Cell stress response impairs de novo NAD+ biosynthesis in the kidney.

The biosynthetic routes leading to de novo nicotinamide adenine dinucleotide (NAD+) production are involved in acute kidney injury (AKI), with a critical role for quinolinate phosphoribosyl transferase (QPRT), a bottleneck enzyme of de novo NAD+ biosynthesis. The molecular mechanisms determining reduced QPRT in AKI, and the role of impaired NAD+ biosynthesis in the progression to chronic kidney disease (CKD), are unknown. We demonstrate that a high urinary quinolinate-to-tryptophan ratio, an indirect indicator of impaired QPRT activity and reduced de novo NAD+ biosynthesis in the kidney, is a clinically applicable early marker of AKI after cardiac surgery and is predictive of progression to CKD in kidney transplant recipients. We also provide evidence that the endoplasmic reticulum (ER) stress response may impair de novo NAD+ biosynthesis by repressing QPRT transcription. In conclusion, NAD+ biosynthesis impairment is an early event in AKI embedded with the ER stress response, and persistent reduction of QPRT expression is associated with AKI to CKD progression. This finding may lead to identification of noninvasive metabolic biomarkers of kidney injury with prognostic and therapeutic implications.

Chemically based transmissible ER stress protocols are unsuitable to study cell-to-cell UPR transmission.

Renal epithelial cells regulate the destructive activity of macrophages and participate in the progression of kidney diseases. Critically, the Unfolded Protein Response (UPR), which is activated in renal epithelial cells in the course of kidney injury, is required for the optimal differentiation and activation of macrophages. Given that macrophages are key regulators of renal inflammation and fibrosis, we suppose that the identification of mediators that are released by renal epithelial cells under Endoplasmic Reticulum (ER) stress and transmitted to macrophages is a critical issue to address. Signals leading to a paracrine transmission of ER stress (TERS) from a donor cell to a recipient cells could be of paramount importance to understand how ER-stressed cells shape the immune microenvironment. Critically, the vast majority of studies that have examined TERS used thaspigargin as an inducer of ER stress in donor cells in cellular models. By using multiple sources of ER stress, we evaluated if human renal epithelial cells undergoing ER stress can transmit the UPR to human monocyte-derived macrophages and if such TERS can modulate the inflammatory profiles of these cells. Our results indicate that carry-over of thapsigargin is a confounding factor in chemically based TERS protocols classically used to induce ER Stress in donor cells. Hence, such protocols are not suitable to study the TERS phenomenon and to identify its mediators. In addition, the absence of TERS transmission in more physiological models of ER stress indicates that cell-to-cell UPR transmission is not a universal feature in cultured cells.

The cellular prion protein is a stress protein secreted by renal tubular cells and a urinary marker of kidney injury.

Endoplasmic Reticulum (ER) stress underlies the pathogenesis of numerous kidney diseases. A better care of patients with kidney disease involves the identification and validation of ER stress biomarkers in the early stages of kidney disease. For the first time to our knowledge, we demonstrate that the prion protein PrPC is secreted in a conventional manner by ER-stressed renal epithelial cell under the control of the transcription factor x-box binding protein 1 (XBP1) and can serve as a sensitive urinary biomarker for detecting tubular ER stress. Urinary PrPC elevation occurs in patients with chronic kidney disease. In addition, in patients undergoing cardiac surgery, detectable urine levels of PrPC significantly increase after cardiopulmonary bypass, a condition associated with activation of the IRE1-XBP1 pathway in the kidney. In conclusion, our study has identified PrPC as a novel urinary ER stress biomarker with potential utility in early diagnosis of ongoing acute or chronic kidney injury.

The cellular prion protein controls the mesenchymal-like molecular subtype and predicts disease outcome in colorectal cancer.

BACKGROUND: Comprehensive transcriptomic analyses have shown that colorectal cancer (CRC) is heterogeneous and have led to the definition of molecular subtypes among which the stem-cell, mesenchymal-like group is associated with poor prognosis. The molecular pathways orchestrating the emergence of this subtype are incompletely understood. In line with the contribution of the cellular prion protein PrPC to stemness, we hypothesize that deregulation of this protein could lead to a stem-cell, mesenchymal-like phenotype in CRC. METHODS: We assessed the distribution of the PrPC-encoding PRNP mRNA in two large CRC cohorts according to molecular classification and its association with patient survival. We developed cell-based assays to explore the impact of gain and loss of PrPC function on markers of the mesenchymal subtype and to delineate the signalling pathways recruited by PrPC. We measured soluble PrPC in the plasmas of 325 patients with metastatic CRC and probed associations with disease outcome. FINDINGS: We found that PRNP gene expression is enriched in tumours of the mesenchymal subtype and is associated with poor survival. Our in vitro analyses revealed that PrPC controls the expression of genes that specify the mesenchymal subtype through the recruitment of the Hippo pathway effectors YAP and TAZ and the TGFß pathway. We showed that plasma levels of PrPC are elevated in metastatic CRC and are associated with poor disease control. INTERPRETATION: Our findings define PrPC as a candidate driver of the poor-prognosis mesenchymal subtype of CRC. They suggest that PrPC may serve as a potential biomarker for patient stratification in CRC. FUNDING: Grant support was provided by the following: Cancéropôle Ile de France (grant number 2016-1-EMERG-36-UP 5-1), Association pour la Recherche sur le Cancer (grant number PJA 20171206220), SATT Ile de France Innov (grant number 415) as well as INSERM.

A comprehensive characterization of the impact of mycophenolic acid on the metabolism of Jurkat T cells.

Metabolic reprogramming is critical for T cell fate and polarization and is regulated by metabolic checkpoints, including Myc, HIF-1α, AMPK and mTORC1. Our objective was to determine the impact of mycophenolic acid (MPA) in comparison with rapamycin (Rapa), an inhibitor of mTORC1, on the metabolism of Jurkat T cells. We identified a drug-specific transcriptome signature consisting of the key enzymes and transporters involved in glycolysis, glutaminolysis or nucleotide synthesis. MPA produced an early and transient drop in the intracellular ATP content related to the inhibition of de novo synthesis of purines, leading to the activation of the energy sensor AMPK. MPA decreases glycolytic flux, consistent with a reduction in glucose uptake, but also in the oxidation of glutamine. Additionally, both drugs reduce aerobic glycolysis. The expression of HIF-1α and Myc, promoting the activation of glycolysis and glutaminolysis, was inhibited by MPA and Rapa. In conclusion, we report that MPA profoundly impacts the cellular metabolism of Jurkat T cells by generating an energetic distress, decreasing the glycolytic and glutaminolytic fluxes and by targeting HIF-1α and Myc. These findings open interesting perspectives for novel combinatorial therapeutic strategies targeting metabolic checkpoints to block the proliferation of T cells.

6-mercaptopurine promotes energetic failure in proliferating T cells.

The anticancer drug 6-mercaptopurine (6-MP) inhibits de novo purine synthesis and acts as an antiproliferative agent by interfering with protein, DNA and RNA synthesis and promoting apoptosis. Metabolic reprogramming is crucial for tumor progression to foster cancer cells growth and proliferation, and is regulated by mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) as well as the oncogenes Myc and hypoxia inducible factor 1α (HIF-1α). We hypothesized that 6-MP impacts metabolic remodeling through its action on nucleotide synthesis. The aim of our study is to provide a comprehensive characterization of the metabolic changes induced by 6-MP in leukemic T cells. Our results indicate that exposition to 6-MP rapidly reduces intracellular ATP concentration, leading to the activation of AMPK. In turn, mTOR, an AMPK target, was inhibited, and the expression of HIF-1α and Myc was reduced upon 6-MP incubation. As a consequence of these inhibitions, glucose and glutamine fluxes were strongly decreased. Notably, no difference was observed on glucose uptake upon exposition to 6-MP. In conclusion, our findings provide new insights into how 6-MP profoundly impacts cellular energetic metabolism by reducing ATP production and decreasing glycolytic and glutaminolytic fluxes, and how 6-MP modifies human leukemic T cells metabolism with potential antiproliferative effects.

A Novel Extrinsic Pathway for the Unfolded Protein Response in the Kidney.

The ribonuclease angiogenin is a component of the mammalian stress response, and functions in both cell-autonomous and non-cell-autonomous ways to promote tissue adaptation to injury. We recently showed that angiogenin regulates tissue homeostasis during AKI associated with endoplasmic reticulum (ER) stress through the production of transfer RNA fragments that interfere with translation initiation and thereby alleviate ER stress. However, whether the paracrine signaling mediated by angiogenin secretion is a genuine component of the ER stress response to kidney injury is unknown. Here, we explored the molecular mechanisms by which angiogenin is secreted upon ER stress, and determined how it modulates the inflammatory microenvironment. In cultured renal epithelial cells, ER stress specifically induced angiogenin secretion under the selective control of inositol-requiring enzyme 1α, a key activator of the unfolded protein response. The transcription factors spliced X-box-binding protein 1 and p65, which are activated by inositol-requiring enzyme 1α upon ER stress, each bound the angiogenin promoter and controlled the amount of angiogenin secreted. Furthermore, p65 promoted angiogenin transcription in an ER stress-dependent manner. Similar to secretion of the ER stress-induced proinflammatory cytokine IL-6, secretion of angiogenin required the ER-Golgi pathway. Notably, incubation of human macrophages with angiogenin promoted macrophage reprogramming toward an activated and proinflammatory phenotype. In patients, angiogenin expression increased upon renal inflammation, and the urinary concentration of angiogenin correlated with the extent of immune-mediated kidney injury. Collectively, our data identify angiogenin as a mediator of the ER stress-dependent inflammatory response and as a potential noninvasive biomarker of AKI.

Intrinsic bevacizumab resistance is associated with prolonged activation of autocrine VEGF signaling and hypoxia tolerance in colorectal cancer cells and can be overcome by nintedanib, a small molecule angiokinase inhibitor.

Colorectal cancer (CRC) is a common tumor type with a high mortality rate, in part due to intrinsic drug resistance. Although bevacizumab, a VEGF-directed neutralizing antibody, is particularly active in this pathology, some patients never respond for reasons not well understood. We here wish to clarify the role of autocrine VEGF signaling in the response of CRC cells to angiogenesis inhibition. Our results show that CRC cells with intrinsic bevacizumab-resistance displayed pronounced upregulation of autocrine HIF-VEGF-VEGFR signaling in response to prolonged bevacizumab exposure whereas the same signaling pathway was downregulated in bevacizumab-sensitive xenografts. Importantly, both bevacizumab-sensitive and -resistant CRC xenografts were sensitive to nintedanib, a small molecule angiokinase inhibitor, which was associated with inhibition of mTORC1. In vitro studies revealed that bevacizumab-resistant cells displayed intrinsically higher HIF-VEGF signaling intensity and hypoxia tolerance compared to their bevacizumab-sensitive counterparts. Interestingly, although nintedanib showed comparable activity toward bevacizumab-sensitive cells under normoxia and hypoxia, the drug was three-fold more toxic to the resistant cells under hypoxia, suggesting that nintedanib attenuated the survival signaling that usually protects these cells from hypoxia-mediated cell death. In conclusion, our findings support a role for autocrine VEGF signaling in the survival of CRC cells to hypoxia and thus to angiogenesis inhibition. We further show that nintedanib, a small molecule angiokinase inhibitor, is active toward CRC models with intrinsic bevacizumab resistance supporting clinical trials of nintedanib in patients that do not respond to bevacizumab, alone or in combination with bevacizumab to increase angiogenesis inhibition.

Acquired irinotecan resistance is accompanied by stable modifications of cell cycle dynamics independent of MSI status.

Irinotecan is a major anticancer agent specifically targeting DNA topoisomerase I. Its cytotoxicity is mediated via a two-step process involving accumulation of reversible DNA­topoisomerase I complexes associated with transient DNA single-strand breaks which subsequently are converted into permanent DNA double-strand breaks by the replication fork during S phase. Irinotecan may be selectively active for treatment of colorectal cancers that show microsatellite instability (MSI) due to deficiencies in mismatch repair enzymes, compared to tumors that are microsatellite stable but show chromosome instability (CIN). Although the clinical activity of irinotecan is principally limited by acquired drug resistance, surprisingly little is known about the influence of prolonged irinotecan exposure on the cell cycle dynamics. We have developed two colon cancer cell lines resistant to SN-38, the active metabolite of irinotecan, one derived from HT-29 (CIN), the other from HCT-116 (MSI). We here show that besides classical resistance mechanisms, SN-38 resistance is accompanied by an increased generation doubling time, a decreased S phase fraction and an increased G2 fraction in vitro as in tumor xenografts for both CIN and MSI models. As a consequence, SN-38-resistant cells and tumors show cross-resistance to the S-phase selective agent 5-fluorouracil. The resistance is accompanied by increased basal levels of γ-H2AX and phospho-Chk2 without notable changes in the levels of phospho-Chk1. Taken together, our results show that prolonged irinotecan exposure is accompanied by stable modifications of cell cycle dynamics which could have profound impact on tumor sensitivity to a wide range of antitumor agents and may influence tumor progression in patients.

EGFR- and VEGF(R)-targeted small molecules show synergistic activity in colorectal cancer models refractory to combinations of monoclonal antibodies.

PURPOSE: Epidermal growth factor receptor (EGFR) and VEGF(R) signaling show extensive cross-talk, providing a rationale for joint targeting of the two pathways. However, combinations of monoclonal antibodies (mAb) targeting EGFR and VEGF showed disappointing activity in patients with colorectal cancer (CRC). We speculated that inhibition of surface receptors and ligands might only partly prevent oncogenic signaling whereas small-molecule tyrosine kinase inhibitors (TKI) would also influence intracellular signaling. EXPERIMENTAL DESIGN: Mice with CRC xenografts were treated with two TKIs, vargatef and afatinib, or with two mAbs, bevacizumab and cetuximab, and their influence on tumor growth, viability, in vivo DNA synthesis, and the presence of phosphorylated EGFR and VEGFR was determined. The activity of the TKIs was further characterized in CRC cells with different KRAS status. RESULTS: Vargatef and afatinib together showed strong tumor growth inhibition toward HT-29 xenografts compared with either drug alone, which was associated with a 5-fold increase in apoptotic tumor cell death. In comparison, bevacizumab and cetuximab together were exclusively cytostatic with no more activity than either drug alone. Exposure to the two TKIs was accompanied by a marked decrease of tumor-associated intracellular phospho-VEGFR1 and phospho-EGFR, whereas similar exposure to the two mAbs had no detectable effect. A synergistic activity of vargatef plus afatinib was observed in all eight CRC cell lines examined, independent of KRAS status. CONCLUSIONS: Our results indicate that attenuation of intracellular EGFR and/or VEGF signaling is required for cytotoxic activity. These findings provide a rationale for trials of the TKIs, even in patients with mutant KRAS.

Trabectedin and its C subunit modified analogue PM01183 attenuate nucleotide excision repair and show activity toward platinum-resistant cells.

PM01183 is a novel marine-derived covalent DNA binder in clinical development. PM01183 is structurally similar to trabectedin (yondelis, ecteinascidin-743) except for the C subunit, and this modification is accompanied by different pharmacokinetics in cancer patients. We here characterize the interaction of PM01183 with the nucleotide excision repair (NER) pathway in comparison with trabectedin. Our results show for the first time that although neither PM01183 nor trabectedin is repaired by NER, both compounds are able to interfere with the NER machinery thereby attenuating the repair of specific NER substrates. We further show that the NER activity is increased in 3 of 4 cellular models with acquired resistance to cisplatin or oxaliplatin, confirming the involvement of NER in the resistance to platinum derivatives. Importantly, both PM01183 and trabectedin show unchanged or even enhanced activity toward all 4 cisplatin- and oxaliplatin-resistant cell lines. We finally show that combinations of PM01183 and cisplatin were mostly synergistic toward both parental and cisplatin-resistant ovarian carcinoma cells as indicated by Chou and Talalay analysis. These data show that the C subunit of trabectedin can be subjected to at least some structural modifications without loss of activity or NER interaction. While PM01183 and trabectedin appear functionally similar in cellular models, it is likely that the differences in pharmacokinetics may allow different dosing and scheduling of PM01183 in the clinic that could lead to novel and/or increased antitumor activity. Taken together, our results provide a mechanistic basis to support clinical trials of PM01183 alone or in combination with cisplatin.

The NER proteins XPC and CSB, but not ERCC1, regulate the sensitivity to the novel DNA binder S23906: implications for recognition and repair of antitumor alkylators.

S23906 belongs to a novel class of alkylating anticancer agents forming bulky monofunctional DNA adducts. A unique feature of S23906 is its "helicase-like" activity leading to the destabilization of the surrounding duplex DNA. We here characterize the recognition and repair of S23906 adducts by the nucleotide excision repair (NER) machinery. All NER-deficient human cell lines tested showed increased sensitivity to S23906, which was particularly pronounced for cells deficient in XPC, CSB and XPA. In comparison, deficiencies in ERCC1 or XPF had lesser impact on the sensitivity to S23906. The sensitivity was, at least in part, linked to the conversion of unrepaired adducts into toxic DNA strand breaks as shown by single cell electrophoresis and gamma-H2AX formation. The pharmacological relevance of these findings was confirmed by the characterization of KB carcinoma cells with acquired S23906 resistance. These cells showed increased NER activity in vivo as well as toward damaged plasmid DNA in vitro. In particular, both global genome NER, as shown by unscheduled DNA synthesis, and transcription-coupled NER, as shown by transcriptional recovery, were up-regulated in the S23906-resistant cells. The increased NER activity was accompanied by up to 5-fold up-regulation of XPC, CSB and XPA proteins without detectable alterations of ERCC1 on the DNA, RNA or protein levels. Our results suggest that S23906 adducts are recognized and repaired by both NER sub-pathways in contrast to other members of this class, that are only recognized by transcription-coupled NER. We further show that NER activity can be up-regulated without changes in ERCC1 expression.

Influence of irofulven, a transcription-coupled repair-specific antitumor agent, on RNA polymerase activity, stability and dynamics in living mammalian cells.

Transcription-coupled repair (TCR) plays a key role in the repair of DNA lesions induced by bulky adducts and is initiated when the elongating RNA polymerase II (Pol II) stalls at DNA lesions. This is accompanied by alterations in Pol II activity and stability. We have previously shown that the monofunctional adducts formed by irofulven (6-hydroxymethylacylfulvene) are exclusively recognized by TCR, without involvement of global genome repair (GGR), making irofulven a unique tool to characterize TCR-associated processes in vivo. Here, we characterize the influence of irofulven on Pol II activity, stability and mobility in living mammalian cells. Our results demonstrate that irofulven induces specific inhibition of nucleoplasmic RNA synthesis, an important decrease of Pol II mobility, coupled to the accumulation of initiating polymerase and a time-dependent loss of the engaged enzyme, associated with its polyubiquitylation. Both proteasome-mediated degradation of the stalled polymerase and new protein synthesis are necessary to allow Pol II recycling into preinitiating complexes. Together, our findings provide novel insights into the subsequent fate of the stalled RNA polymerase II and demonstrate the essential role of the recycling process for transcriptional reinitiation and viability of mammalian cells.

Replication and homologous recombination repair regulate DNA double-strand break formation by the antitumor alkylator ecteinascidin 743.

Adducts induced by the antitumor alkylator ecteinascidin 743 (ET-743, Yondelis, trabectedin) represent a unique challenge to the DNA repair machinery because no pathway examined to date is able to remove the ET adducts, whereas cells deficient in nucleotide excision repair show increased resistance. We here describe the processing of the initial ET adducts into cytotoxic lesions and characterize the influence of cellular repair pathways on this process. Our findings show that exposure of proliferating mammalian cells to pharmacologically relevant concentrations of ET-743 is accompanied by rapid formation of DNA double-strand breaks (DSBs), as shown by the neutral comet assay and induction of focalized phosphorylated H2AX. The ET adducts are stable and can be converted into DSBs hours after the drug has been removed. Loss of homologous recombination repair has no influence on the initial levels of DSBs but is associated with the persistence of unrepaired DSBs after ET-743 is removed, resulting in extensive chromosomal abnormalities and pronounced sensitivity to the drug. In comparison, loss of nonhomologous end-joining had only modest effect on the sensitivity. The identification of DSB formation as a key step in the processing of ET-743 lesions represents a novel mechanism of action for the drug that is in agreement with its unusual potency. Because loss of repair proteins is common in human tumors, expression levels of selected repair factors may be useful in identifying patients particularly likely to benefit, or not, from treatment with ET-743.

Generation of replication-dependent double-strand breaks by the novel N2-G-alkylator S23906-1.

S23906-1, a new DNA alkylating agent that reacts with the exocyclic 2-NH2 group of guanine residues yielding monofunctional adducts, is currently under clinical evaluation in phase I trials. To investigate the mechanism of action of S23906-1, we compared parental KB-3-1 cells and KB/S23-500 cells that are 15-fold resistant to S23906-1. Cell death induced by 1 micromol/L S23906-1 in KB-3-1 cells was associated with their irreversible arrest in the G2-M phases of the cell cycle followed by apoptosis, whereas a proportion of the resistant KB/S23-500 cells were able to exit from the G2 arrest and divide, leading to a significantly lower rate of apoptosis. The attenuated apoptotic response was associated with decreased Chk2 protein phosphorylation, indicating that the DNA damage signaling pathways are more potently activated in the sensitive cells. However, similar rates of adduct formation and repair were measured in both cell lines. Exposure to S23906-1 induced a higher formation of DNA breaks, measured by the comet assay, in sensitive cells. In agreement, a histone H2AX phosphorylation assay revealed that S23906-1 induced double-strand breaks (DSB) in a dose- and time-dependent manner and that these were more persistent in the parental cells. These DSBs were found mainly in S-phase cells and inhibited by aphidicolin, suggesting that they are DNA replication-mediated DSBs. These results suggest that secondary DNA lesions play an important role in the cytotoxicity of this compound and make histone H2AX phosphorylation an attractive marker for monitoring the efficacy of S23906-1.

Characterizations of irofulven cytotoxicity in combination with cisplatin and oxaliplatin in human colon, breast, and ovarian cancer cells.

PURPOSE: This study assessed the cytotoxic effects of irofulven in combination with oxaliplatin and cisplatin in a panel of human cancer cell lines. METHODS: Growth inhibition studies were performed using the human HT29 colon cancer cell line, irofulven-resistant derivative HT29/IF2, breast cancer cell line MCF7, and ovarian cancer line CAOV3. Irofulven-oxaliplatin combinations were compared with irofulven-cisplatin combinations in the same cell lines using similar experimental settings. Cells were exposed for 1 h to irofulven and then for 24 h to oxaliplatin or cisplatin and vice versa. RESULTS: Single agent irofulven displayed cytotoxic effects against human colon HT29 cells, human breast cancer cell lines including MCF7, SKBR3, and ZR-75-1, and human ovarian cancer cell lines CAOV3, OVCAR3, and IGROV1, with OVCAR3 being the most sensitive cancer cell line (IC50: 2.4 microM). In all tested cell lines the oxaliplatin-irofulven combination led to clear evidence of synergistic activity. In HT29 and HT29/IF2, the sequence oxaliplatin followed by irofulven appears to be the most effective whereas in MCF7 cells, irofulven given prior to or simultaneously with oxaliplatin is more effective than the other schedule. The combination displays additive activity toward CAOV3 ovarian cells when irofulven was administered prior to or simultaneously with oxaliplatin and partially synergistic when oxaliplatin was followed by irofulven. In most of the cell lines, the sequence oxaliplatin followed by irofulven appears to be the most effective as compared to other schedules. A combination of irofulven with cisplatin has the same efficacy as with oxaliplatin for the same cell lines. Cell cycle studies show that irofulven increases the proportion of cells in the S phase. Cisplatin-irofulven and oxaliplatin-irofulven combinations block cells in G1/S and potently induce apoptosis. CONCLUSION: Irofulven displays synergistic antiproliferative and pro-apoptotic effects when combined with oxaliplatin over a broad range of concentrations in human colon and breast cancer cells. Acquired resistance to irofulven has limited impact on the effects of cisplatin-irofulven and oxaliplatin-irofulven combinations. Based on these data, irofulven-oxaliplatin and cisplatin-irofulven combinations will be further explored in clinical trials, favoring the use schedules of oxaliplatin given prior to irofulven in patients with cancer.

Irofulven cytotoxicity depends on transcription-coupled nucleotide excision repair and is correlated with XPG expression in solid tumor cells.

BACKGROUND: Irofulven is a novel alkylating agent with promising clinical activity, particularly toward ovarian and hormone-refractory prostate cancers. To facilitate additional clinical development, we have aimed to identify biological markers associated with sensitivity to the compound. METHODS: Fibroblasts derived from patients with xeroderma pigmentosum or Cockayne's syndrome along with a panel of 20 human cancer cell lines (eight different tumor types) were examined to establish the importance of nucleotide excision repair proteins in the sensitivity to irofulven. RESULTS: Human cells deficient in nucleotide excision repair are up to 30-fold more sensitive to the cytotoxic effects of irofulven compared with repair-proficient controls, clearly indicating that nucleotide excision repair plays a crucial role in the sensitivity to the drug. Interestingly, our results show that irofulven-induced lesions are recognized by transcription-coupled repair but not by global genome repair. Another unique feature is the pronounced sensitivity of XPD and XPB helicase-deficient cells to the drug. Comparison of the IC50 values for irofulven, cisplatin, and ecteinascidin 743 with the expression levels of ERCC1, XPD, and XPG genes in different solid tumor cell lines shows no correlation between the expression levels of any of the three nucleotide excision repair proteins and the sensitivity to ecteinascidin 743. In contrast, expression of the XPG endonuclease was correlated with the cytotoxicity for irofulven and, to a lesser degree, for cisplatin. Importantly, XPG expression was also correlated with cellular nucleotide excision repair activity. CONCLUSIONS: Increasing evidence indicates that compromised nucleotide excision repair activity is frequent in several solid tumor types. The results presented here suggest that XPG expression in such tumors may be a useful marker to predict their sensitivity to irofulven.

The antitumor triazoloacridone C-1305 is a topoisomerase II poison with unusual properties.

C-1305 [S-[[3-(dimethylamino)propyl]amino]-8-hydroxy-6H-v-triazolo[4,5,1-de]acridin-6-one] is a triazoloacridone with excellent activity in colon cancer models. The mechanism of C-1305 is unknown, although similarities in the chemical structure between C-1305 and amsacrine suggest common cellular targets. Here, we report that C-1305 is a topoisomerase II poison that is able to induce cleavable complexes with topoisomerase II in vitro as well as in living cells. Even at optimal concentrations, C-1305 is a much weaker inducer of cleavable complexes than amsacrine. Because the cytotoxic activities of the two compounds after continuous drug exposure are comparable, these findings suggest that the low levels of cleavable complexes induced by C-1305 may be unusually toxic. In contrast to amsacrine, the cytotoxicity of C-1305 is strongly time-dependent, with at least 24 h of drug exposure required for optimal cytotoxicity. The p53 tumor suppressor is inactivated in the majority of human tumors, including colorectal cancers. We therefore compared the long-term cytotoxic effects of C-1305, amsacrine, and doxorubicin on human cell lines in which the p53 or p21 pathways have been specifically disrupted by targeted homologous recombination. Disruption of p53 and p21 had minor influence on the cytotoxicity of doxorubicin, whereas p53 but not p21 disruption was associated with increased resistance to amsacrine. In marked contrast, disruption of p53 and p21 was associated with increased sensitivity to C-1305. Taken together, our results show that exposure to C-1305 is accompanied by the formation of low levels of potent cleavable complexes that are selectively toxic toward tumor cells with defective p53 function.

Enhanced antitumor activity of irofulven in combination with 5-fluorouracil and cisplatin in human colon and ovarian carcinoma cells.

Irofulven (6-hydroxymethylacylfulvene, MGI-114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product obtained from the Omphalotus mushroom. Irofulven has demonstrated potent activity against a broad range of solid tumors in both cellular and xenograft models and has shown promising activity in clinical trials. To guide the clinical use of irofulven, the present study used the MTT viability assay to examine the cytotoxic effects obtained by combining irofulven with two other anticancer agents: cisplatin and 5-fluorouracil (5-FU). The study was carried out with HT-29 and HCT-116 colorectal and A2780 ovarian carcinoma cells as well as with their irofulven- (HT-29/IF2, HCT-116/IF27) or cisplatin-resistant (A2780/CP70) variants. The combinations showed strong sequence specificity. Simultaneous exposure to cisplatin and irofulven was at least additive for four cell lines including the cisplatin-resistant A2780/CP70 ovarian cells which exhibit a multifactorial resistance phenotype. Cisplatin followed by irofulven was additive for parental HCT-116 and A2780 cells whereas irofulven followed by cisplatin was antagonistic in all cellular models. Simultaneous exposure to 5-FU and irofulven was at least additive for all six cell lines. 5-FU followed by irofulven was additive for the parental HT-29 and A2780 cells and synergistic for the irofulven-resistant HCT-116 cell line. Irofulven followed by 5-FU was synergistic for the two ovarian cell lines and additive for the two parental colon cell lines. These studies demonstrate that simultaneous exposure to irofulven and cisplatin is at least additive for most cell lines whereas simultaneous exposure to irofulven and 5-FU is additive to synergistic for all the cell lines tested, including the irofulven- and cisplatin-resistant variants. The enhanced cytotoxicity of irofulven in combination with cisplatin and 5-FU support the clinical application of these regimens.

Marked activity of irofulven toward human carcinoma cells: comparison with cisplatin and ecteinascidin.

PURPOSE: To characterize the activities of irofulven, a novelanticancer agent derived from the mushroom natural productilludin S toward human cancer cells. EXPERIMENTAL DESIGN: We have determined the activity spectrum of irofulven toward a human tumor cell panel comprised of 10 different tumor types in comparison with cisplatin and ET-743. We have also evaluated the influence of major resistance mechanisms, such as expression of multidrug resistance-associated drug efflux pumps, cisplatin resistance, loss of p53 function, and absence of mismatch repair on the cytotoxic activity of irofulven. RESULTS: The activity spectrum of irofulven is clearly different from that of ET-743 and cisplatin. Irofulven shows excellent cytotoxicity toward the majority of human carcinoma cell lines tested, but lesser activity toward sarcoma and leukemia cell lines. The cytotoxic activity of irofulven was particularly pronounced toward head and neck, non-small cell lung, colon, and ovary carcinoma cells, as well as toward malignant glioma cell lines. In addition, irofulven displayed good activity toward poorly differentiated, androgen-independent prostate cancer cells and cell lines expressing high levels of the detoxifying enzymes glutathione S-transferase and gamma-glutamyl cysteine synthetase. The cytotoxicity of irofulven was not affected by loss of p53 or mismatch repair function, and the drug was not a substrate for multidrug transporters, such as the P-glycoprotein and multidrug resistance protein 1. CONCLUSIONS: Irofulven has an unusual activity spectrum with strong activity toward tumor cells of epithelial origin. Furthermore, irofulven is not or only marginally affected by resistance mechanisms limiting the efficacy of other alkylating agents.