Aiming to cure leukemia: specific targeting of malignant stem cells with a novel
combination drug therapy
Chronic myeloid leukemia (CML) is a model disease of cancer as its molecular pathogenesis is particularly well known. It originates from the hematopoietic stem cell (HSC) characterized by the presence of BCR-ABL fusion protein. The first effective molecularly targeted treatment of cancer, tyrosine kinase inhibitor (TKI) imatinib mesylate (IM), has become the standard of care for all newly diagnosed patients. This therapy achieves complete hematological response (CHR) in >95% of cases, complete cytogenetic response (CCR) in approximately 80% of cases and is well tolerated. Despite good clinical efficacy, imatinib is currently not considered as a curative treatment and therefore the treatment is life-long. However, the long-term effects of chronic kinase inhibition lasting years or even decades are mostly unknown and therefore the search for curative alternatives is warranted.
Recently, CML stem cells were found to be resistant to TKIs in vitro. The kinetics of disease relapse after IM therapy discontinuation in most patients implies that stem cell resistance is operative in vivo. This also reflects the dysfunctional tumor immune surveillance characteristic for CML. A proportion of patients develop acquired IMresistance, which most often arises as a result of kinase domain mutations in the stem or progenitor cell compartment that affect IM binding. Thus, due to stem cell persistence, lack of immune control and IM resistance it is unlikely that patients will be cured with IM monotherapy. Although the phenotype of CML stem cell is not well characterized, it is likely that the most primitive leukemic stem cells reside in the CD38-negative fraction of CD34-positive hematopoietic stem cells. The effects of TKI therapy on different fractions of stem cells are largely unknown and most of the published studies have been done with leukemic cells obtained from CML patients at diagnosis. Therefore, the knowledge of the impact of TKI therapy on leukemia stem cell pool in vivo is largely missing and needs further studies addressing this question.
The research group of Holyoake (Glasgow, UK) has recently investigated a range of rational drug combinations in an attempt to target the CML stem cell population. The most effective was the combination of a 2nd generation TKI (dasatinib) with a cytotoxic farnesyl transferase inhibitor (FTI) BMS-214662 (Copland ASH2006), which targets the RAS signal transduction pathway. This combination was able to eliminate human primary leukemic stem cells in a long-term culture and appeared selective for CML as compared to normal stem cells. BMS-214662 has been tested in phase I/II studies and showed activity in acute leukemia with minimal toxicity. The concentration required to induce apoptosis of CML stem cells in vitro is readily achievable in vivo following a 24 hour intravenous infusion. CML is known to be highly immunogenic. Donor immune system cures CML after allogeneic stem cell transplantation. A few patients also appear to be operatively "cured" following interferon alpha therapy, although many of these patients still have detectable minimal residual disease (MRD). Recent immunotherapy approaches using BCR-ABL peptide vaccines also suggest that the establishment of immune surveillance by BCRABL- specific cytotoxic T cells can significantly suppress tumor burden.
Any potentially curative strategy for CML would require initiation in early chronic phase (CP), preferably at the time of diagnosis. The consecutive treatment modules should incorporate
1) a potent TKI to rapidly reduce overall tumor bulk and the progenitor population in an effort to reduce the frequency of kinase domain mutations;
2) an effective in vivo purging agent to induce apoptosis within the stem cell pool;
3) some form of effective immunotherapy to establish immune surveillance and control of MRD and
4) a stopping strategy to determine how many cases have been cured.
Aims of the study
1) To set up a clinical study program aiming in the cure of CML by combination of tyrosine kinase inhibitors, leukemia stem cell targeting and immunotherapy
2) Identify leukemia stem cells and evaluate the effect of various therapies on the stem cell compartment
Patients and methods
Patients. The clinical study program combining these different therapy modalities is now planned and is estimated to start during 2008.
Before this new study will begin, newly diagnosed CML patients in chronic phase are continuously recruited to other ongoing study programs with first and second generation tyrosine kinase inhibitors. The stem cell compartment will be analyzed from CML patients in these other study programs as well.
Laboratory methods for stem characterization and evaluation of immune responses The phenotype of CML stem cell is not well characterized, but most primitive leukemic stem cells reside in the CD38-negative fraction of CD34-positive hematopoietic stem cells. The effects of TKI therapy on different fractions of stem cell are largely unknown. As the proportion of stem cells from bone marrow (BM) cells is very small (1-2% of BM cells are CD34 positive and only <5% of these cells are CD38 negative), the analysis of these rare cells is most demanding and requires state-of-the-art facilities and expertise. Our method is based on automated pre-selection of the CD34 population with paramagnetic beads (Miltenyi AutoMACS) and followed by flow cytometric sorting (BD FACSAria). The proportion of the leukemic stem cells will be analyzed by FISH using a CML-specific BCR-ABL probe. The first experiments have been encouraging (example below) and we now aim to standardize and develop the method further, in collaboration with other participating centers. In addition, the effects of the drug to the normal stem cell pool will be studied with hematopoietic colony forming assays. The separated hematopoietic stem cell fractions will be further analyzed with 6-color flow cytometry and 2-dimensional gel electrophoresis in order to find novel leukemia specific surface proteins in hematopoietic stem cells. In addition to stem cell characterization, mechanisms of CML immunity will be studied in detail. This project is closely related to our previous work (STOP study), in which imatinib treatment was discontinued in a proportion of well responding CML patients with close follow-up monitoring. Basic characterization of the cell populations in BM and PB will be done by flow cytometry using a large panel of monoclonal antibodies targeted against various cell surface and intracytoplasmic antigens present in different types of leukocytes (various lymphocytes, dendritic cells, regulatory T-cells and stem cell fraction) in order to find differences among relapsing and non-relapsing patients and possible prognostic markers behind this phenomenon. In addition, functional analysis will be done using tetramers and by STAT analysis discovering signalling pathways in immune effector cells.
Study group and facilities
The studies will be performed at the Hematology Research Unit (Biomedicum, Helsinki) and at the Department of Medicine, Division of Hematology, HUCH. Docent K. Porkka is the principal investigator of ongoing clinical studies in CML in Finland and Dr. S. Mustjoki is responsible for the laboratory studies. In addition, 3 PhD students will participate in the project. This study is done in collaboration with researchers from Nordic CML study group (Prof. Bengt Simonsson, Uppsala; Dr. Henrik Hjorth-Hansen, Trondheim; Dr. Johan Richter, Lund; Dr. Magnús Magnússon, Reykjavik) and with UK CML study groups (Prof. Tessa Holyoake, Glasgow).
Importance of the study
This study has a major academic and clinical impact in the rapidly developing field of targeted cancer therapy. Most patients with hematological malignancies are still treated with conventional cytotoxic agents with suboptimal results and significant therapy related mortality and morbidity. It is crucial to find novel targets for novel therapies aiming in the final goal of the therapy: cure of the disease. The chosen model disease in this study, CML, is ideal in uncovering answers to these key questions, and the results obtained from this study will be directly applicable for the diagnostics, evaluation of the residual disease and development of curative therapeutic strategies in CML.
In addition to the clinical aspects, this study gives excellent opportunities for translational research combining the aspects of basic cancer research of leukemia stem cells. It will give important knowledge of the phenotype and kinetics of leukemia stem cells and is closely related to the research fields of all groups participating in the study. As the study group consists of several international clinical and basic reseachers, it enables the development of international cooperation, which is crucial for the evolution of clinical care in a rare disease like CML in Finland.
Hi, Aiming to target malignant stem cells....
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Posted 07 March 2011 - 09:14 AM
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