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Extra-mitochondrial prosurvival BCL-2 proteins regulate gene transcription by inhibiting the SUFU tumour suppressor

Abstract

Direct interactions between pro- and anti-apoptotic BCL-2 family members form the basis of cell death decision-making at the outer mitochondrial membrane (OMM). Here we report that three anti-apoptotic BCL-2 proteins (MCL-1, BCL-2 and BCL-XL) found untethered from the OMM function as transcriptional regulators of a prosurvival and growth program. Anti-apoptotic BCL-2 proteins engage a BCL-2 homology (BH) domain sequence found in SUFU (suppressor of fused), a tumour suppressor and antagonist of the GLI DNA-binding proteins. BCL-2 proteins directly promote SUFU turnover, inhibit SUFU–GLI interaction, and induce the expression of the GLI target genes BCL-2, MCL-1 and BCL-XL. Anti-apoptotic BCL-2 protein/SUFU feedforward signalling promotes cancer cell survival and growth, and can be disabled with BH3 mimetics—small molecules that target anti-apoptotic BCL-2 proteins. Our findings delineate a chemical strategy for countering drug resistance in GLI-associated tumours and reveal unanticipated functions for BCL-2 proteins as transcriptional regulators.

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Figure 1: Prosurvival BCL-2 proteins collectively promote GLI signalling by suppressing the activity of the tumour suppressor SUFU.
Figure 2: SUFU functions as a BH3-only protein.
Figure 3: Prosurvival BCL-2 proteins promote their own expression by inducing GLI transcriptional activity.
Figure 4: Extra-mitochondrial prosurvival MCL-1 protein regulates SUFU activity.
Figure 5: BH3 mimetics disrupt GLI-dependent transcription in vivo.
Figure 6: BH3 mimetics disable deviant signalling induced by cancer-associated mutations in core HH pathway components.

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Acknowledgements

We thank C. Chen, R. Toftgård, X. Wang, S. Skypek, M. P. Scott, and J. K. Chen for reagents. This work was supported in part by the Welch Foundation (I-1665, L.L.) and CPRIT (RP130212, L.L.). Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA168761 (L.L.), R01CA196851 (L.L.) and P50-CA70907 (Minna). Q.B. was supported by a T32 training grant (5T32CA124334). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. S.Y.C. was supported by the National Basic Research Program of China (2012CB945003).

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X.W., L.-s.Z., J.T., Y.-C.K., J.T.P., R.T., Q.B., C.-w.F., T.M. and L.L. performed the experiments. X.W., L.D.W., M.K., S.Y.C., X.Z., R.B., J.T.O., D.R.G. and L.L. helped with data analysis and discussions. X.W., L.-s.Z., R.B., T.M. and L.L. conceived the study and experimental design. X.W. and L.L. wrote the manuscript.

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Correspondence to Lawrence Lum.

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Integrated supplementary information

Supplementary Figure 1 MCL-1 promotes SUFU turnover and a subset of prosurvival BCL-2 proteins interact with SUFU.

(a) MCL-1 expression status does not alter the levels of Sufu. The abundance of Sufu mRNA was measured by qPCR in Wt, Mcl1-/-, or Mcl1-/- MEFs stably expressing human MCL-1 (n = 3 independent measurements). (b) Loss of MCL-1 expression decreases turn-over of SUFU protein. Lysate from Wt or Mcl1-/- MEFs treated with cycloheximide (CHX) to block translation were subjected to Western blot analysis to determine SUFU, MCL-1, and ACTIN abundance. (c) Quantification of results in “b”. (d) Inhibition of the proteasome with MG132 induces accumulation of SUFU in Wt but not Mcl1-/- MEFs suggesting mitigation of proteasome-mediated SUFU destruction in cells lacking SUFU-interacting prosurvival BCL-2 proteins. (e) Biochemical confirmation of prosurvival Bcl-2 protein expression from transiently transfected cDNAs. (f) Prosurvival BCL-2 proteins that promote GLI activity interact with SUFU. Lysate from HEK293 cells transfected with indicated DNAs were subjected to immunoprecipitation with control (C) or myc (M) antibody. (g) Deletions in the Bcl2 loci in MEFs introduced using CRISPR-Cas9. (h) Overexpression of MCL-1 or BCL-2 decreases SUFU abundance. Lysate from HEK293 cells stably expressing the indicated DNAs were subjected to Western blot analysis. Cells expressing BCL-W (which does not bind SUFU) exhibit little change in SUFU abundance when compared with control. All error bars represent mean ± s.d. Source data are available in Supplementary Table 4 and unprocessed blots in Supplementary Fig. 7.

Supplementary Figure 2 A BH3 sequence in SUFU mediates its interaction with the BCL-2 core of MCL-1.

(a) Raw data for mapping studies aimed at identifying SUFU interaction domains in MCL-1. (b) Summary of MCL-1 mapping studies. Alpha helices are depicted as cylinders. (c) Analysis of MCL-1 mutants for their ability to inhibit SUFU suppression of GLI activity (n = 3 independent measurements). (d) Raw data for mapping studies aimed at identifying MCL-1 interaction domains in SUFU. (e) Summary of SUFU mapping studies. Alpha helices are depicted as cylinders. (f) Mass spectrometric confirmation of the SUFU BH3 peptide used in the NMR studies. (g) Baseline 1H–15N chemical shift perturbations (CSPs) of 150 mM 15N-labeled MCL-1 in 20 mM phosphate buffer, pH 6.8, 4.8% DMSO at 25 °C map within the BC groove (the BH1, BH2 and BH3 regions) and helix 6. (h) Assessment of MCL-1/SUFU complex stoichiometry using a bi-directional IP strategy. The ratio of MCL-1/SUFU protein isolated from HEK293 cells transfected with the indicated DNA remains similar regardless of the IP approach. (i) Silver stain and Western blot analysis of purified MCL-1 and SUFU proteins used in in vitro PKA phosphorylation studies described in j. (j) MCL-1 inhibits PKA-mediated phosphorylation of SUFU in vitro. Purified MCL-1-Fc and SUFU protein were incubated in the presence or absence of PKA and P32-labeled ATP. All error bars represent mean ± s.d. Source data are available in Supplementary Table 4 and unprocessed blots in Supplementary Fig. 7. Statistical significance was calculated using Student’s t-test, P < 0.05, P < 0.01 and P < 0.001.

Supplementary Figure 3 The role of the SUFU BH3 sequence in prosurvival BCL-2 protein-dependent transcription.

(a) HH pathway activation does not alter MCL-1 protein stability. TOP: Luciferase activity of MCL-1-Gaussia luciferase (MCL-1-GL) expressed under the control of a constitutive promoter. BOTTOM: GLI-BS reporter activity (n = 3 independent measurements). (b) MCL1 and BCL2 mRNA are elevated in SHH class medulloblastomas. Comparison of gene expression analysis from 112 SHH subgroup of medulloblastomas and 13 adult cerebellum. (c) C3H10T1/2 but not RMS13 and HAP1 cells elaborate primary cilia. C3H10T1/2, RMS13 and HAP1 cells were stained for acetylated (AC) tubulin, a marker of primary cilia. Scale bar, 10 μm. (d) Absence of change in BCL-2, MCL-1 and BCL-XL expression in wt, SUFUΔBH3, and SUFU null HAP1 cells following exposure to SAG. Note, HAP1 cells are devoid of GLI1 expression. SUFUΔBH3 and SUFU null HAP1 cells were engineered using CRISPR-Cas9. (e) Genomic sequence of the SUFU BH3 encoding region for SUFUΔBH3 and SUFU KO HAP1 cell lines. (f) Genomic sequence of the SUFU BH3 encoding region in the SufuΔBH3 and Sufu null C3H10T1/2 cell line. (g) C3H10T1/2 cells exhibit expression changes in GLI1, BCL-2, MCL-1 and BCL-XL in response to SAG, loss of the SUFU BH3 sequence, or elimination of SUFU altogether. (h) C3H10T1/2 cells expressing SUFUΔBH3 are not transcriptionally responsive to SHH-N or MCL-1 overexpression as measured using the GLI-BS reporter (n = 3 independent measurements). (i) MCL-1 G217E/D218A does not restore homeostatic levels of phosphorylated or total SUFU in Mcl1 null MEFs. (j) A dual luciferase reporter system for monitoring MCL-1/SUFU interactions. (k) MCL-GL and SUFU-FL-Myc protein interactions measured by luciferase activity in Myc antibody associated immuno-precipitant is sensitive to the MCL-1 inhibitors MIM-X and MIM-1 (n = 3 independent measurements). (l) SAG alters SUFU/MCL-1 interactions. SUFU-FL-Myc/MCL-1-GL interaction was measured by luciferase activity in Myc antibody associated immunoprecipitant. All error bars represent mean ± s.d. Source data are available in Supplementary Table 4 and unprocessed blots in Supplementary Fig. 7. Statistical significance was calculated using Student’s t-test, P < 0.001.

Supplementary Figure 4 Engineering cell lines expressing MCL-1ΔTM and BCL-2ΔTM.

(a) Genomic sequence of CRISPR-Cas9-edited C3H10T1/2 Mcl1ΔTM clones. (b) Genomic sequence of CRISPR-Cas9-edited C3H10T1/2 Mcl1 KO clone. (c) Genomic sequence of CRISPR-Cas9-edited C3H10T1/2 Bcl2ΔTM clone.

Supplementary Figure 5 Selective activity of ABT-199 against SUFU mutant proteins.

(a) Distribution of missense SUFU mutations in cancer types (from cBioPortal). (b) Genomic sequence of CRISPR-Cas9-edited RMS13-SUFU KO cells. (c) GLI1 expression in RMS cell lines. (d) Animal weight change data for xenotransplantation studies (n = 5 mice/group). (e) Tumor weight measurements (n = 5 tumors/group). All error bars represent mean ± s.d. Source data are available in Supplementary Table 4 and unprocessed blots in Supplementary Fig. 7.

Supplementary Figure 6 ABT-199 and ABT-263 inhibit various forms of oncogenic GLI activation.

(a) Distribution of mutational spectra associated with Hh pathway components in the SHH subgroup of medulloblastomas. Number of cases evaluated in each age group is indicated in parentheses [Kool et al. Cancer Cell 25, 393–405 (2014)]. (bf) EC50 curves for in vitro drug studies (n = 3 independent measurements). (g) A drug resistant form of SMO (SMO L412F) found in medulloblastoma and meningioma exhibits reduced responsiveness to Vismodegib. C3H10T1/2 cells transfected with indicated expression DNAs and the GLI-BS reporter were treated with Vismodegib for 48 hrs before luciferase activity was measured from cell lysate. (n = 3 independent measurements). (h) ABT-199 inhibits SMO- and SMO L412F-induced GLI activity with equal potency. C3H10T1/2 cells transfected with the GLI-BS reporter were treated with ABT-199 for 48 hrs before luciferase activity was measured from cell lysate. (n = 3 independent measurements). (i) GLI1 expression as a biomarker for cell lines with a prosurvival BCL-2/SUFU regulatory loop. Cell lines with transcriptome profiles inventoried at the Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle/home) were ranked ordered with respect to expression of GLI1, a highly validated target gene of GLI protein transcriptional activity. Corresponding expression of SUFU, BCL2, MCL1, and BCLXL for each cell line reveals nearly ubiquitous expression of SUFU and at least one of the three SUFU interacting prosurvival BCL-2 proteins. Note that this approach for identifying cell lines that potentially exhibit SUFU/prosurvival BCL-2 regulation of GLI activity is able to return RMS13 cells which exhibit GLI1 amplification and confirmed SUFU regulation by prosurvival BCL-2 proteins. All error bars represent mean ± s.d.

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Wu, X., Zhang, Ls., Toombs, J. et al. Extra-mitochondrial prosurvival BCL-2 proteins regulate gene transcription by inhibiting the SUFU tumour suppressor. Nat Cell Biol 19, 1226–1236 (2017). https://doi.org/10.1038/ncb3616

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