Diamanti Et Al Ptl Eliminates Lic in Childhood All

Around 20% of children with acute lymphoblastic leukemia (ALL) relapse due to failure to eradicate the disease. Current drug efficacy studies focus on reducing leukemia cell burden. However, if drugs have limited effects on leukemia-initiating cells (LIC), these cells may expand and eventually cause relapse. Parthenolide (PTL) has been shown to cause apoptosis of LIC in acute myeloid leukemia. Here, we assessed the effects of PTL on LIC populations in childhood ALL. Apoptosis assays demonstrated that PTL was effective against bulk Band T-ALL cells, while the CD34/CD19, CD34/CD7 and CD34 subpopulations were more resistant. However, functional analyses revealed that PTL treatment prevented engraftment of multiple LIC populations in NSG mice. PTL treatment of mice with established leukemias from lowand high-risk cases resulted in survival and restoration of normal murine hemopoiesis. In 3 cases only, disease progression was significantly slowed in mice engrafted with CD34/CD19 or CD34/CD7 and CD34 cells but not prevented, demonstrating that individual LIC populations within patients have different responses to therapy. These observations indicate that PTL may have therapeutic potential in childhood ALL and provide a basis for developing effective therapies that eradicate all LIC populations to prevent disease progression and reduce relapse. DIAMANTI et al PTL ELIMINATES LIC IN CHILDHOOD ALL 3 Introduction Childhood acute lymphoblastic leukemia (ALL) is a heterogeneous disease in terms of karyotype, immunophenotype and blast morphology. Several genome-wide analyses have described additional genetic changes within individual leukemia clones. Consequently, studies which increase our understanding of the biology and evolution of this disease should provide information on leukemogenic pathways for therapeutic targeting. Such studies may help us understand why initial therapies can induce remission but some cases then relapse, especially as many relapses occur in the low-risk groups. ALL cells that can generate leukemias in immune deficient mice, termed leukemia initiating cells (LIC), were initially thought to be rare and have a hierarchical structure. Some of these LIC, particularly CD133/CD19 cells, were resistant to treatment with dexamethasone and vincristine, commonly used in the induction phase of therapy for ALL, so they may survive therapy and eventually cause disease relapse. However, more recent studies using NOD/LtSz-scid IL-2R null (NSG) mice have revealed that both primitive and more differentiated ALL subpopulations can initiate and maintain acute leukemias in this strain. We and others have shown that CD34/CD19, CD34/CD19 and CD34 B cell precursor (BCP) ALL subpopulations contain LIC. In childhood T-ALL we found that CD34/CD7, CD34/CD7 and CD34 subpopulations had LIC properties. Another study described CD7/CD1a and CD7/CD1a LIC from 1 pediatric and 2 adult T-ALL cases and a study of mature/cortical T-ALL found LIC in both CD34/CD7 and CD34/CD7 populations. The in vivo repopulating capacity has been shown to vary depending on the immunophenotype and genotype of the LIC. These findings demonstrate that LIC are more abundant than earlier studies suggested and indicate that leukemia evolution may have a branching structure rather than a hierarchical one. This has significant implications for therapy of leukemias. Elimination of the population, which contains the greatest proportion of LIC, or has the greatest self–renewal potential may just lead to another LIC population evolving and expanding to maintain the disease. Consequently, it will be important to develop therapies that can eliminate all populations with LIC potential to prevent further evolution and recurrence. The challenge is to find a way to specifically target LIC without causing toxicity to normal cells. DIAMANTI et al PTL ELIMINATES LIC IN CHILDHOOD ALL 4 Parthenolide (PTL) has been recently investigated as a potential chemotherapeutic agent for acute myeloid leukemia (AML) and chronic lymphocytic leukemia. PTL is a naturally occurring sesquiterpene lactone used in the treatment of fever, migraines, rheumatoid arthritis and as an anti-inflammatory agent. PTL can induce apoptosis through a range of responses, such as inhibition of nuclear factor molecule kappa B (NF-κB), p53 activation and increase of reactive oxygen species. PTL and DMAPT, an analog of PTL, have been shown to be effective in AML while sparing normal hemopoietic stem cells (HSC). PTL can induce apoptosis in primary B-ALL cells and cell lines. To date there are no reports on the effects of PTL on LIC populations in pediatric ALL. The aim of this study was to evaluate the effects of PTL on childhood ALL cells, especially subpopulations that have LIC activity, including those that have been shown to be resistant to current therapeutic agents. DIAMANTI et al PTL ELIMINATES LIC IN CHILDHOOD ALL 5 Methods Patient cells Bone marrow (BM) cells from children (median age, 6 years 8 months; range, 6 months 19 years) with BCP ALL and T-ALL at presentation or relapse were collected with approval of University Hospitals Bristol NHS Trust. Detailed characteristics of the patient samples are shown in Table 1. Samples were selected on the basis of availability of material for study only. Normal BM (NBM) and peripheral blood (PB) samples were obtained from consented healthy donors. Cells were separated using Ficoll-Hypaque (Sigma-Aldrich, Poole, UK), mononuclear cells (MNC) were suspended in Iscoves modified Dulbeccos medium (IMDM; Invitrogen, Paisley, UK) with 90% fetal calf serum (FCS; Invitrogen) and 10% dimethyl sulfoxide (DMSO; Manor Park Pharmaceuticals, Bristol, UK) and stored in liquid nitrogen. Mean viability of samples on thawing was 83±17% for ALL samples and 71±17% for normal samples. Cell sorting B-ALL cells were stained with antibodies against CD34 (clone 8G12) and CD19 (clone 4G7). T-ALL cells were stained with anti-CD34 and anti-CD7 (clone M-T701). Normal BM and PB samples were stained with anti-CD34 and anti-CD38 (clone HB7). IgG1 antibodies were used as isotype controls (all BD Biosciences, Oxford, UK). Cells were sorted using a Becton Dickinson Influx Cell sorter (BD Biosciences), using Spigot 6.1.9 software, on the basis of fluorescence intensity after gating on 7aminoactinomycin D (7AAD; Sigma-Aldrich) negative cells with low forward and side scatter. Details of the proportions of nucleated cells in the sorted populations are provided in Supplemental Table 1. Sorting was performed using the maximum purity setting and the purity of sorted subfractions from each sample was checked during and after sorting. DIAMANTI et al PTL ELIMINATES LIC IN CHILDHOOD ALL 6 Table 1 Patient characteristics Patient Subtype Karyotype Age at diagnosis Sex Disease status at biopsy MRD Risk Status 1 c-ALL 46XX 5 F Diagnosis ND 2 c-ALL 46XY 6 M Diagnosis ND 3 pre-B t(4;11), +X 0.6 M Diagnosis ND 4 pre-B -9 7 F Diagnosis High 5 c-ALL t(12;21), -3, -6, +10 5 F Diagnosis Low 6 pro-B t(4;11) 0.5 F Relapse High 7 pre-B* +21, +22 3 M Diagnosis Low 8 c-ALL -12p 15 M Diagnosis High 9 c-ALL t(12;21), -12p 6 M Diagnosis IM 10 c-ALL t(12;21) 6 M Diagnosis Low 11 c-ALL t(8;21), +9 19 F Relapse High 12 pre-B Complex 14 M Diagnosis Low 13 c-ALL 46XX 4 F Diagnosis Low 14 pro-B t(12;17) 14 F Diagnosis High 15 c-ALL t(12;21) 4 F Diagnosis Low 16 c-ALL Hyperdiploid 3 F Diagnosis Low 17 pro-B t(4;11) 0.8 F Diagnosis High 18 c-ALL i(9), del(9) 16 M Relapse Low 19 c-ALL +7, +9, -12, (iAMP21) 8 F Diagnosis High 20 c-ALL +3 14 F Diagnosis High 21 pre-B t(12;21) 8 F Diagnosis High 22 pre-B Hyperdiploid 3 M Relapse Low 23 pre-B del 1 2 F Diagnosis Low 24 pre-B Hyperdiploid 2 M Diagnosis Low 25 pre-B +14 2 F Diagnosis Low 26 pre-B t(1;19) 14 M Diagnosis Low 27 pre-B t(9;22) 15 M Diagnosis Low 28 pre-B 46XY 9 M Diagnosis High 29 T-ALL +4, +9 15 M Diagnosis High 30 T-ALL t(11;14) 2 M Relapse Low 31 T-ALL -9p, +9q 6 M Diagnosis High 32 T-ALL +7, -9, t(9;16) 5 M Diagnosis High 33 T-ALL t(1;14), -6 10 M Diagnosis Low 34 T-ALL 46XY 14 M Diagnosis High 35 T-ALL t(6;7) 1 M Diagnosis High 36 T-ALL -6 15 M Diagnosis High 37 T-ALL 46XY 13 M Diagnosis High *ALL secondary to Downs syndrome; † MRD risk status at day 28; IM, intermediate; ND, not determined DIAMANTI et al PTL ELIMINATES LIC IN CHILDHOOD ALL 7 In vivo studies NSG mice were bred and maintained at the University of Bristol Animal Service Unit. Mice were not preconditioned prior to inoculation. Cells were resuspended in 0.3mL IMDM + 5% HAS and injected into the lateral tail veins of 6-8 week old mice. Unsorted cells and sorted cell populations were inoculated at a range of doses to obtain estimates of LIC frequency in each subpopulation. In ex vivo drug sensitivity assays, unsorted cells and sorted subpopulations, from ALL patients and normal donors, were treated with either PTL for 20 to 24 hours or DMSO + phosphate buffered saline (PBS; Invitrogen), as controls, prior to