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[valiantchong]

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.

[valiantchong]

While imatinib and other tyrosine kinase inhibitors (TKIs) are effective in the treatment of chronic myeloid leukemia (CML), patients can fail all of these therapies. One reason for this is that TKIs have only limited activity against CD34+CD38? leukemic stem cells in CML. Recently, we demonstrated that the IL3 receptor (IL3R) is highly expressed on CD34+38- Bcr-Abl(+) CML stem cells and represents an attractive target for therapeutic intervention (ASH 2009 114: Abstract 2172). IL3R overexpression has also been demonstrated on leukemic stem cells of other hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Moreover, IL3R is a clinically validated target, as demonstrated by anti-tumor activity including complete and partial responders with the IL3R targeting agent, SL-401, in a Phase I clinical trial of patients with advanced AML and MDS. However, IL3R has not yet been fully investigated as a therapeutic target in CML. Accordingly, in this study, we examined whether targeting IL3R with SL-401 and SL-501, two recombinant biologic agents, could eradicate CML stem cells. SL-401, which is comprised of human IL3 recombinantly conjugated to diphtheria toxin (DT), has been shown to eradicate AML stem cells in both in vitro and in vivo experimental systems. SL-501 is a second generation IL3R targeting agent that contains an optimized IL3 sequence, which increases its affinity for IL3R. Both SL-401 and SL-501 are directed to IL3R on the leukemic cell surface, thereby triggering receptor-mediated endocytosis, inhibition of protein synthesis, and induction of apoptosis. In this study we demonstrated that SL-401 and SL-501 significantly inhibited the growth of CML cells (p ? 0.00009) and induced apoptosis (p ? 0.003) in 14 primary CML samples. In six primary CML samples, these agents reduced the absolute numbers of viable CD34+/CD38-/CD123+ CML progenitor cells (p ? 0.03) by inducing apoptosis (52.2 ± 9.3% and 65.5 ± 10.7% for SL-401 and SL-501, respectively). To evaluate the effect of these agents on the growth of the most primitive stem cells, CML blasts were pretreated with IL3R targeted agents for 24 h and grown in the Long-Term Culture-Initiating Cell assay. SL-401 and SL-501 significantly reduced formation of hematopoietic colonies from primary CML samples in a dose-dependent manner (colony formation reduced by 54.4-80.5% and 66.6-75.1% for SL-401 and SL-501, respectively; p ? 0.009). Similar results were obtained when growth of CML progenitor cells was assessed in the Colony Forming Cell assay (colony formation reduced by 54.0-70.2% and 71.1-84.4% for SL-401 and SL-501, respectively; p ? 0.0002; N = 6). Notably, the majority of primary samples were obtained from patients resistant to TKIs (12 of 15) and/or harboring an abl mutation including T315I (7 of 15). Moreover, the combination of these IL3R targeted agents with imatinib demonstrated synergistic effects (CI < 1.0) against the KBM5 CML cell line and its TKI resistant KBM5STI subline, which harbors the T315I mutation. In addition, combination of these IL3R directed agents with imatinib further enhanced the apoptotic rate of primary CML cells (N = 5). Importantly, both SL-401 and SL-501 demonstrated high anti-leukemic activity in vivo when NOD/SCID/IL2Rg-KO mice were transplanted with leukemic cells from patients with primary myeloid blast crisis CML (median survival: vehicle = 37 d, SL-401 = 48 d, and SL-501 = 57 d; p = 0.0005). In conclusion, these data indicate that SL-401 and SL-501, two active IL3R directed agents, represent a novel therapeutic modality for the selective targeting of CML stem cells. Combinations of these agents with TKIs may also benefit CML patients who are resistant to TKI treatment by reducing and/or eliminating leukemic stem cells.

[jwbwater]

Hadn't heard of this drug before, nice find.