Functional characterization of cold-shock protein Ybx1 as a potential therapeutic target in Jak2V617F positive myeloproliferative neoplasia (MPN)

• Applicant: Professor Dr. Florian Heidel
• Subject Area: Hematology, Oncology
• Term: since 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 320028127

Janus kinases (JAK) mediate cytokine and hormone responses in hematopoietic cells. JAK2 is one of the most frequently mutated genes in the aging hematopoietic system and in hematopoietic cancers. Notably, JAK mutations constitutively activate downstream signaling and act as driver mutations of myeloproliferative neoplasms (MPN). Unexpectedly, JAK2-mutated cells do not fully depend on JAK-activity: while JAK inhibitors effectively reduce cytokine response and myelo-proliferation, their clinical use has subtle effects on overall disease burden or evolution of persistent clones.This prompted us to investigate the mechanism underlying evolution of JAK inhibitor persistence. In the first funding period of this proposal we applied in-depth phospho-proteome profiling to identify proteins involved in the mRNA processing as JAK2 targets, complemented by RNA interference (RNAi)-based functional validation. Among those, inactivation of the pleiotropic protein YBX1 sensitized the JAK-inhibitor persistent cells to apoptosis. Inactivation of YBX1 results in RNA mis-splicing, retained intron enrichment and disruption of the transcriptional and post-translational control of extracellular signal-regulated kinase (ERK) signaling. Inactivation of the YBX1-ERK axis in combination with pharmacological JAK-inhibition induces apoptosis in JAK2-dependent murine and human cells and leads to in vivo regression of the malignant clone. Collectively, we identified a role of YBX1 critical for the evolution and maintenance of persistent JAK2 clones. Identification of ERK-signaling as a tractable downstream regulator of therapeutic efficacy indicates a novel strategy to influence disease persistence in JAK2-mutated neoplasms.According to our proposed working program we will investigate the mechanistic consequences of differential YBX1-phosphorylation in JAK2-mutated cells. Using phospho-mutants of YBX1 at conserved amino acid residues, we will investigate functional consequences, changes in cellular distribution and binding capacity and transcriptional consequences of differential YBX1 phosphorylation. Effects of YBX1 inactivation on ERK-signaling will be studied by intracellular flow cytometry in cell lines and primary human cells. Transcriptional and post-transcriptional consequences of YBX1 deletion will be assessed by Chromatin- and RNA-immunoprecipitation, analysis of RNA-editing and global transcriptome analyses. So far, specific pharmacologic inhibitors of YBX1 are not available. We aim to identify functionally relevant YBX1 protein domains by genome editing to facilitate development of specific compounds in future projects. Our experimental approach may therefore facilitate therapeutic targeting of the persistent JAK-mutated clone in myeloproliferative neoplasia.

• DFG Programme: Research Grants