Redefining eIF4E's function in Acute Myeloid Leukemia: Implications for engraftment and survival Free

The crosstalk between acute myeloid leukemia (AML) cells and the bone marrow niche is crucial for disease progression. Modeling the specialized microanatomical sites within the bone marrow that support AML cell survival and chemotherapy resistance is essential for understanding leukemic-niche crosstalk. This insight is critical for identifying strategies to disrupt this supportive interaction, thereby enhancing treatment efficacy and reducing relapse rates.

We have a three-dimensional (3D) in vitro model that effectively mimics the leukemic bone marrow niche, particularly the interaction between AML cells and mesenchymal stromal cells (MSCs). This model consists of a spheroid formed from bone marrow MSCs without the use of synthetic scaffolds or exogenous materials. AML cells are co-cultured with the spheroid to assess their colonization capacity into the spheroid, quantified by flow cytometry.

Eukaryotic translation initiation factor 4E (eIF4E) has been shown to play a role in migration, invasion and metastasis in solid tumors. While it is dysregulated in AML, its role in migration to niche and disease progression remains unclear. Thus, we sought to characterize the impact of eIF4E depletion using genetic and pharmacological approaches in both in vitro and in vivo models.

We engineered AML cell lines to disrupt eIF4E expression. We utilized eIF4E^high cell lines (MM6 and THP-1) for eIF4E depletion using CRISPR-Cas9 technology (CRISPR-4E vs. CRISPR-CTRL). NOMO-1 cells were selected for eIF4E overexpression, as its level are comparable to those of healthy CD34⁺ cells, enabling us to upregulate eIF4E (NOMO-4E vs. -CTRL). We corroborated depletion or overexpression of eIF4E in these cells using immunoblot.

We next evaluated the capacity of the cell lines to colonize the 3D in vitro model. We used HS-5 cells to generate the spheroid, as they recapitulate features of MSCs in the human niche. When comparing MM6 CRISPR-4E cells to CRISPR-CTRL cells, we observed a significant reduction (86%) in spheroid colonization by CRISPR-4E cells (p=0.0317). This finding was validated using different CRISPR-Cas9 clones. Similarly, THP-1 CRISPR-4E cells exhibited significantly reduced colonization (77%) compared to their respective CRISPR-CTRL cells (p=0.0079). In contrast, NOMO-1 cells overexpressing eIF4E (NOMO-4E) demonstrated a 2.7-fold higher colonization capacity relative to NOMO-CTRL cells (p<0.0001). These results demonstrate that eIF4E drives the colonization of AML cells into the mesenchymal niche, suggesting that disruption of eIF4E might impact AML cell ability to engraft and control disease progression.

To test the significance of the observations from the 3D in vitro model, we tested the eIF4E-depleted cell lines in immunodeficient mice evaluating their colonization/engraftment to the bone marrow and overall survival. We found a significant decrease in engraftment in mice transplanted with MM6 CRISPR-4E cells (10.1% ± 2.8; n=10) when compared with MM6 CRISPR-CTRL cells (56.5% ± 4.9; n=10), p<0.0001. Similarly, THP-1 CRISPR-4E cells displayed almost 4 times less engraftment when compared with THP-1 CRISPR-CTRL [2.5% ± 0.9 (n=6) vs. 8.9% ± 2.4 (n=6), respectively, p=0.0450]. Consistently, mice transplanted with CRISPR-4E cells had a significant delay in leukemia development and thus had an increased overall survival [median survival (days)]: MM6 CRISPR-4E=48 (n=10) vs. MM6 CRISPR-CTRL=37 (n=10), p<0.0001; THP-1 CRISPR-4E=86 (n=7) vs. THP-1 CRISPR-CTRL=59 (n=10), p=0.0002. We also confirmed these in vivo findings using different CRISPR/Cas9 clones.

To validate the genetic perturbations, we pharmacologically target eIF4E using ribavirin in immunodeficient mice transplanted with eIF4E^high primary AML cells. We found a significant decrease of human leukemia cells in treated mice compared with mice receiving vehicle control [% of human cells in blood (mean ± SE): 0.97 ± 0.28 (n=5) vs. 9.69 ± 2.31 (n=5), respectively, p=0.0079]. Consistently, ribavirin treated mice (n=4) lived approximately 51 days longer than vehicle treated mice (n=5), p=0.038.

Altogether, we demonstrated that the depletion of eIF4E impairs engraftment within the niche and improves overall survival in AML, highlighting an important role for eIF4E controlling migration and invasion also in leukemia. This work opens new avenues for the development of combination therapeutic strategies aimed to disrupt leukemia cell-niche interaction by targeting eIF4E in AML.