The retinoic acid receptor/CaMKII interaction: pharmacologic inhibition of CaMKII enhances the differentiation of myeloid leukemia cells.

Certain myeloid leukemia cells, particularly the acute promyelocytic leukemia (APL) subset, undergo terminal granulocytic differentiation in response to retinoic acid (RA). RA mediates its biologic effects through specific retinoic acid receptors (RARs) which serve as ligand-activated nuclear transcription factors. The Ca(++)/calmodulin-dependent protein kinases (CaMKs) are multifunctional serine/threonine kinases that are regulated by Ca(++) signaling. We have observed significant cross-talk between these Ca(++) and RA signaling pathways that regulates the differentiation of myeloid leukemia cells. We observe that CaMKIIgamma is the CaMK that is predominantly expressed in myeloid cells. This enzyme localizes to the promoter of RAR target genes, physically interacts with and phosphorylates RARalpha and inhibits RAR transcriptional activity. KN-62, a pharmacological inhibitor of the CaMKs, enhances both retinoic acid receptor transcriptional activity as well as the terminal in vitro differentiation of certain myeloid leukemia cell lines including HL-60. However, this compound, as well as related synthetic analogs that enhance HL-60 terminal differentiation, fails to inhibit the growth of HL-60 xenografts in NOD-SCID mice likely because of the unfavorable pharmacokinetics displayed by these compounds. Nevertheless, our observations suggest that CaMKIIgamma may provide a new therapeutic target for the treatment of the RA-responsive human myeloid leukemias.

CaMKII regulates retinoic acid receptor transcriptional activity and the differentiation of myeloid leukemia cells.

Retinoic acid receptors (RARs) are members of the nuclear hormone receptor family and regulate the proliferation and differentiation of multiple different cell types, including promyelocytic leukemia cells. Here we describe a biochemical/functional interaction between the Ca(2+)/calmodulin-dependent protein kinases (CaMKs) and RARs that modulates the differentiation of myeloid leukemia cells. We observe that CaMKIIgamma is the CaMK that is predominantly expressed in myeloid cells. CaMKII inhibits RAR transcriptional activity, and this enzyme directly interacts with RAR through a CaMKII LxxLL binding motif. CaMKIIgamma phosphorylates RARalpha both in vitro and in vivo, and this phosphorylation inhibits RARalpha activity by enhancing its interaction with transcriptional corepressors. In myeloid cell lines, CaMKIIgamma localizes to RAR target sites within myeloid gene promoters but dissociates from the promoter upon retinoic acid-induced myeloid cell differentiation. KN62, a pharmacological inhibitor of the CaMKs, enhances the terminal differentiation of myeloid leukemia cell lines, and this is associated with a reduction in activated (autophosphorylated) CaMKII in the terminally differentiating cells. These observations reveal a significant cross-talk between Ca(2+) and retinoic acid signaling pathways that regulates the differentiation of myeloid leukemia cells, and they suggest that CaMKIIgamma may provide a new therapeutic target for the treatment of certain human myeloid leukemias.

KN-62 analogues as potent differentiating agents of HL-60 cells.

KN-62, an inhibitor of the calmodulin-dependent protein kinases (CaMKs), enhances the terminal differentiation of retinoic acid sensitive human myeloid leukemia cell lines. In an effort to identify additional CaMK inhibitors that exhibit more potent activity in triggering leukemia cell differentiation, we synthesized 45 analogues of KN-62 and determined their ability to induce HL-60 cell differentiation. Sixteen of these novel analogues exhibited significant differentiation-inducing activity, and one analogue, AS-004, was five times more potent than KN-62 in inhibiting proliferation and inducing differentiation of HL-60 cells. Such KN-62 analogues and/or related compounds may prove useful in treating promyelocytic leukemia.

The cytokines IL-3 and GM-CSF regulate the transcriptional activity of retinoic acid receptors in different in vitro models of myeloid differentiation.

The disruption of retinoic acid receptor (RAR) activity that characterizes human acute promyelocytic leukemia (APL) is associated with a block to granulocytic differentiation indicating that RARs are critical regulators of normal myeloid differentiation. Nevertheless, how RAR activity might be regulated in the presumably homogenous concentration of retinoids in blood and bone marrow and how these receptors might interact with specific hematopoietic cytokines to regulate normal myeloid differentiation remain unclear. Here, using several cytokine-dependent in vitro models of myeloid development, it was observed that specific hematopoietic cytokines that normally regulate myeloid lineage commitment and differentiation (interleukin-3 and granulocyte-macrophage colony-stimulating factor) trigger the enhancement of both ligand-dependent and ligand-independent transcriptional activity of both endogenous and exogenous (transiently transfected) RARs. This cytokine-mediated enhancement of RAR activity is not associated with any observed changes in expression of the RARs or their respective coactivators/corepressors. These studies define a previously unknown cytokine-RAR interaction during myelopoiesis and suggest that RAR activation might be a critical downstream event following interleukin-3 and granulocyte-macrophage colony-stimulating factor signaling during myeloid differentiation. This observation of ligand-independent activation of RARs that is mediated by certain cytokines represents a new paradigm with respect to how RAR activity might be modulated during hematopoiesis and also suggests a molecular basis for the differential sensitivity of human acute myelogenous leukemia cells to retinoids.