Exploring Pre-B Cell Receptor Signalling As a Therapeutic Target in Acute Lymphoblastic Leukaemia

Introduction:

Acute lymphoblastic leukaemia (ALL) is a clonal disorder of developing lymphocytes and is the most common malignancy in children and adolescents. While cure rates are high, treatment is associated with significant morbidity and relapsed ALL remains one of the leading causes of cancer-related deaths in children. New, less toxic therapies are clearly needed for refractory ALL.

There are a number of lines of evidence to suggest that ALL cells hijack components of precursor-B cell receptor (Pre-BCR) signalling and this dependency may be amenable to therapeutic exploitation. There are a number of tyrosine kinase inhibitors (TKIs) targeting Pre-BCR signalling that are showing great promise in the clinic for other leukemia subtypes which warrant preclinical evaluation in childhood ALL.[1] These include CAL-101 (PI3K-δ inhibitor), Ibrutinib (BTK inhibitor), Fostamatinib R406 (SYK inhibitor) and Dasatinib (BCR-ABL/SRC inhibitor).

Methods:

TKIs were evaluated in ALL cells, including cell lines (PreB 697 and its glucocorticoid resistant descendant, R3F9; Nalm-6 and Reh) and 25 patient derived xenograft samples (PDX) from 12 predominantly high risk/relapse children ALLs. Resazurin was used to assess cell viability. Flow cytometry was used to detect Pre-BCR expression (µHc, Vpreb and λ5) and functionality using a Calcium flux assay. Phospho-flow cytometry was performed to monitor constitutive phosphorylation and response to Pre-BCR activation and to assess pharmacodynamic drug action (p-AKT, p-BLNK, p-BTK, p-SYK, p-ERK, and p-PLC-ϒ2). GILZ expression was measured by RQ-PCR. Cell cycle and apoptosis were determined by flow cytometry using Propidium Iodide and Annexin V staining.

Results:

ALL cell lines were resistant to CAL-101 (mean GI₅₀ 52.08 µM, range 25 µM-77.83 µM) and Ibrutinib (mean GI₅₀ 15.9 µM, range 11.47 µM-18.3 µM). However, modest sensitivity was seen to R406 (mean GI₅₀ 4.32 µM, range 2.88 µM-5.83 µM) and Dasatinib (mean GI₅₀ 5.33 µM, range 2.45 µM-12.5 µM). CAL-101 and Dasatinib were shown to be cytostatic, causing G1 arrest but no significant apoptosis, while Ibrutinib and R406 were associated with cell cycle arrest and significant apoptosis after 72 hours incubation with GI₅₀ concentrations (16.81±1.71 % and 31.34±5.78 % apoptosis, respectively). Pre-B receptor positive cells were more sensitive to Dasatinib and Fostamatinib. Pharmacodynamic assessment using Phospho-flow cytometry and Western blotting showed inhibition of the relevant targets at the GI₅₀ concentrations. PDX ALL cells were generally more sensitive than the cell lines; CAL-101 (mean GI₅₀ 25.56 µM, range 76 nM-100 µM; 2 out of 12 patient samples <2µM); Ibrutinib (mean GI₅₀ 14.23 µM, range 490 nM-100 µM; 3 out of 12 patient samples <5µM); R406 (mean GI₅₀ 11.52 µM and range 56 nM-25 µM, 4 out of 12 patient samples <4µM); Dasatinib (mean GI₅₀ 25.56 µM, range 76 nM-100 µM; 3 out of 12 patient samples <0.5µM). ALLs sensitive to Dasatinib were Ph+ or Pre-BCR positive and the latter were also sensitive to Fostamatinib.

Synergism was seen after co-treatment of TKIs with the glucocorticoid (GC), Dexamethasone (CAL-101 CI mean 0.7, range 0.056-1.34; Ibrutinib CI mean 0.71, range 0.41-0.97; R406 CI mean 0.27, range 0.1-0.6 and Dasatinib CI mean 0.63, range 0.14-1.29). No synergism was observed in the glucocorticoid receptor negative, Reh cell line. For R406 and Dasatinib, co- exposure was strongly synergistic and was associated with increased apoptosis in PreB 697 and the GC resistant lines, Nalm-6 and R3F9. Synergism was associated with a significant increase in expression of the GR target, GILZ and an enhanced downregulation of R406 and Dasatinib targets (p-SYK for R406 and p-BTK, p-SYK for Dasatinib).

Conclusion:

We have identified significant sensitivity of TKIs impacting on Pre-BCR signalling in ALL cells at clinically relevant concentrations; Pre-BCR positive ALLs were associated with Dasatinib and Fostamatinib sensitivity; Pre-BCR negative ALL cells were also sensitive to some TKIs, although predictive biomarkers remain to be established. Marked synergism was observed in combination with dexamethasone, even in GC resistant cells. In vivo preclinical confirmation of these data may offer new therapies for refractory ALL.

1. Young, R.M. and L.M. Staudt, Targeting pathological B cell receptor signalling in lymphoid malignancies. Nature Reviews Drug Discovery, 2013. 12(3): p. 229-243.