Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1–9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

Histone deacetylases (HDACs) play important roles in inflammatory diseases like asthma and chronic obstructive pulmonary disease (COPD). Unravelling of and interfering with the functions of specific isoenzymes contributing to inflammation provides opportunities for drug development. Here we synthesize proteolysis targeting chimeras (PROTACs) for degradation of class I HDACs in which o-aminoanilide-based class I HDAC inhibitors are tethered to the cereblon ligand pomalidomide. One of these PROTACs, denoted HD-TAC7, showed promising degradation effects for HDAC3 with a DC₅₀ value of 0.32 μM. In contrast to biochemical evidence using siRNA, HD-TAC7 showed a minimal effect on gene expression in LPS/IFNγ-stimulated RAW 264.7 macrophages. The lack of effect can be attributed to downregulation of the NF-κB subunit p65, which is a known side effect of pomalidomide treatment. Altogether, we describe a novel PROTAC that enables selective downregulation of HDAC3 levels, however we note that concomitant downregulation of the NF-κB subunit p65 can confound the biological outcome.

Epigenetic mechanisms are important for the regular development and maintenance of the tissue-specific expression of cytokine genes. One of the crucial cytokines involved in cancer and inflammation is macrophage migration inhibitory factor (MIF), which triggers the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways by binding to CD74 and other receptors. Altered expression of this cytokine and altered activity states of the connected pathways are linked to inflammatory disease and cancer. Therapeutic strategies based on epigenetic mechanisms have the potential to regulate MIF-mediated signaling in cancer and inflammation.

Macrophage migration inhibitory factor (MIF) is involved in protein‐protein interactions that play key roles in inflammation and cancer. Current strategies to develop small molecule modulators of MIF functions are mainly restricted to the MIF tautomerase active site. Here, we use this site to develop proteolysis targeting chimera (PROTAC) in order to eliminate MIF from its protein‐protein interaction network. We report the first potent MIF‐directed PROTAC, denoted MD13, which induced almost complete MIF degradation at low micromolar concentrations with a DC₅₀ around 100 nM in A549 cells. MD13 suppresses the proliferation of A549 cells, which can be explained by deactivation of the MAPK pathway and subsequent induction of cell cycle arrest at the G2/M phase. MD13 also exhibits antiproliferative effect in a 3D tumor spheroid model. In conclusion, we describe the first MIF‐directed PROTAC (MD13) as a research tool, which also demonstrates the potential of PROTACs in cancer therapy.

Macrophage polarization plays a crucial role in inflammatory processes. The histone deacetylase 3 (HDAC3) has a deacetylase‐independent function that can activate pro‐inflammatory gene expression in lipopolysaccharide‐stimulated M1‐like macrophages and cannot be blocked by traditional small‐molecule HDAC3 inhibitors. Here we employed the proteolysis targeting chimera (PROTAC) technology to target the deacetylase‐independent function of HDAC3. We developed a potent and selective HDAC3‐directed PROTAC, P7, which induces nearly complete HDAC3 degradation at low micromolar concentrations in both THP‐1 cells and human primary macrophages. P7 increases the anti‐inflammatory cytokine secretion in THP‐1‐derived M1‐like macrophages. Importantly, P7 decreases the secretion of pro‐inflammatory cytokines in M1‐like macrophages derived from human primary macrophages. This can be explained by the observed inhibition of macrophage polarization from M0‐like into M1‐like macrophage. In conclusion, we demonstrate that the HDAC3‐directed PROTAC P7 has anti‐inflammatory activity and blocks macrophage polarization, demonstrating that this molecular mechanism can be targeted with small molecule therapeutics.

Transcriptional coactivators p300 and CBP catalyze the acetylation of lysine residues in histone proteins. Upregulation of p300 and CBP has been associated with lung, colorectal and hepatocellular cancer, indicating an important role of p300 and CBP in tumorigenesis. Recently, the novel p300 and CBP-selective inhibitor A485 became available, which was shown to inhibit proliferation of 124 different cancer cell lines. Here, we found that downregulation of EP300 or CREBBP enhances apoptosis upon TRAIL stimulation in non-small-cell lung cancer (NSCLC) cells. A485 upregulates pro- and anti-apoptotic genes at the mRNA level, implying an apoptosis-modulating effect in NSCLC cells. However, A485 alone does not induce apoptosis. Interestingly, we observed that the number of apoptotic cells increases upon combined treatment with A485 and TRAIL. Therefore, A485, as a TRAIL-sensitizer, was used in combination with TRAIL in wild type of NSCLC cell lines (HCC827 and H1650) and cells with acquired erlotinib resistance (HCC827-ER and H1650-ER). Our results show that the combination of A485 and TRAIL synergistically increases cell death and inhibits long-term cell proliferation. Furthermore, this combination inhibits the growth of 3D spheroids of EGFR-TKI-resistant cells. Taken together, we demonstrate a successful combination of A485 and TRAIL in EGFR-TKI-sensitive and resistant NSCLC cells.