FDA-approved Small-molecule Inhibitors Research Articles

Inhibition of DUSP18 impairs cholesterol biosynthesis and promotes anti-tumor immunity in colorectal cancer

Tumor cells reprogram their metabolism to produce specialized metabolites that both fuel their own growth and license tumor immune evasion. However, the relationships between these functions remain poorly understood. Here, we report CRISPR screens in a mouse model of colo-rectal cancer (CRC) that implicates the dual specificity phosphatase 18 (DUSP18) in the establishment of tumor-directed immune evasion. Dusp18 inhibition reduces CRC growth rates, which correlate with high levels of CD8+ T cell activation. Mechanistically, DUSP18 dephosphorylates and stabilizes the USF1 bHLH-ZIP transcription factor. In turn, USF1 induces the SREBF2 gene, which allows cells to accumulate the cholesterol biosynthesis intermediate lanosterol and release it into the tumor microenvironment (TME). There, lanosterol uptake by CD8+ T cells suppresses the mevalonate pathway and reduces KRAS protein prenylation and function, which in turn inhibits their activation and establishes a molecular basis for tumor cell immune escape. Finally, the combination of an anti-PD-1 antibody and Lumacaftor, an FDA-approved small molecule inhibitor of DUSP18, inhibits CRC growth in mice and synergistically enhances anti-tumor immunity. Collectively, our findings support the idea that a combination of immune checkpoint and metabolic blockade represents a rationally-designed, mechanistically-based and potential therapy for CRC.

Abstract GS03-09: Characterization and proposed therapeutic exploitation of fusion RNAs in metastatic breast cancers

Abstract BACKGROUND: Large-scale genomic studies such as The Cancer Genome Atlas (TCGA) and Pan-Cancer Analysis of Whole Genomes (PCAWG) show that the breast cancer (BrCa) genome is dominated by structural variation (SV) rather than single base pair mutations, producing a fertile environment for gene fusions. In this study, we implement a rigorous, expression-based approach to create a comprehensive landscape of fusion RNAs in metastatic breast cancer (MBC). We find fusion RNAs—many of which are novel involving known oncogenes—are surprisingly common in the advanced setting and credential their use as base-editing therapeutic targets. METHODS: Two retrospective cohorts of MBC RNA-sequencing data were analyzed— Dana-Farber Cancer Institute CCPM (n = 252 cases, 276 specimens), MichiganCSER (n = 171 cases, 190 specimens)—with a Fusion MetaCaller that integrates 5 unsupervised fusion-finding algorithms. Fusion RNAs identified in at least 2 callers (High-Confidence) and absent in RNA-seq from normal tissue (Cancer-Specific) were classified as HCCS-Fusions. Further removal of common artifactual fusions was performed using public databases. Expression of each HCCS-Fusion was quantified using a supervised method (FusionInspector)—expressed HCCS-Fusions were defined as having a Fusion Fragment Per Million (FFPM) value > 0.1 and at least 10% of read counts mapping to the fusion breakpoint versus the flanking 5’ or 3’ partners’ exons. Normalized gene-level expression abundances were calculated to correlate transcriptomic features (gene expression, PAM50) with fusion RNAs. Recurrent, potentially pathogenic fusion RNAs were annotated using OncoKB and outlier expressed fusions (Q3 FFPM + [1.5 X IQR]) were interrogated. RESULTS: The frequency of HCCS-Fusions differed between subtypes with basal BrCa harboring the most per tumor followed by Her2, LumB and LumA—with a median HCCS-Fusion count of 13, 12, 7, 4 respectively. 64.5% of cases harbored a HCCS-Fusion in an OncoKB cancer-related gene. The most recurrent fusions involving a cancer-related gene were 5’ ESR1 fusions (14 cases)—all with in-frame breakpoints near exon6/7, disrupting ESR1’s ligand binding domain. 13 of 14 ESR1 fusions were called in Luminal B (LumB) metastases (Fisher’s exact enrichment p < 0.005 vs other subtypes)—defining an ESR1 fusion frequency of 6.5% in LumB disease. Beyond ESR1, we identify recurrent, low-frequency (2-4 cases) in-frame kinase fusions involving FGFR2, ADK, TLK2, PRKCA, BRAF, CHKA, CSNK1D, NEK11, TNIK—some potentially targetable with FDA-approved small molecule inhibitors—as well as recurrent, predicted loss-of-function fusion RNAs in NF1, MSI2, USP32, PTEN, and CDH1. Lastly, 33.6% of cases harbored at least one outlier expressed fusion RNA; including highly expressed in-frame fusions involving known BrCa mediators such as ERBB2, BRCA1, ARID1B, RPS6KB1/2, PIK3R3, AXIN1, TGFB1/2, FOXP1, PAK1, and CREBBP. CONCLUSIONS: Taken together, these results demonstrate that fusion RNAs in MBC—some recurrent, many highly expressed and unique to individual tumors—are common. We create the most comprehensive catalog of ESR1 fusions in MBC, better define their frequency, discover their enrichment in LumB-like tumors, and will discuss clinicopathologic and transcriptomic features associated with ESR1 fusion positive disease. We identify druggable fusions that would likely be missed by current testing standards, find recurrent loss-of-function fusion RNAs, and show that over one-third of metastatic cases harbor at least one outlier expressed fusion—many of which involve BrCa-related genes. In summary, we propose that fusion RNAs are a driving and perhaps overlooked mechanism of tumor evolution in therapy-resistant disease and postulate fusion transcripts present a compelling therapeutic opportunity in MBC. Preliminary data targeting fusion RNA breakpoints using a novel RNA base-editing approach will be discussed. Citation Format: Nolan Priedigkeit, Alinés Lebrón-Torres, Janny Liao, Jean-Baptiste Alberge, Stefania Morganti, Jakob Weiss, Jorge Gomez Tejeda Zanudo, Albert Grinshpun, Melissa Hughes, Karla Helvie, Kerry Sendrick, Kyleen Nguyen, Sarah Strauss, Janet Files, Maxwell Lloyd, Nikhil Wagle, Chip Stewart, Eric Winer, Bruce Johnson, Yvonne Li, Rinath Jeselsohn, Sara Tolaney, Daniel Abravanel, Nancy Lin, Heather Parsons, Gad Getz, Steffi Oesterreich, Adrian Lee, Todd Golub. Characterization and proposed therapeutic exploitation of fusion RNAs in metastatic breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-09.

EZH2 inhibition reactivates epigenetically silenced FMR1 and normalizes molecular and electrophysiological abnormalities in fragile X syndrome neurons.

Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here, using a candidate-based shRNA screen, we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors, or FMR1- SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells, neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2, for which an FDA-approved small molecule inhibitor, EPZ6438 (also known as tazemetostat), is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately, EZH2 inhibitors do not efficiently cross the blood-brain barrier, limiting their therapeutic use for FXS. Recently, antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons, and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively, our results establish EZH2 inhibition in general, and EZH2 ASOs in particular, as a therapeutic approach for FXS.

ChemSpaceAL: An Efficient Active Learning Methodology Applied to Protein-Specific Molecular Generation.

The incredible capabilities of generative artificial intelligence models have inevitably led to their application in the domain of drug discovery. Within this domain, the vastness of chemical space motivates the development of more efficient methods for identifying regions with molecules that exhibit desired characteristics. In this work, we present a computationally efficient active learning methodology and demonstrate its applicability to targeted molecular generation. When applied to c-Abl kinase, a protein with FDA-approved small-molecule inhibitors, the model learns to generate molecules similar to the inhibitors without prior knowledge of their existence and even reproduces two of them exactly. We also show that the methodology is effective for a protein without any commercially available small-molecule inhibitors, the HNH domain of the CRISPR-associated protein 9 (Cas9) enzyme. To facilitate implementation and reproducibility, we made all of our software available through the open-source ChemSpaceAL Python package.

Novel Substrate Prediction for the TAM Family of RTKs Using Phosphoproteomics and Structure-Based Modeling.

The TAM family of receptor tyrosine kinases is implicated in multiple distinct oncogenic signaling pathways. However, to date, there are no FDA-approved small molecule inhibitors for the TAM kinases. Inhibitor design and screening rely on tools to study the kinase activity. Our goal was to address this gap by designing a set of synthetic peptide substrates for each of the TAM family members: Tyro3, Axl, and Mer. We used an in vitro phosphoproteomics workflow to determine the substrate profile of each TAM kinase and input the identified substrates into our data processing pipeline, KINATEST-ID, producing a position-specific scoring matrix for each target kinase and generating a list of candidate synthetic peptide substrates. We synthesized and characterized a set of those substrate candidates, systematically measuring their initial phosphorylation rate with each TAM kinase by LC-MS. We also used the multimer modeling function of AlphaFold2 (AF2) to predict peptide-kinase interactions at the active site for each of the novel candidate peptide sequences against each of the TAM family kinases and observed that, remarkably, every sequence for which it predicted a putative catalytically competent interaction was also demonstrated biochemically to be a substrate for one or more of the TAM kinases. This work shows that kinase substrate design can be achieved using a combination of preference motifs and structural modeling, and it provides the first demonstration of peptide-protein interaction modeling with AF2 for predicting the likelihood of constructive catalytic interactions.

P15.13.B DEVELOPMENT OF [18F]AG-120 AS AN IMAGING AGENT FOR THE DETECTION OF THE MUTANT ISOCITRATE DEHYDROGENASE 1 IN GLIOMA BY PET

Abstract BACKGROUND Gliomas are highly invasive and infiltrative solid tumors that present significant clinical challenges in terms of achieving complete surgical resection. These tumors often harbor mutated isocitrate dehydrogenase enzymes (mIDH), making them attractive theranostic targets. Therefore, accurately assessing mIDH status is essential for effective patient management. Currently, the presence of mIDH1 is determined either by invasive biopsy or indirectly by measuring the mIDH-derived oncometabolite 2-hydroxyglutarate (2-HG) using magnetic resonance spectroscopy (MRS). The availability of mIDH-targeted radioligands would allow for PET neuroimaging to detect the mIDH1 protein directly and non-invasively. MATERIAL AND METHODS The study involved obtaining 18F-labeled AG-120 (Ivosidenib), an FDA-approved small molecule inhibitor of mIDH, by Cu-mediated radiofluorination of a corresponding diastereomerically pure stannyl precursor. The researchers studied the internalization of [18F]AG-120 in vitro in U251 human glioblastoma cells stably transfected with IDH1 or IDH1R132H. Brain uptake and metabolism of [18F]AG-120 were investigated in healthy CD-1 mice by dynamic PET-MR imaging and radio-chromatographic analyses of plasma and brain tissue. Dynamic PET-CT imaging studies were performed in nude rats bearing U251-IDH1 or U251-IDH1R132H glioblastoma. RESULTS The (S,S)-enantiomer of [18F]AG-120 was successfully prepared, and the procedure was implemented into a remotely-controlled radiosynthesis module. A considerably higher uptake of activity was observed in U251-IDH1R132H cells compared to U251-IDH1 cells (0.422 vs. 0.014% applied activity/μg protein after 120 min incubation). The percentage of non-metabolized [18F]AG-120 in the mouse 30 min post i.v. injection was 85% in plasma and 91% in the brain, respectively. Dynamic PET studies showed limited blood-brain barrier permeation of [18F]AG-120 (SUV < 0.1 within the first 5 min post i.v. injection), which could be significantly increased by inhibiting the p-glycoprotein 1 with cyclosporine A. PET studies with glioblastoma rats indicated slightly higher tumor-to-blood ratios in IDH1R132H-tumors compared to IDH1-tumors (~1.7 vs. ~1.3 at 40-60 min p.i.). CONCLUSION For the first time, pure (S,S)-[18F]AG-120 has been synthesized. In addition, GMP-compliant production is facilitated by the established automated radiosynthesis. Despite limited BBB permeation, [18F]AG-120 shows target-specific internalization in vitro and high metabolic stability in vivo. Furthermore, the slightly higher accumulation of [18F]AG-120 in IDH1R132H-glioblastoma compared to IDH1-glioblastoma in vivo encourages further evaluation of mIDH inhibitors for the noninvasive detection of IDH1R132H in glioma by PET. SUPPORT European-Regional-Development-Fund ERDF and Sächsische-Aufbaubank SAB (#100364142).

BZW2 Inhibition Reduces Colorectal Cancer Growth and Metastasis.

FDA-approved small-molecule inhibitors of EZH2 can indirectly target BZW2 and because BZW2 functions as an oncogene, these inhibitors could serve as therapeutic agents for colorectal cancer.

Niclosamide-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis.

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.

Molecular modelling and structure-activity relationship of a natural derivative of o-hydroxybenzoate as a potent inhibitor of dual NSP3 and NSP12 of SARS-CoV-2: in silico study

The nsp3 macrodomain and nsp12 (RdRp) enzymes are strongly implicated in the virulent regulation of the host immune response and viral replication of SARS-CoV-2, making them plausible therapeutic targets for mitigating infectivity. Remdesivir remains the only FDA-approved small-molecule inhibitor of the nsp12 in clinical conditions while none has been approved yet for the nsp3 macrodomain. In this study, 69,067 natural compounds from the IBScreen database were screened for efficacious potentials with mechanistic multitarget-directed inhibitory pharmacology against the dual targets using in silico approaches. Standard and extra precision (SP and XP) Maestro glide docking analyses were employed to evaluate their inhibitory interactions against the enzymes. Four compounds, STOCK1N-45901, 03804, 83408, 08377 consistently showed high XP scores against the respective targets and interacted strongly with pharmacologically essential amino acid and RNA residues, in better terms than the standard, co-crystallized inhibitors, GS-441524 and remdesivir. Further assessments through the predictions of ADMET and mutagenicity distinguished STOCK1N-45901, a natural derivative of o-hydroxybenzoate as the most promising candidate. The ligand maintained a good conformational and thermodynamic stability in complex with the enzymes throughout the trajectories of 100 ns molecular dynamics, indicated by RMSD, RMSF and radius of gyration plots. Its binding free energy, MM-GBSA was recorded as –54.24 and –31.77 kcal/mol against the respective enzyme, while its structure-activity relationships confer high probabilities as active antiviral, anti-inflammatory, antiinfection, antitussive and peroxidase inhibitor. The IBScreen database natural product, STOCK1N-45901 (2,3,4,5,6-pentahydroxyhexyl o-hydroxybenzoate) is thus recommended as a potent inhibitor of dual nsp3 and nsp12 of SARS-CoV-2 for further study. Communicated by Ramaswamy H. Sarma

Contribution of Bcl-2 Inhibitor Venetoclax Toward Anti-CD19 CAR T Cell Efficacy in Relapsed/Refractory Diffuse Large B Cell Lymphoma

IntroductionClinical outcomes of anti-CD19 chimeric antigen receptor T-cell (CAR-T CD19) therapy for the treatment of relapsed/refractory Diffuse Large B Cell lymphoma (r/r DLBCL) must be improved, as patients undergoing CAR-T CD19 therapy can expect a cure rate of only 41% and a median survival of just 6.3 months if unresponsive. Of patients presenting with naïve DLBCL, 35% will advance to third-line treatment with CAR-T CD19 therapy. Poor clinical responses to CAR-T CD19 therapy and a small window of survival for unresponsive patients highlights the critical need for improvement of CAR-T CD19 therapy in the setting of r/r DLBCL. Overexpression of anti-apoptotic Bcl-2 protein has been characterized in r/r DLBCL, and contributes to the disease's aggressive phenotype. Aberrant expression of the Bcl-2 protein proves targetable by an FDA-approved small molecule inhibitor which mimics the role of pro-apoptotic BH3 proteins, known as Venetoclax. Here, we investigate the effect of Venetoclax on CAR-T CD19 cell immunophenotype, effector function, and cytotoxicity upon co-culture with a lab-generated model of immortalized rituximab/chemo-resistant B cell lymphomas (Raji4RH) which recapitulate the resistant phenotype of r/r DLBCL seen in patients.MethodsPBMCs were isolated and activated from apheresis samples from consenting adults (healthy donors) using the Miltenyi Biotec TransACT human CD3/CD28 Kit. Cells were transduced with lentiviral vectors encoding for 2nd generation CAR constructs 24 hours post-activation. Transduced cells were cultured in RPMI media supplemented with hIL-2 (1ng/mL) and hIL-7 (10ng/mL). At day 14, cells were cultured with Raji4RH and Venetoclax in either combined or solitary cultures. Co-culture cytotoxicity assays were performed by pre-labeling target cells with permeant dye (Thermo Fisher Scientific CellTrace Blue Proliferation Kit), then incubating with CAR-T CD19 cells (GFP labeled) at various effector:target ratios and Venetoclax concentrations for 24 hours. Sample wells were stained with viability dye and acquired on BD LSR II Cytometer. CAR-T CD19 cell immunophenotype was identified following 24-hour exposure of Venetoclax to CAR T cell populations at various concentrations. Following exposure, samples were stained with viability dye, CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD25, and CD95. Frequencies of T REG, T Central Memory, T naive, and T Stem Cell Memory populations were calculated. CAR-T CD19 cell effector function was elucidated by activating (24 hours), then exposing CAR-T CD19 cells to concentrations of Venetoclax for 24 hours. Brefeldin A was added to culture for the remaining 4 hours of incubation. Cells were collected and stained intracellularly for IFNγ, Perforin, and Granzyme B; and extracellularly for CD3, CD4, and CD8. Sample acquisition for effector function assay was performed on BD LSRII Cytometer.ResultsConcurrent in vitro CAR-T CD19 administration and Venetoclax exposure significantly increases Raji4RH cell death as opposed to single agent cohorts. CAR-T CD19 cell viability is unaffected by Venetoclax at concentrations which confer synergy, and a predominantly CD8+ population is favored. Finally, Venetoclax appears to significantly increase the frequency of T N/T SCM and T CM within exposed cultures, while affording a 21% decrease in T REG frequency. Additionally, the notable increase in T N/T SCM and T CM phenotypes following exposure to Venetoclax did not arise as a consequence of decreased cellular viability.ConclusionThe combination of Venetoclax exposure and αCD19 CAR T cell administration demonstrates synergy when employed in the context of rituximab-resistant lymphoma cells, which may be explained by the effect of the BH3 mimetic's exposure on CAR-T CD19 cell immunophenotype. The ability to manipulate Bcl-2 protein expression within CAR-T CD19 cells affords insight into the role that the Bcl-2 family pathway plays within CAR-T CD19 cell biology. In addition to answering fundamental questions of CAR T cell biology, the combination of Venetoclax and CAR-T CD19 therapy may provide a solution to the observed clinical gap, in which CAR-T CD19 therapy alone cures less than half of all patients who advance to this third line treatment regimen. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bio-O2-07 - Primary cytotoxic T cell lymphomas harbor recurrent targetable alterations in the JAK-STAT pathway

<h3>Background:</h3> Primary cytotoxic T cell lymphomas are a heterogenous collection of ill-defined diseases with aggressive disease course and poor response to conventional therapy. Of these, primary cutaneous CD8+ aggressive epidermotropic T cell lymphoma and cutaneous T cell lymphoma, not otherwise specified, have undergone few to no systematic analyses of its genetic landscape. In this study, we classify these lymphomas based on their histological and clinical features and perform systematic genomic analyses to find novel therapeutic targets. <h3>Methods:</h3> We clinically characterize 35 patients from 10 institutions with cytotoxic T cell lymphoma and performed integrative clinical, pathological, and genetic analyses. We perform Archer™ Heme Fusion on an initial 24 FFPE samples. In addition, we perform DNA and/or RNA sequencing on 11 cases. We functionally validate our genetic findings by transducing alterations into Ba/F3 cells and subsequently performing biochemical assays. In this setting, we assess sensitivity to FDA-approved small molecule inhibitors. We provide orthogonal evidence of transcriptional upregulation by geneset enrichment analysis. <h3>Results:</h3> We find kinase fusions impacting JAK-STAT signaling in all primary cutaneous CD8+ aggressive epidermotropic T cell lymphomas in our cohort. Three of these fusions have not previously been described in cancer. Known fusions have been shown to upregulate JAK-STAT signaling. Upon each transduction, <i>CAPRIN1-JAK2</i> and <i>SELENOI-ABL1</i> cause increased phosphorylation and display IL-3-independent growth compared to control. Upon treatments to small molecule inhibitors, ruxolitinib and imatinib respectively, CAPRIN1-JAK2 and SELENOI-ABL1 expressing cells exhibit sensitivity. Cutaneous T cell lymphoma, not otherwise specified, harbor JAK-STAT hotspot mutations but not kinase fusions. <h3>Conclusions:</h3> All 9 primary cutaneous CD8+ aggressive epidermotropic T cell lymphomas in our series harbor kinase fusions, while the majority of the cutaneous T cell lymphoma, not otherwise specified, cases in our cohort harbor hotspot JAK/STAT mutations. We provide evidence that the of presence these alterations, which are associated with inhibitor response, depend on cytotoxic T cell lymphoma histology.

A Sampling of Highlights from the Literature

Prostaglandin E2 (from WikiMedia Commons)Neutrophils can be immunosuppressive when located in the microenvironment of tumors. GM-CSF in the tumor microenvironment stimulates STAT5-upregulation of fatty-acid transporter protein FATP2 primarily on tumor neutrophils. This increases arachidonic acid uptake and the generation of PGE2, with subsequent suppression of tumor-specific T-cell responses. By combining inhibition of FATP2 with blockade of CTLA-4, tumor growth can be effectively inhibited in two mouse tumor models.Veglia F, …, Gabrilovich DI. Nature 2019 Apr 17;569:73–8.Neo open reading frame peptides (from Koster et al., Fig. 2)Cancer cell genomes can contain frameshift mutations, from which long neopeptides can be translated. Examination of over 10,000 TGCA tumor samples finds 143,444 frame-shift mutations, and 70,000 unique peptides ≥ 10 amino acids. Tumor-drivers are commonly frame-shifted genes. The neo-open-reading-frame peptides (NOPs) converge on common peptide sequences, in that by including only 6 driver genes, 1244 new peptides could be found in 30% of the patients. NOPs offer a new source of commonly shared potential neoantigens that could inform production of an “off-the-shelf” library of vaccine reagents.Koster J and Plasterk RHA. Sci Rep 2019 Apr 29;9:6577.Antibody binding sites on PD-1 (from Fenwick et al., Fig. 2F)Clinically useful mAbs to PD-1 block the PD-L1 binding site and thereby block the inhibition signal. Fenwick and colleagues find two new mAbs that bind to PD-1, but structural analysis indicates that binding is on locations away from the PD-1 binding site. The nonblocking mAbs act through the CD28 coreceptor, restoring signaling through the AKT–NF-κB pathway, and helping further rescue exhausted T cells. Combining both types of antibodies in tumor models enhances tumor clearance over monotherapy.Fenwick C, …, Pantaleo G. J Exp Med 2019 May 23. DOI: 10.1084/jem.20182359.Effects of DDR and PD-L1 inhibition (from Sen et al., Fig. 7E)Immune checkpoint blockade is successful only in some cases of small cell lung cancer (SCLC). Sen et al. find that FDA-approved small molecule inhibitors of CHK1 and PARP, which participate in the DNA damage response, can enhance PD-L1 blockade of the CD8+ T cell antitumor response. This is mediated in part through activation of the STING pathway that leads to increased production of T cell chemoattractants like CXCL10 and CCL5. The results suggest dual therapy could improve immunotherapy in SCLC.Sen T, …, Byers LA. Cancer Discov 2019 May 1;9:646–61.Melanoma-associated lymph node (from Broggi et al., Fig. 6A)Two papers show the promise of using tumor-draining lymph node exudate as the substrate for tumor biomarkers and evaluation of antitumor immune responses. Garcia-Silva et al. make use of extracellular vesicles (EVs) to detect markers of melanoma progression and the status of BRAF mutations. Broggi et al. compare plasma to draining lymph node exudate and find the exudate and EVs to be enriched in metastatic tumor–derived factors with protein signatures that can differentiate early from advanced metastasis and provide insights into tumor interactions with the immune system.García-Silva S, …, Peinado H. J Exp Med 2019 MAY 6;216: 1061–70.Broggi MAS, …, Swartz MA. J Exp Med 2019 May 6;216:1091–107.Moderately differentiated gastric tumor (by Nephron via Wikimedia Commons)The ramifications of PD-1 blockade on regulatory T cells (Tregs) are not clear. Almost 10% of patients with gastric cancer treated with anti–PD-1 respond with rapid progression of their tumors. In these patients, treatment increases the proliferative rate and immune suppressive activity of their Tregs. Tregs from PD-1–deficient mice lack PD-1 and both proliferate strongly and have enhanced immunosuppressive activity of antitumor responses. This association of hyperprogressive disease with PD-1 treatment suggests that Tregs be routinely monitored during checkpoint blockade therapy to identify this patient subset.Kamada T, …, Nishikawa H. Proc Natl Acad Sci U S A 2019 May 14;116:9999–10008.

Nanoemulsion-Based Delivery of Fluorescent PARP Inhibitors in Mouse Models of Small Cell Lung Cancer.

The preclinical potential of many diagnostic and therapeutic small molecules is limited by their rapid washout kinetics and consequently modest pharmacological performances. In several cases, these could be improved by loading the small molecules into nanoparticulates, improving blood half-life, in vivo uptake and overall pharmacodynamics. In this study, we report a nanoemulsion (NE) encapsulated form of PARPi-FL. As a proof of concept, we used PARPi-FL, which is a fluorescently labeled sensor for olaparib, a FDA-approved small molecule inhibitor of the nuclear enzyme poly(ADP-ribose)polymerase 1 (PARP1). Encapsulated PARPi-FL showed increased blood half-life, and delineated subcutaneous xenografts of small cell lung cancer (SCLC), a fast-progressing disease where efficient treatment options remain an unmet clinical need. Our study demonstrates an effective method for expanding the circulation time of a fluorescent PARP inhibitor, highlighting the pharmacokinetic benefits of nanoemulsions as nanocarriers and confirming the value of PARPi-FL as an imaging agent targeting PARP1 in small cell lung cancer.

Homogeneous BTK Occupancy Assay for Pharmacodynamic Assessment of Tirabrutinib (GS-4059/ONO-4059) Target Engagement

Bruton’s tyrosine kinase (BTK) is a clinically validated target for B-cell leukemias and lymphomas with FDA-approved small-molecule inhibitors ibrutinib and acalabrutinib. Tirabrutinib (GS-4059/ONO-4059, Gilead Sciences, Inc., Foster City, CA) is a second-generation, potent, selective, irreversible BTK inhibitor in clinical development for lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL). An accurate pharmacodynamic assay to assess tirabrutinib target coverage in phase 1/2 clinical studies will inform dose and schedule selection for advanced clinical evaluation. We developed a novel duplex homogeneous BTK occupancy assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to measure free and total BTK levels in a multiplexed format. The dual-wavelength emission property of terbium-conjugated anti-BTK antibody served as the energy donor for two fluorescent energy acceptors with distinct excitation and emission spectra. The assay was characterized and qualified using full-length purified recombinant human BTK protein and peripheral blood mononuclear cells derived from healthy volunteers and patients with CLL. We demonstrated assay utility using cells derived from lymph node and bone marrow samples from patients with CLL and DLBCL. Our TR-FRET-based BTK occupancy assay provides accurate, quantitative assessment of BTK occupancy in the clinical trial program for tirabrutinib and is in use in ongoing clinical studies.

Abstract P3-05-21: An integrative approach to the discovery of triple-negative breast cancer markers derived from extracellular vesicles

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype accounting for nearly 20% of all breast cancer (BC) cases with disproportionally poor prognosis. Treatment options are limited by the lack of expression of common BC receptors targeted by FDA-approved small molecule inhibitors, resulting in an unmet need for efficient and cost effective treatments. Immunotherapies are emerging as a powerful alternative to standard chemo- and radiation therapy. Our objectives are to generate vaccines targeting TNBC and to identify circulating biomarkers capable of monitoring their therapeutic efficacy in a human transgenic mouse model (C57BL/6) compatible with EO771.LMB, a murine cell line of TNBC with a reported basal-like phenotype. Our current study characterized the cancer-related proteomic and metabolic signatures derived from extracellular vesicles isolated from EO771.LMB. It has been shown that extracellular vesicles (EV) shed by tumors carry complex cargos comprised of proteins, metabolites and nucleic acids and may exhibit cancer-specific signatures with a potential as prognostic markers or predictors of therapy response. EVs released by TNBC tumors into circulation might provide non-invasive and highly actionable insight into the tumor biology of this aggressive cancer. To further our understanding of cancer-specific EV proteomes, we used mass-spectrometry based proteomics and identified a total of 2265 unique proteins from EVs isolated from conditioned media from EO771.LMB. Our survey across the EV proteome, the expression proteome (5096) and phosphoproteome (2728) identified 6461 unique proteins overall. Within EV sub-fractions, microvesicles and apoptotic bodies were characterized by proteomic cargos different from exosomes. Over 90% of proteins identified in EVs shed by cultured EO771.LMB mapped with proteins curated in ExoCarta. Of interest, exploration of the EV proteome identified 47 GPI-anchored surface proteins. These candidate membrane proteins included Glypican-1 and CD109, previously reported as highly specific to cancer, as well as potential TNBC-specific markers. Our multipronged strategy for deep proteomic profiling of EO771.LMB enhanced identification by ˜25% compared to global whole cell proteomics alone. We also identified ˜3150 metabolites corresponding to lysophosphatidyl choline (35%), fatty-acids, -esters, -amides, -alcohols (30%), glycerophospholipids and sphingolipids (16%), sugar (1%), amino acids and biogenic amines (4%), organic acids and derivatives, cyclic alcohols, aromatic compounds and steroids (13%). Of these, 1450 metabolites were shared between cell line and EVs. A proteogenomic approach expanded identification to onco-proteoforms and tumor-specific peptides not represented in canonical databases. Custom, cell-specific protein databases derived from whole transcriptome sequencing of EO771.LMB elucidated novel protein variants and mutations. Pan-cancer markers vs. TNBC-specific markers will be verified in other murine cell lines, across multiple cancer types and fibroblast controls. Optimal membrane markers for downstream immune-affinity purification from plasma will be proposed, and their ability to diagnose disease or monitor tumor load during treatment will be discussed. Citation Format: Igyártó B, Sharma R, Garcia K, David-Dirgo V, Pathak K, Kroll M, Terraf P, Van Keuren-Jensen K, Pirrotte P. An integrative approach to the discovery of triple-negative breast cancer markers derived from extracellular vesicles [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-21.

Abstract P1-10-09: EPHA2-targeting enhances eicosapentaenoic acid cytotoxicity against triple-negative inflammatory breast cancer via ABCA1 inhibition–mediated membrane rigidity

Abstract Background: Effective treatment options for triple-negative inflammatory breast cancer (TN-IBC), the most aggressive form of breast cancer, are currently lacking. We previously reported that mediators of inflammation promote the growth of TN-IBC xenografts. Eicosapentaenoic acid (EPA), an omega-3 fatty acid (fish oil) with anti-inflammatory properties, is an emerging FDA-approved therapeutic with a favorable toxicology profile. Here we aimed to develop a novel approach to enhance EPA efficacy against TN-IBC by identifying a kinase inhibitor that synergizes with EPA's antitumor activity. Methods and Results: Using a high-throughput siRNA screen in the TN-IBC cell line SUM149PT, we identified inhibition of ephrin type-A receptor 2 (EPHA2), an oncogenic receptor tyrosine kinase, as a target that sensitizes TN-IBC cells to EPA therapy. To determine the clinical relevance of EPHA2, we investigated a meta-analysis of breast cancer mRNA expression data sets and found that high EPHA2 tumor expression, compared with low expressing, correlated significantly with poor overall survival in TN-IBC patients (P = 0.01), while not with other subtypes. Similar findings were observed in vitro, were EPHA2 protein and mRNA overexpression occurred predominantly in the TN subtypes among 49 and 51 breast cancer cell lines (63% and 47%, respectively), highlighting EPHA2 translational potential. Functional expression studies using proliferation and apoptosis assays in vitro, and xenografts in vivo, were performed in two EPHA2-expressing TN-IBC cell lines, SUM149PT and BCX010, to validate EPHA2 as a synergistic combinational target with EPA. EPHA2 gene silencing in combination with EPA significantly reduced cell growth, and enhanced apoptosis, compared with untreated and monotherapy in vitro (P &amp;lt; 0.05), and in vivo (P &amp;lt; 0.001). To translate our findings to the clinic, we validated dasatinib, an FDA-approved small molecule inhibitor of EPHA2, in combination to EPA to significantly enhance apoptosis of TN-IBC cells in vitro (P &amp;lt; 0.05) and in vivo (P &amp;lt; 0.05), compared with untreated and monotherapies. Using membrane fluidity assessment and cholesterol quantification we determined that apoptosis induction after combination therapy was due to increased membrane rigidity and cholesterol concentrations in the plasma membrane of TN-IBC cells (P &amp;lt; 0.05, compared with monotherapies). Finally, we discovered by western blot and gain/loss-of-expression studies that combination therapy inhibited the cholesterol efflux protein ATP-binding cassette sub-family A member 1 (ABCA1), which plays a significant role mediating increased cellular cholesterol (P &amp;lt; 0.05), cell membrane rigidity (P &amp;lt; 0.05), and induction of apoptosis (P &amp;lt; 0.05) in TN-IBC after EPA and EPHA2-targeting combination therapy. Conclusions: This is the first study demonstrating that EPA can enhance conventional targeted therapy against breast cancer. Our study provides molecular and preclinical evidence to support the development of an EPA/EPHA2-inhibition–based phase I clinical trial for patients with EPHA2-positive TN-IBC; our study further suggests the use of EPHA2 and ABCA1 protein expression as biomarkers for patient selection and therapeutic response. Citation Format: Torres-Adorno AM, Vitrac H, Qi Y, Tan L, Levental KR, Fan Y-Y, Yang P, Chapkin RS, Eckhardt BL, Ueno NT. EPHA2-targeting enhances eicosapentaenoic acid cytotoxicity against triple-negative inflammatory breast cancer via ABCA1 inhibition–mediated membrane rigidity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-09.

Non-Canonical Thinking for Targeting ALK-Fusion Onco-Proteins in Lung Cancer.

Anaplastic lymphoma kinase (ALK) gene rearrangements have been identified in lung cancer at 3–7% frequency, thus representing an important subset of genetic lesions that drive oncogenesis in this disease. Despite the availability of multiple FDA-approved small molecule inhibitors targeting ALK fusion proteins, drug resistance to ALK kinase inhibitors is a common problem in clinic. Thus, there is an unmet need to deepen the current understanding of genomic characteristics of ALK rearrangements and to develop novel therapeutic strategies that can overcome ALK inhibitor resistance. In this review, we present the genomic landscape of ALK fusions in the context of co-occurring mutations with other cancer-related genes, pointing to the central role of genetic epistasis (gene-gene interactions) in ALK-driven advanced-stage lung cancer. We discuss the possibility of targeting druggable domains within ALK fusion partners in addition to available strategies inhibiting the ALK kinase domain directly. Finally, we examine the potential of targeting ALK fusion-specific neoantigens in combination with other treatments, a strategy that could open a new avenue for the improved treatment of ALK positive lung cancer patients.

Abstract B29: Inhibition of histone deacetylases 1 and 2 (HDAC1,2) perturbs DNA replication and DNA repair in cancer cells: Implications in mechanism-based therapeutic strategies

Abstract Class I family enzymes, histone deacetylases 1 and 2 (HDAC1,2), are targets of FDA-approved small molecule inhibitors currently used in cancer therapy. HDAC inhibitors impair DNA repair and replication in cancer cells to trigger genotoxic stress and cause cell death. How HDAC1,2 function in genome maintenance processes, DNA replication and repair, in mammalian cells is far from fully understood. Using small molecules to selectively inhibit HDAC1,2 activities, we have systematically defined their direct functions in DNA replication and repair. Our studies show that HDAC1,2 inhibition reduces replication fork velocity and activates the replication stress associated DNA damage independent of its effect on transcription. HDAC1,2 inhibition increases histone H4K16 acetylation (H4K16ac) and nuclease sensitivity of nucleosomes associated with newly replicated DNA, suggesting that HDAC1,2 are required for chromatin compaction during DNA replication. Thus, mechanistically, HDAC1,2 activities are required for maintaining nascent chromatin structure and dynamics required for efficient progression of the replication fork. In addition to H4K16ac, we have found that HDAC1,2 inhibition activities increases H3K27ac and H4K91ac present on the histone tail and core regions, respectively. Increase in these histone acetyl marks perturbs the acetyl-methyl or the acetyl-ubiquityl switch during DNA repair leading to impaired EZH2-mediated H3K27me3-dependent DNA repair signaling and the H4K91ubiquityl-dependent 53BP1-mediated DNA repair signaling in chemoresistant EZH2 gain-of-function diffuse large B-cell lymphoma cells. We are currently investigating the therapeutic benefits of HDAC1,2 inhibition in mouse models of aggressive chemoresistant diffuse large B-cell lymphoma and in primary patient samples. Overall, our studies have uncovered novel functions for HDAC1,2 in genome maintenance processes and provide insight into the mode-of-action of HDAC1,2 inhibitors in overcoming chemoresistance in cancer cells (via targeting the DNA repair process) and in inducing DNA damage and death in the rapidly cycling cancer cells (via targeting DNA replication). Note: This abstract was not presented at the conference. Citation Format: Danielle Johnson, Simon Jones, Steven Quayle, Jeff Shearstone, Srividya Bhaskara. Inhibition of histone deacetylases 1 and 2 (HDAC1,2) perturbs DNA replication and DNA repair in cancer cells: Implications in mechanism-based therapeutic strategies. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B29.

Differential Effects of Tyrosine Kinase Inhibitors on Normal and Oncogenic EGFR Signaling and Downstream Effectors.

Constitutive activation of EGFR due to overexpression or mutation in tumor cells leads to dysregulated downstream cellular signaling pathways. Therefore, EGFR as well as its downstream effectors have been identified as important therapeutic targets. The FDA-approved small-molecule inhibitors of EGFR, gefitinib (Iressa) and erlotinib (Tarceva), are clinically effective in a subset of patients with non-small cell lung cancer (NSCLC) whose tumors harbor activating mutations within the kinase domain of EGFR. The current study examined effects of these drugs in 32D cells expressing native (WT) or oncogenic (L858R) EGFR as well as in cancer cell lines A431 and H3255. Distinct patterns for gefitinib and erlotinib inhibition of EGFR autophosphorylation at individual tyrosines were revealed for wild-type (WT) and L858R EGFR. Phosphorylation of Y845 has been shown to be important in cancer cells and Y1045 phosphorylation is linked to Cbl-mediated ubiquitination and degradation. Dramatic differences were observed by greater potency of these drugs for inhibiting downstream effectors for L858R EGFR including Cbl and STAT5. Selective targeting of Cbl may play a role in oncogene addiction and effects on STAT5 identify features of signaling circuitry for L858R EGFR that contribute to drug sensitivity and clinical efficacy. These data provide new understanding of the EGFR signaling environment and suggest useful paradigms for predicting patient response to EGFR-targeted therapy as well as combination treatments. This study offers fundamental insights for understanding molecular mechanisms of drug sensitivity on oncogenic forms of EGFR and downstream signaling components as well as considerations for further drug optimization and design of combination therapy.

Abstract 1885: Targeting the FOXO1/KLF6 transcriptional network to modulate response to anti-EGFR based therapy

Abstract Epidermal growth factor receptor (EGFR) activation is both a key molecular driver of disease progression and the target of a broad class of molecular agents designed to treat advanced cancer. Nevertheless, resistance develops through several mechanisms including constitutive activation of AKT signaling. Additional molecular characterization of the downstream mediators of EGFR signaling may lead to the development of new classes of targeted molecular therapies to treat resistant disease. Here we identify a transcriptional network involving the KLF6 and FOXO1 tumor suppressor genes that negatively regulate activated EGFR signaling and that can be reactivated using the combination of two FDA approved agents in both cell culture and in vivo models of the disease. In both murine models and patient derived lung adenocarcinoma samples, EGFR activation is associated with FOXO1 mislocalization and decreased KLF6 expression. Furthermore, in a Kras driven mouse model, KLF6 expression is not significantly changed whereas AKT activation seen in the Pten/Mmac1+/− heterozygous mouse model results in FOXO1 mislocalization and decreased KLF6 expression. Consistent with these findings, inhibition of AKT signaling promotes increase in nuclear FOXO1 resulting in transactivation of the KLF6 tumor suppressor gene in lung adenocarcinoma cell lines. Correspondingly, the EGFRL858R mouse model demonstrates spontaneous tumor regression when treated with the anti-EGFR based therapy, erlotinib, an FDA-approved small-molecule inhibitor of EGFR signaling. We analyzed L858R mouse tumors samples treated with erlotinib and found increased KLF6 expression following EGFR inhibition. Conversely, targeted reduction of KLF6 resulted in decreased erlotinib response in both cell culture and in vivo models of disease suggesting a direct link between KLF6 upregulation and the induction of apoptosis by anti-EGFR based therapy. Therefore, we hypothesized that acquired resistance to anti-EGFR based therapies could be overcome by restoring downstream function of the FOXO1/KLF6 transcriptional network. Here we demonstrate that an FDA-approved drug, trifluoperazine hydrochloride (TFP), which has been shown to inhibit FOXO1 nuclear export, restores sensitivity to AKT-driven erlotinib-resistance through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft models. Furthermore, silencing of FOXO1 blunts apoptosis mediated through combination erlotinib and TFP treatment suggesting that this transcriptional network is important for negatively regulating AKT signaling. Combined, these studies define a novel transcriptional network regulating oncogenic EGFR signaling and identify a class of FDA-approved drugs with the potential for rapid clinical translation to restore chemosensitivity to anti-EGFR-based therapy for the treatment of metastatic lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1885. doi:1538-7445.AM2012-1885