Abstract 146: KDM1A inhibition is effective in reducing stemness and treating triple-negative breast cancer
Authors: Zhou Mei , Viswanadhapalli Suryavathi , Li Mengxing , Li Xiaonan , Suzuki Takayoshi , Tekmal Rajeshwar R. , Brenner Andrew , Vadlamudi Ratna K. , Sareddy Gangadhara R. 
Published: August 2018

Background: Triple-negative breast cancer (TNBC) lacks targeted therapy, exhibits an aggressive clinical course and contributes to a disproportional share of the overall mortality from breast cancer. TNBC is characterized by significant heterogeneity, a high proportion of cancer stem cells (CSCs), and resistance to conventional therapies. CSCs are highly tumorigenic, spared by chemotherapy, sustain the tumor growth and enhance the tumor recurrence after conventional therapies. Therefore, eradication of CSCs is critical for the development of efficient therapeutic strategies for TNBC. The lysine-specific histone demethylase 1A (KDM1A) is highly expressed in TNBC and is essential for maintenance of CSCs. However, the role of KDM1A in CSC function remains unknown. The objective of this study is to examine the role of KDM1A in CSCs and test the utility of KDM1A inhibitors using in vivo models.

Methods: We have generated KDM1A knockout cells using the CRISPR/Cas9 system and knockdown cells using KDM1A shRNA lentiviral particles. The effect of KDM1A inhibitors on the cell viability was examined by MTT assays. The effect of KDM1A knockdown and KDM1A inhibition using novel inhibitor NCD-38 on stemness and self-renewal of CSCs was examined using mammosphere formation and extreme limiting dilution assays. Global transcriptional changes modulated by KDM1A inhibition were examined by RNA-sequencing. Mechanistic studies were conducted using Western blot, RT-qPCR, and immunoprecipitation (IP) analysis. Mouse xenograft and patient-derived xenograft models were used for preclinical evaluation of the KDM1A inhibitor.

Results: Western blot and RT-qPCR analysis demonstrated that KDM1A is highly expressed in CSCs compared to non-CSCs. Cell viability assays using various KDM1A inhibitors revealed that novel KDM1A inhibitor NCD-38 is highly potent in reducing cell viability of TNBC cells compared to other inhibitors. NCD-38 effectively reduced the cell viability of therapy-sensitive and therapy-resistant TNBC cells. Flow cytometry analysis revealed that KDM1A knockout or KDM1A inhibitor treatment reduced the CSC population on TNBC cells. Further, KDM1A inhibition reduced the cell proliferation, mammosphere formation and self-renewal ability of CSCs. RNA-seq analysis revealed that KDM1A inhibition modulated pathways that are involved in the stemness of TNBC. IP analysis and GST pulldown assays revealed that KDM1A interacts with TLX and KDM1A inhibition modulated TLX target genes. Knockout of KDM1A reduced the in vivo tumor growth in xenograft models and KDM1A inhibitor NCD-38 treatment significantly reduced the tumor growth in patient-derived xenograft (PDX) models.

Conclusions: Our results establish KDM1A inhibitors as a novel class of drugs for treating TNBC and contribute to the development of a highly promising therapy that can significantly extend patient survival with minimal toxicity.

Citation Format: Mei Zhou, Suryavathi Viswanadhapalli, Mengxing Li, Xiaonan Li, Takayoshi Suzuki, Rajeshwar R. Tekmal, Andrew Brenner, Ratna K. Vadlamudi, Gangadhara R. Sareddy. KDM1A inhibition is effective in reducing stemness and treating triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 146.