Proteomic Correlates and Prognostic Significance of Kidney Injury in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Kidney disease is common in heart failure with preserved ejection fraction (HFpEF). However, the biologic correlates and prognostic significance of kidney injury (KI), in HFpEF, beyond the estimated glomerular filtration rate (eGFR), are unclear. METHODS AND RESULTS: Using baseline plasma samples from the TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist) trial, we measured the following KI biomarkers: cystatin-C, fatty acid-binding protein-3, Beta-2 microglobulin, neutrophil gelatinase-associated lipocalin, and kidney-injury molecule-1. Factor analysis was used to extract the common variability underlying these biomarkers. We assessed the relationship between the KI-factor score and the risk of death or HF-related hospital admission in models adjusted for the Meta-Analysis Global Group in Chronic Heart Failure risk score and eGFR. We also assessed the relationship between the KI factor score and ~5000 plasma proteins, followed by pathway analysis. We validated our findings among HFpEF participants in the Penn Heart Failure Study. KI was associated with the risk of death or HF-related hospital admission independent of the Meta-Analysis Global Group in Chronic Heart Failure risk score and eGFR. Both the risk score and eGFR were no longer associated with death or HF-related hospital admission after adjusting for the KI factor score. KI was predominantly associated with proteins and biologic pathways related to complement activation, inflammation, fibrosis, and cholesterol homeostasis. KI was associated with 140 proteins, which reproduced across cohorts. Findings regarding biologic associations and the prognostic significance of KI were also reproduced in the validation cohort. CONCLUSIONS: KI is associated with adverse outcomes in HFpEF independent of baseline eGFR. Patients with HFpEF and KI exhibit a plasma proteomic signature indicative of complement activation, inflammation, fibrosis, and impaired cholesterol homeostasis.

Circulating biomarker analyses in a longitudinal cohort of patients with IPF.

Idiopathic pulmonary fibrosis (IPF) is an incurable interstitial lung disease characterized by fibrosis. Two FDA-approved drugs, pirfenidone and nintedanib, only modestly prolong survival. In this study, we asked whether levels of select circulating biomarkers in patients with IPF demonstrated changes in response to treatment over time and whether treatment with pirfenidone and nintedanib led to differential biomarker expression. Serial plasma samples from 48 patients with IPF on usual treatment and six healthy volunteers were analyzed to identify differentially expressed blood protein. Hypothesis-driven potential biomarker selection was based on recent literature, internal preclinical data, and the PROLIFIC Consortium (Schafer P. 6th Annual IPF Summit. Boston, MA, 2022) proposed biomarkers of pulmonary fibrosis. We compared our findings to public databases to provide insights into relevant signaling pathways in IPF. Of the 26 proteins measured, we found that 11 (SP-D, TIMP1, MMP7, CYFRA21-1, YKL40, CA125, sICAM, IP-10, MDC, CXCL13) were significantly elevated in patients with IPF compared with healthy volunteers but their levels did not significantly change over time. In the IPF samples, seven proteins were elevated in the treatment group compared with the no-treatment group. However, protein profiles were not distinguishable between patients on pirfenidone versus nintedanib. We demonstrated that most proteins differentially detected in our samples were predicted to be secreted from the lung epithelial or interstitial compartments. However, a significant minority of the proteins are not known to be transcriptionally expressed by lung cells, suggesting an ongoing systemic response. Understanding the contributions of the systemic response in IPF may be important as new therapeutics are developed.NEW & NOTEWORTHY In this study, we confirmed protein expression differences in only a subset of predicted biomarkers from IPF and control subjects. Most differentially expressed proteins were predicted to be secreted from lung cells. However, a significant minority of the proteins are not known to be transcriptionally expressed by lung cells, suggesting an ongoing systemic response. The contributions of the systemic response in IPF may be important as new therapeutics are developed.

Iberdomide in patients with systemic lupus erythematosus: a randomised, double-blind, placebo-controlled, ascending-dose, phase 2a study.

OBJECTIVE: To evaluate safety, pharmacokinetics, pharmacodynamics and efficacy of iberdomide in patients with SLE. Iberdomide is a high-affinity cereblon ligand that targets the hematopoietic transcription factors Ikaros and Aiolos for proteasomal degradation. METHODS: A 12-week, multicentre, double-blind, placebo-controlled, dose-escalation study in active SLE was followed by a 2-year, open-label active treatment extension phase (ATEP) (NCT02185040). In the dose-escalation phase, adults with active SLE were randomised to oral placebo or iberdomide (0.3 mg every other day, 0.3 mg once daily, 0.6 mg and 0.3 mg alternating once daily, or 0.6 mg once daily). Primary endpoints were safety and tolerability. RESULTS: The dose-escalation phase enrolled 42 patients, with 33 completing this phase and 17 patients enrolling into the ATEP. In the dose-escalation phase, the most common treatment-emergent adverse events (TEAEs; iberdomide/placebo groups) were nausea (20.6%/12.5%), diarrhoea (17.6%/12.5%) and upper respiratory tract infection (11.8%/12.5%). Most TEAEs were mild or moderate in severity and more common in the highest dose groups in both study phases. In the dose-escalation phase, Physician's Global Assessment and Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) activity scores improved relative to baseline and placebo in all iberdomide groups, with a trend toward continued score improvements in the ATEP. In the dose-escalation phase, iberdomide treatment resulted in dose-dependent reductions in total B cells and plasmacytoid dendritic cells in blood. Improvements in CLASI activity scores correlated with plasmacytoid dendritic cell depletion. CONCLUSIONS: These proof-of-concept findings suggest a favourable benefit/risk ratio in SLE for iberdomide, a drug with a novel immunomodulatory mechanism of action, supporting further clinical investigation.

Clinical and Proteomic Correlates of Plasma ACE2 (Angiotensin-Converting Enzyme 2) in Human Heart Failure.

ACE2 (angiotensin-converting enzyme 2) is a key component of the renin-angiotensin-aldosterone system. Yet, little is known about the clinical and biologic correlates of circulating ACE2 levels in humans. We assessed the clinical and proteomic correlates of plasma (soluble) ACE2 protein levels in human heart failure. We measured plasma ACE2 using a modified aptamer assay among PHFS (Penn Heart Failure Study) participants (n=2248). We performed an association study of ACE2 against ≈5000 other plasma proteins measured with the SomaScan platform. Plasma ACE2 was not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 was associated with older age, male sex, diabetes mellitus, a lower estimated glomerular filtration rate, worse New York Heart Association class, a history of coronary artery bypass surgery, and higher pro-BNP (pro-B-type natriuretic peptide) levels. Plasma ACE2 exhibited associations with 1011 other plasma proteins. In pathway overrepresentation analyses, top canonical pathways associated with plasma ACE2 included clathrin-mediated endocytosis signaling, actin cytoskeleton signaling, mechanisms of viral exit from host cells, EIF2 (eukaryotic initiation factor 2) signaling, and the protein ubiquitination pathway. In conclusion, in humans with heart failure, plasma ACE2 is associated with various clinical factors known to be associated with severe coronavirus disease 2019 (COVID-19), including older age, male sex, and diabetes mellitus, but is not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 protein levels are prominently associated with multiple cellular pathways involved in cellular endocytosis, exocytosis, and intracellular protein trafficking. Whether these have a causal relationship with ACE2 or are relevant to novel coronavirus-2 infection remains to be assessed in future studies.

Mechanisms Underlying the Clinical Effects of Apremilast for Psoriasis.

Psoriasis is a chronic, systemic, inflammatory disease with manifestations resulting from a dysregulated immune response. In psoriatic skin, expression of all phosphodiesterase 4 (PDE4) isoforms (A-D), part of a family of enzymes known to regulate cyclic adenosine monophosphate levels and immune homeostasis, is elevated compared with healthy controls. Agents that inhibit the enzymatic activity of PDE4, the predominant PDE in most immune cells, exert well-recognized anti-inflammatory effects. Apremilast is a selective PDE4 inhibitor approved for the treatment of adults with moderate to severe plaque psoriasis and/or psoriatic arthritis. In vitro and in vivo investigations have demonstrated the beneficial impact of apremilast treatment on PDE4 activity, inflammatory signal expression, and dermal psoriasiform signs. In patients with moderate to severe psoriasis, treatment with apremilast is associated with significant reductions in plasma levels of interleukin (IL)-17F, IL-17A, IL-22, and tumor necrosis factor-α compared with placebo as early as week 4; decreases in cytokine levels were sustained with continued treatment. Multivariate analyses demonstrated that while changes in IL-17F are the most important predictor of improvement in Psoriasis Area and Severity Index scores, apremilast exerts synergistic attenuating effects among a key group of cytokines involved in the pathology of psoriasis, and these effects correlate with improved skin symptoms. These in vitro and clinical data demonstrate that the beneficial effects of apremilast on known inflammatory mediators are associated with its clinical efficacy. J Drugs Dermatol. 2018;17(8):835-840.

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Aiolos Overexpression in Systemic Lupus Erythematosus B Cell Subtypes and BAFF-Induced Memory B Cell Differentiation Are Reduced by CC-220 Modulation of Cereblon Activity.

BAFF is a B cell survival and maturation factor implicated in the pathogenesis of systemic lupus erythematosus (SLE). In this in vitro study, we describe that soluble BAFF in combination with IL-2 and IL-21 is a T cell contact-independent inducer of human B cell proliferation, plasmablast differentiation, and IgG secretion from circulating CD27+ memory and memory-like CD27-IgD- double-negative (DN) B cells, but not CD27-IgD+ naive B cells. In contrast, soluble CD40L in combination with IL-2 and IL-21 induces these activities in both memory and naive B cells. Blood from healthy donors and SLE patients have similar circulating levels of IL-2, whereas SLE patients exhibit elevated BAFF and DN B cells and reduced IL-21. B cell differentiation transcription factors in memory, DN, and naive B cells in SLE show elevated levels of Aiolos, whereas Ikaros levels are unchanged. Treatment with CC-220, a modulator of the cullin ring ligase 4-cereblon E3 ubiquitin ligase complex, reduces Aiolos and Ikaros protein levels and BAFF- and CD40L-induced proliferation, plasmablast differentiation, and IgG secretion. The observation that the soluble factors BAFF, IL-2, and IL-21 induce memory and DN B cell activation and differentiation has implications for extrafollicular plasmablast development within inflamed tissue. Inhibition of B cell plasmablast differentiation by reduction of Aiolos and Ikaros may have utility in the treatment of SLE, where elevated levels of BAFF and Aiolos may prime CD27+ memory and DN memory-like B cells to become Ab-producing plasmablasts in the presence of BAFF and proinflammatory cytokines.

Phosphodiesterase 4 in inflammatory diseases: Effects of apremilast in psoriatic blood and in dermal myofibroblasts through the PDE4/CD271 complex.

Phosphodiesterases 4 (PDE4) act as proinflammatory enzymes via degradation of cAMP, whereas PDE4 inhibitors play an anti-inflammatory role in vitro and in vivo. In particular, apremilast has been recently approved for the treatment of psoriasis and psoriatic arthritis. However, little is known on the expression pattern of PDE4 in psoriasis. We report that PDE4B and PDE4D mRNA are overexpressed in peripheral blood mononuclear cells (PBMC) from psoriasis, as compared with normal controls, while apremilast reduces PBMC production of a number of pro-inflammatory cytokines and increases the levels of anti-inflammatory mediators. PDE4 expression is up-regulated in psoriatic dermis as compared with normal skin, with particular regard to fibroblasts. This is confirmed in vitro, where both dermal fibroblasts (DF) and, to a greater extent, myofibroblasts (DM) express all PDE4 isoforms at the mRNA and protein level. Because PDE4 interacts with the nerve growth factor (NGF) receptor CD271 in lung fibroblasts, we evaluated the relationship and function of PDE4 and CD271 in normal human skin fibroblasts. All PDE4 isoforms co-immunoprecipitate with CD271 in DM, while apremilast inhibits apoptosis induced by ß-amyloid, a CD271 ligand, in DM. Furthermore, apremilast significantly reduces NGF- and transforming growth factor-ß1 (TGF-ß1)-induced fibroblast migration, and inhibits DF differentiation into DM mediated by NGF or TGF-ß1. Finally, in DM, apremilast significantly reduces cAMP degradation induced by treatment with ß-amyloid. Taken together, these results indicate that PDE4 play an important role in psoriasis. In addition, the study reveals that the PDE4/CD271 complex could be important in modulating fibroblast functions.

Apremilast, a novel phosphodiesterase 4 (PDE4) inhibitor, regulates inflammation through multiple cAMP downstream effectors.

INTRODUCTION: This work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR). METHODS: After apremilast and LPS incubation, intracellular cAMP, TNF-α, IL-10, IL-6 and IL-1α were measured in the Raw264.7 monocytic murine cell line. PKA, Epac1/2 (signaling intermediates for cAMP) and A2AR knockdowns were performed by shRNA transfection and interactions with A2AR and A2BR, as well as with methotrexate were tested in vitro and in the murine air pouch model. Statistical differences were determined using one or two-way ANOVA or Student's t test. The alpha nominal level was set at 0.05 in all cases. A P value of < 0.05 was considered significant. RESULTS: In vitro, apremilast increased intracellular cAMP and inhibited TNF-α release (IC50=104nM) and the specific A2AR-agonist CGS21680 (1µM) increased apremilast potency (IC50=25nM). In this cell line, apremilast increased IL-10 production. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by apremilast. In the murine air pouch model, both apremilast and MTX significantly inhibited leukocyte infiltration, while apremilast, but not MTX, significantly inhibited TNF-α release. The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than with apremilast alone. CONCLUSIONS: The immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because the A2AR is also involved in the anti-inflammatory effects of MTX, the mechanism of action of both drugs involves cAMP-dependent pathways and is therefore partially overlapping in nature.

CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL.

Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros. Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-α, -ß, and -γ production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide.

Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.).

Cereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4(CRBN) . T cell co-stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4(CRBN) substrates responsible for T cell co-stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4(CRBN) is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4(CRBN) with enhanced ubiquitination leading to cereblon-dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression. The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity.

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

Immunomodulatory effects in a phase II study of lenalidomide combined with cetuximab in refractory KRAS-mutant metastatic colorectal cancer patients.

UNLABELLED: This study assessed the immunomodulatory effects in previously treated KRAS-mutant metastatic colorectal cancer patients participating in a phase II multicenter, open-label clinical trial receiving lenalidomide alone or lenalidomide plus cetuximab. The main findings show the T cell immunostimulatory properties of lenalidomide as the drug induced a decrease in the percentage CD45RA(+) naïve T cells 3-fold while increasing the percentage HLA-DR(+) activated T helper cells and percentage total CD45RO(+) CD8(+) memory T cytotoxic cells, 2.6- and 2.1-fold respectively (p<0.0001). In addition, lenalidomide decreased the percentage of circulating CD19(+) B cells 2.6-fold (p<0.0001). Lenalidomide increased a modest, yet significant, 1.4-fold change in the percentage of circulating natural killer cells. Our findings indicate that lenalidomide significantly activates T cells, suggestive of an immunotherapeutic role for this drug in settings of maintenance therapy and tumor immunity. Furthermore, reported for the first time is the effect of lenalidomide in combination with cetuximab on T cell function, including increases in circulating naïve and central memory T cells. In summary, lenalidomide and cetuximab have significant effects on circulating immune cells in patients with colorectal carcinoma. TRIAL REGISTRATION: ClinicalTrials.gov NCT01032291.

Isosteric analogs of lenalidomide and pomalidomide: synthesis and biological activity.

A series of analogs of the immunomodulary drugs lenalidomide (1) and pomalidomide (2), in which the amino group is replaced with various isosteres, was prepared and assayed for immunomodulatory activity and activity against cancer cell lines. The 4-methyl and 4-chloro analogs 4 and 15, respectively, displayed potent inhibition of tumor necrosis factor-α (TNF-α) in LPS-stimulated hPBMC, potent stimulation of IL-2 in a human T cell co-stimulation assay, and anti-proliferative activity against the Namalwa lymphoma cell line. Both of these analogs displayed oral bioavailability in rat.

Lenalidomide efficacy in activated B-cell-like subtype diffuse large B-cell lymphoma is dependent upon IRF4 and cereblon expression.

Durable responses with lenalidomide monotherapy have been reported in patients with non-Hodgkin lymphoma. In relapsed/refractory diffuse large B-cell lymphoma (DLBCL), higher responses were observed in the activated B-cell-like (ABC) subtype than in the germinal centre B-cell-like subtype. Herein, the molecular mechanisms involved in the differential efficacy of lenalidomide in DLBCL subtypes were investigated. Using DLBCL cell lines, lenalidomide treatment was found to preferentially suppress proliferation of ABC-DLBCL cells in vitro and delay tumour growth in a human tumour xenograft model, with minimal effect on non-ABC-DLBCL cells. This tumouricidal effect was associated with downregulation of interferon regulatory factor 4 (IRF4), a hallmark of ABC-DLBCL cells. IRF4 inhibition by lenalidomide induced downregulation of B-cell receptor (BCR)-dependent NF-κB. Whereas IRF4-specific small, interfering RNA mimicked the effects of lenalidomide reducing NF-κB activation, IRF4 overexpression enhanced NF-κB activation and conferred resistance to lenalidomide. These findings indicate the crucial role of IRF4 inhibition in lenalidomide efficacy in ABC cells. Furthermore, lenalidomide-induced IRF4 downregulation required the expression of cereblon, a molecular target of lenalidomide. Taken together, these findings suggest that lenalidomide has direct antitumour activity against DLBCL cells, preferentially ABC-DLBCL cells, by blocking IRF4 expression and the BCR-NF-κB signalling pathway in a cereblon-dependent manner.

Effect of an angiogenesis inhibitor on hepatic tumor perfusion and the implications for adjuvant cytotoxic therapy.

PURPOSE: To determine whether dynamic contrast material-enhanced (DCE) computed tomography (CT) can help identify hepatic tumor perfusion response to vascular remodeling induced by antiangiogenesis treatment in a rabbit model. MATERIALS AND METHODS: The study was approved by the Animal Use Subcommittee of the University Council on Animal Care. DCE CT hepatic perfusion measurements were performed in the livers of 20 rabbits implanted with VX2 carcinoma. Vascular remodeling was induced with thalidomide dissolved in dimethyl sulfoxide and sterile water, starting at a tumor diameter of 0.7 cm±0.1 and continuing until metastatic lung nodules were observed. The control group (n=8) was given an equivalent volume of the vehicle. The therapy group was subdivided into animals that survived for more than 24 days without lung metastasis (responder group, n=5) or those that survived for less than 24 days (nonresponder group, n=7). Data were analyzed with the Kruskal-Wallis or Friedman rank test and reported as medians and interquartile ranges. RESULTS: DCE CT depicted differential perfusion change within the therapy group after treatment. By day 4, hepatic blood volume (HBV) in the responder group decreased by 29.2% (-32.5% to -11.8%) relative to that before treatment and was significantly different from that in the nonresponder (P=.048) and control (P=.011) groups, where HBV remained stable. By day 8, hepatic artery blood flow decreased by 50.0% (-59.08% to -21.05%) relative to that before treatment in the responder group and was significantly different from that in the nonresponder and control groups (P=.030 for both), which remained stable at -3.5% (-8.5% to 28.7%, P=.50) and -10.0% (-33.8% to 10.4%, P=.48), respectively. CONCLUSION: DCE CT can help differentiate responders from nonresponders by their early differential perfusion response to antiangiogenesis therapy.

Lenalidomide downregulates the cell survival factor, interferon regulatory factor-4, providing a potential mechanistic link for predicting response.

Overexpression of the transcription factor interferon regulatory factor-4 (IRF4), which is common in multiple myeloma (MM), is associated with poor prognosis. Patients with higher IRF4 expression have significantly poorer overall survival than those with low IRF4 expression. Lenalidomide is an IMiD immunomodulatory compound that has both tumouricidal and immunomodulatory activity in MM. This study showed that lenalidomide downregulated IRF4 levels in MM cell lines and bone marrow samples within 8 h of drug exposure. This was associated with a decrease in MYC levels, as well as an initial G1 cell cycle arrest, decreased cell proliferation, and cell death by day 5 of treatment. In eight MM cell lines, high IRF4 levels correlated with increased lenalidomide sensitivity. The clinical significance of this observation was investigated in 154 patients with MM. Among MM patients with high levels of IRF4 expression, treatment with lenalidomide led to a significantly longer overall survival than other therapies in a retrospective analysis. These data confirm the central role of IRF4 in MM pathogenesis; indicate that this is an important mechanism by which lenalidomide exerts its antitumour effects; and may provide a mechanistic biomarker to predict response to lenalidomide.

CC-5079: a small molecule with MKP1, antiangiogenic, and antitumor activity.

INTRODUCTION: CC-5079, a small molecule inhibitor of tubulin polymerization and phosphodiesterase-4 activity, was evaluated for antiangiogenic and antitumor activities. MATERIALS AND METHODS: First, CC-5079 in vitro activity on human umbilical vein endothelial cells (HUVECs), fibroblasts, and MC38 were evaluated by proliferation, migration, and invasion assays. Second, CC-5079 effect on microvessel formation was evaluated ex vivo by chick chorioallantoic membrane (CAM), rat aortic rings assays, and with directed in vivo angiogenesis assay (DIVAA). Third, CC-5079 antitumor effect was determined in treatment of C57BL/6 mice with MC38 tumors. Finally, CC-5079 modulation of MKP1 in HUVECs, human fibroblast, and MC38 were determined by RNA isolation for qRT-PCR. RESULTS: At the 0.1 µM concentration CC-5079 significantly inhibited HUVEC, fibroblast, and MC38 proliferation and migration (all P < 0.001). At the 0.1 µM concentration, CC-5079 also inhibited HUVEC invasion (P < 0.05) but not fibroblast. In the CAM and rat aortic ring assays, CC-5079 at 0.1 µM inhibited microvessel formation (P < 0.05). By DIVAA, CC-5079 at 1 mg/kg/d continuous delivered inhibited microvessel formation (P < 0.05). Intraperitoneal CC-5079 was well tolerated and inhibited the growth of subcutaneous MC38 at 100 mg/kg/d (P < 0.01). By qRT-PCR, CC-5079 stimulated MKP1 expression in HUVEC and fibroblast. CONCLUSION: CC-5079 demonstrated stimulation of MKP1, antiangiogenic, and antitumor properties.

Pomalidomide and lenalidomide induce p21 WAF-1 expression in both lymphoma and multiple myeloma through a LSD1-mediated epigenetic mechanism.

Lenalidomide and pomalidomide have both been evaluated clinically for their properties as anticancer agents, with lenalidomide being available commercially. We previously reported that both compounds cause cell cycle arrest in Burkitt's lymphoma and multiple myeloma cell lines by increasing the level of p21(WAF-1) expression. In the present study, we unravel the molecular mechanism responsible for p21(WAF-1) up-regulation using Namalwa cells as a human lymphoma model. We show that the increase of p21(WAF-1) expression is regulated at the transcriptional level through a mechanism independent of p53. Using a combination of approaches, we show that several GC-rich binding transcription factors are involved in pomalidomide-mediated up-regulation of p21(WAF-1). Furthermore, we report that p21(WAF-1) up-regulation is associated with a switch from methylated to acetylated histone H3 on p21(WAF-1) promoter. Interestingly, lysine-specific demethylase-1 (LSD1) silencing reduced both pomalidomide and lenalidomide up-regulation of p21(WAF-1), suggesting that this histone demethylase is involved in the priming of the p21(WAF-1) promoter. Based on our findings, we propose a model in which pomalidomide and lenalidomide modify the chromatin structure of the p21(WAF-1) promoter through demethylation and acetylation of H3K9. This effect, mediated via LSD1, provides GC-rich binding transcription factors better access to DNA, followed by recruitment of RNA polymerase II and transcription activation. Taken together, our results provide new insights on the mechanism of action of pomalidomide and lenalidomide in the regulation of gene transcription, imply possible efficacy in p53 mutated and deleted cancer, and suggest new potential clinical uses as an epigenetic therapy.

The anti-cancer drug lenalidomide inhibits angiogenesis and metastasis via multiple inhibitory effects on endothelial cell function in normoxic and hypoxic conditions.

Lenalidomide (Revlimid) is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk Myelodysplastic Syndromes (MDS) associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of multiple myeloma patients who have received at least one prior therapy. Previous reports suggest that lenalidomide is anti-angiogenic and this property appears to be related to efficacy in patients with MDS. We have investigated the effect of lenalidomide on the formation of microvessels in a novel in vitro angiogenesis assay utilizing human umbilical arterial rings and in a capillary-like cord formation assay using cultured primary endothelial cells. We found that lenalidomide consistently inhibits both sprout formation by arterial rings and cord formation by endothelial cells in a dose-dependent manner. We also found an inhibitory effect of lenalidomide on the associations between cadherin 5, beta-catenin and CD31, adherens junction proteins whose interaction is critical for endothelial cell cord formation. Furthermore, lenalidomide inhibited VEGF-induced PI3K-Akt pathway signaling, which is known to regulate adherens junction formation. We also found a strong inhibitory effect of lenalidomide on hypoxia-induced endothelial cell formation of cords and HIF-1 alpha expression, the main mediator of hypoxia-mediated effects and a key driver of angiogenesis and metastasis. Anti-metastatic activity of lenalidomide in vivo was confirmed in the B16-F10 mouse melanoma model by a >40% reduction in melanoma lung colony counts versus untreated mice. Our results suggest that inhibitory effects on microvessel formation, in particular adherens junction formation and inhibition of hypoxia-induced processes support a potential anti-angiogenic and anti-metastatic mechanism for this clinically active drug.