Anticancer effect of a pyrrole-imidazole polyamide-triphenylphosphonium conjugate selectively targeting a common mitochondrial DNA cancer risk variant in cervical cancer cells.

Cervical cancer remains a major threat to women's health, especially in countries with limited medical resources, and new drugs are needed to improve patient survival and minimize adverse effects. Here, we examine the effects of a triphenylphosphonium (TPP)-conjugated pyrrole-imidazole polyamide (CCC-h1005) targeting the common homoplasmic mitochondrial DNA (mtDNA) cancer risk variant (ATP6 8860A>G) on the survival of cervical cancer cell lines, cisplatin-resistant HeLa cells and patient-derived cervical clear cell carcinoma cells as models of cervical cancer treatment. We found that CCC-h1005 induced death in these cells and suppressed the growth of xenografted HeLa tumors with no severe adverse effects. These results suggest that PIP-TPP designed to target mtDNA cancer risk variants can be used to treat many cervical cancers harboring high copies of the target variant, providing a foundation for clinical trials of this class of molecules for treating cervical cancer and other types of cancers.

Suppression of non-small-cell lung cancer A549 tumor growth by an mtDNA mutation-targeting pyrrole-imidazole polyamide-triphenylphosphonium and a senolytic drug.

Certain somatic mutations in mtDNA were associated with tumor progression and frequently found in a homoplasmic state. We recently reported that pyrrole-imidazole polyamide conjugated with the mitochondria-delivering moiety triphenylphosphonium (PIP-TPP) targeting an mtDNA mutation efficiently induced apoptosis in cancer cells with the mutation but not normal cells. Here, we synthesized the novel PIP-TPP, CCC-021-TPP, targeting ND6 14582A > G homoplasmic missense mutation that is suggested to enhance metastasis of non-small-cell lung cancer A549 cells. CCC-021-TPP did not induce apoptosis but caused cellular senescence in the cells, accompanied by a significant induction of antiapoptotic BCL-XL. Simultaneous treatment of A549 cells with CCC-021-TPP and the BCL-XL selective inhibitor A-1155463 resulted in apoptosis induction. Importantly, the combination induced apoptosis and suppressed tumor growth in an A549 xenografted model. These results highlight the potential of anticancer therapy with PIP-TPPs targeting mtDNA mutations to induce cell death even in apoptosis-resistant cancer cells when combined with senolytics.

FGFR2 loss sensitizes MYCN-amplified neuroblastoma CHP134 cells to CHK1 inhibitor-induced apoptosis.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. In this study, one CHK1i-sensitive neuroblastoma cell line, CHP134, was investigated, which characteristically carries MYCN amplification and a chromosome deletion within the 10q region. Among several cancer-related genes in the chromosome 10q region, mRNA expression of fibroblast growth factor receptor 2 (FGFR2) was altered in CHP134 cells and associated with an unfavorable prognosis of patients with neuroblastoma. Induced expression of FGFR2 in CHP134 cells reactivated downstream MEK/ERK signaling and resulted in cells resistant to CHK1i-mediated cell growth inhibition. Consistently, the MEK1/2 inhibitor, trametinib, potentiated CHK1 inhibitor-mediated cell death in these cells. These results suggested that FGFR2 loss might be prone to highly effective CHK1i treatment. In conclusion, extreme cellular dependency of ERK activation may imply a possible application for the MEK1/2 inhibitor, either as a single inhibitor or in combination with CHK1i in MYCN-amplified neuroblastomas.

Prediction of the differences in tumor mutation burden between primary and metastatic lesions by radiogenomics.

Tumor mutational burden (TMB) is gaining attention as a biomarker for responses to immune checkpoint inhibitors in cancer patients. In this study, we evaluated the status of TMB in primary and liver metastatic lesions in patients with colorectal cancer (CRC). In addition, the status of TMB in primary and liver metastatic lesions was inferred by radiogenomics on the basis of computed tomography (CT) images. The study population included 24 CRC patients with liver metastases. DNA was extracted from primary and liver metastatic lesions obtained from the patients and TMB values were evaluated by next-generation sequencing. The TMB value was considered high when it equaled to or exceeded 10/100 Mb. Radiogenomic analysis of TMB was performed by machine learning using CT images and the construction of prediction models. In 7 out of 24 patients (29.2%), the TMB status differed between the primary and liver metastatic lesions. Radiogenomic analysis was performed to predict whether TMB status was high or low. The maximum values for the area under the receiver operating characteristic curve were 0.732 and 0.812 for primary CRC and CRC with liver metastasis, respectively. The sensitivity, specificity, and accuracy of the constructed models for TMB status discordance were 0.857, 0.600, and 0.682, respectively. Our results suggested that accurate inference of the TMB status is possible using radiogenomics. Therefore, radiogenomics could facilitate the diagnosis, treatment, and prognosis of patients with CRC in the clinical setting.

Mitochondria: Endosymbiont bacteria DNA sequence as a target against cancer.

As the energy factory for the cell, the mitochondrion, through its role of adenosine triphosphate production by oxidative phosphorylation, can be regarded as the guardian of well regulated cellular metabolism; the integrity of mitochondrial functions, however, is particularly vulnerable in cancer due to the lack of superstructures such as histone and lamina folds to protect the mitochondrial genome from unintended exposure, which consequently elevates risks of mutation. In cancer, mechanisms responsible for enforcing quality control surveillance for identifying and eliminating defective mitochondria are often poorly regulated, and certain uneliminated mitochondrial DNA (mtDNA) mutations and polymorphisms can be advantageous for the proliferation, progression, and metastasis of tumor cells. Such pathogenic mtDNA aberrations are likely to increase and occasionally be homoplasmic in cancer cells and, intriguingly, in normal cells in the proximity of tumor microenvironments as well. Distinct characteristics of these abnormalities in mtDNA may provide a new path for cancer therapy. Here we discuss a promising novel therapeutic strategy, using the sequence-specific properties of pyrrole-imidazole polyamide-triphenylphosphonium conjugates, against cancer for clearing abnormal mtDNA by reactivating mitochondrial quality control surveillance.

A PI polyamide-TPP conjugate targeting a mtDNA mutation induces cell death of cancer cells with the mutation.

Mitochondrial DNA (mtDNA) mutations occur frequently in cancer cells, and some of them are often homoplasmic. Targeting such mtDNA mutations could be a new method for killing cancer cells with minimal impact on normal cells. Pyrrole-imidazole polyamides (PIPs) are cell-permeable minor groove binders that show sequence-specific binding to double-stranded DNA and inhibit the transcription of target genes. PIP conjugated with the lipophilic triphenylphosphonium (TPP) cation can be delivered to mitochondria without uptake into the nucleus. Here, we investigated the feasibility of the use of PIP-TPP to target a mtDNA mutation in order to kill cancer cells that harbor the mutation. We synthesized hairpin-type PIP-TPP targeting the A3243G mutation and examined its effects on the survival of HeLa cybrid cells with or without the mutation (HeLamtA3243G cells or HeLamtHeLa cells, respectively). A surface plasmon resonance assay demonstrated that PIP-TPP showed approximately 60-fold higher binding affinity for the mutant G-containing synthetic double-stranded DNA than for the wild-type A-containing DNA. When added to cells, it localized in mitochondria and induced mitochondrial reactive oxygen species production, extensive mitophagy, and apoptosis in HeLamtA3243G cells, while only slightly exerting these effects in HeLamtHeLa cells. These results suggest that PIP-TPPs targeting mtDNA mutations could be potential chemotherapeutic drugs to treat cancers without severe adverse effects.

Matters of data openness and KapWeb, a web tool of multi-cancer survival analysis for cancer survivors.

KapWeb is an interactive tool for the determination of cancer survival rates based on case outcomes compiled from more than half a million records from cancer registries all over Japan, and we believe that both the tool and the call for data openness and transparency are important.

Targeting the mutant PIK3CA gene by DNA-alkylating pyrrole-imidazole polyamide in cervical cancer.

PIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole-imidazole polyamide-seco-CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole-imidazole polyamides. P3AE5K interacted with double-stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K-positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole-imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.

A linear five-ring pyrrole-imidazole polyamide-triphenylphosphonium conjugate targeting a mitochondrial DNA mutation efficiently induces apoptosis of HeLa cybrid cells carrying the mutation.

Somatic mutations in mitochondrial DNA may provide a new avenue for cancer therapy due to their associations to a number of cancers and a tendency of homoplasmicity. In consideration of mitochondrial features and its relatively small genome size, a nucleotide-based targeting approach is a considerably more promising option. To explore the efficacy of short linear N-methylpyrrole-N-methylimidazole polyamide (PI polyamide), we synthesized a five-ring short PI polyamide that provided sequence-specific homing for the A3243G mitochondrial mutation upon conjugation with triphenylphosphonium cation (TPP). This PI polyamide-TPP was able to induce cytotoxicity in HeLamtA3243G cybrid cells, while preserving preferential binding for oligonucleotides containing the A3243G motif from melting temperature assays. The PI polyamide-TPP also localized in the mitochondria in HeLamtA3243G cells and induced mitochondrial reactive oxygen species production, mitophagy and apoptosis in a mutation-specific fashion compared to the wild-type HeLamtHeLa cybrids; normal human dermal fibroblasts were also relatively unaffected to suggest discriminating selectivity for the mutant mitochondria, offering a novel outlook for cancer therapy via mitochondrial homing of short linear PIP-TPPs.

Effects of compound-326, a selective delta-5 desaturase inhibitor, in ApoE knockout mice with two different protocols for atherosclerosis development.

PURPOSE: We previously confirmed its anti-atherosclerotic effects by pre-treatment with compound-326, a selective delta-5 desaturase (D5D) inhibitor, in Western diet-fed ApoE knockout mice. In the present study, we evaluated effects of compound-326 in ApoE knockout mice with two different protocols for atherosclerosis development. METHODS: In a post-treatment protocol, where the compound treatment started after 10 weeks pre-feeding of Western diet, compound-326 (1 and 3 mg/kg/day, p.o. for 12 weeks) significantly reduced the atherosclerotic lesion area in the aorta (24% reduction at 3 mg/kg/day). In another protocol using Paigen diet (containing 12.5% cholesterol and 5% sodium cholate), compound-326 (3 and 10 mg/kg/day, p.o. for 7 weeks) also significantly reduced the lesion area (36% reduction at 3 mg/kg/day). RESULTS: In both protocols, Compound-326 significantly reduced the hepatic ratio of arachidonic acid to dihomo-γ-linolenic acid, blood inflammatory eicosanoid production and plasma soluble intercellular adhesion molecule 1 (sICAM-1) levels, similarly to the previous pre-treatment study. CONCLUSIONS: Compound-326 exerted anti-atherosclerotic effects in ApoE knockout mice with the two different protocols for atherosclerosis development further supporting D5D inhibition as a promising strategy in treating atherosclerosis.

Radiogenomics for predicting p53 status, PD-L1 expression, and prognosis with machine learning in pancreatic cancer.

BACKGROUND: Radiogenomics is an emerging field that integrates "Radiomics" and "Genomics". In the current study, we aimed to predict the genetic information of pancreatic tumours in a simple, inexpensive, and non-invasive manner, using cancer imaging analysis and radiogenomics. We focused on p53 mutations, which are highly implicated in pancreatic ductal adenocarcinoma (PDAC), and PD-L1, a biomarker for immune checkpoint inhibitor-based therapies. METHODS: Overall, 107 patients diagnosed with PDAC were retrospectively examined. The relationship between p53 mutations as well as PD-L1 abnormal expression and clinicopathological factors was investigated using immunohistochemistry. Imaging features (IFs) were extracted from CT scans and were used to create prediction models of p53 and PD-L1 status. RESULTS: We found that p53 and PD-L1 are significant independent prognostic factors (P = 0.008, 0.013, respectively). The area under the curve for p53 and PD-L1 predictive models was 0.795 and 0.683, respectively. Radiogenomics-predicted p53 mutations were significantly associated with poor prognosis (P = 0.015), whereas the predicted abnormal expression of PD-L1 was not significant (P = 0.096). CONCLUSIONS: Radiogenomics could predict p53 mutations and in turn the prognosis of PDAC patients. Hence, prediction of genetic information using radiogenomic analysis may aid in the development of precision medicine.

Effects of Pyrrole-Imidazole Polyamides Targeting Human TGF-ß1 on the Malignant Phenotypes of Liver Cancer Cells.

Synthetic pyrrole-imidazole (PI) polyamides bind to the minor groove of double-helical DNA with high affinity and specificity, and inhibit the transcription of corresponding genes. In liver cancer, transforming growth factor (TGF)-ß expression is correlated with tumor grade, and high-grade liver cancer tissues express epithelial-mesenchymal transition markers. TGF-ß1 was reported to be involved in cancer development by transforming precancer cells to cancer stem cells (CSCs). This study aimed to evaluate the effects of TGF-ß1-targeting PI polyamide on the growth of liver cancer cells and CSCs and their TGF-ß1 expression. We analyzed TGF-ß1 expression level after the administration of GB1101, a PI polyamide that targets human TGF-ß1 promoter, and examined its effects on cell proliferation, invasiveness, and TGF-ß1 mRNA expression level. GB1101 treatment dose-dependently decreased TGF-ß1 mRNA levels in HepG2 and HLF cells, and inhibited HepG2 colony formation associated with downregulation of TGF-ß1 mRNA. Although GB1101 did not substantially inhibit the proliferation of HepG2 cells compared to untreated control cells, GB1101 significantly suppressed the invasion of HLF cells, which displayed high expression of CD44, a marker for CSCs. Furthermore, GB1101 significantly inhibited HLF cell sphere formation by inhibiting TGF-ß1 expression, in addition to suppressing the proliferation of HLE and HLF cells. Taken together, GB1101 reduced TGF-ß1 expression in liver cancer cells and suppressed cell invasion; therefore, GB1101 is a novel candidate drug for the treatment of liver cancer.

Co-Inhibition of the DNA Damage Response and CHK1 Enhances Apoptosis of Neuroblastoma Cells.

Checkpoint kinase 1 (CHK1) is a central mediator of the DNA damage response (DDR) at the S and G2/M cell cycle checkpoints, and plays a crucial role in preserving genomic integrity. CHK1 overexpression is thought to contribute to cancer aggressiveness, and several selective inhibitors of this kinase are in clinical development for various cancers, including neuroblastoma (NB). Here, we examined the sensitivity of MYCN-amplified NB cell lines to the CHK1 inhibitor PF-477736 and explored mechanisms to increase its efficacy. PF-477736 treatment of two sensitive NB cell lines, SMS-SAN and CHP134, increased the expression of two pro-apoptotic proteins, BAX and PUMA, providing a mechanism for the effect of the CHK1 inhibitor. In contrast, in NB-39-nu and SK-N-BE cell lines, PF-477736 induced DNA double-strand breaks and activated the ataxia telangiectasia mutated serine/threonine kinase (ATM)-p53-p21 axis of the DDR pathway, which rendered the cells relatively insensitive to the antiproliferative effects of the CHK1 inhibitor. Interestingly, combined treatment with PF-477736 and the ATM inhibitor Ku55933 overcame the insensitivity of NB-39-nu and SK-N-BE cells to CHK1 inhibition and induced mitotic cell death. Similarly, co-treatment with PF-477736 and NU7441, a pharmacological inhibitor of DNA-PK, which is also essential for the DDR pathway, rendered the cells sensitive to CHK1 inhibition. Taken together, our results suggest that synthetic lethality between inhibitors of CHK1 and the DDR drives G2/M checkpoint abrogation and could be a novel potential therapeutic strategy for NB.

Hydrophobic structure of hairpin ten-ring pyrrole-imidazole polyamides enhances tumor tissue accumulation/retention in vivo.

To examine the hydrophobic structure of PI polyamides on tumor accumulation in vivo, PI polyamide-fluorescein conjugates 1-5 with the distinct number of N-methylimidazole (Im) units were synthesized. There existed an inverse relationship between the Im unit number of the compounds and their hydrophobicity. Compound 1 with one Im unit and 3 with three Im units accumulated and retained preferentially in tumor tissues compared to 5 with five Im units. These results suggest the importance of a PI polyamide's primary structure, which partly contributes to its hydrophobic property, on its accumulation and/or retention in tumor tissues in vivo.

Identification of novel mutations in Japanese ovarian clear cell carcinoma patients using optimized targeted NGS for clinical diagnosis.

OBJECTIVE: Ovarian clear cell carcinoma (OCCC) is an aggressive ovarian cancer with a higher frequency in Japan and often becomes chemorefractory disease. Reliable genetic diagnosis is essential to affirm the success of precision medicine for OCCC treatment. The aim of this study is, therefore, to identify novel mutations in OCCCs and develop a feasible clinical next generation sequencing (NGS) approach using formalin-fixed paraffin-embedded (FFPE) rather than preferable but not always available fresh frozen (FF) samples. METHODS: We optimized and evaluated exome analyses of 409 cancer-related genes using FFPE and FF DNA and analyzed NGS data to identify somatic mutations in Japanese OCCCs. RESULTS: Sufficient and good quality DNAs from FFPE samples were extracted from 18 (FIGO Stage I: 12) out of 29 pairs of matched normal and OCCC for NGS (63%). The fine quality of extracted DNAs depended on the length of storage period (<2years storage). We also identified 45 somatic mutations in 34 genes including unreported variants from those FFPE DNA, in which somatic mutations in the PIK3CA gene was the most common (28%) as previously reported. Seven genes (PIK3CA, ARID1A, CTNNB1, CSMD3, LPHN3, LRP1B, and TP53) were mutated in at least two independent OCCCs. FF samples from 3 out of those 18 OCCCs were available and 13 out of 14 FFPE somatic mutations were confirmed. CONCLUSIONS: We successfully identified novel genetic alterations in Japanese OCCCs and demonstrated a feasible clinical diagnostic procedure using targeted NGS for OCCC FFPE samples.

Unfavorable neuroblastoma prognostic factor NLRR2 inhibits cell differentiation by transcriptional induction through JNK pathway.

The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)-mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA-induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c-Jun, a member of the activator protein-1 (AP-1) family in NB cells. Moreover, the expressions of NLRR2 and c-Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c-Jun reduced NLRR2 expression. We then searched AP-1 binding consensus in the NLRR2 promoter region and confirmed c-Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.

Identification of a novel E-box binding pyrrole-imidazole polyamide inhibiting MYC-driven cell proliferation.

The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and overexpression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E-box (5'-CACGTGT-3') sequence at gene promoters contributes to more than 4000 MYC-dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence-specific DNA-binding pyrrole-imidazole (PI) polyamide, Myc-5, that recognizes the E-box consensus sequence. Bioinformatics analysis revealed that the Myc-5 binding sequence appeared in 5'- MYC binding E-box sequences at the eIF4G1, CCND1, and CDK4 gene promoters. Furthermore, ChIP coupled with detection by quantitative PCR indicated that Myc-5 has the ability to inhibit MYC binding at the target gene promoters and thus cause downregulation at the mRNA level and protein expression of its target genes in human Burkitt's lymphoma model cell line, P493.6, carrying an inducible MYC repression system and the K562 (human chronic myelogenous leukemia) cell line. Single i.v. injection of Myc-5 at 7.5 mg/kg dose caused significant tumor growth inhibition in a MYC-dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of E-box-mediated MYC downstream gene expression and is a model for showing that phenotype-associated MYC downstream gene targets consequently inhibit MYC-dependent tumor growth.

The oncogenic role of GASC1 in chemically induced mouse skin cancer.

Gene amplified in squamous cell carcinoma (SCC) 1 (GASC1), also known as KDM4C/JMJD2C, encodes a histone demethylase that specifically demethylates lysine residues (H3K9, H3K36, and H1.4K26) and plays a crucial role in the regulation of gene expression as well as in heterochromatin formation. GASC1 is located at human chromosome 9p23-24, where frequent genomic amplification is observed in human esophageal cancer, and its aberrant expression is detected in a variety of human cancers, such as breast, colon, and prostate. Therefore, it is highly likely that GASC1 contributes to the genesis and/or development of cancer. However, there is a lack of direct evidence of GASC1 having an oncogenic function. In this study, we aimed to clarify the role of GASC1 in the skin SCC carcinogenesis. For this purpose, we generated Gasc1-heterozygous mice (Gasc1+/-) with reduced expression of Gasc1. On the basis of our results, Gasc1+/- mice displayed a significantly lower incidence and multiplicity of both benign and malignant tumors induced by the two-stage skin carcinogenesis protocol than wild-type mice. In addition, the volume of carcinoma was significantly lower in Gasc1+/- mice. Consistent with these observations, knocking down of Gasc1 resulted in reduced cell viability of SCC cells in vitro. Our findings clearly demonstrated that GASC1 has an oncogenic role in skin carcinogenesis.

Genome-wide screening of aberrant DNA methylation which associated with gene expression in mouse skin cancers.

Epigenetic alteration of genomic DNA is a common and key process in carcinogenesis. There is considerable evidence indicating that some of the somatic alterations occurring during carcinogenesis in humans also involve the same processes as those observed in mice. Therefore, we analyzed mouse skin cancer tissues induced by the 2-stage carcinogenesis model to identify skin tumor-specific differentially methylated regions (ST-DMRs) during the multistep carcinogenesis process. We have previously identified ST-DMRs using the restriction landmark genomic scanning (RLGS) technique and reported that some of the mouse ST-DMRs were also epigenetically modified in human cancers, such as melanoma, neuroblastoma, and brain tumor. These results encouraged us to pursue global methylation screening in mouse skin carcinogenesis. Using the methylated DNA immunoprecipitation (MeDIP) method combined with the NimbleGen promoter plus CpG island (CpGi) array, we identified 615 ST-DMRs. In combination with global gene expression analysis, 91 of these ST-DMRs were shown to be located on or around the genes differentially expressed between normal skin and tumor tissues, including a candidate human tumor suppressor gene Tfap2e. As observed in human colorectal cancers, Tfap2e was methylated at a CpGi located in intron 3 and downregulated in skin tumors. Our results identified aberrant methylated regions that were associated with gene expression regulation during carcinogenesis, which may indicate critical genetic regions also involved in human carcinogenesis. © 2013 Wiley Periodicals, Inc.

Selective Targeting of the KRAS Codon 12 Mutation Sequence by Pyrrole-Imidazole Polyamide seco-CBI Conjugates.

Mutation of KRAS is a key step in many cancers. Mutations occur most frequently at codon 12, but the targeting of KRAS is notoriously difficult. We recently demonstrated selective reduction in the volume of tumors harboring the KRAS codon 12 mutation in a mouse model by using an alkylating hairpin N-methylpyrrole-N-methylimidazole polyamide seco-1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one conjugate (conjugate 4) designed to target the KRAS codon 12 mutation sequence. Herein, we have compared the alkylating activity of 4 against three other conjugates that were also designed to target the KRAS codon 12 mutation sequence. Conjugate 4 displayed greater affinity for the G12D mutation sequence than for the G12V sequence. A computer-minimized model suggested that conjugate 4 could bind more efficiently to the G12D match sequence than to a one-base-pair mismatch sequence. Conjugate 4 was modified for next-generation sequencing. Bind-n-Seq analysis supported the evidence showing that conjugate 4 could target the G12D mutation sequence with exceptionally high affinity and the G12V mutation sequence with much higher affinity than that for the wild-type sequence.