Somatic mutations in the VHL gene, coupled with VHL loss of heterozygosity, drive sporadic clear cell renal cell carcinoma (ccRCC). From structural considerations, we hypothesized that certain mutations in the VHL gene thermodynamically destabilize the folded protein product; these mutations would increase the ratio of unfolded:folded pVHL, and thus cause loss of function. To test this, we used computational structure-based screening followed by biophysical characterization and cellular assays to identify small molecules that bind to the folded (native) conformation of pVHL and stabilize it. These studies led to creation of an agent, CP4.29, that stabilizes the native folded structure of mutant pVHL: this in turn restores wild-type pVHL activities to cells that harbor mutant VHL. These compounds may serve as starting points for further development into an unprecedented new class of kidney cancer drugs. The approach described herein may also serve as a blueprint for developing agents to correct destabilized mutations underlying other human diseases.

Von Hippel-Lindau disease (VHLD) is a rare autosomal dominant disease, occuring in 1 in 35,000 individuals. Overall, individuals with inherited mutations in the VHL tumor suppressor gene are predisposed to a variety of cancer, including high frequencies of clear cell renal cell carcinoma (ccRCC), pancreatic neuroendocrine tumors, and hemangioblastomas, as well as benign cystic conditions and other cancers. The degree of risk for each of these pathological conditions depends on the location and severity of the inherited germline mutation, and the specific VHL protein interactions and functions disrupted. A core VHL protein function is as the targeting subunit of an E3 ligase complex, with protein degradation activity based on interactions with elongins (ELOB, ELOC), Cullin 2 (CUL2), and RBX1. For ccRCC and some other cancers, loss of VHL-dependent degradation of key substrates—the transcription factors hypoxia-inducible factor alpha (HIF-1α and HIF-2α)—and upregulation of HIF-dependent transcripts are critical to promote tumor formation. For this reason, drugs such as the HIF signaling inhibitor belzutifan have emerged as promising clinical agents for treatment of VHLD patients prone to ccRCC. However, other biological consequences of VHL loss are independent of HIFα degradation, and in some cases independent of the VHL ubiquitin ligase activity. Non-canonical activities of VHL include regulation of microtubule stability, mitotic progression, and ciliation, as well as formation of the extracellular matrix (ECM); the degree to which disruption of these activities contributes to VHLD is currently not well understood. This review provides a concise update of the current literature on VHLD pathogenesis, the relationship of VHL structure and protein interactions to the spectrum of phenotypes associated with VHLD, and current and proposed treatment, prevention, and interception of cancer formation for VHLD patients.

BACKGROUND:Osimertinib is a standard therapy for EGFR-mutant NSCLC. However, markers to better identify those at risk for poor outcomes are needed. This is the largest study to date evaluating the impact of EGFR subtype combined with TP53 co-mutation status on survival endpoints with front-line osimertinib. METHODS:Patients from a U.S. clinical-genomic database with advanced EGFR-mutant NSCLC receiving front-line osimertinib were studied. Real-world progression-free survival (rwPFS) and overall survival (OS) were determined using Kaplan-Meier methods, and multivariable Cox regression compared outcomes after accounting for relevant clinical covariates. RESULTS:Of 606 patients, 277 (46%) had EGFR L858R, 384 (63%) had TP53 co-mutations, and 186 (30.7%) had both. Bearing L858R vs. exon 19 deletions (rwPFS: hazard ratio [HR] 1.4, P = 0.001; OS: HR 1.3, P = 0.01) or a TP53 co-mutation vs. wildtype (rwPFS: HR 1.5, P < 0.001; OS: HR 1.6, P < 0.001) predicted inferior outcomes. Especially short median rwPFS (10.1 vs. 21.4 months, HR 2.2, P < 0.001) and OS (21.3 vs. 53.4 months, HR 2.3, P < 0.001) were observed in patients with both markers (L858R/TP53-mutant) as compared to neither (exon 19 deletion/TP53-wildtype). CONCLUSIONS:Having EGFR L858R or a TP53 co-mutation were independent predictors of inferior rwPFS and OS with front-line osimertinib. Patients with both unfavorable alterations had the shortest survival. Risk stratifying using a combination of these markers can assist in identifying patients for novel trials or approved intensified therapies.

Abstract Incomplete tumor resection remains a primary driver of cancer recurrence and would benefit from technologies that enable both accurate tumor delineation and localized ablation of residual disease. Here, we report a compositionally tunable nanotheranostic platform based on sub-50 nm (∼40–50 nm) poly(ethylene glycol)-block-poly(caprolactone) (PEG-PCL) nanoscale micelles coloaded with phthalocyanine (Pc) and naphthalocyanine (Nc) dyes, designed for integrated photoacoustic (PA) imaging, direct visual guidance, and photothermal therapy (PTT). By incorporating a tetra-tert-butyl-substituted zinc phthalocyanine (ZnPc(TB)) and an octabutoxy-substituted copper naphthalocyanine (CuNc(Octa)) into micelles via both coencapsulation and postformulation mixing, we achieved an optimized 80:20 ZnPc(TB):CuNc(Octa) ratio that balances blue tumor coloration with high-contrast PA signals and efficient photothermal conversion. In vivo evaluation in orthotopic triple-negative breast cancer and spontaneous colorectal tumor models demonstrated that these micelles accumulate sufficiently in tumors to enable PA mapping, followed by real-time visual identification of tumor margins under white light, and efficient photothermal ablation. Notably, no difference was observed between the coencapsulated and mixed formulations. Comprehensive hematologic analysis confirmed the platform’s safety, with observed shifts attributable to procedural stress and tumor-associated physiology rather than to treatment-specific effects. These findings establish multichromophore Pc/Nc micelles as a versatile platform in which optical signatures can be rationally tuned to bridge the gap between deep-tissue detection, intraoperative guidance, and tumor eradication, offering a nanoscale, translatable strategy for applications such as intraoperative surgical margin assessment during breast-conserving surgery and endoscopic identification and ablation of colorectal adenomas.

While the emergence of checkpoint inhibitors has changed the treatment landscape of non-small cell lung cancer (NSCLC), most never-smokers derive little benefit. This lack of benefit is due to many never-smokers possessing oncogene-driven cancers (e.g. EGFR, ALK, ROS1, etc.) that have “cold” tumor microenvironments (TME) that poorly respond to anti-PD-1(-L1). However, being female and young are also associated with inferior responses to immunotherapy in several cancer types, possibly due to these patients having more endogenous hormones that may be immunosuppressive. To our knowledge, the impact of gender and age on the efficacy of anti-PD-1(-L1) has not been studied in NSCLCs that occur in never-smokers. Such an investigation is particularly relevant given the frequency of this diagnosis in young women. A nationwide de-identified EHR-derived database (Flatiron Health) was used to evaluate patients without a smoking history with advanced NSCLC treated with single-agent anti-PD-1(-L1). De-identified data originated from approximately 280 US cancer clinics (∼800 sites of care). Kaplan-Meier curves estimated rwPFS (based on clinician documentation of progression) in months (m) with gender and age comparisons evaluated using the log-rank test and multivariable Cox regression. In our cohort of 1535 patients, 1029 (67%) were female, 836 (55%) had a tumor oncogene, 144 (9%) were <55 years of age (yoa), 782 (51%) were 55-75 yoa, and 609 (40%) were >75 yoa. Real-world PFS did not differ between females (2.9 m, 95% CI 2.8-3.1) and males (2.8 m, 95% CI 2.7-3.2) (p=0.52). However, among female patients, those < 55 yoa had shorter rwPFS (2.1 m, 95% CI 1.9-2.7) than females 55-75 yoa (2.9 m, 95% CI 2.7-3.2) and >75 yoa (3.2 m, 95% CI 2.9-3.6) (p=0.001). In a Cox regression model that adjusted for performance status, race, therapy line, and presence of a tumor oncogene, female patients >75 yoa (HR 0.74, 95% CI 0.58-0.95) and 55-75 yoa (HR 0.82, 95% CI 0.65-1.03) were at lower risk for disease progression as compared to females < 55 yoa (reference group). Differences by age in rwPFS were not observed in males. Our analysis revealed that among women without a smoking history, young patients < 55 yoa with NSCLC experience especially poor survival with anti-PD-1(-L1). Research is needed to elucidate the biological mechanisms underlying this disparity. While data on menopausal status were not available for this analysis, most women < 55 yoa are pre- or perimenopausal. The contribution of greater levels of endogenous estrogens to immunosuppression in the lung TME warrants further study. (Supported by a Flatiron-FCCC Academic Partnership Research Grant Award) Hye Sung Kim, Li Zhang, Margie L. Clapper, Jill Hasler, Tia Nicolas, Hossein Borghaei, Martin J. Edelman, Joseph Treat, J. Nicholas Bodor. The impact of gender and age on real-world progression-free survival (rwPFS) in patients without a smoking history with NSCLC treated with checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3377.

Abstract Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) characterized by persistent mucosal inflammation extending throughout the colon. Due to compromised intestinal surface integrity and the prolonged use of medication, individuals with UC often develop a folate deficiency, requiring folic acid (FA) supplementation. Prior findings from this group indicate that FA supplementation, at clinically relevant levels, promotes UC-associated dysplasia. Supplementation with FA resulted in an increased multiplicity of dysplasia and a significant elevation in the expression of several inflammatory genes (e.g. IL-6, Cox-2, Iκκ-β) within the colonic mucosa of mice with UC. Genome-wide expression profiling of normal and dysplastic colonic epithelial cells from mice treated with different doses of FA revealed differentially expressed genes were enriched for members of the ERK and NF-κB pathways. Subsequent in vitro analyses, utilizing isogenic RKO colon adenocarcinoma cells exposed to various doses of FA, demonstrated the downregulation of ERK and p-ERK in wild type (WT) p53+/+ cells, while an increase in expression was observed in p53−/− cells at the protein level. The goal of this study is to investigate the role of the MAPK/ERK pathway in the development of UC-associated neoplasms. An in vitro model that faithfully reproduces the inflamed microenvironment in the context of WT vs. mutant p53, the putative gatekeeper of UC-associated carcinogenesis, is needed to enhance our understanding of early changes in the colon that are induced by FA and drive tumor formation. Colon organoids are being generated by isolating crypts from WT p53+/+ and mutant p53+/515A mice. These organoids are then co-cultured with mouse macrophages (RAW264.7 cells) in inserts to stimulate crosstalk between the macrophages and colonic epithelial cells, as in the inflamed colon. Analyses demonstrate that the macrophages express pivotal inflammatory cytokines characteristic of UC, including IL-1β, IL-10, TNF-α, and IL-6, irrespective of the presence or absence of LPS stimulation. Co-cultured organoids and macrophages will be exposed to different concentrations of FA for various lengths of time, and the impact of treatment on the levels of ERK, p-ERK, and p65 will be assessed. The ability of p-ERK to mediate the transcriptional activation of NF-κB and its downstream effector IL-6 will also be evaluated. Subsequent investigations employing this innovative experimental model will reveal the mechanisms by which FA supplementation promotes the progression of UC-associated dysplasia. Given the potential risks associated with FA supplementation in individuals with UC, there is an urgent need to translate these results to a clinical setting. (Supported by CA262551 and the Timothy and Aurora Hughes Cancer Research Fund). Citation Format: Ariane Rocha Bartolomeu, Wen-Chi Chang, Lisa Vanderveer, Kristen N. Harvey, Mitchell Cheung, Harry S. Cooper, Margie Lee Clapper. Effects of folic acid supplementation on early stages of colitis-associated carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2170.

center dot EGFR-mutant tumors with exon 19 deletions, L858R mutations, or exon 20 insertions were less likely to be PD-L1 high (TPS >= 50%) or TMB high (>= 10 mut/MB) as compared to wild-type (WT) tumors. Among EGFRmutant tumors, those with uncommon alterations in L861Q and G719X had greater rates of TMB high and TP53 co-mutations. center dot Utilizing bulk RNA sequencing data to characterize the immune tumor microenvironment (TME), exon 19 deletion and L858R mutation tumors were found to have lower median percent fractions of CD8+ T cells versus WT. However, a wide range of values for CD8+ T cells was observed and a subset of exon 19 deletion and L858R tumors had higher values comparable to WT. center dot Exon 19 deletion, L858R, and exon 20 insertion tumors were enriched with immunosuppressive M2 macrophages and neutrophils as compared to WT. In contrast, L861Q and G719X tumors had levels of M2 macrophages similar to WT. center dot A small subset of EGFR-mutant tumors, particularly those with uncommon alterations, bear characteristics that may render them more immunogenic. Continued research is needed to evaluate biomarkers, including the specific subtype of EGFR, as predictors of immunotherapy response to better identify the small subgroup of patients with EGFR-mutant disease that benefit from checkpoint inhibitors.

Abstract Introduction: The factors responsible for NSCLC development in never-smokers remain unclear, thus hindering prevention efforts. Our group is one of the first in the U.S. to build a registry of never-smokers with NSCLC to facilitate research to address this global problem. Given the high prevalence of women among cases, we are investigating the contribution of estrogen metabolism to this disease. CYP1B1 converts parent estrogens to the putative carcinogen 4-hydroxyestrogen (4-OHE), a process further accelerated by tobacco smoke exposure. In contrast, CYP1A1 generates 2-hydroxyestrogen (2-OHE), which may be converted to anti-proliferative derivatives. We determined previously that women with EGFR-mutant NSCLC enrolled in the registry have a 2-fold higher ratio of 4-OHEs to 2-OHEs, as compared to cancer-free controls. While EGFR-mutant NSCLCs are typically thought to be diagnosed in never-smokers, prior data indicate that a fraction of patients report some smoking history. The goal of this current study is to determine the smoking prevalence among women with EGFR-mutant NSCLC in our registry and explore the impact of smoking and hormonal history on estrogen metabolism. Methods: The Lung Cancer in Never-Smokers Risk Registry (LCNS-RR) is recruiting patients with NSCLC who are never-smokers (<100 cigarettes/lifetime) and/or have tumors with oncogenes characteristically present in never-smokers (e.g., EGFR, ALK). In addition to donating biospecimens (e.g., blood, urine), participants complete a survey on tobacco and environmental exposures, hormonal history, and other factors. Using UPLC-MS/MS, profiles of urinary estrogens (E1, E2, E3, 4-OHE1, 4-OHE2, 2-OHE1, 2-OHE2) were determined for 32 women with EGFR-mutant NSCLC in the LCNS-RR. The proportion of each estrogen species over total estrogen and the ratio of 4-OHEs to 2-OHEs were calculated. Medians were compared using the Wilcoxon rank-sum test. Results: Among women in this EGFR-mutant cohort, none were current smokers, 16 were former smokers, and 16 were never-smokers. Among the former smokers, median pack-years was 11.6. The ratio of 4-OHEs to 2-OHEs was comparable in former- vs. never-smokers (0.51 vs. 0.49, p-value = 0.956). Twenty-three women (71.8%) had a history of oral contraception use, and 5 (15.6%) reported early menarche (<12 years of age). However, neither of these factors correlated with 4-OHE production. Among 28 post-menopausal women, 7 (25%) reported estrogen use since menopause, an exposure that was not associated with 4-OHE levels. Conclusion: This preliminary analysis suggests that smoking history, use of hormones, or early menarche do not enhance the production of 4-OHEs in women with EGFR-mutant NSCLC. Recruitment to the LCNS-RR is ongoing and will facilitate future analyses in a larger cohort. (Supported by a Ride Hard Breathe Easy Innovation Award) Citation Format: Ahmad Raza, Joseph Treat, Daniel D. Krzizike, Lisa Vanderveer, Mitchell Cheung, Carolyn Zawislak, Lisa Etkins, Eric Ross, Douglas B. Flieder, Martin J. Edelman, Margie L. Clapper, J Nicholas Bodor. Tobacco smoke exposure, hormonal history, and estrogen metabolism in women with EGFR-mutant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 765.

Abstract There has been a consistent rise in the incidence of non-small cell lung cancer (NSCLC) among individuals who have never smoked. Remarkably, over 50% of diagnosed female cases and 15-20% of male cases are never-smokers. Notably, NSCLC in never-smokers exhibits distinctive clinical and pathological features, notably a higher incidence of tumors with EGFR mutations, setting it apart from its counterpart in smokers. Despite the improved disease-free survival observed with EGFR-tyrosine kinase inhibitors, eventual relapse and limited responsiveness to immunotherapy are common, underscoring the urgency to comprehend the molecular mechanisms driving the escalating prevalence of NSCLC in never-smokers and develop targeted preventive and interceptive therapeutic strategies. The preponderance of females among NSCLC patients who never smoked has prompted an exploration into the potential contribution of estrogen to lung tumorigenesis. In contrast to prevailing research focused on estrogen receptor-mediated signaling, our investigations have yielded compelling evidence supporting the involvement of estrogen metabolism in lung carcinogenesis among never-smokers. This group is the first to report that estrogen is metabolized extensively in both mouse and human lungs, resulting in the generation of various derivatives, including the potential carcinogen 4-hydroxyestrogen (4-OHE). Notably, recent findings from our laboratory indicate that prolonged exposure of nonneoplastic human bronchial epithelial cells to 17β estradiol or 4-OHE, at concentrations approximating physiological levels, induces the accumulation of double-strand DNA breaks and cellular transformation. A comprehensive analysis was performed to further elucidate the mechanisms underlying the transformation process. Isolated transformed clones obtained after 9, 18, and 22 wks of exposure to 4-OHE were subjected to karyotyping, F.I.S.H., and RNA and exome sequencing. A longitudinal progression of aberrant chromosomal rearrangements and copy number alterations was observed, that increased with time of 4-OHE exposure. In addition, RNA-Seq analyses revealed the epithelial-mesenchymal transition pathway was highly enriched at the early timepoint (9 wks), while replicative stress-related signaling pathways were induced at all timepoints. To determine if the differentially expressed genes were also dysregulated in clinical lung cancer cases, TCGA lung adenocarcinoma (LUAD) data were examined. Interestingly, many of the genes upregulated after estrogen exposure were similarly overexpressed in the LUAD samples and often associated with a worse prognosis. In conclusion, our data support the role of estrogen metabolites in promoting lung cancer among never-smokers. (Supported by CA217161 and generous contributions from the Hellendall Family Foundation, Gregory G. Lawton and the Timothy and Aurora Hughes Cancer Research Fund.) Citation Format: Mitchell Cheung, Lisa A. Vanderveer, Daniel Krzizike, J. Nicholas Bodor, Joseph Treat, Joseph R. Testa, Margie L. Clapper. Contribution of estrogen metabolites to never-smoking lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1467.

PURPOSE:The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. METHODS:Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). RESULTS:A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/β-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. CONCLUSIONS:High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

Perspective on this Article from Altered Gene Expression in Morphologically Normal Epithelial Cells from Heterozygous Carriers of BRCA1 or BRCA2 Mutations

ADT 061 pharmacokinetics and tissue distribution. ADT 061 was detected in plasma at a concentration of 2.8 µM 0.5 hr after a single oral administration of a dose of 100 mg/kg in 0.5% CMC/0.25% T80 in water to female C57BL/6 mice and reached plasma Cmax of 4.9 µM 4 hr after administration, which is >10 times higher than human colon cancer cell growth inhibition IC50 values. ADT 061 reached a Cmax in colon mucosa within 2 hr after oral administration (62.6 nmol/g) and was still present at concentrations exceeding IC50 values 8 hr after administration (28.9 nmol/g). ADT 061 concentrations in lungs, ovaries, and uterus (1-2 nmol/g) were appreciably lower than colon mucosa levels, while the ADT 061 concentration in the brain was nearly undetectable 2 and 8 hr post treatment (0.1 and 0.2 nmol/g, respectively) (n=4, mean {plus minus} SD).

Prior data suggest driver-mutated NSCLC, especially EGFR and ALK tumors, poorly respond to immunotherapy. However, little research using real-world cohorts have been performed, nor is it clear whether PD-L1 and smoking history are predictive of outcomes in such tumors. This study assessed rwPFS in a large cohort with driver-mutated advanced NSCLC treated with single-agent PD-1/PDL-1 inhibitors. Real-world data from 1746 patients were analyzed and rwPFS with immunotherapy was determined for EGFR, ALK, BRAF, and KRAS tumors. Kaplan–Meier curves characterized rwPFS and correlated with PD-L1 and smoking history. Comparisons were tested using log-rank. Median rwPFS and the percent progression-free at 12 months were greater among KRAS (3.3 months, 21.1

HPLC chromatogram of compound 5.

HPLC chromatogram of ADT 061

The current standard of care for colon cancer surveillance relies heavily on white light endoscopy (WLE). However, dysplastic lesions that are not visible to the naked eye are often missed when conventional WLE equipment is used. Although dye-based chromoendoscopy shows promise, current dyes cannot delineate tumor tissues from surrounding healthy tissues accurately. The goal of the present study was to screen various phthalocyanine (PC) dye-loaded micelles for their ability to improve the direct visualization of tumor tissues under white light following intravenous administration. Zinc PC (tetra-tert-butyl)-loaded micelles were identified as the optimal formulation. Their accumulation within syngeneic breast tumors led the tumors to turn dark blue in color, making them clearly visible to the naked eye. These micelles were similarly able to turn spontaneous colorectal adenomas in Apc+/Min mice a dark blue color for easy identification and could enable clinicians to more effectively detect and remove colonic polyps.

Perspective on this Article from Early Changes in Gene Expression Induced by Tobacco Smoke: Evidence for the Importance of Estrogen within Lung Tissue

Reaction steps for synthesis of ADT 061 (a) 3-(4-methoxyphenyl)-2-methylacrylic acid (compound 1): 4-Methoxybenzaldehyde (219 g, 1.61 mole), propionic anhydride (315g, 2.42 mole), and sodium propionate (155g, 1.61 mole) were stirred at 140 ºC until a clear solution was achieved (~48 h). The solution was cooled to room temperature and poured into 8L of ice water. The precipitation formed was collected by filtration, transferred into a 2L round-bottom flask, and refluxed in 1.5 L of ethanol for 3h. The flask was stored at -20 ºC overnight. Compound 1 was obtained as a colorless crystal (213g) after filtration. Compound 1 is also commercially available from AstraTech (# W18287,95% purity). (b) 3-(4-Methoxyphenyl)-2-methylpropanoic acid (compound 2): p-Methoxy-α-methylcinnamic acid (213g) and palladium on active charcoal (Pd-C, 10%, 2g) were suspended in 1.5 L of 95% ethanol and warmed to 60 ºC in a water bath. The warm suspension was immediately put on a catalytic hydrogenator, treated with hydrogen (40 psi). The reaction was completed within 45 min, as indicated by the complete dissolving of the solid starting material. The catalyst was removed by filtration, and the filtrate was concentrated to give compound 2 as a colorless oil (215g), which was used for the next reaction step without further purification and characterization. Compound 2 is also commercially available from PharmaBlocks (# PBTQ6955, 97% purity). (c) 6-Methoxy-2-methyl-2,3-dihydro-1H-inden-1-one (compound 3): Phosphoryl acid (98%, 500g) and polyphosphoryl acid (PPA, 450g) were pre-warmed separately to 70{degree sign}C in a water bath before being transferred to a 2L, three-necked flask equipped with a thermometer, a mechanical stirrer, and a dropping funnel. The flask was kept in an oil bath at 55-60 {degree sign}C for 1 h until smooth stirring was achieved. Compound 2 (95 g) was added dropwise over a period of 5 min. The temperature was carefully raised to 70-75{degree sign}C for 15 min. The reaction solution was immediately transferred to 6L of ice water and stirred until the PPA was completely dissolved. The mixture was extracted with ethyl ether (1L Ã- 3). The organic layer was dried with sodium sulfate and concentrated. The residue was purified by a silica gel column and eluted with hexane and acetone. Purity was monitored by TLC. Compound 3 was obtained as a clear, colorless oil (76g) that was used for the next reaction step without further purification and characterization. Compound 3 is also commercially available from AstraTech (# 96721, 95% purity). (d) 2-(5-Methoxy-2-methyl-1H-inden-3-yl) acetic acid (compound 4): A mixture of compound 3 (76g, 0.39 mole), cyanoacetic acid (36.6g, 0.43 mole), acetic acid (40 mL) and ammonium acetate (9 g) in 500 mL of toluene was refluxed with stirring for 48h. The liberated water was collected by a Dean-Stark trap. The reaction mixture was cooled and filtered, and the filtrate was concentrated. The residue was dissolved in 250 mL of ethanol. A solution of potassium hydroxide (90 g) in 360 mL of water was added to the solution. The mixture was refluxed overnight under argon. The organic solvents were removed under vacuum, and the remaining aqueous solution was diluted with 500 mL of water, extracted with ethyl ether, boiled with active charcoal for 1h, then filtered. The filtrate was acidified with 6N HCl and sonicated for 2h. The precipitate was collected by filtration, washed with water, refluxed in 180 mL of acetone for 2h, and stored at -20 ºC overnight. The precipitate was collected by filtration and dried under vacuum to give compound 4 as a colorless solid (42g). The mother liquor was concentrated, and a second crop (3.5g) was obtained after repeating the recrystallization procedure. The structure was confirmed by mass spectrometry (M+H: 219.04) and 1H-NMR (DMSO-d6) Î'(ppm): 7.240(d, 1H); 6.804(d, 1H); 6.658(m, 1H); 3.781 (s, 3H); 3.741(s, 2H); 3.250(s, 2H); 2.050(s, 3H). Purity was determined by HPLC (98.7%) (Suppl. Fig. 2a-c). (e) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid (compound 5): Compound 4 (32.7g, 0.150 mole), 3, 4, 5-trimethoxybenzaldehyde (35.3g, 0.180 mole) and sodium methoxide (21g) in 300 mL of anhydrous methanol were refluxed in a 500 mL round-bottomed flask overnight. After cooling, the reaction mixture was diluted with 150 mL of acetone and sonicated for 30 min. The precipitate was collected by filtration and washed twice with acetone, then dissolved in 150 mL of water. The aqueous solution was acidified with 6N HCl and sonicated for 30 min. The precipitate was collected by filtration, washed with water, recrystallized from methanol, and dried under vacuum to afford compound 5 as a yellow crystal (51.7g). The structure was confirmed by mass spectrometry (M+H: 397.11) and 1H-NMR (DMSO-d6) Î'(ppm): 12.377 (s, 1H); 7.429 (d, 1H); 7.139 (s, 1H); 6.877(s, 2H); 6.669 (d, 1H); 6.546(dd, 1H); 3.780 (s, 3H); 3.780(s, 3H); 3.737(s, 6H); 3.545(s, 2H); 21.121(s, 3H). Purity was determined by HPLC (99.64%) (Suppl. Fig. 3a-c). (f) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)-N-(pyridin-3-yl) acetamide (ADT 061): To a 500 mL round-bottomed flask containing compound 5 (39.6g, 0.100 mole) and 300 mL of anhydrous dichloromethane, 1,1'-carbonyldiimidazole (CDI, 20.0g, 0.115 mole) was added in portions and stirred for 15 min at room temperature. 3-Amino-pyridine (10.6g, 0.115 mole) was added, followed by anhydrous pyridine (80 mL). The solution was stirred at 40{degree sign}C overnight, treated with 5g of sodium hydroxide in 20 mL of water for 10 min, then diluted with 300 mL of dichloromethane, and washed with water (250 mL x 3). The solution was concentrated, and the residue was purified with a silica gel column. Recrystallization from methanol and then from ethyl acetate afforded ADT 061 as a yellow crystal (32.7g). The structure of ADT 061 was confirmed by mass spectrometry (M+H: 473.2) and 1H-NMR (DMSO-d6) Î'(ppm): 10.453(sb, 1H); 8.765(d, 1H); 8.276(dd, 1H); 8.044(m, 1H); 7.431(d, 1H); 7.351(dd, 1H); 7.142(s,1H); 6.914(s, 1H); 6.880(s, 2H); 6.542(m, 1H); 3.781 (s, 6H); 3.731(s, 3H); 3.726(s, 3H); 3.696(s, 2H); 2.194(s, 3H). Purity was determined by HPLC (>99.7%) (Suppl. Fig. 4a-c).

Supplementary Methods, Tables 1-3, Figure 1 from Altered Gene Expression in Morphologically Normal Epithelial Cells from Heterozygous Carriers of BRCA1 or BRCA2 Mutations