Small molecule agents for triple negative breast cancer: Current status and future prospects

Highlights

• •A comprehensive summary of small molecule drugs targeting TNBC.
• •Clinical trial progress, resistance mechanisms and solutions for each of the small molecule drugs for TNBC are listed by target.
• •No previous articles have listed every small molecule drug for triple negative breast cancer by target.
• •Offers other new ideas for treating TNBC.

Abstract

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis. The number of cases increased by 2.26 million in 2020, making it the most commonly diagnosed cancer type in the world. TNBCs lack hormone receptor (HR) and human epidermal growth factor 2 (HER2), which limits treatment options. Currently, paclitaxel-based drugs combined with other chemotherapeutics remain the main treatment for TNBC. There is currently no consensus on the best therapeutic regimen for TNBC. However, there have been successful clinical trials exploring large-molecule monoclonal antibodies, small-molecule targeted drugs, and novel antibody-drug conjugate (ADC). Although monoclonal antibodies have produced clinical success, their large molecular weight can limit therapeutic benefits. It is worth noting that in the past 30 years, the FDA has approved small molecule drugs for HER2-positive breast cancers. The lack of effective targets and the occurrence of drug resistance pose significant challenges in the treatment of TNBC. To improve the prognosis of TNBC, it is crucial to search for effective targets and to overcome drug resistance. This review examines the clinical efficacy, adverse effects, resistance mechanisms, and potential solutions of targeted small molecule drugs in both monotherapies and combination therapies. New therapeutic targets, including nuclear export protein 1 (XPO1) and hedgehog (Hh), are emerging as potential options for researchers and become integrated into clinical trials for TNBC. Additionally, there is growing interest in the potential of targeted protein degradation chimeras (PROTACs), degraders of rogue proteins, as a future therapy direction. This review provides potentially valuable insights with clinical implications.

Graphical abstract

Introduction

Breast cancer (BC) stands out as one of the most prevalent malignancies globally, experiencing a staggering increase of 2.26 million cases in 2020, surpassing lung cancer as the most commonly diagnosed cancer worldwide [1]. Notably, BC constitutes 24.5 % of all cancers in women [2]. Various factors contribute to the occurrence of BC, encompassing emotional stimulation, sleep patterns, and dietary habits [3]. As the pace of life accelerates, workloads intensify, and dietary patterns undergo transformation, BC is progressively emerging as a prominent contributor to female mortality. The categorization of BC relies on the expression of hormone receptors (HR), including estrogen receptor (ER) and progesterone receptor (PR), as well as human epidermal growth factor 2 receptor (HER2) and Ki-67 expression. Three primary types are identified: Luminal type BC, encompassing Luminal A and Luminal B; HER2-positive BC; and triple-negative breast cancer (TNBC) [4]. In comparison to other cancer types, early-stage BC generally exhibits a favorable prognosis. According to GLOBOCAN 2020 data, BC accounts for only 6.7 % of all new cancer deaths. Additionally, patients who receive treatment according to guidelines typically have a median progression-free survival (mPFS) of 101 months [1,5], and a 10-year survival rate of over 40 % [6]. However, the treatment of TNBC presents challenges due to the absence of HR and HER2. To date, TNBC remains a formidable challenge in the global landscape of cancer research and treatment.

TNBC constitutes 15–20 % of all BC [7]. It is distinguished by the lack of expression of ER, PR, and HER2 [8]. Due to the absence or low expression of HR and HER2, TNBC is insensitive to endocrine therapy and targeted anti-HER2 drug therapy, resulting in a poor clinical prognosis. Furthermore, TNBC is associated with a heightened risk of distant recurrence and mortality compared to other BC subtypes [9]. Presently, the standard therapeutic approach for TNBC involves the administration of anthracyclines in combination with paclitaxel [10]. However, this chemotherapy regimen exhibits poor selectivity and limited efficacy. Compounding the issue, residual lesions, genetic mutations, amplifications, and immune escape phenomena subsequent to chemotherapy contribute to the development of chemotherapy resistance, further exacerbating the prognosis for TNBC patients [11,12]. Despite previous research indicating that TNBC displays heightened immunogenicity and increased infiltration of immune cells compared to other BC subtypes [13], and that anthracyclines can serve as inducers of immunogenic cell death (ICD) to enhance the immune response in BC, especially when combined with immunosuppressants [14]. However, clinical studies have shown that the combination of immunotherapy with anthracyclines for TNBC yields a low response rate and induces severe adverse reactions [15]. This phenomenon may be attributed to the significant heterogeneity of TNBC [16] and the predominantly immunosuppressive of its tumor microenvironment (TME) [17]. In fact, the existing chemotherapy options with the capability to induce ICD and transform "cold" tumors into "hot" tumors are limited. Moreover, current clinical research on ICD inducers combined with immunotherapy lacks adequate detailing, such as dosage and order of use [18]. Consequently, the optimal utilization of ICD inducers in TNBC remains uncertain. There is a pressing need for further exploration to comprehend the potential of inducing ICD as a strategy to enhance immunotherapy for TNBC. Numerous studies, spanning both fundamental research and clinical trials, have unequivocally illustrated that the synergistic combination of chemotherapy drugs with targeted therapy holds substantial efficacy in surmounting chemotherapy resistance [19]. This integrative approach has demonstrated a tangible enhancement in the progression-free survival (PFS) of patients afflicted with TNBC [20]. Consequently, the amalgamation of chemotherapy and targeted therapy emerges as a promising and prospective avenue for the treatment of TNBC.

Molecular targeted drugs with high efficiency and low toxicity due to their high selectivity, which can be combined with chemotherapy, radiotherapy and immunotherapy for the treatment of TNBC. These targeted drugs can be categorized into large molecule drugs, encompassing monoclonal antibodies, antibody-coupled drugs, among others, and small molecule targeted drugs. Large molecule drugs are typically administered via injection due to their substantial molecular weight, while small molecule drugs, characterized by their comparatively smaller molecular weight, can be conveniently administered orally. Between 1991 and 2022, the U.S. Food and Drug Administration (FDA) has sanctioned eighteen small molecule drugs for BC, but primarily designated for the management of HER2-positive BC. Notably, the development of novel small molecule targeted drugs specifically tailored for TNBC poses a formidable challenge [21]. Small molecule targeted drugs have a broad spectrum of targets, including various kinases, proteasomes, epigenetic regulatory proteins, and other distinct targets (Fig. 1). In recent years, an increasing number of approved or investigational small molecule targeted drugs have been employed in clinical trials for TNBC. This article provides a comprehensive review of small molecule targeted drugs relevant to the treatment of TNBC since the year 2017.

Fig. 1.

Small molecule inhibitor targets for TNBC.

The targets of these small molecule inhibitors will be described in the paragraphs below.

Some of the illustrations were produced by Servier Medical Art (SMART-Servier Medical ART).

Small molecule drugs targeting TNBC

Kinase inhibitors

Protein kinases constitute one of the largest families within the human genome [22], playing a pivotal role in various signal transduction pathways through phosphorylation events. Mutations and aberrant expression of protein kinases are intricately linked to the development of tumors [23]. Protein kinase inhibitors can target various targets in the signaling network to inhibit further tumor progression [24]. These inhibitors can be categorized into tyrosine kinase inhibitors (Table 1) and serine/threonine kinase inhibitors (Table 2), depending on the substrate residues of the protein kinases. Additionally, they can be classified as highly selective single kinase inhibitors and multi-kinase inhibitors (Table 3), based on their targeting selectivity [25]. Single kinase inhibitors inhibit a single target in the cellular signaling network. Multi-kinase inhibitors can compensate for the limitations of single kinase inhibitors by targeting multiple targets to achieve synergistic effects to improve anti-tumor activity and reduce the occurrence of drug resistance [26].

Table 1.

Tyrosine kinase inhibitors.

Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
EGFR inhibitors	Erlotinib(Tarceva)	EGFR	Metastatic TNBC	NCT01650506	Ⅰ	Combined with Metformin	Diarrhea, rash
			Metastatic TNBC	NCT0073340	Ⅱ	Weekly nab-paclitaxel and bevacizumab followed by maintenance targeted therapy with bevacizumab and erlotinib	Neutropenia, atigue, neuropathy
VEGFR inhibitors	Apatinib	VEGFR	Advanced TNBC	NCT03394287	Ⅱ	Combined with apatinib	Elevated aminotransferase, hand-foot syndrome
			Advanced TNBC	NCT04303741	Ⅱ	Combined with apatinib and eribulin	elevated aminotransferase, leukopenia, hand-foot syndrome, neutropenia, alopecia, fatigue
			Rrecurrent/metastatic TNBC	NCT03945604	Ib	Combined with camrelizumab and fuzuloparib	Leukocytopenia, hypertension, neutropenia, elevated aminotransferase
			Refractory metastatic TNBC without gBRCA1/2 mutation	NCT03805399 (FUTURE)	Ib/Ⅱ	Apatinib monotherapy	Fatigue, anemia, thrombocytopenia

Table 2.

Serine/theonine kinase inhibitors.

Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
PI3K inhibitors	Buparlisib (BKM120)	PI3K	Metastatic TNBC	NCT01790932	Ⅱ	Buparlisib monotherapy	Fatigue, nausea, hyperglycemia, anorexia
	Alpelisib (BYL719)	PI3K	Advanced TNBC	NCT01623349	Ib	Combined with olaparib	Fatigue, anorexia, hyperglycemia, nausea
			Metastatic TNBC	NCT02506556	Ⅱ	Alpelisib monotherapy	Hyperglycemia, rash, colitis, diarrhea
AKT inhibitors	Ipatasertib (GDC-0068)	AKT	Early TNBC	NCT02301988	Ⅱ	Combined with paclitaxel	Diarrhea, fatigue, peripheral neuropathy, nausea, rash
			Inoperable locally advanced/metastatic TNBC	NCT02162719 (LOUS)	Ⅱ	Combined with paclitaxel	Diarrhoea, neutropenia
	Capivasertib (AZD5363)	AKT	Metastatic TNBC	NCT02423603 (PAKT)	Ⅱ	Combined with paclitaxel	Diarrhea, infection, neutropenia, rash, fatigue
			Locally advanced/metastatic TNBC	NCT03997123	Ⅲ	Combined with paclitaxel	Diarrhea, infection, neutropenia, rash, fatigue
			TNBC	NCT02208375	Ib	Combined with olaparib	Nausea, anemia, diarrhea, elevated creatinine, fatigue, hyperglycemia
	Uprosertib (GSK2141795)	AKT	Locally advanced TNBC	NCT01138085	Ⅰ	Combined with trametinib	Diarrhea, fatigue, nausea, vomiting, dermatitis acneiform
Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
mTOR inhibitors	Temsirolimus (CCI-779, Torisel)	mTOR	Metaplastic TNBC	NCT00761644	Ⅰ	Combined with liposomal doxorubicin, bevacizumab	Neutropenia, mucositis, hand-foot syndrome, fatigue, anemia
	Everolimus (RAD001, Afinitor)	mTOR	Refractory metastatic TNBC with PI3K/AKT pathway mutation	NCT03805399 (FUTURE)	Ib/Ⅱ	Combined with nab-paclitaxel	Anemia, diarrhea, stomatitis, rash
			Residual TNBC post neoadjuvant chemotherapy	NCT01931163	Ⅱ	Combined with cisplatin	Fatigue, nausea, mucositis
			Metastatic TNBC	NCT02120469	Ⅰ	Combined with eribulin	Neutropenia, leukopenia, lymphopenia
			Metastatic TNBC	NCT02616848	Ib	Combined with eribulin	Neutropenia, leukopenia, lymphopenia
			Metaplastic TNBC	NCT00761644	Ⅰ	Combined with liposomal doxorubicin, bevacizumab	Neutropenia, mucositis, hand-foot syndrome, fatigue, anemia
CDK4/6 inhibitors	Trilaciclib (G1T28)	CDK4/6	Metaplastic TNBC	NCT02978716	Ⅱ	Combined with gemcitabine and carboplatin	Anaemia, neutropenia, thrombocytopenia, nausea
			Metaplastic TNBC	NCT02978716	Ⅱ	Administered prior to gemcitabine plus carboplatin	Neutropenia, thrombocytopenia
ATR inhibitors	Berzosertib (M6620, VX-970)	ATR	Advanced TNBC	NCT02157792	Ib	Ccombined with cisplatin	Nausea, fatigue, neutropenia, vomiting
Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
	Ceralasertib (AZD6738)	ATR	Metastatic TNBC	NCT03330847	Ⅱ	Combined with olaparib	Anaemia, neutropenia, nausea, vomiting, diarrhoea, fatigue, asthenia
			Metastatic TNBC	NCT03801369	Ⅱ	Ceralasertib monotherapy	No results posted
			Metastatic TNBC	NCT05582538 (ATRiBRAVE)	Ⅱ	Combined with durvalumab and nab-paclitaxel	No results posted
CHK1 inhibitors	Prexasertib (LY2606368)	CHK1	BRCA wild-type, advanced TNBC	NCT02203513	Ⅱ	Prexasertib monotherapy	Afebrile neutropenia, anemia, hrombocytopenia
WEE1 inhibitors	Adavosertib (AZD1775)	WEE1	Metastatic TNBC	NCT03012477	Ⅱ	Combined with cisplatin	Nausea, diarrhea, anemia, neutropenia
			Metastatic TNBC	NCT03330847	Ⅱ	Combined with olaparib	Febrile neutropenia, neutropenia, anaemia, vomiting, diarrhoea,nausea
MEK inhibitors	Trametinib (GSK1120212)	MEK	TNBC	NCT01138085	Ⅰ	Combined with uprosertib	Diarrhea, fatigue, vomiting, mucosal inflammation, stomatitis
			Metastatic TNBC	NCT01964924	Ⅱ	Combined with uprosertib	Anemia, diarrhea, mucositis oral, dyspnea
	Cobimetinib (GDC-0973)	MEK	Locally advanced/metastatic TNBC	NCT01562275	Ib	Combined with ipatasertib	Diarrhea, nausea, vomiting, dermatitis acneiform, fatigue
			Locally advanced/metastatic TNBC	NCT02322814 (COLET)	Ⅱ	Combined with chemotherapy, with or without atezolizumab	Diarrhea

Table 3.

Multi-kinase inhibitors.

Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
Cabozantinib	MET, VEGFR2, RET, AXL, FTL3, etc.	TNBC with brain metastases	NCT02260531	Ⅱ	Cabozantinib alone or in combination with trastuzumab	Fatigue, elevated aminotransferase, hyponatremia, thromboembolic events
		Metastatic TNBC	NCT01738438	Ⅱ	Cabozantinib monotherapy	Fatigue, diarrhea, mucositis, palmar-plantar erythrodysesthesia
Sunitinib	VEGF, PDGFR, HGF, etc.	Locally advanced TNBC	NCT00513695	Ⅱ	Sunitinib with paclitaxe followed by doxorubicin and cyclophosphamide	Neutropenia, leukopenia, fatigue, anemia, diarrhea, mucositis
		Locally advanced TNBC	NCT00887575	Ⅰ/Ⅱ	Combined with paclitaxel/carboplatin	Neutropenia, thrombocytopenia, anemia, leukopenia, fatigue, alopecia, constipation, diarrhea, mucositis, dysgeusia, peripheral neuropathy
		Advanced TNBC	NCT00246571	Ⅱ	Sunitinib monotherapy	Nausea, diarrhea, fatigue, asthenia, neutropenia, mucosal inflammation
ENMD-2076	Aurora-A, VEGFR, FGFR	Advanced/metastatic TNBC	NCT01639248	Ⅱ	ENMD-2076 monotherapy	Hypertension, fatigue, diarrhea, nausea
Lapatinib (Tykerb)	EGFR, HER2	Metastatic TNBC	NCT02158507	Not applicable	Combined with veliparib	Fatigue, diarrhea, constipation, insomnia, vomiting, anemia, headache, dizziness, dyspnea, rash
pyrotinib (SHR1258)	EGFR, HER2, HER4	Refractory metastatic TNBC with ERBB2 somatic mutation	NCT03805399 (FUTURE)	Ib/Ⅱ	Combined with capecitabine	Diarrhea, fatigue, hand-foot syndrome
Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
Samotolisib (LY3023414)	PI3K, mTOR	Locally advanced/metastatic TNBC which not amenable to curative intent surgery or radiotherapy	NCT02124148	Ib	Combined with Prexasertib	Leukopenia, neutropenia, thrombocytopenia, nausea

Tyrosine kinase inhibitors

EGFR inhibitors

The human epidermal growth factor receptor (EGFR) belongs to the ErbB family of tyrosine kinase receptor proteins and is located on the cell membrane surface, where it is activated by binding to ligands. Activated EGFR can activate downstream signaling pathways through phosphorylation to induce cell proliferation and differentiation, and EGFR overexpression can lead to malignant proliferation and migration of tumor cells [27]. EGFR overexpression is common in BC, with approximately 78 % of patients with TNBC having EGFR overexpression [28]. Targeting EGFR holds promise for improving the poor clinical prognosis of TNBC, but clinical trials of EGFR tyrosine kinase inhibitors (EGFR-TKI) targeting TNBC have low response rates, so only a subset of patients benefit from them.

Erlotinib (Tarceva)

Previous clinical studies have shown that EGFR inhibitors are widely used in the treatment of malignant tumors [29]. In 2004, the first-generation EGFR inhibitor, erlotinib, was approved by the FDA for non-small cell lung cancer (NSCLC) and pancreatic cancer (PC) [30]. In recent years, some investigators have used erlotinib in clinical trials in TNBC. Erlotinib monotherapy had poor efficacy in TNBC, combining with PI3K/AKT pathway inhibitors could enhance the anti-tumor activity of erlotinib [31]. The combination of anti-EGFR monoclonal antibody or anti-angiogenesis inhibitors can dual target tumor cells, enhance the anti-tumor effect of erlotinib, and effectively prolong the PFS of TNBC patients [32]. A phase 2 study [33] treated fifty-five patients with metastatic triple-negative breast cancer (mTNBC) with paclitaxel plus bevacizumab followed by erlotinib plus bevacizumab maintenance therapy. Fifty-three of the treated patients were evaluable according to RECIST criteria, with a partial remission (PR) rate of 74 %, a stable disease (SD) rate of 10 %, a mPFS of 9.1 months, and a median overall survival (mOS) of 18.1 months. The treatment regimen was safe and well tolerated, while treatment-related adverse events occurred mainly during the induction phase, including most commonly neutropenia, fatigue, and neuropathy.

The EGFR-TKI combination is effective in treating TNBC, but resistance is ultimately inevitable. The occurrence of drug resistance is mainly associated with secondary EGFR mutations (T790M mutation, C797S mutation, etc.), gene amplification and histological transformation [34], [35], [36]. Inhibition of EGFR together with activation of its downstream signaling pathways is one of the potential strategies to overcome drug resistance. Metformin can inhibit MAPK and PI3K/AKT signaling pathway synergistically with EGFR-TKI [37]. However, in a small sample of phase I clinical trials, treatment of mTNBC with metformin in combination with erlotinib had a low clinical benefit rate (CBR), with an mPFS of 60 days and only 25 % of patients achieving SD, with no patients achieving PR and complete remission (CR) [38].

VEGFR inhibitors

Vascular neovascularization of tumors relies on endothelial cells to form new blood vessels from existing normal vessels [39]. Angiogenesis depends on the regulation of vasopromoting endogenous molecules. Vascular epidermal growth factor A (VEGF-A, a member of the VEGF family) is an important regulator of angiogenesis, and VEGF-A binds to vascular epidermal growth factor receptor 1/2 (VFGFR1/2) to drive physiological or pathological angiogenesis [40]. Physiological neovascular sprouts expand into large vessels and then gradually branch into microvessels that provide access to the body's nutrient metabolism. Abnormal vascular proliferation is an important feature of cancer. Excessive activation of the VEGF-VEGFR pathway leads to abnormal vascular proliferation, which provides sufficient nutrients for tumor cell proliferation and becomes a new channel for tumor cell invasion and metastasis [41]. Anti-angiogenic drugs targeting the VEGF-VEGFR signaling pathway have achieved initial results in the treatment of tumors [42].

Apatinib

The VEGFR2 inhibitor apatinib can regulate the immunological microenvironment of tumors by inhibiting angiogenesis, enhancing the anti-tumor activity of anti PD −1 drugs, and slowing tumor growth [43]. Apatinib in combination with camrelizumab showed promising clinical results in several studies. An open-label, two-arm phase II clinical trial included 40 patients with advanced TNBC treated systemically [44], all of whom were treated with camrelizumab and received either continuous 14-day oral treatment with apatinib or intermittent oral treatment with apatinib on days 1 to 7. In this study, no objective response was observed in the intermittent administration group. The objective response rate (ORR) in the continuous-dose group was 43.3 %, and the disease control rate (DCR) was 63.3 %, which was higher than the 40 % DCR in the intermittent-dose group. Continuous dosing was able to extend patients' mPFS from 1.9 months in the intermittent-dosing group to 3.7 months. Another multicenter phase 2 clinical trial compared the efficacy of the monoclonal antibody camrelizumab and apatinib in combination with eribulin in the treatment of 46 heavily pretreated advanced TNBC patients [45]. The ORR of Intentional Treatment (ITT) population in this treatment was 37 %, with a median time to tumor remission (mTTR) and median duration of remission (mDOR) of 1.5 and 8.6 months, respectively. In the ITT population, DCR was 87 %, CBR was 50 %, mPFS was 8.1 months, and mOS was not achieved, with a 1-year OS rate of 68.3 %. The combination of camrelizumab and apatinib with fluzopanib also showed good safety and significant anti-tumor activity [46]. In a phase 1b clinical trial, the combination strategy was used in 32 patients with relapsed or metastatic TNBC. In the evaluable population, the ORR was 6.9 %, DCR reached 62.1 %, mPFS was 5.2 months, and the 1-year OS rate was 64.2 %, with no mOS achieved.

Serine theonine kinase inhibitors

PI3K/AKT/mTOR inhibitor

The phosphatidylinositol 3-kinase (PI3K)/Serine/threonine protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) signaling pathway is essential for cell growth, promotion, transcription, translation, apoptosis, and angiogenesis [47,48]. Abnormal activation of this pathway is closely related to tumor occurrence. Its abnormal activation can inhibit the induction of cell apoptosis, regulate cell cycle transition, promote tumor cell proliferation and migration, and initiate epithelial-mesenchymal transition (EMT) [49]. The main targets of the PI3K/AKT/mTOR signaling pathway are PI3K, AKT, and mTOR. PI3K is activated by binding to growth factor receptors or directly activated by Ras and p110 to catalyze PIP2 to generate PIP3, to bind to AKT and PDK1, which in turn phosphorylate AKT to activate AKT. Activated AKT can further phosphorylate PRAS40 to release its inhibitory effect on mTOR, thereby activating mTOR and regulating protein synthesis, which in turn is involved in apoptosis and autophagy [50,51]. Dysregulation of the PI3K/AKT pathway is common in a variety of cancers. Aberrant expression of PI3K and mTOR proteins, as well as deletion or low expression of PTEN, are associated with poor prognosis of TNBC [52]. Targeting the PI3K/AKT/mTOR pathway has beneficial effects on the treatment of TNBC.

PI3K inhibitors

Buparlisib (BKM120)

The pan-class I PI3K inhibitor buparlisib has shown good efficacy in combination with other drugs in the treatment of HR+/HER2- BC [53]. Chemotherapy, targeted therapy, or oral vitamin C can enhance the anti-tumor activity of the PI3K inhibitor buparlisib and slow tumor recurrence and growth [54], [55], [56]. However, no significant objective responses were observed with buparlisib monotherapy in mTNBC. A single-arm phase 2 clinical trial evaluated the efficacy of buparlisib in mTNBC patients [57]. No PR or CR cases occurred during treatment. The CBR was 12 %, mPFS was 1.8 months, and mOS was 11.2 months.

Alpelisib (BYL719)

As the first PI3K inhibitor used in BC, alpelisib has shown effective clinical activity in combination with the targeted drug olaparib in a phase 1b clinical trial involving 17 cases of recurrent advanced TNBC [58]. Under this treatment regimen, ORR was 18 %, CBR was 35 %, DCR was 53 %, mDOR was 7.4 months, mPFS was 3.6 months, 6-month progression-free survival rate was 31.2 %, and mOS was 11.8 months. However, aparlisib monotherapy may not have anti-tumor activity in mTNBC [59]. A phase 2 clinical cohort study shows that aparlisib monotherapy has favorable anti-tumor activity in patients with ER+/HER2- BC. The ORR in the intent-to-treat (ITT) population was 30 %, while both ORR and CBR were 0 % in mTNBC. However, the sample size of this study was small, and further clinical trials are needed to further evaluate the anti-tumor activity of apalutamide in TNBC.

AKT inhibitors

Ipatasertib (GDC-0068)

Ipatasertib is a highly selective ATP-competitive AKT inhibitor. High AKT levels, PTEN loss, and PIK3CA mutations can increase the sensitivity of tumor cells to ipatasertib [60]. However, in BC cells with PIK3CA wild-type, ipatasertib cannot exert antitumor effects because the signaling pathway is not excessively activated. Therefore, BC patients with wild-type PIK3CA are unlikely to benefit from treatment with ipatasertib [61]. The LOTUS study [62,63] is a double-blind, multicenter, randomized, controlled phase 2 clinical trial involving 124 patients with unresectable, locally advanced or metastatic TNBC. The objective of the study is to observe the antitumor effect of ipatasertib in combination with paclitaxel. The treatment group in this study received ipatasertib plus paclitaxel, while the control group received placebo plus paclitaxel. The results showed that mPFS was higher in the treatment group at 6.2 months compared with 4.9 months in the control group, and that the combination of ipatasertib and paclitaxel increased patients' mOS from 16.9 months to 25.8 months, with the 1-year rate OS increasing from 68 % to 83 %. However, in the FAIRLANE study [64], the addition of ipatasertib to docetaxel had no effect on pathologic pCR. In this study, 132 patients were divided into two groups, both treated with paclitaxel. The treatment group was additionally treated with ipatasertib, and the control group received placebo. The primary endpoint was pCR for the ITT populations and low expression PTEN populations. Ipatasertib showed no significant improvement in pCR in the ITT group. However, both the LOTUS and FAIRLANE studies showed that the addition of ipatasertib was more effective in populations with PIK3CA/AKT1/PTEN mutations.

Capivasertib (AZD5363)

Capivasertib is a pan-AKT inhibitor that targeting AKT1, AKT2, and AKT3. As with ipatasertib, PIK3CA mutations and loss of the PTEN gene are important factors in increasing the antitumor sensitivity of capivasertib [65]. PIK3CA/AKT1/PTEN-mutated populations showed a greater advantage in capivasertib treatment. PAKT [66] is a double blind, randomized, placebo-controlled Phase 2 clinical trial in previously untreated mTNBC. Compared to treatment with paclitaxel and placebo, the addition of the AKT inhibitor capivasertib to paclitaxel can effectively prolong patients' PFS and OS. In the ITT population, the capivasertib group prolonged mOS from 12.6 months in the control group to 19.1 months and mPFS from 4.2 months to 5.9 months. Although the prolongation of mPFS was not statistically significant in the ITT population, mPFS differed significantly between the capivasertib group and the control group in the subgroup with PIK3CA/AKT1/PTEN alteration. In the PIK3CA/AKT1/PTEN subgroup, paclitaxel plus capivasertib prolonged mPFS from 3.7 months to 9.3 months, representing a significant clinical benefit. It should be noted that there were significantly more adverse events in the capivasertib group than in the control group, which mainly manifested as diarrhea, fatigue, and rash. The Phase 3 clinical trial of capivasertib in combination with paclitaxel is ongoing (NCT03997123), and final results are not yet available. Inhibition of AKT signaling increases the sensitivity of metastatic castration-resistant prostate cancer to the PARP inhibitor olaparib [67]. PARP inhibitors may be involved in regulating the PI3K/AKT/mTOR pathway, and AKT inhibitors interact with PARP inhibitors to jointly induce apoptosis, enhancing the antitumor effect of PARP inhibitors [68], but the clinical efficacy of the PARP inhibitor olaparib in combination with capivasertib in the treatment of TNBC is not evident. A phase 1b clinical trial showed that [69] the combination had an ORR of 11 % and a CBR of 22 % in locally advanced or metastatic TNBC.

Uprosertib (GSK2141795)

Uprosertib is a selective ATP-competitive inhibitor targeting AKT1, AKT2, and AKT3. AKT inhibitors show excellent antitumor activity in cells with mutations or deletions in the AKT pathway PI3K or PTEN and low sensitivity in cells with mutations in the MAPK pathway KRAS or BRAF. The combination of uprosertib with the MEK inhibitor trametinib, which targets the MAPK pathway, enhances antitumor activity in mice with KRAS-mutated pancreatic cancer [70]. Although clinical trials have shown that uprosertib monotherapy at a daily dose of 75 mg is safe and well tolerated in patients with solid tumors with PIK3CA mutation or PTEN deletion [71], patients with locally advanced TNBC have difficulty tolerating trametinib 1.5 mg daily combined with 50 mg Uprosertib once daily [72], The initial efficacy of this combination was insufficient, and the mPFS of TNBC patients was only 54 days.

mTOR inhibitors

Temsirolimus (CCI-779, Torisel)

Temsirolimus is a derivative of sirolimus that can target the mTOR pathway, block the cell cycle, and inhibit protein synthesis [73]. The combination of temsirolimus with other therapies can inhibit the progression of TNBC [74]. Compared with non-metaplastic BC, trastuzumab shows greater benefits in metaplastic BC [75]. In a phase 1 clinical trial [76], a combination of toceranib phosphate, liposomal doxorubicin, and bevacizumab was observed to have a CBR of 40 % and an ORR of 21 % in the treatment of TNBC. After examining tissues from 43 patients, it was found that abnormalities in the PI3K pathway may be significantly related to improved ORR. In addition, temsirolimus in combination with the EGFR inhibitor gefitinib can disrupt the hosphorylation of eIF4B, thereby blocking the proliferation of TNBC cells [77]. Follow-up studies have also shown that the mTOR inhibitor everolimus in combination with EGFR inhibitors can effectively block the TNBC cell cycle and enhance cancer cell apoptosis [78], but the protocol has not been clinically applied and requires further investigation.

Everolimus (RAD001, Afinitor)

Another mTOR inhibitor, everolimus, inhibits EGFR-positive and CK5/6-positive basal-like subtypes of TNBC [79], but the clinical efficacy of everolimus monotherapy has not yet been confirmed. Everolimus combined with chemotherapy has shown good results in the treatment of mTNBC. The microtubule depolymerizer eribulin inhibits AKT phosphorylation and inhibits tumor growth synergistically with mTOR inhibitors [80]. A phase 1 clinical trial [81] for mTNBC showed that the CBR of everolimus combined with eribulin was 72 %, mPFS was 2.6 months, and mOS was 8.9 months. In TNBC patients with residual disease after neoadjuvant chemotherapy, everolimus in combination with cisplatin resulted in an ORR of 23 % in RCB I [82].

Mutations in the PI3K/AKT/mTOR pathway are common in TNBC. Overexpression of PAK1 and abnormal activation of downstream AKT by MAPK4 [83] are associated with resistance to PI3K inhibitors [84,85]. The PI3K pathway includes numerous targets that have proven difficult to block completely. Therefore, PI3K/AKT/mTOR inhibitors have limited therapeutic effect in TNBC. The dual action of MEK inhibitors targeting the MAPK pathway and PI3K inhibitors increases the sensitivity of PI3K blockade to inhibit tumor growth [86]. Loss of PIK3CA, PTEN, and P53 is common in TNBC [87]. In TNBC with mutations in PIK3CA, AKT1, BRAF and FGFR4, dual inhibition of PI3K/MEK may have more potent anti-tumor activity [88]. Moreover, the combination of CDK4/6 inhibitors can overcome the adaptive resistance of BC with PIK3CA mutation and improve the sensitivity of PI3K inhibitors [89]. Significant antitumor activities of CDK4/6 inhibitors and PI3K/AKT/mTOR inhibitors may be related to impaired glucose metabolism in cells [90].

CDK4/6 inhibitors

Cyclin-dependent kinase (CDK) is an important component in the regulation of cell proliferation and division. The CDK4/6-Rb pathway controls cell cycle progression by regulating the G1/S checkpoint. Targeting CDK4/6 can block the cell cycle, inhibit tumor cell proliferation and division, and promote their apoptosis [91]. Disruption of the CDK4/6-Rb pathway is common in BC [92]. Targeting CDK and the signaling pathway it mediates can reduce the invasiveness of TNBC and inhibit TNBC metastasis and progression [93,94].

Trilaciclib (G1T28)

Trilaciclib is a potent selective CDK4/6 inhibitor [30] that was originally approved for advanced small cell lung cancer (SCLC) and also shows promising therapeutic prospects in TNBC. Administration of trilaciclib prior to chemotherapy may protect hematopoietic stem and progenitor cells from chemotherapy-induced bone marrow damage and reduce chemotherapy-induced hematologic toxicity [95,96]. The combination of trilaciclib with chemotherapy can increase the number of peripheral lymphocytes, enhance T-cell activation, and improve the tumor immunological microenvironment, leading to further enhancement of the drug's anti-tumor activity [97,98]. A multicenter, open-label, randomized phase 2 trial showed better OS outcomes and safety profile for tralacil in combination with gemcitabine and carboplatin in metaplastic TNBC [99]. In this program, patients' mOS was 20.1 months and mPFS was 8.8 months, which was better than the mOS and mPFS using chemotherapy alone. In addition, the CBR after combination with trilaciclib was 50 %, which was higher than that in the chemotherapy group. Treatment with trilaciclib before gemcitabine and carboplatin is more effective, and the sequential chemotherapy regimen with trilaciclib significantly prolongs mOS in patients with mTNBC [100]. In a phase 2 randomized controlled trial, 102 patients with mTNBC were divided into three groups. The first group of 34 patients received therapy with gemcitabine and carboplatin on days 1 and 8. The second group received pretreatment with trilaciclib on days 1 and 8 before receiving gemcitabine and carboplatin. The third group, like the second group, received trilaciclib alone on days 1 and 8 and then pretreatment with trilaciclib on days 2 and 9 before being treated with gemcitabine and carboplatin. The mOS of patients in group 1 was 12.6 months, while the mOS of group 3 extended to 17.8 months and group 2 did not reach mOS. In conclusion, antitumor activity was increased by pretreatment with trilaciclib before chemotherapy, while there was no significant increase in antitumor activity by the additional administration of trilaciclib alone before chemotherapy.

Activation of the cellular bypass pathway by deletion or mutation of the Rb1 gene, amplification of CDK4 and E2F, and overexpression of the INK4 protein can lead to resistance to CDK4/6 inhibitors [101], [102], [103]. Combining inhibitors that target key cell cycle and activation pathway targets is a potential strategy to overcome CDK4/6 drug resistance.

ATR/CHK1/WEE1 inhibitors

The ATR/CHK1/WEE1 pathway is an important pathway for DNA damage repair. The normal progression of mitosis depends on the precise regulation of the cell cycle. When single-stranded DNA is damaged, the upstream kinase ATR is activated to phosphorylate CHK1, arresting S phase and G2/M phase, allowing time for repair of damaged DNA [104,105]. Upon completion of DNA repair, the key protein WEE1 kinase, which controls the G2/M cell cycle checkpoint, is ubiquitinated and degraded, and the cell continues to undergo mitosis [106]. In normal cells, the G1/S phase checkpoint depends on regulation by P53. Most patients with TNBC have a P53 mutation or deletion. At this point, inhibition of WEE1 kinase inactivates the G2/M phase checkpoint and damaged DNA loses repair time, resulting in mitotic abnormalities of the cell [107,108], so WEE inhibitors can selectively inhibit tumor cells with p53 deletion or mutation.

ATR inhibitors

Ceralasertib (AZD6738)

Ceralasertib inhibits DNA break-induced replication and homologous recombination repair. Ceralasertib acts synergistically with DNA-damaging drugs that block replication forks to increase antitumor efficacy. Administration of ceralasertib together with lower doses of carboplatin or irinotecan may achieve better antitumor efficacy than ceralasertib monotherapy, but administration of ceralasertib prior to chemotherapy has no significant effect [109]. In BRCA-mutated TNBC, ceralasertib in combination with the PARP inhibitor olaparib may also show effective synergistic interactions [110]. Patients with BRCA-mutated TNBC treated with this regimen showed longer mPFS (7.4 months vs. 3.9 months), mDOR (8.1 months vs. 4.1 months), and better ORR (49% vs. 20 %) than patients with non-BRCA-mutated TNBC [111].

Berzosertib (M6620, VX-970)

Berzosertib is an intravenous ATR inhibitor that has shown potent anti-tumour activity in combination with chemotherapy in high-grade serous ovarian cancer and lung cancer [112,113]. The first clinical trial of berzosertib in combination with cisplatin in advanced TNBC was published in 2022 [114]. After using berzosertib in combination with cisplatin in 43 evaluable patients with advanced TNBC, the ORR was 23.4 %, the DOR was 6 months, the mPFS was 4 months, and the mOS was 12.4 months.

CHK1 inhibitors

Prexasertib (LY2606368)

Prexasertib, a second-generation CHK1 inhibitor, can block CHK phosphorylation and activation, induce homologous recombination defects [115], block DNA damage repair, and cause mitotic catastrophe [116]. A single-arm phase 2 clinical trial of prexasertib monotherapy in advanced wild-type BRCA TNBC showed an ORR of 11.1 % and mPFS of 86 days [117]. Recent experiments have shown that EGFR overexpression and activation can promote TNBC cell resistance to prexasertib, and the synergistic anti-tumour effect of prexasertib in combination with EGFR inhibitors may become a new research direction for TNBC treatment [118,119].

WEE1 inhibitors

Adavosertib (AZD1775)

Adavosertib is a highly selective small molecule Wee1 and Wee2 kinase inhibitor [120] that can enhance the activity of chemotherapeutic agents that act on DNA structure and function [121]. Preclinical experiments have already demonstrated the tumour-inhibitory effect of the combination of adavosertib and capecitabine/5-fluorouracil (5FU) in a TNBC model [122]. A phase 2 clinical trial conducted in 2021 showed that [123] the combination of adavosertib and cisplatin demonstrated better short-term clinical efficacy than platinum alone. Among the 34 mTNBC patients enrolled in the trial, the ORR was 26 %, although it did not reach the expected 30 %, but the mPFS was 4.9 months, which was higher than the 3-month mPFS of first-line platinum-based therapy. However, the median disease-free survival (mDFS) in this study was 13 months, which was significantly lower than the mDFS of 21 months in the TBCRC009 trial with platinum as first-line therapy [124]. As can be seen, the therapeutic regimen of adavosertib in combination with chemotherapy needs further research and refinement.

The mechanism of resistance to ATR/CHK1/WEE1 inhibitors is unclear, and deletion of the FAM122A/PABIR1 gene may lead to the occurrence of cross-resistance between ATR inhibitors and CHK inhibitors [125]. But ATR/CHK1/WEE1 inhibitors can destabilize the replication fork, resist DNA damage repair, and overcome cancer therapy with PARP inhibitors [126].

MEK inhibitors

Activation of the RAS-RAF-MEK-ERK pathway plays an important role in tumor cell proliferation and invasion. When Ras protein is activated, the cascade response phosphorylates Raf, further activates MEK and its substrate ERK, and ERK regulates downstream signaling pathways to control cell proliferation, invasion, and apoptosis [127]. Overactivation of the MAPK pathway is widespread in TNBC, and inhibition of MAPK pathway-related molecules can inhibit tumor progression [128,129]. Mutations of the Ras and Raf genes in the MAPK pathway can lead to abnormal activation of the MEK gene and continue to affect downstream signaling pathways, leading to malignant proliferation and invasion of cells, making MEK inhibitors potentially more effective than simple inhibition of the Ras or Raf genes [130].

Trametinib (GSK1120212)

Trametinib is an oral, non-competitive MEK1 and MEK2 inhibitor. It has already been approved by the FDA in combination with BRAF inhibitors for the treatment of metastatic melanoma [131]. In preclinical models, trametinib in combination with the HER inhibitor neratinib can significantly inhibit the TNBC cell proliferation marker Ki001 and promote cell apoptosis to enhance antitumor effects [132]. In TNBC, the PI3K and MAPK signaling pathways are often activated simultaneously [88]. The combined use of MEK inhibitors and AKT inhibitors may show dual inhibitory effects in TNBC cell lines [133], but in a phase 1 clinical trial [72] the MEK inhibitor trametinib and the AKT inhibitor uprosertib were combined in 20 TNBC patients, the mPFS in this trial was only 54 days.

Cobimetinib (GDC-0973)

Cobimetinib in combination with ipatasertib has been shown to be effective in the treatment of TNBC [134], but the efficacy of cobimetinib in combination with chemotherapy and immunotherapy is not significant. In the COLET trial, no significant antitumor activity was observed with cobimetinib plus paclitaxel in patients with mTNBC [135]. Compared with the placebo plus paclitaxel group, the increase in ORR and mPFS with cobimetinib was not significant, and there was no statistical difference. Even when atezolizumab was added to cobimetinib plus paclitaxel, the mPFS observed in the COLET cohort was not significantly different from the mPFS of atezolizumab plus nab-paclitaxel in the IMpassion130 trial.

Abnormal activation of the MAPK pathway in tumor cells can lead to chemotherapy resistance [136], promote immune evasion, and shorten patient survival [137]. Therefore, MEK inhibitors have been developed and manufactured by many pharmaceutical companies. However, according to current data from clinical trials, MEK inhibitors have not shown significant efficacy in TNBC. TNBC resistance to MEK inhibitors prevents TNBC patients from benefiting from MEK inhibitors in the long term. CDK inhibitors targeting the cell cycle can overcome resistance to MEK inhibitors [138], and inhibition of proteasome-induced autophagy can indirectly promote MEK gene expression and increase sensitivity to MEK inhibitors [139]. The dual combination of MEK inhibitors and proteasome inhibitors significantly inhibits tumor growth in TNBC transplant mouse model [140].

Multi-kinase inhibitors

Cabozantinib

Cabozantinib is a small molecule tyrosine kinase inhibitor that simultaneously targets MET, VEGFR2 and other tyrosine kinases such as RET, AXL and FTL3 [141]. Cabozantinib can treat renal cell carcinoma, liver cancer, etc. [142,143]. In mTNBC, cabozantinib monotherapy had shown effective antitumor activity, but in TNBC with brain metastases, monotherapy still has no antitumor activity. In a single-arm phase 2 clinical trial [144], PR occurred in 3 patients after 35 patients received cabozantinib monotherapy. The ORR of this study was 9 %, CBR was 34 %, and mPFS was 2 months. The results showed that high expression of sMET was associated with prolonged mOS and slowed tumor progression. The immune function of patients in this study was activated, and serum CTLs and NK cell levels were increased. Compared to baseline, pain symptoms were relieved in some patients, and pain medications could even be discontinued. Compared with other BC, cabozantinib monotherapy is less effective in patients with TNBC brain metastases. A single-arm phase 2 clinical trial showed [145] that in TNBC patients with brain metastases, CNS ORR was 0 %, 12-week CBR was 13 %, mPFS was 2.4 months, and mOS was 5.1 months.

Sunitinib

Sunitinib is a class of oral small molecule multi-kinase inhibitors that target VEGF, PDGFR, and HGF to enhance tumor immune response and anti-tumor activity [146,147]. Sunitinib monotherapy in advanced TNBC showed poorer efficacy than standard chemotherapies. In a randomized, open-label phase 2 clinical trial [148], patients receiving sunitinib monotherapy had lower mPFS and ORR than patients receiving standard treatment. The pCR when sunitinib was added to neoadjuvant chemotherapy was 27 % [149], but previous research by the team showed that the addition of sunitinib was not related to patients' pCR. Due to the low safety and high toxicity of sunitinib in combination with paclitaxel [150], it is not recommended as first-line treatment in clinical practice [151,152].

ENMD-2076

The aurora kinase A and angiogenesis (VEGFR, FGFR) inhibitor ENMD-2076 has been studied in hematologic tumors, ovarian cancer, and colorectal cancer [153], [154], [155]. A 2018 study showed durable clinical activity of ENMD-2076 in TNBC [156]. Among the 36 patients with locally advanced or metastatic TNBC included in this study, the CBR at 4 and 6 months after ENMD-2076 monotherapy was 27.8 % and 16.7 %, respectively. And the mPFS of all treated patients was 1.84 months.

Lapatinib (Tykerb)

The team led by Erica M. Stringer-Reasor found in preclinical experiments that EGFR inhibitors can promote the antitumor activity of PARP inhibitors in non-BRCA mutated tumors [157]. In a subsequent open-label study [158], the team evaluated the safety, tolerability, and anti-tumor activity of the EGFR and HER2 inhibitor lapatinib in combination with the PARP inhibitor veliparib in mTNBC. Of the 20 patients enrolled in the study, 17 patients were evaluated for efficacy, of which 4 patients reached PR and 2 patients reached SD.

Pyrotinib (SHR1258)

An oral, irreversible pan-ErbB receptor tyrosine kinase inhibitor, pyrotinib can target EGFR, HER2, and HER4. Pyrrolitinib was launched in China in 2018 and approved in combination with capecitabine for patients with HER2-positive advanced BC who had received anthracycline- or paclitaxel-based chemotherapy [159]. The clinical efficacy of pyrotinib in combination with capecitabine in patients with HER2-positive metastatic BC resistant to trastuzumab has also been confirmed in subsequent clinical trials [160]. However, exploratory trials of Pyrotinib in TNBC are rarely reported. In the FUTURE trial, pyrotinib in combination with capecitabine was shown to be effective in treating refractory TNBC, but the sample size is too small and further studies are needed [161].

Samotolisib (LY3023414)

Several clinical trials are ongoing for samotolisib, a dual serine/threonine kinase inhibitor targeting PI3K and mTOR. The combination of samotolisib and prexasertib showed better antitumor activity. In a phase 1b clinical trial, the regimen was used in 16 patients with locally advanced or advanced TNBC, with an ORR of 25 %, DCR of 62.5 %, and mPFS of 2.33 months [162].

Proteasome inhibitors

The ubiquitin-proteasome system (UPS) stands out as one of the principal intracellular pathways for protein degradation, encompassing two fundamental processes: the ubiquitination of substrate proteins and the subsequent degradation of ubiquitin-tagged proteins by the proteasome. Ubiquitinated proteins undergo degradation via the autophagy-lysosome pathway, recognition and degradation by the proteasome complex, or deubiquitination mediated by deubiquitinating enzymes, thereby intricately regulating downstream pathways [163,164]. The proteasome is a complex consisting of several subunits that plays an important role in cell cycle regulation. 80 % of proteins in the human body are degraded by the proteasome complex [165]. Dysfunction within the proteasome complex can exert a profound impact on cell proliferation and growth. Proteasome inhibitors (Table 4), designed to target the proteasome, exhibit the capability to modulate the degradation of substrate proteins associated with pertinent pathways, thereby holding promise for anti-tumor therapeutic interventions.

Table 4.

Proteasome inhibitors.

Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
PARP inhibitors	Pamiparib (BGB-290)	PARP	Advanced TNBC	NCT03333915	Ⅰ/Ⅱ	Pamiparib monotherapy	Asthenia, nausea
			Locally advanced/metastatic TNBC	NCT03575065	Ⅱ	Pamiparib monotherapy	Anemia, white blood cell count decreased, neutrophil count decreased, nausea, vomiting, decreased appetite, diarrhea
	Talazoparib	PARP	Early TNBC	NCT03499353	Ⅱ	Talazoparib monotherapy	Neutropenia, thrombocytopenia, alopecia, dizziness, dyspnea
	Olaparib (Lynparza, AZD2281, KU-0,059,436)	PARP	Advanced/metastatic TNBC	UMIN00009498	Ⅰ/Ⅱ	Combined with eribulin	Neutropenia, leucopenia, anaemia, febrile neutropenia, thrombosis
			Locally advanced TNBC	NCT02681562 (OLTRE)	Ⅱ	Olaparib monotherapy	Unpublished results
Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
			Inflammatory, loco-regionally advanced/metastatic TNBC	NCT03109080	Ⅰ	Combined with radiotherapy	Lymphopenia
			TNBC	NCT01042379 (I-SPY2)	Ⅱ/Ⅲ	Combined with durvalumab and paclitaxel	Neutropenia, fatigue,anemia, neuropathy, diarrhea
			Unselected TNBC	NCT02624973 (PETREMAC)	Ⅱ	Olaparib monotherapy	Fatigue
			Non-BRCA TNBC	ANZCTRN: 12,613,000,924,752 (SOLACE)	Ⅰ	Combined with cyclophosphamide	Lymphocyte count decrease, nausea, vomiting, leukopenia, anaemia, fatigue
			Advanced TNBC	NCT01623349	Ib	Combined with alpelisib	Hyperglycemia, rash, fatigue, nausea, anorexia
			TNBC	NCT02208375	Ib	Combined with capivasertib	Nausea, anemia, diarrhea, elevated creatinine, fatigue, hyperglycemia
			TNBC	NCT02789332 (GeparOLA)	Ⅱ	Combined with paclitaxel	Anaemia, leukopenia, neutropenia, atigue, alopecia, peripheral sensory neuropathy
	Iniparib (SAR240550, BSI-201)	PARP	Metastatic TNBC	NCT01045304	Ⅱ	Combined with gemcitabine and carboplatin	Neutropenia, anemia, leukopenia, thrombocytopenia, nausea, fatigue, constipation, vomiting
	Veliparib (ABT-888)	PARP	TNBC	NCT01042379 (I-SPY2)	Ⅱ	Paclitaxel plus veliparib and carboplatin followed by doxorubicin and cyclophosphamide	Febrile neutropenia, thrombocytopenia, anemia, vomiting
Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
			Early stage TNBC	NCT02032277 (BrighTNess)	Ⅲ	Paclitaxel plus veliparib and carboplatin followed by doxorubicin or cyclophosphamide	Neutropenia, anaemia, thrombocytopenia, febrile neutropenia
			Metastatic TNBC	NCT02158507	Not Applicable	Combined with lapatinib	Fatigue, diarrhea, constipation, insomnia, vomiting, anemia, headache, dizziness, dyspnea, rash
			TNBC	NCT01145430	Ⅰ	Combined with pegylated liposomal doxorubicin	Abdominal pain, fatigue, nausea alopecia, anemia, constipation
			Advanced TNBC	NCT0110429	Ⅰ	Combined with cisplatin and vinorelbine	Neutropenia, anemia, thrombocytopenia
			Metastatic TNBC	NCT01251874	Ⅰ	Combined with carboplatin	Fatigue, vomiting, headache, diarrhea, thrombocytopenia, neutropenia, anemia
			Recurrent/advanced TNBC	NCT01306032	Ⅱ	Combined with cyclophosphamide	Lymphopenia, fatigue
			Advanced/metastatic TNBC	NCT02163694	Ⅲ	Combined with carboplatin and paclitaxel	Neutropenia, nausea, diarrhea

PARP inhibitors

The Poly ADP-ribose polymerase (PARP) enzyme plays a critical role in repairing DNA damage. Two common types of DNA break repair in cells are homologous recombination repair (HRR) to repair DNA double-strand breaks and single-strand break repair involving PARP [166]. When the single-strand break occurs and PARP repair is blocked, DNA causes a double-strand break when it replicates to the broken DNA strand, and homologous recombination (HR) participates in the repair [167]. When both PARP and HRR pathways are disrupted, this can lead to cell genome instability, resulting in synthetic lethal effects and the induction of cell apoptosis [168]. Therefore, PARP inhibitors have a better effect on cells with HR deficiency (such as BRCA gene mutation) [169]. Most TNBC patients have HR deficiency, so PARP inhibitors may be beneficial in TNBC [170]. In recent years, studies have shown that PARP inhibitors also have some effect on TNBC without BACR mutations [58].

Pamiparib (BGB-290)

The CBR of pamiparib monotherapy in patients with advanced TNBC was 0 %, and its antitumor effect was not satisfactory [171]. The clinical sample of this study is small, and only 6 TNBC patients received the study, so the study results may be biased. In a phase 2 clinical trial for patients with germline BRCA1/2 mutations (gBRCA1/2 m), metastatic or locally advanced HER BC that the team conducted later, pamiparib monotherapy showed encouraging efficacy. Among the 68 TNBC patients with gBRAC1/2 mutations enrolled in the study, the ORR was 38.2 %, with an mDOR of 7 months [172]. In addition, parmiparib has brain-penetrating properties [173]. Pamiparib in combination with temozolomide had a good curative effect in the treatment of intracranial tumors [174]. Brain metastases frequently occur in advanced TNBC patients [175], so it is worth looking forward to whether Parmiparib can improve the prognosis of TNBC patients with brain metastases.

Talazoparib

Talazoparib has been shown to be more effective than standard chemotherapy in advanced gBACA-mutated BC [176]. Significant pathologic response was also observed in neoadjuvant treatment of gBRCA-positive, operable TNBC patients treated with talazoparib alone. Of the 19 patients who participated in the evaluation of pathologic response outcomes, TNBC patients with RCB-0/1 accounted for 57 % [177]. The combination of talazopanib and avelumab can also observe durable anti-tumor response in patients with advanced TNBC [178], with mDOR of 1.2 months and ORR of 18.2 %. However, acquired drug resistance limits the clinical efficacy of talazoparib, and combination with PI3K inhibitors may be one of the effective strategies to overcome talazoparib resistance. The PI3K inhibitor LY294002 promotes cell cycle arrest and apoptosis and increases DNA damage to synergistically inhibit proliferation of BRCA1973-mutated TNBC cells [179].

Olaparib (Lynparza, AZD2281, KU-0,059,436)

Olaparib, an oral PARP inhibitor, is more effective than standard therapy in patients with HER -negative metastatic BC and germline BRCA-mutated BC [180]. In 32 unselected primary TNBC patients monotherapied with olaparib, the ORR was 56.3 % after clinical and MRI evaluation [181]. Combination therapy with olaparib also has a good effect in the treatment of TNBC. A multicenter phase 1/2 trial of olaparib plus eribulin in 24 patients with advanced or metastatic TNBC showed that [182] mDOR was 5.5 months, mPFS was 4.2 months and OS was 14.5 months. In the I-SPY2 trial [183] 21 patients with TNBC received durvalumab in combination with olaparib and paclitaxel. Compared with TNBC patients in the control group who were treated with paclitaxel alone, the pCR rate of patients in the treatment group increased from 27 % to 47 %. Olaparib in combination with radiotherapy was well tolerated in TNBC [184]. Low-dose long-term treatment with olaparib may improve the radiosensitivity of TNBC [185], and olaparib may be a promising radiosensitizer for TNBC [186]. Although the clinical benefit of olaparib can be observed in different types of TNBC, the pCR rate is higher in gBRCA carriers than in non-carriers, and BRCA mutation remains an important factor affecting the treatment and prognosis of TNBC [187].

Iniparib (SAR240550, BSI-201)

Iniparib in combination with gemcitabine and carboplatin showed good efficacy in the treatment of mTNBC. The research team enrolled 163 patients with mTNBC in an open-label, randomized phase 2 clinical trial. The trial used two different treatment regimens of iniparib in combination with carboplatin and gemcitabine once weekly or twice weekly. The results showed that the once-weekly iniparib combination regimen was superior, the ORR was 34.1 %, the CBR was 41.5 %, the mPFS was 5.5 months and the mOS was 12.6 months, while the twice-weekly inipalib combination regimen had an ORR of 29.6 %, a CBR of 32.1 %, an mPFS of 4.3 months and an mOS of 12.4 months [188]. Iniparib combination therapy shows good efficacy in advanced or metastatic TNBC, but early TNBC does not appear to benefit [189].

Veliparib (ABT-888)

As a PARP inhibitor, the efficacy of veliparib in combination chemotherapy is variably assessed. A randomized phase 2 clinical trial showed that the addition of the low-dose PARP inhibitor veliparib to cyclophosphamide did not increase ORR, and although patients' PFS improved, this was not statistically significant [190]. When examining the clinical efficacy of veliparib in combination with carboplatin and vinorelbine [191], there were 2 cases of CR, 15 cases of PR, and 21 cases of SD among the 48 patients included in the evaluation. Veliparib in combination with carboplatin and paclitaxel achieved better results in TNBC patients [192]. In the I-SPY 2 trial [193], researchers combined veliparib and carboplatin in patients with HER2-negative BC, 72 patients received paclitaxel and carboplatin in combination with veliparib, and 44 patients were randomly assigned to the control group receiving paclitaxel and carboplatin. After 12 cycles of the above regimen, all patients received intravenous doxorubicin plus cyclophosphamide every 2 to 3 weeks. The efficacy of the subjects was assessed by molecular typing of HER2 and HR after 4 injection doses, and the pCR rate of patients in the TNBC combination treatment group was 51 % and that of the control group was only 26 %, whereas the pCR rate of HR+/HER2- combination treatment patients was 19 % lower than that of TNBC combination treatment patients. It appears that TNBC patients are a beneficial population for veriparib in combination with carboplatin compared to HR+/HER2- patients. However, results from the BrighTNess trial reported in 2018 [194] showed that adding carboplatin and veriparib to paclitaxel effectively increased the rate of pCR in patients, but there was no difference in the effect of combining or not combining veriparib on the rate of pathologic complete remission in patients. In a follow-up study, the team also reported that the addition of carboplatin to paclitaxel reduced the risk of event-free survival (EFS), while the addition of veriparib had no significant effect on EFS [195]. There was no significant effect of different dosing regimens on the efficacy of veriparib. In a multicenter phase 1 clinical trial reported in 2020 [196], 39 patients with mTNBC treated with 7- or 14-day intermittent veriparib or 21-day continuous veriparib in addition to carboplatin dosing had a CBR of 67.4 % and an mPFS of 18.3 weeks, with no significant impact of different veriparib dosing regimens on mPFS.

Reversion mutations reactivate BRCA1/2, and after recovery of HRR, tumor cells regain DNA damage repair function. At this point, tumor cells become resistant to PARP inhibitors [197], and the effect of PARP inhibitors is greatly reduced. Long-term use of PARP inhibitors can also lead to an increase in P-glycoprotein and BC resistance protein, increasing drug efflux and leading to drug resistance [198,199]. In addition, increased structural stability of DNA replication forks or DNA damage repair, resulting in the inability to form lethal DNA damage, is also an important reason for PARP resistance [200,201]. Therefore, combined treatment of tumor cells with inhibitors of DNA damage repair is a viable strategy to overcome drug resistance [202].

Epigenetic inhibitors

Epigenetics, a sub-discipline of genetics, delves into the study of heritable changes in gene expression that occur without alterations in the nucleotide sequence of genes. Its focus revolves around two key components: DNA and histones [203]. The mechanisms underlying epigenetic changes encompass four primary avenues: DNA methylation, histone modifications, chromatin structure remodeling, and RNA modifications [204]. Notably, abnormalities in epigenetic modifications are intricately linked to the onset of various diseases, including tumors, central nervous system disorders, diabetes, among others. In contrast to diseases stemming from genetic mutations, those arising from abnormalities in epigenetic modifications are generally considered reversible [205]. As a result, the advancement of epigenetic research has paved the way for the development of an increasing number of epigenetic inhibitors (Table 5) aimed at the treatment of diverse diseases.

Table 5.

Epigenetic inhibitors.

Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
HDAC inhibitors	Chidamide	HDAC	Relapsed or metastatic TNBC	NCT04192903	Ⅱ	Combined with cisplatin	Neutropenia, thrombocytopenia, leucopenia, vomiting
	Entinostat (MS-275)	HDAC	Newly diagnosed Stage I-IIIC TNBC	NCT03361800	I	Entinostat monotherapy	Unpublished results
			Postmenopausal and operable TNBC	NCT01234532 (0927GCC)	II	Combined with anastrozole	Nausea, fatigue, acid reflux
	Vorinostat (SAHA, VOR)	HDAC	Newly diagnosed operable TNBC	NCT00616967(TBCRC008)	II	Combined with carboplatin and paclitaxel albumin-stabilized nanoparticle formulation	Neutropenia, fatigue, diarrhea, nausea, anorexia
			Relapsed/metastatic TNBC	NCT03742245	I/Ib	Combined with olaparib	Unpublished results
	Panobinostat (LBH589)	HDAC	Advanced/metastatic TNBC	NCT02890069	I	Combined with PDR001	Unpublished results
	Romidepsin (FK-228)	HDAC	Recurrent/advanced TNBC	NCT02393794	Ⅰ/Ⅱ	Combined with cisplatin and romidepsin	Unpublished results

Histone deacetylase (HDAC) inhibitors

The central component of chromatin comprises DNA and nucleosomes, with histones constituting the nucleosomal structure. Histone modifications exert a profound impact on the transcriptional functionality of chromatin DNA. Histone deacetylase (HDAC) serves as a catalytic regulator in the realm of histone modification, playing a pivotal role in this intricate process. The dynamic equilibrium between HDAC and histone acetylase (HAT) intricately governs chromosome modification and gene expression. Perturbations in this balance, characterized by heightened histone deacetylation and diminished acetylation, can induce aberrations in the cell cycle, thereby fostering tumorigenesis [206]. Studies have shown that histone differences are associated with TNBC [206]. HDAC inhibitors may play a role in TNBC by inhibiting HDAC activity, decreasing the level of histone deacetylation, regulating glycolytic metabolism [207], inhibiting angiogenesis and tumor growth [208,209], and inducing tumor autophagy and apoptosis [210].

Chidamide

Chidamide is an oral selective HDAC inhibitor that has achieved remarkable efficacy in hematologic tumors and has been approved for the treatment of lymphoma in many countries [211]. Chidamide can mediate cell autophagy and reverse chemotherapy resistance to increase sensitivity to chemotherapy [212], but chidamide and cisplatin have not achieved significant curative effects in the first-line treatment of mTNBC [213]. The ORR under first-line treatment was 26.67 %, which did not reach the expected 33.33 %. Cell and animal studies have shown that chidamide can reduce the expression of the drug resistance genes RAD51 and MRE11 to reverse the drug resistance of TNBC cells to fluzopanib, inducing apoptosis and enhancing the anti-tumor effect [214]. However, there are no relevant clinical reports.

Entinostat (MS-275)

Entinostat, a drug that targets HADC1, has shown significant efficacy in HR-positive or HER2-positive BC [215,216]. However, its effectiveness is not ideal in TNBC. In a multicenter phase 2 clinical trial, a combination of the epigenetic modifier azacitidine (5-AZA) and the HDAC inhibitor entinostat was administered to 27 hormone-resistant patients and 13 TNBC patients. Unfortunately, the trial did not meet its endpoints, with an ORR of only 4 %. During a follow-up period of 10.4 months, most patients experienced disease progression or death, and no disease remission was observed in the 13 TNBC cases [217]. Despite research exploring entinostat as a monotherapy (NCT03361800) or in combination with endocrine therapy (NCT01234532) for TNBC, both trials were prematurely terminated, and no clinical data could be obtained.

Vorinostat (SAHA, VOR)

Despite numerous clinical studies in BC patients, the clinical efficacy of vorinostat in combination with chemotherapy for TNBC appears to be less promising. A dual-center phase 1/2 clinical trial published as early as 2014 showed that the pCR rate of vorinostat combined with paclitaxel in TNBC patients was only 27 % [218]. The TBCRC008 study, published in 2018, was a multicenter, randomized, double-blind phase 2 clinical trial [219]. It included 62 patients with HER2-negative BC. The aim was to evaluate the pCR rate of these patients after 12 weeks of preoperative treatment with carboplatin combined with albumin-bound paclitaxel, with or without vorinostat. The results showed that the placebo group had a pCR rate of 58.3 %, while the vorinostat group had a lower rate of 41.7 %. This study once again demonstrated the limited benefit of vorinostat in TNBC. Currently, an ongoing phase 1b/2 clinical study is evaluating the combination of the PARP inhibitor olaparib with vorinostat for the treatment of metastatic or refractory HER2-negative BC patients. It will be interesting to see if the results differ from the clinical effectiveness of vorinostat in combination with chemotherapy for TNBC.

Valproic acid (VPA)

Valproic acid can inhibit the growth of TNBC cells, but there are few clinical trials related to TNBC. The VPA-FEC100 trial combined valproic acid with FFC100 to treat metastatic BC. However, due to the unknown molecular classification of metastatic BC, it cannot be confirmed whether valproic acid is effective in treating TNBC. Another clinical trial (NCT01552434) demonstrated that the combination of temsirolimus, bevacizumab, and valproic acid can be utilized for treating metastatic BC. However, the results of this trial have not yet been published, so the clinical efficacy of this treatment for TNBC is uncertain.

Other HDAC inhibitors for the treatment of TNBC are also being investigated in clinical trials. This includes research on panobinostat (LBH589) in combination with the immunosuppressant PDR001 (NCT02890069), as well as ricolinostat (ACY-1215, RICO) in combination with albumin-bound paclitaxel (NCT02632071). Phase 1 clinical trials of paclitaxel in the treatment of metastatic BC have been completed, but the results have not yet been released. At the same time, phase 1/2 trials of Romidepsin (FK-228) combined with the chemotherapy drug cisplatin and the immunotherapy drug nivolumab are also ongoing (NCT02393794). Whether these drugs can achieve clinical efficacy in TNBC is worth anticipating. On the other hand, there are numerous potential HDAC targets under investigation. HDAC3 can regulate the proliferation of cancer stem cells (CSC) and TNBC cells [220]. HDAC3 selective inhibitor compounds 18 and 28 throng modulating the Akt/GSK3β pathway to downregulate the expression of β-catenin, thereby inhibiting the efficacy of CSC subpopulations in TNBC cells [221]. Another HDAC3 inhibitor, I-7ab, can induce P53 expression, promote cell apoptosis, and inhibit TNBC cell activity [222]. HDAC6 has been shown to be associated with TNBC proliferation and metastasis in preclinical studies [223]. Downregulating the expression of HDAC6 in TNBC can induce tumor autophagy [224]. In TNBC preclinical models, both HDAC6 inhibitors YSL-109 and 10 g have exhibited significant anti-proliferative activity [225,226]. The overexpression of HDAC8 in TNBC is associated with TNBC tumor size, lymphatic infiltration, and tumor grade [206]. HDAC8 can activate YY1 or Hippo/YAP signaling to promote the proliferation and migration of TNBC [227,228]. Down-regulation of HDAC8 can reduce the migration and invasion of TNBC and induce apoptosis in cancer cells [229]. Differently, the expression of HDAC7 is decreased in TNBC patients. Low expression of HDAC7 can lead to TNBC cell proliferation and is linked to a poor prognosis for TNBC patients [230]. Activating HDAC7 may be an effective approach to treating TNBC.

Hedgehog (Hh) inhibitors

The Hedgehog (Hh) pathway is a highly conserved signaling pathway associated with embryonic development and consists of Hedgehog ligands (SHH, DHH, IHH), Ptch receptors, SMO proteins, kinesin Kif7, protein kinase A (PKA), and glioma-associated oncogene (GLI) transcription factors [231]. Binding of Hh ligand to the Ptch receptor activates SMO and downstream pathways, leading to activation of GLI protein, and entry of GLI into the nucleus promotes tumor cell division and growth [232]. Overexpression of the Hh pathway is one of the pathological features of TNBC [233], [234], [235]. Abnormal activation of the Hh pathway is associated with angiogenesis, invasive metastasis, and high risk of recurrence in TNBC [236,237]. Hedgehog pathway inhibitors (Table 6) can be divided into Hh acyltransferase (Hhat) inhibitors, SMO inhibitors, and GLI inhibitors according to their mode of action. Among all inhibitors of the Hh pathway, SMO inhibitors are the most intensively studied. Several SMO inhibitors are currently in clinical trials.

Table 6.

Hedgehog (Hh) inhibitors.

Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
Smo inhibitor	Sonidegib (LDE225)	Smo	Advanced TNBC	NCT02027376 (EDALINE)	Ib	Combined with docetaxel	Neutropenia, leukopenia, paresthesia

Sonidegib (LDE225)

Sonidegib is a selective oral SMO inhibitor that increases the sensitivity of tumor cells to docetaxel by downregulating the expression levels of cancer stem cell markers (CSC) [238]. The EDALINE [239] combined the SMO inhibitor sonidegib with docetaxel in 12 patients with TNBC to determine the maximum dose and recommended phase 2 dose (RP2D) of the two drugs in combination. The study showed an ORR of 10 %, an mPFS of 42.5 days, and the mPFS for patients treated with RP2D was 188 days.

Nuclear export protein 1 (XPO1) inhibitors

Nuclear export protein 1 (XPO1), also known as chromosomal region stabilizing protein 1, regulates the export of NSE-containing proteins such as p53, BRCA1, APC, and some RNAs from the nucleus to the cytoplasm, which plays an important role in maintaining cellular stability. When exported to the cytoplasm, these proteins can promote tumor proliferation [240,241]. Overexpression of XPO1 is associated with poor prognosis in BC, and targeting XPO1 (Table 7) may be an effective treatment for TNBC [242,243].

Table 7.

Nuclear export protein 1 (XPO1) inhibitors.

Classification	Name	Target	TNBC Types	Trial Identifier	Phase	Combined medication	Major adverse effects
XPO1 inhibitors	Selinexor (KPT-330)	XPO1	Metastatic TNBC	NCT02402764	Ⅱ	Selinexor monotherapy	Constipation, anorexia, nausea, vomiting, fatigue, dyspnea, thrombocytopenia, blurred vision

Selinexor (KPT-330)

Selinexor is an oral selective nuclear output inhibitor mainly used for the treatment of refractory multiple myeloma [244]. It has been used in some trials for the treatment of mTNBC [245], but clinical outcomes are poor. In 10 patients with mTNBC treated with selinexor monotherapy, the CBR was 30 %. It is worth noting that selinexor can enhance the antitumor activity of olaparib in TNBC regardless of gBRCA mutation status [246].

Other potential targets

Programmed cell death protein 1 (PD-1, CD279)

Programmed cell death protein 1 (PD-1, CD279) plays a crucial role in regulating programmed cell death [247].It inhibits T cell activation by interacting with Programmed cell death-Ligand 1 (PD-L1) or Programmed cell death-Ligand 2 (PD-L2) to enable immune evasion of tumor cells [248]. PD-1 not only regulates T cells, B cells, and other immune cells, but is also expressed in cancer cells [249]. Studies have shown that the expression of PD-1 is upregulated in TNBC patients. In vitro and in vivo experiments have demonstrated that overexpression of PD-1 promotes the growth of TNBC cells and metastasis. Blocking the activation of the PD-1/PD-L1 signaling pathway can inhibit the proliferation, growth, and metastasis of TNBC [250]. Although the PD-1 inhibitor pembrolizumab combined with chemotherapy has been shown to improve the OS of PD-L1-positive metastatic TNBC patients, the clinical benefit of immunotherapy is related to the population [251]. The FUTURE-C-Plus trial utilized the PD-1 monoclonal antibody camrelizumab in combination with angiogenesis inhibitors and paclitaxel for the treatment of advanced TNBC. This study shows that the clinical benefit of PD-1 monoclonal antibody immunotherapy is limited, and patients with CD8 and PD-L1 positive can benefit more from it [252]. Furthermore, adverse reactions associated with immunotherapy are predominantly of grade 3–4 [253], and in severe cases, they can lead to fatal conditions such as fulminant myocarditis [254]. Looking for alternatives to immunotherapy may be necessary. Because small molecule drugs have comparable efficacy to monoclonal antibodies but are safer [255], researchers have begun to focus on developing small molecule inhibitors targeting PD-1. The small molecule chemical Methylene Blue (MB) can block PD-1 signal molecules to enhance the cytotoxicity of CD8+ cytotoxic T lymphocytes (CTL) and play an anti-tumor role [256]. Other small molecule inhibitors targeting PD-1 are also under development. Although there is currently a lack of research related to PD-1 small molecule inhibitors, their safety and effectiveness will lead to ongoing development of these inhibitors.

V-domain Ig suppressor of T-cell activation (VISTA)

V-domain Ig suppressor of T-cell activation (VISTA) is an immune checkpoint protein with the capacity to suppress T cell activity [257], playing a crucial role in anti-tumor immunity. Studies have shown that VISTA is expressed at low levels in TNBC, but it is highly expressed in immune cells within the TME. Elevated levels of VISTA in immune cells are correlated with increased immune infiltration and a more favorable prognosis for TNBC [258]. In a study involving 254 patients, it was found that high expression of VISTA in immune cells was associated with lymph node metastasis-free survival (MFS) and OS in patients with TNBC [259]. Despite its low expression in TNBC, VISTA is intricately linked to the epithelial-mesenchymal transition (EMT) and the induction of immunosuppression in this context. Inhibition of VISTA presents a synergistic effect when combined with the blockade of PD-L1 or Cytotoxic T Lymphocyte-Associated Antigen-4 (CTLA-4), thereby augmenting the efficacy of immunotherapy [260]. Notably, the expression of VISTA has been observed to increase following PD-L1 immunotherapy [258], suggesting that VISTA may be a potential target for drug resistance in immunotherapy. Consequently, targeting VISTA holds promise for enhancing therapeutic outcomes in TNBC cases characterized by a low response rate to PD-1/PD-L1 or exhibiting resistance to PD-1/PD-L1 inhibitors.

Inducible T-Cell costimulator (ICOS, CD278)

Inducible T-Cell costimulator (ICOS, CD278) belongs to the CD28 protein family and is primarily expressed on activated T cells [261]. ICOS expression extends beyond CD8+ effector T cells, encompassing regulatory T cells (Tregs) as well [262]. Upon activation, CD8+ effector T cells release immune-enhancing cytokines such as IFN-γ and TNF-α to bolster tumor immunity [263]. While the proliferation of Tregs, crucial for maintaining the immunosuppressive microenvironment, relies on the interaction of ICOS and Inducible T-cell costimulator ligand (ICOSL) [264]. Hence, promoting the activation of ICOS-expressing CD8+ effector T cells and inducing the depletion of ICOS-expressing Tregs emerge as promising strategies to ameliorate the tumor immune microenvironment and reestablish the tumor immune response [262]. Given the bidirectional regulatory nature of the ICOS/ICOSL signaling pathway, drugs targeting this pathway can be classified as either agonists or antagonists. Research findings indicate the up-regulation of ICOS in more than 20 types of tumor tissues, including BC [265]. The expression level of ICOS in BC correlates with clinical and molecular classifications, with high ICOS expression linked to favorable survival outcomes in TNBC [266]. Currently, the ICOS agonist monoclonal antibody vopratelimab has demonstrated good tolerability in clinical trials involving patients with advanced solid tumors, whether treated with or without immunotherapy. Although the clinical benefit is limited, the ORR of vopratelimab monotherapy was 1.4 %. Even combining with nivolumab, the ORR was only 2.3 %. However, this trial identified ICOS high-expressing CD4 T cells as a potential predictive biomarker for good efficacy, offering a pathway for future clinical research [267].

Interleukin-6 (IL-6)

Interleukin-6 (IL-6) is a crucial inflammatory cytokine that plays a significant role in tumor development. Overexpression of IL-6 in tumor tissues can stimulate the overactivation of JAK/STAT3 signaling pathway, leading to tumor angiogenesis, chemotherapy resistance, and tumor immunosuppression [268], [269], [270], ultimately resulting in a poor prognosis for patients [271]. Regulation of the IL-6/JAK/STAT3 pathway can inhibit tumor proliferation and metastasis, and activate tumor immunity to control the malignant progression of tumors [272,273]. Studies have shown that the activation of the IL-6/JAK/STAT3 pathway is associated with the EMT of TNBC and the migration and metastasis. Blocking the IL-6 signaling pathway has a significant impact on enhancing TNBC tumor immunity and inhibiting the growth and spread of TNBC [274]. IL-6/IL-6R antagonists, JAK inhibitors, and STAT3 inhibitors have all demonstrated significant anti-tumor effects in basic research. Although the IL-6 small molecule inhibitor COB-141 has demonstrated strong inhibition in TNBC cells [275], there are currently no relevant clinical studies. The study of the STAT3 small molecule inhibitor TTI-101 on patients with advanced solid tumors is currently ongoing, and the results have not yet been announced.

Transforming growth factor-β1 (TGF-β1)

EMT is a crucial mechanism for tumor invasion and metastasis. Transforming growth factor-β1 (TGF-β1) plays a key role in regulating EMT [276]. Its expression level in TNBC tissues is significantly higher than that in non-TNBC tissues, which is closely associated with OS and PFS of TNBC patients [277]. TGF-β can drive the pleiotropy of NDRG1 to differentially regulate CSC and EMT of TNBC at different stages to promote metastasis [278]. Inhibiting the activation of the TGF-β1 pathway can prevent the EMT, thus preventing the progression of TNBC [279]. On the other hand, TGF-β1 can regulate the downstream factor hepatic leukemia factor (HLF) to promote the proliferation, metastasis, and chemotherapy resistance of TNBC [280]. At the same time, upregulation of TGF-β1 expression can induce the polarization of M2 macrophages to modulate the TME [281]. Silencing of TGF-β can reshape the immunosuppressive microenvironment and work together with anti-PD-L1 to produce anti-tumor effects [282]. Therefore, inhibiting TGF-β1 in combination with chemotherapy or immunotherapy is an effective approach for controlling the progression of TNBC. A phase 1 clinical study has been conducted on the TGF-β1 small molecule inhibitor galunisertib combined with paclitaxel for the treatment of TNBC patients to assess its effectiveness and safety. The results have not been announced yet. Other clinical trials using TGF-β1 small molecule inhibitors such as LY3200882 and GFH018 are also ongoing [283].

Activated Cdc42-associated kinase 1 (ACK1, TNK2)

Activated Cdc42-associated kinase 1 (ACK1, TNK2) is a non-receptor tyrosine kinase that is overexpressed in various tumor tissues, such as lung cancer, prostate cancer, gastric cancer, and breast cancer and so on. High expression of it is associated with tumor malignant progression and poor prognosis [284]. ACK1 plays a crucial role in the proliferation, invasion, migration, and drug resistance of BC. ACK1 can promote the growth of BC by interacting with the estrogen receptor (ER) /histone demethylase KDM3A complex [285]. Excessive activation of ACK1 can promote the efficient transcription of cell cycle genes such as CCNB1 and CCNB2, making BC cells resistant to CDK4/6 inhibitors [286]. ACK1 inhibitor (R)−9bMS can not only inhibit the proliferation of TNBC [287], but also block the cell cycle and make the growth of drug-resistant HR+/HER2- BC fade [286]. Previous studies have shown that dasatinib monotherapy is ineffective in the treatment of TNBC, the ORR was 4.7 %, the DCR was 9.3 % and the mPFS was only 8.3 weeks after intervention with either 100 mg or 70 mg of dasatinib twice daily [288]. However, preclinical studies have shown that dasatinib can enhance the sensitivity of PARP inhibitors or chemotherapy drugs in TNBC, leading to a synergistic anti-tumor effect [289]. In addition, ceritinib, a small molecule drug used for NSCLC, can be combined with enzalutamide or paclitaxel to block the ACK1/AR pathway and inhibit the growth of TNBC [290]. Therefore, targeting ACK1 is also an option for treating TNBC.

Conclusion: challenges and prospects

TNBC is the most prognostically poor subtype of BC, characterized by a high recurrence rate, a high risk of metastasis, and a short survival time. Due to its specific molecular features, it is insensitive to endocrine therapy. Due to the lack of specific targets, clinical treatment of TNBC is still based on anthracyclines in combination with paclitaxel as first-line therapy, but the emergence of drug resistance makes TNBC less sensitive to chemotherapy, so new effective treatment modalities need to be sought. Targeted drugs based on small molecules have the advantage of being highly selective, low in toxicity, and easy to administer. They can be precisely targeted to specific targets to achieve personalized treatment of TNBC, which is one of the viable methods for the treatment of TNBC. This article summarizes the reports of targeted small molecule drugs that have entered phase 1–3 clinical trials in the past five years. Due to the complexity of advanced or metastatic TNBC, there is often a lack of effective treatment modalities, so clinical drug trials are mostly conducted for patients with advanced or metastatic TNBC, and there are fewer reports of small-molecule targeted drugs for early-stage TNBC. BRCA mutation is the more beneficial type of TNBC treatment, and the PARP inhibitors olaparib or talazoparib are important options for patients with advanced TNBC with BRCA mutation. The OlympiAD trial showed that olaparib improved mPFS in germline BRCA-mutated or HER2-negative metastatic BC, with a subgroup analysis showing a greater PFS benefit in TNBC patients among all HER2-negative patients [291]. The EMBRACA trial showed that talazoparib monotherapy prolonged PFS better than standard monotherapy in patients with advanced BRCA-mutated BC [176]. Fewer drugs are available for TNBC with non-BRCA mutations, and the PARP inhibitor olaparib in combination with cyclophosphamide shows promise for expanded use in TNBC with non-BRCA mutations [292]. Although better clinical efficacy in TNBC can be achieved with targeted small molecule drugs, drug resistance is a perennial problem in tumors, and the development of multi-target drugs and a new generation of drug-resistant mutation inhibitors and combination drugs is a viable way to overcome drug resistance. Three generations of EGFR inhibitors are on the market, and due to the problem of EGFR inhibitor resistance, the fourth generation of EGFR inhibitors targeting Del19/T790M/C797S or L858R/T790M/C797S mutations, such as BLU-945 and EAI045, have been developed and entered clinical trials [293,294]. Resistance to PARP inhibitors is also prevalent in TNBC therapy, as restoration of homologous recombination repair or deficiency of PAR hydrolase (PARG) in BRCA-mutated TNBC cells can abrogate the synthetic lethal effects mediated by PARP inhibitors and lead to resistance to PARP inhibitors [197,295]. The combination of CDK inhibitors targeting tumor immunological microenvironment or other ways besides HRR to enhance synthetic lethal effect is an important way to overcome PARP resistance [202,296,297]. Although the combination of drugs may increase drug resistance and drug sensitivity, it is necessary to pay attention to the safety of drug intake and tolerability for patients, and it is a direction that should be explored in the future to ensure the efficacy of drugs and control adverse drug reactions.

Other new drugs for the treatment of TNBC are under development. TROP2 is highly expressed in TNBC and is associated with poor prognosis in TNBC [298]. Sacituzumab govitecan (SG) is an antibody-drug conjugate (ADC) that targets the antibody-coupled irinotecan metabolite TROP2 and showed stronger antitumor activity than chemotherapy in metastatic or relapsed TNBC in which multiple lines of treatmenthave failed [299]. SG significantly improved PFS and OS in TNBC patients compared with chemotherapy [300]. Targeted protein degradation (TPD) is a new targeted therapeutic approach. Targeted protein degradation chimeras (PROTACs) have low toxicity due to their ability to degrade target proteins not previously inhibited by small molecule drugs and are used in various cancer studies [301]. They can inhibit tumor growth and invasion by targeting and inducing the degradation of target proteins via the UPS [302]. The PROTAC degrader PP-C8 can cooperate with PARP inhibitors to achieve anti-TNBC effects by inducing CDK12 degradation [303]. PROTAC may also exert synergistic antitumor effects by counteracting resistance to small molecule-targeted drugs. PARP PROTAC NN1 and NN3 promote PARP1 degradation for antitumor effects against mutation-induced PARP resistance [304]. PROTAC is effective and shows good antitumor activity even in wild-type BRCA TNBC [305]. PROTAC degrader XL01126 can cross the blood-brain barrier in mice after oral administration, so PROTAC degrader may be a hope for TNBC brain metastasis patients [306]. As PROTAC research progresses, more target-specific PROTAC degraders are being developed that directly degrade target proteins and play a more effective role than conventional small molecule drugs that inhibit the target [307]. PROTAC may become a new star among small molecule targeted drugs, and a new era of treatment may begin for TNBC.

CRediT authorship contribution statement

Yan Ou: Writing – original draft, Conceptualization, Resources, Writing – review & editing. Mengchao Wang: Methodology, Resources, Writing – review & editing. Qian Xu: Writing – review & editing, Conceptualization. Binxu Sun: Writing – review & editing. Yingjie Jia: Conceptualization, Resources, Supervision, Writing – review & editing.

Declaration of competing interest

All authors disclosed no relevant relationships.