Abstract 26: RUNX1 as a transcriptional target of activated Shp2 (PTPN11) in juvenile myelomonocytic leukemia
Authors: Dorantes-Acosta Elisa , Huang Hui , Garcia Sara P. , Stieglitz Elliot , Loh Mignon , Yuan Guo-Cheng , Cantor Alan B. 
Published: August 2018

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ~50% of children still die from their disease. Somatic mutations leading to constitutive activation of the non-receptor tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in &gt;70% of JMML cases. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that the key myelomonocytic transcription factor RUNX1 is inactivated by src-family kinase-mediated tyrosine phosphorylation (Huang et al. Genes Dev 2012;26:1587-1601). Moreover, we demonstrated that Shp2 directly dephosphorylates RUNX1 leading to RUNX1 relative activation. We now show that overexpression of a mutant RUNX1 that is resistant to SFK-mediated tyrosine phosphorylation (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F—“RUNX1-5F”), mimicking constitutive dephosphorylation, causes cytokine-independent growth of Ba/F3 cells. Overexpression of RUNX15F in murine Lin- Sca-1+ c-kit+, (LSK) bone marrow cells leads to a dramatic expansion of Gr1+, Mac-1+ cells, and CFU-M/CFU-GM in vitro and in vivo. These effects are not seen when wild-type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379FD,Y386FD (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells have increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures express higher levels of the direct RUNX1 target genes PU.1 and cyclin D1. RUNX1 haploinsufficiency in a Shp2 LSL-D61Y conditional knock-in JMML mouse model reduces the disease phenotype, and treatment with the RUNX1 inhibitor Ro5-3335 ameliorates cytokine-independent in vitro bone marrow colony growth from this mouse model. Gene expression analysis of CD34+ cells from JMML patients versus controls combined with RUNX1 CD34+ cell ChIP-seq data shows significant enrichment for RUNX1 chromatin occupancy at differentially expressed genes compared to all expressed genes. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML due to an activating Shp2 mutation (Shp2E78G) were lentivirally transduced with doxycylcine-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. RUNX1 is also by direct ERK mediated phosphorylation (which is downstream of RAS/MEK). We propose that RUNX1 activation is an important common downstream consequence of both activated Shp2 and RAS signaling in the pathogenesis of JMML, and that RUNX1 inhibition may represent a novel therapeutic approach for this disorder.

Citation Format: Elisa Dorantes-Acosta, Hui Huang, Sara P. Garcia, Elliot Stieglitz, Mignon Loh, Guo-Cheng Yuan, Alan B. Cantor. RUNX1 as a transcriptional target of activated Shp2 (PTPN11) in juvenile myelomonocytic leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 26.