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Understanding the molecular mechanisms behind formation of melanoma, the deadliest form of skin cancer, is crucial for improved diagnosis and treatment. One key is to better understand the cross-talk between epidermal keratinocytes and pigment-producing melanocytes. Here, using a bigenic mouse model system combining mutant oncogenic NRASQ61K (constitutively active RAS) or mutant activated CDK4R24C/R24C (prevents binding of CDK4 by kinase inhibitor p16INK4A) with an epidermis-specific knockout of the nuclear retinoid X receptor alpha (RXRαep−/−) results in increased melanoma formation after chronic ultraviolet-B (UVB) irradiation compared with control mice with functional RXRα. Melanomas from both groups of bigenic RXRαep−/− mice are larger in size with higher proliferative capacity, and exhibit enhanced angiogenic properties and increased expression of malignant melanoma markers. Analysis of tumor adjacent normal skin from these mice revealed altered expression of several biomarkers indicative of enhanced melanoma susceptibility, including reduced expression of tumor suppressor p53 and loss of PTEN, with concomitant increase in activated AKT. Loss of epidermal RXRα in combination with UVB significantly enhances invasion of melanocytic cells to draining lymph nodes in bigenic mice expressing oncogenic NRASQ61K compared with controls with functional RXRα. These results suggest a crucial role of keratinocytic RXRα to suppress formation of UVB-induced melanomas and their progression to malignant cancers in the context of driver mutations such as activated CDK4R24C/R24C or oncogenic NRASQ61K.Implications: These findings suggest that RXRα may serve as a clinical diagnostic marker and therapeutic target in melanoma progression and metastasis. Mol Cancer Res; 13(1); 186–96. ©2014 AACR.

Antibodies used for experiments. Supplementary Table S2: PCR primers used for genotyping of mouse lines.

Supplementary Figure S5: No change in infiltrating mast cells as determined by CEM staining is observed in melanocytic lesions from either Cdk4R24c or Tyr-NRASQ61K mice with epidermis-specific knockout of Rxra. Assay was performed on bleached 5 J.lm paraffin sections. Infiltrating mast cells are labeled with light blue staining. E=Epidermis, 0= Dermis. Scale bars= 50 micron

Supplementary Figure S8: Chronically UVB-irradiated tumor-adjacent normal skin from Tyr-NRASQBtK or Cdk4R24c mice with epidermis-specific knockout of Rxra show modest increase in CDK4 expression; downregulation of phosphorylated ERK, as well as increased E2F1 expression specifically in Rxraep-/- 1 Cdk4R24c mice. (A-D) Modest increase of CDK4 expression in both epidermis and dermis of Rxraep-/- I Tyr- NRASQ61K and Rxraep-/- 1 Cdk4R24c mice as compared to RxraL2/L2 controls, as determined by immunohistochemistry (5 IJm bleached paraffin sections). E=Epidermis, D= Dermis. Scale bars= 50 IJm. # = no statistical significance , *= p s 0.05, *** = p s 0.001 (E, F) Down regulation in phosphorylated ERK in tumor-adjacent normal skin of Rxraep-/- 1 Tyr-NRASQotK and Rxraep-/- 1 Cdk4R24cmice as compared to RxraL2/L2 controls; as shown by Western Blot (Two biological replicates for each group are shown). (G, H) Analysis of E2F1 protein levels in tumor-adjacent normal skin of Rxraep-/- 1 Tyr-NRASQotK and Rxraep-/- I Cdk4R24c mice as compared to RxraL2/L2 controls; as shown by Western Blot (Two biological replicates for each group are shown). No difference was observed in the Tyr-NRASQotK group, while increased E2F1 levels were observed in Rxraep-/- 1 Cdk4R'

Supplementary methods

Supplementary Figure S2: Chronic-UVB treated and parallel non-treated controls used in this study. (A,B) H&E staining of non-UVB treated skin from parallel age-matched mice (A) and TAN skin from chronic-UVB treated mice (B). In all groups, TAN skin has similar morphology to non-UVB treated skin, except for increased epidermal thickening; and dermal pigmentation which is enhanced further in the RXRaep-/- mice. Mice carrying Cdk4R24c in all groups show sebaceous gland hyperplasia within hair follicles (yellow boxes). E=Epidermis, D=Dermis, scale bar =100 IJm. (C) Epidermis measurements taken from H&E stained samples. In non-UVB treated mice, Tyr-NRAS0 61K or Cdk4R24c combined with Rxraep-/- mutation exhibited slightly thicker epidermis than RxraL2/L2 counterparts [7]. This is attenuated by chronic-UVB treatment, which results in a similarly thickened epidermis across all groups. ** = p

Supplementary Figure S1: Breeding/Genotyping of mouse lines used in this study. (A, B) Breeding crossings used. K14-Cre'g/O I RxraL2/L2 is also known as RXRaep-/-. (C) Semi-quantitative PCR was performed on biopsies containing both epidermal and non-epidermal tissue; to confirm genotypes with regard to presence/absence of K14-Cre recombinase (top), epidermis-specific ere-mediated excision of Rxra exon 4 (middle), and the mutant Cdk4R24c allele (bottom). Presence of K14-Cre recombinase is indicated by a single 349 bp band; animals negative for K14-Cre show no amplification. In K14-Cre positive animals, ere-mediated epidermis-specific excision of Rxra is indicated by a 110 bp (L-) band, while a 203 bp (L2) band is also amplified due to presence of non-epidermal tissue in the biopsy in which excision did not occur. Animals negative for K14-Cre show only the 203 bp (L2) band for Rxra, as no excision occurred in any tissue. Animals homozygous for the mutant Cdk4R24c allele show a single band at 530 bp, while animals homozygous for the WT Cdk4 allele show a single band at 440 bp. Primers used are detailed in the Supplementary Table S2. (D) Tyr-NRAS061K genotype is determined phenotypically 10-12 days postnatal by presence/absence of dark pigmentation of the foot pads.

Supplementary Figure S6: Heat Map and Clustergram of dysregulated genes in melanocytic lesions from Rxraep-/- 1 Tyr-NRASQ61K mice_ Tissue from melanocytic lesions was captured using Laser Capture Microdissection (LCM) (See Figure 4 for details). Isolated RNA was converted to eDNA and gene expression in Rxraep-/- lesions relative to RxraL2/L2 lesions was assayed using an RT-qPCR array (SA Biosciences, PAMM-033Z (Mouse Cancer)). Mean fold change relative to Rxrcf21L2 group is represented by the heat map (A). Three replicate arrays were performed for both RxraL2/L2 and Rxraep-/- groups; magnitude of gene expression for each replicate is shown in the clustergram (B), which correlates genes with similar expression levels.

Supplementary Figure S3: Controls for IHC experiments used in this study. (A) For all IHC studies, a liquid-repellant slide marker pen was used to separate out a single section on each slide that does not receive primary antibody but otherwise carried through the same labeling protocol; for use as a negative control to demonstrate that signals observed were specific. (B) PEP1 antibody (anti-TYRP1) and PEPS antibody (anti-TYRP2) give similar specific staining patterns for melanocytic cells in RxraL2/L2 neonatal skin following irradiation with a single UVB dose. (C) Fluorescent IHC for p-AKT and PTEN on chronically-irradiated TAN skin showing similar staining pattern to chromogenic IHC shown in Figure 5. (D,E) Non-UVB treated skin showed reduced staining intensity of PTEN, p-AKT, and p53 compared to irradiated skin. Unlike skin treated with chronic UVB, no discernable difference in expression pattern between RxraL2/L2 and bi enic Rxraep-/- mice was observed. E=E idermis, D=Dermis, scale bars =1 00 J.Jm.

Supplementary Figure S4: An increase in infiltrating macrophages is observed in melanocytic lesions from Cdk4R24cmice with epidermal-specific knockout of Rxra, but not in Rxraep-/- Tyr-NRASQBtK mice. No change in infiltrating CD4+ T-cells observed in melanocytic lesions from either Cdk4R24c or Tyr-NRASQ61K mice with epidermal-specific knockout of Rxra. (A, B) No change in infiltrating macrophages, as determined by fluorescent immunohistochemistry (I HC) of of 5 J.lm paraffin sections (MAC1 antigen), was observed in Rxraep-/- 1 Tyr-NRAS061K mice as compared to RxraL2/L2 controls. (C, D) An increase in infiltrating macrophages was observed in Rxraep-/- 1 Cdk4R24c mice as compared to RxraL2/L2 controls. (E-H) No change in infiltrating CD4+ T-cells as determined by fluorescent IHC was observed in Rxraep-/- Tyr-NRAS061K or Rxraep-/- Cdk4R24c mice as compared to RxraL 21L2 controls. Scale bars= 50 J.lm. ** = p :5 0.01

This document contains supplemental figures 1-17. Figure legends corresponding to each SF are on the subsequent page. Supplemental Figure 1. MCTS formation across a panel of ovarian cancer cell lines. Supplemental Figure 2. The number of proliferating cells is universally reduced in ovarian cancer cells grown as spheroids. Supplemental Figure 3. HIO cell lines form small spheroids which lack stem-like properties. Supplemental Figure 4. JMJD1A protein expression is enhanced in ovarian cancer cells when grown in 3D. Supplemental Figure 5. Targeted therapies used in ovarian cancer clinical trials induce a variable pattern of expression of stem-like associated genes. Supplemental Figure 6. CellTiter-Glo measured cell viability and drug response in MCTS. Supplemental Figure 7. 3D drug screening in non-tumorigenic cell lines did not identify 3D Hits. Supplemental Figure 8. Secondary screening validated 3D hits across multiple cell lines. Supplemental Figure 9. Dosage response validation of a 2D hit and 2D and 3D hit. Supplemental Figure 10. Licofelone showed enhanced activity in paclitaxel resistant 2D cells. Supplemental Figure 11. Licofelone had more activity against ovarian cancer cell-derived MCTS than the COX-2 specific inhibitor celecoxib. Supplemental Figure 12. Combination index values show strong synergy between with paclitaxel following licofelone pretreatment. Supplemental Figure 13. Pretreatment of ovarian cancer cell-derived MCTS with paclitaxel synergistically enhances licofelone activity. Supplemental Figure 14. Combination of licofelone and paclitaxel leads to increases in apoptosis in ovarian cancer cell-derived MCTS. Supplemental Figure 15. Licofelone can also sensitize ovarian cancer cells grown as spheroids to carboplatin. Supplemental Figure 16. Licofelone treatment decreases mitochondrial metabolism in OVCAR8 spheroids. Supplemental Figure 17. Changes in Cell Proliferation in Tumor Cells Isolated from Recurrent PDX Ascites Fluid.

Expression of ABC transporter genes in 2D and in 3D cultures.

Drug development for first-line treatment of epithelial ovarian cancer (EOC) has been stagnant for almost three decades. Traditional cell culture methods for primary drug screening do not always accurately reflect clinical disease. To overcome this barrier, we grew a panel of EOC cell lines in three-dimensional (3D) cell cultures to form multicellular tumor spheroids (MCTS). We characterized these MCTS for molecular and cellular features of EOC and performed a comparative screen with cells grown using two-dimensional (2D) cell culture to identify previously unappreciated anticancer drugs. MCTS exhibited greater resistance to chemotherapeutic agents, showed signs of senescence and hypoxia, and expressed a number of stem cell–associated transcripts including ALDH1A and CD133, also known as PROM1. Using a library of clinically repurposed drugs, we identified candidates with preferential activity in MCTS over 2D cultured cells. One of the lead compounds, the dual COX/LOX inhibitor licofelone, reversed the stem-like properties of ovarian MCTS. Licofelone also synergized with paclitaxel in ovarian MCTS models and in a patient-derived tumor xenograft model. Importantly, the combination of licofelone with paclitaxel prolonged the median survival of mice (>141 days) relative to paclitaxel (115 days), licofelone (37 days), or vehicle (30 days). Increased efficacy was confirmed by Mantel–Haenszel HR compared with vehicle (HR = 0.037) and paclitaxel (HR = 0.017). These results identify for the first time an unappreciated, anti-inflammatory drug that can reverse chemotherapeutic resistance in ovarian cancer, highlighting the need to clinically evaluate licofelone in combination with first-line chemotherapy in primary and chemotherapy-refractory EOC.Significance: This study highlights the use of an in vitro spheroid 3D drug screening model to identify new therapeutic approaches to reverse chemotherapy resistance in ovarian cancer. Cancer Res; 78(15); 4370–85. ©2018 AACR.

Introduction Monitoring patients with acute myeloid leukemia can be implemented through various techniques such as multiparameter flow cytometry, real-time quantitative polymerase chain reaction, and next-generation sequencing. However, there is scarce studies when comparing the data of next-generation sequencing and flow cytometry for monitoring disease progression, particularly how they might supplement one another when used in tandem. Methods We investigated 107 patients via retrospective analysis using follow-up MFC and NGS data with a total of 717 MFC and 247 NGS studies to compare these methods in monitoring minimal/measurable residual disease. Results 197 instances were MFC+ /NGS+ , 3 were MFC- /NGS- , 44 were MFC- /NGS+ , and 3 are MFC+ /NGS- . The majority of the MFC- /NGS+ cases occurred within 6 months during the post-treatment phase (64%). Among 44 MFC- /NGS+ instances, 13 had similar NGS profiles to their original day 0 diagnosis. The remaining cases showed preleukemic clonal hematopoiesis mutations, "likely pathogenic mutations," or "variants of uncertain significance." Conclusion Our findings show that flow cytometry has its advantages with comparable sensitivity in detecting minimal/measurable residual disease. Next-generation sequencing could be used in an increased and more regular capacity in conjunction with flow cytometry to achieve a more comprehensive surveillance of these patients, resulting in improved outcomes.

INTRODUCTION: Next-generation sequencing (NGS) analysis showed clonal cytopenia of undetermined significance (CCUS) as an immediate precursor to myelodysplastic syndrome (MDS). METHODS: We evaluated and compared morphologic, multiparametric flow cytometry (MFC), and molecular genetic findings in patients with CCUS (n = 37), MDS (n = 75), and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC, n = 24). RESULTS: CCUS patients showed variable MFC abnormalities including >2% CD34+ myeloblasts (5.8%), altered antigen expression on myeloblasts, monocytes, and granulocytes (1.2, 1.5, and 0.2/case), abnormal maturation of myeloblasts (45.8%), decreased hematogones (17.6%), and decreased side scatter (SSC) of granulocytes (11.4%). CCUS patients with high-risk mutations showed significantly more MFC abnormalities. However, CCUS patients with >20% variant allelic fraction (VAF) did not show more MFC aberrations than the rest of the group. MDS patients showed significantly more MFC abnormalities compared with CCUS patients (p = 7.8E-05 – 0.047). Low-grade MDS patients showed significantly fewer MFC abnormalities compared with high-grade MDS or AML-MRC patients (p = 1.89E-05– 0.04). AML-MRC patients showed significantly elevated blast counts, more antigen aberrations, decreased hematogones, and decreased SSC of granulocytes compared with CCUS patients (p = 2.0E-05– 0.01). CCUS patients carried predominantly TET2/DNMT3A/ASXL1 mutations. They harbored fewer mutations in gene coding splicing factors compared with MDS patients (p = .0001–. 02) and fewer mutations in tumor suppressor and transcription factor genes compared with AML-MRC patients (p = .0006– .02). CONCLUSIONS: CCUS is an immediate precursor to low-g rade MDS. The progression from CCUS to MDS to AML-MRC is a stepwise process that requires acquisition of mutations in splicing, transcription factor, and tumor suppressor genes with accumulations of additional MFC abnormalities.

Lung cancer accounts for approximately 14% of all newly diagnosed cancers and is the leading cause of cancer-related deaths. Chimeric RNA resulting from gene fusions (RNA fusions) and other RNA splicing errors are driver events and clinically addressable targets for non-small cell lung cancer (NSCLC). The reliable assessment of these RNA markers by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We describe the development and verification of a system for targeted RNA sequencing for the analysis of challenging, low-input solid tumor biopsies that includes reagents for nucleic acid quantification and library preparation, run controls, and companion bioinformatics software. Assay development reconciled sequence discrepancies in public databases, created predictive formalin-fixed, paraffin-embedded RNA qualification metrics, and eliminated read misidentification attributable to index hopping events on the next-generation sequencing flow cell. The optimized and standardized system was analytically verified internally and in a multiphase study conducted at five independent laboratories. The results show accurate, reproducible, and sensitive detection of RNA fusions, alternative splicing events, and other expression markers of NSCLC. This comprehensive approach, combining sample quantification, quality control, library preparation, and interpretive bioinformatics software, may accelerate the routine implementation of targeted RNA sequencing of formalin-fixed, paraffin-embedded samples relevant to NSCLC.

Melanoma treatment has been revolutionized over the past decade. Long-term results with immuno-oncology (I-O) agents and targeted therapies are providing evidence of durable survival for a substantial number of patients. These results have prompted consideration of how best to define long-term benefit and cure. Now more than ever, oncologists should be aware of the long-term outcomes demonstrated with these newer agents and their relevance to treatment decision-making. As the first tumor type for which I-O agents were approved, melanoma has served as a model for other diseases. Accordingly, discussions regarding the value and impact of long-term survival data in patients with melanoma may be relevant in the future to other tumor types. Current findings indicate that, depending on the treatment, over 50% of patients with melanoma may gain durable survival benefit. The best survival outcomes are generally observed in patients with favorable prognostic factors, particularly normal baseline lactate dehydrogenase and/or a low volume of disease. Survival curves from melanoma clinical studies show a plateau at 3 to 4 years, suggesting that patients who are alive at the 3-year landmark (especially in cases in which treatment had been stopped) will likely experience prolonged cancer remission. Quality-of-life and mixture-cure modeling data, as well as metrics such as treatment-free survival, are helping to define the value of this long-term survival. In this review, we describe the current treatment landscape for melanoma and discuss the long-term survival data with immunotherapies and targeted therapies, discussing how to best evaluate the value of long-term survival. We propose that some patients might be considered functionally cured if they have responded to treatment and remained treatment-free for at least 2 years without disease progression. Finally, we consider that, while there have been major advances in the treatment of melanoma in the past decade, there remains a need to improve outcomes for the patients with melanoma who do not experience durable survival.

BackgroundTumor-infiltrating lymphocyte (TIL) therapy is a personalized cancer treatment which involves generating ex vivo cultures of tumor-reactive T cells from surgically resected tumors and administering the expanded TILs as a therapeutic infusion. Phase 1 of many TIL production protocols use aldesleukin (IL-2) alone to establish TIL cultures (termed “PreREP” (Pre-Rapid Expansion Protocol)); however, this fails to consistently produce TIL cultures from renal cell carcinoma (RCC) in a timely manner. Adding mitogenic stimulation via anti-CD3/anti-CD28 beads along with IL-2 to the fresh tumor digest (FTD) during TIL generation (termed “FTD+ beads”) increases successful TIL culture rates; however, T cells produced by this method may be suboptimal for adoptive transfer. We hypothesize that adherent cell depletion (ACD) before TIL expansion will produce a superior TIL product by removing the immunosuppressive signals originating from adherent tumor and stromal cells. Here we investigate if “panning,” a technique for ACD prior to TIL expansion, will impact the phenotype, functionality and/or clonality of ex vivo expanded RCC TILs.MethodsTumor specimens from 55 patients who underwent radical or partial nephrectomy at the University of Kansas Medical Center (KUMC) were used to develop the panning method and an additional 19 specimens were used to validate the protocol. Next-generation sequencing, immunohistochemistry/immunocytochemistry and flow cytometry were used during method development. The phenotype, functionality and clonality of autologous TILs generated in parallel by panning, PreREP, and FTD+ beads were assessed by flow cytometry, in vitro co-culture assays, and TCRB CDR3 sequencing.ResultsTIL cultures were successfully generated using the panning protocol from 15/16 clear cell, 0/1 chromophobe, and 0/2 papillary RCC samples. Significantly fewer regulatory (CD4+/CD25+/FOXP3+) (p=0.049, p=0.005), tissue-resident memory (CD8+/CD103+) (p=0.027, p=0.009), PD-1+/TIM-3+ double-positive (p=0.009, p=0.011) and TIGIT+ T cells (p=0.049, p=0.026) are generated by panning relative to PreREP and FTD+ beads respectively. Critically, a subset of TILs generated by panning were able to degranulate and/or produce interferon gamma in response to autologous tumor cells and the average tumor-reactive TIL yield was greatest when using the panning protocol.ConclusionsRemoving immunosuppressive adherent cells within an RCC digest prior to TIL expansion allow for the rapid production of tumor-reactive T cells with optimal characteristics for adoptive transfer.

Introduction: Residual disease (RD) after neoadjuvant chemotherapy (NACT) is associated with high risk of recurrence in TNBC. RCB classification is prognostic in patients with RD. Recent studies show that post-NACT circulating cell-free tumor DNA (ctDNA) also provides prognostic information in patients with RD. Most TNBC patients with RD receive adjuvant therapy after surgery (chemotherapy and/or radiation), thus ctDNA status at completion of all adjuvant therapy (end of treatment, EOT) may be a better indicator of long-term prognosis. Furthermore, the impact of EOT ctDNA status on prognosis in context of RCB is of interest. Utilizing data from a prospective registry, the objective of this study was to investigate the impact of EOT ctDNA status and RCB class on outcomes in TNBC patients with RD. We hypothesized that RCB and EOT ctDNA status may provide complementary prognostic information. Methods: Study population included TNBC patients with RD post-NACT and available EOT plasma samples who were enrolled in an IRB-approved multisite prospective registry between 2011 and 2018. EOT samples were collected after completion (1-6 months) of all curative treatment (local and systemic). ctDNA was isolated and subjected to next generation sequencing (QIAseq 275-gene Human Comprehensive Cancer Panel on an Illumina NextSeq 550). Samples demonstrating pathogenic/likely pathogenic variant(s) with 3-40% allelic frequencies were considered ctDNA positive. Variants with allelic frequencies ≥40% were included in ctDNA positive status only if not present in ClinVar8/dbSNP9 as a known germline variant. The impact of EOT ctDNA status and RCB on event-free survival (EFS) and overall survival (OS) were estimated according to the Kaplan-Meier method and compared among groups by log-rank test, followed by Cox regression analysis. Results: For 47 TNBC patients with RD and available EOT plasma sample, the median age was 47 years, and 43% had node-positive disease at diagnosis. RCB class distribution was as follows: RCB I=28%, RCB II=49%, RCB III=15%, RCB unknown=8%. 45% of patients received adjuvant chemotherapy (59% with RCB II-III received adjuvant chemotherapy), and 68% received adjuvant radiation. EOT ctDNA was positive in 34% (16/47) of patients and was associated with higher T stage (p=0.012), TNM stage (p=0.033) and trend toward higher RCB class (p=0.078). ctDNA positivity rates in RCB I, II and III classes were 23%, 30% and 71%, respectively. Among all patients, 3-year EFS and OS were 71% and 73%, respectively. Table 1 provides 3-year EFS and OS by ctDNA status in all patients and by RCB class. ctDNA positive status was associated with inferior EFS and OS. Conclusion: EOT ctDNA positivity was noted in one-third of TNBC patients with residual disease and was highly prognostic, with almost half of patients with ctDNA positivity suffering an EFS event by 3 years. Patients with RCB III had very poor outcome (3-year EFS ≤20%) regardless of ctDNA status. However, in RCB classes I/II, ctDNA provided further prognostic utility, as ctDNA negative patients with RCB I/II had excellent outcomes (3-year EFS >90%). These findings should be confirmed in other studies and provide insights into the role of ctDNA for patient stratification/selection in residual disease adjuvant therapy intensification trials for TNBC. 3-year EFS3-year OSAll patients: ctDNA positive vs ctDNA negative56% vs 78%, HR 3.02 (95% CI: 1.01-9.01), p=0.03856% vs 82%, HR 3.05 (95% CI: 1.02-9.13), p=0.037RCB I/II: ctDNA positive vs ctDNA negative73% vs 92%, HR 4.38, p=0.07873% vs 92%, HR 3.03, p=0.159RCB III: ctDNA positive vs ctDNA negative0% vs 20%, HR 1.67, p=0.5610% vs 20%, HR 1.30, p=0.765 Citation Format: Priyanka Sharma, Shane R Stecklein, Bruce F Kimler, Rachel Yoder, Kelsey Schwensen, Joshua M Staley, Qamar J Khan, Anne P O'Dea, Lauren E Nye, Manana Elia, Jaimie Heldstab, Trisha Home, Stephen Hyter, Kamilla Isakova, Harsh B Pathak, Andrew K Godwin. Impact of post-treatment ctDNA and residual cancer burden (RCB) on outcomes in patients with triple-negative breast cancer (TNBC) and residual disease [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-05.

// Thuy-Vy Do 1 , Jeff Hirst 1 , Stephen Hyter 1 , Katherine F. Roby 2,3 and Andrew K. Godwin 1,3 1 Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA 2 Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS, USA 3 University of Kansas Cancer Center, Kansas City, KS, USA Correspondence to: Andrew K. Godwin, email: // Keywords : Aurora A kinase, ovarian cancer, PARP, alisertib, double-strand break Received : April 07, 2017 Accepted : June 16, 2017 Published : July 05, 2017 Abstract Ovarian cancer is usually diagnosed at late stages when cancer has spread beyond the ovary and patients ultimately succumb to the development of drug-resistant disease. There is an urgent and unmet need to develop therapeutic strategies that effectively treat ovarian cancer and this requires a better understanding of signaling pathways important for ovarian cancer progression. Aurora A kinase (AURKA) plays an important role in ovarian cancer progression by mediating mitosis and chromosomal instability. In the current study, we investigated the role of AURKA in regulating the DNA damage response and DNA repair in ovarian carcinoma cells. We discovered that AURKA modulated the expression and activity of PARP, a crucial mediator of DNA repair that is a target of therapeutic interest for the treatment of ovarian and other cancers. Further, specific inhibition of AURKA activity with the small molecule inhibitor, alisertib, stimulated the non-homologous end-joining (NHEJ) repair pathway by elevating DNA-PKcs activity, a catalytic subunit required for double-strand break (DSB) repair, as well as decreased the expression of PARP and BRCA1/2, which are required for high-fidelity homologous recombination-based DNA repair. Further, AURKA inhibition stimulates error-prone NHEJ repair of DNA double-strand breaks with incompatible ends. Consistent with in vitro findings, alisertib treatment increased phosphorylated DNA-PKcs(pDNA-PKcs T2609 ) and decreased PARP levels in vivo . Collectively, these results reveal new non-mitotic functions for AURKA in the regulation of DNA repair, which may inform of new therapeutic targets and strategies for treating ovarian cancer.

Drug development for first-line treatment of epithelial ovarian cancer (EOC) has been stagnant for almost three decades. Traditional cell culture methods for primary drug screening do not always accurately reflect clinical disease. To overcome this barrier, we grew a panel of EOC cell lines in three-dimensional (3D) cell cultures to form multicellular tumor spheroids (MCTS). We characterized these MCTS for molecular and cellular features of EOC and performed a comparative screen with cells grown using two-dimensional (2D) cell culture to identify previously unappreciated anticancer drugs. MCTS exhibited greater resistance to chemotherapeutic agents, showed signs of senescence and hypoxia, and expressed a number of stem cell–associated transcripts including ALDH1A and CD133, also known as PROM1. Using a library of clinically repurposed drugs, we identified candidates with preferential activity in MCTS over 2D cultured cells. One of the lead compounds, the dual COX/LOX inhibitor licofelone, reversed the stem-like properties of ovarian MCTS. Licofelone also synergized with paclitaxel in ovarian MCTS models and in a patient-derived tumor xenograft model. Importantly, the combination of licofelone with paclitaxel prolonged the median survival of mice (>141 days) relative to paclitaxel (115 days), licofelone (37 days), or vehicle (30 days). Increased efficacy was confirmed by Mantel–Haenszel HR compared with vehicle (HR = 0.037) and paclitaxel (HR = 0.017). These results identify for the first time an unappreciated, anti-inflammatory drug that can reverse chemotherapeutic resistance in ovarian cancer, highlighting the need to clinically evaluate licofelone in combination with first-line chemotherapy in primary and chemotherapy-refractory EOC. Significance: This study highlights the use of an in vitro spheroid 3D drug screening model to identify new therapeutic approaches to reverse chemotherapy resistance in ovarian cancer. Cancer Res; 78(15); 4370–85. ©2018 AACR.

There is a lack of personalized treatment options for women with recurrent platinum-resistant ovarian cancer. Outside of bevacizumab and a group of poly ADP-ribose polymerase inhibitors, few options are available to women that relapse. We propose that efficacious drug combinations can be determined via molecular characterization of ovarian tumors along with pre-established pharmacogenomic profiles of repurposed compounds. To that end, we selectively performed multiple two-drug combination treatments in ovarian cancer cell lines that included reactive oxygen species inducers and HSP90 inhibitors. This allowed us to select cell lines that exhibit disparate phenotypes of proliferative inhibition to a specific drug combination of auranofin and AUY922. We profiled altered mechanistic responses from these agents in both reactive oxygen species and HSP90 pathways, as well as investigated PRKCI and lncRNA expression in ovarian cancer cell line models. Generation of dual multi-gene panels implicated in resistance or sensitivity to this drug combination was produced using RNA sequencing data and the validity of the resistant signature was examined using high-density RT-qPCR. Finally, data mining for the prevalence of these signatures in a large-scale clinical study alluded to the prevalence of resistant genes in ovarian tumor biology. Our results demonstrate that high-throughput viability screens paired with reliable in silico data can promote the discovery of effective, personalized therapeutic options for a currently untreatable disease.

Skin homeostasis is maintained, in part, through regulation of gene expression orchestrated by type II nuclear hormone receptors in a cell and context specific manner. This group of transcriptional regulators is implicated in various cellular processes including epidermal proliferation, differentiation, permeability barrier formation, follicular cycling and inflammatory responses. Endogenous ligands for the receptors regulate actions during skin development and maintenance of tissue homeostasis. Type II nuclear receptor signaling is also important for cellular crosstalk between multiple cell types in the skin. Overall, these nuclear receptors are critical players in keratinocyte and melanocyte biology and present targets for cutaneous disease management.

The long-term overall survival of Ewing sarcoma (EWS) patients remains poor; less than 30% of patients with metastatic or recurrent disease survive despite aggressive combinations of chemotherapy, radiation and surgery. To identify new therapeutic options, we employed a multi-pronged approach using in silico predictions of drug activity via an integrated bioinformatics approach in parallel with an in vitro screen of FDA-approved drugs. Twenty-seven drugs and forty-six drugs were identified, respectively, to have anti-proliferative effects for EWS, including several classes of drugs in both screening approaches. Among these drugs, 30 were extensively validated as mono-therapeutic agents and 9 in 14 various combinations in vitro. Two drugs, auranofin, a thioredoxin reductase inhibitor, and ganetespib, an HSP90 inhibitor, were predicted to have anti-cancer activities in silico and were confirmed active across a panel of genetically diverse EWS cells. When given in combination, the survival rate in vivo was superior compared to auranofin or ganetespib alone. Importantly, extensive formulations, dose tolerance, and pharmacokinetics studies demonstrated that auranofin requires alternative delivery routes to achieve therapeutically effective levels of the gold compound. These combined screening approaches provide a rapid means to identify new treatment options for patients with a rare and often-fatal disease.

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV-induced melanocyte homeostasis. Selective ablation of endothelin-1 (EDN1) in murine epidermis (EDN1(ep-/-) ) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non-cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte-derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV-induced melanocyte activation and recapitulated the phenotype seen in EDN1(ep-/-) mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV-induced melanocyte homeostasis in vivo.

Background Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer death among women in the United States (5 % of cancer deaths). The standard treatment for patients with advanced EOC is initial debulking surgery followed by carboplatin-paclitaxel combination chemotherapy. Unfortunately, with chemotherapy most patients relapse and die resulting in a five-year overall survival around 45 %. Thus, finding novel therapeutics for treating EOC is essential. Connectivity Mapping (CMAP) has been used widely in cancer drug discovery and generally has relied on cancer cell line gene expression and drug phenotype data. Therefore, we took a CMAP approach based on tumor information and clinical endpoints from high grade serous EOC patients. Methods We determined tumor gene expression signatures (e.g., sets of genes) associated with time to recurrence (with and without adjustment for additional clinical covariates) among patients within TCGA (n = 407) and, separately, from the Mayo Clinic (n = 326). Each gene signature was inputted into CMAP software (Broad Institute) to determine a set of drugs for which our signature “matches” the “reference” signature, and drugs that overlapped between the CMAP analyses and the two studies were carried forward for validation studies involving drug screens on a set of 10 EOC cell lines. Results Of the 11 drugs carried forward, five (mitoxantrone, podophyllotoxin, wortmannin, doxorubicin, and 17-AAG) were known a priori to be cytotoxics and were indeed shown to effect EOC cell viability. Conclusions Future research is needed to investigate the use of these CMAP and similar analyses for determining combination therapies that might work synergistically to kill cancer cells and to apply this in silico bioinformatics approach using clinical outcomes to other cancer drug screening studies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3149-5) contains supplementary material, which is available to authorized users.

Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part, through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. We show that absence of keratinocytic RXRα in combination with mutant Cdk4 generated cutaneous melanoma that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf2) were elevated in skin of bigenic mice, while Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes. Significance Malignant melanoma continues to evade modern curative efforts as a result of the complex and elusive nature of metastatic tumors. We demonstrated that RXRα expression is lost in epidermal keratinocytes during melanoma progression in humans. We have presented evidence for the association of keratinocytic nuclear receptor RXRα-mediated paracrine/juxtacrine signaling and the malignant transformation of melanocytic tumors. The present work highlights cooperative effects of aberrant keratinocytic signaling and activated Cdk4 in melanoma metastasis. This mouse model will allow greater understanding of the mechanisms responsible for transition towards a malignant phenotype. Manipulation of these receptor-mediated signaling pathways could hold therapeutic value when combating metastatic disease.

Introduction: Reliable assessment of cancer-associated RNA markers in lung cancer produced by gene-fusions or exon skipping events by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We evaluated a comprehensive system for targeted RNA-Seq that includes reagents for nucleic acid quantification, library prep, run controls, and companion bioinformatics software. The reproducibility of this system was evaluated in a multi-phase study design at 5 independent laboratories. Methods: Total nucleic acid (TNA) was isolated from formalin-fixed, paraffin-embedded (FFPE) residual non-small cell lung cancer (NSCLC) tumor biopsies and cell-lines (RT-112, H596, HCC78). These TNA isolates were used to prepare a set of 30 test samples, including a dilution series to assess assay sensitivity. A non-template control and kit positive control were also included. The sample set was evaluated using the QuantideX® NGS RNA Lung Cancer Kit RUO (Asuragen) and sequenced on the MiSeq® system (Illumina) at Asuragen and 4 independent laboratories. Analyses were conducted using QuantideX® NGS Reporter RUO (Asuragen), a software suite that includes a FASTQ processing pipeline and incorporates pre-analytical QC information into the fusion-caller algorithm and reporting tool. Results: Laboratories were trained on the assay workflow and companion bioinformatics software in less than two days followed by independent library preparation and sequencing workflows. A total of 266 sample libraries were evaluated from inputs down to Conclusions: The accuracy of this novel targeted NGS assay for RNA fusions and splice variants in NSCLC was demonstrated in a multi-site laboratory evaluation using clinically-relevant specimens and low inputs of TNA. The ability of the panel to detect both common and rare gene fusion transcripts and exon skipping events within an integrated wet- and dry-bench workflow provides a foundation for the reliable detection of oncogenic RNA fusions and aberrant splicing events that can respond to current and emerging targeted therapies. This study highlighted the ease of implementation and consistent performance that can be achieved in different laboratories when the process from sample-to-report is highly integrated. Citation Format: Gary J. Latham, Richard Blidner, Brian C. Haynes, Shobha Gokul, Maria L. Aguirre, Stephen Hyter, Ziyan Y. Pessetto, Maria Curtis, Dan Su, Tom Halsey, Victor Weigman, Patrick Hurban, Andrew K. Godwin, Leon C. van Kempen. Accurate and reproducible detection of fusions and exon skipping events in NSCLC-derived samples using a comprehensive, targeted RNA-Seq system across multiple laboratories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1764. doi:10.1158/1538-7445.AM2017-1764

Understanding the molecular mechanisms behind formation of melanoma, the deadliest form of skin cancer, is crucial for improved diagnosis and treatment. One key is to better understand the cross-talk between epidermal keratinocytes and pigment-producing melanocytes. Here, using a bigenic mouse model system combining mutant oncogenic NRASQ61K (constitutively active RAS) or mutant activated CDK4R24C/R24C (prevents binding of CDK4 by kinase inhibitor p16INK4A) with an epidermis-specific knockout of the nuclear retinoid X receptor alpha (RXRαep−/−) results in increased melanoma formation after chronic ultraviolet-B (UVB) irradiation compared with control mice with functional RXRα. Melanomas from both groups of bigenic RXRαep−/− mice are larger in size with higher proliferative capacity, and exhibit enhanced angiogenic properties and increased expression of malignant melanoma markers. Analysis of tumor adjacent normal skin from these mice revealed altered expression of several biomarkers indicative of enhanced melanoma susceptibility, including reduced expression of tumor suppressor p53 and loss of PTEN, with concomitant increase in activated AKT. Loss of epidermal RXRα in combination with UVB significantly enhances invasion of melanocytic cells to draining lymph nodes in bigenic mice expressing oncogenic NRASQ61K compared with controls with functional RXRα. These results suggest a crucial role of keratinocytic RXRα to suppress formation of UVB-induced melanomas and their progression to malignant cancers in the context of driver mutations such as activated CDK4R24C/R24C or oncogenic NRASQ61K. Implications: These findings suggest that RXRα may serve as a clinical diagnostic marker and therapeutic target in melanoma progression and metastasis. Mol Cancer Res; 13(1); 186–96. ©2014 AACR.

Understanding the molecular mechanisms of ultraviolet (UV) induced melanoma formation is becoming crucial with more reported cases each year. Expression of type II nuclear receptor Retinoid-X-Receptor α (RXRα) is lost during melanoma progression in humans. Here, we observed that in mice with melanocyte-specific ablation of RXRα and RXRβ, melanocytes attract fewer IFN-γ secreting immune cells than in wild-type mice following acute UVR exposure, via altered expression of several chemoattractive and chemorepulsive chemokines/cytokines. Reduced IFN-γ in the microenvironment alters UVR-induced apoptosis, and due to this, the survival of surrounding dermal fibroblasts is significantly decreased in mice lacking RXRα/β. Interestingly, post-UVR survival of the melanocytes themselves is enhanced in the absence of RXRα/β. Loss of RXRs α/β specifically in the melanocytes results in an endogenous shift in homeostasis of pro- and anti-apoptotic genes in these cells and enhances their survival compared to the wild type melanocytes. Therefore, RXRs modulate post-UVR survival of dermal fibroblasts in a “non-cell autonomous” manner, underscoring their role in immune surveillance, while independently mediating post-UVR melanocyte survival in a “cell autonomous” manner. Our results emphasize a novel immunomodulatory role of melanocytes in controlling survival of neighboring cell types besides controlling their own, and identifies RXRs as potential targets for therapy against UV induced melanoma., Author Summary Melanoma is the deadliest form of skin cancer. It derives from melanocytes, the melanin-producing cells of our skin, which give our skin its tone in addition to protecting it from harmful effects of ultraviolet radiation (UVR). Changes in the skin microenvironment, such as signaling from other cell types, can influence melanoma progression. While several key genes in melanoma development have been identified, the underlying mechanisms are complex; different combinations of mutations can result in melanoma formation and genetic profiles of tumors can vary greatly among patients. Therefore, identification of novel therapeutic targets is crucial. Our present study uses a tissue-specific gene ablation strategy to characterize a novel role of type II nuclear receptors [Retinoid-X-Receptors (RXRs)] in melanocytes to control UVR-induced skin immune responses and cell survival. Several of these observed changes are risk factors for melanoma progression and identify RXRs as potential drug targets for melanoma diagnosis, prevention, and treatment. This newly-discovered role of retinoid receptor signaling in immune surveillance can be studied in different types of cancer and in other diseases including metabolic syndromes and atherosclerosis. The identified pathway is ideal for targeting using specific ligands or small molecule modulators of RXR signaling in different cell types and tissues.

Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. In the absence of keratinocytic RXRα, in combination with mutant Cdk4, cutaneous melanoma was generated that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf 2 ) was elevated in skin of bigenic mice, whereas Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes.

Chicken ovalbumin upstream promoter-transcription factor-interacting protein 2 (CTIP2) is a transcriptional regulator that is highly expressed in skin during mouse development, as well as in proliferating cells of adult mouse skin. We investigated expression of CTIP2 along with proliferation marker Ki-67 in normal human skin, and in skin from atopic dermatitis (AD), and in allergic contact dermatitis (ACD) samples by immunohistochemistry (IHC). We discovered for the first time that CTIP2 was expressed in proliferating basal and suprabasal layer in normal human epidermis. CTIP2 expression was dramatically increased in the epidermis from the AD and ACD samples compared with normal samples, and was labelled in both proliferating basal and suprabasal layers. Altogether our results suggest that CTIP2 expression could be linked to disease progression and/or maintenance in AD and ACD patients.

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer death among women in the United States (5% of cancer deaths). The standard treatment for patients with advanced EOC is initial debulking surgery followed by carboplatin-paclitaxel combination chemotherapy. Unfortunately, even with modern chemotherapy, most patients relapse and die with the five-year overall survival around 45%. In addition, those patients that initially respond to taxane-platinum therapy eventually develop platinum-resistant tumors and relapse. Thus, finding novel therapeutics for treating EOC is essential. One approach that has been used widely in cancer drug discovery is Connectivity Mapping (CMAP) using gene expression data and drug phenotype data assessed on cancer cell lines (CCL) (i.e. “sensitive” or “resistant” based on dose-response drug data). However, recent publications have highlighted some issues with the use of these standard CCL sets that are often used in these drug screening studies. Therefore, instead of CMAP analysis based on gene expression signatures developed on drug response data collected on a small set of EOC cell lines, we took a novel CMAP approach based on information collected on EOC patients and clinical endpoints. That is, we determined the gene expression signature (e.g., set of genes) that were associated with time to recurrence in data collected on EOC patients within TCGA (n = 518) and a Mayo Clinic study (n = 474). The set of genes associated with time to recurrence (with and without adjustment for additional clinical covariates) was completed separately for TCGA and Mayo Clinic studies, restricting all analyses to EOC tumor of high-grade serous histology. Each of these 4 sets of genes, where genes with hazard ratios (HR) > 1 coded as “positively” associated and genes with HR < 1 coded as “negatively” associated with the phenotype, were inputted into CMAP software (Broad Institute) to determine a set of drugs for which our signature “matches” the “reference” signature. The drugs that overlapped between the CMAP analyses on the two studies were carried forward for validation studies involving drug screens on a set of 10 EOC cell lines. Of the 11 drugs carried forward, we a priori hypothesized that 5 of these drugs would kill EOC cells (mitoxantrone, podophyllotoxin, wortmannin, doxorubicin, and 17-AAG), for which all 5 showed an ability to kill EOC cells in vitro (cell lines were treated using serial dilutions of the drugs (20-0 μM) for 72 h followed by measuring cell viability using the CellTiter-Blue assay). Further research is on-going to understand the functional mechanism by which these candidate drugs might treat EOC and to apply this bioinformatics approach to other cancer drug screening studies. Note: This abstract was not presented at the meeting. Citation Format: Brooke L. Fridley, Rama Raghavan, Gottfried E. Konecny, Chen Wang, Ellen L. Goode, Harsh B. Pathak, Stephen Hyter. A bioinformatics approach to drug discovery: patient based connectivity mapping. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4853. doi:10.1158/1538-7445.AM2015-4853