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Aberrant RHO guanine nucleotide exchange factor (RhoGEF) activation is chief mechanism driving abnormal activation of their GTPase targets in transformation and tumorigenesis. Consequently, a small-molecule inhibitor of RhoGEF can make an anti-cancer drug. We used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like acute lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation–dependent RacGEF, as the target of the small molecule IODVA1. We show that through binding to VAV3, IODVA1 inhibits RAC activation and signaling and increases pro-apoptotic activity in BCR-ABL1-transformed cells. Consistent with this mechanism of action, cellular and animal models of BCR-ABL1-induced leukemia in Vav3-null background do not respond to IODVA1. By durably decreasing in vivo RAC signaling, IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal. Importantly, IODVA1 suppresses the leukemic burden in the treatment refractory pediatric Ph+ and TKI-resistant Ph+ B-ALL patient-derived xenograft models better than standard-of-care dasatinib or ponatinib and provides a more durable response after treatment withdrawal. Pediatric leukemia samples with diverse genetic lesions show high sensitivity to IODVA1 ex vivo and this sensitivity is VAV3 dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapy.

Oncogenic kinase inhibitors show short-lived responses in the clinic due to high rate of acquired resistance. We previously showed that pharmacologically exploiting oncogene-induced proteotoxic stress can be a viable alternative to oncogene-targeted therapy. Here, we performed extensive analyses of the transcriptomic, metabolomic and proteostatic perturbations during the course of treatment of Her2+ breast cancer cells with a Her2 inhibitor covering the drug response, resistance, relapse and drug withdrawal phases. We found that acute Her2 inhibition, in addition to blocking mitogenic signaling, leads to significant decline in the glucose uptake, and shutdown of glycolysis and of global protein synthesis. During prolonged therapy, compensatory overexpression of Her3 allows for the reactivation of mitogenic signaling pathways, but fails to re-engage the glucose uptake and glycolysis, resulting in proteotoxic ER stress, which maintains the protein synthesis block and growth inhibition. Her3-mediated cell proliferation under ER stress during prolonged Her2 inhibition is enabled due to the overexpression of the eIF2 phosphatase GADD34, which uncouples protein synthesis block from the ER stress response to allow for active cell growth. We show that this imbalance in the mitogenic and proteostatic signaling created during the acquired resistance to anti-Her2 therapy imposes a specific vulnerability to the inhibition of the endoplasmic reticulum quality control machinery. The latter is more pronounced in the drug withdrawal phase, where the de-inhibition of Her2 creates an acute surge in the downstream signaling pathways and exacerbates the proteostatic imbalance. Therefore, the acquired resistance mechanisms to oncogenic kinase inhibitors may create secondary vulnerabilities that could be exploited in the clinic.

PURPOSE: Optical redox imaging (ORI), based on collecting the endogenous fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) containing a redox cofactor flavin adenine dinucleotide (FAD), provides sensitive indicators of cellular metabolism and redox status. ORI indices (such as NADH, FAD, and their ratio) have been under investigation as potential progression/prognosis biomarkers for cancer. Higher FAD redox ratio, i.e., (FAD/(FAD+NADH)) has been associated with higher invasive/metastatic potential in tumor xenografts and cultured cells. This study is to examine whether ORI indices can respond to the modulation of oncogene DEK activities that change cancer cell invasive/metastatic potential. PROCEDURES: Using lentiviral shRNA, DEK gene expression was efficiently knocked down in MDA-MB-231 breast cancer cells (DEKsh). These DEKsh cells, along with scrambled shRNA transduced control cells (NTsh), were imaged with a fluorescence microscope. In vitro invasive potential of the DEKsh cells and NTsh cells were also measured in parallel using the transwell assay. RESULTS: FAD and FAD redox ratio in polyclonal cells with DEKsh were significantly lower than that in NTsh control cells. Consistently, the DEKsh cells demonstrated decreased invasive potential than their non-knockdown counterparts NTsh cells. CONCLUSIONS: This study provides direct evidence that oncogene activities could mediate ORI-detected cellular redox state.

Breast cancer (BC) is the second leading cause of cancer deaths among women. DEK is a known oncoprotein that is highly expressed in over 60% of breast cancers and is an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To identify novel oncogenic functions of DEK, we performed RNA-Seq analysis on isogenic Dek-knockout and complemented murine BC cells. Gene ontology analyses identified gene sets associated with immune system regulation and cytokine-mediated signaling and differential cytokine and chemokine expression was confirmed across Dek-proficient versus Dek-deficient cells. By exposing murine bone marrow-derived macrophages (BMDM) to tumor cell conditioned media (TCM) to mimic a tumor microenvironment, we showed that Dek-expressing breast cancer cells produce a cytokine milieu, including up-regulated Tslp and Ccl5 and down-regulated Cxcl1, Il-6, and GM-CSF, that drives the M2 polarization of macrophages. We validated this finding in primary murine mammary tumors and show that Dek expression in vivo is also associated with increased expression of M2 macrophage markers in murine tumors. Using TCGA data, we verified that DEK expression in primary human breast cancers correlates with the expression of several genes identified by RNA-Seq in our murine model and with M2 macrophage phenotypes. Together, our data demonstrate that by regulating the production of multiple secreted factors, DEK expression in BC cells creates a potentially immune suppressed tumor microenvironment, particularly by inducing M2 tumor associated macrophage (TAM) polarization.

Head and neck cancer (HNC) remains the sixth most common malignancy worldwide and survival upon recurrence and/or metastasis remains poor. HNSCC has traditionally been associated with alcohol and nicotine use, but more recently the Human Papilloma Virus (HPV) has emerged as a favorable prognostic risk factor for oropharyngeal HNSCC. However, further stratification with additional biomarkers to predict patient outcome continues to be essential. One candidate biomarker is the DEK oncogenic protein, which was previously detected in the urine of patients with bladder cancer and is known to be secreted by immune cells such as macrophages. Here, we investigated if DEK could be detected in human plasma and if DEK levels correlated with clinical and pathological variables of HNSCC. Plasma was separated from the peripheral blood of newly diagnosed, untreated HNSCC patients or age-matched normal healthy controls and analyzed for DEK protein using ELISA. Plasma concentrations of DEK protein were lower in p16-negative tumors compared to both normal controls and patients with p16-positive tumors. Patients with lower plasma concentrations of DEK were also more likely to have late stage tumors and a lower white blood cell count. Contrary to previously published work demonstrating a poor prognosis with high intratumoral DEK levels, we show for the first time that decreased concentrations of DEK in patient plasma correlates with poor prognostic factors, including HPV-negative status as determined by negative p16 expression and advanced tumor stage.

Aberrant activation of RHO guanine nucleotide exchange factors (RhoGEFs) is a chief mechanism driving abnormal activation of their RhoGTPase targets in transformation and tumorigenesis. Consequently, a small molecule inhibitor of RhoGEF activities can be used as an anti-cancer drug. Herein, we used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation-dependent RacGEF, as the target of the small molecule IODVA1. We show that IODVA1 binds tightly to VAV3, inhibits RAC activation and signaling, and increases pro-apoptotic activity in BCR-ABL1-transformed cells only. Consistent with this mechanism of action, both VAV3-deficient leukemic cells and mouse models of BCR-ABL1 leukemia do not respond to IODVA1. IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal by decreasing RAC signaling in vivo. Importantly, IODVA1 is superior to standard of care dasatinib and ponatinib at prolonging the survival of PDX models of relapsed pediatric Ph+ and TKI-resistant Ph+ B-ALL with commonly found genetic mutations especially after treatment withdrawal. Cells representing pediatric ALL patients with diverse genetic lesions are highly sensitive to IODVA1 ex vivo and this sensitivity is VAV3-dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapeutic agent. Citation Format: Nicolas N. Nassar, Shailaja Hegde, Kark Wunderlich, Yuan Lin, Reza Ahmadian, William Seibel, Yi Zheng, Benjamin E. Mizukawa, Lisa Privette Vinnedge, Jose A. Cancelas. Inhibition of the RAC Activator VAV3 by the small molecule IODVA1 impedes RAC signaling & overcomes resistance to tyrosine kinase inhibition in lymphoblastic leukemia [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 LB198.

We report the synthesis and preliminary characterization of IODVA1, a potent small molecule that is active in xenograft mouse models of Ras-driven lung and breast cancers. In an effort to inhibit oncogenic Ras signaling, we combined in silico screening with inhibition of proliferation and colony formation of Ras-driven cells. NSC124205 fulfilled all criteria. HPLC analysis revealed that NSC124205 was a mixture of at least three compounds, from which IODVA1 was determined to be the active component. IODVA1 decreased 2D and 3D cell proliferation, cell spreading and ruffle and lamellipodia formation through downregulation of Rac activity. IODVA1 significantly impaired xenograft tumor growth of Ras-driven cancer cells with no observable toxicity. Immuno-histochemistry analysis of tumor sections suggests that cell death occurs by increased apoptosis. Our data suggest that IODVA1 targets Rac signaling to induce death of Ras-transformed cells. Therefore, IODVA1 holds promise as an anti-tumor therapeutic agent.

We report the synthesis and preliminary characterization of IODVA1, a potent small molecule that is active in xenograft mouse models of Ras-driven lung and breast cancers. In an effort to inhibit oncogenic Ras signaling, we combined in silico screening with inhibition of proliferation and colony formation of Ras-driven cells. NSC124205 fulfilled all criteria. HPLC analysis revealed that NSC124205 was a mixture of at least three compounds, from which IODVA1 was determined to be the active component. IODVA1 decreased 2D proliferation, 3D spheroid formation, spreading and lamellipodia formation in breast cancer cells. IODVA1 significantly impaired xenograft tumor growth of Ras-driven cancer cells with no observable toxicity. Immuno-histochemistry analysis of tumor sections suggests that cell death occurs by increased apoptosis. Our data suggest that IODVA1 targets a node used by Ras to regulate cell survival, spreading, and movement including lamellipodia formation. Therefore, IODVA1 holds promise as an anti-tumor therapeutic agent.

Breast cancer is the second leading cause of cancer deaths among women. DEK is a known oncoprotein found to be highly expressed in more than 60% breast cancers and is found to be an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To elucidate the oncogenic functions of DEK, we performed RNA-seq analysis on isogenic Dek-knockout and complemented murine breast cancer cells, which indicated dysregulation of immune signaling. Among the target genes identified and confirmed was an upregulation of thymic stromal lymphopoietin (TSLP) in Dek expressing murine breast cancer cells. TSLP was previously shown by Han et al to amplify the alternative (M2) activation of macrophages. M2-like macrophages are tumor promoting, they recycle iron for cell growth and encourage tissue remodeling and repair activities like angiogenesis. To test the immune modulating functions of Dek-expressing breast cancer cells, we treated bone marrow derived macrophages (BMDM) with tumor cell conditioned media. We found that in vitro, Dek expressing cancer cells induced an M2-like polarization of macrophages, as determined by the expression of M2-associated genes, enhanced migration, and iron recycling phenotypes, which was accompanied by inhibited ERK1/2 signaling. We found this phenotypic trend to be true in vivo, as well. In sectioned mammary tumors from MMTV-RontgDek+/+ and MMTV-RontgDek−/− mice, we see lower levels of iron staining in Dek+/+ tumors than in Dek−/− tumors using Prussian blue staining that co-localized with F4/80 macrophage marker staining. This suggested that Dek expression in the tumors induced an iron recycling, M2-like phenotype in tumor associated macrophages. Furthermore, we observed increased TSLP expression and angiogenesis, as determined by CD31 staining, in Dek-expressing tumors in vivo. These findings suggest that tumor Dek expression may promote breast cancer progression by inducing M2-like macrophage polarization in a murine breast cancer model. Citation Format: Miranda Shephard, Nicholas Pease, Lisa M. Privette Vinnedge. Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages [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 2836.

Increasing evidence suggests that the tumorigenic process is driven by various forms of transcriptional and epigenetic defects beyond somatic mutations. We recently described a novel non-genomic phenomenon associated with tumorigenesis that we call Transcription Elongation Defects (TEdeff), which is characterized by high levels of truncated transcripts, intron retention, and loss of gene body exon expression. The TEdeff phenotype exists in more than 20% of all human tumors and is linked to immunotherapy resistance (Modur et al, 2018). The aim of this study was to find a new therapeutic strategy for these types of tumors by targeting cellular pathways that are essential for TEdeff tumor survival. Computational analyses of RNA-sequencing data indicate that TEdeff tumors have extensive overexpression of the cytoplasmic protein homeostasis machinery, including several components of autophagy and proteasome degradation pathways. This strongly suggests that these cells may have increased dependency on cellular protein quality control due to elevated proteotoxic stress. Accordingly, we experimentally show that TEdeff cancer cell lines have a profound accumulation of protein aggregates (aggresomes) that appear to be associated with autophagy for clearance. Importantly, TEdeff cells, but not control cells with proficient transcription elongation (TEprof), are highly sensitive to autophagy inhibitors like chloroquine (CQ), but not proteasome inhibitors, which indicates their increased dependency on autophagy for survival. We also show evidence that the proteasomal degradation system may be defective in TEdeff cells, further supporting the hypothesis that these cells are dependent on alternative protein quality control mechanisms. Mechanistically, we propose that blocking the autophagy pathway maximizes proteotoxic stress, due to the accumulation of toxic aggresomes and proteins generated from defective transcripts, leading to a strong synthetic lethality in TEdeff cells. Therefore, autophagy inhibition may be a potent treatment strategy for TEdeff tumors, which are inherently resistant to current immunotherapies. Citation Format: Belal Muhammad, Lisa Vinnedge, KAkajan Komurov. Autophagy inhibition as a new therapeutic strategy for cancers with transcription elongation defects [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 5836.

Aberrant activation of Rho guanine nucleotide exchange factors (RhoGEFs) is a chief mechanism driving abnormal activation of their RhoGTPase targets in cancer. Thus, small molecule inhibitor of RhoGEF activities can be used as a drug lead to treat leukemia and other malignancies. We have identified an active small molecule, IODVA1, in several xenograft mouse models of cancer including Ras-driven cancers. Here, we used cellular and mouse models of Ph+(BCR-ABL1) B-ALL to identify Vav3, the multi-domain tyrosine phosphorylation-dependent RacGEF as the target of the small molecule IODVA1. IODVA1 specifically reduces the proliferation and survival of p190-BCR-ABL but not of empty vector expressing human CD34+ blood cells. IODVA1 binds tightly to recombinant Vav3 (Kd= 400 nM) but not to Rac1 or to the RhoGEF LARG. In Ba/F3 cells expressing p190-BCR-ABL, IODVA1 inhibits Rac activation and signaling within minutes of exposure. The decrease in Rac activity is not due to the activation of p50GAP or RhoGDI1 and is accompanied by a decrease in the activity of the pro-survival effectors PAK, JNK, and 4EBP and an increase in pro-apoptotic BAD activity. IODVA1prevents leukemia-related death and eliminates the leukemia burden in a BCR-ABL-induced murine model with no apparent toxicity. It eradicates leukemic propagating activity assessed by serial transplantation. Most importantly, IODVA1 increases the survival of a mouse model of TKI-resistant p210-BCR-ABL1(T315I) B-ALL better than ABL1-TKI imatinib and eliminates leukemic burden. p210-T315 leukemic mice survive 45 days after treatment has ended. Vav3- and Rac1/Rac2-deficient leukemic cells do not respond to IODVA1 in colony formation assay consistent with Vav3 being IODVA1's target. A mouse model of BCR-ABL leukemia deficient in Vav3 also does not respond to IODVA1. Cells from PDX models representing pediatric ALL patients including Ph+ (BCR-ABL(T315I) were found to be highly sensitive to IODVA1 ex vivo. Based on the described mechanism of action, we propose that IODVA1 is an allosteric inhibitor of Vav3 that holds promise as an anti-leukemic agent. Disclosures Cancelas: Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Velico: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding; Cellphire: Research Funding.

Dense epithelial breast tissue, as detected by mammography, is correlated with a four- to six-fold increased risk for developing breast cancer (BC). It is currently not possible to distinguish dense breast tissue from that which will develop into cancer. Therefore, the need to identify novel markers will improve breast cancer screening and reduce expensive overdiagnosis and overtreatment. Previous research has failed to define reliable diagnostic biomarkers that specify dense breast tissue from that which has the possibility to transform into malignant tissue. DEK, a chromatin binding and DNA repair protein, is upregulated in the majority of solid cancers including breast cancer. Previous publications from our lab demonstrate, in vitro, that DEK overexpression promotes proliferation, increased motility, and epithelial polarization in part through canonical WNT/β-catenin signaling. In vivo experiments with a DEK knockout murine model display a mitigated tumor development response as compared to wild-type mice. Given that DEK is highly expressed in over 60% of BC, we generated a conditional mammary epithelial specific doxycycline-regulated Dek overexpressing (DEK OE) murine model. We find that DEK OE leads to dense epithelial breast tissue, largely due to lobuloalveolar hyperplasia. Longitudinal findings demonstrate that DEK OE results in transformation of mammary epithelial tissue with microtumor development. DEK OE tissue sections display increased transcription of Wnt genes, with upregulation of downstream WNT signaling targets such as AXIN and β-catenin. Conversely, longitudinal repression of DEK OE with doxycycline displays normal tissue morphology and organization. Our data suggest that DEK OE stimulates hyperplasia of mammary epithelium, due to increased proliferation, similar to dense breast tissue as detected by mammography, and may increase risk for spontaneous transformation and BC development later in life. Citation Format: Megan Johnstone, Jon Cheek, Lisa M. Privette Vinnedge. DEK overexpression in breast epithelial tissue creates a pro-oncogenic environment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3594.

Breast cancer is the most common cancer and the second leading cause of cancer deaths among women. DEK is a known oncoprotein found to be highly expressed in more than 60% breast cancers and is found to be an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To elucidate the oncogenic functions of DEK, we performed RNA-seq analysis on isogenic Dek-knockout and complemented murine breast cancer cells, which indicated dysregulation of immune signaling. Among the target genes identified and confirmed was an upregulation of thymic stromal lymphopoietin (TSLP) in Dek expressing breast cancer cells. TSLP was previously shown by Han et al to amplify the alternative (M2) activation of macrophages. M2-like macrophages are tumor promoting, they recycle iron for cell growth and encourage tissue remodeling and repair activities like angiogenesis. Classically (M1) activated macrophages are tumor suppressing and are typically associated with response to infection. To test the immune modulating functions of Dek-expressing breast cancer cells, we treated bone marrow derived macrophages (BMDM) with tumor cell conditioned media. We found that in vitro, Dek expressing cancer cells induced an M2-like polarization of macrophages, as determined by the expression of M2-associated genes, enhanced migration, and iron recycling phenotypes, which was accompanied by inhibited ERK1/2 sighnaling. We found this phenotypic trend to be true in vivo, as well. In sectioned mammary tumors from MMTV-RontgDek+/+ and MMTV-RontgDek-/- mice, we see lower levels of iron staining in Dek+/+ tumors than in Dek-/- tumors using Prussian blue staining that co-localized with F4/80 macrophage marker staining. This suggested that Dek expression in the tumors induced an iron recycling, M2-like phenotype in tumor associated macrophages. These findings suggest that tumor Dek expression may promote breast cancer progression by inducing M2-like macrophage polarization in a murine breast cancer model. Citation Format: Miranda S. Shephard, Nicholas A. Pease, Jon Cheek, Lisa M. Privette Vinnedge. Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3471.

Self-renewing hematopoietic stem cells and multipotent progenitor cells are responsible for maintaining hematopoiesis throughout an individual’s lifetime. For overall health and survival, it is critically important that the genome stability of these cells is maintained and that the cell population is not exhausted. Previous reports have demonstrated that the DEK protein, a chromatin structural protein that functions in numerous nuclear processes, is required for DNA damage repair in vitro and long-term engraftment of hematopoietic stem cells in vivo. Therefore, we investigated the role of DEK in normal hematopoiesis and response to DNA damaging agents in vivo. Here, we report that hematopoiesis is largely unperturbed in DEK knockout mice compared to wild-type controls. However, DEK knockout mice have fewer radioprotective units but increased capacity to survive repeated sub-lethal doses of radiation exposure compared to wild-type mice. Furthermore, this increased survival correlated with a sustained quiescent state in which DEK knockout multi-potent progenitor (MPP) cells and HPC-1 restricted progenitors, were nearly three-times more likely to be quiescent following irradiation compared to wild-type cells and were significantly more radioresistant during the early phases of myeloid reconstitution. Together, our studies demonstrate that DEK functions in the normal hematopoietic stress response to recurrent radiation exposure.

Checkpoint with FHA and RING finger domains (CHFR) was first recognized as an early mitotic checkpoint protein that delayed the cell cycle in response to microtubule-targeting drugs. It is an E3 ubiquitin ligase that ubiquitinates target proteins to direct them to the proteasome for degradation or to alter their activity. To date, however, the downstream target proteins critical to CHFR's normal cellular functions largely remain unidentified with the exception of the key mitosis regulators, and oncogenes, PLK1 and Aurora A kinases. Rapidly growing evidence in mice, primary human tumors, and mammalian cell culture models indicate that CHFR may also function as a potent tumor suppressor. Interestingly, studies reported to date suggest that CHFR both controls a novel prophase checkpoint early in mitosis and regulates chromosome segregation later in mitosis to maintain genomic stability. In addition, loss of CHFR sensitizes cancer cells to microtubule poisons, altering chemoresponsiveness to taxanes and making it a potential biomarker for chemotherapeutic response. Importantly, CHFR may be one of the few proteins that are required for regulating the cell cycle and maintaining genomic instability to inhibit tumorigenesis.

Altered expression of the human septin gene, SEPT9, and its murine homologue, Sept9, has been implicated in neoplasia. However, their role(s) in oncogenesis remains poorly understood. We found amplification of SEPT9 in 67% of breast cancer cells (BCC) when compared with immortalized human mammary epithelial cells (IHMEC) as well as high levels of SEPT9 expression in the majority (61%) of the BCCs studied, unlike IHMECs. Expression profiling of variant SEPT9 transcripts and translated products revealed that high expression of the variant, SEPT9_v1, in contrast to other variants, was widespread in BCCs (55% of the BCCs) but not in IHMECs. High expression of SEPT9_v1 was also observed in primary breast cancer samples by immunohistochemical studies. We subsequently examined the phenotypic consequences of SEPT9_v1 expression in human breast cells. Retroviral expression of SEPT9_v1 in IHMEC cell culture models showed that SEPT9_v1 accelerated growth kinetics, stimulated cell motility, promoted invasion in Matrigel Transwell assays, increased genomic instability with the development of aneuploidy, and stimulated morphologic changes. Significant cytokinesis defects and disruption of tubulin microfilaments were also observed by immunofluorescence when SEPT9_v1 was ectopically expressed in IHMECs. Furthermore, SEPT9_v1 markedly enhanced neoplastic transformation in Hs578T cells, a BCC with no endogenous expression of the SEPT9_v1 isoform. Small interfering RNA–mediated and short hairpin RNA–mediated inhibition of SEPT9_v1 expression in two BCCs with high levels of endogenous SEPT9_v1 expression inhibited neoplastic growth properties of the cells. Taken together, our findings suggest that increased SEPT9_v1 expression contributes to the malignant pathogenesis of some breast tumors. [Cancer Res 2007;67(18):8554–11]

Checkpoint with FHA and Ring Finger (CHFR) is hypothesized to mediate a delay in cell cycle progression early in mitosis in response to microtubule stress, independent of the spindle assembly checkpoint. As a potential regulator of cell cycle progression, CHFR naturally becomes an interesting target for understanding cancer cells. In recent years, there has been increasing evidence supporting the role of CHFR as a tumor suppressor, most of which report loss of expression, occasionally due to promoter hypermethylation, in cancers compared with patient-matched normal tissues. We studied both a panel of breast cancer cell lines as well as primary tissue samples from breast cancer patients to investigate CHFR as a relevant tumor suppressor in breast cancer and to determine whether CHFR expression was associated with clinical and pathologic variables. We report that 41% of cell lines and 36% of patient samples showed low or negative CHFR protein expression or staining. In addition, lack of CHFR detection was associated with increased tumor size and weakly correlated with estrogen receptor–negative tumors from patients. To study the effects of low CHFR expression in vitro, we stably expressed a short hairpin RNA construct targeting CHFR in two lines of immortalized human mammary epithelial cells. Notably, decreased CHFR expression resulted in the acquisition of many phenotypes associated with malignant progression, including accelerated growth rates, higher mitotic index, enhanced invasiveness, increased motility, greater aneuploidy, and amplified colony formation in soft agar, further supporting the role of CHFR as a tumor suppressor in breast cancer. [Cancer Res 2007;67(13):6064–74]

Purpose: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells. Experimental Design: Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors. Results: We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity. Conclusions: These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes. Clin Cancer Res; 22(8); 2062–73. ©2015 AACR.

There is a long-standing correlation between inflammation, inflammatory cell signaling pathways, and tumor formation. Understanding the mechanisms behind inflammation-driven tumorigenesis is of great research and clinical importance. Although not entirely understood, these mechanisms include a complex interaction between the immune system and the damaged epithelium that is mediated by an array of molecular signals of inflammation—including reactive oxygen species (ROS), cytokines, and NFκB signaling—that are also oncogenic. Here, we discuss the association of the unique DEK protein with these processes. Specifically, we address the role of DEK in chronic inflammation via viral infections and autoimmune diseases, the overexpression and oncogenic activity of DEK in cancers, and DEK-mediated regulation of NFκB signaling. Combined, evidence suggests that DEK may play a complex, multidimensional role in chronic inflammation and subsequent tumorigenesis.

Background: Head and neck squamous cell carcinoma (HNSCC) has traditionally been associated with alcohol and nicotine use, but more recently the Human Papilloma Virus (HPV) has emerged as a favorable prognostic risk factor for oropharyngeal HNSCC. However, further stratification with additional biomarkers to predict patient outcome continues to be essential. One candidate biomarker is the chromatin remodeling DEK protein, which is both an auto-antigen in autoimmune diseases and an oncogene in epithelial tissues. DEK is secreted by stimulated macrophages and neutrophils and was previously detected in the urine of patients with bladder cancer. Previously, we have reported that DEK mRNA and protein is upregulated in HNC tumor tissue and higher DEK levels are associated with poor prognoses in many types of solid tumors. We hypothesized that DEK could be detected in the plasma of HNC patients, either due to secretion from the tumor or as part of the antitumor immune response, and therefore may be a biomarker for disease status. Methods: We recruited 38 newly diagnosed HNSCC patients and 37 age-matched normal healthy controls into the study. Plasma isolated from peripheral blood was subjected to DEK specific ELISA and DEK concentration levels were compared to levels found in normal controls, and to clinical and pathological variables. Results: We show for the first time that DEK can be detected in human plasma. We did not find an association between DEK plasma concentrations and variables including sex, age, race, or drinking/smoking status. However, we detected decreased concentrations of DEK in HNC patients with p16-negative disease and in patients with larger tumor sizes, indicating an association between DEK levels and known prognostic markers. In addition, HNC patients with lower DEK concentrations had a decreased white blood cell count, largely due to differences in lymphocyte and eosinophil counts. This direct association between plasma DEK levels and white blood cell count was independent of p16 status. Conclusions: Together, the data suggest that lower levels of DEK in HNC patient plasma may be predictive of poor outcome. This is in direct contrast to what is observed with intratumoral levels of DEK protein, in which higher levels of DEK expression are an independent factor predicting poor prognosis. Future studies will investigate the role that secreted DEK, such as that found in the plasma, may have in the antitumor immune response. Citation Format: Trisha Wise-Draper, Arun Sendilnathan, Sarah Palackdharry, Nicholas Pease, Julianne Qualtieri, Randy Butler, Nooshin Hashemi Sadraei, John C. Morris, Yash Patil, Keith Wilson, Jonathan Mark, Keith Casper, Vinita Takiar, Adam Lane, Lisa M. Privette Vinnedge. Plasma concentrations of the DEK oncogene correlate with pathological variables in a case-control study of patients with HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 50.

Breast cancer (BC) is one of the leading causes of death for women in the United States. This is due, in part, because women wait too long to get a diagnosis or the disease returns, sometimes years later, due to ineffective treatment. Therefore, it is critically important to identify biomarkers that can better inform clinicians while providing a screening test more amenable to patients than mammograms. We are investigating the feasibility of using DEK protein levels in patient plasma as a biomarker for disease stage and prognosis. DEK is a chromatin-organizing protein that functions in DNA replication and repair, transcription, and mRNA splicing. The DEK oncoprotein is highly upregulated in many types of cancer including breast, head and neck, and melanoma. Using human cell culture and murine models, our previous studies have shown that DEK is functionally important for the promotion of cellular growth, invasion/metastasis, drug resistance, and breast cancer stem cell maintenance. Work by the Markovitz laboratory indicated that DEK is secreted by macrophages as a pro-inflammatory signaling molecule and can be internalized by neighboring cancer cells to promote typical DEK functions. This led to our hypothesis that DEK may be present in the plasma of cancer patients. In fact, the detection of DEK protein in urine is currently being explored as a diagnostic biomarker for bladder cancer. To test this hypothesis, we collected peripheral blood from patients with newly diagnosed, untreated BC (irrespective of the clinical stage) and age- and ethnicity-matched normal healthy controls. Plasma was separated from the samples and subjected to DEK specific ELISA (Cusabio, Wuhan, China). Preliminary results demonstrate that DEK is indeed present in the plasma of BC patients and we are currently comparing these levels to normal healthy controls. Further analyses are ongoing to determine whether DEK levels correlate with pertinent clinical and pathological variables including age, tumor stage, and ER/PR/HER-2 status. In addition, we are examining if DEK levels can predict response to various treatment modalities and risk of relapse. These data will be important to verify DEK plasma measurements as a clinically useful test and may give insight to future personalized and targeted treatment strategies for BC. Citation Format: Arun Sendilnathan, Mahmoud Charif, Elizabeth Shaughnessy, Jaime D. Lewis, Neetu Radhakrishnan, Elyse Lower, Catherine Lanigan, Harriet Kumar, Trisha M. Wise-Draper, Lisa Privette Vinnedge. Investigation of the DEK oncogene as a blood biomarker for breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A51.

SEPT9_v1, the largest transcript of the septin gene family member, SEPT9, encodes a septin isoform implicated in the tumorigenic transformation of mammary epithelial cells. High levels of SEPT9_v1 expression also have been observed in both breast cancer cell lines, primary breast cancers as well as other solid tumor malignancies. We found a novel interaction between SEPT9_v1 and the c-Jun-N-terminal kinase (JNK), a mitogen-activated protein kinase important in cellular stress responses, cell proliferation, and cell survival. We found that up-regulation of SEPT9_v1 stabilizes JNK by delaying its degradation, thereby activating the JNK transcriptome. C-jun kinase assays in mammary epithelial cells expressing SEPT9_v1, compared to controls, exhibited increased JNK/c-Jun transcriptional activity. This increase was associated with increased levels of cyclin D1, a critical component of the proliferative response required for progression through G(1) of the cell cycle in many cell types. These findings demonstrate the first link between a septin protein and the JNK signaling pathway. Importantly, it suggests a novel functional role of SEPT9_v1 in driving cellular proliferation of mammary epithelial cells, a hallmark feature of oncogenesis that is directly relevant to breast cancer.

Head and neck cancer (HNC) remains the sixth most common cancer worldwide with almost 50,000 new diagnoses in the US each year. Although, infection with HPV has emerged as a favorable prognostic factor for head and neck squamous cell carcinoma (HNSCC) leading to de-intensifying treatment strategies, no serum biomarkers currently exist to predict tumor response and/or relapse. One candidate serum biomarker is encoded by the human DEK gene. DEK mRNA and protein is highly upregulated in tissue specimens from several tumor types including HNSCC, breast cancer and melanoma and antibodies to DEK are detected in patients with auto-immune disease (juvenile rheumatoid arthritis, systemic lupus erythematous, etc.). Our previous work has demonstrated that DEK is highly and universally expressed in HNSCC tissue specimens regardless of stage or HPV infection. Additionally, in vitro data has suggested that tumor associated macrophages secrete DEK protein leading to the hypothesis that DEK may be present in the serum of cancer patients. In fact, the detection of DEK protein in urine is currently being explored as a diagnostic biomarker for bladder cancer. Here, we sought to determine if secreted DEK protein can be detected in human plasma and if it could be used as a biomarker for HNSCC. Peripheral blood was collected from either patients with newly diagnosed, untreated, HNSCC or age-matched normal healthy controls. Plasma was separated from the samples and subjected to DEK specific ELISA (Cusabio, Wuhan, China). Plasma DEK levels were compared to tumor stage, response to therapy, and overall tumor burden in patients. Preliminary results demonstrate that DEK is indeed present in the plasma of HNSCC patients and we are currently comparing these levels to normal healthy controls. Further analyses are ongoing to determine whether DEK levels predict response to various treatment modalities, correlate with the body's immune response, and whether DEK presence in the serum will predict residual disease and/or early relapse. These data will be important to verify DEK plasma measurements as a clinically useful test and may give insight to future personalized and targeted treatment strategies for HNSCC. Citation Format: Trisha Wise-Draper, Lisa Privette Vinnedge, Arun Sendilnathan. The DEK oncogene may serve as a predictive plasma biomarker in head and neck cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 584. doi:10.1158/1538-7445.AM2015-584

Expression of estrogen and progesterone hormone receptors indicates a favorable prognosis due to the successful use of hormonal therapies such as tamoxifen and aromatase inhibitors. Unfortunately, 15–20% of patients will experience breast cancer recurrence despite continued use of tamoxifen. Drug resistance to hormonal therapies is of great clinical concern so it is imperative to identify novel molecular factors that contribute to tumorigenesis in hormone receptor positive cancers and/or mediate drug sensitivity. The hope is that targeted therapies, in combination with hormonal therapies, will improve survival and prevent recurrence. We have previously shown that the DEK oncogene, which is a chromatin remodeling protein, supports breast cancer cell proliferation, invasion and the maintenance of the breast cancer stem cell population. In this report, we demonstrate that DEK expression is associated with positive hormone receptor status in primary breast cancers and is up-regulated in vitro following exposure to the hormones estrogen, progesterone, and androgen. Chromatin immunoprecipitation experiments identify DEK as a novel estrogen receptor α (ERα) target gene whose expression promotes estrogen-induced proliferation. Finally, we report for the first time that DEK depletion enhances tamoxifen-induced cell death in ER+ breast cancer cell lines. Together, our data suggest that DEK promotes the pathogenesis of ER+ breast cancer and that the targeted inhibition of DEK may enhance the efficacy of conventional hormone therapies.

CHFR is an E3 ubiquitin ligase and an early mitotic checkpoint protein implicated in many cancers and in the maintenance of genomic stability. To analyze the role of CHFR in genomic stability, by siRNA, we decreased its expression in genomically stable MCF10A cells. Lowered CHFR expression quickly led to increased aneuploidy due to many mitotic defects. First, we confirmed that CHFR interacts with the mitotic kinase Aurora A to regulate its expression. Furthermore, we found that decreased CHFR led to disorganized multipolar mitotic spindles. This was supported by the finding that CHFR interacts with alpha-tubulin and can regulate its ubiquitination in response to nocodazole and the amount of acetylated alpha-tubulin, a component of the mitotic spindle. Finally, we found a novel CHFR interacting protein, the spindle checkpoint protein MAD2. Decreased CHFR expression resulted in the mislocalization of both MAD2 and BUBR1 during mitosis and impaired MAD2/CDC20 complex formation. Further evidence of a compromised spindle checkpoint was the presence of misaligned metaphase chromosomes, lagging anaphase chromosomes, and defective cytokinesis in CHFR knockdown cells. Importantly, our results suggest a novel role for CHFR regulating chromosome segregation where decreased expression, as seen in cancer cells, contributes to genomic instability by impairing the spindle assembly checkpoint.