Canonical Notch Signaling Is Required for Kras^G12D -Induced Myeloproliferative Neoplasm

Notch is a highly conserved signaling pathway that regulates cell-fate specification and tissue homeostasis in various contexts. Depending on the context, Notch signaling could be oncogenic or tumor suppressive. The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function (GOF) NOTCH1 mutations are prevalent in human T cell leukemia, while loss of Notch signaling is reported in myeloid leukemias.

To investigate the role of Notch signaling in T cell acute lymphoblastic leukemia/lymphoma (T-ALL) and myeloproliferative neoplasm (MPN) induced by Ras mutation, we downregulated Notch signaling in oncogenic Kras (Kras^G12D) mouse model, which develops T-ALL and MPN simultaneously. We took two independent genetic approaches to downregulate Notch signaling. Mx1-Cre-mediated conditional expression of dominant-negative Mastermind-like1 (DNMAML) potently inhibits canonical Notch-mediated transcriptional activation, while knocking out Protein O-fucosyltransferase 1 (Pofut1) reduces interactions of Notch receptors and their ligands and downregulates Notch signaling. To study the cell-autonomous functions of downregulating Notch signaling in oncogenic Kras -induced leukemogenesis, we transplanted either bone marrow cells or splenocytes into lethally irradiated mice.

As expected, inhibition of Notch signaling significantly inhibited 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, preserved intact Notch signaling, and acquired GOF Notch1 mutation(s). Surprisingly, we found that downregulation of Notch signaling inhibited MPN development in a cell-autonomous manner. Analysis of donor-derived hematopoiesis revealed that loss of Notch signaling significantly reduced myeloid compartment in Kras recipients with significant decrease of proliferation. Further mechanistic studies indicate that inhibition of Notch signaling significantly upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in Kras^G12D cells. RNA-Seq analysis of control and mutant myeloid progenitors (MPs) demonstrated that Kras^G12D MPs exhibited enhanced oxidative phosphorylation and mitochondria respiration, and this aberrant gene expression pattern was largely restored by DNMAML expression. These metabolic changes were further validated in functional assays. Furthermore, we demonstrated that combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target Notch downstream mechanisms in oncogenic Ras-driven myeloid leukemias.