Downregulating Notch Counteracts Kras^G12D-Induced ERK Activation and Oxidative Phosphorylation in Myeloproliferative Neoplasm

Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions highly depend on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN).

We used genetic approaches to down-regulate Notch signaling in Kras^G12D/+mice, which develop both T-ALL and MPN. Down-regulation 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. To determine whether down-regulating Notch signaling prevents T-ALL and/or promotes MPN in a cell-autonomous manner, we transplanted the same number of Kras^G12D/+, Kras^G12D/+; Rosa26^{GFP-dnMAML1/+}, or Kras^G12D/+; 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 Kras^G12D/+; Rosa26^{GFP-dnMAML1/+} or Kras^G12D/+; Pofut1^-/- bone marrow cells and consequently none of them developed donor-derived MPN-like disease. In contrast, ~20% of the recipient mice transplanted with Kras^G12D/+cells developed a lethal, donor-derived MPN (P=0.02). Because the hematopoietic stem cell (HSC) frequency was significantly lower in Kras^G12D/+; Rosa26^{GFP-dnMAML1/+}bone marrow than that in Kras^G12D/+ bone marrow, we investigated whether the absence of MPN was due to the reduced HSC reconstitution in recipients. To normalize for HSC numbers, we transplanted lethally irradiated mice with same number of Kras^G12D/+ or Kras^G12D/+; Rosa26^{GFP-dnMAML1/+} splenocytes, which contained similar numbers of HSCs mobilized from the bone marrow. Consistent with our previous observation, only 1 out of 21 recipient mice with Kras^G12D/+; Rosa26^{GFP-dnMAML1/+} cells developed a donor-derived (Kras^G12D/+; Rosa26^{GFP-dnMAML1/+}) MPN disease, while 6 out of 12 recipient mice with Kras^G12D/+cells died with donor-derived MPN (P=0.002). Of note, in 3 T-ALL free recipients with Kras^G12D/+; Rosa26^{GFP-dnMAML1/+}cells, although mutant cells were dominant in hematopoietic tissues, none of them displayed MPN phenotypes over the time. Together, our results indicate that blocking Notch signaling inhibits both T-ALL and MPN development in a cell-autonomous manner.

Mechanistically, analysis of donor-derived hematopoiesis revealed that loss of Notch signaling significantly reduced myeloid compartment in Kras recipients with significant decrease of proliferation and cytokine-evoked ERK1/2 activation in Kras myeloid progenitor (MP) cells. RNA-Seq analysis of control and mutant MPs suggest that overexpression of DUSP1, a dual phosphatase that inactivates ERK, might lead to reduced ERK signaling in Kras^G12D/+; Rosa26^{GFP-dnMAML1/+} MPs. Moreover, Kras 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. Our results demonstrate an essential role of Notch in oncogenic Kras-induced MPN through modulating ERK signaling and mitochondrial metabolism.