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, 20 (6), 599-606

Rescue of Hippo Coactivator YAP1 Triggers DNA Damage-Induced Apoptosis in Hematological Cancers

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Rescue of Hippo Coactivator YAP1 Triggers DNA Damage-Induced Apoptosis in Hematological Cancers

Francesca Cottini et al. Nat Med.

Abstract

Oncogene-induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies, including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53-independent, proapoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1-induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine-threonine kinase, STK4. Notably, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a new synthetic-lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels.

Figures

Fig. 1
Fig. 1. MM cells present ongoing DNA damage, driving ABL1 inside the nucleus
(a) Immunofluorescence staining of γ-H2A.X in MM cell lines, PBMCs and MM cells. (b) Western blot analysis of γ-H2A.X in MM cell lines, PBMCs and MM cells. (c) Western blot analysis of p–ATM(Ser1981), p–CHK2(Thr68), p–ATR(Ser428), and p– CHK1(Ser296) and their corresponding un–modified forms in MM cell lines and samples from subjects with MM. (d) Immunofluorescence staining for γ-H2A.X–Alexa Fluor 568, cleaved caspase 3–Alexa 488, and DAPI in H929 cells. As positive control, MM cell lines and MM cells were treated with 5 nM Bortezomib (Btz) for 24 h. (e) Subcellular fractionation of p53–WT and p53–mutant MM cell lines. Cell lysates from cytoplasmic (C) and nuclear (N) fractions were analyzed by western blot for ABL1 expression. α-tubulin and Histone H3 were used as loading controls for C and N fractions, respectively. (f) Immunohistochemical ABL1 staining on one representative sample derived from an individual affected by MM (MM–004). Left panel, 400x. Right panel, 1000x magnification.
Fig. 2
Fig. 2. pATM and pJNK modulate ABL1 nuclear re–localization
(a) MM cell lines were treated with 2–10 μM of ATM kinase inhibitor Ku55933 for 1–2 h in MM.1S and for 2 h in UTMC–2, JJN–3 and KMS–20 cells. Lysates were blotted for ABL1, α- tubulin (C loading control), and p84 (N loading control). (b) MM cell lines were treated with 10 μM of JNK1 inhibitor SP600125 for 2 h. Lysates were blotted for ABL1, α-tubulin (C loading control), and Histone H3 (N loading control). (c) Left panel: Western blot analysis for γ-H2A.X, p–ATM(Ser1981) and p–JNK (Thr183/Tyr185) on total lysates in U266 MM cell line after Dmso or doxorubicin (Doxo; 40 nM) treatment for 72 h. Right panel: Western blot analysis for ABL1, Histone H3, and α- tubulin in U266 cells after Dmso or Doxo (40 nM) treatment for 72 h. (d) Annexin V–FITC/PI staining (left panel) and MTT absorbance assay (right panel) in U266 cells after 48 h of Dmso, Doxo (40 nM) or Doxo plus imatinib (10 μM) treatment. Data are mean values ± SD of triplicates, Student's t test.
Fig. 3
Fig. 3. YAP1 deletions and expression in MM cell lines and samples from subjects with MM
(a) YAP1 mRNA expression in solid tumors and hematological cancers (Oncomine at www.oncomine.org; Wooster Cell Line dataset). (b) Gene dosage comparison across cell lines and representative tumors demonstrating homozygous deletions at the YAP1 locus. Number 1 corresponds to data derived from , 2 from , and 3 from . (c) Survival curve relative to YAP1 expression in individuals affected by MM, obtained from www.canevolve.org, and based on GSE2658. (d) Expression data comparing plasma cells from healthy subjects, MGUS, MM, and cell lines (CL), combining data from GSE5900, GSE2658, and from the MMRC collection (http://www.broadinstitute.org/mmgp), probe set 224895_at. *: P < 0.05; **: P < 0.01; ***: P < 0.001, one-way Anova, Dunn's Multiple Comparison Test. (e) Non–quantitative PCR on genomic DNA from YAP1–deleted MM cell lines KMS–18 and KMS–20 and control cell line (EJM). (f) Western blot analysis of YAP1 in MM cell lines and cells derived from subjects with MM. As positive control, lysates from 293T cells transfected with YAP1–EGFP vector were used (band at 98 kDa).
Fig. 4
Fig. 4. YAP1 re–expression leads to ABL1- dependent reduced proliferation and cell death
(a) YAP1 re–expression in KMS–20 (left) and KMS–18 (right) cells. Western blot analysis at 72h after pLENTI4–YAP1–EGFP or pLENTI4–LACZ transfection. Cell number is evaluated by cell counting with trypan blue exclusion. Data are representative of three independent experiments and mean values ± SD of triplicates, Student's t test. (b) Annexin V–PE/7–AAD staining. (c) YAP1 silencing in UTMC–2 cells using a lentiviral delivery system. Left panel: western blot analysis in pLKO.1–infected cells after 48h 2μg mL−1 puromycin selection. Right panel: Cell number and apoptosis as in (a) and (b) respectively. Data are representative of two independent experiments. (d) Effects of YAP1 re–expression at 72 h after transfection. Western blotting, cell counting and Annexin V–PE/7–AAD staining as in (a) and (b), respectively. (e) YAP1 re–expression in combination with imatinib treatment. Left panel: Cell number evaluated as in (a), in the presence of imatinib (10 μM) or Dmso (from day 0 of transfection). Right panel: Western blot analysis after 48h incubation with Dmso or 10 μM imatinib. Data are mean values ± SD of triplicates, Student's t test. (f) Western blot analysis in KMS–20 transfected cells, at 72 h. (g) mRNA levels of p73–target genes in the same cells. (h) dWW–YAP1 re–expression. Left panel: Western blot analysis for YAP1, at 48h. Middle panel: Cell number as in (a). Right panel: Apoptosis as in (b). Data are mean values ± SD of triplicates, Student's t test.
Fig. 5
Fig. 5. STK4 knock–down triggers YAP1 re-expression, reducing MM cell proliferation and increasing apoptosis in vitro and in vivo
(a) STK4 silencing using lentiviral vector pLKO.1 (after 48h 2μg mL−1 puromycin selection) in MM.1S cells. Western blot for YAP1 and STK4 in STK4–silenced cells (3 representative shRNAs). (b) Cell number evaluated by cell counting with trypan blue exclusion in MM.1S cells silenced for STK4. Left panel, pLKO.1 lentivectors; right panel, inducible pTRIPZ lentivectors. One representative experiment of three is shown. Data are mean values ± SD of triplicates, Student's t test. (c) Combined effects of STK4 knock–down associated with treatment with various compounds. Apoptosis evaluated with Annexin V–FITC/PI staining in stably infected cells after 48h incubation with 5 nM bortezomib (Btz) and 40 nM doxorubicin (Doxo). (d) STK4 silencing by transfection using pLKO.1 scrambled or shRNA #5 vectors. Cell counting as in (b). (e) MM.1S cells, infected with inducible STK4–shRNAs, after 24 h of 2 μg mL−1 doxycycline were transfected by electroporation with pJ3H–STK4–wild type or pJ3H–STK4K59R mutant. Left panel: Cell number evaluated as in (b). Right panel: Western blot after 48h transfection. (f) In vivo evaluation of the effects of STK4 knock–down on MM cells. Left panel: injection scheme and tumor size in three representative mice. Right panel: Growth curve assessing tumor size after injection of MM.1S cells transduced with STK4–specific shRNAs or scrambled–control vectors. Data are mean values ± SD of triplicates, Student's t test. (g) Proposed model for the ABL1/YAP1/p73 axis and effects of STK4 inhibition on YAP1 levels in MM.
Fig. 6
Fig. 6. Lymphoma, leukemia and Waldenström's macroglobulinemia cells present ongoing DNA damage and ABL1 nuclear localization, and undergo apoptosis following STK4-mediated YAP1 increased levels
(a) γ-H2A.X staining of Waldenström's macroglobulinemia (BCWM.1 and MWCL.1), lymphoma (DOHH–2 and RAMOS), B–cell acute lymphoblastic leukemia–B–ALL (RS4;11, CEMO–1, REH, and NALM–6), acute myeloid leukemia–AML (OCI/AML3, KG–1, and HL–60), and T– cell acute lymphoblastic leukemia–T–ALL (Jurkat) cell lines. (b) Western blot analysis for γ-H2A.X and p–ATM. (c) Cell lysates from cytoplasmic (C) and nuclear (N) fractions analyzed by Western blot for ABL1 expression. γ-tubulin and Histone H3 were used as loading controls for C and N fractions, respectively. (d) YAP1 protein expression in ALL, lymphomas, and AML. As positive control, lysates from 293T cells transfected with YAP1–EGFP vector were used (98 kDa–band). (e) Survival curve relative to YAP1 expression in subjects with AML, obtained from www.canevolve.org, based on GSE12417. (f) Western blot analysis at 72 h after transfection. Cell number evaluated with cell counting using trypan blue exclusion. Mean values ± SD of triplicate of two experiments are shown. Data are mean values ± SD of triplicates, Student's t test. (g) Apoptosis evaluated by Annexin V–PE/7–AAD staining after gating on GFP–positive cells. (h) mRNA levels of YAP1, p73, and p73–target genes (BAX, PUMA, p21). (i) STK4 knockdown in OCI/AML3 and Jurkat cell lines. Left panel: Western blot for YAP1 and STK4. Middle panel: Cell number measured as in (f) after transfection with scrambled or pLKO.1 #5 vectors. Right panel: Apoptosis by Annexin V–PE/7–AAD staining.

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