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Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). Although MAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration (FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.

Breast cancer affects women globally; the majority of breast cancer-related mortalities are due to metastasis. Acquisition of a mesenchymal phenotype has been implicated in the progression of breast cancer cells to an invasive, metastatic state. Triple-negative breast cancer (TNBC) subtypes have high rates of metastases, recurrence, and have poorer prognoses compared to other breast cancer types, partially due to lack of commonly targeted receptors. Kinases have diverse and pivotal functions in metastasis in TNBC, and discovery of new kinase targets for TNBC is warranted. We previously used a screening approach to identify intermediate-synthesis nonpotent, nonselective small-molecule inhibitors from the Published Kinase Inhibitor Set that reversed the mesenchymal phenotype in TNBC cells. Two of these inhibitors (GSK346294A and GSK448459A) are structurally similar, but have unique kinase activity profiles and exhibited differential biologic effects on TNBC cells, specifically on epithelial-to-mesenchymal transition (EMT). Here, we further interrogate these effects and compare activity of these inhibitors on transwell migration, gene (qRT-PCR) and protein (western blot) expressions, and cancer stem cell-like behavior. We incorporated translational patient-derived xenograft models in these studies, and we focused on the lead inhibitor hit, GSK346294A, to demonstrate the utility of our comparative analysis as a screening modality to identify novel kinase targets and signaling pathways to pursue in TNBC. This study introduces a new method for discovering novel kinase targets that reverse the EMT phenotype; this screening approach can be applied to all cancer types and is not limited to breast cancer.

The organization of cell cytoskeleton is altered in events of epithelial to mesenchymal transition (EMT), promotion of cell motility, and cancer metastases. EMT is associated with decreased cell-cell adhesion, downregulation of epithelial markers like E-Cadherin, cytokeratins, and occludins, and upregulation of mesenchymal markers such as N-cadherin, vimentin, and various transcription factors such as slug and ZEB. Epithelial to mesenchymal transition is also a consequence of drug resistance and is responsible for cancer metastases. Triple negative breast cancer is highly aggressive cancer and patients show poor prognosis and disease-free survival due to the lack of targeted therapy. Mitogen activated protein kinase pathway, including extracellular activated kinase ERK1/2 and ERK5, and phosphoinositide 3-kinase (PI3K) pathway are known to alter the cytoskeleton through the downstream activation of oncogenes such as FRA-1 and loss of focal adhesions. Of these pathways, the MEK5-ERK5 pathway is understudied in triple negative breast cancer TNBC, and there are few research tools available to selectively inhibit this pathway. The diphenylamine analogs were derived from the parent molecule Mekinist, a FDA approved MEK1/2 inhibitor for melanoma, and modified to gain selectivity towards MEK5. SC-1-151, a type-III allosteric inhibitor of MEK5 is a dual MEK1/2 (98.6%) and MEK5 (59%) inhibitor; the molecule inhibits cell viability and colony formation, and attenuates tumor growth. SC-1-151 was serendipitously identified as a mesenchymal to epithelial transition activator in TNBC cell line MDA-MB-231. E-cadherin protein expression and cell morphology were examined to study MET after the treatment of MDA-MB-231 cells with different structural analogs of SC-1-151 after treatment for 5 days. The compound was further found to induce E-cadherin expression and epithelial phenotype in tamoxifen resistant estrogen positive MCF-7 cell line that underwent EMT. The compound is identified to promote this activity by targeting at least the ERK-FRA1-ZEB1 axis. Alkyl or N-Methyl piperazine substituents on the amide of ring 1 produced similar result as SC-1-151, and substituting the amide group with acid or ester also induced MET. In contrast, ortho-fluoro, para-iodo functional groups of the arene ring 2, when replaced with a meta-bromo substituent did not induce MET. We aim to test the compounds on EGF treated MDA-MB-468 cells to observe the attenuation of EGF induced EMT. Future studies will be performed to determine the specific protein interactions of the promising compounds. Citation Format: Bhatt AB, Wright TD, Anna K, Gupta M, Chakrabarty S, Flaherty PT, Hoang V, Burow M, Cavanaugh JE. Study of diphenylamine analogs as inducers of mesenchymal to epithelial transition in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-08-07.

Human bone marrow‐derived mesenchymal stem/stromal cells (BM‐MSCs) represent promising stem cell therapy for the treatment of type 2 diabetes mellitus (T2DM), but the results of autologous BM‐MSC administration in T2DM patients are contradictory. The purpose of this study was to test the hypothesis that autologous BM‐MSC administration in T2DM patient is safe and that the efficacy of the treatment is dependant on the quality of the autologous BM‐MSC population and administration routes. T2DM patients were enrolled, randomly assigned (1:1) by a computer‐based system into the intravenous and dorsal pancreatic arterial groups. The safety was assessed in all the treated patients, and the efficacy was evaluated based on the absolute changes in the hemoglobin A1c, fasting blood glucose, and C‐peptide levels throughout the 12‐month follow‐up. Our data indicated that autologous BM‐MSC administration was well tolerated in 30 T2DM patients. Short‐term therapeutic effects were observed in patients with T2DM duration of, Human bone marrow‐derived mesenchymal stem/stromal cells (BM‐MSCs) represent promising stem cell therapy for the treatment of type 2 diabetes, but the results of autologous BM‐MSC admniistration in T2DM patients are contradicted. Our data indicated that autologous BM‐MSC administration was well tolerated in 30 T2DM patients. The potential therapeutic effects of the treatments were observed in patients with less than 10 years of T2DM and a BMI

Background Bronchopulmonary dysplasia (BPD) is a severe condition in premature infants that compromises lung function and necessitates oxygen support. Despite major improvements in perinatal care minimizing the devastating effects, BPD remains the most frequent complication of extreme preterm birth. Our study reports the safety of the allogeneic administration of umbilical cord-derived mesenchymal stem/stromal cells (allo-UC-MSCs) and the progression of lung development in four infants with established BPD. Methods UC tissue was collected from a healthy donor, followed by propagation at the Stem Cell Core Facility at Vinmec Research Institute of Stem Cell and Gene Technology. UC-MSC culture was conducted under xeno- and serum-free conditions. Four patients with established BPD were enrolled in this study between May 25, 2018, and December 31, 2018. All four patients received two intravenous doses of allo-UC-MSCs (1 million cells/kg patient body weight (PBW) per dose) with an intervening interval of 7 days. Safety and patient conditions were evaluated during hospitalization and at 7 days and 1, 6 and 12 months postdischarge. Results No intervention-associated severe adverse events or prespecified adverse events were observed in the four patients throughout the study period. At the time of this report, all patients had recovered from BPD and were weaned off of oxygen support. Chest X-rays and CT scans confirmed the progressive reductions in fibrosis. Conclusions Allo-UC-MSC administration is safe in preterm infants with established BPD. Trial registration This preliminary study was approved by the Vinmec International Hospital Ethics Board (approval number: 88/2019/QĐ-VMEC; retrospectively registered March 12, 2019).

Conventional mitogen-activated protein kinase (MAPK) family members regulate diverse cellular processes involved in tumor initiation and progression, yet the role of ERK5 in cancer biology is not fully understood. Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. ERK5 signaling contributes to drug resistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). More recently a role for ERK5 in regulation of the extracellular matrix (ECM) has been proposed, and here we investigated the necessity of ERK5 in TNBC tumor formation. Depletion of ERK5 expression using the CRISPR/Cas9 system in MDA-MB-231 and Hs-578T cells resulted in loss of mesenchymal features, as observed through gene expression profile and cell morphology, and suppressed TNBC cell migration. In vivo xenograft experiments revealed ERK5 knockout disrupted tumor growth kinetics, which was restored using high concentration Matrigel™ and ERK5-ko reduced expression of the angiogenesis marker CD31. These findings implicated a role for ERK5 in the extracellular matrix (ECM) and matrix integrity. RNA-sequencing analyses demonstrated downregulation of matrix-associated genes, integrins, and pro-angiogenic factors in ERK5-ko cells. Tissue decellularization combined with cryo-SEM and interrogation of biomechanical properties revealed that ERK5-ko resulted in loss of key ECM fiber alignment and mechanosensing capabilities in breast cancer xenografts compared to parental wild-type cells. In this study, we identified a novel role for ERK5 in tumor growth kinetics through modulation of the ECM and angiogenesis axis in breast cancer.

Background: Bronchopulmonary dysplasia (BPD) is a severe condition in premature infants that compromises theirlung function and necessitatesoxygen support. Despite major improvements in perinatal care minimizing the devastating effects, BPD remains the most frequent complication of extreme preterm birth. Our study reports the safety ofthe allogeneic administration of umbilical cord-derived mesenchymal stem/stromal cells (allo-UC-MSCs) and the preliminary efficacy of the treatment in four infants with established BPD.Methods: UC tissue was collected from a healthy donor, followed by propagation at the Stem Cell Core Facility at Vinmec Research Institute of Stem Cell and Gene Technology. UC-MSC culture was conducted under xeno-free and serum-free conditions. Four patients with established BPD were enrolled in this study between May 25, 2018, and December 31, 2018. All four patients received two intravenous doses of allo-UC-MSCs (1 million cells/kg patient body weight (PBW) per dose) with an intervening interval of 7 days. Safety and efficacy were evaluated during hospitalization and at 7 days and 1, 6 and 12 postdischargemonths.Results: No transplantation-associated severe adverse events or prespecified adverse events were observed in the four patients throughout the study period. At the time of this report, all patients had recovered from BPD and been weaned off of oxygen support. Chest X-rays and CT scans confirmed the dramatic reduction infibrosis.Conclusions: Allo-UC-MSC transplantation is safe and might improve respiratory function anddecrease lung fibrosis in preterm infants with established BPD.Trial registration: This preliminary study was approved by Vinmec International Hospital Ethics Board, approval number: 88/2019/QĐ-VMEC, registered 12 March 2019 - retrospectively registered.

Claudin-low triple negative breast cancer (CL-TNBC) is a clinically aggressive molecular TNBC subtype characterized by a propensity to metastasize, recur and acquire chemoresistance. CL-TNBC has a diverse intra- and extracellular composition and microenvironment, and currently there are no clinically approved targeted therapies. Histone deacetylase inhibitors (HDACi) have been investigated as therapeutic agents targeting invasive TNBC phenotypes. However, further studies are required to evaluate HDAC inhibition in CL-TNBC. Here, we utilize a novel CL- TNBC patient-derived xenograft model to study the various and diverse therapeutic potential targets within CL-TNBC tumors. To evaluate effects of the pan-HDACi panobinostat on metastasis and the mesenchymal phenotype of CL-TNBC, we utilize immunohistochemistry staining and qRT-PCR in in vitro, ex vivo and in vivo studies. Further, we evaluate pan-HDAC inhibition on stem-like subpopulations using 3D mammosphere culture techniques and quantification. Finally, we show that pan- HDACi suppresses collagen expression in CL-TNBC. In this study, we provide evidence that pan-HDAC inhibition has effects on various components of the CL-TNBC subtype, and we demonstrate the potential of our novel CL-TNBC PDX model in therapeutic discovery research.

Epithelial to mesenchymal transition (EMT) is a cellular program that converts non-motile epithelial cells into invasive mesenchymal cells. EMT is implicated in cancer metastasis, chemo-resistance, cancer progression, and generation of cancer stem cells (CSCs). Inducing mesenchymal to epithelial transition (MET), the reverse phenomenon of EMT, is proposed as a novel strategy to target triple negative and tamoxifen-resistant breast cancer. Triple negative breast cancer (TNBC) is characterized by the loss of hormone receptors, a highly invasive mesenchymal phenotype, and a lack of targeted therapy. Estrogen receptor-positive breast cancer can be targeted by tamoxifen, an ER antagonist. However, these cells undergo EMT over the course of treatment and develop resistance. Thus, there is an urgent need to develop therapeutic interventions to target these aggressive cancers. In this study, we examined the role of novel diphenylamine analogs in converting the mesenchymal phenotype of MDA-MB-231 TNBC cells to a lesser aggressive epithelial phenotype. Using analog-based drug design, a series of diphenylamine analogs were synthesized and initially evaluated for their effect on E-cadherin protein expression and changes incell morphology, which was quantified by measuring the spindle index (SI) value. Selected compound 1 from this series increases the expression of E-cadherin, a primary marker for epithelial cells, and decreases the mesenchymal markers SOX2, ZEB1, Snail, and vimentin. The increase in epithelial markers and the decrease in mesenchymal markers are consistent with a phenotypic switch from spindle-like morphology to cobblestone-like morphology. Furthermore, Compound 1 decreases spheroid viability, cell migration, and cell proliferation in triple negative BT-549 and tamoxifen-resistant MCF-7 breast cancer cells. ispartof: FRONTIERS IN ONCOLOGY vol:9 ispartof: location:Switzerland status: published

Background Triple-negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options. Experimental preclinical models that recapitulate their tumors of origin can accelerate target identification, thereby potentially improving therapeutic efficacy. Patient-derived xenografts (PDXs), due to their genomic and transcriptomic fidelity to the tumors from which they are derived, are poised to improve the preclinical testing of drug-target combinations in translational models. Despite the previous development of breast and TNBC PDX models, those derived from patients with demonstrated health-disparities are lacking. Methods We use an aggressive TNBC PDX model propagated in SCID/Beige mice that was established from an African-American woman, TU-BcX-2 K1, and assess its metastatic potential and drug sensitivities under distinct in vitro conditions. Cellular derivatives of the primary tumor or the PDX were grown in 2D culture conditions or grown in mammospheres 3D culture. Flow cytometry and fluorescence staining was used to quantify cancer stem cell-like populations. qRT-PCR was used to describe the mesenchymal gene signature of the tumor. The sensitivity of TU-BcX-2 K1-derived cells to anti-neoplastic oncology drugs was compared in adherent cells and mammospheres. Drug response was evaluated using a live/dead staining kit and crystal violet staining. Results TU-BcX-2 K1 has a low propensity for metastasis, reflects a mesenchymal state, and contains a large burden of cancer stem cells. We show that TU-BcX-2 K1 cells have differential responses to cytotoxic and targeted therapies in 2D compared to 3D culture conditions insofar as several drug classes conferred sensitivity in 2D but not in 3D culture, or cells grown as mammospheres. Conclusions Here we introduce a new TNBC PDX model and demonstrate the differences in evaluating drug sensitivity in adherent cells compared to mammosphere, or suspension, culture. Electronic supplementary material The online version of this article (10.1186/s12885-019-5401-2) contains supplementary material, which is available to authorized users.

Triple-negative breast cancers (TNBCs) constitute approximately 12% of all breast cancer cases and are approximately twice as prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.3% in 2017), and New Orleans has among the highest incidences of TNBC in the country. Louisiana patients also have a high incidence of co-morbidities that affect breast cancer biology and outcomes, including type 2 diabetes and obesity. TNBCs have an aggressive clinical presentation due to high rates of metastasis, recurrence and chemoresistance. There are currently no clinically approved targeted therapies for TNBC; cytotoxic chemotherapy is the first-line treatment for TNBC, and recurrent, chemoresistant cancers are usually fatal. TNBCs are molecularly heterogeneous, consisting of at least four molecular subgroups, and immunologically heterogeneous. Both molecular and immunologic properties are associated with clinical outcomes and are seriously understudied in patients under-represented in biomedical research. Patient-derived xenografts (PDXs), as well as patient-derived organoids (PDO), are currently the best model for translational oncology therapeutic research because they accurately recapitulate the complex architecture and heterogenous genetic and molecular composition of solid cancers. To date, the majority of TNBC research has been based on Caucasian patients, although incidence rates of TNBC are higher in African-American cohorts. Our collaborative team aims to overcome this obstacle by establishing and characterizing TNBC PDX models that represent this understudied cohort. We currently have ten TNBC PDX models representing different patient ethnicities, responsiveness to chemotherapies, as well as different TNBC molecular subtypes and metastatic behavior. We dissect and evaluate the various individual components (tumor cell biology, stroma, immune, extracellular matrix) of TNBC tumors. We utilize these models in vivo, ex vivo and in vitro to examine how unique kinases and targeted inhibitors affect the distinct tumor characteristics. In addition to in vivo treatment studies, we generated cell lines and PDOs and we utilize novel techniques such as tissue decellularization to examine extracellular matrix components. We also analyze mechanistically relevant transcript (qRT-PCR) and protein (Western blot, immunohistochemistry) expression patterns that are unique to each PDX model to evaluate the effects of targeted therapies. We work with surrounding laboratories in the greater New Orleans area (Tulane, LSU, Xavier) that are also focused on therapeutic discovery of TNBC in a collaborative effort to provide translational models for their projects. Our aim is to leverage novel PDX models from understudied patients with a range of clinical and molecular presentations to guide the selection of therapeutically targetable pathways and therapeutic agents in specific molecular subtypes of TNBC. Citation Format: Margarite D. Matossian, Steven Elliott, Hope E. Burks, Maryl Wright, Rachel A. Sabol, Van T. Hoang, Deniz A. Ucar, Alex Alfortish, Jovanny Zabaleta, Fokhrul Hossain, Tiffany Chang, Henri Wathieu, Nicholas Pashos, Bruce Bunnell, Krzysztof Moroz, Arnold Zea, Adam Riker, Steven D. Jones, Elizabeth C. Martin, Lucio Miele, Bridgette M. Collins-Burow, Matthew E. Burow. Applications of patient-derived triple-negative breast cancer xenografts that represent understudied patients in Louisiana in targeted therapeutic research [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C110.

Triple negative breast cancers (TNBCs) are a clinically and biologically aggressive breast cancer (BC) subtype; TNBC tumors have higher rates of metastasis, relapse and acquired/inherent drug resistance. Incidence and mortality rates of TNBC are stratified based on patient ethnicity - patients with African ancestry have higher mortality rates and diagnoses of invasive cancers compared to patients representing other ethnicities. Louisiana has a high proportion of African-American residents (32.7% in 2018), and New Orleans has among the highest incidences of TNBC in the country. Many of our patients present with TNBC tumors that are partially or completely resistant to neoadjuvant chemotherapies. There are currently no clinically approved targeted therapies for TNBC. Current therapeutic discovery focused TNBC research does not aptly address the knowledge gap regarding ethnic disparity in TNBC incidence/mortality rates and TNBC biology. To date, most TNBC-related research and knowledge has been acquired from Caucasian patients, although patients with African and Hispanic ancestries represent the majority of TNBC cases. Patient-derived xenografts (PDXs) are extensively used in BC research, as they mimic complex microanatomy, oncoarchitecture, and cell-cell/cell-stroma interactions of tumors. Here, we demonstrated the unique composition of PDX tumors is not dramatically affected by serial transplantation in mice, based on molecular phenotypes (examined using qRT-PCR and RNA sequencing) and the oncoarchitecture of the extracellular matrix (based on cryogenic scanning electron microscopy). Using these models in basic research facilitates translation of laboratory findings to the clinical setting, and dramatically enhanced drug discovery research. We have established over twelve TNBC PDX models, 90% of which represent patients of African ancestry, and most of which are resistant to neoadjuvant regimens. We focus on dissecting and evaluating kinase inhibitor/targeted drug response to various individual components (tumor cell biology, stroma, immune, extracellular matrix) of chemotherapy resistant TNBC tumors. Histone deacetylase inhibitors (DACi) are a promising therapeutic agent in TNBC systems; they have been shown to suppress tumorigenesis and metastasis in TNBC through suppression of the mesenchymal phenotype in cell line-based studies. In this study we utilized various TNBC PDX models (TU-BcX-2K1, -2O0, 4IC, -4M4, -4QAN, -4QX) to assess these findings in more translational systems. Interestingly, we showed that DACi effect on tumorigenesis and metastasis varied depending on specific TNBC PDXs utilized. These data implicate specific genes/signaling pathways exist in individual patient tumors that can predict tumor responsiveness to DACi. Preliminary data using the NCI oncology drug set implicated the MEK1/2 pathway contributed to sensitization of TNBC cells. Furthermore, we found a disconnect in gene expressions that were previously shown to be affected by DACi therapy (CDH1, VIM, ZEB1, ZEB2) in various derivations of PDX models (cells, PDX-Os, ex vivo, in vivo). These findings demonstrate that testing various derivations of PDX models is crucial to parsing out specific mechanisms of targeted therapies. Our methods presented here to assess targeted drug response and drug resistance using PDX models can be applied to any area of cancer research and is not limited to breast cancer. Citation Format: Margarite Matossian, Steven Elliott, Maryl Wright, Tiffany Chang, Madlin Alzoubi, Henri Wathieu, Rachel Sabol, Alex Alfortish, Hope Burks, Van Hoang, Deniz Ucar, Gabrielle Windsor, Thomas Yan, Jovanny Zabaleta, Fokhrul Hossain, Bruce Bunnell, Krzysztof Moroz, Arnold Zea, Adam Riker, Steven Jones, Elizabeth Martin, Lucio Miele, Bridgette Collins-Burow, Matthew Burow. Effect of histone deacetylase inhibitors on patient-derived neoadjuvant chemotherapy resistant triple negative breast cancer xenografts that represent understudied patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-17.

The transcriptional regulator far upstream element binding protein 1 (FUBP1) acts as an oncoprotein in solid tumor entities and plays a role in the maintenance of hematopoietic stem cells. However, its potential function in leukemia is unknown. In murine models of chronic (CML) and acute myeloid leukemia (AML) induced by BCR-ABL1 and MLL-AF9, respectively, knockdown of Fubp1 resulted in prolonged survival, decreased numbers of CML progenitor cells, decreased cell cycle activity and increased apoptosis. Knockdown of FUBP1 in CML and AML cell lines recapitulated these findings and revealed enhanced DNA damage compared to leukemia cells expressing wild type FUBP1 levels. FUBP1 was more highly expressed in human CML compared to normal bone marrow cells and its expression correlated with disease progression. In AML, higher FUBP1 expression in patient leukemia cells was observed with a trend toward correlation with shorter overall survival. Treatment of mice with AML with irinotecan, known to inhibit topoisomerase I and FUBP1, significantly prolonged survival alone or in combination with cytarabine. In summary, our data suggest that FUBP1 acts as cell cycle regulator and apoptosis inhibitor in leukemia. We demonstrated that FUBP1 might play a role in DNA repair, and its inhibition may improve outcome in leukemia patients.

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH+ cells, What's new? To understand the precise disease‐driving mechanisms in acute myeloid leukemia (AML), comparison of patient‐matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. This study demonstrates the relevance of aldehyde dehydrogenase (ALDH) for the prospective identification of AML cases in which separation of functionally normal HSC from LSC is possible. Increased activity of this biomarker also characterizes a subgroup of patients with adverse outcome, which might be helpful in risk stratification prior to therapy. Overall, this study demonstrates functional heterogeneity of leukemia cells and suggests divergent roles for ALDH activity in normal HSC versus leukemia‐initiating cells.

BACKGROUND: Triple negative breast cancer (TNBC) subtypes are clinically aggressive and are treated with targeted therapeutics commonly used in other BC subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research have limitations. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by xenograft methods. METHODS: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. RESULTS: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including specific cell populations within the tumor, the extracellular matrix, and cancer stem-like cell populations. CONCLUSIONS: Here we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

Triple negative breast cancers (TNBCs) have high recurrence and metastasis rates. Acquisition of a mesenchymal morphology and phenotype in addition to driving migration is a consequential process that promotes metastasis. Although some kinases are known to regulate a mesenchymal phenotype, the role for a substantial portion of the human kinome remains uncharacterized. Here we evaluated the Published Kinase Inhibitor Set (PKIS) and screened a panel of TNBC cell lines to evaluate the compounds' effects on a mesenchymal phenotype. Our screen identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology, which was then prioritized to twelve compounds based on gene expression and migratory behavior analyses. We selected the most active compound and confirmed mesenchymal reversal on transcript and protein levels with qRT-PCR and Western Blot. Finally, we utilized a kinase array to identify candidate kinases responsible for the EMT reversal. This investigation shows the novel application to identify previously unrecognized kinase pathways and targets in acquisition of a mesenchymal TNBC phenotype that warrant further investigation. Future studies will examine specific roles of the kinases in mechanisms responsible for acquisition of the mesenchymal and/or migratory phenotype.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial-to-mesenchymal transition (EMT) is a key contributor in the metastatic process. We previously showed the pan-deacetylase inhibitor LBH589 induces CDH1 expression in TNBC cells, suggesting regulation of EMT. The purpose of this study was to examine the effects of LBH589 on the metastatic qualities of TNBC cells and the role of EMT in this process. A panel of breast cancer cell lines (MCF-7, MDA-MB-231, and BT-549), drugged with LBH589, was examined for changes in cell morphology, migration, and invasion in vitro. The effect on in vivo metastasis was examined using immunofluorescent staining of lung sections. EMT gene expression profiling was used to determine LBH589-induced changes in TNBC cells. ZEB overexpression studies were conducted to validate requirement of ZEB in LBH589-mediated proliferation and tumorigenesis. Our results indicate a reversal of EMT by LBH589 as demonstrated by altered morphology and altered gene expression in TNBC. LBH589 was shown to be a more potent inhibitor of EMT than other HDAC inhibitors, SAHA and TMP269. Additionally, we found that LBH589 inhibits metastasis of MDA-MB-231 cells in vivo. These effects of LBH589 were mediated in part by inhibition of ZEB, as overexpression of ZEB1 or ZEB2 mitigated the effects of LBH589 on MDA-MB-231 EMT-associated gene expression, migration, invasion, CDH1 expression, and tumorigenesis. These data indicate therapeutic potential of LBH589 in targeting EMT and metastasis of TNBC.

We previously demonstrated that leukemia cell lines expressing CD44 and hematopoietic progenitor cells (HPC) from umbilical cord blood (CB) showed rolling on hyaluronic acid (HA)-coated surfaces under physiological shear stress. In the present study, we quantitatively assessed the interaction of HPC derived from CB, mobilized peripheral blood (mPB) and bone marrow (BM) from healthy donors, as well as primary leukemia blasts from PB and BM of patients with acute myeloid leukemia (AML) with HA. We have demonstrated that HPC derived from healthy donors showed relative homogeneous rolling and adhesion to HA. In contrast, highly diverse behavioral patterns were found for leukemia blasts under identical conditions. The monoclonal CD44 antibody (clone BU52) abrogated the shear stress-induced rolling of HPC and leukemia blasts, confirming the significance of CD44 in this context. On the other hand, the immobile adhesion of leukemia blasts to the HA-coated surface was, in some cases, not or incompletely inhibited by BU52. The latter property was associated with non-responsiveness to induction chemotherapy and subsequently poor clinical outcome.

Aims: mesenchymal stromal/stem cells (MSCs) is one of the most promising cell therapies to treat immune related diseases such as GvHD, Crohn's disease, osteoarthritis as well as to support function of heart, liver, and central nervous system. Many advanced clinical trials has been taking place to investigate the safety and efficacy of MSC therapy, however, it remains challenging to compare the results of these trials due to variations in culture conditions and tissue origins. These variations pose the urgent need of a standardized large-scale production and quality control of these cells become more urgent, as the use of MSCs are spreading around the globe and disease backgrounds. Numerous culture protocols for MSCs are available using FBS-containing, human platelet lysate/plasma supplementing, or serum-free xeno-free condition, either commercial or self-prepared media. In this study, we have established a standardized expansion protocol that can be used for MSCs derived from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC). Methods: we tested commercial xeno-free, serum-free media (MesenCult™-ACF Plus Medium/Stem Cell Technologies, MSC NutriStem® XF Medium/BI, Stempro® MSC SFM Medium/ThermoFisher, PowerStem MSC1 Medium/PAN, and StemMACS™ MSC Expansion Media/Miltenyi) in order to identify a common culture condition for all of these MSC sources. The cells were analysed for their population doubling time from P2 to P6, expression of surface markers including the positive markers CD73, CD90, and CD105, and the negative markers CD34, CD45, CD11b, CD19, and HLA-DR. Karyotype was conducted at early and later passage (P3 and P6, respectively). MSCs were tested for their differentiation capacity into osteoblast/osteocyte, adipocyte, and chondrocyte lineages. Bacterial and fungal sterility and mycoplasma test were performed and endotoxin concentration was determined before the cells were transfused into patients. Results: using UC for the primary screening, we identified two medium candidates that supported the isolation and culture of UC-MSCs. MSCs derived from BM and AD are capable of expansion in these media as well. However, one candidate induced an increased HLA-DR expression so that it failed to fulfill the minimal criteria of MSCs (Dominici et al., 2006). The last one showed the best results for MSCs from all tested tissues. Under our established protocol, MSCs grew exponentially until P6, which was the highest analyzed passage. The cells expressed CD73, CD90, CD105 in more than 95% cells and showed less than 2% of negative markers (CD34, CD45, CD11b, CD19, and HLA-DR). Karyotype analysis confirmed no chromosomal aberrations of the cultured cells. The cells were able to differentiate into osteoblast/osteocyte, adipocyte, and chondrocyte lineages. We are now conducting phase 1 clinical trial to address the safety and feasibility of UC-MSCs in the treatment of bronchopulmonary dysplasia and chronic obstructive pulmonary disease. Conclusion: we have established a standardized protocol for the xeno-free, serum-free culture of MSCs from different sources. BM-, AD-, and UC-derived MSCs expand in large-scale and maintain their cellular characteristics, karyotype integrity, and can be applied for cell therapy. Disclosures No relevant conflicts of interest to declare.

Present evidence indicates that acute myeloid leukemia (AML) is a stem cell disease. Leukemia stem cells (LSC) might originate from malignant transformation of normal hematopoietic stem cells (HSC), or alternatively, from progenitors in which the acquired mutations have re-installed a dysregulated self-renewal program. Since LSC, similar to their normal counterparts, divide extreme slowly, this might account for the ineffectiveness of conventional chemotherapy in inducing long-term cure. The present review will focus on the detection of LSC, their cellular and molecular biology, their genetic heterogeneity and on correlative studies that have demonstrated the clinical significance of estimating LSC burden. For long-term cure of AML, it is of importance to define LSC candidates and to understand their biology compared to normal HSC. Finally, we will discuss the perspectives of developing treatment strategies for eradication of LSC.

Estrogen receptor alpha (ERα) signaling pathways are frequently disrupted in breast cancer and contribute to disease progression. ERα signaling is multifaceted and many ERα regulators have been identified including transcription factors and growth factor pathways. More recently, microRNAs (miRNAs) are shown to deregulate ERα activity in breast carcinomas, with alterations in both ERα and miRNA expression correlating to cancer progression. In this study, we show that a high expression of Argonaute 2 (AGO2), a translation regulatory protein and mediator of miRNA function, correlates with the luminal B breast cancer subtype. We further demonstrate that a high expression of AGO2 in ERα+ tumors correlates with a poor clinical outcome. MCF-7 breast cancer cells overexpressing AGO2 (MCF7-AGO2) altered ERα downstream signaling and selective ERα variant expression. Enhanced ERα-36, a 36 kDa ERα isoform, protein and gene expression was observed in vitro. Through quantitative polymerase chain reaction (qPCR), we demonstrate decreased basal expression of the full-length ERα and progesterone receptor genes, in addition to loss of estrogen stimulated gene expression in vitro. Despite the loss, MCF-7-AGO2 cells demonstrated increased estrogen stimulated tumorigenesis in vivo. Together with our clinical findings on AGO2 expression and the luminal B subtype, we suggest that AGO2 is a regulator of altered ERα signaling in breast tumors.

Acute myeloid leukemia (AML) is considered to be a disease of stem cells. A rare defective stem cell population is purported to drive tumor growth. Similarly to their normal counterparts, leukemic stem cells (LSC) divide extreme slowly. This may explain the ineffectiveness of conventional chemotherapy in combatting this disease. Novel treatment strategies aimed at disrupting the binding of LSC to stem cell niches within the bone marrow might render the LSC vulnerable to chemotherapy and thus improving treatment outcome. This review focuses on the detection of LSC, our current knowledge about their cellular and molecular biology, and LSC interaction with the niche. Finally, we discuss the clinical relevance of LSC and prospective targeted treatment strategies for patients with AML.

Triple-negative breast cancers (TNBCs) constitute approximately 12% of all breast cancers, and are approximately twice more prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.5% in 2015), and among the highest incidences of TNBC in the country. Louisiana patients also have a high incidence of comorbidities that affect breast cancer biology and outcomes, including type 2 diabetes and obesity. TNBCs have an aggressive clinical presentation due to high rates of metastasis, recurrence, and chemoresistance, and targeted therapy remains elusive. Discovery of novel therapeutic targets, including a subset of previously uncharacterized kinases in TNBC could provide important insights into future targeted therapies. However, current models utilized in target discovery research are limited by the inability to accurately recapitulate the complex architecture and heterogenous genetic and molecular composition of breast cancer. Furthermore, immortalized cell lines have been selected in a two-dimensional environment and may have lost important epigenetic features of original tumors they derived from. Recently, our laboratory has successfully established four TNBC patient-derived xenograft (PDX) models representing different patient ethnicities, responsiveness to chemotherapies, and different TNBC molecular subtypes and metastatic behavior. Our primary objective was to dissect and evaluate the various individual components (tumor cell biology, stroma, immune, extracellular matrix) that drive complex interactions within TNBC tumors. We utilize these models in vivo, ex vivo, and in vitro to examine how unique kinases and small molecule inhibitors affect the distinct tumor characteristics. In addition to in vivo treatment studies we generated cell lines and mammospheres (TU-BcX-2K1, TU-BcX-2O0, TU-BcX-49S, TU-BcX-4IC), and we utilize novel techniques such as tissue decellularization to examine extracellular matrix components. We also analyze mechanistically relevant transcript (qRT-PCR) and protein (Western blot, immunohistochemistry) expression patterns that are unique to each PDX model to evaluate the effect of small-molecule inhibitors on these transcripts and proteins. Our aim is to leverage novel patient-derived models from understudied patients with a range of clinical presentations to guide the selection of therapeutically targetable pathways and molecules in specific molecular subtypes of TNBC. Citation Format: Margarite Matossian, Hope Burks, Tiffany Chang, Annie Bowles, Rachel Sabol, Van Hoang, Bruce Bunnell, Alex Alfortish, Krzysztof Moroz, Arnold Zea, Adam Riker, Steven Jones, Ayse D. Ucar Bilyeu, Fokhrul Hossain, Kristin Miller, Elizabeth Martin, Benjamen O’Donnell, Nicholas Pashos, Lucio Miele, Matthew Burow, Bridgette Collins-Burow. Application of patient-derived models from understudied patient populations to discover therapeutically targetable pathways in triple-negative breast cancer systems [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A01.

The transcriptional regulator FUBP1 (Far Upstream Element Binding Protein 1) acts as an oncoprotein in hepatocellular carcinoma (HCC) and is important for hematopoietic stem cell (HSC) self-renewal and erythroid maturation in mice. In this study, we investigated the transcriptional network by which FUBP1 controls hematopoiesis and elucidated the relevance of FUBP1 for human erythropoiesis. Furthermore, we shed light on the role of FUBP1 in leukemia initiating cells. Searching for upstream-regulators of FUBP1, we identified E-boxes as potential TAL1 binding sites in the FUBP1 promoter. Indeed, we demonstrated the regulation of FUBP1 expression by TAL1 in human primary CD34+ donor cells. In chromatin immunoprecipitation (ChIP) experiments, the binding of TAL1 to the FUBP1 promoter increased during erythroid differentiation, correlating with up-regulated FUBP1 and TAL1 expression. Activation of the FUBP1 promoter by TAL1 binding was confirmed in luciferase assays. We observed a reduction in erythroid colony-forming units and glycophorin A positive cells derived from erythroid differentiated human CD34+ cells upon knockdown of FUBP1, supporting the hypothesis that FUBP1 is required for efficient erythropoiesis. In the transduction/transplantation leukemia mouse models for BCR-ABL1+ CML and MLL-AF9+ AML, we observed that Fubp1 knockdown resulted in reduced total cell and progenitor cell numbers. In CML, Fubp1 knockdown cells showed lower cell cycle activity and increased apoptosis. Consistently, CML and AML mice transplanted with Fubp1 knockdown cells survived longer than control mice that received transduced bone marrow expressing wildtype FUBP1 levels. Furthermore, pharmacological treatment of AML mice with the FUBP1 inhibitor irinotecan prolonged their survival significantly as a single drug or in combination with Ara-C. Analysis of FUBP1 expression in bone sections derived from CML and AML patients, and from healthy donors by immunohistochemistry showed no increased FUBP1 expression in leukemic samples, but we noticed a shorter overall survival in those AML patients with strong FUBP1 expression. In CML patients, FUBP1 levels correlate with the disease stage. Thus, elevated expression of FUBP1 might be an indicator for the aggressiveness of leukemia. Our data identify TAL1 as an FUBP1 upstream-regulator and confirm the importance of FUBP1 for HSC self-renewal and erythroid maturation, not only in murine but also in human cells. Furthermore, FUBP1 acts as an oncogenic factor in leukemia. Our findings might provide important evidence for the potential use of FUBP1 in clinical settings, e.g. as a molecular target for the treatment of leukemia patients and as a modulator for the production of red cells. Citation Format: Marlene Steiner, Van T. Hoang, Jasmin Yillah, Katharina Gerlach, Jörn Lausen, Hans-Michael Kvasnicka, Thomas Oellerich, Hanibal Bohnenberger, Daniela Krause, Martin Zörnig. The role of FUBP1 in the hematopoietic system and leukemia [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 1500.

Background Hand, foot and mouth disease (HFMD) has become a major public health problem across the Asia-Pacific region, and is commonly caused by enterovirus A71 (EV-A71) and coxsackievirus A6 (CV-A6), CV-A10 and CV-A16. Generating pathogen whole-genome sequences is essential for understanding their evolutionary biology. The frequent replacements among EV serotypes and a limited numbers of available whole-genome sequences hinder the development of overlapping PCRs for whole-genome sequencing. We developed and evaluated a non-ribosomal random PCR (rPCR) and next-generation sequencing based assay for sequence-independent whole-genome amplification and sequencing of HFMD pathogens. A total of 16 EV-A71/CV-A6/CV-A10/CV-A16 PCR positive rectal/throat swabs (Cp values: 20.9–33.3) were used for assay evaluation. Results Our assay evidently outperformed the conventional rPCR in terms of the total number of EV-A71 reads and the percentage of EV-A71 reads: 2.6 % (1275/50,000 reads) vs. 0.1 % (31/50,000) and 6 % (3008/50,000) vs. 0.9 % (433/50,000) for two samples with Cp values of 30 and 26, respectively. Additionally the assay could generate genome sequences with the percentages of coverage of 94–100 % of 4 different enterovirus serotypes in 73 % of the tested samples, representing the first whole-genome sequences of CV-A6/10/16 from Vietnam, and could assign correctly serotyping results in 100 % of 24 tested specimens. In all but three the obtained consensuses of two replicates from the same sample were 100 % identical, suggesting that our assay is highly reproducible. Conclusions In conclusion, we have successfully developed a non-ribosomal rPCR and next-generation sequencing based assay for sensitive detection and direct whole-genome sequencing of HFMD pathogens from clinical samples. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0580-9) contains supplementary material, which is available to authorized users.

Triple negative breast cancers (TNBCs) constitute 12% of all breast cancers, and is approximately twice more prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.5% in 2015), and thus hosts a higher population of TNBC patients. TNBCs have an aggressive phenotype that is elusive to the targeted therapeutics used to treat other breast cancer subtypes. The claudin-low molecular subtype has higher rates of metastases and recurrence. Certain kinase families have been extensively studied as regulators of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Discovery of novel kinase targets within the subset of uncharacterized kinases could provide important insight into future targeted therapies. However, current models utilized in target discovery research are limited by the inability to accurately recapitulate the complex stromal architecture and heterogenous genetic and molecular composition of breast cancer. Furthermore, immortalized cell lines are limited to a 2D environment and over time acquire mutations that may not reflect the primary tumor. Recently, our laboratory has successfully established two TNBC patient-derived xenograft (PDX) models derived from African-American patients, and generated cell lines (TU-BCx-2K1, TU-BCx-2O0) and mammospheres. One of these models, 2O0, presents tumor architecture, cellular composition, genomic (qRT-PCR) and protein (western blot) expressions that are concordant with a claudin-low subtype. Furthermore, we show that both TNBC models metastasize to the lungs, and exhibit molecular characteristics consistent with mesenchymal phenotypes. We utilized these translational PDX models to screen a library of small molecule inhibitors that represent a variety of kinase pathways to identify novel therapeutic targets and/or pathways that are specific to TNBC subtypes. We found in a preliminary cell morphology screen using three TNBC cell lines (MDA-MB-231, BT549, MDA-MB-157), two small molecule inhibitors that increased epithelial marker (CDH1) gene expression, suppressed mesenchymal (VIM, c-FOS, SNAI1, ZEB1) expression and/or suppressed cellular motility in transwell migration assays. We observed after ex vivo treatments with our PDX tumors the two compounds increase the epithelial marker CDH1 expression, and suppress mesenchymal markers (VIM, MMP2) expressions. We confirm these findings in the TU-BCx-2K1 cell line. Kinase array data revealed candidate kinases responsible for the observed EMT changes in the two compounds of interest (NEK5, NEK9, NEK1 potentially affect cell motility; SRC-family kinases, TAOK2, STK10 potentially affect EMT gene changes); we plan to utilize the PDX cell lines to characterize these kinases in EMT. We aim to ultimately discover novel therapeutic targets specific to different TNBC molecular subtypes. Citation Format: Margarite D. Matossian, Hope E. Burks, Annie C. Bowles, Rachel A. Sabol, Van T. Hoang, Bruce Bunnell, William J. Zuercher, David H. Drewry, Carrow Wells, Krzysztof Moroz, Matthew E. Burow, Bridgette Collins-Burow. Triple negative breast cancer patient-derived xenografts as a translational model for discovery of novel therapeutic targets [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 1117. doi:10.1158/1538-7445.AM2017-1117

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with higher rates of metastasis and recurrence than other classified subtypes. The decreased survival rates seen in TNBC can be attributed in part to the lack of targeted therapeutics. HDAC inhibitors (HDACi) have emerged as a potential adjuvant therapy for patients with triple negative disease. Previously, our lab has identified Panobinostat, a pan-DAC inhibitor, as a successful therapy for the reversal of the metastatic phenotype in TNBC. However, this therapy lacks specificity and may have increased side effects as a result. In this study, we aim to dissect the necessary HDACs responsible for driving and maintaining the metastatic phenotype of TNBC, with the goal of informing specific HDAC targeted therapies. Here we show that Romidepsin, an HDAC 1 and 2 specific inhibitor, is capable of recapitulating aspects of Panobinostat treatment in TNBC both in vitro and in a patient derived xenograft model in vivo. To parse the necessity and role of HDAC1 and HDAC2 individually, we created knockdown cell lines and analyzed them for changes in gene expression changes and their respective associated biological processes, including proliferation, migration, mammosphere formation, tumor growth and metastasis. We found that HDAC2 knockdown was able to significantly repress migration and proliferation in TNBC cells and their associated gene expression signatures in vitro. Furthermore, HDAC2 knockdown resulted in decreased tumor growth and metastasis an in vivo mouse model. These results illustrate the efficacy of Romidepsin in inhibition of oncogenic processes and implicate HDAC2 as a potential target for therapeutic intervention in TNBC. Citation Format: Hope E. Burks, Steven Elliott, Van T. Hoang, Margarite D. Matossian, Bridgette Collins Burow, Matthew E. Burow. Inhibition of HDAC2, but not HDAC1, abrogates triple negative breast cancer progression 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 5079. doi:10.1158/1538-7445.AM2017-5079

Mounting evidences indicate that leukemic cells in patients with acute myeloid leukemia (AML) are derived from leukemia stem cells (LSC). In analogy to normal hematopoietic stem cells (HSC), LSC remain mostly dormant and are hence resistant to conventional chemotherapy. Residual, physiological HSC exist alongside with LSC, with heterogeneous dominance of LSC over HSC in individual patients. We have devised a flow cytometric method for the identification and separation of these two stem cell populations based on surface antigen markers such as CD34, CD38, lineage aberrant markers, and aldehyde dehydrogenase (ALDH) enzyme activity.

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the MAPK/extracellular signal-regulated kinases (MEK) pathways has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). Here we proposed to investigate dual inhibition of MEK1/2 and MEK5 as a more efficacious method for intervention to target mesenchymal and highly metastatic breast cancer cells than MEK1/2 or MEK5 alone through the use of a novel pan-MEK inhibitor SC-151. Interestingly, TNBC cells demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET) and exhibited a significant decrease in migration potential following pan-MEK inhibition. Additionally, immuno-compromised mice inoculated with MDA-MB-231 cells and treated with SC-151 demonstrated decreased tumor volumes compared to vehicle-treated animals. To parse the roles of MEK1/2 and MEK5 in EMT and tumorigenesis, we used the CRISPR/Cas9 approach to knock out ERK5 expression in the TNBC cell line MDA-MB-231. Similar to biological changes induced by pan-MEK inhibition, loss of ERK5 promoted epithelial characteristics in TNBC cells at the morphological and molecular level and impaired tumor formation in vivo. Treatment of ERK5-ko cells with SC-151 further enhanced these effects in vitro, suggesting that MEK1/2 and MEK5 play distinct roles in maintaining the mesenchymal phenotype. Further analysis revealed that constitutive activation of MEK5 abrogated the effects of SC-151 on the reversal of EMT, highlighting the requirement for MEK5 inhibition in MET induction. Taken together, these findings show that while the MEK5-ERK5 pathway may be sufficient in EMT regulation, MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Thus, dual MEK inhibition exerts optimal effects in the reversal of EMT. These data present a novel compound and viable therapeutic strategy to target both MEK1/2 and MEK5 in phenotypically mesenchymal and clinically aggressive breast cancer cells, warranting further investigation into mechanisms by which MEK1/2 and MEK5 individually modulate the EMT axis. Additionally, as MEK inhibition has been shown to sensitize resistant cancer cells to targeted therapies, synergistic and sensitizing effects of SC-151 combined with inhibitors of alternative signaling pathways as well as kinases upstream of MEK will be examined. Citation Format: Van T. Hoang, Steven Elliott, Elizabeth C. Martin, Lyndsay V. Rhodes, Hope E. Burks, Margarite Matossian, Suravi Chakrabarty, Darlene Monlish, Theresa B. Phamduy, Lowry Curley, Muralidharan Anbalagan, Brian G. Rowan, Doug Chrisey, Jane E. Cavanaugh, Patrick T. Flaherty, Bridgette M. Collins-Burow, Matthew E. Burow. Induction of mesenchymal-to-epithelial transition through pan-MEK inhibition in triple-negative breast cancer. [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 1596.

Triple negative breast cancer (TNBC) is associated with poor survival, metastatic recurrence, increased mortality, and has few viable therapeutic options. Given the high mortality associated with metastasis of this disease, discovery of therapeutics targeting the metastasis axis is imperative. Kinases have been demonstrated to have an essential role in regulation of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Elucidating specific signaling pathways that promote EMT has the potential to provide novel targeting strategies in the treatment of TNBC. Here we demonstrate a partially non-selective polo-like kinase 1 (PLK1) inhibitor, GSK346294, is capable of EMT reversal in phenotypically mesenchymal TNBC cell lines, as evidenced by enhanced E-cadherin expression and loss of cellular migration potential. Structurally analogous but highly specific PLK inhibitors did not exhibit similar EMT changes, which led us to investigate off-target kinases inhibited by GSK346294. Follow-up kinase profiling studies revealed members of the NEver-in-mitosis-related Kinase family (NEK5 and NEK9) as additional kinases inhibited by GSK346294, leading us to investigate their individual roles in the progression of EMT. NEK5 overexpression in a non-invasive breast cancer cell line increased both migration and invasion in trans-well assays. Additionally, enhanced NEK5 expression altered the TNBC gene expression profile, enhancing expression of genes associated with growth factor signaling (MET, EGFR) and the mesenchymal gene signature (SLUG, VIM). To date, no studies exist which delineate the regulatory roles of NEK5 and NEK9 in EMT or cellular invasion/motility. Further investigation into the function of NEK5 and/or NEK9 in the metastatic progression may provide novel therapeutic targets for the treatment of TNBC, the implication of which could impact the treatment and management of neoplastic disease and metastasis beyond breast cancer. Citation Format: Margarite D. Matossian, Steven Elliott, Van T. Hoang, Hope E. Burks, Theresa B. Phamudy, Doug Chrisey, William J. Zuercher, David H. Drewry, Carrow Wells, Bridgette Collins-Burow, Matthew E. Burow. Targeting an unconventional kinase-invasion axis in breast cancer metastasis. [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 684.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), has been identified as a tumor suppressor in many cancers including breast. Low LKB1 expression has been associated with poor prognosis of breast cancer patients, and we report here a significant association between loss of LKB1 expression and reduced patient survival specifically in the basal subtype of breast cancer. Owing to the aggressive nature of the basal subtype as evidenced by high incidences of metastasis, the purpose of this study was to determine if LKB1 expression could regulate the invasive and metastatic properties of this specific breast cancer subtype. Induction of LKB1 expression in basal-like breast cancer (BLBC)/triple-negative breast cancer cell lines, MDA-MB-231 and BT-549, inhibited invasiveness in vitro and lung metastatic burden in an orthotopic xenograft model. Further analysis of BLBC cells overexpressing LKB1 by unbiased whole transcriptomics (RNA-sequencing) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). In addition, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating reversal of the EMT phenotype in the MDA-MB-231 cells. We further demonstrated marked inhibition of matrix metalloproteinase 1 expression and activity via regulation of c-Jun through inhibition of p38 signaling in LKB1-expressing cells. Taken together, these data support future development of LKB1 inducing therapeutics for the suppression of invasion and metastasis of BLBC.

The mitogen-activated protein kinase (MAPK) pathway has well-established roles in cellular processes including proliferation, differentiation, and regulation of cell fate, namely survival and apoptosis. In breast cancer, constitutive activation of the MAPK/extracellular signal-regulated kinases (ERK) pathways have been linked to chemoresistance and metastatic progression through distinct mechanisms including the activation of epithelial-to-mesenchymal transition (EMT). Our previous studies have shown that overexpression of MEK5 promotes EMT markers and induces the progression to a mesenchymal phenotype. Here, we tested the effects of a novel MEK1/2 and MEK5 inhibitor, SC-1-151, and other known MAPK signaling inhibitors (PD184,352 (MEK1/2), AZD6244 (MEK1/2), BIRB796 (p38)) on a panel of mesenchymal and highly metastatic breast cancer cell lines. While the MEK1/2 and p38 inhibitors decreased cell viability across cell lines, only the dual inhibition of MEK1/2 and MEK5 though the use of SC-1-151 demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET). Furthermore, the cells exhibited a significant decrease in migration potential following SC-1-151 treatment. Further analysis of the effects of SC-1-151 in the triple-negative breast cancer cell lines revealed an alteration of the genes associated with EMT, notably a decrease in expression of Fra-1, a transcription factor downstream of MAPK. Immuno-compromised mice inoculated with the MDA-MD-231 cell line and treated with SC-1-151 demonstrated decreased tumor volumes compared to vehicle-treated animals at day 30 post cell injection, implicating the role of MEK inhibition on tumorigenesis. These data demonstrate the need for a better understanding of the dual role of MEK1/2 and MEK5 signaling in breast cancer, and suggest that inhibition of the MEK1/2 and MEK5 signaling pathways leads to a decrease in EMT and cell migration. Citation Format: Van T. Hoang, Steven Elliott, Elizabeth C. Martin, Lyndsay V. Rhodes, Henry C. Segar, Hope Burks, Suravi Chakrabarty, Darlene Monlish, Theresa B. Phamduy, Doug Chrisey, Jane E. Cavanaugh, Patrick Flaherty, Bridgette M. Collins-Burow, Matthew E. Burow. Dual role of MEK1/2 and MEK5 in the reversal of epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1052. doi:10.1158/1538-7445.AM2014-1052

The basal sub-type, which shares features with triple-negative breast cancer (TNBC), is among the most lethal breast cancer subtype, characterized by a highly aggressive and metastatic phenotype. Although pathways that may represent targets for novel therapeutic intervention for basal like breast cancer (BLBC) have begun to be elucidated, the ability to define and selectively target the invasive and metastatic phenotype of basal-type/TNBC remains a major challenge facing the breast cancer field. Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is a known tumor suppressor in many cancers including breast. Low LKB1expression has been observed in breast cancer patients and we report a significant association between loss of LKB1 expression and poor prognosis specifically in the basal sub-type of breast cancer. Induction of LKB1 expression in BLBC cell lines inhibited invasiveness in vitro as well as lung and brain metastatic burden in an orthotopic xenograft tumor model. Further analysis of BLBC cell lines overexpressing LKB1 by next generation sequencing (RNA-seq) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extra cellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). We further demonstrated marked inhibition of matrix metalloproteinase 1 (MMP-1) expression and activity via regulation AP-1 family member cJun. Additionally, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating a reversal of the EMT phenotype in a triple-negative breast cancer cell line MDA-MB-231. We have demonstrated a role for LKB1 expression in the regulation of cell invasion and metastasis in addition to tumorigenesis. Taken together these data support future development of therapeutic agents to induce the LKB1 signaling pathway in BLCB/triple-negative breast cancer. Citation Format: Lyndsay V. Rhodes, Chandra R. Tate, Hope E. Burks, Van T. Hoang, Diari Gilliam, Elizabeth C. Martin, Steven Elliott, David FB Miller, Aaron Buechlein, Douglas Rusch, Haixu Tang, Kenneth P. Nephew, Matthew E. Burow, Bridgette M. Collins-Burow. The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1571. doi:10.1158/1538-7445.AM2014-1571

While the precise molecular mechanisms underlying metastasis remain unclear, epithelial-to-mesenchymal transition (EMT), the loss of an epithelial cell phenotype and acquisition of a mesenchymal cell phenotype, has been implicated in cancer cell invasion and dissemination. The ZEB family of transcription factors, which includes ZEB1 and ZEB2, has been demonstrated to mediate this transition by downregulating the expression of genes associated with an epithelial phenotype. We sought to investigate the effects of direct ZEB family overexpression on EMT in estrogen receptor-positive (ER+) breast cancer cell systems. We overexpressed ZEB1 or ZEB2 in the epithelial, ER+, luminal A breast cancer cell lines MCF-7 and ZR75. Overexpression of individual ZEB1 and ZEB2 levels were confirmed and localization of the ZEB factors to the nucleus was confirmed by confocal microscopy in both cell lines. ZEB2 overexpressing cells, but not ZEB1 overexpressing cells, showed increased migration and invasion in vitro compared to the vector control in both MCF-7 and ZR75 cell lines, suggesting differential function of the two ZEB family members. Additionally, MCF-7-ZEB2 xenografts exhibited increased lung metastasis compared to MCF-7-vector cells. To elucidate the effects of ZEB on our ER+ cell line we performed next generation deep sequencing on MCF-7 -vector, ZEB1 and ZEB2 overexpressing cells. Analysis of total gene regulation using the NCI Pathway Interaction Database demonstrated an increase in genes associated with RhoA activity, an important mediator of cell motility, in ZEB2 overexpressing cells. However, the ZEB overexpressing cells show no change in morphology and canonical EMT markers remained unchanged between cell lines, suggesting a potential post-translational modification affecting ZEB1 and ZEB2 function. Together these results indicate that ZEB factors drive motility in breast cancer cells but are incapable of promoting a complete EMT in ER+ cells, warranting further investigation into the mechanisms involved in ZEB action. Elucidating the pathways involved in ZEB family function is an important step in understanding the mechanisms underlying metastasis and has the potential to yield new therapeutic targets. Citation Format: Hope E. Burks, Lyndsay Rhodes, Elizabeth Martin, Theresa Phamduy, Steven Elliot, Van Hoang, Henry Segar, Aaron Buechlein, Douglas Rusch, Dave Miller, Melody Baddoo, Erik Flemington, Kenneth Nephew, Douglas Chrisey, Bridgette Collins-Burow, Matthew Burow. ZEB2 promotes cell motility and metastasis in ER+ breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1034. doi:10.1158/1538-7445.AM2014-1034

Separation of leukemic stem cells (LSC) and residual hematopoietic stem cells (HSC) from the same individual patient with acute myeloid leukemia (AML) is essential for a proper understanding of the leukemic driving mechanisms. We have studied the role of aldehyde dehydrogenase (ALDH) for this purpose and have defined the functional properties of ALDHbright cells in specific subgroups of AML. We have examined the ALDH activity by flow cytometry in bone marrow samples (BM) from 14 healthy donors and 73 patients with de novo AML. The median frequency of cells with high ALDH activity (ALDHbright cells) in the healthy subjects was 1.92% with a range from 0.58 to 3.16%. For patients with AML, the median number of ALDHbright cells was 0.25% with a broad range from 0.004 to 33.57%. Whereas the majority of patients with AML (n = 56) had low frequencies of ALDHbright cells (median 0.11%; range 0.004 – 1.77%; defined as ALDH-low AML), 17 patients had relatively numerous ALDHbright cells (median 9.01; range 3.54 – 33.57%; defined as ALDH-numerous AML). In both groups, ALDHbright cell populations were highly enriched for CD34+CD38- cells. The ALDHbright cells derived from ALDH-low AML did not contain chromosomal and molecular aberrations characteristic of the original leukemia, and were able to induce multi-lineage hematopoiesis in NSG mouse models. Thus, genetically and functionally normal HSC could be successfully isolated in the ALDHbright subset, whereas LSC were enriched in ALDHdimCD34+CD38- subset for patients with ALDH-low AML. For 17 patients with ALDH-numerous AML, the ALDHbright subset was consistently contaminated with LSC. In clinical follow-ups, patients with ALDH-numerous AML showed resistance to induction chemotherapy and were characterized by a very poor long-term outcome that was comparable to patients with high-risk cytogenetic or molecular genetic markers. In four patients with ALDH-numerous AML we demonstrated that the ALDHbrightCD34+CD38- subset contained chemotherapy-resistant clones with repopulating ability. Furthermore, such ALDHbright cells were characterized by a lower cell-cycle activity and an increased resistance to cytarabine in comparison with ALDHdim blasts in in vitro assays. Our data have provided evidence that LSC and residual HSC can be separated using ALDH in patients with low frequencies of ALDHbright cells. In patients with ALDH-numerous AML, the ALDHbright subset is associated with leukemic features both in vitro and in animal models. Thus our data demonstrated the feasibility of appropriate comparisons of LSC versus HSC from the same patient with specific subtypes of AML and the impact of LSC properties on clinical outcome. Disclosures: Buss: Novartis: Travel support Other; Micromet/Amgen: Reimbursements for participation in a clinical study , Reimbursements for participation in a clinical study Other. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genzyme: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.