Delineating the novel HDAC3-SIAH2 signaling node and its impact on oncogenic JAK2 in leukemic cells

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Constitutively active mutants of the tyrosine kinase (JAK2, point mutation JAK2V617F) causally contribute to the development of myeloproliferative neoplasms. The transformation of blood cells with JAK2V617F triggers replication stress through increased cell proliferation and aberrant gene expression patterns. Epigenetic modulators of the histone deacetylase (HDAC) family are appreciated targets for novel pharmacological intervention strategies. We want to understand why HDAC inhibitors (HDACi) are very toxic for leukemic cells with mutant JAK2. According to recent results, HDACs are key regulators for the activation of checkpoint kinases in cells with replication stress and DNA damage. Moreover, HDACs critically influence protein degradation via the ubiquitin-proteasome-system. Consequently, HDACi can modulate the growth of tumor cells via these parameters. Our novel data suggest that HDACi accelerate the proteasomal degradation of JAK2V617F, which leads to a depletion of the growth-promoting oncogene. Our hypothesis is that this process relies on an induction of the E3 ubiquitin-ligase SIAH2 at the protein level. Such an accumulation of SIAH2 appears to be specifically regulated through HDAC3 as a gatekeeper and this might involve the control of SIAH2 by acetylation. Therefore, we aim to molecularly define the interaction of SIAH2 with JAK2V617F, the poly-ubiquitination of JAK2V617F, and a control of SIAH2 by acetylation in various cell systems. We aim to clarify whether molecularly defined SIAH degron motifs and proteins of the SOCS family are involved in the ubiquitination and proteasomal degradation of tyrosine-phosphorylated and unphosphorylated JAK2V617F and wild-type JAK2. Moreover, we want to investigate whether SIAH2 destabilizes JAK2V617F with its known interaction partner, the E2 ubiquitin-conjugase UBCH8. Published data show that the phosphorylation of SIAH2 by checkpoint kinases after DNA damage inactivates SIAH2. However, we find that SIAH2 remains active against JAK2V617F after HDACi-induced replication stress/DNA damage. This finding indicates that we describe for the first time an HDAC-dependent control of SIAH2. Within this context, we want to address whether SIAH2 targets checkpoint kinases. For our analyses, we want to follow hypotheses-driven approaches and we aim to create new CRISPR-Cas9-based cell systems to test our theories stringently. Unbiased proteomics will define the SIAH2-dependent proteome. Our analyses concerning the role of HDAC3 and its inhibition for the survival of leukemic cells with mutant JAK2 will reveal if HDAC3 is indeed a best suited pharmacological target structure and whether the accumulation of SIAH2 is a pharmacodynamic marker for the efficacy of HDACi. Patients with JAK2V617F-positive leukemia might profit from such insights.