| Summary: | The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 (<i>Id1</i>) and inhibitor of differentiation 3 <i>(Id3)</i> (referred to as <i>Id</i>) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying <i>Id</i> control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either <i>Id</i> depletion or <i>Id1</i> expression in order to identify <i>Id</i> targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 (<i>Kif11</i>) and Aurora Kinase A (<i>Aurka</i>), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in <i>Id</i> depletion conditions, <i>Kif11</i> and <i>Aurka</i> knockdown leads to a G2/M arrest, suggesting a novel <i>Id</i> cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of <i>Kif11</i> to block the <i>Id1–Kif11</i> axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the <i>Id/Kif11</i> pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting <i>Id</i> targets such as <i>Kif11</i> in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the <i>Id1–Kif11</i> molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.
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