![SFPQ-ABL1 is sufficient to transform IL-3–dependent cell lines but is a relatively weaker driver of proliferation compared with BCR-ABL1. (A) Schematic of SFPQ-ABL1 fusion transcript and resultant chimeric protein. (B) Schematic of BCR-ABL1 fusion transcript, identified in a Ph+ patient from our RNA-seq cohort and resultant chimeric protein. (C) Sanger sequencing result of SFPQ-ABL1 breakpoint polymerase chain reaction product amplified from patient complementary DNA, confirming fusion of exon 9 of SFPQ to exon 4 of ABL1. (D) western blot analysis of Abl and Phospho-CrkL (P-CrkL) (Y207) expression after 4-hour IL-3 withdrawal in BCR-ABL1– and SFPQ-ABL1–expressing Ba/F3 cells (representative western blot image is shown, n = 3). (E) Quantification of western blot analysis (shown in panel D) of P-CrkL expression relative to ABL1 fusion expression. Protein intensities were normalized to actin loading control. Data are presented as mean ± standard error of the mean (SEM) (n = 3). (F) Viability analysis of BCR-ABL1– and SFPQ-ABL1–expressing Ba/F3 cells after 48-hour IL-3 withdrawal. Viability was determined by propidium iodide (PI) exclusion, measured by flow cytometry. Data are presented as mean ± SEM (n = 6). (G) Proliferation of BCR-ABL1– and SFPQ-ABL1–expressing Ba/F3 cells after 48-hour IL-3 withdrawal. Proliferation was measured by luminescence relative to MSCV empty vector control at 24 hours (Day 1) using the CellTiter-Glo 2.0 reagent (Promega, Madison, WI). Groups were compared by using unpaired Student t tests with Holm-Šídák correction for multiple comparisons (error bars show mean ± SEM, n = 6). ***P < .001, ****P < .0001. (H) Viability analysis of BCR-ABL1– and SFPQ-ABL1–expressing Ba/F3 cells treated with a dose titration of imatinib, dasatinib, ponatinib, or ruxolitinib. Data are normalized to vehicle control (0.001% dimethyl sulfoxide [DMSO]; not shown on graphs) and nonlinear regression analysis was performed to fit dose–response curves. Data are presented as mean ± SEM (n = 3 for imatinib and ruxolitinib treatments, n = 8 for dasatinib and ponatinib).](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/6/7/10.1182_bloodadvances.2021006076/5/advancesadv2021006076f1a.png?Expires=1724940686&Signature=yKcyqIJjpCUB8plL2jrmuDb23rDiJfAlULjorSwhQCH6PAsw26I5mYzO-UtSlIEmhHfW7JiK~4a9LoneoszZbK1mx5BLqRKWQAtTSXbIK1dmNK5xCYJQJcMC3lsnmzSJ8o-TEygvEcSkuhxHBPvdyOQ8e-ticQPWtNRwKjcPW1U691Y-5u~f40VoX0MrgA1jtCMkvulHN93X5LgVn6zK01qlQBBZDrtqhNAg4mwc2ryU-1Y4WHAEflMzLWT9~NBG3v7GDhzFvjUY8gpE6HMDq49u27jn5MpPH33~VB-h0gn3VHdFkbLSEDoA-oI3ydW8JSOg0RfBWkoB3hQ4-Ew04g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
SFPQ-ABL1 is sufficient to transform IL-3–dependent cell lines but is a relatively weaker driver of proliferation compared with BCR-ABL1. (A) Schematic of SFPQ-ABL1 fusion transcript and resultant chimeric protein. (B) Schematic of BCR-ABL1 fusion transcript, identified in a Ph+ patient from our RNA-seq cohort and resultant chimeric protein. (C) Sanger sequencing result of SFPQ-ABL1 breakpoint polymerase chain reaction product amplified from patient complementary DNA, confirming fusion of exon 9 of SFPQ to exon 4 of ABL1. (D) western blot analysis of Abl and Phospho-CrkL (P-CrkL) (Y207) expression after 4-hour IL-3 withdrawal in BCR-ABL1– and SFPQ-ABL1–expressing Ba/F3 cells (representative western blot image is shown, n = 3). (E) Quantification of western blot analysis (shown in panel D) of P-CrkL expression relative to ABL1 fusion expression. Protein intensities were normalized to actin loading control. Data are presented as mean ± standard error of the mean (SEM) (n = 3). (F) Viability analysis of BCR-ABL1– and SFPQ-ABL1–expressing Ba/F3 cells after 48-hour IL-3 withdrawal. Viability was determined by propidium iodide (PI) exclusion, measured by flow cytometry. Data are presented as mean ± SEM (n = 6). (G) Proliferation of BCR-ABL1– and SFPQ-ABL1–expressing Ba/F3 cells after 48-hour IL-3 withdrawal. Proliferation was measured by luminescence relative to MSCV empty vector control at 24 hours (Day 1) using the CellTiter-Glo 2.0 reagent (Promega, Madison, WI). Groups were compared by using unpaired Student t tests with Holm-Šídák correction for multiple comparisons (error bars show mean ± SEM, n = 6). ***P < .001, ****P < .0001. (H) Viability analysis of BCR-ABL1– and SFPQ-ABL1–expressing Ba/F3 cells treated with a dose titration of imatinib, dasatinib, ponatinib, or ruxolitinib. Data are normalized to vehicle control (0.001% dimethyl sulfoxide [DMSO]; not shown on graphs) and nonlinear regression analysis was performed to fit dose–response curves. Data are presented as mean ± SEM (n = 3 for imatinib and ruxolitinib treatments, n = 8 for dasatinib and ponatinib).