Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Nov 15;11(22):6761.
doi: 10.3390/jcm11226761.

Preclinical Evaluation of a Novel Small Molecule Inhibitor of LIM Kinases (LIMK) CEL_Amide in Philadelphia-Chromosome Positive (BCR::ABL+) Acute Lymphoblastic Leukemia (ALL)

Jeannig Berrou  1 , Mélanie Dupont  1 , Hanane Djamai  1 , Emilie Adicéam  1 , Véronique Parietti  2 , Anna Kaci  1 , Emmanuelle Clappier  3 , Jean-Michel Cayuela  1   3 , André Baruchel  1   4 , Fabrice Paublant  5 , Renaud Prudent  5 , Jacques Ghysdael  6 , Claude Gardin  1   7 , Hervé Dombret  1   8 , Thorsten Braun  1   7
Affiliations

Preclinical Evaluation of a Novel Small Molecule Inhibitor of LIM Kinases (LIMK) CEL_Amide in Philadelphia-Chromosome Positive (BCR::ABL+) Acute Lymphoblastic Leukemia (ALL)

Jeannig Berrou et al. J Clin Med. .

Abstract

Ph+ (BCR::ABL+) B-ALL was considered to be high risk, but recent advances in BCR::ABL-targeting TKIs has shown improved outcomes in combination with backbone chemotherapy. Nevertheless, new treatment strategies are needed, including approaches without chemotherapy for elderly patients. LIMK1/2 acts downstream from various signaling pathways, which modifies cytoskeleton dynamics via phosphorylation of cofilin. Upstream of LIMK1/2, ROCK is constitutively activated by BCR::ABL, and upon activation, ROCK leads to the phosphorylation of LIMK1/2, resulting in the inactivation of cofilin by its phosphorylation and subsequently abrogating its apoptosis-promoting activity. Here, we demonstrate the anti-leukemic effects of a novel LIMK1/2 inhibitor (LIMKi) CEL_Amide in vitro and in vivo for BCR::ABL-driven B-ALL. The IC50 value of CEL_Amide was ≤1000 nM in BCR::ABL+ TOM-1 and BV-173 cells and induced dose-dependent apoptosis and cell cycle arrest in these cell lines. LIMK1/2 were expressed in BCR::ABL+ cell lines and patient cells and LIMKi treatment decreased LIMK1 protein expression, whereas LIMK2 expression was unaffected. As expected, CEL_Amide exposure caused specific activating downstream dephosphorylation of cofilin in cell lines and primary cells. Combination experiments with CEL_Amide and BCR::ABL TKIs imatinib, dasatinib, nilotinib, and ponatinib were synergistic for the treatment of both TOM-1 and BV-173 cells. CDKN2Ako/BCR::ABL1+ B-ALL cells were transplanted in mice, which were treated with combinations of CEL_Amide and nilotinib or ponatinib, which significantly prolonged their survival. Altogether, the LIMKi CEL_Amide yields activity in Ph+ ALL models when combined with BCR::ABL-targeting TKIs, showing promising synergy that warrants further investigation.

Keywords: ALL; BCR::ABL; CEL_Amide; LIM kinase; tyrosine kinase inhibitors.

PubMed Disclaimer

Conflict of interest statement

T.B. and H.D. received research funding from CELLIPSE Company. R.P. and F.P. are employed by CELLIPSE company. All other authors have no conflict of interest.

Figures

Figure 1
Figure 1
In vitro effect of LIMKi on viability, apoptosis, and cell cycle in BCR::ABL1+ ALL cell lines. IC50 was determined by MTS assay after exposure to increasing doses of CEL_Amide (78–10,000 nM) in indicated cell lines at 72 h. Results are shown as mean ± SEM from triplicate (n = 3) (A). Apoptosis of BV-173 and TOM-1 cell lines after 48 and 72 h treatment with different doses of CEL_Amide (250–1500 nM). Apoptotic cells were defined as Annexin V+ with or without PI uptake. Results are shown as mean ± SEM from duplicate (n = 3) (B). Cell cycle modifications at 48 h induced by increasing CEL_Amide doses (500–1500 nM) in BV-173 and TOM-1 cell lines (n = 3) (C).
Figure 2
Figure 2
LIMK1 and LIMK2 basal expression and in vitro effect of LIMKi on LIMK1, LIMK2, cofilin, and phospho-cofilin protein expression in BCR::ABL1+ ALL cell lines and patient cells. LIMK1 and LIMK2 basal gene expression in BV-173 and TOM-1 cell lines were determined by RT-qPCR. Results are shown as mean ± SEM from triplicates (n = 3) (A). Western blot showing protein changes of LIMK1, LIMK2, cofilin, and phospho-cofilin in BV-173 and TOM-1 cell lines after 72 h exposure to increasing CEL_Amide doses (250–1500 nM). GAPDH was used as a loading control. One representative experiment out of three is shown (B). Western blot showing basal LIMK1 and phospho-cofilin expression in healthy donors (n = 3) and BCR::ABL+ ALL patients (n = 4) ex vivo (C). Densitometry for LIMK1, LIMK2, cofilin, and phospho-cofilin proteins were reported as a ratio with GAPDH.
Figure 3
Figure 3
Drug combinations of LIMKi with BCR::ABL-targeting TKI inhibitors in BCR::ABL1+ ALL cell lines. BV-173 and TOM-1 cell lines were exposed for 72 h to increasing doses of different BCR::ABL inhibitors, including imatinib, dasatinib, and nilotinib or ponatinib, in combination with LIMKi CEL_Amide. The dose-response matrix was made according to the Loewe model. Results are shown from duplicates of 3 independent experiments (A). Apoptosis was measured in BV-173 and TOM-1 cell lines after 48 and 72 h exposure to CEL_Amide (1500 nM), nilotinib (25 nM), and ponatinib (1 nM), alone or in combination. Results are shown as mean ± SEM from duplicates (n = 3) (B).
Figure 4
Figure 4
Effects of LIMKi in vivo. To evaluate LIMK1/2 inhibition by CEL_Amide in vivo, C57BL/6J mice were engrafted with GFP+ CDKN2Ako/BCR::ABL1+ B-ALL mice cells. Mice were treated with vehicle (group 1; n = 5); CEL_Amide alone (group 2; n = 5); nilotinib or ponatinib (group 3; n = 10); or a combination of CEL_Amide and nilotinib or ponatinib (group 4; n = 10) (A). Kaplan–Meyer survival curves for mice according to treatment combination of CEL_Amide with nilotinib (B). Kaplan–Meyer survival curves for mice according to treatment combination of CEL_Amide with ponatinib (C).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Samra B., Jabbour E., Ravandi F., Kantarjian H., Short N.J. Evolving therapy of adult acute lymphoblastic leukemia: State-of-the-art treatment and future directions. J. Hematol. Oncol. 2020;13:70. doi: 10.1186/s13045-020-00905-2. - DOI - PMC - PubMed
    1. Moorman A.V., Chilton L., Wilkinson J., Ensor H.M., Bown N., Proctor S.J. A population-based cytogenetic study of adults with acute lymphoblastic leukemia. Blood. 2010;115:206–214. doi: 10.1182/blood-2009-07-232124. - DOI - PubMed
    1. Thomas X., Thiebaut A., Olteanu N., Danaila C., Charrin C., Archimbaud E., Fiere D. Philadelphia chromosome positive adult acute lymphoblastic leukemia: Characteristics, prognostic factors and treatment outcome. Hematol. Cell Ther. 1998;40:119–128. - PubMed
    1. Foà R., Bassan R., Vitale A., Elia L., Piciocchi A., Puzzolo M.-C., Canichella M., Viero P., Ferrara F., Lunghi M., et al. Dasatinib–Blinatumomab for Ph-Positive Acute Lymphoblastic Leukemia in Adults. N. Engl. J. Med. 2020;383:1613–1623. doi: 10.1056/NEJMoa2016272. - DOI - PubMed
    1. Jain N., Maiti A., Ravandi F., Konopleva M., Daver N., Kadia T., Pemmaraju N., Short N., Kebriaei P., Ning J., et al. Inotuzumab ozogamicin with bosutinib for relapsed or refractory Philadelphia chromosome positive acute lymphoblastic leukemia or lymphoid blast phase of chronic myeloid leukemia. Am. J. Hematol. 2021;96:1000–1007. doi: 10.1002/ajh.26238. - DOI - PMC - PubMed

Grants and funding