Page 141 - 2021_04-Haematologica-web

Activation of this complex occurs through the SRC family kinase (SFK) members lymphocyte cell-specific protein- tyrosine kinase (LCK) and FYN. They are critical modula- tors of T-cell development and activation.17 LCK phospho- rylates the plasma membrane-associated TCR complex18 and ZAP70.19 ZAP70 in turn phosphorylates the linker for activation of T cells (LAT) leading to the activation of downstream signaling cascades. The overall activity of LCK is regulated by the phosphorylation status of the acti- vating and inhibitory tyrosine residues 394 and 505, respectively.20 LCK activation correlates with Y416SRC (also Y394LCK) phosphorylation, as the latter over-rides the inhibitory effects of Y505 phosphorylation.21
We hypothesized, therefore, that T-ALL continues to rely on proliferative and survival stimuli inherent to the TCR signaling pathway, which, if inhibited, may enhance GC sensitivity. A targeted shRNA screen directed against components of the TCR signaling initiation complex iden- tified a crucial role for LCK in T-ALL proliferation, both in vitro and in vivo. The anti-proliferative effects of LCK knockdown could be replicated by using the small mole- cule inhibitor dasatinib (DAS). Drug synergy was observed using DAS in combination with dexamethasone (DEX) on patient-derived xenograft (PDX) cell survival in vitro. Mirroring the design of early phase human trials, a murine phase II-like trial demonstrated significantly impaired leukemia progression in vivo using combination treatment. Our results present a clear rationale for using DAS in conjunction with DEX to enhance conventional chemotherapeutic treatment and revert GC resistance in pediatric T-ALL patients.
Methods
Patient samples
The patient-derived material was collected as part of diagnostic investigations of patients at the Great North Children’s Hospital, Department of Paediatric Haematology and Oncology, Newcastle upon Tyne, UK. The material was collected and stored with informed consent obtained from all subjects in accordance with the Declaration of Helsinki.
Samples with explicit written consent for in vivo studies were requested from the Newcastle Biomedicine Biobank, Newcastle University, UK, and used according to approvals given by the Newcastle Biomedicine Biobank (NHB application NHB-008) and the local institutional review board Newcastle & North Tyneside Ethics Committee (REC reference: 07/H0906/109).
DEX and DAS impair in vivo T-ALL propagation culture, the plates were developed as above. Drug synergy was
determined using Combenefit software (v.2.021).22
Phase II-like murine trial
For each of the ten PDX samples, 8x106 cells were intrafemoral- ly (IF) injected into four NSG mice (40 mice in total) under isoflu- rane anesthesia. The four NSG mice derived from one PDX sam- ple were matched for gender and age. T-ALL engraftment in mouse peripheral blood was monitored weekly by tail vein bleeds (20 mL blood/mouse). The four mice of each PDX were random- ized to receive control vehicle, DAS (35 mg/kg), DEX (1 mg/kg) or DEX/DAS combination by intraperitoneal (IP) injection upon engraftment, defined as ≥0.5% peripheral blood hCD45+/hCD7+ cells. The median treatment duration of these mice was 15 days, depending on their clinical status. When any of the four mice dis- played signs of ill health or weight loss, all four mice derived from this PDX were killed at the same time to assess leukemia engraft- ment in bone marrow, blood, spleen, liver and CNS. Spleen size and weight were recorded. Statistical analyses were performed using RStudio (Boston, MA, USA) with linear mix model. The final analysis excluded the four mice derived from patient sample LK214, as all mice succumbed to T-ALL before treatment was ini- tiated.
See the Online Supplementary Appendix for further details of the methods used.
Results
A targeted shRNA screen of T-cell receptor pathway components identifies an essential role for lymphocyte cell-specific kinase in T-cell acute lymphoblastic leukemia cell line and patient-derived xenograft proliferation in vitro
To explore the importance of the pTCR/TCR signaling complex in proliferation and survival of malignant T cells, we performed a limited shRNA screen targeting six genes with three shRNAs per gene, including LCK, ZAP70, PTCRA, FYN, CD3E and LAT in four T-ALL cell lines (HPB- ALL, CUTLL1, MOLT4, SUPT1), and included 18 control shRNAs (see Online Supplementary Methods and Online Supplementary Table S1A). In silico analysis using the Cancer Cell Line Encyclopedia (CCLE) demonstrated that these six genes are highly expressed in a panel of T-ALL cell lines (Online Supplementary Figure S1A). LCK and PTCRA expres- sion was confirmed by targeted gene expression analysis in T-ALL cell lines and patient samples (Figure 1B) (Online Supplementary Figure S1B). The limited shRNA screen revealed the shLCK#3 construct targeting LCK was the only construct significantly depleted in all four cell lines, when compared with base line shRNA integration, under- lining an important role for LCK in T-ALL cell line prolifer- ation and/or survival (Figure 1A and Online Supplementary Figure S1C and D). The shLCK#1, shZAP70#1 and shPTCRA#2 constructs were lost in 3 out of 4 cell lines. Constructs targeting FYN, CD3E, or LAT were significantly depleted in one cell line only, suggesting that these mole- cules do not play an universal role in T-ALL cell prolifera- tion. ShRNAs against essential ribosomal genes were pre- dictably depleted, whilst all three shRNA constructs target- ing the tumor suppressor PTEN were enriched as expected. Repeated sampling at 16, 30 and 40 days after transduction demonstrated progressive depletion of shRNA constructs targeting LCK and ZAP70 (Online Supplementary Table S2 and Online Supplementary Figure S1E). PDX LK203 showed
Drug matrix assays
Dasatinib (9 nM – 30 mM) (DC Chemicals, Shanghai, China) was titrated on T-ALL cell lines (4x104/well) in 96-well plates (Corning, NY, USA). Cell viability was assessed after 3 days using Cell Counting Kit 8 (NBS Biologicals, Cambridgeshire, UK). Absorbance was measured at OD450 nm using a POLARstar Omega plate reader (BMG LABTECH, Bucks, UK). IC50 values were determined by GraphPad Prism. Assays were performed in triplicate and at least three independent repeats were performed.
For DAS/DEX combination treatments DAS (80 nM – 50 mM) and DEX (0.09 nM – 600 nM) were titrated in 2-dimensions on T-ALL cell lines (4x104 cells per well in 96-well plate) or ex vivo expanded PDX cells (8x104 cells per well in 96-well plate). Ex vivo expansion was achieved after co-culture with OP9-DL1 for 1 week, after which cells were separated from their feeders by repetitive transfer and subsequently plated. After 72 hours (h) of
haematologica | 2021; 106(4)
1057