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Supplementary Figure 3 describes the ability of X8768 expressing bacterial luciferase to colonize tumors and peripheral tissues, as well as how HA degradation affects vasculature in PANC-1 tumors.

Supplementary Table 1 describes the parental strains and mutations used to generate attenuated ST strains.

Supplementary Figure 1 describes BHs expression in uninduced ST strains as well as the predicted localization of BHs. Viability of bHs-expressing ST strains is analyzed.

Supplementary Figure 2 describes the ability of X8768-BHs to degrade HA from fixed human PC-3 prostate cancer cells.

Supplementary Figure 4 shows that treatment with X8768-BHs does not degrade HA is non-tumor tissues, and does not affaect total cellular content of tumor tissue where HA was degraded.

Pancreatic cancer is considered one of the most lethal cancers in the US. It contributes to an estimated 47,000 deaths annually and is predicted to surpass prostate, breast and colorectal cancers as the leading cause of cancer-related death. Although major advancements in cancer treatment have improved outcomes for many cancer types, survival rate for pancreatic cancer has not improved in nearly four decades despite tremendous effort. One attribute of pancreatic cancer that is considered a major barrier to effective treatment is the formation of fibrotic tissue around tumor cells known as desmoplasia. A number of promising approaches have been developed to deplete fibrotic components in pancreatic tumors to enhance drug delivery, some of which have been tested in clinical trials of advanced, unresectable pancreatic cancer. Here, we discuss previous efforts, shortcomings and new considerations for developing more effective agents to eliminate desmoplasia.

In pancreatic ductal adenocarcinoma (PDAC), the extracellular matrix (ECM) surrounding cancer cells forms a barrier that often limits the ability of chemotherapeutic drugs and cytotoxic immune subsets to penetrate and eliminate tumors. The dense stromal matrix protecting cancer cells, also known as desmoplasia, results from the overproduction of major ECM components such as collagens and hyaluronic acid (HA). Although candidate drugs targeting ECM components have shown promise in increasing penetration of chemotherapeutic agents, severe adverse effects associated with systemic depletion of ECM in peripheral healthy tissues limits their use at higher, more effective doses. Currently, few strategies exist that preferentially degrade ECM in tumor tissue over healthy tissues. In light of this, we have developed an attenuated, tumor-targeting Salmonella typhimurium (ST) expressing functional bacterial hyaluronidase (bHs-ST), capable of degrading human HA deposited within PDAC tumors. Our data show that bHs-ST (i) targets and colonizes orthotopic human PDAC tumors following systemic administration and (ii) is efficiently induced in vivo to deplete tumor-derived HA, which in turn (iii) significantly increases diffusion of Salmonella typhimurium within desmoplastic tumors. BHs-ST represents a promising new tumor ECM–targeting strategy that may be instrumental in minimizing off-tumor toxicity while maximizing drug delivery into highly desmoplastic tumors.

Cutaneous melanoma is the most common skin cancer with an incidence that has been rapidly increasing in the past decades. Melanomas are among the most immunogenic tumors and, as such, have the greatest potential to respond favorably to immunotherapy. However, like many cancers, melanomas acquire various suppressive mechanisms, which generally act in concert, to escape innate and adaptive immune detection and destruction. Intense research into the cellular and molecular events associated with melanomagenesis, which ultimately lead to immune suppression, has resulted in the discovery of new therapeutic targets and synergistic combinations of immunotherapy, targeted therapy and chemotherapy. Tremendous effort to determine efficacy of single and combination therapies in pre-clinical and clinical phase I-III trials has led to FDA-approval of several immunotherapeutic agents that could potentially be beneficial for aggressive, highly refractory, advanced and metastatic melanomas. The increasing availability of approved combination therapies for melanoma and more rapid assessment of patient tumors has increased the feasibility of personalized treatment to overcome patient and tumor heterogeneity and to achieve greater clinical benefit. Here, we review the evolution of the immune system during melanomagenesis, mechanisms exploited by melanoma to suppress anti-tumor immunity and methods that have been developed to restore immunity. We emphasize that an effective therapeutic strategy will require coordinate activation of tumor-specific immunity as well as increased recognition and accessibility of melanoma cells in primary tumors and distal metastases. This review integrates available knowledge on melanoma-specific immunity, molecular signaling pathways and molecular targeting strategies that could be utilized to envision therapeutics with broader application and greater efficacy for early stage and advanced metastatic melanoma.

Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK–protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein–protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers., The ERK signalling pathway is activated in many cancers, however ERK1 and ERK2 are difficult to target pharmacologically. Here, the authors identify a small molecule inhibitor that binds covalently to the D-recruitment site of ERK and induces cell death and reduces tumour growth in mice.

Therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) can be attributed, in part, to a dense extracellular matrix containing excessive collagen deposition. Here, we describe a novel Salmonella typhimurium (ST) vector expressing the bacterial collagenase Streptomyces omiyaensis trypsin (SOT), a serine protease known to hydrolyze collagens I and IV, which are predominantly found in PDAC. Utilizing aggressive models of PDAC, we show that ST-SOT selectively degrades intratumoral collagen leading to decreases in immunosuppressive subsets, tumor proliferation and viability. Ultimately, we found that ST-SOT treatment significantly modifies the intratumoral immune landscape to generate a microenvironment that may be more conducive to immunotherapy.

Therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) can be attributed, in part, to a dense extracellular matrix containing excessive collagen deposition. Here, we describe a novel Salmonella typhimurium (ST) vector expressing the bacterial collagenase Streptomyces omiyaensis trypsin (SOT), a serine protease known to hydrolyze collagens I and IV, which are predominantly found in PDAC. Utilizing aggressive models of PDAC, we show that ST-SOT selectively degrades intratumoral collagen leading to enhancement of immune checkpoint blockade (ICB) therapy in tumor-bearing mice. Ultimately, we found that ST-SOT treatment significantly modifies the intratumoral immune landscape to generate a microenvironment more conducive to ICB.

Simple Summary The deposition of fibrotic tissue within pancreatic tumors acts as a physical barrier to therapeutic treatment. Collagen constitutes a large part of this barrier and serves as an ideal target to improve delivery and efficacy of anti-cancer treatments. This study characterizes a novel bacterial-based agent engineered to degrade collagens present only in pancreatic tumor tissue. Treatment using our collagen-degrading bacteria in mouse models of pancreatic cancer resulted in significant decreases in intratumoral collagen content and pro-tumor immune cell subsets, ultimately enhancing the efficacy of immunotherapy. These results support the idea that overcoming fibrosis in pancreatic cancer can dramatically improve therapeutic outcomes. Abstract Therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) can be attributed, in part, to a dense extracellular matrix containing excessive collagen deposition. Here, we describe a novel Salmonella typhimurium (ST) vector expressing the bacterial collagenase Streptomyces omiyaensis trypsin (SOT), a serine protease known to hydrolyze collagens I and IV, which are predominantly found in PDAC. Utilizing aggressive models of PDAC, we show that ST-SOT selectively degrades intratumoral collagen leading to decreases in immunosuppressive subsets, tumor proliferation and viability. Ultimately, we found that ST-SOT treatment significantly modifies the intratumoral immune landscape to generate a microenvironment that may be more conducive to immunotherapy.

Therapeutic options for non-small cell lung cancer (NSCLC) treatment have changed dramatically in recent years with the advent of novel immunotherapeutic approaches. Among these, immune checkpoint blockade (ICB), using monoclonal antibodies, has shown tremendous promise in a small proportion of patients. In order to better predict patients that will respond to ICB treatment, biomarkers such as tumor-associated CD8+ T cell frequency, tumor checkpoint protein status and mutational burden have been utilized, however, with mixed success. In this study, we hypothesized that significantly altering the suppressive tumor immune landscape in NSCLC could potentially improve ICB efficacy. Using sub-therapeutic doses of our Salmonella typhimurium-based therapy targeting the suppressive molecule indoleamine 2,3-dioxygenase (shIDO-ST) in tumor-bearing mice, we observed dramatic changes in immune subset phenotypes that included increases in antigen presentation markers, decreased regulatory T cell frequency and overall reduced checkpoint protein expression. Combination shIDO-ST treatment with anti-PD-1/CTLA-4 antibodies enhanced tumor growth control, compared to either treatment alone, which was associated with a significant intratumoral influx of CD8+ and CD4+ T lymphocytes. These results suggest that the success of ICB therapy may be more accurately predicted by taking into account multiple factors such as potential for antigen presentation and frequency of suppressive immune subsets in addition to markers already being considered. Alternatively, combination treatment with agents such as shIDO-ST could be used to create a more conducive tumor microenvironment for improving response rates to immunotherapy.

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We developed a novel vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we used this novel vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. Mice immunized with these sMVA vectors developed robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a novel vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We demonstrate the construction of a vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we use this vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. We show that mice immunized with these sMVA vectors develop robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate., Chiuppesi et al. demonstrate the use of a synthetic poxvirus-based platform to rapidly generate multi-antigenic vaccine candidates expressing spike and nucleocapsid antigens of SARS-CoV-2. Immunization of mice stimulates potent antigen-specific humoral and cellular immune responses, including neutralizing antibodies.

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We developed a novel vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we used this novel vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. Mice immunized with these sMVA vectors developed robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a novel vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.

Tumors evolve a variety of mechanisms to escape immune detection while expressing tumor-promoting molecules that can be immunogenic. Here, we show that transposable elements (TE) and gene encoded, tumor-associated antigens (TAA), which can be both highly immunogenic and tumor-promoting, are significantly upregulated during the transition from pre-malignancy to malignancy in an inducible model of pancreatic ductal adenocarcinoma (PDAC). Coincident with the increased presence of TEs and TAAs was the downregulation of gene transcripts associated with antigen presentation, T cell recruitment and intrinsic anti-viral responses, suggesting a unique strategy employed by PDAC to possibly augment tumorigenesis while escaping detection by the immune system. In vitro treatment of mouse and human PDAC cell lines with the DNA methyltransferase inhibitor 5-azacytidine (Aza) resulted in augmented expression of transcripts for antigen presentation machinery and T cell chemokines. When immunocompetent mice implanted with PDAC were therapeutically treated with Aza, we observed significant tumor regression that was not observed in immunocompromised mice, implicating anti-tumor immunity as the principal mechanism of tumor growth control. Analysis of PDAC tumors, immediately following Aza treatment in immunocompetent mice, revealed a significantly greater infiltration of T cells and various innate immune subsets compared to control treatment, suggesting that Aza treatment enhances tumor immunogenicity. Thus, augmenting antigen presentation and T cell chemokine expression using DNA methyltransferase inhibitors could be leveraged to potentiate adaptive anti-tumor immune responses against PDAC.

In pancreatic ductal adenocarcinoma (PDAC), the extracellular matrix (ECM) surrounding cancer cells forms a barrier that often limits the ability of chemotherapeutic drugs and cytotoxic immune subsets to penetrate and eliminate tumors. The dense stromal matrix protecting cancer cells, also known as desmoplasia, results from the overproduction of major ECM components such as collagens and hyaluronic acid (HA). Although candidate drugs targeting ECM components have shown promise in increasing penetration of chemotherapeutic agents, severe adverse effects associated with systemic depletion of ECM in peripheral healthy tissues limits their use at higher, more effective doses. Currently, few strategies exist that preferentially degrade ECM in tumor tissue over healthy tissues. In light of this, we have developed an attenuated, tumor-targeting Salmonella typhimurium (ST) expressing functional bacterial hyaluronidase (bHs-ST), capable of degrading human HA deposited within PDAC tumors. Our data show that bHs-ST (1) targets and colonizes orthotopic human PDAC tumors following systemic administration and (2) is efficiently induced in vivo to deplete tumor-derived HA, which in turn (3) significantly increases diffusion of ST within desmoplastic tumors. BHs-ST represents a promising new tumor ECM-targeting strategy that may be instrumental in minimizing off-tumor toxicity while maximizing drug delivery into highly desmoplastic tumors.

Serotonin (5-hydroxytryptamine, 5-HT) is a critical local regulator of epithelial homeostasis in the breast and exerts its actions through a number of receptors. Dysregulation of serotonin signaling is reported to contribute to breast cancer pathophysiology by enhancing cell proliferation and promoting resistance to apoptosis. Preliminary analyses indicated that the potent 5-HT1B/1D serotonin receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like molecule, induced cell death in breast cancer cell lines. Thus, we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer cells.

Triple negative breast cancers (TNBC) have poor prognosis compared to other breast cancer subtypes and represent 15-20% of breast cancers diagnosed. Unique targets and new molecularly-targeted therapies are urgently needed for this subtype. Despite high expression of Epidermal Growth Factor Receptor, inhibitors such as lapatinib have not shown therapeutic efficacy in TNBC patients. Herein, we report that treatment with the covalent JNK inhibitor, JNK-IN-8, synergizes with lapatinib to cause cell death, while these compounds as single agents have little effect. The combination significantly increases survival of mice bearing xenografts of MDA-MB-231 human TNBC cells. Our studies demonstrate that lapatinib treatment increases c-Jun and JNK phosphorylation indicating a mechanism of resistance. Combined, these compounds significantly reduce transcriptional activity of Nuclear Factor kappa B, Activating Protein 1, and Nuclear factor erythroid 2-Related Factor 2. As master regulators of antioxidant response, their decreased activity induces a 10-fold increase in reactive oxygen species that is cytotoxic, and is rescued by addition of exogenous antioxidants. Over expression of p65 or Nrf2 also significantly rescues viability during JNK-IN-8 and lapatinib treatment. Further studies combining JNK-IN-8 and lapatinib may reveal a benefit for patients with TNBC, fulfilling a critical medical need.

Mutations in pathways that enhance the activity of ERK1 and ERK2 are frequently present in human cancers, reflecting their important roles in tumor initiation, and progression. This notion is reinforced by observations in BRAF V600E mutant melanoma where the majority of the mechanisms of resistance to FDA-approved combination therapies targeting BRAF and MEK involve reactivation of ERK1 and ERK2. Recently, the direct targeting of the ERK enzymes using ATP-competitive inhibitors has emerged as an attractive strategy to overcome acquired resistance to current combination therapies. The ERK enzymes employ unique mechanisms of molecular recognition to engage protein components of the MAPK pathway. Here we report the potent targeting of an ERK-protein docking interaction by a small molecule thiotriazole, which abrogates ERK signaling in vivo. The thiotriazole binds covalently to a highly conserved cysteine residue within the D-recruitment site of ERK1/2 with more than a 100-fold discrimination over other MAPKs (e.g. JNK1/2, p38MAPKs and ERK5). Treatment of various BRAF-inhibitor naive or inhibitor-resistant melanoma cell lines expressing BRAF V600E with the thiotriazole for 2 hours induces dose-dependent inhibition of ERK activation and downstream signaling. Inhibition is maintained for up to 5 hours following thiotriazole washout and induces apoptosis and growth inhibition. Treatment of mice carrying a BRAFV600E A375 melanoma xenograft with the thiotriazole blocked tumor growth. Transient expression of a mutant form of ERK2, which is resistant to the thiotriazole, promotes survival of A375 and HEK 293 cells treated with thiotriazole. This study lays the foundation for developing a new modality for the treatment of solid tumors driven by excessive ERK signaling. Citation Format: Tamer S. Kaoud, William H. Johnson, Nancy D. Ebelt, Andrea Piserchio, Diana Zamora-Olivares, Sabrina V. Ravenstein, Jacey R. Pridgen, Ramakrishna Edupuganti, Rachel Sammons, Micael Cano, Mangalika Warthaka, Pengyu Ren, Eric V. Anslyn, Kenneth Y. Tsai, Ranajeet Ghose, Kevin N. Dalby. Targeting multi-functional ERK-protein complexes in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3872.

Disseminated cancer cells rely on intricate interactions among diverse cell types in the tumor-associated stroma, vasculature, and immune system for survival and growth. Ubiquitous expression of c-Jun N-terminal kinase (jnk) genes in various cell types permits their control of metastasis. In early stages of metastasis, JNKs affect tumor-associated inflammation and angiogenesis as well as tumor cell migration and intravasation. Within the tumor stroma, JNKs are essential for the release of growth factors that promote epithelial-to-mesenchymal transition (EMT) in tumor cells. JNK3, the least ubiquitous isoform, facilitates angiogenesis by increasing endothelial cell migration. Importantly, JNK expression in tumor cells integrates stromal signals to promote tumor cell invasion. However, JNK isoforms differentially regulate migration toward the endothelial barrier. Once tumor cells enter the bloodstream, JNKs increase circulating tumor cell (CTC) survival and homing to tissues. By promoting fibrosis, JNKs improve CTC attachment to the endothelium. Once anchored, JNKs stimulate EMT to facilitate tumor cell extravasation and enhance the secretion of endothelial barrier disrupters. Tumor cells attract barrier-disrupting macrophages by JNK-dependent transcription of macrophage chemoattractant molecules. In the secondary tissue, JNKs are instrumental in the premetastatic niche and stimulate tumor cell proliferation. JNK expression in cancer cells stimulates tissue-remodeling macrophages to improve tumor colonization. However, in T-cells, JNKs alter cytokine production that increases tumor surveillance and inhibits the recruitment of tissue-remodeling macrophages. Therapeutically targeting JNKs for metastatic disease is attractive considering their promotion of metastasis; however, specific JNK tools are needed to determine their definitive actions within the context of the entire metastatic cascade.

Despite intensive research into novel treatment strategies for cancer, it remains the second most common cause of death in industrialized populations. Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal prognosis. Currently, surgical resection offers the best chance for extended survival, yet recurrence remains high and is associated with poor outcome. Systemic treatment has evolved from non-specific, cytotoxic chemotherapy to the use of cancer-targeting agents, profoundly changing treatment approaches in the metastatic and adjuvant settings. One promising approach, highlighted in this review, uses the inherent capacity of Salmonella to colonize and eliminate solid tumors.

Tumors are much stiffer than healthy tissue, and progressively stiffen as the cancer develops. Tumor stiffening is largely the result of extracellular matrix (ECM) remodeling, for example, deposition and crosslinking of collagen I. Well established in vitro models have demonstrated the influence of the microenvironment in regulating tissue homeostasis, with matrix stiffness being a particularly influential mediator. Non-malignant MCF10A mammary epithelial cells (MECs) lose their epithelial characteristics and become invasive when cultured in stiff microenvironments, leading to the hypothesis that tumor stiffening could contribute directly to disease progression. However, previous studies demonstrating MCF10A invasion have been performed in gels with constant mechanical properties, unlike the dynamically stiffening tumor microenvironment. Here, we employ a temporally stiffening hydrogel platform to demonstrate that matrix stiffening induces invasion from and proliferation in MCF10A mammary acini. After allowing MCF10A acini to form in soft hydrogels for 14 days, the gels were stiffened to the level of a malignant tumor, giving rise to a proliferative and invasive phenotype. Cells were observed to collectively migrate away from mammary acini while maintaining cell–cell contacts. Small molecule inhibition of PI3K and Rac1 pathways was sufficient to significantly reduce the number and size of invasive acini after stiffening. Our results demonstrate that temporal matrix stiffening can induce invasion from mammary acini and supports the notion that tumor stiffening could be implicated in disease progression and metastasis.

Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression. DOI: http://dx.doi.org/10.7554/eLife.07270.001, eLife digest Damaged DNA threatens the normal activity of living cells, so cells use a number of mechanisms to ensure that this damage is repaired. When DNA is damaged, an enzyme called ATM activates several other proteins that ultimately lead to the DNA being repaired. Problems with detecting and repairing damaged DNA have been linked to cancer. Thus, these pathways, including the activity of ATM, were previously thought to only be involved in cancer suppression. Now, Chen et al. report a new cancer-promoting role for ATM. The experiments reveal that reducing the amount of ATM in cancer cells actually made them less able to migrate and less invasive. Likewise, human breast cancer cells in which the levels of ATM had been depleted formed fewer lung tumors than normal breast cancer cells when they were transplated into mice. Oxidative stress—a build-up of harmful chemicals inside cells—is a signature feature of cancer cells and is known to be another signal that activates ATM. Chen et al. found that activating ATM through oxidative stress, but not by DNA damage, encouraged the cancer cells to migrate and become invasive. Further analysis of cellular responses following ATM activation by oxidative stress revealed that this enzyme regulates the production of a small protein called interleukin-8. This protein is an important pro-inflammatory molecule that has been implicated in cancer, in particular, in helping cancer cells to migrate to other tissues. When interleukin-8 was added to ATM-depleted cancer cells, it rescued their defects in spreading and invasiveness, thereby providing strong evidence that interleukin-8 is a biologically important target of ATM. Clinical data confirmed that breast cancer cells that had also spread to the patient's lungs often produced high levels of interleukin-8. Together, these findings suggest that ATM could be a potential target for anti-cancer therapies, as inhibiting this enzyme would inhibit interleukin-8, and in turn slow the progression and spread of cancer. DOI: http://dx.doi.org/10.7554/eLife.07270.002

// Michael A. Cantrell 1, * , Nancy D. Ebelt 1, * , Adam D. Pfefferle 3, 4 , Charles M. Perou 3, 4, 5 , Carla Lynn Van Den Berg 1, 2 1 Institute of Cellular & Molecular Biology, College of Pharmacy, University of Texas at Austin, Dell Pediatric Research Institute, Austin, TX 78723, USA 2 Division of Pharmacology &Toxicology, College of Pharmacy, University of Texas at Austin, Dell Pediatric Research Institute, Austin, TX 78723, USA 3 Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599, USA 4 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599, USA 5 Department of Genetics, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599, USA * These authors have contributed equally to this work Correspondence to: Carla Lynn Van Den Berg, e-mail: carla.vandenberg@austin.utexas.edu Keywords: breast cancer, JNK2, p53, Notch1, BRCA1 Received: March 13, 2015 Accepted: April 13, 2015 Published: April 25, 2015 ABSTRACT Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis.

Mitogen-activated protein kinase kinase-4 (MKK4) has been reported to either enhance or suppress oncogenesis. Evidences of its pro-oncogenic activities in breast, pancreatic, lung and skin cancer have been reported. Although the mechanism of its possible tumorigenic role is still unclear. Recent studies in glioblastoma and lung carcinoma have suggested important roles for the constitutive activation of its downstream substrate JNK2. JNKs require phosphorylation by both MKK4 and MKK7 to be fully activated. The JNK2 isoform shows a unique propensity among the JNKs to autophosphorylate in vitro. Here we show that JNK2 autophosphorylation contributes to the proliferation and migration of NSCLC cells under conditions of low serum through an MKK4-dependent mechanism. This suggests a pro-oncogenic role of MKK4 in NSCLC through a JNK2 autophosphorylation-dependent pathway. Evidence in several cell lines, including mouse embryonic stem cells lacking MKK4 or MKK7 suggests that autophosphorylation alone activates JNK2 weakly in cells. However, JNK2 autophosphorylates on Thr-183, to create a pool of JNK2 primed for activation by MKK4, which phosphorylates JNK2 on Tyr-185 to activate it. Under conditions of low serum the down-regulation/overexpression of MKK4 or JNK2 but not MKK7 or JNK1 suppresses/promotes proliferation of multiple NSCLC cell lines, through STAT3. Under the same conditions, A549 cell migration is inhibited upon down regulation of either MKK4 or JNK2. Further supporting the notion that active JNK2 results primarily from autophosphorylation of Thr-183, pan-JNK ATP competitive inhibitors (JNK-IN-8 and SP600125) showed a dose-dependent dephosphorylation of JNK2 in NSCLC cell lines and inhibited their proliferation under conditions of low serum. Moreover, the downregulation of MKK4 or inhibition of JNK2 autophosphorylation by SP600125 inhibited nuclear localization of JNK2 in A549 cells cultured in low serum. To further understand the pro-oncogenic role of MKK4 in the A549 cell line, we investigated tumor growth and lung metastasis in A549 Xenografts in which either JNK2 or MKK4 were stably knocked down. Either JNK2 or MKK4 down regulation showed a similar suppression of tumor growth and lung metastasis if compared to cells expressing an shRNA control. Sequencing of MKK4 in the A549 cells revealed no genomic deletion or somatic mutations. Taken together, the data suggest that MKK4 may exhibit pro-oncogenic properties in NSCLC by activating Thr-183-autophosphorylated JNK2. Targeting this pathway may reduce or block lung tumor progression and/or metastasis. Note: This abstract was not presented at the meeting. Citation Format: Tamer Saad Kaoud, Nancy D. Ebelt, Lili Du, Sabrina Van Ravenstein, Kenneth Y. Tsai, Kevin N. Dalby. MKK4/JNK2 down-regulation in NSCLC suppresses tumor growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1899. doi:10.1158/1538-7445.AM2017-1899

Background: Specification of the murine mammary placode in embryonic development involves pathways that are dysregulated in murine and human breast cancers. Beginning with secretion of Neuregulin 3 (Nrg3) and Wingless-Integrated (Wnt) 10b by the dermal mesenchyme, at embryonic day (E) 10.5, a mammary placode will condense over 48 hours due to signaling through the canonical Wnt pathway as well as through the avian ERythroBlastosis oncogene B (ErbB) receptors 4 and 2. By E15.5 placodes will invade the underlying mesenchyme to form the primary duct. The Wnt and ErbB pathways are also known to cause or promote cancer when dysregulated. Methods and Materials: Number four mammary glands of female, virgin mice (jnk1+/+ or jnk1-/-) were harvested and stained with carmine alum to observe ducts. Wildtype mice were injected with 4T1.2 cells with shRNA targeted to Jnk1 (shJnk1) or non-targeting shRNA (GipZ). Tumors were palpated for growth changes and lungs were stained with India ink to observe lung metastases. RNA from tumors was compared using microarray fro expression of cancer related genes. Results: We have observed that adult glands from jnk1-/- mice contain multiple primary ducts whereas jnk1+/+ mice contain only one, indicating a possible defect during embryonic placode formation. This phenotype mimics what has been previously reported in models of ectopic NRG3 expression. In a 4T1.2 murine mammary cancer model, shJnk1 results in increased tumor growth as well as increased lung metastasis when compared to cells transfected with GipZ. In the tumors grown from the 4T1.2shJnk1 and 4T1.2GipZ cells, a microarray analysis revealed increases in Nrg3, Wnt targets and ligands, and the ErbB2 and Wnt target ETV5 in 4T1.2shJnk1 cells. An increase in the expression of ETV5, Bcl-9, Cox-2 (an ETV5 target), and ErbB2 (a target of ETV5) in 4T1.2shJnk1 cells vs 4T1.2GipZ cells has been confirmed by RT-PCR and western blotting for ErbB2 (Figure 2). I plan to determine how JNK1 affects the Wnt and ErbB pathways in mouse embryos and mammary tumor models. Since Wnt and ErbB pathways are necessary for placode formation, it is likely that these pathways, which are upregulated in our cancer model, are involved in the embryonic phenotype. I hypothesize that JNK1 loss causes increased signaling through these pathways, resulting in the formation of ectopic mammary placodes as well as a more aggressive tumor phenotype. If my hypothesis is supported by these studies then future studies will evaluate if low jnk1 expression is associated with a higher risk of developing breast cancer or a worse prognosis using publically available microarray datasets derived from human tumor samples. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-13.

Development and screening of small molecule compounds for anti-cancer activity has been of prime interest to the scientific community following the success of targeted, large anti-cancer molecules such as therapeutic antibodies. Small molecules pass more easily through cell membranes and may cross the blood-brain barrier. KD06 is a small molecule triptan-like compound whose parent molecule binds and inhibits serotonin receptors. This compound increases apoptosis of the triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-157 via caspase activation. Treatment with KD06 also causes increased autophagy as well as activation of ER stress responses. The growth of tumor xenografts of MDA-MB-231 cells in nude mice are significantly inhibited by twice weekly treatment with 30mg/kg KD06. Analysis of signaling changes by KD06 using reverse phase protein array (RPPA) revealed significant decreases in Akt/mTOR signaling leading to decreased activation of the translation initiation factor 4E-BP1. Other notable changes included decreased expression of proteins important for mitosis such as Cyclin B1, Aurora B and PLK1, and increased phosphorylation of EGFR and increased expression of PDGFR. Analysis of PIP3 and ATP levels showed no change after treatment with KD06, indicating that decreased signaling through Akt/mTOR is not likely due to PI3K inhibition or AMPK activation. Immunofluorescence with KD06 treated cells revealed a change in cell shape after 4 hours of treatment that was reminiscent of cells treated with microtubule binding drugs. Akt localization was affected. These results imply that KD06 may have anti-cancer activity through its effect on microtubule dynamics, inhibiting proper localization and signaling of molecules important for survival and protein translation such as Akt and mTOR. Citation Format: Nancy D. Ebelt, Clint D J Taveres, Xuemei Xie, Youssef W. Naguib, Jiney Jose, Tinashe B. Ruwona, Ashwini K. Devkota, Jihyun Park, Tamer S. Kaoud, Eric V. Anslyn, Jeffrey T. Chang, Zhengrong Cui, Chandra Bartholomeusz, Kevin N. Dalby. KD06 is a novel anti-cancer drug that causes cell death in triple-negative breast cancer cell lines and tumor xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3774.

Acquired drug resistance, especially mechanisms associated with the reactivation of the MAPK (RAF/MEK/ERK) pathway represent a major challenge to current treatments of melanoma. Recently, targeting ERK has evolved as a potentially attractive strategy to overcome this resistance. Several ERK inhibitors have already entered clinical trials. Most of the available ERK inhibitors are reversible inhibitors that either act through an allosteric mechanism, or by targeting the ATP binding site. Taking advantage of our understanding of ERK-docking interactions we tried to discover an irreversible substrate-selective inhibitor that targets the protein-binding site of ERK. Here, we report the discovery of a covalent inhibitor of ERK that targets its protein-docking site. Protein NMR, Mass spectroscopy, mutagenesis and molecular docking studies indicate a covalent interaction of the inhibitor with a conserved cysteine residue, Cys-159. Extensive biochemical studies provide an estimate of its kinetic parameters and its kinase-selectivity profile. The new ERK inhibitor inhibits ERK activation, as well as its ability to phosphorylate downstream substrates (e.g. p90RSK and Elk-1) in HEK293T and A375 melanoma cells. The targeting of ERK in HEK293T cells was confirmed using a chemical-genetic approach where the ERK2 C159A mutant was used to rescue the effects of this compound on ERK2 signaling and cell proliferation. Currently, we are testing the effect of the compound on tumor growth inhibition in an A375 melanoma cancer xenografts model. This covalent inhibitor represents a potentially valuable lead molecule whose development may result in a novel class of pharmacologically useful ERK inhibitors for targeting resistant forms of melanoma. Citation Format: Tamer S. Kaoud, William H. Johnson, Nancy D. Ebelt, Andrea Piserchio, Mangalika Warthaka, Micael Cano, Rachel Sammons, Qiantao Wang, Pengyu Ren, Ranajeet Ghose, Kevin N. Dalby. Discovery of a covalent inhibitor of ERK docking-interactions that inhibits A375 melanoma cells proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3771.

Oncogenes induce cell proliferation leading to replicative stress, DNA damage and genomic instability. A wide variety of cellular stresses activate c-Jun N-terminal kinase (JNK) proteins, but few studies have directly addressed the roles of JNK isoforms in tumor development. Herein, we show that jnk2 knockout mice expressing the Polyoma Middle T Antigen transgene developed mammary tumors earlier and experienced higher tumor multiplicity compared to jnk2 wildtype mice. Lack of jnk2 expression was associated with higher tumor aneuploidy and reduced DNA damage response, as marked by fewer pH2AX and 53BP1 nuclear foci. Comparative genomic hybridization further confirmed increased genomic instability in PyV MT/jnk2-/- tumors. In vitro, PyV MT/jnk2-/- cells underwent replicative stress and cell death as evidenced by lower BrdU incorporation, and sustained chromatin licensing and DNA replication factor 1 (CDT1) and p21(Waf1) protein expression, and phosphorylation of Chk1 after serum stimulation, but this response was not associated with phosphorylation of p53 Ser15. Adenoviral overexpression of CDT1 led to similar differences between jnk2 wildtype and knockout cells. In normal mammary cells undergoing UV induced single stranded DNA breaks, JNK2 localized to RPA (Replication Protein A) coated strands indicating that JNK2 responds early to single stranded DNA damage and is critical for subsequent recruitment of DNA repair proteins. Together, these data support that JNK2 prevents replicative stress by coordinating cell cycle progression and DNA damage repair mechanisms.

Basal-like and claudin-low breast cancers have the worst prognosis and represent 15-20% of breast cancers diagnosed each year. Endocrine and molecularly targeted therapies, such as trastuzumab, are ineffective due to lack of ER expression or HER2 amplification in the tumors. They also have a high frequency of p53 mutations, low BRCA1 expression and high EGFR expression. Our lab has shown that high expression of JNK2 in human basal-like breast cancers leads to significantly decreased disease-free survival. JNK2 also promotes basal-like tumor progression in mice by increasing EGFR-mediated migration through facilitating internalization of EGFR, upregulating EMT gene expression and promoting metastasis. The ATP-competitive JNK inhibitor SP600125 has been commonly used by researchers to elucidate JNK-specific mechanisms, but this inhibitor was found to have high affinity to many other intracellular kinases. A new JNK inhibitor, JNK-IN-8, has been developed that binds covalently to all three JNK gene products and is more selective than the SP600125 compound (Zhang, et al. 2012). Using this inhibitor, we have found that basal-like breast cancer cell lines can become sensitized to lapatinib. This combination is synergistic and causes apoptotic cell death, while as single agents at these concentrations, these drugs have little effect on cell viability. Treatment with either lapatinib or JNK-IN-8 decreases transcriptional activity of NF-κB significantly, but combination of the two drugs reduces NF-κB activity to an almost negligible amount compared to vehicle treatment. Combination treatment also led to a 6-fold increase in ROS production that may be activating apoptosis. We hypothesize that inhibition by both JNK-IN-8 and lapatinib cause independent decreases in NF-κB activation that, when combined, cause a synergistic decrease in NF-κB-mediated survival mechanisms. Use of the JNK-IN-8 inhibitor with lapatinib in humans may increase survival in patients with basal-like or claudin-low breast cancers. Zhang, T. et al. "Discovery of potent and selective covalent inhibitors of JNK". Chem Biol. 2012 Jan 27;19(1):140-54. Citation Format: Ebelt ND, Van Den Berg CL. The covalent JNK inhibitor, JNK-IN-8, synergizes with lapatinib to cause cell death in basal-like breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-14.

Murine and human mammary cancers often show dysregulation of important signaling pathways including the canonical Wingless-iNTegrated (Wnt) and avian ERythroBlastosis oncogene B (ErbB) pathways. Transgenic expression of Wnt ligands causes transformation of normal mammary cells in mice, and Wnt10b is frequently upregulated in human breast cancers. Overexpression of ErbB ligands and amplification of receptors have also been implicated in human breast cancer. JNK1 is a tumor suppressor in the skin and intestinal epithelium, and JNKs are known to integrate ErbB and Wnt pathways as well as others to control cell growth and differentiation. In a murine mammary cancer model where 4T1.2 cells were injected into the mammary gland, reducing JNK1 levels by expressing shRNA (shJNK1) resulted in increased tumor growth and lung metastasis compared to mice injected with control vector- (pSM2) expressing cells. A microarray analysis comparing gene expression between shJNK1 and control tumors revealed 2 and 2.5-fold increases in the ErbB pathway genes Nrg3 and ETV5, 2-fold increases in the Wnt genes Bcl-9 and Wnt10a, and a 1.6-fold increase in the EMT gene Twist1. RT-PCR analysis of in vitro grown 4T1.2 cell lines transfected with shJNK1 confirmed increased expression of Bcl-9 and ETV5, as well as the ETV5 target Cox-2. ErbB2 protein was also overexpressed. The shJNK1 cells showed upregulation of pERK in response to Heregulin1 (an Erbb2/3 ligand) and Fibroblast Growth Factor (FGF) 1, which further amplifies canonical Wnt signaling. In a p53-/− tumor model, ETV5, Bcl-9, and Cox-2 were still upregulated in jnk1-/−compared to wildtype tumor cells, indicating this effect is p53-independent. In the normal mammary gland, a 4-fold increase in ETV5 and a 5-fold increase in Twist1 were found in jnk1-/− mice compared to wildtype, further indicating that this effect is dependent on JNK1 alone. The ErbB and Wnt pathways are known to upregulate EMT and stem-cell related genes, however, the involvement of JNK1 in these effects is a novel hypothesis. Thus far, our data suggest that JNK1 deficiency targets these oncogenic pathways to contribute to a more aggressive tumor phenotype due to heightened EMT and “stem-cellness”. Further studies using cell sorting and differentiation assays will determine whether normal jnk1-/− glands contain a higher fraction of stem cells than wildtype glands. Inhibition of EMT genes and/or ETV5 in the p53-/−;jnk1-/− cancer cells will determine whether the tumor growth or metastasis phenotypes are dependent on these genes. EMT and stem-cell genes are frequently expressed in human breast cancer subtypes that exhibit low survival rates, and EMT is known to be linked to increased metastasis. Some of these sub-types, such as claudin-low tumors, currently have no molecularly-targeted treatments, therefore it is important to determine what proteins critically contribute to these phenotypes so that efficient and effective treatments can be developed. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-01.

The classification of patient tumors by clinical subtype has gained great interest due to the implications for prognosis and treatment. For example, it is known that the five-year survival rate of patients diagnosed with the basal subtype of breast cancer is considerably lower than those with luminal subtypes because they lack expression of common drug targets. For this reason, it is important to elucidate the molecular mechanisms responsible for differentiation of mammary stem cell populations into basal and luminal cell lineages. Data presented in this poster outline a novel pathway whereby a ubiquitously expressed protein kinase, JNK2, influences the differentiation of basal, myoepithelial cells (MEps) while suppressing luminal cell (LEp) populations. Gross anatomical observation of whole-mounted mammary glands from wildtype (jnk2wt) and jnk2-/- (jnk2ko) mice revealed that JNK2 increases the rate of pubertal development. Loss of jnk2 increased the rate of ductal invasion, branching, and terminal end bud (TEB) count. Although a slight increase in proliferation was seen in jnk2ko glands, data suggested that it was not due to growth factor signaling. Instead, it was noticed that jnk2ko glands possess 35% fewer MEps (p=0.0078) with a corresponding increase in LEp populations (p=0.100). LEps are known to proliferate at a higher rate than MEps. The differentiation phenotype was corroborated through 3D culture of primarymammary epithelial cells (MECs). The rate of growth of 3D MEC acini was 31.5% higher in jnk2ko cultures than in jnk2wt (P Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-15.

Basal-like breast cancers represent 15-20% of breast cancers diagnosed each year. These tumors appear earlier in life and have the worst prognosis due to high prevalence of metastasis. Endocrine and molecularly targeted therapies such as trastuzumab or lapatinib are ineffective against this tumor subtype. A high frequency of p53 mutations, low BRCA1 expression along with high epidermal growth factor receptor (EGFR) expression provide some insight into what types of targeted therapies may be useful for basal-like tumors in the future. We have recently shown that JNK2 mediates the expression of p53 and BRCA1 in response to EGFR activation to promote epithelial to mesenchymal transition and metastasis. Further, high JNK2 expression is associated with poorer survival in patients with basal-like breast cancer. These findings suggest that the JNK pathway may be a promising target for basal-type breast cancer treatment. Commonly used competitive ATP inhibitors of JNK have suffered from lack of specificity for JNK. However, the Gray laboratory recently developed highly specific covalent inhibitors of JNK (Zhang, et al. 2012). The agent showing the best properties is JNK-IN-8. Our data show that treatment of human and mouse basal-like, breast cancer cell lines with JNK-IN-8 sensitizes them to lapatinib. Single agents had minor affects on cell viability and cell cycle regulation, but combination treatment led to G2/M arrest and endoreduplication along with synergistic apoptotic responses. Further, the expression and electrophoretic mobility of mitotic arrest deficient 2 (MAD2) (a protein mediating chromosomal segregation) decreased after combination treatment which could explain the chromosomal duplication abnormalities observed. Combination treatment using lapatinib and JNK-IN-8 modulates extracellular signal-regulated kinase (ERK) phosphorylation unlike either drug alone suggesting that both JNK and ERK target G2/M check point proteins, and when perturbed, sensitize basal-type breast cancer cells to targeted therapies. We conclude that use of specific JNK inhibitors in tumors that are resistant to lapatinib, or perhaps trastuzumab, may sensitize them to treatments with these drugs, and may also prevent resistance to these drugs in tumors that were initially responsive. Considering that JNKs are nodes in receptor tyrosine kinase and many other signaling pathways, JNK-IN-8 treatment may improve the efficacy of other targeted therapies as well. Zhang, T. et al. “Discovery of potent and selective covalent inhibitors of JNK”. Chem Biol. 2012 Jan 27;19(1):140-54. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-17.

The classification of patient tumors by clinical subtype has gained wide acceptance due to the implications for prognosis and treatment. However, recent studies have cast doubt on previously advocated normal mammary origins of these subtypes. Thus, the link between the normal mammary gland and mammary tumors is more complex than expected. C-Jun N-Terminal Kinase-2 (JNK2) is a protein that is involved in numerous developmental processes and our previous work has shown it to be important for DNA damage response in mammary tumors. In attempt to gain insight into the link between mammary development and tumorigenesis, we compared normal mammary glands of JNK2 knockout (jnk2ko) mice to jnk2ko mammary tumors expressing or lacking wildtype p53 (p53ko). These studies showed that jnk2ko glands possess 35% fewer basal cells (p=0.0078) with a corresponding increase in luminal epithelial cell populations (p=0.100). This luminal response is corroborated by in vitro 3D assays of primary mammary epithelial cells (MECs) where luminal cell differentiation is normalized by inhibition of Notch signaling. Expression notch-1, a well-known regulator of MEC differentiation, is increased jnk2ko mammary glands. Increased expression of the Notch-1 target gene, hes-1, was also seen (p=0.005). Histology revealed that increased expression of active Notch-1 is localized to the mammary stem cell niche, the terminal end bud. Similar to the normal gland, jnk2ko mammary tumors possessing wildtype p53 exhibit decreased proportions of basal cells (p=0.0002) and increased proportions of luminal cells (p=0.0411) relative to wildtype. Jnk2ko cell lines derived from these tumors show decreased expression of notch-1 (p=0.0018) and hes-1 (p=0.0602) following introduction of JNK2. Luciferase assays comparing activity of the notch-1 promoter to a notch-1 promoter with mutated p53 response elements revealed a dependence of increased notch-1 promoter activity in jnk2ko cells on the p53 response element. P53ko tumor cells, by contrast, do not exhibit alterations in notch-1 promoter activity in the absence of p53 response elements, regardless of JNK2 status. QPCR showed that loss of JNK2 in normal mammary glands and tumors causes increased p53 expression—thus providing a potential mechanism. In support that Notch upregulation in the absence of JNK2 is dependent upon p53, normal glands lacking p53 show no differences in lineage differentiation. P53ko tumors also show no differences in basal lineage differentiation, however, increases in luminal differentiation are maintained in the absence of JNK2. Consistent with increased luminal differentiation, jnk2ko caused decreased expression of markers involved in the epithelial to mesenchymal transition phenotype. This data suggests that JNK2 is important not only for lineage differentiation in normal mammary glands, but in mammary tumors and that the effect is dependent on both Notch1 and p53. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-01.