TKIs might hinder effects of allo-SCT in CML

Inge Jedema, PhD

Barcelona—Researchers have discovered a subgroup of quiescent leukemic stem cells that are resistant to tyrosine kinase inhibitors (TKIs), alloreactive T cells, and natural killer (NK) cells.

This finding indicates that TKIs do not act synergistically with immunological interventions, and the agents might actually hinder the immune response of allogeneic stem cell transplant.

Inge Jedema, PhD, and her colleagues at Leiden University Medical Center in the Netherlands came to these conclusions after extensive study of CD34+ chronic myeloid leukemia (CML) cells. The researchers decided to investigate these cells because the need for life-long treatment so common in CML indicates a small population of leukemic precursor cells escape treatment. 

“Are these cells susceptible to donor T cells mediating the graft vs leukemia effect after allogeneic stem cell transplantation?” Dr Jedema asked. “To address this question, we have developed quantity flow cytometric analysis methods to determine the phenotype as well as proliferative status of CML precursor cells.”

The team first isolated CD34+ CML precursor cells from the peripheral blood or bone marrow of patients with CML and labeled them with fluorescent dyes so single cell proliferation could be monitored. The researchers then used a “cocktail” of cytokines to induce cell proliferation. They found that the majority of cells diluted the dye, but a subpopulation of cells remained fluorescent.

The next step was to expose the CD34+ CML cells to either imatinib (at doses ranging from 1μM to 100μM) or dasatinib (at doses ranging from 0.01nM to 50nM). Though both TKIs did kill the bulk of cells¾and these effects were observed at all dose levels—a subpopulation of CD34+ non-defining CML precursor cells remained. In fact, when the team compared the TKI-treated samples to non-treated controls, they found that the cells had actually increased following treatment with dasatinib and imatinib.

“So there really seems to be a subpopulation of CML precursor cells which is not attacked by TKIs,” Dr Jedema said. “So our research question was, can these cells… be therapeutically targeted with high affinity, minor antigen in our case, specific cytotoxic T lymphocytes?”

To answer this question, Dr Jedema and her colleagues introduced the lymphocytes into samples of non-treated, imatinib-treated, or dasatinib-treated CML cells. But again, the subpopulation of quiescent precursor stem cells remained in all samples.

When the team performed phenotypic analysis, they found the quiescent cells had a significantly lower expression of HLA-A2 than the other CML cells. Unfortunately, attempts to overcome this characteristic with interferon gamma treatment and exogenous peptide loading proved ineffectual, and the quiescent cells remained.

“Then we thought we would be smart,” Dr Jedema said. “We said, if there’s a lower expression of HLA-A2, people working in the NK field always say that there’s an amazing cell concept. So we thought maybe [the quiescent cells are] not a good target for T cells, but maybe they can, due to the amazing cell concept, be very good targets for NK cells.”

So the team isolated NK cells from a fully HLA-matched stem cell donor and made cytotoxic NK cells by adding high-dose IL-2 and IL-15. While the bulk of CML cells were susceptible to the NK cells, the quiescent stem cell population proved resistant yet again.

Dr Jedema presented this research at the 15th Congress of the European Hematology Association, which took place June 10-13.