Homing in on CML stem cells
by Kai-Jye Lou, Staff Writer
Researchers at Lund University have found a cell surface marker called IL-1 receptor accessory protein that could be used to isolate and kill chronic myelogenous leukemia stem cells.1Cantargia AB, a biotech founded based on the findings, is now developing a mAb to attack CML at its root by killing the stem cells, which respond poorly to standard CML treatments and are believed to spawn the leukemic cells that characterize the disease.
The majority of CML cases are characterized by cells carrying the Philadelphia chromosome, which contains the oncogenic BCR-ABL fusion protein. Tyrosine kinase inhibitors (TKIs) like Gleevec imatinib are very effective at putting the disease into remission and prolonging patient survival by inhibiting the BCR-ABL tyrosine kinase. However, CML patients require chronic daily treatment with TKIs, and interruption or discontinuation of therapy almost invariably results in disease relapse.
Novartis AG markets Gleevec to treat multiple cancers, including CML. The company also markets a second-generation TKI, Tasigna nilotinib, for Philadelphia chromosome-positive (Ph+) CML. Bristol-Myers Squibb Co. markets another second-generation TKI, Sprycel dasatinib, to treat CML and Ph+ acute lymphoblastic leukemia (ALL) patients who are resistant or intolerant to prior therapy, including Gleevec.
Mature and differentiated CML cells, which are readily killed by TKIs, are spawned from a small population of self-renewing stem cells that carry the Philadelphia chromosome.2 Earlier studies have shown that these CML stem cells are partially resistant to TKIs and are likely to be the culprits in disease relapse.3, 4, 5
The problem is that CML stem cells have been difficult to isolate and study because there were no cell surface markers that would allow for prospective separation of normal hematopoietic stem cells from CML stem cells.
Although the Philadelphia chromosome is the putative marker for CML, the chromosome and its associated BCR-ABL tyrosine kinase are located within the cell. In general, intracellular molecules cannot be used for prospective stem cell isolation and are difficult to target with therapeutic antibodies.
The Lund researchers have now identified a potential cell surface marker by carrying out a microarray-based gene expression profiling study comparing samples from CML patients and healthy controls. The study pinpointed IL-1 receptor accessory protein (IL-1RAP) as a cell surface marker that is upregulated in CML cells but not in normal bone marrow cells.
"We were actively looking for cell surface markers that are upregulated on leukemic cells and targetable for diagnostic and therapeutic purposes," said Thoas Fioretos, a professor and senior consultant in the Department of Clinical Genetics at the university.
His group analyzed IL-1RAP expression in a stem cell-enriched subpopulation of cells isolated from five CML patients and found that 99.9% of cells in the IL-1RAP+ group carried the Philadelphia chromosome. In contrast, only 2.9% cells in the IL-1RAP? group had the chromosome.
After showing that IL-1RAP is a highly specific marker on the surface of CML stem cells, the Lund researchers carried out a preliminary evaluation of IL-1RAP as a potential therapeutic target.
In both stem cell-enriched cell populations from patients and human leukemia cell lines, a polyclonal rabbit antibody targeting human IL-1RAP increased antibody-dependent cellular cytotoxicity (ADCC) against Ph+ cells compared with that against Ph? cells. Importantly, the antibody did not induce ADCC in normal bone marrow cells isolated from the stem cell-enriched subpopulation.
Results were published in the Proceedings of the National Academy of Sciences.
"We have identified a marker on the cell surface that could make it easy to sort out candidate CML stem cells from a population of normal hematopoietic stem cells," said Fioretos, who is corresponding author on the paper. "We found that if you sort the stem cell-enriched cell population based on IL-1RAP, you could get a cell population where almost every cell contains the Philadelphia chromosome, which characterizes CML. By performing long-term cultures, we should demonstrate that the sorted population of cells contains candidate CML stem cells."
The study "is an interesting example of cell surface target discovery using microarrays," said Thomas Cirrito, director of operations at Stemline Therapeutics Inc. "What they've presented here is that the IL-1 receptor accessory protein appears to be a cell surface marker for Philadelphia chromosome-positive CML stem cells, which would make it a valuable tool for prospectively isolating this population of cells."
Stemline's lead program is a biologic that targets the IL-3 receptor, which is overexpressed on leukemic blasts and leukemia stem cells in multiple hematological malignancies including CML, acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). The compound is in a Phase I/II trial in patients with advanced AML and MDS. In in vitro and in vivo CML models, including samples from patients with TKI-resistant disease, SL401 has been shown to induce apoptosis in both CML blasts and CML stem cells.