ß-Catenin signaling in alveolar macrophages enhances lung metastasis through a TNF-dependent mechanism.

The main cause of malignancy-related mortality is metastasis. Although metastatic progression is driven by diverse tumor-intrinsic mechanisms, there is a growing appreciation for the contribution of tumor-extrinsic elements of the tumor microenvironment, especially macrophages, which correlate with poor clinical outcomes. Macrophages consist of bone marrow-derived and tissue-resident populations. In contrast to bone marrow-derived macrophages, the transcriptional pathways that govern the pro-metastatic activities of tissue-resident macrophages (TRMs) remain less clear. Alveolar macrophages (AMs) are a TRM population with critical roles in tissue homeostasis and metastasis. Wnt/ß-catenin signaling is a hallmark of cancer and has been identified as a pathologic regulator of AMs in infection. We tested the hypothesis that ß-catenin expression in AMs enhances metastasis in solid tumor models. Using a genetic ß-catenin gain-of-function approach, we demonstrated that (a) enhanced ß-catenin in AMs heightened lung metastasis; (b) ß-catenin activity in AMs drove a dysregulated inflammatory program strongly associated with Tnf expression; and (c) localized TNF-α blockade abrogated this metastatic outcome. Last, ß-catenin gene CTNNB1 and TNF expression levels were positively correlated in AMs of patients with lung cancer. Overall, our findings revealed a Wnt/ß-catenin/TNF-α pro-metastatic axis in AMs with potential therapeutic implications against tumors refractory to the antineoplastic actions of TNF-α.

Inhibiting the biogenesis of myeloid-derived suppressor cells enhances immunotherapy efficacy against mammary tumor progression.

While immune checkpoint inhibitors (ICIs) have transformed the therapeutic landscape in oncology, they are effective in select subsets of patients. Efficacy may be limited by tumor-driven immune suppression, of which 1 key mechanism is the development of myeloid-derived suppressor cells (MDSCs). A fundamental gap in MDSC therapeutics is the lack of approaches that target MDSC biogenesis. We hypothesized that targeting MDSC biogenesis would mitigate MDSC burden and bolster tumor responses to ICIs. We tested a class of agents, dihydroorotate dehydrogenase (DHODH) inhibitors, that have been previously shown to restore the terminal differentiation of leukemic myeloid progenitors. DHODH inhibitors have demonstrated preclinical safety and are under clinical study for hematologic malignancies. Using mouse models of mammary cancer that elicit robust MDSC responses, we demonstrated that the DHODH inhibitor brequinar (a) suppressed MDSC production from early-stage myeloid progenitors, which was accompanied by enhanced myeloid maturation; (b) augmented the antitumor and antimetastatic activities of programmed cell death 1-based (PD-1-based) ICI therapy in ICI-resistant mammary cancer models; and (c) acted in concert with PD-1 blockade through modulation of MDSC and CD8+ T cell responses. Moreover, brequinar facilitated myeloid maturation and inhibited immune-suppressive features in human bone marrow culture systems. These findings advance the concept of MDSC differentiation therapy in immuno-oncology.

Mitigating the prevalence and function of myeloid-derived suppressor cells by redirecting myeloid differentiation using a novel immune modulator.

BACKGROUND: Immune suppression is common in neoplasia and a major driver is tumor-induced myeloid dysfunction. Yet, overcoming such myeloid cell defects remains an untapped strategy to reverse suppression and improve host defense. Exposure of bone marrow progenitors to heightened levels of myeloid growth factors in cancer or following certain systemic treatments promote abnormal myelopoiesis characterized by the production of myeloid-derived suppressor cells (MDSCs) and a deficiency in antigen-presenting cell function. We previously showed that a novel immune modulator, termed 'very small size particle' (VSSP), attenuates MDSC function in tumor-bearing mice, which was accompanied by an increase in dendritic cells (DCs) suggesting that VSSP exhibits myeloid differentiating properties. Therefore, here, we addressed two unresolved aspects of the mechanism of action of this unique immunomodulatory agent: (1) does VSSP alter myelopoiesis in the bone marrow to redirect MDSC differentiation toward a monocyte/macrophage or DC fate? and (2) does VSSP mitigate the frequency and suppressive function of human tumor-induced MDSCs? METHODS: To address the first question, we first used a murine model of granulocyte-colony stimulating factor-driven emergency myelopoiesis following chemotherapy-induced myeloablation, which skews myeloid output toward MDSCs, especially the polymorphonuclear (PMN)-MDSC subset. Following VSSP treatment, progenitors and their myeloid progeny were analyzed by immunophenotyping and MDSC function was evaluated by suppression assays. To strengthen rigor, we validated our findings in tumor-bearing mouse models. To address the second question, we conducted a clinical trial in patients with metastatic renal cell carcinoma, wherein 15 patients were treated with VSSP. Endpoints in this study included safety and impact on PMN-MDSC frequency and function. RESULTS: We demonstrated that VSSP diminished PMN-MDSCs by shunting granulocyte-monocyte progenitor differentiation toward monocytes/macrophages and DCs with heightened expression of the myeloid-dependent transcription factors interferon regulatory factor-8 and PU.1. This skewing was at the expense of expansion of granulocytic progenitors and rendered the remaining MDSCs less suppressive. Importantly, these effects were also demonstrated in a clinical setting wherein VSSP monotherapy significantly reduced circulating PMN-MDSCs, and their suppressive function. CONCLUSIONS: Altogether, these data revealed VSSP as a novel regulator of myeloid biology that mitigates MDSCs in cancer patients and reinstates a more normal myeloid phenotype that potentially favors immune activation over immune suppression.

Myeloid-driven mechanisms as barriers to antitumor CD8+ T cell activity.

The adaptive immune system is essential for host defense against pathogenic challenges, and a major constituent is the CD8+ cytotoxic T cell. Ordinarily, CD8+ T cells are endowed with a unique ability to specifically recognize and destroy their targets. However, in cases where disease emerges, especially in cancer, the efficacy of the CD8+ T cell response is frequently counterbalanced in a 'tug-of-war' by networks of tumor-driven mechanisms of immune suppression. As a result, antitumor CD8+ T cell activity is hampered, which contributes to clinical manifestations of disease. It is now well-recognized that prominent elements of that network include myeloid-derived suppressor cells (MDSC) and macrophages which assume tumor-supportive phenotypes. Both myeloid populations are thought to arise as consequences of chronic inflammatory cues produced during the neoplastic process. Numerous preclinical studies have now shown that inhibiting the production, trafficking and/or function of these immune suppressive myeloid populations restore antitumor CD8+ T cell responses during both immune surveillance or in response to immune-targeted interventions. Correlative studies in cancer patients support these preclinical findings and, thus, have laid the foundation for ongoing clinical trials in patients receiving novel agents that target such myeloid elements alone or in combination with immunotherapy to potentially improve cancer patient outcomes. Accordingly, this review focuses on how and why it is important to study the myeloid-T cell interplay as an innovative strategy to boost or reinvigorate the CD8+ T cell response as a critical weapon in the battle against malignancy.

IFN regulatory factor-8 expression in macrophages governs an antimetastatic program.

High macrophage infiltration in cancer is associated with reduced survival in animal models and in patients. This reflects a shift in the macrophage response from a tumor-suppressive to tumor-supportive program governed by transcriptional events regulated by the inflammatory milieu. Although several transcription factors are known to drive a prometastatic program, those that govern an antimetastatic program are less understood. IFN regulatory factor-8 (IRF8) is integral for macrophage responses against infections. Using a genetic loss-of-function approach, we tested the hypothesis that IRF8 expression in macrophages governs their capacity to inhibit metastasis. We found that: (a) metastasis was significantly increased in mice with IRF8-deficient macrophages; (b) IRF8-deficient macrophages displayed a program enriched for genes associated with metastasis; and (c) lower IRF8 expression correlated with reduced survival in human breast and lung cancer, as well as melanoma, with high or low macrophage infiltration. Thus, a macrophagehiIRF8hi signature was more favorable than a macrophagehiIRF8lo signature. The same held true for a macrophageloIRF8hi vs. a macrophageloIRF8lo signature. These data suggest that incorporating IRF8 expression levels within a broader macrophage signature or profile strengthens prognostic merit. Overall, to our knowledge, our findings reveal a previously unrecognized role for IRF8 in macrophage biology to control metastasis or predict outcome.

Novel tropolones induce the unfolded protein response pathway and apoptosis in multiple myeloma cells.

Tropolones are small organic compounds with metal-directing moieties. Tropolones inhibit the proliferation of cancer cell lines, possibly through their effects on metalloenzymes such as select histone deacetylases (HDACs). Pan-HDAC inhibitors are therapeutically beneficial in the treatment of multiple myeloma, however there is interest in the use of more selective HDAC inhibitor therapy to minimize adverse side effects. We hypothesized that tropolones might have anti-myeloma activities. To this end, a series of novel α-substituted tropolones were evaluated for effects on multiple myeloma cells. While all tested tropolones showed some level of cytotoxicity, MO-OH-Nap had consistently low IC50 values between 1-11 µM in all three cell lines tested and was used for subsequent experiments. MO-OH-Nap was found to induce apoptosis in a concentration-dependent manner. Time course experiments demonstrated that MO-OH-Nap promotes caspase cleavage in a time frame that was distinct from the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Furthermore, MO-OH-Nap- and SAHA-treated cells possess unique gene expression patterns, suggesting they promote apoptosis via different mechanisms. In particular, MO-OH-Nap increases the expression of markers associated with endoplasmic reticulum stress and the unfolded protein response. Synergistic cytotoxic effects were observed when cells were treated with the combination of MO-OH-Nap and the proteasome inhibitor bortezomib. However, treatment with MO-OH-Nap did not abrogate the bortezomib-induced increase in aggresomes, consistent with an HDAC6-independent mechanism for the observed synergy. Collectively, these finding support further investigation into the usefulness of α-substituted tropolones as anti-myeloma agents.