P-280: Elucidating the Kinase-Independent Autoregulatory Loop of PIM2 in Multiple Myeloma

Introduction: PIM2, an oncogenic serine/threonine kinase, is significantly overexpressed in multiple myeloma (MM), correlating with disease progression and poor prognosis. Our laboratory has identified a novel kinase-independent (Ki) function of PIM2 that may play a crucial role in maintaining its own expression and promoting MM cell survival. Preliminary data suggest that PIM2 can autoregulate its expression through a feedback loop involving the transcription factor MYC, a mechanism potentially critical for MM pathogenesis and resistance to existing therapies.



Methods: To investigate the potential mechanism of PIM2 Ki autoregulation in MM cell lines, we utilized luciferase reporter assays, chromatin immunoprecipitation (ChIP), and pharmacological inhibition of PIM2. Luciferase assays were performed using the full-length PIM2 promoter to assess transcriptional activity in response to PIM2 Ki inhibitors (JP1 and JP2) and a PIM2 kinase-dependent (Kdep) inhibitor (AZD1208). Sequential promoter truncation luciferase assays identified the minimal promoter region for PIM2 expression. Site-directed mutagenesis was used to identify critical cis-regulatory elements (CREs) regulating PIM2 kinase-independent expression. ChIP-qPCR was employed to evaluate MYC occupancy at the PIM2 promoter under different PIM2 inhibitor treatments.


Results: Luciferase assays revealed a significant decrease in PIM2 promoter activity upon treatment with the PIM2 Ki inhibitor JP1, but not with the Kdep inhibitor AZD1208. Sequential promoter truncation luciferase studies identified a region of approximately 2000 base pairs regulating PIM2 expression. Single base pair mutation using site-directed mutagenesis revealed a single SP1 binding site regulating PIM2 kinase-independent autoregulation. ChIP-qPCR demonstrated enrichment of MYC at the PIM2 promoter, which was disrupted by JP1 treatment and increased by AZD1208 treatment. Furthermore, pharmacological inhibition of MYC using the small molecule inhibitor 10058-F4 resulted in decreased PIM2 protein levels, similar to the effects of JP1 and JP2. These findings suggest that MYC may directly bind to the PIM2 promoter and complex with SP1, facilitating a PIM2 Ki autoregulatory loop that maintains aberrant PIM2 overexpression in MM cells.


Conclusions: Our preliminary data support the hypothesis of a novel PIM2 kinase-independent autoregulatory mechanism involving MYC and SP1 in MM. Disruption of this potential PIM2 Ki autoregulatory loop using selective inhibitors (JP1 and JP2) decreases PIM2 expression and promoter activity, suggesting it as a potential therapeutic target. Further investigation into the specific cis-regulatory elements and binding sites governing PIM2 Ki autoregulation may uncover new vulnerabilities to exploit in MM treatment.