MLL-Menin Inhibition Reverses Pre-Leukemic Progenitor Self-Renewal Induced By NPM1 Mutations and Prevents AML Development

Nucleophosmin (NPM1) mutant acute myeloid leukemia (AML) is the most common type of cytogenetically normal AML. The mutant NPM1 gene (NPM1c) co-occurs with other mutations such as DNMT3A^R882 and is associated with distinctive HOX gene expression. While DNMT3A mutations are detected in the most primitive stem cell compartment, NPM1 mutations are found in committed progenitors and differentiated myeloid cells but are absent from the stem cell compartment. This suggests that NPM1c may induce self-renewal in myeloid progenitors as a critical step in the development of AML.

To address this hypothesis, we used a conditional NPM1c mutant knock-in mouse model (NPM1c^flox-cA/+). We activated NPM1c in hematopoietic cells by treatment of NPM1c^flox-cA/+, Mx1-Cre mice with pIpC. NPM1c expression induced a stem cell gene expression program including HoxA, HoxB and Meis1 in granulocyte-macrophage progenitors (GMPs) and multipotent progenitors (MPPs) in just 4 weeks. Functionally, progenitors expressing NPM1c showed enhanced self-renewal in serial replating assays (GMP, 4 rounds; MPPs, >8 rounds), in comparison to their wildtype counterparts that could not be replated. Progenitors isolated from DNMT3a^R878H/+ (homolog to human R882H) mutant knock-in mice showed no enhanced Hox expression or self-renewal properties while NPM1c^flox-cA/+/DNMT3a^R878H/+ double mutant cells performed similarly to NPM1c single mutants. To determine if NPM1c directly induced stem cell gene expression in progenitor cells, Cre-negative NPM1c^flox-cA/+GMPs and MPPs were transduced with retroviral Cre. This led to activation of HoxA/HoxB/Meis1 expression and enhanced replating capacity, thus demonstrating activation of stem cell like programs and activity in progenitor cells.

We further evaluated the self-renewal potential of myeloid progenitors expressing NPM1c in vivo by transplantation. Wildtype GMPs engrafted at low levels 4 weeks post-transplant (~1% blood chimerism) and were undetectable at later time points. In contrast, NPM1c positive GMPs engrafted at higher percentages (average ~10%) and about half of the recipients showed >1% engraftment for over 12 weeks. These pre-leukemic, NPM1c-expressing GMPs engrafted in secondary recipients, indicating that they had been transformed to long-term myeloid restricted progenitors (LT-GMPs). We observed AML in primary (~8-month post-transplant) as well as secondary (~3-month post- transplant) LT-GMP engrafting mice. These results demonstrate that NPM1c has the ability to initiate self-renewal in committed myeloid progenitor cells as a step in the progression to AML.

We have previously shown that inhibition of the interaction between the histone methyltransferase MLL1 and Menin (MEN1) reverses leukemogenic gene expression in NPM1c AML. We assessed whether MLL-MEN1 inhibition could inhibit NPM1c induced progenitor self-renewal and prevent AML development. We used a recently developed selective and orally bioavailable Menin-MLL inhibitor (VTP-50469) to target MLL1/MEN1 in pre-AML and AML in vivo. To test whether NPM1c^flox-cA/+ and NPM1c^flox-cA/+/DNMT3a^R878H/+ double mutant LT-GMPs could be eradicated before overt signs of AML were observed, we treated secondary LT-GMP mice with VTP-50469 for 6 weeks with no evidence of toxicity. Remarkably, LT-GMP engraftment was rapidly reduced to <1% in the blood. Mutant cells remained undetectable for at least 2 months post-treatment while untreated mice succumbed AML. Gene expression analysis of VTP-50469 treated LT-GMPs in vivo showed a rapid loss of stem cell gene expression such as Meis1 (-2.6 fold) and Mecom/Evi-1 (-3 fold) after 5 days of treatment. VTP-50469 was also effective against NPM1c mouse AMLs as well as human PDX models as shown by dramatic reduction of leukemic engraftment and increased survival in inhibitor treated mice. In summary, NPM1 mutations induce stem cell gene expression and self-renewal in myeloid progenitors during progression to AML. Targeted inhibition of MLL1/MEN1 reverses this stem cell signature and self-renewal in myeloid progenitors and effectively reverses aberrant self-renewal in pre-leukemic cells before the development of frank AML. These findings highlight the importance of progenitor self-renewal during progression to AML and also suggests a strategy for intervention/prevention in patients with DNMT3A mutations and clonal hematopoiesis that acquire NPM1c mutations.