Histopathological features of bone marrow in patients with arthritis and T-cell large granular lymphocyte (T-LGL) lymphocytosis

Patient 1 with rheumatoid arthritis (RA) and T-LGL leukemia. Staining for CD57 demonstrates intrasinusoidal linear arrays and interstitial clusters of T cells (EnVision stain, ×100). Granzyme B highlights cytotoxic granules in these cells (EnVision stain, ×200). Patient 10 with polyclonal T-LGL lymphocytosis. Staining for CD8 shows dispersed T cells (EnVision stain, ×200). Patient 9 with unclassified arthritis, T-LGL leukemia, and and gene rearrangements. CD3 staining shows interstitial and nodular infiltration of T cells (EnVision stain, ×100). Patient 9. The lymphoid nodule contains few CD20B cells (EnVision stain, ×200). Patient 7 with RA and T-LGL leukemia. A decreased count of granulocytic precursors (myeloperoxydase) is shown (EnVision stain, ×200). IGKV, immunoglobulin kappa variable; IGLV, immunoglobulin lambda variable<p><b>Copyright information:</b></p><p>Taken from "Characteristics of T-cell large granular lymphocyte proliferations associated with neutropenia and inflammatory arthropathy"</p><p>http://arthritis-research.com/content/10/3/R55</p><p>Arthritis Research & Therapy 2008;10(3):R55-R55.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2483444.</p><p></p

Ethidium bromide-stained polyacrylamide gel showing polymerase chain reaction products derived from gene rearrangements in patients with rheumatoid arthritis and T-cell large granular lymphocyte (T-LGL) proliferations

Polyclonal expansion of T-LGLs in patient 10. Lane 1: gene rearrangement–negative, polyclonal smear (tube A); lane 2: gene rearrangement-negative, polyclonal smear (tube B); lane 3: gene rearrangement-negative, polyclonal smear (tube C); lane 4: standard 50 base pairs (bp); lane 5: gene rearrangement-negative, polyclonal smear (tube A); lane 6: gene rearrangement-negative, polyclonal smear (tube B); and lane 7: gene rearrangement-negative, polyclonal smear. Monoclonal expansion in polyclonal background in patient 7. Lane 1: gene rearrangement: monoclonal product 180 bp (i) in tube A; lane 2: gene rearrangement: monoclonal product 210 bp (ii) in polyclonal background (tube B); lane 3: gene rearrangement: monoclonal product 160 bp (iii); lane 4: (tube A) gene rearrangement-negative, polyclonal smear; lane 5: standard 50 bp; lane 6: (tube B) gene rearrangement-negative, polyclonal smear; and lane 7: (tube C) gene rearrangement-negative, polyclonal smear. Monoclonal gene rearrangements in patient 1 with T-LGL leukemia. Lane 1: gene rearrangement-negative (tube A); lane 2: gene rearrangement-positive, monoclonal product 250 bp (iv) in tube B; lane 3: (tube C): gene rearrangement-negative, polyclonal smear; lane 4: standard 50 bp; lane 5: gene rearrangement-positive, monoclonal product 230 bp (v) in tube A; lane 6: gene rearrangement-positive, monoclonal product 180 bp (vi) in tube B; and lane 7: gene rearrangement-negative, polyclonal smear. TCR, T-cell receptor.<p><b>Copyright information:</b></p><p>Taken from "Characteristics of T-cell large granular lymphocyte proliferations associated with neutropenia and inflammatory arthropathy"</p><p>http://arthritis-research.com/content/10/3/R55</p><p>Arthritis Research & Therapy 2008;10(3):R55-R55.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2483444.</p><p></p

MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy

<div><p>Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased <i>in vitro</i> dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.</p></div

Overexpression of a constitutively active MEK1 mutant (MEK-Q56P) in GC-sensitive RS4;11 cells induces resistance to DEX.

<p>(A) RS4;11 cells were retrovirally transduced with MEK-Q56P or empty control. Cells were lysed and ERK1/2 phoshorylation status was assessed by immunoblotting. (B-C) Control cells and MEK-Q56P—transduced cells were incubated with DEX (0.05, 2 or 30 μg/ml) for 72h. Thereafter, cell death was assessed by annexinV/PI staining and flow cytometry analysis. Absolute, averaged numbers of apoptotic cells in two independent experiments are indicated in (C). Error bars represent SD. P value was calculated using Student’s t-test.</p

BCN1 knockdown reduces SEL-mediated sensitization to DEX.

<p>(A) SEMK2 cells were retrovirally transduced with BCN1-specific shRNA or scrambled control (SCR). After selection of stable transfectants, BCN1 knockdown was confirmed by western blot. (B,C) Cells with BCN1 knockdown or control cells were incubated with DEX, (0.05 μg/ml or 2 μg/ml) in the presence or absence of MEK1/2 inhibitor, selumetinib (SEL, 200 nM) for 72h. Apoptosis was assessed by annexinV/PI staining followed by flow cytometry analysis. Representative dot-plots from FACS analysis are shown in B; averaged results from 3 independent experiments with SD are indicated in (C) Statistical difference in responses between mock- and shBCN1-transduced cells were determined using 2-sided Student’s t-test. (D) Beclin-1 expression in cells treated with DEX, SEL or their combination. SEMK2 cells were incubated for 24 h with DEX (0.05 μg/ml), SEL (200 nM) or combination of DEX+SEL and BCN1 expression level was assessed by western blot.</p

MEK1/2 inhibitor, selumetinib, intensifies DEX induced LC3 conversion, MDC staining and GFP-LC3 relocalization in GC-resistant SEMK2 ALL cells.

<p>(A) GC-sensitive (RS4;11) and GC-resistant (SEMK2) cells were incubated with DEX (0.05 μg/mL) in the presence or absence of MEK1/2 inhibitor, selumetinib (SEL, 200 nM) for 24h. When indicated, cells were pretreated for 3 h with 50 μM or 100 μM of chloroquine (CQ). Thereafter, LC3 processing was assessed by immunoblotting. Densitometric analyses of LC3II/I are indicated below the blots. (B) SEMK2 and RS4;11 cells were cultured as described above for 24h, stained with MDC (50 μM) and analyzed by fluorescence microscopy. (C) SEMK2 cells were stably transduced with GFP-LC3 and incubated with DEX, SEL or combination of DEX+SEL for 24h. GFP-LC3 relocalization from diffuse cytoplasmic in control cells to a massive dotty pattern in DEX+SEL treated cells indicates LC3 recruitment to autophagosome membranes. In the lower panel, the percentage of cells with GFP-LC3 dots was quantified by counting the number of cells with > 3 dots and divided by a total number of GFP positive cells in 5 random non-overlapping fields. Pictures were taken at 630 × magnification. P value was calculated using Student’s t-test. (D) Induction of autophagy markers by the DEX and SEL co-treatment involves mTOR suppression. SEMK2 and RS4;11 cells were incubated with DEX in the presence or absence of SEL and lysed. 4E-BP1 phoshorylation status was assessed by immunoblotting. (E) GC-resistant (SEMK2) and—sensitive (RS4;11) cells were incubated with mTOR inhibitor rapamycin (100nM) in the presence or absence of DEX (0.05 μg/mL) for 24h. Thereafter, LC3 processing was assessed by immunoblotting and quantified. Densitometric analyses of LC3II/I are indicated below the blots. (F) Cells were treated as in (E) for 72h. Thereafter, cell numbers were assessed by counting 6 independent fields in Burker’s chamber. Data represent two independent experiments. P-values were calculated using 2-sided Student’s t-test.</p

Viability of ALL cell lines incubated with SEL.

<p>ALL cell lines were incubated with SEL at the indicated doses for 72h and cell death was assessed by annexinV/PI staining followed by flow cytometry analysis. Error bars indicate SD from at least 3 independent repeats.</p

Schematic diagram showing a postulated mechanism of MEK inhibition—induced sensitization to GCs via modulation of autophagy.

<p>Schematic diagram showing a postulated mechanism of MEK inhibition—induced sensitization to GCs via modulation of autophagy.</p

MEK1/2 inhibitor, selumetinib sensitizes GC-resistant ALL cells to dexamethasone.

<p>(A) GC-resistant cells exhibit coordinate upregulation of MAPK/ERK pathway components. GSEA plots show relative upregulation of MAPK/ERK cascade components in GC-resistant ALL cells in two independent datasets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155893#pone.0155893.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155893#pone.0155893.ref026" target="_blank">26</a>]. Relative expression of pathway components is visualized by the heat map. FDR- false discovery rate. (B) ALL cell lines with active MAPK/ERK pathway (SEMK2, 697, CCRF-CEM) and ALL cells with undetectable expression of MAPK/ERK pathway (RS4;11) were incubated for 4h with MEK1/2 inhibitor, selumetinib (SEL, 200 nM). Thereafter, phosphorylation status of ERK1/2 and its substrate p90RSK were assessed by immunoblotting. (C) ALL cell lines were incubated with DEX (0.05 μg/ml 2 μg/ml and 30 μg/ml), SEL (200 nM) or combination of DEX+SEL for 72h and cell death was assessed by annexinV/PI staining followed by flow cytometry analysis. * p<0.05, ** p<0.01, ns- not significant. Error bars represent SD of three independent experiment. P values were calculated using Student’s t-test. (D) Cells were incubated as in (C) and used to determine the phosphorylation status of ERK1/2 by immunoblotting.</p

BIM and GR expression in cells incubated with DEX, SEL or their combination.

<p>GC- resistant and -sensitive SEMK2 and RS4;11 were incubated for 24 h with DEX (0.05–2 μg/ml), SEL (200 nM) or combination of DEX+SEL. BIM (A) and GR (B) expression levels were assessed by western blot.</p