Targeting Isoprenylcysteine Carboxyl Methyltransferase to Overcome Resistance and Improve Responses in Chronic Myeloid Leukemia.
Authors: Tian Sun Wen , Ng Bee Ling , Yang Li Yi , Wang Mei , Hwang William , Melo Junia V. , Druker Brian J. , Casey Patrick J , Chuah Charles 
Published: October 2019

Abstract
Abstract 3273
Poster Board III-1
A strategy to overcome resistance of chronic myeloid leukemia (CML) to Bcr-Abl tyrosine kinase inhibitors (TKI) is by targeting alternative pathways. Increasing evidence suggests that activation of the Ras family of small GTPases is important in leukaemogenic transformation by Bcr-Abl. Ras and Rho GTPases play important roles in the initiation and progression of cancer by disrupting the normal regulation of specific intracellular signalling pathways, promoting cell cycle progression and resistance to apoptosis. The biological function of Ras and Rho GTPases is, to a large extent, dependent on prenylation, a three-step post-translational modification process. The first step is catalysed by farnesyl transferase or geranylgeranyl transferase and the final step is catalysed by isoprenylcysteine carboxyl methyltransferase (Icmt). We have found inhibition of Icmt by a novel selective inhibitor, cysmethynil, enhanced the anti-proliferative effect of imatinib in CML cell lines. The combination of cysmethynil and imatinib was synergistic in inducing apoptosis in the imatinib-sensitive K562 and Bafp210 CML cell lines and apoptosis was further increased when the drugs were given sequentially, with cysmethynil added after imatinib. This combination was also effective in the imatinib-resistant Bcr-Abl- and P-glycoprotein-overexpressing LAMA84 cell line. The synergy was also observed when cysmethynil was combined with dasatinib in both the imatinib-sensitive Bafp210 and imatinib-resistant Bafp210 Y253F mutant but not in the Bafp210 T315I mutant cell lines. The combination therapy was more effective than single agent in inducing apoptosis in treatment-naive primary CML CD34+ cells and was not toxic to cord blood CD34+ cells (Figure 1).
Figure 1 Effect of imatinib and cysmethynil on apoptosis in CD34+ cells. Abbreviations: IM + Cys, imatinib and cysmethynil added concurrently; IM −> Cys, cysmethynil added 9 hours after imatinib; Cys −> IM, imatinib added 9 hours after cysmethynil Figure 1. Effect of imatinib and cysmethynil on apoptosis in CD34+ cells. Abbreviations: IM + Cys, imatinib and cysmethynil added concurrently; IM −> Cys, cysmethynil added 9 hours after imatinib; Cys −> IM, imatinib added 9 hours after cysmethynil
Imatinib decreases p-CrkL through its inhibition of Bcr-Abl kinase. Interestingly, in both K562 and Bafp210, cysmethynil also decreases p-CrkL and when combined with imatinib, p-CrkL levels are reduced further (Figure 2). CrkL activation is dependent on a multimeric protein complex containing PI-3 kinase, docking protein 2, CrkL, Vav and the Rho GTPase, Rac. We hypothesise that inhibition of CrkL phosphorylation in the CML cell lines by cysmethynil is through the inhibition of Rac prenylation. This may be responsible for the synergistic activity observed when cysmethynil is combined with imatinib. In conclusion, our preliminary results suggest that the combination of a Bcr-Abl TKI and an Icmt inhibitor exerts a selective apoptotic effect in CML cells and this combination should be explored further as a useful tool to overcome resistance and improve responses in CML.
Figure 2 Effect of imatinib and cysmethynil on p-CrkL in Bafp210. Figure 2. Effect of imatinib and cysmethynil on p-CrkL in Bafp210.

Disclosures:
Druker: MolecularMD: Equity Ownership; Novartis Pharmaceuticals: ; Bristol-Myers Squibb:.