Trioxide In Acute Promyelocytic Leukemia Research Articles

AML-282 Venetoclax Upregulates Mitochondrial Stress Response to Overcome the Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

AML-282 Venetoclax Upregulates Mitochondrial Stress Response to Overcome the Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

POSTER: AML-282 Venetoclax Upregulates Mitochondrial Stress Response to Overcome the Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

POSTER: AML-282 Venetoclax Upregulates Mitochondrial Stress Response to Overcome the Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

An effective and chemotherapy-free strategy of all-trans retinoic acid and arsenic trioxide for acute promyelocytic leukemia in all risk groups (APL15 trial)

The combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has been demonstrated to have comparable effectiveness or better to ATRA and chemotherapy (CHT) in non-high-risk acute promyelocytic leukemia (APL). However, the efficacy of ATRA-ATO compared to ATRA-ATO plus CHT in high-risk APL remains unknown. Here we performed a randomized multi-center non-inferiority phase III study to compare the efficacy of ATRA-ATO and ATRA-ATO plus CHT in newly diagnosed all-risk APL to address this question. Patients were assigned to receive ATRA-ATO for induction, consolidation, and maintenance or ATRA-ATO plus CHT for induction followed by three cycles of consolidation therapy, and maintenance therapy with ATRA-ATO. In the non-CHT group, hydroxyurea was used to control leukocytosis. A total of 128 patients were treated. The complete remission rate was 97% in both groups. The 2-year disease-free, event-free survival rates in the non-CHT group and CHT group in all-risk patients were 98% vs 97%, and 95% vs 92%, respectively (P = 0.62 and P = 0.39, respectively). And they were 94% vs 87%, and 85% vs 78% in the high-risk patients (P = 0.52 and P = 0.44, respectively). This study demonstrated that ATRA-ATO had the same efficacy as the ATRA-ATO plus CHT in the treatment of patients with all-risk APL.

The Development and Clinical Applications of Oral Arsenic Trioxide for Acute Promyelocytic Leukaemia and Other Diseases.

Appreciation of the properties of arsenic trioxide (ATO) has redefined the treatment landscape for acute promyelocytic leukaemia (APL) and offers promise as a treatment for numerous other diseases. The benefits of ATO in patients with APL is related to its ability to counteract the effects of PML::RARA, an oncoprotein that is invariably detected in the blood or bone marrow of affected individuals. The PML::RARA oncoprotein is degraded specifically by binding to ATO. Thus ATO, in combination with all-trans retinoic acid, has become the curative treatment for ATO. The multiple mechanisms of action of ATO has also paved the way for application in various condition encompassing autoimmune or inflammatory disorders, solid organ tumours, lymphomas and other subtypes of AML. The development of oral formulation of ATO (oral ATO) has reduced costs of treatment and improved treatment convenience allowing widespread applicability. In this review, we discuss the mechanisms of action of ATO, the development of oral ATO, and the applications of oral ATO in APL and other diseases.

Clinical Profile, Complications and Outcomes of Patients with Acute Promyelocytic Leukemia: Indian Perspective

Background:The introduction of all-trans-retinoic-acid (ATRA) and arsenic trioxide (ATO) has changed acute promyelocytic leukaemia (APL) from a highly fatal malignancy to a major success in the sphere of haemato-oncology, with long-term survival exceeding 90% in some studies [1]. While literature from the developed world boasts of outstanding outcomes of patients with APL, there is a paucity of data on management strategies, complications and outcomes from the developing world in general and India in particular [2]. With a large population residing in rural areas, high infection rates and limited access to healthcare, we aim to delineate the challenges faced in curing APL in the developing world. MethodsWe retrospective collected data from medical records of all patients diagnosed with APL between January 2016 to December 2020 in the department of Hematology. A presumptive diagnosis of APL was made based on the presence of abnormal promyelocytes in peripheral blood or bone marrow and flow cytometry (CD13, CD33 positive, HLADR, CD34 negative). The diagnosis was confirmed by the presence of PML-RARα fusion gene by RT-PCR. Data on baseline characteristics, therapy, complications and outcome was collected. Overall survival (OS) was defined from the date of diagnosis to death or date of last follow up. Disease free survival (DFS) was defined from the date of first remission to date of first relapse, death or date of last follow up. Results64 patients were treated during the study period. Baseline characteristics, therapy administered and induction outcomes are mentioned in Table 1. 1.Complications:56.25% patients developed differentiation syndrome (DS). The incidence was higher in Sanz high-risk group as compared to Sanz-low risk group (68.8% vs 43.2%, p value 0.044).The incidence of infection at presentation was similar in Sanz high-risk and low-risk groups (55.2% vs 59.4%, p value 0.7). The incidence of new episode of febrile neutropenia during therapy was 62.5% and was higher in Sanz high-risk group as compared to low-risk group (75% vs 50%, p value 0.039). On multivariate analysis, development of infection during induction was a significant predictor of death or relapse (p value 0.02 HR:0.3; 95% CI 0.12-0.84).17.2% patients developed pseudotumor cerebri. All of them were treated with acetazolamide, 6 patients' symptoms persisted despite acetazolamide and the dose of ATRA was lowered to 25mg/m2.3 patients developed thrombosis. One patient had superior sagittal sinus thrombosis at presentation and two patients developed central line related thrombosis during therapy.15.6% patients developed hepatotoxicity of which 3 had an underlying chronic liver disease. The remaining had a normal liver echotexture on ultrasonography and viral markers were normal; the liver dysfunction was attributed to drug induced liver injury.One patient who received AIDA protocol developed PSVT and improved with adenosine. Another patient who received ATRA+ATO+Daunorubicin developed accelerated hypertension and was managed symptomatically.1.Outcomes75% patients attained complete remission after induction. 9 patients died from infection, 6 from bleeding and one from DS.At a median follow up of 26 months, 2 patients belonging to high-risk group had relapsed. They received reinduction with ATRA and ATO; one succumbed to pancreatitis during induction, the other attained CR2 and is on maintenance therapy.The 4-year DFS and OS was 71.25% (95% CI 59.9 to 82.5) and 73.13% (95% CI 62.2 to 84.1) respectively. Patients in Sanz low risk group had a better 4-year DFS (84.9% vs 58.3%, p value 0.026) and OS (84.9% vs 62.5%, p value 0.044) as compared to the Sanz high-risk group. There was a trend towards improved outcomes in high-risk patients with the use of ATRA+ATO+Daunorubicin. ConclusionAPL patients in India have a higher incidence of DS, pseudotumor cerebri, hepatotoxicity, baseline infections & new infections during induction complicating therapy. CR rate, DFS, OS were comparable to other population-based studies. ATRA, ATO and daunorubicin combination is the preferred protocol for treating high-risk patients. REFERENCES1. Iland HJ et al. All-trans-retinoic acid, idarubicin, and IV arsenic trioxide as initial therapy in acute promyelocytic leukemia (APML4). Blood. 2012;120(8):1570-802. Lo-Coco F et al. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med. 2013;369(2):111-21. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Blood Parameters in Treatment with Arsenic Trioxide in Acute Promyelocytic Leukemia: A Systematic Review

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). APL is famed with some special blood coagulation disorders such as disseminated intravascular coagulation (DIC). The therapeutic methods of APL contain All Trans Retinoic Acid (ATRA), arsenic trioxide (ATO) or/and chemotherapy. Many studies have been done on APL blood disorders and its treatment. These studies have shown different results. In this systematic article, we tried to review the effect of ATO therapy with or without ATRA and chemotherapy on DIC parameters (D-dimer, Prothrombin Time, Activated Partial Thrombin Time, Platelet count) in APL patients. The result of included studies demonstrated that although ATO can reduce the number of malignant cells in the bone marrow and peripheral blood, it does not have enough potential to attenuate the danger of high score DIC that is usual in APL patients and should be better to be used with other therapeutic methods.

Activation of PPARγ intensified the effects of arsenic trioxide in acute promyelocytic leukemia through the suppression of PI3K/Akt pathway: Proposing a novel anticancer effect for pioglitazone.

The indulgent success of arsenic trioxide (ATO) in the induction of complete remission in acute promyelocytic leukemia (APL) patients has accommodated this agent into the therapeutic protocols. However, the intrusion of unfavorable side effects had put an unanswered question on the way of the application of this agent; whether the benefits of ATO may outweigh its drawbacks. In this study, we found that when ATO is accompanied by an activator of peroxisome proliferator-activated receptors gamma (PPARγ), even the lower concentrations could induce significant inhibitory effects on the survival of NB4 through diminishing the ability of the cells to replicate DNA in the S phase of cell cycle. We also found that through suppression of the PI3K pathway, the combination of pioglitazone and ATO provided a signal through which the induction of apoptotic cell death was enhanced probably via the elevation of reactive oxygen species (ROS). With respect to the tight connection between PI3K pathway and autophagy system and given to the inhibitory effect of pioglitazone-plus-ATO on PI3K, we found that the combination of these agents not only suppressed the expression of autophagy-related genes, but also their efficacy was augmented when autophagy was inhibited in NB4; clarifying the encouraging role of autophagy in the survival maintenance of APL cells. In conclusion, given the significant efficacy as well as the safety profile of pioglitazone in potentiating the anticancer effects of chemotherapeutic drugs, the present study suggests it as a promising agent to be used in adjuvant strategy for the treatment of APL.

Abstract B33: Synergistic effect of gefitinib and arsenic trioxide in acute promyelocytic leukemia

Abstract Gefitinib and erlotinib are well-known epidermal growth factor receptor (EGFR) inhibitors approved by the Food and Drug Administration for the treatment of non-small cell lung cancer (NSCLC). Interestingly, patients with synchronous NSCLC and acute myeloid leukemia (AML) treated with erlotinib presented regression of both neoplasms, although AML myeloblasts were shown to lack expression of EGFR. In addition, preclinical studies have shown that gefitinib alone or combined with arsenic trioxide (ATO) or all-trans retinoic acid (ATRA) was able to induce differentiation in the EGFR-negative cell lines of acute promyelocytic leukemia (APL), which is a subtype of AML characterized by the t(15;17)/PML/RARA rearrangement. Our previous study demonstrated that EGFR gene expression levels were associated with prognostic outcomes of APL patients treated according to the International Consortium on APL protocol. However, the EGFR levels were not assessed at protein level. Two phase 3 trials reported that ATRA and ATO therapy was associated with excellent outcomes in APL. The two drugs induced the degradation of the PML/RARA oncoprotein through different mechanisms, with ATRA acting through the proteasome pathway, and ATO functioning through the PML-transformation related protein 53 (Trp53) axis. The purpose of this study was to investigate whether gefitinib or erlotinib has synergistic effects with ATO or ATRA in the treatment of APL. To this end, the APL cell lines NB4 and NB4-R2 were treated with gefitinib (C22H24ClFN4O; Selleck Chemicals, #S7786) and erlotinib (C22H23N3O4; Selleck-Chemicals, #S1025) alone or combined with ATO (As2O3; Sigma-Aldrich, #202673). The stock solutions of gefitinib and erlotinib were prepared in DMSO while ATO was dissolved in 1M NaOH solution. Then each drug was diluted with RPMI medium to the desired final concentration. The median effective dose (ED50) for gefitinib was calculated to be 20.97 and 27.06 µM for NB4 and NB4-R2, respectively. ATO was a potent inducer of apoptosis with an ED50 of 2.27 µM for NB4 and 1.73 µM for NB4-R2 cells. ED50 of ATRA (C20H28O2; Sigma-Aldrich, #R2625) was not calculated because ATRA is a potent differentiation inducer but did not induce cell death of APL blasts. Due to the high value of ED50 for NB4 (78.97 µM) and NB4-R2 (111.36 µM) after 24 h of erlotinib treatment, the analysis of this drug was discontinued. The interaction between gefitinib and ATO demonstrated a moderate synergism for NB4 and NB4-R2 with a combination index values of 0.88 and 0.83, respectively. These in vitro results encouraged us to investigate the in vivo effects of gefitinib alone or in combination with ATO and/or ATRA. To this second end, we developed a syngeneic transplant murine model using leukemic cells obtained from transgenic mice hCG-PML/RARA, which develop a form of leukemia that closely recapitulates the human disease. The engraftment of leukemic cells from hCG-PML-RARα transgenic mice transplanted to wild-type littermate recipients was evaluated by morphology and flow cytometry analysis of the peripheral blood (PB), bone marrow (BM), and spleen. Recipient mice were euthanized and evaluated for signs of engraftment 14 (n=3) and 20 (n=3) days after transplantation. Of note, the morphologic analysis revealed >20% of blasts among nucleated cells in the BM samples. The flow cytometric analysis showed the presence of CD11b+ CD117+ cells in PB (median 10.50, range 8.81-12.20), BM (median 18.30, range 17.80-18.80), and spleen (median 16.45, range 16.00-16.90). These results demonstrate that this rapid and robust murine model is useful to assess the efficacy of EGFR inhibitors in APL. Taken together, our preliminary in vitro results suggest that gefitinib and ATO may have potential application for the APL treatment and that this current syngeneic transplant mouse model of APL is suitable to test this hypothesis. Citation Format: Luciana Yamamoto Almeida, Cleide Lúcia Araújo Silva, Isabel Weinhaüser, Larissa Ananias Cândido, Priscila Santos Scheucher, Camila Cristina Oliveira Menezes Bonaldo, Bárbara Amélia Aparecida Santana, Ana Sílvia Gouvêa Lima Yamada, Eduardo Magalhães Rego. Synergistic effect of gefitinib and arsenic trioxide in acute promyelocytic leukemia [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B33.

Direct Speciation Analysis of Arsenic in Whole Blood and Blood Plasma at Low Exposure Levels by Hydride Generation-Cryotrapping-Inductively Coupled Plasma Mass Spectrometry.

A method for analysis of toxicologically important arsenic species in blood plasma and whole blood by selective hydride generation with cryotrapping (HG-CT) coupled either to atomic absorption spectrometry (AAS) with a quartz multiatomizer or to inductively coupled plasma mass spectrometry (ICPMS) has been validated. Sample preparation, which involved only 5 times dilution with addition of Triton X-100, Antifoam B, and l-cysteine, suppressed excessive foaming in a hydride generator. Calibration slopes for whole blood and blood plasma spiked with arsenate, monomethylarsonate, and dimethylarsinate at 0.25-1 μg L-1 As and 0.025-0.1 μg L-1 As for AAS and ICPMS detection, respectively, did not differ from slopes in aqueous solutions. HG-CT-AAS was used to analyze samples with elevated levels of arsenic species-blood plasma from patients treated with arsenic trioxide for acute promyelocytic leukemia and whole blood from mice fed an arsenic-containing diet. A good agreement between results of the direct analysis and analysis after mild digestion in phosphoric acid proved the good efficiency of the direct HG-CT procedure for the arsenic species in these types of biological samples. In the next step, plasma and whole blood from healthy donors that were spiked with the plasma from leukemia patients at levels of 0.15-0.4 μg L-1 As were analyzed by direct HG-CT-ICPMS. Good recoveries for all species even at these low levels (88-104%) were obtained. Limits of detection in blood and plasma were 0.014 μg L-1 for inorganic arsenic and below 0.002 μg L-1 As for methylated arsenic species. Thus, the ultrasensitive direct HG-CT-ICPMS method is uniquely suited for analyses of blood plasma and whole blood from individuals at low exposure levels.

Metabolic Rewiring Drives Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

The focus of ATO resistance in acute promyelocytic leukemia (APL) has centered on mutations in PML-RARA gene (Blood 2011, NEJM 2014). However such mutations are rare and cannot explain the majority of relapses seen in the clinic. To evaluate the mechanisms of ATO resistance, we generated ATO resistant NB4 sub clone NB4-EVAsR1 (A216V - VAF-91.7%) in our laboratory. We also had another ATO resistant cell line (UF1) which does not have the A216V mutation. In an expression array we noted that redox signaling, AMPK signaling and energy metabolism pathways were significantly dysregulated in the ATO resistant cell lines compared to naïve NB4 cells.Towards validating the microarray data and to characterize the ATO resistant cell lines we measured the basal levels of reactive oxygen species (ROS), glutathione(GSH), mitochondrial membrane potential (MMP), glucose uptake and their sensitivity to glycolytic inhibitor 2-Deoxy glucose (2-DG) in comparison to naïve NB4 cells. We observed that resistant cell lines have significantly lower ROS, MMP, glucose uptake (Fig 1a) and increased GSH. We also observed that the resistant cell lines were significantly less susceptible to treatment with 2-DG in comparison to naïve NB4 cells (Fig 1b) suggesting that resistant cell lines were less dependent on glycolysis. ATO has been reported to directly inhibit the glycolytic pathway, this effect is believed to contribute to its cytotoxic effect (PNAS 2015). However, we did not observe any cytotoxic synergy between ATO and 2-DG on naïve NB4 cells and neither did this combination restore sensitivity to ATO in the resistant cell lines (Fig 1b).Next we assessed the sensitivity of these resistant cell lines to oxidative phosphorylation (OXPHOS) inhibitors. We used an uncoupler (FCCP at 10uM) of OXPHOS which promotes uncoupled respiration by deregulating the proton gradient which drives ATP synthesis via ATP synthase. We observed that the FCCP treatment alone did not reduced the viability of naïve NB4 cells. Similarly, viability of ATO resistant cell lines also did not reduce significantly suggesting the ability of these cells to uncouple their metabolic pathway from OXPHOS to glycolysis when inhibited. However, when FCCP was combined with ATO it significantly restored the sensitivity of the resistant cell lines to ATO (Fig 1c). The same combination did not have any additive effect on naïve NB4 cells. The combination not only restored the sensitivity of the ATO resistant cell lines but also sensitized the conventionally ATO resistant cell lines such U937 (Fig 1c) and THP1. In spite of the profound effect on leukemic cells we also observed a significant bystander effect on the normal peripheral blood mononuclear cells (Fig 1c). The data suggests that the sensitivity of these resistant cell lines could be potentially restored by combining ATO with an OXPHOS uncoupler. A number of molecules that are FDA approved and used in the clinic also have OXPHOS uncoupling activity and could potentially be evaluated for their synergistic activity with ATO in leukemia. This data also draws attention to possible severe systemic off-target toxicity of such combinations which may be inadvertently used in the clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Arsenic Trioxide in Acute Promyelocytic Leukemia and Associated Bone Marrow Changes

Arsenic Trioxide in Acute Promyelocytic Leukemia and Associated Bone Marrow Changes

Dose-adjusted arsenic trioxide for acute promyelocytic leukaemia in chronic renal failure.

To determine the potential for arsenic trioxide (ATO) to be safely and effectively incorporated into induction therapy of newly diagnosed acute promyelocytic leukaemia (APL) in patients with severe chronic renal failure (CRF) by reduction of the ATO dosage to compensate for reduced renal elimination of arsenic in CRF. Two of the four CRF patients with APL in the study were dialysis-dependent, and two had eGFRs of 18 and 19mL/min/1.73m(2) . ATO dosage schedules were adjusted to obtain comparable whole-blood arsenic levels to those in APL patients with normal renal function who achieved molecular remission (MR) while receiving 10mg ATO daily for 28d. Average ATO administered per day in CRF patients ranged from 36 to 50% of the ATO administered to APL patients with normal renal function. No clinically significant cardiac, hepatic or other toxicities were detected. RT-PCR-negative MR was achieved after one treatment course in two patients and after two courses in the others. Relapse-free survival is 155, 60, 43 and 5months. The observations in this pilot study have demonstrated whole-blood arsenic levels can provide a guide to adjustments of ATO dosage schedules that permit safe and effective therapeutic outcomes in APL patients with severely compromised renal function.

Retinoic Acid and Arsenic Trioxide for Acute Promyelocytic Leukemia

BackgroundAll-trans retinoic acid (ATRA) with chemotherapy is the standard of care for acute promyelocytic leukemia (APL), resulting in cure rates exceeding 80%. Pilot studies of treatment with arsenic trioxide with or without ATRA have shown high efficacy and reduced hematologic toxicity.MethodsWe conducted a phase 3, multicenter trial comparing ATRA plus chemotherapy with ATRA plus arsenic trioxide in patients with APL classified as low-to-intermediate risk (white-cell count, ≤10×109 per liter). Patients were randomly assigned to receive either ATRA plus arsenic trioxide for induction and consolidation therapy or standard ATRA–idarubicin induction therapy followed by three cycles of consolidation therapy with ATRA plus chemotherapy and maintenance therapy with low-dose chemotherapy and ATRA. The study was designed as a noninferiority trial to show that the difference between the rates of event-free survival at 2 years in the two groups was not greater than 5%.ResultsComplete remission was achieved in all 77 patients in the ATRA–arsenic trioxide group who could be evaluated (100%) and in 75 of 79 patients in the ATRA–chemotherapy group (95%) (P=0.12). The median follow-up was 34.4 months. Two-year event-free survival rates were 97% in the ATRA–arsenic trioxide group and 86% in the ATRA–chemotherapy group (95% confidence interval for the difference, 2 to 22 percentage points; P<0.001 for noninferiority and P=0.02 for superiority of ATRA–arsenic trioxide). Overall survival was also better with ATRA–arsenic trioxide (P=0.02). As compared with ATRA–chemotherapy, ATRA–arsenic trioxide was associated with less hematologic toxicity and fewer infections but with more hepatic toxicity.ConclusionsATRA plus arsenic trioxide is at least not inferior and may be superior to ATRA plus chemotherapy in the treatment of patients with low-to-intermediate-risk APL. (Funded by Associazione Italiana contro le Leucemie and others; ClinicalTrials.gov number, NCT00482833.)

Role of Arsenic Trioxide in Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia that is characterized by distinct clinical, morphological, cytogenetic, and molecular abnormalities. It is associated with a striking risk of early hemorrhagic death due to disseminated intravascular coagulation and hyperfibrinolysis. The prognosis of APL has improved dramatically following the introduction of all-trans retinoic acid (ATRA) and its combination with anthracycline-based chemotherapy during induction and consolidation. Patients with high-risk APL, defined by a white cell count >10 × 10(9)/L at diagnosis, also appear to benefit from the addition of intermediate- or high-dose cytarabine during consolidation. Arsenic trioxide (ATO) has proved to be even more effective than ATRA as a single agent, and is now routinely used for the treatment of the 20%-30% of patients who manifest disease relapse after initial treatment with ATRA and chemotherapy. ATO has a toxicity profile that differs considerably from that of both ATRA and cytotoxic chemotherapy, and accordingly presents its own specific challenges during treatment. Optimizing a strategy for the incorporation of ATO into initial therapy is currently the focus of several cooperative group trials, with an emphasis on minimizing or even eradicating the use of chemotherapy. ATRA plus ATO without chemotherapy appears to be adequate during induction and consolidation for patients with standard-risk APL, but triple therapy that includes limited anthracycline or gemtuzumab ozogamicin (GO) during induction is required for high-risk APL. Uncertainty still exists regarding the minimum amount of chemotherapy and number of consolidation cycles necessary, the optimal scheduling of ATO, and the potential utility of oral ATO administration. Although prolonged oral maintenance therapy is usually included in most current APL treatment protocols, its value remains controversial, and the superior anti-leukemic efficacy of ATO-based therapy may facilitate its elimination in the future.

Mechanism of Synergy Between Bortezomib and Arsenic Trioxide in Acute Promyelocytic Leukemia and Clinical Efficacy in Relapsed Patients

AbstractAbstract ▪3607▪This icon denotes a clinically relevant abstractBortezomib (Bo) has been shown to have direct cytotoxicity against malignant promyelocytes. There remain concerns about combining it with ATO. A major concern is that proteasomal inhibition by Bo could decrease ATO induced PML-RARA degradation and reduce its efficacy. In an in-vitro experiment we noted a significant synergistic cytotoxic effect when Bo (80ng/ml; a pharmacologically relevant concentration) was combined with ATO (1– 6uM) on NB4 cells (n = 12), P=0.03 (Fig 1a). We also noted that co-culture of NB4 or primary APL cells with stromal cells (Hs-5 cell line) protects them from ATO induced apoptosis and Bo can overcome this protective effect (Fig 1b). We undertook a series of experiments to address the mechanism of these observations.We observed an increased activation of NF-κB pathway in malignant promyelocytes (NB4 cells) co-cultured with stromal cells. There was a synergistic inhibition of NF-κB when a combination of ATO and Bo was used (Fig 1c). Considering potent inhibition of NF-κB pathway and the known effect of this on reactive oxygen species (ROS), we checked the level of ROS in the malignant promyelocytes and observed that there was an increase in ROS levels when compared to ATO or Bo alone treated cells (n=5; treatment for 6 hours) (Fig 1d). When the ROS was abrogated by pre-treating the NB4 cells with N-Acetyl Cysteine (NAC; 5mM for 2 hrs) there was a protective effect against the ATO combined with Bo induced apoptosis at 24 hours (mean increase of 73% from 65.5%; p=0.03; n=4).Degradation of PML-RARA was seen in ATO or Bo alone treated cells at 24 hours and a similar degradation was also seen when ATO and Bo were combined (Fig 1f; n=3). Bo at the concentration used (80ng/ml) was able to block 26s proteasome complex, as a result accumulation of ubiquitinated products was seen at 24 hours followed by degradation at the end of 48 hours (Fig 1e). We then looked at autophagy as a possible alternate mechanism to explain PML-RARA degradation when a combination of ATO and Bo was used in the setting of effective proteasomal inhibition. At 24 hours, there was a evidence of induction in autophagy as shown by LC3II formation using western blot technique and this was maximum at the end of 48 hours, this time point coincides with time at which maximum PML-RARA degradation happens in this experimental setting (Fig 1f). We have also observed that there is an accumulation of p62 (ubiquitinated protein binding protein) at 24 hours and degraded by 48 hours suggests that accumulated ubiquitinated products were cleared by autophagy via p62 (Fig 1f). The induction of autophagy was further validated by real time-PCR where autophagy genes atg5 and beclin1 levels were significantly increased by 3.1 fold (p=0.01) and 2.1 fold (p=0.03) respectively. Blocking autophagy by 3-methyl adenine or chloroquine did not enhance survival but paradoxically appeared to further enhance cell death (n= 3; p=0.02) though there was partial inhibition in the degradation of PML-RARA (data not shown).We have also observed that there was a synergistic and more rapid increase in apoptosis when ATO and Bo were combined as evidenced by an enhanced degradation of caspase3 (Fig 1f).Preliminary phase I data in 4 patients is summarized in table 1. Patients received Bo in induction and in consolidation once a week for 4 weeks along with ATO. Two cases, due to financial constraints, could not have a stem cell transplant (SCT) in molecular remission. In summary the combination of these drugs was well tolerated and durable CR3 and CR2 was obtained even in cases that did not have a SCT in remission. None of these cases have relapsed at a median follow up of 14.5 months.In conclusion blocking 26s proteasomal complex by Bo does not alter the efficacy of ATO, instead Bo synergizes with ATO. The mechanism of this synergy is multi-factorial and appears to be predominantly due to increase in ROS activity and increased apoptosis. Additionally stromal cell mediated protection against ATO induced apoptosis is overcome by addition of Bo. Observed degradation of PML-RARA by ATO+Bo could be explained by induction of autophagy in these cells. [Display omitted] Table 1:Summary of cases and duration of CR prior and post remission induction with arsenic trioxide and bortezomibCaseAgeSexRelapse numberDuration of last CR (months)Prior autologous SCTPost remission SCTDuration of current CR (months)RS25MR219YesNo19BJ31MR115NoYes (auto)15TK35MR224YesYes (MUD)14SS34FR119NoNo13 Disclosures:No relevant conflicts of interest to declare.

Exploring Old Drugs for the Treatment of Hematological Malignancies

Drug discovery is costly and time-consuming, but it will become easier and simpler if a drug could be developed from an old one with well-documented investigations associated with pharmacology, pharmacokinetics, toxicology and clinical safety. In terms of hematological malignancies, several successful drugs have been discovered and developed from old ones such as arsenic trioxide for acute promyelocytic leukemia and thalidomide for multiple myeloma. In this review, we discussed the latest advancement in exploring old drugs for the treatment of hematological malignancies.

Chemokine induction by all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia: triggering the differentiation syndrome

AbstractIn acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) and/or arsenic trioxide can induce a differentiation syndrome (DS) with massive pulmonary infiltration of differentiating leukemic cells. Because chemokines are implicated in migration and extravasation of leukemic cells, chemokines might play a role in DS. ATRA stimulation of the APL cell line NB4 induced expression of multiple CC-chemokines (CCLs) and their receptors (> 19-fold), resulting in increased chemokine levels and chemotaxis. Induction of CCL2 and CCL24 was directly mediated by ligand-activated retinoic acid receptors. In primary leukemia cells derived from APL patients at diagnosis, ATRA induced chemokine production as well. Furthermore, in plasma of an APL patient with DS, we observed chemokine induction, suggesting that chemokines might be important in DS. Dexamethasone, which efficiently reduces pulmonary chemokine production, did not inhibit chemokine induction in APL cells. Finally, chemokine production was also induced by arsenic trioxide as single agent or in combination with ATRA. We propose that differentiation therapy may induce chemokine production in the lung and in APL cells, which both trigger migration of leukemic cells. Because dexamethasone does not efficiently reduce leukemic chemokine production, pulmonary infiltration of leukemic cells may induce an uncontrollable hyperinflammatory reaction in the lung.

Efficacy of arsenic trioxide for acute promyelocytic leukemia: a systematic review and meta-analysis

To systematically review the efficacy and safety of arsenic trioxide (ATO) in treatment of acute promyelocytic leukemia (APL). The Cochrane Library (Issue 1, 2009), Cochrane Central Register of Controlled Trials (from 1970 to January 2009), MEDLINE (from 1978 to October 2008), EMBASE (from 1950 to March 2009), Chinese Biological Medical Literature Database (from 1978 to December 2008), China National Knowledge Infrastructure (CNKI, from 1994 to December 2008), and China Medical Academic Conference Database (from 1994 to December 2008) were electronically searched. We also searched the Meta-Register of controlled trials, Conference Proceedings of American Society of Hematology (from 1946 to December 2008) and Conference Proceedings of American Society of Clinical Oncology (from 1946 to December 2008) on the internet for grey literature. The related journals in the library of Third Military Medical University were hand-searched. The randomized controlled trials (RCTs) of ATO in treatment of APL were included. We adopted complete remission, overall survival rate, disease free survival rate, time to complete remission, relapse rate, mortality and adverse reactions as outcome indicators. Data were entered and analyzed with the Cochrane review manager software 5.0 (RevMan 5.0). After merger of the included trials, five eligible RCTs with 328 cases were included. All the RCTs focused on the comparison of all-trans retinoic acid (ATRA) plus ATO regimen with ATRA monotherapy. Meta-analysis showed that the effect indexes for time to complete remission, two-year disease free survival rate, relapse rate, incidence of edema and incidence rate of QT interval prolongation were -1.20 [-1.68, -0.72], 8.64 [1.66,45.00], 0.21 [0.09,0.47], 4.16 [1.46,11.79] and 22.10 [2.75,177.49], respectively. The influences on other outcome indicators such as complete remission and leukocytosis were statistically non-significant. ATO can prolong disease free survival and reduce the time to complete remission and relapse rate of newly diagnosed APL patients, and increase the incidence of edema and prolongation of corrected QT interval during the treatment. Due to limitation of the included trials, this conclusion needs to be validated by further studies.

Curing APL: Differentiation or Destruction?

In a recent issue of Nature Medicine, Nasr et al. show that the effectiveness of all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia is independent of their ability to cause differentiation. Targeted destruction of the PML-RARalpha oncoprotein appears key to eliminating the cells from which relapse can arise.

Therapeutic advances in leukemia and myelodysplastic syndrome over the past 40 years.

Major therapeutic progress has been accomplished in leukemia and myelodysplastic syndrome (MDS) over the past 40 years, which may not be fully appreciated by the larger medical community. The objective of this review was to briefly highlight the treatment breakthroughs in leukemia and MDS. Therapeutic progress happened through better understanding of disease pathophysiologies and rational development of targeted agents, like imatinib mesylate in chronic myeloid leukemia (CML), and through astute, empirical discoveries in the clinic, like all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia (APL) and chlorodeoxyadenosine in hairy cell leukemia (HCL). Today, the 5- to 10-year survival rates in patients with APL and HCL exceed 80%. In patients with CML, imatinib therapy has been associated with estimated 5- to 7-year survival rates from 85% to 90%. In patients with adult acute lymphocytic leukemia, modern intensive regimens have improved the 5-year survival rates from 20% up to 40%. In patients with chronic lymphocytic leukemia, chemoimmunotherapy recently produced high rates of quality responses and improved long-term outcome. In younger patients with acute myeloid leukemia (AML), the 5-year survival rates range from 40% to 50%, although elderly AML remains a therapeutic challenge. In patients with MDS, it was recently demonstrated that epigenetic therapy with hypomethylating agents improved survival. Much therapeutic progress has been witnessed in leukemia and MDS, and much more is expected to occur soon.