Breast cancer incidence is the highest incidence of malignant tumors globally [1]. Surgery, chemotherapy, radiotherapy, and endocrine therapy are methods employed for the treatment of breast cancer [2]. In recent years, advances in therapeutic techniques have significantly increased the cure rate of early-stage breast cancer, but the remission rate of advanced breast cancer is still low. In particular, TNBC has a poorer prognosis than other subtypes [3]. TNBC is defined as highly aggressive breast cancer with negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2), accounting for 11.2 % of breast cancer cases [4]. The greatest challenges to TNBC treatment are the susceptibility to tumor metastasis and poorer prognosis [5]. Restricted by the lack of effective therapeutic targets, the clinical therapeutics for TNBC are largely dependent on cytotoxic agents including paclitaxel (PTX) and anthracyclines. Still if resistance occurs, it faces the situation of no drug availability [6]. Therefore, there is an urgent need to validate new precision medicine strategies for treatment of TNBC.

The identification of new targets and the remining of classical drug targets are common approaches to pursue new precision medicine strategies (PMS). The new target identification is a systematic project that is not only time-consuming and labor-intensive but also carries huge uncertainties and risks [7]. The remining of classical drug targets with clinically established druggability is usually spotlighted. It is well known that the JAK2/STAT3 signaling pathway has been clinically well-characterized as a therapeutic target in inflammation-related diseases [8,9]. Moreover, the aberrations in the JAK2/STAT3 pathway are closely related to TNBC proliferation, invasion, and metastasis [[10], [11], [12]]. Excitingly, it has been shown that JAK2/STAT3 pathway inhibition not only suppresses phenotype of TNBC but also reverses PTX resistance [13,14]. Unfortunately, the failure of ruxolitinib (a dual JAK1/2 inhibitor)-involved clinical trials has greatly slowed down the progress of JAK2-targeted therapeutic strategies for TNBC (Fig. 1) [15]. We speculated that the aberrant activation of JAK2/STAT3 phosphorylation transduction represents only one node in the mechanistic network of TNBC progression. Hence, whether JAK2 can be an effective therapeutic target for TNBC urgently needs to be demonstrated.

Deubiquitinating enzymes (DUBs) are responsible for removing ubiquitin chains linked to protein substrates and play a vital role in regulating both protein abundance and function [16]. Their aberrant activity or expression can contribute to tumor development [17]. USP21 is a subtype of ubiquitin-specific proteases (USP) in the subfamily of DUB [18]. Studies have shown that highly expressed USP21 can promote TNBC cell proliferation, migration, and invasion [19,20], but the mechanisms involved are largely unidentified. In addition, SOCS3 binds to CUL5 through Elongin B, Elongin C and Rbx2 to form a functional CRL5 E3 ligase [[21], [22], [23]], which is activated by binding to NEDD8 [24]. Activated CRL5 binds to the substrate protein JAK2 to form a protein complex that promotes ubiquitylation of JAK2, thus facilitating the degradation of the JAK2 proteasome [21,25]. However, the deubiquitinating enzyme USP21 can detach NEDD8 from CRL5 and inactivate CRL5, thus preventing the ubiquitinated degradation of JAK2 protein [26]. We further speculated that USP21 may promote the phenotype and resistance of TNBC by stabilizing JAK2 via mediating its de-ubiquitination (Fig. 1). As a result, the above speculation urgently needs a USP21/JAK2/STAT3 axis-targeting probe as a tool molecule to authenticate the proposed mechanism.

In our previous work dedicated to the development of anti-malignant drugs, we discovered a drug lead NP16 that can modulate the JAK2/STAT3 signaling pathway [27]. However, the enzyme-based assay suggested that NP16 exhibited weak JAK2 inhibitory activity with a IC50 value of >10 μM (Table S1). These findings led us to hypothesized that NP16 may effectively regulate JAK2 signaling through other manners, such as de-ubiquitination regulation. Hence, we carried out a structural modification on NP16 and synthesized 54 N-anthraniloyl tryptamine derivatives (NP16 analogues). The cellular-based anti-TNBC structure-activity relationship studies identified a superior analogue, 13c, with nanomolar levels of cytotoxic activity against two TNBC cells, MDA-MB-231 and HCC-1806. In vitro phenotypic evaluation and in vivo antitumor efficacy validation further confirmed the excellent anti-TNBC potency of compound 13c. Importantly, mechanistic studies have clarified that 13c could act as a molecular probe to recognize and regulate the signal transduction of USP21/JAK2/STAT3 axis. This study delivered a candidate molecule 13c with a novel chemotype and mechanism of action for TNBC therapy.