Abstract P6-10-08: Evaluation of potent novel second-generation MELK-selective inhibitor in aggressive breast cancers
Authors: Mughees Mohd , Tacam Moises , Tan Alex , Gonzalez Hector , Villodre Emilly S. , Debeb Bisrat , Tripathy Debu , Bartholomeusz Geoffrey , Lee Juhyeon , Dalby Kevin , Bartholomeusz Chandra 
Published: March 2023

Background Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are aggressive breast cancer subtypes with poor clinical outcomes due to the lack of well-validated and actionable targets and the onset of chemo-resistant metastasis. TNBC and IBC account for 15-20% and 2-4% of all breast cancer diagnoses but result in 30% and 8%–10% of breast cancer-related deaths, respectively. MELK (maternal embryonic leucine zipper kinase) is a potential therapeutic target in both TNBC and triple-negative IBC (TN-IBC). We have shown that 1) MELK expression is higher in basal tumors (mostly TNBC) and in IBC than in non-IBC, 2) high MELK expression is an independent prognostic factor for poor overall survival (P=0.0002) and poor distant metastasis-free survival (P=0.008) in breast cancer, and 3) MELK knockout leads to significantly lower metastatic burden and prolonged survival in a xenograft model of TNBC (unpublished data). MELK inhibitor (MELKi) OTS167 is in clinical trials but it lacks specificity and cross-reacts with other essential kinases. It is critical to discover novel, selective inhibitors with good bioavailability that target MELK and minimize adverse effects. We have developed a second-generation small-molecule MELK inhibitor 30e that is a highly potent and slow-binding ATP-competitive inhibitor of MELK. 30e exhibits potent cellular inhibition of MELK (IC50&lt; 10 nM), as assessed using a live cell NanoBRET assay, and is highly selective as assessed by live-cell KiNativ profiling experiments in MDA-MB-231 TNBC cells. Methods We screened a panel of TNBC and IBC cell lines for MELK expression by western blot and qPCR analysis. We assessed the effects of MELK inhibition with the 30e on migration and invasion. The role of MELK in regulating cancer stem cell phenotypes was assessed using the mammosphere assay and flow cytometry (CD44+/CD24-/low surface marker). MELK’s protumorigenic role was determined in vitro using a soft agar assay, and the anti-tumor activity of 30e was assessed in vivo using a TN-IBC orthotopic xenograft mouse model. Results We found that TNBC had high mRNA levels of MELK (62%) compared to non-TNBC cell lines (25%), which correlated with MELK protein levels. 30e inhibited cell viability in TNBC and IBC cells in a dose-dependent manner and with IC50s ranging from 0.45 to 1.76 μM (P≤0.05). However, no significant effect was observed when normal MCF-10A breast cells were treated with 30e (IC50&gt;20 μM). 30e also significantly reduced colony formation ability. Further, we observed that 30e reduced mammosphere formation efficiency and the CD44+/CD24-/low subpopulation in both TNBC and IBC cells in a dose-dependent manner (P≤0.05), suggesting the potential of 30e to inhibit the stem cell population in vitro. The soft agar assay also showed a significant decrease in colony formation in the TNBC and IBC cells after treatment with 30e, an indirect indicator of in vivo tumorigenicity. To assess the efficacy of 30e treatment on tumor growth, we evaluated this inhibitor as a single agent in a SUM-149 (TN-IBC) orthotopic mouse model. The mice were treated with intraperitoneal injections daily with 3 doses (2.5, 5, and 10 mg/kg) of 30e. Our data show that 30e suppressed tumor growth in a dose-dependent manner (P&lt; 0.007). Conclusions and Future Directions Our data demonstrate that a novel, potent and specific MELK inhibitor inhibits tumor growth by suppressing the cancer stem cell phenotype. MELK is a promising target for aggressive cancers such as TNBC and IBC. We are evaluating how MELK inhibition modulates the tumor microenvironment, which is a critical component of breast cancer response to treatment. We performed a cytokine antibody array and are validating the top targets. Further, we will evaluate 30e in combination with standard-of-care treatment for toxicity and inhibition of metastasis.

Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Hector Gonzalez, Emilly S. Villodre, Bisrat Debeb, Debu Tripathy, Geoffrey Bartholomeusz, Juhyeon Lee, Kevin Dalby, Chandra Bartholomeusz. Evaluation of potent novel second-generation MELK-selective inhibitor in aggressive breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-08.