Fig 2: Optimisation of lipid nanoparticle mediated RUNX1/ETO knockdown.Measurement of LNPs/siRNAs diameter (a) and polydispersity (b) was performed after 1:100 dilution in PBS, each dot represents independent LNPs formulation. c, d Kasumi-1 cells were treated for 30 min with endocytosis inhibitors, then washed with PBS, resuspended in fresh medium, and exposed to Dil-labeled LNPs. The Dil fluorescence was measured by flow cytometry (c) 60 min and 24 h post-LNPs treatment (n = 4), and by fluorescence microscopy (d), the LNPs appear in red in the cytoplasm surrounding the DAPI-positive nucleus. e quantification of RUNX1/ETO expression in Kasumi-1 on day 3 relative to GAPDH (n = 1). Cells were treated with either 0.2 or 2 µg/ml LNPs/siRNAs for either 15 min, 60 min, 4 h or 24 h then cells were washed thrice with PBS to remove access LNPs. f gene enrichment analysis of Kasumi-1 and SKNO-1 following RUNX1/ETO knockdown by siRNA-LNPs treatment or electroporation (n = 3). Cells were either treated with 2 µg/ml siRNA-LNPs for 24 h then washed thrice with PBS and cultivated for further 2 days, or cells were electroporated with 200 nM siRNA. RNA-seq was performed on day 3.

Fig 3: A chemically modified siRNA provides prolonged activity.a The t(8;21) fusion transcript RUNX1/ETO has a unique breakpoint targeted with siRE spans the fusion site, swapping two nucleotides in siRE generates a mismatch control siMM. b Chemically modified siRNAs (siRE-mod, siMM-mod) are generated by the introduction of 2’-deoxy- (2’-H), 2’-fluoro (2’-F) and 2’-methoxy (2’-Ome) ribose modifications and 3’-terminal phosphorothioate (PS). c Western blotting of RUNX1-ETO, RUNX1 and GAPDH in Kasumi-1 following RUNX1/ETO knockdown using either siRE (top) and siRE-mod (bottom). Cells were electroporated once on day 0 and cell lysates collected after 3 and 7 days. d–f Kasumi-1 cells were electroporated sequentially on days 0 and 3 with either 200 nM siMM, 200 nM siRE, 100 nM siRE-mod or no oligos (mock), d Proliferation curve of Kasumi-1 cells following RUNX1/ETO knockdown (n = 4). ln(cell number), natural logarithm of the cell number; t(d), time in days. e Western blotting showing RUNX1/ETO, RUNX1, p-RB1 T821, RB1 and GAPDH in Kasumi-1 cells on days 6, f Senescence-associated β-galactosidase (SAβGal) staining (n = 3). g Semi-solid colony formation units of Kasumi-1 cells following RUNX1/ETO knockdown, cells were seeded on day 1 following the first electroporation and colonies were counted on day 8 and replated (n = 3). h RUNX1/ETO expression level in t(8;21)-AMLs blast 3 days after electroporation with 200 nM siMM, 200 nM siRE or 100 nM siRE-mod. Significance was tested by unpaired Student’s t tests (d, f, g).

Fig 4: (A) Volcano plot showing relationship between magnitude of gene expression change (log2 of fold-change; y-axis) and statistical significance of this change [-log10 of adjusted p value; x-axis] in a comparison of tumors and adjacent non-tumor tissues in GSE71989 (left) and GSE28735 (right) cohorts. Red points represent differentially expressed genes (with cut-off FDR < 0.05) with magnitude of change ≥2. (B) Gene expression of RUNX1 in two independent cohorts of PDAC. RUNX1 is increased in tumors compared with non-tumor tissues in GSE71989 (left) and GSE28735 (right) cohorts. Box plots represent gene expression level with relative intensity (log2) of microarray data. Bars indicate median value. Student t test. (C) Average relative RUNX1 expression level in PDAC compared with that in normal tissues. Expression of RUNX1 was measured by qRT-PCR and normalized by GADPH. (D) Immunohistochemical staining for RUNX1 protein in normal pancreas and PDAC tissues, brown granule in nucleus showed low or high expressed RUNX1 with lower magnification images (5×, left) and its expanded views (20×, right). (E) Protein expression in 3 paired PDAC and the adjacent normal pancrea tissues. RUNX1 protein expression level was determined by immunoblotting with GADPH as a control. (F) Kaplan-Meier curves for OS in PDAC patients with RUNX1 expession. Gene expression of RUNX1 is divided into high and low expression groups using a median cutoff. Log-rank P value is indicated in the graphs.

Fig 5: RUNX1 directly transactivates EGFR expression(A and B) Tsc2−/− MEFs (A) or Tsc1−/− MEFs (B) were transfected with siRNAs targeting RUNX1 or the control siRNAs (siNC) for 48 h. (C and D) Tsc2−/− MEFs (C) or Tsc1−/− MEFs (D) were treated with or without Ro5-3335 for 24 h. (E) Tsc2+/+ MEFs were transduced with Lv-RUNX1 or Lv lentiviruses. In (A) to (E), the mRNA level of EGFR of the indicated cells was analyzed by qRT-PCR. (F) Schematic representation of the putative RUNX1-binding site in the promoter of mouse EGFR gene. (G and H) HEK 293T cells (G) or Tsc2+/+ MEFs (H) were co-transfected with pEGFR-Luc or pEGFRmut-Luc plus pcDNA 3.1-RUNX1 or control pcDNA 3.1 and the internal control plasmid pRL-TK. Relative luciferase activity was detected 24 h after transfection. (I) Tsc2+/+, Tsc2−/−, and rapamycin-treated Tsc2−/− MEFs were subjected to a ChIP analysis of RUNX1-binding site in EGFR promoter region using an anti-RUNX1 antibody. Normal rabbit IgG antibody served as negative control. qRT-PCR was performed to amplify regions surrounding the putative RUNX1-binding region (PBR) and a nonspecific RUNX1-binding region (NBR). The data were plotted as the ratio of immunoprecipitated DNA subtracting nonspecific binding to IgG versus total input DNA (%). Representative data from three independent experiments are shown. Data indicate mean ± SD of triplicate samples. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.