Collaboration of Jak-Stat pathway mutations and haploinsufficiency of tumor suppressor genes in hematological malignancies
NETWORK CONTRIBUTION
Myeloproliferative neoplasms (MPN) are a group of chronic hyperproliferative myeloid malignancies frequently initiated by oncogenic mutations in the Jak-Stat pathway, such as Jak2
V617F, Jak2
exon12 or MPL
W515L/K. Activation of Stat5 has been shown to mediate the hyperproliferative effect of the Jak2 and MPL mutations. MPNs thus represent an important model disease to study the Jak-Stat pathway. Appropriate MPN mouse models are available, providing a good link to the work on Jak-Stat signaling in the development and progression of hematopoietic cancer. Besides oncogenic activations of the Jak-Stat pathway, our genetic studies of MPN have identified frequent focal deletions of genes with an unclear role in hematopoietic cancer. In our clinical studies, these deletions are found to be associated with leukemic transformation of chronic phase MPN patients. Deletions frequently occur in transcription factors such as CUX1, FOXP1, ETV6, and IKZF1. We hypothesize that deletions in these candidate tumor suppressor genes (TSG) collaborate with oncogenes in the Jak-Stat pathway, thereby resulting in hyperactivation of tyrosine phosphorylated Stat5 (pYStat5).
We are aiming to understand whether specific TSG loss and hyperactivity of the Jak-Stat pathway causes progression to acute myeloid leukemia (AML). We will functionally validate the genetic lesions detected in MPN-patients with respect to their effects on hematopoiesis in transgenic mice. We will attempt to simulate the clinical observation that deletions of these TSG in the presence of Jak2
V617F triggers leukemic transformation and follow this up with an extensive phenotypic comparative analysis. We will investigate gene expression data for signatures associated with activation of the Jak-Stat pathway and other core cancer pathways. Furthermore, we will explore the synergies and specific effects of cancer-associated defects to delineate the regulatory networks associated with the mutations in primary murine hematopoietic cells. Finally, we will explore the preclinical question of how murine models of MPN or AML respond to specific drugs targeting the Jak-Stat pathway.
The proposed project part will start with a characterization of the hematopoietic phenotype of mice carrying hemizygous deletions of CUX1, FOXP1, ETV6, and IKZF1 on a wild-type or Jak2
V617F knock-in background. The primary hematopoietic cells with various combinations of oncogenes and TSG mutations will also be characterized for cellular functions in ex vivo cultures. Furthermore, we will use RNA-Seq to profile the changes in gene expression associated with the Jak2
V617F driver mutation, TSG haploinsufficiency and their combinations.
Goals and Key Hypotheses:
Our research is based on the
following hypotheses:
(1) IKZF1, CUX1, ETV6 and FOXP1 display a distinct hematopoietic phenotype and modify the MPN model expressing the Jak2V617F oncoprotein.
(2) Mice with combined activation of Jak-Stat and haploinsufficient loss of TSG-MPN display MPN phenotypes with shortened disease latency and more severe progression.
(3) Loss of TSG-MPN increases the activity of the Jak-Stat signalling axis.
Our specific
goals are as follows:
(1) Characterize the hematopoietic phenotype of mice carrying hemizygous deletions of CUX, FOXP1, ETV6 and IKZF1 on a (a) wild-type and (b) Jak2
V617F knock-in background.
(2) Validate the clinical observations that deletions of TSG in the presence of Jak2
V617F trigger leukemic transformation in primary hematopoietic cells for cellular functions in
ex vivo cultures and for clonal outgrowth as well as drug sensitivity; and examine engraftment and drug sensitivity
in vivo.
(3) Study the changes in gene expression and expression signatures associated with activation of the Jak-Stat pathway in primary murine hematopoietic cells as a result of the Jak2
V617F oncogene, TSG haploisufficiency and their combinations.
Report on funding period 1 (03/2006-03/2010)
SFB-P05PK: Inflammation: Integration of Interferon and Stress
Signals (Pavel Kovarik)
We showed that simultaneous activation of stress and IFN signaling causes increased promoter recruitment of Cdk9 and, consequently, induction of genes that are not induced by IFNs alone. One of them, tristetraprolin (TTP), was characterized in more detail. The IFN-mediated induction of TTP, an mRNA-destabilizing and anti-inflammatory gene, limits the pro-inflammatory function of IFNs by suppressing the production of cytokines. Further analysis revealed that TTP is also targeted by Stat3 in IL-10 responses and that TTP is needed for the full anti-inflammatory function of IL-10. Thus, TTP was established as novel Jak-Stat player with strong anti-inflammatory properties. The function of TTP in cytokine responses will be studied in vivo using TTPfl/fl animals that were generated in the project. In a mechanistical study of IFN-induced serine phosphorylation of the Stat1 transactivation domain (TAD) on Ser727 the group demonstrated that this modification occurs only on Stat1 molecules bound to the target gene promoters. This so far unique chromatin-associated signaling event indicates that the Stat1 TAD kinase is part of a chromatin-linked feedback control of transcription. Further characterization of the kinase and of its effects on the transcription cycle is currently in progress.
Proposal for funding period 2 (03/2010-03/2013)
SFB-P05PK (Pavel Kovarik) Chromatin-associated Stat signaling, Stat stoichiometry
PK investigates mechanistical aspects of both canonical and non-canonical Jak-Stat signaling thereby contributing to the SFB Research Areas ‘JakStat in Infection and Immunity’ and ‘Non-canonical Mechanisms of Jak-Stat Action’. The main focus is the analysis of mechanisms and key factors of the chromatin-associated signaling events that regulate serine phosphorylation of the Stat1 transactivating domain and Stat1 tyrosine dephosphorylation in responses to IFNs. These studies address a new concept of activation and inactivation of Stat1 by chromatin-linked feedback control. Furthermore, the effect of physiological changes in the cellular stoichiometry of unphosphorylated Stat1, Stat2 and Stat3 on the composition and abundance of individual Stat dimers, and consequently on cytokine responses, will be investigated. The projects will provide the SFB consortium with new key players and molecular principles for studies of Jak-Stat signaling in animal models of infection and cancer.
The proposed project will mainly address two issues of Jak-Stat signaling: 1) chromatin-associated signaling such as serine phosphorylation of the Stat transactivation domain (TAD) and dephosphorylation of Stats, and 2) the role of Stat stoichiometry in cytokine responses.
The first sub-project (SP1: chromatin-associated Stat signaling) is based on our findings in the first funding period that the final activating modification of Stat1 by IFNs, i.e. the serine phosphorylation of Stat1 TAD, occurs after the recruitment of Stat1 to chromatin. This so far unique chromatin-linked signaling event implicates that the TAD serine kinase is associated with the transcription complexes and it is involved in feedback control of transcription. We will identify the Stat1 TAD serine kinase and examine its precise role in the Stat1-dependent transcription process. The global contribution of the TAD kinase to the IFN-regulated transcription responses will be further investigated using a ChIP-Seq approach that will allow a correlation analysis between the promoter occupancy of the serine kinase and the IFN-induced gene expression. This will complement efforts by SFB-P10VS and SFB-P03TD to elucidate the role of the Stat1 TAD phosphorylation in NK cell-dependent tumor surveillance. Given the degree of the TAD conservation, the TAD kinases of other Stats are likely to be similar to the Stat1 TAD kinase. We will address the identity of the Stat3 and Stat5 TAD kinases since these Stats are in the research focus of SFB-P04RE, SFB-P06WM and SFB-P07RM so that new interactions may arise with these SFB partners. In the SP1 we will also examine the mechanism of the tyrosine-dephosphorylation of Stat1. Our preliminary data indicate that the tyrosine dephosphorylation is also a chromatin-tethered signaling event. Altogether, SP1 will address several key Jak-Stat signaling events that all occur on chromatin-bound Stats. The molecular characterization of these regulatory steps will improve our knowledge about how the so far poorly understood chromatin-linked feedback signaling controls the initiation and termination of the transcription cycle.
In the SP2 (Stat stoichiometry) we will study how the cellular Stat ratios, with emphases on Stat1 and Stat2, govern cytokine responses (collaboration with SFB-P08BSMM). Our data indicate that the ratios of cellular Stats significantly determine the amount and composition of preformed Stat dimers. For example, excessive amounts of Stat2 would sequester Stat1 leaving only small amount of Stat1 available for the formation of Stat1 homodimers. Consequently, IFN signaling is affected. The role of Stat stoichiometry is of particular importance for pathological conditions like inflammation/infection or cancer when the expression of e.g. Stat1 and Stat3, respectively, is often up-regulated. The consequences of these changes in Stat ratios on the cytokine responses have so far not been studied.
CONTACT
CeMM Research Center for Molecular Medicine
Austrian Academy of Sciences
Lazarettgasse 14, AKH BT 25.3
A-1090 Vienna, Austria
rkralovics@cemm.oeaw.ac.at
phone: +43(0)1 40160-70 027
