Downregulating Notch Signaling in KrasG12D/+ Mice Inhibits Both T-Cell Leukemia and Myeloproliferative Neoplasm in a Cell-Autonomous Manner
Authors: Kong Guangyao , Chang Yuan-I , Ranheim Erik A. , Zhou Yun , Liu Yangang , Lambert Paul , Zhou Lan , Stanley Pamela , Pear Warren S. , Zhang Jing 
Published: October 2019

Abstract
Notch signaling is implicated in diverse functions in hematopoiesis, including stem cell maintenance, cell fate specification, cell proliferation, and apoptosis. Aberrant Notch signaling is associated with the pathogenesis of various hematopoietic malignancies. NOTCH1 mutations that lead to elevated intracellular Notch1 activity are identified in 50-60% of patients with acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) and in 100% of oncogenic Ras-induced T-ALL in mice. Although Notch1 mutations contribute to the malignant transformation of normal T-cells to T-cell leukemia/lymphoma initiating cells in the KrasG12D/+-induced T-ALL model, it remains unclear whether Notch1 signaling is necessary for T-ALL genesis. Moreover, it remains controversial whether loss of Notch signaling promotes myeloproliferative neoplasms (MPN) in a cell-autonomous manner.
To address these questions, we used genetic approaches to downregulate Notch signaling in KrasG12D/+ mice, which develop both T-ALL and MPN. Downregulation of Notch signaling in hematopoietic cells is achieved through Mx1-Cre–mediated conditional expression of Rosa26-GFP-dnMAML1, which inhibits canonical Notch signaling, or conditional knockout of Pofut1, which catalyzes O-fucosylation of Notch receptors and modulates Notch receptor ligand interactions. We found that either overexpression of dnMAML1 or deletion of Pofut1 significantly enhanced MPN phenotypes and shortened the survival of KrasG12D/+ mice. However, several pieces of evidence suggest that downregulation of Notch signaling in non-hematopoietic cells might influence MPN development. First, 100% of compound mice developed atopic dermatitis-like disease that is shown to promote MPN in a cell non-autonomous manner. Second, ~30% of endothelial cells were GFP-positive (expressing dnMAML1) in KrasG12D/+; Rosa26GFP-dnMAML1/+ mice.
To determine whether downregulating Notch signaling prevents T-ALL and/or promotes MPN in a cell-autonomous manner, we transplanted the same number of KrasG12D/+, KrasG12D/+; Rosa26GFP-dnMAML1/+, or KrasG12D/+; Pofut1-/- bone marrow cells (CD45.2+) along with congeneic competitor cells (CD45.1+) into lethally irradiated mice (CD45.1+). As expected, inhibiting Notch signaling significantly blocked T-cell development and completely prevented T-ALL development in recipients; T-ALL that developed in a fraction of recipient mice were derived from rare donor cells that expressed oncogenic Kras and preserved intact Notch signaling. Surprisingly, we found that the percentage of donor-derived myeloid cells was significantly lower in recipients transplanted with KrasG12D/+; Rosa26GFP-dnMAML1/+ or KrasG12D/+; Pofut1-/- bone marrow cells and consequently none of them developed donor-derived MPN-like disease. In contrast, ~20% of the recipient mice transplanted with KrasG12D/+ cells developed a lethal, donor-derived MPN (P=0.02). Because the hematopoietic stem cell (HSC) frequency was significantly lower in KrasG12D/+; Rosa26GFP-dnMAML1/+ bone marrow than that in KrasG12D/+ bone marrow, we investigated whether the absence of the MPN was due to the reduced HSC reconstitution in recipients. To normalize for HSC numbers, we transplanted lethally irradiated mice with same number of KrasG12D/+ or KrasG12D/+; Rosa26GFP-dnMAML1/+ splenocytes, which contained similar numbers of HSCs mobilized from the bone marrow. Consistent with our previous observation, only 1 out of 12 recipient mice with KrasG12D/+; Rosa26GFP-dnMAML1/+ cells developed a donor-derived (KrasG12D/+; Rosa26GFP-dnMAML1/+) MPN disease, while 6 out of 12 recipient mice with KrasG12D/+ cells died with donor-derived MPN (P=0.02). Together, our results indicate that blocking Notch signaling inhibits both T-ALL and MPN development in a cell-autonomous manner. We are currently working on the underlying mechnisms.

Disclosures
No relevant conflicts of interest to declare.