DNA-damaging Drugs Research Articles

Design, synthesis and evaluation of novel thienopyridazine derivatives as Chk1/2 inhibitors

In order to search for novel checkpoint kinase 1/2 (Chk1) inhibitors, we have designed and synthesized a series of new compounds incorporating thienopyridazine core. Bioevaluation showed that compounds 10j, 10i, 13e and 10o exhibited relatively good inhibitory activity. Notably, compound 10o displayed high selectivity against a panel of kinases and inhibited Chk1/2 signaling pathway stimulated by DNA damage drugs in cellular level. Molecular docking of 10o to the ATP-binding site of Chk1 kinase domain indicated the existence of polar interactions between 10o and the ATP-ribose-binding residues of Chk1. In mouse HT-29 xenografts, a synergistic effect was observed. Co-treatment by CPT-11 and 10o significantly diminished the tumor volume, indicating the great potential of 10o as a candidate of Chk1/2 inhibitor.

Miscoding and DNA Polymerase Stalling by Methoxyamine-Adducted Abasic Sites.

Apurinic/apyrimidinic (AP) sites appear in DNA spontaneously and as intermediates of base excision DNA repair. AP sites are noninstructive lesions: they strongly block DNA polymerases, and if bypassed, the nature of the incorporated dNMP is mostly guided by the interactions within the polymerase-DNA active site. Many DNA polymerases follow the "A-rule", preferentially incorporating dAMP opposite to natural AP sites. Methoxyamine (MX), a small molecule, efficiently reacts with the aldehyde moiety of natural AP sites, thereby preventing their cleavage by APEX1, the major human AP endonuclease. MX is currently regarded as a possible sensitizer of cancer cells toward DNA-damaging drugs. To evaluate the mutagenic potential of MX, we have studied the utilization of various dNTPs by five DNA polymerases of different families encountering MX-AP adducts in the template in comparison with the natural aldehydic AP site. The Klenow fragment of Escherichia coli DNA polymerase I strictly followed the A-rule with both natural AP and MX-adducted AP sites. Phage RB69 DNA polymerase, a close relative of human DNA polymerases δ and ε, efficiently incorporated both dAMP and dGMP. DNA polymerase β mostly incorporated dAMP and dCMP, preferring dCMP opposite to the natural AP site and dAMP opposite to the MX-AP site, while DNA polymerase λ was selective for dGMP, apparently via the primer misalignment mechanism. Finally, translesion DNA polymerase κ also followed the A-rule for MX-AP and additionally incorporated dCMP opposite to a natural AP site. Overall, the MX-AP site, despite structural differences, was similar to the natural AP site in terms of the dNMP misincorporation preference but was bypassed less efficiently by all polymerases except for Pol κ.

Caffeic acid phenethyl ester targets ubiquitin-specific protease 8 and synergizes with cisplatin in endometrioid ovarian carcinoma cells

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.

Elucidating the role of the kinase activity of endothelial cell focal adhesion kinase in angiocrine signalling and tumour growth.

A common limitation of cancer treatments is chemotherapy resistance. We have previously identified that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) sensitises tumour cells to DNA-damaging therapies, reducing tumour growth in mice. The present study addressed the kinase activity dependency of EC FAK sensitisation to the DNA-damaging chemotherapeutic drug, doxorubicin. FAK is recognised as a therapeutic target in tumour cells, leading to the development of a range of inhibitors, the majority being ATP competitive kinase inhibitors. We demonstrate that inactivation of EC FAK kinase domain (kinase dead; EC FAK-KD) in established subcutaneous B16F0 tumours improves melanoma cell sensitisation to doxorubicin. Doxorubicin treatment in EC FAK-KD mice reduced the percentage change in exponential B16F0 tumour growth further than in wild-type mice. There was no difference in tumour blood vessel numbers, vessel perfusion or doxorubicin delivery between genotypes, suggesting a possible angiocrine effect on the regulation of tumour growth. Doxorubicin reduced perivascular malignant cell proliferation, while enhancing perivascular tumour cell apoptosis and DNA damage in tumours grown in EC FAK-KD mice 48 h after doxorubicin injection. Human pulmonary microvascular ECs treated with the pharmacological FAK kinase inhibitors defactinib, PF-562,271 or PF-573,228 in combination with doxorubicin also reduced cytokine expression levels. Together, these data suggest that targeting EC FAK kinase activity may alter angiocrine signals that correlate with improved acute tumour cell chemosensitisation. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Halofuginone Sensitizes Lung Cancer Organoids to Cisplatin via Suppressing PI3K/AKT and MAPK Signaling Pathways.

Lung cancer is the leading cause of cancer death worldwide. Cisplatin is the major DNA-damaging anticancer drug that cross-links the DNA in cancer cells, but many patients inevitably develop resistance with treatment. Identification of a cisplatin sensitizer might postpone or even reverse the development of cisplatin resistance. Halofuginone (HF), a natural small molecule isolated from Dichroa febrifuga, has been found to play an antitumor role. In this study, we found that HF inhibited the proliferation, induced G0/G1 phase arrest, and promoted apoptosis in lung cancer cells in a dose-dependent manner. To explore the underlying mechanism of this antitumor effect of halofuginone, we performed RNA sequencing to profile transcriptomes of NSCLC cells treated with or without halofuginone. Gene expression profiling and KEGG analysis indicated that PI3K/AKT and MAPK signaling pathways were top-ranked pathways affected by halofuginone. Moreover, combination of cisplatin and HF revealed that HF could sensitize the cisplatin-resistant patient-derived lung cancer organoids and lung cancer cells to cisplatin treatment. Taken together, this study identified HF as a cisplatin sensitizer and a dual pathway inhibitor, which might provide a new strategy to improve prognosis of patients with cisplatin-resistant lung cancer.

CBIO-22. PARP INHIBITION SYNERGIZES WITH DNA DAMAGING DRUGS IN PEDIATRIC CNS TUMORS

Abstract BACKGROUND Central nervous system (CNS) tumors are the second most common childhood cancer. Despite innovations in surgery and chemo-/radiotherapy, CNS tumors remain the major cause of cancer-related death in children. Previous sequencing analyses in a pediatric cancer cohort identified BRCA and DSB repair signatures as potentially targetable events. Based on these findings, we propose the use of PARP inhibitors (PARPi) for aggressive CNS tumor subtypes, including high-grade glioma (HGG), medulloblastoma (MB) and ependymoma (EPN). METHODS We tested multiple PARPi in tumor cell lines (n=8) as well as primary patient-derived models (n=11) of pediatric HGG, MB, EPN and atypical teratoid/rhabdoid tumors (ATRTs). Based on PARPi sensitivity, selected models were further exposed to a combination of PARPi and DNA-damaging/modifying agents. The mode of action was investigated using Western blot and flow cytometry. RESULTS We show that a fraction of pediatric MB, EPN and ATRT demonstrate sensitivity towards PARP inhibition, which is paralleled by susceptibility to the DNA damaging drugs cisplatin and irinotecan. Interestingly, talazoparib, the most potent PARPi, showed synergistic cytotoxicity with DNA-damaging/modifying drugs. In addition, cell cycle blockade and increased DNA damage combined with reduced DNA repair signaling, such as activation of the ATR/Chk1 pathway were observed. Corroboratively, talazoparib exhibited a synergistic anti-cancer effect in combination with inhibitors of ATR, a major regulator of DNA damage response. CONCLUSION/OUTLOOK To sum up, we demonstrate that PARP inhibition synergizes with DNA damaging anti-cancer compounds or DNA repair inhibitors and, thus, represents a promising therapeutic strategy for a defined subgroup of pediatric high-risk CNS tumors patients. More in depth characterization of the underlying molecular events will most likely allow the identification of predictive biomarkers for most efficient implementation of this strategy into clinical application.

TCTP protein degradation by targeting mTORC1 and signaling through S6K, Akt, and Plk1 sensitizes lung cancer cells to DNA-damaging drugs

Translationally controlled tumor protein (TCTP) is expressed in many tissues, particularly in human tumors. It plays a role in malignant transformation, apoptosis prevention, and DNA damage repair. The signaling mechanisms underlying TCTP regulation in cancer are only partially understood. Here, we investigated the role of mTORC1 in regulating TCTP protein levels, thereby modulating chemosensitivity, in human lung cancer cells and an A549 lung cancer xenograft model. The inhibition of mTORC1, but not mTORC2, induced ubiquitin/proteasome-dependent TCTP degradation without a decrease in the mRNA level. PLK1 activity was required for TCTP ubiquitination and degradation and for its phosphorylation at Ser46 upon mTORC1 inhibition. Akt phosphorylation and activation was indispensable for rapamycin-induced TCTP degradation and PLK1 activation, and depended on S6K inhibition, but not mTORC2 activation. Furthermore, the minimal dose of rapamycin required to induce TCTP proteolysis enhanced the efficacy of DNA-damaging drugs, such as cisplatin and doxorubicin, through the induction of apoptotic cell death in vitro and in vivo. This synergistic cytotoxicity of these drugs was induced irrespective of the functional status of p53. These results demonstrate a new mechanism of TCTP regulation in which the mTORC1/S6K pathway inhibits a novel Akt/PLK1 signaling axis and thereby induces TCTP protein stabilization and confers resistance to DNA-damaging agents. The results of this study suggest a new therapeutic strategy for enhancing chemosensitivity in lung cancers regardless of the functional status of p53.

EMP3 negatively modulates breast cancer cell DNA replication, DNA damage repair, and stem-like properties

Enhanced DNA damage repair capacity attenuates cell killing of DNA-damaging chemotherapeutic agents. In silico analysis showed that epithelial membrane protein 3 (EMP3) is associated with favorable survival, and negatively regulates cell cycle S-phase. Consistently, loss and gain of function studies demonstrated that EMP3 inhibits breast cancer cell S-phage entry, DNA replication, DNA damage repair, and stem-like properties. Moreover, EMP3 blocks Akt-mTOR signaling activation and induces autophagy. EMP3 negatively modulates BRCA1 and RAD51 expression, indicating EMP3 suppresses homologous recombination repair of DNA double-strand breaks. Accordingly, EMP3 sensitizes breast cancer cells to the DNA-damaging drug Adriamycin. EMP3 downregulates YTHDC1, a RNA-binding protein involved in m6a modification, which at least in part mediates the effects of EMP3 on breast cancer cells. Taken together, these data indicate that EMP3 is a putative tumor suppressor in breast cancer, and EMP3 downregulation may be responsible for breast cancer chemoresistance.

The trans cell cycle effects of PARP inhibitors underlie their selectivity toward BRCA1/2-deficient cells.

PARP inhibitor (PARPi) is widely used to treat BRCA1/2-deficient tumors, but why PARPi is more effective than other DNA-damaging drugs is unclear. Here, we show that PARPi generates DNA double-strand breaks (DSBs) predominantly in a trans cell cycle manner. During the first S phase after PARPi exposure, PARPi induces single-stranded DNA (ssDNA) gaps behind DNA replication forks. By trapping PARP on DNA, PARPi prevents the completion of gap repair until the next S phase, leading to collisions of replication forks with ssDNA gaps and a surge of DSBs. In the second S phase, BRCA1/2-deficient cells are unable to suppress origin firing through ATR, resulting in continuous DNA synthesis and more DSBs. Furthermore, BRCA1/2-deficient cells cannot recruit RAD51 to repair collapsed forks. Thus, PARPi induces DSBs progressively through trans cell cycle ssDNA gaps, and BRCA1/2-deficient cells fail to slow down and repair DSBs over multiple cell cycles, explaining the unique efficacy of PARPi in BRCA1/2-deficient cells.

Research progress on the association between environmental pollutants and the resistance mechanism of PARP inhibitors in ovarian cancer.

The occurrence and progression of ovarian cancer are closely related to genetics and environmental pollutants. Poly(ADP-ribose) polymerase (PARP) inhibitors have been a major breakthrough in the history of ovarian cancer treatment. PARP is an enzyme responsible for post-translational modification of proteins and repair of single-stranded DNA damage. PARP inhibitors can selectively inhibit PARP function, resulting in a synthetic lethal effect on tumor cells defective in homologous recombination repair. However, with large-scale application, drug resistance also inevitably appears. For PARP inhibitors, the diversity and complexity of drug resistance mechanisms have always been difficult problems in clinical treatment. Herein, we mainly summarized the research progress of DNA damage repair and drug resistance mechanisms related to PARP inhibitors and the impact of environmental pollutants on DNA damage repair to aid the development prospects and highlight urgent problems to be solved.

Inhibition of Protein Synthesis Induced by CHK1 Inhibitors Discriminates Sensitive from Resistant Cancer Cells.

The DNA-damage-activated checkpoint protein CHK1 is required to prevent replication or mitosis in the presence of unrepaired DNA damage. Inhibitors of CHK1 (CHK1i) circumvent this checkpoint and enhance cell killing by DNA-damaging drugs. CHK1i also elicit single-agent cytotoxicity in a small subset of cell lines. Resolving the mechanisms underlying the single-agent activity may permit patient stratification and targeted therapy against sensitive tumors. Our recent comparison of three CHK1i demonstrated that they all inhibited protein synthesis only in sensitive cells. LY2606368, the most selective of these CHK1i, was used in the current study. Comparison across a panel of cell lines demonstrated that sensitive cells died upon incubation with LY2606368, whereas resistant cells underwent growth inhibition and/or cytostasis but failed to die. Sensitive cells exhibited inhibition of protein synthesis, elevated DNA damage, impaired DNA repair, and subsequently death. The consequence of CHK1 inhibition involved activation of cyclin A/CDK2 and MUS81, resulting in DNA damage. This damage led to activation of AMPK, dephosphorylation of 4E-BP1, and inhibition of protein synthesis. Inhibition of MUS81 prevented activation of AMPK, while inhibition of AMPK enhanced DNA repair and cell survival. The activation of AMPK may involve a combination of LKB1 and CaMKKβ. This study raises questions concerning the potential importance of the inhibition of protein synthesis in response to other drugs, alone or in combination with CHK1i. It also highlights the importance of clearly discriminating among growth inhibition, cytostasis, and cell death, as only the latter is likely to result in tumor regression.

Wild-type IDH2 protects nuclear DNA from oxidative damage and is a potential therapeutic target in colorectal cancer.

Although the role of isocitrate dehydrogenase (IDH) mutation in promoting cancer development has been well-characterized, the impact of wild-type IDH on cancer cells remains unclear. Here we show that the wild-type isocitrate dehydrogenase 2 (IDH2) is highly expressed in colorectal cancer (CRC) cells, and plays an unexpected role in protecting the cancer cells from oxidative damage. Genetic abrogation of IDH2 in CRC cells leads to reactive oxygen species (ROS)-mediated DNA damage and an accumulation of 8-oxoguanine with DNA strand breaks, which activates DNA damage response (DDR) with elevated γH2AX and phosphorylation of ataxia telangiectasia-mutated (ATM) protein, leading to a partial cell cycle arrest and eventually cell senescence. Mechanistically, the suppression of IDH2 results in a reduction of the tricarboxylic acid (TCA) cycle activity due to a decrease in the conversion of isocitrate to α-ketoglutarate (α-KG) with a concurrent decrease in NADPH production, leading to ROS accumulation and oxidative DNA damage. Importantly, abrogation of IDH2 inhibits CRC cell growth in vitro and in vivo, and renders CRC cells more vulnerable to DNA-damaging drugs. Screening of an FDA-approved drug library has identified oxaliplatin as a compound highly effective against CRC cells when IDH2 was suppressed. Our study has uncovered an important role of the wild-type IDH2 in protecting DNA from oxidative damage, and provides a novel biochemical basis for developing metabolic intervention strategy for cancer treatment.

A375 melanoma cells are sensitized to cisplatin-induced toxicity by a synthetic nitro-flavone derivative 2-(4-Nitrophenyl)-4H-chromen-4-one through inhibition of PARP1.

Cisplatin has been extensively used in therapeutics for its broad-spectrum anticancer activity and frequently used for thetreatment of solid tumors. However, it presents several side-effects and several cancers develop resistance. Combination therapy of cisplatin with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors has been effective in increasing its efficacy at lower doses. In this work, we have shown that the nitro-flavone derivative, 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO), can improve the sensitivity of cancer cells to cisplatin through inhibition of PARP1. The effect of 4NCO on cisplatin toxicity was studied through combination therapy in both exponential and density inhibited A375 melanoma cells. Combination index (CI) was determined from isobologram analysis. The mechanism of cell killing was assessed by lactate dehydrogenase (LDH) assay. Temporal nicotinamide adenine dinucleotide (NAD+) assay was done to show the inhibition of PARP1. We also performed in silico molecular modeling studies to know the binding mode of 4NCO to a modeled PARP1-DNA complex containing cisplatin-crosslinked adduct. The results from both in silico and in cellulo studies confirmed that PARP1 inhibition by 4NCO was most effective in sensitizing A375 melanoma cells to cisplatin. Isobologram analysis revealed that 4NCO reduced cell viability both in exponential and density inhibited A375 cells synergistically. The combination led to cell death through apoptosis. The synthetic nitro-flavone derivative 4NCO effectively inhibited the important nuclear DNA repair enzyme PARP1 and therefore, could complement the DNA-damaging anticancer drug cisplatin in A375 cells and thus, could act as a potential adjuvant to cisplatin in melanoma therapy.

Enhanced Antitumor Response to Immune Checkpoint Blockade Exerted by Cisplatin-Induced Mutagenesis in a Murine Melanoma Model.

Sequencing data from different types of cancers including melanomas demonstrate that tumors with high mutational loads are more likely to respond to immune checkpoint blockade (ICB) therapies. We have previously shown that low-dose intratumoral injection of the chemotherapeutic DNA damaging drug cisplatin activates intrinsic mutagenic DNA damage tolerance pathway, and when combined with ICB regimen leads to tumor regression in the mouse YUMM1.7 melanoma model. We now report that tumors generated with an in vitro cisplatin-mutagenized YUMM1.7 clone (YUMM1.7-CM) regress in response to ICB, while an identical ICB regimen alone fails to suppress growth of tumors generated with the parental YUMM1.7 cells. Regressing YUMM1.7-CM tumors show greater infiltration of CD8 T lymphocytes, higher granzyme B expression, and higher tumoral cell death. Similarly, ex-vivo, immune cells isolated from YUMM1.7-CM tumors-draining lymph nodes (TDLNs) co-incubated with cultured YUMM1.7-CM cells, eliminate the tumor cells more efficiently than immune cells isolated from TDLNs of YUMM1.7 tumor-bearing mice. Collectively, our findings show that in vitro induced cisplatin mutations potentiate the antitumor immune response and ICB efficacy, akin to tumor regression achieved in the parental YUMM1.7 model by ICB administered in conjunction with intratumoral cisplatin injection. Hence, our data uphold the role of tumoral mutation burden in improving immune surveillance and response to ICB, suggesting a path for expanding the range of patients benefiting from ICB therapy.

Abstract 366: Identification of a clinically significant pan-cancer biomarker for homologous recombination deficiency

Abstract [Background] Genomic alterations in BRCA1/2 and the ‘genomic scar' signatures are associated with homologous recombination DNA repair deficiency (HRD) and serve as therapeutic biomarkers for sensitivity to platinum and PARP inhibitors in breast and ovarian cancers. However, the clinical significance of these biomarkers in other homologous recombination repair (HRR) pathway genes or in other cancer types is not fully understood. [Method] From the TCGA and the AACR GENIE project databases, we obtained clinical and genomic data of patients with multiple solid cancers, evaluated both locus-specific LOH in HRR pathway gene mutations and two genomic scar signature scores (HRD score and mutational signature 3), identified potential HRD cases, and examined the efficacy of DNA-damaging drugs in these HRD cases. [Results] Germline and somatic gene mutations in the HRR pathway genes with locus-specific LOH were significantly enriched in cases having high genomic scar scores, whereas those without the LOH were not enriched. Somatic mutations without the LOH were remarkably enriched in hypermutated cases and considered to be passenger mutations. The mutation rate and frequency with LOH in the HRR pathway genes varied widely by cancer type. When stratifying patients by gender and presence of TP53 mutation, the correlation between the two genomic scar scores and the resulting optimal cutoff values for biallelic HRR pathway gene alterations differed greatly. The potential HRD cases that were selected by the two genomic scar cutoffs and biallelic HRR pathway gene alterations showed significantly high gene expression-based HRD scores. In Cox proportional hazard multivariate analyses with covariates of age, stage, gender, HRD, and TP53 mutation, HRD was a good prognostic factor in the group with DNA-damaging agents, but a poor one in the group without the agents. [Conclusion] Increased attention on locus-specific LOH in HRR pathway gene alterations and the differences in genomic scar signatures stratified by gender and TP53 mutation is newly warranted in order to properly assess HRD in a pan-cancer manner. The results in the present study proved the clinical significance of HRD across cancer types and may contribute to the development of personalized medicine based on HRD. Citation Format: Shiro Takamatsu, JB Brown, Koji Yamanoi, Ken Yamaguchi, Junzo Hamanishi, Takashi Kobayashi, Masaki Mandai, Noriomi Matsumura. Identification of a clinically significant pan-cancer biomarker for homologous recombination deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 366.

DNA Damaging Agents Cause a Sub-Cellular Re-Localisation of Nucleophosmin in an npm1 Mutated Acute Myeloid Leukemia (AML) Cell Line and in Primary AML Cells Carrying an npm1 Mutation

Nucleophosmin (NPM) is a ubiquitously expressed phosphoprotein involved in many cellular functions including ribosome biogenesis, p53 activation, cell proliferation and genetic stability and is considered essential for cellular activity and survival. NPM contains a number of motifs which mediate interactions with a range of binding partners as well as influencing its cellular localisation. Primarily, NPM is located to the nucleolus due to a C-terminal nucleolar localisation signal (NoLS), however it is able to translocate through the nucleoplasm into the cytoplasm due to the presence of a bipartite nuclear localisation signal (NLS) and two nuclear export signals (NES).The most commonly mutated gene in Acute Myeloid Leukemia (AML) is npm1, occurring in approximately 35% of all AML patients. All npm1 mutations characterised to date occur within the C-terminal domain and alter the normal nucleo-cytoplasmic shuttling of NPM resulting in an abnormal cytoplasmic localisation of the protein. The mutations are always heterozygous, however mutant NPM oligomerises with wild type NPM resulting in an accumulation of both forms of the protein within the cytoplasm, leading to abnormal cellular functioning.Utilising an AML cell line containing a heterozygous npm1 mutation (OCI-AML3), we have shown that nucleolar localisation of NPM can be restored following short term exposure (4 - 24 hours) of the cells to the DNA damaging agent etoposide. Western blot analysis of the levels of NPM within cytoplasmic and nuclear fractions isolated from OCI-AML3 cells showed a significant decrease in cytoplasmic NPM following treatment relative to untreated cells (p=0.0045; N=3). Further analysis of OCI-AML3 cells using confocal microscopy identified an increase in nucleolar NPM following treatment relative to untreated controls (Figure 1). This post treatment increase of nucleolar NPM was also observed in primary leukemic cells isolated from patients with npm1 mutated AML.Similar results were observed with the DNA damaging agent cytarabine, however the potent anti-leukemic multi-kinase inhibitor, SB1317 had no effect on the sub-cellular localisation of NPM suggesting that the response is specific to DNA damage.In order to identify whether the nucleolar re-localisation of NPM following DNA damage could be abrogated by inhibiting the cellular response to DNA damage, OCI-AML3 cells were co-treated with etoposide and the PARP-1 DNA repair inhibitor, olaparib for 24 hours. Confocal microscope analysis of co-treated cells revealed that the sub-cellular distribution of NPM was predominantly cytoplasmic, with very little NPM-nucleolus co-localisation, i.e. resembling that of untreated cells.In summary, our data show that DNA damage caused by chemotherapeutic drugs results in a sub-cellular re-localisation of NPM in npm1 mutated AML cells, with the post-treatment NPM localisation resembling that of npm1 wildtype cells. In a chemotherapeutic context utilising DNA-damaging agents however, this re-localisation of NPM may not be desirable considering the involvement of NPM in DNA repair pathways. Re-localisation of NPM can be prevented however, by inhibiting the activity of other molecules involved in DNA damage response pathways, specifically PARP-1. Therefore these results suggest that the treatment of npm1 mutated AML with DNA damaging drugs should be considered within this context. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Azithromycin enhances the cytotoxicity of DNA-damaging drugs via lysosomal membrane permeabilization in lung cancer cells.

Cancer cells use autophagy for growth, survival, and cytoprotection from chemotherapy. Therefore, autophagy inhibitors appear to be good candidates for cancer treatment. Our group previously reported that macrolide antibiotics, especially azithromycin (AZM), have potent autophagy inhibitory effects, and combination treatment with tyrosine kinase inhibitors or proteasome inhibitors enhances their anti–cancer activity. In this study, we evaluated the effect of combination therapy with DNA‐damaging drugs and AZM in non–small‐cell lung cancer (NSCLC) cells. We found that the cytotoxic activities of DNA‐damaging drugs, such as doxorubicin (DOX), etoposide, and carboplatin, were enhanced in the presence of AZM in NSCLC cell lines, whereas AZM alone exhibited almost no cytotoxicity. This enhanced cell death was dependent on wild‐type‐p53 status and autophagosome‐forming ability because TP53 knockout (KO) and ATG5‐KO cells attenuated AZM‐enhanced cytotoxicity. DOX treatment upregulated lysosomal biogenesis by activating TFEB and led to lysosomal membrane damage as assessed by galectin 3 puncta assay and cytoplasmic leakage of lysosomal enzymes. In contrast, AZM treatment blocked autophagy, which resulted in the accumulation of lysosomes/autolysosomes. Thus, the effects of DOX and AZM were integrated into the marked increase in damaged lysosomes/autolysosomes, leading to prominent lysosomal membrane permeabilization (LMP) for apoptosis induction. Our data suggest that concomitant treatment with DNA‐damaging drugs and AZM is a promising strategy for NSCLC treatment via pronounced LMP induction.

Detecting Attomolar DNA-Damaging Anticancer Drug Activity in Cell Lysates with Electrochemical DNA Devices.

Here, we utilize electrochemical DNA devices to quantify and understand the cancer-specific DNA-damaging activity of an emerging drug in cellular lysates at femtomolar and attomolar concentrations. Isobutyl-deoxynyboquinone (IB-DNQ), a potent and tumor-selective NAD(P)H quinone oxidoreductase 1 (NQO1) bioactivatable drug, was prepared and biochemically verified in cancer cells highly expressing NQO1 (NQO1+) and knockdowns with low NQO1 expression (NQO1-) by Western blot, NQO1 activity analysis, survival assays, oxygen consumption rate, extracellular acidification rate, and peroxide production. Lysates from these cells and the IB-DNQ drug were then introduced to a chip system bearing an array of DNA-modified electrodes, and their DNA-damaging activity was quantified by changes in DNA-mediated electrochemistry arising from base-excision repair. Device-level controls of NQO1 activity and kinetic analysis were used to verify and further understand the IB-DNQ activity. A 380 aM IB-DNQ limit of detection and a 1.3 fM midpoint of damage were observed in NQO1+ lysates, both metrics 2 orders of magnitude lower than NQO1- lysates, indicating the high IB-DNQ potency and selectivity for NQO1+ cancers. The device-level damage midpoint concentration in NQO1+ lysates was over 8 orders of magnitude lower than cell survival benchmarks, likely due to poor IB-DNQ cellular uptake, demonstrating that these devices can identify promising drugs requiring improved cell permeability. Ultimately, these results indicate the noteworthy potency and selectivity of IB-DNQ and the high sensitivity and precision of electrochemical DNA devices to analyze agents/drugs involved in DNA-damaging chemotherapies.

BKM120 sensitizes glioblastoma to the PARP inhibitor rucaparib by suppressing homologous recombination repair

PARP inhibitors have been approved for the therapy of cancers with homologous recombination (HR) deficiency based on the concept of “synthetic lethality”. However, glioblastoma (GBM) patients have gained little benefit from PARP inhibitors due to a lack of BRCA mutations. Herein, we demonstrated that concurrent treatment with the PARP inhibitor rucaparib and the PI3K inhibitor BKM120 showed synergetic anticancer effects on GBM U251 and U87MG cells. Mechanistically, BKM120 decreased expression of HR molecules, including RAD51 and BRCA1/2, and reduced HR repair efficiency in GBM cells, therefore increasing levels of apoptosis induced by rucaparib. Furthermore, we discovered that the two compounds complemented each other in DNA damage response and drug accumulation. Notably, in the zebrafish U87MG-RFP orthotopic xenograft model, nude mouse U87MG subcutaneous xenograft model and U87MG-Luc orthotopic xenograft model, combination showed obviously increased antitumor efficacy compared to each monotherapy. Immunohistochemical analysis of tumor tissues indicated that the combination obviously reduced expression of HR repair molecules and increased the DNA damage biomarker γ-H2AX, consistent with the in vitro results. Collectively, our findings provide new insight into combined blockade of PI3K and PARP, which might represent a promising therapeutic approach for GBM.

Molecular mechanisms underlying synergistic anticancer effects of spliceosome inhibitor combined with cisplatin

Recently, several splicing inhibitors (Spliceostatin A, Pladienolide B, H3B-8800) have been proposed as effective and promising chemotherapeutic agents. These drugs have been shown to be effective in treating various types of cancer, such as chronic lymphocytic leukemia, breast adenocarcinoma, prostate cancer, ovarian adenocarcinoma and colorectal adenocarcinoma (Bonnal, Vigevani, and Valcárcel 2012; Webb, Joyner, and Potter 2013; Sciarrillo et al. 2019; Jiménez-Vacas et al. 2019). These molecules effectively eliminate cancer cells both in vivo and in vitro, but multiple side effects have been identified in phase I clinical trials due to the compounds' high toxicity (Eskens et al. 2013). We believe that optimal scheme of combined chemotherapy can reduce the concentration of splicing inhibitors and reduce their toxicity. Based on bioinformatic analysis, we have demonstrated that DNA-damaging drugs lead to a decrease in the number of spliceosomal proteins in the cell (Anufrieva, 2018). According to our results, decreased abundance of spliceosomal proteins can contribute to cancer cell survival after therapy. A decrease in the presence of spliceosomal proteins in the cell triggers the cell cycle arrest and serves as signal about the DNA damage. We have shown that spliceosome inhibitor (Pladienolide B) can suppress such response to DNA damaging chemotherapy. Based on survival analysis and apoptosis assessment, we found that Pladienolide B significantly increases the sensitivity of cancer cells to Cisplatin.Using flow cytometry and immunofluorescence assay for DNA repair marker (phospho-ATM) we showed that sequential treatment with spliceosome inhibitor and DNA-damaging agent leads to therapeutic synergy in vitro due to reduced DNA repair efficiency. Further, we performed proteomic profiling of ovarian cancer cells SKOV3 after sequential treatment of Pladienolide B and Cisplatin. It was shown that the action of the two drugs resulted in a massive reduction in the abundance of proteins involved in DNA repair as well as proteins involved in TP53 activation. Apparently, the spliceosome inhibitor Pladienolide B leads to the activation of compensatory mechanisms in the cell resulting in increased expression of genes involved in mRNA splicing. Thus, we showed that sequential action of Pladienolide B and Cisplatin reduces the efficiency of DNA repair in the cell. This synergistic effect could allow to reduce the therapeutic doses of these drugs and avoid the side effects found in patients during therapy with splicing inhibitor drugs.