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Supplemental Figure 1. Gene set enrichment analysis. Supplemental Figure 2. TCF21 expression analysis. Supplemental Figure 3. TCF21 expression in cell lines. Supplemental Figure 4. Loss-of-function and gain-of-function studies of TCF21 Supplemental Figure 5. CD24 and CD44 expression in TCF21 OE UM-UC3 and UM-UC13 cells. Supplemental Figure 6. GSEA analysis of common pathways altered between the three cell lines. Supplemental Figure 7. Heatmap of hierarchical clustering of luminal markers showing differential expression after TCF21 upregulation in UC13 LN (A) and UC13 MET (B) cells.

Supplementary Figures S1-S3 from Effects of mTOR Inhibitor Everolimus (RAD001) on Bladder Cancer Cells

Purpose: We investigated the effect of the mTOR inhibitor RAD001 (everolimus) on human bladder cancer (BC) cells in vitro and in vivo.Experimental Design: The effect of RAD001 on the growth of UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 BC cells were assessed by crystal violet and [3H]thymidine incorporation assays. Flow cytometric cell-cycle analyses were done to measure the apoptotic cell fraction. Protein synthesis was measured using tritium-labeled leucine incorporation assays. The effects of RAD001 on the mTOR pathway were analyzed by Western blotting. To test the effects of RAD001 in vivo, UM-UC-3, UM-UC-6, and UM-UC-9 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with RAD001 or placebo. Tumors were harvested for immunohistochemical analysis.Results:In vitro, RAD001 transiently inhibited BC cell growth in a dose-dependent manner. This effect was augmented by re-treatment of cells after 3 days. UM-UC-14 cells were the most sensitive to RAD001, whereas UM-UC-9 cells were the least sensitive. After re-treatment with RAD001, only sensitive cell lines showed G1-phase arrest, with no evidence of apoptosis. RAD001 significantly inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of protein synthesis through the S6K and 4EBP1 pathways seems to be the main mechanism for the RAD001-induced growth inhibition. However, inhibition of angiogenesis was the predominant mechanism of the effect of RAD001 on UM-UC-9 cells.Conclusions: The mTOR inhibitor RAD001 inhibits growth of BC cells in vitro. RAD001 is effective in treating BC tumors in an in vivo nude mouse model despite the heterogeneity of in vitro responses. Clin Cancer Res; 17(9); 2863–73. ©2011 AACR.

Supplementary Figure 1 from Responses in Mantle Cell Lymphoma Cells to SNS-032 Depend on the Biological Context of Each Cell Line

SNS-032 is a potent inhibitor of cyclin-dependent kinases (Cdk) 2, 7, and 9 that regulate the cell cycle and transcription. Our studies in indolent primary chronic lymphocytic leukemia cells showed that SNS-032 inhibited transcription, diminished the antiapoptotic protein Mcl-1, and induced apoptosis. The present study focuses on evaluating this compound in four proliferating mantle cell lymphoma lines (Jeko-1, Granta 519, Mino, and SP-53). Consistent with its action against Cdk9 and Cdk7, SNS-032 inhibited the phosphorylation of RNA pol II in all four lines and blocked RNA synthesis. The transcripts and protein levels of short-lived proteins decreased, including cyclin D1 and Mcl-1. Cell growth was inhibited in a concentration-dependent manner in all lines. Apoptosis was induced in JeKo-1, Mino, and SP-53 cells without disrupting cell cycle distribution. However, apoptosis was limited in Granta cells; rather, there was a significant reduction of clonogenic survival. Small interfering RNA was used to specifically knock down Mcl-1 and cyclin D1 in JeKo-1 and Granta cells. Knocking down Mcl-1 induced significant apoptosis in Jeko-1 cells but not Granta cells. Reducing cyclin D1, rather than Mcl-1, was associated with loss of clonogenic survival in Granta cells. Thus, these results indicated that mantle cell lymphoma cell lines have distinct mechanisms sustaining their survival, and the mechanism of action of SNS-032 is dependent on the biological context of an individual line. Cancer Res; 70(16); 6587–97. ©2010 AACR.

Aim: Multiple myeloma (MM) is a hematological malignancy of antibody-producing mature B cells or plasma cells. The proteasome inhibitor, bortezomib, was the first-in-class compound to be FDA approved for MM and is frequently utilized in induction therapy. However, bortezomib refractory disease is a major clinical concern, and the efficacy of the pan-histone deacetylase inhibitor (HDACi), panobinostat, in bortezomib refractory disease indicates that HDAC targeting is a viable strategy. Here, we utilized isogenic bortezomib resistant models to profile HDAC expression and define baseline and HDACi-induced expression patterns of individual HDAC family members in sensitive vs. resistant cells to better understanding the potential for targeting these enzymes. Methods: Gene expression of HDAC family members in two sets of isogenic bortezomib sensitive or resistant myeloma cell lines was examined. These cell lines were subsequently treated with HDAC inhibitors: panobinostat or vorinostat, and HDAC expression was evaluated. CRISPR/Cas9 knockdown and pharmacological inhibition of specific HDAC family members were conducted. Results: Interestingly, HDAC6 and HDAC7 were significantly upregulated and downregulated, respectively, in bortezomib-resistant cells. Panobinostat was effective at inducing cell death in these lines and modulated HDAC expression in cell lines and patient samples. Knockdown of HDAC7 inhibited cell growth while pharmacologically inhibiting HDAC6 augmented cell death by panobinostat. Conclusion: Our data revealed heterogeneous expression of individual HDACs in bortezomib sensitive vs. resistant isogenic cell lines and patient samples treated with panobinostat. Cumulatively our findings highlight distinct roles for HDAC6 and HDAC7 in regulating cell death in the context of bortezomib resistance.

Acute myeloid leukemia (AML) with a mutation in the fms-like tyrosine kinase receptor 3 gene (FLT3) confers poor survival for pediatric patients, indicating a significant need to improve treatment efficacy. A significant percentage of pediatric patients consume diets high in fat and sugar content. These macronutrients modulate oxidative stress yet their impact on AML treatment is unexplored. Studies have implicated a role for BMAL1 rhythmicity, a circadian clock protein, in leukemogenesis. However, BMAL1 has not been studied in FLT3 mutant AML. Given that both nutrition and circadian rhythmicity can be modulated by behavior, we hypothesized that modulating redox and BMAL1 status through diet composition and timing would enhance treatment efficacy in an orthotopic mouse model. In all in vivo experiments, mice were injected via tail vein with 100,000 MOLM13 luciferase labelled cells, a FLT3 AML human cell line. After engraftment was confirmed 3 days later, mice were put on diet modification regimens. In three subsequent experiments, distinct diet interventions were delivered: low fat (30%)/low sucrose (10%), low sucrose alone (10%), or an isocaloric time feeding regimen (TRF) restricted to mice’s active phase (7 PM to 7 AM). Standard chow, high sucrose diet (45%) and ad libitum feeding were controls, respectively. In each experiment, doxorubicin (2 mg/kg) or PBS was injected twice weekly via tail vein. Tumor burden was monitored via bioluminescent imaging. Reactive oxygen species (ROS) levels were assessed with flow cytometry, and changes in circadian, redox, and apoptotic protein expression were measured by western blot and mass cytometry. On day 17, in the presence of doxorubicin, mice on a low fat/low sucrose diet showed significantly lower tumor burden compared to control. Doxorubicin induced oxidative stress was reduced in the mice fed the modified diet as demonstrated by a decrease in ROS levels in peripheral blood mononuclear cells. Even in the absence of doxorubicin treatment, the modified diet group showed significantly lower tumor burden on day 17. For mice on a low sucrose diet, changes in redox and circadian protein expression were observed on day 17. Of note, total BMAL1 protein showed a two-fold increase in spleen from the low sucrose group. In the timed feeding experiments, we observed TRF mice had significantly reduced leukemic burden on day 14. Importantly, doxorubicin efficacy was not compromised by TRF. In addition, a significant increase in total BMAL1 protein expression was observed in liver from these TRF mice on day 14. These results point to a novel finding: diet composition and timing slows progression of FLT3 AML growth in vivo and are accompanied by BMAL1 modulation. In future studies, we aim to further define the role of circadian rhythmicity and diet composition in AML progression and identify methods to complement treatment to positively impact survival outcomes for patients diagnosed with AML. Citation Format: Megan Rodriguez, Mary Figueroa, Brianna Murphy, Tiewei Cheng, Baharan Fekry, Kendra Allton, Michelle Barton, Marina Konopleva, Kristin Mahan, Joya Chandra. Effects of diet composition and timing on treatment for FLT3 positive acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 726.

Little is known regarding the subclone evolution process in advanced bladder cancer, particularly with respect to the genomic alterations that lead to the development of metastatic lesions. In this project, we identify gene expression signatures associated with metastatic bladder cancer through mRNA expression profiling of RNA isolated from 33 primary bladder cancer and corresponding lymph node (LN) metastasis samples. Gene expression profiling (GEP) was performed on RNA isolated using the Illumina DASL platform. We identified the developmental transcription factor TCF21 as being significantly higher in primary bladder cancer compared with LN metastasis samples. To elucidate its function in bladder cancer, loss- and gain-of-function experiments were conducted in bladder cancer cell lines with high and low expression of TCF21, respectively. We also performed GEP in bladder cancer cell lines following TCF21 overexpression. We identified 2,390 genes differentially expressed in primary bladder cancer and corresponding LN metastasis pairs at an FDR cutoff of 0.1 and a fold change of 1. Among those significantly altered, expression of TCF21 was higher in the primary tumor compared with LN metastasis. We validated this finding with qPCR and IHC on patient samples. Moreover, TCF21 expression was higher in luminal cell lines and knockdown of TCF21 increased invasion, tumor cell dissemination, and metastasis. In contrast, overexpression of TCF21 in highly metastatic basal bladder cancer cell lines decreased their invasive and metastatic potential. Implications: TCF21 is differentially overexpressed in primary bladder cancer compared with matched LN metastasis, with in vitro and in vivo studies demonstrating a metastasis suppressor function of this transcription factor.

PURPOSE: Amongst the epigenetically-targeted therapies, targeting of the histone deacetylases (HDACs) has yielded numerous drugs for clinical use in hematological malignancies, but none as yet for acute lymphocytic leukemia (ALL). Single agent activity of HDAC inhibitors (HDACi) has been elusive in ALL, and has prompted study of combinatorial strategies. Because several HDACi raise levels of intracellular oxidative stress, we evaluated combinations of two structurally distinct HDACi with the redox active compound adaphostin in acute lymphocytic leukemia (ALL). METHODS: The HDACi vorinostat and entinostat were tested in combination with adaphostin in human ALL cell lines. DNA fragmentation, caspase activation, mitochondrial disruption and levels intracellular peroxides, superoxide and glutathione were measured in cells treated with the HDACi/adaphostin combinations. Antioxidant blockade of cell death induction and gene expression profiling of cells treated with vorinostat/adaphostin versus entinostat/adaphostin combinations was evaluated. RESULTS: Both combinations synergistically induced apoptotic DNA fragmentation, which was preceded by an increase in superoxide levels, a reduction in mitochondrial membrane potential, and an increase in caspase-9 activation. The antioxidant N-acetylcysteine (NAC) blocked superoxide generation and prevented reduction of mitochondrial membrane potential. NAC decreased DNA fragmentation and caspase activity in cells treated with adaphostin and vorinostat, but not in those treated with adaphostin and entinostat. Gene expression arrays revealed differential regulation of several redox genes prior to cell death induction. CONCLUSIONS: A redox modulatory agent, adaphostin, enhances efficacy of two HDACi, vorinostat or entinostat, but via different mechanisms indicating a point of divergence in the mechanisms of synergy between the two distinct HDACi and adaphostin.

Epithelial-to-mesenchymal transition (EMT) has been implicated in the progression of bladder cancer. To study its contribution to bladder cancer metastasis, we established new xenograft models derived from human bladder cancer cell lines utilizing an orthotopic "recycling" technique that allowed us to isolate and examine the primary tumor and its corresponding circulating tumor cells (CTC's) and metastatic lesions. Using whole genome mRNA expression profiling, we found that a reversible epithelial-to-mesenchymal transition (EMT) characterized by TGFβ pathway activation and SNAIL expression was associated with the accumulation of CTCs. Finally, we observed that conditional silencing of SNAIL completely blocked CTC production and regional/distant metastasis. Using this unique bladder cancer xenograft model, we conclude that metastasis is dependent on a reversible EMT mediated by SNAIL.

SummaryMuscle-invasive bladder cancers (MIBCs) are biologically heterogeneous and have widely variable clinical outcomes and responses to conventional chemotherapy. We discovered three molecular subtypes of MIBC that resembled established molecular subtypes of breast cancer. Basal MIBCs shared biomarkers with basal breast cancers and were characterized by p63 activation, squamous differentiation, and more aggressive disease at presentation. Luminal MIBCs contained features of active PPARγ and estrogen receptor transcription and were enriched with activating FGFR3 mutations and potential FGFR inhibitor sensitivity. p53-like MIBCs were consistently resistant to neoadjuvant methotrexate, vinblastine, doxorubicin and cisplatin chemotherapy, and all chemoresistant tumors adopted a p53-like phenotype after therapy. Our observations have important implications for prognostication, the future clinical development of targeted agents, and disease management with conventional chemotherapy.

Purpose: We investigated the effect of the mTOR inhibitor RAD001 (everolimus) on human bladder cancer (BC) cells in vitro and in vivo. Experimental Design: The effect of RAD001 on the growth of UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 BC cells were assessed by crystal violet and [3H]thymidine incorporation assays. Flow cytometric cell-cycle analyses were done to measure the apoptotic cell fraction. Protein synthesis was measured using tritium-labeled leucine incorporation assays. The effects of RAD001 on the mTOR pathway were analyzed by Western blotting. To test the effects of RAD001 in vivo, UM-UC-3, UM-UC-6, and UM-UC-9 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with RAD001 or placebo. Tumors were harvested for immunohistochemical analysis. Results: In vitro, RAD001 transiently inhibited BC cell growth in a dose-dependent manner. This effect was augmented by re-treatment of cells after 3 days. UM-UC-14 cells were the most sensitive to RAD001, whereas UM-UC-9 cells were the least sensitive. After re-treatment with RAD001, only sensitive cell lines showed G1-phase arrest, with no evidence of apoptosis. RAD001 significantly inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of protein synthesis through the S6K and 4EBP1 pathways seems to be the main mechanism for the RAD001-induced growth inhibition. However, inhibition of angiogenesis was the predominant mechanism of the effect of RAD001 on UM-UC-9 cells. Conclusions: The mTOR inhibitor RAD001 inhibits growth of BC cells in vitro. RAD001 is effective in treating BC tumors in an in vivo nude mouse model despite the heterogeneity of in vitro responses. Clin Cancer Res; 17(9); 2863–73. ©2011 AACR.

Histone deacetylase inhibitors (HDACis) have emerged as novel therapeutic agents for cancer. Currently, four HDACis are approved by the Food and Drug Administration (FDA) to treat various hematologic malignancies. Panobinostat (LBH-589, trade name Farydak®, developed and marketed by Novartis) is a potent pan-HDACi with demonstrated anticancer activities against multiple myeloma, a B-cell malignancy, at a low nanomolar range in preclinical settings, and in 2015 was granted FDA approval for the treatment of relapsed and refractory multiple myeloma. Here, we review the development of HDACis, the unique features of panobinostat, and the rationale for developing panobinostat in a combination setting for the treatment of multiple myeloma. We also review the completed and ongoing clinical trials testing the efficacy of panobinostat in combination therapies and highlight future therapeutically relevant strategies.

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Fibroblast growth factor receptors (FGFRs) are activated by mutation and overexpressed in bladder cancers (BCs), and FGFR inhibitors are currently being evaluated in clinical trials in BC patients. However, BC cells display marked heterogeneity in their responses to FGFR inhibitors, and the biological mechanisms underlying this heterogeneity are not well defined. Here we used a novel inhibitor of FGFRs 1-3 and RNAi to determine the effects of inhibiting FGFR1 or FGFR3 in a panel of human BC cell lines. We observed that FGFR1 was expressed in BC cells that also expressed the "mesenchymal" markers ZEB1 and vimentin, whereas FGFR3 expression was restricted to the E-cadherin- and p63-positive "epithelial" subset. Sensitivity to the growth-inhibitory effects of BGJ-398 was also restricted to the "epithelial" BC cells and it correlated directly with FGFR3 mRNA levels but not with the presence of activating FGFR3 mutations. In contrast, BGJ-398 did not strongly inhibit proliferation but did block invasion in the "mesenchymal" BC cells in vitro. Similarly, BGJ-398 did not inhibit primary tumor growth but blocked the production of circulating tumor cells (CTCs) and the formation of lymph node and distant metastases in mice bearing orthotopically implanted "mesenchymal" UM-UC3 cells. Together, our data demonstrate that FGFR1 and FGFR3 have largely non-overlapping roles in regulating invasion/metastasis and proliferation in distinct "mesenchymal" and "epithelial" subsets of human BC cells. The results suggest that the tumor EMT phenotype will be an important determinant of the biological effects of FGFR inhibitors in patients.

SNS-032 is a potent inhibitor of cyclin-dependent kinases (Cdk) 2, 7, and 9 that regulate the cell cycle and transcription. Our studies in indolent primary chronic lymphocytic leukemia cells showed that SNS-032 inhibited transcription, diminished the antiapoptotic protein Mcl-1, and induced apoptosis. The present study focuses on evaluating this compound in four proliferating mantle cell lymphoma lines (Jeko-1, Granta 519, Mino, and SP-53). Consistent with its action against Cdk9 and Cdk7, SNS-032 inhibited the phosphorylation of RNA pol II in all four lines and blocked RNA synthesis. The transcripts and protein levels of short-lived proteins decreased, including cyclin D1 and Mcl-1. Cell growth was inhibited in a concentration-dependent manner in all lines. Apoptosis was induced in JeKo-1, Mino, and SP-53 cells without disrupting cell cycle distribution. However, apoptosis was limited in Granta cells; rather, there was a significant reduction of clonogenic survival. Small interfering RNA was used to specifically knock down Mcl-1 and cyclin D1 in JeKo-1 and Granta cells. Knocking down Mcl-1 induced significant apoptosis in Jeko-1 cells but not Granta cells. Reducing cyclin D1, rather than Mcl-1, was associated with loss of clonogenic survival in Granta cells. Thus, these results indicated that mantle cell lymphoma cell lines have distinct mechanisms sustaining their survival, and the mechanism of action of SNS-032 is dependent on the biological context of an individual line. Cancer Res; 70(16); 6587–97. ©2010 AACR.

Overexpression of TGFbeta inducible early gene (TIEG1) mimics TGFbeta action and induces apoptosis. In this study, we found that TIEG1 was significantly up-regulated during apoptosis induced by homoharringtonine or velcade. Overexpression of TIEG1 could induce apoptosis in K562 cells and promote apoptosis induced by HHT or velcade. TIEG1-induced apoptosis was shown to involve Bax and Bim up-regulation, Bcl-2 and Bcl-XL down-regulation, release of cytochrome c from mitochondria into the cytosol, activation of caspase 3 and disruption of the mitochondrial membrane potential (DeltaPsim). We concluded that TIEG1 is a key regulator which induces and promotes apoptosis through the mitochondrial apoptotic pathway.

4538 Background: Muscle-invasive bladder cancers (MIBCs) are a heterogeneous group of tumors that display widely variable clinical outcomes and responses to conventional chemotherapy. Methods: We used whole genome mRNA expression profiling and unsupervised hierarchical cluster analyses on a cohort of 73 flash frozen primary tumors to identify 3 distinct subsets of muscle-invasive bladder cancer (MIBC). We confirmed the existence of these 3 subsets in a second cohort of 57 formalin-fixed, paraffin-embedded (FFPE) MIBCs and in 2 other public datasets. Analysis of primary tumors and mechanistic studies in human bladder cancer cell lines identified tumors that respond to FGFR inhibitors or chemotherapy. Results: The first subset was driven by an active "basal" EGFR-STAT3-p63 transcriptional network, and was associated with poor clinical outcomes. High miR-200c expression stratified the survival of these basal tumors. The second subset was characterized by active p53 pathway activation, and tumors and cell lines with these features were resistant to cis-platinum based chemotherapy. The third subset expressed "luminal" markers and active estrogen receptor (ER) and PPARγ signaling, and luminal cell lines were sensitive to fibroblast growth factor receptor (FGFR) inhibition. Conclusions: Molecular subtyping of MIBCs can be used to identify lethal cancers and enrich for tumors that will respond to FGFR inhibitors or conventional chemotherapy.

Activating mutations in the type-3 receptor of fibroblast growth factors (FGFR3) are present in a large subset of human bladder cancers. In addition, the epidermal growth factor receptor (EGFR) is overexpressed as a function of progression in bladder cancer, and EGFR inhibitors are being evaluated in Phase II clinical trials. However, in previous studies we found that human bladder cancer cells display marked heterogeneity in their sensitivities to EGFR inhibitors and demonstrated that epithelial-to-mesenchymal transition (EMT) is involved in resistance. Nonetheless, many “epithelial” bladder cancer cells are resistant to EGFR inhibitors, suggesting that proliferation in these cells may be driven by another growth factor receptor. To more precisely define this biological heterogeneity, we screened a panel of human urothelial cancer cells using small molecule EGFR or FGFR inhibitors and correlated responses to baseline whole genome mRNA expression profiles. A panel of 25 human urothelial cancer cells was tested for the anti-proliferative effects of AZD4547 by MTT. We identified 5 cell lines(RT4, RT112, UM-UC1, UM-UC14, SW780) strongly inhibited by ≤ 1 μM of the drug. Similarly, we identified 4 cell lines (UM-UC4, UM-UC5, UM-UC9, and UM-UC16) were sensitive to ≤ 1 μM gefitinib. Strikingly, cells displayed mutually exclusive sensitivities to AZD4547 or gefitinib, and combinations of the drugs didn't further increase growth arrest. Furthermore, AZD4547 sensitivity was not closely linked to FGFR3 mutational status: 4/5 of the sensitive cell lines expressed wild-type FGFR3, and gefitinib-sensitive UM-UC16 cells expressed an activating (S249C) mutation. All cells sensitive to either AZD4547 or gefitinib were confined to the “epithelial” subgroup which was characterized by high expression of E-cadherin and low expression of Zeb-1. Using Illumina whole genome arrays to comparing gene expression profile between FGFR sensitive and EGFR sensitive cells, we found that active PPAR signaling, particularly high baseline expression of the PPAR target gene FABP4, characterized the FGFR3-dependent cells. We further investigated whether PPAR could regulate FABP4 expression in FGFR dependent cells through knocking down specific genes of interests by siRNA. We found that among three isoforms of PPARs, PPARδ has more effect than PPARδ, ≤ to down-regulate FABP4 in UM-UC14 and RT4. In summary, sensitivity to the anti-proliferative effects of EGFR and FGFR inhibitors is confined to the “epithelial” subset of human bladder cancer cells. Within the epithelial subset, sensitivity to EGFR or FGFR inhibitors is mutually exclusive. Mutational activation of FGFR3 doesn't correlate well with sensitivity to FGFR inhibition. Rather, FGFR-dependent cells express high levels of FABP4 and other genes associated with PPAR signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4578. doi:1538-7445.AM2012-4578