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Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.
Intrinsic resistance of unknown mechanism impedes the clinical utility of inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) in malignancies other than breast cancer. Here, we used melanoma patient-derived xenografts (PDXs) to study the mechanisms for CDK4/6i resistance in preclinical settings. We observed that melanoma PDXs resistant to CDK4/6i frequently displayed activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, and inhibition of this pathway improved CDK4/6i response in a p21-dependent manner. We showed that a target of p21, CDK2, was necessary for proliferation in CDK4/6i-treated cells. Upon treatment with CDK4/6i, melanoma cells up-regulated cyclin D1, which sequestered p21 and another CDK inhibitor, p27, leaving a shortage of p21 and p27 available to bind and inhibit CDK2. Therefore, we tested whether induction of p21 in resistant melanoma cells would render them responsive to CDK4/6i. Because p21 is transcriptionally driven by p53, we coadministered CDK4/6i with a murine double minute (MDM2) antagonist to stabilize p53, allowing p21 accumulation. This resulted in improved antitumor activity in PDXs and in murine melanoma. Furthermore, coadministration of CDK4/6 and MDM2 antagonists with standard of care therapy caused tumor regression. Notably, the molecular features associated with response to CDK4/6 and MDM2 inhibitors in PDXs were recapitulated by an ex vivo organotypic slice culture assay, which could potentially be adopted in the clinic for patient stratification. Our findings provide a rationale for cotargeting CDK4/6 and MDM2 in melanoma.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists.
Breast cancer cells frequently home to the bone, but the mechanisms controlling tumor colonization of the bone marrow remain unclear. We report significant enrichment of bone-disseminated estrogen receptor positive human MCF7 cells by 17 β-estradiol (E2) following intracardiac inoculation. Using flow cytometric and quantitative PCR approaches, tumor cells were detected in >80% of MCF7 tumor-inoculated mice, regardless of E2, suggesting that E2 is not required for MCF7 dissemination to the bone marrow. Furthermore, we propose two additional models in which to study prolonged latency periods by bone-disseminated tumor cells: murine D2.0R and human SUM159 breast carcinoma cells. Tumor cells were detected in bone marrow of up to 100% of D2.0R and SUM159-inoculated mice depending on the detection method. These findings establish novel models of bone colonization in which to study mechanisms underlying tumor cell seeding to the marrow and prolonged latency, and provide highly sensitive methods to detect these rare events.
Cancer immunotherapies that remove checkpoint restraints on adaptive immunity are gaining clinical momentum but have not achieved widespread success in breast cancers, a tumor type considered poorly immunogenic and which harbors a decreased presence of tumor-infiltrating lymphocytes. Approaches that activate innate immunity in breast cancer cells and the tumor microenvironment are of increasing interest, based on their ability to induce immunogenic tumor cell death, type I IFNs, and lymphocyte-recruiting chemokines. In agreement with reports in other cancers, we observe loss, downregulation, or mutation of the innate viral nucleotide sensor retinoic acid-inducible gene I (RIG-I/) in only 1% of clinical breast cancers, suggesting potentially widespread applicability for therapeutic RIG-I agonists that activate innate immunity. This was tested using an engineered RIG-I agonist in a breast cancer cell panel representing each of three major clinical breast cancer subtypes. Treatment with RIG-I agonist resulted in upregulation and mitochondrial localization of RIG-I and activation of proinflammatory transcription factors STAT1 and NF-κB. RIG-I agonist triggered the extrinsic apoptosis pathway and pyroptosis, a highly immunogenic form of cell death in breast cancer cells. RIG-I agonist also induced expression of lymphocyte-recruiting chemokines and type I IFN, confirming that cell death and cytokine modulation occur in a tumor cell-intrinsic manner. Importantly, RIG-I activation in breast tumors increased tumor lymphocytes and decreased tumor growth and metastasis. Overall, these findings demonstrate successful therapeutic delivery of a synthetic RIG-I agonist to induce tumor cell killing and to modulate the tumor microenvironment These findings describe the first in vivo delivery of RIG-I mimetics to tumors, demonstrating a potent immunogenic and therapeutic effect in the context of otherwise poorly immunogenic breast cancers. .
©2018 American Association for Cancer Research.
Female nude mice (J:NU-Foxn1nu; age, 6 wk) were injected with 1 million MCF7 human breast cancer cells in the fourth mammary fat pads and received a 21-d sustained-release estrogen pellet (0.25 mg) subcutaneously in the dorsum of the neck. All mice were maintained in sterile housing and provided sterile water and irradiated rodent chow. Approximately 6 wk after implantation, 4 of the 30 mice showed clinical signs of depression and dehydration. The 2 animals most severely affected were euthanized and presented for necropsy. The urinary bladders of these animals were distended with variable sized white, opaque uroliths. Urinalysis revealed coccal bacteria, erythrocytes, neutrophils and struvite crystals. Urine cultures from both necropsied animals grew heavy, pure growths of Staphylococcus xylosus. The organism was sensitive to all antibiotics tested except erythromycin (intermediate). Analysis of the uroliths revealed 100% struvite composition. Remaining mice in the study were evaluated clinically for hydration status, the ability to urinate, and the presence of palpable stones in the urinary bladder; one additional mouse had a firm, nonpainful bladder (urolithiasis suspected). Given the sensitivity of the organisms cultured from urine samples, the remaining mice were placed on enrofloxacin in the drinking water (0.5 mg/mL). All remaining mice completed the study without further morbidity or mortality. Previous studies have reported the association of estrogen supplementation with urinary bladder pathology, including infection and urolithiasis. Here we present a case of urolithiasis and cystitis in nude mice receiving estrogen supplementation that was associated with Staphylococcus xylosus, which previously was unreported in this context. When assessing these nude mice for urolithiasis, we found that visualizing the stones through the body wall, bladder palpation, and bladder expression were helpful in identifying affected mice.
Although ribosomal protein S6 kinase A3 (RSK2) activation status positively correlates with patient responses to antiestrogen hormonal therapies, the mechanistic basis for these observations is unknown. Using multiple and models of estrogen receptor-positive (ER) breast cancer, we report that ERα sequesters active RSK2 into the nucleus to promote neoplastic transformation and facilitate metastatic tumor growth. RSK2 physically interacted with ERα through its N terminus to activate a proneoplastic transcriptional network critical to the ER lineage in the mammary gland, thereby providing a gene signature that effectively stratified patient tumors according to ERα status. ER tumor growth was strongly dependent on nuclear RSK2, and transgenic mice engineered to stably express nuclear RSK2 in the mammary gland developed high-grade ductal carcinoma Mammary cells isolated from the transgenic model and introduced systemically successfully disseminated and established metastatic lesions. Antiestrogens disrupted the interaction between RSK2 and ERα, driving RSK2 into the cytoplasm and impairing tumor formation. These findings establish RSK2 as an obligate participant of ERα-mediated transcriptional programs, tumorigenesis, and divergent patient responses to antiestrogen therapies. Nuclear accumulation of active RSK drives a protumorigenic transcriptional program and renders ER breast cancer susceptible to endocrine-based therapies. .
©2018 American Association for Cancer Research.
An estimated 40,000 deaths will be attributed to breast cancer in 2016, underscoring the need for improved therapies. Evading cell death is a major hallmark of cancer, driving tumor progression and therapeutic resistance. To evade apoptosis, cancers use antiapoptotic Bcl-2 proteins to bind to and neutralize apoptotic activators, such as Bim. Investigation of antiapoptotic Bcl-2 family members in clinical breast cancer datasets revealed greater expression and more frequent gene amplification of as compared with or (Bcl-xL) across three major molecular breast cancer subtypes, Luminal (A and B), HER2-enriched, and Basal-like. While Mcl-1 protein expression was elevated in estrogen receptor α (ERα)-positive and ERα-negative tumors as compared with normal breast, Mcl-1 staining was higher in ERα tumors. Targeted Mcl-1 blockade using RNAi increased caspase-mediated cell death in ERα breast cancer cells, resulting in sustained growth inhibition. In contrast, combined blockade of Bcl-2 and Bcl-xL only transiently induced apoptosis, as cells rapidly acclimated through Mcl-1 upregulation and enhanced Mcl-1 activity, as measured using Mcl-1/Bim proximity ligation assays. Importantly, gene expression levels correlated inversely with sensitivity to pharmacologic Bcl-2/Bcl-xL inhibition in luminal breast cancer cells, whereas no relationship was seen between the gene expression of or and sensitivity to Bcl-2/Bcl-xL inhibition. These results demonstrate that breast cancers rapidly deploy Mcl-1 to promote cell survival, particularly when challenged with blockade of other Bcl-2 family members, warranting the continued development of Mcl-1-selective inhibitors for targeted tumor cell killing. Mcl-1 levels predict breast cancer response to inhibitors targeting other Bcl-2 family members, and demonstrate the key role played by Mcl-1 in resistance to this drug class. .
©2016 American Association for Cancer Research.
Breast cancer is a complex disease, characterized by gene deregulation. There is less systematic investigation of the capacity of long intergenic non-coding RNAs (lincRNAs) as biomarkers associated with breast cancer pathogenesis or several clinicopathological variables including receptor status and patient survival. We designed a two-stage study, including 1,000 breast tumor RNA-seq data from The Cancer Genome Atlas (TCGA) as the discovery stage, and RNA-seq data of matched tumor and adjacent normal tissue from 50 breast cancer patients as well as 23 normal breast tissue from healthy women as the replication stage. We identified 83 lincRNAs showing the significant expression changes in breast tumors with a false discovery rate (FDR) < 1% in the discovery dataset. Thirty-seven out of the 83 were validated in the replication dataset. Integrative genomic analyses suggested that the aberrant expression of these 37 lincRNAs was probably related with the expression alteration of several transcription factors (TFs). We observed a differential co-expression pattern between lincRNAs and their neighboring genes. We found that the expression levels of one lincRNA (RP5-1198O20 with Ensembl ID ENSG00000230615) were associated with breast cancer survival with P < 0.05. Our study identifies a set of aberrantly expressed lincRNAs in breast cancer.
Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-molecule ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis experiments, provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function.