In normal healthy tissues and cells, phospholipids are asymmetrically distributed within the plasma membrane, where anionic lipids such as Phosphatidylserine (PS) and Phosphatidylinositol (PI) are typically restricted to the inner surface, whereas other lipids such as Sphingolipids and Cholesterol are localized on the outer membrane surface. In dying cells that are committed to apoptosis, PS asymmetry collapses, and PS is irreversibly externalized to the outer membrane where it serves as one of emblematic signals that flags dying cells for phagocytic uptake, a process referred to as efferocytosis in recent years.
In addition to cells that die by apoptosis, PS is also externalized on stressed viable cells, such as tumor endothelial cells, and on certain activated viable cells such as T cells and monocytes. In contrast to PS mediated phagocytosis of dying cells, externalization of PS on viable cells is important for the resolution of inflammation and a return of cells to a non-activated state.
While externalization of PS on dying cells and stressed/activated cells has important homeostatic functions to maintain healthy tissues and suppress inflammation, dysregulated sustained PS externalization in both solid cancers (tumors are referred to as “wounds that never heal”) and in chronic viral-infected cells becomes pathophysiological by sustaining a prolonged immunosuppressive signal that suppresses host anti-tumor immunity and host anti-viral immunity. Constitutive PS externalization leads to durable immune evasion and disease progression.
Based on this idea that constitutively externalized PS presents a global immunosuppressive signal in cancer and infectious disease, we employseveral pre-clinical approaches to target PS with the goals to enhance host immunity. Our efforts include (i) testing PS-targeting monoclonal antibodies such as Bavituximab, IN11, and 11.31, (ii) evaluating antibodies that target PS-receptors such as Mertk and Axl (TAM receptors), (iii) exploring second generation PS-targeting biologicals that fuse PS binding domains to cytokines and interferons (Gas-IFNs), and (iv) cloning novel anti-phospholipid antibodies from patients with cancer and virus infections as potential novel therapeutics.
We are also interested to understand stress-mediated signaling pathways that lead to activation PS externalization in the tumor microenvironment and during viral infections. Efforts include characterization of lipid scramblases, namely TMEM16F (a calcium activated scramblase), and Xkr8 (a caspase activated scramblase) to assess signaling pathways leading to PS externalization and whether knockouts of scramblases impinge on tumor immunity.
Our ultimate goals aim to better understand chronic PS externalization in pathological states of cancer and chronic infection, and in doing so, reverse immunosuppression to enhance appropriate host immune responses.
TAM receptors (abbreviated Tyro-3, Axl, and Mertk) comprise a family of homologous type Receptor Tyrosine Kinases (RTK) that have important roles in both homeostatic and patho-physiological signal transduction. The main ligands of Mertk are Vitamin K-modified endogenous proteins Gas6 and Protein S (ProS1), heterobifunctional modular proteins that bind TAMs via two carboxyl-terminal laminin-like globular (LG) domains, and an N-terminal Gla domain that binds externalized phosphatidylserine (PS) on stressed viable and caspase-activated apoptotic cells. Our studies indicate that Vitamin K-dependent 𝛾-carboxylation on the N-terminal Gla domain of Gas6 and Protein S is absolutely required for PS binding and TAM activation, implying that TAMs are preferentially active in tissues where there is high externalized PS, such as the tumor microenvironment (TME) and acute virally infected tissues.
Once stimulated, TAMs receptors, particularly Axl, can provide a survival advantage for cancer cells, drive compensatory proliferation, and promote aggressive features of cancer cells such as EMT and metastasis. Concomitantly, on monocytes and tumor-associated macrophages, TAMs, particularly Mertk, promote efferocytosis and acts as an inhibitory receptor that impairs host anti-tumor immunity, functioning akin to a myeloid checkpoint inhibitor.
Our current efforts aim to investigate TAMs expressed on cancer cells as well as TAMs on macrophages subvert host immunity. Our recent studies showing that targeting TAMs in cancer show enhanced host anti-tumor immunity support important roles of TAMs in immune-oncology that can be therapeutically targeted.
The SH2 and SH3 domain containing protein Crk is one of the most versatile genes in eukaryotic cells, binding many signaling proteins that are involved in cell proliferation and cytoskeletal reorganization. The clinical significance of Crk in human cancer has been enumerated in recent years, as Crk is frequently over-expressed in several different cancers, including breast, ovarian, gastric, lung, glioblastoma, and sarcomas and siRNA-mediated targeting of Crk reverses the malignant and metastatic features of these cancers.
We have recently observed that Crk expression drives up-regulation of PD-L1 suggesting that Crk, in addition to acting as a proto-oncogene, may also be involved in intrinsic pathways of immune escape. These findings have led to a new urgency to understand the mechanisms by which Crk promotes malignant transformation in the hope that new information can be exploited to develop therapeutics, particularly for tumors with a predisposition towards invasion, metastasis, and immune escape.