Introduction of the T315I gatekeeper mutation of BCR/ABL1 into a Philadelphia chromosome-positive lymphoid leukemia cell line using the CRISPR/Cas9 system

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome‐positive acute lymphoblastic leukemia through upregulation of integrin α6

“…SU/SR acquired the imatinib‐resistant phenotype as a result of T315I mutation (Figure 3B). In the KOPN55bi and KOPN55biR pair, 28 KOPN55biR with T315I mutation was established from KOPN55bi by homologous recombination using the CRISPR/Cas9 system (Figure 3B). When treated with imatinib, consistent with their phosphorylation status of BCR::ABL1 protein (Figure S11), the cell surface expression levels of CD49f were downregulated in the parental SU‐Ph2 and KOPN55bi but not in the T315I‐acquired SU/SR and KOPN55biR (Figure 3C; Figure S12A,B).…”

“…Following the instructions provided by the respective manufacturer, each cell line was preincubated with 25 μg/mL of anti‐CD49f (#313614, BioLegend), 30 12.5 μg/mL of anti‐CD29 (#921304, BioLegend), 31 or 10 μg/mL of anti‐CD104 (#325‐820, Ancell) neutralizing antibodies or isotype control rat IgG2α,κ (#400502, BioLegend) (CD49f and CD29 blocking) or mouse IgG (#6602398, Beckman Coulter) (CD104 blocking) for 30 min. To verify the effect of imatinib on laminin adhesion, parental KOPN55bi cells or T315I‐acquired KOPN55biR cells 28 were preincubated with 0.5 μM of imatinib for 7 days. After washing with PBS, the cells were resuspended in the medium at 0.5 × 10 6 /mL, and 100 μL of the cells was plated into each well of the laminin‐coated 96‐well plate in triplicate and allowed to attach to laminin for 3 h. After 3‐hour incubation of the cells on the plate with or without laminin coating, the plate was mechanically shaken using a plate mixer (Mikura Orbis Personal Single Plate Shaker, Mikura, West Sussex, UK) at 1300 rpm for 15 s. Then, the numbers of floating cells in the supernatant were evaluated after Trypan blue exclusion staining under an optical microscope.…”

“…As listed in Table 1, KOPN55biR with the T315I mutation was established from KOPN55bi by homologous recombination using the CRISPR/Cas9 system. 28 All cell lines were maintained in RPMI1640 medium supplemented with 10% fetal calf serum in a humidified atmosphere of 5% CO 2 at 37 C.…”

“…Two pairs of imatinib‐sensitive parental cell lines (SU‐Ph2 and KOPN55bi) and their T315I‐acquired sublines (SU/SR and KOPN55biR) were cultured in the presence or absence (control) of 0.5 μM of imatinib for 12 h. Each cell line was also cultured in the presence of 0.01% of DMSO for 12 h as Mock. Immunoblotting was performed with the anti‐ABL (BD Pharmingen #554148, 1:1000 dilution), anti‐Phsoph‐Tyr 204‐cABL (Cell Signaling #C4285, 1:1000 dilution), and anti‐α‐Tubulin (Sigma #T‐5168, 1:1000 dilution) antibodies as previously reported 28 …”

Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line

“…The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is useful to induce specific mutations into the intrinsic gene by homologous directed repair (HDR). 12 , 13 However, there are several limitations: Cas9 enzyme generates harmful DNA double‐strand breaks (DSBs), which might induce cell death if repaired improperly. Moreover, in the cellular processes of DSB repair, the nonhomologous end joining (NHEJ) pathway, which results in unexpected insertion/deletion (indel), is dominant.…”