Tumor-associated macrophages (TAMs) play a pivotal role in shaping the tumor microenvironment (TME) and influencing the outcomes of immunotherapy. However, most drug screening strategies emphasize tumor cell cytotoxicity and neglect immune effector modulation. Here, we describe a macrophage-centric phenotypic screening platform to identify selective HDAC6 inhibitors that reprogram TAMs toward an antitumor phenotype. Building on the HDAC6 inhibitor SS-208, we synthesized a novel class of tetrazolone-based compounds with potent selectivity and minimal cytotoxicity. Among these, SM-06-09 emerged as a lead candidate, showing subnanomolar HDAC6 inhibition, enhanced macrophage phagocytosis, antigen presentation, and T-cell activation in vitro. In a syngeneic melanoma model, SM-06-09 suppressed tumor growth and promoted M1-like TAM polarization. Combination with anti-PD-1 therapy further enhanced immune infiltration, increased effector memory and central memory T-cells, and improved antitumor efficacy. This study establishes a functional screening framework for identifying immunomodulatory compounds and supports the clinical potential of macrophage-targeted HDAC6 inhibitors as adjuncts to immune checkpoint blockade.

Immune cells can significantly influence the therapeutic outcomes of anti-cancer therapies. Particularly, tumor-associated macrophages (TAMs) mainly exhibit an anti-inflammatory M2-like phenotype and are well recognized for their pro-tumoral activity, which supports tumor growth and suppresses anti-tumor immune responses. Nonetheless, TAMs exhibit remarkable plasticity, allowing for their reprogramming toward an anti-tumoral M1-like phenotype through targeted epigenetic modulations. Our group has previously reported several highly selective histone deacetylase 6 inhibitors (HDAC6is) that promote the polarization of TAMs toward the M1-like phenotype while suppressing the M2-like pro-tumoral phenotype. Despite this promising discovery, the current HDAC6is face several drawbacks, including efficacy at micromolar concentrations, which results in poor isoform selectivity and potential off-target effects. To address these limitations, we developed an advanced screening pipeline for HDAC6is on macrophage functions, and the antitumor benefits associated with their treatment, alone or in combination with anti-PD1 in the immunocompetent SM1 murine melanoma model. Based on an in silico screening of 980 HDAC6is derivatives and a macrophage-based screening described previously, we identified two candidates, SM-05-947 and SM-06-09, that showed superior properties compared to their parent molecules. The screening included the determination of cytotoxicity, specificity of HDAC inhibition, and the modulation of macrophage phenotype. Using bone marrow-derived macrophages, the treatment-associated changes in macrophage functions were characterized. Following dose determination, we studied their antitumor effects on SM1 melanoma tumors when administered as a single agent or combined with anti-PD1 antibodies. The tumor microenvironment was further characterized by immunotyping using flow cytometry, CyToF, and sc-RNA sequencing. The functional assays provided further evidence of increased phagocytosis of tumor cells and improved antigen cross-presentation with HDAC6 inhibition. Specifically, the presence of SIINFEKL peptide in the context of MHC class I in macrophages treated with HDAC6is showed a significant increase in antigen presentation by M1 macrophages. Administration of SM-06-09 and SM-05-947 as a single agent significantly reduced tumor growth when compared to the vehicle group. The two combination groups of HDAC6is and anti-PD1 antibodies further significantly improved these antitumor effects, as observed by flow cytometry, CyToF, and sc-RNA sequencing analysis. In conclusion, these findings provide valuable insights and pave the way toward the development of next-generation HDAC6is with maximum efficiency, minimal toxicity, and enhanced anticancer potential of TAMs. Nithya Gajendran, Manasa Suresh, David Quiceno, Sonia Sebaoui, Xintang Li, Marie Durr, Sebastian J. Marquez Rodriguez, Mario A. Noboa, Sruthi Mohan, Barbora Havlinova, Julia Kudlacova, Annie Heuer, Satish Kumar Reddy Noonepalle, Cyril Barinka, Duncan J. Wardrop, Alejandro Villagra. Advancing cancer immunotherapy through the development of a novel selective HDAC6 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4216.

Tumor-associated macrophages (TAMs) exhibit hybrid phenotypes ranging between M1-like (anti-tumor) and M2-like (pro-tumor) macrophages. In most tumor types, a higher M1/M2 ratio favors antitumor immunity. Therefore, strategies to enhance the M1/M2 ratio can potentially alter the tumor microenvironment (TME) towards antitumor immunity. These strategies include reprogramming resident TAMs, altering the fate of tumor-infiltrating monocytes towards M1-like macrophages, and finally, adoptive cell therapy (ACT) with M1-like macrophages. A significant challenge with the above strategies is to retain the antitumor properties of M1-like macrophages in an overwhelmingly immunosuppressive TME. In this study, we demonstrate a macrophage-based ACT where treatment with HDAC6 inhibitors (HDA6is) retains the M1-like characteristics while significantly suppressing the tumor growth in syngeneic SM1 murine melanoma and with radiation therapy (RT). We reprogrammed GFP-expressing antitumor M1 macrophages ex-vivo with HDAC6 inhibitors to lock into the M1 phenotype and administered intratumorally as ACT in the syngeneic SM1 murine melanoma model and humanized NSG-SGM3 melanoma model. We performed histological analysis of tumors for macrophage markers, immune phenotyping of infiltrated immune cells, single-cell secretome analysis of tumor macrophages, and single-cell RNA-seq analysis of CD45+ immune cell populations. Initially, tail vein injected (i.v) administration of luciferase-expressing monocytes in combination with HDAC6i reached the tumors and diminished SM1 tumor growth compared to the control group. On the contrary, luciferase-expressing M1 macrophages failed to reach the tumor as visualized by IVIS imaging, suggesting that i.v is not an effective route for macrophage therapy. Whereas, intratumor HDAC6i treated M1 macrophage ACT resulted in diminished tumor growth. Single-cell (Sc) RNA-seq analysis of CD45+ sorted immune cells revealed a significant increase in the M1/M2 macrophage ratio in concurrence with flow cytometry data. NicheNet cell-cell interaction analysis indicated that M1-like TAMs activated infiltrating T-cells and monocytes through ligand-receptor interactions. Cell fate analysis of infiltrated monocytes indicated differentiation toward M1-like macrophages in ACT tumors. Flow cytometry analysis indicated an increase in CD8 effector T-cells. Sc-secretome analysis of F4/80 sorted tumor macrophages by the Isoplexis platform revealed polyfunctionality of HDAC6-treated M1 macrophages. Histological examination of tumor sections for macrophage phenotypic markers suggested that transplanted macrophages retained the M1 phenotype post-ACT in both SM1 murine and NSG-SGM3 melanoma tumor models. Our study comprehensively demonstrated the potential of reprogramming macrophages with HDAC6 inhibitors as a viable macrophage cell therapy for treating solid tumors. Satish Kumar Reddy Noonepalle, Maria Gracia Hernandez, Nima Aghdam, Nithya Gajendran, Manasa Suresh, Xintang Li, Abishek Sehgal, David Quiceno, Michael Berrigen, Tessa Knox, Karen Tan, Eduardo Sotomayor, Katherine Chiappinelli, Duncan Wardrop, Anelia Horvath, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Alejandro Villagra. HDAC6 inhibitors reprogram macrophages in antitumor-adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2253.

Abstract The interplay between tumor cells and immune cells within the tumor microenvironment (TME) dictates protumor or antitumor immune responses. Tumor-associated macrophages (TAMs) respond to signals of the TME and exhibit a spectrum of phenotypes ranging between M1 (antitumor) and M2 (protumor) macrophages. In most tumor types, including melanoma, the balance between M1 and M2 macrophages is critical with a higher M1/M2 ratio favoring antitumor immunity. Therefore, strategies enhancing the M1/M2 ratio can significantly alter the TME towards antitumor immunity. In this study, we administered luciferase and GFP-expressing M1 macrophages as an adoptive cell therapy (ACT) through intravenous (i.v) and intratumor routes respectively, into mice bearing SM1 murine melanoma tumors to determine the effective treatment modality. We reprogrammed antitumor M1 macrophages ex-vivo with HDAC6 inhibitors to lock into the M1 phenotype and administered intratumorally as ACT in the syngeneic SM1 murine melanoma and humanized NSG-SGM3 melanoma models. We performed histological analysis of tumors for macrophage markers, immune phenotyping of infiltrated immune cells, single-cell secretome analysis of tumor macrophages, and single-cell RNA-seq analysis of CD45+ immune cell populations to demonstrate the benefit of macrophage ACT. Tail vein injected, luciferase-expressing M1 macrophages localized in the lungs and spleen, failed to reach the tumor as visualized by IVIS imaging, suggesting that i.v administration is ineffective for macrophage therapy. On the contrary, intratumor M1 macrophage ACT resulted in diminished tumor growth. Single-cell RNA-seq analysis of the CD45+ sorted tumor-associated immune cell population revealed distinct macrophage subsets and a significant M1/M2 macrophage ratio increase. NicheNet cell-cell interaction analysis indicated that M1-like TAMs activated infiltrating T-cells and monocytes through ligand-receptor interactions. Trajectory analysis of infiltrated monocytes indicated differentiation toward inflammatory macrophages in ACT tumors. Flow cytometry analysis corroborated that ACT increased the M1/M2 macrophage ratio and an increase in CD8 effector T-cells. Furthermore, HDAC6i-treated macrophages increased antigen cross-presentation. Single-cell secretome analysis of F4/80+ TAMs by the Isoplexis platform revealed polyfunctionality of HDAC6-treated M1 macrophages secreting inflammatory cytokine Tnfa and T-cell recruiting chemokine Cxcl10. Histological examination of tumor sections for macrophage phenotypic markers suggested that transplanted macrophages retained the M1 phenotype post-ACT in both SM1 murine and NSG-SGM3 melanoma tumor models. We demonstrated the potential of reprogramming macrophages ex vivo with HDAC6 inhibitors as a feasible macrophage cell therapy to treat solid tumors. Citation Format: Satish Kumar Reddy Noonepalle, Nithya Gajendran, Manasa Suresh, Xintang Li, Maria D. Hernandez, Christian Zevallos Delgado, Nima Aghdam, Michael Berrigen, Tessa Knox, Karen Tan, Marie Durr, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman H. Lee, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Maho Shibata, Alejandro Villagra. Reprogramming tumor microenvironment with intratumor macrophage adoptive cell therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5247.

BACKGROUND:Macrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies. METHODS:In this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT. RESULTS:Macrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME. CONCLUSIONS:In summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.

Tumor-associated macrophages (TAMs) play a critical role in shaping the tumor microenvironment (TME). Responding to numerous cues from various cell types present in the tumor, TAMs often exhibit a spectrum of phenotypes, often ranging between M1 (inflammatory) and M2 (tumor-supportive) macrophages. Therefore, the M1/M2 macrophage ratio often determines tumor suppression or tumor growth. Higher TAMs infiltration is usually associated with poor prognosis in several cancer types due to their predisposition towards M2 function. Therefore, reprogramming TAMs to the M1 phenotype is a promising avenue to be explored. In this study, we reprogrammed bone marrow-derived macrophages (BMDMs) ex-vivo by treating them with HDAC6 inhibitor (HDAC6i) followed by polarization to M1 phenotype and directly implanted them into immunocompetent tumors. We performed immunohistochemistry of tumors for macrophage markers, immunophenotyping of tumor-infiltrated immune cells by flow cytometry, gene expression analysis by quantitative PCR, and immunoblot analyses. We also performed single-cell analyses, including secretome with Isoplexis platform and CD45+ tumor-infiltrated immune cells by single-cell RNA-seq analysis. To further underscore the translatability of macrophage-based cell therapy, we treated humanized NSG-SGM3 mice bearing melanoma PDX tumors with HDAC6i-treated human M1 macrophages. Adoptive cell therapy (ACT) with HDAC6i-treated M1 macrophages resulted in significant tumor suppression and prolonged survival compared to other cohorts in the study. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered them resistant to M2 polarization. Inhibition of HDAC6 suppressed STAT3 activation and subsequent M2 marker Arg1 expression, further underscoring the role of HDAC6 in macrophage plasticity. M2 re-polarization assay further corroborated that HDAC6i-treated M1 macrophages were resistant to change into M2 phenotype. Single-cell secretome analysis revealed polyfunctionality as HDAC6-treated M1 macrophages were capable of secreting inflammatory cytokines such as Tnfa and t-cell recruiting Cxcl10. Histological analysis of tumor sections for macrophage markers and single-cell transcriptomic analysis of tumor-infiltrated immune cells further corroborated the M1/M2 ratio the increase observed by flow cytometry. Pro-inflammatory gene expression signature from scRNA-seq analysis also correlated with better survival in the SKCM dataset. In both immuno-competent SM1 murine melanoma and humanized NSG-SGM3 melanoma models, ACT enhanced anti-tumor immunity by increasing the M1/M2 ratio and infiltration of CD8 effector T-cells shifting the balance towards anti-tumor immunity. For the first time, we demonstrate that reprogramming macrophages with class-specific HDAC inhibitors is a viable cell therapy option to treat solid tumors. Citation Format: Satish Kumar Reddy Noonepalle, Maria Gracia Hernandez, Christian Zevallos Delgado, Nima Aghdam, Nithya Gajendran, Tessa Knox, Karen Tan, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman Lee, Anatoly Dritschilo, Rohan Fernandes, Maho Shibata, Karthik Musunuri, Alejandro Villagra. Reprogramming macrophages with HDAC6 inhibitors for anti-cancer macrophage-based cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 900.

An efficient, one-pot method for the preparation of 5-(1-hydroxyalkyl)tetrazoles is reported. N-Morpholinomethyl-5-lithiotetrazole, generated by the deprotonation of 4-(N-tetrazolylmethyl)morpholine with LiHMDS, undergoes addition to ketones and aldehydes (both aromatic and aliphatic) to form 5-(1-hydroxyalkyl)tetrazoles in a high yield, after acidic workup. The reported protocol displays a broad substrate scope and functional group tolerance, avoids the use of cyanide- or azide-based reagents, and provides access to sterically congested and unsaturated tetrazoles, which are difficult to access by other means.

A novel approach to the diazatricyclic madangamine ABC ring system and the synthesis of an advanced, differentially protected intermediate for the synthesis of madangamine D is reported. Central to the success of this approach is the iodine(III)-mediated intramolecular oxamidation of an unsaturated O-methyl hydroxamate, a π-N+-type cyclization which proceeds in high yield and with complete regioselectivity to generate the 2-azabicyclo[3.3.1]nonane (morphan) system encompassing rings A and C.

Hydrogen sulfide is produced from l-cysteine by the action of both cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) and increasingly has been found to play a profound regulatory role in a range of physiological processes. Mounting evidence suggests that upregulation of hydrogen sulfide biosynthesis occurs in several disease states, including rheumatoid arthritis, hypertension, ischemic injury, and sleep-disordered breathing. In addition to being critical tools in our understanding of hydrogen sulfide biology, inhibitors of CSE hold therapeutic potential for the treatment of diseases in which increased levels of this gasotransmitter play a role. We describe the discovery and development of a novel series of potent CSE inhibitors that show increased activity over the benchmark inhibitor and, importantly, display high selectivity for CSE versus CBS.

The viral envelope protein hemagglutinin (HA) plays a critical role in influenza entry and thus is an attractive target for novel therapeutics. The small molecule tert-butylhydroquinone (TBHQ) has previously been shown to bind to HA and inhibit HA-mediated entry with low micromolar potency. However, enthusiasm for the use of TBHQ has diminished due to the compound's antioxidant properties. In this work we show that the antioxidant properties of TBHQ are not responsible for the inhibition of HA-mediated entry. In addition, we have performed a structure-activity relationship (SAR) analysis of TBHQ derivatives. We find that the most promising compound, 3-tert-butyl-4-methoxyphenol, exhibits enhanced potency (IC50 = 0.6 μM), decreased toxicity (CC50 = 340 μM), and increased stability (t1/2 > 48 h). Finally, we have characterized the binding properties of 3-tert-butyl-4-methoxyphenol using NMR and molecular dynamics to guide future efforts for chemical optimization.

AbstractReview: [electronic structure and the reactivity of alkyl, aryl, alkylaryl and heteroatm‐stabilized nitrenium ions; 225 refs.

The recent outbreak of H7N9 influenza in China is of high concern to public health. H7 hemagglutinin (HA) plays a critical role in influenza entry and thus HA presents an attractive target for antivirals. Previous studies have suggested that the small molecule tert-butyl hydroquinone (TBHQ) inhibits the entry of influenza H3 HA by binding to the stem loop of HA and stabilizing the neutral pH conformation of HA, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure and immunogenicity, H7 is a related Group 2 HA. In this work we show, using a pseudovirus entry assay, that TBHQ inhibits H7 HA-mediated entry, as well as H3 HA-mediated entry, with an IC50 ~ 6 µM. Using NMR, we show that TBHQ binds to the H7 stem loop region. STD NMR experiments indicate that the aromatic ring of TBHQ makes extensive contact with the H7 HA surface. Limited proteolysis experiments indicate that TBHQ inhibits influenza entry by stabilizing the H7 HA neutral pH conformation. Together, this work suggests that the stem loop region of H7 HA is an attractive target for therapeutic intervention and that TBHQ, which is a widely used food preservative, is a promising lead compound.

The development of a mild, base-free method for the generation of alkylidenecarbenes is reported. Treatment of 5-hydroxyalkyl-1H-tetrazoles with carbodiimides generates products arising from the 1,2-rearrangement or [1,5]-C-H bond insertion of a putative alkylidenecarbene. Formation of this divalent intermediate is proposed to occur by way of a tetraazafulvene, which undergoes extrusion of 2 mol of dinitrogen. Details of this methodology, its application to the synthesis of combretastatin A-4, and an improved route to 5-hydroxyalkyl-1H-tetrazoles are described.

AbstractA general route is developed to unsaturated O‐alkyl hydroxamate precursors which are subjected to a chemoselective oxidative cyclization process.

The 1,2-diamine moiety is a ubiquitous structural motif present in a wealth of natural products, including non-proteinogenic amino acids and numerous alkaloids, as well as in pharmaceutical agents, chiral ligands and organic reagents. The biological activity associated with many of these systems and their chemical utility in general has ensured that the development of methods for their preparation is of critical importance. While a wide range of strategies for the preparation of 1,2-diamines have been established, the diamination of alkenes offers a particularly direct and efficient means of accessing these systems. The purpose of this review is to provide an overview of all methods of direct alkene diamination, metal-mediated or otherwise.

The embodiment of lactam rings within a wealth of physiologically active natural products and pharmaceutical agents ensures that the development of synthetic methods, which facilitate the preparation of these saturated N - heterocycles, is of critical importance. Herein the development of a versatile method for the synthesis of 4 to 8-membered α-vinyl and α-(2-silylvinyl) lactams involving the iodine(III)-mediated oxidative cyclization of unsaturated O -alkyl hydroxamates, which encompass an allylsilane, is reported. Importantly, the outcome of this transformation can be effectively controlled through variation of the substitution pattern at the silicon center. While allyltrimethylsilanes undergo ring closure with desilylation to form α-vinyl lactams, the corresponding triisopropyl and triphenylsilanes cyclize without loss of the larger silyl group to form E- vinylsilanes with excellent stereoselectivity. From a mechanistic standpoint, it is proposed that this reaction proceeds via concerted alkene addition of a singlet nitrenium ion (or its equivalent) to form a bicyclic N -acyl- N -alkoxyaziridinium ion, which undergoes eliminative ring opening.

AbstractThe tetrazoles are prepared via an optimized literature procedure starting from aldehydes, ketones or ynones.