Patient-Derived Stem Cell Surrogates of Acute Lymphoblastic Leukemia Are Sensitive towards TRAIL-Induced Apoptosis Which Is Determined at the Level of Receptor-Proximal Signaling

Abstract 2133

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and a promising future cytotoxic drug for cancer treatment. TRAIL induces apoptosis in tumor cells with no apparent toxicity in patients. Here, we studied the effect and signaling mechanisms of TRAIL on patient-derived acute lymphoblastic leukemia (ALL) cells and stem cell surrogates.

In 171 fresh primary acute leukemia tumor cells, TRAIL induced apoptosis in 28 % of samples and survival in 13 % of samples, while TRAIL had no effect on the remaining majority of samples (59 %). After amplification of 20 samples in NOD/SCID mice, xenograft ALL-cells were isolated from spleens of diseased mice and used for in vitro assays. In the 20 xenograft ALL-samples, TRAIL induced apoptosis in 5 samples (20 %), had no effect on 12 samples (60 %) and induced survival on 3 samples (15 %) and thus in similar frequencies as in primary tumor cells. TRAIL-induced apoptosis in vitro remained unaltered upon passaging of cells through several generations of mice. To study the effect of TRAIL on leukemia stem cell surrogates, frequency of leukemia-initiating cells (LICs) was determined by limiting dilution analysis. Fresh xenograft ALL-cells were treated with and without TRAIL in vitro and were transplanted into 25 mice per treatment group in different absolute cell numbers. After 3 months, engraftment was determined in all mice by FACscan and immunohistochemistry of bone marrow, spleen and liver, and LIC frequency was calculated. TRAIL induced 60 % apoptosis in leukemia bulk cells and 75 % apoptosis in LICs suggesting that TRAIL induced significant apoptosis in LICs of this xenograft sample.

We next asked which signaling mechanisms determine apoptosis sensitivity of patient-derived ALL cells towards TRAIL. The first signaling steps upon stimulation with TRAIL consist in binding of TRAIL to its receptors and recruitment of the adapter molecule and the proximal initiator caspase Caspase-8 to form the so called “death inducing signaling complex” (DISC). The expression pattern of intracellular DISC proteins was similar between TRAIL-sensitive and TRAIL-resistant samples. In contrast to other tumor entities, expression of pro-apoptotic TRAIL-receptors correlated well to TRAIL-induced apoptosis in patient-derived ALL cells. TRAIL-sensitive, but not TRAIL-resistant samples showed significant binding of TRAIL to the surface of living cells and cytosolic activation of Caspase-3. Accordingly, DISC immune precipitations showed that TRAIL bound TRAIL-receptor 1 and 2 which recruited FADD leading to cleavage of Caspase-8 and FLIP at the DISC in sensitive, but not resistant samples. Taken together our data indicate that in patient-derived ALL cells, TRAIL-sensitivity is regulated at the receptor-proximal signaling pathway, where sufficient binding between TRAIL and its receptors is required to enable recruitment of FADD, activation of Caspase-8 and induction of apoptosis.

Our data suggest that sensitivity for TRAIL-induced apoptosis is regulated at the receptor-proximal TRAIL signaling pathway in patient-derived ALL cells. Characterization of the receptor-proximal TRAIL signaling pathway might thus allow the prediction of TRAIL function for potential individualized treatment strategies. As TRAIL is able to induce apoptosis in LICs as leukemia stem cell surrogates, TRAIL might represent an interesting future drug for the treatment of ALL.