Signaling lymphocytic activation molecule family receptors as potential immune therapeutic targets in solid tumors.

Recently, cancer immunotherapy has revolutionized cancer treatment. Various forms of immunotherapy have a manageable safety profile and result in prolongation of overall survival in patients with solid tumors, but only in a proportion of patients. Various factors in the tumor microenvironment play critical roles and may be responsible for this lack of therapeutic response. Signaling lymphocytic activation molecule family (SLAMF) members are increasingly being studied as factors impacting the tumor immune microenvironment. SLAMF members consist of nine receptors mainly expressed in immune cells. However, SLAMF receptors have also been detected in cancer cells, and they may be involved in a spectrum of anti-tumor immune responses. Here, we review the current knowledge of the expression of SLAMF receptors in solid tumors and tumor-infiltrating immune cells and their association with patient outcomes. Furthermore, we discuss the therapeutic potential of targeting SLAMF receptors to improve outcomes of cancer therapy in solid tumors. We believe the research on SLAMF receptor-targeted strategies may enhance anti-cancer immunity in patients with solid tumors and improve clinical outcomes.

Sulfamate Acetamides as Self-Immolative Electrophiles for Covalent Ligand-Directed Release Chemistry.

Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of "turn-on" probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.

CD74 as a regulator of transcription in normal B cells.

CD74 is receptor for the cytokine macrophage migration inhibitory factor (MIF). MIF binding to CD74 induces a signaling cascade resulting in the release of its cytosolic intracellular domain (CD74-ICD) that serves as a transcriptional regulator in chronic lymphocytic leukemia (CLL) cells. In the current study, we investigated the transcriptional and regulatory function of CD74-ICD in normal B cells. We show that following activation, CD74-ICD forms a complex in the cytosol with transcription factors, like PAX5, and binds the chromatin at a significantly higher number of sites compared with its binding in CLL cells. The expression of a major portion of these bound genes is shut down in the malignant cells. The CD74-ICD:PAX5 complex binds the promoter areas of a tumor-suppressor gene, DMTF1, and downregulates its expression through inhibition of transcription. These findings can help identify novel therapeutic pathways that are regulated during oncogenic transformation and are targets for future treatments.

CXCR4 and CD74 together enhance cell survival in response to macrophage migration-inhibitory factor in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small, mature CD5+ B lymphocytes in the blood, marrow, and lymphoid organs. Cell survival depends on interaction with the leukemic microenvironment. However, the mechanisms controlling CLL cell survival are still incompletely understood. Macrophage migration-inhibitory factor (MIF), a pro-inflammatory and immunoregulatory chemokine-like cytokine, interacts with CXCR4, a major chemokine receptor, as well as with CD74/invariant chain, a single-pass type II receptor. In this study, we analyzed the roles of CXCR4, CD74, and MIF in CLL. Mononuclear cells from patients with hematological malignancies were analyzed for coexpression of CXCR4 and CD74 by flow cytometry. Strong co- and overexpression of CXCR4 and CD74 were observed on B cells of CLL patients (n = 10). Survival and chemotaxis assays indicated that CXCR4 and CD74 work together to enhance the survival and migration of malignant cells in CLL. Blockade of the receptors, either individually or in combination, promoted cell death and led to an abrogation of MIF-driven migration responses in murine and human CLL cells, suggesting that joint activation of both receptors is crucial for CLL cell survival and mobility. These findings indicate that the MIF/CXCR4/CD74 axis represents a novel therapeutic target in CLL.

Polysome-CAGE of TCL1-driven chronic lymphocytic leukemia revealed multiple N-terminally altered epigenetic regulators and a translation stress signature.

The transformation of normal to malignant cells is accompanied by substantial changes in gene expression programs through diverse mechanisms. Here, we examined the changes in the landscape of transcription start sites and alternative promoter (AP) usage and their impact on the translatome in TCL1-driven chronic lymphocytic leukemia (CLL). Our findings revealed a marked elevation of APs in CLL B cells from Eµ-Tcl1 transgenic mice, which are particularly enriched with intra-genic promoters that generate N-terminally truncated or modified proteins. Intra-genic promoter activation is mediated by (1) loss of function of 'closed chromatin' epigenetic regulators due to the generation of inactive N-terminally modified isoforms or reduced expression; (2) upregulation of transcription factors, including c-Myc, targeting the intra-genic promoters and their associated enhancers. Exogenous expression of Tcl1 in MEFs is sufficient to induce intra-genic promoters of epigenetic regulators and promote c-Myc expression. We further found a dramatic translation downregulation of transcripts bearing CNY cap-proximal trinucleotides, reminiscent of cells undergoing metabolic stress. These findings uncovered the role of Tcl1 oncogenic function in altering promoter usage and mRNA translation in leukemogenesis.

CD74 is a regulator of hematopoietic stem cell maintenance.

Hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that have the capacity for self-renewal and differentiate into all blood cell lineages. These cells are the most useful cells for clinical transplantations and for regenerative medicine. So far, it has not been possible to expand adult hematopoietic stem cells (HSCs) without losing their self-renewal properties. CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), and its mRNA is known to be expressed in HSCs. Here, we demonstrate that mice lacking CD74 exhibit an accumulation of HSCs in the bone marrow (BM) due to their increased potential to repopulate and compete for BM niches. Our results suggest that CD74 regulates the maintenance of the HSCs and CD18 expression. Its absence leads to induced survival of these cells and accumulation of quiescent and proliferating cells. Furthermore, in in vitro experiments, blocking of CD74 elevated the numbers of HSPCs. Thus, we suggest that blocking CD74 could lead to improved clinical insight into BM transplant protocols, enabling improved engraftment.

The survival and function of IL-10-producing regulatory B cells are negatively controlled by SLAMF5.

B cells have essential functions in multiple sclerosis and in its mouse model, experimental autoimmune encephalomyelitis, both as drivers and suppressors of the disease. The suppressive effects are driven by a regulatory B cell (Breg) population that functions, primarily but not exclusively, via the production of IL-10. However, the mechanisms modulating IL-10-producing Breg abundance are poorly understood. Here we identify SLAMF5 for controlling IL-10+ Breg maintenance and function. In EAE, the deficiency of SLAMF5 in B cells causes accumulation of IL10+ Bregs in the central nervous system and periphery. Blocking SLAMF5 in vitro induces both human and mouse IL-10-producing Breg cells and increases their survival with a concomitant increase of a transcription factor, c-Maf. Finally, in vivo SLAMF5 blocking in EAE elevates IL-10+ Breg levels and ameliorates disease severity. Our results suggest that SLAMF5 is a negative moderator of IL-10+ Breg cells, and may serve as a therapeutic target in MS and other autoimmune diseases.

CD84 is a regulator of the immunosuppressive microenvironment in multiple myeloma.

Multiple myeloma (MM) is characterized by an accumulation of malignant plasma cells (PCs) within the BM. The BM microenvironment supports survival of the malignant cells and is composed of cellular fractions that foster myeloma development and progression by suppression of the immune response. Despite major progress in understanding the biology and pathophysiology of MM, this disease is still incurable and requires aggressive treatment with significant side effects. CD84 is a self-binding immunoreceptor belonging to the signaling lymphocyte activation molecule (SLAM) family. Previously, we showed that CD84 bridges between chronic lymphocytic leukemia cells and their microenvironment, and it regulates T cell function. In the current study, we investigated the role of CD84 in MM. Our results show that MM cells express low levels of CD84. However, these cells secrete the cytokine macrophage migration inhibitory factor (MIF), which induces CD84 expression on cells in their microenvironment. Its activation leads to an elevation of expression of genes regulating differentiation to monocytic/granulocytic-myeloid-derived suppressor cells (M-MDSCs and G-MDSCs, respectively) and upregulation of PD-L1 expression on MDSCs, which together suppress T cell function. Downregulation of CD84 or its blocking reduce MDSC accumulation, resulting in elevated T cell activity and reduced tumor load. Our data suggest that CD84 might serve as a novel therapeutic target in MM.

Transcription Dynamics Regulate Poly(A) Tails and Expression of the RNA Degradation Machinery to Balance mRNA Levels.

Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.

Golgi organization is regulated by proteasomal degradation.

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.

Bone marrow dendritic cells support the survival of chronic lymphocytic leukemia cells in a CD84 dependent manner.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B lymphocytes. The microenvironment of the CLL cells is a vital element in the regulation of the survival of these malignant cells. CLL cell longevity is dependent on external signals, originating from cells in their microenvironment including secreted and surface-bound factors. Dendritic cells (DCs) play an important part in tumor microenvironment, but their role in the CLL bone marrow (BM) niche has not been studied. We show here that CLL cells induce accumulation of bone marrow dendritic cells (BMDCs). Depletion of this population attenuates disease expansion. Our results show that the support of the microenvironment is partly dependent on CD84, a cell surface molecule belonging to the Signaling Lymphocyte Activating Molecule (SLAM) family of immunoreceptors. Our results suggest a novel therapeutic strategy whereby eliminating BMDCs or blocking the CD84 expressed on these cells may reduce the tumor load.

Circulating Picomolar Levels of CCL2 Downregulate Ongoing Chronic Experimental Autoimmune Encephalomyelitis by Induction of Regulatory Mechanisms.

Multiple sclerosis is an inflammatory disease of the CNS characterized by neurologic impairment resulting from primary demyelination and axonal damage. The pathogenic mechanisms of disease development include Ag-specific T cell activation and Th1 differentiation, followed by T cell and macrophage migration into the CNS. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells. We previously demonstrated that picomolar levels of CCL2 strongly restrict the development of inflammation in models of inflammatory bowel disease. Moreover, CCR2 deficiency in T cells promotes a program inducing the accumulation of Foxp3+ regulatory T cells while decreasing the levels of Th17 cells in vivo. In the current study, the effect of picomolar levels of CCL2 on the autoimmune inflammatory response associated with a multiple sclerosis-like disease in mice was analyzed. We found that low dosages of CCL2 were effective in suppressing MOG-induced experimental autoimmune encephalomyelitis (EAE), and they downregulated chronic EAE. The modulation of EAE by CCL2 was associated with downregulation of Th1/Th17 cells and upregulation of TGF-ß and induction of regulatory CD4+Foxp3 T cells. Most strikingly, these low levels of CCL2 induced formation of highly functional regulatory T cells. Thus, this study strongly supports the potential use of CCL2 as a regulatory mediator for treating inflammatory autoimmune diseases.

SLAMF9 regulates pDC homeostasis and function in health and disease.

SLAMF9 belongs to the conserved lymphocytic activation molecule family (SLAMF). Unlike other SLAMs, which have been extensively studied, the role of SLAMF9 in the immune system remained mostly unexplored. By generating CRISPR/Cas9 SLAMF9 knockout mice, we analyzed the role of this receptor in plasmacytoid dendritic cells (pDCs), which preferentially express the SLAMF9 transcript and protein. These cells display a unique capacity to produce type I IFN and bridge between innate and adaptive immune response. Analysis of pDCs in SLAMF9-/- mice revealed an increase of immature pDCs in the bone marrow and enhanced accumulation of pDCs in the lymph nodes. In the periphery, SLAMF9 deficiency resulted in lower levels of the transcription factor SpiB, elevation of pDC survival, and attenuated IFN-α and TNF-α production. To define the role of SLAMF9 during inflammation, pDCs lacking SLAMF9 were followed during induced experimental autoimmune encephalomyelitis. SLAMF9-/- mice demonstrated attenuated disease and delayed onset, accompanied by a prominent increase of immature pDCs in the lymph node, with a reduced costimulatory potential and enhanced infiltration of pDCs into the central nervous system. These results suggest the crucial role of SLAMF9 in pDC differentiation, homeostasis, and function in the steady state and during experimental autoimmune encephalomyelitis.

SLAMF receptors on normal and malignant B cells.

The Signaling Lymphocyte Activation Molecule family (SLAMF) is a collection of nine surface receptors expressed mainly on hematopoietic cells, and was found to modulate the behavior of immune cells. SLAMF receptors are expressed on B cells in health and disease. Each SLAM receptor has a unique differential expression pattern during the development and activation of B cells. Furthermore, recent findings have revealed a principal role for this family of receptors in B cell malignancies, emphasizing their importance in the control of malignant cell survival, cell to cell communication within the tumor microenvironment, retention in the supporting niches and regulation of T cell anti-tumor response. This review summarizes the latest studies regarding SLAMF expression and behavior in B cells and in B cell pathologies, and discusses the therapeutic potential of these receptors.

CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of mature B lymphocytes in the peripheral blood, lymphoid tissues, and bone marrow. CLL is characterized by profound immune defects leading to severe infectious complications. T cells are numerically, phenotypically, and functionally highly abnormal in CLL, with only limited ability to exert antitumor immune responses. Exhaustion of T cells has also been suggested to play an important role in antitumor responses. CLL-mediated T cell exhaustion is achieved by the aberrant expression of several inhibitory molecules on CLL cells and their microenvironment, prominently the programmed cell death ligand 1/programmed cell death 1 (PD-L1/PD-1) receptors. Previously, we showed that CD84, a member of the SLAM family of receptors, bridges between CLL cells and their microenvironment. In the current study, we followed CD84 regulation of T cell function. We showed that cell-cell interaction mediated through human and mouse CD84 upregulates PD-L1 expression on CLL cells and in their microenvironment and PD-1 expression on T cells. This resulted in suppression of T cell responses and activity in vitro and in vivo. Thus, our results demonstrate a role for CD84 in the regulation of immune checkpoints by leukemia cells and identify CD84 blockade as a therapeutic strategy to reverse tumor-induced immune suppression.

LPS-mediated cell surface expression of CD74 promotes the proliferation of B cells in response to MIF.

Macrophage migration inhibitory factor (MIF) is a chemokine-like inflammatory cytokine, which plays a pivotal role in the pathogenesis of inflammatory and cardiovascular diseases as well as cancer. We previously identified MIF as a novel B cell chemokine that promotes B cell migration through non-cognate interaction with the CXC chemokine receptor CXCR4 and CD74, the surface form of MHC class II invariant chain. In this study, we have analyzed the regulation of the MIF receptors under inflammatory conditions by investigating the impact of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) on CD74 and CXCR4 expression in B lymphocytes. We found that both LPS and TNF-α stimulation of primary B cells and the human B myeloma cell line RPMI-8226 enhanced protein expression as well as mRNA levels of CD74 in a time- and dose-dependent manner. By contrast, no effect on CXCR4 expression was observed. Selective inhibition of IκBα phosphorylation significantly attenuated LPS-induced expression of CD74, suggesting the contribution of NF-κB signaling pathways to the regulation of CD74 expression. Importantly, individual or simultaneous blockade of MIF or CD74 using specific neutralizing antibodies markedly affected B cell proliferation after LPS exposure. Taken together, our findings unveil a connection between the pro-proliferative activity of MIF/CD74 signaling in B cells and inflammation, offering novel target mechanisms in inflammatory cardiovascular or autoimmune pathogenesis.

T Cells Regulate Peripheral Naive Mature B Cell Survival by Cell-Cell Contact Mediated through SLAMF6 and SAP.

The control of lymphoid homeostasis is the result of a very fine balance between lymphocyte production, proliferation, and apoptosis. In this study, we focused on the role of T cells in the maintenance/survival of the mature naive peripheral B cell population. We show that naive B and T cells interact via the signaling lymphocyte activation molecule (SLAM) family receptor, SLAMF6. This interaction induces cell type-specific signals in both cell types, mediated by the SLAM-associated protein (SAP) family of adaptors. This signaling results in an upregulation of the expression of the cytokine migration inhibitory factor in the T cells and augmented expression of its receptor CD74 on the B cell counterparts, consequently enhancing B cell survival. Furthermore, in X-linked lymphoproliferative disease patients, SAP deficiency reduces CD74 expression, resulting in the perturbation of B cell maintenance from the naive stage. Thus, naive T cells regulate B cell survival in a SLAMF6- and SAP-dependent manner.