The Formin mDia1 Regulates Acute Lymphoblastic Leukemia Engraftment, Migration, and Progression in vivo

Abstract

Leukemias typically arise in the bone marrow and then spread to the blood and into other tissues. To disseminate into tissues, leukemia cells migrate into the blood stream and then exit the circulation by migrating across vascular endothelial barriers. Formin proteins regulate cytoskeletal remodeling and cell migration of normal and malignant cells. The Formin mDia1 is highly expressed in transformed lymphocytes and regulates lymphocyte migration. However, the role of mDia1 in regulating leukemia progression in vivo is unknown. Here, we investigated how mDia1 mediates the ability of leukemia cells to migrate and disseminate in vivo. For these studies, we used a mouse model of Bcr-Abl pre-B cell acute lymphoblastic leukemia. Our data showed that mDia1-deficient leukemia cells have reduced chemotaxis and ability to complete transendothelial migration in vitro. In vivo, mDia1 deficiency reduced the ability of leukemia cells to engraft in recipient mice. Furthermore, leukemia dissemination to various tissues and leukemia progression were inhibited by mDia1 depletion. Finally, mDia1 depletion in leukemia cells resulted in prolonged survival of recipient mice in a leukemia transfer model. Overall, our data show that the Formin mDia1 mediates leukemia cell migration, and drives leukemia engraftment and progression in vivo, suggesting that targeting mDia1 could provide a new method for treatment of leukemia.

Keywords: Diaph1; cytoskeleton; formins; leukemia engraftment; mDia1; transendothelial migration.

Figure 1

mDia1 knock-down does not affect B-ALL cell viability and proliferation. B-ALL cells were transduced with two independent shRNA constructs targeting mDia1 (mDia1 KD1 and mDia1 KD2) or with a control shRNA. The transduced cells were sorted based on the co-expression of a fluorescent marker (ZsGreen or DsRed) on the shRNA vector. (A) mDia1 is depleted in B-ALL cells transduced with mDia1-specifc shRNAs. Left panels, Western blot analysis of cell lysates from control and mDia1 knock-down (KD) cells. Tubulin staining is shown for normalization purposes. Right panel, quantification of KD levels in the two mDia1 KD cell lines. (B) B-ALL cell apoptosis is not increased in mDia1 KD cells. Left, representative flow cytometry staining for Annexin V of control and mDia1 KD cells. Right, quantification of the frequency of apoptotic cells. (C) mDia1 KD does not impair B-ALL proliferation. In vitro proliferation of B-ALL cells over the course of 6 days. Data in (A,C) are the average of at least 4 independent experiments; data in (B) are the average of at least 3 independent experiments. Error bars are the SEM.

Figure 2

mDia1 deficiency impairs transendothelial migration of B-ALL cells. Fluorescently-labeled control and mDia1 KD B-ALL cells were introduced into flow chambers with a monolayer of bEnd.3 endothelial cells in the presence of CXCL12 and then maintained under a shear flow of 2 dyne/cm² and imaged for 30 min using a spinning-disk confocal microscope. Phase contrast and fluorescence images were captured during time-lapse imaging every 15–25 s as indicated. (A) Representative images of a control B-ALL cell undergoing transendothelial migration (TEM). Top panels, overlay of ZsGreen fluorescence and phase contrast images; bottom panels, phase contrast images. As the control cell completes transendothelial migration the light ring around the cell body in the Phase contrast channel (bottom panels) disappears step-wise. The white arrow indicates a cellular protrusion initiating the transendothelial migration process. Time is in min:sec. (B) Representative images of an mDia1 KD B-ALL cell attempting and failing to complete transendothelial migration (depicted as in A). C. Similar adhesion of control and mDia1 KD B-ALL cells to the endothelial monolayer. (D–F) Control and mDia1 KD cells have equivalent crawling behavior on the endothelial cell monolayer. (G) mDia1 depletion does not significantly alter B-ALL detachment from the endothelial monolayer. (H) mDia1 KD cells take a similar amount of time to initiate TEM. (I) mDia1 deficiency significantly reduces the capacity of B-ALL cells to complete transendothelial migration. Data in (A,B) are representative of 4 independent experiments; data in (C–I) are the average of 4 independent experiments, with at least 61 cells/group for each experiment (C,D,G–I) or at least 27 cells/group for each experiment (E,F). Error bars are the SEM.

Figure 3

mDia1 depletion reduces the ability of B-ALL cells to undergo chemotaxis. (A) Left, representative flow cytometry staining for CXCR4 of control and mDia1 KD B-ALL cells. Right, quantification of CXCR4 surface expression on control and mDia1 KD cells. (B) Quantification of the percentage of chemotactic migration with or without CXCL12 through 5 μm pore transwell membranes of control and mDia1 KD B-ALL cells. Data in (A) are from 4 independent experiments; data in (B) are the average of 5 independent experiments. Error bars are the SEM.

Figure 4

Depletion of mDia1 decreases engraftment of B-ALL cells. Differentially dye-labeled control and mDia1 KD B-ALL cells were intra-venously co-transferred at a 1:1 ratio in CD45.1+ recipient mice. The number of B-ALL cells in the blood, bone marrow, and spleen of recipient mice was determined 24 h post-transfer by flow cytometry. (A) Representative flow cytometry plots of differentially-labeled control and mDia1 KD cells recovered from the indicated tissues. The transferred B-ALL cells were identified by gating on CD45.1-negative/ZsGreen-positive cells (not shown). (B) Quantification of the ratio and number of control and mDia1 KD B-ALL cells in the indicated tissues. A ratio below 1.0, indicated by the horizontal red line, shows reduced numbers of the mDia1 KD B-ALL cells. Data in (A) are representative of 4 experiments; data in (B) are the average of 4 experiments each with 2 mice/group. Error bars are the SEM.

Figure 5

mDia1 deficiency reduces leukemia progression in vivo and prolongs survival. Control or mDia1 KD B-ALL cells were transferred intra-venously into CD45.1+ recipient mice. (A,B) Every 3 days, the number of transferred B-ALL cells was quantified by flow cytometry in randomly selected pairs of recipient mice (1 control and 1 mDia1 KD). The transferred B-ALL cells were identified by gating on CD45.1-negative/ZsGreen-positive cells. (A) Representative flow cytometry plots of control and mDia1 KD B-ALL cells recovered from recipient mice in the indicated tissues. (B) Quantification of the frequency of control and mDia1 KD cells in the indicated tissues over time. (C) Reduced spleen colonization by mDia1-deficient B-ALL cells. As a readout of spleen colonization by the leukemia, the weight of the spleen in randomly selected pairs of recipient mice was determined every 3 days. (D) mDia1 depletion in leukemia cells prolongs survival. Using the above experimental set up, the recipient mice were monitored daily for signs of leukemia and euthanized once signs of morbidity were detected. Data in (A) are representative of 3 experiments; data in (B,C) are the average of 3 experiments each with 2 mice/group/time-point; data in (D) are pooled from 3 independent experiments each with cohorts of 5 mice/group/experiment. Error bars are the SEM.