Key Points• Human and mouse macrophages share partially conserved gene and protein expression programs in the resting or M2 activated state. • TGM2 is a novel M2 marker consistently induced in human and mouse M2 macrophages.The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4-activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma. (Blood. 2013;121(9):e57-e69)

SummaryMultinucleated giant cells (MGCs) form by fusion of macrophages and are presumed to contribute to the removal of debris from tissues. In a systematic in vitro analysis, we show that IL-4-induced MGCs phagocytosed large and complement-opsonized materials more effectively than their unfused M2 macrophage precursors. MGC expression of complement receptor 4 (CR4) was increased, but it functioned primarily as an adhesion integrin. In contrast, although expression of CR3 was not increased, it became functionally activated during fusion and was located on the extensive membrane ruffles created by excess plasma membrane arising from macrophage fusion. The combination of increased membrane area and activated CR3 specifically equips MGCs to engulf large complement-coated targets. Moreover, we demonstrate these features in vivo in the recently described complement-dependent therapeutic elimination of systemic amyloid deposits by MGCs. MGCs are evidently more than the sum of their macrophage parts.

BackgroundInterference with post-translational modifications such as methylation and acetylation of DNA and histones may enhance the intrinsic anti-tumor capacity of the immune system. Using the iOTarg genetic screening platform, salt-inducible kinase 3 (SIK3) was recently identified as a novel immune checkpoint that controls T cell-mediated apoptosis in tumor cells. SIK3, a serine/threonine kinase of the AMPK family, regulates pro-survival gene expression in tumor cells through epigenetic modulation of the NF κB-driven gene landscape via histone deacetylase 4 (HDAC4), causing the tumor to evade T cell-mediated killing.MethodsIn turn, SIK3 knockout or knockdown abates downstream pro-survival signaling and sensitizes a panel of murine and human tumor cells to death receptor-mediated apoptosis.OMX-0407, an orally available, single-digit nanomolar inhibitor of SIK3 was shown to effectively reduce TNF-induced HDAC4 phosphorylation and downstream NF-κB activity in a dose-dependent manner, thereby enhancing caspase-mediated apoptosis in murine and human tumor cell lines. Decreased intratumoral NF-κB activity was demonstrated in vivo with an MC38 NF κB-luc reporter cell line.ResultsInhibition of the pro-tumorigenic NF-κB pathway using OMX-0407 monotherapy translated into significant tumor growth inhibition (TGI) as well as prolonged survival in the highly infiltrated syngeneic murine colorectal carcinoma model MC38 (76% TGI). Moreover, OMX-0407 repolarized the tumor microenvironment (TME) by strongly reducing the number of regulatory T cells (T-regs) and M2-polarized macrophages in the tumor bed, while not affecting the peripheral T cell compartment. Thereby, exposure to OMX-0407 achieved a pronounced pro-inflammatory TME, characterized by a rise in activated cytotoxic T lymphocytes (CTL) and an increased CTL-to-T-reg ratio.Using the breast cancer mouse model EMT6, which represents an immune-excluded, cold tumor phenotype, we demonstrated that despite the minimal anti-tumor efficacy of OMX-0407 and anti-PD-1 monotherapy, respectively, upon combination treatment, both therapies synergize by combining apoptosis sensitization with a reduction in immunosuppressive TME and an increase in cytotoxic T cell activity. Combination treatment resulted in partial and complete tumor remissions in 60% of the animals, along with a significant prolongation of overall survival.ConclusionsIn summary, OMX-0407, a first-in-class oral SIK3 inhibitor, demonstrates potent monotherapy efficacy in a pro-inflammatory tumor setting by reshaping the immune compartment and sensitizing tumor cells to death receptor-mediated apoptosis. The ability of OMX-0407 to remodel an immunosuppressed TME in a generally cold tumor setting, harbors great clinical potential for OMX-0407 combination therapy with anti-PD-1/PD-L1 immune checkpoint blockade, specifically in patients with high unmet medical need who are resistant to current immune checkpoint inhibitor monotherapy.

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Post-translational modifications of DNA and histones can improve the intrinsic antitumor capacity of the immune system. Using the iOTarg screening platform, salt-inducible kinase 3 (SIK3) was identified as a novel epigenetic modulator in cancer therapy. SIK3, a serine/threonine kinase of the AMP-activated protein kinase family, is known for regulating the NF-κB driven gene landscape through phosphorylation of class IIa histone deacetylases (HDACs) and CREB-regulated transcriptional coactivators causing the tumor to evade death receptor-mediated killing. Moreover, salt inducible kinases were recently described to regulate cell cycle checkpoints and thereby promote cancer cell proliferation. We demonstrate that SIK3 knockdown abates downstream pro-survival signaling and induces cell death in a distinct panel of tumor cell lines. OMX-0407, an orally available, single-digit nanomolar inhibitor of SIK3 was shown to effectively reduce TNF-induced HDAC4 phosphorylation and downstream NF-κB activity in a dose-dependent manner, thereby enhancing apoptosis in murine and human tumor cell lines. OMX-0407 dose-dependent suppression of intratumoral NF-κB activity was shown in vivo in an MC38 NF-κB-luc reporter cell line. Using OMX-0407 monotherapy, this translated to significant tumor growth inhibition as well as prolonged survival in the highly immune infiltrated syngeneic murine colorectal carcinoma model MC38. Besides its direct inhibitory effects on cancer cells, OMX-0407 repolarizes the tumor microenvironment (TME) by strongly decreasing regulatory T cells (T-regs) and M2-polarized macrophages in the tumor bed, while not affecting the peripheral T cell compartment. Thus, exposure to OMX-0407 leads to a distinct pro-inflammatory TME, characterized by an expansion of activated cytotoxic T lymphocytes (CTL) and an increased CTL-to-T-reg ratio. Using an immune-excluded tumor phenotype as seen in the breast cancer mouse model EMT6, we demonstrated that OMX-0407 and anti-PD-1 act synergistically by combining the sensitization towards cell death with a reduction in immunosuppressive TME and an increase in cytotoxic T cell activity, despite having only minimal anti-tumor efficacy as monotherapy. Thereby, partial or complete tumor remission in 60% of the animals and extension of overall survival were achieved. In summary, OMX-0407, a first-in-class oral SIK3 inhibitor, demonstrates potent monotherapy efficacy in a pro-inflammatory tumor setting by reshaping the immune compartment and accelerating tumor cell death. The ability of OMX-0407 to remodel an immunosuppressed TME in a generally cold tumor setting, harbors great clinical potential for OMX-0407 combination therapy with anti-PD-1/PD-L1 immune checkpoint blockade, specifically in patients with high unmet medical need who are resistant to current immune checkpoint inhibitor monotherapy. Citation Format: Christina Hartl, Ilona-Petra Maser, Tillmann Michels, Ronny Milde, Vanessa Klein, Philipp Beckhove, Nisit Khandelwal, Hannes Loferer, Stefan Bissinger. OMX-0407, a highly potent SIK3 inhibitor, sensitizes tumor cells to cell death and eradicates tumors in combination with PD-1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3708.

Tumor-associated macrophages (TAMs) are important players to maintain immunosuppression within the tumor microenvironment (TME) and attenuate the function of effector immune cells to promote tumor survival. High density of TAMs is well-recognized as a feature of tumor progression and poor prognostic factor across various tumor types. Therefore, therapeutic strategies to dampen TAMs’ suppressive potential in TME and reprogram them towards a pro-inflammatory phenotype are being increasingly appreciated as being paramount for an effective immunotherapy. Nevertheless, the key mediators of TAM-induced immunosuppression remain largely unknown, identification of which can lead to the development of novel immunotherapeutic strategies aimed at re-educating the immunosuppressive TAMs to an anti-tumor phenotype. To systematically interrogate the genes responsible for the immune-suppressive phenotype of TAMs, we developed a functional, high-throughput genetic screening platform (iOTarg™) based on human primary monocyte-derived M2-like macrophages. Using CRISPR, we knocked out a proprietary library of well-expressed and druggable genes (1400 genes) in two independent donor-derived M2-like macrophages and cocultured them with activated autologous effector T cells (TCs). We achieved nearly complete knockout (KO) efficacy over a range of control genes. Functional impact of individual KO on macrophage viability and phenotype, as well as on TC activity, was measured using multiparametric assay readouts. Initial hits were reconfirmed in three additional donors in a secondary screen that integrated an additional tumor lysis readout. As expected, knockout of CSF1R, which is essential for macrophage maintenance, resulted in a dramatic loss of macrophage cell viability, whereas KO of established TAM markers, TREM2 and Clever-1, induced a change in macrophage phenotype. Furthermore, KO of immune-inhibitory receptor LILRB2 reduced the M2-like phenotype and restored TC activity. Inhibition of M2 activation of macrophages and subsequent increase in TC activation culminated in strong tumor cell killing for a subset of genes, highlighting an untapped repertoire of novel TAM-associated immune-checkpoint targets. Taken together, we report for the first time a highly sophisticated target discovery platform that addresses the functional role of any TAM-expressed gene in regulating macrophage viability, phenotype, T cell activity and even its gross impact on tumor cell lysis, all done in a high-throughput format employing CRISPR-edited primary human immune cells and multi-parametric functional immunological assays. As a result, we could confirm well-known targets in clinical testing, as well as identify additional novel targets that could lead to first-in-class, TME-based therapeutics and expansion of treatment options in immune oncology. Citation Format: Kritika Sudan, Tillmann Michels, Carmen Amerhauser, Claudia Tschulik, Leonie Majunke, Lucille Albert, Valentina Volpin, Adriana Turqueti-Neves, Ronny Milde, Nisit Khandelwal. A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment [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 3471.