Journal: bioRxiv

Article Title: Cholesterol Biosynthesis is a Targetable Vulnerability of CEBPA -mutant Acute Myeloid Leukemia

doi: 10.64898/2026.02.20.706425

Figure Lengend Snippet: (A) Experimental setup of expansion and electroporation of bone marrow (BM)-derived CD34 + hematopoietic stem and progenitor cells (HSPCs). HSPCs were electroporated with Cas9 ribonucleoproteins (RNPs) and studied in vitro in colony-forming-cell (CFC) assays and co-cultures on a layer of mouse stromal-5 (MS5) cells. (B) Insertion/deletion (indel) frequency 24 hour (hr) after electroporation of individual BM samples. sg; sgRNA. (C) Western blot of CEBPA (96hr), TET2 (24hr), GATA2 (24hr), and WT1 (24hr) after electroporation. Vinculin was used as loading control. Indel frequency of the relevant gene is indicated below each lane. (D) Colony count of BM-derived CD34 + HSPCs after plating (round 1), replating (round 2), and replating twice (round 3). From hereon: A; AAVS1, p30; CEBPA-p30, G; GATA2 knockdown (KD), T; TET2 knockout (KO), and W; WT1 KO. (E) Indel frequency of CFCs shown in panel D. Indel frequency was determined 24hr after electroporation (t=0) and after each round of colony analysis. In case of double-mutants (dashed lines), the gene between brackets is shown. (F) Cumulative cell counts of edited HSPCs grown on a stromal layer of MS5 cells. Arrows indicate when cells were replated onto a fresh layer of MS5 cells. (G) Percentage of CD34 + cells in the supernatant harvested at day 7 (left) and day 15 (right). (H-I) Percentage of myeloblasts/myelocytes (H) and segmented neutrophils (I) in the supernatant harvested at day 15. (J-K) Cumulative cell counts of two independent HSPC co-cultures. Supernatant and adherent cells of A, C, T, and C+T (J) or C+T and C+W (K) cultures were harvested after 4 weeks or 13 weeks respectively and transplanted intravenously (i.v.) into NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mice. Bar plots represent mean ± SD. Error bars in growth curves represents mean ±SD of technical triplicates. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following primary antibodies were used: CEBPA (#2295, Cell Signaling), TET2 (#A304-247A, Bethyl Laboratories), GATA2 (#4595, Cell Signaling), WT1 (CAN-R9(IHC)-56-2, Abcam), and Vinculin (#13901, Cell Signaling).

Techniques: Electroporation, Derivative Assay, In Vitro, Western Blot, Control, Knockdown, Knock-Out

Journal: bioRxiv

Article Title: Cholesterol Biosynthesis is a Targetable Vulnerability of CEBPA -mutant Acute Myeloid Leukemia

doi: 10.64898/2026.02.20.706425

Figure Lengend Snippet: (A) Schematic showing sgRNA binding sites in each condition ( , ). bZIP; Basic Leucine Zipper, NHEJ; non-homologous enjoining, TAD; transactivation domain. (B) CEBPA-p42 and CEBPA-p30 expression in U937 cells 4 hours (hr), 24 hr, and 96 hr after electroporation. Vinculin was used as loading control. Insertion/deletion (indel) frequency of the relevant gene are indicated below each lane. (C) Indel frequency of single-mutant (left) and double-mutant (right) bone marrow (BM) hematopoietic stem/progenitor cells (HSPCs) co-cultured on mouse stromal-5 (MS5) cells as shown in . In case of double-mutants, the gene between brackets is shown. From here on: A; AAVS1, p30; CEBPA-p30, G; GATA2 knockdown (KD), T; TET2 knockout (KO), and W; WT1 KO. (D) Cumulative cell counts of edited HSPCs grown on a stromal layer of MS5 cells at days 7, 21, and 48. Lines indicate mean of technical triplicates. (E) Representative gating strategy for neutrophilic differentiation in supernatant cells harvested at day 15 of co-culture. (F) Cumulative cell counts of supernatant cells harvested at day 69 from co-cultures shown in . A portion of these cells were subsequently grown in liquid culture. Error bars represent mean ±SD of technical triplicates. (G-H) Representative gating strategy (G) and percentage of myeloblasts/myelocytes (left) and segmented neutrophils (right) (H) in W+p30 cells harvested from co-cultures (day 83) and liquid cultures (day 14). Bar plots represent mean ± SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following primary antibodies were used: CEBPA (#2295, Cell Signaling), TET2 (#A304-247A, Bethyl Laboratories), GATA2 (#4595, Cell Signaling), WT1 (CAN-R9(IHC)-56-2, Abcam), and Vinculin (#13901, Cell Signaling).

Techniques: Binding Assay, Expressing, Electroporation, Control, Mutagenesis, Cell Culture, Knockdown, Knock-Out, Co-Culture Assay

Journal: bioRxiv

Article Title: Cholesterol Biosynthesis is a Targetable Vulnerability of CEBPA -mutant Acute Myeloid Leukemia

doi: 10.64898/2026.02.20.706425

Figure Lengend Snippet: (A) Representative gating strategy used to sort BM-derived hCD45+CD3-CD19-CD34+ HSPCs (scRNA-seq and proteomics) and hCD45+CD3-CD19-cells (scRNA-seq) from NSGS mice for each experimental condition. FMO; fluorescence minus one. (B) Indel frequency within BM-derived hCD45+CD34+CD3-CD19-HSPCs from NSGS mice. GATA2-mutant samples indicated by a red dot were excluded from analysis due to low GATA2 editing efficiency.

Article Snippet: The following primary antibodies were used: CEBPA (#2295, Cell Signaling), TET2 (#A304-247A, Bethyl Laboratories), GATA2 (#4595, Cell Signaling), WT1 (CAN-R9(IHC)-56-2, Abcam), and Vinculin (#13901, Cell Signaling).

Techniques: Derivative Assay, Fluorescence, Mutagenesis