Page 147 - Haematologica May 2020

denominated as leukemic stem cells (LSC).4,10-14 In CD34+ AML, stem cell characteristics were shown to be present in all four CD34/CD38 phenotypic compartments, though the CD34+CD38– fraction was the most LSC-enriched.15 Moreover, a high LSC load at diagnosis was shown to be a significant adverse prognostic factor.16-19 Unfortunately, current chemotherapeutic regimens were shown to per- form inadequate towards LSC eradication14 and they induce important toxicity.5,6,20 Also hematopoietic stem cell transplantation, performed in high-risk (HR) patients or as salvage therapy, carries a high mortality and morbid- ity risk,2,5 highlighting the need for alternative treatments. Thus, identifying LSC aberrations is crucial to tackle the high relapse rate and to develop therapeutic targeting strategies for LSC elimination, while ensuring salvage of normal hematopoietic stem cells (HSC).
ethical committee, in accordance with the Declaration of Helsinki. Buffy coats from donors were obtained from the Red Cross (Mechelen, Belgium) and used for CTL isolation and the prepara- tion of feeder cell medium.
Flow cytometry analysis and cell sorting
Cell pellets were surface stained (Online Supplementary Table S2), followed by 20 min incubation at 4°C and washing with PBS+2% BSA. For cell-sorting, labeled cells were resuspended in medium and sorted on a FACSAria III with red, blue, and violet lasers (BD Biosciences). For flow cytometry (FCM) analysis, cells were resus- pended in PBS+2% BSA and analyzed on a LSR II or a FACSCanto II, equipped with four or three solid-state lasers, respectively (both BD Biosciences). All scatters were devoid of doublets based on FSC-H/FSC-A, and propidium iodide (PI) was used to exclude dead cells. Sorting strategies are described in Online Supplementary Data 2.2. Regarding FCM-based cytotoxicity and cytokine assays (Online Supplementary Data 2.9), living cells were selected using a LIVE/DEAD staining (1:10000 dilution, ThermoFisher Scientific) instead of PI. Target cells were stained with a Violet CellTraceTM (VT) Cell Proliferation Kit (5 mM, 1:10000 dilution, ThermoFisher Scientific) prior to incubation with TCR-engineered CTL. After incubation and before surface staining, Flow-CountTM Fluorospheres (1:20 diluted, Beckman Coulter) were added to each well to enable target quantification (measurement of minimum 1000Fluorospheres/well).
Transcript expression
Details on micro-array profiling, RNA isolation, cDNA synthe- sis, quantitative polymerase chain reaction (qPCR) conditions and primers can be found in Online Supplementary Data 2.3, 2.4, 2.5 and Online Supplementary Table S3. qPCR data analysis was performed according to state-of-the-art methods.38,39 Relative quantity (RQ) values were normalized against housekeeping genes GAPD, HPRT1 and TBP. For TARP expression, normalized relative quanti- ties were calibrated (calibrated normalized relative quantity, CNRQ) versus a single calibrator to allow interrun comparison. For the investigation of the subcellular localization of TARP, delta (d) Ct between cytoplasmic and nuclear compartments were calculat- ed and compared to MALAT1 and TBP expression. Functional TCRG gene rearrangements were excluded if sufficient material remained using DNA TCRG GeneScan analysis40 and/or TRGV(J)C qPCR (Online Supplementary Table S4).
Protein detection
Details on western blotting and confocal microscopy are pro- vided in Online Supplementary Data 2.6.
Viral transduction of acute myeloid leukemia cell lines and generation of T-cell receptor-transgenic cytotoxic T cells
All transfer and helper plasmids used, and procedures for trans- formation, plasmid isolation, transfection and transduction are described in Online Supplementary Data 2.7 and 2.8.
Six AML cell lines (HL-60, Kg-1a, MOLM-13, HL-60-Luc, MOLM-13-Luc and MV4;11-Luc) were transduced with HLA- A*0201 MHC-I encoding retrovirus, hereafter defined as A2+. Transgenic TARP overexpression (OE) cell lines were generated for OCI-AML3 and THP-1, next to mock controls. TARP was knocked down in four TARP-high AML cell lines (HL-60, Kg-1a, MV4;11 and THP-1) using three different shRNA, next to mock controls.
TARP-TCR engineered CTL were generated by transduction with lentiviral (LV) or retroviral (RV) particles encoding a TCRA8- T2A-TCRB12 sequence directed against the HLA-A*0201-restrict-
Targeted therapy has led to remarkable progress in the survival rates of multiple cancers. The introduction of tyrosine kinase inhibitors in the treatment of chronic myeloid leukemia (CML) accomplished a major break- through, and CD19-directed chimeric antigen receptor (CAR) therapy has led to an enormous improvement in survival in relapsed/refractory pediatric ALL.21,22 These successes paved the way for the exploration of the clinical applicability of targeting antibodies and CAR- or T-cell receptor (TCR)-transgenic cytotoxic T cells (CTL) in AML.2,23-28 Although an increasing number of LSC-specific membrane markers have been identified over recent years,18,23,29,30 only a few reports address the molecular abnormalities of LSC compared to HSC,15,31-37 especially in pedAML.
Here, we identified the T-cell receptor (TCR)γ chain alternate reading frame protein (TARP) as an AML-specific target, expressed in the LSC and blasts of pediatric and adult AML, while absent in their normal counterparts. TARP transcript expression was associated with FLT3-ITD in pedAML. In addition, we provide in vitro evidence that TARP may serve as a novel immunotherapeutic target in AML for TARP-TCR engineered CTL.
Methods
Patients
We retrospectively selected diagnostic material from 13 pedAML and 17 adult AML patients based on the sample avail- ability, LSC load, CD34 positivity, FLT3 mutational status, and HLA-status (Table 1 and Online Supplementary Table S1). At diag- nosis, mononuclear cells (MNC) were isolated from bone marrow (BM) or peripheral blood (PB) by Ficoll density gradient (Axis- shield) and cryopreserved in 90% fetal calf serum (FCS) and 10% dimethylsulfoxide (DMSO). Samples were thawed, followed by 30 minutes (min) incubation at room temperature (RT) in 20 mL RPMI with 20% FCS, 200 mL DNase I (1 mg/mL, grade II bovine pancreas), and 200 mL MgCl2 (1 M) (Sigma-Aldrich). After incuba- tion, cells were spinoculated (10 min, 400 rpm) and washed once more with RPMI/20% FCS.
In addition, we prospectively collected material from 15 healthy subjects. Normal bone marrow (NBM, n=6) was collected from posterior iliac crest of pediatric patients (4-18 years) undergoing scoliosis surgery. Umbilical cord blood (CB, n=7) was obtained after normal vaginal deliveries at full term. Mobilized peripheral blood stem cells (mPBSC, n=2) were collected by apheresis of adult donors pre-allotransplant. All patients or their guardians gave their informed consent and approval was obtained by the
haematologica | 2020; 105(5)
TARP as target in acute myeloid leukemia
1307