PRMT5 Inhibition Enhances Elimination of FLT3-ITD AML Stem Cells in Combination with TKI Treatment

Introduction: The FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) is a commonly observed molecular abnormality in AML and is associated with increased risk of relapse and poor survival. FLT3 tyrosine kinase inhibitors (TKIs) have shown benefit in the treatment of FLT3-ITD AML but do not eliminate primitive leukemia stem cells and are limited by frequent relapse and resistance. There is considerable interest in understanding mechanisms of FLT3-ITD AML stem cell persistence to develop strategies to enhance their elimination. Here we evaluated the role of epigenetic regulators in maintenance of human FLT3-ITD AML cells.

Methods: We performed an epigenetic screen using a well-curated collection of epigenetic probes (Epigenetics Probes Collection, Structural Genomics Consortium, n=41). FLT3-ITD (MOLM-13 and MV4-11), and FLT3-WT AML cell lines (OCI-AML3) were exposed to inhibitors for 7 days and CellTiter-Glo and Annexin V labeling were used to evaluate response. We further studied the effects of the PRMT5 inhibitors, GSK-591and LLY-283, with and without FLT3 TKIs, Quizartinib and Giltertinib, on FLT3-ITD+ and FLT3-WT AML cell lines. Synergy between PRMT5 inhibitors and TKIs was analyzed with Calcusyn software using the median effects method of Chou and Talalay. We performed in vivo analysis of the effects of PRMT5 and FLT3 kinase inhibition in a FLT3-ITD Tet2 deleted genetic mouse model of FLT3-ITD AML. Finally, we studied in vitro and in vivo effects of PRMT5 and FLT3 inhibition on primary CD34+ cells from FLT3-ITD AML patients.

Results: Screening with epigenetic probes identified PRMT5 arginine methyl transferase inhibitors, GSK591 and LLY-283, amongst the probes with the greatest inhibitory effects on growth and survival of FLT3-ITD MOLM-13 and MV4-11 AML cells. In contrast, inhibitors of PRMT1, PRMT3. PRMT4 and PRMT7 were associated with significantly less growth inhibition of FLT3-ITD AML cell lines. FLT3-WT OCI-AML3 AML cells were also sensitive to PRMT5 inhibition. However, ectopic FLT3-ITD expression in FLT3-WT THP1 cells led to enhanced sensitivity to PRMT5 inhibition. The combination of GSK-591 or LLY-283 with either of the FLT3 TKIs Quizartinib or Giltertinib resulted in synergistically enhanced inhibition of FLT3-ITD+ MOLM-13 and MV4-11 AML cells, compared to TKI or PRMT5 inhibitor alone. On the other hand, FLT3-WT cells were insensitive to TKI alone, and the combination of PRMT5 inhibitor and TKI did not lead to synergistic enhancement of inhibition. We next evaluated the effect of PRMT5 inhibition in a genetic mouse model of FLT3-ITD AML. AML mice were treated with vehicle (control), Giltertinib, LLY283 or the combination of Giltertinib and LLY283 for 2 weeks. AML cells from LLY-283 treated mice showed significantly reduced symmetric dimethylarginine in SmB protein. Significant inhibition of mature AML cells and of AML stem and progenitor cells was seen in the bone marrow and spleen of mice treated with LLY283, and significantly enhanced inhibition was seen with combination of LLY283 and Giltertinib (Figure 1a). Finally, we evaluated the effect of TKI (Giltertinib or Quizartinib), LLY283 or the combination on primary FLT3-ITD AML CD34+ cells with diverse co-mutations (n=8). LLY283 significantly inhibited FLT3-ITD AML CD34+ cell growth and survival in vitro by itself, and the combination of TKI and LLY283 resulted in significantly increased inhibition compared to TKI alone, including both CD34+CD38- and CD34+CD38+ cell populations (Figure 1b). In addition, in vivo treatment of immunodeficient NRGS mice xenografted with human FLT3-ITD AML cells with LLY283 or the combination of Giltertinib and LLY283 led to significant depletion of AML cells in BM and spleen.

Conclusion: An epigenetic probe screen identified PRMT5 as a key regulator of FLT3-ITD AML cell viability. We show an important role for PRMT5 in maintenance of murine and human FLT3-ITD AML stem cells and in their persistence following FLT3 TKI treatment. Treatment with a PRMT5 inhibitor in combination with a FLT3 TKI could be a promising approach to enhance elimination of FLT3-ITD AML stem cells.

No relevant conflicts of interest to declare.