The Indenoisoquinoline LMP517: A Novel Antitumor Agent Targeting both TOP1 and TOP2.

The camptothecin derivatives topoisomerase I (TOP1) inhibitors, irinotecan and topotecan, are FDA approved for the treatment of colorectal, ovarian, lung and breast cancers. Because of the chemical instability of camptothecins, short plasma half-life, drug efflux by the multidrug-resistance ABC transporters, and the severe diarrhea produced by irinotecan, indenoisoquinoline TOP1 inhibitors (LMP400, LMP776, and LMP744), which overcome these limitations, have been developed and are in clinical development. Further modifications of the indenoisoquinolines led to the fluoroindenoisoquinolines, one of which, LMP517, is the focus of this study. LMP517 showed better antitumor activity than its parent compound LMP744 against H82 (small cell lung cancer) xenografts. Genetic analyses in DT40 cells showed a dual TOP1 and TOP2 signature with selectivity of LMP517 for DNA repair-deficient tyrosyl DNA phosphodiesterase 2 (TDP2)- and Ku70-knockout cells. RADAR assays revealed that LMP517, and to a lesser extent LMP744, induce TOP2 cleavage complexes (TOP2cc) in addition to TOP1ccs. Histone γH2AX detection showed that, unlike classical TOP1 inhibitors, LMP517 targets cells independently of their position in the cell cycle. Our study establishes LMP517 as a dual TOP1 and TOP2 inhibitor with therapeutic potential.

The Indenoisoquinoline TOP1 Inhibitors Selectively Target Homologous Recombination-Deficient and Schlafen 11-Positive Cancer Cells and Synergize with Olaparib.

PURPOSE: Irinotecan and topotecan are used to treat a variety of different cancers. However, they have limitations, including chemical instability and severe side effects. To overcome these limitations, we developed the clinical indenoisoquinolines: LMP400 (indotecan), LMP776 (indimitecan), and LMP744. The purpose of the study is to build the molecular rationale for phase II clinical trials. EXPERIMENTAL DESIGN: CellMinerCDB (http://discover.nci.nih.gov/cellminercdb) was used to mine the cancer cell lines genomic databases. The causality of Schlafen11 (SLFN11) was validated in isogenic cell lines. Because topoisomerase I (TOP1)-mediated replication DNA damage is repaired by homologous recombination (HR), we tested the "synthetic lethality" of HR-deficient (HRD) cells. Survival and cell-cycle alterations were performed after drug treatments in isogenic DT40, DLD1, and OVCAR cell lines with BRCA1, BRCA2, or PALB2 deficiencies and in organoids cultured from prostate cancer patient-derived xenografts with BRCA2 loss. We also used an ovarian orthotopic allograft model with BRCA1 loss to validate the efficacy of LMP400 and olaparib combination. RESULTS: CellMinerCDB reveals that SLFN11, which kills cells undergoing replicative stress, is a dominant drug determinant to the clinical indenoisoquinolines. In addition, BRCA1-, BRCA2-, and PALB2-deficient cells were hypersensitive to the indenoisoquinolines. All 3 clinical indenoisoquinolines were also synergistic with olaparib, especially in the HRD cells. The synergy between LMP400 and olaparib was confirmed in the orthotopic allograft model harboring BRCA1 loss. CONCLUSIONS: Our results provide a rationale for molecularly designed clinical trials with the indenoisoquinolines as single agents and in combination with PARP inhibitors in HRD cancers expressing SLFN11.

Novel Fluoroindenoisoquinoline Non-Camptothecin Topoisomerase I Inhibitors.

Contrary to other anticancer targets, topoisomerase I (TOP1) is targeted by only one chemical class of FDA-approved drugs: topotecan and irinotecan, the derivatives of the plant alkaloid, camptothecin. The indenoisoquinolines LMP400, LMP744, and LMP776 are novel noncamptothecin TOP1 inhibitors in clinical trial, which overcome the limitations of camptothecins. To further improve metabolic stability, their methoxy groups have been replaced by fluorine, as in the fluoroindenoisoquinolines NSC 781517 (LMP517), NSC 779135 (LMP135), and NSC 779134 (LMP134). We tested the induction and stability of TOP1 cleavage complexes (TOP1cc), and the induction and persistence of DNA damage measured by histone H2AX phosphorylation (γH2AX) compared with their parent compounds LMP744 and LMP776 in leukemia CCRF-CEM and colon carcinoma HCT116 cells. The fluoroindenoisoquinolines induced TOP1cc and γH2AX at nanomolar concentrations, and at higher levels than the parent indenoisoquinolines. The fluoroindenoisoquinoline LMP135 showed greater antitumor activity than topotecan in small-cell lung cancer cell H82 xenografts. It was also more potent than topotecan in the NCI-60 cancer cell line panel. Bioinformatics tools (http://discover.nci.nih.gov/cellminercdb) were used to investigate the following: (i) the correlations of fluoroindenoisoquinolines activity with other drugs, and (ii) genomic determinants of response in the NCI-60. The activity of the fluoroindenoisoquinolines was mostly correlated with camptothecin derivatives and the parent indenoisoquinolines, consistent with TOP1 targeting. Genomic analyses and activity assays in CCRF-CEM SLFN11-deleted cells showed that SLFN11 expression is a dominant determinant of response to LMP135. This study shows the potential value of the fluoroindenoisoquinolines for further development as novel anticancer agents targeting TOP1. Mol Cancer Ther; 17(8); 1694-704. ©2018 AACR.

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy.

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.

Targeting the p38 MAPK pathway inhibits irinotecan resistance in colon adenocarcinoma.

Despite recent advances in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. To better elucidate the molecular mechanisms involved in resistance to irinotecan (and its active metabolite SN38), we established SN38-resistant clones derived from HCT-116 and SW48 cell lines. These clones show various levels (6- to 60-fold) of resistance to SN-38 and display enhanced levels of activated MAPK p38 as compared with the corresponding parental cells. Because four different isoforms of p38 have been described, we then studied the effect of p38 overexpression or downregulation of each isoform on cell sensivity to SN38 and found that both α and ß isoforms are involved in the development of resistance to SN38. In this line, we show that cell treatment with SB202190, which inhibits p38α and p38ß, enhanced the cytotoxic activity of SN38. Moreover, p38 inhibition sensitized tumor cells derived from both SN38-sensitive and -resistant HCT116 cells to irinotecan treatment in xenograft models. Finally, we detected less phosphorylated p38 in primary colon cancer of patients sensitive to irinotecan-based treatment, compared with nonresponder patients. This indicates that enhanced level of phosphorylated p38 could predict the absence of clinical response to irinotecan. Altogether, our results show that the p38 MAPK pathway is involved in irinotecan sensitivity and suggest that phosphorylated p38 expression level could be used as a marker of clinical resistance to irinotecan. They further suggest that targeting the p38 pathway may be a potential strategy to overcome resistance to irinotecan-based chemotherapies in colorectal cancer.

The PGD2 pathway, independently of FGF9, amplifies SOX9 activity in Sertoli cells during male sexual differentiation.

Activation by the Y-encoded testis determining factor SRY and maintenance of expression of the Sox9 gene encoding the central transcription factor of Sertoli cell differentiation are key events in the mammalian sexual differentiation program. In the mouse XY gonad, SOX9 upregulates Fgf9, which initiates a Sox9/Fgf9 feedforward loop, and Sox9 expression is stimulated by the prostaglandin D2 (PGD2) producing lipocalin prostaglandin D synthase (L-PGDS, or PTDGS) enzyme, which accelerates commitment to the male pathway. In an attempt to decipher the genetic relationships between Sox9 and the L-Pgds/PGD2 pathway during mouse testicular organogenesis, we found that ablation of Sox9 at the onset or during the time window of expression in embryonic Sertoli cells abolished L-Pgds transcription. By contrast, L-Pgds(-/-) XY embryonic gonads displayed a reduced level of Sox9 transcript and aberrant SOX9 protein subcellular localization. In this study, we demonstrated genetically that the L-Pgds/PGD2 pathway acts as a second amplification loop of Sox9 expression. Moreover, examination of Fgf9(-/-) and L-Pgds(-/-) XY embryonic gonads demonstrated that the two Sox9 gene activity amplifying pathways work independently. These data suggest that, once activated and maintained by SOX9, production of testicular L-PGDS leads to the accumulation of PGD2, which in turn activates Sox9 transcription and nuclear translocation of SOX9. This mechanism participates together with FGF9 as an amplification system of Sox9 gene expression and activity during mammalian testicular organogenesis.