Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.
◥Purpose: Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2-selective inhibitor, has had success in the clinic, another family member, Bcl-x L , has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-x L inhibitor that broadens the therapeutic activity while minimizing Bcl-x L -mediated thrombocytopenia.Experimental Design: We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-x L and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time.Results: We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-x L , and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2-and Bcl-x L -dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2-selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-x L -dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models.Conclusions: AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-x L inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.
We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with 3-9 progressive gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (3 gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the use of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective “multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.
Secreted interorgan communication factors encode key regulators of homeostasis. However, long-standing questions surround their origins/destinations, mechanisms of interactions, and the number of proteins involved. Progress has been hindered by the lack of methodologies for these factors' largescale identification and characterization, as conventional approaches cannot identify low-abundance factors and the origins and destinations of secreted proteins. We established an in vivo platform to investigate secreted protein trafficking between organs proteome-wide, whereby engineered promiscuous biotin ligase BirA*G3 (a relative of TurboID) biotinylates all proteins in a subcellular compartment of one tissue, and biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Using this platform, we identified 51 putative muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, of which 60-70% have human orthologs. We demonstrate, in particular, that conserved fat body-derived novel interorgan communication factors CG31326, CG2145, and CG4332 promote muscle activity. Our results indicate that the communication network of secreted proteins is vast, and we identified systemic functions for a number of these factors.This approach is widely applicable to studies in interorgan, local and intracellular protein trafficking networks, non-conventional secretion, and to mammalian systems, under healthy or diseased states. One Sentence SummaryWe developed an in vivo platform to investigate protein trafficking between organs proteomewide, provide a resource for interorgan communication factors, and determined conserved adipokines that affect muscles. Main TextLocal tissue homeostasis is becoming increasingly well-understood. However, the physiological importance and presence of secreted interorgan communication factors is only beginning to be documented from experiments in Drosophila and vertebrates. Secreted factors acting directly or indirectly between organs encode key regulators of systemic homeostasis (1). These factors traffic, or translocate, intracellularly from their production sites within cells (2) to distal organs through blood (1). For instance, Droujinine et al 3 adipokines including leptin and adiponectin encode adipose tissue-derived systemic metabolic regulators(1). In addition, myokines such as irisin (cleaved form of FNDC5) and interleukin-6 are secreted by muscles to control metabolism in adipose tissue (1). Despite their importance, identification of interorgan communication factors is technically challenging, and a number of published results were later determined to be irreproducible or controversial (1,(3)(4)(5). Also, origins and/or destinations of factors including glucagon-like peptide 1 (GLP-1), ghrelin, leptin, cholecystokinin (CCK), and growth differentiation factor 11 (GDF-11) need to be clarified (1, 5). Moreover, because large-scale screening
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