The role of RKIP in myeloid leukemogenesis

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasia caused by malignant transformation of hematopoietic stem- and progenitor cells (HSPCs). Constitutive activation of the RAS-mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) module is a key event in oncogenic transformation and frequently occurs in the pathogenesis of this disorder. We recently described loss of RAF kinase inhibitor protein (RKIP), a physiologic inhibitor of the RAS- MAPK/ERK pathway, in more than 20% of AML patients and observed its coexistence with oncogenic mutations in RAS. Further in-vitro analyses demonstrated that ectopic overexpression of RKIP inhibits RAS-MAPK/ERK signaling as well as RAS driven malignant transformation. Importantly, preliminary results pinpoint a role of deregulated microRNA expression patterns as a potential cause of RKIP silencing in AML. In the proposed project we will therefore test the following two hypotheses: i) RKIP is a pivotal modulator of RAS-MAPK/ERK signaling in the myeloid hematopoietic system and its loss aggravates RAS driven myeloid leukemogenesis. ii) Loss of RKIP in AML is caused by aberrant miRNA expression. In a first phase of the project, we will determine the effects of RKIP loss on RAS- MAPK/ERK signaling as well as its impact on proliferation and self renewal of HSPC in mice with a complete knockout of RKIP (RKIP-/-). Briefly, we will use HSPC of RKIP-/- animals to characterize the activation status of the RAS-MAPK/ERK pathway, as well as to determine their capacity for proliferation/self-renewal in colony formation assays and serial-transplantation experiments, respectively. In a second step, we will determine the role of RKIP in RAS driven myeloid leukemogenesis by crossing RKIP-/- mice to animals carrying an inducible oncogenic mutation in KRAS in bone marrow cells (Mx1-Cre KRASG12D). Induction of the KRAS mutation results in development of a lethal myeloproliferative disease with a penetrance of 100%, however, second genetic hits are required for transformation into AML. In more detail, we will initially delineate if RKIP loss results in shortened survival and/or morphologic transformation to AML in these mice. Subsequently, we will use transformed myeloid cells for analysis of RAS-MAPK/ERK signaling, proliferation and self-renewal employing flow cytometry, colony formation assays and serial- transplantation experiments, respectively. In a final project phase we will determine the role of microRNAs in the development of RKIP loss in AML. Therefore, five candidate microRNAs, that have been identified by the means of microRNA chip profiling in AML patient samples, will be examined in functional in-vitro studies using a broad range of AML cell lines. Taken together, the results obtained in this project will enable us to delineate the causes and consequences of RKIP loss in physiologic hematopoiesis and myeloid leukemogenesis, which should ultimately lead to the development of targeted treatment approaches.

Acute myeloid leukemia (AML) is an aggressive cancer of the blood (or hematopoietic) system, which is caused by malignant transformation of hematopoietic stem and progenitor cells (HSPCs). This can be caused by aberrations in intracellular signal transduction cascades, which are normally present to regulate essential cellular functions, including the growth and lifespan of these cells. RAF kinase inhibitor protein (RKIP) is a negative regulator of cellular signaling. Our group has described loss of RKIP expression as a frequent event in AML previously. In this project, we now aimed to extend our knowledge about RKIP in AML and to delineate the functional effects of RKIP loss on the hematopoietic system. Therefore, we used a series of primary leukemia patient samples, in-vitro laboratory techniques in immortalized AML cell lines, and a set of leukemia mouse models with and without artificial inactivation of RKIP. We demonstrate that loss of RKIP expression is indeed of relevance for leukemia development, as it induced leukemic effects in the in-vitro experiments, and as it aggravated the leukemia development in mice. Mechanistically, we could show that this is most likely caused by increased activation of intracellular signaling, which induces the growth of HSPCs on the one hand, and damages the normal blood cell differentiation on the other hand. Interestingly, although RKIP loss aggravated the leukemia development in mice with additional aberrations in cellular signaling, it failed to induce leukemias when it was present as a single pathologic aberration. This indicates that RKIP loss acts as an amplifier rather than an inducer of leukemogenesis. This conclusion was further strengthened by the analysis of human patient specimens, where RKIP loss preferentially co-occurred with these additional pathologic events. In the second part of the project, we screened for reasons behind the development of RKIP loss in AML. We thereby discovered that RKIP loss is caused by increased expression of a specific micro-RNA (miR-23a). Micro-RNAs are small fragments of RNA and do not code for genes or proteins. They initially have been considered as cellular debris. However, it is now evident that they play a seminal role in maintaining the cellular homeostasis, as they are central regulators of gene expression profiles. In this project, we demonstrate that miR-23a binds and downregulates RKIP in AML and that this causes the pathologic effects observed after inactivation of RKIP. Indeed, analysis of human patient specimens revealed that AML patients do not only exhibit decreased levels of RKIP but also increased expression of this miRNA. Taken together, our project identifies RKIP as a novel player in leukemogenesis and discovers the reasons behind the development of RKIP loss in AML. These data will help to develop novel and innovative therapeutic approaches for patients affected by myeloid leukemias.