Hideyuki Yamada’s research while affiliated with Chiba University and other places

Microtubule-targeting agents (MTAs) have been successfully translated from basic research into clinical therapies and have been widely used as first- and second-line chemotherapy drugs for various cancers. However, current MTAs exhibit positive responses only in subsets of patients and are often accompanied by side effects due to their impact on normal cells. This underscores an urgent need to develop novel therapeutic strategies that enhance MTA efficacy while minimizing toxicity to normal tissues. In this study, we demonstrate that inhibition of the p38-MK2 (MAP kinase-activated protein kinase 2) pathway sensitizes cancer cells to MTA treatment. We utilize CMPD1, a dual-target inhibitor, to concurrently suppress the p38-MK2 pathway and microtubule dynamicity. In addition to established role as an MK2 inhibitor, we find that CMPD1 rapidly induces microtubule depolymerization, preferentially at the microtubule plus-end, leading to the inhibition of tumor growth and cancer cell invasion in both in vitro and in vivo models. Notably, 10 nM CMPD1 is sufficient to induce irreversible mitotic defects in cancer cells, but not in non-transformed RPE1 cells, highlighting its high specificity to cancer cells. We further validate that a specific p38-MK2 inhibitor significantly potentiates the efficacy of sub-clinical concentrations of MTA. In summary, our findings suggest that the p38-MK2 pathway presents a promising therapeutic target in combination with MTAs in cancer treatment.

Microtubule-targeting agents (MTAs) have been successfully translated from basic research into clinical therapies and have been widely used as first- and second-line chemotherapy drugs for various cancers. However, current MTAs exhibit positive responses only in subsets of patients and are often accompanied by side effects due to their impact on normal cells. This underscores an urgent need to develop novel therapeutic strategies that enhance MTA efficacy while minimizing toxicity to normal tissues. In this study, we demonstrate that inhibition of the p38-MK2 (MAP kinase-activated protein kinase 2) pathway sensitizes cancer cells to MTA treatment. We utilize CMPD1, a dual-target inhibitor, to concurrently suppress the p38-MK2 pathway and microtubule dynamicity. In addition to established role as an MK2 inhibitor, we find that CMPD1 rapidly induces microtubule depolymerization, preferentially at the microtubule plus-end, leading to the inhibition of tumor growth and cancer cell invasion in both in vitro and in vivo models. Notably, 10 nM CMPD1 is sufficient to induce irreversible mitotic defects in cancer cells, but not in non-transformed RPE1 cells, highlighting its high specificity to cancer cells. We further validate that a specific p38-MK2 inhibitor significantly potentiates the efficacy of sub-clinical concentrations of MTA. In summary, our findings suggest that the p38-MK2 pathway presents a promising therapeutic target in combination with MTAs in cancer treatment.

Microtubule-targeting agents (MTAs) have been successfully translated from basic research into clinical therapies and have been widely used as first- and second-line chemotherapy drugs for various cancers. However, current MTAs exhibit positive responses only in subsets of patients and are often accompanied by side effects due to their impact on normal cells. This underscores an urgent need to develop novel therapeutic strategies that enhance MTA efficacy while minimizing toxicity to normal tissues. In this study, we demonstrate that inhibition of the p38-MK2 (MAP kinase-activated protein kinase 2) pathway sensitizes cancer cells to MTA treatment. We utilize CMPD1, a dual-target inhibitor, to concurrently suppress the p38-MK2 pathway and microtubule dynamicity. In addition to established role as an MK2 inhibitor, we find that CMPD1 rapidly induces microtubule depolymerization, preferentially at the microtubule plus-end, leading to the inhibition of tumor growth and cancer cell invasion in both in vitro and in vivo models. Notably, 10 nM CMPD1 is sufficient to induce irreversible mitotic defects in cancer cells, but not in non-transformed RPE1 cells, highlighting its high specificity to cancer cells. We further validate that a specific p38-MK2 inhibitor significantly potentiates the efficacy of sub-clinical concentrations of MTA. In summary, our findings suggest that the p38-MK2 pathway presents a promising therapeutic target in combination with MTAs in cancer treatment.

Eribulin (ERI), clinically utilized for locally advanced or metastatic breast tumors, has shown potential links to the immune system. Notably, the cGAS-STING pathway, a key component of innate immunity, has gained prominence. Yet, limited reports explore ERI's effects on the cGAS-STING pathway. Additionally, the nuclear presence of cGAS remains poorly understood. This study uniquely delves into ERI’s impact on both the cytosolic cGAS-STING pathway and nuclear cGAS. ERI enhances nuclear localization of cGAS, resulting in hyper-activation of the cGAS-STING pathway in triple-negative breast cancer cells. Reduction of cGAS heightened both cell proliferation and ERI sensitivity. In clinical data using ERI in a neo-adjuvant setting, patients with low cGAS cases exhibited reduced likelihood of achieving pathological complete response after ERI treatment. These findings illuminate the potential of cGAS and IFNβ as predictive biomarkers for ERI sensitivity, providing valuable insights for personalized breast cancer treatment strategies.

Background: Eribuliln (ERI), a microtubule polymelization, is approved for locally advanced or metastatic breast cancer. However, its effects on drug-naive cancers are not well understood. In EMBRACE trial, ERI improves overall survival (OS) of the patients with metastatic breast cancer. This result might suggest that ERI could be involved in the immune system. In particular, there are a few reports on the effects of ERI on the innate immune system. This study aimed to investigate how ERI influences the innate immune system, focusing on the cyclic-GMP-AMP synthase (cGAS), a DNA sensor that triggers the production of type-I interferons. Methods: Clinical samples from the JONIE-3 trial were analyzed using immunohistochemistry (IHC). 121 patients were assigned to 2 different neoadjuvant chemotherapy (NAC) groups receive ERI (Group E) or paclitaxel (Group P) followed by FEC. The patients of both groups were performed biopsy before and after chemotherapy. We performed IHC on 56 samples and examined for association with pathological complete response (pCR), which was defined as no invasive redidual tumor tissue in the breast. Additionally, 5 different cell lines were established to evaluate the acute and chronic effects of ERI treatment. The cell lines were as follows: no treatment (control), PTX for short time (PTX short), PTX for long time (PTX long), ERI for short time (ERI short) and ERI for long time (ERI long). We evaluated the acute effects of short-term dosing, while the chronic effects of long-term dosing, which mimic resistance to treatment. Protein expression of the cGAS-STING pathway was examined, along with cGAS and IFNβ expression levels and their impact on cell division in the above cell lines. Then at the cellular level, each cell lines were evaluated for differences in cGAS and IFNβ expression and their effects on cell division. The differences in cGAS expression between cytoplasmic and nuclear fractions were verified by the cell fractuation assay. In addition, mitotic abnormalities and cell proliferation were also assessed. Results: In the clinical trial, ERI did not significantly differ from paclitaxel in terms of pathological complete response (pCR). However, high cGAS expression in Group E (ERI) correlated with increased pCR rates, while no such correlation was observed in Group P (PTX) (Figure). Additionally, High IFNβ expression in Group E also correlated with increased pCR rates, differed from Group P. In vitro, ERI upregulated cGAS, STING, pIRF3, and IFNβ protein expression compared to PTX. Notably, ERI induced elevated cGAS expression in the nucleus, as confirmed by immunofluorescence and cell fracturation assays. Additionally, PTX and ERI differed in their ability to cause mitotic abnormalities. PTX induced more micronuclei cells than ERI, on the other hand ERI induced more micronuclei cells and mitotic slippage. These results were also verified by live cell imaging. Finally, the knockdown of cGAS resulted in accelerated cell proliferation. Conclusion: ERI promoted chromosomal instability, leading to increased cGAS expression, particularly in the nucleus. These findings contribute to our understanding of ERI's effects on the innate immune response in triple-negative breast cancer, potentially paving the way for improved therapeutic strategies. The differences in intensity scores between GroupP and GroupE in the immunostaining of cGAS High cGAS expression in Group E correlated with increased pCR rates (p=0.0375), while no such correlation was observed in Group P (p=0.2983). Citation Format: Hideyuki Yamada, Mamoru Takada, Aussie Suzuki, Yu Muhan, Takeshi Nagashima, Hiroshi Fujimoto, Junta Sakakibara, Masaharu Kasuya, Takahiko Kawate, Daishu Miura, Masato Suzuki, Masaru Miyashita, Kazutaka Narui, Yoshie Hasegawa, Takashi Ishikawa, Masayuki Otsuka. Eribulin Induces Chromosomal Instability and Enhances cGAS Expression in the Nucleus of Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-05.

Background Luminal subtype recurrence risk classifications for breast cancer have been updated and put into practice with the development of treatment. Despite this, it is noteworthy that from 2008 to 2016, early breast cancer (EBC) of luminal subtype has been reported to have little improvement in survival probability. For early-stage luminal breast cancer, several clinical trials, such as monarchE, NATALEE (which is not available in Japan) or POTENT, were being conducted. In order to avoid overtreating individuals who might not benefit from adjuvant therapy, those at high risk of recurrence ought to be accurately picked up. In monarchE and POTENT trials, abemaciclib and S-1 have shown to be effective in adjuvant therapies for luminal breast cancer although it is unrevealed if the eligible patients for each criterion are at higher risk of recurrence than ineligible patients. Besides, some of the eligibility for POTENT overlaps with those for monarchE, the patients who met POTENT but did not meet monarchE criteria at how much risk of recurrence still remains unknown. Here, we investigated recurrence risk according to the criteria of each trial in Japanese patients in real world. Methods We reviewed the records of 1209 patients who received surgery for stage I–III breast cancer from January 2016 to May 2022 and selected 637 analytic cohort patients and retrospectively analyzed the recurrence-free survival (RFS) of the patients using the Kaplan–Meier method. High-recurrence-risk was defined according to monarchE trial and POTENT trial. Patients’ RFS was the primary endpoint. Results The 5-year RFS for all luminal breast cancer patients was 94.87% at Chiba University Hospital. Among monarchE eligible patients, the 5-year RFS was 82.49% and cohort 1 and cohort 2 eligible patients was 78.62% and 92.18% respectively, which were statistically lower than monarchE non-eligible patients (98.58%) (p < 0.0001, p = 0.0216, respectively). Even though the 5-year RFS rate for POTENT eligible patients (91.16%) was lower than POTENT non-eligible patients (99.13%) (p < 0.0001), while excluding those who met the monarchE criteria, the prognosis of POTENT eligible patients (5-year RFS rate 97.76%) had no significant differences from the patients with POTENT non-eligible breast cancer (p =0.0660). Our results suggested that the eligible patients of both monarchE and POTENT were associated with poor prognoses so the criteria set are considered to be appropriate. However, although POTENT criteria suggested a reasonable capacity for recurrence prediction, there was no dramatic difference in recurrence between POTENT non-eligible patients and the patients who are POTENT eligible but are not monarchE eligible. This might offer justification for reconsidering the use of S-1 in monarchE non-eligible patients. With the use of clinicopathological factors, it may assist in identifying individuals with high recurrence risk who would benefit from lengthier adjuvant chemotherapy regimens. Conclusion MonarchE criteria accurately identifies patients at high risk of relapse but the relapse in the patients who only qualified for POTENT is statistically almost the same as that in POTENT non-eligible patients. Citation Format: Muhan Yu, Mamoru Takada, Hideyuki Yamada, Hiroshi Fujimoto, Junta Sakakibara, Hiroto Yamamoto, Takeshi Nagashima, Masayuki Otsuka. Risk analysis of the differences in eligibility criteria between monarchE and POTENT clinical trials [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-17-07.

Background: Microtubule-targeting agents (MTAs) have been widely utilized in cancer treatment, but the development of novel MTAs has faced limitations. CMPD1, a highly selective inhibitor of MAP kinase-activated protein kinase 2 (MK2) with dual functions as a microtubule inhibitor, shows promise as a targeted therapy for triple-negative breast cancer (TNBC). This study aimed to investigate the effects of CMPD1, targeting MK2 and acting as a microtubule inhibitor, on cell cycle progression, microtubule dynamics, and TNBC-related processes. Methods: CMPD1 was evaluated in non-transformed retinal pigment epithelial cells (RPE1) and TNBC cell lines, including MDA-MB-231 and MDA-MB-468 cells. Time-lapse microscopy and immunofluorescence staining were employed to observe the effects of CMPD1 on mitotic progression, cellular morphology, activation of the spindle assembly checkpoint (SAC), and microtubule dynamics. Anchorage-independent growth assays and in vivo studies using a TNBC xenograft model were performed to assess the impact of CMPD1 on colony formation, tumor growth, and metastasis. Results: CMPD1 treatment induced prometaphase arrest and mitotic slippage in both non-transformed and TNBC cells. This arrest was associated with SAC activation, evidenced by increased levels of Mad1 at kinetochores. CMPD1 exhibited robust microtubule depolymerization, primarily targeting microtubule plus-ends and inhibiting microtubule polymerization in vitro. Furthermore, CMPD1 disrupted the organization of the microtubule network in interphase cells and perturbed the mitotic spindle during mitosis. Additionally, CMPD1 treatment resulted in the formation of cytoplasmic protrusions, membrane deformations, and dynamic movement of clustered mitotic chromosomes. These morphological alterations were indicative of the impact of CMPD1 on cellular structures and processes during mitosis. Notably, CMPD1 demonstrated significant efficacy in inhibiting anchorage-independent growth in TNBC cells, highlighting its potential as an anti-TNBC agent. In the TNBC xenograft model, CMPD1 exhibited superior anti-tumor activity compared to the standard chemotherapeutic agent Taxol. Tumor growth was markedly suppressed, and the formation of metastatic lesions was significantly reduced upon CMPD1 treatment. Furthermore, CMPD1 treatment effectively inhibited TNBC cell migration and invasion, likely through its modulation of microtubule dynamics. The disruption of microtubule dynamics by CMPD1 impaired the ability of TNBC cells to migrate and invade surrounding tissues, which are crucial processes in cancer metastasis. Importantly, CMPD1 showed high selectivity for TNBC cells, exerting minimal cytotoxic effects on normal cells, and could be efficiently washed out. This selectivity and washout ability enhance its potential as a targeted therapeutic agent for TNBC treatment. Conclusions: CMPD1, a highly selective MK2 inhibitor and microtubule inhibitor, exerts profound effects on cell cycle progression, microtubule dynamics, and TNBC-related processes. It induces prometaphase arrest, mitotic slippage, and microtubule depolymerization, resulting in altered cellular morphology. CMPD1 displays high selectivity for TNBC cells, while sparing normal cells and allowing efficient washout, making it a promising therapeutic agent for TNBC treatment. Its inhibition of anchorage-independent growth, suppression of tumor growth in vivo, and ability to impair TNBC cell migration and invasion further underscore its potential as a targeted anti-TNBC therapy. Citation Format: Mamoru Takada, Yu-Chia Chen, Yu Muhan, Hideyuki Yamada, Junta Sakakibara, Hiroshi Fujimoto, Takeshi Nagashima, Masayuki Otsuka, Motoki Takaku, Aussie Suzuki. Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-10.

Eribulin (ERI), clinically utilized for locally advanced or metastatic breast tumors, has shown potential links to the immune system. Notably, the cGAS-STING pathway, a key component of innate immunity, has gained prominence. Yet, limited reports explore ERI's effects on the cGAS-STING pathway. Additionally, the nuclear presence of cGAS remains poorly understood. This study uniquely delves into ERI's impact on both the cytosolic cGAS-STING pathway and nuclear cGAS. ERI enhances nuclear localization of cGAS, resulting in hyper-activation of the cGAS-STING pathway in triple-negative breast cancer cells. Reduction of cGAS heightened both cell proliferation and ERI sensitivity. In clinical data using ERI in a neo-adjuvant setting, patients with low cGAS cases exhibited reduced likelihood of achieving pathological complete response after ERI treatment. These findings illuminate the potential of cGAS and IFNβ as predictive biomarkers for ERI sensitivity, providing valuable insights for personalized breast cancer treatment strategies.

Background Although targeted treatments against human epidermal growth factor receptor 2 (HER2) have improved survival in patients with metastatic HER2-positive breast cancer, long and repeated treatment is time-consuming and costly for patients. To reduce these burdens, we developed ex vivo gene-transduced adipocytes that secrete anti-HER2 antibodies and evaluated their anti-tumor effects.Methods Ceiling culture-derived proliferative adipocytes (ccdPA) secreting anti-HER2 antibody against domain IV receptors: TRA-ccdPA, and domain II receptors: PER-ccdPA, were constructed using a plasmid lentivirus. Delivery of secreted antibody and its specific binding to HER2 breast cancer were evaluated in vitro and in vivo. To optimize antibody production from ccdPA, different conditions of ccdPA implantation were examined. Anti-tumor efficacy was evaluated in HER2-positive-cancer-inoculated nude mice.ResultsAnti-HER2 antibody against domain II was identified in supernatants from PER-ccdPAs. The optimal method to achieve the highest concentration of antibody in mouse sera was injecting differentiated ccdPA cells into the mammary fat pad. Antibody in supernatants from PER-ccdPAs bound to the surface of HER2-positive breast cancer cells similar to pertuzumab. Antibodies in mouse sera were delivered to HER2-positive breast cancer tumors and tumor necrosis was observed microscopically. One-time administration of combined TRA-ccdPAs and PER-ccdPAs produced antibody continuously in mouse sera, and anti-tumor effects were maintained for the duration of this study in xenograft models. Furthermore, combination therapy significantly suppressed tumor growth compared with a single administration.Conclusion Ex vivo gene-transduced adipocytes might be useful for cell-based gene therapy. This system may be a platform for various antibody therapies.

Background: In monarchE and Postoperative Therapy with Endocrine and TS-1 (POTENT) trials, abemaciclib and S-1 have, respectively, shown to be effective as adjuvant therapies for luminal breast cancer (BC), although whether patients who meet the criteria are at high risk of recurrence compared to non-eligible patients is still unknown. Here, we investigated recurrence risk according to the criteria of each trial in Japanese patients. Methods: We reviewed the records of 992 patients who received surgery at Chiba University Hospital for stage I-III BC from January 2017 to May 2022 and selected 553 analytic cohort patients and retrospectively analyzed the relapse-free survival of the patients as the primary endpoint. High-recurrence risk was defined according to monarchE trial and POTENT trial. Results: The 5-year RFS for monarchE cohort 1 and cohort 2 eligible patients were 77.78% and 89.33%, respectively, which were significantly lower than monarchE non-eligible patients (98.31%; p < 0.0001). However, the 5-year RFS rate for POTENT eligible patients (90.51%) was lower than for POTENT non-eligible patients (98.75%; p = 0.0001); excluding those who met the monarchE criteria, the prognosis of POTENT eligible patients had no significant differences from the prognosis of patients with POTENT non-eligible BC (p = 0.3100). Conclusion: MonarchE criteria accurately identify patients who are prone to relapse. Moreover, although POTENT criteria also suggested a reasonable capacity for recurrence prediction, there was no significant difference in recurrence between POTENT non-eligible patients and the patients who were POTENT but not monarchE eligible. This might offer justification for reconsidering the use of S-1 in monarchE non-eligible patients.