Abstract 3849: The partial tandem duplication of Mll (Mll PTD) is a gain-of-function in the absence of Mll wildtype (Mll WT) in adult mouse hematopoiesis.

Cytogenetically normal acute myeloid leukemia (CN AML) patients with a MLL PTD have a poor prognosis compared to CN AML patients with MLL WT. We previously reported that the MLL WT gene in MLL PTD+ CN AML is epigenetically silenced. To investigate in vivo significance of the MLL PTD in the absence of MLL WT on hematopoiesis and leukemogenesis, we generated homozygous Mll PTD/PTD and hemizygous Mll PTD/- mice. These mice died in utero or as neonates, respectively, precluding further study on adult hematopioesis and leukemogenesis. In the current study, we crossed Mll PTD/WT mice with Mll-conditional knock-out (cKO) animals to produce Mll PTD/cKO mice. Like the Mll PTD/WT mice, the Mll PTD/cKO mice survive to adulthood, as both models express Mll WT and PTD. As previously reported by Jude et al, (Cell Stem Cell, 2007) Cre activation in hematopoietic cells of mice carrying the Mll cKO allele resulted in an intragenic deletion in Mll (deltaN allele) that when expressed, the protein was unable to translocate to the nucleus and thereby unable to exert normal Mll function. DNA PCR confirmed the correct Mll PTD/deltaN genotype was generated. Mll deltaN/deltaN mice develop bone marrow (BM) failure (marked hypocellularity) at a median of 18 days post-Cre activation. Comparatively, Mll PTD/deltaN mice (n=5) survived beyond 52 weeks post-Cre activation (P PTD/deltaN animals exhibit equivalent white blood cell counts and hematocrit when compared to Mll WT/WT , Mll PTD/WT and Mll PTD/cKO controls. To evaluate whether Mll PTD function was maintained in bone marrow, qRT-PCR analysis of the Mll transcriptional target Hoxa9 at 12 days post-Cre activation revealed a 20-fold reduction in Hoxa9 mRNA levels in Mll deltaN/deltaN animals and ∼4-5 fold increase in Mll PTD/deltaN and Mll PTD/cKO BM (P=0.025) compared to WT BM. Mll PTD/cKO animals, with two functional copies of Mll, had nearly equivalent overexpression of HoxA9 as seen in Mll PTD/deltaN mice that maintain only one functional copy of Mll, supporting our previous hypothesis that the Mll PTD acts as a gain-of-function mutation. In conclusion, these data demonstrate for the first time that Mll PTD alone can support adult hematopoiesis. In conclusion, these data demonstrate for the first time that Mll PTD alone can support adult hematopoiesis and even though Hoxa9 is upregulated, another oncogene is required for leukemogenesis. Absence of Mll WT is not sufficient to promote acute leukemogenesis in Mll PTD+ mice, but appears to be a contributory factor. We recently reported that Mll PTD/WT mice crossed with Flt3 ITD/WT mice, the latter of which also do not develop AML (Lee et al. Cancer Cell, 2006), generated double mutant mice that do develop AML. Leukemic blasts from these mice exhibit a reduction in Mll WT expression (Zorko et al, Blood, 2012), recapitulating what is seen in human MLL PTD+ CN AML. Citation Format: Nicholas A. Zorko, Daniel A. Yanes, W. Courtland Lewis, Daniel L. Brook, Kelsie M. Bernot, Ronald F. Siebenaler, Susan P. Whitman, Elshafa H. Ahmed, Kathleen K. McConnell, John Nemer, Patricia Ernst, Gang Huang, Guido Marcucci, Michael A. Caligiuri. The partial tandem duplication of Mll (Mll PTD) is a gain-of-function in the absence of Mll wildtype (Mll WT) in adult mouse hematopoiesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3849. doi:10.1158/1538-7445.AM2013-3849