3038 – UM171 PROMOTES EX VIVO EXPANSION OF HUMAN HSCS BY TARGETING THE EPIGENETIC MODULATOR COREST FOR DEGRADATION

Abstract

Numerous strategies have been proposed to facilitate the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs) for clinical benefit using combinations of growth factors and small molecule compounds. Notably, UM171, a pyrimidoindole derivative, was shown to significantly expand the number of umbilical cord blood (UCB) HSPCs, and currently UM171 expanded UCB HSPCs are being tested in clinical trials with promising initial results. However, the precise target of UM171, and the molecular mechanisms of its function, have not yet been reported. We have found that inhibition of the epigenetic regulator Lysine-specific histone demethylase 1A (LSD1) induces a rapid expansion of human cord blood derived CD34+ cells and promotes in vitro propagation of long-term repopulating HSCs by preventing differentiation. Early transcriptional changes triggered by LSD1 inhibition in HSCs were strikingly similar to the gene signature induced upon UM171 treatment. To further explore this and identify direct or immediate indirect protein targets of UM171, we performed thermal proteome profiling (TPP) which measures, the target stabilization or destabilization upon drug binding. We identified five candidate proteins that were significantly altered by UM171, one of which was RCOR1, a core member and main scaffold protein of the LSD1 containing CoREST complex. We could validate the CoREST complex as target of UM171 in HSPCs by Western blot showing that UM171 treatment almost completely abolished both RCOR1 and LSD1 protein levels following only three hours' exposure. CRISPR/Cas9 depletion of RCOR1, resulted in expansion of CD34+ cells similar to LSD1 inhibition and UM171, indicating that the CoREST complex is a principal target of UM171. Further, addition of the proteasome inhibitor Bortezomib along with UM171 prevented the reduction of RCOR1 in a dose dependent manner, suggesting the involvement of the ubiquitin proteasomal system (UPS). Indeed, we found that both RCOR1 and LSD1 were rapidly poly-ubiquitinated upon UM171 treatment. We thus identified a mechanistic basis for the HSC expansion molecule UM171 whereby it targets the CoREST complex for poly-ubiquitination and subsequent degradation.