![Validation of MSI2 as a putative therapeutic target in patient-derived T-ALL xenograft models and ICN-1-induced murine T-ALL models. (A) MSI2 RNA immunoprecipitation and MYC quantitative PCR of reverse-transcribed cDNA from 5 T-ALL patient-derived xenograft (PDX) samples. Experiments were performed in triplicate. Data are presented as mean values ± SEM. (B) Relative MYC mRNA expression levels normalized to GAPDH as control for the 5 T-ALL PDX samples from panel A. (C) Western blot assay validation of MYC and MSI2 expression in the 5 T-ALL PDX samples from panels A-B. Experiments were performed in triplicate. Representative chemiluminescence images are shown. (D) MSI2 RNA immunoprecipitation and MYC qPCR of reverse-transcribed cDNA after treatment with MSI2 RNA binding inhibitor Ro 08-2750 (10 μM or 20 μM) for 12 hours for T-ALL primary cells from PDX T410 (mentioned in panels A-C) compared with the no-treatment vehicle subgroup. Data are presented as mean values ± SEM. P values were estimated using the 2-tailed Student t test; ∗P < .05 and ∗∗P < .01. (E) Evaluation of apoptosis in T-ALL primary cells from PDX T410 (mentioned in panels A-C) after treatment with Ro 08-2750 (2.5 μM, 5 μM, or 10 μM) for 48 hours compared with the no-treatment vehicle subgroup by annexin V and DAPI staining. Experiments were performed in triplicate. Data are presented as mean values ± SEM. Representative scatterplots are shown. (F) Comparison of measurable residual disease (MRD) levels at end of induction between patients with higher MSI2 mRNA expression (MSI2high, fragments per kilobase of transcript per million mapped reads [FPKM] of ≥20.5) and those with lower MSI2 mRNA levels (MSI2low, FPKM of <20.5) from 2 independent T-ALL cohorts, the TARGET-TALL cohort and the CCCG2015-TALL cohort. P values were estimated using the 2-sided Mann-Whitney U test; ∗P < .05. (G) Schematic for establishment of ICN-1–induced murine T-ALL model (C57BL/6) by intravenous injection of 2 × 106 cells per mouse into lethally irradiated mice, then transplanting GFP+ T-ALL cells into the second generation once blast percentage reached 80% in the peripheral blood. For in vivo efficacy observation, treatment was initiated 4 days after injection. Two groups were assigned vehicle or Ro 08-2750 10 mg/kg by intraperitoneal injection (IP) on days 4, 7, 10, 13, and 16. For biological experiments, 3 in vivo treatment groups, vehicle, 10 mg/kg per day Ro 08-2750 IP (1 dose), and 10 mg/kg per day Ro 08-2750 IP for 2 days, were prepared after the GFP+ T-ALL blast percentage reached 30% to 40% in the peripheral blood. MSI2 RIP-qPCR for Myc was performed on sorted BM GFP+ T-ALL cells. (H) Leukemia-free survival estimated for Ro 08-2750 treatment (red) or vehicle (blue) subgroups of ICN-1–induced T-ALL model. P values were estimated using the Cox regression model; the exact P value is provided in the figure. Each treatment group included 7 mice. (I) MSI2 RNA immunoprecipitation and Myc qPCR of reverse-transcribed cDNA from GFP+ T-ALL cells after treatment with Ro 08-2750 (10 mg/kg) for 24 and 48 hours for murine ICN-1 T-ALL model in vivo. Data are presented as mean values ± SEM. P values were estimated using the 2-tailed Student t test; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/143/4/10.1182_blood.2023020490/1/blood_bld-2023-020490-gr6.jpeg?Expires=1744615828&Signature=Vfy73~mbHpppo-r2GLnNOxwEyPfxIWkil4AW-AVulNKko24diaVlCzQkdLqEHVoTfejlnil8e7A2oxXdYy5R422iGQdQxnBYKenKON1r1wHPjyAXup06lRlsdkTQNnkBJCQ7uKB1ZFT29B90fz~SLgRz~pzB0zO8-loneXtLgwwcDMsi4CYdQimSNJ2LF95jIwtxDauuUh7a3nRiYwdnzuHDkvLsNz5W1CeDw61v3Ej4DN5ROYeE4P~znP1FrVwj1AdiMGJ9ZXrkdF0qgl4tuamXcE7TF9tWmFSDB~lK~rGRbq3B8dxGywtM-jTVYBTyNtnLze0elr5SGNKUZniX0Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Validation of MSI2 as a putative therapeutic target in patient-derived T-ALL xenograft models and ICN-1-induced murine T-ALL models. (A) MSI2 RNA immunoprecipitation and MYC quantitative PCR of reverse-transcribed cDNA from 5 T-ALL patient-derived xenograft (PDX) samples. Experiments were performed in triplicate. Data are presented as mean values ± SEM. (B) Relative MYC mRNA expression levels normalized to GAPDH as control for the 5 T-ALL PDX samples from panel A. (C) Western blot assay validation of MYC and MSI2 expression in the 5 T-ALL PDX samples from panels A-B. Experiments were performed in triplicate. Representative chemiluminescence images are shown. (D) MSI2 RNA immunoprecipitation and MYC qPCR of reverse-transcribed cDNA after treatment with MSI2 RNA binding inhibitor Ro 08-2750 (10 μM or 20 μM) for 12 hours for T-ALL primary cells from PDX T410 (mentioned in panels A-C) compared with the no-treatment vehicle subgroup. Data are presented as mean values ± SEM. P values were estimated using the 2-tailed Student t test; ∗P < .05 and ∗∗P < .01. (E) Evaluation of apoptosis in T-ALL primary cells from PDX T410 (mentioned in panels A-C) after treatment with Ro 08-2750 (2.5 μM, 5 μM, or 10 μM) for 48 hours compared with the no-treatment vehicle subgroup by annexin V and DAPI staining. Experiments were performed in triplicate. Data are presented as mean values ± SEM. Representative scatterplots are shown. (F) Comparison of measurable residual disease (MRD) levels at end of induction between patients with higher MSI2 mRNA expression (MSI2high, fragments per kilobase of transcript per million mapped reads [FPKM] of ≥20.5) and those with lower MSI2 mRNA levels (MSI2low, FPKM of <20.5) from 2 independent T-ALL cohorts, the TARGET-TALL cohort and the CCCG2015-TALL cohort. P values were estimated using the 2-sided Mann-Whitney U test; ∗P < .05. (G) Schematic for establishment of ICN-1–induced murine T-ALL model (C57BL/6) by intravenous injection of 2 × 106 cells per mouse into lethally irradiated mice, then transplanting GFP+ T-ALL cells into the second generation once blast percentage reached 80% in the peripheral blood. For in vivo efficacy observation, treatment was initiated 4 days after injection. Two groups were assigned vehicle or Ro 08-2750 10 mg/kg by intraperitoneal injection (IP) on days 4, 7, 10, 13, and 16. For biological experiments, 3 in vivo treatment groups, vehicle, 10 mg/kg per day Ro 08-2750 IP (1 dose), and 10 mg/kg per day Ro 08-2750 IP for 2 days, were prepared after the GFP+ T-ALL blast percentage reached 30% to 40% in the peripheral blood. MSI2 RIP-qPCR for Myc was performed on sorted BM GFP+ T-ALL cells. (H) Leukemia-free survival estimated for Ro 08-2750 treatment (red) or vehicle (blue) subgroups of ICN-1–induced T-ALL model. P values were estimated using the Cox regression model; the exact P value is provided in the figure. Each treatment group included 7 mice. (I) MSI2 RNA immunoprecipitation and Myc qPCR of reverse-transcribed cDNA from GFP+ T-ALL cells after treatment with Ro 08-2750 (10 mg/kg) for 24 and 48 hours for murine ICN-1 T-ALL model in vivo. Data are presented as mean values ± SEM. P values were estimated using the 2-tailed Student t test; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.
