Recruitment of MLL1 Complex Is Essential for SETBP1 to Induce Myeloid Transformation
Authors: Nguyen Nhu , Gudmundsson Kristbjorn Orri , Soltis Anthony R. , Oakley Kevin , Han Yufen , Maciejewski Jaroslaw P. , Ernst Patricia , Dalgard Clifton L. , Du Yang 
Published: November 2021

Abstract
Abnormal activation of SETBP1 due to overexpression or missense mutations occurs frequently in various myeloid neoplasms and associates with poor prognosis. Direct activation of Hoxa9/Hoxa10/Myb transcription by SETBP1 and its missense mutants is essential for their transforming capability; however, the underlying mechanisms for such activation remain elusive. We found that knockdown of Mll1 in mouse myeloid progenitors immortalized by SETBP1 or its missense mutant SETBP1(D/N) caused significant reduction in the mRNA levels of Hoxa9/Hoxa10/Myb, suggesting that Mll1 is critical for their transcriptional activation induced by SETBP1 and its missense mutants. Physical association of MLL1 with SETBP1/SETBP1(D/N) was readily detected by co-immunoprecipitation in nuclear extracts of these cells, further suggesting that they may form a complex in myeloid cells to activate transcription. This complex formation is likely mediated by direct interactions between SETBP1/SETBP1(D/N) and MLL1 as both SETBP1 and SETBP1(D/N) are capable of interacting with multiple regions of MLL1 in binding assays using proteins synthesized by in vitro transcription and translation. To better understand the extent of SETBP1/SETBP1(D/N)-MLL1 interaction in regulating gene transcription, we carried out both ChIP-seq and RNA-seq analysis in mouse Lin -Sca-1 +c-Kit + (LSK) cells transduced by pMYs retrovirus expressing SETBP1 or SETBP1(D/N) or empty pMYs virus. These analyses revealed extensive overlap in genomic occupancy for MLL1 and SETBP1/SETBP1(D/N) and their cooperation in activating many oncogenic transcription factor genes in addition to Hoxa9/Hoxa10/Myb, including additional HoxA genes (Hoxa1, Hoxa3, Hoxa5, Hoxa6, and Hoxa7), Myc, Eya1, Mef2c, Meis1, Sox4, Mecom, and Lmo2. A large group of ribosomal protein genes were also found to be directly activated by MLL1 and SETBP1/SETBP1(D/N), identifying ribosomal biogenesis as another significant pathway induced by their cooperation. To further assess the requirement for MLL1 in SETBP1-induced transformation using a genetic approach, we also generated SETBP1/SETBP1(D/N)-induced immortalized myeloid progenitors and AMLs using LSK cells from Mll1 conditional knockout mice. Mll1 deletion in immortalized progenitors significantly decreased SETBP1/SETBP1(D/N)-induced transcriptional activation and their colony-forming potential. More importantly, Mll1 deletion significantly extended the survival of mice transplanted with SETBP1/SETBP1(D/N)-induced AMLs, indicating that Mll1 is essential for the maintenance of such leukemias in vivo. We further found that pharmacological inhibition of MLL1 complex using a WDR5 inhibitor OICR-9429 efficiently abrogated SETBP1/SETBP1(D/N)-induced transcriptional activation and transformation. Thus, MLL1 complex plays a critical role in Setbp1-induced transcriptional activation and transformation and represents a promising target for treating myeloid neoplasms with SETBP1 activation.

Disclosures
Maciejewski: Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.