120 Background: Various oral therapies exist and have improved outcomes for men with prostate cancer. These agents are expensive and put patients at risk for financial toxicity. Although assistance mechanisms exist to offset these costs, navigating through assistance can be complicated and places excess burdens on patients. We conducted a survey of men prescribed oral therapies for prostate cancer to assess financial toxicity and perceptions on the process of obtaining oral cancer medications for prostate cancer. Methods: Patients with prostate cancer who had been prescribed abiraterone, enzalutamide, apalutamide or darolutamide at our institution in the six months prior to survey distribution were identified. Those with an email address were eligible for inclusion and sent a survey. Financial toxicity was assessed using the FACIT-COST score. Perceptions of the process of obtaining medication were assessed with a novel measure, which included questions regarding affordability, delays in care and ease of obtaining medications. We assessed bivariate associations between financial variables and financial toxicity measures. Results: Of 238 patients identified, 188 had a listed email address. Response rate was 28% of eligible patients (n = 53). Among respondents, 53% reported household income < $100,000 and 32% > $100,000, 15% declined to answer. A total of 41% utilized commercial insurance, 23% Medicare D, 28% an alternative Medicare program, 2% Medicaid and 4% had no prescription coverage. Monthly costs were between $100 and $1000 for 15% of patients and > $1000 for 6%. A total of 25% of patients considered their medication somewhat or very unaffordable; 13% were somewhat or not at all confident about affording their medication in the future; 6% reported delays in starting; 15% found the initial process of obtaining their medication somewhat or very stressful; 9% reported ongoing stress about affording their medications. Those with monthly cost > $100 were less likely to consider their medication affordable (p = 0.001). Those with commercial insurance were least likely to perceive delays in care (p = 0.024). Primary insurance type but no other variable was associated with difference in COST score with those who had commercial insurance reporting less financial toxicity than those with Medicare. Conclusions: The costs of oral prostate cancer medications are a concern for many men with prostate cancer. In a survey at our institution, many patients have high costs, despite the availability of financial assistance. In addition, many reported distress and uncertainty related to the process of obtaining their prostate cancer medications. Our survey was limited by small sample size and low response rate utilizing email as primary contact. We expect that many patients without access to email would be those at higher risk of financial toxicity. Further studies will aim to recruit patients directly in clinics.

Background Low-density lipoprotein receptor-related protein 1b (encoded by LRP1B) is a putative tumor suppressor, and preliminary evidence suggests LRP1B-mutated cancers may have improved outcomes with immune checkpoint inhibitors (ICI). Methods We conducted a multicenter, retrospective pan-cancer analysis of patients with LRP1B alterations treated with ICI at Duke University, Johns Hopkins University (JHU) and University of Michigan (UM). The primary objective was to assess the association between overall response rate (ORR) to ICI and pathogenic or likely pathogenic (P/LP) LRP1B alterations compared with LRP1B variants of unknown significance (VUS). Secondary outcomes were the associations with progression-free survival (PFS) and overall survival (OS) by LRP1B status. Results We identified 101 patients (44 Duke, 35 JHU, 22 UM) with LRP1B alterations who were treated with ICI. The most common tumor types by alteration (P/LP vs VUS%) were lung (36% vs 49%), prostate (9% vs 7%), sarcoma (5% vs 7%), melanoma (9% vs 0%) and breast cancer (3% vs 7%). The ORR for patients with LRP1B P/LP versus VUS alterations was 54% and 13%, respectively (OR 7.5, 95% CI 2.9 to 22.3, p=0.0009). P/LP LRP1B alterations were associated with longer PFS (HR 0.42, 95% CI 0.26 to 0.68, p=0.0003) and OS (HR 0.62, 95% CI 0.39 to 1.01, p=0.053). These results remained consistent when excluding patients harboring microsatellite instability (MSI) and controlling for tumor mutational burden (TMB). Conclusions This multicenter study shows significantly better outcomes with ICI therapy in patients harboring P/LP versus VUS LRP1B alterations, independently of TMB/MSI status. Further mechanistic and prospective validation studies are warranted.

70 Background: Numerous oral anticancer drugs approved for metastatic prostate cancer are expensive and put patients at risk for financial toxicity. Mechanisms available to mitigate these costs are complex to navigate. Furthermore, the ability to access these resources may not be the same for every insurance. We examined the out-of-pocket (OOP) liability of patients prescribed abiraterone or enzalutamide, the assistance mechanisms used, and the impact on timing of therapy. Methods: Patients with prostate cancer who were prescribed abiraterone or enzalutamide at our comprehensive cancer center between January 1, 2017 and March 31, 2019 were identified. Patients who filled prescriptions via an external pharmacy were excluded. Data regarding demographics, out of pocket costs and assistance mechanisms were evaluated. Results: We identified 219 patients. Of these, 33% had commercial insurance, 61% had Medicare part D, 3% had Medicaid and 8% had no insurance. Most patients (74%) were prescribed abiraterone and 26% enzalutamide. Among those with Medicare Part D, almost one third received significant financial assistance: 11% free drug from the manufacturer and 18% foundational grants. Patients with commercial insurance received a copay card from manufacturers 21% of the time. Patients with commercial insurance paid an average of $57 OOP; 94% with initial cost < $100. Patients with Medicare part D paid an average of $582 OOP; 22% with initial cost > $1000. Commercially insured patients started treatments in under 10 days from the prescription date 51% of the time vs 39% for Medicare patients. In contrast, 11% of commercially insured patients required > 30 days to fill vs 19% on Medicare. Only 1% of commercially insured patients were unable to fill due to cost issues compared to 7% of patients on Medicare. Conclusions: Financial assistance mechanisms such as grants and free drug programs help alleviate some financial burden for patients, but many still experience high OOP costs. These burdens appear to be disproportionately experienced by patients with Medicare Part D. We observed that patients with Medicare Part D had higher OOP costs for oral prostate cancer therapies. Those who required assistance often experienced delays in obtaining medication. Further evaluation is planned into how high costs and delays in treatment impacts prostate cancer treatment and patient care. [Table: see text]

61 Background: Numerous oral anticancer drugs approved for metastatic prostate cancer are expensive and put patients at risk for financial toxicity. Mechanisms to mitigate these costs are complex to navigate and are variable for different patient populations. We examined the impact of high costs and insurance type on time on therapy and reason for discontinuation of oral medication in patients with prostate cancer. Methods: We identified patients with prostate cancer who filled prescriptions for abiraterone or enzalutamide at our comprehensive cancer center 1/1/17-3/31/19, or who were prescribed one of these therapies but could not fill due to cost. Those with commercial insurance or Medicare part D were included. Clinical and demographic data was extracted for evaluation. Primary outcome variables included initial out-of-pocket cost, time on treatment, and failure to fill medication due to cost. Analysis using chi squared, T-tests and ANOVA was performed to assess associations. Results: We identified 193 patients. Of these, 34% had commercial insurance and 66% had Medicare part D. Mean out-of-pocket expense for the first fill was $62 for patients with commercial insurance and $582 for patients with Medicare Part D. Among those who did not fill due to cost, 9 had Medicare Part D and 1 had commercial insurance. The mean time on therapy for those with commercial insurance was 452 days vs 442 days for those with Medicare Part D. At time of data analysis, 50% of those with commercial insurance vs 45% of those with Medicare Part D remained on treatment. Of those who discontinued, 50% of those with commercial insurance did so due to progression or death vs 44% with Medicare part D. 14% with commercial insurance and 15% with Medicare part D discontinued to due patient preference or toxicities. 1 patient with Medicare part D and no patients with commercial insurance discontinued due to cost. There was no difference in time on therapy by out-of-pocket expense (average 444 days for out-of-pocket < $100 vs 477 days for out-of pocket > $100; p = 0.6) although those with an initial out of pocket expense > $100 were more likely not to fill their medication (24% vs 1%, p < 0.01). Conclusions: Costs remain high for oral prostate cancer treatments. Our findings suggest disproportionate financial burden among patients with Medicare Part D causing a significant number of patients to forego recommended therapy. While high costs did lead to failure to fill, we did not observe significant differences in time on treatment for patients with Medicare part D or with high out-of-pocket expense. Additionally, there was not a significant difference in stated reason for stopping therapy. Further research on patient perceptions of financial assistance programs and medication costs are planned.

3007 Background: Low-density lipoprotein receptor-related protein 1b (LRP1b) is a putative tumor suppressor and one of the most frequently altered genes in cancer. Our prior single-center work suggested that LRP1B alterations may enrich for responses to immune checkpoint inhibitors (ICI) in solid tumors including prostate cancer; however, validation of these findings is needed. Methods: We conducted a multicenter, retrospective analysis of patients with LRP1B alterations (on tissue-based next-generation sequencing panels) treated with ICI at Duke, Johns Hopkins (JHU), and University of Michigan (UM). The primary objective was to assess the association between objective response rate (ORR) to ICI and pathogenic LRP1B alterations, defined as deletions or truncating alterations, when compared with LRP1B variants of undetermined significance (VUS), defined as missense mutations not predicted to be pathogenic in COSMIC. Missense changes with a COSCMIC FATHMM score of > 0.8 were categorized separately as likely pathogenic. Summary statistics, ORR, progression free survival (PFS), and overall survival (OS) were calculated. Results: 101 patients (44 Duke, 35 JHU, 22 UM) with LRP1B alterations were treated with ICI. Median age was 61 (range 32-82). The most common tumor types by alteration (pathogenic or likely pathogenic/VUS%) were lung (33/47%), GI (17/13%), prostate (11/7%), sarcoma (2/9%), melanoma (11/0%), and others (26/24%). 93% of patients received single-agent PD-(L)1 inhibition. The ORR for patients with either pathogenic/likely pathogenic alterations, or VUS alterations was 57% and 18%, respectively. After excluding MSI-high or TMB-high ( > 10 mut/Mb) tumors, ORR was 14/25 (56%) and 6/36 (17%), respectively. Pathogenic or likely pathogenic LRP1B alterations were associated with longer PFS (HR 0.39, 95% CI 0.24-0.63) and OS (HR 0.58, 95% CI 0.36-0.95). Conclusions: This multicenter study shows impressive and durable objective response rates to ICI for patients harboring pathogenic LRP1B alterations when compared to those with LRP1B VUS, independent of TMB/MSI status. Further mechanistic insights and prospective validation studies are warranted. [Table: see text]

11528 Background: Ewing Sarcoma (ES) afflicts 225 children and 180 adults in the US annually, but the vast majority of research is in ped pts. In multiple series, adults have poorer outcomes. The aim of this study is to evaluate clinical features that correlate with overall survival (OS)/progression-free survival (PFS). Methods: ES pts at University of Michigan from 2007-15 were identified using an institutional database. Charts were reviewed for demographic and clinical data. Ped pts were defined as age ≤18 at diagnosis. Two-sample t-tests or Chi-squared tests/Fisher’s exact tests were used for comparisons. Survival outcomes were analyzed using Kaplan-Meier methods and Cox proportional hazards regression models. Results: Seventy-eight ES pts (26 peds, 52 adult) were included in analysis. Factors evaluated in multivariate analysis model included localized disease, age, surgery, radiation, tumor size, cumulative doxorubicin (DOX), osseous primary and no. of first-line chemo cycles. Localized disease correlated with improved PFS (HR = 0.20, p = < 0.0001) and marginally with OS (HR = 0.386, p = 0.0580). Five-yr OS was higher in ped pts vs adults (81% vs. 49%, p = 0.0218). Ped pts received more cycles of first-line chemo (14.0 vs 11.8, p = 0.003), which positively correlated with OS (HR = 0.78, p = 0.002) and PFS (HR = 0.82, p = 0.024). Other differences included higher cumulative DOX dose and increased incidence of extraosseous ES in adults, but these did not correlate with OS/PFS. Conclusions: In our series, younger age correlated with improved OS. Ped pts were more likely to have osseous ES and less DOX, but only increased number of first-line chemo cycles was associated with improved outcomes. Further evaluation of potential tissue biomarkers to differentiate the two groups and early progressors is planned. Variable Peds (n = 26) Adult (n = 52) p-value Localized disease, n (%) 17 (65.4) 33 (63.5) 0.867 Mean no. of first-line chemo cycles 14 11.8 0.003 Cumulative DOX (mg/m2) 357.9 445.6 < 0.0001 Osseous primary, n (%) 21 (84) 22 (45.8) 0.002 Mean tumor size, cm (range) 8.2 (3.2 – 19.3) 9.6 (1.8 – 23.5) 0.384 Surgical Resection, n (%) 19 (73.1) 28 (53.8) 0.142 Progression during first line rx, n (%) 1 (4.2) 7 (15.2) 0.249

To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3fl/fl;Nf1fl/fl;DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials.

Osteosarcoma (OS) is the most common type of malignant bone tumor. Despite aggressive multimodal treatments, including surgical resection, chemotherapy and adjunctive immunotherapies, patients with OS with high-grade malignancy have a poor five-year survival rate that has remained unchanged over the past two decades, highlighting the urgent requirement for novel therapeutic approaches. Signal transducers and activators of transcription 3 (STAT3) has been implicated as an oncogene and therapeutic target in a variety of neoplastic diseases. The aim of the present study was to determine whether inhibition of the janus kinase 2 (JAK2)/STAT3 pathway by FLLL32, a specific JAK2/STAT3 inhibitor, is able to provide a potential therapy for OS. FLLL32 inhibited OS cell growth in vitro and delayed OS growth in an OS xenograft nude mouse model. STAT3 knockdown by short hairpin RNA delayed OS formation in vivo. Thus, the JAK2/STAT3 pathway is important in OS formation. Efficacy of the FLLL32 pharmacological inhibitor in delaying OS growth suggests that targeting JAK2/STAT3 may be a potential therapeutic strategy for patients with OS.

To identify genes and signaling pathways that drive peripheral nerve tumor initiation and growth beyond the Ras-MAPK pathway we used unbiased insertional mutagenesis screening. We identified Stat3 as a potential driver of Neurofibromatosis type 1 neurofibroma. Targeted genetic deletion of Stat3 in Schwann cell precursors (SCPs) and Schwann cells (SCs) largely prevented neurofibroma formation, and self-renewal of tumor initiating cells. Genetic gain- and loss-of-function identified EGFR as the major upstream regulator of P-Stat3 in mouse and human neurofibroma SCP and in neurofibroma initiation; IL-6 reinforced EGFR/Jak/Stat signaling. Preclinical tests of a Jak2/Stat3 inhibitor reduced established neurofibroma growth, supporting an additional role for Stat3 in benign nerve tumor maintenance. Unexpectedly, downstream of Stat3, we identified β-catenin, and β-catenin expression rescued phenotypic effects of Stat3 loss in SCPs. Phosphorylated STAT3 (Y705) and β-catenin were strongly correlated in NF1 human plexiform neurofibromas. The data support testing of JAK/STAT inhibition and Wnt/ β-catenin pathway inhibition in neurofibroma therapeutic trials. Supported by: NIH R01 NS28840 to N.R. and NIH P50 NS057531 to N.R. and D.L.), a DAMD New Investigator Award (W81XWH-11-1-0259) and an Ohio State University Comprehensive Cancer Center Pelotonia Idea Grant (to J.W.). The American Cancer Society (IRG-67-003-44) supported J.R.F.

Colon tumors are a major cause of cancer death, yet their molecular intricacies are not fully understood. We demonstrate that the histone demethylases JMJD2A, JMJD2B and JMJD2C are overexpressed in colon cancer cell lines, whereas another related protein, JMJD2D, is not. Interestingly, despite their high homology, the intracellular localization of JMJD2A-C is different in colon and other cancer cells, with JMJD2A being present comparably in the cytoplasm and nucleus, JMJD2B more prevalent in the nucleus and JMJD2C strongly associated with chromatin. This suggests that each of these three proteins performs different, non-redundant functions. Moreover, we show that JMJD2C (also called KDM4C) forms complexes with β-catenin, an oncoprotein whose overexpression is crucial for the development of most colonic tumors. In addition, JMJD2C downregulation reduced both growth and clonogenic capacity of HCT-116 colon cancer cells. Further, JMJD2C was required for efficient expression of the growth stimulatory proteins FRA1 and cyclin D1 as well as the survival factor BCL2. Lastly, we identified derivatives of curcumin as in vitro inhibitors of JMJD2 enzymes, suggesting that these curcuminoids could be useful for decreasing JMJD2 activity in vivo. In conclusion, our data highlight that overexpression of JMJD2C confers a pro-growth effect on colon cancer cells and, therefore, its inhibition by curcuminoids or other small molecules could be beneficial as an adjuvant therapy for colon cancer patients.

Meeting abstracts Cholangiocarcinoma (CC) is a universally lethal disease that responds poorly to chemo- and immunotherapy. Patients with CC typically display elevated systemic interleukin-6 (IL-6) and systemic expansion of myeloid-derived suppressor cells (MDSCs). Interestingly, both of these

Abstract Biliary cancer (BC), or cholangiocarcinoma originates from the malignant transformation of epithelial cells in the bile ducts. This deadly cancer is typically refractory to standard therapies and has a 3 year survival rate of only 10%. Therefore, novel treatment strategies are desparately needed against this malignancy. One important feature of BC cells is their ability to secrete interleukin-6 (IL-6) in an autocrine manner. This characteristic affords them an opportunity to activate numerous pro-oncogenic signaling pathways including MAPK, and STAT3 within the tumor cell, while simultaneously promoting immunologic changes in patients with advanced disease. We hypothesized that inhibitors of Signal-Transducer and Activator of Transcription-3 (STAT3) pathway may elicit a dual effect by promoting apoptosis of human BC cell lines, and limiting the secretion of immunosuppressive cytokines from these cells. A panel of human BC cell lines with various genotypic profiles was utilized, and all demonstrated secretion of IL-6 (range 5149-68pg/mL) and had constitutively phosphorylated STAT3 as determined by western blot. Similar to positive control conditions with IL-6 + GM-CSF (10 ng/mL each), exposure of human peripheral blood mononuclear cells to 5% or 10% BC culture supernatants induced in vitro differentiation into myeloid derived suppressor cells (MDSC). These data indicate that BC cells have robust activation of the IL-6/STAT3 signaling axis, and that culture supernatants contain factors capable of promoting expansion of immune suppressive cell subsets. A novel small molecule inhibitor FLLL100, could directly inhibit Tyr705 phosphorylation within the SH2 domain of STAT3, and induce apoptosis in the BC cell lines regardless of genotypic profile. Both growth inhibitory (by MTT assay) and pro-apoptotic effects (by Annexin V/PI staining) were observed within 24-48 hours of drug exposure at micromolar concentrations. Apoptosis was confirmed after drug exposure by assessment of PARP cleavage by immunoblot. Exposure of BC cell lines to FLLL100 resulted in significantly reduced secretion of immunomodulatory cytokines including IL-6 (p<0.05) and GM-CSF (p<0.05) in culture supernatants. Together, these data indicate that the IL-6/STAT3 signaling axis plays a role in human BC survival and that targeting this pathway can limit immune suppressive factors derived from BC cell lines. Citation Format: Jennifer Yang, Kaitlin Keenan, Thomas Mace, Tanios Bekaii-Saab, James Fuchs, Eric Schwartz, Chenglong Li, Jiayuh Lin, Pui-Kai Li, Gregory Lesinski. STAT3 inhibitors elicit direct anti-tumor effects against human biliary caner cell lines and limit release of immune suppressive cytokines in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4073. doi:10.1158/1538-7445.AM2014-4073

Leishmaniasis is a neglected, parasitic disease transmitted through the bite of infected sandflies. In an effort to identify novel compounds demonstrating activity against the parasite, a multidisciplinary research effort involving natural product isolation, chemical synthesis, and biological evaluation was initiated. Recently, our group reported the isolation of several sterols from the roots of Pentalinon andrieuxii, a plant native to Mexico, that show potent antileishmanial activity both in vitro and in vivo. Analysis of these compounds indicated three distinct structural subtypes and afforded preliminary structure-activity relationship (SAR) data. Semi-synthetic approaches toward these structural classes from readily available, commercial starting materials have been carried out to prepare the molecules in quantities sufficient to enable detailed mechanistic studies and SAR studies. Syntheses of pentalinonsterol and ergosta-4,24(28)-dien-3-one, members of the first two classes, respectively, have already been accomplished and the synthesis of an advanced intermediate of 6,7-dihydroneridienone, the compound displaying the most potent antileishmanial activity and a representative member of the third class possessing, has also been achieved. These synthetic efforts have also facilitated the preparation of a number of structurally related analogues and probes designed to explore the mechanism of action of this class of natural product.

Malignant peripheral nerve sheath tumors (MPNSTs) develop sporadically or in the context of neurofibromatosis type 1. Epidermal growth factor receptor (EGFR) overexpression has been implicated in MPNST formation, but its precise role and relevant signaling pathways remain unknown. We found that EGFR overexpression promotes mouse neurofibroma transformation to aggressive MPNST (GEM-PNST). Immunohistochemistry demonstrated phosphorylated STAT3 (Tyr705) in both human MPNST and mouse GEM-PNST. A specific JAK2/STAT3 inhibitor FLLL32 delayed MPNST formation in an MPNST xenograft nude mouse model. STAT3 knockdown by shRNA prevented MPNST formation in vivo. Finally, reducing EGFR activity strongly reduced pSTAT3 in vivo. Thus, an EGFR–STAT3 pathway is necessary for MPNST transformation and establishment of MPNST xenografts growth but not for tumor maintenance. Efficacy of the FLLL32 pharmacological inhibitor in delaying MPNST growth suggests that combination therapies targeting JAK/STAT3 might be useful therapeutics.

Leishmaniasis is a parasitic disease transmitted through the bite of infected sandflies. Although therapeutics are currently available for the treatment of the disease, the emergence of drug resistance in the parasites suggests a continued need for the discovery of novel chemical entities that act via new or different mechanisms of action. In an effort to identify compounds which may be effective for the treatment of leishmaniasis, a multidisciplinary research effort involving natural product isolation, chemical synthesis, and biological evaluation was initiated. Several sterols were isolated from the roots of Pentalinon andrieuxii, a plant native to Mexico, that show potent antileishmanial activity both in vitro and in vivo. A structural survey of the active compounds indicated three distinct structural subtypes. Semi-synthetic approaches toward these structural classes from affordable, commercially available starting materials have been designed to prepare the molecules in quantities sufficient to enable detailed mechanistic studies and structure-activity relationship studies. Representative examples from two of the classes, pentalinonsterol and ergosta-4,24(28)-dien-3-one, have been synthesized from 5-pregnen-3β-ol-20-one and lithocholic acid, respectively. These routes have also facilitated the preparation of a number of structurally related analogues.

Abstract Neurofibromatosis type 1 (NF1) is a very common inherited disease, affecting 1:3500 individuals worldwide. Nearly all (95%) of NF1 patients develop benign neurofibromas and malignant peripheral nerve sheath tumors(MPNSTs). Currently, their prevention is not possible, partially because the molecular mechanisms of tumorigenesis and the molecules that mark benign neurofibroma formation are poorly understood. This study is to test the relevance of EGFR expression to neurofibroma formation, and to identify possible additional pathways and genes that might contribute to neurofibroma formation. We bred the Nf1 flox/flox;DhhCre mice, 100% of which form neurofibromas (Wu et al., 2008), to CNP-hEGFR mice and to Wa2 mice, an EGFR hypomorphic allele. To test the role of EGFR in tumorigenesis, we also used sleeping beauty (SB) insertional mutagenesis to obtain quadruple transgenic mice (Rosa26-lsl-SB11;T2/Onc; Nf1flox/flox;DhhCre). To define neurofibroma initiation and progression genes, we used Pyrosequencing to identify common insertion sites (CISs) that had more SB insertions that are most likely to harbor disease-related genes. We used ingenuity pathway analysis to predict pathways and genes that might contribute to neurofibroma formation. We used a “neurofibroma sphere” culture system, a method used for detecting self-renewing stem/progenitor cells, to determine inhibitory effects of a STAT3 inhibitor (FLLL32). We immunostained human and mouse sections with anti-pSTAT3 (tyr705) to determine STAT3 activation status. We found that mouse neurofibroma number and size increased in Nf1flox/flox;DhhCre mice with hEGFR expressed in nerve Schwann cells. Diminished EGFR signaling in Nf1 flox/flox;DhhCre, Wa2/+ mice decreased neurofibroma number, not size. We used insertional mutagenesis to identify other modifiers of neurofibroma tumorigenesis. Analysis of CISs identified hubs involving GSK3B, TNF, and STAT3. STAT3 was the most significant changed pathway. Inhibition of STAT3 by shRNA or a specific STAT3 inhibitor FLLL32 blocked human neurofibroma-sphere formation. Immunohistochemistry identified STAT3(p705) in human and mouse neurofibromas and MPNSTs. FLLL32 inhibited cell proliferation and stimulated cell death as well as reduced neurofibroma growth in vivo in the Nf1flox/flox;DhhCre mouse neurofibromas. STAT3 knockdown by shRNA prevented MPNST formation in vivo. Finally, reducing EGFR activity strongly reduced pSTAT3 in vivo. Thus, an EGFR-STAT3 pathway regulates neurofibroma number and neurofibroma growth, and promotes transformation. STAT3 inhibitors may be useful in NF1 therapeutics. (*This work was supported by the National Institutes of Health (R01 NS28840 to N.R. and P50 NS057531 to N.R. and D.L.) and an Ohio State University Comprehensive Cancer Center pelotonia idea award to J.W.) 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 2937. doi:1538-7445.AM2012-2937

Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in osteosarcoma, and hence, may serve as a therapeutic target. In order to target STAT3, we tested two new STAT3 inhibitors, LLL12 and FLLL32. LLL12 and FLLL32 inhibit STAT3 phosphorylation and STAT3 downstream targets. LLL12 and FLLL32 also inhibit IL-6 induced STAT3 phosphorylation. The inhibition of STAT3 by LLL12 and FLLL32 resulted in the induction of apoptosis, reduction of plating efficiency, and migration in osteosarcoma cells. Furthermore, LLL12 and FLLL32 inhibited SJSA osteosarcoma cells and OS-33 tumor growth in murine xenografts. These results provide evidence that constitutive STAT3 signaling is required for osteosarcoma survival and migration in vitro and tumor growth in vivo. Blocking persistent STAT3 signaling by LLL12 and FLLL32 may be a novel therapeutic approach for osteosarcoma.

The Janus kinase-2 (Jak2)-signal transducer and activator of transcription-3 (STAT3) pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC) and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3), and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma.