Recruitment of MLL1 Complex Is Essential for SETBP1 to Induce Myeloid Transformation

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.