Myosin-IIA regulates leukemia engraftment and brain infiltration in a mouse model of acute lymphoblastic leukemia

Abstract

Leukemia dissemination (the spread of leukemia cells from the bone marrow) and relapse are associated with poor prognosis. Often, relapse occurs in peripheral organs, such as the CNS, which acts as a sanctuary site for leukemia cells to escape anti-cancer treatments. Similar to normal leukocyte migration, leukemia dissemination entails migration of cells from the blood circulation into tissues by extravasation. To extravasate, leukemia cells cross through vascular endothelial walls via a process called transendothelial migration, which requires cytoskeletal remodeling. However, the specific molecular players in leukemia extravasation are not fully known. We examined the role of myosin-IIA a cytoskeletal class II myosin motor protein, in leukemia progression and dissemination into the CNS by use of a mouse model of Bcr-Abl-driven B cell acute lymphoblastic leukemia. Small hairpin RNA-mediated depletion of myosin-IIA did not affect apoptosis or the growth rate of B cell acute lymphoblastic leukemia cells. However, in an in vivo leukemia transfer model, myosin-IIA depletion slowed leukemia progression and prolonged survival, in part, by reducing the ability of B cell acute lymphoblastic leukemia cells to engraft efficiently. Finally, myosin-IIA inhibition, either by small hairpin RNA depletion or chemical inhibition by blebbistatin, drastically reduced CNS infiltration of leukemia cells. The effects on leukemia cell entry into tissues were mostly a result of the requirement for myosin-IIA to enable leukemia cells to complete the transendothelial migration process during extravasation. Overall, our data implicate myosin-IIA as a key mediator of leukemia cell migration, making it a promising target to inhibit leukemia dissemination in vivo and potentially reduce leukemia relapses.

Keywords: Bcr-Abl; cytoskeleton; dissemination; extravasation; migration.

Figure 1.. B-ALL cell proliferation and apoptosis are not altered by MyoIIA KD.

B-ALL leukemia cells were transduced with retroviral vectors coexpressing control shRNA or MyoIIA-specific shRNA 6592 (MyoIIA KD) and ZsGreen. (A) Depletion of MyoIIA in ZsGreen⁺-sorted MyoIIA KD cells compared with control shRNA-transduced cells was confirmed by densitometry analysis of Western blots stained with an isoform-specific MyoIIA antibody. Densitometry values were normalized to the relative protein loading measured by tubulin levels in each sample. Typical KD levels of MyoIIA were between 80% and 90%. (B and C) Expression of MyoIIB and MyoIIC in B-ALL leukemia cells. COS7 cells and PC12 cell lysates were used as positive controls for MyoIIB and MyoIIC expression, respectively. At most, only trace levels of MyoIIB and MyoIIC were detected by Western blot in B-ALL leukemia cells, and KD of MyoIIA did not result in increased expression of these other class II myosin isoforms. (D) MyoIIA KD B-ALL cells proliferate similarly to control B-ALL cells. In vitro growth curves of control and MyoIIA KD B-ALL leukemia cells. B-ALL cells were set at a concentration of 2.5 × 10⁵/ml and diluted every 2 d by a 1:10 factor. The B-ALL cells were cultured for up to 10 d, and growth curves were generated from calculating total cell numbers over the entire growth period. (E) MyoIIA KD does not affect steady-state apoptosis of B-ALL cells. B-ALL leukemia cells were cultured for 48 h at 37°C and then stained with APC-Annexin V and 7-AAD. The percentage of apoptotic cells was determined by quantifying the Annexin V-positive population using flow cytometry. ns, Not significant. (A) Data are representative of at least 3 experiments. (B and C) Data are representative of 2 experiments. (D and E) Data are the means ± sem averaged from at least 3 independent experiments.

Figure 2.. Depletion of MyoIIA in B-ALL cells prolongs survival and slows leukemia progression.

C57BL/6 CD45.1⁺ recipient mice were intravenously adoptively transferred with 5 × 10⁴ ZsGreen⁺ CD45.2⁺ control (black circles) or MyoIIA KD (shRNA 6592; gray squares) B-ALL cells. (A) Depletion of MyoIIA in B-ALL cells results in prolonged survival of leukemia recipient mice. Recipient mice were monitored daily for signs of leukemia progression and euthanized as soon as signs of morbidity became apparent. The median survival of control B-ALL recipient mice was 14 d, whereas the median survival of MyoIIA KD B-ALL recipient mice was 21.5 d (the 2 survival curves are statistically different, with P < 0.0001). (B–F) Progression of MyoIIA KD B-ALL leukemia is reduced compared with control B-ALL leukemia. Randomly selected leukemia recipient mice were euthanized, 3, 6, or 9 d post-transfer of 5 × 10⁴ control or MyoIIA KD B-ALL cells. The frequency of B-ALL cells among the recovered live cells from each organ analyzed was quantified by flow cytometry. B-ALL cells were identified by ZsGreen and CD45.2 expression vs. endogenous leukocytes, which are CD45.1⁺. **P < 0.01, ***P < 0.001, ****P < 0.0001. (A) Data are pooled from 4 independent experiments with cohorts of 5 mice/group each. (B–F) Data are the means ± sem of 4 independent experiments with 1 recipient mouse/group/time point.

Figure 3.. MyoIIA depletion reduces B-ALL cell engraftment.

Leukemia engraftment is reduced in MyoIIA KD B-ALL cells. Differentially dye-labeled control and MyoIIA KD (shRNA 6592) B-ALL cells (2.5 × 10⁶ each) were cotransferred at a 1:1 ratio by tail-vein injection into C57BL/6 CD45.1⁺ mice. At 8 and 24 h post-transfer, the recipient mice were euthanized, and the number and ratio of control and MyoIIA KD B-ALL cells were determined in the blood, bone marrow (BM), and spleen by flow cytometry. B-ALL cells were identified by ZsGreen and CD45.2 expression vs endogenous leukocytes, which are CD45.1⁺. A ratio below 1.0 (horizontal black line) indicates reduced numbers of MyoIIA KD cells. **P < 0.01, ***P < 0.001 compared with the injected ratio.

Figure 4.. MyoIIA inhibition impairs the ability of B-ALL cells to extravasate in vivo and infiltrate the brain.

ZsGreen⁺ CD45.2⁺ control or MyoIIA KD (shRNA 6592) B-ALL cells (2 × 10⁷ each) were transferred intravenously into wild-type, congenically marked C57BL/6 CD45.1⁺ recipient mice. Eighteen hours after transfer, to mark the transferred B-ALL cell present in the vasculature, the recipient mice were intravenously injected with CD19-APC antibodies and then euthanized 4 min later. B-ALL cells (ZsGreen⁺ CD45.1⁻), recovered from the brain that stained for CD19-APC, were considered to still be arrested in the vasculature, whereas CD19-APC-negative B-ALL cells recovered from the brain were considered as having fully extravasated and entered the brain parenchyma. (A) Representative flow cytometric analysis of B-ALL cell extravasation and infiltration into the brain. Live cells were gated by forward- and side-scatter and by a single-cell gate (not shown), and then transferred B-ALL cells were gated using a ZsGreen⁺ CD45.1⁻ gate (left). B-ALL cells that were arrested in the vasculature vs. cells that had fully infiltrated the brain were distinguished by CD19⁺ or CD19⁻ gates, respectively (right). (B) MyoIIA deficiency inhibits the capacity of B-ALL cells to complete extravasation in vivo. (Left) Frequency of control and MyoIIA KD B-ALL cells arrested in the brain vasculature that have not infiltrated the brain (ZsGreen⁺ CD45.1⁻ CD19⁺). (Right) Quantification of the total number of control and MyoIIA KD B-ALL cells that fully extravasated and infiltrated the brain (ZsGreen⁺ CD45.1⁻ CD19⁻). (C) MyoIIA inhibition by blebbistatin treatment in vivo impairs leukemia cell extravasation into the brain. One hour before B-ALL cell transfer, C57BL/6 CD45.1⁺ recipient mice were treated with vehicle or blebbistatin (2.5 mg/kg). Twenty-four hours after B-ALL cell transfer, the recipient mice were intravenously injected with CD19-APC antibodies and then euthanized 4 min later. Data were analyzed as in B. (Left) Frequency of B-ALL cells arrested in the brain vasculature of vehicle or blebbistatin-treated mice. (Right) Quantification of the total number of B-ALL cells that fully infiltrated the brain of vehicle or blebbistatin-treated mice. (A) Data are representative of 4 independent experiments. (B and C) Data are the means ± sem of 4 and 5 independent experiments, respectively.

Figure 5.. MyoIIA-inhibited B-ALL cells are defective in chemotaxis to CXCL12.

(A) The percentage of chemotactic migration through 5 μm pore Transwells, with or without 1 μg/ml CXCL12 of control, MyoIIA KD (shRNA 6592), and blebbistatin-treated control B-ALL cells, was measured using flow cytometry in the presence of counting beads. (B–E) Control and MyoIIA KD B-ALL cells have similar surface expression of the chemokine receptor CXCR4 and of adhesion molecules. Control and MyoIIA KD B-ALL cells were stained for CXCR4 and the integrins CD11a, CD29, and CD49d, as indicated and analyzed by flow cytometry. (A) Data are the means ± sem of 3 independent experiments. (B–E) Data are representative of 2 independent experiments.

Figure 6.. MyoIIA-deficient B-ALL cells are impaired in completing TEM.

ZsGreen⁺ control or MyoIIA-depleted (shRNA 6592) B-ALL cells were perfused into flow chambers containing bEnd.3 brain endothelial cell monolayers and kept under shear flow (2 dyne/cm²) for up to 30 min. During this time, phase contrast and green fluorescence images were acquired every 15 s using a spinning-disk confocal microscope. Time in minutes:seconds. (A) Selected time points of a representative control B-ALL cell during transmigration. This transmigrating leukemia cell undergoes TEM, evidenced by a step-wise darkening in the phase-contrast channel during the time lapse. The red arrow points to the formation of membrane protrusions under the endothelial monolayer; the green arrow points to the cell completing TEM, as shown by the disappearance of the phase ring. (B) Selected time points of a representative MyoIIA KD B-ALL cell attempting transmigration. The red arrow points to the formation of membrane protrusions under the endothelial monolayer; however, this MyoIIA KD cell never completes TEM, as evidenced by the preservation of the phase ring. (C–F) Of the B-ALL cells that adhered to the endothelial monolayer for at least 5 min, the percentage of B-ALL cells that detached, crawled over the endothelial cells, attempted TEM (evidenced by extension of membrane protrusions underneath the endothelial monolayer), and completed TEM was calculated. (C) MyoIIA KD does not significantly affect leukemia cell detachment from the endothelial cell monolayer. (D) Control and MyoIIA KD B-ALL cells have similar crawling over the endothelial monolayer. (E) MyoIIA KD does not affect the ability of B-ALL cells to attempt TEM. (F) MyoIIA depletion significantly impairs the ability of B-ALL leukemia to complete TEM. (A and B) Data are representative of 3 independent experiments. (C–F) Data are the means ± sem of 3 independent experiments.