Targeting the WT1^mut-Mir-193a Transcriptional Axis with INT-1B3, a Novel Lipid Nanoparticle-Formulated Mir-193a-3p Mimic in Acute Myeloid Leukemia

Wilms Tumor 1 mutations (WT1^mut) occur in 10% of acute myeloid leukemia (AML) patients with normal karyotype (CN-AML) and are associated with poor prognosis. Treatment of this subgroup still relies on standard polychemotherapy as no druggable molecular pathway has been identified. Considering that AML patients with WT1^mut are characterized by a DNA hypermethylation signature, we hypothesized that epigenetic silencing of downstream genes support the growth of WT1^mut AML cells and could represent druggable targets.

Analysis of publicly available methylation and miRNA expression data of human AML cell lines revealed increased DNA methylation at the miR-193a locus and reduced levels of miR-193a transcript in WT1^mut AML cells, suggesting transcriptional repression of this miRNA. MiR-193a is located within a large CpG island and has been previously implicated as a tumor suppressor in AML patients, however, its regulation and impact in CN-AML is unknown. MiRNA-Seq data of 300 CN-AML patients subsequently confirmed repression of miR-193a transcript levels in patients with WT1^mut. Moreover, lentiviral overexpression of WT1^mut in human AML cell lines conferred low miR-193a levels, whereas overexpression of WT1^wt increased miR-193a even in the presence of endogenous WT1^mut, highlighting the role of WT1^wt as a transcriptional activator of miR-193a in AML cells.

To model the potential tumor suppressor role of miR-193a in CN-AML, we took advantage of a primary murine AML transplantation model based on the combined overexpression of Hoxa9 and Meis1 (H9M), which are frequently upregulated in CN-AML. Enforced expression of miR-193a significantly reduced H9M cell viability as well as c-kit expression in vitro, and delayed AML onset in vivo. In addition, overexpression of miR-193a in multiple human AML cell lines and primary patient samples impaired AML cell growth and colony-forming capacity while promoting monocytic differentiation, underscoring its potent tumor suppressor role in AML cells.

MiRNA-Seq analysis revealed that both 5p and 3p arms of miR-193a were expressed in AML patients suggesting that both arms are functionally relevant. To further define the target range of each arm, we performed RNA-Seq in human AML cells transfected with miR-193a-3p or 5p mimics, or lentivirally transduced with miR-193a expressing both arms. Subsequent gene set enrichment analysis showed downregulation of the MAPK pathway and activation of pro-inflammatory responses, suggesting that miR-193a, in particular miR-193a-3p modulates the MAPK pathway and exogenous interactions between AML cells and the immune system.

Therapeutic modulation of miRNA levels in cancer patients has been an attractive concept due to their ability to impact on multiple pathways, but technical limitations in their delivery and tissue enrichment have prevented translation from the preclinical stage to patients. To overcome these hurdles in AML patients, we tested a novel lipid-nanoparticle (LNP) formulation of miR-193a-3p (INT-1B3), which is currently being investigated in a phase I clinical trial for solid tumor patients (NCT04675996). In H9M cells, INT-1B3 induced the downregulation of the MAPK pathway and immune activation, confirming the effects of miR-193a-3p. To evaluate the effect of INT-1B3 on leukemogenesis in vivo, we engrafted H9M cells in a murine host with an intact immune system, allowing INT-1B3 to exert its immunomodulatory effects. Biweekly i.v. treatments for 8 weeks of INT-1B3 or PBS after H9M cell transplantation prevented AML formation in the INT-1B3 arm, whereas all mice in the control arm succumbed to AML, highlighting the potent anti-leukemic activity of this miRNA based therapeutic.

In summary, we uncover a previously unknown regulatory mechanism between WT1^mut and miR-193a in AML cells, which provides a pre-clinical rationale for a miR-193a based therapeutic for AML patients.

Döhner:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Research Funding; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kronos: Research Funding. Eisfeld:Karyopharm Therapeutics: Other: Spouse is current company employee. Klusmann:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees.