CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation.

Unbalanced protein homeostasis (proteostasis) networks are frequently linked to tumorigenesis, making cancer cells more susceptible to treatments that target proteostasis regulators. Proteasome inhibition is the first licensed proteostasis-targeting therapeutic strategy, and has been proven effective in hematological malignancy patients. However, drug resistance almost inevitably develops, pressing for a better understanding of the mechanisms that preserve proteostasis in tumor cells. Here we report that CD317, a tumor-targeting antigen with a unique topology, was upregulated in hematological malignancies and preserved proteostasis and cell viability in response to proteasome inhibitors (PIs). Knocking down CD317 lowered Ca2+ levels in the endoplasmic reticulum (ER), promoting PIs-induced proteostasis failure and cell death. Mechanistically, CD317 interacted with calnexin (CNX), an ER chaperone protein that limits calcium refilling via the Ca2+ pump SERCA, thereby subjecting CNX to RACK1-mediated autophagic degradation. As a result, CD317 decreased the level of CNX protein, coordinating Ca2+ uptake and thus favoring protein folding and quality control in the ER lumen. Our findings reveal a previously unrecognized role of CD317 in proteostasis control and imply that CD317 could be a promising target for resolving PIs resistance in the clinic.

Regulating Histone Deacetylase Signaling Pathways of Myeloid-Derived Suppressor Cells Enhanced T Cell-Based Immunotherapy.

Immunotherapy has emerged as a promising approach to combat immunosuppressive tumor microenvironment (TME) for improved cancer treatment. FDA approval for the clinical use of programmed death receptor 1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors revolutionized T cell-based immunotherapy. Although only a few cancer patients respond to this treatment due to several factors including the accumulation of immunosuppressive cells in the TME. Several immunosuppressive cells within the TME such as regulatory T cells, myeloid cells, and cancer-associated fibroblast inhibit the activation and function of T cells to promote tumor progression. The roles of epigenetic modifiers such as histone deacetylase (HDAC) in cancer have long been investigated but little is known about their impact on immune cells. Recent studies showed inhibiting HDAC expression on myeloid-derived suppressor cells (MDSCs) promoted their differentiation to less suppressive cells and reduced their immunosuppressive effect in the TME. HDAC inhibitors upregulated PD-1 or PD-L1 expression level on tumor or immune cells sensitizing tumor-bearing mice to anti-PD-1/PD-L1 antibodies. Herein we discuss how inhibiting HDAC expression on MDSCs could circumvent drawbacks to immune checkpoint inhibitors and improve cancer immunotherapy. Furthermore, we highlighted current challenges and future perspectives of HDAC inhibitors in regulating MDSCs function for effective cancer immunotherapy.

Synergistic Tumor Cytolysis by NK Cells in Combination With a Pan-HDAC Inhibitor, Panobinostat.

Histone deacetylases (HDAC) are frequently overexpressed in tumors, and their inhibition has shown promising anti-tumor effects. However, the synergistic effects of HDAC inhibition with immune cell therapy have not been fully explored. Natural killer (NK) cells are cytotoxic lymphocytes for anti-tumor immune surveillance, with immunotherapy potential. We showed that a pan-HDAC inhibitor, panobinostat, alone demonstrated anti-tumor and anti-proliferative activities on all tested tumors in vitro. Additionally, panobinostat co-treatment or pretreatment synergized with NK cells to mediate tumor cell cytolysis. Mechanistically, panobinostat treatment increased the expression of cell adhesion and tight junction-related genes, promoted conjugation formation between NK and tumor cells, and modulates NK cell-activating receptors and ligands on tumor cells, contributing to the increased tumor cytolysis. Finally, panobinostat therapy led to better tumor control and synergized with anti-PD-L1 therapy. Our data highlights the anti-tumor potential of HDAC inhibition through tumor-intrinsic toxicity and enhancement of NK -based immunotherapy.

Upregulation of V-ATPase by STAT3 Activation Promotes Anoikis Resistance and Tumor Metastasis.

Most cancer mortality results from metastatic tumor cells and not the localized tumor. Overcoming anoikis is one of the most important steps for detached tumor cells to migrate and metastasize. However, the molecular mechanisms remain to be fully deciphered. Herein, our study revealed upregulation of vacuolar ATPase (V-ATPase) in cancer cells during ECM detachment plays a key role in anoikis evasion. V-ATPase is an enzyme complex that utilizes energy from ATP hydrolysis to maintain cellular homeostasis and had been reported to enhance cancer progression. In this study, V-ATPase inhibition sensitized human cervical cancer, breast cancer, and murine melanoma cells to anoikis via increased ROS production, accumulation of misfolded protein, and impaired pulmonary metastasis in vivo. Scavenging ROS restored anoikis resistance and clearance of misfolded protein accumulation in the tumor cells. Mechanistically, STAT3 upregulates V-ATPase expression while blockade of STAT3 activity repressed V-ATPase expression in these tumor cells as well as sensitized cells to anoikis, increased ROS production, and misfolded protein accumulation. Altogether, our data demonstrate an unreported role of STAT3 in mediating the upregulation of V-ATPase to promote anoikis resistance, thus provides an alternative option to target cancer metastasis.

Mechanisms for Modulating Anoikis Resistance in Cancer and the Relevance of Metabolic Reprogramming.

The attachment of cells to the extracellular matrix (ECM) is the hallmark of structure-function stability and well-being. ECM detachment in localized tumors precedes abnormal dissemination of tumor cells culminating in metastasis. Programmed cell death (PCD) is activated during tumorigenesis to clear off ECM-detached cells through "anoikis." However, cancer cells develop several mechanisms for abrogating anoikis, thus promoting their invasiveness and metastasis. Specific factors, such as growth proteins, pH, transcriptional signaling pathways, and oxidative stress, have been reported as drivers of anoikis resistance, thus enhancing cancer proliferation and metastasis. Recent studies highlighted the key contributions of metabolic pathways, enabling the cells to bypass anoikis. Therefore, understanding the mechanisms driving anoikis resistance could help to counteract tumor progression and prevent metastasis. This review elucidates the dynamics employed by cancer cells to impede anoikis, thus promoting proliferation, invasion, and metastasis. In addition, the authors have discussed other metabolic intermediates (especially amino acids and nucleotides) that are less explored, which could be crucial for anoikis resistance and metastasis.

Lipidomics data showing the effect of lipofermata on myeloid-derived suppressor cells in the spleens of tumor-bearing mice.

The regulation of myeloid-derived suppressor cells (MDSCs) function is key for effective tumor immunotherapy. Recent lipidomics data revealed that MDSCs accumulate lipid species thereby promote their immunosuppressive activity on T cells. However, genetic manipulation of fatty acid transport protein 2 in mice reduced lipid accumulation in polymorphonuclear MDSCs. Herein we present for the first time lipidome of splenic MDSCs from B16F10 melanoma-bearing mice treated with FATP2 inhibitor - lipofermata compared to the control group. B16F10 were subcutaneously injected into the left flank of wild-type C57BL/6 mice, either lipofermata or vehicle was administered to the mice every day starting from day 7 post-tumor injection for 2 weeks. CD11b+Gr1+ cells from the spleen referred to as MDSCs were sorted on a flow cytometer machine for lipid extraction. Lipid was extracted using methyl­tert­butyl ether as previously described with slight modification, followed by liquid chromatography-mass spectrophotometry lipid profiling using a Q-Exactive instrument coupled with HPLC. The raw scans were identified and quantified with LipidSearch while raw data for various lipid species available on the Mendeley Data repository [1]. The lipid profiles reveal change in lipid species following blockade of FATP2 expression in MDSCs compared to the control. These data were collected in connection to a co-submitted paper [2].

Regulation of ROS in myeloid-derived suppressor cells through targeting fatty acid transport protein 2 enhanced anti-PD-L1 tumor immunotherapy.

Despite the remarkable success and efficacy of immune checkpoint blockade (ICB) therapy against the PD-1/PD-L1 axis, it induces sustained responses in a sizeable minority of cancer patients due to the activation of immunosuppressive factors such as myeloid-derived suppressor cells (MDSCs). Inhibiting the immunosuppressive function of MDSCs is critical for successful cancer ICB therapy. Interestingly, lipid metabolism is a crucial factor in modulating MDSCs function. Fatty acid transport protein 2 (FATP2) conferred the function of PMN-MDSCs in cancer via the upregulation of arachidonic acid metabolism. However, whether regulating lipid accumulation in MDSCs by targeting FATP2 could block MDSCs reactive oxygen species (ROS) production and enhance PD-L1 blockade-mediated tumor immunotherapy remains unexplored. Here we report that FATP2 regulated lipid accumulation, ROS, and immunosuppressive function of MDSCs in tumor-bearing mice. Tumor cells-derived granulocyte macrophage-colony stimulating factor (GM-CSF) induced FATP2 expression in MDSCs by activation of STAT3 signaling pathway. Pharmaceutical blockade of FATP2 expression in MDSCs by lipofermata decreased lipid accumulation, reduced ROS, blocked immunosuppressive activity, and consequently inhibited tumor growth. More importantly, lipofermata inhibition of FATP2 in MDSCs enhanced anti-PD-L1 tumor immunotherapy via the upregulation of CD107a and reduced PD-L1 expression on tumor-infiltrating CD8+T-cells. Furthermore, the combination therapy blocked MDSC's suppressive role on T- cells thereby enhanced T-cell's ability for the production of IFN-γ. These findings indicate that FATP2 plays a key role in modulating lipid accumulation-induced ROS in MDSCs and targeting FATP2 in MDSCs provides a novel therapeutic approach to enhance anti-PD-L1 cancer immunotherapy.

Targeting Oxidative Phosphorylation-Proteasome Activity in Extracellular Detached Cells Promotes Anoikis and Inhibits Metastasis.

Metastasis arises owing to tumor cells' capacity to evade pro-apoptotic signals. Anoikis-the apoptosis of detached cells (from the extracellular matrix (ECM)) is often circumvented by metastatic cells as a result of biochemical and molecular transformations. These facilitate cells' ability to survive, invade and reattach to secondary sites. Here, we identified deregulated glucose metabolism, oxidative phosphorylation, and proteasome in anchorage-independent cells compared to adherent cells. Metformin an anti-diabetic drug that reduces blood glucose (also known to inhibit mitochondrial Complex I), and proteasome inhibitors were employed to target these changes. Metformin or proteasome inhibitors alone increased misfolded protein accumulation, sensitized tumor cells to anoikis, and impaired pulmonary metastasis in the B16F10 melanoma model. Mechanistically, metformin reduced cellular ATP production, activated AMPK to foster pro-apoptotic unfolded protein response (UPR) through enhanced expression of CHOP in ECM detached cells. Furthermore, AMPK inhibition reduced misfolded protein accumulation, thus highlight relevance of AMPK activation in facilitating metformin-induced stress and UPR cell death. Our findings provide insights into the molecular biology of anoikis resistance and identified metformin and proteasome inhibitors as potential therapeutic options for tumor metastasis.

Blockade of myeloid-derived suppressor cell function by valproic acid enhanced anti-PD-L1 tumor immunotherapy.

Regardless of the remarkable clinical success of immune checkpoint blockade (ICB) against PD-1/PD-L1 pathway, this approach has encountered drawbacks in most patients due to the activation of tumor immunosuppressive factors such as myeloid-derived suppressor cells (MDSCs). Histone deacetylase (HDAC) inhibitors combat ICB resistance by attenuating the immunosuppressive function of MDSCs and increasing PD-L1 expression on tumor cells. However, whether an HDAC inhibitor - valproic acid (VPA) suppression of MDSCs function could enhance PD-L1 blockade-mediated tumor immunotherapy remains unknown. Here we report that VPA and anti-PD-L1 antibody combined treatment promoted the polarization of bone marrow-derived precursor cells into M-MDSCs. Interestingly, the combination treatment of VPA and anti-PD-L1 antibody activated IRF1/IRF8 transcriptional axis in MDSCs leading to blockade of their immunosuppressive function by downregulating the expression of IL-10, IL-6, and ARG1 while re-activating CD8+ T-cells for the production of TNFα to further enhance anti-tumor immunity. These observations provide further rationale for the combination therapy of VPA with anti-PD-L1 antibody in preclinical settings.