Combinatorial Genetic Uncovers DOCK1/RAC2 As Specific Targets for the Treatment of NPM1;Cohesin Mutated AML

Acute myeloid leukemia (AML) is an aggressive malignancy of the blood and bone marrow resulting from the accumulation of multiple serially acquired mutations. The mutational complexity of the disease makes AML difficult to treat, contributing to a low 5-year survival rate. A better understanding of how different mutations interact with one another to influence disease characteristics is critical and may result in the development of targeted therapies to improve patient outcome. The most common recurrent somatic mutation in AML affects the gene NPM1 and occurs in 25-30% of patients. This mutation results in the aberrant cytoplasmic localization of the protein and is termed NPM1cA (Falini et al. 2005, Cancer Genome Atlas Research Network 2013). NPM1cA is considered to be a driver of AML, however Npm1 ^cA/+ mice only develop disease after a long latency (median 18 months), suggesting that other cooperating mutations are required for AML development (Vassiliou et al. 2011).

Approximately 50% of patients with an NPM1cA mutation also harbor a mutation in one of four members of the cohesin complex (STAG2, SMC3, SMC1A, and RAD21). Mutations in cohesin genes are mutually exclusive and result in haploinsufficiency of the complex. As cohesin is known to regulate gene expression by facilitating promoter-enhancer interactions and three-dimensional genome organization, we wished to determine how cohesin mutation influences AML biology and gene expression in the presence of NPM1cA. We utilized the inducible Npm1 ^cAflox/+and Smc3 ^flox/+mouse models to examine this genetic interaction. We and others have shown that cohesin mutations result in enhanced hematopoietic stem and progenitor cell (HSPC) self-renewal (Mazumdar et al. 2015, Viny et al. 2015, Mullenders et al. 2015, Galeev et al. 2016, Fisher et al. 2017). Consistent with this, Npm1 ^cA/+;Smc3 ^Δ/+ HSPCs show enhanced self-renewal in vitro over HSPCs harboring either single mutation. Despite a shared role in self-renewal, Npm1 ^cA/+;Smc3 ^Δ/+mice developed AML with similar latency as Npm1 ^cA/+mice. Interestingly, however, Npm1 ^cA/+;Smc3 ^Δ/+ HSPCs exhibited dysregulation of a unique set of genes compared to cells from either single mutant, suggesting that the Npm1 ^cA;cohesin mutational combination uniquely alters the transcriptional environment. Further, Npm1 ^cA/+;Smc3 ^Δ/+ leukemias had a completely unique spectrum of acquired mutations compared to Npm1 ^cA/+ leukemias, suggesting that the addition of Smc3 haploinsufficiency to Npm1 ^cA/+alters AML evolution and that different driver mutations result in the accumulation of very different somatic mutations.

Among the uniquely upregulated genes in Npm1 ^cA/+;Smc3 ^Δ/+ HSPCs is Dock1, a guanine nucleotide exchange factor (GEF) for Rac1/2. As high Dock1 expression has been associated with low overall and disease-free survival in multiple cohorts of AML patients (Lee et al. 2017, Zhang et al. 2019), we hypothesized that Dock1 would be a novel target for the treatment of Npm1cA; cohesin ^mut leukemias. Consistent with this hypothesis, We found that knockdown of Dock1 resulted in decreased growth and adhesion and increased apoptosis in an Npm1 ^cA/+;Smc3 ^Δ/+leukemic line, but not in an Npm1 ^cAsingle mutant line. Higher Rac activity was also observed in Npm1 ^cA/+;Smc3 ^Δ/+ vs. Npm1 ^cA/+ leukemic lines. We found that DN Rac2 specifically impacted both the growth and apoptosis of an Npm1 ^cA/+;Smc3 ^Δ/+line, suggesting that Dock1 functions primarily through Rac2 to regulate survival. Importantly, the Dock1/Rac pathway is specifically targetable in Npm1 ^cA/+;Smc3 ^Δ/+ AMLs in vitro and in vivo, as knockdown of Dock1 resulted in prolonged latency in a Npm1 ^cA/+;Smc3 ^Δ/+ transplant model and slowed the growth and enhanced apoptosis of an Npm1 ^cA/+;Smc3 ^Δ/+, but not an Npm1 ^cA/+, leukemic line. Further, small molecule inhibitors of Dock and Rac had similar effects. Our results suggest that Dock1/Rac2 represent unique targets for the treatment of patients harboring the NPM1cA;cohesin mutational combination and provide validity to the concept that combinatorial genetics can uncover novel precision oncology targets.