Non-Canonical PRC1.1 is required for the activity of Menin-MLL1 Inhibitors in NUP98-Rearranged AML [RNA-seq]

Translocations involving the Nucleoporin 98 (NUP98) gene predict a poor prognosis for patients with acute myeloid leukemia. These translocations generate NUP98-fusion proteins which interact with the mixed-lineage leukemia (MLL1/KMT2A) chromatin modifying enzyme and its binding partner Menin. The mechanisms by which the NUP98-fusion proteins maintain gene expression remain unclear. We demonstrate that the NUP98-fusion-Menin-KMT2A complex is essential for maintenance of H3K27Ac and active transcription at key loci such as Meis1 and the Hox cluster, among others. However, eviction of the complex from chromatin is insufficient to completely silence gene expression. Accumulation of the PRC1.1 complex and deposition of H2aK119Ub and H3K27Me3 is required to transition to a transcriptionally repressive chromatin state. Furthermore, we demonstrate that loss of PRC1.1 function leads to resistance to small molecule Menin inhibitors. Therefore, a critical function of the NUP98-fusion-Menin-KMT2A complex is to antagonize repressive chromatin complexes. These same mechanisms are critical for efficacy of newly developed therapeutics. NUP98-rearranged murine leukemia cell lines were engineered to constitutively express CRISPR-Cas9 and sgRNA targeting individual components of PRC1.1 (Bcor, Kdm2b, Rnf2, or Pcgf1) or the sgluciferase control. Vectors containing sgRNA also contained an RFP fluorescent marker. Cell lines were established by sorting RFP+ cells, and CRISPR editing was confirmed by Sanger sequencing with the Synthego ICE algorithm. RNA-seq libraries were constructed from RNA extracted from established cell lines at steady state.