Increased Frequency of Cells with Activated Ribosomal Protein S6 at Diagnosis Associates with MRD Positivity and Relapse in Childhood BCP ALL

Numerous studies have demonstrated the association between minimal residual disease (MRD) positivity and risk of eventual relapse for children with B cell precursor acute lymphoblastic leukemia (BCP ALL).  Yet despite varied methods to detect MRD (flow cytometry, PCR, sequencing), we still lack knowledge of the biology of drug resistant cells that mediate relapse. 

Performing single cell proteomic analysis using mass cytometry in a clinically annotated cohort of diagnostic samples, we have identified a phosphorylated ribosomal protein S6 (prpS6) signaling phenotype associated with MRD positivity and relapse.  This phenotype has been observed in matched diagnostic and MRD samples and notably, in matched diagnosis/relapse pairs, we have observed an enrichment for this population at relapse. 

Methods Diagnostic BCP ALL samples from patients treated on the AIEOP protocols or healthy bone marrow were assayed either in basal condition or after ex vivo treatment with tyrosine kinase inhibitors (Dasatinib, BEZ-235).  Samples were analyzed using mass cytometry for expression of 40 functional and/or phenotyping proteins.  Mass cytometry data was processed and analyzed using Cytobank. Statistical analysis was performed using GraphPad Prism software.  Significance was determined using student’s t-test or one way ANOVA.

Results Of the nine functional proteins measured in this study, rpS6 demonstrated, in resting cells from patients, the most variable levels of phosphorylation. Across the cohort the frequency of cells with activated rpS6 in the basal state varied between 4%-86% (median 24.7%), depending on the patient.  This level was significantly higher than in healthy bone marrow controls (median 10.7%; p=0.0009). In BCP ALL samples the cells with activated rpS6 expressed higher levels of CD19 (p=0.0001), IgH (p=0.0059), and PAX5 (p=0.0021) when compared to cells without activated rpS6, suggesting a more mature phenotype for the prpS6⁺ blasts. These cells also had significant activation of pCREB (p<0.0001). Treatment with BEZ-235, a PI3K/TORk inhibitor, or Dasatinib, dual ABL/SRC kinase inhibitor, was able to significantly decrease the frequency of cells with activated prpS6 by inhibiting the phosphorylation of rpS6 after 30-minute ex vivo treatment.

Patients with intermediate or high risk MRD status (per clinical protocol definitions) had an increased frequency of cells with activated rpS6 at diagnosis compared with patients with standard risk MRD (30% vs 13.9%; p=0.02). For patients for whom long term follow up data was available (n=31), patients that went on to relapse (n=9) compared to those in remission (n=22) had a statistically significant increased frequency of cells with activated prpS6 (p=0.0391).

To determine whether activation of prpS6 was important for cell survival, we identified two cells lines amongst 7 BCP ALL cell lines that contained this high basal prpS6 subpopulation: NALM6 and SUP-B15.  Treatment of these cells with Dasatinib in combination with the BEZ-235 or the MEKi PD0325901 resulted in decreased viability (6%-25% in TKI treated vs. 84% in untreated) and decreased activation of prpS6 and pCREB at 72 hours. These results support the hypothesis that the prpS6 high phenotype is important for cell survival.

Given the association of activated rpS6 at diagnosis with detectable MRD and relapse, we examined an additional cohort of 9 matched samples from diagnosis Day 8 and Day 15 post induction therapy.  In these samples, it was noted that by Day 8, there was a significant enrichment for cells with activated rpS6 (p=0.0009). In paired diagnosis-relapse samples, in 5 of 6 pairs, there was an enrichment for cells with activated rpS6 at relapse, all suggesting that rpS6 is associated with cells that are drug resistant. Mean prpS6 was significantly higher at relapse compared to diagnosis (p=0.035). Further studies will be critical to determine whether the rpS6 signature is proximal to causal events driving resistance.

Significance We describe biochemical features of a cellular population observed at diagnosis across genetic subtypes associated with MRD positivity and relapse that is enriched in early MRD and relapse samples in BCP ALL.  Ribosomal protein S6 is a target of PI3K, MEK/ERK signaling and the B cell receptor pathway. Inhibition of this signaling results in cell death in model cell lines and suggests a role for therapy directed at this target in BCP ALL.