1 Control of Pim 2 kinase stability and expression in transformed human hematopoietic cells

The oncogenic Pim2 kinase is overexpressed in several hematological malignancies such as multiple myeloma and acute myeloid leukemia and constitutes a strong therapeutic target candidate. Like other Pim kinases, Pim2 is constitutively active and is believed to be essentially regulated through its accumulation. We show that in leukemic cells, the three Pim2 isoforms have dramatically short halflives although the longer isoform is significantly more stable than the shorter isoforms. All isoforms present a cytoplasmic localization and their degradation was neither modified by broad-spectrum kinase or phosphatase inhibitors such as staurosporine or okadaic acid nor by specific inhibition of several intracellular signaling pathways including Erk, Akt and mTORC1. Pim2 degradation was inhibited by proteasome inhibitors but Pim2 ubiquitination was not detected even by blocking both proteasome activity and protein deubiquitinases. Moreover, Pyr41, an ubiquitin-activating enzyme (E1) inhibitor, did not stabilize Pim2, strongly suggesting that Pim2 was degraded by the proteasome without ubiquitination. In agreement, we observed that purified 20S proteasome particles could degrade Pim2 molecule in vitro. Pim2 mRNA accumulation in UT7 cells was controlled by erythropoietin through STAT5 transcription factors. In contrast, the translation of Pim2 mRNA was not regulated by mTORC1. Overall, our results suggest that Pim2 is only controlled by its mRNA accumulation level. Catalytically active Pim2 accumulated in proteasome inhibitor-treated myeloma cells. We show that Pim2 inhibitors and proteasome inhibitors such as bortezomib, have additive effects to inhibit the growth of myeloma cells, suggesting that Pim2 could be an interesting target for the treatment of multiple myeloma. Summary statement: The constitutively active kinase Pim2 overexpressed in transformed cells constitutes an attractive target in cancer. We show that both Pim2 degradation realized by the proteasome in an ubiquitin-independent manner and Pim2 translation are constitutive. Short title: Ubiquitin-independent Pim2 degradation by the proteasome