Lipid nanoparticles (LNPs) largely rely on ionizable lipids to yield successful nucleic acid delivery via electrostatic disruption of the endosomal membrane. Here, we report the identification and evaluation of ionizable lipids containing a thiophene moiety (Thio-lipids). The Thio-lipids can be readily synthesized via the Gewald reaction, allowing for modular lipid design with functional constituents at various positions of the thiophene ring. Through the rational design of ionizable lipid structure, we prepared 47 Thio-lipids and identified some structural criteria required in Thio-lipids for efficient mRNA (messenger RNA) encapsulation and delivery in vitro and in vivo. Notably, none of the tested lipids have a pH-response profile like traditional ionizable lipids, potentially due to the electron delocalization in the thiophene core. Placement of the tails and localization of the ionizable headgroup in the thiophene core can endow the nanoparticles with the capability to reach various tissues. Using high-throughput formulation and barcoding techniques, we optimized the formulations to select two top lipids— 20b and 29d —and investigated their biodistribution in mice. Lipid 20b enabled LNPs to transfect the liver and spleen, and 29d LNP transfected the lung and spleen. Unexpectedly, LNP with lipid 20b was especially potent in mRNA delivery to the retina with no acute toxicity, leading to the successful delivery to the photoreceptors and retinal pigment epithelium in non-human primates.

Genomic medicines have significant therapeutic potential to cure rare genetic diseases and cancer, but currently, most treatments rely on ex vivo cell editing, which is time consuming, costly, and suffers from logistic and manufacturing challenges. In contrast, in vivo delivery of genomic medicines would unlock new therapeutic areas and provide broader patient access. However, the success of in vivo delivery is dependent on the development of safe delivery vehicles capable of transporting the genomic medicines to their intended target tissues and cells. In recent years, lipid nanoparticles (LNPs) have emerged as versatile delivery solutions for RNA therapeutics but have seldom been used fo r in vivo delivery beyond liver and muscle tissue. Challenges in extrahepatic delivery of LNPs include adequate tissue-specific targeting to achieve therapeutic levels of genome editing while avoiding anti-drug immune responses. We have developed novel LNPs designed to deliver RNA payloads to either T cells or hematopoietic stem cells (HSCs) in vivo. Using our novel T-cell LNPs, we have demonstrated single-dose in vivo delivery of GFP mRNA in two humanized mouse models. In the spleen of humanized NSG mice (pre-engrafted with human HSCs 12-weeks prior to study), we achieved GFP expression in 24% of human CD3 + T cells. In another model, NSG mice infused with human T cells 14-days prior to study, GFP expression was observed in 76% of T cells in the spleen. Single-dose administration of this T-cell LNP in rhesus macaque resulted in 40% GFP expression in peripheral blood T cells. Furthermore, we did not observe any detectable GFP expression in human or rhesus B cells, suggesting this LNP system may be suitable for the development of in vivo CAR-T therapies for B cell malignancies. Separately, we treated humanized NBSGW mice (pre-engrafted with human HSCs for 12 weeks) with a single dose of our HSC LNPs delivering GFP mRNA. Expression of GFP was observed in approximately 90% of bone marrow hematopoietic stem and progenitor cells (HSPCs defined as Lin -CD34 +CD38 - cells) and approximately 95% of long-term bone marrow HSC-enriched population (HSCs defined as Lin -CD34 +CD38 -CD90 +CD45RA - cells). Single-dose intravenous administration of the same LNP in cynomolgus macaques led to GFP expression in 61% of HSPCs and 62% of HSCs in the bone marrow. Gene editing presents a greater challenge than reporter mRNA delivery due to the increased size and complexity of the RNA cargos. Toward this goal, we went on to demonstrate that our HSC LNP platform can deliver Cas9 mRNA, with a beta-2 microglobulin (B2M) sgRNA, to human HSCs in humanized NBSGW mice resulting in the knockout (KO) of the B2M gene through the introduction of insertion or deletion mutations. Likewise, following administration of a B2M editing T-cell LNP in humanized NSG mice, we demonstrated B2M KO in splenic human CD3 + T cells. Together, these in vivo delivery results indicate our LNP delivery platform is capable of efficiently delivering RNA payloads to different hematopoietic cell types in blood and tissues, providing a potential delivery platform for in vivo genome editing.

The FDA approval of autologous Chimeric Antigen Receptor (CAR) T-cell therapies have added a powerful tool in the armamentarium to treat relapsed and refractory leukemias and lymphomas. However, significant challenges limit these therapies from accommodating current patient demand including supply chain limitations and wait times associated with availability of GMP quality viral vectors and long needle-to-needle time from initiation of treatment to receiving the final drug product. As of yet other competitive therapies including bi-specific antibodies and allogeneic CAR-T have not achieved the same level of therapeutic efficacy as conventional autologous T cell therapies highlighting the critical unmet need for same day treatment options for autologous CAR-T products. RNA Gene Writers leverage target-primed reverse transcription (TPRT) biochemistry evolved from non-LTR retrotransposon mobile genetic elements to modify the genetic information in cells using RNA templates and without the need to introduce DNA breaks. Moreover, RNA Gene Writers can be engineered to catalyze a variety of editing reactions, such as the introduction of gene-length DNA sequences, substitutions, insertions, and deletions. These edits can be achieved with all-RNA delivery in primary cells and in vivo, eliminating the necessity for viral vectors and DNA template-based gene editing. LNP-RNA delivery of RNA Gene Writers resulted in the integration and expression of transgenes in >60% of primary human T cells in vitro. Furthermore, our LNPs deliver RNA to T cells in vivo, with 80% reporter expression in a humanized mouse model and 45% in non-human primates (NHP). We have demonstrated LNP delivery of RNA Writers into primary human T cells with an RNA template encoding a CAR cassette can achieve >20% CAR+ T cells without detriments to cell viability or proliferative capacity and have demonstrated ability to mediate tumor cell killing in vitro. Moreover, RNA Gene Writer derived CAR-T cells can be introduced into mouse xenograft models to clear antigen specific tumors in vivo. CAR mRNA can also be packaged into proprietary LNP formulations and delivered to primary human T cells in a mixture of lymphocytes commonly found in patient leukapheresis to generate CAR-T cells in vitro. This opens the possibility of using RNA Gene Writers to develop a same-day CAR-T treatment. In addition, the modularity of our RNA Gene Writing technology allows multiplex editing to co-introduce multiple genetic changes including generation of universal and more potent CAR-T cells through the knock-out of B2M and TRAC at comparable levels (>80% double knockout) to Cas9 nucleases. Further, we show that we can achieve both edits simultaneously in 80% of T cells that have achieved CAR transgene insertion via another RNA Gene Writer enzyme. Importantly, we have demonstrated successful B2M and TRAC double knock-out without inducing translocations (undetectable vs 8% translocations with Cas9). RNA Gene Writing technology is uniquely positioned for same-day delivery of all-RNA components toenable a wide range of editing applications, from gene knock-out to gene integration. The ability to package the RNA Gene Writer system into LNPs unlocks the potential for the genetic engineering of autologous T cell therapies to enable same-day delivery a current limitation of conventional lentiviral-based CAR-T therapies.

Lipid nanoparticle (LNP)–based mRNA delivery holds promise for the treatment of inherited retinal degenerations. Currently, LNP-mediated mRNA delivery is restricted to the retinal pigment epithelium (RPE) and Müller glia. LNPs must overcome ocular barriers to transfect neuronal cells critical for visual phototransduction, the photoreceptors (PRs). We used a combinatorial M13 bacteriophage–based heptameric peptide phage display library for the mining of peptide ligands that target PRs. We identified the most promising peptide candidates resulting from in vivo biopanning. Dye-conjugated peptides showed rapid localization to the PRs. LNPs decorated with the top-performing peptide ligands delivered mRNA to the PRs, RPE, and Müller glia in mice. This distribution translated to the nonhuman primate eye, wherein robust protein expression was observed in the PRs, Müller glia, and RPE. Overall, we have developed peptide-conjugated LNPs that can enable mRNA delivery to the neural retina, expanding the utility of LNP-mRNA therapies for inherited blindness.

Protein kinases are key regulators of cellular signaling and play a critical role in oncogenesis. Inhibitors of protein kinases are pursued by both industry and academia as a promising target for cancer therapy. Within the protein kinases, the ATP site has produced more than 40 FDA‐approved drugs. The ATP site is broadly composed of a hinge region, gatekeeper residues, DFG‐loop, ribose pocket, and other hydrophobic regions. The hinge region in the ATP site can be used for designing potent inhibitors. In this review, we discuss some representative studies that will highlight the interactions of heterocyclic compounds with hinge regions of different kinases like BRAF kinase, EGRF kinase, MAP kinase, and Mps1 kinase.

Since the discovery of DNA intercalating agents (by Lerman, 1961), a growing number of organic, inorganic, and metallic compounds have been developed to treat life-threatening microbial infections and cancers. Fused-heterocycles are amongst the most important group of compounds that have the ability to interact with DNA. DNA intercalators possess a planar aromatic ring structure that inserts itself between the base pairs of nucleic acids. Once inserted, the aromatic structure makes van der Waals interactions and hydrogen-bonding interactions with the base pairs. The DNA intercalator may also contain an ionizable group that can form ionic interactions with the negatively charged phosphate backbone. After the intercalation, other cellular processes could take place, leading ultimately to cell death. The heterocyclic nucleus present in the DNA intercalators can be considered as a pharmacophore that plays an instrumental role in dictating the affinity and selectivity exhibited by these compounds. In this work, we have carried out a revision of small organic molecules that bind to the DNA molecule via intercalation and cleaving and exert their antitumor activity. A general overview of the most recent results in this area, paying particular attention to compounds that are currently under clinical trials, is provided. Advancement in spectroscopic techniques studying DNA interaction can be examined in-depth, yielding important information on structure-activity relationships. In this comprehensive review, we have focused on the introduction to fused heterocyclic agents with DNA interacting features, from medicinal point of view. The structure-activity relationships points, cytotoxicity data, and binding data and future perspectives of medicinal compounds have been discussed in detail.

Lipid nanoparticles (LNPs) are a type of lipid vesicles that possess a homogeneous lipid core. These vesicles are widely used in small-molecule drug and nucleic acid delivery and recently gained much attention because of their remarkable success as a delivery platform for COVID-19 mRNA vaccines. Nonetheless, the utility of transient protein expression induced by mRNA extends far beyond vaccines against infectious diseases─they also hold promise as cancer vaccines, protein replacement therapies, and gene editing components for rare genetic diseases. However, naked mRNA is inherently unstable and prone to rapid degradation by nucleases and self-hydrolysis. Encapsulation of mRNA within LNPs protects mRNA from extracellular ribonucleases and assists with intracellular mRNA delivery.In this Account, we discuss the core features of LNPs for RNA delivery. We focus our attention on LNPs designed to deliver mRNA; however, we also include examples of siRNA-LNP delivery where appropriate to highlight the commonalities and the dissimilarities due to the nucleic acid structure. First, we introduce the concept of LNPs, the advantages and disadvantages of utilizing nucleic acids as therapeutic agents, and the general reasoning behind the molecular makeup of LNPs. We also briefly highlight the most recent clinical successes of LNP-based nucleic acid therapies. Second, we describe the theory and methods of LNP self-assembly. The common idea behind all of the preparation methods is inducing electrostatic interactions between the nucleic acid and charged lipids and promoting nanoparticle growth via hydrophobic interactions. Third, we break down the LNP composition with special attention to the fundamental properties and purposes of each component. This includes the identified molecular design criteria, commercial sourcing, impact on intracellular trafficking, and contribution to the properties of LNPs. One of the key components of LNPs is ionizable lipids, which initiate electrostatic binding with endosomal membranes and facilitate cytosolic release; however, the roles of other lipid components should not be disregarded, as they are associated with stability, clearance, and distribution of LNPs. Fourth, we review the attributes of LNP constructs as a whole that can heavily influence RNA delivery. These attributes are LNP size, charge, internal structure, lipid packing, lipid membrane hydration, stability, and affinity toward biomacromolecules. We also discuss the specific techniques used to examine these attributes and how they can be adjusted. Finally, we offer our perspective on the future of RNA therapies and some questions that remain in the realm of LNP formulation and optimization.

The title sterically congested piperazine derivative, C 20 H 27 FN 2 O 2 , was prepared using a modified Bruylants approach. A search of the Cambridge Structural Database identified 51 compounds possessing an N-tert- butyl piperazine substructure. Of these only 14 were asymmetrically substituted on the piperazine ring and none with a synthetically useful second nitrogen. Given the novel chemistry generating a pharmacologically useful core, determination of the crystal structure for this compound was necessary. The piperazine ring is present in a chair conformation with di-equatorial substitution. Of the two N atoms, one is sp 3 hybridized while the other is sp 2 hybridized. Intermolecular interactions resulting from the crystal packing patterns were investigated using Hirshfeld surface analysis and fingerprint analysis. Directional weak hydrogen-bond-like interactions (C—H...O) and C—H...π interactions with the dispersion interactions as the major source of attraction are present in the crystal packing.

mRNA vaccines have evolved from being a mere curiosity to emerging as COVID-19 vaccine front-runners. Recent advancements in the field of RNA technology, vaccinology, and nanotechnology have generated interest in delivering safe and effective mRNA therapeutics. In this review, we discuss design and self-assembly of mRNA vaccines. Self-assembly, a spontaneous organization of individual molecules, allows for design of nanoparticles with customizable properties. We highlight the materials commonly utilized to deliver mRNA, their physicochemical characteristics, and other relevant considerations, such as mRNA optimization, routes of administration, cellular fate, and immune activation, that are important for successful mRNA vaccination. We also examine the COVID-19 mRNA vaccines currently in clinical trials. mRNA vaccines are ready for the clinic, showing tremendous promise in the COVID-19 vaccine race, and have pushed the boundaries of gene therapy.

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.

Abstract 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.

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.

Epithelial to mesenchymal transition is an important cellular adaptation that helps cancer cells acquire a spindle-like phenotype from a cuboidal phenotype, degrade the extracellular matrix, invade the neighboring tissues, and metastasize to other organs and form secondary tumor. Cellular plasticity is governed by growth factors that act in a paracrine manner to activate downstream oncogenes and regulate the activity of epigenetic factors, which facilitate phenotypic switch from epithelial to mesenchymal, ultimately leading to increased cell migration and invasion. The intracellular phosphorylation cascade that is downstream of growth factor receptors plays an important role in transmitting the signal from the extracellular environment into the nucleus, thereby completing the loop required to elicit a cellular response. Triple negative breast cancer (TNBC), characterized by loss of hormone receptors is a highly aggressive form of cancer and patients show poor prognosis and disease-free survival due to lack of targeted therapy. In contrast, estrogen positive breast cancer can be targeted by estrogen receptor antagonists or CDK4/6 inhibitors, but drug resistance and relapse is often associated with epithelial to mesenchymal transition and poor patient outcome. Hence, targeting the mesenchymal phenotype with small molecule inhibitors is an emerging strategy to attenuate the invasive and aggressive nature of cancer cells. The MEK5-ERK5 pathway is understudied in triple negative breast cancer and there are few research tools available to selectively inhibit this pathway. Diphenylamine derivatives, synthesized as putative MEK5 inhibitors from parent MEK1/2 inhibitor trametinib, are effective in inducing mesenchymal to epithelial transition in MDA-MB-231 triple negative breast cancer cells, as indicated by an increase in E-cadherin expression, which is a marker of epithelial phenotype, and a decrease in spindle index, an important cell shape determinant. Compound 1, a dual MEK1/2 (98%) and MEK5 (59%) inhibitor was further characterized in functional assays; Compound 1 was found to significantly inhibit cell viability, proliferation, migration, spheroid viability, and colony formation in MDA-MB-231 cells. Compound 1 is effective in reversing the mesenchymal phenotype of MDA-MB-231, BT-549, and tamoxifen resistant-MCF-7 breast cancer cells. Signaling crosstalk and drug resistance in cancer limits the applicability of monotherapy. Our current work is focused on treating diverse breast cancer cells with combination of novel compound 1 and paclitaxel, ipatasertib, JQ-1, and LBH589 to induce synthetic lethality at lower concentration, restore drug sensitivity, and enhance selectivity. Citation Format: Akshita B. Bhatt, Thomas D. Wright, Katie Anna, Mohit Gupta, Suravi Chakrabarty, Van Hoang, Matthew Burow, Patrick T. Flaherty, Jane E. Cavanaugh. Novel diphenylamine analogs induce mesenchymal to epithelial transition and enhance the sensitivity of breast cancer cells to conventional chemotherapeutic agents [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 1879.

Quinoline and isoquinoline motifs are commonly encountered in natural products of diverse origins. These moderately basic fused-heterocyclic rings containing natural products are adorned with remarkable biological activities with clinical use in various diseases demonstrating nature elegance and creativity. Therefore, these privileged rings have attracted profound interest from the scientific community. In this perspective, we have discussed medicinal chemistry perspective of the natural products containing quinoline and isoquinoline scaffolds.

Abstract Triple negative breast cancer (TNBC) is a molecularly heterogeneous, clinically aggressive disease group that is highly prevalent among African-Americans and younger patients. Standard chemo/radio therapy often produces clinical responses, but recurrence and metastasis are unfortunately common. Metastatic disease is generally incurable. Chemo/radiotherapy has been shown to induce EMT and enrich a chemo-resistant cancer stem-like cell (CSC) population in TNBC. CSCs are thought to drive disease recurrence. Notch signaling is critical for maintenance of TNBC CSC. Expression of Notch1 and its ligand Jagged1 are correlated with poor prognosis. Efforts to pharmacologically target Notch with Gamma Secretase Inhibitors (GSIs) have been impaired by the systemic toxicity of the GSIs, and by the fact that Notch1 also plays a key role in anti-tumor adaptive immunity. Therapeutic agents that indirectly and selectively target Notch signaling in breast cancer cells would be a potentially attractive strategy. However, no such agents have been identified to date. We have found that the MAPK5-ERK5 kinase pathway, which contains at least two druggable targets, functions as a master regulator of Notch signaling in TNBC cells. ERK5 knockout TNBC cells have dramatically decreased expression of Notch receptors, ligands and transcriptional targets. In vivo, these cells form barely detectable tumors that do not metastasize and express lower levels of Notch1 and its ligand Jagged1. Using in silico screening, we identified a class of compounds that selectively target MAP2K5 (MEK5) and decrease the phosphorylation of MAPK7 (ERK5). We selected compound SC-181 for further study. Consistent with ERK5KO cells, pharmacological suppression of ERK5 phosphorylation with SC-181 decreased Notch1 and Jagged1 mRNAs and proteins. SC-181 reversed EMT and reduced the CD44hi/CD24lo CSC population in TNBC cells, but had no effect on T-cell proliferation. SC-181 decreased the number and size of mammospheres in a concentration-dependent manner. Overexpression of the Notch1 intracellular domain (N1IC) in ERK5KO cells rescues their phenotype, dramatically increasing the CSC fraction and promoting EMT. Our results suggest that targeting the MEK5-ERK5 pathway is a promising new strategy to selectively modulate Notch signaling in TNBC CSC without compromising tumor immunity. Citation Format: Ucar DA, Matossian MD, Hoang-Barnes VT, Hossain FM, Gupta M, Burks HE, Wright TD, Cavanaugh J, Flaherty P, Burow ME, Miele L. A novel druggable target upstream of Notch: MEK5/ERK5 signaling regulates Jagged-1 and Notch1 expression in triple negative breast cancer stem cells [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 P2-03-04.

Abstract Triple negative breast cancer (TNBC) is a molecularly heterogeneous, clinically aggressive disease group that is highly prevalent among African-Americans and younger patients. Standard chemo/radio therapy often produces clinical responses, but recurrence and metastasis are unfortunately common. Metastatic disease is generally incurable. Chemo/radiotherapy has been shown to induce EMT and enrich a chemo-resistant cancer stem cell-like (CSC) population in TNBC. CSCs are thought to drive disease recurrence. Notch signaling, particularly Notch1, is critical for maintenance of TNBC CSC. Expression of Notch1 and its ligand Jagged1 are correlated with poor prognosis. Efforts to pharmacologically target Notch directly have been impaired by the systemic toxicity of the Gamma Secretase Inhibitors (GSI) used, and by the fact that Notch1 also plays a key role in anti-tumor adaptive immunity. Therapeutic agents that target Notch signaling in breast cancer cells indirectly and selectively are a potentially attractive strategy. However, no such target has been identified to date. We have found that the MAPK5-ERK5 kinase pathway, which contains at least two druggable targets, functions as a master regulator of Notch signaling in TNBC cells. ERK5 knockout TNBC cells have dramatically decreased expression of Notch receptors, ligands and targets. In vivo, these cells form barely detectable tumors that do not metastasize and express lower levels of Notch1 and its ligand Jagged1. Using in silico screening method, we have identified a small molecule compound that targets MAP2K5 (MEK5) and decreases phosphorylation of MAPK7 (ERK5). Expression of ERK5 is associated with poor prognosis in TNBC. Consistent with ERK5KO cells, suppression of ERK5 phosphorylation decreased the amount of Notch1 and Jagged1 protein and mRNAs. More importantly, a selective MEK5 inhibitor, SC-181, reversed EMT and reduced the CD44hi/CD24lo CSC population in TNBC cells without suppressing T-cell proliferation. Treatment with nanomolar concentration of this compound decreased the number and size of mammospheres in a dose- dependent manner. Our preliminary results suggest that targeting the MEK5-ERK5 pathway is a promising strategy to selectively target Notch signaling in TNBC CSC without systemic Notch inhibition. Citation Format: Deniz A. Ucar-Bilyeu, Margarite D. MATOSSIAN, VAN Hoang Barnes, Fokhrul M. Hossain, Mohit Gupta, HOPE E. BURKS, THOMAS D. WRIGHT, Jane Cavanaugh, Patrick Flaherty, Matthew E. Burow, Lucio Miele. Targeting notch one notch above [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 967.

Abstract Triple negative breast cancer (TNBC) is characterized by the loss of hormone receptors and high invasive potential. TNBC cells express high levels of the mesenchymal markers vimentin, fibronectin, N-cadherin, and cadherin11, which are involved in cell invasion and metastasis, and low levels of the epithelial markers E-cadherin, occludins and cytokeratins, which are involved in cellular contact and stability of the cell membrane. Mitogen activated protein kinases, including ERK5 and ERK1/2 are important pro-survival proteins and are known to be upregulated in most cancers. Of these pathways, the MEK5-ERK5 pathway is understudied in triple negative breast cancer, and there are few research tools available to selectively inhibit this pathway. The diphenylamine moiety was derived from the parent molecule Trametinib, a FDA approved MEK1/2 inhibitor for melanoma. Structural modifications were made on the lead molecule to gain selectivity towards MEK. SC-1-151, one of the compounds from this diphenylamine series was identified as a dual MEK1/2 (98.6% inhibition) and MEK5 (59% inhibition) inhibitor. Additionally, we have shown that SC-1-151 attenuates tumor growth in severe combined immunodeficient (SCID) xenograft mice and causes a mesenchymal to epithelial transition (MET) in MDA-MB-231 TNBC cells. The goal of this research is to determine the structural features of diphenylamines that are responsible for MET. E-cadherin and cadherin-11 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 for 5 days. 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. Therefore, our data suggest the necessary functional groups for inducing MET include the hydrogen atom on the amine linkage, and the iodine atom on the arene ring 2. Future studies will be performed to determine the specific protein interactions of the promising compounds. Citation Format: Akshita B. Bhatt, Thomas D. Wright, Katie Anna, Mohit Gupta, Patrick Flaherty, Van Hoang, Matthew Burow, Jane E. Cavanaugh. Structure-activity relationship of diphenylamine derivatives to target epithelial to mesenchymal transition in triple negative breast cancer [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 4204.