Birge, R. B. | Search bioRxiv

bioRxiv2025

Phospholipid Scramblases TMEM16F and Xkr8 regulate distinct features of Phosphatidylserine (PS) externalization and immune regulation in the tumor microenvironment to regulate tumor growth

The phospholipid scramblases Xkr8 and TMEM16F externalize phosphatidylserine (PS) by distinct mechanisms. Xkr8, is activated by caspase-mediated proteolytic cleavage, and in synergy with inactivation of P4-ATPase flippases, results in the irreversible externalization of PS on apoptotic cells and an "eat-me" signal for efferocytosis. In contrast, TMEM16F is a calcium activated scramblase that reversibly externalizes PS on viable cells via the transient increase in intracellular calcium in live cells. The tumor microenvironment (TME) is abundant with exposed PS, resulting from prolonged oncogenic and metabolic stresses and high apoptotic indexes of tumors. Such chronic PS externalization in the TME has been linked to host immune evasion from interactions of PS with inhibitory PS receptors such as TAM and TIM receptors. Here, in an effort to better understand the contributions of apoptotic vs live cell PS-externalization to tumorigenesis and immune evasion, we employed an E0771 orthotopic breast cancer model and genetically ablated Xkr8 and TMEM16F using CRISPR/Cas9. While neither the knockout of Xkr8 nor TMEM16F showed defects in cell intrinsic properties related to proliferation, tumor-sphere formation, and growth factor signaling, both knockouts suppressed tumorigenicity in immune-competent mice, but not in NOD/SCID or RAG-KO immune-deficient strains. Mechanistically, Xkr8-KO tumors suppressed macrophage-mediated efferocytosis, and TMEM16F-KO suppressed ER stress/calcium-induced PS externalization. Our data support an emerging idea in immune-oncology that constitutive PS externalization, mediated by scramblase dysregulation on tumor cells, supports immune evasion in the tumor microenvironment. This links apoptosis/efferocytosis and oncogenic stress involving calcium dysregulation, contributing to PS-mediated immune escape and cancer progression.

cancer biology↗

bioRxiv2025

Phosphatidylserine (PS)-targeting chimeric Interferon (IFN) fusion proteins for anti-tumor applications

In viable healthy cells, membrane phospholipids are asymmetrically distributed across the lipid bilayer, whereby the anionic phospholipid phosphatidylserine is virtually all distributed on the inner leaflet of the plasma membrane. During apoptosis, phospholipid asymmetry collapses and PS is externalized to the external leaflet where it serves as an "eat-me" signal for efferocytosis, the process whereby dying cells are engulfed and degraded by phagocytes. PS is also externalized on viable activated tumor endothelial cells, stromal cells and cancer cells in the tumor microenvironment reflecting a pathophysiological state of solid cancers that function to suppress host anti-tumor immunity. Several strategies have been envisioned to target dysregulated PS in the tumor microenvironment including PS binding proteins such as Annexin V and PS-targeting monoclonal antibodies (Bavituximab) with promising preclinical results. Here, in an attempt to enhance the efficacy of PS-targeting therapeutics, we have generated a series of recombinant chimeric fusion proteins that fuse type I and type III IFNs (IFN-{beta}-IFN-{lambda}) into a single polypeptide chain separated by a short linker. The IFN-{beta}-IFN-{lambda} fusion proteins retain functions of both type I and type III IFNs but show combined effects to improve biological function as well as enhance anti-tumor activities. To localize IFNs to sites of externalized PS, we next fused the IFN-{beta}-IFN-{lambda} chimeric protein to the PS-targeting gamma-carboxyglutamic acid-rich (Gla) domain of Growth Arrest Specific factor 6 (Gas-6), rendering these IFN biologics as PS targeting modalities. Gas6-IFN-{beta}-IFN-{lambda} proteins selectively bind PS as evident by solid-phase ELISA assays as well as bind PS-positive cells, including apoptotic cells and cells that express CDC50 subunit mutant of the ATP11C flippase. In vivo, Gas6-IFN-{beta}-IFN-{lambda} retain strong anti-tumor activities in a syngeneic model when expressed ectopically in a E0771 breast cancer model and B16-F10 melanoma models. Collectively, we report on the generation and utility of a series of novel in class IFN fusion proteins that target the immune stimulatory features of IFNs to the PS externalization in the tumor microenvironment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/634764v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@6fecceorg.highwire.dtl.DTLVardef@23d0d0org.highwire.dtl.DTLVardef@1621a39org.highwire.dtl.DTLVardef@1a7aa6f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Gas6-IFN--IFN-{lambda} (VitK) have tri-functional activities, acting on a diverse set of cell types to induce an anti-tumor affect. The Gas6 domain aids in homing to the PS rich tumor microenvironment, binding to apoptotic or live stressed PS positive tumor cells. The Gla domain directly binds to PS, whereas the EGF domains help in oligomerization and signal amplification resulting from intermolecular disulphide bonds. The IFN- domain acts on immune cells such as dendritic cells and macrophages, inducing an interferon response, whereas the IFN-{lambda} domain acts on the tumor epithelial cells, inducing tumor intrinsic anti-tumor activity. C_FIG

cancer biology↗