Correction to: CXCR5 overexpression in HL-60 cells enhances chemotaxis toward CXCL13 without anticipated interaction partners or enhanced MAPK signaling.

There is a typographical error in the primer sequence published in this article.

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.

CXCR5 overexpression in HL-60 cells enhances chemotaxis toward CXCL13 without anticipated interaction partners or enhanced MAPK signaling.

CXCR5 is a serpentine receptor implicated in cell migration in lymphocytes and differentiation in leukocytes. It causes MAPK pathway activation and has known membrane partners for signaling. CXCR5 mRNA is reportedly expressed in neutrophils following isolation, but its role in this cellular context is unknown. CXCR5 is also expressed in HL-60 cells, a human acute myeloid leukemia line, following treatment with all-trans retinoic acid, which induces differentiation toward a neutrophil-like state. CXCR5 is necessary for this process; differentiation was crippled in CXCR5 knockout cells and enhanced in cells ectopically expressing it. Since CXCR5 has various membrane protein partners, we investigated whether CXCR5-driven all-trans retinoic acid-induced differentiation depends on its association with such partners. Pursuing this, we generated HL-60 cells overexpressing the protein. We found that CXCR5 drove migration toward its ligand, CXCL13, and probed for interactions with several candidates using flow cytometry-based Förster resonance energy transfer. Surprisingly, we did not detect interactions with any candidates, including three reported in other cellular contexts. Additionally, we observed no significant changes in all-trans retinoic acid-induced differentiation; this may be due to the stoichiometry of CXCR5 and partner receptors or CXCL13. The anticipated membrane partnerings were surprisingly apparently unnecessary for downstream CXCR5 signaling and all-trans retinoic acid-induced 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.

Probing the requirement for CD38 in retinoic acid-induced HL-60 cell differentiation with a small molecule dimerizer and genetic knockout.

CD38 is an ectoenzyme and receptor with key physiological roles. It metabolizes NAD+ to adenosine diphosphate ribose (ADPR) and cyclic ADPR, regulating several processes including calcium signalling. CD38 is both a positive and negative prognostic indicator in leukaemia. In all-trans retinoic acid (RA)-induced differentiation of acute promyelocytic leukaemia and HL-60 cells, CD38 is one of the earliest and most prominently upregulated proteins known. CD38 overexpression enhances differentiation, while morpholino- and siRNA-induced knockdown diminishes it. CD38, via Src family kinases and adapters, interacts with a MAPK signalling axis that propels differentiation. Motivated by evidence suggesting the importance of CD38, we sought to determine whether it functions via dimerization. We created a linker based on the suicide substrate arabinosyl-2'-fluoro-2'-deoxy NAD+ (F-araNAD+), dimeric F-araNAD+, to induce homodimerization. CD38 homodimerization did not affect RA-induced differentiation. Probing the importance of CD38 further, we created HL-60 cell lines with CRISPR/Cas9-mediated CD38 truncations. Deletion of its enzymatic domain did not affect differentiation. Apart from increased RA-induced CD11b expression, ablation of all but the first six amino acids of CD38 affected neither RA-induced differentiation nor associated signalling. Although we cannot discount the importance of this peptide, our study indicates that CD38 is not necessary for RA-induced differentiation.

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.

Artificial muscle actuators in biorobotic fish fins.

Artificial muscle technologies offer the possibility of designing robotic systems that take full advantage of biological architectures. Of current artificial muscle technologies, nickel titanium (Ni-Ti) shape memory alloys are among a few that are readily usable by engineering labs without specialized skills in material science and/or chemistry. Ni-Ti actuators are now being used to replace servomotors in biorobotic fins. This has significantly reduced the volume that is required for actuators, and will enable several fins to be integrated into a multi finned, flexible bodied, biorobotic fish.