The roles of TPL in hematological malignancies

ABSTRACT Triptolide (TPL) is a diterpenoid isolated from the traditional Chinese medicine Tripterygium wilfordii. It has powerful antitumor, immunosuppressive and anti-inflammatory properties. Recent studies have shown that TPL can induce apoptosis of hematological tumor cells, inhibit their proliferation and survival, promote autophagy and ferroptosis, and enhance the efficacy of traditional chemotherapy and targeted therapies. Various molecules and signaling pathways, such as NF-κB, BCR-ABL, and Caspase, are involved in inducing apoptosis of leukemia cells. To solve the water solubility and toxic side effects of TPL, low-dose TPL (IC20) combined with chemotherapy drugs and various TPL derivatives have entered preclinical studies. This review discusses advances in molecular mechanism, the development and utilization of structural analogues of TPL in hematologic tumors in the past two decades, and clinical applications.


Introduction
Triptolide (TPL, PG 490, LDTT-2, C20H24O6, molecular weight 360.4) is a diterpenoid tricyclic oxide from the Chinese herbal medicine Tripterygium wilfordii, which was first described in 1972 [1].Previous studies have shown that TPL has a wide range of anti-inflammatory and immunosuppressive activities and has been used to treat autoimmune diseases [2][3][4].Many studies have shown that TPL has antitumor effects [5][6][7][8][9].However, most of these studies concentrated on solid tumors, and relatively few studies on hematological malignancies.This review summarizes the research progress of TPL in hematological malignancies and the molecular mechanisms involved.
1 The effect of TPL as a single drug

TPL induces apoptosis in hematological tumor cells
Apoptosis is the main antitumor mechanism of TPL.Multiple signaling pathways and mechanisms are involved in mediating the proapoptotic effect of TPL (Figure 1).XIAP (X-linked inhibitor of apoptosis protein) is a natural intracellular caspase inhibitor and is highly expressed in a variety of leukemia cell lines and acute myelocytic leukemia (AML) blasts [10,11].TPL can induce caspase-dependent apoptosis in several AML cell lines and AML blasts by reducing the expression of XIAP and MCL-1 [12], and promoting cytochrome C release to upregulate caspase-dependent apoptotic pathways [13,14].The p53 signaling pathway is also involved in this process [15].MDM2 is an intracellular negative regulator of p53.TPL induces AML cell apoptosis by reducing MDM2 expression to disrupt MDM2-p53 homeostasis [16], thereby promoting p53 activation and upregulating the expression of the downstream death receptor DR5 [17].AML cells are relatively resistant to tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL), which can be overcome by TPL by downregulating XIAP and activating the P53 signaling pathway [18,19].Therefore, TPL combined with TRAIL may achieve a better effect in treating AML.
ROCK is a serine/threonine protein kinase composed of ROCK1 and ROCK2.Caspase-3 can cause the cleavage and activation of ROCK1 and lead to the phosphorylation of MLC and MYPT [20,21].TPL can selectively induce leukemia cell apoptosis through the ROCK1/MLC pathway [22,23].In mouse leukemia xenotransplantation models, TPL can inhibit tumor growth through ROCK1 activation and MLC phosphorylation [24].In addition, TPL can also inhibit the proliferation of AML cell lines by inhibiting the NF-κB pathway and activating the MAPK pathway to upregulate caspase-dependent apoptosis [25].In myeloproliferative disorder (MPD) cells, TPL can induce tumor cell apoptosis by inhibiting JAK transcription [26].In lymphocytic leukemia cell lines, TPL promotes apoptosis by inhibiting the NF-κB pathway and downregulating the expression of microRNA-16-1* [27].
TPL has excellent antitumor effects in specific types of hematological tumors.In chronic myelogenous leukemia (CML) cell lines, TPL can inhibit the proliferation of CML cells and reduce the expression level of BCR-ABL [28].TPL can overcome imatinib resistance in T315I mutant cells by inhibiting BCR-ABL transcription and inducing apoptosis [29].In t (8; 21) AML, the development of the disease requires the formation of the AML1-ETO fusion gene and C-KIT mutation.TPL can inhibit these two processes and suppress the proliferation of tumor cells by inhibiting the JAK-STAT pathway and promoting apoptosis [30].
In multiple myeloma (MM), TPL can inhibit the growth of bone marrow-derived mesenchymal stem cells (BMMSCs) from MM patients by decreasing IL-6, IL-1β and SCF mRNA expression [56,58,59], and improve the bone marrow hematopoietic microenvironment by inhibiting the proliferation of MM cells [59].TPL inhibits Mcl-1 mRNA transcription and promotes its protein degradation through the ubiquitinproteasome pathway [87], and induces MM cell apoptosis by upregulating the expression of RIZ1 [88] and protein tyrosine phosphatase 1 (SHP-1), and down-regulating antiapoptotic factors through the STAT3 pathway [89].In addition, TPL induces apoptosis in MM cells by blocking the aggregation of trimethylation of histone H3 lysine 4 (H3K4me3) in the promoter region of c-Myc and VEGFA [90], and inhibiting the transcription factor NF-κB [91].
In Burkitt lymphoma (BL), where the loss of SIRT3 is associated with high aggressiveness [70], TPL promotes apoptosis by upregulating SIRT3 expression and activating the SIRT3/GSK-3β/Bax pathway.In addition, TPL inhibits tumor growth in mouse BL xenografts in a dose-dependent manner [71].In B lymphoma cells, TPL inhibits tumor cell growth by reducing the expression of human telomerase reverse transcriptase (hTERT) through the down-regulation of the expression levels of EBV-positive B lymphoma cell-specific protein 1 (SP1) and c-Myc transcription factor [92]. TPL inhibits the proliferation of Raji cells, a B-cell non-Hodgkin's lymphoma (B-NHL) cell line, by blocking the SDF-1/CXCR4 axis [93].Low-dose TPL promotes apoptosis in B lymphoma cells by increasing phosphorylated histone H2AX levels, DNA fragmentation, and activation of caspase-3 [94].In T cell lymphoblastic lymphoma (T-LBL), TPL exerts an antitumor effect by increasing the expression of Caspase-3 and Bax, downregulating the expression of Bcl-2 [95], and inhibiting invasion and EMT of Jurkat cells through the regulation of PI3 K/AKT/mTOR pathway [95].

TPL inhibits the proliferation of hematological tumor cells by inducing cell cycle arrest
TPL can inhibit tumor cell proliferation through cell cycle arrest (Figure 1).RPB1, the largest subunit of RNAPII, maintains genome integrity and promotes DNA repair through its interaction with Williams syndrome transcription factor (WSTF) (RPB1-WSTF) [45].TPL can induce apoptosis by causing DNA damage through the downregulation of WSTF-RPB1.In vitro and in vivo experiments showed that TPL inhibits the proliferation of AML cell lines and causes cell cycle arrest in G1 phase [87].Compared with solid tumors and other types of leukemia, AML cell lines are more sensitive to low-dose TPL-induced cell cycle arrest [96].Raman spectroscopy data show that TPL can inhibit the proliferation of leukemic T lymphocytes by arresting them in the G1 and S phases [97].
In multiple myeloma (MM), RPMI8226 cells treated with TPL showed a significant increase in the proportion of cells in the G0/G1 phase compared with the control, suggesting that TPL treatment can induce G0/G1 phase cell cycle arrest in these cells [31].In contrast, in MM cell line U266, TPL arrests the cells in the G2/M phase [32,98].TPL-induced cell cycle arrest is achieved by inhibiting the methylation of histone proteins in both cell types.
TPL can also induce cell cycle arrest in murine leukemia cells.TPL can also impair the survival ability of K562/A02 cells and overcome their resistance to daunorubicin (DOX) by inhibiting the expression of miR-21 and thus reducing the level of PENT [32].

TPL promotes autophagy in hematological tumor cells
Studies have shown that autophagy plays an important role in tumorigenesis and treatment sensitivity [33,34], and there is an interaction between apoptosis and autophagy [35][36][37][38].It was shown that TPL induces both apoptosis and autophagy in WEHI-3 murine leukemia cells, evident by the characteristic changes in apoptotic proteins, the expression level of the autophagy marker LC-3, and the increased expression of autophagy molecules related to apoptosis, Atg5, Atg7, and Atg12 [39].The results indicate that the inhibitory effect of TPL on WEHI-3 may involve the interaction between apoptosis and autophagy, as shown in Figure 1.

TPL promotes ferroptosis in hematological tumor cells
Recent studies have shown that TPL promotes ferroptosis in leukemic cells [99].TPL-induced ferroptosis in DOXresistant leukemic cells is achieved by inhibiting the expression of Nrf2, resulting in increased ROS level, lipid oxidation, and decreased glutathione peroxidase 4 (GPX4).Notably, low-dose TPL treatment could resensitize DOX-resistant leukemic cells [99], which is consistent with the inhibitory effect of TPL on Nrf2 target gene expression in lung cancer cells [100].
2 Combined effects of TPL and chemotherapy drugs 2.1 TPL in combination with traditional chemotherapy drugs DOX and idarubicin (IDA), both of which are anthracyclines, have a broad killing effect on various solid and hematologic tumors [40,41].Cytosine arabinoside (Ara-c) is the most commonly used chemotherapy drug in the treatment of AML [42,43].These three drugs are widely used in the treatment of hematologic tumors, and the '7 + 3' regimen consisting of DOX/IDA and Ara-c has become the primary chemotherapy for AML [44].
The combination of TPL with chemotherapeutic agents exerts antitumor effects through multiple mechanisms of action (Figure 2).IDA/DOX combined with TPL can induce apoptosis of leukemia cells by downregulating HIF-1a and Nrf2 [45,46].HIF-1a and Nrf2 are transcription factors.HIF-1a plays an important role in the adaptive response of tumor cells to a hypoxic environment, and it is also significant for the survival of leukemia stem cells [47].Nrf2 is an antioxidant transcription factor [48][49][50].The inhibition of HIF-1a expression can directly promote tumor cell apoptosis [51], while Nrf2 reduction can also induce tumor cell apoptosis caused by the accumulation of ROS [52].TPL combined with chemotherapy drugs can induce leukemia cell apoptosis through DNA damage [101].
The cotreatment of TPL and DOX/Ara-c in NALM-6 cells can effectively inhibit DNA damage checkpoints 1 and 2, leading to intense DNA damage and tumor cell apoptosis [53].TPL can inhibit the expansion and survival of leukemia cells.DOX is a DNA-damaging drug that induces G2 phase arrest [54,55].In the study by Bing Z. Carter et al., the treatment of OCI-AML3 cells with DOX or gemtuzumab ozogamicin (GO) alone produced significant G2 phase arrest after 48 h.However, when TPL and DOX/GO were combined, this cell arrest was abolished, leading to cell death [12].This indicates that TPL can induce cell death by breaking the DOX/GO-induced G2 phase arrest.

TPL in combination with targeted therapy
Targeted therapy is an important means of tumor treatment that has made rapid progress in recent years and is widely used in hematological malignancies [57].This effect can be improved when combined with TPL (Figure 2) [13,14,60].Venetoclax is a BCL-2 inhibitor approved by the FDA for treating chronic lymphocytic leukemia (CLL) and AML [61].The combination of TPL and Venetoclax can exert anti-leukemic effects by regulating the expression of Bcl-2 family proteins and activating the endogenous apoptosis pathway [13].Sorafenib is a multitargeted tyrosine kinase inhibitor that is mainly used for the treatment of AML with FLT3-ITD mutations [62].However, it is difficult to achieve durable remission, and long-term use is associated with drug resistance [63,64].Lowdose TPL (IC20) combined with sorafenib can enhance the apoptosis rate of FLT3-ITD AML cells by reducing the expression of FLT3, STAT5, and P-STAT5, and the two drugs show a good synergistic effect [65].HSP-90 is overexpressed in AML cells and closely related to the proliferation of leukemia cells [66,67].Inhibition of HSP-90 expression can degrade the BCR-ABL fusion protein and induce apoptosis in Ph + Acute lymphoblastic leukemia (ALL) cells [68].BIIB021 is a selective inhibitor of HSP-90 that is used for the treatment of a wide range of tumors [69].In vitro experiments showed that TPL combined with BIIB021 could disrupt the dynamic balance between P53 and MDM2 in MOLT4 T-ALL cells, resulting in excessive activation of P53 and upregulation of the expression of Bak and Bim to induce tumor cell apoptosis [16].When combined with tyrosine kinase inhibitor imatinib, TPL can induce apoptosis in CML and acute promyelocytic leukemia (APL) cells by inhibiting the expression of HIF-1a and Nrf2 [48].TPL cooperates with Nutlin-3a to induce mitochondria-mediated apoptosis in vitro and in vivo.In addition, there is a synergistic effect in some cases of p53 deletion.Nutlin-3a upregulates the expression of p53 downstream targets PUMA and p21, while TPL reduces the mRNA levels of XIAP and Mcl-1.These two drugs have synergistic effects when used together [102].
The synergistic effect of TPL and bortezomib (BTZ) on MM cells was achieved by reducing the expression of Apg-1 and HSF1 and increasing the expression of cleaved-caspase3 [103].In addition, the combination of TPL and BTZ can also overcome cell adhesion-mediated drug resistance [103].TPL combined with dexamethasone can promote apoptosis in MM cells by decreasing the expression of phospho-AKT and increasing Bad and P53 through the phosphatidylinositol 3-kinase (PI3 K) PI3k/Akt/NF-κB pathway [104].In addition, TPL can overcome dexamethasone resistance by increasing the expression of the glucocorticoid receptor (GR) through the downregulation of miR142-5p and miR181a [105].
The combined treatment of TPL and As₂O₃ can induce apoptosis in myelodysplastic syndromes (MDS) SKM-1 cells by increasing the expression of ROS, Bax, and caspase 3, and decreasing the expression of Bcl-2 [106].

Targeted delivery of TPL using specific antibodies
CD26 is a type II glycoprotein that has been used as a poor prognostic factor, and CD26-positive cells have been found to be highly expressed in various hematologic tumors [107][108][109].Humanized anti-CD26 monoclonal antibody (YS110) has been coupled to TR1, a modified version of TPL at the sulfhydryl (SH) group, to produce an antibody-drug conjugate Y-TR1.Y-TR1 binds to CD26 on the cell surface and can cause cell death through immune-mediated cytotoxicity [110,111].Y-TR1 significantly inhibits the proliferation of CD26-positive tumor cells without affecting CD26negative cells.YS110 can be internalized to the nucleus to inhibit cell proliferation by suppressing the transcription of POLR2A, a subunit of RNA polymerase II.Y-TR1 inhibits cell proliferation by inhibiting general transcription factor IIH (TFIIH), which is required for the transcription of RNA polymerase II (Pol II) [111,112].
Table 1 summarizes the important features of TPL combination therapy.

Application of TPL structural analogues
Due to its poor water solubility and toxic side effects, the clinical application of TPL is limited to a certain extent.Therefore, a number of water-soluble structural TPL analogues with low toxicity have been developed [72,73].At present, there are nearly ten types of TPL structural analogues (Figure 3), including F60008, MRx102, and minnelide [74].Among them, F60008 (PG490-88) [75], LLDT-246 [76], LLDT-288 [77], and LLDT-8 [78,79] are more commonly used in solid tumors.Minnelide, a prodrug of TPL, has entered phase 2 clinical trials in patients with advanced pancreatic cancer and has shown significant activity in preclinical models of pancreatic cancer [80].Minnelide has also shown good efficacy and tolerability in clinical trials of advanced gastrointestinal (GI) malignancies [81].
A clinical trial of minnelide for leukemia is currently underway (phase 1, NCT03760523).This clinical trial will establish the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) for minnelide monotherapy in patients with relapsed/refractory (R/ R) AML who are ineligible for intensive chemotherapy.
Currently, the TPL structural analogues that have been used for the treatment of AML include MRx102 (18-benzoyloxy19-benzoylfuran triptolide) and minnelide.MRx102, which is soluble in water, is highly cytotoxic to human leukemia cells in vitro at nanomolar concentrations and reduces the viability of CD34+ AML mother cells.In xenotransplantation tumors with MV4-11 human AML cells, MRx102 (1.35 mg/kg/ day) reduced tumor volume by 99%.It can inhibit tumor growth and metastasis by inducing cell apoptosis and inhibiting RNA transcription by downregulating  the antiapoptotic proteins XIAP and Mcl2 and inhibiting the Wnt signaling pathway [82][83][84].Minnelide (14-O-phosphooxymethyl triptolide disodium salt), a water-soluble prodrug of TPL, is converted into the active form of TPL in vivo to exert its biological activity.Studies have shown that minnelide has an antileukemic effect in various leukemia models at a dose below the maximum tolerated dose (MTD) without affecting normal hematopoietic stem cells.Experiments in mice have shown that low-dose minnelide has an effect on both AML blasts and THP-1-expressing cells [60].Low-dose TPL(IC20) significantly reduces the colony formation ability of leukemia cell lines and the expression of stem cell markers [13,14,53,65,80,101], resulting in downregulation of c-Myc and leukemia cell cycle arrest at the G1/S phase [85,86].
Intriguingly, a recently developed water-soluble prodrug of TPL, TP-P1, is synthesized by linking TPL to cyclic amino acids through glycolic acid.Compared with minnelide, TPL was released more rapidly from TP-P1 into plasma.While maintaining its safety profile, 25 μg/kg TP-P1 could effectively inhibit tumor growth in an AML mouse model, while at a dose of 100 μg/kg, TP-P1 could eliminate tumor [113].TP-P1 can also significantly enhance the efficacy of FLT3 inhibitors [113].With the improved synthesis efficiency of TP-P1, the cost was greatly reduced [113].Therefore, TP-P1 has the potential to become a prodrug for treating AML.

Conclusions and prospects
An increasing number of studies have shown that TPL has a significant anti-AML effect.In response to the poor water solubility and toxic side effects of TPL, a number of TPL analogues have been developed.Combination therapies and targeted drug delivery systems further improved the efficacy.However, considering the complexity of the mechanism of action of TPL with different targets and signaling pathways, further studies are needed to understand how TPS exerts it effect in various types of hematological malignancies and different subtypes of AML.It is expected that these studies will provide a safe and effective new treatment strategy for personalized treatment of hematological malignancies.

Figure 3 .
Figure 3.The most well-studied structural analogue of TPL.

Table 1 .
The target and mechanism of action for combination therapy for treating hematological tumors.