all three cell lines had statistically significant decreases in cell and nuclear size indicating maturation. By day 13 of culture, Wright-Giemsa staining showed that the majority of the cells were orthochromatic erythroblasts or enucleate reticulocytes. Reducing cell culture costs is needed for in vitro manufacturing of red blood cells to be economically feasible. These results show that a c-Kit activating mutations in human erythroblasts removes the cost of SCF and reduces the cost of Epo while still allowing for terminal maturation and enucleation. Disclosures No relevant conflicts of interest to declare.

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El Hoss, Sara, Sylvie Cochet, Auria Godard, Hongxia Yan, Michaël Dussiot, Giacomo Frati, Bénédicte Boutonnat-Faucher, et al. "Fetal Hemoglobin Rescues Ineffective Erythropoiesis in Sickle Cell Disease." Blood 136, Supplement 1 (November 5, 2020): 14–15. http://dx.doi.org/10.1182/blood-2020-137477.

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Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

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Heatley, Susan L., Elyse C. Page, Laura N. Eadie, Barbara J. McClure, Jacqueline Rehn, David T. Yeung, Michael Philip Osborn, Tamas Revesz, Maria Kirby, and Deborah L. White. "Modeling Relapsed, Refractory Acute Lymphoblastic Leukemia from a Child with Neurofibromatosis." Blood 138, Supplement 1 (November 5, 2021): 1317. http://dx.doi.org/10.1182/blood-2021-150086.

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Abstract Neurofibromatosis type 1 (NF-1) is an autosomal dominant disorder affecting approximately 1:3000 individuals globally. While approximately 50% are familial, with over 3000 causative germline variants in the neurofibromatosis (NF1) gene identified, the remainder occur sporadically. These mutations lead to haploinsufficiency of NF1 and neurofibromin, a tumor suppressor and important negative regulator of RAS signaling. Children with NF-1 have a higher risk of developing juvenile myelomonocytic leukemia and acute myeloid leukemia, but rarely develop acute lymphoblastic leukemia (ALL). A 9-year-old male presented in 2015 with persistent migratory subcutaneous swellings and multiple bony aches with lytic lesions on bone imaging. He had a high white cell count with eosinophilia (WCC 43.4 x 10 9/L, eosinophils 23.87 x 10 9/L) with no circulating blasts, 10% marrow blasts (CD10+/CD19+/CD34+) and was CNS negative. Although previously undiagnosed, NF-1 was clinically suspected due to typical skin changes. He was diagnosed with iAMP21 ALL and NF-1 was confirmed with the identification of a germline NF1 donor splice site mutation (c.1845G>A:p.L615=). Bone marrow cells were sorted by flow cytometry on CD19 positivity and underwent transcriptomic sequencing. This revealed a P2RY8-CRLF2 gene fusion, with no other clinically relevant variants, while a custom Taqman low density array indicated high-risk B-ALL subtype Ph-like ALL. Multiplex ligation-dependent probe amplification (MLPA) confirmed iAMP21 and also identified IKZF1 exon 2-3 and BTG1 deletions. Treatment followed the high-risk B-ALL arm of the AEIOP-BFM ALL2009 protocol due to persistent end-consolidation MRD in addition to iAMP21 and the Ph-like phenotype. He relapsed three years later off treatment and was refractory to both salvage chemotherapy and blinatumomab. The iAMP21, P2RY8-CRLF2 gene fusion, IKZF1 exon 2-3 and BTG1 deletions remained detectable. Whole exome sequencing of CD19 positive samples from diagnosis, relapse and mesenchymal stem cells (germline control) was performed, identifying a NF1 c.7400dupT:p.L2467 frameshift (fs) mutation only at relapse. To understand the implications of NF1 p.L2467fs, the P2RY8-CRLF2 gene fusion was first transduced into the interleukin 3 (IL3) dependent murine pro-B cell line Ba/F3. P2RY8-CRLF2 alone is not transforming and is thought to be a secondary event in iAMP21 ALL, providing an ideal model to study the cumulative effect of the NF1fs. The NF1fs was then introduced to the P2RY8-CRLF2 cells by CRISPR/Cas9. A proliferation assay was performed without IL3 and demonstrated the P2RY8-CRLF2+NF1fs cell line was IL3 independent, indicative of leukemic transformation, whereas all other lines were not (vs Ba/F3, p = 0.001). Neurofibromin can be constitutively phosphorylated at the c-terminus, negatively regulating NF1-GAP activity, suppressing RAS signaling and inducing cell cycle arrest. Therefore, to demonstrate loss of function due to the c-terminus NF1 p.L2467fs and increased RAS signaling, western blotting for pERK was performed. Significant upregulation of pERK was confirmed in P2RY8-CRLF2+NF1fs in comparison to Ba/F3 control cells (p=0.007) (Figure 1). The MEK inhibitors trametinib and mirdametinib are in clinical trials for NF-1 patients and have shown efficacy in ALL models with RAS mutations. In a 3-day cell death assay, only P2RY8-CRLF2+NF1fs demonstrated sensitivity to trametinib (LD 50 P2RY8-CRLF2 = >6.4 µM, NF1fs = >6.4 µM, P2RY8-CRLF2+NF1fs =1.7µM; p < 0.001) and mirdametinib (LD 50 P2RY8-CRLF2 = >16 µM, NF1fs = >16 µM, P2RY8-CRLF2+NF1fs = 8.3 µM; p < 0.0001) (Figure 2). Here, we have demonstrated a LOF NF1fs mutation using an in-vitro model of ALL. Germline NF1 haploinsufficiency and a second hit NF1 mutation in ALL is limited to one report of monozygotic twins with neurofibromatosis. We propose that NF1 p.L2467fs caused bi-allelic LOF and therefore contributed to relapse in this patient. An understanding of the genomic complexities that lead to relapse may also inform personalized treatment strategies. While this patient subsequently achieved remission with inotuzomab and underwent successful stem cell transplantation, the sensitivity to MEK inhibitors is an exciting development for neurofibromatosis patients with ALL. Figure 1 Figure 1. Disclosures Yeung: Amgen: Honoraria; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. White: BMS: Honoraria, Research Funding; Novartis: Research Funding.

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Takata, Katsuyoshi, Daisuke Ennishi, Ali Bashashati, Saeed Saberi, Elena Viganò, Shannon Healy, Julie S. Nielsen, et al. "Somatic PRAME Deletions Are Associated with Decreased Immunogenicity, Apoptosis Resistance and Poor Outcomes in Diffuse Large B-Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 667. http://dx.doi.org/10.1182/blood-2018-99-113516.

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Abstract Background: The current standard of care in diffuse large B-cell lymphoma (DLBCL) consists of chemotherapy and therapeutic monoclonal antibodies that have significantly improved patient outcomes over the past 15 years. However, a large proportion of patients suffer from refractory or relapsed disease. Therefore, the development of new therapeutic strategies for this subgroup of patients, who are threatened by a high chance of disease-related death, represents an important unmet clinical need. Methods: We enrolled into our study 347 de novo DLBCL patients uniformly treated with R-CHOP from the BC Cancer population-based cohort between September 2000 and January 2012. RNAseq and high-resolution copy number analysis were performed and correlated with clinical outcome data and tumor microenvironment composition. We also performed functional studies to investigate PRAME-mediated memory T-cell responses and gene expression changes. Results: We discovered novel, highly focal deletions of 22q11.22, including the PRAME gene in 13% (44/338) of the cases. The deletions cluster in a narrow chromosomal region that includes a very small number of genes (VpreB1, ZNF280A/B, PRAME, GGTLC2, miR-650). Of clinical importance, 22q11.22 deletions were found significantly more frequently in germinal centre B-cell-like (GCB) type DLBCL (17% (31/180) vs. activated B-cell-like (ABC) type: 8% (8/98), P < 0.01), and were also significantly associated with worse outcome, which was specifically observed in GCB-DLBCL (5-year disease specific survival, non-PRAME-deleted: 84.5% vs. PRAME-deleted: 67.2%, P = 0.026). Homozygous deletions were more strongly associated with poor outcome than heterozygous deletions. Interestingly, 90% of PRAME-deleted cases were Ig-lambda restricted (P < 0.001). PRAME is a prominent member of the cancer testis antigen (CTA) family of proteins that are expressed in various types of cancers, but not in normal tissues, including normal mature B-cells, apart from male germinal cells. Due to the cancer-specific expression of CTAs, these molecules are considered promising targets for cancer immunotherapy using cytotoxic T-cells and tumor vaccination approaches. To determine the association with tumor microenvironment composition, we analyzed CD4/CD8 flow cytometry data from DLBCL patient samples. The numbers of CD4 and CD8-positive T cells were significantly lower in PRAME-deleted cases compared to wild type (CD4: P < 0.001, CD8: P = 0.013). Notably, RNAseq analysis revealed that the HLA-A*0201 genotype was seen significantly more often in PRAME deleted cases (PRAME wt: 2.5% vs. PRAME deleted: 10.8%, P = 0.005). In order to functionally characterize its interaction with the immune microenvironment, we utilized enzyme-linked immunoSpot (ELISPOT) assays to investigate memory T-cell reactions of patient-derived T cells to PRAME antigens using patient-derived peripheral blood mononuclear cells (PBMC) and measured IFN-g production (7 control healthy donors, 4 PRAME-deleted and 4-wild type patients). While T cells from PRAME-replete patients had no reaction to PRAME antigens, PRAME-deleted patient-derived T-cells had significant reactions to 4 independent PRAME peptides. These data suggest that PRAME-deleted tumor cells can escape from cytotoxic T-cell attack to gain growth advantage. Next, we performed PRAME knock-out (KO) experiments using CRISPR/Cas9 genome editing to clarify the cell autonomous effects of PRAME deletions. Using 2 different cell lines (Karpas422 and SUDHL-4), we found TNFSF10 (TRAIL) expression was significantly down-regulated in homozygous PRAME-KO cell lines compared to wild type. The soluble form of TRAIL (sTRAIL) was also reduced, as measured with enzyme-linked immunosorbent assays. These results suggest that PRAME downregulated cells may contribute to cell survival via TRAIL and sTRAIL reduction. Conclusion: We identified recurrent PRAME deletions and characterized their clinical and functional role in DLBCL. Our findings contribute to the understanding of cell-autonomous and extrinsic roles of PRAME deletions in lymphomagenesis and may lead to the discovery of new therapeutic avenues to simultaneously treat the tumor and the host. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Janssen: Research Funding; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Tioma: Research Funding; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

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Grzegorski, Steven, Divyani Paul, James H. Morrissey, and Jordan A. Shavit. "Genetic Duplication of Tissue Factor Leads to Partial Specialization of Function in Zebrafish." Blood 134, Supplement_1 (November 13, 2019): 486. http://dx.doi.org/10.1182/blood-2019-131980.

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Tissue factor (TF) is a critical factor for hemostasis in response to tissue injury. Among mouse knockouts of procoagulant factors, those lacking TF have the most severe phenotype, with complete lethality by midgestation. Furthermore, complete loss of TF has never been described in humans. Together, these suggest additional roles in embryonic development beyond coagulation. Zebrafish are a small freshwater teleost fish with a well described hemostatic system, including conservation of the coagulation cascade. Zebrafish are prolific breeders that reproduce through external fertilization, with subsequent rapid and transparent development, allowing studies not possible in mammals. Due to an ancient genomic event, 30-40% of the teleost genome is duplicated, resulting in two TF paralogs (TFa and TFb) with unknown functions. Here we use CRISPR/Cas9 to produce null alleles of TFa and TFb and uncover partial subspecialization of these duplicates. It has been shown previously that both TFs are expressed before the initiation of blood circulation, between 24 and 48 hours post fertilization, yet complete loss of TFa and TFb yielded no gross abnormalities. Embryos and larvae were able to develop normally through juvenile stages but succumbed to hemorrhage by early adulthood at 9 weeks of age. Surprisingly, a single allele of either TFa or TFb was able to rescue survival in the context of complete loss of the other gene. To evaluate for hemostatic effects of TF deficiency, laser-mediated endothelial injury was used in the venous and arterial systems at 3 and 5 days post fertilization (dpf), respectively. Loss of TFb alone at 3 dpf resulted in no observable hemostatic defects. Conversely, loss of TFa led to a 50% increase in the time to venous occlusion (TTO), which was exacerbated by concomitant loss of one allele of TFb. Total TF deficiency led to a complete inability to form occlusive venous thrombi, indicating that both TFs can trigger coagulation but TFa is able to completely compensate for the loss of TFb. Concordant with these data, loss of TFb resulted in transcriptional upregulation of TFa but not vice versa. Interestingly, the roles are reversed in the arterial vasculature. Loss of TFa had no effect, loss of TFb lead to a 60% reduction in the number of occlusive thrombi, and complete deficiency resulted in no arterial occlusion. Combined with the venous results, these data point to differentiated roles of TFa and TFb in the venous and arterial systems. In order to test whether these differences were functional, recombinant TF (rTF) molecules were expressed in E. coli, purified, and incorporated into 80% phosphatidylcholine/20% phosphatidylserine liposomes. Ex vivo tube-tilt clotting assays were performed by using each rTF to activate citrated plasma from lake trout. rTFa triggered stable clot formation within 1-2 minutes of recalcification. rTFb usually failed to induce clot formation, with occasional delayed fibrin thrombi that appeared to be grossly disorganized and were easily disrupted following agitation. Taken with the in vivo data, this hints at an altered kinetic profile, with TFa being a more potent cofactor for factor VIIa in low flow (venous) settings. The laboratory is an artificially safe environment, so a synthetic chemical stress test was performed on 3 dpf larvae. Prolonged treatment with cortisol and epinephrine led to the development of cardiac tamponade in larvae with complete TF deficiency (61%), but similar results were only found at low levels in wild type siblings (2-5%). The same assay in prothrombin mutants also revealed a high rate of tamponade (75%), but lower levels in fibrinogen-deficient larvae (20%). These data suggest an extrahemostatic risk factor for tamponade that is modified by prothrombin and tissue factor levels and is independent of fibrin formation. Our results intimate that TFa and TFb have overlapping procoagulant functions but differential kinetic profiles in venous vs arterial systems. We also find that the duplication provides a layer of quantitative regulation and creates a titratable level for regulation of hemostatic and extrahemostatic roles of TF. Overall, this novel model provides new structural and physiologic information about TF function in vivo, including potential previously unknown roles in perivascular development, cardiovascular stability, remodeling and/or regeneration. Disclosures Morrissey: PrevThro Pharmaceuticals: Equity Ownership; Cayuga Pharmaceuticals: Equity Ownership; Kerafast, Inc: Research Funding; Issued and pending patent applications relating to medical uses of polyphosphate and polyphosphate inhibitors: Patents & Royalties. Shavit:Bayer: Consultancy; Sanofi: Consultancy; Shire/Takeda: Consultancy; Spark Therapeutics: Consultancy; CSL-Behring: Consultancy; Novo Nordisk: Consultancy.

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Rimando, Joseph, Michael P. Rettig, Matt Christopher, Julie K. Ritchey, Miriam Y. Kim, John Muth, Jan Davidson, and John F. DiPersio. "Flotetuzumab and Other Cellular Immunotherapies Upregulate MHC Class II Expression on Acute Myeloid Leukemia Cells in Vitro and In Vivo." Blood 136, Supplement 1 (November 5, 2020): 22–23. http://dx.doi.org/10.1182/blood-2020-133891.

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Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative therapy for patients with high-risk and refractory acute myeloid leukemia (AML). Unfortunately, up to 50 percent of patients relapse after allo-HCT.Recent research has shown that 30-50 percent of AML samples from patients relapsing after allo-HCT have downregulation of MHC class II (MHC-II) expression, which may promote immune effector evasion and disease relapse. These studies also report that interferon gamma (IFNγ) can restore MHC-II expression. IFNγ has never been systemically administered after allo-HCT and would likely cause significant and potentially life-threatening toxicities. Reinduction of MHC-II expression may lead to re-engagement of immune effectors, restoration of the graft-versus-malignancy effect, and disease control. We hypothesized that T cell immunotherapies targeting AML cells will lead to T cell activation, localized IFNγ release, and upregulation of MHC-II on AML cells. Methods: For in vitro experiments, THP1 cells (THP1s), which have intermediate MHC-II expression, or primary human AML samples with low MHC-II expression from a patient relapsing after allo-HCT (AML-low cells) were co-cultured with or without T-cell immunotherapy and with or without human MHC-mismatched CD3+ T cells. The following T-cell immunotherapies were tested: flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule; a CD33 x CD3 bispecific molecule (Creative Biolabs, Shirley, NY); and CD123-directed chimeric antigen receptor (CAR) T cells. THP1 IFNγ receptor-1 (IFNγR1) knockout cell lines were generated using CRISPR-Cas9. MHC-II expression was measured by flow cytometry and IFNγ concentrations via Luminex immunoflourescence assay. In order to rescue THP1s from FLZ-induced death and allow for longitudinal evaluation, a transwell plate system was used, placing THP1s, human CD3+ T cells, and FLZ in the top well and THP1s in the bottom well. This allowed for diffusion of IFNγ but not human T cells to the bottom wells, permitting MHC-II upregulation while limiting death. The upper and lower wells were coincubated together for 24 hours prior to harvesting of the THP1s in the lower well for longitudinal studies and mixed-lymphocyte reactions. For in vivo experiments, NOD-scid IL2Rgammanull mice expressing human IL-3, GM-CSF, and SCF (NSG-S) were irradiated with 250 rads and injected with 10e6 primary AML-low cells per mouse. After 5.5 weeks, mice were divided into the following groups: 1) untreated control; 2) FLZ only (2mg/kg); 3) human mismatched T cells only (10e7 T cells per mouse); 4) FLZ and T cells. Results: In vitro co-culture of THP1 or AML-low cells with FLZ and T cells led to significantly increased MHC-II expression at 48 hours when compared with the control, FLZ only, and T cell only groups (Figure 1A-B). Co-culture of THP1s with the CD123 CAR-T cells led to similar results. Although co-incubation with a CD33 x CD3 bispecific led to a similar result, the MHC-II upregulation was not nearly as dramatic as that seen with CD123 targeting agents. Using a transwell system to rescue THP1s from FLZ-mediated toxicity, FLZ-induced MHC-II upregulation on THP1s peaked at 48-72 hours (similar kinetics to what is seen with IFNγ alone). These THP1s with upregulated MHC-II activated third-party donor mismatched human CD4+ T cells to a greater extent than untreated THP1s controls. Co-cultures of THP1s with CD4+ T cells and FLZ induced the secretion of very high concentrations of IFNγ, and blockade of IFNγ signaling through knockout of IFNγR1 led to abrogation of the effect (Figure 1C-D). Finally, in an in vivo model, NSG-S mice injected with AML-low samples and treated with FLZ and T cells showed significant upregulation of MHC-II expression on the AML cells. Single cell RNA-sequencing of AML cells purified from these mice is ongoing. Conclusions: Use of FLZ and other T-cell immunotherapies targeting AML antigens led to both direct AML killing as well as significant upregulation of MHC-II expression on AML cells both in vitro and in vivo. The effect appears to be mediated primarily by IFNγ. T-cell immunotherapies represent a promising treatment approach for AML patients relapsing after allo-HCT and may lead to enhanced immune recognition in the 30-50% of patients who relapse after allo-HCT. Based on these results, a clinical trial treating patients relapsing after allo-HCT with FLZ is planned. Disclosures Christopher: Boulder Bioscience: Patents & Royalties: IP around the use of interferon gamma to treat stem cell transplant. Kim:Tmunity: Patents & Royalties: methods for gene editing in hematopoietic stem cells to enhance the therapeutic efficacy of antigen-specific immunotherapy (Licensed by University of Pennsylvania); Neoimmune Tech: Patents & Royalties: use of long-acting IL-7 analogs to enhance CAR T cells (licensed by Washington University). Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Davidson:MacroGenics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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Hersey, Sarah, Steve Keller, Joel Mathews, Lindsay King, Abbas Bandukwala, Flora Berisha, Mary Birchler, et al. "2021 White Paper on Recent Issues in Bioanalysis: ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry (Part 2 – Recommendations on Biomarkers/CDx Assays Development & Validation, Cytometry Validation & Innovation, Biotherapeutics PK LBA Regulated Bioanalysis, Critical Reagents & Positive Controls Generation)." Bioanalysis, May 17, 2022. http://dx.doi.org/10.4155/bio-2022-0080.

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The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “context of use” [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

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Loo, Lina, Shannon Harris, Mark Milton, Meena, Wibke Lembke, Flora Berisha, Sylvie Bertholet, et al. "2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 – Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization)." Bioanalysis, May 17, 2022. http://dx.doi.org/10.4155/bio-2022-0081.

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The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “Context of Use – COU”); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

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Zoppo, Marina, Nicole Okoniewski, Stanislav Pantelyushin, Johannes vom Berg, and Kristin Schirmer. "A ribonucleoprotein transfection strategy for CRISPR/Cas9‐mediated gene editing and single cell cloning in rainbow trout cells." Cell & Bioscience 11, no. 1 (June 3, 2021). http://dx.doi.org/10.1186/s13578-021-00618-0.

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Abstract Background The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example. Results Ribonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively. Conclusions The designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function.

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Kamali, Elahe, Fatemeh Rahbarizadeh, Zohreh Hojati, and Morten Frödin. "CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells." BMC Biotechnology 21, no. 1 (January 29, 2021). http://dx.doi.org/10.1186/s12896-020-00665-4.

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Abstract Background The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient. Results Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12–14% for the two genes, which translated into 7–8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis. Conclusion Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.

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Petković, Igor, Johannes Bischof, Thomas Kocher, Oliver Patrick March, Bernadette Liemberger, Stefan Hainzl, Dirk Strunk, et al. "COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa." Frontiers in Medicine 9 (August 25, 2022). http://dx.doi.org/10.3389/fmed.2022.976604.

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BackgroundEpidermolysis bullosa (EB), a severe genetic disorder characterized by blister formation in skin, is caused by mutations in genes encoding dermal-epidermal junction proteins that function to hold the skin layers together. CRISPR/Cas9-induced homology-directed repair (HDR) represents a promising tool for editing causal mutations in COL17A1 in the treatment of junctional epidermolysis bullosa (JEB).MethodsIn this study, we treated primary type XVII collagen (C17)-deficient JEB keratinocytes with either Cas9 nuclease or nickase (Cas9n) ribonucleoproteins (RNP) and a single-stranded oligonucleotide (ssODN) HDR template in order to correct a causal pathogenic frameshift mutation within the COL17A1 gene.ResultsAs analyzed by next-generation sequencing of RNP-nucleofected keratinocytes, we observed an HDR efficiency of ∼38% when cells were treated with the high-fidelity Cas9 nuclease, a mutation-specific sgRNA, and an ssODN template. The combined induction of end-joining repair and HDR-mediated pathways resulted in a C17 restoration efficiency of up to 60% as assessed by flow cytometry. Furthermore, corrected JEB keratinocytes showed a significantly increased adhesive strength to laminin-332 and an accurate deposition of C17 along the basement membrane zone (BMZ) upon differentiation into skin equivalents.ConclusionHere we present a gene editing approach capable of reducing end joining-generated repair products while increasing the level of seamless HDR-mediated gene repair outcomes, thereby providing a promising CRISPR/Cas9-based gene editing approach for JEB.

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Chirikian, Orlando, William R. Goodyer, Elda Dzilic, Vahid Serpooshan, Jan W. Buikema, Wesley McKeithan, HaoDi Wu, et al. "CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes." Scientific Reports 11, no. 1 (February 4, 2021). http://dx.doi.org/10.1038/s41598-021-81860-x.

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AbstractGenerating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3′ untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

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Journal articles on the topic 'CRISPR/Cas9, flow cytometry, order of event'

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