Transcriptome of Expressed Genes in CD34+ and CD66b+ Chronic Myeloid Leukemia Cells and Its Potential Role in the Transport of Kinase Inhibitors

Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterized by Phildaelphia chromosome and by formation of BCR-ABL fusion. Some studies have shown that residual cells are part of the leukemic cells undifferentiated compartment. In 2005, Michor et al. (Nature 435: 1267–1270), through mathematical model, concluded that imatinib efficiently reduces the differentiated leukemic cells population, but it has not the same effect on the cell population that drives this disease, the CD34+ leukemic stem cells, which can be kept alive during the treatment. However, Mahon et al. (Lancet Oncol 11: 1029–35, 2010) described a cohort in which patients remained disease free for 18 months after discontinued treatment. This finding is an indication that the leukemia stem cells (LSC) are not totally insensitive to kinase inhibitors (KIs).

Identify the expressed genes in CD34+ and CD66b+ cells as candidates for KIs transport.

Samples of bone marrow (BM) and peripheral blood (PB) were obtained from five patients with CML treated with imatinib in optimal response in according to European LeukemiaNet criteria. Cells Isolation and RNA extraction. CD34+ cells were isolated from BM of five patients with CML. Mature CD66b+ PB cells were isolated from the same patients. SOLiD sequencing. cDNA was sequenced according to the manufacturer's protocols for the SOLiD. Transcriptome. Two libraries were constructed for this purpose and approximately 120 millions of beads were deposited on a half slide for each library and sequenced using the Opti Fragment Library Sequencing kit-Master Mix 50 on a SOLiD machine (Ver 3+). To characterize the genes showing differential regulation, we analyzed the Gene Ontology (GO) annotation associated with transporters genes exhibiting a greater than 2-fold difference in expression by RNA-seq. Analysis using the functional annotation clustering feature of DAVID.

We have sequenced 14.133 genes in CD34+ pool cells and 3.379 genes in CD66b+ pool cells, with 2.883 genes expressed in both. Of these, 1.201 genes from membrane transport were functionally annotated, and we have found 560 genes expressed exclusively in CD34+, and 99 genes in CD66b+, and 542 genes in both, as shown below.

Regarding imatinib transportation, two major classes of transporters are widely recognized for its importance in drug influx and efflux inside the cell, the ATP-biding cassette transporters (ABC family) and Solute Carrier family (SLC family). Studies have demonstrated that the organic cation transporter 1 (OCT-1, also known as SLC22A1) is the major active influx transporter for imatinib in CML cells, in our study we find no evidence of OCT-1 in any of our samples, which may indicate that this is not the channel for the influx of drugs into cells. Within ABC family is already recognized the important function of at least three genes in multidrug-resistance cancer through the mechanism of drug efflux through the membrane, they are: ABCB1 (MDR1); ABCG2 (Breast Cancer Resistance Protein – BCRP) e ABCC1 (MRP1). These genes were found exclusively in the CD34+ lineage of CML patients, reinforcing the theory that HSC are resistant to treatment with imatinib, through the expression of efflux channels.

The efflux channel genes exclusively expressed in CD34+ cells represents a major barrier to maintaining optimal response to KIs in long-term treatment of CML patients. Those genes should be investigated to achieve the development of drugs with potential to block the efflux channels and improve outcome for cancer patients.

Lemos:Novartis Oncology: Research Funding.