Identification of [1,2,4]Triazolo[4,3-a]pyrazine PARP1 inhibitors with overcome acquired resistance activities.

Poly(ADP-ribose) polymerase 1 (PARP1) inhibitors can selectively kill homologous recombination (HR) deficient cancer cells and elicit anticancer effect through a mechanism of synthetic lethality. In this study, we designed, synthesized and pharmacologically evaluated a series of [1,2,4]triazolo[4,3-a]pyrazine derivatives as a class of potent PARP1 inhibitors. Among them, compounds 17m, 19a, 19c, 19e, 19i and 19k not only displayed more potent inhibitory activities (IC50s < 4.1 nM) than 9 and 1 against PARP1, but also exhibited nanomolar range of antiproliferative effects against MDA-MB-436 (BRCA1-/-, IC50s < 1.9 nM) and Capan-1 (BRCA2-/-, IC50s < 21.6 nM) cells. Notably, 19k significantly inhibited proliferation of resistant Capan-1 cells (IC50s < 0.3 nM). Collectively, the newly discovered PARP1 inhibitors act as a useful pharmacological tool for investigating the mechanism of acquired resistance to PARP1 inhibitors, and may also represent promising therapeutic agents for the treatment of HR deficient cancers with the potential to overcome the acquired resistance.

Thioparib inhibits homologous recombination repair, activates the type I IFN response, and overcomes olaparib resistance.

Poly-ADP-ribose polymerase (PARP) inhibitors (PARPi) have shown great promise for treating BRCA-deficient tumors. However, over 40% of BRCA-deficient patients fail to respond to PARPi. Here, we report that thioparib, a next-generation PARPi with high affinity against multiple PARPs, including PARP1, PARP2, and PARP7, displays high antitumor activities against PARPi-sensitive and -resistant cells with homologous recombination (HR) deficiency both in vitro and in vivo. Thioparib treatment elicited PARP1-dependent DNA damage and replication stress, causing S-phase arrest and apoptosis. Conversely, thioparib strongly inhibited HR-mediated DNA repair while increasing RAD51 foci formation. Notably, the on-target inhibition of PARP7 by thioparib-activated STING/TBK1-dependent phosphorylation of STAT1, triggered a strong induction of type I interferons (IFNs), and resulted in tumor growth retardation in an immunocompetent mouse model. However, the inhibitory effect of thioparib on tumor growth was more pronounced in PARP1 knockout mice, suggesting that a specific PARP7 inhibitor, rather than a pan inhibitor such as thioparib, would be more relevant for clinical applications. Finally, genome-scale CRISPR screening identified PARP1 and MCRS1 as genes capable of modulating thioparib sensitivity. Taken together, thioparib, a next-generation PARPi acting on both DNA damage response and antitumor immunity, serves as a therapeutic potential for treating hyperactive HR tumors, including those resistant to earlier-generation PARPi.

Regulation of CCL2 by EZH2 affects tumor-associated macrophages polarization and infiltration in breast cancer.

Tumor associated macrophages (TAMs) play an important role in tumorigenesis, development and anti-cancer drug therapy. However, very few epigenetic compounds have been elucidated to affect tumor growth by educating TAMs in the tumor microenvironment (TME). Herein, we identified that EZH2 performs a crucial role in the regulation of TAMs infiltration and protumoral polarization by interacting with human breast cancer (BC) cells. We showed that EZH2 inhibitors-treated BC cells induced M2 macrophage polarization in vitro and in vivo, while EZH2 knockdown exhibited the opposite effect. Mechanistically, inhibition of EZH2 histone methyltransferase alone by EZH2 inhibitors in breast cancer cells could reduce the enrichment of H3K27me3 on CCL2 gene promoter, elevate CCL2 transcription and secretion, contributing to the induction of M2 macrophage polarization and recruitment in TME, which reveal a potential explanation behind the frustrating results of EZH2 inhibitors against breast cancer. On the contrary, EZH2 depletion led to DNA demethylation and subsequent upregulation of miR-124-3p level, which inhibited its target CCL2 expression in the tumor cells, causing arrest of TAMs M2 polarization. Taken together, these data suggested that EZH2 can exert opposite regulatory effects on TAMs polarization through its enzymatic or non-enzymatic activities. Our results also imply that the effect of antitumor drugs on TAMs may affect its therapeutic efficacy, and the combined application with TAMs modifiers should be warranted to achieve great clinical success.

Author Correction: G9a regulates breast cancer growth by modulating iron homeostasis through the repression of ferroxidase hephaestin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting Epigenetic Crosstalk as a Therapeutic Strategy for EZH2-Aberrant Solid Tumors.

Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.

Identification of compound D2923 as a novel anti-tumor agent targeting CSF1R.

Colony-stimulating factor 1 receptor (CSF1R) plays a critical role in promoting tumor progression in various types of tumors. Here, we identified D2923 as a novel and selective inhibitor of CSF1R and explored its antitumor activity both in vitro and in vivo. D2923 potently inhibited CSF1R in vitro kinase activity with an IC50 value of 0.3 nM. It exhibited 10- to 300-fold less potency against a panel of kinases tested. D2923 markedly blocked CSF-1-induced activation of CSF1R and its downstream signaling transduction in THP-1 and RAW264.7 macrophages and thus inhibited the in vitro growth of macrophages. Moreover, D2923 dose-dependently attenuated the proliferation of a small panel of myeloid leukemia cells, mainly by arresting the cells at G1 phase as well as inducing apoptosis in the cells. The results of the in vivo experiments further demonstrated that D2923 displayed potent antitumor activity against M-NFS-60 xenografts, with tumor growth inhibition rates of 50% and 88% at doses of 40 and 80 mg/kg, respectively. Additionally, D2923 was well tolerated with no significant body-weight loss observed in the treatment groups compared with the control. Furthermore, a western blot analysis and the immunohistochemistry results confirmed that the phosphorylation of CSF1R in tumor tissue was dramatically reduced after D2923 treatment, and this was accompanied by the depletion of macrophages in the tumor. Meanwhile, the expression of the proliferation marker Ki67 was also markedly decreased in the D2923 treatment group compared with the control group. Taken together, we identified D2923 as a novel and effective CSF1R inhibitor, which deserves further investigation.

SOMCL-085, a novel multi-targeted FGFR inhibitor, displays potent anticancer activity in FGFR-addicted human cancer models.

Aberrant fibroblast growth factor receptor (FGFR) activation is found across a diverse spectrum of malignancies, especially those lacking effective treatments. SOMCL-085 is a novel FGFR-dominant multi-target kinase inhibitor. Here, we explored the FGFR-targeting anticancer activity of SOMCL-085 both in vitro and in vivo. Among a panel of 20 tyrosine kinases screened, SOMCL-085 potently inhibited FGFR1, FGFR2 and FGFR3 kinase activity, with IC50 values of 1.8, 1.9 and 6.9 nmol/L, respectively. This compound simultaneously inhibited the angiogenesis kinases VEGFR and PDGFR, but without obvious inhibitory effect on other 12 tyrosine kinases. In 3 representative human cancer cell lines with different mechanisms of FGFR activation tested, SOMCL-085 (20-500 nmol/L) dose-dependently inhibited FGFR1-3 phosphorylation and the phosphorylation of their key downstream effectors PLCγ and Erk. In 7 FGFR aberrant human cancer cell lines, regardless of the mechanistic complexity of FGFR over-activation, SOMCL-085 potently inhibited FGFR-driven cell proliferation by arresting cells at the G1/S phase. In the FGFR1-amplified lung cancer cell line H1581 xenograft mice and FGFR2-amplified gastric cancer cell line SNU16 xenograft mice, oral administration of SOMCL-085 (25, 50 mg·kg-1·d-1) for 21 days substantially suppressed tumor growth without affecting their body-weight. These results suggest that SOMCL-085 is a potent multi-target FGFR inhibitor that inhibits the FGFR-dependent neoplastic phenotypes of human cancer cells in vitro and in vivo.

G9a regulates breast cancer growth by modulating iron homeostasis through the repression of ferroxidase hephaestin.

G9a, a H3K9 methyltransferase, shows elevated expression in many types of human cancers, particularly breast cancer. However, the tumorigenic mechanism of G9a is still far from clear. Here we report that G9a exerts its oncogenic function in breast cancer by repressing hephaestin and destruction cellular iron homeostasis. In the case of pharmacological inhibition or short hairpin RNA interference-mediated suppression of G9a, the expression and activity of hephaestin increases, leading to the observed decrease of intracellular labile iron content and the disturbance of breast cancer cell growth in vitro and in vivo. We also provide evidence that G9a interacts with HDAC1 and YY1 to form a multi-molecular complex that contributes to hephaestin silencing. Furthermore, high G9a expression and low hephaestin expression correlate with poor survival of breast cancer are investigated. All these suggest a G9a-dependent epigenetic program in the control of iron homeostasis and tumor growth in breast cancer.G9a is a histone methyltransferase highly expressed in several cancers including breast cancer. Here the authors propose a mechanism through which G9a promotes breast cancer by regulating iron metabolism through the repression of ferroxidase hephaestin.

Combining 53BP1 with BRCA1 as a biomarker to predict the sensitivity of poly(ADP-ribose) polymerase (PARP) inhibitors.

Over half of patients with BRCA1-deficient cancers do not respond to treatment with poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we report that a combination of 53BP1 and BRCA1 may serve as a biomarker of PARP inhibitor sensitivity. Based on the mRNA levels of four homologous recombination repair (HR) genes and PARP inhibitor sensitivity, we selected BRCA1-deficient MDA-MB-436 cells to conduct RNA interference. Reducing expression of 53BP1, but not the other three HR genes, was found to lower simmiparib sensitivity. Additionally, we generated 53BP1-/-/BRCA1-/- clonal variants by the transcription activator-like effector nuclease (TALEN) technique and found that depleting 53BP1 impaired PARP inhibitor sensitivity with a 36.7-fold increase in their IC50 values. Consistent with its effect on PARP inhibitor sensitivity, 53BP1 loss alleviated cell cycle arrest and apoptosis and partially restored HR function. Importantly, 53BP1 depletion dramatically reduced the ability of PARP inhibitors to suppress tumor growth in vivo. The inhibition rate of simmiparib was 74.16% for BRCA1-deficient MDA-MB-436 xenografts, but only 7.79% for 53BP1/BRCA1-deficient xenografts. Re-expressing 53BP1 in the dual-deficient cells restored PARP inhibitor sensitivity and the levels of HR regulators. Considering that at least 10% of BRCA1-deficient breast and ovarian cancers have reduced expression of 53BP1, using a combination of 53BP1 with BRCA1 as a biomarker for patient selection should reduce the number of patients undergoing futile treatment with PARP inhibitors.

Marine-derived chromopeptide A, a novel class I HDAC inhibitor, suppresses human prostate cancer cell proliferation and migration.

Histone deacetylases (HDACs), especially HDAC1, 2, 3 and 4, are abundantly expressed and over-activated in prostate cancer that is correlated with the poor prognosis. Thus, inhibition of HDAC activity has emerged as a potential alternative option for prostate cancer therapy. Chromopeptide A is a depsipeptide isolated from the marine sediment-derived bacterium Chromobacterium sp. HS-13-94; it has a chemical structure highly similar to FK228, a class I HDAC inhibitor that is approved by FDA for treating T-cell lymphoma. In this study, we determined whether chromopeptide A, like FK228, acted as a class I HDAC inhibitor, and whether chromopeptide A could inhibit the growth and migration of human prostate cancer in vitro and in vivo. HDAC enzyme selectivity and kinetic analysis revealed that chromopeptide A selectively inhibited the enzymatic activities of HDAC1, 2, 3 and 8 in a substrate non-competitive manner with comparable IC50 values for each HDAC member as FK228 in vitro. Importantly, chromopeptide A dose-dependently suppressed the proliferation of human prostate cancer cell lines PC3, DU145 and LNCaP with IC50 values of 2.43±0.02, 2.08±0.16, and 1.75±0.06 nmol/L, respectively, accompanied by dose-dependent inhibition of HDAC enzymatic activity in PC3 and DU145 cells. Chromopeptide A (0.2-50 nmol/L) caused G2/M phase arrest and induced apoptosis in the prostate cancer cell lines. Moreover, chromopeptide A dose-dependently inhibited the migration of PC3 cells. In mice bearing PC3 prostate cancer xenografts, intravenous injection of chromopeptide A (1.6, 3.2 mg/kg, once a week for 18 d) significantly suppressed the tumor growth, which was associated with increased expression levels of Ac-H3 and p21 in tumor tissues. Our results identify chromopeptide A as a novel class I HDAC inhibitor and provide therapeutic strategies that may be implemented in prostate cancer.

Poly(ADP-ribose)polymerase (PARP) inhibition and anticancer activity of simmiparib, a new inhibitor undergoing clinical trials.

Poly(ADP-ribose)polymerase (PARP)1/2 inhibitors have been proved to be clinically effective anticancer drugs. Here we report a new PARP1/2 inhibitor, simmiparib, displaying apparently improved preclinical anticancer activities relative to the first approved inhibitor olaparib. Simmiparib inhibited PARP1/2 approximately 2-fold more potently than olaparib, with more than 90-fold selectivity over the other tested PARP family members. Simmiparib and olaparib caused similar cellular PARP1-DNA trapping. Simmiparib selectively induced the accumulation of DNA double-strand breaks, G2/M arrest and apoptosis in homologous recombination repair (HR)-deficient cells. Consistently, simmiparib showed 26- to 235-fold selectivity in its antiproliferative activity against HR-deficient cells over the corresponding isogenic HR-proficient cells. Notably, its antiproliferative activity was 43.8-fold more potent than that of olaparib in 11 HR-deficient cancer cell lines. Simmiparib also potentiated the proliferative inhibition of several conventional anticancer drugs. Simmiparib reduced the poly(ADP-ribose) formation in HR-deficient cancer cells and xenografts. When orally administered to nude mice bearing xenografts, simmiparib revealed excellent pharmacokinetic properties. Simmiparib caused approximately 10-fold greater growth inhibition than olaparib against HR-deficient human cancer cell- or tissue-derived xenografts in nude mice. Collectively, these findings support the undergoing clinical trials of simmiparib.

Novel PARP1/2 inhibitor mefuparib hydrochloride elicits potent in vitro and in vivo anticancer activity, characteristic of high tissue distribution.

The approval of poly(ADP-ribose) polymerase (PARP) inhibitor AZD2281 in 2014 marked the successful establishment of the therapeutic strategy targeting homologous recombination repair defects of cancers in the clinic. However, AZD2281 has poor water solubility, low tissue distribution and relatively weak in vivo anticancer activity, which appears to become limiting factors for its clinical use. In this study, we found that mefuparib hydrochloride (MPH) was a potent PARP inhibitor, possessing prominent in vitro and in vivo anticancer activity. Notably, MPH displayed high water solubility (> 35 mg/ml) and potent PARP1/2 inhibition in a substrate-competitive manner. It reduced poly(ADP-ribose) (PAR) formation, enhanced γH2AX levels, induced G2/M arrest and subsequent apoptosis in homologous recombination repair (HR)-deficient cells. Proof-of-concept studies confirmed the MPH-caused synthetic lethality. MPH showed potent in vitro and in vivo proliferation and growth inhibition against HR-deficient cancer cells and synergistic sensitization of HR-proficient xenografts to the anticancer drug temozolomide. A good relationship between the anticancer activity and the PARP inhibition of MPH suggested that PAR formation and γH2AX accumulation could serve as its pharmacodynamic biomarkers. Its high bioavailability (40%~100%) and high tissue distribution in both monkeys and rats were its most important pharmacokinetic features. Its average concentrations were 33-fold higher in the tissues than in the plasma in rats. Our work supports the further clinical development of MPH as a novel PARP1/2 inhibitor for cancer therapy.

c-Myc alterations confer therapeutic response and acquired resistance to c-Met inhibitors in MET-addicted cancers.

Use of kinase inhibitors in cancer therapy leads invariably to acquired resistance stemming from kinase reprogramming. To overcome the dynamic nature of kinase adaptation, we asked whether a signal-integrating downstream effector might exist that provides a more applicable therapeutic target. In this study, we reported that the transcriptional factor c-Myc functions as a downstream effector to dictate the therapeutic response to c-Met inhibitors in c-Met-addicted cancer and derived resistance. Dissociation of c-Myc from c-Met control, likely overtaken by a variety of reprogrammed kinases, led to acquisition of drug resistance. Notably, c-Myc blockade by RNA interference or pharmacologic inhibition circumvented the acquired resistance to c-Met inhibition. Combining c-Myc blockade and c-Met inhibition in MET-amplified patient-derived xenograft mouse models heightened therapeutic activity. Our findings offer a preclinical proof of concept for the application of c-Myc-blocking agents as a tactic to thwart resistance to kinase inhibitors.

HSP90 inhibitor AUY922 abrogates up-regulation of RTKs by mTOR inhibitor AZD8055 and potentiates its antiproliferative activity in human breast cancer.

mTOR inhibition led to activation of upstream receptor tyrosine kinases (RTKs) and AKT, which may attenuate the efficacy of mTOR kinase inhibitors. We sought to discover efficient drug combination with mTOR inhibitors by elucidating the survival feedback loops induced by mTOR inhibition in breast cancer. The feedback signaling upon treatment of mTOR inhibitor AZD8055 was determined and the combinatorial activity of AZD8055 and HSP90 inhibitor AUY922 in cell signaling and proliferation were detected. Treatment of breast cancer T47D cells with AZD8055 induced activation of AKT and phosphatidylinositol 3-kinase (PI3K), which was accompanied with increase in expression of multiple upstream proteins including EGFR, HER2, HER3 and IRS-1. Different RTKs were revealed to be responsible for the reactivation of AKT by AZD8055 in different breast cancer cell lines. Down-regulation of these proteins differentially enhanced the antiproliferative activity of AZD8055. AZD8055 and AUY922 displayed synergistic effect against a panel of human breast cancer cells irrespective their genotype, which was associated with enhanced cell cycle arrest and inhibition of DNA synthesis. AUY922 destabilized multiple tested tyrosine kinases and abrogated activation of AKT induced by AZD8055. AZD8055 also inhibited up-regulation of HSP70 and HSP27 upon AUY922 treatment. Cotreatment of these two drugs demonstrated synergistic activity against triple negative MDA-MB-468 xenograft without enhanced toxicity. The combination of AZD8055 and AUY922 demonstrated synergistic activity against various types of breast cancer and established a mechanistic rationale for a combination approach using catalytic mTOR kinase inhibitor and HSP90 inhibitor in the treatment of breast cancer.

[Evaluation of right ventricle function by tissue Doppler imaging in patients with acute organophosphate poisoning].

OBJECTIVE: To evaluate the right ventricular functions of patients with acute organophosphate poisoning patient (AOPP) by measuring the velocities of tricuspid annular motion through Doppler tissue imaging (DTI). METHODS: Tissue Doppler velocity mode was employed to observe tricuspid annulus movement in 78 AOPP patients and 32 healthy adults. After tricuspid annular pulsed-wave rate of E/A were measured by traditional echocardiographic, the peak values of tricuspid annular early diastolic velocity (E(m)) and late diastolic velocity (A(m)) were analyzed by DTI. RESULTS: E/A > E(m)/A(m) > 1 was observed in health control group as well as the mild-moderate AOPP; E(m)/A(m) < E/A < 1 was observed in the severe AOPP group. There was significant difference between two groups. The right ventricular E/A correlated positively with E(m)/A(m) (P < 0.001). Pulmonary arterial pressure (PAP) correlated positively with right ventricular E/Em, Tei Index (P < 0.001) and negatively with right ventricular S(m), E(m) and E(m)/A(m) (P < 0.001). CONCLUSION: The right ventricle functions, in terms of contraction and relaxation, decrease in severe AOPP patients versus health control and mild-moderate groups. TDI is a useful tool to assess the right ventricle functions of AOPP patients.

[Application of fluorescence in situ hybridization in prenatal diagnosis of complex chromosomal abnormalities].

OBJECTIVE: To investigate the application of fluorescence in situ hybridization (FISH) technique in prenatal diagnosis of complex chromosomal abnormalities. METHODS: Eleven prenatal diagnosis cases (8 from amniocentesis and 3 from cord blood) with complex chromosomal abnormalities detected by routine G-banding, were further analyzed by FISH. RESULTS: The FISH technique confirmed the results of balanced chromosome rearrangements detected by G-banding, and clarified the structure of the derivative chromosomes in the 3 amniocentesis samples and the origin of the mark chromosomes in the 2 cord blood samples. CONCLUSION: FISH can be used to diagnose the complex chromosomal abnormalities accurately in prenatal diagnosis, and can provide very useful genetic information for clinical diagnosis and treatment.

[Molecular and cytogenetic study on 18 cases of amenorrhea: the use of fluorescence in situ hybridization and high resolution-comparative genomic hybridization].

OBJECTIVE: To explore the use of fluorescence in situ hybridization (FISH) and high resolution-comparative genomic hybridization (HR-CGH) techniques in amenorrhea study. METHODS: After routine gynecologic examination, ultrasonography and endocrine examination, 17 cases of primary amenorrhea and 1 case of secondary amenorrhea were analysed by using chromosomal diagnoses including multiplex FISH and HR-CGH analyses. RESULTS: Among 17 cases of primary amenorrhea, 7 revealed a 46,XX karyotype; 10 cases (58.8%) had abnormal karyotype, including 3 cases of 46,XY females, 2 cases of Turner's syndrome with 45,X and 45,X/46,XX, and other 5 cases with abnormal structure of X chromosome (including partial monosomy of X,X isochromosome and X/Y mosaic). The karyotype of the patient with secondary amenorrhea was translocation between X chromosome and euchromosome. CONCLUSION: The using of FISH and HR-CGH can correctly diagnose the patients' karyotypes, and provide absolutely necessarily medical genetic data for clinical diagnosis and therapy.