Fig 2: A Combination treatment of duvelisib plus dexamethasone or ibrutinib leads to sustained inhibition of pAKT. Western blot analysis of DOHH2 (top) and SU-DHL-4 (bottom) treated with duvelisib (1 μM), dexamethasone (500 nM), ibrutinib (100nM) or the combination of duvelisib plus dexamethasone or ibrutinib for 6 hours or 24 hours and stained with pAKT (S473). B. mTOR inhibition can suppress re-phosphorylation of pAKT. SU-DHL-4 cells treated with duvelisib (1 μM), the pan-PI3K inhibitor GDC-0941(500 nM) or the ERK inhibitor SCH-772984 (500 nM) resulted in re-phosphorylation of pAKT (S473) at 24 hours, while combination with the PI3K/mTOR inhibitor PF-04691502 (500 nM) prevented re-phosphorylation of pAKT at 24 hours. C. Re-phosphorylation of pAKT after duvelisib treatment is dependent on mTORC2. DOHH2 (top) or SU-DHL-4 (bottom) treated with duvelisib (1 μM), mTOR1/2 inhibitor AZD-8055 (200 nM) or the mTORC1 inhibitor rapamycin (100 nM) for 1 hour (left) or following a 24 hour treatment with duvelisib (1 μM)(right). D. Duvelisib combinations with dexamethasone and ibrutinib reduce activation of downstream effectors. DOHH2 (top) and SU-DHL-4 (bottom) were treated with duvelisib, dexamethasone and ibrutinib as in Fig 3A. Western blot staining for downstream effectors pPRAS40 (T246), pP70S6K (T389) and pS6 (S235/236). E. Combination of duvelisib with dexamethasone or ibrutinib leads to inhibition of cap-dependent translation. DOHH2 (left) and SU-DHL-4 (right) treated as in Fig 3A and western blot stained for p4EBP1 (S65), c-MYC, peIF4E (s209) and cleaved PARP. The total eIF4E blots demonstrate that equal amounts of protein were loaded per lane.

Fig 3: Differential expression of ALK2 and ALK3 following LR-AMH treatment is associated with the expression of cell survival and apoptosis markers in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN ovarian cancer cells. Quantification of the western blot analysis of AMH signaling (pSMAD1/5), ALK2 and ALK3 expression, apoptosis induction (cleaved caspase-3 and PARP) and pAKT levels following incubation with 1.6 and 25 nM LR-AMH for 6 h. Data are presented as the fold-change relative to control (no LR-AMH) by bars corresponding from left to right to 0, 1.6 and 25 nM for each protein expression. n>3. Raw data are presented in Fig. S5. AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; p, phosphorylated; c, cleaved; PARP, poly(ADP-ribose) polymerase.

Fig 4: lncRNA GAS5 plays a critical role in the MM1-mediated induction of apoptosis. (a) Effect of lncRNA GAS5 depletion on cell apoptosis. Left panel: A375 cells were transfected with siRNA against lncRNA GAS5 (si-lncRNA GAS5) at 50 nM or with nontargeting siRNA(−). After 48 h, the transfected cells were treated with 75 μM of MM1 for 24 h. The levels of lncRNA GAS5 were assessed by real-time RT-PCR and expressed as relative to that of non-MM1-treated control (Left panel). The cleavage of PARP and caspase-9 was analyzed by western blotting (Right panel). β-actin served as an internal control. (b and c) Effect of lncRNA GAS5 overexpression on cell proliferation and apoptosis. A375 cells were transfected with pCDNA3.1-lncRNA GAS5 or the empty vector (control). After 48 h, the transfected cells were assayed for expression of lncRNA GAS5 (b, left panel), for viability by MTT method (b, right panel), and for the induction of apoptosis by phosphatidylserine exposure with Annexin V-FITC using flow cytometry (c). Data are expressed as the mean±S.D. of three independent experiments. Symbols: **P<0.01; and ***P<0.001, as analyzed by unpaired t-tests

Fig 5: DYRK2 reduces sensitivity to proteotoxic stress via HSF1.A Equal number of MDA-MB-468 WT (Left panel) or DYRK2-KO (Right panel) were seeded. After 24 h, cells were exposed to 45 °C (HS) for 45 min followed by recovery at 37 °C (cells labelled as 45 °C) or they were left at 37 °C (cells labelled as 37 °C). Number of cells at each point were analysed using the Alamar Blue assay. Data represent means ± SD (n = 3). B Control (WT) and DYRK2-KO MDA-MB-468 cells were incubated at 37 °C (−) or at 45 °C for the indicated times, followed by recovery at 37 °C. On the next day, cells were lysed and the levels of apoptosis were analysed by western blotting using an antibody that recognises cleaved PARP. The corresponding quantifications of PARP protein levels are shown in Supplementary Fig. S5A. C DYRK2-KO MDA-MB-468 infected with virus encoding for either empty vector (Ctrl), DYRK2-WT (R. WT) or the DYRK2 HSF1-interaction deficient mutant (R. BR1 + 2) were incubated at 37 °C or at 45 °C for 45 min followed by recovery at 37 °C. On the next day, cells were lysed and the levels of apoptosis were analysed by western blotting using an antibody that recognises cleaved PARP. The corresponding quantifications of PARP protein levels are shown in Supplementary Fig. S5B. MDA-MB-468 (D) or HSF1-KO MDA-MB-468 (E) cells transfected with either siControl or siDYRK2 were incubated at 37 °C or at 45 °C for the indicated times, followed by recovery at 37 °C. On the next day, cells were lysed and the levels of apoptosis were analysed by western blotting using an antibody that recognises cleaved parp. The corresponding quantifications of PARP protein levels are shown in Supplementary Fig. S5C, D. See also Fig. S5.