High Risk Acute Lymphoblastic Leukemia with Rapid NOD/SCID Engraftment Is Characterized by High Protein Expression of CYCLIN B, Beta-CATENIN, ANNEXIN I and Decreased PKC Alpha Activation
Authors: Seyfried Felix , Accordi Benedetta , Queudeville Manon , Eckhoff Sarah Mirjam , Milani Gloria , Galla Luisa , Giordan Marco , Kraus Johann M , Basso Giuseppe , Kestler Hans Armin , Kronnie Geertruy te , Debatin Klaus-Michael , Meyer Lueder Hinrich 
Published: October 2019

Abstract
Abstract 1457
Acute lymphoblastic leukemia (ALL) is the most frequent form of childhood cancer. Although therapy efforts have achieved cure rates of above 80%, about every fifth patient encounters relapse, which is associated with poor outcome. Risk stratification based on prognostic factors is critical for selecting appropriate treatment intensity to improve therapy efficacy and reduce toxicity. However, in spite of new strategies for risk stratification, the majority of relapsed patients are initially stratified into non-high risk groups pointing out the need to identify new prognostic markers that might refer to novel therapeutic targets. We have recently shown in a NOD/SCID/huALL mouse xenotranplant model that early relapse of pediatric B-cell precursor (BCP-) ALL patients is reflected by rapid engraftment (time to leukemia/ TTLshort) of primary ALL cells.
In this study we aimed to identify differently expressed and activated proteins in xenograft ALL of distinct engraftment subgroups employing a Reverse Phase Protein Array (RPPA) strategy. Levels of total or activated proteins were analyzed in patient derived xenograft BCP-ALL samples characterized with respect to their NOD/SCID engraftment phenotype (TTLshort n= 7, TTLlong n= 9). The proteome of each sample was immobilized on nitrocellulose coated glass slides and overall protein expression or phosphorylation of 51 key signaling molecules was detected by incubation with the corresponding specific antibodies. Protein expression and/ or activation was quantified and compared between the different engraftment subgroups (TTLshort/ early relapse versus TTLlong/ good prognosis). RPPA results were validated by Western Blot analyses.
The association of NOD/SCID/huALL engraftment and clinical patient outcome was analyzed and revealed a significantly inferior survival of patients with the TTLshort in contrast to the TTLlong phenotype also in this subgroup of patients (Kaplan Meier, log rank, P=.003).
Upon comparison of the protein expression data (Shrinkage t-test, P <.05, fold change ≥ 1.5) in the TTL subgroups the three proteins CYCLIN B, beta-CATENIN and ANNEXIN I were identified to be overexpressed in TTLshort. In addition, an increased activation of protein-kinase C alpha (phosporylated serine 657, p-PKC alpha S657) was detected in TTLlong leukemia samples.
CYCLIN B is a positive regulator of the cell cycle and highly expressed in the G2/M phase. Consistent with its pro-proliferative function, CYCLIN B was identified to be up-regulated in leukemia samples with rapid NOD/SCID/huALL growth (TTLshort/ early relapse phenotype) (P=.013). Interestingly, the level of CYCLIN B protein expression correlates negatively to TTL (Spearman, rs=-.516, P=.041), suggesting a direct association of leukemia cell cycle progression and leukemia engraftment in the NOD/SCID/huALL xenograft model. Beta-CATENIN, a molecule involved in WNT-signaling and cell adhesion but also associated with apoptosis inhibition and cell growth in other hematological malignancies, was found to be over-expressed in TTLshort (P=.006). Furthermore, beta-CATENIN protein levels were reversely correlated to TTL (Spearman, rs=-.557, P=.025). Additionally, ANNEXIN I, involved in the regulation of inflammation and apoptosis and reported to be up-regulated in hairy cell leukemia, showed high protein levels in TTLshort (P=.033). Protein-kinase C alpha was previously reported to induce apoptosis in an ALL- cell line and showed increased activation in non-relapsing pediatric T-ALL patient samples. In line with this, we found higher levels of PKC alpha activation in the TTLlong -group (P= 8.5e–9), indicating an association with good prognosis.
Taken together, this study identified differentially expressed proteins in prognostic subgroups of BCP-ALL patients with distinct clinical outcomes, which can be further evaluated as new prognostic markers and therapeutic targets.

Disclosures:
Debatin: Apogenix GmbH: Patents & Royalties, Research Funding.