Relapsed Acute Promyelocytic Leukemia Patients Research Articles

Stromal-Cells Downregulate MiR-23a-5p Levels in Myeloid Leukemic Cells to Activate Protective-Autophagy Against Chemotherapeutic Agents

Role of stromal micro-environment in drug resistance has been extensively reported for several cancers. We have demonstrated earlier that there is significant microenvironment mediated drug resistance (EMDR) to arsenic trioxide (ATO) in acute promyelocytic leukemia (APL) and that this was predominantly driven by up-regulation of the NF-kB pathway in the malignant cell (Ganesan S et al. Leukemia 2016). In our current study we have probed the molecular mechanism of EM-DR in further detail. Towards this we evaluated the potential role played by miRNA in EMDR to ATO in APL. Using NGS based small RNA sequencing we identified hsa-miR-23a-5p to be significantly downregulated (adjusted P value <0.01; figure 1a) in NB4 cells upon co-culture with HS-5 stromal cells. We noted that this miRNA is negatively regulated by NF-kB pathway and inversely correlated with NF-kB status in relapsed APL patients (n=8; matched samples). These results were consistent with our earlier reported observations that NF-kB pathway is dysregulated and enhances drug resistance to ATO. Further, we observed that low expression of miR-23a-5p observed in the relapsed patient's samples showed a trend towards an increased association with subsequent second relapse.We also observed miR-23a-5p mimics were able to restore the sensitivity of NB4 cells to ATO in the presence of stromal cells (figure 1b), while NB4/GFP-MAD cells (NF-kB inhibited cell line showing high expression of miR-23a-5p ) were not protected from ATO on stromal co-culture. Looking for the targets of miR-23a-5p , TargetScan analysis data demonstrated a clustering of targets involving toll like receptor genes (TLR) and autophagy pathway genes (TLRs - TLR2, TLR3, TLR4 and TLR5 and autophagy pathway genes - AMBRA1, ATG13, ATG9 and ATG2 ). Previously reported data clearly demonstrated the ability of TLR genes to activate autophagy (Delgado MA. et al. EMBO J 2008). Based on this data we evaluated the role of autophagy in EM-DR. We noted that there was an upregulation of autophagy pathway in leukemic cells upon co-culture with stroma (where miR-23a-5p was downregulated). Re-introducing miR-23a-5p in the leukemic cells targeted autophagy pathway genes which was validated through western blot (figure 1c). We also observed that inhibiting autophagy by chemical inhibitors (bafilomycin A1 and hydroxychloroquine) along with ATO was able to overcome EM-DR to ATO (figure 1d). The in-vivo efficacy of combining ATO along with hydroxychloroquine was further validated in an APL mouse model where we were able to demonstrate a significant decrease in tumor burden on day20 when compared to controls and significantly prolonged the survival of APL mice treated with the combination in comparison to either drug alone.Similar results were obtained with AML cells, where the stromal cells conferred a protective effect over U937 cells against cytarabine (AraC) and daunorubicin (Dnr). The downregulation of miR-23a-5p was also observed in U937 cells upon co-culture. We also observed that re-introducing miR-23a-5p in these cells restored sensitivity to AraC and Dnr. Our results thus illustrates a complex molecular cross-talk between stromal cells and leukemic cells in protecting against the cytotoxic effects of various chemotherapeutic agents in myeloid leukemia. This data along with that reported earlier by us illustrates multiple levels of regulation of the NF-kB pathway and resistance to chemotherapeutic drugs by stromal cell co-culture. Our work thus suggests that miR-23a-5p mimics or autophagy-inhibitors could potentially be effective adjuvants along with chemotherapy/ATO in the treatment of myeloid malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Resource Utilization Using Micro-Costing Method and Cost Effectiveness Analysis of Treatment of Acute Promyelocytic Leukemia with Generic Arsenic-Trioxide

We have previously reported on the challenges of treating AML in India, the major challenge being the cost of treatment (Chepsy et al. BJH 2015). With conventional treatment of AML it is recognized that the major part of this cost is not related to the cost of the individual chemotherapeutic drugs, but rather from the high cost of supportive care to take patients through repeated cycles of prolonged neutropenia induced by conventional chemotherapy. In contrast with arsenic trioxide (ATO) based regimens in acute promyelocytic leukemia (APL), when expensive patented ‘innovator’ ATO was used, the reported 3 year direct pharmacy cost of drugs was higher with an ATO+ATRA regime compared with a conventional ATRA+chemotherapy regimen (Euro 46,700 Vs 6,700), though the cost of supportive care was a third of conventional therapy (Kruse M et al. PLoS One. 2015). There is limited data on the cost effectiveness of using generic ATO, as available in India, in the treatment of APL (10 mg vial costs US $7 in India versus US $ 676 in North America)In a single center, retrospective study, 30 newly diagnosed APL (ND-APL) patients, treated with ATO and 16 relapsed APL (R-APL) patients, treated on a phase II trial with a ATO based regimen followed by an autologous stem cell transplant (regimens as previously reported by us; Mathews et al. Blood 2012 and Mathews et al. Blood 2016 abstract) were evaluated. In the absence of patients being treated at our center with a conventional ATRA+chemotherapy regimen (APL-CT) the cost of treatment for APL with such a regimen was assumed to be similar to the cost that would be incurred to treat a non-M3 AML patient at our center with a conventional AML chemotherapy schedule. Incremental cost effectiveness ratio was calculated using weighted costs with weighing factor obtained from survival data for 60 patients from our database for APL ATO group and from the AIDA 2000 trial for APL-CT group.A three phase micro-costing method (figure 1) used four cost centers involved in providing patient care: (i) chemotherapy administration (ii) supportive care (iii) treatment of complications (iv) laboratory services. For each of these cost centers there were four cost drivers: (i) drugs / reagents (ii) personnel (iii) ward (iv) instruments / overheads. The actual cost of cost drivers was found out by micro-costing and summed up to obtain the unit cost of each cost center (m). Each time a patient used a cost center this unit cost would be incurred. Next, the frequency of utilization (n) of each cost center was found out from the patient database and multiplied by the unit cost of the cost center to obtain the total cost of treatment in each protocol (m x n) (For illustration and international comparison all cost were converted to US$).In ND-APL patients, the mean cost of induction and consolidation was $5078.5±1581 and $902±212 respectively while in APL-CT it was $9812.1±3239 and $11803.1±4872 respectively (exchange rate 1 US$=61.87 Indian rupees). Our data illustrated that APL-CT group incurred higher costs mainly due to the greater duration of in-patient care that was required in this group and since they had higher rate of infectious complications (Table 1). During maintenance phase, in ND-APL, there were no in-patient stays and the entire chemotherapy was given on outpatient basis and the average cost was $2074±146 and the cost of following up these patients for an additional three years was $704.6±289. The mean lifetime cost of treatment of ND-APL using ATO was $8759.2±2078 while using APL-CT was $24393±5528. In R-APL patients, the total cost of re-induction and consolidation was $3498±1220 and $2274±372 respectively. The mean cost of an autologous-SCT was $4318±159. The average lifetime cost of treatment of relapsed APL patients using ATO based regimen followed by an autologous SCT was $12269.7±4915. The weighted cost per living for ATO and CT was estimated to be $12426 and $28330 respectively. The incremental cost effectiveness ratio showed ATO to be cheaper by $13359 for each life saved compared to conventional chemotherapy.ATO based regimen in ND-APL and R-APL are associated with significant reduction in resource utilization and costs of treatment and is also cost-effective as compared to conventional ATRA+chemotherapy. With the world still debating the best treatment for APL, generic ATO based regimen offers a significant opportunity for resource constrained countries to treat all patients with a diagnosis of APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Crosstalk between hnRNP K and SET in ATRA-induced differentiation in acute promyelocytic leukemia.

HnRNP K protein is a heterogeneous nuclear ribonucleoprotein which has been proposed to be involved in the leukemogenesis of acute promyelocytic leukemia (APL), as well as in differentiation induced by all‐trans retinoic acid (ATRA). We previously demonstrated a connection between SET and hnRNP K function in head and neck squamous cell carcinoma (HNSCC) cells related to splicing processing. The objective of this study was to characterize the participation of hnRNP K and SET proteins in ATRA‐induced differentiation in APL. We observed higher (5‐ to 40‐fold) levels of hnRNP K and SET mRNA in APL patients at the diagnosis phase compared with induction and maintenance phases. hnRNP K knockdown using short‐hairpin RNA led to cell death in ATRA‐sensitive NB4 and resistant NB4‐R2 cells by apoptosis with SET cleavage. In addition, hnRNP K knockdown increased granulocytic differentiation in APL cells, mainly in NB4‐R2 with ATRA. hnRNP K knockdown had an effect similar to that of treatment with U0126 (an meiosis‐specific serine/threonine protein kinase/ERK inhibitor), mainly in NB4‐R2 cells. SET knockdown in APL cells revealed that apoptosis induction in cells with hnRNP K knockdown occurred by SET cleavage rather than by reduction in SET protein. Transplantation of NB4‐R2 cells into nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has higher potential against tumor progression when compared to ATO. Therefore, hnRNP K/SET and ERK are potential therapeutic targets for both antineoplastic leukemia therapy and relapsed APL patients with ATRA resistance.

Arsenic trioxide therapy predisposes to herpes zoster reactivation despite minimally myelosuppressive therapy

Acute Promyelocytic Leukemia (APL) is a unique subtype of acute myeloid leukemia that is highly responsive to minimally myelosuppressive therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). We and others have observed a higher than expected incidence of herpes zoster reactivation in APL patients treated with ATO. Memorial Sloan Kettering Cancer Center (MSKCC) has been using ATO since 1997 in all relapsed APL patients, and more recently has included it in our front-line APL regimens. Here we present a retrospective analysis of the factors contributing to herpes zoster reactivation among APL patients.

Primaquine phosphate induces the apoptosis of ATRA-resistant acute promyelocytic leukemia cells by inhibition of the NF-κB pathway.

As a subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) is characterized by a chromosomal translocation, most of which result in the production of a PML-RAR alpha fusion protein. Although the overall survival rate of APL patients has improved dramatically due to all-trans retinoic acid (ATRA) treatment, ATRA-resistance remains a clinical challenge in the management of APL. Therefore, alternative agents should be considered for ATRA-resistant APL patients. Here, we report that antimalaria drug primaquine phosphate (PRQ) exhibits an anti-leukemia effect on both ATRA-sensitive cell line NB4 and ATRA-resistant APL cell lines, NB4-LR2, NB4-LR1, and NB4-MR2. Moreover, PRQ significantly inhibited primary colony formation of untreated or relapsed APL patients. Further study showed that PRQ could induce the apoptosis of APL cells by inhibiting NF-κB signaling pathway. The in vivo study showed that PRQ significantly inhibited NB4-LR2 xenograft tumors growth. These results suggest that PRQ is a potential therapeuticagentfor ATRA-resistant APL patients.

Efficacy of Arsenic Trioxide Combined with ATRA and Chemotherapy for Relapsed Acute Promyelocytic Leukemia Patients

To investigate the efficacy and safety of arsenic trioxide combined with ATRA and chemo- therapy for treatment of relapsed acute promyelocytic leukemia (APL) patients. The clinic data of 25 patients with relapse APL treated in our hospital from 1996 to 2013 were collected and analyzed. Among the 25 patients, 15 patients suffered first-time hematological relapse (HR), and the other 10 patients showed first-time molecular relapse (MR). The patients with first-time replase were treated with ATO+ATRA+Anthracycline re-induction chemotherapy. The clinical features, complete remission (CR) rate, overall survival (OS), disease-free survival (DFS) and adverse events after re-induction therapy were analyzed. Fourteen of 15 hematological relapsed patients achieved the second-time hematological complete remission (CR2) after re-induction therapy except one patient died of bleeding complication during the re-induction. 8 of 14 patient showed molecular complete remission (CRm) after two cycles of therapy with this regimen. Totally, eleven out of the 14 HR patients were alive without disease till the last follow-up, and 3 of the 14 HR patients died because of bleeding complications. All of the 10 molecular relapsed patients received the second CRm after treated by the regimen. Among these 10 patients, 6 patients suffered only once relapse and continued with the molecular CR2 status, and for the other 4 patients with more than two-relapses, only 1 survived untill 89.3 months after achieved second-time CRm, and other 3 patients died because of bleeding complications. For relapsed APL patients, the treatment with ATO+ATRA+chemotherapy regimen after relapse still shows encouraging efficacy, no matter whether or not the application of ATO in the previous regimens. In addition, patients with more than two molecular relapses show a poor prognosis.

Management of Relapsed Acute Promyelocytic Leukemia Post ATO Upfront Therapy: Open-Labeled Phase II Study Evaluating Role of Proteasome Inhibition

▪Introduction: There is limited data on the best treatment strategy for patients who relapse after upfront arsenic trioxide (ATO) based therapy for acute promyelocytic leukemia (APL). We had recently reported that there was significant micro-environment mediated drug resistance (EM-DR) to ATO which we noted was predominantly mediated by upregulation of the NF-κB pathway. We also noted that this upregulation was more prominent in relapsed APL and that EMDR to ATO could be overcome by the use of proteasome inhibitors (Leukemia 2016 - In Press). Pre-clinical mouse model and preliminary clinical data validated these observations. There has also been a recent concern of mutations in the oncogenic PML-RARA gene following treatment with ATO, with reports suggesting a third of relapsed APL patients having such mutations and that the clinical outcomes in this subset with conventional therapy is poor [NEJM. 2014;370(19): 1864-6].Methods: In this prospective, open-label, non-randomized, phase 2, single center study, we enrolled APL patients in first hematological relapse. Inclusion criteria included presence of t(15; 17) and exposure to ATO as part of up front therapy. Enrolled patients received conventional therapy for relapse, included ATO and ATRA in induction, consolidation. Anthracycline (mitoxantrone) was used only in induction. Additionally, patients received two doses of bortezomib (1.4mg / m2 / dose) in induction and consolidation on days 2 and 5 and one dose in maintenance (10 days/month x 6 months) on day 2. Primary end point was safety (graded according to the NCI Common Terminology Criteria for Adverse Events version 4.0) and the key secondary end points were proportion in complete molecular remission at the end of induction and event free survival (EFS). The study is ongoing and continues to recruit patients. This trial is registered with ClinicalTrials.gov, number NCT01950611.Results: Between Sep 2013 and Mar 2016, 18 patients met eligibility criteria and were enrolled in this study. All 18 patients achieved CHR at a median of 45 days (range: 42-63). Seventeen patients (94.4%) were RT-PCR negative at the end of induction. The median time complete molecular remission was 35 days (range: 21-56 days). None of the patients had any major bleeding or thrombotic events in induction. With the exception of one patient who had Grade IV neurotoxicity which required treatment and recovered after temporarily discontinuing treatment during induction none of the other patients had any Grade III/IV non-hematological toxicity. Other non-hematological events were ≤ Grade II, usually transient and judged to be unrelated to the interventional agent. These included mild headache in 8 (33.3%) and one case (5.5%) each with diarrhea, mild rash, redness of eyes, oral ulcers, vertigo and paresthesia. Auto-SCT was offered to all patients in molecular remission post consolidation. Eight patients (44.4%) underwent an autologous SCT post consolidation while the rest opted to receive maintenance therapy. One patient who opted for maintenance therapy relapsed 6 months after completing maintenance. At an actuarial median follow up of 24 months (range: 4-35) the 3 year KM estimate of EFS was 92.3±7.4% and the cohort had 100% survival at last follow up. This clinical profile and outcome data was compared with a historical cohort that was treated with the same protocol with the exception of bortezomib and the comparative data is summarized in table 1 and figures 1A and 1B.Conclusion: Addition of proteasome inhibitors with ATO is well tolerated. In combination with conventional agents, proteasome inhibitors can potentially improve the clinical outcome of patients with relapsed APL treated with upfront ATO. Our preliminary data also suggests that this combination can obviate the need for an auto-SCT in this setting. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tamibarotene Compared to All-Trans Retinoic Acid (ATRA) As Add-on to Arsenic Trioxide (ATO) in Subjects with Relapsed Acute Promyelocytic Leukemia (APL)

BackgroundCombination therapy with ATRA and ATO is a standard therapy for relapsed APL. Despite the high complete remission (CR) rate with ATRA-ATO combination therapy, patients administered this combination for relapsed disease may acquire tolerance to ATRA, which reduces the CR rate in relapsed APL patients. Tamibarotene is a novel synthetic retinoid created in Japan with a several-fold-higher differentiation-inducing effect than ATRA, as well as low affinity for cellular retinoic acid-binding protein and absence of binding affinity for RARγ, unlike ATRA. It has been used for treating patients relapsed after ATRA therapy since 2005 in Japan. We conducted a phase III, randomized, open-label, parallel-group, active-controlled, multicenter clinical trial to compare tamibarotene-ATO and ATRA-ATO combination therapies in relapsed APL patients.MethodsA Phase III, randomized, open-label, parallel-group, active-controlled, multicenter, study of 56 days treatment duration will evaluate the efficacy and safety of 6mg/m2/day Tamibarotene versus 25mg/m2/day ATRA as add-on to 0.15mg/kg/day ATO in subjects with relapsed APL. 71 subjects were randomly assigned to 1 of 2 treatment groups: ATRA-ATO (control group) or tamibarotene-ATO (study group).ResultsThe number of subjects for efficacy analysis constituting a full analysis set (FAS) of all subjects administered study drugs, including drop-outs, was 35 each in the control and study groups. The number of subjects in each per protocol set (PPS), excluding drop-outs, was 27 and 33 in the control and study groups, respectively. CR rate for the FAS was significantly higher in the study group (80.00% vs. 54.29%; P = 0.0220, Table 1). In contrast, CR rate for the PPS was 84.85% and 70.37% for the study and control groups, respectively. The intergroup difference was 14.48% with a 97.1% confidence interval of -9.06 to 38.01%, which fulfilled requirements for non-inferiority of Δ = 0.10. These results verified the non-inferiority of the study to the control group. Of the patients who attained CR, the number who showed complete molecular remission (CRm) was higher in the study group, suggesting that a higher quality of remission was possibly attained in the study group (Table 2).Serious adverse events occurred in 3 control group patients (intracerebral hemorrhage, 2 and retinoic acid syndrome, 1) and 1 patient in the study group (type I respiratory failure). There was no significant intergroup difference in the incidences of serious adverse events. The most commonly reported adverse reactions included hypertriglyceridemia, hypercholesterolemia, rash, and elevation of ALT and AST levels; there was no significant intergroup difference in the incidences of any of these adverse reactions. Other adverse reaction findings were similar between the groups. Therefore, the tolerability observed in the study group was considered to be similar to that in the control. The only adverse finding that showed an appreciable intergroup difference was leukocytosis, which occurred at a significantly lower frequency in the study group than in the control group. Therefore, the therapeutic regimen of the study group might reduce the risk of retinoic acid syndrome, an important adverse reaction in treatment of APL, when the association of leukocytosis with development of retinoic acid syndrome is considered.ConclusionThis controlled clinical trial demonstrated the efficacy and safety of combination therapy with tamibarotene and ATO in patients with relapsed APL. Tamibarotene-ATO combination therapy was considered not inferior in efficacy and safety to the standard combination of ATRA and ATO, and may be a useful new combination regimen for treating relapsed APL. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 3811: Arsenic trioxide induces cell cycle arrest, apoptosis through interaction of DAXX and degradation of MDM2 in acute leukemia cells

Abstract Arsenic trioxide (ATO) alone or combination with all trans retinoic acid (ATRA) has been successfully used in the treatment of all age group of acute promyelocytic leukemia (APL) patients. Recent human clinical trails result of ATO and their combination with ATRA shown complete remission in both de novo and relapsed APL patients. However, the detailed molecular mechanisms of its anti-leukemic action through cell cycle arrest and apoptosis are poorly known. We have used APL cell line and transgenic mice model to elucidate the anti-cancer properties of arsenic trioxide. We hypothesized that ATO arrests cell cycle and apoptosis through activation of p53 and interaction of death domain-associated protein (DAXX), which disrupted the MDM2-DAXX-HAUSP interactions. It also promoted MDM2 self-ubiquitination and degradation. To test the hypothesis, we used western blotting, confocal imaging and other molecular techniques to identify detailed cellular and molecular mechanisms of ATO action in APL cells and transgenic mice hepatocytes as well as bone marrow cells. We found that the expression levels of p53 and p21 increased significantly, whereas MDM2, DAXX and HAUSP decreased dose dependent manner. Our immunoprecipitation (IP) studies shown that they are well associated each other in cells and ATO disrupts associations. After 21 days treatment of ATO different doses (1.25, 2.5, 5.0 and 7.5 mg/kg body wt) in transgenic mice, we have collected liver tissue and isolated hepatocytes and bone marrow. In liver tissue, we found p53 nicely activated dose dependent ATO treated mice. On the basis of these findings, we conclude that ATO induces cell cycle arrest and apoptosis in APL cell line as well as mice hepatocytes through p53 activation, MDM2 self-ubiquitination and disruption of MDM2-DAXX-HAUSP interaction. It is novel target for treatment of APL patients through designing of new anti-leukemic drugs. Keywords: Arsenic trioxide, HL-60 cells, cell cycle arrest, apoptosis, mitochondrial pathway, Mice hepatocytes and bone marrow. Acknowledgements: This research was financially supported by National Institutes of Health NCRR Grant No. 5G12RR013459 and MIMHD Grant No. 8G12MD007581, through the RCMI-Center for Environmental Health at Jackson State University. Citation Format: Sanjay Kumar, Paul B. Tchounwou. Arsenic trioxide induces cell cycle arrest, apoptosis through interaction of DAXX and degradation of MDM2 in acute leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3811. doi:10.1158/1538-7445.AM2015-3811

Salvage Chemotherapy Followed By Autologous Peripheral Stem Cell Transplantation for Relapsed Acute Promyelocytic Leukemia; A Single Center Experience

Background: After the addition of differentiating agents such as all-transretinoic acid (ATRA) to conventional chemotherapy, treatment outcomes of acute promyelocytic leukemia (APL) had been markedly improved. However, about 10% of patients with APL still have experienced relapse and have to receive salvage chemotherapy. Although arsenic trioxide (ATO) plays an important role in salvage chemotherapy as well as induction chemotherapy, the optimal treatment strategy remains unclear, especially for patients in whom autologous peripheral stem cell transplantation (AutoHCT) is planned. We tried to find the role of salvage chemotherapy including ATO-based regimen in relapsed APL patients, and want to share our experience in these patients.Patients and Methods: Among 77 patients diagnosed with APL from 2005 to 2014 at Yonsei University Severance Hospital, 11 (14.3 %) relapsed patients were enrolled. Clinical data of these patients was retrospectively reviewed. Except one patient refused further treatment at the time of relapse, 10 patients were included in the final analysis for treatment outcome.Results: All patients received remission induction chemotherapy with modified PETHEMA regimen, and achieved complete remission (CR) confirmed at the molecular level. The median duration of the first CR was 38.8 months (range 28.0–88.0 months). As pre-transplant salvage chemotherapy, earlier 3 patients received conventional chemotherapy regimen without ATO, but later 7 patients received ATO-based regimen after ATO was available in our country. There was no statistical difference in median duration to the second CR from salvage chemotherapy according to the regimen (68 days in conventional chemotherapy patients vs. 60 days in ATO-based chemotherapy, P>0.05). However, 3 patients receiving conventional regimen without ATO did not received AutoHCT because they died due to treatment-related complications during additional salvage chemotherapies. In contrast, 7 patients receiving ATO-based regimen received AutoHCT successfully, and all patients remains alive without further relapse. The median duration from the time of second CR to AutoHCT was 3.8 months (range 1.3–7.6 months), and median follow up duration after AutoHCT was 8.3 months (range 0.6–45.9 months). In these 7 patients receiving ATO-based regimens, duration of ATO treatment was variable (median 72 days, range 42-113 days), and a range of total dose of ATO given before AutoHCT was also variable (median 701.23 mg, range 420-1242.8 mg). Total dose and treatment duration of ATO was mainly determined by the time of molecular CR achieved again. The peripheral hematopoietic stem cells were harvested when molecular complete remission was confirmed, and median number of stem cells harvested was 5.14 x 10^6 cells/kg of recipient (range 2.90–7.76 x 10^6 cells/kg). The yield of harvest peripheral stem cells and the outcomes of followed AutoHCT were not significantly related to the total dose or treatment duration of pre-transplant ATO-based salvage therapy.Conclusion: In this study, ATO-based chemotherapy followed by AutoHCT was safe and excellent salvage treatment strategy for relapsed APL patients. The achievement of second molecular CR might be more important in this treatment strategy than total dose or treatment duration of ATO-based salvage therapy before AutoHCT. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2291: Arsenic trioxide induces cell cycle arrest, apoptosis and MAPKinase signaling cascade in acute promyelocytic leukemia cells

Abstract Arsenic trioxide (ATO) has recently been successfully used in the treatment of all-trans retinoic acid resistant relapsing acute promyelocytic leukemia (APL) patients both alone or combination with other drugs. Its use as induction and consolidation therapy has resulted in complete remission rate of both de novo and relapsed APL patients. However, the detailed molecular mechanisms of its therapeutic action are poorly known. We have used human leukemia (HL-60) cells as a model to elucidate the anti-cancer properties of arsenic trioxide. We hypothesized that ATO arrests cell cycle progression of HL-60 cells at G1 phase and leading to cell death by intrinsic pathway of apoptotic signaling. To test the hypothesis, we used western blotting, confocal imaging and spectrofluoremetric techniques to identify detailed cellular and molecular mechanisms of ATO action in HL-60 cells. We found that the expression levels of pro-apoptotic molecules (Bax) and cytochrome C were up-regulated, while that of anti-apoptotic Bcl2 protein was down-regulated at 4 and 6mg/mL ATO doses significantly. We also found that ATO stimulates signaling molecules like P38, JNK in a dose dependent manner in HL-60 cells. Oxidative stress and related imbalance of pro- and anti-apoptotic molecules lead to change of mitochondrial membrane potential and opening of the mitochondrial membrane transition pores. Our confocal imaging data shows a clear translocation of Bax from cytosol to mitochondria and of cytochrome c from mitochondria to cytosol in ATO-treated cells. A dose-dependent decrease/drop-down in mitochondrial membrane potential was also observed in ATO-treated cells, as evidenced by JC-1 leveling and spectrofluoremetric potential recording. On the basis of these findings, we conclude that mitochondrial pathway of apoptosis plays a key role in ATO pharmacology and should be further assessed as a drug target in APL chemotherapy. Citation Format: Sanjay Kumar, Clement Yedjou, Paul B. Tchounwou. Arsenic trioxide induces cell cycle arrest, apoptosis and MAPKinase signaling cascade in acute promyelocytic leukemia cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2291. doi:10.1158/1538-7445.AM2014-2291

MicroRNA-181a-mediated downregulation of AC9 protein decreases intracellular cAMP level and inhibits ATRA-induced APL cell differentiation.

AC9 is one of the adenylate cyclase (AC) isoforms, which catalyze the conversion of ATP to cAMP, an important second messenger. We previously found that the integration of cAMP/PKA pathway with nuclear receptor-mediated signaling was required during all-trans retinoic acid (ATRA)-induced maturation of acute promyelocytic leukemia (APL) cells. Here we showed that AC9 could affect intracellular cAMP level and enhance the trans-activity of retinoic acid receptor. Knockdown of AC9 in APL cell line NB4 could obviously inhibit ATRA-induced differentiation. We also demonstrated that miR-181a could decrease AC9 expression by targeting 3′UTR of AC9 mRNA, finally controlling the production of intracellular cAMP. The expression of miR-181a itself could be inhibited by CEBPα, probably accounting for the differential expression of miR-181a in NB4 and ATRA-resistant NB4-R1 cells. Moreover, we found that AC9 expression was relatively lower in newly diagnosed or relapsed APL patients than in both complete remission and non-leukemia cases, closely correlating with the leukemogenesis of APL. Taken together, our studies revealed for the first time the importance of miR-181a-mediated AC9 downregulation in APL. We also suggested the potential value of AC9 as a biomarker in the clinical diagnosis and treatment of leukemia.

Outcomes and prognostic factors of first relapsed acute promyelocytic leukemia patients undergoing salvage therapy with intravenous arsenic trioxide and chemotherapy

Arsenic trioxide (ATO) is an effective therapy for relapsed acute promyelocytic leukemia (APL) patients; however, the optimal treatment strategy remains unclear, and knowledge of the prognostic factors is still limited. We retrospectively analyzed the outcomes of 64 consecutive first relapsed APL patients (12 with molecular relapse and 52 with hematologic relapse). Patients received re-induction with intravenous ATO-based regimens. Patients who achieved a CR2 were offered further courses of alternating ATO/conventional chemotherapy with or without stem cell transplantation (SCT). With a median follow-up of 27 months (range, 6-57) in the molecular relapsed subgroup, the 3-year relapse-free survival (RFS) and overall survival (OS) rates were 81.5% and 100%, respectively. With a median follow-up of 38 months (range, 0-129) in the hematologic relapse group, the 3-year RFS and OS rates were 57.1% and 72.1%, respectively. Furthermore, in the hematologic relapse group, we compared the outcome between relapsed patients after previous ATO therapy (n = 20) with those who did not receive prior ATO therapy (n = 32). The CR2 rate was 80% (16/20) vs. 93.8% (30/32), (p = 0.189). However, the relapse rate was 68.8% (11/16) vs. 33.3% (10/30), (p = 0.03). The 4-year OS rate was 62.4% vs. 71.2%, (p = 0.816), and the 4-year RFS rate was 29.8% vs. 66.2% (p = 0.023). The results indicate that, irrespective of frontline therapy with ATO, salvage therapy with an ATO-based regimen remains effective. However, the long-term survival for those patients who received previous ATO-based treatment was inferior compared to those who did not receive prior ATO. In addition, the alternating ATO/chemotherapy strategy can be a post-remission treatment option in a subset of patients.

Role of hematopoietic stem cell transplantation for relapsed acute promyelocytic leukemia: A retrospective analysis of JALSG‐APL97

For patients with relapsed acute promyelocytic leukemia (APL), all-trans retinoic acid-based salvage regimens can achieve second complete remission (CR2), but the optimal post-remission strategy for APL patients after CR2 remains unclear. Hematopoietic stem cell transplantation (HSCT) during CR2 might be effective, but data on the role of HSCT for APL patients after CR2 are limited in Japan. We retrospectively analyzed outcomes for 57 relapsed APL patients who achieved CR2 in the JALSG APL97 study. Of those, six received autologous (auto)-HSCT, 21 received allogeneic (allo)-HSCT, and 30 received various regimens other than HSCT. The 5-year event-free survival (EFS) rate, overall survival (OS) rate and cumulative incidence of relapse (CIR) were 50.7%, 77.4% and 51.0% in the non-HSCT group, 41.7%, 83.3% and 58.3% in the auto-HSCT group and 71.1%, 76.2% and 9.8% in the allo-HSCT group, respectively. Both the EFS rate and CIR were significantly better in the allo-HSCT group than in other groups. Allo-HSCT appears effective in APL patients in CR2, with a low relapse rate beyond a relatively early transplantation-related mortality (19%). Among older patients (age ≥40 years), the 5-year OS was significantly better in the non-HSCT group than in the HSCT group (78.0% vs 40.5%; P = 0.04). Further prospective studies with larger patient numbers are required to confirm the impact of HSCT alone and in combination with arsenic trioxide on outcomes for patients with APL in CR2.

Aquaporin 9, a promising predictor for the cytocidal effects of arsenic trioxide in acute promyelocytic leukemia cell lines and primary blasts

A close correlation between the cytocidal effects of arsenic trioxide (ATO) and aquaporin-9 (AQP9) expression levels has been proposed, yet detailed studies are still needed to confirm this association. Thus, in the present study, the correlation between the expression levels of AQP9 and sensitivity to ATO was investigated using two acute promyelocytic leukemia (APL) cell lines, NB4 and HT93A, as well as primary APL cells from newly diagnosed and relapsed APL patients. A substantially higher sensitivity to ATO-mediated induction of apoptosis was observed in the NB4 cells when compared to that in the HT93A cells. In addition, markedly higher expression levels of AQP9, as assessed using flow cytometry, along with more intracellular arsenic accumulation, were observed in the NB4 cells. More importantly, similar to APL cell lines, the trend of expression levels of AQP9 correlated closely with the differential sensitivity to ATO-mediated induction of apoptosis in primary APL cells. In contrast, no correlation was observed between ATO sensitivity associated with AQP9 expression levels and the expression profiles of cell surface markers as well as chromosomal alterations. These results provide direct evidence that the expression levels of AQP9, rather than other biomarkers such as cell surface markers and chromosomal alterations, correlate closely with the sensitivity to ATO in both APL cell lines and primary blasts. These findings suggest that the AQP9 expression status of APL patients is a predictive marker for the successful outcome of ATO treatment, since AQP9 plays a pivotal role in various arsenite-mediated biological effects on normal and cancer cells. Moreover, flow cytometry may be a new convenient and valuable tool for analyzing the AQP9 status of APL patients compared to current methods such as western blotting.

The efficacy and safety of arsenic trioxide with or without all-trans retinoic acid for the treatment of acute promyelocytic leukemia: A meta-analysis

Arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA) could induce apoptosis and differentiation in acute promyelocytic leukemia (APL) cells, respectively, thus the possibility of synergism between them was raised. This meta-analysis assessed the effectiveness and safety of ATO combined with ATRA in the treatment of APL. Compared with ATO alone, induction therapy with ATO/ATRA significantly increased the complete remission (CR) rate (RR: 1.08, 95% CI: 1.00–1.17, P = 0.04), shortened the time to achieve CR (WMD: −6.51, 95% CI: −11.32 to −1.70, P = 0.008), and improved the molecular remission rate after consolidation therapy (RR: 1.74, 95% CI: 1.14–2.66, P = 0.01) and the 1-year disease-free survival rate (RR: 1.22, 95% CI: 1.00–1.50, P = 0.05). There were no statistically significant differences between two treatments in terms of early death and main adverse events. These results suggested that ATO/ATRA could synergistically improve the overall outcome of newly diagnosed and relapsed APL patients, supporting the use of ATO/ATRA as an effective treatment for all APL patients previously untreated with ATO.

Comparison of Clinical Outcomes of Relapsed APL Patients Induced With ATO and Consolidated With Either an Autologous SCT or ATO Based Treatment Regimen

Arsenic trioxide (ATO) is the most effective agent in inducing remission among patients with relapsed acute promyelocytic leukemia (APL). After induction of remission in relapsed patients with ATO the best consolidation regimen remains to be defined. Since January 2000, 37 patients with relapsed APL were treated at our center. The median age was 34 years (range: 6 – 57) and there were 20 (54.1%) males. The initial treatment was conventional ATRA plus chemotherapy in 23 (62.2%) and single agent ATO in 14 (37.8%).

Arsenic Trioxide Augments Chk2/p53-mediated Apoptosis by Inhibiting Oncogenic Wip1 Phosphatase

The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damage-induced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.

Myeloperoxidase Is Required for the Combined Apoptotic Effects of Arsenic Trioxide with Ascorbic Acid or Epigallocatechin-3-Gallate in Myeloid Leukemia Cells.

Arsenic trioxide (As2O3) is a drug used world-wide that selectively causes the death of acute promyelocytic leukemia (APL) cells by novel mechanisms and induces complete clinical remission in 90% of patients without significant toxicity. The outstanding success of As2O3 therapy in relapsed APL patients has not been replicated by an equivalent success in other types of acute myeloid leukemia (AML). We have studied As2O3 mechanism(s) of action and identified agents to use in combination to improve the use of As2O3 as a treatment for other types of AML. As2O3 produces higher levels of H2O2 and apoptosis in APL NB4 cells than in other AML cells at therapeutic concentrations of 1-2 uM. As2O3 does not induce apoptosis in HL-60 cells but is synergistic with ascorbic acid (AA) or epigallocatechin-3-gallate (EGCG). Both AA and EGCG produce H2O2 which is augmented by the addition of As2O3. Apoptosis induction by As2O3 in combination with AA or EGCG is inhibited by catalase and the antioxidant N-acetylcysteine. Myeloperoxidase (MPO), a major neutrophil enzyme, augments H2O2-induced apoptosis by converting it into more potent reactive oxygen species. HL-60, NB4, SKNO-1 and PLB985 cells which express high level of MPO, but not U937 and K562 cells without expression of MPO, are responsive to As2O3 plus AA or EGCG-induced apoptosis. HP-100 cells, a subclone of HL-60 cells without MPO expression, are resistant to both combination treatments. MPO stable transfection sensitizes K562 cells to apoptosis following treatment with As2O3 and AA or EGCG. NADPH oxidase is an enzyme complex which generates O2− and H2O2 in neutrophils. In APL NB4 cells, As2O3 induces expression of NADPH oxidase members and is thought to participate in H2O2 production required for As2O3 response. X-CGD cells, a subclone of PLB-985 with targeted disruption of the gp91phox gene (Zhen et al, PNAS 90:9832, 1993) and loss of NADPH oxidase activity but with high levels of MPO activity, are responsive to treatment with As2O3 and AA or EGCG. These results suggest that AA and EGCG enhance As2O3-induced apoptosis through a synergistic production of H2O2 which is independent NADPH oxidase activation in non-APL AML cells. MPO augments the apoptotic effect of H2O2 produced by As2O3 plus AA or EGCG and it could be used as a marker to predict the sensitivity of AML cells to both combination treatments.

Comparison of Clinical Outcomes of Relapsed APL Patients Induced with ATO and Consolidated with Either an Autologous SCT or ATO Based Treatment Regimen.

Arsenic trioxide (ATO) is the most effective agent in inducing remission among patients with relapsed acute promyelocytic leukemia (APL). After induction of remission in relapsed patients with ATO the best consolidation regimen remains to be defined. Since January 2000, 33 patients with relapsed APL were treated at our center. The median age was 35 years (range: 6 – 57) and there were 16 (48.5%) males. The initial treatment was conventional ATRA plus chemotherapy in 22 (66.7%) and single agent ATO in 11 (33.3%). The median duration of first remission was 19.4 months (range: 2.9 – 81.2). Relapse was treated with single agent ATO in 22 (66.7%), with ATO+ATRA in 5(15.2%) and with ATO+ATRA+anthracycline in 6 (18.2%). Thirty one (94%) achieved CR while one patient died of sepsis in induction and one died from progressive disease. After remission induction a consolidation course of the same agents used in induction was administered (ATO and ATRA were administered for 28 days), 29 (88%) achieved molecular remission after this course. All patients who achieved molecular remission were offered an autologous SCT. Nine patients opted to have an autologous SCT and the remaining patients were scheduled to receive monthly cycles of ATO as a single agent (n=15) or ATO+ATRA (n=7) for 6 months. The age, sex, duration of first CR, induction and consolidation regimen used to treat relapse was comparable between the patients that underwent an autologous SCT and the remaining patients. Patients who underwent an autologous SCT were conditioned with a conventional Bu/Cy regimen and received PBSC graft with a median cell dose of 5 x 106 MNC/Kg (range: 3.54 – 9.24). There were no treatment related deaths following the autologous SCT, one patient had an isolated CNS relapse 6 months post transplant. There were no other relapses or deaths in this group. Among the remaining patients who achieved CR but did not have an autologous SCT (n=22), 14 relapsed and all except one of these patients eventually died of progressive disease at median period of 1.6 months from second relapse (range: 0 – 42.6). At a median follow up of 24 months, the 3-year Kaplan-Meier estimate of EFS and OS among those who received an autologous SCT vs. those that did not was 88.9±10.5 vs. 28.8±9.7 (log rank: P=0.019) (Figure 1) and 100±00 vs. 36.2±10.4 (log rank: P=0.009) respectively. Following remission induction with ATO based regimens in patients with relapsed APL, consolidation with an autologous SCT, in those who achieve a molecular remission, is associated with a significantly superior clinical outcome in comparison to alternative ATO/ATRA based consolidation and maintenance regimens. [Display omitted]