Pre-Clinical Analysis Of The Novel Antifolate Pralatrexate In Multiple Myeloma Reveals Functional Biomarkers Correlated With Response

Multiple myeloma (MM) is a neoplasia of plasma cells and is the second most common hematologic malignancy. Current combination therapies for MM typically include proteasome inhibitors (PIs) and/or immunomodulatory drugs (ImiDs). Although these regimens are effective, relapse and resistance to these agents are inevitable. There is a need to develop new classes of drugs with activity against MM and to identify biomarkers associated with response to these agents, in order to tailor salvage therapy for individuals most likely to respond and minimize exposure to therapies that are unlikely to be of benefit. We investigated these principles with the novel antifolate pralatrexate (PDX) in laboratory models of MM. Antifolates such as methotrexate (MTX) are not commonly used in the treatment of MM; however, combination chemotherapy regimens containing MTX have demonstrated clinical efficacy. In addition, case reports of the application of intrathecal MTX have shown activity against MM in the central nervous system. PDX was designed to be a preferred substrate for the reduced folate carrier (RFC) and folylpolyglutamate synthetase (FPGS) as compared to MTX, and it is approved for the treatment of peripheral T cell lymphoma. PDX induced intrinsic apoptosis with greater potency than MTX in a subset of human myeloma cell lines (HMCLs). Interestingly, other HMCLs were resistance to antifolate-induced apoptosis. This dichotomy among HMCLs provided a platform to identify functional biomarkers associated with sensitivity or resistance to PDX-induced apoptosis.

We examined expression of the genes that are determinants of antifolate actions: RFC, FPGS, gamma-glutamyl hydrolase (GGH), and dihydrofolate reductase (DHFR) in sensitive and resistant HMCLs. Higher pre-treatment levels of RFC mRNA by qRT-PCR correlated with increased sensitivity to PDX and higher rates of RFC-mediated transport. We then quantified mRNA for folate metabolism genes by qRT-PCR in total RNA from CD138-selected MM cells from patients and detected a broad range of expression for RFC, indicating that further studies with primary MM cells are warranted to assess RFC expression as a biomarker of antifolate sensitivity. Interestingly, PDX-resistant HMCLs also demonstrated dose-dependent increases in DHFR protein, one of the major antifolate targets, after treatment whereas PDX-sensitive HMCLs did not. Messenger RNA expression was unchanged under these conditions, suggesting a translational or post-translational mechanism for this activity. Sensitivity to PDX-induced apoptosis was not suppressed by co-incubation with recombinant human interleukin-6 or by co-culture with HS-5 bone marrow stromal cells. Finally, we used a previously validated bioluminescent xenograft mouse model to examine the effects of PDX in vivo. PDX-sensitive MM.1s cells stably-transduced with a dual cassette construct expressing both green fluorescent protein (GFP) and luciferase were introduced by tail-vein injection into NOD/Shi-scid/IL-2Rγ^null (NOG) mice. These cells engrafted in the marrow of the femur, pelvis, vertebral bodies, and sternum, recapitulating human disease, and tumor burden was quantified by in vivo imaging after luciferin injection. PDX treated mice demonstrated reductions in tumor burden compared to vehicle treated controls.

These results demonstrate that the novel antifolate PDX has potent activity against a subset of HMCLs in vitro and in vivo. Baseline mRNA expression of RFC and post-treatment protein levels of DHFR correlated with response in HMCLs. These findings support further clinical studies to assess the potential role of PDX in the treatment of MM coupled with correlative studies to validate the clinical utility of the biomarkers identified in HMCLs. PDX may be an effective addition to individualized therapy for relapsed multiple myeloma.