Journal: The Journal of Experimental Medicine
Article Title: MYC-mediated resistance to trametinib and HCQ in PDAC is overcome by CDK4/6 and lysosomal inhibition
doi: 10.1084/jem.20221524
Figure Lengend Snippet: c-MYC expression drives cell cycle progression. (A) Tet-On c-MYC–expressing PDX220 or Panc 10.05 cells treated with vehicle or 2 μg/ml Dox for 3, 5, or 7 d were assessed for cell cycle. Bar graphs show representative data from two independent experiments for PDX220 and one experiment for Panc 10.05. (B–D) c-MYC T58A reactivates the cell cycle in the face of T/CQ co-treatment, but not P/CQ co-treatment. Percentage of cells in each cell cycle phase in (B) Panc 10.05 ( n = 1 from one independent experiment), (C) HPAF-II ( n = 3 from one experiment), or (D) PDX220 cells ( n = 1 from three independent experiments) expressing Tet-On c-MYC T58A are depicted. Cells were treated for 7 d with DMSO (Ctrl), 2 μg/ml Dox, 10–100 nM trametinib and 10–20 μM CQ (T/CQ), D/T/CQ, 2.5 μM palbociclib and 10–20 μM CQ (P/CQ) or D/P/CQ and cell cycle phase was quantitated. Bars represent mean percent cell cycle phase ± SEM. *, P < 0.05; **, P < 0.05; ns, not significant by Student’s two-sided t test. (E) CQ co-treatment does not significantly affect cell cycle status in trametinib- or palbociclib-treated PDAC cells. PDX220 Tet-On c-MYC cells were treated with vehicle control (Ctrl), CQ (20 μM), trametinib (T, 100 nM), palbociclib (P, 2.5 μM), T/CQ, or P/CQ for 7 d and processed for cell cycle analysis ( n = 1 from two independent experiments, mean ± SEM). ns, not significant by Student’s two-tailed t test. (F) Panc 10.05 cells expressing Tet-On c-MYC were treated for 7 d with DMSO (Ctrl), CQ (20 μM), trametinib (T, 100 nM), or palbociclib (P, 2.5 μM) or the combinations (T/CQ and P/CQ), and with or without Dox (D, 2 μg/ml; n = 1). (G) Cell cycle profiles were assessed in MIA-PaCa2 cells after treatment with palbociclib with and without CQ co-treatment. A Student’s two-tailed t test was utilized in order to determine statistical significance of G 0 /G 1 phase between the DMSO control and the experimental groups as well as between the experimental groups in one experiment ( n = 3). ns, not significant; ***, P < 0.001. (H–J) c-MYC T58A expression elevates pRB, cyclinB1, and cyclinA2 expression in the face of trametinib and CQ, but not P/CQ co-treatment. Representative immunoblot analysis of Tet-On c-MYC T58A expressing Panc 10.05 (H), HPAF-II (I), and PDX220 (J) cells that were treated as in B–D (from three independent experiments). Lysates were blotted for c-MYC, pERK1/2, ERK1/2, pRB, RB, E2F1, CCNB1, CCNA2, CCND2, CCNE1, CDK6, p27 KIP1 , p21 CIP1 , and ACTB. (K and L) c-MYC WT elevates pRB, cyclinB1, and cyclinA2 expression in the face of T/CQ, but not P/CQ. Representative immunoblot analysis of Tet-On c-MYC HPAF-II and PDX220 cells that were treated as above (from two independent experiments for HPAF-II and n = 1 experiment for PDX220). Lysates were blotted for c-MYC, pERK1/2, ERK1/2, pRB, RB, E2F1, CCNB1, CCNA2, CCND2, CCNE1, CDK2, CDK4, CDK6, p27 KIP1 , p21 CIP1 , and ACTB. Source data are available for this figure: .
Article Snippet: Antibodies used are as follows (used at 1:1,000 unless noted otherwise): 1:2,000 phospho-T202/Y204 p44/42 MAPK (ERK1/2; CST:4370), total ERK1/2 (CST:4696); p-RB 807/811 1:1,000 (CST 8516S); RB (CST:9309S); c-MYC (CST:5605S); E2F1 (CST:3742S); Cyclin B1 (CST:12231S); Cyclin A2 (CST:91500S); Cyclin D2 (CST:3741S); Cyclin E1 (CST:20808S); CDK2 (CST:2546S); CDK4 (CST:12790S); CDK6 (CST:3136S); p27 KIP1 , (CST:3686S); p21 WAF1/CIP1 (CST:2947S); 1:1,000 Vinculin (CST:13901); 1:2,000 GAPDH (CST:97166S); 1:5,000 β-actin (CST:4970S).
Techniques: Expressing, Cell Cycle Assay, Two Tailed Test, Western Blot