Deficiency of Wiskott-Aldrich syndrome protein has opposing effect on the pro-oncogenic pathway activation in nonmalignant versus malignant lymphocytes

Abstract

Immunodeficiency is associated with cancer risk. Accordingly, hematolymphoid cancers develop in Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder (PID) resulting from the deficiency of WAS-protein (WASp) expressed predominantly in the hematolymphoid cell lineages. Despite the correlation between WASp deficiency and hematolymphoid cancers, the molecular mechanism underlying the oncogenic role of WASp is incompletely understood. Employing the WASp-sufficient and WASp-deficient cell-pair model of human T and B lymphocytes, we show that WASp deficiency differentially influences hyperactivation versus inhibition of both CDC42:ERK1/2 and NF-κB:AP-1 pro-oncogenic signaling pathways in nonmalignant versus malignant T and B lymphocytes. Furthermore, WASp deficiency induces a cell-type specific up/down-modulation of the DNA-binding activities of NF-κB, AP-1, and multiple other transcription factors with known roles in oncogenesis. We propose that WASp functions as a putative "tumor-suppressor" protein in normal T and B cells, and "oncoprotein" in a subset of established T and B cell malignancies that are not associated with the NPM-ALK fusion.

Fig. 1. The up/or down modulatory effect of WASp deficiency on CDC42-GTP levels is context-dependent.

a Western blot analyses of the indicated isogenic T cell and B cell pairs, nonmalignant (normal) or malignant (B cell lymphomas; T cell leukemia), expressing endogenous WASp (WT control) or lacking WASp (WAS gene knock-out by CRISPR/Cas9, WKO) sequentially re-probed with the indicated antibodies. The data are representative of at least three independent experiments performed at different intervals. b Gel densitometric quantitation of the Western blot bands from (a) shown as mean + SD for the WASp-deficient sample relative to their corresponding WT sample, after normalizing the values to their respective loading control (β-actin, shown as 1.0). Western blot data are representative of 3–4 independent assays. c Quantitation of active CDC42-GTP levels in the indicated cell pairs, WT and WASp-deficient, using the ELISA assay kit (G-LISA). Top panel, shows the actual experimental values as mean + SD, n = 3–4 experiments. (*) denotes p values < 0.001 determined by unpaired, two-tailed Student’s t test with Welch’s correction. Positive (CDC42 protein) and negative (no protein, background signal) controls are also shown. Bottom panel, shows the relative abundance or paucity of active CDC42-GTP fraction normalized to total CDC42 expression (shown as % increase or decrease) in WASp-deficient sample compared to WT control (shown as 1.0).

Fig. 2. The up/or down modulatory effect of WASp deficiency on MAPK/ERK and PI3K/AKT pathways is also context-dependent.

a Diagrammatic depiction of the key downstream effectors of the CDC42, MAPK, PI3K, and mTOR signaling pathways funneling into NF-κB/AP-1 activation that influences oncogenicity. b and c Western blots of the whole cell lysates from nonmalignant (b) or malignant (c) T and B cells, WT and WASp-deficient, sequentially re-probed with the indicated antibodies. Isogenic T cell pair (WT and WKO), heterologous B cells (WT is from a normal donor; WAS03, WAS68 are from 2 WAS patients). Small “p” denotes phosphorylation at the residues shown in parenthesis. The data are representative of at least three experiments conducted at different intervals. Gel densitometric quantitation of the Western blot bands from b and c are shown under the corresponding images as mean + SD for the WASp-deficient sample relative to their corresponding WT sample (WT = 1.0), after normalizing the values to their respective loading control (β-actin).

Fig. 3. WASp deficiency alters the constitutive DNA-binding activities of NF-κB, AP-1, and multiple other transcription factors in a context-dependent manner.

a The DNA-binding activity of the indicated transcription factors to their respective DNA-sequences (radioactive DNA-probes) monitored by electrophoretic mobility shift assay (EMSA) is shown for the indicated cell types, malignant and nonmalignant. Verification of the NF-κB and AP-1 EMSA-bands was performed by super-shift and competition assays, shown in Supplementary Fig. 1B. The data are representative of at least three independent EMSA assays for each factor. b Shows the tabulated summary of the ELISA (CDC42-GTP) and Western blot results (pERK, pAKT) (from Figs. 1 and 2) along with the DNA-binding activities of NF-κB and AP-1 (from Fig. 3a). Increased activation is denoted by up arrow in green, decreased activation by down arrow in red, and unchanged by “unch”. A similar set of data showing modulation of other transcription factors by WASp-deficiency is summarized in Supplementary Fig. 2. c Cartoon summarizing the key findings of the above table, which forms the basis to propose a model for WASp’s double-role in oncogenesis in nonmalignant versus malignant lymphocytes. Symbols ↑ or ↓ denotes increased or decreased activation/activity of the indicated factor in the indicated cell type.

Fig. 4. Pharmacological inhibition of hyperactivated CDC42-GTP and ERK pathways in nonmalignant WASp-deficient T and B cells decreases the DNA-binding activity of transcription factors and cell-growth.

a EMSA assays show changes in the DNA-binding activity of three transcription factors (NF-κB, AP-1, CREB) induced by chemically inhibiting (+) or not inhibiting (−) different effectors of the indicated signaling pathways in WASp-deficient cells. Data are representative of 2–3 independent experiments. b Western blot showing the effect of suppressing CDC42-GTP by ML141 on pERK1/2 and pAKT activation in the indicated WASp-deficient T and B cells. Gel densitometric quantitation of the Western blot bands are shown as mean + SD for the WASp-deficient sample relative to their corresponding WT sample (WT = 1.0), after normalizing the values to their respective loading control (β-actin). c MTS cell proliferation assay to monitor percentage of cell-growth inhibition induced by different signaling factor inhibitors (inh), individually or in combination. The data are shown as mean ± SD, n = 3 independent experiments. p value, two-way ANOVA comparing WT against WASp-deficient; ns nonsignificant.