Tubeimoside-1 induces TFEB-dependent lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting mTOR

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) cascade is an effective therapeutic target for immune checkpoint blockade (ICB) therapy. Targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity. Using flow cytometry-based assay, we identify tubeimoside-1 (TBM-1) as a promising antitumor immune modulator that negatively regulates PD-L1 level. TBM-1 disrupts PD-1/PD-L1 interaction and enhances the cytotoxicity of T cells toward cancer cells through decreasing the abundance of PD-L1. Furthermore, TBM-1 exerts its antitumor effect in mice bearing Lewis lung carcinoma (LLC) and B16 melanoma tumor xenograft via activating tumor-infiltrating T-cell immunity. Mechanistically, TBM-1 triggers PD-L1 lysosomal degradation in a TFEB-dependent, autophagy-independent pathway. TBM-1 selectively binds to the mammalian target of rapamycin (mTOR) kinase and suppresses the activation of mTORC1, leading to the nuclear translocation of TFEB and lysosome biogenesis. Moreover, the combination of TBM-1 and anti-CTLA-4 effectively enhances antitumor T-cell immunity and reduces immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our findings reveal a previously unrecognized antitumor mechanism of TBM-1 and represent an alternative ICB therapeutic strategy to enhance the efficacy of cancer immunotherapy.     

Epigenetic strategies synergize with PD-L1/PD-1 targeted cancer immunotherapies to enhance antitumor responses

Immunotherapy strategies targeting the programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) pathway in clinical treatments have achieved remarkable success in treating multiple types of cancer. However, owing to the heterogeneity of tumors and individual immune systems, PD-L1/PD-1 blockade still shows slow response rates in controlling malignancies in many patients. Accumulating evidence has shown that an effective response to anti-PD-L1/anti-PD-1 therapy requires establishing an integrated immune cycle. Damage in any step of the immune cycle is one of the most important causes of immunotherapy failure. Impairments in the immune cycle can be restored by epigenetic modification, including reprogramming the environment of tumor-associated immunity, eliciting an immune response by increasing the presentation of tumor antigens, and by regulating T cell trafficking and reactivation. Thus, a rational combination of PD-L1/PD-1 blockade and epigenetic agents may offer great potential to retrain the immune system and to improve clinical outcomes of checkpoint blockade therapy.     

Predictive effect of PD-L1 expression for immune checkpoint inhibitor (PD-1/PD-L1 inhibitors) treatment for non-small cell lung cancer: A meta-analysis

BackgroundProgrammed death-ligand-1 (PD-L1) is a well-known predictive biomarker in non-small cell lung cancer (NSCLC) patients, however, its accuracy remains controversial. Here, we investigated the correlation between PD-L1 expression level and efficacy of its inhibitors, and hence assessed the predictive effect of PD-L1 expression.MethodsStudies that evaluated the efficacy of programmed death-1 (PD-1)/ PD-L1 inhibitors in advanced NSCLC patients according to tumor PD-L1 expression levels were searched for on Medline, Cochrane Library, and Embase. The pooled risk ratio (RR) and 95% confidence intervals (95% CIs) were calculated for the objective response rate (ORR) with overall survival (OS) and progression-free survival (PFS) were measured in terms of hazard ratio (HR) and the corresponding 95% CIs.Results1432 NSCLC patients from six randomized controlled trials (RCTs) were included and three PD-1/PD-L1 inhibitors (atezolizumab, nivolumab, and pembrolizumab) were used to treat the patients. A significantly higher ORR was observed in the high PD-L1 expression group compared to the low expression group (0.35 [95% CI, 0.30–0.40] vs 0.11 [95% CI, 0.09–0.14]). The results of the subgroup analysis, grouped by the type of drugs and antibodies which assess immune checkpoint inhibitors were identical with the pooled result. However, our study showed that PD-L1 expression was neither prognostic nor predictive of overall survival (OS) or progression-free survival (PFS) in patients treated with PD-1/PD-L1 inhibitors compared to chemotherapy.ConclusionsPD-L1 can be a predictive biomarker for ORR. Nevertheless, PD-L1 expression is not a good predictive tool for OS and PFS.     

Proposed mechanisms for the extracellular release of PD-L1 by the anticancer saponin platycodin D

Platycodin D (PTD) is an oleanane-type terpenoid saponin, isolated from the plant Platycodon grandiflorus. PTD displays multiple pharmacological effects, notably significant anticancer activities in vitro and in vivo. Recently, PTD was shown to trigger the extracellular release of the immunologic checkpoint glycoprotein PD-L1. The reduction of PD-L1 expression at the surface of cancer cells leads to interleukin-2 secretion and T cells activation. In the present review, we have analyzed the potential origin of this atypical PTD-induced PD-L1 release to propose a mechanistic explanation. For that, we considered all published scientific information, as well as the physicochemical characteristics of the natural product (a modeling analysis of PTD and the related saponin β -escin is provided). On this basis, we raise the hypothesis that the capacity of PTD to induce PD-L1 extracellular release derives from two main mechanisms: (i) a drug-promoted shedding of membrane PD-L1 by metalloproteases or more likely, (ii) a cholesterol binding-related effect, that would lead to perturbation of membrane raft domains, limiting the recruitment of proteins like TLR4. The drug-induced membrane effects (frequently observed with saponin drugs), associated with a production of interferon-γ,can favor the release of proteins like PD-L1 into membrane vesicles. Our analysis supports the hypothesis that PTD is a cholesterol-dependent lipid raft-modulating agent able to promote the formation of PD-L1 containing extracellular vesicles. The anticancer potential of PTD and its capacity to modulate the functioning of the PD-1/PD-L1 checkpoint should be further considered.     

Novel phthalimides regulating PD-1/PD-L1 interaction as potential immunotherapy agents

Programmed cell death 1(PD-1)/programmed cell death ligand 1(PD-L1) have emerged as one of the most promising immune checkpoint targets for cancer immunotherapy. Despite the inherent advantages of small-molecule inhibitors over antibodies, the discovery of small-molecule inhibitors has fallen behind that of antibody drugs. Based on docking studies between small molecule inhibitor and PD-L1 protein, changing the chemical linker of inhibitor from a flexible chain to an aromatic ring may improve its binding capacity to PD-L1 protein, which was not reported before. A series of novel phthalimide derivatives from structure-based rational design was synthesized. P39 was identified as the best inhibitor with promising activity, which not only inhibited PD-1/PD-L1 interaction (IC₅₀ = 8.9 nmol/L), but also enhanced killing efficacy of immune cells on cancer cells. Co-crystal data demonstrated that P39 induced the dimerization of PD-L1 proteins, thereby blocking the binding of PD-1/PD-L1. Moreover, P39 exhibited a favorable safety profile with a LD₅₀ > 5000 mg/kg and showed significant in vivo antitumor activity through promoting CD8⁺ T cell activation. All these data suggest that P39 acts as a promising small chemical inhibitor against the PD-1/PD-L1 axis and has the potential to improve the immunotherapy efficacy of T-cells.     

Factors affecting tumor responders and predictive biomarkers of toxicities in cancer patients treated with immune checkpoint inhibitors

Cancer immunotherapy has brought a great revolution in the treatment of advanced human cancer. Immune checkpoint inhibitors (ICIs) that target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death protein 1 pathway (PD-1/PD-L1) have been widely administrated in the past years and demonstrated promising in a variety of malignancies. While some patients show benefit from ICIs, others do not respond or even develop resistance to these therapies. Among the responders, the treatments are consequently accompanied with immune-related adverse effects (irAEs), which are diverse in their effected organs, degree of severity and timing. Some of the toxicities are fatal and result in discontinuance of immunotherapy. The toxicity profile from anti-CTLA-4 to anti-PD-1/PD-L1 immunotherapies is distinct from those caused by conventional anticancer therapies, though their presentation may be similar. In order to better help clinicians recognize, monitor and manage irAEs in a growing population of cancer patients who are receiving ICI therapy, this article summarizes the FDA approved ICIs and focuses on (1) existing toxic evidence related to ICIs, (2) occurrence of irAEs, (3) factors influencing tumor responders treated with ICIs, (4) predictive biomarkers of irAEs, and (5) new potential mechanisms of resistance to ICI therapy.     

Blocking PD-1/PD-L1 by an ADCC enhanced anti-B7-H3/PD-1 fusion protein engages immune activation and cytotoxicity

Antibody therapy based on PD-1/PD-L1 blocking or ADCC effector has produced significant clinical benefit for cancer patients. We generated a novel anti-B7-H3 antibody (07B) and engineered the Fc fragment to enhance ADCC. To improve efficacy and tumor selectivity, we developed anti-B7-H3/PD-1 bispecific fusion proteins that simultaneously engaged tumor associate marker B7-H3 and immune suppressing ligand PD-L1 as well as enhanced ADCC to promote potent and highly selective tumor killing. Fusion proteins were designed by fusing human PD-1 extra domain to 07B in four different formats and showed good binding capacity to both targets. Indeed, the affinity of fusion proteins to B7-H3 is over 10,000 fold higher compared to that of the analogous PD-L1 and the blocking of fusion proteins to PD-L1 was worse but it greatly enhanced when bound to B7-H3, thus achieving directly PD-L1-blockade to B7-H3-expressing tumor cells. Importantly, IL-2 production was enhanced by fusion proteins from staphylococcal enterotoxin B (SEB) stimulated PBMC. Similarly, cytokines induced by fusion proteins was enhanced when co-cultured with stimulated CD8⁺ T cells and B7-H3/PD-L1 transfected raji cells. Additionally, fusion proteins improved activation to CD16a by Fc modification and delivered selective cytotoxicity to B7-H3 expressing tumor cells. In conclusion, fusion proteins blocked the PD-1/PD-L1 signal pathway and significantly increased potency of ADCC in a B7-H3-directed manner, thereby selectively activating CD8⁺ T cells and enhancing natural killing towards tumor. This novel fusion protein with its unique targeting preference may be useful to enhance efficacy and safety of immunotherapy for B7-H3-overexpressing malignancies.     

First small-molecule PROTACs for G protein-coupled receptors: inducing α1A-adrenergic receptor degradation

Proteolysis targeting chimeras (PROTACs) are dual-functional hybrid molecules that can selectively recruit an E3 ubiquitin ligase to a target protein to direct the protein into the ubiquitin-proteasome system (UPS), thereby selectively reducing the target protein level by the ubiquitin-proteasome pathway. Nowadays, small-molecule PROTACs are gaining popularity as tools to degrade pathogenic protein. Herein, we present the first small-molecule PROTACs that can induce the α_1A-adrenergic receptor (α_1A-AR) degradation, which is also the first small-molecule PROTACs for G protein-coupled receptors (GPCRs) to our knowledge. These degradation inducers were developed through conjugation of known α₁-adrenergic receptors (α₁-ARs) inhibitor prazosin and cereblon (CRBN) ligand pomalidomide through the different linkers. The representative compound 9c is proved to inhibit the proliferation of PC-3 cells and result in tumor growth regression, which highlighted the potential of our study as a new therapeutic strategy for prostate cancer.     

The co-expression characteristics of LAG3 and PD-1 on the T cells of patients with breast cancer reveal a new therapeutic strategy

Many studies have shown a special interaction between LAG3 and PD-1 in T cell inhibition, while the co-expression and effect of LAG3 and PD-1 on T cells in breast cancer patients are still not very clear. Here, with strict exclusion criteria, 88 patients with breast cancer and 18 healthy controls were enrolled. The percentages of LAG3⁺PD-1⁺ T cells in their peripheral blood (PBL) and tumor infiltrating T cells (TIL) were analyzed by flow cytometry, which showed an increase in TILs but no difference in PBLs and presented differences in TILs in different molecular subtypes (P < 0.05). In triple-negative breast cancer (TNBC), the highest percentages were observed, while in ER+/PR+ breast cancer, the lowest percentages were observed; however, these percentages were not different in different clinical stages (P > 0.05). Immunohistochemical staining showed that the expression of their ligands, PD-L1, MHC class II molecular and FGL1, was inconsistent in different molecular subtypes and clinical stages. Analysis of the functions of T cells with different phenotypes showed that the proliferation and secretion capacity of LAG3⁺PD-1⁺ T cells was obviously exhausted, with more than a two-fold of decrease compared with the groups of single positive LAG3 or PD-1 (P < 0.05). Finally, in a mouse model of TNBC, the dual blockade of LAG3 and PD-1 was indicated to achieve a better anti-tumour effect than either one alone (P < 0.05), which may provide a new strategy for the immunoregulatory treatment of patients with TNBC in the future.     

Synergistic antitumor activity of artesunate and HDAC inhibitors through elevating heme synthesis via synergistic upregulation of ALAS1 expression

Artemisinin and its derivatives (ARTs) were reported to display heme-dependent antitumor activity. On the other hand, histone deacetylase inhibitors (HDACi) were known to be able to promote heme synthesis in erythroid cells. Nevertheless, the effect of HDACi on heme homeostasis in non-erythrocytes remains unknown. We envisioned that the combination of HDACi and artesunate (ARS) might have synergistic antitumor activity through modulating heme synthesis. In vitro studies revealed that combination of ARS and HDACi exerted synergistic tumor inhibition by inducing cell death. Moreover, this combination exhibited more effective antitumor activity than either ARS or HDACi monotherapy in xenograft models without apparent toxicity. Importantly, mechanistic studies revealed that HDACi coordinated with ARS to increase 5-aminolevulinate synthase (ALAS1) expression, and subsequent heme production, leading to enhanced cytotoxicity of ARS. Notably, knocking down ALAS1 significantly blunted the synergistic effect of ARS and HDACi on tumor inhibition, indicating a critical role of ALAS1 upregulation in mediating ARS cytotoxicity. Collectively, our study revealed the mechanism of synergistic antitumor action of ARS and HDACi. This finding indicates that modulation of heme synthesis pathway by the combination based on ARTs and other heme synthesis modulators represents a promising therapeutic approach to solid tumors.     

Targeting autophagy in colorectal cancer: An update on pharmacological small-molecule compounds

Autophagy, an evolutionarily highly conserved cellular degradation process, plays the Janus role (either cytoprotective or death-promoting) in colorectal cancer, so the targeting of several key autophagic pathways with small-molecule compounds may be a new therapeutic strategy. In this review, we discuss autophagy-associated cell death pathways and key cytoprotective autophagy pathways in colorectal cancer. Moreover, we summarize a series of small-molecule compounds that have the potential to modulate autophagy-associated cell death or cytoprotective autophagy for therapeutic purposes. Taken together, these findings demonstrate the Janus role of autophagy in colorectal cancer, and shed new light on the exploitation of a growing number of small-molecule compounds to target autophagy in future cancer drug discovery.     

Breast cancer cells promote CD169+ macrophage-associated immunosuppression through JAK2-mediated PD-L1 upregulation on macrophages

Macrophages are recognized as one of the major cell types in tumor microenvironment, and macrophage infiltration has been predominantly associated with poor prognosis among patients with breast cancer. Using the murine models of triple-negative breast cancer in CD169-DTR mice, we found that CD169⁺ macrophages support tumor growth and metastasis. CD169⁺ macrophage depletion resulted in increased accumulation of CD8⁺ T cells within tumor, and produced significant expansion of CD8⁺ T cells in circulation and spleen. In addition, we observed that CD169⁺ macrophage depletion alleviated tumor-induced splenomegaly in mice, but had no improvement in bone loss and repression of bone marrow erythropoiesis in tumor-bearing mice. Cancer cells and tumor associated macrophages exploit the upregulation of the immunosuppressive protein PD-L1 to subvert T cell-mediated immune surveillance. Within the tumor microenvironment, our understanding of the regulation of PD-L1 protein expression is limited. We showed that there was a 5-fold higher relative expression of PD-L1 on macrophages as compared with 4T1 tumor cells; coculture of macrophages with 4T1 cells augmented PD-L1 levels on macrophages, but did not upregulate the expression of PD-L1 on 4T1 cells. JAK2/STAT3 signaling pathway was activated in macrophages after coculture, and we further identified the JAK2 as a critical regulator of PD-L1 expression in macrophages during coculture with 4T1 cells. Collectively, our data reveal that breast cancer cells and CD169⁺ macrophages exhibit bidirectional interactions that play a critical role in tumor progression, and inhibition of JAK2 signaling pathway in CD169⁺ macrophages may be potential strategy to block tumor microenvironment-derived immune escape.     

In-vitro and in-vivo anti-breast cancer activity of synergistic effect of berberine and exercise through promoting the apoptosis and immunomodulatory effects

PurposeBreast cancer is the most common reason of cancer death in women. Berberine (BBR), a main alkaloid in Coptis chinensis, exerted anti-cancer activities. Exercise is a new immunotherapy treatment against cancer. However, it is unclear whether exercise has effects on breast cancer and whether exercise has synergistic anti-cancer effect when co-treated with BBR. Thus, it is assumed that exercise might exert an anti-cancer effect through the immune way.MethodThe anti-tumor effect of exercise and BBR in vivo was studied in mice. The MTT method, hoechst staining and cell morphology were performed to determine the synergistic effect of exercise and BBR on breast cancer in vitro. At the same time, Western blotting, intestinal microbial and SCFA detection, Q-PCR and other methods were used to study the anti-cancer molecular mechanism.ResultsThe study found that exercise and BBR co-treatment significantly slowed the progression of breast cancer in 4T1 tumor-bearing mice (p < 0.01). Compared with the TC group, the infiltration of NK cells increased in the combined group of BBR and exercise (p < 0.01), and the expression of immune factors and cytokines was also regulated. At the same time, the synergistic effect significantly increased the level of short chain fatty acids (SCFA). SCFA can promote apoptosis of 4T1 cells and change the inflammatory factors in vitro. The expression of bcl-2 and XIAP was reduced in tumor tissues, and the expression of Fas, Fadd, Bid, Cyto-C, and Caspase-3/8/9 was also increased in vitro experiments (p < 0.05).ConclusionsThese results indicate that the synergistic treatment of exercise and BBR can improve the immune system, regulate intestinal microbial metabolite (SCFA), activate the mitochondrial apoptosis pathway and Fas death receptor apoptosis pathway, and thus play an anticancer role. This may provide a new method for the treatment of breast cancer.     

Targeting regulated cell death (RCD) with small-molecule compounds in cancer therapy: A revisited review of apoptosis,autophagy-dependent cell death and necroptosis

Evasion of regulated cell death (RCD), mainly referring to apoptosis, autophagy-dependent cell death, necroptosis, and other subroutines, is one of the well-established hallmarks of cancer cells. Accumulating evidence has revealed several small-molecule compounds that target different subroutines of RCD in cancer therapy. In this review, we summarize key pathways of apoptosis, autophagy-dependent cell death and necroptosis in cancer, and describe small-molecule compounds that target these pathways and have potential as therapeutics. These inspiring findings light the way towards the discovery of more ‘magic bullets’ that could work individually or cooperatively to target precisely the three RCD subroutines and so improve cancer treatment.     

Epigenetically silenced PD-L1 confers drug resistance to anti-PD1 therapy in gastric cardia adenocarcinoma

ObjectivesThe anti-PD-1/PD-L1 therapy has been demonstrated safe and effective for cancer patients. However, our previous data showed that it had no obvious effects on gastric cardia adenocarcinoma (GCA). Thus, we investigated how the expression level of the PD-L1 was affected by the anti-PD-1 therapy, because it has been demonstrated that the PD-L1 level affects the therapeutic efficient of the anti-PD-1 therapy.Materials and methodsThe mRNA and protein levels of PD-L1 in the GCA tissues and corresponding normal tissues were determined by qPCR and ELISA. Promoter methylation was analyzed by bisulfite sequencing. Finally the methylation of PD-L1 promoter was confirmed in the mice.ResultsThe level of PD-L1 was up-regulated in the GCA tissues when compared to the adjacent non-tumor tissues. The anti-PD1 therapy could reduce the PD-L1 levels in patients with cancer recurrence. The promoter of PD-L1 was more hypermethylated in the secondary GCA after the anti-PD-1 therapy when compared with the adjacent non-tumor tissues or the primary GCA without the anti-PD-1 therapy. Furthermore, the promoter methylation of PD-L1 could be induced by the anti-PD-1 therapy in the mice model. Finally, the anti-PD-1 plus DNA hypomethylating agent azacytidine could significantly suppressed the tumor growth better than the anti-PD-1 therapy.ConclusionsHere we demonstrated that the unresponsiveness of GCA to the anti-PD-1 therapy might result from the promoter methylation and down-regulation of PD-L1. The anti-PD-1 plus azacytidine might be a more promising approach to treat GCA.     

Magnetic systems for cancer immunotherapy

Immunotherapy is a rapidly developing area of cancer treatment due to its higher specificity and potential for greater efficacy than traditional therapies. Immune cell modulation through the administration of drugs, proteins, and cells can enhance antitumoral responses through pathways that may be otherwise inhibited in the presence of immunosuppressive tumors. Magnetic systems offer several advantages for improving the performance of immunotherapies, including increased spatiotemporal control over transport, release, and dosing of immunomodulatory drugs within the body, resulting in reduced off-target effects and improved efficacy. Compared to alternative methods for stimulating drug release such as light and pH, magnetic systems enable several distinct methods for programming immune responses. First, we discuss how magnetic hyperthermia can stimulate immune cells and trigger thermoresponsive drug release. Second, we summarize how magnetically targeted delivery of drug carriers can increase the accumulation of drugs in target sites. Third, we review how biomaterials can undergo magnetically driven structural changes to enable remote release of encapsulated drugs. Fourth, we describe the use of magnetic particles for targeted interactions with cellular receptors for promoting antitumor activity. Finally, we discuss translational considerations of these systems, such as toxicity, clinical compatibility, and future opportunities for improving cancer treatment.     

PD-1/PD-L1抑制剂联合其他免疫检查点抑制剂治疗三阴性乳腺癌的研究进展#br#

三阴性乳腺癌（TNBC）具有全身转移率高、对常规治疗不敏感以及容易产生耐药性等特点,导致患者的预后较差。随着对机体免疫系统抗肿瘤机制及TNBC免疫特点的不断探究,以程序性细胞死亡蛋白1（PD-1）和程序性死亡蛋白配体1（PD-L1）为代表的免疫检查点抑制剂为TNBC提供了新的治疗方案,但PD-1/PD-L1抑制剂单药治疗的效果不甚理想。本文就PD-1/PD-L1抑制剂联合其他具有不同机制的免疫检查点抑制剂在TNBC患者中的应用进行综述。     

Antioxidant and anti-inflammatory activities of lycopene against 5-fluorouracil-induced cytotoxicity in Caco2 cells

5-fluorouracil (5FU) is widely used to treat colorectal cancer (CC) and its main mechanisms of anticancer action are through generation of ROS which often result in inflammation. Here, we test the effect of Lycopene against 5FU in Caco2 cell line. Caco2 cells were exposed to 3 µg/ml of 5FU alone or with 60, 90, 120 µg/ml of lycopene. This was followed by assessment of cytotoxicity, oxidative stress, and gene expression of inflammatory genes. Our findings showed that Lycopene and 5FU co-exposure induced dose-dependent cytotoxic effect without compromising the membrane integrity based on the LDH assay. Lycopene also significantly enhanced 5FU-induced SOD activity and GSH level compared to control for all mixture concentrations (p < 0.01). Lycopene alone and combination with 5FU-induced expression of IL-1β, TNF-α, and IL-6. Furthermore, IFN-γ expression was significantly enhanced by only mixture of lycopene (90 µg/ml) and 5FU (p < 0.05). In conclusion, Lycopene supplementation with 5FU therapy resulted in improvement in antioxidant parameters such as catalase and GSH levels giving the cell capacity to cope with 5FU-mediated oxidative stress. Lycopene also enhanced IFN-γ expression in the presence of 5FU, which may activate antitumor effects further enhancing the cancer killing effect of 5FU.     

Identification of a novel PAK1 inhibitor to treat pancreatic cancer

Pancreatic cancer is one of the most aggressive cancers with poor prognosis and a low 5-year survival rate. The family of P21-activated kinases (PAKs) appears to modulate many signaling pathways that contribute to pancreatic carcinogenesis. In this work, we demonstrated that PAK1 is a critical regulator in pancreatic cancer cell growth. PAK1-targeted inhibition is therefore a new potential therapeutic strategy for pancreatic cancer. Our small molecule screening identified a relatively specific PAK1-targeted inhibitor, CP734. Pharmacological and biochemical studies indicated that CP734 targets residue V342 of PAK1 to inhibit its ATPase activity. Further in vitro and in vivo studies elucidated that CP734 suppresses pancreatic tumor growth through depleting PAK1 kinase activity and its downstream signaling pathways. Little toxicity of CP734 was observed in murine models. Combined with gemcitabine or 5-fluorouracil, CP734 also showed synergistic effects on the anti-proliferation of pancreatic cancer cells. All these favorable results indicated that CP734 is a new potential therapeutic candidate for pancreatic cancer.