BRD4-specific PROTAC inhibits basal-like breast cancer partially through downregulating KLF5 expression.

Interest in the use of proteolysis-targeting chimeras (PROTACs) in cancer therapy has increased in recent years. Targeting bromodomain and extra terminal domain (BET) proteins, especially bromodomain-containing protein 4 (BRD4), has shown inhibitory effects on basal-like breast cancer (BLBC). However, the bioavailability of BRD4 PROTACs is restricted by their non-selective biodegradability and low tumor-targeting ability. We demonstrated that 6b (BRD4 PROTAC) suppresses BLBC cell growth by targeting BRD4, but not BRD2 and BRD3, for cereblon (CRBN)-mediated ubiquitination and proteasomal degradation. Compound 6b also inhibited expression of Krüppel-like factor 5 (KLF5) transcription factor, a key oncoprotein in BLBC, controlled by BRD4-mediated super-enhancers. Moreover, 6b inhibited HCC1806 tumor growth in a xenograft mouse model. The combination of 6b and KLF5 inhibitors showed additive effects on BLBC. These results suggest that BRD4-specific PROTAC can effectively inhibit BLBC by downregulating KLF5, and that 6b has potential as a novel therapeutic drug for BLBC.

Discovery of Pyrimidinediamine Derivatives as Potent Methuosis Inducers for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a leading malignancy among women that currently lack effective targeted therapeutic agents, and the limitations of treatment have prompted the emergence of new strategies. Methuosis is a novel vacuole-presenting cell death modality that promotes tumor cell death. Hence, a series of pyrimidinediamine derivatives were designed and synthesized through evaluation of their abilities that inhibit proliferation as well as induce methuosis against TNBC cells. Among them, JH530 showed excellent anti-proliferative activities and vacuolization capacity in TNBC. The mechanism research indicated that JH530 caused cell death through inducing methuosis of cancer cells. Furthermore, JH530 inhibited tumor growth remarkably in the HCC1806 xenograft model without an apparent decrease in body weight. Overall, JH530 is a methuosis inducer that displayed remarkable suppression of TNBC growth in vitro and in vivo, which provides a basis for the future progress of more small molecules for TNBC treatment.

A novel methuosis inducer DZ-514 possesses antitumor activity via activation of ROS-MKK4-p38 axis in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most malignant tumors with poor prognosis. Methuosis is a new type of nonapoptotic cell death characterized by the accumulation of cytoplasmic vacuoles. In this study, we synthesized and screened a series of N-phenyl-4-pyrimidinediamine derivatives in TNBC cells, finding that DZ-514 was the best compound with high toxicity independent of the inhibition of BCL6. DZ-514 decreased cell viability, inhibited cell cycle progression, and induced caspase-independent cell death in TNBC cells. Interestingly, DZ-514 induced cytoplasm vacuolation, which could be blocked by Baf A1, the V-ATPase inhibitor. Furthermore, we found that DZ-514-induced vacuoles were derived from macropinosomes rather than autophagosomes. Most importantly, methuosis induced by DZ-514 was partially mediated by activating the ROS-MKK4-p38 axis. Finally, we demonstrated that DZ-514 significantly inhibited tumor growth in an HCC1806 xenograft mouse model. These findings revealed that the novel methuosis inducer DZ-514 could be developed for TNBC treatment.

RNF126-Mediated MRE11 Ubiquitination Activates the DNA Damage Response and Confers Resistance of Triple-Negative Breast Cancer to Radiotherapy.

Triple-negative breast cancer (TNBC) has higher molecular heterogeneity and metastatic potential and the poorest prognosis. Because of limited therapeutics against TNBC, irradiation (IR) therapy is still a common treatment option for patients with lymph nodes or brain metastasis. Thus, it is urgent to develop strategies to enhance the sensitivity of TNBC tumors to low-dose IR. Here, the authors report that E3 ubiquitin ligase Ring finger protein 126 (RNF126) is important for IR-induced ATR-CHK1 pathway activation to enhance DNA damage repair (DDR). Mechanistically, RNF126 physically associates with the MRE11-RAD50-NBS1 (MRN) complex and ubiquitinates MRE11 at K339 and K480 to increase its DNA exonuclease activity, subsequent RPA binding, and ATR phosphorylation, promoting sustained DDR in a homologous recombination repair-prone manner. Accordingly, depletion of RNF126 leads to increased genomic instability and radiation sensitivity in both TNBC cells and mice. Furthermore, it is found that RNF126 expression is induced by IR activating the HER2-AKT-NF-κB pathway and targeting RNF126 expression with dihydroartemisinin significantly improves the sensitivity of TNBC tumors in the brain to IR treatment in vivo. Together, these results reveal that RNF126-mediated MRE11 ubiquitination is a critical regulator of the DDR, which provides a promising target for improving the sensitivity of TNBC to radiotherapy.

Histone Deacetylase Inhibitors (HDACi) Promote KLF5 Ubiquitination and Degradation in Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) accounts for approximately 15% of all breast cancer cases, and patients with BLBC have a low survival rate. Our previous study demonstrated that the KLF5 transcription factor promotes BLBC cell proliferation and tumor growth. In this study, we demonstrated that the histone deacetylase inhibitors (HDACi), suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA), increased KLF5 acetylation at lysine 369 (K369), downregulated KLF5 protein expression levels, and decreased cell viability in BLBC cell lines. HDACi target KLF5 for proteasomal degradation by promoting KLF5 protein ubiquitination. K369 acetylation of KLF5 decreases the binding between KLF5 and its deubiquitinase, BAP1. These findings revealed a novel mechanism by which HDACi suppress BLBC, and a novel crosstalk between KLF5 protein acetylation and ubiquitination.