Takabe, Kazuaki

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive yet immunogenic subtype of breast cancer (BC) and the only one with FDA-approved immune checkpoint inhibitor (ICI) - pembrolizumab. Currently, PD-L1 (22C3) is the companion biomarker for predicting pembrolizumab benefit, but its utility is limited by spatial/temporal heterogeneity, assay variability, and modest correlation with treatment response. Chemokines such as CXCL9, CXCL10, and CCL5 recruit CD8+ T cells into the tumor microenvironment and have been associated with improved response to ICI, but their predictive value alongside PD-L1 remains unclear. Here, we examined the relationship between these chemokines, PD-L1, and pembrolizumab efficacy in TNBC. Methods: We analyzed 3662 TNBC tumors profiled by NGS (592, NextSeq; WES/WTS, NovaSeq; Caris Life Sciences). Immune cells were estimated using WTS deconvolution (Quantiseq). PD-L1 positivity was defined as >10% CPS (PharmDx 22C3). CXCL9/CXCL10/CCL5-high (H) and -low (L) TNBC were classified by RNA expression above or below 50th percentile. Real-world median overall survival (mOS) was derived from insurance claims and calculated from start of pembrolizumab to last contact using Kaplan-Meier. Statistical significance was assessed by chi-square and Mann-Whitney U with multiple comparison adjustments (q<0.05). Results: TNBC expressed higher levels of CXCL9, CXCL10, and CCL5 (N = 3662, median TPM: 5.3, 14.8, and 15.1) compared to HER2+ (N = 1344; 4.8, 10.1, 10.6) and HR+HER2- BC (N = 6097; 2.7, 6.6, 9.4), all q<0.05. Moderate correlations were observed between PD-L1 CPS and each chemokine (r = 0.41, p<.01). PD-L1-positive tumors had better mOS (24.3 months (m) vs 18.6 m, HR 0.75, 95% CI 0.6-0.92, p=0.007) compared to PD-L1-negative TNBC post-pembrolizumab. Among PD-L1-negative tumors, those with CXCL9-H had better mOS (26.9 m vs 15.8 m for CXCL9-L, HR 0.61, 95% CI 0.45-0.82, p=0.001) post-pembrolizumab. Among PD-L1-positive tumors, CXCL10-H and CCL5-H was associated with improved mOS (CXCL10-H: 25.7 m vs. 20.7 m for CXC10-L, p = 0.03; CCL5-H: 26.7 vs. 18.5 m for CCL5-L, p = 0.03) post-pembrolizumab. PD-L1 negative CXCL9-H, PD-L1 positive CXCL10-H and PD-L1 positive CCL5-H groups showed higher infiltration of CD8+ T cell, B cells, M1/M2 macrophages, Tregs. They also exhibited higher T cell inflamed score, IFNy score and MAPK activation score vs. CXCL9-L, CXCL10-L and CCL5-L groups (Table). Conclusions: In TNBC, a chemokine signature comprising CXCL9, CXCL10 and CCL5 refines PD-L1 based prediction of pembrolizumab benefit. Specifically, high CXCL10 or CCL5 expression in PD-L1-positive tumors, and high CXCL9 expression in PD-L1-negative tumors, were associated with improved survival post pembrolizumab and immune-rich microenvironment. These findings support the use of chemokine profiling to complement PD-L1 and enhance patient selection for ICI therapy in TNBC. Citation Format: S. Gandhi, S. Deshmukh, S. Wu, J. Xiu, G. Sledge Jr., D. Trapani, J. Leone, S. Chumsri, M. Lustberg, C. Hong, S. Yao, K. Takabe, M. Ernstoff, M. Opyrchal, P. Chalasani, C. Paulos, G. Lesinski, A. Madabhushi, S. Badve, K. Kalinsky, P. Kalinski. Chemokine signatures improve PD-L1-based prediction of pembrolizumab response in triple-negative breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-08-14.

Background: Mechanically gated PIEZO channels lead to an influx of cations, activation of additional Ca2+ channels, and cell depolarization. This study aimed to investigate PIEZO2’s role in breast cancer. Methods: The clinical relevance of PIEZO2 expression in breast cancer patient was analyzed in a publicly available dataset. Utilizing PIEZO2 overexpressed breast cancer cells, and in vitro and in vivo experiments were conducted. Results: High expression of PIEZO2 was correlated with a worse survival in triple-negative breast cancer (TNBC) but not in other subtypes. Increased PEIZO2 channel function was confirmed in PIEZO2 overexpressed cells after mechanical stimulation. PIEZO2 overexpressed cells showed increased motility and invasive phenotypes as well as higher expression of SNAIL and Vimentin and lower expression of E-cadherin in TNBC cells. Correspondingly, high expression of PIEZO2 was correlated with the increased expression of epithelial–mesenchymal transition (EMT)-related genes in a TNBC patient. Activated Akt signaling was observed in PIEZO2 overexpressed TNBC cells. PIEZO2 overexpressed MDA-MB-231 cells formed a significantly higher number of lung metastases after orthotopic implantation. Conclusion: PIEZO2 activation led to enhanced SNAIL stabilization through Akt activation. It enhanced Vimentin and repressed E-cadherin transcription, resulting in increased metastatic potential and poor clinical outcomes in TNBC patients.

Abstract Background: PIM2 is overexpressed in hematopoietic and solid cancers, and is involved in multiple signaling pathways. In hematologic malignancies, PIM2 has been shown to be a pro-survival factor and demonstrated to be a possible therapeutic target. However, the role of PIM2 as a therapeutic target in solid cancers has not been fully elucidated. Methods: PIM2 expression was compared between normal and cancer tissues in The Cancer Genome Atlas (TCGA) datasets. Pan-PIM inhibitor treatment, as well as PIM2 overexpression/knockdown technique was utilized to investigate the PIM2 role in solid cancers. Multiple cancer models were utilized for in vitro and in vivo experiments to investigate if PIM2 as a therapeutic target and examine mechanism of anti-tumor activity by JP11646. Results: PIM2 was overexpressed in several types of cancer tissues, including breast, esophageal, head-neck, kidney, liver and endometrial cancers in TCGA. PIM2 overexpression promoted tumor growth in in vivo breast cancer mouse model. PIM2 inhibition by siRNA as well as pan-PIM inhibitor JP11646 activated apoptosis as seen by increased cleaved PARP. JP11646 treatment inhibited cancer cell proliferation in a dose-dependent manner in vitro in various types of solid cancers, including head-neck, ovarian, breast, prostate, liver, pancreatic, colorectal and non-small cell lung cancers. Interestingly, treatment with pan-PIM inhibitor JP11646 resulted in selective downregulation of PIM2 protein expression, but not PIM1 or PIM3 in breast cancer cell lines, BT549 and MDA-MB-231. The treatment also resulted in downregulation of phosphorylated downstream targets, 4EBP1 and TSC2 in breast cancer cells. However, additional proteasome inhibitor treatment blocked PIM2 downregulation and apoptosis activation was not seen. The result suggests that proteasome complex is required for PIM2 degradation which is necessary to induce apoptosis. JP11646 treatment inhibited tumor growth in breast, liver, pancreas, and non-small cell lung cancer in vivo xenograft models and was compared to multiple standard of care treatments showing advantage in lung tumors. Conclusion: We found that PIM2 promoted cancer progression in solid tumors. We show novel mechanism of JP11646 targeting PIM2 for degradation and induction of apoptosis. We show that PIM2 may serve as a novel therapeutic target for multiple types of solid cancers with enhanced therapeutic effect through protein degradation rather than kinase inhibition alone. Citation Format: Eriko Katsuta, Kazuaki Takabe, Alex Adjei, Mateusz Opyrchal. Novel mechanism of PIM2 inhibition leads to significant effect on solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6426.

Abstract Introduction: Breast cancer is the most common female malignancy. Despite improvements in therapeutic options, metastatic disease remains incurable with limited treatments especially in triple-negative breast cancer (TNBC). Clinical outcomes in patients with TNBC continue to be worse as compared to other breast cancer subtypes. Therefore, novel therapeutic targets for TNBC are in urgent need. PIEZO2, which is a transmembrane calcium channel, is known to function as a channel-type mechanosensor in various aspects of mechanotransduction. Recently there have been reports that PIEZO2 may be involved in carcinogenesis and cancer progression. However, the role of PIEZO2 in breast cancer has not been elucidated. We hypothesized that PIEZO2 plays significant roles in progression of TNBC. Methods: To investigate the role of PIEZO2 in human breast cancer patients, The Cancer Genome Atlas (TCGA) breast cancer cohort, in which there are mRNA expression data from RNA-seq and clinical data, was utilized. PIEZO2 expression level was compared among breast cancer subtypes. Patients’ survival was compared based on PIEZO2 expression in each subtype. We used gene set enrichment analysis (GSEA) algorithm to evaluate pathways associated with PIEZO2 expression. To further elucidate the mechanisms of PIEZO2 roles, TNBC cell line, MDA-MB-231 was utilized for in vitro experiments. PIEZO2 expression was downregulated by small interfering RNA (siRNA). Cell viability was quantified by MTT assay. Protein expression was compared by western blot. Results: Breast cancer showed the highest PIEZO2 expression level, among various types of cancers in TCGA. In the breast cancer cohort, PIEZO2 expression level was lower in the TNBC subtype compared to other subtypes (p<0.001). Thus we hypothesized that PIEZO2 has different roles in each subtype. Survival analyses revealed that patients with high expression of PIEZO2 tumors showed significantly worse overall survival (p=0.016) in the TNBC cohort. However, there was no significant difference in the hormone receptor (HR)-positive (p=0.555) and the HER2-positive (p=0.380) cohorts. To explore underlying mechanisms how PIEZO2 high expressing tumors associate with worse prognosis in TNBC, GSEA was conducted. PIEZO2 high expressing tumors enriched epithelial-mesenchymal transition (EMT; NES=1.90, p=0.012) and Angiogenesis (NES=1.91, p<0.001) gene sets, as well as TAZ/YAP gene set (NES=1.57, p=0.028). PIEZO2 knockdown by siRNA significantly suppressed cell viability. This finding was validated by PIEZO inhibitor GSMTx-4 treatment. Further, expression levels of EMT related proteins, such as Vimentin and Snail, were downregulated in the PIEZO2 knockdown MDA-MB-231 cells compared to control cells. Conclusion: We describe a novel signaling pathway mediated by PIEZO2 leading to worse clinical outcomes in patients with TNBC by activation of EMT. Further work is ongoing to validate PIEZO2 as a novel therapeutic target for TNBC. Citation Format: Eriko Katsuta, Marija Vujcic, Shipra Gandhi, Arthur Beyder, Kazuaki Takabe, Mateusz Opyrchal. Mechanosensing calcium channel PIEZO2 accelerates epithelial-mesenchymal transition in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-07-04.

Abstract Background: Aurora A (AURKA) is a mitotic kinase responsible for centrosome segregation and mitotic spindle formation. In normal cells, expression of AURKA is highly regulated and is predominantly restricted to G2/M phases of the cell cycle. Unlike healthy cells, cancer cells overexpress AURKA through all phases of the cell cycle resulting in the acquisition of alternate non-mitotic functions. Little is known about cellular functions regulated by AURKA and its interaction with other signaling molecules. Here, we report a novel interaction between AURKA and the mitogen-activated protein kinase (MAPK) pathway at the level of MEK1 in breast cancer cells. This interaction may serve as a novel target as well as demonstrate by an additive cytotoxic effect of AURKA- and MEK1/2-specific inhibitors against estrogen positive (ER+) and triple negative breast cancer (TNBC) cells. Results: We show that treatment of ER+ HER2- MCF-7, ER- HER2+ SKBR3 and ER- HER2- BT549 cells with AURKA specific inhibitors alisertib, MK8745 and Aurora A Inhibitor I resulted in over 2-fold increase in relative levels of poMEK1/2 and poERK1/2 compared to untreated controls. The activation of the MAPK pathway was rapid with changes seen within 5 min after treatment with AURKA inhibitors and was sustained for at least 48 hours. Treatment with the pan RAF inhibitor TAK-632 did not diminish alisertib-induced poERK and poMEK1/2. Alternatively, treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated alisertib-induced phosphorylation of MEK1/2 and ERK1/2. In situ proximity ligation and pull down assays demonstrated AURKA and MEK1/2 direct interaction. In vitro kinase assay showed direct phosphorylation of MEK1 by AURKA. Combined treatment of alisertib and PD0325901 in vitro revealed significant additive cytotoxic effect in MCF-7 and BT549 cells when compared to either agent used alone (p< 0.008 and p<0.011; p <0.04 and p<0.028) with early trend toward significance in survival in a BT549 xenograft breast cancer in vivo model. Conclusions: Our data shows a novel AURKA-MEK1 interaction in breast cancer cells. In depth in vivo analysis is ongoing. The results reveal a promising new strategy for the treatment TNBC patients using a combination of AURKA and MEK1/2 inhibitors. Citation Format: Gandhi S, Gil M, Khoury T, Takabe K, Puzanov I, Gelman I, D'Assoro A, Opyrchal M. A novel interaction of AURKA with MAPK pathway in breast cancer cells as a potential therapeutic target [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-17.

Abstract Background: Aurora-A (AURKA) belongs to the family of serine-threonine kinases that are crucial for cell cycle control. In particular, AURKA controls centrosome function, mitotic spindle formation, and proper chromosome segregation. In contrast to the well-defined function of AURKA in normal cells, the role of AURKA in malignant transformation and cancer progression is still under investigation. Here, we report that AURKA directly interacts with MEK1/2 kinase and induces chemotaxis of breast cancer cells. Results: We demonstrated that overexpression of AURKA in luminal ER+ MCF-7 cells (MCF-7AURKA) resulted in over 10-fold increase in the number of cells displaying chemotactic response upon stimulation with growth factors when compared to parental MCF-7 cells. Both AURKA inhibitor MLN8237 and MEK1/2 inhibitor PD0325901 reduced migratory properties of MCF-7AURKA cells. In contrast, treatment with the pan RAF inhibitor TAK-632 did not affect chemotactic response of MCF-7AURKA cells. We demonstrated colocalization of AURKA and MEK1/2 immunofluorescence signals in MCF-7AURKA and triple-negative BT549 cells. In situ proximity ligation assay (PLA) revealed the presence of AURKA-MEK1/2 complexes in MCF-7AURKA and BT549 cells, and the notion that AURKA and MEK1/2 formed cellular complexes was confirmed with co-immunoprecipitation experiments. In vitro kinase assays with recombinant AURKA and MEK1 proteins showed that AURKA kinase could directly phosphorylate MEK1. Incubation of MCF-7AURKA and BT549 cells with the AURKA-specific inhibitors MLN8237 or MK8745 resulted in over a 2-fold increase in the level of pERK1/2 compared to the untreated controls. Treatment with the pan RAF inhibitor TAK-632 did not diminish MLN8237-induced pERK1/2, whereas treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated MLN8237-induced activation of ERK1/2. Conclusions: Our data present a novel AURKA-MEK1/2 interaction that contributes to the increased chemotaxis of breast cancer cells. The results suggest that AURKA negatively regulates MEK1 through a novel, direct phosphorylation event, possibly leading to constitutive activation of MEK2 and increased chemotaxis. The combined treatment of AURKA and MEK1/2 inhibitors represents a new therapeutic approach to decrease metastatic potential of breast cancer cells and delay tumor progression. The in-depth analysis of the AURKA-MEK1/2 complexes as well as in vivo studies testing the efficacy of combining AURKA and MEK1/2 inhibitors on patient-derived xenografts are currently in progress. Citation Format: Malgorzata Gil, Archana Chidambaram, Thaer Khoury, Kazuaki Takabe, Igor Puzanov, Irwin H. Gelman, Antonino B. D’Assoro, Evanthia Galanis, Mateusz Opyrchal. AURKA interaction with MEK1/2 complex and its role in promoting breast cancer cell metastasis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A60.

Abstract Background: Aurora A (AURKA) is a mitotic kinase responsible for centrosome segregation and mitotic spindle formation. In normal cells, expression of AURKA is highly regulated and is predominantly restricted to G2/M phases of the cell cycle. Unlike healthy cells, cancer cells overexpress AURKA through all phases of the cell cycle resulting in the acquisition of alternate non-mitotic functions. Little is known about cellular functions regulated by AURKA and its interaction with other signaling molecules. Here, we report a novel interaction between AURKA and the mitogen-activated protein kinase (MAPK) pathway in wild type BRAF breast cancer cells as well as demonstrate an additive cytotoxic effect of AURKA- and MEK1/2-specific inhibitors against estrogen positive (ER+) and triple negative (ER-, PR-, HER2-) breast cancer cells. Results: We show that treatment of ER+ HER2- MCF-7, ER- HER2+ SKBR3 and ER- HER2- BT549 cells with AURKA specific inhibitors alisertib, MK8745 and Aurora A Inhibitor I resulted in over 2-fold increase in the levels of both pMEK1/2 and pERK1/2 compared to the untreated controls. The activation of the MAPK pathway was rapid with changes seen within 5 min after treatment with AURKA inhibitors and was sustained for at least 48 hours. No differences in phosphorylation of MEK1/2 or ERK1/2 were observed in BRAFG464V triple negative MDA-MB-231 cells. Treatment with AURKA inhibitors resulted in downregulation of pAURKA and a significant increase in levels of total AURKA protein. The pull-down assay with Ras-binding domain coated agarose beads followed by western blot analysis with anti-pan-RAS Ab revealed no changes in active GTP-bound RAS in alisertib-treated MCF-7 cells compared to the untreated control. Consistently, no significant changes were observed in RAS-inducible phosphorylation of RAF1 activation site at Ser338 as demonstrated by western blot. Treatment with the pan RAF inhibitor TAK-632 did not diminish alisertib-induced pERK and pMEK1/2. Alternatively, treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated alisertib-induced phosphorylation of MEK1/2 and ERK1/2. Furthermore, combined treatment of alisertib and PD0325901 in vitro revealed significant additive cytotoxic effect in MCF-7 and BT549 cells when compared to either agent used alone (p< 0.008 and p<0.011; p <0.04 and p<0.028). Conclusions: Our data suggests that AURKA is a RAF1-independent negative regulator of MAPK activity in breast cancer cells. The in-depth analysis of the AURKA-MEK1/2 interaction is currently under investigation. The results reveal a promising new strategy for the treatment of wild type BRAF, TNBC patients using a combination of AURKA and MEK1/2 inhibitors. Citation Format: Malgorzata Gil, Archana Chidambaram, Thaer Khoury, Kazuaki Takabe, Igor Puzanov, Irwin Gelman, Antonio D’Assoro, Mateusz Opyrchal. Inhibition of AURKA induces Raf1-independent activation of MAPK pathway in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2123. doi:10.1158/1538-7445.AM2017-2123