Tyrosine Kinase Inhibitor Independent Gene Expression Signature in CML Offers New Targets for LSPC Eradication Therapy

Abstract

Tyrosine kinase inhibitors (TKI) have revolutionised the treatment of CML. However, TKI do not eliminate the leukaemia stem cells (LSC), which can re-initiate the disease. Thus, finding new therapeutic targets in CML LSC is key to finding a curative treatment. Using microarray datasets, we defined a list of 227 genes that were differentially expressed in CML LSC compared to the healthy controls but were not affected by TKI in vitro. Two of them, CD33 and PPIF, are targeted by gemtuzumab–ozogamicin and cyclosporin A, respectively. We treated CML and the control CD34+ cells with either drug with or without imatinib to investigate the therapeutic potential of the TKI-independent gene expression programme. Cyclosporine A, in combination with imatinib, reduced the number of CML CFC compared with non-CML controls, but only at supra-therapeutic concentrations. Gemtuzumab–ozogamicin showed an EC50 of 146 ng/mL, below the plasma peak concentration of 630 ng/mL observed in the AML patients and below the EC50 of 3247 ng/mL observed in the non-CML cells. Interestingly, gemtuzumab–ozogamicin seems to promote cell cycle progression in CML CD34+ cells and demonstrated activation of the RUNX1 pathway in an RNAseq experiment. This suggests that targeting the TKI-independent genes in CML LSC could be exploited for the development of new therapies in CML.

GAS2 Upregulation Is a Targetable Vulnerability in Chronic Myeloid Leukemia

“…Another interesting therapeutic approach to eliminate LSCs has been to target genes the mRNA expression of which is not affected by TKI treatment. For example, Gómez-Castañeda et al showed that CD33 and PPIF upregulation was a signature of TKI-treated LSCs and could be targeted in vitro by gemtuzumab-ozogamicin (an anti-CD33 conjugated monoclonal antibody) and cyclosporin A, respectively [18]. In general, further investigation and novel approaches are still needed to address the persistence of LSCs in CML that have potential to translate to the clinic.…”

“…Cell numbers were counted by hemocytometer using trypan blue dye exclusion [18] (Sigma-Aldrich Co., Ltd., Gillingham, Dorset, UK), or resazurin assay [19] using 10 µL of 0.5 µM resazurin (Sigma-Aldrich) per 100 µL of sample according to manufacturer's instructions (Sigma-Aldrich). For experiments involving treatment, 2 × 10 5 cells/mL K562 and 1 × 10 5 cells/mL KCL-22 cells were plated and cultured for 72 h with concentrations ranging from 0.3 to 60 µM XK469 (racemic free acid, NSC 697887, Sigma-Aldrich) and/or 10 to 3000 nM IM (Stratec Scientific Ltd., Cambridge, Ely, UK), both diluted stocks reconstituted in DMSO.…”