Unraveling the Kinase-Independent Autoregulatory Mechanism of PIM2 in Multiple Myeloma
Authors: Schorr Christopher Rolf , Petrusca Daniela Nicoleta , Carlson Louise , Hardwick Joyce , Reinke Julia Grace , Lee Kelvin P. 
Published: December 2024

Introduction: Multiple Myeloma (MM) is an incurable hematological malignancy characterized by uncontrolled plasma cells in the bone marrow. Despite recent therapeutic advances, MM poses a significant health burden with a 5-year survival rate below 60%. Treatment failure in MM is primarily driven by drug resistance, highlighting the critical need for a comprehensive exploration of MM cell pro-survival mechanisms.
One emerging pro-survival mediator and exciting therapeutic target in MM is the oncogenic serine/threonine kinase PIM2 (Proviral Integration site in Moloney Murine Leukemia Virus 2). PIM2 is a part of the PIM family of kinases (PIM1, PIM2, and PIM3) which are overexpressed across a wide range of hematologic and solid cancers. In MM patients, PIM2 overexpression correlates with advanced disease progression and poor prognosis. PIM2 kinase-dependent (Kdep) functions promote MM pathogenesis through diverse signaling pathways, including mRNA translation, mTORC1 regulation, DNA damage response, cMYC stabilization, anti-apoptosis, and cell cycle progression. While ATP-competitive PIM2 kinase inhibitors have shown limited efficacy in clinical trials, our laboratory has recently developed and reported on JP11646 (JP1), a first-in-class PIM2-selective non-ATP competitive inhibitor (JR Nair, 2017). Intriguingly, JP1 demonstrates a superior MM cell-killing potency and efficacy compared to other PIM kinase inhibitors, likely due to a decrease in PIM2 mRNA and protein expression not observed with ATP-competitive inhibitors.
In summary, we have identified a novel kinase-independent (Ki) function of PIM2 that appears essential for maintaining its expression, as well as promoting MM cell survival. Our findings suggest PIM2 autoregulates its own expression through a feedback loop involving the MYC transcription factor and SP1 representing a novel way to target MM therapeutically.
Methods: In this study, we investigated PIM2 Ki autoregulation in MM cell lines using luciferase reporter assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and pharmacological inhibition with JP1, JP2, and AZD. PIM2 promoter activity was measured using luciferase assays with PIM2 Ki inhibitors (JP1, JP2) and a kinase-dependent (Kdep) inhibitor (AZD1208). Sequential promoter truncation and site-directed mutagenesis identified key regulatory regions and elements. ChIP-qPCR evaluated MYC, SP1, and PIM2 occupancy at the PIM2 promoter under various inhibitor treatments. In order to perform ChIP on PIM2, a recombinant FLAG tag was added to both the N-terminal and C-terminal end of a PIM2 lentiviral expression vector. EMSA and supershift assays were performed to evaluate SP1 binding to the PIM2 promoter and complex formation with PIM2 and MYC.
Results: Luciferase assays showed decreased PIM2 promoter activity with JP1 treatment, but not with AZD. We identified a ~1,000 bp region regulating PIM2 expression and a critical SP1 binding site mediating Ki autoregulation. ChIP-qPCR demonstrated MYC enrichment at the PIM2 promoter, disrupted by JP1 and enhanced by AZD. MYC inhibition with 10058-F4 reduced PIM2 protein levels, mimicking JP1 and JP2 effects. EMSA and supershift assays confirmed SP1 binding to the PIM2 promoter. SP1 inhibition with terameprocol (TMP) decreased PIM2 protein levels, as shown by Western blot analysis. These results collectively suggest that MYC and SP1 directly bind the PIM2 promoter, forming a complex that facilitates a PIM2 Ki autoregulatory loop maintaining PIM2 overexpression in MM cells.
Conclusions: Our data support a novel PIM2 Ki autoregulatory mechanism involving MYC and SP1 in MM. Disrupting this loop with selective inhibitors (JP1, JP2) reduces PIM2 expression and promoter activity, highlighting a new therapeutic target to decrease PIM2 expression. The identification of specific regulatory elements governing PIM2 Ki autoregulation reveals a new vulnerability in MM that may be exploited for treatment. Further investigation of this complex regulatory network may lead to innovative strategies for targeting PIM2 overexpression in MM therapy.