Functional interactions between EVI1 and atRA in LSCs

The ecotropic viral integration site 1 (EVI1) gene codes for a transcription factor that affects multiple cellular processes in a context dependent manner. Apart from its physiological roles, EVI1 acts as an oncogene in acute myeloid leukemia (AML) and other malignancies: it is overexpressed in ~10% of patients with AML, and its aberrant expression is associated with poor response to current therapeutic approaches, resulting in short remission duration and decreased survival. All-trans retinoic acid (atRA) is a derivative of vitamin A with important roles in numerous biological processes. It influences cellular physiology primarily by binding to and activating nuclear receptors that regulate the transcription of target genes through direct interactions with DNA elements in their regulatory regions. atRA promotes the differentiation of both healthy and malignant myeloid cells, and this property is thought to explain its great therapeutic effectiveness in acute promyelocytic leukemia (APL), a subtype of AML characterized by rearrangements of the retinoic acid receptor gene that partially disrupt the function of its protein product. Recent studies from the applicant`s laboratory have shown that EVI1 enhanced transcriptional regulation as well as cell cycle arrest, differentiation, and apoptosis in response to atRA in malignant myeloid cells. Nevertheless, and in stark contrast to the situation in APL, no clear benefit of the addition of atRA to conventional chemotherapy could so far be established in non-APL AML, whether associated with overexpression of EVI1 or not. A possible explanation for the lack of effectiveness of atRA in this context lies in the fact that atRA promotes the differentiation of partially matured myeloid cells, but stimulates the expansion of hematopoietic and probably also leukemic stem cells (LSCs), i.e., the rarely dividing, most immature, pluripotent cells that maintain both normal and malignant hematopoiesis. In the work proposed in this application, the interaction between EVI1 and atRA with respect to the maintenance and expansion of LSCs cells shall be investigated and compared to the effects of atRA on APL LSCs. Two different murine model systems, in which AML can occur both in the presence and absence of Evi1 overexpression, shall be employed, along with a mouse model for APL. LSC numbers, activity, and proliferation shall be measured using several complementary state of the art assays. Genes synergistically regulated by EVI1 and atRA in LSCs shall be identified using RNA based next generation sequencing, and begun to be characterized for their functional contribution to the biological effects jointly elicited by these factors. A better understanding of the effects of atRA on AML LSCs, and of its interactions with EVI1 in this context, will provide a basis on which its impact as a therapeutic agent in AML can be interpreted, and might instruct adaptations of the treatment regimen that allow its potential therapeutic effectiveness in this disease entity to be realized.

Background and goals: Acute myeloid leukemia (AML) is an aggressive malignancy of blood cells that is often fatal despite of intensive therapy. Like all tumors, it is caused by acquired (tumor-cell restricted) changes in the composition or the activity ("expression") of genes. Based on the specific identity of these changes, AML subgroups can be defined that respond differently to different treatments. One of these subgroups, acute promyelocytic leukemia, responds particularly well to all-trans retinoic acid (atRA). atRA is an endogenous substance; its therapeutic application is associated with relatively mild side effects. Despite of intensive research and some promising data, however, its therapeutic usefulness in other AML subgroups has remained unclear. A neglected aspect in this context was the fact that AML is a stem cell-driven disease, i.e., leukemogenesis, therapy resistance, and relapse are caused by a small subpopulation of cells, so-called leukemic stem cells (LSCs). Understanding the efficacy of therapeutic agents therefore requires an understanding of their effects on LSCs. At the inception of this project, the effects of atRA on AML LSCs had been investigated only indirectly in a single publication. The goal of this project was therefore to investigate the impact of atRA on AML LSCs, specifically focussing on an AML subgroup characterized by high expression of EVI1. EVI1 plays important roles in normal blood stem cells. Aberrant EVI1-expression ("overexpression") in ~10% of patients with AML ist associated with a particularly poor response to current chemotherapy. Results: A mouse model was used in which AML is caused by MLL-AF9, one of the aberrant genes observed in human AML. Both in human and murine MLL-AF9-driven AML, additional overexpression of EVI1 may be present or not. In the MLL-AF9 model, atRA stimulated LSC activity, i.e., this otherwise anti-leukemic agent acted in a leukemia-promoting manner in this context. However, this effect was dependent on the additional expression of Evi1; in its absence, atRA did not affect LSCs. An antagonist (a substance with opposite biological effect) inhibited the activity of LSCs from MLL-AF9-driven, Evi1-overexpressing AML, and prolonged survival of mice with this disease. Comprehensive gene expression analyses on LSCs showed that EVI1 and atRA also interacted with respect to the regulation of target genes. For two such target genes, Notch4 und Il2ra, important functions in leukemogenesis and/or LSCs were shown. In a second mouse model, driven by the altered genes Flt3-ITD and Npm1c, atRA inhibited LSC activity, but this effect was cancelled by overexpression of Evi1. Conclusion: Depending on the nature of the AML-specific genetic changes, atRA can have either pro- or anti-leukemic effects on AML LSCs. This complexity was unexpected, but represents a first important step towards a genetically tailored use of atRA in AML ("precision medicine").