RNA activation of CEBPA improves leukemia treatment

Abstract

Acute myeloid leukemia (AML) is a highly aggressive blood cancer marked by impaired differentiation and uncontrolled proliferation of myeloid cells. This phenotype is often driven by dysregulated expression of the transcription factor C/EBPα (encoded by CEBPA), especially in high-risk subtypes with FLT3 mutations. We hypothesized that RNA activation (RNAa) of CEBPA could reduce the growth of FLT3-mutated AML, and synergize with currently approved FLT3 inhibitors, thereby offering an alternative treatment strategy for a deadly disease. Our study shows that MTL-CEBPA, a chemically modified small activating RNA encapsulated in NOV340 liposomes, selectively targets myeloid cells, boosts CEBPA expression, and promotes a non-proliferative, mature state in FLT3-mutated AML cells. Importantly, MTL-CEBPA enhances the efficacy of commonly prescribed FLT3 inhibitor, gilteritinib, both in vitro and in vivo. All together, these findings support RNAa of CEBPA as a potential adjuvant therapy for FLT3-mutated AML.

Keywords: CEBPA; FLT3-ITD; MT: Oligonucleotides: Therapies and Applications; acute myeloid leukemia; drug delivery; myeloid differentiation; nucleic acid therapeutics; small activating RNA; transcription factors; tyrosine kinase inhibitors.

Graphical abstract

Figure 1

NOV340 liposomes deliver RNA into AML cells in vitro via macropinocytosis (A) Graphic of Cy3-MTL-CEBPA and controls. (B) Delivery of fluorescently tagged CEBPA-51 saRNA in myeloid leukemia and lymphoid leukemia cell lines. Two-way ANOVA with Holm-Sidak multiple comparison test; n = 3. Data represented as mean (SD). (C) Schematic of endocytosis inhibitors used for in vitro uptake inhibition assay. (D) Representative histograms of Cy3 fluorescence in AML cell lines after treatment with clathrin-mediated endocytosis inhibitor, Pitstop 2, or macropinocytosis inhibitors, EIPA or imipramine. (E) Changes in uptake of Cy3-MTL-CEBPA following treatment with endocytosis inhibitors in THP-1, MOLM-14, and KG1a cells. Two-way ANOVA with Holm-Sidak multiple comparison test; n = 3. Data represented as mean (SD).

Figure 2

Cy3-MTL-CEBPA is taken up into human leukemic cells in vivo (A) Delivery of Cy3-MTL-CEBPA to hCD45+ leukemic cells in AML1 PDX model. Multiple unpaired student t tests; n = 2–3. Data represented as mean (SD). (B) Representative histogram of Cy3 fluorescence in human CD45+ cells isolated from bone marrow and spleen of AML1 PDX model. (C) Delivery of Cy3-MTL-CEBPA to hCD45+ leukemic cells in AML2 PDX model. Multiple unpaired student t tests; n = 2–3. Data represented as mean (SD). (D) Representative histogram of Cy3 fluorescence in human CD45+ cells isolated from bone marrow and spleen of AML2 PDX model.

Figure 3

MTL-CEBPA upregulates CEBPA in AML in vitro models (A) Changes in CEBPA mRNA expression in THP-1, KG1a, or MOLM-14 cell lines 72-h post-MTL-CEBPA or control (MTL-FLUC) treatment. Two-tailed unpaired student t tests; n = 3. Data represented as mean (SD). (B) Representative western blots of THP-1 and MOLM-14 cell lines 96 h post-treatment. (C) Upregulation of CEBPA mRNA in ex vivo AML3 patient sample 48 h post MTL-CEBPA or control (MTL-FLUC) treatment. Two-tailed unpaired student t test; n = 3. Data represented as mean (SD).

Figure 4

MTL-CEBPA upregulates CEBPA following systemic administration in an in vivo model of AML (A) Schematic for in vivo upregulation experiment. (B) Change in CEBPA and SPI1 mRNA expression in leukemic cells isolated from MOLM-14-xenograft mouse model. Two-tailed unpaired student t test; n = 3 (each data point represents the pooled samples of two mice). Data represented as mean (SD). (C) Western blot of C/EBPα expression in isolated leukemic cells from MOLM14-xenograft mouse model (n = 1; each sample is a pool of six mice).

Figure 5

MTL-CEBPA sensitizes MOLM-14 cells to the anti-proliferative effects of FLT3 inhibitors (A) Heatmap of FLT3 inhibitors showing IC₅₀ values when combined with MTL-CEBPA or control (MTL-FLUC) in MOLM-14-Ven-Luc cells. The fold change in the IC50 (control vs. MLT-CEBPA) for each FLT3 inhibitor is listed next to the heatmap. (B) Difference in gilteritinib IC₅₀ in MOLM-14-Ven-Luc cells treated with MTL-CEBPA or control (MTL-FLUC). Two-way unpaired student t test; n = 3. Data represented as mean (SD). (C) Growth curves for MOLM-14 cells following treatment with 10 μg/mL of MTL-CEBPA or control (MTL-FLUC) with and without 5 nM gilteritinib. Two-way ANOVA with Holm-Sidak multiple comparison test; n = 6. Data represented as mean (SEM). (D) Percent viability of cells on day 6. Two-way unpaired student t test; n = 6. Data represented as mean (SD). (E) Decrease in c-Myc expression overtime after treatment with 10 μg/mL MTL-CEBPA. One-way ANOVA with Holm-Sidak multiple comparison test; n = 2–3. Data represented as mean (SD). (F) Representative western blot of c-Myc expression over time in MOLM-14 cells. (G) Fold change in CD11b MFI and (H) CD14 MFI in MOLM-14 cells 96 h following treatment with 10 μg/mL MTL-CEBPA or control (untreated). Two-way unpaired student t test; n = 2–3. Data represented as mean (SD).

Figure 6

MTL-CEBPA enhances anti-leukemic effects of gilteritinib in vivo (A) Schematic depicting treatment schedule for therapeutic evaluation of MTL-CEBPA in MOLM-14-Ven-Luc cell-line-derived xenotransplantation model. (B) Change in leukemic burden (as measured by luciferase bioluminescence) in MOLM-14-xenograft mice following treatment with MTL-CEBPA + gilteritinib or PBS + gilteritinib. One-tailed unpaired student t test; n = 10–11. (C) Representative images obtained from in vivo bioluminescence imaging.