TPEN Selectively Eliminates Lymphoblastic B Cells from Pediatric Acute Lymphoblastic Lleukemia Patients

B-acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by abnormal proliferation and accumulation of immature B-lymphoblast arrested at various differentiation stages. Despite some advances in treatment, there is still an important percentage of pediatric patients with precursor-B ALL who relapsed. Therefore, alternative therapies are needed to improve cure rates for pediatric patients. TPEN is a pro-oxidant agent capable of selectively inducing apoptosis in leukemia cells. Consequently, TPEN has been suggested as a potential agent for oxidative therapy. However, it is not yet known whether TPEN can selectively destroy leukemia cells in a more disease-like milieu e.g., bloodstream and bone marrow (BM) in vivo. In this investigation, we report for the first time that TPEN significantly induces apoptosis in CD34+/CD19+ cells from whole bone marrow de novo B-ALL (n=5) and refractory B-ALL (n=6) patients by oxidative stress (OS, n=8). We found that TPEN significantly increased not only positive cell counts for the oxidation of the stress sensor protein DJ-1 as a sign of the formation of H2O2, but also significantly increased positive cell counts for the proapoptotic protein TP53, PUMA, and CASPASE-3 as indicative of apoptosis in B-ALL cells irrespective of diagnostic status (de novo or refractory) and sex. Understanding the TPEN-induced cell death in leukemia cells provides insight into more effective therapeutic prooxidant-inducing anticancer agents.


Introduction
B-acute lymphoblastic leukemia (B-ALL) is a neoplasm of immature B-cell precursors characterized by abnormal proliferation and accumulation of immature lymphoblast arrested at pre-pro-B cell or pro-B cell stages [1]. B-ALL typically affects children younger than 6 years but also in older children and in adult populations [2,3]. The diagnosis is established by immunophenotyping, commonly by flow cytometry, which shows immature B lineage. Many cases of B-ALL harbor recurrent chromosomal abnormalities, which are critical determinants of prognosis [4]. Despite intensive classical chemotherapy and other available targeted therapies [5], about 20% of children with precursor-B ALL will relapse (e.g., [6]). Therefore, alternative therapies are needed to improve cure rates for pediatric patients with B-cell relapsed leukemia.
To gain insight into this issue, we wanted to determine whether TPEN treatment induces apoptosis in B-ALL cells in WBM from refractory patients. To this aim, we evaluated the effect of TPEN on CD34+/ CD19+ leukemic cells (de novo B-ALL n=5, refractory B-ALL n=6) with selected OS markers (e.g., oxidation of D-1 protein, oxDJ-1), and cell death (e.g., BH3-only protein PUMA, transcription factor TP53, execution protein CASP3) by using flow cytometry analysis. We found that TPEN induces apoptosis in B-ALL cells via the generation of H2O2, switch on of TP53 and PUMA, activation of CASP-3, and switch off of antioxidant protein DJ-1. Taken together these observations suggest that TPEN efficiently eliminates B-ALL cells in pediatric patients with de novo and/or refractory B-ALL.

Discussion
Previous studies have shown TPEN as a pro-oxidant agent capable of inducing selective pro-apoptotic leukemia cell death through oxidative stress mechanism, involving generating H2O2, oxidation of stress sensor protein DJ-1, activation of transcription factor TP53 and pro-apoptotic effector BH-3 only protein PUMA, mitochondrial damage, activation of proapoptotic executor protein CASPASE-3, and disassembly of the nuclei in leukemia cell model (e.g., [25,26]) and in ex vivo isolated refractory leukemia cells from ALL bone marrow samples [27]. Consequently, TPEN has been suggested as a potential agent for oxidative therapy. However, it is not yet known whether TPEN can selectively destroy leukemia cells in a more disease-like milieu e.g., bloodstream and bone marrow (BM). BM is an important microenvironment in which deranged lymphocyte proliferation (e.g., B-ALL) occurs [28]. Since BM is routinely examined for diagnosis and disease prognosis, it represents an important source of biological material and clinical information. Here, we report for the first time that TPEN specifically induces apoptosis in de novo (n=5, i.e., patients with no clinical history of prior B-ALL disorder, or exposure to potentially leukemogenic therapies or agents) and refractory leukemia cells (n=6) from ex vivo whole bone marrow (n= 11) according to the detection of selected OS-signal and cell death markers in eight (n=8) CD34+/CD19+ (B-ALL) patients. We found that TPEN significantly increased not only the oxidation of stress sensor protein DJ-1 as indication of the formation of H2O2 and OS, but also increased the cell signal positive for the proapoptotic protein TP53, PUMA, and CASPASE-3 as indicative of apoptosis cell death. Therefore, in agreement with previous work [25,27], TPEN provokes apoptosis in lymphoblastic cells, e.g., in T-ALL and B-ALL cells through activation of intrinsic apoptotic pathway [12]. This investigation, however, differs from previous ones. First, TPEN induces apoptosis in ex vivo WBM B-ALL cells in a dose-independent manner. Indeed, the effective dose (ED50%) TPEN on ex vivo B-ALL cells was between 100 M-500 M whereas in vitro the ED50% TPEN on leukemia cells and ex vivo isolated T-ALL/B-ALL cells was 5 M [25,27]. Indeed, the TPEN concentration should rise in about 20-to 100fold to be effective in ex vivo. Clearly, the toxicity of TPEN on cells is lessened by BM milieu. Therefore, although TPEN induces apoptosis in leukemia cells by OS-mechanism, the cytotoxic concentrations of TPEN vary hugely when applied to isolated versus non-isolated cell conditions. This observation should be taken into account when data migrate from preclinical experimental procedures to clinical trials. Interestingly, TPEN has been reported to be harmless to mice (e.g., at 5 mg/kg daily for 4 months [29] or at 10 or 15 mg/kg body weight for seven successive days [30]. Unfortunately, pharmacokinetic and pharmacodynamic studies of TPEN in humans are still lacking. Therefore, further studies are needed to establish if TPEN can be therapeutically applied to patients with leukemia. How does TPEN provoke apoptosis in B-ALL? Mounting evidence suggests that TPEN induces cell death by generation of H2O2 by acting as class 5 mitocan [31] or by forming a TPEN-copper complex [19]. In both incidences, TPEN chelates active metals such as iron and copper from mitochondrial complex I-III to convert oxygen (O2) into anion superoxide radicals (O2.-), which in turn dismutate into H2O2. Whatever the mechanism, we found a significantly increased in the oxidized OS-sensor protein DJ-1 as a probe of the specific and selective oxidation residue Cys106-SH (thiol) of DJ-1 into Cys106-SO3 (sulfonate) by H2O2 [32]. In addition, H2O2 can also indirectly trigger the activation of the transcription factor TP53 through MAPK kinases [33]. Consequently, TP53 activates PUMA [34,35]. Effectively, we observed a significantly increased in both proteins TP53 and PUMA according to flow cytometry. Taken together these observations suggest that TPEN/H2O2 induce apoptosis via activation of TP53 and PUMA. Outstandingly, PUMA represents one of the most potent initiator pro-apoptotic BH3-only proteins [36]. Indeed, PUMA cooperates with direct activator proteins to promote mitochondrial outer membrane permeabilization [37] and activation of caspases, e.g., CASPASE-3, which play a central role in the execution-phase of cell apoptosis [38]. In this regard, we have consistently detected an association of increased positive cells CD34/CD19/CASP3 with positive cells CD34/CD19/DJ-1Cys106-SO3. These data suggest that stressed CD34+/CD19+ cells by TPEN/H2O2 ended up dismantled by CASP3. Interestingly, CASP3+ and PUMA+ showed similar percentage of activation in CD34+/CD19+ cells. Taken together, these observations imply that TPEN induces apoptosis in a cascade-like mechanism: TPEN/H2O2 >>> TP53> PUMA>> CASP3 >cell death.  Cells without primary antibodies served as negative control. For assessment, it was acquired 20,000 events and quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software.

Statistical analysis
Statistical analyses were performed using the GraphPad Prism 6 scientific software (GraphPad, Software, Inc. La Jolla, CA, USA). One-way ANOVA with a Tukey post hoc and Student t tests was used to compare the differences between the experimental groups. A P-value <0.05 (*), <0.01 (**) and <0.001 (***) was considered statistically significant.

Conclusions
We have demonstrated that TPEN as pro-oxidant agent is capable of triggering apoptosis in ex vivo whole bone marrow cells from B-ALL pediatric patients independent of diagnosis (e.g., de novo and refractory B-ALL) and sex through a similar OS mechanism previously shown in Jurkat cell model of ALL [25] as well as ex vivo peripheral bloodisolated ALL cells [27]. Our present data support the view that TPEN can be safely used in the clinic as salvage therapy for acute lymphocytic leukemia. Funding: This research was funded by "Fundación Alfonso Moreno Jaramillo" grant #2017-16748 and grant #2018-20454.
Institutional Review Board Statement: All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee for Research Act # # 17-10-697 and HPTU 08/2018 from HPTU, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed Consent Statement: Informed consent was obtained from pediatric ex vivo B-ALL collected in the study.
Data Availability Statement: All data used during the study appear in the submitted article.

Conflicts of Interest:
The authors declare no conflict of interest. The founding sponsors had no role