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Simple Summary: The inhibitors of BET proteins represent a promising class of therapeutic agents that target the oncogenic activity of MYC and repress DLBCL cell migration, but the mechanism of such repression remains elusive. Herein, we found that BET inhibitor JQ1 abrogated the amoeboid movement of DLBCL cells through a small GTPase-driven mechanism, including both restrained RAS signaling and MYC-mediated suppression of GTP-RhoA activity. BET inhibition drastically increased the expression of a GTPase regulatory protein, the IQ motif containing GTPase activating protein 3 (IQGAP3), in DLBCL. Proteomics-based re-stratification identified a specific subgroup of DLBCL patients whose tumors harbored an enhanced PI3K activity and had an inferior survival, whereas a lower IQGAP3 expression level further portended a very dismal outcome for those patients. The inhibitors of both BET and RAS (through attenuated PI3K signaling) activities effectively ameliorated the outspread of in vivo DLBCL tumors, indicating the potential of their synergism in the treatment of specific DLBCL subtypes. Phenotypic heterogeneity and molecular diversity make diffuse large B-cell lymphoma (DLBCL) a challenging disease. We recently illustrated that amoeboid movement plays an indispensable role in DLBCL dissemination and inadvertently identified that the inhibitor of bromodomain and extra-terminal (BET) proteins JQ1 could repress DLBCL migration. To explore further, we dissected the impacts of BET inhibition in DLBCL. We found that JQ1 abrogated amoeboid movement of DLBCL cells through both restraining RAS signaling and suppressing MYC-mediated RhoA activity. We also demonstrated that BET inhibition resulted in the upregulation of a GTPase regulatory protein, the IQ motif containing GTPase activating protein 3 (IQGAP3). IQGAP3 similarly exhibited an inhibitory effect on RAS activity in DLBCL cells. Through barcoded mRNA/protein profiling in clinical samples, we identified a specific subgroup of DLBCL tumors with enhanced phosphatidylinositol-3-kinase (PI3K) activity, which led to an inferior survival in these patients. Strikingly, a lower IQGAP3 expression level further portended those with PI3K-activated DLBCL a very dismal outcome. The inhibition of BET and PI3K signaling activity led to effective suppression of DLBCL dissemination in vivo. Our study provides an important insight into the ongoing efforts of targeting BET proteins as a therapeutic approach for DLBCL. [ABSTRACT FROM AUTHOR]

Simple Summary: Patients with advanced intrahepatic cholangiocarcinoma (iCCA) have a very poor prognosis, and no targeted therapy is approved for advanced iCCA. A therapeutic strategy for wild-type p53 cancers is the reactivation of p53 by inhibition of its the negative regulators, MDM2, and WIP1. In the present study, we used HDM201 (an MDM2-p53 binding antagonist) to increase p53 stabilization and upregulate the expression of downstream targets (p21 and MDM2) in RBE and SK-Hep-1 liver adenocarcinoma cell lines. The survival rate and clonogenicity decreased after HDM201 treatment in a dose-dependent manner. Combined treatment with HDM201 and GSK2830371 (WIP1 inhibitor) increased p53 phosphorylation, leading to sustained p53 activation. This combination treatment resulted in G2/M phase arrest and promoted cytotoxicity compared with MDM2 inhibitor monotherapy. Furthermore, increased expression of p53 signaling pathway target genes were identified following combination treatment with HDM201 and GSK2830371, suggesting potential roles for this combination strategy in iCCA therapy. Background: Intrahepatic cholangiocarcinoma (iCCA) is an adenocarcinoma arising from the intrahepatic bile duct. It is the second most common primary liver cancer and has a poor prognosis. Activation of p53 by targeting its negative regulators, MDM2 and WIP1, is a potential therapy for wild-type p53 cancers, but few reports for iCCA or liver adenocarcinoma exist. Methods: Both RBE and SK-Hep-1 liver adenocarcinoma cell lines were treated with the HDM201 (Siremadlin) MDM2-p53 binding antagonist alone or in combination with the GSK2830371 WIP1 phosphatase inhibitor. Cell proliferation, clonogenicity, protein and mRNA expression, cell cycle distribution, and RNA sequencing were performed to investigate the effect and mechanism of this combination. Results: GSK2830371 alone demonstrated minimal activity on proliferation and colony formation, but potentiated growth inhibition (two-fold decrease in GI50) and cytotoxicity (four-fold decrease in IC50) by HDM201 on RBE and SK-Hep-1 cells. HDM201 increased p53 protein expression, leading to transactivation of downstream targets (p21 and MDM2). Combination with GSK2830371 increased p53 phosphorylation, resulting in an increase in both p53 accumulation and p53-dependent trans-activation. G2/M arrest was observed by flow cytometry after this treatment combination. RNA sequencing identified 21 significantly up-regulated genes and five downregulated genes following p53 reactivation by HDM201 in combination with GSK2830371 at 6 h and 24 h time points compared with untreated controls. These genes were predominantly known transcriptional targets regulated by the p53 signaling pathway, indicating enhanced p53 activation as the predominant effect of this combination. Conclusion: The current study demonstrated that GSK2830371 enhanced the p53-dependent antiproliferative and cytotoxic effect of HDM201 on RBE and SK-Hep-1 cells, providing a novel strategy for potentiating the efficacy of targeting the p53 pathway in iCCA. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma is the most common primary malignant tumor of the bile duct. The current standard first-line treatment for advanced or metastatic cholangiocarcinoma is gemcitabine and cisplatin. However, few effective treatment choices exist for refractory cholangiocarcinoma, and additional therapeutic drugs are urgently required. Our previous work demonstrated that the ALDH isoform 1A3 plays a vital role in the malignant behavior of cholangiocarcinoma and may serve as a new therapeutic target. In this study, we found a positive correlation between ALDH1A3 protein expression levels and the cell migration abilities of three cholangiocarcinoma cell lines, which was verified using ALDH1A3-overexpressing and ALDH1A3-knockdown clones. We also used ALDH1A3-high and ALDH1A3-low populations of cholangiocarcinoma cell lines from the library of integrated network-based cellular signatures (LINCS) program and assessed the effects of ruxolitinib, a commercially available JAK2 inhibitor. Ruxolitinib had a higher cytotoxic effect when combined with gemcitabine. Furthermore, the nuclear translocation STAT1 and STAT3 heterodimers were markedly diminished by ruxolitinib treatment, possibly resulting in decreased ALDH1A3 activation. Notably, ruxolitinib alone or combined with gemcitabine led to significantly reduced tumor size and weight. Collectively, our studies suggest that ruxolitinib might suppress the ALDH1A3 activation through the JAK2/STAT1/3 pathway in cholangiocarcinoma, and trials should be undertaken to evaluate its efficacy in clinical therapy. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) is a malignant tumor with aggressive biological behavior. Immune checkpoints such as cytotoxic T-lymphocyte antigen 4 (CTLA4) and antiprogrammed death 1 (PD-1) are critical immune-checkpoint molecules that repress T-cell activation. The DNA vaccine potential against CTLA4 and PD-1 in CCA is unknown. We used a thioacetamide (TAA)-induced intrahepatic cholangiocarcinoma (iCCA) rat model to investigate the DNA vaccine potential against CTLA4, PD-1, and PD-L1. We detected PD-L1 expression in CCA and CD8+ T-cell infiltration during CCA progression in rats. We validated antibody production, carcinogenesis, and CD8+ T-cell infiltration in rats receiving DNA vaccination against PD-1, PD-L1, or CTLA4. In our TAA-induced iCCA rat model, the expression of PD-L1 and the infiltration of CD8+ T cells increased as in rat CCA tumorigenesis. PD-1 antibodies in rats were not increased after receiving PD-1 DNA vaccination, and CCA tumor growth was not suppressed. However, in rats receiving PD-L1–CTLA4 DNA vaccination, CCA tumor growth was inhibited, and the antibodies of PD-L1 and CTLA4 were produced. Furthermore, the number of CD8+ T cells was enhanced after PD-L1–CTLA4 DNA vaccination. DNA vaccination targeting CTLA4–PD-L1 triggered the production of specific antibodies and suppressed tumor growth in TAA-induced iCCA rats. [ABSTRACT FROM AUTHOR]

Patients with advanced biliary tract cancer (BTC) inevitably experience progression after first-line, gemcitabine-based chemotherapy, due to chemo-resistance. The genetic alterations of DNA damage repair (DDR) genes are usually determined in BTC tumors. In this study, we found that the POLQ mRNA levels are downregulated and the ataxia-telangiectasia mutated (ATM) inhibitor AZD0156 was more sensitive in gemcitabine-resistant BTC sublines than in the parental cell lines. The knockdown of DNA polymerase θ does not affect cell proliferation, but its combination with the ATM inhibitor facilitated cell death in gemcitabine-resistant and gemcitabine-intensive BTC cells. Moreover, in the DNA damage caused by photon, hydrogen peroxide, or chemotherapy drugs, synthetic lethal interactions were found in combination with ATM inhibition by AZD0156 and DNA polymerase θ depletion, resulting in increased DNA damage accumulation and micronucleus formation, as well as reduced cell survival and colony formation. Collectively, our results reveal that ATM acts as a potential target in gemcitabine-resistant and DNA polymerase θ-deficient BTC. [ABSTRACT FROM AUTHOR]

Gemcitabine-based chemotherapy is the current standard treatment for biliary tract cancers (BTCs) and resistance to gemcitabine remains the clinical challenge. TP53 mutation has been shown to be associated with poor clinicopathologic characteristics and survival in patients with BTCs, indicating that p53 plays an important role in the treatment of these cancers. Herein, we comprehensively reviewed previous BTC preclinical research and early clinical trials in terms of p53, as well as novel p53-targeted treatment, alone or in combination with either chemotherapy or other targeted therapies in BTCs. Preclinical studies have demonstrated that p53 mutations in BTCs are associated with enhanced gemcitabine resistance, therefore targeting p53 may be a novel therapeutic strategy for treatment of BTCs. Directly targeting mutant p53 by p53 activators, or indirectly by targeting cell cycle checkpoint proteins (Chk1, ataxia telangiectasia related (ATR), and Wee1) leading to synthetic lethality, may be potential future strategies for gemcitabine-resistant p53 mutated BTCs. In contrast, for wild-type p53 BTCs, activation of p53 by inhibition of its negative regulators (MDM2 and wild-type p53-induced phosphatase 1 (WIP1)) may be alternative options. Combination therapies consisting of standard cytotoxic drugs and novel small molecules targeting p53 and related signaling pathways may be the future key standard approach to beat cancer. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors (ICIs) have become the standard of care in various cancers, although their predictive tools have not yet completely developed. Here, we aimed to exam the role of 70-gene chromosomal instability signature (CIN70) in cancers, and its association with previous predictors, tumor mutation burden (TMB), and microsatellite instability (MSI), for patients undergoing ICIs, as well as the possible predictive value for ICIs. We examined the association of CIN70 with TMB and MSI, as well as the impact of these biomarkers on the survival of 33 cancer cohorts from The Cancer Genome Atlas (TCGA) databank. The predictive value of the ICIs of CIN70 in previously published reports was also validated. Using the TCGA dataset, CIN70 scores were frequently (either positively or negatively) associated with TMB, but were only significantly associated with MSI status in three types of cancer. In addition, our current study showed that all TMB, MSI, and CIN70 had their own prognostic values for survival in patients with various cancers, and that they could be cancer type-specific. In two validation cohorts (melanoma by Hugo et al. and urothelial cancer by Snyder et al.), no significant difference of CIN70 scores was found between responders and non-responders (p-value = 0.226 and 0.108, respectively). In addition, no overall survival difference was noted between patients with a high CIN70 and those with a low CIN70 (p-value = 0.106 and 0.222, respectively). In conclusion, the current study, through a comprehensive bioinformatics analysis, demonstrated a correlation between CIN70 and TMB, but CIN70 is not the predictor for cancer patients undergoing ICIs. Future prospective studies are warranted to validate these findings. [ABSTRACT FROM AUTHOR]