ALBERT

Description: TET2 is one of the most commonly mutated genes in myeloid neoplasia. Somatic TET2 mutations (TET2MT) cause complete or partial loss of enzymatic activity. TET2 (along with TET1/3) are Fe2+ and αKG-dependent DNA-dioxygenases that catalyze the oxidation of 5mC→5hmC→5fC→5caC. Ultimately, 5hmC generated by TET2-dioxygenase passively prevents maintenance methylation due to DNA methyltransferase's inability to recognize 5hmC. Alternatively, demethylation may also be a result of base excision repair of fC and caC. TET2MT can serve as therapeutic targets because they are often initiating lesions and present in a large fraction of patients. In this study, a comprehensive analysis of the configurations of TET2MT in myeloid neoplasia including MDS (n=1809) and AML (n=808), showed a remarkable exclusivity with 2-HG producing neomorphic IDH1/2MT (Fig.A). TET2 expression in 97 healthy and 909 MDS/MPN or AML patients from two independent studies showed that IDH1/2MT cases have significantly higher TET2 expression and were also mutually exclusive in cases with lower TET2 expression. Doxycycline inducible expression of IDH1MT led to profound growth inhibition of both a natural TET2MT cell line SIG-M5 and engineered TET2-/- K562, while the effect to parental K562 was mild (Fig.B-E). These observations suggest that mutual exclusivity of TET2MT and IDH1/2MT is due to synthetic lethality of TET2MT cell caused by 2-HG production, rather than redundancy of the consequences of IDH1/2MT and TET2MT. In TET2MT cell 2-HG further inhibit the residual TET-activity (TET1/3) and may cause synthetic lethality to cells with affected TET2 function. SIG-M5 cells expresses significant amount of TET3 while negligible levels of TET1. The reliance on relative compensation through residual TET3 activity has been confirmed in cells by inducible TET3 knockdown. We hypothesized that transient suppression of the residual DNA dioxygenase activity with inhibitors may selectively eliminate TET2-deficient clones. The known TET inhibitors 2-HG, N-oxalylglycine (NOG) and dimethyl methyl fumarate (DMF) lack specificity, pharmacologic properties and potency. Based on the results of in silico docking simulations, we designed and synthesized 16 aKG derivatives. Among them, TETi76 showed best inhibition effect in both TET activity and cell growth of TET2 low expressing cell. TETi76 binds to the α-KG co-factor site of TET2 that principally involves H1801, H1381 and S1898. These amino acids are conserved in all three TET enzymes. To test the in vitro efficacy and specificity of TETi, we used several human myeloid cell lines that harbor loss of function TET2 mutations or constitutively express low TET2 levels as well as bone marrow derived from Tet2+/+, Tet2+/- and Tet2-/- mice (Fig.F-G). Results showed that cells with low 5hmC level were more sensitive to TETi76 treatment. Specificity of TETi76 was further confirmed by RNAseq analyses of TETi76 treated K562, TET2-/- K562 and parental control cells. Moreover, TETi treatment did not appear to affect the function of α-KG-dependent histone dioxygenases. Mechanistically, treatment of SIG-M5 cells with TETi76 induced early and late stages of apoptotic cell death, a finding further confirmed by PARP1 and caspase-3 cleavage. RNAseq analyses of SIGM5 cells after treatment with TETi demonstrated a significant down-regulation of genes involved in transcription and peptide elongation, consistent with the consequences of TET inhibition. Interestingly, we also observed significant up-modulation of oxidative stress response pathway genes consistent with the inhibition of dioxygenases. In particular, TETi76 treatment induces 8-fold increase of oxidative stress sensor NQO1 a NRF2 target gene. To further probe the effects of TETi76 on TET2 deficient cells, Tet2MT/Tet2WT BM cells were co-cultured at fixed ratios to mimic the evolving Tet2MT clones. TETi76 effectively eliminated otherwise dominating Tet2MT cells (Fig.H). To determine the in vivo effects of TETi e.g., on elimination of Tet2MT clones, we performed bone marrow competitive reconstitution assays in PEP mice. TETi treatment selectively restricted the proliferative advantage of Tet2MT HSC compared to vehicle control where, as expected, TET2 mutant clones took over the WT cells. In clinical applications, TET inhibitors may constitute a new class of agents to be used in a targeted fashion in TET2 mutant neoplasia. Figure. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Abazeed:Bayer AG: Honoraria, Other: Travel Support, Research Funding; Siemens: Research Funding. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Description: Taking a mechanistically unbiased approach to the discovery of new myeloma drugs we developed a three organ system assay that selected anti-myeloma compounds from a primary 30,000 small molecule ATP-based screen in one sandwich setup for tolerability by normal bone marrow, stability towards liver enzymes, and activity in the context of cell barriers, bone marrow stromal support, and short, kidney-clearance-like exposure (fig.1). The most promising compound (CCF642) emerging from this test had IC50s around 500 nM in all 8 MM cell lines tested including a proteasome inhibitor refractory line while the IC50 was not reached in 5 healthy bone marrow samples at doses up to 6750 nM. Similar sensitivity as in MM was seen in 7 lymphoma cell lines. Investigation of its mechanism of action revealed that CCF642 immediately stimulates protein ubiquitination which is followed by degradation of major myeloma survival factors (c-MYC, IRF4, NF-κB) and apoptosis, while normal bone marrow cells increase ubiquitination to a much lesser degree and only transiently without undergoing cell death. Experiments with an active biotinylated analog showed that it preferentially enters myeloma cells with cytoplasmic accumulation and over 10-fold lower entry into normal bone marrow. Immunoblots revealed that it becomes covalently attached to proteins in a time- and distribution pattern that parallels ubiquitination responses. In vitro, it bound to ubiquitin activating enzyme UBA1 at the active site cysteine in a similarly stable and reversible way as ubiquitin. Selective alkylation of cysteine sulfhydryl groups inhibited binding of biotinylated CCF642 to UBA1, while addition of ATP and ubiquitin led to its dissociation from UBA1. CCF642 may therefore at least in part act as a small molecule ubiquitin analog. Accordingly CCF642 binding to target proteins of UBA1 containing HeLa fraction II was increased by addition of ATP and blocked in the presence of EDTA. Injected twice a week IP at 30mg/kg it suppressed systemically engrafted luciferase expressing 5TGM1 mouse myeloma cells in syngeneic mice comparable to bortezomib given at the MTD for this mouse strain (0.5mg/kg SC twice a week) with equal prolongation of survival. Results support use of our sandwich assay to select myeloma drug candidates for in vivo testing and reveal a new UBA1 interacting small molecule ubiquitination enhancer with promise for clinical translation. Figure 1: Sandwich Three Organ System Assay Figure 1:. Sandwich Three Organ System Assay Disclosures No relevant conflicts of interest to declare.

Description: Regulation of topoisomerase II (TOPO II) isozymes  and β is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO II and TOPO IIβ proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II ( and β) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIβ protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIβ protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 μmol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 μmol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 μmol/L CPT was significantly lower (P = .003 to P 〈 .001) in RA-treated versus control cells. Results suggest that TOPO IIβ protein levels are posttranscriptionally regulated and that degradation of TOPO IIβ is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II ( + β) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIβ may have a specific role in transcription of genes involved in differentiation with RA treatment. © 1998 by The American Society of Hematology.

Description: Human hematopoietic system produces various types of differentiated and short-lived cells with specialized functions, which require continuous replenishment through the function of hematopoietic stem cells (HSC). HSC failure is a common distal endpoint of various pathogenic mechanisms in almost all bone marrow failure (BMF) syndromes and associated diseases. Hematopoietic growth factor cocktails (HGF) used in expanding bone marrow cells e.g., to increase cellularity of the HSC grafts, lead to differentiation and decreased HSC count. Theoretically, when used in vivo, they may act on progenitors rather than HSC and lead to stimulation of clonal outgrowth. Our current ability to stimulate HSC self-renewal to provide reconstitution of long-term hematopoiesis is limited. Nicotinamide adenine dinucleotide (NAD+) serves as an essential cofactor and substrate for a number of critical cellular processes. NAD+ depletion may occur in response to DNA damage due to free radical/ionizing radiation attack, resulting in significant activation of NAD+ consuming PARPs. Because of their long lifespan, maintenance of the genomic integrity of HSCs by efficient and accurate DNA repair to reduce the risk of BMF and cellular transformation is essential. NAD+ is also required for the maintenance of sirtuins activity, important class III HDAC essential for the prevention of senescence. Aging or chronic immune activation and inflammatory cytokine production result in upmodulation of NAD+ degrading enzyme CD38 that rapidly depletes cellular and extracellular levels of NAD+. Various lines of evidence suggest that regulation of CD38 NADase activity is essential for maintenance of physiologic NAD+ levels. Enhancing NAD+ level can profoundly reduce oxidative cell damage in catabolic tissue, including blood. Consequently, promotion of intracellular NAD+ by preventing NAD+ catabolism represents a promising therapeutic strategy for degenerative diseases in general, and BMF and associated diseases in particular. Therefore; CD38, a major NAD+ degrading enzyme, can be an excellent therapeutic target to increase the cellular levels of NAD+ and consequently improve the function of HSC. Here we report the development of inhibitors of CD38 NADase activity that extends the self-renewal and proliferative life span of HSC. We used structure-guided virtual screening followed by docking simulation to develop CD38 inhibitors. The compounds were synthesized using rational chemical synthesis and characterized by high-resolution mass spectroscopy and C13 & H1NMR. HPLC based assays were performed to assess the ability of compounds to inhibit NAD+ degradation by recombinant CD38. Using an iterative approach of synthesis characterization and activity, we selected the most potent compound, designated as ccf1172, for further studies. Docking simulations, surface plasmon resonance, and HPLC based assays demonstrate that ccf1172 binds (KD=12 nM) and inhibits CD38 (IC50=10 nM) (Fig.1B, C&D). To further characterize the ability of ccf1172, colony forming assays (CFU-A) and long-term culture-initiating cell assays (LTCIC-A) were performed with cord blood, human and murine bone marrows. No GF-like activity was observed, but in combination with GF mix ccf1172 increased the number of erythroid and myeloid colonies (n=9) in dose-dependent manner with a maximal effect seen at 100 nM in a serial replating assay. Significant extension of proliferative life span of hematopoietic progenitors (n=5) were observed (Fig 1E). When we studied the ability of CD38 inhibitor to expand LTCICs in stromal cultures (n=3) as best in vitro surrogates of HSC, ccf1172 increased LTCIC numbers 2.6-fold at 10 nM. The effect did not require the presence of accessory cells as ccf1172 treatment resulted in ~2-fold increase in CD34+Lin-/CD45+ cells in stem cell culture media supplemented with growth factors over a period of 25 days (Fig 1F). The CD38 inhibitor demonstrated cytotoxic effects on nine different leukemic cell lines with IC 50 ranging from 1 to 5 µM while no effect was observed on normal bone marrow. Here, we demonstrate that CD38 inhibition may be a potential therapeutic principle for ex vivo and in vivo expansion of HSC. Decreasing levels of NAD+ have been linked to aging and stem cell dysfunction, as a key aspect of various BMF syndromes. The strategy of CD38 inhibition to preserve NAD+ is innovative and relevant therapeutic strategy. Disclosures Saunthararajah: Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy.

Description: Discovery of many somatic lesions in leukemia enables development of targeted therapies. TET2 is one of the most frequently mutated genes in MDS/related disorders and is also present in a significant proportion of CHIP (clonal hematopoiesis of indeterminate potential) carriers. TET2 mutations (TET2mt) are mostly loss of function and occur in biallelic, heterozygous and hemi/homozygous configurations. TET2 encodes for Fe2+-dependent DNA dioxygenase that utilizes 2-ketoglutarate (αKG) for oxidation of 5-methylcytosine (mC) that results in demethylation either actively, by base excision repair of further oxidation products (fC or caC), or passively, via replication, due to DNA methyltransferase's inability to recognize 5-hydroxymethylcytosine (hmC). TET2mt are good targets for drug discovery because they often initiate the clonal evolution and are present in a large fraction of patients. However, except for ascorbic acid (AA) applied to augment TET2 activity and hypomethylating agents to which TET2mtmay be more susceptible, no specific therapies have been conceptualized for TET2mt disease. Synthetic lethality can be applied to genetic loci affected by loss of function mutations never occurring in homo/hemi/homozygous configuration. However, many tumor suppressor genes (TSG) are similar to TET2, where biallelic inactivation promotes oncogenicity and thus synthetic lethality would not be directly applicable. However, TET2 has TET1/TET3 homologs and a transient inhibition of TET enzymes could still yield a synthetic lethality (particularly in TET2mt). The idea for the proposed therapeutic strategy of TET inhibition was conceptualized based on in vivo observations of mutual exclusivity of TET2mt and IDH1/2 mutations which produces 2HG as a bone-fide natural TET inhibitor. Indeed, in our study of 485 TET2mt cases only 9 carried IDH1/2mt (mostly tiny subclones). Conversely, among 157 IDH1/2mtcases, there were only 11TET2mt (p=5.5x10-9) of which 4 were non-deleterious missense alterations or had a small clonal burden. To further support our hypothesis, we knocked in IDH1mt controlled by the doxycycline-inducible promoter into TET2mt cells. Induction of IDH1R132C or IDH1R132H (or IDH2mt), expression resulted in rapid cytotoxicity in TET2mt, and no growth perturbation was observed for TET2wt cells. Similar results have been observed in mice, where knockdown of TET3 in TET2 background shortened the life span. These observations led us to the idea of developing an aKG antagonist TET specific inhibitors (TETi). Using a structure-guided targeted discovery approach we designed, synthesized and characterized TETi, which demonstrated dose dependent inhibition of dioxygenase activity in a cell-free system. Using an iterative approach of design synthesis and activity, we selected the most potent 'hit', designated as TETi76, for further evaluation. Esterified TETi76 decreased 5hmC production and selectively induced cell death in TET2mt leukemia cell lines, SIGM5 (TET2-/-) and OCI-AML5 (TET2+/-), while a minimal effect was observed in K562 and CMK which are TET2+/+. The LD50 of TETi was 250-fold lower than 2HG in TET2mt cells. In TET2-/-engineered K562, TETi76 also showed cytotoxicity leaving a therapeutic window as compared with wild type K562. Normal bone marrows were resistant to TETi76 in clonogenic assay. In vitro mixing experiments in which Tet2mt/Tet2wt were subjected to methocult cultures at fixed ratios to mimic evolving Tet2mt clones, CD45.2 (either Tet2+/-or Tet2-/-) outcompeted CD45.1 marrow in a control setting, while treatment with TETi76 led to a gradual elimination of mutant marrow. This result was further recapitulated in vivo. In a competitive repopulation transplantation model using graft consist of a mixture of CD45 isotypes mismatched Tet2+/- or Tet2-/-and Tet2+/+marrow cells, TETi76 treatment selectively eliminated Tet2 deficient marrow. Our data have several important implications. It is likely that compensatory function from other TETs or remaining allele is needed for survival and elimination of dioxygenase appears to be lethal. This explains the exclusivity of TET2/IDH1/2 mutations. Dioxygenase inhibitors may have therapeutic applicability for selective elimination of TET2mt cells in MDS or potentially as a preventive measure in CHIP. TETi may represent a novel class of antileukemic agents. Disclosures Nazha: MEI: Consultancy. Maciejewski:Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: 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.

Description: 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.