Investigating the role of rho kinase in the survival of CML cells


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Leena Mukherjee1,Cassie Clarke1,Heather Jorgensen1,Mhairi Copland1,Mike Olson2,Tessa Holyoake1
1University of Glasgow,2Ryerson University

Abstract

Background

Tyrosine kinase inhibitors (TKI) form the cornerstone of chronic myeloid leukaemia (CML) therapy.  However, stem cell persistence, resistance and advanced disease remain.  The rho kinase (ROCK) pathway is important in invasion and metastasis.  In-house microarray data shows the ROCK pathway is upregulated in CML CD34+ cells and does not normalise despite TKI treatment.

Method

CML primary patient samples and immortalised cell lines were treated in vitro with ROCK inhibitor (RI) H-1152 alone and in combination with nilotinib (nil), a TKI, to investigate the effects of targeting the ROCK pathway. 

Results

In TKI sensitive and resistant KCL22 and Ba/F3 CML immortalised cell lines, there is increased growth inhibition with H-1152 in combination with nil versus nil alone (p<0.0001; n=3) with synergism between compounds (CI =0.658).  Apoptosis is increased with RI+nil versus nil alone (p<0.0001; n=3).   

Western blotting (WB) indicates proteins downstream of ROCK remain expressed despite nil treatment.  These are undetectable following RI treatment.  Lentiviral knockdown (KD) of ROCK1 in KCL22WT cell lines corroborates this - ROCK inhibition alone affects cell survival, with a 39% decrease in cell viability (p=0.006) compared to scrambled control.  The combination of ROCK KD and nil was more lethal than nil alone, with a 31% decrease in cell viability in KD cells versus transfected controls (p=0.04) treated with nil.

In primary samples, WB shows CML CD34+ cells have a higher ROCK expression than normal CD34+ cells.  CML CD34+ cells are more sensitive to RI than non-CML CD34+ cells (n=2), giving a clear therapeutic window, as shown by an 89 and 29% increase in growth inhibition and apoptosis respectively, and a 48% decrease in colony formation following treatment with RI.  Primary CML CD34+ cells treated with RI+nil are unable to form colonies in colony forming assay.

Conclusion

ROCK is a promising new target in both TKI sensitive and resistant CML.