Extended Data Figure 8: Analysis and functional validation of Wnt signalling as a key driver of BET resistance. | Nature

Extended Data Figure 8: Analysis and functional validation of Wnt signalling as a key driver of BET resistance.

From: Transcriptional plasticity promotes primary and acquired resistance to BET inhibition

Extended Data Figure 8

a, Protein levels of TCF4 and IGF2BP1 in K-562 cells transduced with pRT3GEN expressing indicated shRNAs after 7 days of doxycycline treatment, compared to Ren.713 and wild-type (wt) control samples. b, Competitive proliferation assay of JQ1-sensitive MOLM-13 cells expressing GFP-linked TCF4 cDNA or empty vector. Plotted is the relative fraction of GFP+ cells 72 h after JQ1 treatment using the indicated doses (n = 3, means ± s.e.m., ***P < 0.001; **P < 0.01; *P < 0.05 as determined by Student’s t-test). Cells overexpressing TCF4 exhibit a dose-dependent competitive advantage under JQ1 treatment. c, Protein levels of TCF4 after overexpression of TCF4 cDNA subcloned into pMSCV-PGK-NeoR-IRES-GFP in MOLM-13 after 4 weeks of G418 (0.5 mg ml−1) selection, compared to MOLM-13 transduced with empty control vector and to TCF4 protein levels in K-562 cells. d, ChIP qRT–PCR analysis of TCF7L2 binding to AXIN2, SP5, MYC promoter and the PVT1 enhancer element in K-562 cells at indicated time points after treatment with 200 nM JQ1 (n = 2 biological replicates, mean ± s.e.m.). TCF7L2 binding increases gradually over time at promoters of Wnt target genes and the PVT1 enhancer at the MYC locus. e, Protein levels of Apc in 3T3 murine fibroblast cells 7 days after infection with the indicated shRNAs cloned into pLMP compared to Ren.713 and wild-type control samples. f, Relative Axin2 mRNA expression levels, determined by qRT–PCR normalized to B2m, after expression of the indicated shRNAs targeting Apc. g, Competitive proliferation assays of MLL–AF9;NrasG12D leukaemia cells transduced with pLMP constructs expressing shRNAs targeting Apc, in combination with JQ1 (50 nM) or DMSO over time (continuation from Fig. 4d). h, Top, bioluminescent imaging of mice transplanted with 1 × 105 MLL–AF9;NrasG12D leukaemia cells expressing constitutively active Ctnnb1 (Ctnnb1.4x). Treatment with JQ1 (50 mg kg−1 per day) or DMSO carrier started at day 1 after injection. Bottom, Kaplan–Meier survival curves of control and JQ1-treated mice demonstrate decreased survival rates in mice treated with JQ1 (n = 5). Statistical significance was calculated using the log-rank test. i, Competitive proliferation assays of BCR/ABLp210;p53−/− and MLL/ENL;NrasG12D leukaemia cells transduced with constitutively active Ctnnb1 (Ctnnb1.4x) or empty vector control in the absence or presence of JQ1. Measurements started 4 days after transduction together with JQ1 treatment (50 nM). Shown is the fraction of GFP+ cells (relative to initial) over time.

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