Introduction: Retinoic acid (RA), the active metabolite of vitamin A, influences biological processes by activating the retinoic acid receptor (RAR). RARs are ligand-controlled transcription factors that function as heterodimer with retinoid X receptors (RXRs) and regulate expression of target genes affecting homeostasis of cellular differentiation and death. The success of RAR activation in the treatment of acute promyelocytic leukemia (APL), particularly by the use of all-trans retinoic acid (ATRA) has stimulated considerable interest in the development of small molecules that can modulate RAR and RXR in AML cells. RA can also activate the pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ) in a context dependent manner. In the aqueous intracellular milieu, RA is transported by the cellular retinoid-binding protein CRABP-II, or by the fatty-acid-binding protein FABP5, depending on the ratio of FABP5 to CRABP-II and cellular levels of RA. In cells, expressing high CRABP-II and low FABP5, RA activates the RAR, whereas in the presence of the reverse ratio, RA activates PPARβ/δ. These two different modes of RA delivery leads to opposite cellular outcomes. Cells harboring high level of CRABP-II, RA is delivered to RAR leading to differentiation and growth arrest. However, FABP5 high expressing cells, RA is delivered to PPARβ/δ resulting in survival and proliferation. Therefore, preventing FABP5 from such antagonism may be a novel therapeutic strategy for AML with high FABP5. Here, we report the development of a therapeutic approach using a specific FABP5 inhibitor (iFABP5) that facilitates the delivery of RA to tumor suppressor RAR.
Methods: Genetic as well as pharmacologic models in AML cell lines were used to validate the therapeutic target by decoupling PPARβ/δ and RAR/RXR pathways. Knockdown and overexpression of key regulators of these pathways were rationally used to understand pro-survival effects of FABP5 overexpression. The efficacy of small molecule pharmacophore were evaluated in vitro in cell free and cell culture model systems as well as in vivo small animal model systems as a proof of therapeutic concept.
Results: We analyzed AML samples from different cohorts (Beat AML and TCGA) for the expression levels of genes involved in RAR and PPARβ/δ pathways. Most of the AML patients have upregulation of pro-survival PPARβ/δ pathway genes including FABP5 and downregulation of differentiating promoting RAR pathway genes including critical RA chaperone CRABP2. On the otherhand, ATRA sensitive APL patients and cell lines have lower expression of FABP5 compared to AML. We established FABP5 as a therapeutic target for ATRA sensitization in AML cell line THP1. Genetic ablation of FABP5 using shRNA sensitizes THP1 cells to ATRA treatment compared to scramble control cells. We used in silico approach to develop a novel small molecule iFABP5 that binds to recombinant FABP5 (Kd= 2x10-7) with a 56-fold higher affinity compared to ATRA (Kd=11x10-6 Mole) in a cell free fluorescence reporter binding assay using 8-Anilino-1-naphthalenesulfonic acid (ANS). Efficacy of iFABP5 as a single agent and in combination with ATRA was evaluated in THP1, HL-60, K562, and OCI-AML5 cells. The effect of the combination was most pronounced in THP-1 cells. The iFABP5 synergizes with ATRA and induces the differentiation at 72 hours as assessed by both CD11b and CD14 levels. Treatment of cells with single agent iFABP5 leads to downregulation of PPARβ/δ target genes and upregulation of RAR target genes consistent with the genetic ablation of shFABP5. THP1 cells stably expressing shRNA targeting FABP5 has significant growth perturbation in a NSG mouse model compared to non-targeting shRNA. Pre-clinical in vivo evaluation of iFABP5 in combination with ATRA is ongoing as a therapeutic proof of concept.
Conclusion: The lack of effective ATRA response in AML might be due to an aberrant activation of pro-survival PPARβ/δ pathway, which negatively affects ATRA-regulated gene expression and its antileukemic activity. Reprogramming of the RA delivery to RAR pathway with the addition of a small molecular inhibitor of FABP5 could potentially restore therapeutic effects of ATRA in FABP5 over expressing cases of AML subtypes.
Disclosures
Maciejewski: Novartis: Consultancy; Alexion: Consultancy.
Effects of Simvastatin on Retinoic Acid System in Primary Human Endometrial Stromal Cells and in a Chimeric Model of Human Endometriosis
