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. 2024 Aug 8;16(16):2791.
doi: 10.3390/cancers16162791.

SKF-96365 Expels Tyrosine Kinase Inhibitor-Treated CML Stem and Progenitor Cells from the HS27A Stromal Cell Niche in a RhoA-Dependent Mechanism

Audrey Dubourg  1 , Thomas Harnois  1 , Laetitia Cousin  1 , Bruno Constantin  1 , Nicolas Bourmeyster  1   2
Affiliations

SKF-96365 Expels Tyrosine Kinase Inhibitor-Treated CML Stem and Progenitor Cells from the HS27A Stromal Cell Niche in a RhoA-Dependent Mechanism

Audrey Dubourg et al. Cancers (Basel). .

Abstract

Background: A major issue in Chronic Myeloid Leukemia (CML) is the persistence of quiescent leukemia stem cells (LSCs) in the hematopoietic niche under tyrosine kinase inhibitor (TKI) treatment.

Results: Here, using CFSE sorting, we show that low-proliferating CD34+ cells from CML patients in 3D co-culture hide under HS27A stromal cells during TKI treatment-a behavior less observed in untreated cells. Under the same conditions, Ba/F3p210 cells lose their spontaneous motility. In CML CD34+ and Ba/F3p210 cells, while Rac1 is completely inhibited by TKI, RhoA remains activated but is unable to signal to ROCK. Co-incubation of Ba/F3p210 cells with TKI, SKF-96365 (a calcium channel inhibitor), and EGF restores myosin II activation and amoeboid motility to levels comparable to untreated cells, sustaining the activation of ROCK. In CFSE+ CD34+ cells containing quiescent leukemic stem cells, co-incubation of TKI with SKF-96365 induced the expulsion of these cells from the HS27A niche.

Conclusions: This study underscores the role of RhoA in LSC behavior under TKI treatment and suggests that SKF-96365 could remobilize quiescent CML LSCs through reactivation of the RhoA/ROCK pathway.

Keywords: Chronic Myeloid Leukemia; RhoGTPases; SKF-96365; leukemia niche; leukemia stem cells; tyrosine kinase inhibitors.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Imatinib treatment modifies CML-patient CD34+ and Ba/F3p210 cell mobility. (a) CML patients’ CD34+ cells and HS27A cells were stained with 5 µM CFSE and 5 µM CTV, respectively. After 5 h, CFSE-stained CD34+ cells were added to adherent CTV-HS27A cells with or without 5 µM imatinib. After 3 days of co-culture, CFSE-CD34+ and CTV-HS27A were sorted according to CFSE intensity: low CFSE (high proliferation) and high CFSE (low proliferation). (b) The percentage of CD34+ cells hiding under HS27A and the average time spent under HS27A were determined through a time-lapse videomicroscopy analysis (N = 2 patients, n = 5). (c) Time-lapse images (t = 0; t = 3 min; t = 9 min and t = 12 min) extracted from JuliStage recordings of CFSE-stained CD34+ cells from CML patients seeded on CTV-stained HS27A stromal cells in imatinib (5 µM) conditions. Scale bar: 10 µm. Arrowhead shows CD34+ cell hiding under HS27A cells. (d) Cells (0.5 × 105 Ba/F3p210 and Ba/F3p210S509A) were included in 2.5 mg/mL liquid Matrigel diluted in culture medium and cells were incubated with 5 µM imatinib. Each type of displacement (immobile cells, rolling/crawling, or amœboid displacement) was determined through time-lapse videomicroscopy analysis (n > 3). Right panel: spider graph showing the accumulated tracks of analyzed individual cell motility for Ba/F3p210 cells treated or not with imatinib 5 µM. t-test *** p ≤ 0.001.
Figure 2
Figure 2
RhoGTPase signaling in imatinib conditions. (a) Western blot analysis of phosphorylated and total Vav, activated (GTP-Rac1), and total Rac1 in Ba/F3p210 cells. Coimmunoprecipitation of Vav and P-Vav, with p210BCR-ABL. Ba/F3p210 cell lysates were immunoprecipitated using anti-ABL IgG and immunoblotted as indicated. Western blot analysis of activated (GTP-Rac1) and total Rac1 in Ba/F3p210 cells obtained via GST-pulldown. (b) Western blot analysis of activated (GTP-RhoA) and total RhoA in Ba/F3p210 cells obtained via GST-pulldown. Affinity-binding assay of phosphorylated and total p210BCR-ABL with negative dominant RhoA (RhoAG17A). Ba/F3p210 cell lysates were immunoprecipitated with sepharose-bound GST-fused with RhoA-G17A and immunoblotted as indicated. (c) Coimmunoprecipitation of ROCK1 and RhoA with p210BCR-ABL. Ba/F3p210 cell lysates were immunoprecipitated with anti-ABL IgG and immunoblotted as indicated. Western blot analysis of phosphorylated MYPT1 and tubulin in Ba/F3p210 cells. (d) Western blot analysis of activated (GTP-RhoA or GTP-Rac1) and total RhoA or Rac1 in CML patient CD34+ cells obtained via GST-pulldown. Western blot analysis of phosphorylated MYPT1 and β-tubulin in CML patient CD34+ cells. Each experiment was carried out 3 times. Ba/F3p210 and CD34+ patient cells were treated with 1 µM ((d) top), 5 µM imatinib (a–c), or as indicated (d).
Figure 3
Figure 3
SKF-96365 induces amœboid movements in immobile imatinib-treated Ba/F3p210 cells. (a) Cells (0.5 × 105 Ba/F3p210 and Ba/F3p210S509A) were included in 2.5 mg/mL liquid Matrigel diluted in culture medium and incubated with or without 5 µM imatinib and/or 40 µM SKF-96365. Each type of displacement (immobile cells, rolling/crawling, or amœboid displacement) was determined through time-lapse videomicroscopy analysis. The right panel shows the analysis of amoeboid contractions in immobile cells (n > 3). t-test ** p ≤ 0.01; *** p ≤ 0.001; ns: non-significant. (b) Western blot analysis of phosphorylated and total p210BCR-ABL in Ba/F3p210 cells during imatinib and SKF-96365 treatment. (c) Western blot analysis of phosphorylated and total Vav, activated (GTP-Rac1), and total Rac1 in Ba/F3p210 cells during imatinib and SKF-96365 treatment. (d) Upper panel: Western blot analysis of activated (GTP-RhoA) and total RhoA or Rac1 in Ba/F3p210 cells obtained via GST-pulldown. Lower panel: Western blot analysis of phosphorylated MYPT1 and β-tubulin in CML patient CD34+ cells. (e) Coimmunoprecipitation of RhoA with p210BCR-ABL. Ba/F3p210 cell lysates were immunoprecipitated with anti-ABL IgG and immunoblotted as indicated.
Figure 4
Figure 4
EGF in conjunction with SKF-96365 restores Ba/F3p210 cell motility inhibited by imatinib. (a) Cells (0.5 × 105 Ba/F3p210 and Ba/F3p210S509A) were included in 2.5 mg/mL liquid Matrigel diluted in culture medium and incubated with or without 5 µM imatinib and/or 40 µM SKF-96365 and/or EGF 10 ng/mL. Each type of displacement (immobile cells, rolling/crawling, or amœboid displacement) was determined through time-lapse videomicroscopy analysis (n > 3). Right panel: spider graph showing the accumulated tracks of analyzed individual cell motility for Ba/F3p210 cells treated or not with imatinib 5 µM. t-test ** p ≤ 0.01; *** p ≤ 0.001. (b) Analysis of velocity (µm/s) (left) and accumulated distance (µm) (right) of Ba/F3p210 cells treated with SKF-96365 alone or imatinib+ SKF-96365 and EGF. Data were obtained using TrackMate plugins. * p < 0.05; ** p < 0.01; ns: non-significant. (c) Western blot analysis of phosphorylated p210BCR-ABL in Ba/F3p210 cells under the indicated conditions. (d) Upper panel: Western blot analysis of activated (GTP-RhoA) and total RhoA in Ba/F3p210 cells obtained via GST-pulldown. Lower panel: Western blot analysis of phosphorylated MYPT1 and actin in Ba/F3p210 cells. (e) Western blot analysis of activated (GTP-Rac1) and total Rac1, and phosphorylated and total Vav in Ba/F3p210 cells under the indicated conditions.
Figure 4
Figure 4
EGF in conjunction with SKF-96365 restores Ba/F3p210 cell motility inhibited by imatinib. (a) Cells (0.5 × 105 Ba/F3p210 and Ba/F3p210S509A) were included in 2.5 mg/mL liquid Matrigel diluted in culture medium and incubated with or without 5 µM imatinib and/or 40 µM SKF-96365 and/or EGF 10 ng/mL. Each type of displacement (immobile cells, rolling/crawling, or amœboid displacement) was determined through time-lapse videomicroscopy analysis (n > 3). Right panel: spider graph showing the accumulated tracks of analyzed individual cell motility for Ba/F3p210 cells treated or not with imatinib 5 µM. t-test ** p ≤ 0.01; *** p ≤ 0.001. (b) Analysis of velocity (µm/s) (left) and accumulated distance (µm) (right) of Ba/F3p210 cells treated with SKF-96365 alone or imatinib+ SKF-96365 and EGF. Data were obtained using TrackMate plugins. * p < 0.05; ** p < 0.01; ns: non-significant. (c) Western blot analysis of phosphorylated p210BCR-ABL in Ba/F3p210 cells under the indicated conditions. (d) Upper panel: Western blot analysis of activated (GTP-RhoA) and total RhoA in Ba/F3p210 cells obtained via GST-pulldown. Lower panel: Western blot analysis of phosphorylated MYPT1 and actin in Ba/F3p210 cells. (e) Western blot analysis of activated (GTP-Rac1) and total Rac1, and phosphorylated and total Vav in Ba/F3p210 cells under the indicated conditions.
Figure 5
Figure 5
SKF-96365 expels low-proliferating CD34+ cells from the HS27A niche. (a) CML patient CD34+ cells and HS27A cells were stained with 5 µM CFSE and 5 µM CTV, respectively. After 5 h, CFSE-stained CD34+ cells were added to adherent CTV-HS27A cells with or without 5 µM imatinib. After 3 days of co-culture, CFSE-CD34+ and CTV-HS27A were sorted according to CFSE intensity: low CFSE (high proliferation) and high CFSE (low proliferation). CFSE-sorted CD34+ cells from CML patients were reseeded on previously cultured CTV-stained HS27A cells and cultured for 2 days under the indicated conditions. Then, videomicroscopy was performed using JuliStage in fluorescent mode during 6 h recording. (b) The percentage of CD34+ cells hiding under HS27A cells and the average time spent under HS27A cells were determined through time-lapse videomicroscopy analysis (N = 2 patients, n = 5). (c) Analysis of the hiding behavior of low-proliferating CD34+ cells from CML patients during the recording time. Two behaviors were identified corresponding to a short-duration stay under HS27A cells (SD; <30 min) and a long-duration stay (LD; 30 to >300 min) (see Supplementary Figure S5 for details).
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