XIAP represents a promising therapeutic target for relapsed/refractory ALL (2022)
Figure 4

Figure 4: Silencing XIAP sensitizes ALL cells towards Vincristine in vivo

A Schematic representation of lentiviral vectors. Control (CTRL) and XIAP-targeting shRNAs were expressed in the 3´untranslated region of dsRED in the background of a miR30 cassette under control of the Pol II promoter. mTagBFP and eGFP were used to mark each cell population; the control shRNA expressing plasmid was transduced into mTagBFP+, the XIAP KD plasmid into eGFP+ cells.

B shRNA expression control. Equal expression levels of ds-RED in the sh-CTRL/mTagBFP and sh-XIAP/eGFP subpopulation indicates identical expression levels of control and XIAP shRNA. One representative histogram out of 3 independent experiments is shown.

C Expression of IAP. Western blot analysis of XIAP in NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP under the SFFV promoter; GAPDH was used as loading control. One representative immunoblot out of 3 independent experiments is shown.

D Western blot analysis of NALM-6 cells with moderate (69%, EF1α promoter) or strong (92%, SFFV promoter) KD of XIAP; GAPDH was used as loading control. The XIAP KD levels (% KD) were quantified relative to sh-CTRL. One representative immunoblot out of 3 independent experiments is shown.

E Schematic representation of competitive in vivo assays. Cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into mice (+/- in vivo treatment); at indicated time points, mice were sacrificed, cells isolated from bone marrow and analyzed.

F Moderate knockdown of XIAP sensitizes towards chemotherapy. NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice. 4 days after injection, mice were either treated with PBS (n=4), or with Vincristin (VCR, n=8), 0.3mg/kg i.v. once a week for 3 consecutive weeks. 7 days after last treatment, mice were sacrificed, and relative proportion of sh-CTRL and sh-XIAP subpopulations re-isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** p ≤0.0001 by unpaired Student's t-test with Welch's correction.

G Strong knockdown of XIAP impairs NALM-6 growth in vivo. NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice (n=8). 38 or 46 days after injection, mice were sacrificed, and relative proportion of sh-CTRL and sh-XIAP subpopulations re-isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** p ≤0.0001 by unpaired Student's t-test.

H PARP cleavage in NALM-6 cells with strong XIAP inhibition. NALM-6 cells were lentivirally transduces with sh-CTRL or sh-XIAP (SFFV). 4 days later, cells were lysed and relative cleavage level of PARP (%) was analyzed by protein immunoassay (Simple Western). Mean ± SD of 3 independent experiments is shown; ** p <0.01 by unpaired Student's t-test.

I XIAP reconstitution in NALM-6 cells. NALM-6 cells were transduced with shCTRL, shXIAP.1 alone or shXIAP.1 together with the XIAP overexpression vector. At day 0, transduced cells were mixed in a 1:1 ratio with parental (untransduced) NALM-6 cells. Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time. Mean ± SD of 3 independent experiments is shown; ** p <0.01 by unpaired Student's t-test.

Source data are available online.