Loss of Wild-Type Kras is Associated with Enhanced Fitness and Increased Mitogen-Activated Protein Kinase Pathway Addiction in K-RasG12D Acute Myeloid Leukemia

Abstract

Functional analysis of therapeutic outlier responses in cancer therapy can identify unexpected synthetic lethal interactions and uncover biomarkers that predict enhanced sensitivity to targeted agents. We performed preclinical trials of the MEK inhibitor PD0325901 (PD901) in primary murine acute myeloid leukemias (AMLs) generated using retroviral insertional mutagenesis in KrasG12D “knock-in” mice. Similar to previous observations Nf1 and Nras mutant leukemias, treatment with PD901 prolonged survival in six independent leukemias harboring diverse retroviral integrations. Intriguingly, one outlier leukemia, clone Kras.101, demonstrated a greater than 4-fold improvement in median overall survival when treated with the MEK inhibitor. Importantly, this AML demonstrated a novel retroviral integration at relapse, indicating clonal evolution during treatment. The relapsed clone displayed phenotypic resistance upon transplantation into secondary recipients and retreatment with PD901. We performed a detailed analysis of the paired PD901-sensitive/resistant Kras.101 clones to identify the mediators of mitogen-activated protein kinase pathway (MAPK) addiction. Functional studies demonstrated that the novel dominant retroviral integration in the PD901 resistant clone did not directly alter drug response. However, genome-wide analysis of copy number alterations revealed a gain of chromosome 6 involving the Kras locus in the resistant clone, which was confirmed by spectral karyotyping and fluorescence in situ hybridization. Surprisingly, the PD901-sensitive leukemia harbored two copies of oncogenic KrasG12D due to somatic uniparental disomy, while the resistant clone harbored two mutant and one wild-type Kras alleles. This observation and paired single nucleotide polymorphism analysis indicated that the PD901-resistant leukemia was evolutionarily ancestral to the drug sensitive clone. Consistent with this hypothesis, competitive repopulation experiments with fluorescently labeled leukemia cells demonstrated increased fitness of the drug sensitive leukemia in the absence of the MEK inhibitor and over-expression of wild type K-Ras in the drug-sensitive leukemia reduced fitness in vivo. Finally, utilizing a tetracycline-inducible system, we found that wild-type Kras expression enhances the fitness of the Kras.101 clone only in the presence of the MEK inhibitor. Together, these data suggest that loss of wild-type Kras imparts a fitness advantage at the expense of increased MAPK pathway addiction, which results in enhanced sensitivity to MEK inhibition in leukemias driven by oncogenic K-Ras. DisclosuresNo relevant conflicts of interest to declare.