Leflunomide Inhibits c-Myc Signaling and Impairs Myeloma Cell Growth

Introduction:

Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide (Lef), a commercially available oral immunosuppressive agent that has been FDA-approved for treatment of rheumatoid arthritis was evaluated as a potential MM therapeutic. In vitro anti-MM activity of Lef has previously been reported; however, the molecular mechanisms of action of Lef in MM are incompletely understood. Inhibition of Src kinase-family members and blocking of dihydroorotate dehydrogenase, an enzyme involved in pyrimidine synthesis are among the few known targets of Lef. To identify novel direct targets of Lef and to further our understanding of Lefs' mode of action on MM cells, we performed in vitro kinase screening assays and RNA/miRNA sequencing analysis of cells treated with or without Lef.

Methods and Results:

Our pre-clinical studies with teriflunomide (Ter), the active metabolite of Lef, showed that it inhibited growth of MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226, OPM-2, L-363) and primary MM patients' (CD138+) plasma cells at IC50s ranging from 30 uM to 140 uM. Pharmacokinetics analysis accompanying a phase 1 dose-escalation clinical trial in MM at our institute has demonstrated that Ter serum concentrations of 500 uM can be obtained. We also found that Ter induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <150 uM. At higher in vitro concentrations (100 uM to 200 uM), Ter induces apoptosis in MM cells. Moreover, Ter synergized with dexamethasone as well as lenalidomide in in vitro growth inhibition of MM cell line MM.1S. In vivo mouse studies with Lef as single agent or in combination with the immune modulatory drug lenalidomide or with the proteasome inhibitor ixazomib are currently ongoing and results will be presented at the meeting. To identify novel direct targets of Ter, a screening assay described by Anastassiadis et al. (PMCID: PMC3230241) was applied to test the inhibitory activity of Ter on 600 known kinases. We identified 55 kinases that were inhibited >50% at 200 uM Ter. Subsequent dose-response kinase assays identified, among others, Aurora A and PIM family members, all of which are involved in MM pathogenesis, as novel direct targets of Lef, with IC50s ranging from 35 uM to 53 uM. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon Lef exposure, 4 MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary MM cells were exposed to 200 µM Ter or DMSO control for 24h before extraction, purification and sequencing of mRNA and microRNA. RNA-sequencing analysis and DAVID functional annotation revealed that similar changes were present between patient samples and cell lines. Pathways that were predominantly affected by Ter included purine/pyrimidine metabolism, DNA-replication and cell-cycle regulation. Gene set enrichment analysis revealed that c-Myc, an oncogene that contributes to the malignant phenotype of MM and a known promising target in MM, signaling was impaired upon Ter treatment, in MM.1S cells. Differential expression analysis, real-time PCR and western blot analysis showed that c-Myc mRNA, c-Myc protein and c-Myc-regulated miRNAs belonging to the oncomir-1 cluster miR-17-92 were all down-regulated upon Ter exposure in the majority of the MM cells. Since PIM family members are involved in c-Myc signaling in cancer cells, we performed in silico molecular docking studies using Ter/PIM. Our results suggest that the active metabolite of Lef, Ter, can directly bind to the PIM family of proteins by binding to the ATP-binding site, thereby inhibiting its kinase activity. Since pan-PIM inhibitors such as PIM447 have been reported to be cytotoxic for myeloma cells due to decrease in c-Myc levels, these results suggest that decrease in c-Myc induced by Ter may be due to PIM inhibition.

Conclusion:

Leflunomide impairs in vitro MM cell growth - at least in part - through inhibition of c-Myc signaling. A clinical trial with leflunomide in MM has been initiated and a separate abstract with encouraging initial results has been submitted for presentation at this meeting.