Fig 2: (A) Western blot analysis of JAG1 and JAG2 in a panel of 6 MM cell lines (30 µg each). The bands of JAG1 full length (JAG1-FL) and of JAG1-ICD as well as of JAG2 have been detected. α-tubulin was used to normalize the protein extracts loading. The image is representative of two different experiments with similar results. (B) Xenograft models of advanced MM were generated as reported in the scheme injecting U266SCR or U266KD cells in NSG (n = 4/5 mice/group). Tumor engraftment was measured via (C) ELISA for λ Ig human chain in mice serum or (D) via flow cytometry analysis of hCD319+ MM cells in the flushed BM cells. Mean values ± SD are shown. Statistical analyses were performed via a two-tailed t-test (* = p < 0.05). (E) Representative immunohistochemistry analyses of mouse femurs show the different growth of U266SCR and U266KD cells (human λ Ig chain in brown) after doxycycline treatment (scale bar 2.50 mm). (F) Representative images of immunohistochemical analysis of human λ Ig light chain, Ki67, JAG2 and HES6 (scale bar 100 µm). Photographs were acquired using Nano-Zoomer 2.0. (G) Graphs showing the percentage of human λ Ig light chain, Ki67, JAG2 and HES6-positive cells in the femurs of mice bearing U266SCR or U266KD cells. Statistical analyses were performed via unpaired t-test; * = p < 0.05, ** = p < 0.01. (H) Correlation analysis between the percentage of positive cells for human JAG2 and those positive for human λ Ig light chain, Ki67 or HES6. Pearson’s correlation coefficient (r) and the p-value (calculated using a two tailed t-tests) are reported for the correlation analyses.

Fig 3: (A) Kaplan–Meier survival curves in the CoMMpass global dataset including 767 MM samples: overall survival (OS) on the left and progression-free survival (PFS) on the right. MM cases were stratified in high and low JAG2 gene expression groups, based on the median expression level across the dataset. Log-rank test p-value measuring the global difference between survival curves and number of samples at risk in each group across time are reported. (B) Forest plot of cox regression multivariate analysis considering all features with adjusted p-value < 0.05 in univariate analysis with regard to OS (left) and PFS (right) in 497 MM cases. Hazard Ratio, 95% Confidence Interval and Log-rank p-value are indicated in the plot for each variable. Significant p-value: * ≤ 0.05; ** ≤ 0.01.

Fig 4: Candidate genes regulated by 4SC-202 in human SJSA-1 OS cells. Selected differentially expressed genes in (A) the Wnt/ß-catenin signaling pathway and (B) the Notch signaling pathway. (C) Representative Western blot analysis of JAG2, NOTCH4, NOTCH3, and β-ACTIN in OS cells treated with or without 4SC-202. Selected differentially expressed genes in (D) cell cycle, (E) apoptosis, (F) cancer stem cells (CSCs) and multidrug resistance (MDR), and (G) immunotherapy. Average FPKM values from RNA-seq analysis were used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are presented as mean (SD) of triplicate samples treated with or without 4SC-202.

Fig 5: Effect of a SMI (MO-I-1100) on PC tumor growth in subcutaneous (s.c.) tumor model of nude mice(a) ASPH transfected MIA PaCa2 cells had high tumorigenicity compared to MIA PaCa2 cells transfected with empty vector. Growth curve on 8 animals in each group indicated enhanced tumor development induced by ASPH overexpression. (b) represents tumor growth curves derived from MIA PaCa2 cells stably transfected with empty vector or a WT-ASPH expression construct as delivered by lentivirus. Note that anti-tumor effects exhibited by MO-I-1100 were observed only in tumors with exogenous ASPH overexpression. Each group had 10 animals. Effect of MO-I-1100 on Notch signaling in vivo has been observed. Tumors taken from the growth curve that were responsive to MO-I-1100 treatment depicted in (b) were analyzed for Notch signaling compared to untreated control. There were 3 tumors taken from the MO-I-1100 treatment group and 3 from the untreated control. (c) demonstrates that the expression of the Notch1 ICN is substantially downregulated by the SMI of APSH β-hydroxylase activity by IHS. (d) represents reduction in the expression of JAG2, as well as Notch activated genes HES1 and PCNA following MO-I-1100 treatment. Similar inhibitory effects of MO-I-1100 on PC tumor growth in vivo were observed in (e) HPAFII and (f) AsPC-1 cells induced s.c. tumors. These human PC cell lines had high endogenous expression of ASPH as shown in Fig. 1a. *p < 0.05; **p < 0.01.