High TCL1 expression and intact T-cell receptor signaling define a hyperproliferative subset of T-cell prolymphocytic leukemia

Abstract

The T-cell leukemia 1 (TCL1) oncoprotein is overexpressed by chromosomal rearrangement in the majority of cases of T-cell prolymphocytic leukemia (T-PLL). In vitro, TCL1 can modulate the activity of the serine-threonine kinase AKT, a downstream effector of T-cell receptor (TCR) signaling. In a series of 86 T-PLL tumors, we show that expression of TCR, and levels of TCL1 and activated AKT are adverse prognostic markers. High-level TCL1 in TCR-expressing T-PLL is associated with higher presenting white blood cell counts, faster tumor cell doubling, and enhanced in vitro growth response to TCR engagement. In primary tumors and TCL1-transfected T-cell lines, TCR engagement leads to rapid recruitment of TCL1 and AKT to transient membrane activation complexes that include TCR-associated tyrosine kinases, including LCK. Pharmacologic inhibition of AKT activation alters the localization, stability, and levels of these transient TCL1-AKT complexes and reduces tumor cell growth. Experimental introduction and knockdown of TCL1 influence the kinetics and strength of TCR-mediated AKT activation. We propose that in T-PLL, TCL1 represents a highly regulated, targetable modulator of TCR-mediated AKT growth signaling.

Figure 1

The molecular feature of TCL1 expression is associated with a hyperproliferative clinical subgroup of T-PLL. Kaplan-Meier analysis of overall survival in T-PLL patients showed an inverse association with presenting white blood cell (WBC) count (A), pretreatment lymphocyte doubling time (LDT, B), and TCL1 immunohistochemical expression (C). The TCL1⁺ subset of T-PLL showed higher presenting WBC counts (D) and shorter pretreatment LDT (E).

Figure 2

The T-cell receptor signaling pathway is functional in T-PLL and responses are influenced by TCL1 levels. (A) Detection of surface T-cell receptor (sTCR) in T-PLL cells by flow cytometry for sCD3 and TCRα/β (67/82 sTCR⁺ versus 15/82 sTCR⁻ cases) correlated with shorter overall survival (OS). (B) Expression of the Ser473-phosphorylated activated form of AKT, as analyzed by Western blot, also significantly correlated with poor OS. (C) TCR engagement stimulated growth in T-PLL tumor cells only in cases expressing sTCR, whereas ConA/IL2 or PMA/ionomycin stimulated growth in nearly all cases (MTT assay values normalized to unstimulated control). (D) In sTCR⁺ T-PLL, the degree of growth induction by TCR engagement (MTT assay) was higher in those tumors that strongly expressed TCL1 (solid dots) compared with TCL1-low/negative tumors (white circles). (E) Western blot analysis reveals that T-PLL with higher TCL1 levels was associated with a faster induction of pERK1/2 and activated AKT (left versus right panels).

Figure 3

TCL1 and AKT are recruited to membrane complexes following TCR engagement. (A) Laser confocal microscopy on unstimulated primary T-PLL cultures showed a predominantly cytoplasmic distribution of TCL1 and AKT without apparent colocalization (ctr, top panel); only rare “activated” cells show TCL1-AKT colocalization. TCR engagement using anti-CD3/28 precoated plates leads to rapid focal recruitment along with colocalization of TCL1 and AKT in a uniform perimembraneous pattern (middle panel), which was also seen with ConA/IL2 stimulation (bottom panel). (B) Continuous TCR engagement in sTCR⁺/sCD3⁺ T-PLL shifted localization of TCL1 to discrete membrane complexes that also included pAKT-S473 and LCK. Staining was done using anti-TCL1 (FITC: green) in combination with anti-AKT1/2 or anti-LCK (Cy3: red), or a TCL1 antiserum (Cy3: red) with a monoclonal anti–pAKT-S473 (FITC: green). Merged pictures are shown for low-power fields; asterisk indicates location of cell shown in enlarged panels for individual fluorochromes and merged image.

Figure 4

Inhibitors of AKT signaling alter AKT-TCL1 complex formation and T-PLL growth in culture. (A) AKT complexes were isolated from primary T-PLL lysates by immunoprecipitation using an immobilized anti-AKT antibody, and showed increased phosphoactivated (pS473) AKT and TCL1 as well as increased amounts of ZAP70 and LAT following 30 minutes of TCR engagement. Increased AKT kinase activity following TCR stimulation was found by elevated phospho-GSK3α/β substrate levels (numbers indicate fold changes following stimulation compared with DMSO control). Preincubation of T-PLL tumor cells with the PI3K inhibitor LY294002 or the AKT inhibitor A443654 abolished the TCR-stimulated phosphorylation of AKT and GSK3α/β target and decreased the amount of TCL1 complexed with immunoprecipitated AKT. A443654 also led to an overall increase in pAKT-S473. (B) LY294002 at 20 μM, A443654 at 0.5μM, and QLT0267 at 40 μM reduced the level of proliferation in unstimulated T-PLL cultures at baseline and following TCR cross-linking compared with the DMSO vehicle control (MTT assay at 48 hours).

Figure 5

TCL1 introduction into leukemic T cells influences AKT phosphorylation and kinase activity. (A) Transfection of TCL1 into the sTCR⁺ mature T-cell leukemia line HH augmented baseline and TCR-induced AKT phosphoactivation, and increased phosphorylation of the AKT target GSK3α/β (2.6-fold at baseline and 1.3- to 4.2-fold TCR induced). Similar to primary T-PLL tumor cells, TCR cross-linking also increased the amount of TCL1 complexed with AKT by 2.2-fold. Preincubation of tumor cells with the AKT inhibitor A443654 at 0.5 μM leads to slightly decreased GSK3α/β phosphorylation, and a compensatory pAKT increase (kinase assay and Western blot following AKT immunoprecipitation). Indicated are fold changes over the unstimulated DMSO vehicle control. (B) Transient knockdown of TCL1 in the HH-TCL1 cell line reduced baseline levels of activated pAKT in immunoprecipitated complexes compared with control HH-TCL1 (down to 0.7-fold) or cells transfected with scrambled siRNA (assayed at 72 hours). TCL1 knockdown also reduced levels of activated pAKT following TCR engagement for 30 minutes and 2 hours at these time points (ie, 3.3- to 2.2-fold after 30 minutes of simulation).