403P - Stemness-targeted therapies to inhibit cancer cell plasticity in triple negative breast cancer

Background

Despite recent advances in the treatment of triple negative breast cancer (TNBC) many patients are still suffering from early recurrence, therapy resistance, and organ metastasis 1 to 3 years post-chemotherapy. TNBC cells with stem cell-like properties are resistant to standard of care chemotherapy and are responsible for early tumor relapse and poor outcome. Up-regulation of PD-L1 expression in Cancer Stem Cells (CSCs) has been associated with immune evasion capacity and promotion of their stem-like properties. In this study we identify two druggable stemness target, Aurora-A Kinase (AURKA) and NOTCH3, with positive feedback on intra-tumoral PD-L1 expression.

Methods

MDA-MB 231, MDA-MB 231 LM and SUM149-PT cell lines were used both in 2D and 3D environment. Unique PDX-derived TNBC cells were established from metastatic PDX models. TNBC cells were treated with Alisertib (AURKA inhibitor, 50nM) or AV353 (NOTCH3 inhibitor, 200ng). Immunofluorescence and immunoblot were used to compare different biomarker expression. ALDH was measured using Aldefluor kit. Apoptotic cells were quantified in real-time using IncuCyte S3. In vivo studies; 1x10⁶ MDA MB 231/LM cells were injected into the mammary fat pad of NSG-female mice and treated with Alisertib or AV353. Control groups were treated with placebo.

Results

Our study shows that AURKA and NOTCH3 pharmacological targeting reduced nuclear/undruggable PD-L1, that is not achieved clinically by using FDA-approved immune checkpoint inhibitors (ICIs). Significantly, following stemness-targeted therapy with alisertib or AV353, reduction of intra-tumoral PD-L1 resulted in inhibition of tumor growth, decreased levels of CD44 expression, increased CD8+ T-cell tumor infiltration, impairment in the enrichment of ALDH^high CSCs and enhanced sensitivity to chemotherapy. In vivo pharmacological targeting of AURKA and NOTCH3 signaling pathways induced inhibition of tumor growth, apoptosis, and impairment of organ metastases.

Conclusions

This study demonstrates that selective AURKA and NOTCH3 pharmacological targeting results in the inhibition of cancer cell plasticity that is linked to reduction of undruggable nuclear PD-L1 in TNBC cells.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Antonino D'Assoro.

Funding

Mayo Clinic / University of Minnesota.

Disclosure

All authors have declared no conflicts of interest.