Sensitivity of Pediatric Acute Leukemia Cells to Bortezomib and Epoxyketone-Based Proteasome Inhibitors: Correlations with Proteasome Subunit Expression
Authors: Niewerth Denise , Franke Niels , Jansen Gerrit , van Meerloo Johan , Assaraf Yehuda , Kirk Christopher J , Chan Elena T , Zweegman Sonja , Kaspers Gertjan J. , Cloos Jacqueline 
Published: October 2019

Abstract
Abstract 1513
Good response to glucocorticoids (GC) has favorable prognostic value for the survival of children with acute lymphoblastic leukemia (ALL). Hence, GC-resistant and relapsed ALL patients may benefit from GC-sensitization strategies. For this purpose, the reversible proteasome inhibitor (PI) Bortezomib (BTZ) is currently being evaluated in clinical trials in combination with Dexamethasone (DEX) and other drugs. Despite the encouraging results of BTZ in several hematological malignancies, emergence of resistance to BTZ may be a limiting factor to its efficacy. Therefore, the aim of our study was to examine the differential sensitivity of pediatric leukemia cells to BTZ and DEX, as compared to second generation PIs designed to overcome BTZ resistance. These include the epoxyketone-based irreversibly binding PIs Carfilzomib (CFZ), its orally bioavailable analog ONX 0912, and the immunoproteasome inhibitor ONX 0914. The drug concentration required for 50% cell death (LC50) was determined in pediatric patient samples (29 ALL and 12 AML) after 4 days drug exposure using the MTT cytotoxicity assay. Furthermore, the sensitivity to PIs was correlated with protein expression levels of the constitutive proteasome subunits beta5, beta1 and beta2, and the (immuno) proteasome subunits beta5i and beta1i.
ALL cells were significantly more sensitive for BTZ than AML cells (median LC50: 6.0 nM vs 14.2 nM, respectively, p=0.002), and also markedly more sensitive to Dex (median LC50: 23.0 nM vs. >600 nM, p<0.001). Sensitivity profiles for the PIs CFZ, ONX 0912 and ONX 0914 are presented in Table 1. Collectively, ALL cells were significantly more sensitive than AML cells for all these 3 PIs with irreversible binding properties. LC50 concentrations for CFZ were comparable to those of BTZ. In descending order, ONX 0912 and ONX 0914 displayed lower potencies than BTZ/CFZ, but LC50 concentrations were still in the low nanomolar range.Table 1.Difference in in vitro sensitivity to proteasome inhibitors and proteasome subunit expression between pediatric ALL and AML patientsAcute Lymphoblastic LeukemiaAcute Myeloid LeukemiaANOVA p-valueNMedian LC50 (nM)RangeNMedian LC50 (nM)RangeDrugs    BTZ296.03.0–46.11114.210.1–61.00.002    CFZ274.10.08–8.71020.86.0–30.80.000    ONX 09122719.27.6–80.91093.755.7–3940.000    ONX 09142744.68.4–1171024889.2–6780.000    DEX2723.00.50–>60012600.0164–>6000.000Subunit expressionRatio*Ratio*    beta5280.760.00–30.0106.02.2–23.90.080    beta5i2762.58.5–3661055.010.6–3400.714    beta1282.40.00–28.11011.70.92–26.10.029    beta1i2835.15.42–1061017.77.2–49.50.032    beta2284.80.38–23.41020.47.4–39.10.000    beta2iN.D.N.D.N.D.N.D.N.D.N.D.N.D.N.D.: Not Determined. * Ratio proteasome subunit / β-actin based on loading of 15 ug total protein (Western blot analysis)
For ALL, LC50 concentrations for CFZ and ONX 0912 were significantly correlated (r=0.449, p=0.019). Interestingly, for AML, a significant correlation was observed between BTZ and CFZ LC50 concentrations (r=0.900, p=0.001), suggestive for overlapping activities.
Expression of constitutive proteasome subunits is higher in AML cells than ALL cells. Within ALL samples, constitutive proteasome subunit expression did not correlate with LC50 concentrations for each of the PIs. Within AML patients, however, beta 5 expression significantly correlated with BTZ LC50 (r=0.980, p<0.001). A trend towards a significant correlation was observed for BTZ LC50 and beta 1 (r=0.550, p=0.125) and beta 2 expression (r=0.500, p=0.17). Next, LC50 concentrations of CFZ correlated significantly with beta 5 (r=0.783, p=0.013) and beta 1 (r=0.817, p=0.007) expression. Finally, both in ALL and AML samples, no correlations were revealed for immunoproteasome subunits expression and LC50 concentrations for BTZ, CFZ, ONX 0912 and ONX 0914.
In conclusion, ALL cells were more sensitive to PIs than AML, which may be due lower constitutive proteasome unit expression. Pediatric leukemia cells display marked sensitivity to BTZ and second generation PIs, but lack cross-resistance between BTZ and several second generation PIs. Together, for second generation PIs, these data may hold promise for circumvention of BTZ resistance and further exploration of efficacy assessments in combination with other drugs, in particular GCs.

Disclosures:
No relevant conflicts of interest to declare.