Plasma Hsp90 Level as a Marker of Early Acute Lymphoblastic Leukemia Engraftment and Progression in Mice

<div><p>Current monitoring of acute lymphoblastic leukemia (ALL) in living mice is based on FACS analysis of blood hCD45+ cells. In this work, we evaluated the use of human IGFBP2, B2M or Hsp90 as soluble markers of leukemia. ELISA for B2M and IGFBP2 resulted in high background levels in healthy animals, precluding its use. Conversely, plasma levels of Hsp90 showed low background and linear correlation to FACS results. In another experiment, we compared Hsp90 levels with percentage of hCD45+ cells in blood, bone marrow, liver and spleen of animals weekly sacrificed. Hsp90 levels proved to be a superior method for the earlier detection of ALL engraftment and correlated linearly to ALL burden and progression in all compartments, even at minimal residual disease levels. Importantly, the Hsp90/hCD45+ ratio was not altered when animals were treated with dexamethasone or a PI3K inhibitor, indicating that chemotherapy does not directly interfere with leukemia production of Hsp90. In conclusion, plasma Hsp90 was validated as a soluble biomarker of ALL, useful for earlier detection of leukemia engraftment, monitoring leukemia kinetics at residual disease levels, and pre-clinical or mouse avatar evaluations of anti-leukemic drugs.</p></div

ELISA of plasma Hsp90 levels for earlier engraftment confirmation.

<p>(A) Experimental workflow. Cryopreserved primary ALL cells were thawed and injected into 3 mice for expansion. Fresh xenograph ALL cells were then injected into 6 to 12 secondary animals for experiments. For RS4;11 and TALL-1, cultured cells were directly injected in animals for the experiments. Animals were monitored weekly and sacrificed as indicated. (B) Kinetic of Hsp90 plasma levels over time, following ALL injection. Data points represent mean of 3 animals. The cut-off Hsp90 value (0.1 ng/mL) is indicated. Asterisks represent starting week of sequential sacrifice of animals (Time Point 1). Some groups did not have enough animals for the experiment to be carried out until the third week of sacrifice. (C) Percentage of ALL cells (hCD45+) in bone marrow (BM) and peripheral blood (PB) at the different time points. Data points represent mean of 3 animals. Dotted line represents the usual cut-off (0.5%) for ALL detection by flow cytometry in blood samples. Different ALL cells are represented by colors. Circles; BCP-ALL. Triangles, T-ALL.</p

Chemotherapy effects on biomarkers levels.

<p>Plasma biomarkers levels (ELISA) and matched percentage of ALL cells (hCD45+) in peripheral blood (PB) from animals under chemotherapy treatment. Samples were collected for analysis at the beginning (C1 = day 5), middle (C2 = day 19) and end (C3 = day 33) of treatment. Colored bars represent mean ± SD of biomarker levels (left Y axis) for individual mice samples analyzed in duplicate. Different colors represent different treatments. The PI3K inhibitor used was AS605240. Black bars represent percentages of ALL cells in peripheral blood (right Y axis). The dotted line in the Hsp90 graphic represents the ELISA cut-off value. Red arrows highlight lower than expected ELISA values, when compared to levels found in untreated animals with similar percentage of ALL cells.</p

Correlation between plasma Hsp90 level and percentage of ALL cells in the different tissues analyzed.

<p>One representative case of three BCP-ALL or T-ALL analyzed is shown. For complete data refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.s002" target="_blank">S2 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.s003" target="_blank">S3 Fig</a> ELISA Hsp90 and flow cytometry hCD45+ data were transformed to log10 and analyzed by Pearson’s correlation. Correlations between ALL in peripheral blood and in the different tissues are shown for comparisons. Dotted line represents cut-off values for ALL detection by flow cytometry (0.5%) or Hsp90 levels (0.1 ng/mL). Data points correspond to individual samples. Numbers near each data point represent time point of sampling (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.g002" target="_blank">Fig 2</a>). PB; peripheral blood. BM; bone marrow. Circles, BCP-ALL. Triangles, T-ALL.</p

Analysis of three potential leukemia plasma biomarkers.

<p>(A) Peripheral blood B2M, IGFBP-2 and Hsp90 levels (ELISA) in NOD/SCID mice transplanted with a primary human T-ALL or healthy controls. Animals were divided into groups according to the percentage of ALL cells (hCD45+) in peripheral blood by FACS. Data points correspond to individual samples, and horizontal bars correspond to median. *P<0.05; Mann-Whitney U test. (B) Correlation between Hsp90 levels and % ALL cells in peripheral blood. Data points correspond to individual samples. Data were transformed to log10 and analyzed by Pearson’s correlation. (C) Cut-off value of Hsp90 (dotted line) as determined by adding 2 times SD to mean. Animals transplanted with different BCP-ALL (n = 3) and T-ALL (n = 2), sacrificed at the earliest time point (time point 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129298#pone.0129298.g002" target="_blank">Fig 2</a>) were included in the analysis. Leukemia engraftment was confirmed by FACS analysis. Data points correspond to individual samples. PB, peripheral blood; SD, standard deviation.</p

SB225002 and <i>GLIPR1</i> knockdown effects on ROS generation in ALL cells.

<p><b>(A)</b> Reactive oxygen species production in B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) cells treated with DMSO (vehicle; 0.1%) and SB225002 [5 μM and 10 μM]. Cells were treated for 24 h. <b>(B)</b> ROS generation in <i>GLIPR1</i>-knockdown (G-KD) <i>versus</i> control scramble (S) cells treated with DMSO (vehicle; 0.1%) or SB225002 [10 μM] for REH and RS4;11 or SB225002 [5 μM] for Jurkat and TALL-1 for 24 h. <b>(C)</b> Effect of N-Acetyl Cysteine (NAC; a ROS scavenger) pre-treatment on the survival of <i>GLIPR1</i>-knockdown (G-KD) <i>versus</i> control scramble (S) ALL cell lines upon SB225002 treatment for 48 h. Cells were pre-incubated or not with NAC [10 mM] for 3h prior to the SB225002 treatment. SB225002 was used at [10 μM] (REH and RS4;11) or [5 μM] (Jurkat and TALL-1). S = scramble transfection control; G-KD = cells infected with <i>GLIPR1</i>-shRNA lentiviral particles (Sigma-Aldrich). P values were calculated using two-tailed Student’s t-test.</p

SB225002 induces cell death in ALL cell lines.

<p>Effect of SB225002 [100 to 1.5625 μM] on the survival and proliferation of <b>(A)</b> B-ALL and T-ALL cell lines. <b>(B)</b> Effect of SB225002 [5 and 10 μM] on the survival and proliferation of normal PHA-stimulated human lymphocytes. <b>(C)</b> Cell cycle analysis of B-ALL (REH and RS4;11), T-ALL (Jurkat and TALL-1) and normal human PHA-stimulated lymphocytes treated with DMSO (vehicle; 0.1%) and the following concentrations of SB225002: REH and RS4;11 [10 μM]; Jurkat and TALL-1 [3.125 μM]; PHA-stimulated lymphocytes [10 μM]. Representative PI-staining histograms of cells treated with vehicle (clear area) or SB225002 (shaded area) are shown. <b>(D)</b> Annexin-V and propidium iodide flow cytometry analyses of B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) treated with DMSO (vehicle; 0.1%) and SB225002 [10 μM]. Cells were treated for 24 h (for cell cycle and Annexin-V analyses) and 48 h (for MTT analysis). ALL = acute lymphoblastic leukemia; PI = propidium iodide; Lym = PHA-stimulated lymphocytes; C or Ctr = DMSO (vehicle control); SB = SB225002 treatment.</p

Connectivity Map and Ingenuity Pathway Analysis using the SB225002-derived gene expression signature.

<p><b>(A)</b> Connectivity Map (C-Map) analysis using the gene expression signature of Jurkat cells treated with SB225002 [IC₅₀] for 9 h. Compounds colored as black bars in each respectively C-Map plot. Compounds are color-coded as follows: blue, PI3K/mTOR inhibitors; green, HSP90 inhibitors; red, tubulin inhibitors. <b>(B)</b> Signaling pathways activated in Jurkat cells in response to 6 h of SB225002 [IC₅₀] treatment. The statistical threshold (line without boxes) represents the cut-off for significance on the log scale (<i>y</i>-axis, left side). The ratio (line with boxes) of the number of significant genes from the data set that mapped to a pathway divided by the total number of genes from the pathway is also shown (<i>y</i> axis, right side). <b>(C)</b><i>JUN</i>, <b>(D)</b><i>p53</i> and <b>(E)</b><i>TNF</i> pathways are modulated in Jurkat cells after 6 h of SB225002 [IC₅₀] treatment. Analyses were performed using the Ingenuity Pathways Analysis package (Ingenuity Systems).</p

Modulation of <i>CX3CR1</i> and <i>GLIPR1</i> expression in ALL cells upon SB225002 treatment.

<p><b>(A)</b><i>CX3CR1</i> and <b>(B)</b><i>GLIPR1</i> gene expression analysis by quantitative PCR (Q-PCR) and Western blot in Jurkat cells treated with SB225002 [IC₅₀] or DMSO (vehicle control; 0.1%). Treatments were performed for 3h, 6 h, 9 h or 12 h, as indicated. In the Q-PCR analysis, expression values were calculated considering vehicle control (DMSO) as 100%. β-actin was used as loading control in Western blot analysis. Control = DMSO (vehicle control); SB = SB225002 treatment.</p

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by <i>N</i>-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340)

<div><p>Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by <i>N</i>-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.</p></div