Targeting CRABP-II Overcomes Pancreatic Cancer Drug Resistance By Reversing Lipid Raft Cholesterol Accumulation and AKT Survival Signaling

Background: Resistance to standard therapy is a major reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Developing novel therapy to overcome PDAC drug-resistance is urgently needed. CRABP-II was highly expressed in all PDAC but not expressed in normal pancreatic tissues and chronic pancreatitis. CRABP-II was shown to promote PDAC migration and metastasis while its potential role in promoting PDAC drug-resistance was not known.Methods: A paired cohort of human primary and relapsing PDAC tissues was assessed for CRABP-II expression by immunohistochemistry. CRISPR/cas9 gene editing was used to establish CRABP-II knockout cell lines and MTT assays were performed to assess gemcitabine sensitivity in vitro. Cleaved caspase-3/PARP blots and Annexin V staining were conducted to detect cell apoptosis. Gene expression microarray, Q-PCR, western blots, Co-IP and RNA-IP were used to study the molecular function of CRABP-II. Sucrose gradient ultracentrifugation was applied to isolate lipid rafts and LC-MS-MS was used to assess cholesterol content. Both subcutaneous and orthotopic PDX models were established to examine the efficacy of SNIPER-11 and the synergistic effect between SNIPER-11 and gemcitabine in vivo. Results: A higher expression of CRABP-II was found in relapsing PDAC tissue and was associated with poor prognosis. Gemcitabine-resistant cell lines exhibited increased level of CRABP-II, while CRABP-II knockout resensitized PDAC cells to gemcitabine. Mechanistically, aberrant expression of CRABP-II increased the stability of SREBP-1c mRNA through cooperation with HuR and upregulated the downstream genes of SREBP-1c to favor cholesterol uptake and accumulation in lipid rafts. Increased lipid raft cholesterol accumulation facilitated ATK survival signaling and PDAC drug resistance. The small compound SNIPER-11 treatment effectively induced CRABP-II protein degradation, induced apoptosis, and suppressed tumor growth. Combination of SNIPER-11 and gemcitabine significantly reduced the lipid raft cholesterol content in PDX and profoundly inhibited tumor progression. Conclusions: These findings identified CRABP-II as a novel regulator of cholesterol metabolism and suggested that CRABP-II is a selective target for overcoming PDAC drug resistance.

Fatty Acid Binding Protein FABP5: A Novel Therapeutic Target in Acute Myeloid Leukemia

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.

Targeting Antagonists of Retinoic Acid Signaling in Acute Myeloid Leukemia

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 heterodimers with retinoid X receptors (RXRs) to regulate homeostasis of cellular growth. The success of RAR modulation 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. Recent studies have demonstrated that RA can also activate the peroxisome proliferator-activated receptor β/δ (PPARβ/δ). 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. 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 (Fig 1). These two different modes of RA delivery due to the different ratio of these two cargos leads to opposite cellular outcomes. Cells harboring high level of CRABP-II, RA is delivered to RAR leading to apoptosis, growth arrest, and anticancer activity. However, when FABP5 expression is high, RA is delivered to PPARβ/δ resulting in survival, proliferation, and tumor growth. In both cases, retinoid X receptor (RXR) is the indispensable partner of the nuclear receptor involved. Therefore, preventing FABP5 from such antagonism may be a novel therapeutic strategy for AML. Here we report the development of a therapeutic strategy based on a highly specific FABP5 inhibitor (iFABP5) (Fig 2) that will allow the delivery of RA by CRABP-II to activate tumor suppressor function of RAR and RXR. Methods: An iterative approach of design synthesis and activity were employed to select the most potent hit, iFABP5, for further experiments. Expression levels were analyzed by western blot analysis and qRT-PCR. Colony forming assays were used to analyze iFABP5 activity against AML cell lines. Flow cytometry based cell differentiation assay were performed to assess the efficacy of iFABP5 and ATRA combination treatment. Results: The analysis of TCGA data set revealed that a certain class of AML patients (pts) (Trisomy 8 AML) have low levels of CRABP-II and high levels of FABP5, presumably due to gene duplication, that in part explains the inability of ATRA to induce terminal differentiation in AML cells. To test our hypothesis, we screened AML and APL patient (pt) bone marrow cells and found that a number of AML pts bone marrow have high FABP5 and low CRABP-II protein levels while the ATRA responding APL pts has opposite ratio determined by western blotting. Therefore, low CRABP-II and high FABP5 levels in a subset of AML pts lead to the activation of pro-survival PPARβ/δ pathway that promotes proliferation and opposes the differentiation. We also analyzed AML and APL pt samples along with different AML cell lines for mRNA expression using qRT-PCR. High FABP5 levels were observed in the majority of the AML cell lines. Efficacy of novel small molecule FABP5 inhibitor as a single agent and in combination with ATRA was evaluated in HL-60 cells. The FABP5 inhibitor iFABP5 was found to increase differentiation at 72 hours as assessed by both CD11b and CD14 levels in HL-60 cells. To confirm that iFABP5 is targeting FABP5 and indirectly targeting the PPARβ/δ pathway, levels of RAR and PPARβ/δ target genes were evaluated in the absence and presence of iFABP5. Changes in the gene expression of RAR and PPARβ/δ target genes in the presence and absence of iFABP5 were also examined in shFABP5, shPPARβ/δ, and shCRABP2 versus wild type cells. Conclusion: We demonstrated that a small molecule inhibitor of FABP5 synergizes with ATRA and induces the differentiation in AML cells. High FABP5 levels (mRNA and protein) were observed in the majority of the AML cell lines. Hence, FABP5 can be a therapeutic target in AML. Utilizing virtual screening and structurally guided design, we developed a small molecule FABP5 inhibitor that induces monocytic differentiation as observed by increased CD14 surface expression as a single agent and in combination with ATRA. FABP5 is not only a strong target to treat AML pts but also an excellent approach for developing a novel therapeutic for pts where FABP5 expression and activity is high. Disclosures Maciejewski: Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy. Carraway:Agios: Consultancy, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; FibroGen: Consultancy; Jazz: Speakers Bureau; Novartis: Speakers Bureau.

Differential CRABP-II and FABP5 expression patterns and implications for medulloblastoma retinoic acid sensitivity

This study demonstrates the highly variable expression patterns of CRABP-II and FABP5 in medulloblastoma cells and their correlation with RA sensitivities.