Hiroyuki Yoda

Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole–imidazole polyamide conjugate

Despite extensive efforts to target mutated RAS proteins, anticancer agents capable of selectively killing tumour cells harbouring KRAS mutations have remained unavailable. Here we demonstrate the direct targeting of KRAS mutant DNA using a synthetic alkylating agent (pyrrole-imidazole polyamide indole-seco-CBI conjugate; KR12) that selectively recognizes oncogenic codon 12 KRAS mutations. KR12 alkylates adenine N3 at the target sequence, causing strand cleavage and growth suppression in human colon cancer cells with G12D or G12V mutations, thus inducing senescence and apoptosis. In xenograft models, KR12 infusions induce significant tumour growth suppression, with low host toxicity in KRAS-mutated but not wild-type tumours. This newly developed approach may be applicable to the targeting of other mutant driver oncogenes in human tumours.

Runt-related transcription factor 2 attenuates the transcriptional activity as well as DNA damage-mediated induction of pro-apoptotic TAp73 to regulate chemosensitivity

Although runt‐related transcription factor 2 (RUNX2) is known to be an essential key transcription factor for osteoblast differentiation and bone formation, RUNX2 also plays a pivotal role in the regulation of p53‐dependent DNA damage response. In the present study, we report that, in addition to p53, RUNX2 downregulates pro‐apoptotic TAp73 during DNA damage‐dependent cell death. Upon adriamycin (ADR) exposure, human osteosarcoma‐derived U2OS cells underwent cell death in association with an upregulation of TAp73 and various p53/TAp73‐target gene products together with RUNX2. Small interfering RNA‐mediated silencing of p73 resulted in a marked reduction in ADR‐induced p53/TAp73‐target gene expression, suggesting that TAp73 is responsible for the ADR‐dependent DNA damage response. Immunoprecipitation and transient transfection experiments demonstrated that RUNX2 forms a complex with TAp73 and impairs its transcriptional activity. Notably, knockdown of RUNX2 stimulated ADR‐induced cell death accompanied by a massive induction of TAp73 expression, indicating that RUNX2 downregulates TAp73 expression. Consistent with this notion, the overexpression of RUNX2 suppressed ADR‐dependent cell death, which was associated with a remarkable downregulation of TAp73 and p53/TAp73‐target gene expression. Collectively, our present findings strongly suggest that RUNX2 attenuates the transcriptional activity and ADR‐mediated induction of TAp73, and may provide novel insights into understanding the molecular basis behind the development and/or maintenance of chemoresistance. Thus, we propose that the silencing of RUNX2 might be an attractive strategy for improving the chemosensitivity of malignant cancers.

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53 -mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations.

Improvement of gemcitabine sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells by RUNX2 depletion-mediated augmentation of TAp73-dependent cell death.

Pancreatic cancer exhibits the worst prognostic outcome among human cancers. Recently, we have described that depletion of RUNX2 enhances gemcitabine (GEM) sensitivity of p53-deficient pancreatic cancer AsPC-1 cells through the activation of TAp63-mediated cell death pathway. These findings raised a question whether RUNX2 silencing could also improve GEM efficacy on pancreatic cancer cells bearing p53 mutation. In the present study, we have extended our study to p53-mutated pancreatic cancer MiaPaCa-2 cells. Based on our current results, MiaPaCa-2 cells were much more resistant to GEM as compared with p53-proficient pancreatic cancer SW1990 cells, and there existed a clear inverse relationship between the expression levels of TAp73 and RUNX2 in response to GEM. Forced expression of TAp73α in MiaPaCa-2 cells significantly promoted cell cycle arrest and/or cell death, indicating that a large amount of TAp73 might induce cell death even in the presence of mutant p53. Consistent with this notion, overexpression of TAp73α stimulated luciferase activity driven by p53/TAp73-target gene promoters in MiaPaCa-2 cells. Similar to AsPC-1 cells, small interfering RNA-mediated knockdown of RUNX2 remarkably enhanced GEM sensitivity of MiPaCa-2 cells. Under our experimental conditions, TAp73 further accumulated in RUNX2-depleted MiaPaCa-2 cells exposed to GEM relative to GEM-treated non-silencing control cells. As expected, silencing of p73 reduced GEM sensitivity of MiPaCa-2 cells. Moreover, GEM-mediated Tyr phosphorylation level of TAp73 was much more elevated in RUNX2-depleted MiaPaCa-2 cells. Collectively, our present findings strongly suggest that knockdown of RUNX2 contributes to a prominent enhancement of GEM sensitivity of p53-mutated pancreatic cancer cells through the activation of TAp73-mediated cell death pathway, and also provides a promising strategy for the treatment of patients with pancreatic cancer bearing p53 mutation.

Depletion of runt-related transcription factor 2 (RUNX2) enhances SAHA sensitivity of p53-mutated pancreatic cancer cells through the regulation of mutant p53 and TAp63

Suberoylanilide hydroxamic acid (SAHA) represents one of the new class of anti-cancer drugs. However, multiple lines of clinical evidence indicate that SAHA might be sometimes ineffective on certain solid tumors including pancreatic cancer. In this study, we have found for the first time that RUNX2/mutant p53/TAp63-regulatory axis has a pivotal role in the determination of SAHA sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells. According to our present results, MiaPaCa-2 cells responded poorly to SAHA. Forced depletion of mutant p53 stimulated SAHA-mediated cell death of MiaPaCa-2 cells, which was accomapanied by a further accumulation of γH2AX and cleaved PARP. Under these experimental conditions, pro-oncogenic RUNX2 was strongly down-regulated in mutant p53-depleted MiaPaCa-2 cells. Surprisingly, RUNX2 silencing augmented SAHA-dependent cell death of MiaPaCa-2 cells and caused a significant reduction of mutant p53. Consistent with these observations, overexpression of RUNX2 in MiaPaCa-2 cells restored SAHA-mediated decrease in cell viability and increased the amount of mutant p53. Thus, it is suggestive that there exists a positive auto-regulatory loop between RUNX2 and mutant p53, which might amplify their pro-oncogenic signals. Intriguingly, knockdown of mutant p53 or RUNX2 potentiated SAHA-induced up-regulation of TAp63. Indeed, SAHA-stimulated cell death of MiaPaCa-2 cells was partially attenuated by p63 depletion. Collectively, our present observations strongly suggest that RUNX2/mutant p53/TAp63-regulatory axis is one of the key determinants of SAHA sensitivity of p53-mutated pancreatic cancer cells.

Abstract 3775: Mutated cancer cell-specific cell death activity of alkylating Pyrrole-Imidazole polyamide conjugates targeting a variety of oncogenic driver gene mutations

Cancer may be recognized as non-self by antibiotics such as minor groove binders, which show self / non-self recognition partially due to preferential DNA sequence recognition and distinguish from other non-self bacteria. We learned minor groove binders produced from Streptomyces and synthesized Pyrrole-Imidazole polyamide indol-seco CBI conjugate to alkylate specific sites in the cancer genome. Although a tremendous amount of studies has been made to directly target oncogenic drivers, such as RAS and MYC, yet no drug is clinically available because of difficulties to develop RAS- or Myc-targeted anti-cancer therapeutics due to the smooth 3D surface topology. One of major limitations of targeting the RAS pathway may be intrinsic or acquired resistance as seen in the other molecular target therapy. New approaches that directly target driver genes may provide a more direct route to helps address unmet medical needs for refractory cancer conquest. We therefore synthesized several Pyrrole-Imidazole polyamide conjugates, each of which specifically alkylated KRAS codon 12 mutant DNA (G12D or G12V), amplified MYCN or mutated DNA of PI3K E542K mutation. All three conjugates reduced expression of the mutated oncogenic-protein by RNA transcription inhibition and induced cancer cell death at low dose (1 to 50 nM). Low dose tail vein injections of conjugates-targeting KRS or MYCN also demonstrated significant anti-tumor effects on xenograft models of human tumors harboring oncogenic mutated driver with minimum host toxicity, but not in xenografts harboring wild type or non-recognized mutations. We also performed a series of biological searches for toxicities by applying Modified SHIRPA (behavioral and functional analysis of mouse phenotype) to test any pathological phenotypes and examinations of blood and urine in ICR mice. Modified SHIRPA screening, blood chemistry, blood cell analysis and urea tests exhibited no toxicologically significant changes. Additionally, we examine pharmacokinetics of PI polyamide conjugates In vivo using LC-mass and fluorescent imaging of tumor-bearing mice. Intriguingly, 48 hours after the administration the highest fluorescence intensity was observed in the tumor-cell nuclear and almost no fluorescent intensity in all other organs, tissues and cells. These data suggest that sequence-dependent alkylating approach using antibiotic mimics of alkylating PI-polyamide conjugates, may open a new strategy not only targeting point mutation of driver oncogene but also targeting key driver gene in the cancer amplicon. This approach should be used for future ‘ Precision cancer medicine’. Citation Format: Hiroki Nagase, Kiriko Hiraoka, Takahiro Inoue, Hiroyuki Yoda, Krishnamurthy Sakthisri, Jason Lin, Takayoshi Watanabe, Nobuko Koshikawa, Atsushi Takatori. Mutated cancer cell-specific cell death activity of alkylating Pyrrole-Imidazole polyamide conjugates targeting a variety of oncogenic driver gene mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3775.

Abstract 4687: A novel alkylating pyrrol-imidazole polyamide, KR12, specifically recognizes mutant KRAS genes and potently induces cell death

RAS mutations are found in around 30% of all human cancers, with KRAS being the most frequently activated RAS family of oncogenes. Although extensive efforts to develop attractive chemotherapeutic drugs targeting KRAS mutations with clinical benefit have been made, these experimental trials have often resulted in unsuccessful. Recently, we have successfully produced for the first time a novel alkylating agent (termed KR12) conjugated with the sequence-specific Pyrrole-Imidazole polyamide (PI polyamide), which was expected to have an ability to bind to base sequences of KRAS mutations at codon 12 (G12D and G12V). According to our results, KR12-treated colon cancer-derived LS180 cells carrying a KRAS G12D heterozygous mutation underwent remarkable G2/M cell cycle arrest, cellular senescence and subsequent p53-dependent apoptotic cell death in association with a massive down-regulation of mutant KRAS expression as examined by quantitative real-time RT-PCR and immunoblotting. In the present study, we have further assessed the sequence-specificity of KR12 in detail by using an in vitro gel mobility shift assay, binding affinity assays with surface plasmon resonance (Biacore system) and also determined IC50 of KR12 in a variety of colon cancer-derived cells with the distinct KRAS status. Gel mobility shift assay demonstrated that the mobility of the oligonucleotide containing KRAS mutation was significantly retarded in the presence of KR12 but not in the absence of KR12. In agreement, KR12 specifically bound to the oligonucleotide containing KRAS mutation with high affinity as examined by Biacore system in vitro. Notably, ligation-mediated PCR analysis (LM-PCR) revealed that KR12 indeed alkylate the adenine residue next to KRAS at codon G12D in LS180 cells. These observations imply that KR12 is capable to bind to and alkylate the expected KRAS sequence in vitro and in cells. Next, we have investigated the sensitivity to KR12 in a variety of colon cancer-derived cells. Based on our standard WST cell survival assay demonstrated that KRAS mutation-bearing SW480 (G12V), SW620 (G12V), and LS180 (G12D) cells exhibited a significantly higher sensitivity to KR12 as compared with HT-29 (WT), Caco-2 (WT), DLD-1 (G13D) and SW1463 (G12C) cells. Taken together, our present results strongly suggest that KR12 is a novel sequence-specific alkylating agent targeting KRAS G12D as well as G12V mutation, and thus might be a promising anti-cancer drug for the treatment of patients bearing malignant cancers with KRAS mutations. Citation Format: Kiriko Hiraoka, Takahiro Inoue, Hiroyuki Yoda, Atsushi Takatori, Takayoshi Watanabe, Nobuko Koshikawa, Toshinori Ozaki, Hiroki Nagase. A novel alkylating pyrrol-imidazole polyamide, KR12, specifically recognizes mutant KRAS genes and potently induces cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4687. doi:10.1158/1538-7445.AM2015-4687

Abstract 4691: KRAS mutation specific alkylating pyrrole-imidazole polyamide (KR12) suppresses mutant KRAS expression and inhibits tumor growth by showing accumulation in KRAS mutant xenografts

Constitutive active mutations of KRAS are detected in 35-40% of human colon cancers, and almost all of them are the constitutive active missense mutations at codon 12 (80%) or codon 13 (20%). Consistent with these observations, the presence of KRAS mutations has been shown to be associated with malignant properties of tumors as well as a poor clinical outcome of the patients bearing these tumors. Unfortunately, yet no effective anti-cancer drug(s) specifically targeting KRAS mutations have been developed. Hence, we synthesized an alkylating agent conjugated with the Pyrrole-Imidazole polyamide (KR12: PI-polyamide-seco-CBI), which recognized KRAS G12D or G12V mutations at codon12. We have previously found that KR12 has anti-tumor effects in vitro and in vivo. However, it still remains elusive whether KR12 exerts its selective toxicity towards colon cancer cells by penetrating into the tumor tissues and inhibiting the expression of mutant KRAS gene in mouse model of human cancer. To address this issue, we decided to examine the distribution of KR12 using FITC labeled PI polyamide. In vivo imaging of tumor-bearing mice after single intravenous administration demonstrated that the highest fluorescence intensity was seen in the tumor sites 24 hours after injection with showing nuclear localization. Quantitative RT-PCR revealed that KR12 decreased the mutated KRAS expression in tumor tissues obtained from KRAS-heterozygous-mutated-LS180-xenografted mice (G12D heterozygous mutation). Since KR12 showed long lasting accumulation in xenografts we compared the effect of single and multiple administration of KR12. Once a week injection for five to eight weeks resulted in significant suppression of tumor growth in homozygous mutant SW480 (G12V homozygous mutation) xenografts. Surprisingly, both single and multiple treatments of KR12 induced massive tumor volume reduction without affecting body weight gain. These data suggest that KR12 accumulation in colon xenograft tumor tissues may emphasize drug local effect and minimize systemic adverse effect. Citation Format: Takahiro Inoue, Kiriko Hiraoka, Yusei Suzuki, Hiroyuki Yoda, Takayoshi Watanabe, Atsushi Takatori, Nobuko Koshikawa, Toshinori Ozaki, Hiroki Nagase. KRAS mutation specific alkylating pyrrole-imidazole polyamide (KR12) suppresses mutant KRAS expression and inhibits tumor growth by showing accumulation in KRAS mutant xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4691. doi:10.1158/1538-7445.AM2015-4691