Statins markedly potentiate aminopeptidase inhibitor activity against (drug-resistant) human acute myeloid leukemia cells.

Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells. Methods: U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and in vitro non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation. Results: A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents; and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation; this was experimentally confirmed by impaired Rheb prenylation by simvastatin. Conclusion: These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.

Targeting histone methylation to reprogram the transcriptional state that drives survival of drug-tolerant myeloid leukemia persisters.

Although chemotherapy induces complete remission in the majority of acute myeloid leukemia (AML) patients, many face a relapse. This relapse is caused by survival of chemotherapy-resistant leukemia (stem) cells (measurable residual disease; MRD). Here, we demonstrate that the anthracycline doxorubicin epigenetically reprograms leukemia cells by inducing histone 3 lysine 27 (H3K27) and H3K4 tri-methylation. Within a doxorubicin-sensitive leukemia cell population, we identified a subpopulation of reversible anthracycline-tolerant cells (ATCs) with leukemic stem cell (LSC) features lacking doxorubicin-induced H3K27me3 or H3K4me3 upregulation. These ATCs have a distinct transcriptional landscape than the leukemia bulk and could be eradicated by KDM6 inhibition. In primary AML, reprogramming the transcriptional state by targeting KDM6 reduced MRD load and survival of LSCs residing within MRD, and enhanced chemotherapy response in vivo. Our results reveal plasticity of anthracycline resistance in AML cells and highlight the potential of transcriptional reprogramming by epigenetic-based therapeutics to target chemotherapy-resistant AML cells.

IgA Complexes in Plasma and Synovial Fluid of Patients with Rheumatoid Arthritis Induce Neutrophil Extracellular Traps via FcαRI.

Autoantibodies, including rheumatoid factor (RF), are an important characteristic of rheumatoid arthritis (RA). Interestingly, several studies reported a correlation between the presence of IgA autoantibodies and worse disease course. We demonstrated previously that triggering the IgA Fc receptor (FcαRI) on neutrophils results in neutrophil recruitment and the release of neutrophil extracellular traps (NETs). Because this can lead to tissue damage, we investigated whether IgA immune complexes in plasma and synovial fluid of RA patients activate neutrophils. RF isotypes were measured with ELISA, and immune complexes were precipitated using polyethylene glycol 6000. Isolated neutrophils were incubated with immune complexes, and activation and release of NETs were determined in the presence or absence of FcαRI-blocking Abs. Plasma and SF of RA patients contained IgM, IgG, and IgA RFs. Patient plasma IgA RF and IgM RF showed a strong correlation. No uptake of IgM and minimal endocytosis of IgG immune complexes by neutrophils was observed, in contrast to avid uptake of IgA complexes. Incubation of neutrophils with immune complexes resulted in the production of reactive oxygen species, as well as the release of NETs, lactoferrin, and chemotactic stimuli. Importantly, activation of neutrophils was reduced when FcαRI was blocked. Neutrophils were activated by IgA immune complexes, which suggests that neutrophils play a role in inducing joint damage in RA patients who have IgA autoantibody complexes, thereby increasing the severity of disease. Blocking FcαRI inhibited neutrophil activation and, as such, may represent an additional attractive novel therapeutic strategy for the treatment of RA.

Multifactorial resistance to aminopeptidase inhibitor prodrug CHR2863 in myeloid leukemia cells: down-regulation of carboxylesterase 1, drug sequestration in lipid droplets and pro-survival activation ERK/Akt/mTOR.

Aminopeptidase inhibitors are receiving attention as combination chemotherapeutic agents for the treatment of refractory acute myeloid leukemia. However, the factors determining therapeutic efficacy remain elusive. Here we identified the molecular basis of acquired resistance to CHR2863, an orally available hydrophobic aminopeptidase inhibitor prodrug with an esterase-sensitive motif, in myeloid leukemia cells. CHR2863 enters cells by diffusion and is retained therein upon esterase activity-mediated conversion to its hydrophilic active metabolite drug CHR6768, thereby exerting amino acid depletion. Carboxylesterases (CES) serve as candidate prodrug activating enzymes given CES1 expression in acute myeloid leukemia specimens. We established two novel myeloid leukemia sublines U937/CHR2863(200) and U937/CHR2863(5uM), with low (14-fold) and high level (270-fold) CHR2863 resistance. The latter drug resistant cells displayed: (i) complete loss of CES1-mediated drug activation associated with down-regulation of CES1 mRNA and protein, (ii) marked retention/sequestration of the prodrug, (iii) a substantial increase in intracellular lipid droplets, and (iv) a dominant activation of the pro-survival Akt/mTOR pathway. Remarkably, the latter feature coincided with a gain of sensitivity to the mTOR inhibitor rapamycin. These finding delineate the molecular basis of CHR2863 resistance and offer a novel modality to overcome this drug resistance in myeloid leukemia cells.

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors.

BACKGROUND: In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence. METHODS: Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC). RESULTS: Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10-12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive ß5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2-4 fold increase in the mRNA and protein levels of the constitutive ß5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC. CONCLUSIONS: These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.

Inactivating PSMB5 mutations and P-glycoprotein (multidrug resistance-associated protein/ATP-binding cassette B1) mediate resistance to proteasome inhibitors: ex vivo efficacy of (immuno)proteasome inhibitors in mononuclear blood cells from patients with rheumatoid arthritis.

Bortezomib (BTZ), a registered proteasome inhibitor (PI) for multiple myeloma, has also been proposed as a potential antirheumatic agent. Its reported side effects, however, make it unappealing for long-term administration, and resistance may also develop. To overcome this, second-generation PIs became available. Here, we investigated whether a novel class of peptide epoxyketone-based PIs, including carfilzomib, N-((S)-3-methoxy-1-(((S)-3-methoxy-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-2-methylthiazole-5-carboxamide (ONX0912), and (S)-3-(4-methoxyphenyl)-N-((S)-1-((S)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanamide (ONX0914), might escape two established BTZ-resistance mechanisms: 1) mutations in the proteasome ß5 subunit (PSMB5) targeted by these PIs, and 2) drug efflux mediated by ATP-binding cassette transporters. THP1 myeloid sublines with acquired resistance to BTZ (54- to 235-fold) caused by mutations in the PSMB5 gene displayed marked cross-resistance but less pronounced cross-resistance to carfilzomib (9- to 32-fold), ONX0912 (39- to 62-fold), and ONX0914 (27- to 97-fold). As for ATP-binding cassette transporter-mediated efflux, lymphoid CEM/VLB cells with P-glycoprotein (Pgp)/multidrug resistance 1 overexpression exhibited substantial resistance to carfilzomib (114-fold), ONX0912 (23-fold), and ONX0914 (162-fold), whereas less resistance to BTZ (4.5-fold) was observed. Consistently, ß5 subunit-associated chymotrypsin-like proteasome activity was significantly less inhibited in these CEM/VLB cells. Ex vivo analysis of peripheral blood mononuclear cells from therapy-naive patients with rheumatoid arthritis revealed that, although basal Pgp levels were low, P-glycoprotein expression compromised the inhibitory effect of carfilzomib and ONX0914. However, the use of P121 (reversin 121), a Pgp transport inhibitor, restored parental cell inhibitory levels in both CEM/VLB cells and peripheral blood mononuclear cells. These results indicate that the pharmacologic activity of these PIs may be hindered by drug resistance mechanisms involving PSMB5 mutations and PI extrusion via Pgp.

Adipokines as novel biomarkers in paediatric systemic lupus erythematosus.

OBJECTIVES: Patients with SLE are at risk for premature atherosclerosis and metabolic syndrome. Cytokines produced by adipocytes, adipokines, are important in glucose/lipid metabolism and the development of atherosclerosis. The objectives of this study were to evaluate leptin, adiponectin and ghrelin concentrations in paediatric SLE (pSLE) and to correlate these concentrations with measures of disease activity, serum lipid concentrations, measures of insulin resistance and serum homocysteine concentrations. METHODS: Two hundred and eight samples from 105 patients with pSLE and 77 samples from 77 healthy paediatric controls were evaluated by ELISA to measure leptin, adiopnectin and ghrelin. Students' t-test was used for analysis. Concentrations of adipokines were correlated with disease activity, serum lipids, insulin resistance and homocysteine. RESULTS: Overall 35 SLE patients (34%) had an abnormally elevated leptin level. The only significant correlation of leptin concentrations was with homocysteine concentrations but not disease activity, prednisone dose, lipids or insulin resistance. There was no difference in the mean adiponectin concentrations between the control and patient groups and none of the patient samples were below the normal lower limit while seven were elevated. There was a significant correlation of adiponectin concentrations with prednisone dose, lipid concentrations and insulin resistance but not with disease activity or homocysteine. Elevated ghrelin concentrations were found in 20% of the pSLE patients. The only correlation of ghrelin concentrations was with homocysteine. CONCLUSIONS: Adipokines are novel biomarkers in pSLE. They may represent cardiovascular risk and are not just surrogate markers for disease activity, therapy or serum lipids. Their correlation with atherosclerosis needs to be explored.