Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phospholipase C-β1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition.

Anthrax lethal toxin (LeTx) is a cytotoxic virulence factor that causes cell cycle arrest and cell death in various cell types. However, susceptibility to the cytotoxic effects varies depending on cell types. In proliferating monocytes, LeTx has only transient cytotoxic effects due to activation of the phosphoinositide 3-kinase (PI3K)-AKT-mediated adaptive responses. To date, the mechanism of LeTx in activating PI3K-AKT signaling axis is unknown. This study shows that the histone deacetylase 8 (HDAC8) is involved in activating PI3K-AKT signaling axis through down-regulating the phosphatase and tensin homolog 1 (PTEN) in human monocytic THP-1 cells. The HDAC8-specific activator TM-2-51 and inhibitor PCI-34051 enhanced and prevented, respectively, AKT activation and cell cycle progression in LeTx-treated cells. Furthermore, HDAC8 induced tri-methylation of histone H3 lysine 27 (H3K27me3), which is known to suppress PTEN expression, through at least in part down-regulating the H3K27me3 eraser Jumonji Domain Containing (JMJD) 3. Importantly, the JMJD3-specific inhibitor GSK-J4 induced AKT activation and protected cell cycle arrest in LeTx-treated cells, regardless the presence of HDAC8 activity. Collectively, this study for the first time demonstrated that HDAC8 activity determines susceptibility to cell cycle arrest induced by LeTx, through regulating the PI3K-PTEN-AKT signaling axis.

The programmed cell death protein 1 (PD-1) plays a critical role in cancer immune evasion. Blocking the PD-1-PD-L1 interaction by monoclonal antibodies has shown remarkable clinical efficacy in treating certain types of cancer. However, antibodies are costly to produce, and antibody-based therapies can cause immune-related adverse events. To address the limitations associated with current PD-1/PD-L1 blockade immunotherapy, we aimed to develop peptide-based inhibitors of the PD-1/PD-L1 interaction as an alternative means to PD-1/PD-L1 blockade antibodies for anti-cancer immunotherapy. Through the functional screening of peptide arrays encompassing the ectodomains of PD-1 and PD-L1, followed by the optimization of the hit peptides for solubility and stability, we have identified a 16-mer peptide, named mL7N, with a remarkable efficacy in blocking the PD-1/PD-L1 interaction both in vitro and in vivo. The mL7N peptide effectively rejuvenated PD-1-suppressed T cells in multiple cellular systems designed to recapitulate the PD-1/PD-L1 interaction in the context of T-cell receptor signaling. Furthermore, PA-mL7N, a chimera of the mL7N peptide coupled to albumin-binding palmitic acid (PA), significantly promoted breast cancer cell killing by peripheral blood mononuclear cells ex vivo and significantly curbed tumor growth in a syngeneic mouse model of breast cancer. Our work raises the prospect that mL7N may serve as a prototype for the development of a new line of peptide-based immunomodulators targeting the PD-1/PD-L1 immune checkpoint with potential applications in cancer treatment.

Macrophages undergo different cellular states upon activation that can be hyporesponsive (tolerated) or hyperresponsive (primed or trained) to subsequent stimuli. Epigenetic modifications are known to play key roles in determining these cellular states. However, little is known about the role of signaling pathways that lead to these epigenetic modifications. Here, we examined the effects of various inhibitors targeting key signaling pathways induced by lipopolysaccharide (LPS) on tolerance and priming in murine macrophages. We found that a prolonged inhibition (>18 h) of the mitogen-activated protein kinase (MEK)1/2—extracellular signal-regulated kinase (ERK)1/2 signaling axis reversed tolerance and primed cells in expressing interleukin (IL)-1β and other inflammatory cytokines such as IL-6, tumor necrosis factor (TNF)α, and CXCL10. The ectopic expression of catalytically active and inactive MEK1 mutants suppressed and enhanced IL-1β expression, respectively. A transcriptomic analysis showed that cells primed by the MEK1/2 inhibitor U0126 expressed higher levels of gene sets associated with immune responses and cytokine/chemokine production, but expressed lower levels of genes with cell cycle progression, chromosome organization, and heterochromatin formation than non-primed cells. Of interest, the mRNA expressions of the histone 3 lysine 9 (H3K9) methyltransferase Suv39h1 and the H3K9 methylation reader Cbx5 were substantially suppressed, whereas the H3K9 demethylase Kdm7a was enhanced, suggesting a role of the MEK1/2-ERK signaling axis in H3K9 demethylation. The H3K9 trimethylation levels in the genomic regions of IL-1β, TNFα, and CXCL10 were decreased by U0126. Also, the H3K9 methyltransferase inhibitor BIX01294 mimicked the U0126 training effects and the overexpression of chromobox homolog (CBX)5 prevented the U0126 training effects in both RAW264.7 cells and bone-marrow-derived macrophages. Collectively, these data suggest that the prolonged inhibition of the MEK1/2-ERK signaling axis reverses tolerance and primed macrophages likely through decreasing the H3K9 methylation levels.

Macrophages undergo different cellular states upon activation that can be hyporesponsive (tolerated) or hyperresponsive (primed or trained) to subsequent stimuli. Epigenetic modifications are known to play a key role in determining these cellular states. However, little is known about the role of signaling pathways that lead to these epigenetic modifications. Here, we examined the effects of various inhibitors targeting key signaling pathways induced by lipopolysaccharide (LPS) on tolerance and priming in the murine macrophages. We found that a prolonged inhibition (>18 h) of the MEK1/2-ERK signaling axis reversed tolerance and primed cells in expressing IL-1β and other inflammatory cytokines such as IL-6, TNFα, and CXCL10. Ectopic expression of catalytically active and inactive MEK1 mutants suppressed and enhanced IL-1β expression, respectively. Primed cells by the MEK1/2 inhibitor U0126 expressed higher levels of gene sets associated with immune responses and cytokine/chemokine production but lower levels of genes with cell cycle progression, chromosome organization, and heterochromatin formation than non-primed cells. Of interest, expression of the histone 3 lysine 9 (H3K9) methyltransferase Suv39h1 and the H3K9 methylation reader Cbx5 was substantially suppressed, whereas the H3K9 demethylase Kdm7a was enhanced, suggesting a role of the MEK1/2-ERK signaling axis in H3K9 demethylation. The H3K9 trimethylation levels in the genomic regions of IL-1β, TNFα, and CXCL10 were decreased by U0126. Also, the H3K9 methyltransferase inhibitor BIX01294 mimicked and overexpression of Cbx5 prevented the U0126 training effects in both RAW264.7 cells and bone marrow-derived macrophages. Collectively, these data suggest that prolonged inhibition of the MEK1/2-ERK signaling axis reverses tolerance and primed macrophages likely through decreasing H3K9 methylation levels.

Background: Targeting tumor-associated macrophages (TAMs) is a promising immunotherapy for cancers, but current strategies are limited due to strategic caveats. PU.1 is a transcription factor required for macrophage generation and differentiation. To date, the effect of PU.1 inhibition on solid tumors is unknown. Methods: This study examines the anti-tumor effect of PU.1 inhibition and its mechanism using the small-molecule DB2313 in mouse melanoma and breast tumor models. Results: We found that inhibition of PU.1 by DB2313 suppresses B16-OVA melanoma and 4T1 breast tumor growth in mice. In the melanoma tumor model, DB2313 enhanced tumor recruitment of CD4⁺ T helper 1 (Th1) and cytotoxic T/natural killer (NK) cells by targeting TAMs. Transcriptome and targeted gene expression analyses revealed that PU.1 inhibition by DB2313 and small-interference RNAs enhances CXCL9 expression in bulk tumors, TAMs, and bone marrow-derived macrophages. The anti-tumor effects of DB2313 were abolished by depleting macrophages with clodronate or inhibiting the CXCL9-CXCR3 chemokine axis using CXCL9- or CXCR3-neutralizing antibodies. Conclusions: These results suggest that pharmacological inhibition of PU.1 suppresses tumor growth by at least promoting the infiltration of lymphocytes into tumors through the CXCL9-CXCR3 chemokine axis. Our study establishes a framework for developing TAM-modulating immunotherapies by targeting the transcriptional factor PU.1.