SUMMARY We previously reported that the viability of some breast cancers depends on histone deacetylase 6 (HDAC6). We also developed a biomarker (HDAC6-score, validated with a NY CLIA certified test) to identify cancers that depend on HDAC6 function. By analyzing over 3,000 primary breast cancers, we have recently found that ~30% of all breast cancers can benefit from targeted therapy against HDAC6. Thus, we designed a clinical trial in partnership with Acetylon/Celgene to investigate the leading HDAC6 inhibitor (HDAC6i, Ricolinostat) plus nab-paclitaxel as breast cancer therapy (NCT02632071). Notably, we have observed that this regimen is well tolerated, and that clinical activity is identified in patients with metastatic disease. Molecularly, we have found that HDAC6 de- acetylates cMyc and that inhibition of HDAC6 promotes hyperacetylation of cMyc and its degradation by the proteasome. Furthermore, we linked the reduction of Myc expression due to HDAC6 inhibition to the anticancer activity of HDAC6i (manuscript accepted in Nature Cancer).Objective: In this grant proposal, our ultimate goal is to understand the molecular mechanism that mediates the anticancer activity of HDAC6i/s and to use this information to improve HDAC6-based breast cancer regimens. In this grant proposal, we will pursue these objectives by two independent but complementary aims. Aim 1. Define the clinical space for HDAC6 inhibitors in breast cancer treatment we hypothesize that including HDAC6i/s in therapeutic regiments for HR+ and HER2+ patients with high HDAC6 scores will have a superior therapeutic impact. - Aim 1a. Evaluate the response of high and low HDAC6-score HR+/HER2- cancer cells to a combination of HDAC6i/s and the standard of care systemic therapy. - Aim 1b. -Aim 1b. Evaluate the response of HDAC6-score high and low HER2+ cancer cells to a combination of HDAC6i/s and standard-of-care systemic therapy. -Aim 1c. Molecular characterization of tumor response at a tumor-tumor microenvironment (TME) level using sc- RNAseq and spatial transcriptomics. Aim 2. Investigate the mechanisms of resistance to HDAC6i/s in breast cancer cells. Despite the success of targeted therapies, resistance to treatment can emerge. Here, we will perform both, candidate- driven and unbiased comprehensive analysis of tissue samples from patients in our trial plus cell lines and PDOs to catalog and functionally test the molecular differences between sensitive and resistance cancers. - Aim 2a. Profile the post-translational modifications (PTMs) of c-MYC in HDAC6i-resistant and sensitive BCs. - Aim 2b. Evaluate the steady-state levels of proteasomal activity in HDAC6i-resistant and sensitive BCs. - Aim 2c. Network analysis of HDAC6i/s sensitive and resistant BC cells. - Aim 2d. Increase the specificity of the HDAC6 score with resistance data.

NARRATIVE We previously reported that the viability of some breast cancers depends on histone deacetylase 6 (HDAC6). In this grant proposal, our ultimate goal is to define the clinical space for HDAC6 inhibitors in breast cancers (aim 1) and understand the molecular mechanism that mediates the anticancer activity of HDAC6i/s to use this information to improve HDAC6-based breast cancer regimens. We will pursue these objectives by Investigating the mechanisms of resistance to HDAC6i/s (aim 2).