Extra-Nuclear Dislocation of PU.1 but Not CEBPA by Mutated NPM1 in AML Explains Lineage-Associations, ATRA Therapeutic Potential and DNMT3A Secondary Mutation.

Abstract 2427

In AML, there is robust linkage between genetic abnormalities and particular lineage-morphologies. One example is the linkage between NPM1 mutation and monocytic morphology: 51% of NPM1-mutated AML (n=47) in the TCGA database are monocytic (M4 or M5) (p=0.007, Chi-test). AML is the culmination of years of neoplastic evolution, and a reasonable postulate is that lineage associations are not epiphenomenom but optimal phenotype with respect to initiating genetic events: MYC, the ancient driver and coordinator of cell growth and division, is most highly activated and stabilized with lineage-commitment, and leukemia cells, including leukemia initiating/stem cells, are lineage-committed not only by surface phenotype, but also with high expression of lineage-specifying transcription factors (e.g., PU.1, CEBPA). However, sustained MYC activity also requires suppression of lineage maturation genes which are potent MYC-antagonists (e.g., CEBPE). We hypothesized that mutated NPM1 most efficiently impairs activation of monocytic maturation genes, creating selective pressure for neoplastic evolution towards this lineage.

PU.1 and CEBPA are essential drivers of monocyte and granulocyte commitment and maturation respectively. OCI-AML3 is an AML cell line containing mutated NPM1; fractionation into cytoplasmic and nuclear fractions and Western blot confirmed NPM1 dislocation into the cytoplasm. Similar to NPM1, PU.1 was detected in cytoplasmic but not nuclear fractions. Strong protein-protein interaction between NPM1 and PU.1 was confirmed by bidirectional immunoprecipitation/Western blot analysis, suggesting that mutated NPM1 caused the PU.1 dislocation. Unlike PU.1, CEBPA was expressed and localized in the nucleus at high levels. In OCI-AML2 cells containing wild-type NPM1, both PU.1 and CEBPA protein were localized in the nucleus. PU.1 but not CEBPA absence from the nucleus of OCI-AML3 suggests limited possibility of monocytic, but potential for granulocytic maturation, especially since PU.1 may usually antagonize retinoic acid (ATRA) mediated gene activation (Gauthier et al): ATRA at a clinically relevant concentration (0.5 microM) terminated OCI-AML3 proliferation accompanied by >50% upregulation in CD11b expression and morphologic granulocyte maturation (nuclear condensation and segmentation). CD14 (monocyte marker) expression decreased (>80%) simultaneously. NPM1 mutated AML has normal cytogenetics and limited mutations, underscoring that key late-differentiation MYC-antagonist genes are genetically intact but epigenetically suppressed. Accordingly, the addition of the epigenetic drug decitabine at non-cytotoxic concentrations augmented ATRA-induced granulocytic maturation. Vitamin D3 at clinically relevant concentration (1 nM) did not decrease cell proliferation or induce morphologic maturation.

Another implication of the potent block in cell cycle exit via monocytic but not granulocytic pathways is selective pressure for secondary mutations that favor monocyte lineage-commitment. DNMT3A interacts with polycomb proteins that repress lineage-programs and DNMT3A inactivating mutation is frequent in NPM1 mutated AML (51% of TCGA cases). Suggesting DNMT3A loss of function releases a monocytic program in an AML cellular context, significant inverse correlation between DNMT3A and PU.1 expression (Corr. Coeff. −0.43, p<0.0001) was observed in TCGA intermediate risk AML. Both in AML in general and more specificially in NPM1 mutated AML, DNMT3A mutation was significantly associated with M4 or M5 (14/23 [61%]) versus M1 morphology (4/13 [30%], p<0.001 Chi-test), higher peripheral blood absolute monocyte counts (median 3.96 IQR18.8 v 0.37 IQR7.4, p=0.04 Wilcoxon test) and bone marrow non-specific esterase positivity (14/21 [67%] v 7/19 [37%], p=0.06 Chi-test). Interestingly, DNMT3A mutations are not observed in AML containing primary genetic events that potently repress granulocytic but not necessarily monocytic maturation (RUNX1-ETO).

Lineage-phenotype in AML reflects selection for secondary mutations that cooperate with initiating events to restrict opportunities for cell cycle exit by maturation. These considerations in NPM1 mutated AML provide a mechanism-basis for frequent DNMT3A secondary mutations, efficacy of ATRA (also observed clinically: Schlenk et al;Burnett et al), and for combination ATRA/decitabine therapy.