Hotspots of Aberrant Enhancer Activity in Fibrolamellar Carcinoma Reveal Candidate Oncogenic Pathways and Therapeutic Vulnerabilities.

Fibrolamellar carcinoma (FLC) is a rare, therapeutically intractable liver cancer that disproportionately affects youth. Although FLC tumors exhibit a distinct gene expression profile, the chromatin regulatory landscape and the genes most critical for tumor cell survival remain unclear. Here, we use chromatin run-on sequencing to discover ∼7,000 enhancers and 141 enhancer hotspots activated in FLC relative to nonmalignant liver. Bioinformatic analyses reveal aberrant ERK/MEK signaling and candidate master transcriptional regulators. We also define the genes most strongly associated with hotspots of FLC enhancer activity, including CA12 and SLC16A14. Treatment of FLC cell models with inhibitors of CA12 or SLC16A14 independently reduce cell viability and/or significantly enhance the effect of the MEK inhibitor cobimetinib. These findings highlight molecular targets for drug development, as well as drug combination approaches.

MiR-29 Regulates de novo Lipogenesis in the Liver and Circulating Triglyceride Levels in a Sirt1-Dependent Manner.

MicroRNAs (miRNAs) are known regulators of lipid homeostasis. We recently demonstrated that miR-29 controls the levels of circulating cholesterol and triglycerides, but the mechanisms remained unknown. In the present study, we demonstrated that systemic delivery of locked nucleic acid inhibitor of miR-29 (LNA29) through subcutaneous injection effectively suppresses hepatic expression of miR-29 and dampens de novo lipogenesis (DNL) in the liver of chow-fed mice. Next, we used mice with liver-specific deletion of Sirtuin 1 (L-Sirt1 KO), a validated target of miR-29, and demonstrated that the LNA29-induced reduction of circulating triglycerides, but not cholesterol, is dependent on hepatic Sirt1. Moreover, lipidomics analysis revealed that LNA29 suppresses hepatic triglyceride levels in a liver-Sirt1 dependent manner. A comparative transcriptomic study of liver tissue from LNA29-treated wild-type/floxed and L-Sirt1 KO mice identified the top candidate lipogenic genes and hepatokines through which LNA29 may confer its effects on triglyceride levels. The transcriptomic analysis also showed that fatty acid oxidation (FAO) genes respond differently to LNA29 depending on the presence of hepatic Sirt1. Overall, this study demonstrates the beneficial effects of LNA29 on DNL and circulating lipid levels. In addition, it provides mechanistic insight that decouples the effect of LNA29 on circulating triglycerides from that of circulating cholesterol.

Acute suppression of insulin resistance-associated hepatic miR-29 in vivo improves glycemic control in adult mice.

MicroRNAs (miRNAs) are important posttranscriptional regulators of metabolism and energy homeostasis. Dysregulation of certain miRNAs in the liver has been shown to contribute to the pathogenesis of Type 2 diabetes (T2D), in part by impairing hepatic insulin sensitivity. By small RNA-sequencing analysis, we identified seven hepatic miRNAs (including miR-29b) that are consistently aberrantly expressed across five different rodent models of metabolic dysfunction that share the feature of insulin resistance (IR). We also showed that hepatic miR-29b exhibits persistent dysregulation during disease progression in a rat model of diabetes, UCD-T2DM. Furthermore, we observed that hepatic levels of miR-29 family members are attenuated by interventions known to improve IR in rodent and rhesus macaque models. To examine the function of the miR-29 family in modulating insulin sensitivity, we used locked nucleic acid (LNA) technology and demonstrated that acute in vivo suppression of the miR-29 family in adult mice leads to significant reduction of fasting blood glucose (in both chow-fed lean and high-fat diet-fed obese mice) and improvement in insulin sensitivity (in chow-fed lean mice). We carried out whole transcriptome studies and uncovered candidate mechanisms, including regulation of DNA methyltransferase 3a (Dnmt3a) and the hormone-encoding gene Energy homeostasis associated (Enho). In sum, we showed that IR/T2D is linked to dysregulation of hepatic miR-29b across numerous models and that acute suppression of the miR-29 family in adult mice leads to improved glycemic control. Future studies should investigate the therapeutic utility of miR-29 suppression in different metabolic disease states.Enho; insulin resistance; liver; microRNA-29 (miR-29); UCD-T2DM.

Retinoic acid and 6-formylindolo(3,2-b)carbazole (FICZ) combination therapy reveals putative targets for enhancing response in non-APL AML.

In non-acute promyelotic leukemia (APL)- non myelocytic leukemia (AML), identification of a signaling signature would predict potentially actionable targets to enhance differentiation effects of all-trans-retinoic acid (RA) and make combination differentiation therapy realizable. Components of such a signaling machine/signalsome found to drive RA-induced differentiation discerned in a FAB M2 cell line/model (HL-60) were further characterized and then compared against AML patient expression profiles. FICZ, known to enhance RA-induced differentiation, was used to experimentally augment signaling for analysis. FRET revealed novel signalsome protein associations: CD38 with pS376SLP76 and caveolin-1 with CD38 and AhR. The signaling molecules driving differentiation in HL-60 cluster in non-APL AML de novo samples, too. Pearson correlation coefficients for this molecular ensemble are nearer 1 in the FAB M2 subtype than in non-APL AML. SLP76 correlation to RXRα and p47phox were conserved in FAB M2 model and patient subtype but not in general non-APL AML. The signalsome ergo identifies potential actionable targets in AML.

The c-Raf modulator RRD-251 enhances nuclear c-Raf/GSK-3/VDR axis signaling and augments 1,25-dihydroxyvitamin D3-induced differentiation of HL-60 myeloblastic leukemia cells.

Differentiation therapy is used in cancer treatment. Epidemiologic studies showed that higher vitamin D levels are associated with reduced cancer risks. However, the therapeutic doses needed for differentiation are accompanied by hypercalcemia and intolerable pathological sequelae. In the present work we evaluated if RRD-251, a small-molecule, can enhance vitamin D3-induced differentiation of leukemic cells, in the hope of decreasing the needed vitamin D3-dose. We demonstrate that RRD-251 enhances vitamin D3-induced differentiation of leukemic cells, the enrichment of the c-Raf kinase in the nucleus, the binding of nuclear c-Raf to GSK-3, increased phosphorylation of GSK-3 ser 21/9 inhibitory sites, and the binding of GSK-3 to VDR, where GSK-3 inhibition is known to enhance transcriptional activation by VDR. Enhancement of D3-induced p-GSK-3 ser 21/9 by RRD-251 was associated with enhanced Akt-GSK-3 binding, Akt being a known GSK-3 inhibitor, and diminished Erk1/2 binding. Diminishing Erk interaction with GSK-3 was associated with enhanced interaction with Vav1, a known driver of myeloid differentiation. This is redolent of an ATRA/c-Raf/GSK-3/RARα axis we previously reported, although the phosphorylation effects to enhance transcriptional activation on RARα vs VDR diverge. Taken together this indicates potential therapeutic significance for a c-Raf/GSK-3/VDR or RARα axis for leukemic myelo-monocytic differentiation.

Src family kinase inhibitor bosutinib enhances retinoic acid-induced differentiation of HL-60 leukemia cells.

The acute promyelocytic leukemia (APL) has been treated with all-trans retinoic acid (RA) for decades. While RA has largely been ineffective in non-APL AML subtypes, co-treatments combining RA and other agents are currently in clinical trials. Using the RA-responsive non-APL AML cell line HL-60, we tested the efficacy of the Src family kinase (SFK) inhibitor bosutinib on RA-induced differentiation. HL-60 has been recently shown to bear fidelity to a subtype of AML that respond to RA. We found that co-treatment with RA and bosutinib enhanced differentiation evidenced by increased CD11b expression, G1/G0 cell cycle arrest, and respiratory burst. Expression of the SFK members Fgr and Lyn was enhanced, while SFK activation was inhibited. Phosphorylation of several sites of c-Raf was increased and expression of AhR and p85 PI3K was enhanced. Expression of c-Cbl and mTOR was decreased. Our study suggests that SFK inhibition enhances RA-induced differentiation and may have therapeutic value in non-APL AML.

Potential for subsets of wt-NPM1 primary AML blasts to respond to retinoic acid treatment.

Acute myeloid leukemia (AML) has high mortality rates, perhaps reflecting a lack of understanding of the molecular diversity in various subtypes and a lack of known actionable targets. There are currently 12 open clinical trials for AML using combination therapeutic modalities including all-trans retinoic acid (RA). Mutant nucleophosmin-1, proposed as a possible marker for RA response, is the criterion for recruiting patients in three active RA phase 3 clinical trials. We tested the ability of RA alone or in combination with either bosutinib (B) or 6-formylindolo(3,2-b) carbazole (F) to induce conversion of 12 de novo AML samples toward a more differentiated phenotype. We assessed levels of expression of cell surface markers associated with differentiation, aldehyde dehydrogenase activity, and glucose uptake activity. Colony formation capacity was reduced with the combined treatment of RA and B or F, and correlated with modulation of a c-Cbl/Lyn/c-Raf-centered signalsome. Combination treatment was in most cases more effective than RA alone. Based on their responses to the treatments, some primary leukemic samples cluster closer to HL-60 cells than to other primary samples, suggesting that they may represent a hitherto undefined AML subtype that is potentially responsive to RA in a combination differentiation therapy.

An Effective Model of the Retinoic Acid Induced HL-60 Differentiation Program.

In this study, we present an effective model All-Trans Retinoic Acid (ATRA)-induced differentiation of HL-60 cells. The model describes reinforcing feedback between an ATRA-inducible signalsome complex involving many proteins including Vav1, a guanine nucleotide exchange factor, and the activation of the mitogen activated protein kinase (MAPK) cascade. We decomposed the effective model into three modules; a signal initiation module that sensed and transformed an ATRA signal into program activation signals; a signal integration module that controlled the expression of upstream transcription factors; and a phenotype module which encoded the expression of functional differentiation markers from the ATRA-inducible transcription factors. We identified an ensemble of effective model parameters using measurements taken from ATRA-induced HL-60 cells. Using these parameters, model analysis predicted that MAPK activation was bistable as a function of ATRA exposure. Conformational experiments supported ATRA-induced bistability. Additionally, the model captured intermediate and phenotypic gene expression data. Knockout analysis suggested Gfi-1 and PPARg were critical to the ATRAinduced differentiation program. These findings, combined with other literature evidence, suggested that reinforcing feedback is central to hyperactive signaling in a diversity of cell fate programs.

RRD-251 enhances all-trans retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells.

All-trans-retinoic acid (RA) is known to induce terminal granulocytic differentiation and cell cycle arrest of HL-60 cells. Responding to an RA-induced cytosolic signaling machine, c-Raf translocates to the nucleus, providing propulsion for RA-induced differentiation. This novel mechanism is not understood, but presumably reflects c-Raf binding with nuclear gene regulatory proteins. RRD-251 is a small molecule that prevents the interaction of c-Raf and RB, the retinoblastoma tumor suppressor protein. The involvement of c-Raf and RB in RA-induced differentiation motivates interest in the effects of combined RA and RRD-251 treatment on leukemic cell differentiation. We demonstrate that RRD-251 enhances RA-induced differentiation. Mechanistically, we find that nuclear translocated c-Raf associates with pS608 RB. RA causes loss of pS608 RB, where cells with hypophosphorylated S608 RB are G0/G1 restricted. Corroborating the pS608 RB hypophosphorylation, RB sequestration of E2F increased with concomitant loss of cdc6 expression, which is known to be driven by E2F. Hypophosphorylation of S608 RB releases c-Raf from RB sequestration to bind other nuclear targets. Release of c-Raf from RB sequestration results in enhanced association with GSK-3 which is phosphorylated at its S21/9 inhibitory sites. c-Raf binding to GSK-3 is associated with dissociation of GSK-3 and RARα, thereby relieving RARα of GSK-3 inhibition. RRD-251 amplifies each of these RA-induced events. Consistent with the posited enhancement of RARα transcriptional activity by RRD-251, RRD-251 increases the RARE-driven CD38 expression per cell. The RA/c-Raf/GSK-3/RARα axis emerges as a novel differentiation regulatory mechanism susceptible to RRD-251, suggesting enhancing RA-effects with RRD-251 in therapy.

Induced myelomonocytic differentiation in leukemia cells is accompanied by noncanonical transcription factor expression.

Transcription factors that drive non-neoplastic myelomonocytic differentiation are well characterized but have not been systematically analyzed in the leukemic context. We investigated widely used, patient-derived myeloid leukemia cell lines with proclivity for differentiation into granulocytes by retinoic acid (RA) and/or monocytes by 1,25-dihyrdroxyvitamin D3 (D3). Using K562 (FAB M1), HL60 (FAB M2), RA-resistant HL60 sublines, NB4 (FAB M3), and U937 (FAB M5), we correlated nuclear transcription factor expression to immunophenotype, G1/G0 cell cycle arrest and functional inducible oxidative metabolism. We found that myelomonocytic transcription factors are aberrantly expressed in these cell lines. Monocytic-lineage factor EGR1 was not induced by D3 (the monocytic inducer) but instead by RA (the granulocytic inducer) in lineage bipotent myeloblastic HL60. In promyelocytic NB4 cells, EGR1 levels were increased by D3, while Gfi-1 expression (which promotes the granulocytic lineage) was upregulated during D3-induced monocytic differentiation in HL60, and by RA treatment in monocytic U937 cells. Furthermore, RARα and VDR expression were not strongly correlated to differentiation. In response to different differentiation inducers, U937 exhibited the most distinct transcription factor expression profile, while similarly mature NB4 and HL60 were better coupled. Overall, the differentiation induction agents RA and D3 elicited cell-specific responses across these common FAB M1-M5 cell lines.

Retinoid Chemoprevention: Who Can Benefit?

Acute promyelocytic leukemia (APL) is a treatment success story. From a highly deadly disease it was turned into a highly curable disease by the introduction of differentiation-induction therapy with all-trans retinoic acid (ATRA) in the 1990's. During the last quarter of century, ATRA and other retinoids were used for the treatment and prevention of other cancers and even other diseases. The results were less spectacular, but nevertheless important. Progress has been made toward understanding the mechanism of action of retinoids in different physiological and pathological contexts. For some diseases, specific genetic backgrounds were found to confer responsiveness to retinoid therapy. Therapies that include retinoids and other modalities are very diverse and used both for combined targeting of multiple pathways and for diminishing toxicity.

Resveratrol and Malignancies.

Carcinogenesis is a multifactorial process, frequently encompassing 3 stages: initiation, promotion and progression. It is characterized by multiple deviations from normal both at the cell and organism levels. Although most people have a small number of cells that present deviations from normal, most of those cells will not cause cancer. However, some will. What tips the balance between normal and abnormal is the subject of intense scientific research as well as unfounded speculations. Chronic inflammation is one of the risk factors for cancer. Resveratrol is consumed by the population as a dietary supplement in the hope of decreasing the risk of inflammation and cancer and other chronic diseases such as diabetes and vascular diseases. There is a discrepancy between the doses used in the animal studies showing that resveratrol decreases all three stages of carcinogenesis, and the doses ingested by the population either as supplements or in the diet. While there is health benefit from using high resveratrol doses, it might be also of practical and scientific benefit to focus future effort in understanding the effects of normal dietary resveratrol levels.

6-Formylindolo(3,2-b)Carbazole (FICZ) Modulates the Signalsome Responsible for RA-Induced Differentiation of HL-60 Myeloblastic Leukemia Cells.

6-Formylindolo(3,2-b)carbazole (FICZ) is a photoproduct of tryptophan and an endogenous high affinity ligand for aryl hydrocarbon receptor (AhR). It was previously reported that, in patient-derived HL-60 myeloblastic leukemia cells, retinoic acid (RA)-induced differentiation is driven by a signalsome containing c-Cbl and AhR. FICZ enhances RA-induced differentiation, assessed by expression of the membrane differentiation markers CD38 and CD11b, cell cycle arrest and the functional differentiation marker, inducible oxidative metabolism. Moreover, FICZ augments the expression of a number of the members of the RA-induced signalsome, such as c-Cbl, Vav1, Slp76, PI3K, and the Src family kinases Fgr and Lyn. Pursuing the molecular signaling responsible for RA-induced differentiation, we characterized, using FRET and clustering analysis, associations of key molecules thought to drive differentiation. Here we report that, assayed by FRET, AhR interacts with c-Cbl upon FICZ plus RA-induced differentiation, whereas AhR constitutively interacts with Cbl-b. Moreover, correlation analysis based on the flow cytometric assessment of differentiation markers and western blot detection of signaling factors reveal that Cbl-b, p-p38α and pT390-GSK3ß, are not correlated with other known RA-induced signaling components or with a phenotypic outcome. We note that FICZ plus RA elicited signaling responses that were not typical of RA alone, but may represent alternative differentiation-driving pathways. In clusters of signaling molecules seminal to cell differentiation, FICZ co-administered with RA augments type and intensity of the dynamic changes induced by RA. Our data suggest relevance for FICZ in differentiation-induction therapy. The mechanism of action includes modulation of a SFK and MAPK centered signalsome and c-Cbl-AhR association.

The AhR agonist VAF347 augments retinoic acid-induced differentiation in leukemia cells.

In binary cell-fate decisions, driving one lineage and suppressing the other are conjoined. We have previously reported that aryl hydrocarbon receptor (AhR) promotes retinoic acid (RA)-induced granulocytic differentiation of lineage bipotent HL-60 myeloblastic leukemia cells. VAF347, an AhR agonist, impairs the development of CD14(+)CD11b(+) monocytes from granulo-monocytic (GM) stage precursors. We thus hypothesized that VAF347 propels RA-induced granulocytic differentiation and impairs D3-induced monocytic differentiation of HL-60 cells. Our results show that VAF347 enhanced RA-induced cell cycle arrest, CD11b integrin expression and neutrophil respiratory burst. Granulocytic differentiation is known to be driven by MAPK signaling events regulated by Fgr and Lyn Src-family kinases, the CD38 cell membrane receptor, the Vav1 GEF, the c-Cbl adaptor, as well as AhR, all of which are embodied in a putative signalsome. We found that the VAF347 AhR ligand regulates the signalsome. VAF347 augments RA-induced expression of AhR, Lyn, Vav1, and c-Cbl as well as p47(phox). Several interactions of partners in the signalsome appear to be enhanced: Fgr interaction with c-Cbl, CD38, and with pS259c-Raf and AhR interaction with c-Cbl and Lyn. Thus, we report that, while VAF347 impedes monocytic differentiation induced by 1,25-dihydroxyvitamin D3, VAF347 promotes RA-induced differentiation. This effect seems to involve but not to be limited to Lyn, Vav1, c-Cbl, AhR, and Fgr.

GW5074 and PP2 kinase inhibitors implicate nontraditional c-Raf and Lyn function as drivers of retinoic acid-induced maturation.

The multivariate nature of cancer necessitates multi-targeted therapy, and kinase inhibitors account for a vast majority of approved cancer therapeutics. While acute promyelocytic leukemia (APL) patients are highly responsive to retinoic acid (RA) therapy, kinase inhibitors have been gaining momentum as co-treatments with RA for non-APL acute myeloid leukemia (AML) differentiation therapies, especially as a means to treat relapsed or refractory AML patients. In this study GW5074 (a c-Raf inhibitor) and PP2 (a Src-family kinase inhibitor) enhanced RA-induced maturation of t(15;17)-negative myeloblastic leukemia cells and rescued response in RA-resistant cells. PD98059 (a MEK inhibitor) and Akti-1/2 (an Akt inhibitor) were less effective, but did tend to promote maturation-uncoupled G1/G0 arrest, while wortmannin (a PI3K inhibitor) did not enhance differentiation surface marker expression or growth arrest. PD98059 and Akti-1/2 did not enhance differentiation markers and have potential, antagonistic off-targets effects on the aryl hydrocarbon receptor (AhR), but neither could the AhR agonist 6-formylindolo(3,2-b)carbazole (FICZ) rescue differentiation events in the RA-resistant cells. GW5074 rescued early CD38 expression in RA-resistant cells exhibiting an early block in differentiation before CD38 expression, while for RA-resistant cells with differentiation blocked later, PP2 rescued the later differentiation marker CD11b; but surprisingly, the combination of the two was not synergistic. Kinases c-Raf, Src-family kinases Lyn and Fgr, and PI3K display highly correlated signaling changes during RA treatment, while activation of traditional downstream targets (Akt, MEK/ERK), and even the surface marker CD38, were poorly correlated with c-Raf or Lyn during differentiation. This suggests that an interrelated kinase module involving c-Raf, PI3K, Lyn and perhaps Fgr functions in a nontraditional way during RA-induced maturation or during rescue of RA induction therapy using inhibitor co-treatment in RA-resistant leukemia cells.

A 3D in situ cell counter reveals that breast tumor cell (MDA-MB-231) proliferation rate is reduced by the collagen matrix density.

Many cell types require the biophysical and biochemical cues within the 3D extracellular matrix (ECM) to exhibit their true physiologically relevant behavior. As a result, cell culture platforms have been evolving from traditional 2D petridish plates into 3D biomatrices, and there is a need for developing analytic tools to characterize 3D cell culture. The existing cell counting method, using a hemocytometer or coulter counter, requires that cells are suspended in fluids prior to counting. This poses a challenge for 3D cell culture as cells are embedded in a 3D biomatrix. We use a facile 3D cell counting method that overcomes this limitation and allows for in situ cell counting in a 3D cell culture using equipment that is commonly available in a biology lab. Using a breast tumor cell line, MDA-MB-231, as a model system, we demonstrated that MDA-MB-231 cells (1) grow slower within a 3D collagen matrix than on a 2D substrate for an extended growth time (a week) with a comparable, initial cell-to-cell distance, (2) their cell growth rate decreases with the increase of collagen concentration, showing a linear growth rate rather than an exponential growth rate. Further work using flow cytometry showed that the observed growth rate reduction was consistent with the retardation of the transition to S (synthesis) phase in the cell cycle. This work demonstrates the validity of the 3D cell counting method and the importance of cell-ECM interactions in cell proliferation.

Retinoic acid therapy resistance progresses from unilineage to bilineage in HL-60 leukemic blasts.

Emergent resistance can be progressive and driven by global signaling aberrations. All-trans retinoic acid (RA) is the standard therapeutic agent for acute promyelocytic leukemia, but 10-20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2) HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17)-negative cells. Wild-type (WT) HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D3 (D3). Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D3-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or "precommitment" phase) or subsequently (the late or "lineage-commitment" phase). HL-60 were treated with RA or D3 for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D3 was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D3 responsiveness (unilineage maturation block), followed by bilineage maturation block and progressive signaling defects, notably the reduced expression of Vav1, Fgr, and c-Raf.

6-Formylindolo (3,2-b)carbazole (FICZ) enhances retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells.

BACKGROUND: The aryl hydrocarbon receptor (AhR) ligand 6-Formylindolo(3,2-b)carbazole (FICZ) has received increasing attention since its identification as an endogenous AhR ligand and a photoproduct of tryptophan. FICZ and its metabolites have been detected in human fluids. We recently reported that AhR promotes retinoic acid (RA)-induced granulocytic differentiation of HL-60 myeloblastic leukemia cells by restricting the nuclear abundance of the stem cell associated transcription factor Oct4. The standard clinical management of acute promyelocytic leukemia (APL) is differentiation induction therapy using RA. But RA is not effective for other myeloid leukemias, making the mechanism of RA-induced differentiation observed in a non-APL myeloid leukemia of interest. To our knowledge, this is the first study regarding the influence of FICZ on RA-induced differentiation in any type of leukemic blasts. METHODS: Using flow cytometry and Western blotting assays, we determined the effects of FICZ on RA-induced differentiation of HL-60 human leukemia cells. All experiments were performed in triplicate. The groups RA and FICZ + RA were compared using the Paired-Samples T-Test. Western blot figures present the typical blots. RESULTS: We demonstrate that FICZ enhances RA-induced differentiation, assessed by the expression of the membrane differentiation marker CD11b; cell cycle arrest; and the functional differentiation marker, inducible-oxidative metabolism. FICZ causes changes in signaling events that are known to drive differentiation, and notably augments the RA-induced sustained activation of the RAF/MEK/ERK axis of the mitogen-activated protein kinase (MAPK) cascade. FICZ also augments expression of the known MAPK signaling regulatory molecules c-Cbl, VAV1, pY458 p85 PI3K, Src-family kinases (SFKs), and IRF-1, a transcription factor associated with this putative signalsome that promotes RA-induced differentiation. Moreover, FICZ in combination with RA also increases expression of AhR and even more so of both Cyp1A2 and p47phox, which are known to be transcriptionally regulated by AhR. pY1021 PDGFRß, a marker associated with retinoic acid syndrome was also increased. CONCLUSIONS: Our data suggest that FICZ modulates intracellular signaling pathways and enhances RA-induced differentiation.

The Src-family kinase inhibitor PP2 rescues inducible differentiation events in emergent retinoic acid-resistant myeloblastic leukemia cells.

Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47(phox) expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid's biological effects in WT HL60 cells.

Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells.

All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation.