Immunorecognition of leukemic stem cells by NK cells : the role of HDAC inhibitors in NKG2D ligand-mediated anti-tumor responses in acute myeloid leukemia

The diagnosis of acute myeloid leukemia (AML) is associated to a poor long-term
outcome due to frequent relapse despite intensive chemotherapy, radiation and
hematopoietic stem cell transplantation (HSCT) as well as continuous advances in
treatment modalities. Relapses might be caused by leukemic stem cells (LSC).
According to a recently emerging concept, LSC display many features of normal
hematopoietic stem cells (HSC) like quiescence and self renewal capacity and
therefore are poorly accessible for conventional therapies which primarily reach the
rapidly proliferating cells. Additionally, LSC are apparently able to escape from
immunorecognition and thereby sustain the disease. NK cells, as the main innate
immune effectors against tumor cells, are able to recognize and kill malignant cells
when triggered by cell surface expression of a multitude of activating ligands.
The best-described receptor-ligand pair in humans is NKG2D and its ligands, ULBP
and MICA/B. Furthermore, NCR is an important family of activating receptors on
NK cells, whose ligands are not yet known. The regulation of NK cells is completed
by several inhibitory receptors (KIR) specific for different HLA class I molecules on
potential target cells. While preceding work in our lab was describing the interaction
between NK cells and leukemic blasts of AML, there is no information available on
the recognition of LSC by NK cells.
In this study we aimed to elucidate the interaction of NK cells with LSC of AML.
The cell surface expression of ligands for activating and inhibitory NK cell receptors
on LSC was in focus of these studies. Moreover, we applied a pharmacological
approach to treat the patient-derived primary AML leukemic cells and examined the
consequences for cell surface expression of NK cell-specific ligands. By employing
hematopoietic colony forming assays, cytotoxicity assays as well as in vivo
NOD/SCID xenotransplantation we aimed to functionally assess the implications of
the upregulation of activating ligands for NK cell immunorecognition of LSC.
In initial experiments, we demonstrated that activating ligands for the
NKG2D receptor and NCR receptors on NK cells are absent or only weakly expressed
on the surface of patient derived AML blasts. This expression could be increased by
pharmacological means applying bryostatin-1, a modulator of PKC activity.
Upregulation of cell surface expression of NKG2D ligands on AML blasts led to increased immunorecognition by NK cells in cytotoxicity assays. Subsequently, we
demonstrated that similarly to total blasts, LSC of AML as judged by the phenotype
CD45dimCD34+CD38-, did not express ULBP and MICA/B on their surface.
To pharmacologically increase their expression, we employed the HDAC inhibitor
valproic acid (VA), a drug acting through epigenetic modification of gene expression
and having long-term records in different clinical applications. This treatment with
VA proved to be of importance for the immunorecognition by NK cells. In the
functional assays we employed NK cells selected for the KIR-HLA class I mismatch
in order to circumvent inhibitory signals inactivating the NK cells. Serial replating
colony forming unit (CFU) assays with LSC after treatment with VA and after
coincubation with KIR-HLA mismatched NK cells demonstrated an efficient
reduction in colony formation capacity upon this synergistic treatment.
The cytotoxicity assays with VA-treated LSC as targets and KIR-HLA mismatched
NK cells as effectors revealed interindividual differences among patient samples,
reflecting a complex regulation of NK cell activation and immunorecognition.
Altogether, a direct interaction of NK cells and LSC could be demonstrated in vitro.
In the in vivo setting, by transplantation of AML cells intrafemurally into NOD/SCID
mice with consecutive treatment of VA and HLA-mismatched NK cells, we were able
to achieve a stable engraftment of human AML in the mouse bone marrow. However,
the combined treatment with VA and NK cells was not influencing the content of
malignant cells as compared to untreated mice. The ongoing studies aim at
optimization of AML treatment with NK cell-based immunotherapy in the preclinical
NOD/SCID transplantation model.
Taken together, these results showed the potential of VA as an applicable
anti-neoplastic drug to enhance immunorecognition of LSC of AML by NK cells,
mediated by increased cell surface expression of activating ligands. The functional
consequences of an enhanced immunorecognition by NK cells in abolishing the
colony forming capacity of patient derived LSC are promising beneficial effects for
innovative AML treatments in future.