Delineating the novel HDAC1/HDAC2-SIAH2 signaling node and its impact on ATR-CHK1 in leukemic cells with oncogenic JAK2

• Applicant: Professor Dr. Oliver Holger Krämer
• Subject Area: Hematology, Oncology ; Pharmacology ; Toxicology, Laboratory Medicine
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 445785155

Mammalian cells express 18 histone deacetylases (HDACs). Knowledge on the exact functions of individual HDACs for the development and progression of tumors can allow the rational use of specific histone deacetylase inhibitors (HDACi) for cancer treatment. Such HDACi cause fewer toxic effects than pan-HDACi. The data that we collected in the last funding period reveal that an inhibition of HDAC1 and HDAC2 is sufficient to induce apoptosis of leukemic cells carrying JAK2V617F as causal oncogene. We show that HDAC1 and HDAC2 control the stability of the E3 ubiquitin ligase SIAH2, that SIAH2 is decisive for the stability of JAK2V617F, and that SIAH2 controls leukemic cell survival through this mechanism. A SIAH interaction motif in the catalytic domain of JAK2V617F determines its SIAH2-mediated proteasomal degradation. The elimination of JAK2V617F by SIAH2 is linked to an overproduction of reactive oxygen species (ROS), DNA replication stress, and cytotoxic DNA damage. As underlying mechanism, we show that HDAC1 and HDAC2 are gatekeepers for the expression of the mitochondrial chaperone SMIM20, through their impact on SIAH2 and the JAK2V617F-STAT signaling pathway. SMIM20 stabilizes the electron transport chain of mitochondrial oxidative phosphorylation to ensure ROS homeostasis. We submitted these data for peer-review and publication, published a review article, and two articles in which we characterize the activities of novel HDACi in leukemic cells with JAK2V617F. We further reveal that SIAH2 controls the protein levels of the kinases ATR and CHK1. ATR and its substrate CHK1 are among the first proteins that cells activate upon DNA replication stress/DNA damage. We demonstrate that leukemic cells with JAK2V617F are highly susceptible to pro-apoptotic effects of ATR inhibitors. We aim to analyze the HDACi-induced loss of ATR and CHK1 with the hypothesis of an UBCH8-SIAH2-mediated proteasomal degradation. We want to define the molecular structures for the interaction of SIAH2 with ATR-CHK1 and how the acetylation of SIAH2 and the phosphorylation of ATR and CHK1 affect their stabilities. In addition, we suggest to functionally characterize SMIM20 by CRISPR-Cas9 loss- and gain-of-function experiments. In the last funding period, we also collected hints for further new target proteins of SIAH2. We aim to identify new, direct targets of SIAH2 with proximity-dependent biotinylation in living cells. To test our data on the interplay between ATR, CHK1, HDAC1, HDAC2, and SIAH2 in further cell systems and to increase the translational impact of our studies, we propose experiments with JAK2V617F-positive primary murine and human cells.

• DFG Programme: Research Grants