Unraveling Vulnerabilities in Endocrine Therapy-Resistant HER2+/ER+ Breast Cancer.

Breast tumors overexpressing human epidermal growth factor receptor (HER2) confer intrinsic resistance to endocrine therapy (ET), and patients with HER2/estrogen receptor-positive (HER2+/ER+) breast cancer (BCa) are less responsive to ET than HER2-/ER+. However, real-world evidence reveals that a large subset of patients with HER2+/ER+ receive ET as monotherapy, positioning this treatment pattern as a clinical challenge. In the present study, we developed and characterized 2 in vitro models of ET-resistant (ETR) HER2+/ER+ BCa to identify possible therapeutic vulnerabilities. To mimic ETR to aromatase inhibitors (AIs), we developed 2 long-term estrogen deprivation (LTED) cell lines from BT-474 (BT474) and MDA-MB-361 (MM361). Growth assays, PAM50 subtyping, and genomic and transcriptomic analyses, followed by validation and functional studies, were used to identify targetable differences between ET-responsive parental and ETR-LTED HER2+/ER+ cells. Compared to their parental cells, MM361 LTEDs grew faster, lost ER, and increased HER2 expression, whereas BT474 LTEDs grew slower and maintained ER and HER2 expression. Both LTED variants had reduced responsiveness to fulvestrant. Whole-genome sequencing of aggressive MM361 LTEDs identified mutations in genes encoding transcription factors and chromatin modifiers. Single-cell RNA sequencing demonstrated a shift towards non-luminal phenotypes, and revealed metabolic remodeling of MM361 LTEDs, with upregulated lipid metabolism and ferroptosis-associated antioxidant genes, including GPX4. Combining a GPX4 inhibitor with anti-HER2 agents induced significant cell death in both MM361 and BT474 LTEDs. The BT474 and MM361 AI-resistant models capture distinct phenotypes of HER2+/ER+ BCa and identify altered lipid metabolism and ferroptosis remodeling as vulnerabilities of this type of ETR BCa.

Development and characterization of a prototypic pan-amyloid clearing agent - a novel murine peptide-immunoglobulin fusion.

Introduction: Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues. Amyloid accumulation causes organ dysfunction and is not generally cleared by the immune system. Current treatment focuses on reducing amyloid precursor protein synthesis and slowing amyloid deposition. However, curative interventions will likely also require removal of preexisting amyloid deposits to restore organ function. Here we describe a prototypic pan-amyloid binding peptide-antibody fusion molecule (mIgp5) that enhances macrophage uptake of amyloid. Methods: The murine IgG1-IgG2a hybrid immunoglobulin with a pan amyloid-reactive peptide, p5, fused genetically to the N-terminal of the immunoglobulin light chain was synthesized in HEK293T/17 cells. The binding of the p5 peptide moiety was assayed using synthetic amyloid-like fibrils, human amyloid extracts and amyloid-laden tissues as substrates. Binding of radioiodinated mIgp5 with amyloid deposits in vivo was evaluated in a murine model of AA amyloidosis using small animal imaging and microautoradiography. The bioactivity of mIgp5 was assessed in complement fixation and in vitro phagocytosis assays in the presence of patient-derived amyloid extracts and synthetic amyloid fibrils as substrates and in the presence or absence of human serum. Results: Murine Igp5 exhibited highly potent binding to AL and ATTR amyloid extracts and diverse types of amyloid in formalin-fixed tissue sections. In the murine model of systemic AA amyloidosis, 125I-mIgp5 bound rapidly and specifically to amyloid deposits in all organs, including the heart, with no evidence of non-specific uptake in healthy tissues. The bioactivity of the immunoglobulin Fc domain was uncompromised in the context of mIgp5 and served as an effective opsonin. Macrophage-mediated uptake of amyloid extract and purified amyloid fibrils was enhanced by the addition of mIgp5. This effect was exaggerated in the presence of human serum coincident with deposition of complement C5b9. Conclusion: Immunostimulatory, amyloid-clearing therapeutics can be developed by incorporating pan-amyloid-reactive peptides, such as p5, as a targeting moiety. The immunologic functionality of the IgG remains intact in the context of the fusion protein. These data highlight the potential use of peptide-antibody fusions as therapeutics for all types of systemic amyloidosis.

Unraveling Vulnerabilities in Endocrine Therapy-Resistant HER2+/ER+ Breast Cancer.

Background: Breast tumors overexpressing human epidermal growth factor receptor (HER2) confer intrinsic resistance to endocrine therapy (ET), and patients with HER2/ estrogen receptor-positive (HER2+/HR+) breast cancer (BCa) are less responsive to ET than HER2-/ER+. However, real-world evidence reveals that a large subset of HER2+/ER+ patients receive ET as monotherapy, positioning this treatment pattern as a clinical challenge. In the present study, we developed and characterized two distinct in vitro models of ET-resistant (ETR) HER2+/ER+ BCa to identify possible therapeutic vulnerabilities. Methods: To mimic ETR to aromatase inhibitors (AI), we developed two long-term estrogen-deprived (LTED) cell lines from BT-474 (BT474) and MDA-MB-361 (MM361). Growth assays, PAM50 molecular subtyping, genomic and transcriptomic analyses, followed by validation and functional studies, were used to identify targetable differences between ET-responsive parental and ETR-LTED HER2+/ER+ cells. Results: Compared to their parental cells, MM361 LTEDs grew faster, lost ER, and increased HER2 expression, whereas BT474 LTEDs grew slower and maintained ER and HER2 expression. Both LTED variants had reduced responsiveness to fulvestrant. Whole-genome sequencing of the more aggressive MM361 LTED model system identified exonic mutations in genes encoding transcription factors and chromatin modifiers. Single-cell RNA sequencing demonstrated a shift towards non-luminal phenotypes, and revealed metabolic remodeling of MM361 LTEDs, with upregulated lipid metabolism and antioxidant genes associated with ferroptosis, including GPX4. Combining the GPX4 inhibitor RSL3 with anti-HER2 agents induced significant cell death in both the MM361 and BT474 LTEDs. Conclusions: The BT474 and MM361 AI-resistant models capture distinct phenotypes of HER2+/ER+ BCa and identify altered lipid metabolism and ferroptosis remodeling as vulnerabilities of this type of ETR BCa.

Collagen inhibits phagocytosis of amyloid in vitro and in vivo and may act as a 'don't eat me' signal.

BACKGROUND: Systemic amyloidosis refers to a group of protein misfolding disorders characterized by the extracellular deposition of amyloid fibrils in organs and tissues. For reasons heretofore unknown, amyloid deposits are not recognized by the immune system, and progressive deposition leads to organ dysfunction. METHODS: In vitro and in vivo phagocytosis assays were performed to elucidate the impact of collagen and other amyloid associated proteins (eg serum amyloid p component and apolipoprotein E) had on amyloid phagocytosis. Immunohistochemical and histopathological staining regimens were employed to analyze collagen-amyloid interactions and immune responses. RESULTS: Histological analysis of amyloid-laden tissue indicated that collagen is intimately associated with amyloid deposits. We report that collagen inhibits phagocytosis of amyloid fibrils by macrophages. Treatment of 15 patient-derived amyloid extracts with collagenase significantly enhanced amyloid phagocytosis. Preclinical mouse studies indicated that collagenase treatment of amyloid extracts significantly enhanced clearance as compared to controls, coincident with increased immune cell infiltration of the subcutaneous amyloid lesion. CONCLUSIONS: These data suggest that amyloid-associated collagen serves as a 'don't eat me' signal, thereby hindering clearance of amyloid. Targeted degradation of amyloid-associated collagen could result in innate immune cell recognition and clearance of pathologic amyloid deposits.

Targeted isolation and cultivation of uncultivated bacteria by reverse genomics.

Most microorganisms from all taxonomic levels are uncultured. Single-cell genomes and metagenomes continue to increase the known diversity of Bacteria and Archaea; however, while 'omics can be used to infer physiological or ecological roles for species in a community, most of these hypothetical roles remain unvalidated. Here, we report an approach to capture specific microorganisms from complex communities into pure cultures using genome-informed antibody engineering. We apply our reverse genomics approach to isolate and sequence single cells and to cultivate three different species-level lineages of human oral Saccharibacteria (TM7). Using our pure cultures, we show that all three Saccharibacteria species are epibionts of diverse Actinobacteria. We also isolate and cultivate human oral SR1 bacteria, which are members of a lineage of previously uncultured bacteria. Reverse-genomics-enabled cultivation of microorganisms can be applied to any species from any environment and has the potential to unlock the isolation, cultivation and characterization of species from as-yet-uncultured branches of the microbial tree of life.

Expression and function of protein A in Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and the most frequent cause of canine pyoderma. Protein A, a potent virulence factor in S. aureus is encoded by the spa gene. S. pseudintermedius possesses genes seemingly analogous to spa, but the expression and the characteristics of their products have not been directly determined. The purpose of this study was to test isolates from major clonal groups for the presence of spa gene orthologs, quantitate their expression levels, and to characterize protein A in S. pseudintermedius. From the data, it was observed that S. pseudintermedius isolates express genes analogous to spa in S. aureus. Isolates representing major clonal populations in the United States and Europe, ST68 and ST71 respectively, bound significantly higher amounts of canine IgG than isolates with other genetic backgrounds, suggesting that these isolates have a higher density of protein A on their surface. Also, canine IgG bound to protein A on S. pseudintermedius via its Fc region similar to protein A from S. aureus. The mRNA profile differed based on the bacterial sequence types and correlated to the density of protein A on the bacterial surface. Protein A was also found to be secreted during the exponential growth phase. Phagocytosis experiments with S. pseudintermedius show that blocking of protein A enhanced phagocytosis in whole blood, neutrophils and in DH82 canine macrophage-like cell line. Taken together, the results demonstrate that S. pseudintermedius produces protein A that shares S. aureus protein A's ability to bind the Fc region of immunoglobulins and may serve as a potential virulence factor by evading the host immune system.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

Complete Genome Sequence of Staphylococcus aureus Strain Wood 46.

Here, we report the first complete genome sequence of the Staphylococcus aureus strain Wood 46. Wood 46 has played an important role in understanding the virulence and pathogenesis of S. aureus infections. This report will assist efforts in vaccine development against methicillin-resistant S. aureus (MRSA) infections.