Fatty Acid Binding Protein 6 (FABP6) Represents a Novel Target for Multiple Myeloma

Multiple myeloma (MM) requires innovative approaches, as it commonly evades therapies and re-emerges aggressively. We showed that MM is dependent on fatty acid metabolism and hypothesize that studying the cellular mechanisms governing lipid metabolism will provide major insights into the pathology of MM and reveal novel targets. Fatty acid binding proteins (FABPs) play an important role in the modulation of lipid metabolism and signal transduction: they are noncatalytic fatty acid, bile acid, and cannabinoid-binding proteins with cargo-dependent and independent functions. FABP6 is a hormone-responsive member that has been reported to inhibit solid tumor progression via effects on cell proliferation, angiogenesis, invasion, and more, but its role in myeloma is not known. However, recent Kaplan-Meier analysis of the MMRF CoMMpass data demonstrates worse overall survival (OS) and progression free survival (PFs) in patients with high FABP6. Herein, we explored the role of FABP6 and FABP5, which can have FABP6-compensatory functions, in myeloma cell phenotypes to elucidate molecular mechanisms by which FAPB5/6 regulate MM growth and progression.

By qRT-PCR analysis, we measured FABP3, FABP4, FABP5 and FABP6 gene expression in 7 MM cell lines (MMCLs) (KMS27, KMS34, H929, MM.1S, OPM2, RPMI8226 and U266) using hRPLP0 as a housekeep gene, and protein expression by immunoblotting. Genetic FABP knockdown (KD) was performed in 3 MMCLs, and one patient sample, using short hairpin (sh) RNAs and stable cell lines were derived after 2 weeks of puromycin selection. The TRIPZshRNA inducible lentivirus expression system containing a red fluorescent protein (RFP) reporter was used to generate stable, doxycycline (DOX)-inducible cells with knockdowns as follow: FABP5 (shFABP5), FABP6 (using 3 different shRNAs: shFABP6/58 shFABP6/59, and shFABP6/61), or non-targeting controls (NC). Real-time Glo cell viability assays were used to detect effects of FABP5/6 KD on cell growth. Seahorse metabolic assays and RNAsequencing of KD and control cells are ongoing.

FABP6 mRNA expression was high in 3 (KMS27, KMS34 and H929) of 7 MMCLs, moderate in two, and low in two, while a high level of FABP5 mRNA was seen in all MMCLs. In contrast, high FABP3 expression was seen only in RPMI8226. Protein levels of FABP5 and FABP6 mirrored gene levels. KD of FABP5 and FABP6 genes in stable cell lines were confirmed by expression of RFP and qRT-PCR (at 72 hours), after DOX treatment. Significant KD of FABP6 and FABP5 was first assessed by RFP imaging, and gene expression was confirmed by qRT-PCR analysis.

KMS27 showed significant, large-fold decreases of FABP6 with all three FABP6 shRNAs [shFABP6/58 (0.1071 ± 0.01), shFABP6/59 (0.0059 ± 0.01) and shFABP6/61 (0.174 ± 0.01), compared with shNC (1.09 ± 0.1)]. Similarly, decrease of FABP6 mRNA were obtained with 2 shRNAs in H929 [shFABP6/58 (0.2498 ± 0.01) and shFABP6/59 (0.58 ± 0.01) compared with shNC (1.001 ± 0.089)]. FABP6 KD was also effective in MM.1S cells (shFABP6/58 (0.17 ± 0.08), FABP6/59 (0.068 ± 0.08), shFABP6/61 (0.2598±0.08), shNC (1.01 ± 0.08)). FABP5 KD was significant in MM.1S and H929 compared with respective controls as well. There was significantly lower cell viability in KMS27 cells with FABP5 and FAPB6 KD with all shRNAs on days 3 to day 6, and in H929 cells with shFABP5 and shFABP6/59. Moreover, decreased cell viability was also observed in primary patient CD138+ cells treated with shFABP5 (p=0.0290) and shFABP6/58 (p=0.014) after 72 hours.

To assess the potential for targeting the FABP6/FABP5 in myeloma, since no specific FABP6 inhibitors are available, we tested a pan-FABP inhibitor (BMS309403) using intratumoral drug delivery, to overcome previously observed delivery and stability challenges with this compound. Using two cohorts of mice (8- or 20-week-old SCID-Beige females), we subcutaneously inoculated 1x10^6 MM.1S^gfp+luc+ cells, randomized by mouse weight, and subcutaneously treated with either BMS309403 (5 mg/kg) or vehicle (three or five times weekly). In both cohorts, bioluminescent imaging (BLI) analysis revealed significantly lower tumor burden in BMS309403-treated versus vehicle-treated mice. MMCLs with FABP6 and FABP5 KD will next be assessed in vivo. Taken together, in vitro, in vivo, and patient data all suggest that FABP5 and FABP6 are novel targets in MM and suggest that FA metabolism is a newly-identified vulnerability that should be exploited for MM therapy.