Pb1789: rac1 inhibition is feasible and affects cell migration in a zebrafish larvae model of aml, investigated using a software tool for single-cell visualization

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Ras-related C3 botulinum toxin substrate 1 (Rac1) is a GTPase signaling molecule implicated downstream of membrane receptors CXCR4 and CD44, which attract and mediate contact of the leukemic cells to the bone marrow niche. Rac1 inhibition with the lead compound EHOP-016 has a potent antiproliferative effect (IC50 of 3µM) on patient-derived leukemic cells. Zebrafish larvae have emerged as a relevant model to study how compounds affect the behavior and distribution of AML cells in vivo. A software tool for processing confocal images has been developed to visualize single-cell fluorescent leukemic cells from the AML cell line MOLM-13 in this model. Aims: Investigate the feasibility of Rac1 inhibition with EHOP-016 in AML therapy using zebrafish larvae injected with MOLM-13 as a model system. Test the performance of the software tool by exploring how Rac1 inhibition affects the mobility of MOLM-13 cells and their re-distribution from the vascular system to the caudal hematopoietic tissue (CHT) in the larvae. Methods: The antiproliferative effect of EHOP-016 on MOLM-13 was measured with a 3H-Thymidine incorporation assay; the cell migration was studied using a transwell migration assay. LD50 and maximum tolerated dose (MTD) of EHOP-016 in the zebrafish larvae were established by administering the compound in water from the 2nd day post-fertilization (2 dfp) and daily monitoring for signs of toxicity. Larvae were injected intravenously with MOLM-13 cells and exposed to the MTD of EHOP-016. A time-lapse series of images were acquired over 12 hours to study cell mobility to the CHT. Finally, larvae injected with MOLM-13 cells were treated with EHOP-016 alone or in combination with Daunorubicin (DNR) (i.v. administration) for three days from 2 dpf. A software tool for ImageJ, coupled with the images, was used to segment single fluorescent MOLM-13 cells and quantify the position within the larvae. Results: IC50 of the MOLM-13 cells treated with EHOP-016 for 48 hours was 6.1 µM. The cell migration was reduced by 22% with 6µM and 68% with 8µM (20h). The MTD of EHOP-016 in the zebrafish larvae was 20µM. The software tool could precisely quantitate cell counts in the time-lapse images of the CHT and map MOLM-13 cell distribution at specific time points in predefined regions of the larvae. The time-lapse series demonstrated that EHOP-016 treated cells were less attracted to the CHT than the untreated control (Figure 1, Mann-Whitney test, p=0.02), Zebrafish larvae treated with EHOP-016 for three days had a high ratio of cells (compared to cell count 2 dpf) migrating to the middle region of the larvae and the dorsal part of the tail, mainly consisting of larger blood vessels. The ratio of cells in the CHT was stable. The cells treated only with DNR resided primarily in the CHT at 5 dpf. The combined treatment with EHOP-016 and DNR resulted in higher cell ratios at the dorsal part of the tail and in the CHT. The cells migrating to the vessels in the middle region of the larvae when treated with EHOP-016 alone were not present in the combined treatment. Summary/Conclusion: The inhibition of Rac1 with EHOP-016 is feasible in water in a zebrafish larvae model injected with MOLM-13 cells. At an MTD of 20µM, EHOP-016 affects the migration ability and attraction of the MOLM-13 cells to the bone marrow niche of the larvae. The cells mainly reside in larger blood vessels in the middle or tail’s dorsal regions. The software tool successfully investigated a new lead compound and demonstrated that EHOP-016 warrants further research in AML therapy Figure: Timelapse-series of the CHT (square, counting region) of zebrafish larvae injected with red fluorescent MOLM-13 cells. The images are from 0h, 6h, or 12h after adding EHOP-016 20µM in water or not (control).Keywords: Signaling molecules, AML, Cell migration, Zebra fish