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Abstract Background Cancer cells can overexpress CD47, an innate immune checkpoint that prevents phagocytosis upon interaction with signal regulatory protein alpha (SIRPα) expressed in macrophages and other myeloid cells. Several clinical trials have reported that CD47 blockade reduces tumor growth in hematological malignancies. However, CD47 blockade has shown modest results in solid tumors, including melanoma. Our group has demonstrated that histone deacetylase 6 inhibitors (HDAC6is) have immunomodulatory properties, such as controlling macrophage phenotype and inflammatory properties. However, the molecular and cellular mechanisms controlling these processes are not fully understood. In this study, we evaluated the role of HDAC6 in regulating the CD47/SIRPα axis and phagocytosis in macrophages. Methods We tested the role of HDAC6is, especially Nexturastat A, in regulating macrophage phenotype and phagocytic function using bone marrow-derived macrophages and macrophage cell lines. The modulation of the CD47/SIRPα axis and phagocytosis by HDAC6is was investigated using murine and human melanoma cell lines and macrophages. Phagocytosis was evaluated via coculture assays of macrophages and melanoma cells by flow cytometry and immunofluorescence. Lastly, to evaluate the antitumor activity of Nexturastat A in combination with anti-CD47 or anti-SIRPα antibodies, we performed in vivo studies using the SM1 and/or B16F10 melanoma mouse models. Results We observed that HDAC6is enhanced the phenotype of antitumoral M1 macrophages while decreasing the protumoral M2 phenotype. In addition, HDAC6 inhibition diminished the expression of SIRPα, increased the expression of other pro-phagocytic signals in macrophages, and downregulated CD47 expression in mouse and human melanoma cells. This regulatory role on the CD47/SIRPα axis translated into enhanced antitumoral phagocytic capacity of macrophages treated with Nexturastat A and anti-CD47. We also observed that the systemic administration of HDAC6i enhanced the in vivo antitumor activity of anti-CD47 blockade in melanoma by modulating macrophage and natural killer cells in the tumor microenvironment. However, Nexturastat A did not enhance the antitumor activity of anti-SIRPα despite its modulation of macrophage populations in the SM1 tumor microenvironment. Conclusions Our results demonstrate the critical regulatory role of HDAC6 in phagocytosis and innate immunity for the first time, further underscoring the use of these inhibitors to potentiate CD47 immune checkpoint blockade therapeutic strategies.

Background: Cancer cells can overexpress CD47, an innate immune checkpoint that prevents phagocytosis upon interaction with signal regulatory protein alpha (SIRPα) expressed in macrophages and other myeloid cells. Several clinical trials have reported that CD47 blockade reduces tumor growth in hematological malignancies. However, CD47 blockade has shown modest results in solid tumors, including melanoma. Our group has demonstrated that histone deacetylase 6 inhibitors (HDAC6is) have immunomodulatory properties, such as controlling macrophage phenotype and inflammatory properties. However, the molecular and cellular mechanisms controlling these processes are not fully understood. In this study, we evaluated the role of HDAC6 in regulating the CD47/SIRPα axis and phagocytosis in macrophages. Methods: We tested the role of HDAC6is, especially Nexturastat A, in regulating macrophage phenotype and phagocytic function using bone marrow-derived macrophages and macrophage cell lines. The modulation of the CD47/SIRPα axis and phagocytosis by HDAC6is was investigated using murine and human melanoma cell lines and macrophages. Phagocytosis was evaluated via coculture assays of macrophages and melanoma cells by flow cytometry and immunofluorescence. Lastly, to evaluate the antitumor activity of Nexturastat A in combination with anti-CD47 or anti-SIRPα antibodies, we performed in vivo studies using the SM1 and/or B16F10 melanoma mouse models. Results: We observed that HDAC6is enhanced the phenotype of antitumoral M1 macrophages while decreasing the protumoral M2 phenotype. In addition, HDAC6 inhibition diminished the expression of SIRPα, increased the expression of other pro-phagocytic signals in macrophages, and downregulated CD47 expression in mouse and human melanoma cells. This regulatory role on the CD47/SIRPα axis translated into enhanced antitumoral phagocytic capacity of macrophages treated with Nexturastat A and anti-CD47. We also observed that the systemic administration of HDAC6i enhanced the in vivo antitumor activity of anti-CD47 blockade in melanoma by modulating macrophage and natural killer cells in the tumor microenvironment. However, Nexturastat A did not enhance the antitumor activity of anti-SIRPα despite its modulation of macrophage populations in the SM1 tumor microenvironment. Conclusions: Our results demonstrate the critical regulatory role of HDAC6 in phagocytosis and innate immunity for the first time, further underscoring the use of these inhibitors to potentiate CD47 immune checkpoint blockade therapeutic strategies. [ABSTRACT FROM AUTHOR]

In this study, we describe poly (lactic-co-glycolic) acid (PLGA)-based nanoparticles that combine photothermal therapy (PTT) with epigenetic therapy for melanoma. Specifically, we co-encapsulated indocyanine green (ICG), a PTT agent, and Nexturastat A (NextA), an epigenetic drug within PLGA nanoparticles (ICG-NextA-PLGA; INAPs). We hypothesized that combining PTT with epigenetic therapy elicits favorable cytotoxic and immunomodulatory responses that result in improved survival in melanoma-bearing mice. We utilized a nanoemulsion synthesis scheme to co-encapsulate ICG and NextA within stable and monodispersed INAPs. The INAPs exhibited concentration-dependent and near-infrared (NIR) laser power-dependent photothermal heating characteristics, and functioned as effective single-use agents for PTT of melanoma cells in vitro. The INAPs functioned as effective epigenetic therapy agents by inhibiting the expression of pan-histone deacetylase (HDAC) and HDAC6-specific activity in melanoma cells in vitro. When used for both PTT and epigenetic therapy in vitro, the INAPs increased the expression of co-stimulatory molecules and major histocompatibility complex (MHC) Class I in melanoma cells relative to controls. These advantages persisted in vivo in a syngeneic murine model of melanoma, where the combination therapy slowed tumor progression and improved median survival. These findings demonstrate the potential of INAPs as agents of PTT and epigenetic therapy for melanoma.

In this study, we describe poly (lactic-co-glycolic) acid (PLGA)-based nanoparticles that combine photothermal therapy (PTT) with epigenetic therapy for melanoma. Specifically, we co-encapsulated indocyanine green (ICG), a PTT agent, and Nexturastat A (NextA), an epigenetic drug within PLGA nanoparticles (ICG-NextA-PLGA; INAPs). We hypothesized that combining PTT with epigenetic therapy elicits favorable cytotoxic and immunomodulatory responses that result in improved survival in melanoma-bearing mice. We utilized a nanoemulsion synthesis scheme to co-encapsulate ICG and NextA within stable and monodispersed INAPs. The INAPs exhibited concentration-dependent and near-infrared (NIR) laser power-dependent photothermal heating characteristics, and functioned as effective single-use agents for PTT of melanoma cells in vitro. The INAPs functioned as effective epigenetic therapy agents by inhibiting the expression of pan-histone deacetylase (HDAC) and HDAC6-specific activity in melanoma cells in vitro. When used for both PTT and epigenetic therapy in vitro, the INAPs increased the expression of co-stimulatory molecules and major histocompatibility complex (MHC) Class I in melanoma cells relative to controls. These advantages persisted in vivo in a syngeneic murine model of melanoma, where the combination therapy slowed tumor progression and improved median survival. These findings demonstrate the potential of INAPs as agents of PTT and epigenetic therapy for melanoma. [ABSTRACT FROM AUTHOR]

High grade serous ovarian cancer (HGSOC) is the most common and aggressive ovarian cancer subtype with the worst clinical outcome due to intrinsic or acquired drug resistance. Standard treatment involves platinum compounds. Cancer development and chemoresistance is often associated with an increase in histone deacetylase (HDAC) activity. The purpose of this study was to examine the potential of HDAC inhibitors (HDACi) to increase platinum potency in HGSOC. Four HGSOC cell lines with different cisplatin sensitivity were treated with combinations of cisplatin and entinostat (class I HDACi), panobinostat (pan-HDACi), or nexturastat A (class IIb HDACi), respectively. Inhibition of class I HDACs by entinostat turned out superior in increasing cisplatin potency than pan-HDAC inhibition in cell viability assays (MTT), apoptosis induction (subG1), and caspase 3/7 activation. Entinostat was synergistic with cisplatin in all cell lines in MTT and caspase activation assays. MTT assays gave combination indices (CI values) < 0.9 indicating synergism. The effect of HDAC inhibitors could be attributed to the upregulation of pro-apoptotic genes (CDNK1A, APAF1, PUMA, BAK1) and downregulation of survivin. In conclusion, the combination of entinostat and cisplatin is synergistic in HGSOC and could be an effective strategy for the treatment of aggressive ovarian cancer. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is a hematological malignancy of plasma cells that produce a monoclonal immunoglobulin protein. Despite significant advances in the treatment of MM, challenges such as resistance to therapy remain. Currently, inhibition of histone deacetylases (HDACs) is emerging as a potential method for treating cancers. Numerous HDAC inhibitors are being studied for the use in monotherapy or in conjunction with other agents for MM. In the present study, we investigated the anti-myeloma effect of Nexturastat A (NexA), a novel selective HDAC6 inhibitor. We found that NexA impaired MM cells viability in a dose- and time-dependent manner. NexA also provoked a cell cycle arrest at the G1 phase in MM cells . Furthermore, NexA promoted apoptosis of MM cells via transcriptional activation of the p21 promoter, which may through its ability to up-regulate the H3Ac and H4Ac levels. Additionally, NexA could overcome bortezomib (BTZ) resistance in MM cells, and NexA in combination with BTZ had stronger efficacy. We also confirmed that NexA inhibited tumor growth in murine xenograft models of MM. These interesting findings provided the rationale for the future advancement of this novel HDAC6 inhibitor as a potential therapeutic anti-myeloma agent., (© 2019 The Author(s).)

Only a handful of therapies offer significant improvement in the overall survival in cases of melanoma, a cancer whose incidence has continued to rise in the past 30 years. In our effort to identify potent and isoform-selective histone deacetylase (HDAC) inhibitors as a therapeutic approach to melanoma, a series of new HDAC6 inhibitors based on the nexturastat A scaffold were prepared. The new analogues 4d , 4e , and 7b bearing added hydrophilic substituents, so as to establish additional hydrogen bonding on the rim of the HDAC6 catalytic pocket, exhibit improved potency against HDAC6 and retain selectivity over HDAC1. Compound 4d exhibits antiproliferative effects on several types of melanoma and lymphoma cells. Further studies indicates that 4d selectively increases acetylated tubulin levels in vitro and elicits an immune response through down-regulating cytokine IL-10. A preliminary in vivo efficacy study indicates that 4d possesses improved capability to inhibit melanoma tumor growth and that this effect is based on the regulation of inflammatory and immune responses.