Journal: bioRxiv

Article Title: Mutant ASXL1 Drives Transcriptional Activation and Repression in Human Hematopoiesis

doi: 10.64898/2026.01.13.699315

Figure Lengend Snippet: A. Schematic comparing ASXL1 knockout with sgRNAs in exon 2 of the gene to ASXL1 truncation with sgRNAs in exon 12 near the canonical hotspot. B. Edited cells from A were grown in liquid culture under multi-lineage differentiation conditions for 7 days, followed by FACS for CD34 . C. Edited cells were grown in methylcellulose for 14 days and then counted by a blinded observer. Cells were replated and cultured for an additional 14 days and recounted. D . Representative images from C . E. FACS was used to measure engraftment in NSGS mice (hCD45) and myeloid (CD33) vs lymphoid (CD3+CD19) output. F . CD34+ cord blood cells expressing mutant ASXL1 or edited at the AAVS1 locus (as in ) were cultured under multi-lineage differentiation conditions for 4 days, followed by RNA-seq (n=3/group). G . Colony-forming assay after FOS knockout by CRISPR. H . Gene set overrepresentation analysis for mutant ASXL1 up and down gene sets using the transcription factor perturbation expression gene set collection (EnrichR). I . Gene set overrepresentation analysis of mutant ASXL1 up genes using DRUG-seqr. *=p<0.05, **=p<0.01, ***=p<0.001, ****= p<0.0001 as measured by a Student’s T-Test or one-way ANOVA with a Holm-Sidak post-test.

Article Snippet: CD34+ cells were then re-isolated using MACS (Miltenyi Biotec, CD34 MicroBead Kit, human, Cat. #130-046-702) and following the manufacturer’s protocol.

Techniques: Knock-Out, Cell Culture, Expressing, Mutagenesis, RNA Sequencing, CRISPR

Journal: bioRxiv

Article Title: Mutant ASXL1 Drives Transcriptional Activation and Repression in Human Hematopoiesis

doi: 10.64898/2026.01.13.699315

Figure Lengend Snippet: (Related to ) A. Results from ICE analysis of CRISPR indels in cord blood CD34+ after Cas9 RNP electroporation and ASXL1 truncation. Indel score is the % of indels that result in an FS or are >21 bp. B. ASXL1 mutant or AAVS1 -edited cells with or without BAP1 co-deletion were cultured in multi-lineage differentiation media for 7 days, and CD34 abundance was measured using FACS. C. ASXL1 mutant or AAVS1 -edited cells with or without BAP1 knockout were plated in methylcellulose and counted by a blinded observer after 14 days. Cells were replated and counted again after 14 days. D . Representative Images from C. E. ASXL1 mutant or AAVS1 -edited cells were treated win increasing concentrations of the BAP1 inhibitor iBAP and cultured in multi-lineage differentiation media for 7 days. CD34 abundance was measured using FACS. F. ASXL1 mutant or AAVS1 -edited cells were treated with iBAP and plated in methylcellulose and counted by a blinded observer after 14 days. Cells were replated and counted again after 14 days. D . Representative Images from C. *=p<0.05, ***=p<0.001, ****= p<0.0001 as measured by a two-way ANOVA with a Holm-Sidak post-test.

Article Snippet: CD34+ cells were then re-isolated using MACS (Miltenyi Biotec, CD34 MicroBead Kit, human, Cat. #130-046-702) and following the manufacturer’s protocol.

Techniques: CRISPR, Electroporation, Mutagenesis, Cell Culture, Knock-Out

Journal: bioRxiv

Article Title: Mutant ASXL1 Drives Transcriptional Activation and Repression in Human Hematopoiesis

doi: 10.64898/2026.01.13.699315

Figure Lengend Snippet: A. Schematic of HiBIT tag knock-in at C632 or the native stop codon of ASXL1 . B. Protein quantification of HiBiT-tagged endogenous ASXL1 by HiBiT lytic assay in CD34+ HSPCs described in A (***=p<0.001). C. Anti-FLAG western blot of HEK cells transiently transfected with constructs coding for FLAG-tagged WT or mutant ASXL1 treated with 20 μg/mL cycloheximide. D. HiBiT-tagged transiently transfected ASXL1 by HiBiT lytic assay in HEK cells. Luminescent signal normalized to backbone abundance using qPCR, controlling for minor differences in transfection efficiency. E. Change in protein levels in HEK cells transiently transfected with HiBiT-tagged ASXL1 by HiBiT lytic assay after treatment with 20 μg/mL cycloheximide (n=3 independent transfections). Signal is normalized to the zero-hour time point to demonstrate relative changes. F. Change in protein levels in HEK cells transiently transfected with HiBiT-tagged ASXL1 by HiBiT lytic assay after treatment with 5 μM/mL MG132 (n=3 independent transfections). Signal is normalized to the zero-hour time point to demonstrate relative changes. ***=p<0.001 as measured by a one-way ANOVA with a Holm-Sidak post-test.

Article Snippet: CD34+ cells were then re-isolated using MACS (Miltenyi Biotec, CD34 MicroBead Kit, human, Cat. #130-046-702) and following the manufacturer’s protocol.

Techniques: Knock-In, Western Blot, Transfection, Construct, Mutagenesis

Journal: bioRxiv

Article Title: Mutant ASXL1 Drives Transcriptional Activation and Repression in Human Hematopoiesis

doi: 10.64898/2026.01.13.699315

Figure Lengend Snippet: A. Quantification of the impact of target gene knockout on CD34 abundance by first calculating the log2-normalized fold change in CD34 abundance due to knockout of the target gene, then calculating the difference in the log2FC between the AAVS1 and ASXL1-mut cells. B. CD34 abundance relative to AAVS1 for top screen hits. C. CUT&Tag for RPB1 (large subunit of RNAPII) in AAVS1 vs ASXL1 -mut CD34+ cells. D. Signal tracks for total and Serine 2 phosphorylated RPB1 in AAVS1 vs ASXL1 -mut CD34+ cells. E. CD34 abundance by flow cytometry after 7 days of drug treatment in differentiation media (*=p<0.05, ****= p<0.0001). F. Colony formation assay with JQ1 treatment. G. Representative images from F. *=p<0.05, **=p<0.01, ****= p<0.0001 as measured by two-way ANOVA with a Holm-Sidak post-test.

Article Snippet: CD34+ cells were then re-isolated using MACS (Miltenyi Biotec, CD34 MicroBead Kit, human, Cat. #130-046-702) and following the manufacturer’s protocol.

Techniques: Gene Knockout, Knock-Out, Flow Cytometry, Colony Assay

Journal: bioRxiv

Article Title: Mutant ASXL1 Drives Transcriptional Activation and Repression in Human Hematopoiesis

doi: 10.64898/2026.01.13.699315

Figure Lengend Snippet: A. scATAC-seq was performed on CD34+ cord blood cells after 3 days of culture in differentiation medium and was mapped to a healthy bone marrow reference dataset. B. Footprint depth scores for TF footprints at MECOM binding sites (from ChIP-seq) that overlap with ASXL1 binding sites (ASXL1-bound, n=1584 sites) in HSCs. Mutant-specific footprints were identified as having a depth score <0 in ASXL1 -Mut cells and >1 in ASXL1 -WT cells. C. Single locus ASXL1 -mutant specific MECOM footprints are shown in ASXL1 -mutant and ASXL1 wild-type cells (n=249 sites) defined based on differential footprint depth score. D. GO analysis was performed on MECOM footprints specific to the ASXL1 -mutant condition using GREAT. E. ASXL1 mutant or AAVS1 -edited cells with or without MECOM co-deletion were cultured in multi-lineage differentiation media for 7 days, and CD34 abundance was measured using FACS. F. ASXL1 mutant or AAVS1 -edited cells with or without MECOM knockout were plated in methylcellulose and counted by a blinded observer after 14 days. No difference in growth between conditions was observed after the initial plating, as in . Cells were replated and counted again after 14 days. G . Representative Images from F. *=p<0.05, ***=p<0.001, ****= p<0.0001 as measured by a two-way ANOVA with a Holm-Sidak post-test.

Article Snippet: CD34+ cells were then re-isolated using MACS (Miltenyi Biotec, CD34 MicroBead Kit, human, Cat. #130-046-702) and following the manufacturer’s protocol.

Techniques: Binding Assay, ChIP-sequencing, Mutagenesis, Cell Culture, Knock-Out

Journal: bioRxiv

Article Title: Mutant ASXL1 Drives Transcriptional Activation and Repression in Human Hematopoiesis

doi: 10.64898/2026.01.13.699315

Figure Lengend Snippet: A. Overview of the Priori regulon analysis algorithm, which leverages interactions from the Pathway Commons Database to predict transcription factor activity using expression data. B. Differential TF activity score analysis in ASXL1 -mutant (n=63) and wild-type AML (n=567) using the Beat AML Cohort. C. Distribution of MECOM activity scores in ASXL1 -mutant and ASXL1 -WT AML samples. None of the ASXL1 -mutant samples had rearrangements of the MECOM locus. MECOM mRNA counts showed a similar distribution. D. Differential drug sensitivity analysis between ASXL1 -mutant and wild-type AML using the Beat AML cohort. E. Reference healthy bone marrow scATAC-seq UMAP with cell type annotations. F. Cell type proportions in ASXL1 -mutant and ASXL1 -wild type cases (n=2/group). G. ASXL1 -mutant and wild-type AML cells projected onto the reference UMAP. H. Peak annotation for differentially accessible regions in ASXL1 mutant and wild-type cells in all stem and progenitor cells (HSC, CMP-LMPP, GMP, GMP-Neu). I. MECOM motif enrichment in differentially accessible peaks from H.

Article Snippet: CD34+ cells were then re-isolated using MACS (Miltenyi Biotec, CD34 MicroBead Kit, human, Cat. #130-046-702) and following the manufacturer’s protocol.

Techniques: Activity Assay, Expressing, Mutagenesis