Abstract PD5-05: PD5-05 DAPK3 modulates migration and invasion of triple negative breast cancers
Authors: Wang Junkai , Kim Beom-Jun , Anurag Meenakshi , Yu Xin , Qi Xiaoli , Wang Jin , Zhang Bing , Cheng Chonghui , Ellis Matthew 
Published: March 2023

Abstract
Triple negative breast cancer (TNBC) is associated with poorer prognosis compared to other subtypes of breast cancer due to aggressive metastatic behavior and lack of targeted therapies. Human kinases are a large family of proteins that play critical roles in multiple biological pathways, and aberrant expression or mutations of kinases drive cancer progression and metastasis. Therefore, we applied Mass Spectrometry (MS)-based Kinase-inhibitor pulldown (KiP) technology to profile unique kinase signatures associated with TNBC and prioritize novel regulators for this subtype. Human kinases were captured by a mixture of nine FDA-approved kinase inhibitors, each conjugated separately to sepharose beads to profile all the major branches of the kinome tree. We performed KiP assay in 16 patient-derived xenografts (PDXs) [PMC5379071] consisting of different molecular subtypes of breast cancers. Differential expression analysis identified Death Associated Protein Kinase 3 (DAPK3) as a significantly upregulated kinase in TNBC PDXs which somehow remains understudied. Furthermore, RNA-seq and proteomics data with the same set of PDXs revealed that the upregulation of DAPK3 expression in TNBC was exclusively at protein levels, not RNA levels. Consistently, DAPK3 protein levels were significantly elevated in TNBC cell lines in DepMap dataset. Genomic knockout of DAPK3 in SUM159 and MDA-MB-231 cells dramatically inhibited migration and invasion measured by the transwell assay. We also observed that DAPK3 knockout reduced the phosphorylation of Myosin Light Chain 2 (MLC2) at T18/S19 site, which was shown to regulate cancer cell invasion [PMC4195943]. DAPK3 belong to DAPK family protein, and has an N-terminus kinase domain with high homology to its family proteins, and a C-terminus leucine-zipper domain. While re-expression of wild-type (WT) DAPK3 cDNA can rescue the migration and invasion phenotype caused by DAPK3 knockout, kinase-dead mutant (D161N) and leucine-zipper-domain-deleted mutant (ΔLZ) cannot rescue the phenotype. Moreover, not only D161N, but also ΔLZ mutant cannot restore the phosphorylation level of MLC2 as DAPK3 WT does. Therefore, both kinase activity and leucin-zipper domain of DAPK3 are critical for the phosphorylation of its substrate MLC2 and thus migration and invasion phenotype. By unbiased Immunoprecipitation-Mass Spectrometry (IP-MS) method, we identified Leucine-Zipper Protein 1 (LUZP1) as one of the strongest interactors of DAPK3. Interestingly, LUZP1 also has a leucine-zipper domain on its N-terminus. Further study showed that DAPK3 binds to LUZP1 via its leucine-zipper domain as expected. Moreover, we observed a tight correlation between DAPK3 and LUZP1 protein expressions in the proteomics data of PDXs and DepMap cell lines (Pearson correlation coefficient is 0.90 and 0.81, respectively). However, the correlation at RNA levels is not significant. Besides, we found that DAPK3 protein stability, but not RNA, is dependent on LUZP1 presence in TNBC cells, strongly implying the roles of LUZP1 on the post-transcriptional regulation of DAPK3. To summarize, we demonstrated that DAPK3, as a novel TNBC-enriched protein, modulates migration and invasion possibly via MLC2 phosphorylation. Both kinase activity and leucine-zipper domain of DAPK3 is necessary for its functionality. We also found LUZP1 is a strong interactor and a potential regulator of DAPK3 in TNBC biology.
Citation Format: Junkai Wang, Beom-Jun Kim, Meenakshi Anurag, Xin Yu, Xiaoli Qi, Jin Wang, Bing Zhang, Chonghui Cheng, Matthew Ellis. PD5-05 DAPK3 modulates migration and invasion of triple negative 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 PD5-05.