Activation Of Checkpoint Kinase Research Articles

Combined HDAC8 and checkpoint kinase inhibition induces tumor-selective synthetic lethality in preclinical models.

The elevated level of replication stress is an intrinsic characteristic of cancer cells. Targeting the mechanisms that maintain genome stability to further increase replication stress and thus induce severe genome instability has become a promising approach for cancer treatment. Here, we identify histone deacetylase 8 (HDAC8) as a drug target whose inactivation synergizes with the inhibition of checkpoint kinases to elicit substantial replication stress and compromise genome integrity selectively in cancer cells. We showed that simultaneous inhibition of HDAC8 and checkpoint kinases led to extensive replication fork collapse, irreversible cell-cycle arrest, and synergistic vulnerability in various cancer cells. The efficacy of the combination treatment was further validated in patient tumor-derived organoid (PDO) and xenograft mouse (PDX) models, providing important insights into patient-specific drug responses. Our data revealed that HDAC8 activity was essential for reducing the acetylation level of structural maintenance of chromosomes protein 3 (SMC3) ahead of replication forks and preventing R loop formation. HDAC8 inactivation resulted in slowed fork progression and checkpoint kinase activation. Our findings indicate that HDAC8 guards the integrity of the replicating genome, and the cancer-specific synthetic lethality between HDAC8 and checkpoint kinases provides a promising replication stress-targeting strategy for treating a broad range of cancers.

Inflammasome protein scaffolds the DNA damage complex during tumor development.

Inflammasome sensors activate cellular signaling machineries to drive inflammation and cell death processes. Inflammasomes also control the development of certain diseases independently of canonical functions. Here, we show that the inflammasome protein NLR family CARD domain-containing protein 4 (NLRC4) attenuated the development of tumors in the Apcmin/+ mouse model. This response was independent of inflammasome signaling by NLRP3, NLRP6, NLR family apoptosis inhibitory proteins, absent in melanoma 2, apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1 and caspase-11. NLRC4 interacted with the DNA-damage-sensing ataxia telangiectasia and Rad3-related (ATR)-ATR-interacting protein (ATRIP)-Ewing tumor-associated antigen 1 (ETAA1) complex to promote the recruitment of the checkpoint adapter protein claspin, licensing the activation of the kinase checkpoint kinase-1 (CHK1). Genotoxicity-induced activation of the NLRC4-ATR-ATRIP-ETAA1 complex drove the tumor-suppressing DNA damage response and CHK1 activation, and further attenuated the accumulation of DNA damage. These findings demonstrate a noninflammatory function of an inflammasome protein in promoting the DNA damage response and mediating protection against cancer.

Dynamic structural remodeling of LINC01956 enhances temozolomide resistance in MGMT-methylated glioblastoma.

The mechanisms underlying stimuli-induced dynamic structural remodeling of RNAs for the maintenance of cellular physiological function and survival remain unclear. Here, we showed that in MGMT promoter-methylated glioblastoma (GBM), the RNA helicase DEAD-box helicase 46 (DDX46) is phosphorylated by temozolomide (TMZ)-activated checkpoint kinase 1 (CHK1), resulting in a dense-to-loose conformational change and an increase in DDX46 helicase activity. DDX46-mediated tertiary structural remodeling of LINC01956 exposes the binding motifs of LINC01956 to the 3' untranslated region of O6-methylguanine DNA methyltransferase (MGMT). This accelerates recruitment of MGMT mRNA to the RNA export machinery and transportation of MGMT mRNA from the nucleus to the cytoplasm, leading to increased MGMT abundance and TMZ resistance. Using patient-derived xenograft (PDX) and tumor organoid models, we found that treatment with the CHK1 inhibitor SRA737abolishes TMZ-induced structural remodeling of LINC01956 and subsequent MGMT up-regulation, resensitizing TMZ-resistant MGMT promoter-methylated GBM to TMZ. In conclusion, these findings highlight a mechanism underlying temozolomide-induced RNA structural remodeling and may represent a potential therapeutic strategy for patients with TMZ-resistant MGMT promoter-methylated GBM.

Endothelial activation and fibrotic changes are impeded by laminar flow-induced CHK1-SENP2 activity through mechanisms distinct from endothelial-to-mesenchymal cell transition.

The deSUMOylase sentrin-specific isopeptidase 2 (SENP2) plays a crucial role in atheroprotection. However, the phosphorylation of SENP2 at T368 under disturbed flow (D-flow) conditions hinders its nuclear function and promotes endothelial cell (EC) activation. SUMOylation has been implicated in D-flow-induced endothelial-to-mesenchymal transition (endoMT), but the precise role of SENP2 in counteracting this process remains unclear. We developed a phospho-specific SENP2 S344 antibody and generated knock-in (KI) mice with a phospho-site mutation of SENP2 S344A using CRISPR/Cas9 technology. We then investigated the effects of SENP2 S344 phosphorylation under two distinct flow patterns and during hypercholesteremia (HC)-mediated EC activation. Our findings demonstrate that laminar flow (L-flow) induces phosphorylation of SENP2 at S344 through the activation of checkpoint kinase 1 (CHK1), leading to the inhibition of ERK5 and p53 SUMOylation and subsequent suppression of EC activation. We observed a significant increase in lipid-laden lesions in both the aortic arch (under D-flow) and descending aorta (under L-flow) of female hypercholesterolemic SENP2 S344A KI mice. In male hypercholesterolemic SENP2 S344A KI mice, larger lipid-laden lesions were only observed in the aortic arch area, suggesting a weaker HC-mediated atherogenesis in male mice compared to females. Ionizing radiation (IR) reduced CHK1 expression and SENP2 S344 phosphorylation, attenuating the pro-atherosclerotic effects observed in female SENP2 S344A KI mice after bone marrow transplantation (BMT), particularly in L-flow areas. The phospho-site mutation SENP2 S344A upregulates processes associated with EC activation, including inflammation, migration, and proliferation. Additionally, fibrotic changes and up-regulated expression of EC marker genes were observed. Apoptosis was augmented in ECs derived from the lungs of SENP2 S344A KI mice, primarily through the inhibition of ERK5-mediated expression of DNA damage-induced apoptosis suppressor (DDIAS). In this study, we have revealed a novel mechanism underlying the suppressive effects of L-flow on EC inflammation, migration, proliferation, apoptosis, and fibrotic changes through promoting CHK1-induced SENP2 S344 phosphorylation. The phospho-site mutation SENP2 S344A responds to L-flow through a distinct mechanism, which involves the upregulation of both mesenchymal and EC marker genes.

Hydroxyurea and inactivation of checkpoint kinase MEC1 inhibit transcription termination and pre-mRNA cleavage at polyadenylation sites in budding yeast

The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. The transcription changes triggered by DDR depend on the nature of DNA damage, activation of checkpoint kinases, and the stage of cell cycle. The transcription changes can be localized and affect only damaged DNA, but they can be also global and affect genes that are not damaged. While the purpose of localized transcription inhibition is to avoid transcription of damaged genes and make DNA accessible for repair, the purpose and mechanisms of global transcription inhibition of undamaged genes are less well understood. We show here that a brief cell treatment with hydroxyurea (HU) globally inhibits RNA synthesis and transcription by RNA polymerase I, II, and III (RNAPI, RNAPII, and RNAPIII). HU reduces efficiency of transcription termination and inhibits pre-mRNA cleavage at the polyadenylation (pA) sites, destabilizes mRNAs, and shortens poly(A) tails of mRNAs, indicating defects in pre-mRNA 3′ end processing. Inactivation of the checkpoint kinase Mec1p downregulates the efficiency of transcription termination and reduces the efficiency of pre-mRNAs clevage at the pA sites, suggesting the involvement of DNA damage checkpoint in transcription termination and pre-mRNA 3′ end processing.

Abstract 2667: PARG inhibition augments CHK1 inhibitor-induced replication stress and synergistically kills ovarian cancer cells

Abstract Ovarian cancer (OC) is one of the leading causes of cancer-related deaths in women in the United States. PARP inhibitors showed promising clinical responses; however, most of the patients eventually relapse and succumb to the resistant disease. Currently, it lacks effective therapies to prevent and treat OC, which warrants novel drugs and combination therapies to improve patients' prognoses. Epithelial OCs are heavily dependent on ATR-CHK1-mediated DNA damage checkpoint signaling for survival, which is also regulated by hedgehog/GLI1 (Hh) signaling in cancer cells. However, both Hh/GLI1 and CHK1 inhibitors failed to show promising results in clinical trials as single agents. Poly(ADP-ribose) glycohydrolase (PARG) is the critical enzyme that removes Poly-ADP ribosylated (PARylated) proteins upon replication stress. We hypothesized that inhibition of PARG could amplify CHK1 inhibitor-induced replication stress by trapping PARylated proteins on the chromatin and compromising DNA repair and causing a metabolic and mitotic catastrophe. We evaluated CHK1 inhibitor prexasertib in combination with PARG inhibitor (PARGi) to target metabolic and DNA repair crosstalk to effectively kill OC cells and to overcome resistance. Our studies demonstrate that prexasertib treatment induces PARylation by inducing replication stress. Similarly, PARG inhibition by (PDD00017273) fails to remove PARylation and activates checkpoint kinase 1 (CHK1) by phosphorylating at S296, S317, and S345 in OC cells. Based on these observations, we hypothesized that a combination of CHK1 and PARG inhibition would augment oncogenic replication stress in OC cells and cause increased DNA damage and cell death. Interestingly, our evaluation of prexasertib and PARGi combination treatment by clonogenic survival and MTT assays showed synergistic killing in a panel of OC cells, including in isogenic chemoresistant models as well as 3D organoid models of primary OC cells compared to individual drugs. As predicted, combined treatment increased DNA double-strand breaks as demonstrated by COMET assays, and increased γH2AX foci, pRPA32 foci, perturbed S/G2 phase arrest, and nuclear disintegration relative to individual-drug treatment. Furthermore, significant depletion of NAD+ levels was seen in PARGi-treated OC cells compared to untreated cells. Together, these results indicate that this combination treatment cause persistence of heavy PARylation at DNA damage sites abrogates cell cycle checkpoint mechanisms, exhausts cellular NAD+ levels, and inhibits DNA repair leading to the metabolic and mitotic collapse of cancer cells and synergistic lethality in OC cells. Currently, prexasertib is under clinical trials and our studies will provide novel mechanistic insight into the therapeutic potential of this combination and provides preclinical evidence to develop further this combination therapy in treating OC. Citation Format: Ganesh Acharya, Chinnadurai Mani, Mark B. Reedy, Komaraiah Palle. PARG inhibition augments CHK1 inhibitor-induced replication stress and synergistically kills ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2667.

A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

Telomeric ends form a loop structure (T-loop) necessary for the repression of ATM kinase activation throughout the normal cell cycle. However, cells undergoing a prolonged mitotic arrest are prone to lose the T-loop, resulting in Aurora B kinase-dependent mitotic telomere deprotection, which was proposed as an anti-tumor mechanism that eliminates precancerous cells from the population. The mechanism of mitotic telomere deprotection has not been elucidated. Here, we show that WRN, a RECQ helicase family member, can suppress mitotic telomere deprotection independently of its exonuclease and helicase activities. Truncation of WRN revealed that N-terminus amino acids 168–333, a region that contains a coiled-coil motif, is sufficient to suppress mitotic telomere deprotection without affecting both mitotic Aurora B-dependent spindle checkpoint and ATM kinase activity. The suppressive activity of the WRN168–333 fragment is diminished in cells partially depleted of TRF2, while WRN is required for complete suppression of mitotic telomere deprotection by TRF2 overexpression. Finally, we found that phosphomimetic but not alanine mutations of putative Aurora B target sites in the WRN168–333 fragment abolished its suppressive effect. Our findings reveal a non-enzymatic function of WRN, which may be regulated by phosphorylation in cells undergoing mitotic arrest. We propose that WRN enhances the protective function of TRF2 to counteract the hypothetical pathway that resolves the mitotic T-loop.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Abstract LB192: Demethylation, DNA damage and antitumor activity of DNA methyltransferase inhibitors in models with intact and disrupted DNMT1 gene

Abstract DNA methyltransferase (DNMT) 1 is an enzyme responsible for maintaining DNA methylation pattern during DNA replication. However, its role on the methylation of tumor suppressor genes and DNA damage in relation to antitumor activity of DNMT inhibitors remains controversial. Here, we investigated the effects of DNMT inhibitors in human colorectal and breast cancer cell lines and animal models with intact (DNMT1+/+) and disrupted DNMT1 (DNMT1-/-) gene. Treatment with 5 µM of 4’-thio-5-aza-2’-deoxycytidine (aza-TdC), decitabine and azacytidine all inhibited DNMT1 expression and cell growth (94.0%/97.1%, 39.9%/58.6% and 69.2/91.3), and induced apoptosis (66.9%/62.5%, 30.9%/23.8% and 66.2/43.1%) in DNMT1+/+ HCT116/MCF7 cells; in contrast, the DNMT inhibitors had less/little activities in DNMT1-/- HCT116/MCF7 cells (P<0.001 each). γH2AX and G2/M cell-cycle arrest were induced by the inhibitors and greater degree of induction was produced by aza-TdC than decitabine in DNMT1+/+ cells (P<0.001), with activation of checkpoint kinase 1 and upregulation of p21. Both demethylation and increased acetylation of CDKN2A and CDKN2B gene promoters together with re-expression of p16INK4A and p15INK4B proteins were detected in DNMT1-/- versusDNMT1+/+ (demethylation only) cells upon exposure to decitabine for 72h. Compared with saline treatment, tumor growth was significantly inhibited by aza-TdC (90%; n=18 mice) and decitabine (62%; n=18) in DNMT1+/+ colorectal xenograft tumors, alongside decreasing expression of DNMT1 (51% and 44%) and an increase in p21 (85% and 48%). Similarly, tumor growth delays (86%; n=15 and 57%; n=14) were associated with the reduction of DNMT1 and augmentation of p21 in bladder PDX tumors. The findings suggest that DNMT inhibitors induce DNA damage and higher anti-tumor activity of aza-TdC is proportional to a greater magnitude of induction of DNA damage, p21 and cell cycle arrest without reactivating the expression of p16INK4A and p15INK4B. The gene re-expression coupled to demethylation and acetylation corresponds to the less anti-tumor activity by decitabine in DNMT1-/- cells. Thus, it is the DNA damage effects, rather than re-expression of the tumor suppressors, that are associated with antitumor activity of the DNMT inhibitors in cancer cells and/or xenograft and PDX tumors. Citation Format: Angelo B. Laranjeira, Melinda Hollingshead, Dat Nguyen, Robert Kinders, Michael Difilippantonio, James H. Doroshow, Sherry X. Yang. Demethylation, DNA damage and antitumor activity of DNA methyltransferase inhibitors in models with intact and disrupted DNMT1 gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB192.

Artesunate improves venetoclax plus cytarabine AML cell targeting by regulating the Noxa/Bim/Mcl-1/p-Chk1 axis

Venetoclax plus cytarabine therapy is approved for elderly acute myeloid leukemia (AML) patients and needs further improvement. We studied the mechanisms of venetoclax plus cytarabine treatment and searched for a third agent to enhance their effects. Cytarabine induces S phase arrest-mediated DNA damage with activation of DNA replication checkpoint kinase 1 (Chk1) through phosphorylation, while venetoclax induces B cell lymphoma 2 (Bcl-2)-interacting mediator of cell death (Bim)-mediated apoptotic DNA damage. Myeloid cell leukemia-1 (Mcl-1) plays negative roles in both events by sequestering Bim and accelerating Chk1 phosphorylation. Venetoclax releases Bim from Bcl-2 with increased Bim binding to Mcl-1. Artesunate, an antimalaria drug, induces Noxa to replace Bim from Mcl-1 and induces synergistic apoptosis with venetoclax accompanied with Mcl-1 reduction. Silencing Mcl-1 or adding venetoclax/artesunate diminishes the cytarabine resistance pathway p-Chk1. The triple combination exhibits S phase arrest with enhanced DNA damage, improves AML colony formation inhibition, and prolongs survival of two mice xenograft models compared to the venetoclax/cytarabine dual combination. Artesunate serves as a bridge for venetoclax and cytarabine combination by Noxa and Bim-mediated apoptosis and Mcl-1 reduction. We provide a new triple combination for AML treatment by targeting the Noxa/Mcl-1/Bim axis to reverse Mcl-1/p-Chk1 resistance of cytarabine therapy.

ChK1 activation induces reactive astrogliosis through CIP2A/PP2A/STAT3 pathway in Alzheimer's disease.

Cancerous Inhibitor of PP2A (CIP2A), an endogenous PP2A inhibitor, is upregulated and causes reactive astrogliosis, synaptic degeneration, and cognitive deficits in Alzheimer's disease (AD). However, the mechanism underlying the increased CIP2A expression in AD brains remains unclear. We here demonstrated that the DNA damage-related Checkpoint kinase 1 (ChK1) is activated in AD human brains and 3xTg-AD mice. ChK1-mediated CIP2A overexpression drives inhibition of PP2A and activates STAT3, then leads to reactive astrogliosis and neurodegeneration in vitro. Infection of mouse brain with GFAP-ChK1-AAV induced AD-like cognitive deficits and exacerbated AD pathologies in vivo. In conclusion, we showed that ChK1 activation induces reactive astrogliosis, degeneration of neurons, and exacerbation of AD through the CIP2A-PP2A-STAT3 pathway, and inhibiting ChK1may be a potential therapeutic approach for AD treatment.

Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants.

The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication, and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin-binding protein Barrier-to-Autointegration Factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to the deformation of NL structure, and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate the contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf misregulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored the expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained misexpressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in the repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global misexpression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found upregulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.

Replication stress response defects are associated with response to immune checkpoint blockade in nonhypermutated cancers.

Treatment with immune checkpoint blockade (ICB) has resulted in durable responses for a subset of patients with cancer, with predictive biomarkers for ICB response originally identified largely in the context of hypermutated cancers. Although recent clinical data have demonstrated clinical responses to ICB in certain patients with nonhypermutated cancers, previously established ICB response biomarkers have failed to accurately identify which of these patients may benefit from ICB. Here, we demonstrated that a replication stress response (RSR) defect gene expression signature, but not other proposed biomarkers, is associated with ICB response in 12 independent cohorts of patients with nonhypermutated cancer across seven tumor types, including those of the breast, prostate, kidney, and brain. Induction or suppression of RSR deficiencies was sufficient to modulate response to ICB in preclinical models of breast and renal cancers. Mechanistically, we found that despite robust activation of checkpoint kinase 1 signaling in RSR-deficient cancer cells, aberrant replication origin firing caused exhaustion of replication protein A, resulting in accumulation of immunostimulatory cytosolic DNA. We further found that deficient RSR coincided with increased intratumoral dendritic cells in both mouse cancer models and human tumors. Together, this work demonstrates that the RSR defect gene signature can accurately identify patients who may benefit from ICB across numerous nonhypermutated tumor types, and pharmacological induction of RSR defects may further expand the benefits of ICB to more patients.

An Adenovirus early region 4 deletion mutant induces G2/M arrest via ATM activation and reduces expression of the mitotic marker phosphorylated (ser10) histone 3

Adenovirus (Ad) type 5 (Ad5) early region 4 (E4) proteins inhibit the DNA damage response (DDR) including activation of the DDR kinase ATM and its substrates, which can induce G2/M cell cycle arrest. Infection with Ad5 or the E4 deletion mutant H5dl1007 (1007) resulted in the accumulation of post G1 cells with > 2 N cellular DNA content. A greater fraction of cells with 4 N DNA content was observed in 1007 infections compared to Ad5; this population was dependent on activation of ATM. G2/M checkpoint kinases, phosphorylated Chk2 (pChk2), and phosphorylated Cdk1 (pCdk1) were upregulated in 1007 infections, and 1007 showed reduced levels of the mitosis marker phosphorylated (Ser10) histone 3 compared to Ad5. Our results show that E4 mutant activation of ATM induces G2/M arrest via activation of checkpoint kinases, thereby contributing to viral-mediated regulation of the cell cycle.

Mouse model of radiation-induced premature ovarian insufficiency reveals compromised oocyte quality: implications for fertility preservation

Research questionWhat is the impact of radiation exposure on oocyte quality and female fertility? DesignPrepubertal mice underwent whole-body irradiation with a single dose (0.02, 0.1, 0.5, 2, 8 Gy) of gamma- or X-rays. Oocytes were quantified in irradiated (n = 36) and sham-treated (n = 8) mice. After a single exposure to 2 Gy, formation of DNA double-strand breaks (n = 10), activation of checkpoint kinase (Chk2) (n = 10) and dynamics of follicular growth (n = 18) were analysed. Fertility assessment was performed in adult irradiated mice and controls from the number of pups per mouse (n = 28) and the fetal abortion rate (n = 24). Ploidy of mature oocytes (n = 20) was analysed after CREST immunostaining, and uterine sections were examined. ResultsRadiation exposure induced a massive loss of primordial follicles with LD50 below 50 mGy for both gamma and X-rays. Growing follicles survived doses up to 8 Gy. This difference in radiosensitivity was not due to a different amount of radio-induced DNA damage, and Chk2 was activated in all oocytes. Exposure to a 2 Gy dose abolished the long-term fertility of females due to depletion of the ovarian reserve. Detailed analysis indicates that surviving oocytes were able to complete folliculogenesis and could be fertilized. This transient fertility allowed irradiated females to produce a single litter albeit with a high rate of fetal abortion (23%, P = 0.0096), related to altered ploidy in the surviving oocytes (25.5%, P = 0.0035). ConclusionsThe effects of radiation on surviving oocyte quality question natural conception as a first-line approach in cancer survivors. Together, the data emphasize the need for fertility preservation before radiation exposure and call for reassessment of the use of cryopreserved oocytes.

Fms-like tyrosine kinase 3-internal tandem duplications epigenetically activates checkpoint kinase 1 in acute myeloid leukemia cells

It is not clear how Fms-like tyrosine kinase 3-internal tandem duplications (FLT3-ITD) regulates checkpoint kinase 1 (CHK1) in acute myeloid leukemia (AML). In this study, we investigated the regulatory effect of FLT3-ITD on CHK1. Our results showed that CHK1 was highly expressed in FLT3-ITD positive AML. The overall survival rate and disease-free survival rate of AML patients with high CHK1 level were lower than those of patients with low CHK1 level. Mechanistically, FLT3-ITD recruited p300 to the CHK1 promoter and subsequently acetylated H3K27, thereby enhancing the transcription of CHK1. Interfering with the expression of CHK1 significantly inhibited the cell proliferation and induced cell apoptosis in FLT3-ITD positive MV4-11 cells. In addition, CHK1 knockdown promoted the sensitivity of MV4-11 cells to the epigenetic inhibitors JQ1 and C646. This study discovers a new therapeutic target for FLT3-ITD + AML and provided evidence for the combination of epigenetic inhibitors for AML treatment.

19-(Benzyloxy)-19-oxojolkinolide B (19-BJB), an ent-abietane diterpene diepoxide, inhibits the growth of bladder cancer T24 cells through DNA damage.

Diterpenoids jolkinolide A and B, were first isolated from Euphorbia fischeriana. In our previous research, 19-(Benzyloxy)-19-oxojolkinolide B (19-BJB), a derivative of jolkinolides, was synthesized as a novel ent -abietane diterpene diepoxide. In this study, 19-BJB showed strong in vitro activity against bladder cancer cell lines. DNA damage which was observed through the interaction of 19-BJB with nucleotide chains and affected DNA repair resulted in the activation of checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) in bladder cancer cell lines. In vivo testing in nude mice also proved that 19-BJB revealed a potential inhibitory effect on tumor growth. Additionally, the 3D-QSAR models of jolkinolides were established. Briefly, we proved that 19-BJB could potentially be used as a drug to inhibit the growth of bladder tumor.

Natural podophyllotoxin analog 4DPG attenuates EMT and colorectal cancer progression via activation of checkpoint kinase 2

Epithelial–mesenchymal transition (EMT) is critical for the metastatic dissemination of cancer cells and contributes to drug resistance. In this study, we observed that epithelial colorectal cancer (CRC) cells transiently exposed to 5-fluorouracil (5-FU) (a chemotherapeutic drug for CRC) as well as 5-FU-resistant cells (5-FU-R) develop EMT characters as evidenced by activation of Vimentin and augmented invasive properties. On the other hand, 4DPG (4′-demethyl-deoxypodophyllotoxin glucoside), a natural podophyllotoxin analog attenuates EMT and invadopodia formation abilities of HCT-116/5-FU-R and SW-620/5-FU-R cells. Treatment with 4DPG restrains Vimentin phosphorylation (Ser38) in 5-FU-R cells, along with downregulation of mesenchymal markers Twist1 and MMP-2 while augmenting the expression of epithelial markers E-cadherin and TIMP-1. Moreover, 4DPG boosts the tumor-suppressor protein, checkpoint kinase 2 (Chk2) via phosphorylation at Thr68 in a dose-dependent manner in 5-FU-R cells. Mechanistically, SiRNA-mediated silencing of Chk2, as well as treatment with Chk2-specific small-molecule inhibitor (PV1019), divulges that 4DPG represses Vimentin activation in a Chk2-dependent manner. Furthermore, immunoprecipitation analysis unveiled that 4DPG prevents complex formation between Vimentin and p53 resulting in the rescue of p53 and its nuclear localization in aggressive 5-FU-R cells. In addition, 4DPG confers suitable pharmacokinetic properties and strongly abrogates tumor growth, polyps formation, and lung metastasis in an orthotopic rat colorectal carcinoma model. In conclusion, our findings demonstrate 4DPG as a targeted antitumor/anti-metastatic pharmacological lead compound to circumvent EMT-associated drug resistance and suggest its clinical benefits for the treatment of aggressive cancers.

Topsentinol L Trisulfate, a Marine Natural Product That Targets Basal-like and Claudin-Low Breast Cancers.

Patients diagnosed with basal-like breast cancer suffer from poor prognosis and limited treatment options. There is an urgent need to identify new targets that can benefit patients with basal-like and claudin-low (BL-CL) breast cancers. We screened fractions from our Marine Invertebrate Compound Library (MICL) to identify compounds that specifically target BL-CL breast cancers. We identified a previously unreported trisulfated sterol, i.e., topsentinol L trisulfate (TLT), which exhibited increased efficacy against BL-CL breast cancers relative to luminal/HER2+ breast cancer. Biochemical investigation of the effects of TLT on BL-CL cell lines revealed its ability to inhibit activation of AMP-activated protein kinase (AMPK) and checkpoint kinase 1 (CHK1) and to promote activation of p38. The importance of targeting AMPK and CHK1 in BL-CL cell lines was validated by treating a panel of breast cancer cell lines with known small molecule inhibitors of AMPK (dorsomorphin) and CHK1 (Ly2603618) and recording the increased effectiveness against BL-CL breast cancers as compared with luminal/HER2+ breast cancer. Finally, we generated a drug response gene-expression signature and projected it against a human tumor panel of 12 different cancer types to identify other cancer types sensitive to the compound. The TLT sensitivity gene-expression signature identified breast and bladder cancer as the most sensitive to TLT, while glioblastoma multiforme was the least sensitive.

Cytotoxic and genotoxic effects of epigenetic inhibitors on bladder cancer cells

Urothelial carcinoma (UC) are characterized by frequent mutations and incorrect expression of epigenetic regulator proteins. Thus epigenetic inhibitors (Epi-I) may be suitable as new therapeutic approaches or supplements to standard therapy. Previous work demonstrated additional cytotoxic effects of Epi-I which may result from disturbed DNA synthesis and DNA damage response (DDR). These were systematically investigated to identify options for novel combination therapies. UC cell lines VMCUB-1 and UM-UC-3 were treated with GSK126 (EZH2), GSK-J4 (inhibitor of histone demethylases such as UTX and JMJD3), PLX51107 (bromodomain protein (BET) inhibitor) and the class I HDAC inhibitor romidepsin. In addition to the effects on short and long-term proliferation, effects on cell cycle and apoptosis induction were investigated using flow cytometry and Western blot analyses. Induction of DNA damage and DDR were analysed using immunocytochemistry and Western blot. Synergy of combination treatment was evaluated by the Chou-Talalay method. RNA of UCCs treated with romidepsin and PLX51107 was subjected to RNA sequencing. Normal HBLAK control cells were used as a control. All Epi-I reduced cell viability and colony formation in both cell lines. Cells responded most weakly to GSK126, while PLX51107 had most significant effects on apoptosis induction and regulation. The apparently aneugenic mode of action of GSKJ4 induced mitotic disorders recognizable by accumulation of micronuclei, "lagging chromosomes" and chromosome bridges and altered expression of DDR markers. Although the clastogenic mode of action of romidepsin and PLX51107 resulted in accumulation of DNA double-strand breaks, no activation of checkpoint kinases was found. On the contrary, expression of CHK1 and factors of homologous recombination was downregulated. GSK126 was cytotoxic without induction of DNA damage. Combination treatments of PLX51107 and inhibitors of DDR or chemotherapeutic compounds were highly synergistic with tolerable toxicity in benign control cells. The genotoxic mode of action of Epi-I, particularly of HDAC and BET proteins, enables new approaches for a combination therapy with inhibitors of DNA repair mechanisms or standard chemotherapy.