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Supplementary Figure S1. Generation of ENZR Xenografts and Cell Lines. Supplementary Figure S2. AR non-driven ENZR cells display a NE differentiation signature. Supplementary Figure S3. BRN2 expression is associated with PCa progression and a NE phenotype in human tumors. Supplementary Figure S4. BRN2 expression is inversely related to AR activity and is required for ENZ-induced NE marker expression in CRPC. Supplementary Figure S5. BRN2 overexpression induces NE marker expression and reciprocally regulates AR. Supplementary Figure S6. BRN2 activity and markers of NE differentiation are suppressed by androgens in CRPC and ENZR cells. Supplementary Figure S7. SOX2 expression is induced by ENZ and suppressed by androgens. Supplementary Figure S8. BRN2 is required for neuroendocrine marker expression and aggressive growth of CRPC cells in vitro.

Supplementary methods, figure legends, and primer lists.

RNAseq of ENZ-resistant cells.

Supplementary Table S1. Summary of ENZ resistant cells' characterization

Rhabdomyosarcoma, consisting of alveolar (aRMS) and embryonal (eRMS) subtypes, is the most common type of sarcoma in children. Currently, there are no targeted drug therapies available for rhabdomyosarcoma. In searching for new molecular therapeutic targets, we carried out genome-wide small interfering RNA (siRNA) library screens targeting human phosphatases (n = 206) and kinases (n = 691) initially against an aRMS cell line, RH30. Sixteen phosphatases and 50 kinases were identified based on growth inhibition after 72 hours. Inhibiting polo-like kinase 1 (PLK1) had the most remarkable impact on growth inhibition (∼80%) and apoptosis on all three rhabdomyosarcoma cell lines tested, namely, RH30, CW9019 (aRMS), and RD (eRMS), whereas there was no effect on normal muscle cells. The loss of PLK1 expression and subsequent growth inhibition correlated with decreased p-CDC25C and Cyclin B1. Increased expression of WEE 1 was also noted. The induction of apoptosis after PLK1 silencing was confirmed by increased p-H2AX, propidium iodide uptake, and chromatin condensation, as well as caspase-3 and poly(ADP-ribose) polymerase cleavage. Pediatric Ewing's sarcoma (TC-32), neuroblastoma (IMR32 and KCNR), and glioblastoma (SF188) models were also highly sensitive to PLK1 inhibition. Finally, based on cDNA microarray analyses, PLK1 mRNA was overexpressed (>1.5 fold) in 10 of 10 rhabdomyosarcoma cell lines and in 47% and 51% of primary aRMS (17 of 36 samples) and eRMS (21 of 41 samples) tumors, respectively, compared with normal muscles. Similarly, pediatric Ewing's sarcoma, neuroblastoma, and osteosarcoma tumors expressed high PLK1. We conclude that PLK1 could be a promising therapeutic target for the treatment of a wide range of pediatric solid tumors including rhabdomyosarcoma. [Mol Cancer Ther 2009;8(11):3024–35]

Supplementary Figure 2 from Small interfering RNA library screen of human kinases and phosphatases identifies polo-like kinase 1 as a promising new target for the treatment of pediatric rhabdomyosarcomas

Supplementary Figure 1 from Small interfering RNA library screen of human kinases and phosphatases identifies polo-like kinase 1 as a promising new target for the treatment of pediatric rhabdomyosarcomas

Supplementary Figure S1. GSEA analyses of subtype-specific genes (PCS1, PCS2 and PCS3) in 42DENZR and 42FENZR cells. Supplementary Figure S2. A) Expression of FOXM1 in different cell lines. Total proteins were extracted from LNCaP, 16DCRPC and ENZR 42D and 42F cells and western blot was performed using FOXM1 antibody (Abcam, 1:1000). Vinculin (1:5000) was used as a loading control. FOXM1 band densitometry was normalized to vinculin. B) Upstream regulator analysis of FOXM1 in PCS1 signature and 42DENZR and 42FENZR cells assessed by IPA (red: target gene overexpression, green: target gene downregulation, orange arrow: target gene is known as induced and blue arrow: target gene known as reduced by pathway activation). C) Expression of FOXM1 pathway expression in metastatic prostate cancer patients analysed using Genesapiens database (20). Supplementary Figure S3. A) Effect of Mon and Thiostrepton effect on FOXM1 transcriptional activity, 42DENZR cells were treated with 100 nM of Mon and Thiostrepton for 24 hours and FOXM1 luciferase activity was performed. B) Monensin reduces FOXM1 protein level. Monensin effect (0, 1, 10, 50 and 100 nM) on FOXM1 protein was addressed using Western blot in 42DENZR cells. Total protein were extracted and western blot was performed using FOXM1 antibody (1:1000). Vinculin (1:5000) was used as a loading control. C) Effect of Mon on cell cycle. Flow cytometry was performed to analyse cell cycle population after Mon treatment for 0 to 72h, percentage of sub-G1 cells in response to Mon in 16DCRPC, 42DENZR and 42FENZR cells was evaluated. D) DARTS assay in presence and absence of Mon in pronase treated samples indicated Mon binding to FOXM1. GAPDH was used as a control. E) Analysis of the effect of Mon on FOXM1 pathway by IPA Upstream regulator analysis of Mon exposed 42DENZR cells (green: target gene downregulation, blue arrow: target gene known as reduced by pathway inactivation). Supplementary Figure S4. Portion of high CD49b (CD49b+/CD24-) cells in 16DENZR, 42DENZR and 42FENZR cells. B) Effect of Mon and C) FOXM1 silencing by FOXM1 siRNA on portion of CD49b+/CD24- cells. Supplementary Figure S5. The effect of FOXM1 inhibition on ALDH activity. A) Portion of ALDH active cells in 16DENZR, 42DENZR and 42FENZR cells. B) The effect of Mon and C) FOXM1 silencing on ALDH activity in 42DENZR and 42FENZR cells. D) Cell proliferation of ALDHHigh and ALDHLow cells (separated from 42DENZR and 42FENZR cells using FACS and Aldefluor ALDH assay) in response to Mon measured by CTG cell proliferation assay. E) FOXM1 protein expression in 2 ALDHHigh and ALDHLow 42DENZR and 42FENZR cells assessed by western blot of FOXM1 and vinculin (loading control). Supplementary Figure S6. A) Mouse body weight (g) in response to Vehicle and Monensin (Mo) treatments for 3.5 weeks. B) Effect of Mon on ALDH activity in 42DENZR tumor xenografts in vivo assessed by Aldefluor ALDH assay and FACS. Supplementary Figure S7. A) The expression of PCS1 signature is enriched in CRPCNeuroendocrine patients. Fold changes of genes in PCS1 signature in CRPC-NE compared to CRPC-Adeno prostate cancer patients are presented as a heatmap. B) The expression of PCS1 signature is enriched in Trp53 and Pten (NPp53 mice) that fail to respond to abiraterone and differentiate to neuroendocrine prostate cancer. Fold changes of genes in PCS1 signature in NPp53 abiraterone-"exceptional non-responders" compared to NPp53 vehicle are presented as a Heatmap. C) PCS1 signature in wild type (WT), single (SKO, (PBCre4:Pten f/f :Rb1 f/+ )), double (DKO, PBCre4:Pten f/f :Rb1 f/f ) and triple knock out (TKO, PBCre4:Pten f/f :Rb1 f/f :Trp53 f/f ) mice . Fold changes of genes in PCS1 signature in SKO, DKO and TKO mice compared to WT mice are presented as a heatmap.

Purpose: Prostate cancer was recently classified to three clinically relevant subtypes (PCS) demarcated by unique pathway activation and clinical aggressiveness. In this preclinical study, we investigated molecular targets and therapeutics for PCS1, the most aggressive and lethal subtype, with no treatment options available in the clinic.Experimental Design: We utilized the PCS1 gene set and our model of enzalutamide (ENZR) castration-resistant prostate cancer (CRPC) to identify targetable pathways and inhibitors for PCS1. The findings were evaluated in vitro and in the ENZR CRPC xenograft model in vivo.Results: The results revealed that ENZR CRPC cells are enriched with PCS1 signature and that Forkhead box M1 (FOXM1) pathway is the central driver of this subtype. Notably, we identified Monensin as a novel FOXM1-binding agent that selectively targets FOXM1 to reverse the PCS1 signature and its associated stem-like features and reduces the growth of ENZR CRPC cells and xenograft tumors.Conclusions: Our preclinical data indicate FOXM1 pathway as a master regulator of PCS1 tumors, namely in ENZR CRPC, and targeting FOXM1 reduces cell growth and stemness in ENZR CRPC in vitro and in vivo. These preclinical results may guide clinical evaluation of targeting FOXM1 to eradicate highly aggressive and lethal PCS1 prostate cancer tumors. Clin Cancer Res; 23(22); 6923–33. ©2017 AACR.

Treatment with androgen receptor pathway inhibitors (ARPIs) in prostate cancer leads to the emergence of resistant tumors characterized by lineage plasticity and differentiation toward neuroendocrine lineage. Here, we find that ARPIs induce a rapid epigenetic alteration mediated by large-scale chromatin remodeling to support activation of stem/neuronal transcriptional programs. We identify the proneuronal transcription factor ASCL1 motif to be enriched in hyper-accessible regions. ASCL1 acts as a driver of the lineage plastic, neuronal transcriptional program to support treatment resistance and neuroendocrine phenotype. Targeting ASCL1 switches the neuroendocrine lineage back to the luminal epithelial state. This effect is modulated by disruption of the polycomb repressive complex-2 through UHRF1/AMPK axis and change the chromatin architecture in favor of luminal phenotype. Our study provides insights into the epigenetic alterations induced by ARPIs, governed by ASCL1, provides a proof of principle of targeting ASCL1 to reverse neuroendocrine phenotype, support luminal conversion and re-addiction to ARPIs.

We use the experimental data of Gaetani and Grove (1995) on rare earth element (REE) partitioning between clinopyroxene and melt in the system CaO-MgO-Al2O3-SiO2 to constrain the energetics of the coupled substitution REEAl[CaSi]-1. The data show that the partition coefficients DYb and DCe, and the ratio DYb/DCe increase with the Ca-Tschermaks content of the clinopyroxene. The compositional dependence of DYb/DCe can be reconciled with a simple model of lattice strain around the misfit Ce3+ and Yb3+ cations in the crystal, while the compositional dependence of DYb and DCe can be eliminated by accounting for configurational entropy in pyroxene and melt using ionic activity models for the component REEMgAlSiO6. For pyroxene we assume ideal mixing on M1 and M2 sites with local charge balance on the T sites. For melt we adopt an ideal two-lattice mixture of cations and pyroxene-like six-oxygen anions. The ionic approach is validated by a more rigorous determination of the equilibrium constant for the formation of REEMgAlSiO6 pyroxene from its molten component oxides using available thermochemical data for MgO, Al2O3 and SiO2 and Henry's law for REEO1.5. In complex systems the simple ionic approach provides a useful means of predicting the compositional dependence of partition coefficients.

We have used modified Born theory to extend the Shock-Helgeson-Sverjensky equation of state of aqueous silica to 20 kbar and 900°C. An important requirement of this equation of state is the dielectric constant of the solvent (ε{lunate}), which has been experimentally measured only to 5 kbar and 550°C. Our extension of the Shock-Helgeson-Svetjensky equation relies, therefore, on molecular dynamics simulations which we have previously shown to reproduce the experimental data at densities between 0.25 and 1.0 g cm-3 and temperatures up to 1000°C (pressure ranging from 0.5 to 20 kbar). A combination of recent solubility measurements and of simulated values of e provide the basis for a quantitative description of the thermodynamic properties of aqueous silica over most of the range of geologic interest from 1 bar to 20 kbar and 25-900°C. © 1995.