OTT-MAL Activates the Notch Signaling Transcription Factor RBPJ and Cooperates with Mutant MPL to Induce Acute Megakaryoblastic Leukemia
Authors: Mercher Thomas , Raffel Glen D. , Moore Sandra A , Cornejo Melanie G , Baudry-Bluteau Dominique , Cagnard Nicolas , Jesneck Jonathan , Pikman Yana , Cullen Dana E. , Williams Ifor , Akashi Koichi , Shigematsu Hirokazu , Bourquin Jean-Pierre , Giovannini Marco , Vainchenker William , Levine Ross L , Lee Benjamin H. , Bernard Olivier , Gilliland D. Gary 
Published: April 2020

Abstract
Acute megakaryoblastic leukemia (AMKL) is a heterogeneous subtype of acute myeloid leukemia (AML) and is more frequent in children than in adults. The molecular basis of AMKL is poorly understood in adults, whereas two major molecular subtypes are recognized in pediatric AMKL. The first group occurs in infants and is associated with the t(1;22)(p13;q13) chromosomal translocation resulting in expression of the OTT-MAL fusion protein (a.k.a. RBM15-MKL1). The second group is represented by Down syndrome (DS) patients with transient myeloproliferative disease and AMKL, who present with acquired GATA-1 mutations. We generated a murine knock-in model of OTT-MAL expression and observed that expression of OTT-MAL results in abnormal megakaryopoiesis during embryonic and adult development, but rarely causes AMKL. We hypothesized that transformation to AMKL associated with OTT-MAL requires cooperating mutations and used a candidate gene approach with previously reported activating alleles of signaling molecules. Our data indicate that OTT-MAL cooperates only with a limited subset of activated signaling molecules, including MPLW515L, to generate a fully penetrant and rapidly fatal disease closely resembling human AMKL. OTT has homology with SHARP, which inhibits Notch signaling through direct interaction with RBPJ. In light of our recent observation that canonical Notch signaling specifies megakaryocyte fate of hematopoietic stem cells (Mercher et al, Cell Stem Cell, in press), we investigated whether OTT-MAL could interfere with the Notch pathway. We observed that OTT-MAL interacts with RBPJ and aberrantly increases its transcriptional activity in vitro and in vivo. The N-terminal portion of OTT is required for interaction of OTT-MAL with RBPJ and the transactivation domain of MAL is necessary for activation of RBPJ-mediated transcription, suggesting that OTT-MAL aberrantly recruits transcriptional co-activators to regulatory elements of genes normally inhibited by OTT/RBPJ. As a confirmation of clinical relevance, we have observed that several Notch pathway genes are specifically upregulated in human AMKL patient cells associated with the t(1;22)(p13;q13) as compared to DS-AMKL. Together our data suggest that aberrant activation of RBPJ by OTT-MAL results in abnormal commitment of hematopoietic progenitors to the megakaryocytic lineage, and is the basis for the specific association between t(1;22)(p13;q13) and AMKL. These results provide important biological insights into a novel mechanism of leukemogenesis of the megakaryocyte lineage, and indicate that concomitant activation of RBPJ (Notch signaling) and MPL (cytokine signaling) transforms cells of the megakaryocytic lineage. Furthermore, these findings suggest that specific targeting of these pathways could be of therapeutic value for human AMKL.