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 patients

Acute Lymphoblastic Leukemia
Acute Myeloid Leukemia
ANOVA p-value
NMedian LC50 (nM)RangeNMedian LC50 (nM)Range
Drugs        
    BTZ 29 6.0 3.0–46.1 11 14.2 10.1–61.0 0.002 
    CFZ 27 4.1 0.08–8.7 10 20.8 6.0–30.8 0.000 
    ONX 0912 27 19.2 7.6–80.9 10 93.7 55.7–394 0.000 
    ONX 0914 27 44.6 8.4–117 10 248 89.2–678 0.000 
    DEX 27 23.0 0.50–>600 12 600.0 164–>600 0.000 
Subunit expression  Ratio*   Ratio*   
    beta5 28 0.76 0.00–30.0 10 6.0 2.2–23.9 0.080 
    beta5i 27 62.5 8.5–366 10 55.0 10.6–340 0.714 
    beta1 28 2.4 0.00–28.1 10 11.7 0.92–26.1 0.029 
    beta1i 28 35.1 5.42–106 10 17.7 7.2–49.5 0.032 
    beta2 28 4.8 0.38–23.4 10 20.4 7.4–39.1 0.000 
    beta2i N.D. N.D. N.D. N.D. N.D. N.D. N.D. 
Acute Lymphoblastic Leukemia
Acute Myeloid Leukemia
ANOVA p-value
NMedian LC50 (nM)RangeNMedian LC50 (nM)Range
Drugs        
    BTZ 29 6.0 3.0–46.1 11 14.2 10.1–61.0 0.002 
    CFZ 27 4.1 0.08–8.7 10 20.8 6.0–30.8 0.000 
    ONX 0912 27 19.2 7.6–80.9 10 93.7 55.7–394 0.000 
    ONX 0914 27 44.6 8.4–117 10 248 89.2–678 0.000 
    DEX 27 23.0 0.50–>600 12 600.0 164–>600 0.000 
Subunit expression  Ratio*   Ratio*   
    beta5 28 0.76 0.00–30.0 10 6.0 2.2–23.9 0.080 
    beta5i 27 62.5 8.5–366 10 55.0 10.6–340 0.714 
    beta1 28 2.4 0.00–28.1 10 11.7 0.92–26.1 0.029 
    beta1i 28 35.1 5.42–106 10 17.7 7.2–49.5 0.032 
    beta2 28 4.8 0.38–23.4 10 20.4 7.4–39.1 0.000 
    beta2i N.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.

Author notes

*

Asterisk with author names denotes non-ASH members.