Multiple Myeloma Progression Is Accelerated By Microenvironmental-Derived Fatty Acid Binding Proteins 4 and 5 (FABP4/FABP5) in a Murine Model

Multiple myeloma (MM) is an incurable cancer of monoclonal plasma cells that largely accumulate within bone marrow (BM) with high rates of patient relapse. The fatty acid binding proteins (FABPs) are a family of ten proteins which serve as metabolic-modulating molecules and fatty acid transporters. We previously found associations between myeloma outcomes and higher tumor cell expression of FABP5 and FABP6. Herein, we interrogated the role of microenvironmentally-derived FABPs in MM progression.

First, a novel strain of mice containing a double knockout of Fabp4/FABP4 and Fabp5/FABP5 were purchased, bred, and characterized (C57BL/Ntac-Fabp4/5^dko). qRT-PCR results revealed a ~50% reduction in the expression of each gene in double heterozygous (het) mice, and no expression in double homozygous KO mice (dKO), as confirmed by adipose tissue histology, compared to wild type (WT) mice. Increases in fat mass and decreases in lean mass were observed with NMR in dKO versus WT mice at 7-9 weeks of age in both sexes (p<0.0001-p<0.001). Tibial bone parameters did not differ between dKO and WT mice, expect that cortical bone tissue mineral density and minimum moment of inertia were significantly lower in dKO vs WT mice in females, 12-15 weeks old only, (by μCT analysis of (two-way ANOVA followed by the Sidak test). Immune cells were assessed in spleens and BM by flow cytometry, which revealed increased tolerogenic dendritic cells (p<0.001) and decreased total number of B cells (p<0.05), including decreased mature and immature B cell populations in BM from het Fabp4/5^dko vs WT mice. Corresponding data from homozygous dKO are forthcoming.

Next, WT and dKO mice (~15 weeks old; 10 WT females, 11 dKO females, 6 WT males, 6 dKO males) were injected i.v. with Vk*MYC murine myeloma cells (1x10^6 cells/mouse). Within each sex, there were no genotype-based differences in body weight throughout the duration of the study. Tumor burden was monitored through the appearance of gamma bands in serum protein electrophoresis (SPEP) and “tumor incidence” criteria were defined by at least one of the following: M-spike presence, ELISA IgG signal, macroscopic tumor presence during gross morphology exam at sacrifice, or spleen or BM tumor presence quantified as >10% CD138⁺ cells by flow cytometry.

Tumor incidence was increased in WT versus to dKO mice in both sexes (50% incidence in WT females versus 36% incidence in dKO females; 83.3% incidence in WT males versus 16.7% dKO males). Overall, WT mice were more than twice as likely to develop myeloma than dKO mice (62.5% WT versus 29.4% dKO). Weekly SPEP results demonstrated that tumors developed more rapidly in male mice compared to females. While spleen weights were not significantly different between the genotypes, we did observe some mice with enlarged spleens (including 1 with splenic rupture at the time of sacrifice) or tumors growing in the abdominal cavity, in both groups. In females, we did not detect any mice with tumor reaching the the technical definition of myeloma tumor burden in the spleen (≥10% plasma cells), however 5 mice did meet that cutoff in BM. In the surviving males, one WT mouse exhibited splenic tumor at approximately 30% of the live cells quantified (mouse with splenic rupture), with only 1 mouse (dKO) reaching the definition of BM myeloma tumor burden. As expected, the mice with extramedullary tumor growth did not exhibit large amounts of tumor cells in the spleen or bone marrow.

To explore why FABP4/5 dKO mice had less tumor incidence, we tested serum leptin and adiponectin levels; we observed no genotype-driven differences in these. Next, since FABP4 and FABP5 are expressed by many immune cells, we performed a complete blood chemistry analysis on blood samples at the time of sacrifice and found no significant differences in circulating reticulocytes, neutrophils, lymphocytes, monocytes, eosinophils, or basophils. BM cellular analysis of immune and tumor cells is underway in tumor-bearing mice.

Overall, these data suggest that FABPs in the microenvironment play a role in MM progression and that blocking these, or other fatty acid-related proteins could lead to better outcomes for MM patients. In mice with genetic knockout of Fabp4 and Fabp5, we observed significant differences in bone and immune parameters, both of which could contribute to myeloma growth. This study provides additional rationale supporting targeting the FABP family as be a promising future myeloma therapy.