Fig. 4: High NAMPT-dependence is associated with NE phenotypes.

a Schematic showing establishment of TR-6TF, a human organoid that can be differentiated into a NE lineage following doxycycline (Doxy)-dependent induction of 6 transcription factors (TF). 6TF: ASCL1, NEUROD1, NKX2-5, POU3F2, SOX2 and TP73. GEKO, gene knock-out. b, c qRT-PCR analyses of TR-6TF organoids. Shown are levels of transcripts encoding NE markers (b) and the PKM1/PKM2 mRNA ratio (c). n = 4 (Doxy-treated group for SYP) or 3 (all others) biological replicates in (b). n = 4 (untreated) or 7 (Doxy-treated) biological replicates in (c). P = 0.0032 (NCAM); P = 0.0036 (SYP) (b). P = 0.0012 (c). d qRT-PCR analyses of TR-6TF organoids. Shown are KYNU and HAAO transcript levels. n = 4 (untreated) or 8 (Doxy-treated) biological replicates. P <0.0001 (KYNU); P <0.0001 (HAAO). e, f NAD (e) and ATP (f) levels in TR-6TF organoids cultured in the presence or absence of Doxy and then treated or untreated 48 h with FK866. n = 3 or 4 biological replicates. P < 0.0001 (w/ vs wo FK866, wo Doxy); P <0.0001 (wo vs w/ Doxy, w/ FK866); P <0.0001 (wo vs w/ FK866, w/ Doxy) (e). P = 0.5724 (wo vs w/ FK866, wo Doxy); P = 0.4892 (wo vs w/ Doxy, wo FK866); P = 0.0064 (wo vs w/ FK866, w/ Doxy) (f). g Classification of prostate cancer (PCa) lines divided into groups based on histopathological types (SCPC, CRPC, or not), androgen receptor (AR) expression and a neuroendocrine (NE) signature. The PKM1/PKM2 ratio was determined by qRT-PCR analysis. Scores are averages of at least two duplicates. Two SCLC lines at the bottom (87-5 and Lu-139) are shown for comparison. h Calculation of the PKM1/PKM2 ratio in NE-type (NEPCa) versus other PCas. Shown are results of 3 NEPCa and 5 other PCa cell lines. P = 0.0265. i Fold-change in the number of cells in PCa and non-transformed lines cultured 4 days with 20 nM FK866. Dashed line represents cell number at day 0. n = 2 (KUCaP13 and LASCLC-01), 7 (PC-3) or 3 (all others) biological replicates. j Comparison of results shown in (i) between NEPCa and other PCas. Shown are results of 3 NEPCa and 5 other PCa cell lines. P = 0.0038. k Comparison of changes in cell number after 4 days of FK866 treatment in SCLC (results shown in Fig. 2c), NEPCa and non-transformed lines (i). Shown are results from 8 SCLC, 3 NEPCa and 4 non-transformed cell lines. P = 0.2459 (SCLC vs NEPCa); P <0.0001 (SCLC vs non-transformed); P <0.0001 (NEPCa vs non-transformed). Data are presented as mean plus the SEM. *P<0.05, **P<0.01, ****P<0.0001 as determined by one-way ANOVA with a post hoc test (e, f, k) or by two-tailed t test (b, c, d, h, j). ns., not significant. Source data are provided as a Source Data file.