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Disruption of KDM6A, a histone lysine demethylase, is one of the most common somatic alternations in bladder cancer. Insights into how KDM6A mutations affect the epigenetic landscape to promote carcinogenesis could help reveal potential new treatment approaches. Here, we demonstrated that KDM6A loss triggers an epigenetic switch that disrupts urothelial differentiation and induces a neoplastic state characterized by increased cell proliferation. In bladder cancer cells with intact KDM6A, FOXA1 interacted with KDM6A to activate genes instructing urothelial differentiation. KDM6A-deficient cells displayed simultaneous loss of FOXA1 target binding and genome-wide redistribution of the bZIP transcription factor ATF3, which in turn repressed FOXA1-target genes and activated cell-cycle progression genes. Importantly, ATF3 depletion reversed the cell proliferation phenotype induced by KDM6A deficiency. These data establish that KDM6A loss engenders an epigenetic state that drives tumor growth in an ATF3-dependent manner, creating a potentially targetable molecular vulnerability.Significance:A gain-of-function epigenetic switch that disrupts differentiation is triggered by inactivating KDM6A mutations in bladder cancer and can serve as a potential target for novel therapies.

The ketogenic diet (KD) is hypothesized to impact tumor progression by altering tumor metabolism. In this study, we assessed the impact of an unrestricted KD on epithelial ovarian cancer (EOC) tumor growth, gene expression, and metabolite concentration in a mouse model. ID8 EOC cells, which were syngeneic with C57Bl/6J mouse strain and transfected with luciferase (ID8-luc), were injectedand monitored for tumor development. Female mice were fed either a strict KD, a high fat/low carbohydrate (HF/LC) diet, or a low fat/high carbohydrate (LF/HC) diet (n = 10 mice per group) ad libitum. EOC tumor growth was monitored weekly, and tumor burden was determined based on luciferase fluorescence (photons/second). At the endpoint (42 days), tumors were collected and processed for RNA sequencing. Plasma and tumor metabolites were evaluated using LC-MS. The KD-fed mice exhibited a statistically significant increase in tumor progression in comparison to the HF/LC- and LF/HC-fed groups (9.1 vs. 2.0 vs. 3.1-fold, respectively, p < 0.001). The EOC tumors of the KD-fed mice exhibited significant enrichment of the peroxisome proliferator-activated receptor (PPAR) signaling and fatty acid metabolism pathways based on the RNA sequencing analysis when compared to the LF/HC- and HF/LC-fed mice. Thus, unrestricted KD diet enhanced tumor progression in our mouse EOC model. KD was associated with the upregulation of fatty acid metabolism and regulation pathways, as well as enrichment of fatty acid and glutamine metabolites.

Figure S1. HMG-CoAR protein is over-expressed and its degradation dysregulated in omCRPC cell lines. Figure S2. Total cholesterol significantly increased in omCRPC cell lines. Figure S3. Cellular free-to-total cholesterol ratio minimally affected by PCa progression to castration-resistance. Figure S4. (prev. page) AMPK activity is hampered in omCRPC cells through Ser-485/491 phosphorylation by constitutively activated Akt. Figure S5. SIM and MET individual treatment dose-response plots. Figure S6. (prev. page) Synergistic quantification of combination SIM and MET treatment in C4-2B3 and C4-2B4 omCRPC cells using Chou-Talalay method. Figure S7. Viability effect of deuterium oxide on C4-2B3 and C4-2B4 omCRPC cells and standard curves for cholesterol and deuterium quantization. Figure S8. Schematic of in vivo experimental design. Figure S9. Low- and high-dose SIM and MET combination treatment inhibited C4-2B4 primary tumor growth in an in vivo model of CRPC. Figure S10. (prev. page) SIM and MET treatment does not adversely affect mouse body weight. Figure S11. SIM and MET treatment does not increase plasma alanine aminotransferase (ALT) levels. Figure S12. SIM and MET treatment does not induce pathology or injury to liver. Figure S13. SIM treatment does not induce pathology of muscle. Figure S14. SIM lactone-to-beta-hydroxyacid hydrolysis. Figure S15. (prev. page) Statin derivatization. Figure S16. SIM acid total ion chromatograph and mass spectrum. Figure S17. SIM lactone total ion chromatograph and mass spectrum. Figure S18. Rosuvastatin total ion chromatograph and mass spectrum. Figure S19. SIM acid calibration curves. Figure S20. MET and phenformin derivatization. Figure S21. MET total ion chromatograph and mass spectrum. Figure S22. Phenformin total ion chromatograph and mass spectrum. Figure S23. MET calibration curves. Figure S24. Comparable metabolic aberrations within Akt-AMPK-HMG-CoAR network observed in other in vitro models of DTXresistant, hormone-refractory bone metastatic prostate and breast cancer. Figure S25. Synergistic combination SIM and MET significantly more effective than DTX at cell viability inhibition in multiple paradigms of DTX-resistant and hormone refractory osseous metastatic cell lines of prostate and breast cancer. Figure S26. (prev. page) Synergistic quantification of combination SIM and MET in PC-3 D12 DTX-resistant bone metastatic androgen-independent PCa, MDA-MB-231 triple-negative breast cancer, and MDA-MB-231(SA) bone metastatic triple-negative breast cancer cells using Chou-Talalay method. Table S1. Primary antibodies for immunoblotting. Table S2. Concentration of SIM (microM) and MET (mM) alone or in (1:500) combination necessary for cell viability inhibition in C4-2B3 and C4-2B4 omCRPC cells. Fa, fraction of cells affected. Table S3. Concentration of SIM acid and MET in the terminal plasma and ventral prostate (VP) tissue amongst the six mouse treatment groups. Table S4. 1:500 SIM and MET combination synergistically inhibits cell viability in bone metastatic prostate and breast cancer cell lines. Table S5. Concentration of SIM (microM) and MET (mM) alone or in (1:500) combination necessary for cell viability inhibition in PC-3 D12 DTX-resistant bone metastatic androgen-independent PCa cells, MDA-MB-231 triple-negative breast cancer cells, and MDAMB-231(SA) highly bone metastatic triple-negative breast cancer cells. Supplementary Materials and Methods.

Docetaxel chemotherapy remains a standard of care for metastatic castration-resistant prostate cancer (CRPC). Docetaxel modestly increases survival, yet results in frequent occurrence of side effects and resistant disease. An alternate chemotherapy with greater efficacy and minimal side effects is needed. Acquisition of metabolic aberrations promoting increased survival and metastasis in CRPC cells includes constitutive activation of Akt, loss of adenosine monophosphate-activated protein kinase (AMPK) activity due to Ser-485/491 phosphorylation, and overexpression of 3-hydroxy-3-methylglutaryl–Coenzyme A reductase (HMG-CoAR). We report that combination of simvastatin and metformin, within pharmacologic dose range (500 nmol/L to 4 μmol/L simvastatin and 250 μmol/L to 2 mmol/L metformin), significantly and synergistically reduces C4-2B3/B4 CRPC cell viability and metastatic properties, with minimal adverse effects on normal prostate epithelial cells. Combination of simvastatin and metformin decreased Akt Ser-473 and Thr-308 phosphorylation and AMPKα Ser-485/491 phosphorylation; increased Thr-172 phosphorylation and AMPKα activity, as assessed by increased Ser-79 and Ser-872 phosphorylation of acetyl-CoA carboxylase and HMG-CoAR, respectively; decreased HMG-CoAR activity; and reduced total cellular cholesterol and its synthesis in both cell lines. Studies of C4-2B4 orthotopic NCr-nu/nu mice further demonstrated that combination of simvastatin and metformin (3.5–7.0 μg/g body weight simvastatin and 175–350 μg/g body weight metformin) daily by oral gavage over a 9-week period significantly inhibited primary ventral prostate tumor formation, cachexia, bone metastasis, and biochemical failure more effectively than 24 μg/g body weight docetaxel intraperitoneally injected every 3 weeks, 7.0 μg/g/day simvastatin, or 350 μg/g/day metformin treatment alone, with significantly less toxicity and mortality than docetaxel, establishing combination of simvastatin and metformin as a promising chemotherapeutic alternative for metastatic CRPC. Mol Cancer Ther; 13(10); 2288–302. ©2014 AACR.

Supplementary Figure 4

Supplementary Fig S4. FACT is involved in the expression of regulatory stem cell transcription factors in TICs. A and B, CD44high cells derived from H446 and CD133high cells derived from H526 were treated with 500 nM (H446) or 1µM (H526) CBL0137 for 24 h. The mRNA levels of SOX2 were analyzed by qPCR (A), and SOX2 protein levels were determined by Western analysis (B).

Mass spectrometric analysis of proteins that bound to B-CCF642 in MM1.S cells

Disruption of KDM6A, a histone lysine demethylase, is one of the most common somatic alternations in bladder cancer. Insights into how KDM6A mutations affect the epigenetic landscape to promote carcinogenesis could help reveal potential new treatment approaches. Here, we demonstrated that KDM6A loss triggers an epigenetic switch that disrupts urothelial differentiation and induces a neoplastic state characterized by increased cell proliferation. In bladder cancer cells with intact KDM6A, FOXA1 interacted with KDM6A to activate genes instructing urothelial differentiation. KDM6A-deficient cells displayed simultaneous loss of FOXA1 target binding and genome-wide redistribution of the bZIP transcription factor ATF3, which in turn repressed FOXA1-target genes and activated cell-cycle progression genes. Importantly, ATF3 depletion reversed the cell proliferation phenotype induced by KDM6A deficiency. These data establish that KDM6A loss engenders an epigenetic state that drives tumor growth in an ATF3-dependent manner, creating a potentially targetable molecular vulnerability.Significance:A gain-of-function epigenetic switch that disrupts differentiation is triggered by inactivating KDM6A mutations in bladder cancer and can serve as a potential target for novel therapies.

Supplementary Figure 3

This Figure reports Kaplan-Meier plot correlating survival of 3951 breast cancer patients with CSF-1 expression levels

S2: Inactivation of bortezomib by liver homogenate; S3: CCF642 effect on survival of normal bone marrow, myeloid neoplasia, myeloma and lymphoma cells; S4: Biotin-stained Western blot after treatment of MM1.S cells with B-CCF642

ATF3 target genes downregulated in K2 that harbor FOXA1 peaks in RT4

Reagent and chemical library screening details, liver homogenate preparation, CCF642 animal treatment formulation, chemical synthesis schemes, details of proteomic analysis, PDI activity assay and PDI mutant primer sequences

Supplementary Figure 5

Supplementary Figure 7

Supplementary Table S1

Legends to Supplementary Figures S1-4

Supplementary Figure 2

Multiple myeloma cells secrete more disulfide bond–rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell–based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrow–sparing compound, exhibited a submicromolar IC50 in 10 of 10 multiple myeloma cell lines. An active biotinylated analog of CCF642 defined binding to the PDI isoenzymes A1, A3, and A4 in MM cells. In vitro, CCF642 inhibited PDI reductase activity about 100-fold more potently than the structurally distinct established inhibitors PACMA 31 and LOC14. Computational modeling suggested a novel covalent binding mode in active-site CGHCK motifs. Remarkably, without any further chemistry optimization, CCF642 displayed potent efficacy in an aggressive syngeneic mouse model of multiple myeloma and prolonged the lifespan of C57BL/KaLwRij mice engrafted with 5TGM1-luc myeloma, an effect comparable to the first-line multiple myeloma therapeutic bortezomib. Consistent with PDI inhibition, CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis-inducing calcium release. Overall, our results provide an illustration of the utility of simple in vivo simulations as part of a drug discovery effort, along with a sound preclinical rationale to develop a new small-molecule therapeutic to treat multiple myeloma. Cancer Res; 76(11); 3340–50. ©2016 AACR.

Structure, H-NMR and HRMS of CCF642, B-CCF642 and CCF642-COOH

List of TCGA cases used in ATAC-seq analysis

Interplay between tumor cells and host cells in the tumor microenvironment dictates the development of all cancers. In breast cancer, malignant cells educate host macrophages to adopt a protumorigenic phenotype. In this study, we show how the integrin-regulatory protein kindlin-2 (FERMT2) promotes metastatic progression of breast cancer through the recruitment and subversion of host macrophages. Kindlin-2 expression was elevated in breast cancer biopsy tissues where its levels correlated with reduced patient survival. On the basis of these observations, we used CRISPR/Cas9 technology to ablate Kindlin-2 expression in human MDA-MB-231 and murine 4T1 breast cancer cells. Kindlin-2 deficiency inhibited invasive and migratory properties in vitro without affecting proliferation rates. However, in vivo tumor outgrowth was inhibited by >80% in a manner associated with reduced macrophage infiltration and secretion of the macrophage attractant and growth factor colony-stimulating factor-1 (CSF-1). The observed loss of CSF-1 appeared to be caused by a more proximal deficiency in TGFβ-dependent signaling in Kindlin-2–deficient cells. Collectively, our results illuminate a Kindlin-2/TGFβ/CSF-1 signaling axis employed by breast cancer cells to capture host macrophage functions that drive tumor progression. Cancer Res; 77(18); 5129–41. ©2017 AACR.

Supplementary Figure 1

Supplementary Figure 8

Traditional treatments of small-cell lung cancer (SCLC) with cisplatin, a standard-of-care therapy, spare the tumor-initiating cells (TIC) that mediate drug resistance. Here we report a novel therapeutic strategy that preferentially targets TICs in SCLC, in which cisplatin is combined with CBL0137, an inhibitor of the histone chaperone facilitates chromatin transcription (FACT), which is highly expressed in TICs. Combination of cisplatin and CBL0137 killed patient-derived and murine SCLC cell lines synergistically. In response to CBL0137 alone, TICs were more sensitive than non-TICs, in part, because CBL0137 increased expression of the tumor suppressor NOTCH1 by abrogating the binding of negative regulator SP3 to the NOTCH1 promoter, and in part because treatment decreased the high expression of stem cell transcription factors. The combination of cisplatin and CBL0137 greatly reduced the growth of a patient-derived xenograft in mice and also the growth of a syngeneic mouse SCLC tumor. Thus, CBL0137 can be a highly effective drug against SCLC, especially in combination with cisplatin.Significance: These findings reveal a novel therapeutic regimen for SCLC, combining cisplatin with an inhibitor that preferentially targets tumor-initiating cells. Cancer Res; 78(9); 2396–406. ©2018 AACR.

FRA-1 forms activator protein-1 complexes in association with members of the JUN family and drives gene transcription. FRA-1 has been implicated in the development of airway squamous metaplasia and is frequently overexpressed in squamous cell carcinomas of the esophagus and stomach. We and others have shown a high level of persistent induction of FRA-1 by lung carcinogens, such as cigarette smoke and asbestos, in pulmonary epithelial cells. However, the exact roles of FRA-1 in regulating lung epithelial cell growth and invasion are poorly understood. To examine this aspect, we have stably overexpressed FRA-1 in human type-II–like alveolar malignant cell line (A549) and a nonmalignant bronchial epithelial cell line (BEAS-2B). FRA-1 greatly enhanced the rate of proliferation, motility, and invasion of A549 and BEAS-2B cells. In athymic nude mice, FRA-1, but not the control vector, rapidly enhanced tumor formation and metastasis by A549 cells. In contrast, FRA-1 failed to promote tumor formation by BEAS-2B. We suggest that FRA-1 can promote motility, invasion, and anchorage-independent growth of lung epithelial cells in vitro, but is insufficient for tumor formation. [Cancer Res 2007;67(13):6204–11]

Infection of arteriovenous fistula (AV) used for hemodialysis (HD) is associated with massive bleeding, sepsis development, formation of metastatic infectious foci, and a high risk of AV loss. Urgent management of an infected AV is crucial for successful treatment and AV salvage.We present the use of partial resection as a successful method of dealing with late AV infection in two cases. In case 1, the resection was performed due to an infection of the native arteriovenous fistula (AVF) with two defects above the drainage vein aneurysms. In case 2, partial resection and replacement of the prosthetic arteriovenous fistula (AVG) were done due to an infection of HD puncture site. The AVs remained patent in both cases, with no further signs of infection postoperatively and, most importantly, without the need to use a temporary HD catheter.The establishment of a new AV is limited by the quality of the venous and arterial systems. All surgical, interventional and non-surgical means should be used to safely maintain the created AV patent. Provided that the requirements of an early indication are met, partial resection of the AV is the method of choice for AV infections and allows us to avoid using a permanent dialysis catheter in our patients.