Enhanced tyrosine kinase inhibitor response on chronic myeloid leukemia cells upon MS4A3 mRNA delivery Free

Chronic myeloid leukemia (CML) is caused by BCR::ABL1, a constitutively active tyrosine kinase that induces profound expansion of the myeloid cell compartment, while terminal differentiation is largely maintained. Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 are highly effective in CML, reflecting the dependence of CML progenitor cells (LPCs) on BCR::ABL1 kinase activity. However, most patients harbor residual leukemia, suggesting that TKI-resistant CML stem cells (LSCs) survive despite continued inhibition of BCR::ABL1. We have previously shown that membrane spanning four A3 (MS4A3) promotes myeloid differentiation of CML CD34⁺ cells by enhancing cellular response to IL-3 and GM-CSF. As MS4A3 is downregulated in primitive CML cells in a BCR::ABL1 independent manner, we hypothesize that re-establishing MS4A3 expression may overcome TKI resistance by promoting differentiation of TKI resistant LSCs into TKI-sensitive LPCs [1]. To test this, we have developed a CD34-targeted lipid nanoparticle (LNP) that encapsulates MS4A3 mRNA to increase expression of MS4A3 in LSPCs.

We synthesized LNPs by mixing an ionizable lipid, cholesterol, a helper lipid, and a poly(ethylene glycol)-lipid in ethanol and MS4A3 or enhanced green fluorescence protein (EGFP) mRNAs in acidic media with a microfluidic mixing chip. LNPs were characterized for their average diameter, polydispersity index (PDI) and zeta potential using dynamic light scattering (DLS). We determined mRNA loading at different amine to phosphate (N/P) ratios using agarose gel electrophoresis. We next synthesized CD34 targeted LNPs by modifying the surface with thiolated CD34 antibodies through thiol-maleimide conjugation, using isotype-matched nonspecific anti-human IgG as control (termed CD34-LNP and ISO-LNP, respectively). We used the RPMI-8402 B lymphoblastic leukemia cell line (100% CD34⁺) and CD34⁺ cells from chronic phase CML patients for in vitro internalization studies. We assessed time dependent internalization of CD34-LNPs using Cy5-tagged LNPs, determined intracellular uptake by flow cytometry, and measured the effect of LNPs on cell viability by CCK8 assay. We analyzed CML cells transfected with EGFP or MS4A3 mRNA encapsulated LNPs for expression of the respective protein by flow cytometry and tested the effect of MS4A3 mRNA with and without added imatinib in colony forming assays of primary human CD34⁺ CML cells.

The average hydrodynamic diameter of LNPs was 90.9 ± 1.0 nm and increased to 129.3 ± 6.3 post-modification with antibodies, indicating LNPs within the desired size range. Approximately 65% of the CD34⁺ RPMI-8402 cells showed LNP uptake after 2 h incubation with CD34-LNPs, while only 45% had internalized ISO-LNP (p=0.01). Selective uptake was maintained over more prolonged incubation times (up to 24 h). Similarly, CML CD34⁺ cells demonstrated 3.8-fold higher uptake of CD34-LNPs compared to ISO-LNPs at 2 h (p=0.01). We validated EGFP and MS4A3 protein expression upon mRNA delivery using the CD34-LNPs in RPMI-8402 cells. EGFP expression was first detected after 24 h and showed the highest fluorescence intensity with approximately 100% of cell positivity on day 5. The MS4A3 expression was detectable after 72 h with approximately 40% of cell positivity and a 2-fold increase in expression. Next, we delivered MS4A3 mRNA into CML CD34⁺ cells. A 2-fold increase of MS4A3 protein expression was detected on day 5 upon mRNA treatment. In the clonogenic assays, MS4A3-LNPs and imatinib (0.5 µM) alone reduced colony formation by 19% and 50%, respectively, while the combination reduced colonies by 82% (p=0.001), consistent with our overarching hypothesis.

(i) Synthesis of CD34 targeted LNPs is feasible, with significantly increased uptake compared to non-targeted LNP. (ii) Targeted MS4A3 mRNA delivery increases MS4A3 protein expression in cell lines and primary CML cells. (iii) CD34-LNP-induced MS4A3 expression enhances imatinib toxicity toward primary CML cells. Studies are underway to validate the results using additional endpoints including cell cycle distribution, differentiation and apoptosis. Delivery of MS4A3 mRNA to CML patients may improve TKI response.

This project was funded by the National Institutes of Health (NIH) under grant no. R01CA268496.

References

• Zhao, H., et al., MS4A3 promotes differentiation in chronic myeloid leukemia by enhancing common β-chain cytokine receptor endocytosis. 2022. 139(5): p. 761-778.