Fig 2: Perturbation of the neddylation pathway disrupts the neuroendocrine signature in high-grade neuroendocrine cancers. (A) Immunoblot analysis for NEDD8, showing the levels of neddylated cullins and for neuroendocrine regulators in the MLN4924-sensitive ex vivo PDX models JHU-LX108 and MSK-LX227C. Cells were treated for 48 h with DMSO or MLN4924 (100 nM). ß-ACTIN was used as loading control. (B) Immunoblot analysis for the levels of neuroendocrine regulators and neddylation pathway members upon inducible knockdown of NEDD8 or RBX1 in the ex vivo PDX model MSK-LX227C. Samples were supplemented with 1 µg/mL doxycycline and collected at days 0, 3, and 4. GAPDH was used as loading control. (C–E) Proliferation assay results comparing on-dox and off-dox MSK-LX227C shGFP (C), shNEDD8 (D), or shRBX1 (E) knockdown cell lines. Samples were assayed at days 1, 4, and 7 with readout determined by CellTiter-Glo. (F) Immunoblot analysis for the levels of INSM1 upon the inducible knockdown of INSM1 in MSK-LX227C ex vivo cells. Samples were supplemented with 1 µg/mL doxycycline and collected at day 0, 6, and 10. GAPDH was used as loading control. (G) Proliferation assay results comparing on-dox and off-dox MSK-LX227C shINSM1 knockdown cells. Samples were assayed at days 1, 4, 7, and 10 with readout determined by CellTiter-Glo. (H) Immunoblot analysis for the levels of INSM1 in MSK-LX227C ex vivo cells constitutively overexpressing INSM1 (phINSM1) or an empty vector control (phEmpty). Cells were treated for 96 h with DMSO or MLN4924 (25 nM). GAPDH was used as loading control. (I) Dose response curve for MLN4924-treated ex vivo MSK-LX227C cells overexpressing INSM1 or empty vector control. Percent survivability determined at 72 h by CellTiter-Glo. Data are means ± SEM (n = 4 biological replicates). (J) Proliferation of ex vivo MSK-LX227C cells overexpressing INSM1 or empty vector control cultured with MLN4924 (25 nM). Samples were assayed at days 1, 4, and 7. Readout was determined at each time point by CellTiter-Glo. (K) Proliferation of ex vivo MSK-LX227C cells overexpressing INSM1 or empty vector control cultured in standard growth media. Samples were assayed at days 1, 4, and 7. Readout was determined at each time point by CellTiter-Glo. For C–E,G, J, and K, data are means ± SEM (n = 3 biological replicates in all groups). Relative cell growth determined by normalizing average luminescence (RLU) values for the initial time point to 1. (ns) Not significant, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001, by unpaired Student's t-test.

Fig 3: Abrogation of the COP9 signalosome and other pathways provides resistance to MLN4924. (A) Response to MLNL4924 treatment (72-h) upon CHUK, IRAK1, MIOS, or COPS4 invalidation in MSK-LX227C ex vivo cells. IC50s are shown. Data are means ± SEM, from at least n = 7 biological replicates. (****) P < 0.0001, unpaired Student's t-test. (B) Representative immunoblot analysis for NEDD8, showing the levels of neddylated cullins and for COPS4 in MSK-LX227C cells treated with DMSO or MLN4924 (100 nM) for 96 h. Parental MSK-LX227C ex vivo cells were compared with cells infected with an inducible shRNA targeting GFP or COPS4. On-dox cells were supplemented with 1 µg/mL doxycycline. GAPDH was used as loading control. (C) Quantification of relative levels of neddylated cullin protein abundance from multiple replicate immunoblot experiments described in B, showing combined parental MSK-LX227C and shGFP data compared with the combined data of two independent COPS4 shRNAs. Data are means ± SEM (n = 7 biological replicates). (**) P < 0.01, (****) P < 0.0001, unpaired Student's t-test. (D) Representative immunoblot analysis for the levels of neuroendocrine regulators and pro-apoptotic proteins for samples described in B. GAPDH was used as loading control. (E) Quantification of the relative levels of cleaved Caspase 7, cleaved PARP, INSM1, and ASCL1 protein abundance for multiple replicate immunoblot experiments described in D. Data are means ± SEM (n = 3 biological replicates). (**) P < 0.01, (***) P < 0.001, unpaired Student's t-test. For C and E, relative abundance levels calculated by normalization to individual GAPDH loading controls with the untreated parental MSK-LX227C set to a relative abundance of 1.

Fig 4: Expression of INSM1 in resected neuroendocrine carcinoma (NEC) tissues and SS-2 cells. A, Localization of INSM-1 in the surgically resected NEC tumors. B, A single band corresponding to INSM-1 was detected in SS-2 cells. C, INSM-1 was detected in nuclei of SS-2 cells. D, Targeting of INSM-1 did not affect the levels of chromogranin A and synaptophysin mRNAs. Magnification: A, ×200; C, ×600. Numbers 1 and 2 indicate samples derived from two independently harvested SS-2 cells

Fig 5: Representative sections of LCNEC stained with hematoxylin and eosin (H&E) and immunostained with YAP1, INSM1, NeuroD1 and DLL3 expression level. The five images in each line correspond to one patient. High YAP1 expression was associated with low expression of INSM1 and DLL3 (B1, C1, D1), vice versa (B2, C2, D2)