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Polo-like kinase 1 (PLK1) is an essential cell-cycle regulator that is frequently overexpressed in various human cancers. To determine whether Plk1 overexpression drives tumorigenesis, we established transgenic mouse lines that ubiquitously express increased levels of Plk1. High Plk1 levels were a driving force for different types of spontaneous tumors. Increased Plk1 levels resulted in multiple defects in mitosis and cytokinesis, supernumerary centrosomes, and compromised cell-cycle checkpoints, allowing accumulation of chromosomal instability (CIN), which resulted in aneuploidy and tumor formation. Clinically, higher expression of PLK1 positively associated with an increase in genome-wide copy-number alterations in multiple human cancers. This study provides in vivo evidence that aberrant expression of PLK1 triggers CIN and tumorigenesis and highlights potential therapeutic opportunities for CIN-positive cancers. Significance: These findings establish roles for PLK1 as a potent proto-oncogene and a CIN gene and provide insights for the development of effective treatment regimens across PLK1-overexpressing and CIN-positive cancers.

Background: MicroRNA-195 (miR-195) exerts the tumor suppressive functions in various cancers, including breast cancer, by targeting and inhibiting the expressions of the cell cycle and cell proliferation associated genes. In this study, we hypothesized that miR-195 low expressing tumors associate with high proliferative characteristics and poor survival. Material and Methods: We obtained the clinicopathological data and survival information of breast cancer patients from two large publicly available databases; The Cancer Genome Atlas (TCGA) and The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC). Total of 755 and 1287 patients’ data were obtained from TCGA and METABRIC respectively. Survival analysis, Overall survival (OS) and Disease-free survival (DFS) was performed by comparing the high and low expression groups. CYT score, xCell, and other immunological factors were used to evaluate intratumoral immune cell composition. Also, gene set enrichment analysis (GSEA) was performed between miR-195 high and low expression groups. Results: The patients were divided into miR-195 high and low groups by utilizing median cutoff. Advanced grades were significantly associated with lower expression of miR-195 in ER positive/HER2 negative (ER+/HER2) subtype with both TCGA and METABRIC cohorts (p Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Eriko Katsuta, Nobuhisa Matsuhashi, Manabu Futamura, Kazuhiro Yoshida, Kazuaki Takabe. Low expression MicroRNA-195 enriched the cell cycle and cell proliferation gene sets and is associated with advanced grades and worse overall survival of ER positive breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-24.

Cell cycle progression is a critical component of cell proliferation, and continuous proliferation is one of the hallmarks of cancer. Components regulating the E2F pathway have been identified in nearly every human malignancy and many of them including E2F transcription factors themselves play major roles in cancer progression, metastasis and treatment response of breasts cancer. Their activity therefore is expected to reflect tumor aggressiveness and responsiveness to therapy. We scored 3,905 tumors of nine breast cancer cohorts for this activity based on their 200 gene expression for the Hallmark E2F targets gene set. As expected, tumors with a high score had increased expression of cell proliferation-related genes, including G2M checkpoint, MYC targets v1 and v2, MITOTIC spindle, MTORC1 signaling, UNFOLDED protein response, and DNA repair in both the TCGA and METABRIC cohorts (false discovery rate < 0.001 in both cohorts). A high score was significantly associated with greater MKI67 expression (p < 0.001 in both cohorts), histological grade (p < 0.001 in both cohorts), and AJCC pathological stage (p < 0.001 in both cohorts). And Indel and single nucleotide variation neoantigen loads were associated with a high E2F pathway score (p = 0.006 and 0.047 respectively). Furthermore, the E2F pathway score correlated positively with copy number alteration (Spearman r = 0.55, p < 0.001). Intra-tumoral genome heterogeneity and proliferation score were significantly associated with the E2F pathway score as well (p < 0.001). The high E2F pathway score group demonstrated significantly higher fractions of not only pro-cancerous regulatory T cells, helper T cell (Th2), but also anti-cancerous CD4 memory T cell, helper T cell (Th1), and M1 macrophage as well as B cells compared to the low score group in the TCGA cohort (p < 0.001). Similar trends were observed in the METABRIC cohort. Furthermore, metastatic tumors had higher E2F scores than the primary tumors from which they arose especially luminal and normal subtype (p = 0.006 and p < 0.001). Interestingly, metastases with a high E2F score were associated with significantly worse PFS in the whole cohort, as well as patient sub-groups with local recurrence and with liver metastasis (p = 0.018, p = 0.025 and p = 0.027). Additionally, the E2F pathway score was significantly decreased with good response to chemotherapy (p < 0.001) and demonstrated higher pCR rate after neoadjuvant therapies in the high E2F pathway score group of estrogen receptor (ER)-positive/HER2-negative cancer (p < 0.001). The E2F score was significantly associated with expression of cyclin-dependent kinase (CDK)-related genes, including CCNE1, CDKN2A, CDKN2D, CDK2, CDK4 and CDK6 (all p < 0.001), and immune checkpoint molecule-related genes including PD-L1/2, CTLA4, IDO1, LAG3 and TIGIT (p = 0.013, 0.014, < 0.001, < 0.001, < 0.001 and 0.002, respectively). These results were validated by other cohort. Finally, the E2F score was strongly correlated with sensitivity to CDK inhibition in ER+/HER2- breast cancer cell-lines (Spearman r = 0.90, p = 0.04). In conclusion, the E2F score is a marker of breast cancer aggressiveness and predicts responsiveness of ER+/HER2- patients to neoadjuvant chemotherapy and possibly to CDK and immune checkpoint inhibitors. Citation Format: Masanori Oshi, Hideo Takahashi, Yoshihisa Tokumaru, Ryusei Matsuyama, Itaru Endo, Kazuaki Takabe. The E2F cell cycle pathway score to predict treatment response among patients with breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-35.

Background: Adhesion G protein-coupled receptors (GPCRs) form the second largest GPCR subfamily and control many cellular processes involved in cancer. ADGRF1 is an adhesion GPCR overexpressed and gene amplified in HER2+ and triple-negative breast cancer (BC). We have previously found a role of ADGRF1 in promoting tumorigenicity and metastatic processes in cell-based studies in HER2+ BC. The goal of this study was to evaluate the effects of ADGRF1 overexpression in vivo in HER2+ BC. Methods: BT474 stable cell lines were generated using lentiviral system containing ADGRF1 cDNA using PHAGE system, overexpressing ADGRF1 in Doxycycline (Dox)-inducible manner. Mice (N=8/group) were injected with various numbers of cells with (+) or without (-) Dox (10K, 30K, 100K, 300K, 1 million) to assess for tumor initiation and growth. Transcriptomic and proteomic analysis of ADGRF1-overexpressing BT474 cells was also performed using RNAseq and RPPA analysis, respectively. The candidates were validated using western blotting and IHC staining. Cell cycle analysis was conducted by measuring DNA content with propidium iodide using flow cytometry. Results: Dox-induced overexpression of ADGRF1 in mice led to inhibition of tumor growth compared to no Dox treated tumors. Furthermore, fewer mice injected with Dox-treated cells formed tumors. Our RNA-Seq data revealed downregulation of genes involved in cell cycle regulation, which included E2F targets, G2M checkpoint pathways, and MYC targets. Likewise in RPPA analysis, several proteins in the cell cycle pathway (FOXO1, AuroraA, Ki67, Ezh2, CHAF1, DEPTOR, Rb1, Phospho-Rb (S780), and Hexokinase II) were downregulated by ADGRF1 overexpression. Validation of RNAseq and RPPA candidates demonstrated lower number of Ki67+ cells and reduced NF-kB expression, and inhibition of STAT3 phosphorylation. Furthermore, we found that ADGRF1 overexpression by Dox also led to cell cycle arrest at GO/G1 phase. Conclusion: Our results suggest that ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer. These results are in contrast to the in vitro data demonstrating a role of ADGRF1 in promoting tumorigenicity and metastatic processes in HER2+ BC. Understanding the mechanism of this difference is critical in investigating ADGRF1 as a drug target in breast cancer. Citation Format: Raksha Bhat, Noor Mazin Abdulkareem, Lanfang Qin, Carmine De Angelis, Hala Yasser, Hariprasad Thangavel, Jennifer Torres, Vidyalakshmi Sethunath, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Martin Shea, Rachel Schiff, Meghana Trivedi. ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-07.

Background: Estradiol (E2) is known to promote breast cancer. However, several small-scale prospective clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)- resistant cases of hormone receptor-positive postmenopausal breast cancer. Considering that the practicality and safety of E2 treatments are still undefined, the objective of this study is to uncover the mechanisms of E2-induced tumor regression, which will lead to a better design of E2 therapies. Methods: While most of our ER+ breast cancer patient-derived xenograft (PDX) models are positively regulated by E2, an estrogen-suppressive model (named GS3) was established from an AI resistant ER+/PR−/HER2− brain metastatic breast cancer. E2 pellets (1mg each) or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC), RNA sequencing, and reverse phase protein array (RPPA) analyses of PDX specimens were conducted to decipher molecular changes after E2 treatments. Importantly, since the cancer tissue has a very heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist (MPP), not by ERβ antagonist (PHTPP). IHC showed that PR expression appeared, Ki-67 and CEACAM5 (CEA) expressions decreased, and the number of apoptotic cells significantly increased after E2 treatment. RNA sequencing indicated that estrogen-regulated genes, e.g., PGR, EGR3, PDZK1, and GREB1, were up-regulated, while tumor growth was repressed by E2. Single cell RNAseq analysis demonstrated that cells from E2-treated and Placebo-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes (HVGs). Importantly, after one-week, many cells of E2-treated PDXs were arrested within the G1 phase of the cell cycle (G1, 60%; S, 19%; G2M, 21%), whereas, cells of placebo-treated samples advanced to the S and G2M phases (G1, 47%; S, 27%; G2M, 26%). Gene Set Variation Analysis of cells from E2-treated PDXs revealed enrichment of genes associated with reactive oxygen species (ROS) mechanism. E2 increased cells expressing pro-apoptotic genes, such as TP53, BOK,IL24, and PGLYRP2. Gene expressions including mitogen-activated protein kinase (MAPK) signaling pathway, such as GADD45A, MAP3K5 (ASK1), MAPK9 (JNK2), and especially MAPKAPK2 were also increased in E2 treated PDXs. Conclusions: E2/ERα-mediated suppression of GS3 tumor growth was accompanying the inhibition of the cell cycle progression and increased expression of pro-apoptotic genes. It is known that classical MAPK pathway induces cell proliferation, meanwhile, JNK MAPK pathway induces p53 signaling pathway and apoptosis. We hypothesize that E2 induces ROS and promotes apoptosis of GS3 due to JNK MAPK pathway activation. This could be an unexpected outcome of AI resistance. Using this valuable estrogen-down-regulated ER+ breast cancer PDX and informative integrative omics analyses, critical molecular mechanisms on E2-mediated apoptosis are being revealed in our laboratory. The ongoing research using E2-sensitive PDXs will identify key markers to select AI resistant patients who are expected to respond effectively to estrogen-induced apoptosis treatments as well as to evaluate the efficacy of drug combination with estrogen, including CDK 4/6 inhibitors. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest and apoptosis in aromatase inhibitor-resistant breast cancer: Insights from single cells analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-08-01.

In breast cancer, both incidence and mortality have been increasing. Triple negative breast cancer (TNBC), one of breast cancer subtypes, is a heterogeneous nature that is negative for estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor type 2 (HER2). Due to the lack of expression of therapeutic targets, new therapeutic strategy and elucidation the mechanism of action in TNBC are strongly desired. Histone acetyltransferases E1A binding protein (p300) and CREB binding protein (CBP) are coactivators of a large number of essential transcription factors that contribute to cell survival and proliferation. p300/CBP (CREBBP) interacts with transcription factors and other proteins through histone acetyltransferase (HAT) and regulates several gene expression. Dysregulation of CREBBP HAT activity could contribute to various diseases, including cancer. It is reported that the high expression of p300 correlates with poor prognosis in breast cancers. However, the mechanism of CREBBP HAT activity remains unclear. We, therefore, focused on CREBBP and clarified its important mechanism in TNBC. In breast cancer cell lines, p300 expression was higher than the normal mammary epithelial cell (MCF10A) in mRNA and protein levels and positively correlated with that of CBP. To evaluate the roles of p300 and CBP in cell survival and proliferation, the loss of function studies was conducted. In TNBC cell line (MDA-MB-231), cell survival and proliferation significantly reduced in p300 knockdown cells. The reduction in cell survival and proliferation was also observed using p300 inhibitor (L002). To examine the mechanism of action for the reduction of cell survival and proliferation in p300 inhibition, cell cycle assay and apoptosis assay were performed. We found that the cell-cycle progression was arrested at the cell cycle G2/M phase and apoptosis induction in p300 knockdown cells. These results suggest that cell cycle G2/M phase arrest and the late stage of apoptosis are caused by p300 inhibition and p300 inhibition enhances the anti-tumor effect. To identify a mechanism responsible for the reduction of cell survival and proliferation in p300 inhibition, antibody array was performed. As a result of this assay and TCGA patients cohorts analysis, we identified survivin (BIRC5), a member of the inhibitor of apoptosis (IAP) family, as a functional novel biomarker of TNBC with significantly higher expression. These results suggest that suppression of BIRC5 through CREBBP inhibitor result in G2/M phase cell cycle arrest and apoptotic cell death in vitro and in vivo. In conclusion, CREBBP-BIRC5 transcriptional complex could be a novel therapeutic target for TNBC. Citation Format: Sunao Tanaka, Natsuki Wariishi, Nobuko Kawaguchi-Sakita, Souichi Adachi, Masakazu Toi, Yasuhiko Kamikubo. The importance of p300/CBP (CREBBP)-survivin (BIRC5) for cell cycle and apoptosis in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-06.

Breast Cancer is the main cause of cancer-associated mortality in women worldwide. The estrogen receptors (ER's) play an important role in normal mammary gland development, as well as in breast cancer. Estrogen Receptor α is expressed in 70% of breast cancers, where it contributes to increased cell proliferation and decreased cell death. Endocrine therapies such as anti-estrogens and aromatase inhibitors target ERα signaling and improve outcomes of these patients. Syngeneic, immunocompetent mouse models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of breast cancer. In contrast to the tumor-inducing role of ERα, ERβ has been shown to have tumor suppressive activities in various cancer, including the breast cancer. Compounds that selectively activate ERβ hold promise because they could potentially avoid the unwanted effects of ERα activation, while exploiting the tumor-suppressive function of ERβ. In the present study, we assessed the antitumor effects of ERβ agonists using three different syngeneic mouse models; D2A1 (BALB/c) and MM51 (FVB) syngeneic models and ex-vivo culture of highly metastatic cell line E0771 (C57/B6). Effect on in vitro cancer cell growth was evaluated by cell proliferation and clonogenic assays. Cell cycle distribution was analysed by flow cytometry. Our results demonstrate that ERβ agonists LY500307 and S-Equol not only inhibited the growth of all three mouse mammary tumor cell lines, but also reduced the colony formation ability. ERβ agonists also induced the cell-cycle arrest in time and dose-dependent manner. In mechanistic studies, ERβ agonists LY500307 and S-Equol, modulated the protein levels of cyclin-dependent kinases (CDKs) (4, 6, and 2), cyclins (D1 and E), in a differential manner in these three cell lines. Our in vivo studies of D2A1 and MM51 cells demonstrates that ERβ agonist LY500307 inhibited the tumor growth and the effect was more pronounce in combination with aromatase inhibitor letrozole. Ex-vivo model of E0771 cells showed that LY500307 has potential to dramatically reduce the proliferation of mouse mammary tumor growth.Together, these results identify potential molecular targets and anticancer effects of ERβ agonists in mouse mammary tumors. Citation Format: Ramasamy K, Samayoa C, Krishnegowda NK, Thurlapati A, Vadlamudi RK, Tekmal RR. Estrogen receptor β agonists inhibits syngeneic mammary tumor growth through cell-cycle arrest by modulating cell-cycle regulators [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-04-01.

Nucleotide supply is essential for DNA replication in proliferating cells, including cancer cells. Ribose-phosphate diphosphokinase 1 (PRPS1) is a key enzyme to produce the consensus precursor of nucleotide synthesis. PRPS1 participates in the pentose phosphate pathway (PPP) by catalyzing the phosphoribosylation of D-ribose 5-phosphate (R-5P) to 5-phosphoribosyl-1-pyrophosphate. Therefore, PRPS1 not only controls purine biosynthesis and supplies precursors for DNA and RNA biosynthesis but also regulates PPP through a feedback loop of the PRPS1 substrate R-5P. However, it is still elusive whether PRPS1 enhances nucleotide synthesis during cell-cycle progression. In this study, we explore the role and activation mechanism of PRPS1 in cell-cycle progression of colorectal cancer, and observed a peak in its enzymatic activity during S phase. CDK1 contributes to upregulation of PRPS1 activity by phosphorylating PRPS1 at S103; loss of phosphorylation at S103 delayed the cell cycle and decreased cell proliferation. PRPS1 activity in colorectal cancer samples is higher than in adjacent tissue, and the use of an antibody that specifically detects PRPS1 phosphorylation at S103 showed consistent results in 184 colorectal cancer tissues. In conclusion, compared with upregulation of PRPS1 expression levels, increased PRPS1 activity, which is marked by S103 phosphorylation, is more important in promoting tumorigenesis and is a promising diagnostic indicator for colorectal cancer. Significance: These findings show that the enzymatic activity of PRPS1 is crucial for cell-cycle regulation and suggest PRPS1 phosphorylation at S103 as a direct therapeutic target and diagnostic biomarker for colorectal cancer.

Inevitable tumor recurrence and a poor median survival are frustrating reminders of the inefficacy of our current standard of care for patients with newly diagnosed glioblastoma (GBM), which includes surgery followed by radiotherapy and chemotherapy with the DNA alkylating agent temozolomide. Because resistance to genotoxic damage is achieved mainly through execution of the DNA damage response (DDR) and DNA repair pathways, knowledge of the changes in DNA repair and cell-cycle gene expression that occur during tumor development might help identify new targets and improve treatment. Here, we performed a gene expression analysis targeting components of the DNA repair and cell-cycle machineries in cohorts of paired tumor samples (i.e., biopsies from the same patient obtained at the time of primary tumor operation and at recurrence) from patients treated with radiotherapy or radiotherapy plus temozolomide. We identified and validated a 27-gene signature that resulted in the classification of GBM specimens into three groups, two of which displayed inverse expression profiles. Each group contained primary and recurrent samples, and the tumor at relapse frequently displayed a gene expression profile different from that of the matched primary biopsy. Within the groups that exhibited opposing gene expression profiles, the expression pattern of the gene signature at relapse was linked to progression-free survival. We provide experimental evidence that our signature exposes group-specific vulnerabilities against genotoxicants and inhibitors of the cell cycle and DDR, with the prospect of personalized therapeutic strategies. Significance: These findings suggest that classification of GBM tumors based on a DNA repair and cell-cycle gene expression signature exposes vulnerabilities to standard-of-care therapies and offers the potential for personalized therapeutic strategies.

Preclinical and clinical development of agents that inhibit cell-cycle progression have brought an understanding of the feasibility of targeting various cell-cycle regulators in patients with cancer. Small molecule inhibitors targeting key proteins that participate in cell-cycle progression including the cyclin-dependent kinases and checkpoint kinases induce cell-cycle arrest and apoptosis in neoplastic cells. Early phase I studies demonstrate targeted inhibitors can be administered safely in adult and pediatric cancer patients, but these agents generally show limited clinical benefits as single agents. In this review, we discuss biological mechanisms that support dual combination strategies of cell-cycle inhibition with chemotherapeutic agents that are anticipated to achieve rationally targeted therapies for cancer patients. The rationale for evaluating these combination strategies is that DNA damage renders tumors highly responsive to irreversible cell-cycle arrest therapy. This approach is predicted to generate less intensive therapies and to maximize the efficacy of individual agents against solid tumors and hematologic malignancies. Cancer Res; 78(2); 320–5. ©2018 AACR.

Purpose: Leptomeningeal metastasis (LM) is a detrimental complication of non–small cell lung cancer (NSCLC) and associated with poor prognosis. However, the underlying mechanisms of the metastasis process are still poorly understood. Experimental Design: We performed next-generation panel sequencing of primary tumor tissue, cerebrospinal fluid (CSF), and matched normal controls from epidermal growth factor receptor (EGFR) mutation-positive NSCLC patients with LM. Results: The status of EGFR-activating mutations was highly concordant between primary tumor and CSF. PIK3CA aberrations were high in these patients, implicating an association with LM risk. Intriguingly, low overlapping of somatic protein-changing variants was observed between paired CSF and primary lesions, exhibiting tumor heterogeneity and genetic divergence. Moreover, genes with CSF-recurrent genomic alterations were predominantly involved in cell-cycle regulation and DNA-damage response (DDR), suggesting a role of the pathway in LM development. Conclusions: Our study has shed light on the genomic variations of NSCLC-LM, demonstrated genetic heterogeneity and divergence, uncovered involvement of cell-cycle and DDR pathway, and paved the way for potential therapeutic approaches to this unmet medical need. Clin Cancer Res; 24(1); 209–16. ©2017 AACR.

CDK7 is remarkable as a key regulator of both cell cycle progression and gene expression. CDK7 promotes cell cycle progression by phosphorylating cell cycle CDKs in the T-loop, thus stimulating their activities. Additionally, phosphorylation of RNA polymerase II (PolII) by CDK7 is required for transcription initiation. Deregulation of cell cycle and transcription processes is common to most cancer types, so CDK7 inhibitors offer considerable promise as cancer therapeutics. We previously reported the identification of the first selective CDK7 inhibitor, BS-181, and demonstrated its ability to inhibit breast cancer cell growth in vitro and in vivo (Ali et al 2009 Cancer Res). Screening of more than one thousand analogues has allowed development of a clinical candidate CDK7 inhibitor, named ICEC0942. ICEC0942 selectively inhibits CDK7 with an IC50 of 40nM. In vitro analyses reveal that ICEC0942 inhibits hormone receptor positive and triple-negative breast cancer cell lines, with GI50 values ranging between 0.2-0.3 μM. Growth inhibition is accompanied by inhibition of CDK7 targets, including CDK1, CDK2 and PolII phosphorylation. In xenograft studies using several cancer cell lines, the drug shows substantial anti-tumor effects, with a notable lack of toxicity at efficacious doses. In the combination setting with tamoxifen, ICEC0942 completely blocks growth of ER-positive tumor xenografts, indicative of potential for co-treatment with hormonal agents. Extensive ADMET and PK/PD studies confirm the suitability of ICEC0942 as a cancer drug and have shown that ICEC0942 is orally bioavailable. Moreover, xenograft tumor studies have allowed definition of surrogate biomarkers of tumor response. Taken together, our findings confirm CDK7 as an important drug target for ER-positive and -negative breast cancer and identify ICEC0942 as a prototype drug with utility as a single agent or in the combination setting. Our findings also point to the potential value of CDK7 inhibition by ICEC0942 in other cancer types that have characteristics of transcription factor addiction and/or cell cycle deregulation. Development of ICEC0942 was made possible through funding by EPSRC, Cancer Research UK and Cancer Research Technologies. Citation Format: Ali S, Patel H, Periyasamy M, Sava G, Bondke A, Slafer BW, Kroll SHB, Barbazanges M, Starkey R, Ottaviani S, Harrod A, Aboagye EO, Buluwela L, Fuchter MJ, Barrett AGM, Coombes RC. ICEC0942, a new oral selective inhibitor of the cell cycle and transcriptional regulator CDK7 for the treatment of estrogen receptor positive and negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-05.

Increasing evidence suggests the presence of minor cell subpopulations in prostate cancer that are androgen independent and poised for selection as dominant clones after androgen deprivation therapy. In this study, we investigated this phenomenon by stratifying cell subpopulations based on transcriptome profiling of 144 single LNCaP prostate cancer cells treated or untreated with androgen after cell-cycle synchronization. Model-based clustering of 397 differentially expressed genes identified eight potential subpopulations of LNCaP cells, revealing a previously unappreciable level of cellular heterogeneity to androgen stimulation. One subpopulation displayed stem-like features with a slower cell doubling rate, increased sphere formation capability, and resistance to G2–M arrest induced by a mitosis inhibitor. Advanced growth of this subpopulation was associated with enhanced expression of 10 cell-cycle–related genes (CCNB2, DLGAP5, CENPF, CENPE, MKI67, PTTG1, CDC20, PLK1, HMMR, and CCNB1) and decreased dependence upon androgen receptor signaling. In silico analysis of RNA-seq data from The Cancer Genome Atlas further demonstrated that concordant upregulation of these genes was linked to recurrent prostate cancers. Analysis of receiver operating characteristic curves implicates aberrant expression of these genes and could be useful for early identification of tumors that subsequently develop biochemical recurrence. Moreover, this single-cell approach provides a better understanding of how prostate cancer cells respond heterogeneously to androgen deprivation therapies and reveals characteristics of subpopulations resistant to this treatment. Significance: Illustrating the challenge in treating cancers with targeted drugs, which by selecting for drug resistance can drive metastatic progression, this study characterized the plasticity and heterogeneity of prostate cancer cells with regard to androgen dependence, defining the character or minor subpopulations of androgen-independent cells that are poised for clonal selection after androgen-deprivation therapy. Cancer Res; 78(4); 853–64. ©2017 AACR.

Research into the biology of soft tissue sarcomas has uncovered very few effective treatment strategies that improve upon the current standard of care which usually involves surgery, radiation, and chemotherapy. Many patients with large (>5 cm), high-grade sarcomas develop recurrence, and at that point have limited treatment options available. One challenge is the heterogeneity of genetic drivers of sarcomas, and many of these are not validated targets. Even when such genes are tractable targets, the rarity of each subtype of sarcoma makes advances in research slow. Here we describe the development of a synergistic combination treatment strategy that may be applicable in both soft tissue sarcomas as well as sarcomas of bone that takes advantage of targeting the cell cycle. We show that Rb-positive cell lines treated with the CDK4/6 inhibitor palbociclib reversibly arrest in the G1 phase of the cell cycle, and upon drug removal cells progress through the cell cycle as expected within 6–24 hours. Using a long-term high-throughput assay that allows us to examine drugs in different sequences or concurrently, we found that palbociclib-induced cell-cycle arrest poises Rb-positive sarcoma cells (SK-LMS1 and HT-1080) to be more sensitive to agents that work preferentially in S–G2 phase such as doxorubicin and Wee1 kinase inhibitors (AZD1775). The synergy between palbociclib and AZD1775 was also validated in vivo using SK-LMS1 xenografts as well as Rb-positive patient-derived xenografts (PDX) developed from leiomyosarcoma patients. This work provides the necessary preclinical data in support of a clinical trial utilizing this treatment strategy. Mol Cancer Ther; 16(9); 1751–64. ©2017 AACR.

All cancers are characterized by defects in the systems that ensure strict control of the cell cycle in normal tissues. The consequent excess tissue growth can be countered by drugs that halt cell division, and, indeed, the majority of chemotherapeutics developed during the last century work by disrupting processes essential for the cell cycle, particularly DNA synthesis, DNA replication, and chromatid segregation. In certain contexts, the efficacy of these classes of drugs can be impressive, but because they indiscriminately block the cell cycle of all actively dividing cells, their side effects severely constrain the dose and duration with which they can be administered, allowing both normal and malignant cells to escape complete growth arrest. Recent progress in understanding how cancers lose control of the cell cycle, coupled with comprehensive genomic profiling of human tumor biopsies, has shown that many cancers have mutations affecting various regulators and checkpoints that impinge on the core cell-cycle machinery. These defects introduce unique vulnerabilities that can be exploited by a next generation of drugs that promise improved therapeutic windows in patients whose tumors bear particular genomic aberrations, permitting increased dose intensity and efficacy. These developments, coupled with the success of new drugs targeting cell-cycle regulators, have led to a resurgence of interest in cell-cycle inhibitors. This review in particular focuses on the newer strategies that may facilitate better therapeutic targeting of drugs that inhibit the various components that safeguard the fidelity of the fundamental processes of DNA replication and repair. Clin Cancer Res; 23(13); 3232–40. ©2017 AACR.

Background: TGFBR1*6A is a polymorphic variant of the Transforming Growth Factor Beta receptor 1 (TGFBR1) that has three GCG synonymous deletions in its signal peptide. It is a low penetrance breast cancer susceptibility allele (OR, 1.15; 95% CI, 1.01 to 1.31) postulated to enhance tumor formation. In this study, a novel breast cancer mouse model carrying humanized TGFBR1*6A (hTGFBR1*6A) knock-in was used to investigate TGFBR1*6A influence on breast cancer formation, and dissect its molecular pathogenicity. Methods: Humanized mouse models of the two most common TGFBR1*6A genotypes - homozygous TGFBR1*6A/6A and heterozygous TGFBR1*9A/6A, and wild-type TGFBR1 were created by replacing murine Tgfbr1 exon 1 with human TGFBR1*6A or TGFBR1 exon 1. The three genotypes were bred with MMTV.Neu mice to study breast cancer development. Between 29-35 nulliparous females from each genotype were monitored for tumor formation (≥1 tumor), multiple tumor formation (≥2 tumors) and survival. Tumor cells were isolated from the hTGFBR1 and hTGFBR1*6A mice and validated for similar Neu protein expression. They were assessed for TGF-β/FoxO1 signaling as it relates to cell cycle progression and proliferation. Results: After 548 days, the mice harboring hTGFBR1*6A developed significantly more tumors (p=0.003 for one/more tumors, and p=0.0009 for two/more tumors) than wild-type mice. About 70% hTGFBR1*6A/6A and 28.6% hTGFBR1*9A/6A variants compared to 18.5% hTGFBR1 wild-type mice developed two (2) or more tumors. The hTGFBR1*6A mice also had shorter survival when compared with the wild-type mice (Log rank X2=7.77, p=0.02). The hTGFBR1*6A/6A and hTGFBR1*9A/6A mice survived for 178-210 and 208-282 days respectively compared to 198-342 days among the wild-type. The isolated hTGFBR1*6A tumor cells formed three (3) times more colonies, and exhibited higher growth rate and G1/S cell cycle transition than wild-type tumor cells. Interestingly, the hTGFBR1*6A cells were less sensitive to TGF-β anti-proliferative effects despite showing marked increase in TGF-β/SMAD signaling. Analyses of the TGF-β/FoxO1 growth inhibition pathway showed decreased FoxO1, p21 and p27 gene and protein expression, which was not increased significantly by TGF-β treatment. This suggests that TGFBR1*6A constitutively inhibits the TGF-β/FoxO1 cell cycle tumor suppressor arm. Conclusions: Altogether, this study provides new evidence in support of TGFBR1*6A as a breast cancer susceptibility allele. It also identifies deregulation of TGF-β/FoxO1 cell cycle inhibition as a potential mechanism underlying its increased cellular proliferation. Citation Format: Kojo Agyemang, Michael Pennison, Minghui Wang, Allan Johansen, Hugo Jimenez, Carl Blackman, Ravi Singh, Ralph D'Agostino, Paul Grippo, Antonio Di Cristofano, Boris Pasche. TGFBR1*6A enhances breast cancer susceptibility and promote cell cycle transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 806.

Invasive lobular breast carcinoma (ILC) is a low grade and mostly chemo-refractory luminal-type breast cancer that has been linked to sustained proliferative quiescence and long-term latency relapses (15-20 years). Loss of E-cadherin causes metastatic lobular breast cancer, partly through acquisition of anchorage independence. It is however still unknown how ILC cells control the balance between proliferative indolence and cell cycle re-entry at the metastatic site. We show here that E-cadherin loss leads to upregulation of Id2 through p120-catenin/Kaiso-dependent transcriptional derepression. Anchorage independent conditions further exacerbate p120-driven Id2 expression, leading to a sustained G0/G1 cell cycle arrest through binding of cytosolic Id2 to hypo-phosphorylated Rb. Intriguingly, we find that E-cadherin inactivation causes increased sensitivity to CDK4/6 inhibition in mouse and human breast cancer cell lines and primary tumor organoids. Finally, we find that Id2 expression is elevated in human ILC when compared to ductal breast cancers. Based on these data, we propose that combined E-cadherin loss and cytosolic Id2 expression can be used for the differential diagnosis of ILC and represent a candidate predictive biomarker pair for cell cycle targeting drug efficacy. Citation Format: Max Antonius Klaus Rätze, Thijs Koorman, Thijmen Sijnesael, Blessing Bassey-Archibong, Robert van de Ven, Lotte Enserink, Daan Visser, Sridevi Jaksani, Elvira Bakker, François Richard, Andrew Tutt, Rebecca Steele, Stephen Pettitt, Christopher J. Lord, Amanda Fitzpatrick, Clare Isacke, Paul J. van Diest, Christine Desmedt, Juliet M. Daniel, Patrick W.B. Derksen. E-cadherin loss drives Id2-dependent dampening of cell cycle progression and predicts increased susceptibility to CDK4/6 inhibition in lobular breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB246.

CCT is a protein folding complex that interacts with known oncoproteins, but its role as a driver of oncogenesis is unknown. Elucidating interactions of CCT with cell cycle regulators could reveal the oncogenic potential of the chaperonin. CCT is multi-subunit complex composed of two stacked rings formed by eight distinct subunits (CCT1-8). While different CCT subunits are associated with cancer and other diseases, we found that the CCT2 subunit is upregulated in breast cancer and other cancers and that its expression inversely correlates with patients' overall and progression free survival. Mining datasets from TCGA revealed that amplification of CCT2 is the predominant genomic alteration occurring in cancer patients and that CCT2 is frequently co-amplified or co-expressed with other oncogenes such as MDM2, FRS2, YEATS4, and CDK4 found in the 12q13-15 amplicon. Genetic alteration of CCT2 significantly associates with worse clinical outcome for cancer patients. To investigate the functional outcomes of CCT2 overexpression in breast cancer, spheroid culture was used. A CCT2-FLAG lentiviral vector was used to transduce T47D and MCF7 luminal breast cancer cells and cells grown in ultra-low attachment plates to induce spheroid growth. Expression of CCT2 increased growth of spheroids as compared to lentiviral control cells. In contrast, CCT2 depletion impaired spheroid formation, instead causing formation of loose aggregates. CCT2 overexpression increased the proliferative potential of spheroid cells and promoted progression though the G1/S phase of the cell cycle in synchronized cells by increasing expression of cell cycle regulators like Cyclin D1, CDK2 and CDK4. After 8 days in spheroid culture, CCT2 overexpressing cancer cells resumed attachment when transferred into 2D culture conditions; these cells acquire anchorage dependent and independent growth capability, increased actin expression, and proliferated. These characteristics of CCT2 overexpressing cells may be indicative of supporting a more invasive and metastatic phenotype. In support, we discovered that, along with cell cycle genes, CCT2 overexpressed cells increased expression of MYC, suggesting the CCT2 could be a novel regulator of MYC expression. Our results contribute new knowledge on the role of CCT2 in uncontrolled breast cancer progression and proliferation by regulating cell cycle genes. The recurrent amplification of CCT2 in different cancer types along with other oncogenes like MYC, MDM2 and CDK4 support it is role as a potential oncogene and novel therapeutic target for breast cancer. Citation Format: Heba H. Ghozlan, Adrian Showalter, Annette R. Khaled. Chaperonin-Containing TCP1 complex (CCT) promotes breast cancer by targeting key cell cycle regulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1967.

Background: Uncontrolled proliferation is a hallmark of cancer cells. Many cell cycle checkpoints and regulators are altered in tumors which in turn results in the accumulation of genetic aberrations. So, this study aimed to identify the mutational profile of cell cycle checkpoints and regulators in tissue biopsies in Egyptian Colorectal Cancer patients. Material and Methods: Sixty two fresh tissues samples were collected from Egyptian patients with CRC. The libraries were performed using Qiaseq UMI-based targeted panel.The detected genetic variants with an average coverage of 500x were annotated using Ingenuity Variant analysis (IVA) and pathway analysis was performed using Ingenuity Pathway analysis (IPA). Results: Data analysis revealed that TP53& ATM genes; the checkpoints at G1/S and G2/M phases, were highly mutated. They harbored 12 and 9 pathogenic mutations respectively, which cause loss of functionality. The most frequently detected pathogenic variants were c.121delG (58%) in the TP53 & c.640delT (29%) in the ATM. While CHECK2; the checkpoint at G2/M phase, harbored 4 pathogenic variants; c.430delC (16%) was the most detected one. Also, two pathogenic variants were detected in the M phase checkpoint BUB1B and the most frequently detected one was c.1669delA (37%). We also detected pathogenic variants involved in the G1/S phase regulators; SMAD4 (c.1241delT (23%)), CDKN2A (c.238delG (18%)), STK11 (c.340delG (11%)), and CDH1 (c.1955delT (19%)). Other pathogenic variants involved in G2/M phase regulators were also detected; BRCA1 (c.3214delC (16%)) & EP300 (c.4408delA (19%)). Moreover, we detected 2 pathogenic variants in M phase regulator; CDC27 (c.1998delA (7%) & c.2070delA (5%)). Conclusion: Our data showed the deregulated pattern of cell cycle checkpoints and regulators that are crucial for understanding colorectal cancer progression in Egyptian patients. Also, it may help in developing personalized targeted therapies that efficiently target the cell cycle genes mutations, thereby limits the CRC progression in those patients. Citation Format: Amira Salah El-Din Youssef, Mai M. Lotfy, Auhood Nassar, Abdel-Rahman N. Zekri. The mutational pattern of cell cycle checkpoints and regulators in Egyptian colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1964.

Background:Cyclin dependent kinase 4/6 inhibitors (CDK 4/6i) are standard of care for hormone receptor positive, HER2 negative metastatic breast cancer (MBC), alone or in combination with endocrine therapy. Combination with paclitaxel (P) is possible with an intermittent, alternating dosing schedule in order to synchronize (sync) the cell cycle (cc) with a short burst of CDKi. To optimize this strategy, we measured cc biomarkers before, during and after a 4 day run in of ribociclib (R) in patients (pts) receiving R with P in a phase I clinical trial (NCT02599363). Methods:Eligible pts with Rb-positive MBC of any subtype and measurable disease were treated with 200, 400 or 600 mg of R on Days (D) 2-5, 9-12 and 16-19, and P 80mg/m2 on D1, 8, 15 and 22 in 28-day cycles (C). Skin biopsies, blood samples and FLT-PET CT were assessed at baseline (pre- R burst), D-3 (post R burst) and C1D1 P (after cc re-entry). Skin biopsies were analyzed for Ki-67 (Dako Santa Clara, CA), phospho-Rb (Ser807/811; Cell Signaling, Danvers, MA) and Rb (clone 1F8, Thermo Scientific, Waltham, MA) by IHC. Serum and plasma were analyzed for thymidine kinase (TK, Biovica, Sweden) and R pharmacokinetics (PK, WuXi, China) respectively.P-Rb and Rb results were binned for intensity and pattern of staining.Intensity was defined as None (0), Weak (W, 1+), Moderate (M, 2+), Strong (S, 3+). Pattern was defined as: Rare (R, 50%). SUV max was averaged over the 5 brightest lesions.Results:13 pts enrolled to the Phase I trial. Nine pts had at least 1 skin biopsy; 8 had ≥2. Five pts had at least one FLT-PET. Biomarker results are summarized in table. Ki-67 (skin) and TK and FLT (tumor) drop after 4 days of R treatment, when R PK levels are highest. Decreases in P-Rb, were less consistent, observed in 4/8 (50%) pts. While R PK levels drop after 2 days off treatment, only 2/8 (25%) subjects had higher cell cycle measurements on C1D1 compared to baseline.Conclusions:CC synchronization with intermittent dosing is possible, and allows safe administration of P. More time off R is needed for cc re-entry. Future trials should give more time off of R before P delivery. Pt ID (Dose(mg))TKi-67 (%)P-RbIntensity- PatternRbIntensity- PatternR PKR PKTK (Du/L)FLT(SUV*)LEE011(ng/mL)LEQ803(ng/mL)002 (200)B22M-FM-D002045n/a004 (200)B8M-RM-D00190311.6D-34NM-D69.612.01459.55.2C1D15M-RM-D16.96.5165310005 (200)B11M-RW-F0083212.4D-32M-RW-D83.013.8122.55.1C1D161M-FM-F17.56.18234.514.8006 (200)Bn/an/an/a009030.512.3D-3n/an/an/a15412.37429.59.6013 (200)B19M-FM-D008566.0D-33M-FW-F15815.8767.55.2007 (400)B8M-FM-D003070.5n/aD-31NM-D33230.82009.5n/a010 (400)B23M-FM-D001964.7D-30.5NM-F35929.322.74.1C1D14M-RM-D78.516.4 Citation Format: Amy Clark, Azadeh Elmi, Nicholas P. McAndrew, Paul Wileyto, Natalie Shih, Michael Feldman, Mark Rosen, Jessica Savage, Robin Holmes, Nancy Dinubile, Theresa Berger, Erin Schubert, Alice Matthai, Melissa Volpe, Payal Shah, Susan Domchek, David Mankoff, Angela DeMichele. Cell cycle synchronization: Biomarker analysis in a phase I trial of alternating ribociclib and paclitaxel in advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB052.

Overcoming checkpoints to cell cycle control is the basis for tumorigenesis and malignant growth. Therefore, models that recapitulate clinically relevant cell cycle deregulation enhance our understanding of defined tumors subsets. Specifically, pancreatic ductal adenocarcinomas (PDAs) frequently delete the 9p21 locus which contains the cyclin dependent kinase (CDK) inhibitors CDKN2A (p16, p14) and CDKN2B (p15), as well as methylthioadenosine phosphorylase (MTAP), a metabolic gene required for methionine salvage from methylthioadenosine. No model currently exists that accurately represents loss of the entire locus in a relevant disease context. Moreover, the contribution of MTAP to tumor progression remains largely unknown. Therefore, we have developed a novel genetically engineered mouse model (GEMM) of PDA which combines loss of the orthologous murine 9p21 region (4qC4) with activated KRAS [Pdx-Cre; LSL-KrasG12D; 9p21L/L (K9C)], which results in rapid adenocarcinoma formation and subsequent mortality in mice homozygous for 9p21 deletion. Single-cell RNA sequencing revealed a remarkable level of inter- and intra-tumoral heterogeneity, including a significant immune and stromal component that contribute to tumor growth and progression. Additionally, K9C derived cell lines are responsive to Pfizer's first-in class CDK2/4/6 selective inhibitor while displaying de novo resistance to CDK4/6 inhibitor Palbociclib. Allograft and single-cell RNA sequencing experiments corroborated these findings and implicate Myc in contributing to CDK2/4/6i sensitivity. Furthermore, phenotypic-based screens revealed synthetic-lethal hits with 9p21 loss, indicating ample opportunities for combination strategies in this select patient population. Thus, we show that the K9C model recapitulates salient aspects of PDA and is amenable to novel therapeutic intervention strategies that may aid in improving the outcomes of patients with this precise genetic background. *All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and use committee Citation Format: Christina Adams, Lynn Wang, Tim S. Wang, Nichol Miller, Elizabeth McMillan, Monica Ramstetter, John Chionis, Koleen Eisele, Jonathan Almaden, Timothy Affolter, Smitha Pillai, Todd VanArsdale, Chris Dillon, Stephen G. Dann. A novel mouse model of pancreatic cancer reveals new insights into cell cycle deregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2960.

Background: Papillary thyroid carcinoma (PTC) is characterized by varied prognosis between gender and cancer subtype, but the cause for gender and subtype-based dissimilarities is unclear. Women are more frequently diagnosed with PTC, while men suffer more advanced-staged PTC. Additionally, tall cell and poorly differentiated variants are more aggressive than classical and follicular variants of PTC. While the intratumor microbiome has become increasingly implicated in cancer development, the microbial landscape of PTC is essentially uninvestigated. Past studies have shown how microbes within tumor tissue can contribute to or inhibit tumor growth by regulating gene expression and immune and cancer pathways. For example, a study published by Bahmani S. et. al. found that the absence of folate-producing Bifidobacterium and Lactobacillus was correlated with hypomethylation at regions of the p53 gene in colorectal cancer. We hypothesized that intratumor microbiome composition distinctly alters the immune landscape by preventing or inducing methylation of critical genes and may therefore predict cancer progression between PTC subtypes and between patient genders. Methods: Raw whole-transcriptome RNA-sequencing data for PTC and adjacent normal tissue was downloaded from The Cancer Genome Atlas, and microbial reads were extracted using Pathoscope 2.0 software. The Kruskal-Wallis test was performed to identify differentially abundant microbes between tumor and normal samples. Level 3 normalized DNA methylation 450k sequencing was downloaded from the GDC portal. We converted B-values to M-values and then performed the probe-wise differential methylation analysis on the matrix of M-values in limma to obtain t-statistics and p-values for each CpG site. The Kruskal-Wallis test was used to correlate microbe abundance to extent of methylation. R Results: Overall, PTC tumor tissue significantly lacked microbes that are populated in adjacent normal tissue. Tissue from different genders and subtypes was characterized by dysbiosis of specific microbe species, and microbes distinctly abundant in tall cell and male patient cohorts were correlated with greater methylation of tumor suppressors. The most significantly methylated sites occurred on chromosomes 1 and 17 and specifically at cell cycle related genes. Lower microbe abundance (Anabaena sp. K119, Trueperella pyogenes, Frankia sp.) in tumor tissue was predominantly correlated with greater extent of methylation at known tumor suppressor genes including NEURL1B, POLE, and SRCIN1. Conclusions: We identified microbes that are uniquely abundant in specific PTC types and are predominantly negatively correlated with methylation of tumor suppressors and cell cycle-related genes. Our results may provide a basis for developing specialized prebiotic and probiotic treatments for varied PTC tumors. Citation Format: Aditi Gnanasekar, Grant Castaneda, Anjali Iyangar, Shruti Magesh, Jaideep Chakladar, Wei T. Li, Lindsay M. Wong, Weg M. Ongkeko. Absence of intratumor microbes induces methylation of tumor suppressors and cell cycle-related genes in papillary thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1784.

14-3-3σ has been implicated in the development of chemo and radiation resistance and in poor prognosis of multiple human cancers. While it has been postulated that 14-3-3σ contributes to these resistances via inhibiting apoptosis and arresting cells in G2–M phase of the cell cycle, the molecular basis of this regulation is currently unknown. In this study, we tested the hypothesis that 14-3-3σ causes resistance to DNA-damaging treatments by enhancing DNA repair in cells arrested in G2–M phase following DNA-damaging treatments. We showed that 14-3-3σ contributed to ionizing radiation (IR) resistance by arresting cancer cells in G2–M phase following IR and by increasing non-homologous end joining (NHEJ) repair of the IR-induced DNA double strand breaks (DSB). The increased NHEJ repair activity was due to 14-3-3σ–mediated upregulation of PARP1 expression that promoted the recruitment of DNA-PKcs to the DNA damage sites for repair of DSBs. On the other hand, the increased G2–M arrest following IR was due to 14-3-3σ–induced Chk2 expression. Implications: These findings reveal an important molecular basis of 14-3-3σ function in cancer cell resistance to chemo/radiation therapy and in poor prognosis of human cancers. Mol Cancer Res; 15(4); 418–28. ©2017 AACR.

Anti-microtubule agents are widely used to treat ovarian cancers in the first line and recurrent setting either in combination with platinum or as single agents. However, the majority of patients will experience a recurrence, and most will die with drug resistant disease. We investigated co-targeting the actin cytoskeleton in combination with anti-microtubule agents to increase efficacy of treatment in epithelial ovarian cancers and potentially overcome resistance mechanisms. We examined the presence of actin/tropomyosin 3.1 (Tpm3.1) filaments in a large cohort of clinical specimens from patients with epithelial ovarian cancer of all histotypes using immunohistochemistry. Combinatorial effects of an anti-Tpm3.1 compound (ATM) with both vinorelbine and paclitaxel were evaluated in three ovarian cancer cell lines using cell viability and apoptosis assays. The mechanisms of synergy of both combinations were established using live-cell imaging, fluorescent microscopy, and pathway analysis. We found that Tpm3.1 is abundant and overexpressed in 97% of ovarian cancers examined (558 of 577) representing all histotypes of epithelial ovarian cancer. High levels of Tpm3.1 were also present in all sites sampled and similar at primary diagnosis and at recurrence. ATM displayed both single agent activity as well as synergy with both anti-microtubule drugs to reduce cell viability in all ovarian cancer cell lines tested, including one with platinum resistance. Only vinorelbine, however, synergised with ATM in the induction of apoptosis. Vinorelbine-induced mitotic arrest was significantly prolonged by ATM with elevated activity of the spindle assembly checkpoint, leading to almost one third of total cells dying in mitosis. In contrast, ATM showed minor impact on paclitaxel-induced mitotic defects. Both combinations resulted in a substantial increase in cells arrested in the subsequent G1 phase with a large decrease of both cyclin D1 and E1 as compared to single agents. Upregulation of p21Cip and p27Kip were associated with both combinations. In summary, targeting Tpm3.1-associated actin filaments in combination with anti-microtubule drugs is a promising treatment strategy that should be tested in clinical trials and is potentially applicable to all histotypes of ovarian cancer. Citation Format: Xing Xu, Yao Wang, Nicole Bryce, Katrina Tang, Nicola S. Meagher, Eun Young Kang, Linda E. Kelemen, Martin Köbel, Susan J. Ramus, Michael Friedlander, Caroline E. Ford, Edna C. Hardeman, Peter W. Gunning. Combined targeting of actin/tropomyosin and microtubules underlies a potential treatment strategy of epithelial ovarian cancer with cell-cycle dependent synergy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1045.

Combining cyclin-dependent kinase (CDK) inhibitors with endocrine therapy improves outcomes for metastatic estrogen receptor positive (ER+), HER2 negative, breast cancer patients. However, the value of this combination in potentially curable earlier stage patients is not clear. Using single cell transcriptomic profiling, we examined the evolutionary trajectories of early stage breast cancer tumors using serial tumor biopsies from a clinical trial of preoperative endocrine therapy alone (letrozole) or in combination with the cell cycle inhibitor ribociclib. Applying hierarchical regression and Gaussian process mathematical modelling, we classified each tumor by whether it shrinks or persists with therapy and determined cancer phenotypes related to evolution of resistance and cell cycle transcriptional rewiring. We found that all patients’ tumors undergo subclonal evolution during therapy, irrespective of the clinical response. However, tumors subjected to endocrine therapy alone showed reduced diversity over time, those facing combination therapy exhibited increased diversity. Despite different diversity, single nuclei RNA sequencing uncovered common phenotypic changes in tumor cells that persist following treatment. In these tumors, accelerated loss of estrogen signaling is convergent with up-regulation of the JNK pathway, while persistent tumors that maintain estrogen signaling during therapy show potentiation of CDK4/6 activation consistent with ERBB4 and ERK signaling up-regulation. Cell cycle reconstruction identified that these tumors can rebound during combination therapy treatment, indicating stronger selection and promotion of a proliferative state. These results indicate that combination therapy in early stage ER+ breast cancers with ER and CDK inhibition drives rapid evolution of resistance via a shift from estrogen signaling to alternative growth factor receptor mediated proliferation and JNK signaling activation, concordant with a bypass in the G1 checkpoint. Citation Format: JI Griffiths, J Chen, PA Cosgrove, A O'Dea, P Sharma, CX Ma, M Trivedi, K Kalinsky, KB Wisinski, R O'Reagan, I Makhoul, LM Spring, A Bardia, FR Adler, AL Cohen, JT Chang, QJ Khan, AH Bild. Convergent evolution of resistance pathways during early stage breast cancer treatment with combination cell cycle (CDK) and endocrine signaling inhibitors [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP012.

Background: Estrogen such as estradiol (E2) is known to promote ER+ breast cancer. However, several clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)-resistant cases of ER+ postmenopausal breast cancer. The objective of this study is to uncover the mechanisms of E2-induced tumor regression, leading to an unconventional treatment of AI resistance. Methods: An E2-suppressive patient-derived xenograft model (named GS3) was established from an AI resistant ER+/PR-/HER2- brain metastatic breast cancer. Placebo or E2 pellets were implanted in mice carrying GS3 for evaluating the effects of E2. Immunohistochemistry (IHC) and RNA sequencing of GS3 were conducted to decipher molecular changes after E2 treatments. Since the cancer tissue has a heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist, not by ERβ antagonist. IHC showed that ER, Ki-67 and CEA expressions decreased and PR expression appeared after E2 treatment. Gene set enrichment analysis (GSEA) using RNAseq results showed that the E2 response gene sets were significantly up-regulated after E2 treatment. However, the cell cycle gene sets and the TNFA/NFKB gene set were down-regulated. GS3 gained an E2 independence after three cycles of intermittent E2 treatment (E2 pellet on/off every 4 weeks; Int-E2). Interestingly, the cell cycle and TNFA/NFKB gene sets were up-regulated after Int-E2 treatment. Single-cell RNAseq analysis revealed that cells from one-week E2-treated and Placebo-treated GS3 were placed in different clusters based on principle component analysis of Highly Variable Genes. Although E2 response genes were up-regulated, the percent of ESR1+ cells decreased after E2 treatment (41.3% vs. 31.5%). The number of cells arrested at the G1 phase increased (+12.5%) after E2 treatment. GSEA using genes expressed in only ESR1+ cells showed that the TNFA/NFKB gene set was significantly down-regulated after E2 treatment. Meanwhile, GSEA using genes expressed in only ESR1– cells showed that cell cycle gene sets were significantly down-regulated. Single-cell trajectory analysis disclosed three major branches; 1) common E2 and placebo, 2) E2, and 3) placebo. In the E2 only branch, the cell cycle arrested at the G1 phase, the E2 response gene sets were up-regulated, and the NFKB gene set was down-regulated in ESR1+ cells. Significantly, E2 response gene sets were also up-regulated and cell cycle genes were down-regulated even in ESR1– cells. In the placebo branch, E2 response gene sets were not up-regulated and cell cycle genes were not down regulated. A group of MKI67+ cells (at G2M phase), including some ESR1+ cells, were present in both E2-treated and placebo-treated tumors. Conclusions: E2-induced suppression is an unexpected outcome of AI resistance. In these cases, elimination of estrogen by AI results in maintaining tumor growth. Analysis of GS3 PDX has revealed that estrogen can induce cell cycle arrest and the expression of estrogen-regulated genes. Our results also suggest the cross-talk between ESR1+ and ESR1- cells as well as potential roles of the TNFA/NFKB pathway. Our findings point out the need of markers for such patients who can benefit from E2 treatment after AI resistance, and measurements of ER and PR expression are not sufficient. An intermittent treatment strategy does not sustain the effect of estrogen-mediated suppression. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest as an unexpected outcome of aromatase inhibitor-resistance: Insights from single-cell trajectory analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-02.

Angiogenesis plays a crucial role in tumor development and metastasis, and many cancer cells upregulate vascular endothelial growth factor-A (VEGF-A) expression to promote angiogenesis. Several clinical trials of anti-angiogenic agents have been conducted in advanced and recurrent endometrial cancer, but only bevacizumab and cediranib have demonstrated activity as single agents. Bevacizumab is a monoclonal antibody against VEGF-A, whereas cediranib is a small molecule angiokinase inhibitor. However, subsequent studies of bevacizumab+chemotherapy failed to improve outcomes compared to chemotherapy alone. We recently demonstrated that chemotherapy plus nintedanib, another angiokinase inhibitor, promotes catastrophic cell death in a xenograft model of endometrial cancer (Ebeid, et al., Nat Nanotech 2019). This effect was specific to cells with loss of function, i.e., null mutations in TP53. Here we compared the efficacy of cediranib vs. bevacizumab in p53-null gynecologic cancer models and determined the mechanism for differential sensitivity. We first performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab vs. cediranib in two p53-null cell gynecologic cancer cell lines. Several signaling events were similar between bevacizumab and cediranib, including phosphorylation of IGF1R, Abl, p70S6K and proteins in the ERK/MAPK and Wnt signaling pathways. We next tested the impact of the anti- angiogenic agents on cell viability using cancer cell lines and over 20 patient-derived organoid cultures of endometrial or ovarian cancer. Neither bevacizumab nor cediranib alone had a notable effect on cell viability, even at 1-10 µM concentrations. By contrast, cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. This effect was most pronounced in p53-null models. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib+chemotherapy, consistent with abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Unexpectedly, the endometrial cancer cell lines were unresponsive to stimulation with VEGF-A, the target of bevacizumab. This was confirmed by the lack of ERK phosphorylation, a downstream signaling event expected after VEGF-A treatment, and was potentially caused low expression of VEGFR1 and 2. Based on these data, we conclude that an anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy. We hypothesize that the mechanism relates to cediranib inhibition of the tumor vasculature as well as its ability to optimize the impact of chemotherapeutic agents during the cell cycle. Future clinical studies should test this hypothesis by studying the combination of standard chemotherapy with cediranib in advanced and recurrent endometrial cancer. Citation Format: Jianling Bi, Andreea M. Newtson, Eric J. Devor, Yuping Zhang, Kristina W. Thiel, Kimberly K. Leslie. Anti-angiogenic tyrosine kinase inhibitor cediranib is superior to bevacizumab in endometrial cancer due to differential effects on cell cycle regulation [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO046.

Alterations in the cyclin-dependent kinase (CDK)-retinoblastoma (RB) machinery disrupt cell-cycle regulation and are being targeted in drug development. To understand the cancer types impacted by this pathway, we analyzed frequency of abnormalities in key cell-cycle genes across 4,864 tumors using next-generation sequencing (182 or 236 genes; Clinical Laboratory Improvement Amendments laboratory). Aberrations in the cell-cycle pathway were identified in 39% of cancers, making this pathway one of the most commonly altered in cancer. The frequency of aberrations was as follows: CDKN2A/B (20.1% of all patients), RB1 (7.6%), CCND1 (6.1%), CCNE1 (3.6%), CDK4 (3.2%), CCND3 (1.8%), CCND2 (1.7%), and CDK6 (1.7%). Rates and types of aberrant cell-cycle pathway genes differed between cancer types and within histologies. Analysis of coexisting and mutually exclusive genetic aberrations showed that CCND1, CCND2, and CCND3 aberrations were all positively associated with CDK6 aberrations [OR and P values, multivariate analysis: CCND1 and CDK6 (OR = 3.5; P < 0.0001), CCND2 and CDK6 (OR = 4.3; P = 0.003), CCND3 and CDK6 (OR = 3.6; P = 0.007)]. In contrast, RB1 alterations were negatively associated with multiple gene anomalies in the cell-cycle pathway, including CCND1 (OR = 0.25; P = 0.003), CKD4 (OR = 0.10; P = 0.001), and CDKN2A/B (OR = 0.21; P < 0.0001). In conclusion, aberrations in the cell-cycle pathway were very common in diverse cancers (39% of 4,864 neoplasms). The frequencies and types of alterations differed between and within tumor types and will be informative for drug development strategies. Mol Cancer Ther; 15(7); 1682–90. ©2016 AACR.

Significant endocrine therapy–resistant tumor proliferation is present in ≥20% of estrogen receptor–positive (ER+) primary breast cancers and is associated with disease recurrence and death. Here, we uncover a link between intrinsic endocrine therapy resistance and dysregulation of the MutL mismatch repair (MMR) complex (MLH1/3, PMS1/2), and demonstrate a direct role for MutL complex loss in resistance to all classes of endocrine therapy. We find that MutL deficiency in ER+ breast cancer abrogates CHK2-mediated inhibition of CDK4, a prerequisite for endocrine therapy responsiveness. Consequently, CDK4/6 inhibitors (CDK4/6i) remain effective in MutL-defective ER+ breast cancer cells. These observations are supported by data from a clinical trial where a CDK4/6i was found to strongly inhibit aromatase inhibitor–resistant proliferation of MutL-defective tumors. These data suggest that diagnostic markers of MutL deficiency could be used to direct adjuvant CDK4/6i to a population of patients with breast cancer who exhibit marked resistance to the current standard of care. Significance: MutL deficiency in a subset of ER+ primary tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy–resistant tumors. Therefore, markers of MutL dysregulation could guide CDK4/6 inhibitor use in the adjuvant setting, where the risk benefit ratio for untargeted therapeutic intervention is narrow. Cancer Discov; 7(10); 1168–83. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047

Rosmarinic acid (RA) is a polyphenolic compound with various pharmacological properties, including anti-inflammatory, immunomodulatory, anti-mutagenic, antioxidant, and anticancer activities. This study evaluated the effects of RA in two racially different triple-negative breast cancer (TNBC) cell lines. One cell line was derived from Caucasian origin (MDA-MB-231), and another one was derived from an African American origin (MDA-MB-468). The apoptotic effect of RA was performed using An Annexin V-FITC Apoptosis Detection Kit, while the Propidium Iodide Flow Cytometry Kit was used to detect the cell cycle distribution. Gene expression profiling was established using Human Apoptosis PCR array. The results obtained show that RA significantly caused cytotoxic and antiproliferative effects in both cell lines in a dose- and time-dependent manner. It was noted that RA induced cell cycle arrest-related apoptosis and altered the expression of many apoptosis-involved genes differently. In MDA-MB-231 cells, RA arrested the cells in the G0/G1 phase. In contrast, RA causes S-phase arrest in MDA-MB-468 cells, leading to a 2-fold increase in the apoptotic effect compared to MDA-MB-231 cells. Furthermore, in MDA-MB- 231 cells, RA significantly upregulated the mRNA expression of three genes: Harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL-2 interacting protein 3 (BNIP3). In contrast, in the MDA-MB-468 cell line, RA induced a significant transcription activation in three genes, including TNF, growth arrest, and DNA damage-inducible 45 alpha (GADD45A), and BNIP3. Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MDA-MB-231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) and baculoviral IAP repeat-containing 5 (BIRC5) in MDA-MB-468 cells. In conclusion, the data suggest that the polyphenol RA may have a potential role in TNBC therapies, particularly in African American cells. Citation Format: Karam F.A. Soliman, Samia S. Messeha, Najla O. Zarmouh, Abrar Asiri. Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-132.

Cancer cells forming primary tumors are heterogeneous and respond in various ways to the stimuli from tumor microenvironment. Transforming growth factor-β (TGF-β) has been implicated in progression of various types of epithelial cancer. TGF-β is known to induce epithelial-mesenchymal transition (EMT) leading to increased invasiveness and thus metastasis. In addition, it has been reported that TGF-β arrests the epithelial cell in early G1 phase. While TGF-β elicits multiple effects on epithelial cancer cells, heterogeneity of tumor mass results in a different response depending on the cell state, thus it remains to be elucidated whether the cells exhibiting TGF-β-induced EMT and high motility are also under the cell cycle arrest. To examine the correlation between TGF-β-induced motility and cell cycle arrest at a single cell level, we have utilized oral squamous carcinoma cell lines, carrying fluorescent ubiquitination-based cell cycle indicator (Fucci) system. Using quantitative RT-PCR, immunoblotting and FACS analysis, we determined the expression of cell cycle-related genes and characterized the population of cells residing in certain cell cycle phases upon TGF-β treatment. Cells treated with TGF-β were also tested for their migratory ability to compare motility of cells residing in different cycle phases. Surprisingly, cells residing in G1 phase were characterized by higher motility than cells residing in S/G2/M phases suggesting a correlation between TGF-β-dependent cell cycle progression and migration. Moreover, increased motility of cells residing in G1 phase did not resulted from the progression of EMT. These results were also confirmed by our in vivo studies. In orthotropic xenograft model, cells under G1 arrest represented the majority of cells forming metastatic lymph nodules, in contrast to primary tumor mass comprising almost of equal number of cells residing in S/G2/M and G1 phases. We also performed cDNA microarray analyses of G1 and S/G2/M phase SAS-Fucci cells and identified gene whose expression was associated with both TGF-β-induced migration and cell cycle arrest. In conclusion our data suggested that cells under G1 cell cycle arrest are motile, but this increased motility is not a result of TGF-β-dependent EMT. Our data also strengthen the importance of targeting both, proliferating cancer cells and cell cycle-arrested cancer cells for the effective anti-cancer therapy. Citation Format: Katarzyna A. Inoue, Kazuki Takahashi, Maki Saito, Atsushi Kaida, Akinari Sugauchi, Toshihiro Uchihashi, Yasuhiro Yoshimatsu, Susumu Tanaka, Masahiko Miura, Mikihiko Kogo, Tetsuro Watabe. Oral squamous carcinoma cells under TGF-β-induced cell cycle arrest represent highly motile and invasive population [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-040.

Introduction; Tumor Treating Fields (TTFields) is a novel non-invasive anticancer treatment modality utilizing alternating electric fields. TTFields is FDA approved for glioblastoma patients after demonstrating a 5 months increase in overall survival, and for unresectable mesothelioma patients after the STELLAR trial showed an overall survival of 18 months without increasing the toxicity. While the effectiveness of TTFields is initially attributed to the effects of TTFields during cell division, studies are ongoing analysing its interphase effects and impact on the tumor (micro)environment. Our aim is to further exploit the working mechanism of TTFields by analysing its effect on the cell cycle and identify targets to increase its efficacy. Material and Methods; To analyse the effect of TTFields on the cell cycle, A172 glioblastoma cells were synchronized in S-phase by double thymidine block. Six hours post-release, cells were subjected to TTFields treatment (200kHz; 4,5V/cm). Cell cycle analysis was performed by flow cytometry (PI, α-phospho-histone H3Ser10). Efficacy of combination strategies were tested in 3 different glioblastoma cells lines (A172, U251 and SNB-19). Efficacy was quantified by colony formation assay (CFA) testing the cell reproductive death after treatment (i.e. only viable cells after treatment are replated to analyse the colony forming capacity). Results: TTFields treatment of synchronized A172 cells showed that TTFields causes an accumulation of cells in G2 phase and delayed entry into mitosis with less than 1% of the cells in mitosis 4 hours after TTFields exposure (versus 5% of mitotic cells in the control arm, p Conclusion: The underlying mechanism of the G2 checkpoint activation and the synergistic effect of TTFields plus G2 checkpoint targeting agents is subject of ongoing research. By any means, the combination of TTFields and G2 checkpoint targeting agents causes a synergistic treatment effect and is a very promising strategy. Citation Format: Paul Slangen, Mariska van Geldorp, Mark de Gooijer, Olaf van Tellingen, Gerben Borst. Increasing TTFields treatment efficacy by targeting G2 cell cycle checkpoint [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 92.

Melanoma is the fifth most common cancer in the United States that has less than a 25% 5-year survival rate once the cancer has metastasized beyond the lymph nodes. Recent exome studies of cutaneous melanoma have identified RAC1P29S as the third most prevalent hotspot mutation in 4-9% of sun-exposed melanoma. RAC1 alteration in cancer is correlated to poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. As a member of the Ras superfamily of small GTPases, RAC1 functions as a master regulator of actin cytoskeleton dynamics and influences proliferation, cell migration, motility, and polarity. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are becoming increasingly evident, the RAC1-driven biological mechanisms contributing to melanomagenesis remain elusive. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis has identified the G2-M cell cycle phase transition as a hyperactive pathway in RAC1P29S-expressing melanocytes. RAC1P29S expression promotes increased kinase activity of the PAK-PLK1-AURORA A axis and increased transition into M-phase as indicated by FACS analysis. Interestingly, RAC1P29S-expressing melanocytes also possess increased DNA damage via γH2AX foci, suggesting a compromise in G2-M checkpoint integrity and an altered DNA damage response. Here, we assess the influence of RAC1P29S on the PAK-PLK1-AURORA A axis and its role in promoting G2-M transition. In addition, we investigate the DNA damage response and cell cycle regulation in the presence of cytotoxic agents. Collectively, these results suggest novel therapeutic pathways to target in RAC1P29S-driven melanoma. Citation Format: Alexa C. Cannon, Jonathan Chernoff. RAC1P29S expression in melanocytes is associated with increased DNA damage and altered cell cycle progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2587.

The purpose of this study is to develop a comprehensive mathematical model of DNA double-strand break (DSB) repair in a cell cycle-dependent manner to analyze the improved radiosensitivity of prostate cancer (PCa) cells to ionizing radiation (IR) when radiation treatment is combined with androgen deprivation therapy (ADT). The effectiveness of the combination treatment, which is currently a standard treatment for PCa, depend on DSB repair capacity of the cells as these are the main lesions following IR. It is reported in the literature that the major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), are both impaired after ADT, which then resulted in increased radiosensitivity and better IR treatment outcomes in PCa. In our previous work, we have developed quantitative models for NHEJ and HR individually to analyze the mechanism of the effect of ADT on IR treatment outcomes through impaired repair dynamics. The present work combines these two models in a cell cycle-dependent manner in order to develop a comprehensive model to analyze the repair dynamics after IR only and IR+ADT treatments. NHEJ is the major pathway, whereas HR is restricted to S- or G2-phases of the cell cycle after DNA replication has been completed and we have incorporated the cell cycle dependent contributions of the NHEJ and HR models in our comprehensive model. The literature data used in the development of these previous models were from both in vitro experiments as well as clinical data from PCa patients. In our comprehensive model, we have used the data from the literature to determine the distribution of the initial DSBs for cells in different cell cycle phases as the damage depends on the cell cycle phase at the time of radiation. We have incorporated the data on the percent contributions of NHEJ and HR repair in different cell cycle phases into the model and calculated the repair outcomes from NHEJ and HR models according to these ratios. Cell cycle arrest is implemented in relation to the amount of remaining DSBs in each cell cycle phase after repair. Using the number of unrepaired DSBs, we have calculated the proportion of cells that would progress to the next cell cycle phase as well as the proportion for which the cell death mechanism is triggered. The simulation results showed that ADT combined with IR has enhanced the treatment outcome. The cell survival rate was lower for the combination treatment case (55.7% compared to 62.1 % of IR only case). The cell proliferation was also significantly slower (85-95h doubling time compared to 35-45h of IR only case). The results agreed well with the experimental data that showed 30h and 95.5 h of doubling times for IR only and IR+ADT treatment respectively. The comprehensive model outcomes show that impaired NHEJ and HR dynamics as a result of ADT have the potential to enhance the IR treatment outcomes for PCa patients. Citation Format: Mengdi Qian, Alexandru Almasan, Evren Gurkan-Cavusoglu. Cell cycle-dependent, comprehensive mathematical modeling of the role of DNA repair in response to radiotherapy for prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5499.

Dysregulation of cellular proliferation and evasion of apoptosis are key contributors to colon cancer (CoCa) development. Chemotherapeutic agents such as 5-Fluorouracil (5FU) and Oxaliplatin (OxT) which are frequently offered to treat CoCa are known to target cells in S and G2 phase of cell cycle. Cancer cells often overcome the effects of 5FU and OxT by deregulating cell cycle in response to these drugs. Therefore, to improve the cytotoxic effect of these chemotherapeutic agents it is important to keep CoCa cells in S and G2 phase. Hence, understanding the molecular mechanisms involved in CoCa cell-cycle progression and chemotherapeutic response could provide strategies for developing effective treatment options. Hence, in this study, we determined the effect of natural agent “Emodin” on CoCa cell-cycle regulation and its ability to increase the efficacy of 5FU and OxT by cell cycle regulation. The effects of Emodin, 5FU, and OxT as a single agent or in combination on CoCa (DLD-1, COLO-201, HCT116 p53WT, and HCT116 p53-/-) cell viability were determined by MTT assays. Combination index of Emodin, 5FU, and OxT were evaluated using Calcusyn. Emodin-induced changes in molecules involved in cell-cycle regulation and proliferation were analyzed via RT-PCR and confirmed by western blot (WB) analysis. Cell-cycle distribution with Emodin treatment alone and in combination with 5FU and OxT were assessed via flow cytometry and WB. Collectively, our data shows that Emodin alone was able to decrease cell survival and induced apoptosis in CoCa cells. Furthermore, cytotoxic ability of 5FU and OxT was increased when combined with Emodin as compared to when these chemotherapeutic drugs were given as single agents Emodin-induced cell-cycle regulation resulted in increased expression of tumor suppressor genes such as p21, p27 and p53, modulated cyclin/cyclin-dependent kinase expression, and induced cell-cycle arrest. Emodin also negatively impacted several signaling pathways associated with cell growth and differentiation. Overall, this study demonstrates that Emodin could be a promising adjuvant to either 5-Fluorouracil or Oxaliplatin in the treatment of CoCa. Citation Format: Ian T. Saunders, Dexter Ezeagwu, Naresh Kumar, Hina Mir, Shailesh Singh. Emodin enhances efficacy of Oxaliplatin and 5 Fluorouracil in colon cancer by promoting cell cycle arrest [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2428.

A majority of cutaneous melanomas harboring oncogenic driver BRAF-V600E gene mutations initially demonstrate superior responses to targeted therapy with BRAF inhibitors (BRAFi), MEK inhibitors (MEKi), or the combination of both. Unfortunately, tumor regrowth and acquired resistance to BRAF and MEK inhibitors is inevitable, and new therapies are required to treat these patients. To determine acquired resistance mechanisms, we performed Whole Exome Sequencing (WES) on BRAF-V600 mutant cutaneous melanomas from two patients each at two timepoints; (1) prior to BRAFi/MEKi combination therapy, and (2) upon tumor regrowth following therapy. Following BRAFi/MEKi resistance, both patients acquired an activating mutation in NRAS and loss of the tumor suppressor CDKN2A. We hypothesized the acquired mechanism of resistance was through reactivation of MAPK pathway, via NRAS mutations, and activation of the cell cycle pathway, via loss of CDKN2A. Further, we predicted that these resistant patents samples would be sensitive to the combination of inhibiting the MAPK and cell cycle pathway. Both BRAFi/MEKi resistant patient samples were established as patient-derived xenograft (PDX) models to test the in vivo efficacy of trametinib (MEKi) and palbociclib (CDK4/6i) as single agents and in combination. In both PDX models, the combination of MEKi and CDK4/6i resulted in significant tumor reduction as compared to the vehicle control and single agents. Using immunohistochemistry, we observed that only in the combination treated tumors there was a significant decrease in tumor proliferation, measured by Ki-67, and MAPK pathway activity, measured by phos-ERK. To study the mechanism of response to the combination of MEKi and CDK4/6i in the context of BRAFi acquired resistance, we generated an in vitro resistance model using a panel of BRAF-V600 mutant cutaneous melanoma cell lines constantly treated with the BRAF inhibitor, vemurafenib, to generate a resistant cell population. We observed that parental cells are more sensitive to single agent MEKi, and more resistant to single agent CDK4/6i, as compared to BRAFi resistant cells. In line with this, we found that MEKi in parental cells decreased both phos-ERK and phos-RB, suggesting that the parental cell lines are dependent on the MAPK pathway, and that the cell cycle pathway is under MAPK pathway regulation. However, in BRAFi resistant lines, MEKi only slightly reduced phos-ERK, and did not significantly decrease phos-RB levels. Suggesting that, in BRAFi resistant lines, the cell cycle pathway is not regulated by the MAPK pathway. Decreased phos-ERK and phos-RB levels are only observed with the combination of MEKi and CDK4/6i. Furthermore, we found that the BRAFi resistant cells are more sensitive to a low concentration of CDK4/6i in combination with MEKi, as compared to the parental cells. We found that parental cells required a higher concentrations of CDK4/6i in combination with MEKi to reduce cell proliferation. Whereas, the BRAFi resistance cells are the opposite and require a lower concentration of CDK4/6i, and a higher concentration of MEKi, to have a durable response. Taken together, this data supports the observation that upon BRAFi resistance, the cell cycle pathway is uncoupled from the MAPK pathway, and cell cycle reactivation may represent a common acquired resistance mechanism to MEKi. Citation Format: Kelsey W. Nassar, Jennifer Hintzsche, Stacey Bagby, Christophe Langouet-Astrie, Jenny Samson, Carol Amato, Nicholas Gordon, Mayumi Fujita, Rebecca Schweppe, William Robinson, Aik-Choon Tan. Uncoupling of the cell cycle pathway from the MAPK pathway in BRAF/MEK inhibitor resistant melanoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B076. doi:10.1158/1535-7163.TARG-19-B076

p28 is an anionic cell-penetrating peptide of 28 amino acids that activates wild-type and mutated p53, leading subsequently to selective inhibition of CDK2 and cyclin A expression and G2–M cell-cycle arrest. In this study, we investigated the cytotoxic effects of p28 treatment alone and in combination with DNA-damaging and antimitotic agents on human cancer cells. p28 enhanced the cytotoxic activity of lower concentrations (IC20-50) of DNA-damaging drugs (doxorubicin, dacarbazine, temozolamide) or antimitotic drugs (paclitaxel and docetaxel) in a variety of cancer cells expressing wild-type or mutated p53. Mechanistic investigations revealed that p28 induced a post-translational increase in the expression of wild-type or mutant p53 and p21, resulting in cell-cycle inhibition at the G2–M phase. The enhanced activity of these anticancer agents in combination with p28 was facilitated through the p53/p21/CDK2 pathway. Taken together, these results highlight a new approach to maximize the efficacy of chemotherapeutic agents while reducing dose-related toxicity. Cancer Res; 76(8); 2354–65. ©2016 AACR.

Purpose: Highly aggressive and resistant to chemotherapy, pancreatic cancers are the fourth leading cause of cancer-related deaths in the western world. The absence of effective chemotherapeutics is leading researchers to develop novel drugs or repurpose existing chemicals. Alexidine dihydrochloride (AD), an orally bioavailable bis-biguanide compound, is an apoptosis stimulating reagent. It induces mitochondrial damage by inhibiting a mitochondrial-specific protein tyrosine phosphatase, PTPMT1. The aim of this study was to test AD as a novel compound to induce apoptosis in a human pancreatic adenocarcinoma cell lines, Panc-1, MIA PaCa-2, AsPC-1, and Psn-1. Methods: After IC50 value of the AD was determined by cytotoxicity assay, apoptosis was observed by a variety of methods including the detection of early apoptosis marker Annexin V and the proteomic profile screening by apoptosis array. Multicaspase and mitochondrial depolarization were measured and changes in cell cycle were analyzed. Results: AD is found to initiate apoptosis by activating the intrinsic pathway and inhibit cell cycle in pancreatic cancer cell lines. Conclusions: As a conclusion, considering its anti-cancer properties and bioavailability, Alexidine dihydrochloride can be considered as a potential candidate against pancreatic adenocarcinomas. Citation Format: Ezgi Kasikci, Esra Aydemir, Bekir Mustafa Yogurtcu, Fikrettin Sahin, Omer Faruk Bayrak. Repurposing of alexidine dihydrochloride as an apoptosis-initiator and cell cycle inhibitor in human pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-031.

Somatic missense mutations targeting MYC's coding sequence are found in >50% of Burkitt lymphomas and in a small fraction of diffuse large B-cell lymphomas. These mutations typically arise in translocated MYC alleles and cluster at specific protein residues, particularly Threonine 58 (T58). Previous studies in non-B cell models suggested that MYC mutations enhance oncogenic properties by escaping MYC-induced apoptosis. However, the functional impact of these mutations on the biology of germinal center (GC) B cells, where these mutations arise, has never been investigated. In GC B cells, MYC expression is induced by positive selection signals during cognate interactions with T follicular helper cells, concurrent with activation of PI3K and mTOR signaling. PI3K and mTOR are activated upon CD40 and B-cell receptor co-engagement and are critically required for positive selection and clonal expansion of GC B cells. Using newly generated MYC T58A knock-in mouse models, we found that MYC T58A mutation allows B cells to bypass a cell cycle entry checkpoint dependent on PI3K and mTOR activities. Exposure of wild-type (WT) B cells to rapamycin or, less markedly, to PI3K inhibitors, prevented the proliferation upon CD40 and IL4 stimulation ex vivo (a cytokine combination mimicking T-cell help) whereas MYC T58A mutant B cells divided multiple times and maintained adequate rates of active biosynthesis. Similarly, administration of rapamycin to immunized WT mice collapsed formed GCs while MYC T58A GCs maintained normal size and proliferation rates in vivo. These phenotypes were in part explained by the effects of PI3K/mTOR signaling on GSK3-beta, a kinase that normally phosphorylates MYC T58 to control protein turnover. Differences in metabolic and gene expression profiles between MYC WT and T58A cells offered additional insights into the mechanisms by which this mutation bypasses PI3K/mTOR checkpoint. Finally, we found that GC B cell-specific overexpression of MYC T58A, resulted in abnormally enlarged GCs upon immunization, a phenotype common to lymphoma models. Altogether, these results indicate that MYC somatic mutations confer competitive advantages to B cells by bypassing a strict requirement for upstream signals during cell cycle entry, which may support the clonal expansion of MYC mutant B cells during GC responses. These results have important translational implications, particularly given the increasing interest in the potential use of PI3K and/or mTOR inhibitors in the treatment of lymphomas. Citation Format: Jongkuen Lee, David Dominguez-Sola. Functional bypass of cell cycle entry checkpoints by MYC T58A mutation in germinal center-derived lymphomas [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-36.

Epidemiological and interventional human studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce cancer incidence and mortality. However, while NSAIDs are chemopreventive against cancer, they are not potent enough to be chemotherapeutic, and their long-term use may lead to significant gastrointestinal (GI), cardiovascular (CV) and renal side effects. In our search for a “better and safer NSAID”, we developed enhanced or e-NSAIDs. These are NSAIDs such as aspirin and naproxen, modified to release nitric oxide (NO) and hydrogen sulfide (H2S), both gaseous signaling molecules of biological relevance. NO and H2S have similar properties to prostaglandins within the GI mucosa, thus modulating the local mucosal defense systems, leading to reduced GI toxicity. Also, NO and H2S have protective roles in the CV and renal systems. NO and H2S signaling pathways regulate many mammalian physiological processes through post-translational cysteine persulfidation or nitrosylation. NO and H2S have a rich in vivo chemistry in which the molecules are transformed into short lived, bioactive species. They also react together to form highly bioactive persulfides. While both gasotransmitters have a complex role in tumor biology, their downstream effects have a profound impact on tumor growth and metastasis. e-NSAIDs and NBS-1120 in particular, have potent anti-tumor activity, demonstrated in in vivo models of colorectal, breast and pancreatic cancer. The activity of NBS-1120 is superior to the parent aspirin, NO releasing aspirin or H2S releasing aspirin. NBS-1120 has displayed the unexpected property of synergism: the sum of the parts (Aspirin+NO+H2S) does not equal the whole (NBS-1120). Here we report that NBS-1120 inhibits FOXM1, an important protein that plays a critical role in the cell cycle, is closely linked to p53 and controls the expression of cell cycle regulatory proteins. FOXM1's association with many solid tumors, its interactions with numerous signaling pathways, and its emerging role as a master regulator of DNA damage response and genotoxic agent resistance make it an important target for anticancer therapies. We describe the effects of NBS-1120 on p53, β-catenin, oxidative stress, NF-ΚB and COX1/2 through inhibition of FOXM1 by protein persulfidation and nitrosylation. Further, we report that the unique activity of e-NSAIDs is related to the simultaneous release of NO and H2S and their subsequent rapid reaction to form the highly bioactive polysulfides H2S2 and H2S3. NBS-1120 induced rapid polysulfide formation, however no evolution of H2S was detected. These data suggest that release of NO and H2S from NBS-1120 results in a very rapid reaction to form polysulfides. Mechanistically, the rapid formation of polysulfides may account for the potent activity of NBS-1120 compared to NSAIDs that release either NO or H2S alone. Citation Format: Jan J. Scicinski, Kenneth Olsen, Janet Stephens, Khosrow Kashfi. NBS-1120: An e-NSAID with potent anti-cancer activity targets cell cycle proteins by generating polysulfides [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6381.

Mutations in RAS family are observed in 6-96% of cancer. In acute myeloid leukemia (AML), mutations in NRAS/KRAS are seen in 10-20% of patients. NRAS proteins are a GDP-GTP regulator and have an important role in proliferative signaling networks such as PI3K and MAPK. However, the role of the magnitude of RAS activation in proliferation and cell cycle progression is not fully understood. To define the role of oncogenic NRAS dose in malignant behavior, we used a genetically engineered THP-1 cell line where the expression of NRASG12V is under the control of a tetracycline-responsive element. In this cell line, doxycycline (Dox) treatment induces expression of NRASG12V in a dose-dependent manner. THP-1 is a NRASG12D-dependent human AML cell line. We used CRISPR/Cas9 to knock out the endogenous NRASG12D and introduced a tetracycline-inducible NRASG12V. We established two modified cell lines: B11 and G6. When grown in the absence of Dox, B11 and G6 are both slow-growing and display high levels of G1 arrest. Growth in 100-1,000 ng/ml of Dox induces NRASG12V expression at levels comparable to native NRASG12D expression in unmodified THP-1 (at the protein and RNA level). To decipher the contribution of NRASG12V dose to cell cycle status, we treated B11 and G6 cell lines with three doses of Dox (0, 1,000, and 10,000 ng/ml) and performed mass cytometry (CyTOF) analysis. Native levels of NRASG12V (1,000 ng/ml Dox) led to maximal cell cycle progression with minimal G1 arrest. Interestingly higher levels of NRASG12V (Dox 10,000 ng/ml) led to reduction in cell cycle progression and an increase in G1 arrest. These data suggest that optimal levels of NRASG12V are required to optimally mediate leukemic activity. Using mass cytometry to assess signaling pathway activation, we also found that optimal NRASG12V maximally activated STAT and p38 pathways, suggesting that these may be critical mediators of NRASG12V-mediated cell cycle progression and self-renewal. These data reveal a dose-dependent role of oncogenic NRAS in mediating the proliferation and demonstrate that excessive oncogenic signaling may inhibit leukemogenic activity. To identify the whole downstream of NRAS signals, CRISPR knockout library, and CRISPR activation library were introduced into B11 and removing Dox to collect clone that can proliferate without NRAS signaling. As a result, 21 NRAS signal-related candidate genes were obtained from the knockout library and 28 from the activation library. DOHH is one of the candidates for CRISPR activation library screening. NRAS activation with Dox can induce the mRNA expression for DOHH in B11. DOHH is newly identified as a downstream of NRAS signaling. Taken together, the doxycycline-NRAS-controllable system can be applied for multiple-application to investigate the whole RAS signaling system. Citation Format: Morito Kurata, Marie L. Antony, Klara E. Noble, Susan K. Rathe, Haruka Hirakouchi, Kouhei Yamamoto, Masanobu Kitagawa, Zohar Sachs, David A. Largaespada. Dose-dependent NRASG12V-mediated signaling controls cell cycle progression and leukemogenic signaling in a CRISPR/Cas9-modified human AML cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2582.

Chronic inflammation is considered an important risk factor in the etiology of neoplastic diseases, including pancreatic cancer (PC). Naturally occurring triterpeinoid - oleanolic acid (OA) was shown to possess anti-inflammatory and anti-tumorigenic activities in the in vitro as well as in vivo models. The limited bioavailability of OA justifies searching for its more available derivatives. In this study novel oleanolic acid oxime derivatives (OAO) differing in substitution group at position C-17 were evaluated in the context of their possible modulating effect of NF-κB signaling pathway and subsequently cell cycle arrest and proliferation of pancreatic cancer cells - line PSN-1. PSN-1 cells were incubated with OA and OAO derivatives at the concentrations of 10µM, 20µM and 30 µM selected basing on the results of MTT assay for 24h. The activation of NF-κB was assessed by measuring the level of binding of its active subunits p65 and p50 to specific DNA sequence using the ELISA assay, and their translocation from cytosol to nucleus by Western blot. For genes expression evaluation (NF-κB, COX-2, ERK1/2 and JNK) the RT-PCR (mRNA) and bead-based sandwich type immunoassay on the Luminex®MAGPIX instrument (protein) was applied. Muse®Cell Analyzer was used to flow cytometric assessment of cells proliferation based on Ki67 expression and the cell cycle progression was assessed by propidium iodide staining. The OAO-morpholide derivative (18) and OAO-benzyl ester derivative (14) reduced the most efficiently the NF-κB activation and expression resulting in a diminished level of COX-2 protein in the cytosolic fraction. The expression of JNK and ERK1/2, the elements of MAPK signaling cascade, was not significantly affected by any of the tested OAO derivatives. Treatment with OAO-morpholide derivative (18) also induced the cell cycle arrest leading to accumulation of the cells in S/G2 phase. None of the tested OAO derivatives affected significantly the cells proliferation measured in term of Ki67 expression. These results indicate that the OAO derivatives, particularly morpholide conjugates are more potent suppressors of NF-κB signaling pathway than parent OA. The specificity of OAO structure substitution seems to be crucial for the NF-κB modulation and subsequent possible anti-inflammatory effect. Downregulation of NF-κB by these compounds does not seem to be connected with the suppression of MAPK signaling pathways. Funding: This work was supported by Polish National Science Centre, grant 2016/21/B/NZ7/01758. Citation Format: Maria Narożna, Violetta Krajak-Kuźniak, Robert Kleszcz, Wanda Maria Baer-Dubowska. Oleanolic acid oxime derivatives modulate NF-κB signaling pathway leading to cell cycle arrest in pancreatic cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 127.

In this study, we aimed to explore and validate the prognostic value of PLA2G4A expression in patients with non-M3/NPM1 wildtype (WT) acute myeloid leukemia (AML) using two independent datasets. Data from the Cancer Genome Atlas-Acute Myeloid Leukemia (TCGA-LAML) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET)-AML were used to assess the prognostic value PLA2G4A in NPM1-WT AML cases. A single-cell RNA-seq dataset that included 96 AML cells from a recurrent AML case was used to check the correlation between PLA2G4A expression and the functional state of AML cells. Results showed that non-M3 AML cases had significantly increased PLA2G4A expression compared to normal peripheral blood samples. Multivariate analysis showed that high PLA2G4A expression was independently associated with shorter OS in 97 non-M3/NPM1-WT AML cases in TCGA-LAML (HR: 1.932, 95%CI: 1.092-3.419, p=0.024). The prognostic value was validated based on 128 non-M3/NPM1-WT AML cases in TARGET-AML (HR: 1.894, 95%CI: 1.065-3.369, p=0.030). Single-cell RNA-seq data suggested that PLA2G4A expression was positively correlated with cell cycle progression of AML cells. Therefore, PLA2G4A expression might serve as an independent prognostic marker in OS in patients with non-M3/NPM1 WT AML. Its upregulation might confer malignant phenotype of AML cells at least partly by promoting cell cycle progression. Future molecular studies are required to explore other molecular pathways involved. Citation Format: Hansong Bai, Zeng Ming. Pla2g4a: A new cell cycle related gene in patients with non-M3/NPM1 wildtype acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 94.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Among the 18.1 million of total cancer patients last year, 2.1 million of the new cases were of LC worldwide. The poor prognosis, higher incidence, and mortality rate make this disease a devastating type of cancer. Cell cycle inhibition is one of the prominent choices among researchers in cancer treatment. Different cell cycle inhibitors are developed by scientists where the cyclin-dependent kinase (CDK) inhibitors are among the most frequently researched. Just recently, 3 CDK inhibitors received FDA-approval for treatment of specific breast cancer subtypes, but no CDK inhibitors currently exist for LC patient treatment. Thus, there is a need for molecules that regulate cell cycle progression and CDK expression. Micro RNAs (miRNAs) are gene-expression regulatory molecules. Among several miRs, we selected miR-143 and miR-506 to regulate CDK1 and CDK4/6, respectively. qPCR and RNA-sequencing indicated that this combinatorial miR therapy caused downregulation of the respective CDK1, 4, and 6 genes in H358 and H1975 lung cancer cell lines. This RNA inhibition corresponded to protein levels downregulation, as detected by western blot analysis. Further investigation found that this treatment caused significant halt of the cell cycle progression, which is markedly stronger than the commercially available CDK inhibitors, Ribociclib and Flavopiridol. We also identified the combined miR-143 and miR-506 caused significant increase of the apoptotic cell population at both 24 and 48 h post-treatment, as detected by an Annexin V/PI analysis. Interestingly, low or no apoptotic impact was observed in HFL1 normal lung fibroblast cells by the combinatorial miR treatment, indicating a cancer-cell specific activity. Upstream pathway analysis through RNA-sequencing using Ingenuity Pathway Analysis software (IPA; Qiagen) indicated a potential ERK-mediation on the activity of the combinatorial miR treatment. Our analysis of the ERK pathway using MAPK (ERK) inhibitors indicated an ERK-independent cell cycle inhibitory activity of the two miRs. Through further analysis of the RNA-Seq data, we observed a potential inhibition of the cellular migration and invasion activity of the cells, which was confirmed by a cell migration assay. In summary, combinatorial treatment of miR-143 and miR-506 has a significant impact on the cell cycle, overall survival, and migration of LC cells, which still merits further study. Citation Format: A K M Nawshad Hossian. Combinatorial micro RNA therapy can be used as potential cell cycle inhibitors to treat lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3710.

Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood. Relapse is associated with poor prognosis, requiring further investigation on high-risk leukemias. MicroRNAs (miRNAs) are involved in the regulation of physiological processes. Deregulated miRNAs have been described to play a role in the initiation and progression of leukemia. Here, we investigated miRNA expression profiles comparing leukemia samples of patients with good and poor outcome, also characterized by slow or rapid engraftment, respectively, upon transplantation into NOD/SCID mice. We found downregulation of the miR-497~195 cluster in B-cell precursor (BCP)-ALL patient-derived xenograft samples with rapid engraftment and early relapse. Accordingly, in an independent cohort, patients with a high expression of miR-497 or miR-195 showed higher event-free survival. We identified promoter methylation as the mechanism responsible for miR-497~195 downregulation, as it was associated with low miR-497~195 levels in xenografts and treatment with the demethylating agent Decitabine increased miR-497~195 expression in BCP-ALL cell lines. To study the role of miR-497~195 in BCP-ALL, we stably overexpressed the cluster in three xenograft samples. MiR-497~195 overexpression impaired engraftment of leukemic cells in NOD/SCID mice, as compared to control cells, prolonging recipient survival. Gene expression profiling and ex vivo analysis of miR-497~195 overexpressing xenograft cells showed that inhibition of cell proliferation is a mechanism responsible for the miRNA cluster tumor suppressive role. Mechanistically, CDK4 and CCND3 were downregulated at mRNA and protein levels upon miR-497~195 overexpression, leading to decreased RB1 phosphorylation, reduced transcription of E2F target genes, and inhibition of the entry in S phase. CDK4 mediated G1/S transition is also inhibited by CDKN2A and CDKN2B, which are deleted in 20% of BCP-ALL. Deletion of CDKN2A/B and concomitant low miR-497~195 expression were particularly associated with a high proportion of early relapse cases in our cohort, indicating that impaired regulation of G1/S transition is critical for ALL aggressiveness. Importantly, targeting this pathway with the CDK4/CDK6 inhibitor Palbociclib impaired ex vivo growth of BCP-ALL xenograft samples, indicating a possible therapeutic strategy. Altogether, we showed that the tumor-suppressive cluster miR-497~195 is downregulated by epigenetic repression in BCP-ALL and that low expression is associated with early relapse and rapid engraftment. Low miR-497~195 expression might cooperate with deletion of CDKN2A/B promoting tumor progression, through loss of CCND3/CDK4-dependent control of cell cycle regulation. Citation Format: Elena Boldrin, Enrico Gaffo, Judith Boer, Salih Demir, Julia Zinngrebe, Rainer Claus, Christoph Plass, Monique L. den Boer, Klaus-Michael Debatin, Geertruy te Kronnie, Stefania Bortoluzzi, Lüder Hinrich Meyer. MicroRNA-497~195 cluster suppresses acute lymphoblastic leukemia growth by targeting CCND3/CDK4 and inhibiting cell cycle progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2541.

Key words: CDK20, E4BP4, MDSC, Immunosuppression, T cell immunity The breakthrough in understanding the inhibition of negative immune regulation has paved way for cancer immunotherapy. The immune-checkpoint blockade (ICB) therapy has produced promising and yet modest objective response rates in some solid cancers such as hepatocellular carcinoma (HCC), which have been attributable to the strong immunosuppressive tumor microenvironment (TME). Accumulating evidence has demonstrated that myeloid-derived suppressor cell (MDSC), an immature myeloid population with potent T cell-suppressive activity, is remarkably associated with poor prognosis and ICB resistance of cancer patients. While targeting MDSC can blunt T cell activity, new approach is directed towards driving differentiation of MDSC into antigen presentation cell crucial for T cell priming and activation. Therefore, decoding the molecular pathways that sustain the immunosuppressive MDSCs will be instrumental in deriving strategies for greater anti-tumor immune responses. Given their pivotal roles in cell cycle and transcriptional regulation, deregulation of cyclin-dependent kinases (CDKs) has become a hallmark of several cancer types. We have recently shown that hepatoma-intrinsic CDK20, or more commonly known as cell cycle-related kinase (CCRK), mitigates anti-tumor T cell responses by expanding MDSCs within TME, while tumoral CCRK depletion diminishes MDSC-mediated immunosuppression leading to improved ICB efficacy. As emerging evidence highlights the key roles of CDKs in immune cell signaling and identity, our new findings from tumor-bearing mice and healthy human blood-derived cell models uncovered specific over-expression of CCRK in MDSCs but not lymphocytes. Notably, blockade of MDSC-intrinsic CCRK induced its differentiation into mature macrophage which amplified T cell responses in vitro and in vivo, resulting in reduced tumorigenicity. Mechanistically, CCRK inhibition suppressed signal transducer and activator of transcription 3 (STAT3) signaling to revert E4-binding protein 4 (E4BP4)-dependent interleukin-10 (IL-10)/IL-12 imbalance and arginase I expression for maintenance of immunosuppression. As we also showed CCRK over-expression in HCC patient-derived MDSCs, the findings of this study not only unravel mechanistic insights in MDSC maintenance and differentiation, but also offer a novel therapeutic kinase-target to resolve ICB resistance, thereby conferring durable eradication of solid tumors. Acknowledgement: This project was supported by the University Grants Committee through the General Research Fund 14108219 and the Collaborative Research Fund C4045-18W. The authors declare no conflict of interests. Citation Format: Jingying Zhou, Alfred Sze Lok Cheng. Targeting myeloid cell-cycle related kinase signaling amplifies anti-tumor T cell responses via inducing myeloid derived suppressor cell differentiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 400.

Background: 14-3-3 protein family have many diverse functions in cellular responses including cell cycle regulation, apoptosis and malignant transformation in a broad range of organisms. 14-3-3 sigma induces G2 arrest enabling the repair of damaged DNA. Especially 14-3-3 sigma is frequently lost in tumor cells and surrounding tissues, indicate that becomes lost at an early stage in invasive cancer. The purpose of this study is to identify the role of HGF upregulated 14-3-3 sigma associated with cancer cell proliferation and invasion in gastric cancer and to identify the role of 14-3-3 sigma level in vivo by analysis 14-3-3 serum level in gastric cancer patients who underwent gastrectomy. Methods: In our study we used cell culture, Northern blotting, cDNA microarray analysis, western blotting, RT-PCR, Zymography, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 14-3-3 sigma knock-down with short hairpin RNA (shRNA), electrophoresis mobility-shift assay, chromatin immunoprecipitation assay, Standard two chamber invasion assay. And we conducted the 14-3-3 sigma ELISA (enzyme-linked immunosorbent assay) of 37 blood samples of patients, who underwent gastrectomy for gastric cancer. Results: First, we confirmed that the expression level of 14-3-3 sigma was up-regulated by HGF(hepatocyte growth factor) in gastric cancer cell. To identify associated pathway of HGF-induced 14-3-3 sigma about MMP-1 (matrix metalloproteinase-1) expression, the cells were treated with MEK (Mitogen-activated protein kinase kinase) inhibitor(PD098059) and then analyzed by Western blotting. The HGF-mediated MMP-1 protein level was decreased with PD098059. The role for 14-3-3 sigma associated with MMP-1 was determined by knock down cell of 14-3-3 sigma. 14-3-3 sigma-sh RNA cells showed a decreased level of MMP-1, pERK and pp38. HGF-mediated cell proliferation and in vitro invasion was decreased in 14-3-3 sigma knock down cell. We also identified that 14-3-3 sigma serum level was statistically significant decreased after gastrectomy in gastric cancer patients with stage 2 or 3. (p value < 0.05) Conclusions: Our results support the hypothesis that 14-3-3 sigma plays an important role in the cell proliferation in gastric cancer, and knock down this domain, it can be preventing to gastric cancer progression. And 14-3-3 sigma serum level is associated with status of treatment locally advanced gastric cancer. Citation Format: Jiyoon Jung, Kyunghee Lee, sungae Koh, sangwoon Kim. The significant role of 14-3-3 sigma including signal transduction, cell cycle regulation, and malignant transformation in human gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-031.

Triple-negative breast cancer (TNBC) persists as a clinical challenge, with targeted therapeutic options remaining limited for the subtype due to few targetable tumor-specific vulnerabilities. While basal-like TNBC tumors express epidermal growth factor receptor (EGFR), the target of multiple clinically efficacious therapies, TNBC showed little to no response to anti-EGFR therapy in clinical trials. Using a genome-wide synthetic lethal pooled barcoded shRNA screen, we identified Death Effector Domain-containing protein (DEDD), upregulated in over 60% of TNBC tumors, as a driver of mitogen-independent G1/S cell cycle transition in TNBC under EGFR inhibition. This cell cycle role of DEDD depends on cytoplasmic localization and is independent of nuclear DEDD, which maintains roles in apoptosis. Cytosolic DEDD promotes G1/S transition through direct interaction with Rb family proteins, encouraging their degradation. Further, DEDD interacts with heat shock 71kDa protein 8 (HSC70) to augment cyclin D1 expression and drive transition to S phase. These interactions of overexpressed cytosolic DEDD with the G1/S checkpoint mechanism present a vulnerability of TNBC to cell cycle inhibition. Frequent Rb-loss in TNBC has prevented clinical testing of CDK4/6 inhibitors in TNBC, however. Yet, we found TNBC with DEDD overexpression to be sensitive to a combination of CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo, independent of Rb status. This novel vulnerability presented by DEDD overexpression in TNBC suggests promising therapeutic potential for CDK4/6 inhibitor use in combinatorial regimens. Citation Format: Yingjia Ni, Keon R. Schmidt, Barnes A. Werner, Jenna K. Koenig, Ian H. Guldner, Patricia M. Schnepp, Xuejuan Tan, Lan Jiang, Misha Host, Longhua Sun, Erin N. Howe, Junmin Wu, Laurie E. Littlepage, Harikrishna Nakshatri, Siyuan Zhang. Functional genome-wide screening identifies triple-negative breast cancer cell cycle-dependent vulnerability driven by genomic gain of death effector domain-containing protein [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4841.

Pediatric germ cell tumors (GCTs) account for approximately 6% of childhood cancers. GCTs are heterogeneous but are grouped together due to a common cell of origin, the primordial germ cell. GCTs are predicted to have high heritability, therefore, genetic association studies are paramount in understanding GCT etiology. Genome-wide association (GWA) studies commonly ignore the aggregate effect of SNPs/genes with weaker individual associations. Pathway-based approaches provide the opportunity to interrogate joint SNP effects in pathways of interest. The cell cycle control pathway is a plausible candidate pathway given the knowledge that alterations in the the mitotic-meiotic switch may lead to genetic instability and initiate GCT development. In this analysis we evaluated a set of 94 genes associated with cell cycle control using data from a case-parent triad study of pediatric GCT. Genes involved in cell cycle control were identified using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We also included additional genes based on reports in the literature suggesting a role in cell cycle control in GCTs, including STRA832, 33, CYP26B134, CKS1B35, and E2F636. Variants within 25,000 base pairs upstream or downstream of the gene were selected for inclusion resulting in a total of 2,846 SNPs included in the analysis. Genotyping data were generated using the Illumina HumanCore Exome Array for 867 GCT cases and their unaffected parents. Preliminary results evaluating individual effects of SNPs in the pathway were conducted using the transmission disequilibrium test (TDT). We did not observe any individual SNP associations that reached genome-wide significance; however, suggestive associations were observed for SNPs located in CDKN2C (p=0.000208), MCM7 (p=0.000787), RB1 (p=0.000798), RBL2 (p=0.00250) and TP53 (p=0.00257). Replication of these findings will be conducted using data from a record linkage case-control study from neonatal biobanks in California and Michigan (N=837 cases and 1057 controls). In addition, we will utilize pathway-based approaches to jointly evaluate the effects of genes in this pathway, including Gene Set Enrichment Analysis for Genome wide association study (i-GSEA4GWAS), MetXcan for functional significance, and Family-based rare-variant association analysis (PedGene). In conclusion, our results suggest that genes within the cell-cycle control pathway play a role in the etiology of pediatric GCT. Citation Format: Aubrey K. Hubbard, Nathan Pankratz, Spencer T. Kelley, John J. Meredith, Jenny N. Poynter. The association between the cell cycle control pathway and pediatric germ cell tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1209.

EGR and AP1 transcription factors are rapidly upregulated in acute myeloid leukemia cells responding to treatment with phorbol esters. Phorbol ester induced transcriptome changes are accompanied by phenotypic changes including cell cycle arrest, myeloid differentiation, and ultimately apoptosis. Here we present results documenting the transcriptome alterations of the phorbol ester response in HL-60 cells by DNA microarray and RNA-seq analyses, and explore the specific roles of EGR and AP1 in determining the evolving transcriptional program. Overexpression of egr1 or c-fos recapitulates many of the phorbol ester-induced transcriptome alterations and affects cell proliferation, differentiation and survival. Identification of changes in EGR1 and FOS/AP1 target gene expression supports the hypothesis that EGR and AP1 are involved in a genetic reprogramming of AML cells leading to loss of the leukemic phenotype and recovery of apoptotic competency. We also provide a comprehensive bioinformatic analysis of AML patient sample data provided by The Cancer Genome Atlas and the published study from Tyner et al., Nature 562:526-531 (2018) accessible through the Beat AML data viewer (Vizome) regarding expression of egr1. Egr1 high expression correlates with improved overall survival and a particular mutational signature distinct from patients with low egr1 expression. We define a mutational and transcriptional signature for AML patients based on egr1 expression levels that correlates with clinical outcomes. Citation Format: Michael Roberts, Lauren Kageler, Kayla Bendinelli, Shannon Bonner, Ashir Borah, Jeffrey Forrester. The role of EGR1 and AP1 in acute myeloid leukemia cell reprogramming toward cell cycle arrest and apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4678.