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

## TL;DR

This study shows that SKF-96365 can expel quiescent leukemia stem cells from their protective niche during tyrosine kinase inhibitor treatment, potentially improving CML therapy.

## Contribution

The novel finding is that SKF-96365, via RhoA activation, can remobilize quiescent CML stem cells under tyrosine kinase inhibitor treatment.

## Key findings

- TKI-treated CML stem and progenitor cells regain motility when exposed to SKF-96365.
- SKF-96365 induces expulsion of quiescent CML stem cells from the HS27A stromal niche.
- RhoA activation is essential for this effect, independent of BCR-ABL's tyrosine kinase activity.

## Abstract

Tyrosine kinase inhibitors (TKIs), particularly imatinib, have afforded Chronic Myeloid Leukemia (CML) patients long-term remission. This revolution in leukemia treatment is unfortunately accompanied by the fact that treatment interruption, in most cases, leads to the re-emergence of BCR-ABL-leukemia cells, caused by leukemia stem cells, that are insensitive to TKI treatment. Moreover, TKIs paradoxically induce a quiescent state in leukemia stem and progenitor cells. Current CML studies focus on these quiescent leukemia stem cells in order to reactivate them and restore TKI sensitivity. We show here that under TKI treatment, leukemia stem cells retrieve motility in a reconstituted niche when incubated with SKF96365, a calcium channel inhibitor, and that this effect is dependent on RhoA activation by BCR-ABL independently of its tyrosine kinase activity. Altogether, these results suggest that SKF-96365 or its derived molecules could be interesting compounds for targeting quiescent CML stem and progenitor cells under TKI treatment.

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.