Against Acute Lymphoblastic Leukemia Research Articles

Use of Microbial L-asparaginase in Cancer Treatment -A Detail Review

Abstract The exceptional anticancer effects of l-asparaginases, which hydrolyze plasma l-asparagine and l-glutamine, have drew a lot of interest in recent years. This enzyme is effective against acute lymphoblastic leukemia (ALL) and lymphosarcoma, as well as ALL in children, neoplasia, and various other cancer .The l-asparaginase anticancer application technique has achieved amazing advances in modern oncology by depleting plasma l-asparagine to suppress cancer cell development, notably in youngsters. As bacterial alternative enzyme sources for anticancer therapy, high levels of l-asparaginase enzyme production by Escherichia coli, Erwinia species, Streptomyces, and Bacillus subtilis species are very desirable. Purification of L-asparaginase by Ammonium Sulphate precipitation and various chromatographic technique methods, and improvement of enzyme purity by dialysis confirmation of molecular weight with SDS page.

CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia

BackgroundChimeric antigen receptor natural killer (CAR-NK) cells represent a promising advancement in CAR cell therapy, addressing limitations observed in CAR-T cell therapy. However, our prior study revealed challenges in CAR-NK cells targeting CD19 antigens, as they failed to eliminate CD19+ Raji cells in NSG tumor-bearing mice, noting down-regulation or loss of CD19 antigen expression in some Raji cells. In response, this study aims to enhance CD19 CAR-NK cell efficacy and mitigate the risk of tumor recurrence due to target antigen escape by developing CD19 and CD20 (CD19/CD20) dual-targeted CAR-NK cells.MethodsInitially, mRNA encoding anti-CD19 CARs (FMC63 scFv-CD8α-4-1BB-CD3ζ) and anti-CD20 CARs (LEU16 scFv-CD8α-4-1BB-CD3ζ) was constructed via in vitro transcription. Subsequently, CD19/CD20 dual-targeted CAR-NK cells were generated through simultaneous electrotransfection of CD19/CD20 CAR mRNA into umbilical cord blood-derived NK cells (UCB-NK).ResultsFollowing co-electroporation, the percentage of dual-CAR expression on NK cells was 86.4% ± 1.83%, as determined by flow cytometry. CAR expression was detectable at 8 h post-electric transfer, peaked at 24 h, and remained detectable at 96 h. CD19/CD20 dual-targeted CAR-NK cells exhibited increased specific cytotoxicity against acute lymphoblastic leukemia (ALL) cell lines (BALL-1: CD19+CD20+, REH: CD19+CD20−, Jurkat: CD19−CD20−) compared to UCB-NK, CD19 CAR-NK, and CD20 CAR-NK cells. Moreover, CD19/CD20 dual-targeted CAR-NK cells released elevated levels of perforin, IFN-γ, and IL-15. Multiple activation markers such as CD69 and cytotoxic substances were highly expressed.ConclusionsThe creation of CD19/CD20 dual-targeted CAR-NK cells addressed the risk of tumor escape due to antigen heterogeneity in ALL, offering efficient and safe 'off-the-shelf' cell products. These cells demonstrate efficacy in targeting CD20 and/or CD19 antigens in ALL, laying an experimental foundation for their application in ALL treatment.

Glutathione levels are associated with methotrexate resistance in acute lymphoblastic leukemia cell lines.

Methotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use. To better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX. MTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested. Altogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.

MicroRNAs and the Diagnosis of Childhood Acute Lymphoblastic Leukemia: Systematic Review, Meta-Analysis and Re-Analysis with Novel Small RNA-Seq Tools.

Simple SummaryMicroRNAs (miRNAs) have been under the spotlight for the last three decades. These non-coding RNAs seem to be dynamic regulators of mRNA stability and translation, in addition to interfering with transcription. Circulating miRNAs play a critical role in cell-to-cell interplay; therefore, they can serve as disease biomarkers. Meta-analysis of published data revealed that the CC genotype of rs4938723 in pri-miR-34b/c and the TT genotype of rs543412 in miR-100 confer protection against acute lymphoblastic leukemia (ALL) in children. Reanalysis of small RNA-seq data with novel tools identified significantly overexpressed members of the miR-128, miR-181, miR-130 and miR-17 families and significantly lower expression of miR-30, miR-24-2 and miR143~145 clusters, miR-574 and miR-618 in pediatric T-ALL cases compared with controls. Inconsistencies in methodology and study designs in most published material preclude reproducibility, and further cohort studies need to be conducted in order to empower novel tools, such as ALLSorts and RNAseqCNV.MicroRNAs (miRNAs) have been implicated in childhood acute lymphoblastic leukemia (ALL) pathogenesis. We performed a systematic review and meta-analysis of miRNA single-nucleotide polymorphisms (SNPs) in childhood ALL compared with healthy children, which revealed (i) that the CC genotype of rs4938723 in pri-miR-34b/c and the TT genotype of rs543412 in miR-100 confer protection against ALL occurrence in children; (ii) no significant association between rs2910164 genotypes in miR-146a and childhood ALL; and (iii) SNPs in DROSHA, miR-449b, miR-938, miR-3117 and miR-3689d-2 genes seem to be associated with susceptibility to B-ALL in childhood. A review of published literature on differential expression of miRNAs in children with ALL compared with controls revealed a significant upregulation of the miR-128 family, miR-130b, miR-155, miR-181 family, miR-210, miR-222, miR-363 and miR-708, along with significant downregulation of miR-143 and miR-148a, seem to have a definite role in childhood ALL development. MicroRNA signatures among childhood ALL subtypes, along with differential miRNA expression patterns between B-ALL and T-ALL cases, were scrutinized. With respect to T-ALL pediatric cases, we reanalyzed RNA-seq datasets with a robust and sensitive pipeline and confirmed the significant differential expression of hsa-miR-16-5p, hsa-miR-19b-3p, hsa-miR-92a-2-5p, hsa-miR-128-3p (ranked first), hsa-miR-130b-3p and -5p, hsa-miR-181a-5p, -2-3p and -3p, hsa-miR-181b-5p and -3p, hsa-miR-145-5p and hsa-miR-574-3p, as described in the literature, along with novel identified miRNAs.

Extracellular expression of Saccharomyces cerevisiae’s L-asparaginase II in Pichia pastoris results in novel enzyme with better parameters

L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae’s L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26–362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg−1. The resultant enzyme is 40% more stable than commercially available Escherichia coli’s ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi.

Genetic Association Analysis Between KIR2DS4 and Its Variant KIR1D With Pediatric Acute Lymphoblastic Leukemia

IntroductionThe killer immunoglobulin-like receptor (KIR) gene family encodes receptors of natural killer (NK) cells and some T cell subsets and is thereby involved in the regulation of anti leukemia effects. Every human being possesses an individual pattern of KIR genes, which can be assigned to either KIR haplotype A (which lacks KIR2DL2, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1) or haplotype B (which contains at least one of the activating KIRs, KIR2DL2 or KIR2DL5). KIR2DS4 is the only activating receptor with a short cytoplasmic tail, which can be found in individuals with KIR haplotype A. Interestingly, besides the cell membrane-anchored receptor, a truncated soluble protein (designed as KIR1D) is produced once the 22 bp deletion in exon 5 of KIR2DS4 is present. Previously, Giebel et al., Leukemia 2008, reported the association of KIR1D with leukemia, in particular with chronic myelogenous leukemia (CML). The aim of our study was to clarify whether the KIR1D allelic variant plays any role for innate immunosurveillance against acute lymphoblastic leukemia (ALL) and whether KIR1D subclassification is helpful for donor selection in the case of pediatric ALL before undergoing haploidentical hematopoietic cell transplantation (HHCT). Patients and Methods312 pediatric patients with ALL and 222 healthy controls were analyzed for the presence of KIR2DS4 and its variant KIR1D and then further subdivided into the following groups: 2DS4-/1D+ (homozygous for the deletion variant KIR1D), 2DS4+/1D+ (heterozygous), 2DS4+/1D- (two intact KIR2DS4 alleles) and 2DS4-/1D- (no KIR2DS4 allele). Furthermore, we investigated the influence of the KIR2DS4 variants on event-free and relapse of 69 patients with ALL receiving HHCT. The 22 bp deletion of KIR2DS4 was determined by PCR and agarose gel electrophoresis using primers described by Alves et al., Tissue Antigens 2009. ResultsOf the 312 patients with ALL, n=160 (51.3%) were identified as 2DS4-/1D+, n=101 (32.4%) as 2DS4+/1D+, n=35 (11.2%) as 2DS4+/1D- and n=16 (5.1%) as 2DS4-/1D-. The frequencies in the control group were n=136 (61.3%) for 2DS4-/1D+, n=55 (24.8%) for 2DS4+/1D+, n=25 (11.3%) for 2DS4+/1D- and n=6 (2.7%) for 2DS4-/1D-. In our study, the occurrence of 2DS4+ alleles, as well as 1D+ alleles were similar for patient and control group (p>0.050, respectively).In order to discover any possible impact of the different KIR2DS4 variants on outcome after HHCT, a retrospective analysis using cumulative incidence adjusted for competing risk (CICR, Scrucca et al., Bone marrow transplantation 2007) was performed. As previously reported by our group, patients with ALL transplanted from a KIR B haplotype donor were found to have lower relapse rates (Oevermann et al., ASH 2011). In the presented study, n=34 (49.3%) stem cell donors showed 2DS4-/1D+, n=20 (29.0%) 2DS4+/1D+, n=5 (7.2%) 2DS4+/1D-, n=10 (14.5%) 2DS4-/1D-. We found that the various KIR2DS4 variants did not influence incidence of relapse rate (2DS4-/1D+ 42.5%, 2DS4+/1D+ 55.0%, 2DS4+/1D- 20.0%, 2DS4-/1D- 30.0%, p=0.514), as well as event-free survival of ALL patients (2DS4-/1D+ 45.7%, 2DS4+/1D+ 40.0%, 2DS4+/1D- 60.0%, 2DS4-/1D- 50.0%, p=0.870). ConclusionWe conclude from our results that KIR1D seems to have no meaningful influence on immunosurveillance against ALL. In contrast to KIR haplotype B donor selection, the preference of a KIR1D positive donor has no additional beneficial impact on the outcome of patients with ALL receiving HHCT. Disclosures:No relevant conflicts of interest to declare.

The XPO1 Inhibitor KPT-8602 Synergizes with Dexamethasone in Acute Lymphoblastic Leukemia.

KPT-8602 (Eltanexor) is a second-generation exportin-1 (XPO1) inhibitor with potent activity against acute lymphoblastic leukemia (ALL) in preclinical models and with minimal effects on normal cells. In this study, we evaluated whether KPT-8602 would synergize with dexamethasone, vincristine, or doxorubicin, three drugs currently used for the treatment of ALL. First, we searched for the most synergistic combination of KPT-8602 with dexamethasone, vincristine, or doxorubicin in vitro in both B-ALL and T-ALL cell lines using proliferation and apoptosis as a readout. Next, we validated this synergistic effect by treatment of clinically relevant B- and T-ALL patient-derived xenograft models in vivo. Finally, we performed RNA-sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to determine the mechanism of synergy. KPT-8602 showed strong synergism with dexamethasone on human B-ALL and T-ALL cell lines as well as in vivo in three patient-derived ALL xenografts. Compared with single-drug treatment, the drug combination caused increased apoptosis and led to histone depletion. Mechanistically, integration of ChIP-seq and RNA-seq data revealed that addition of KPT-8602 to dexamethasone enhanced the activity of the glucocorticoid receptor (NR3C1) and led to increased inhibition of E2F-mediated transcription. We observed strong inhibition of E2F target genes related to cell cycle, DNA replication, and transcriptional regulation. Our preclinical study demonstrates that KPT-8602 enhances the effects of dexamethasone to inhibit B-ALL and T-ALL cells via NR3C1- and E2F-mediated transcriptional complexes, allowing to achieve increased dexamethasone effects for patients.

Abstract 4181: The XPO1 inhibitior, eltanexor, exhibits potent in vivo activity against a broad range of pediatric acute lymphoblastic leukemia subtypes

Abstract Introduction: Exportin 1 (XPO1/CRM1) plays a central role in the export of proteins from the nucleus including those with tumor suppressor and growth regulatory activity. Eltanexor (KPT-8602) is a second-generation Selective Inhibitor of Nuclear Export (SINE) that specifically blocks XPO1 cargo interactions and is under clinical evaluation for adults with cancer (NCT02649790). In previous Pediatric Preclinical Testing Consortium (PPTC) studies, eltanexor showed potent in vivo activity against acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs). In an effort to understand whether eltanexor exhibited subtype-specific activity in pediatric ALL we carried out a single-mouse trial (SMT) against an intended 90 pediatric ALL PDXs. Methods: Pediatric ALL PDXs, an orthotopic model of the disease, were inoculated into NSG mice in an SMT format (a single mouse inoculated with a single PDX was treated with a single drug). Engraftment and drug responses were assessed by weekly enumeration of the % human leukemic blasts in the peripheral blood (%huCD45+/HLA-ABC+). Treatment commenced when the median %huCD45+/HLA-ABC+ exceeded 1%, and mice received eltanexor at 12.5 mg/kg per oral daily x 5 for 4 weeks. The baseline level of the %huCD45+/HLA-ABC+ in each mouse served as its own control. PDX responses to treatment were assessed by time to event, the maximum decrease in %huCD45+/ABC+ at any point after treatment initiation, and by stringent Objective Response Measures (ORMs) modeled after the clinical setting (Houghton et al, Pediatr Blood Cancer, 2007, 49:928-40). Results: Eltanexor was well tolerated with a mean maximum weight loss of 5.5% across all mice. SMT testing was successful in all 90 pediatric ALL PDXs representative of B-ALL (typical B-ALL, Philadelphia chromosome-positive ALL [Ph+-ALL] and Ph-like ALL), T-ALL (typical T-ALL and early T-cell precursor ALL [ETP-ALL]), and MLL-rearranged ALL (MLLr-ALL). Event-free survival (EFS) of mice ranged from 2.6-137 days, and 61 (68%) of mice experienced EFS that extended beyond the treatment window. Regressions of ≥50% from baseline were observed in 56 (62%) of mice, and these were most pronounced in T-ALL (14/18, 78%) and Ph-like ALL (11/19, 58%). ORMs classified as Complete Response (CR) or Maintained CR (MCR) were elicited in 43 (48%) of PDXs, and again these were more common in T-ALL (12/18, 67%). The ALL subtypes that exhibited the poorest responses to eltanexor were Ph+-ALL and ETP-ALL, although with relatively small sample size (n=3 and 6, respectively). ORMs in the SMT correlated highly with historical conventional drug testing (R=0.94, P<0.001, n=12). Conclusions: Eltanexor exhibits potent single-agent in vivo activity against PDXs derived from a broad range of ALL subtypes including T-ALL and Ph-like ALL and warrants further investigation in pediatric ALL. (Supported by NCI Grants CA199222 & CA199000) Citation Format: Richard B. Lock, Kathryn Evans, Connor D. Jones, Stephen W. Erickson, Beverly A. Teicher, TJ Unger, Yosef Landesman, Malcolm A. Smith. The XPO1 inhibitior, eltanexor, exhibits potent in vivo activity against a broad range of pediatric acute lymphoblastic leukemia subtypes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4181.

Direct Delivery of piggyBac CD19 CAR T Cells Has Potent Anti-tumor Activity against ALL Cells in CNS in a Xenograft Mouse Model

The anti-CD19 chimeric antigen receptor (CAR) T cells showed excellent effect against acute lymphoblastic leukemia (ALL) in bone marrow (BM) in clinical trials. However, it remains to be elucidated whether the CD19 CAR T cell therapy is effective for ALL cells in central nervous system (CNS) because the patients with isolated or advanced CNS disease were excluded from clinical trials of systemic intravenous (i.v.) delivery of CAR T cells. Therefore, the preclinical evaluation for the efficacy of CAR T cell therapy against ALL cells in CNS is essential for clinical application. We evaluated the effect and adverse reaction of CD19 CAR T cells against ALL in CNS using a xenograft mouse model by i.v. or intra-cerebroventricular (i.c.v.) delivery of CAR T cells. Injection of piggyBac CD19 CAR T cells by i.v. had partial effects, whereas all CAR T i.c.v.-delivered mice had eliminated ALL in CNS. Although some CAR T i.c.v.-delivered mice showed transient changes of clinical symptoms during the first few days after treatment, none of CAR T i.c.v.-delivered mice displayed fatal adverse events. In this study, we demonstrated that direct delivery into CNS of CAR T cells is a possible therapeutic approach with the xenograft mouse model.

Ergolide, a potent sesquiterpene lactone induces cell cycle arrest along with ROS-dependent apoptosis and potentiates vincristine cytotoxicity in ALL cell lines

Ethnopharmacological relevanceInula oculus christi belongs to the family of Asteraceae and it was traditionally wide used in treatment of kidney stones and urethra infection; besides, recently the potent sesquiterpene lactones isolated from inula species has gained increasing attention in cancer treatments. This study investigates the anti-cancer properties and underlying mechanism of ergolide isolated from Inula oculus christi against leukemic cell lines. MethodsViability, metabolic activity and proliferation evaluated using different index of MTT assay such as IC50 and GI50. Human erythrocytes were used to evaluate hemolytic activity. Flow-cytometry was used to detect and measure ROS level, and the induction of apoptosis and autophagy were evaluated using Annexin V/PI, Acridine Orange staining, respectively. Moreover, qRT-PCR was performed to examine the expression of a large cohort of crucial regulatory genes. Tunel assay was also carried out to assess morphologically ergolide effects. ResultsErgolide did not exert ant cytotoxicity against non-tumorous cells and did not cause noticeable hemolysis. It also caused ROS production during early hours after treatment of cells which was then followed by cell cycle arrest in G0/G1 phase and autophagy induction. Using N-acetyl-L-cysteine (NAC), we found that ergolide could not increase ROS and induce autophagy and moreover repressed cell death, indicating that ergolide induce cell death through ROS-dependent manner by altering the expression of pro apoptotic related genes. Autophagy inhibition also potentiated ergolide-induced cell death. Furthermore, ergolide intensified vincristine cytotoxicity against acute lymphoblastic leukemia (ALL) cell lines revealed robust synergistic properties of ergolide with VCR. ConclusionHere we showed that ergolide could be considered as a potent natural compound against leukemic cells by inducing cell cycle arrest followed by dose-dependent cell death. Based on results, Autophagy response in a result of ROS accumulation acted as a survival pathway and blocking this pathway could noticeably increase ergolide cytotoxicity on ALL cell lines.

A Phase I/Pilot Study of CPX-351 [Daunorubicin and Cytarabine Liposome for Injection (Vyxeos®)] for Children, Adolescents and Young Adults with Recurrent or Refractory Acute Leukemia

▪Background: CPX-351 (Vyxeos®) is a liposomal nanoparticle of cytarabine and daunorubicin in a 5:1 molar ratio currently FDA approved for treatment of adults with newly-diagnosed therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes. The agent also has potent activity against acute lymphoblastic leukemia (ALL) in murine xenograft models [H. Carol et al. Pediatr Blood Cancer 2015;62: 65-71]. We conducted the first phase I/pilot study to evaluate the safety and describe the efficacy of CPX-351 in children and adolescents with relapsed or refractory (R/R) acute leukemia (NCT01943682).Methods: In the Dose Exploration Phase participants age 12 months to 21 years were eligible to receive a single course of CPX-351 administered once daily on Days 1, 3, and 5. Enrollment was initiated at the recommended dose for study in adults (Dose Level 1: 100 Units/m2/dose= cytarabine 100 mg/m2 and daunorubicin 44 mg/m2 per dose) with provisions to explore either a single dose escalation to 134 Units/m2/dose (Dose Level 2) or de-escalation to 67 Units/m2/dose. Response was determined after Day 28. The upper age for eligibility was increased to 30 years and an additional 18 subjects were enrolled into the Expanded Phase of the study.Results: Nine children and adolescents with R/R AML (n=6) or ALL (n=3) were enrolled and evaluable for toxicity in the Dose Exploration Phase. All 9 experienced Grade 3 or 4 hematologic toxicity including 8 with fever with neutropenia or infection. Dose Limiting Toxicity (DLT) was not observed in the 4 patients enrolled at Level 1 but Grade 3 pain/headache lasting greater than 48 hours did occur in 2 of the 5 patients treated at Dose Level 2. One patient with AML was unevaluable for response in this phase due to removal from protocol therapy to receive bone marrow transplantation while in aplasia. All others were evaluable for response. One patient with ALL had stable disease, 2 with AML achieved a complete remission (CR), and 1 with AML achieved a CR in blood and marrow but developed low level disease in the cerebral spinal fluid (CSF).One patient with mixed phenotype acute leukemia and 17 with R/R AML were subsequently enrolled into the Expanded Phase of the study to receive CPX-351 at Dose Level 1. CPX-351 treatment was well tolerated in this population with a similar toxicity profile observed in the Dose Exploration Phase. Dose limiting prolonged aplasia occurred in one patient but no other DLTs were observed. The response has yet to be determined in one patient. To date, seven patients with AML (41%) achieved a CR or complete remission with incomplete blood recovery (CRi). Another patient achieved a CR in marrow but with low level disease detected in the CSF similar to the patient described in the Dose Exploration phase. The remaining 9 patients failed to achieve a clinical response. Pharmacokinetic (PK) parameters of total cytarabine and daunorubicin were analyzed for 21 of these pediatric patients and the results were similar to those observed in adults. However, due to the small sample size of each age group, these interim PK results will need to be validated as data in pediatric patients become available from future clinical studies.Conclusions: CPX-351 100 Units/m2/dose on Days 1, 3, and 5 was well tolerated in this pediatric population and has promising activity in children and young adults with R/R AML. Additional CNS directed therapy is recommended for pediatric patients at high risk for failure in the CNS. DisclosuresPhillips:Novartis AG: Consultancy. An:Jazz Pharmaceuticals: Employment.

Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain.

Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5–10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.

L-Tetrahydropalmatine Induces Apoptosis in EU-4 Leukemia Cells by Down-Regulating X-Linked Inhibitor of Apoptosis Protein and Increases the Sensitivity Towards Doxorubicin.

L-Tetrahydropalmatine (L-THP) is a tetra-hydro protoberberine isoquinoline alkaloid. The phyto-compounds bearing isoquinoline alkaloids have been reported to show a potential effect against a number of human cancers cell lines including leukemia. We hypothesized that L-THP, being an isoquinoline alkaloid, could be a potential molecule against acute lymphoblastic leukemia (ALL), in this study, we evaluate L-THP against p53 deficient leukemia EU-4 cell lines in vitro. For the study, p53 null leukemia EU-4 cells were used and treated with LTHP. The extent of apoptosis and viability of cells were determined. Expression of apoptosis related proteins such as XIAP and MDM2 was done by western blot and PCR studies. The expression of MDM2 and XIAP was knocked down by small interfering RNA (siRNA). Outcomes of the study suggested that L-THP caused p53-indipendent apoptosis mediated by XIAP in EU-4 cells. The treatment of L-THP caused a decrease in the levels of XIAP protein with increasing dose and time. L-THP caused down-regulation of XIAP protein via inhibiting the expression of MDM2 and involving proteasomedependent pathway. Also, the outcomes of experiments suggested increased sensitivity of leukemia cells towards doxorubicin due to the inhibition of XIAP by L-THP or by siRNA. Findings of the study confirm that L-THP resulted in p53 independent apoptosis via down-regulating XIAP protein by inhibiting MDM2 associated with proteasome-dependent pathway and increased sensitivity of EU-4 cells against doxorubicin. L-THP caused activation of caspase and resulted in apoptosis, L-THP may be a novel molecule for inducing apoptosis specifically in p53 null leukemia EU-4 cells.

Abstract 1375: Loss of C-MYC and chromatin acetylation induce epigenetic reprogramming in acute lymphoblastic leukemia

Abstract Epigenetic modifications play a key role in establishing and maintaining gene expression. In cancer, they are highly altered and responsable of gene expression deregulation. Epigenetic drugs have the ability to reset cancer cell epigenome producing cancer cell differentiation and apoptosis. In a drug screening initiative, we recently reported a series of FDA-approved drugs with unsuspected epigenetic and anticancer activities. Here, we tested in a secondary screen the activity of these drugs against acute lymphoblastic leukemia (ALL) cell lines (MOLT-4 and NALM-6). We found that Proscillaridin A, a cardiac glycoside used for heart failure treatment, was the most active with IC50 values in the low nanomolar range, suggesting drug repositioning potential. Proscillaridin treatments induced a significant decrease in RNA and protein levels of C-MYC, a master oncogenic driver in ALL. Shortly after proscillaridin A treatment, C-MYC exhibited a 75% reduction in lysine acetylation, a post-translational modification known to prevent its degradation. Loss of acetylation was associated with down-regulation of lysine acetyltransferases CBP, P300 and Tip60, which also correlated with a reduction in histone 3 and 4 acetylation levels (H3K14ac, H3K9ac, H3K27ac and H4K5ac). Preliminary analysis with si-RNA experiments reveal that independent HAT activities are not responsible of C-MYC downregulation. RNA sequencing and gene set enrichment analysis in proscillaridin-treated ALL cells (5 nM for 48h) showed that genes associated with cell differentiation and apoptosis pathways were up-regulated whereas down-regulated genes were associated with C-MYC target genes. Altogether, our findings show that acetylation through lysine acetyltransferase down-regulation simultaneously induces loss of C-MYC and H3 acetylation leading to epigenetic reprogramming in ALL cells. This drug repositioning strategy, using proscillaridin A, has the potential to reprogram cancer cells that are driven by MYC overexpression or hyperactivation. Citation Format: Elodie M. Da Costa, Gregory Armaos, Simon Jacques-Ricard, Annie Beaudry, Pascal St-Onge, Maxime Caron, Daniel Sinnett, Serge McGraw, Noël J. Raynal. Loss of C-MYC and chromatin acetylation induce epigenetic reprogramming in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1375. doi:10.1158/1538-7445.AM2017-1375

The Second-Generation Exportin-1 Inhibitor KPT-8602 Demonstrates Potent Activity against Acute Lymphoblastic Leukemia.

Purpose: Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports different cargo proteins out of the nucleus. Inducing nuclear accumulation of these proteins by inhibiting XPO1 causes cancer cell death. First clinical validation of pharmacological inhibition of XPO1 was obtained with the Selective Inhibitor of Nuclear Export (SINE) compound selinexor (KPT-330) demonstrating activity in phase-II/IIb clinical trials when dosed 1 to 3 times weekly. The second-generation SINE compound KPT-8602 shows improved tolerability and can be dosed daily. Here, we investigate and validate the drug-target interaction of KPT-8602 and explore its activity against acute lymphoblastic leukemia (ALL).Experimental Design: We examined the effect of KPT-8602 on XPO1 function and XPO1-cargo as well as on a panel of leukemia cell lines. Mutant XPO1 leukemia cells were designed to validate KPT-8602's drug-target interaction. In vivo, anti-ALL activity was measured in a mouse ALL model and patient-derived ALL xenograft models.Results: KPT-8602 induced caspase-dependent apoptosis in a panel of leukemic cell lines in vitro Using CRISPR/Cas9 genome editing, we demonstrated the specificity of KPT-8602 for cysteine 528 in the cargo-binding groove of XPO1 and validated the drug target interaction. In vivo, KPT-8602 showed potent anti-leukemia activity in a mouse ALL model as well as in patient-derived T- and B-ALL xenograft models without affecting normal hematopoiesis.Conclusions: KPT-8602 is highly specific for XPO1 inhibition and demonstrates potent anti-leukemic activity supporting clinical application of the second-generation SINE compound for the treatment of ALL. Clin Cancer Res; 23(10); 2528-41. ©2016 AACR.

IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning

Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed.The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5.We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system.To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively)Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006).Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p<0.0001). 19m28mz-P2A-mIL18 CAR T cells were detected in peripheral blood for up to 30 days after injection, whereas the 19m28mz CAR T cells were not detectable at any time point. In addition, 19m28mz-P2A-mIL18 CAR T cells were capable of inducing B cell aplasia for greater than 70 days, whereas 19m28mz treatment was not capable of inducing B cell aplasia. In vivo serum cytokine analysis demonstrated that 19m28mz-P2A-mIL18 CAR T cells, as compared to 19m28mz, significantly increased the levels of IFN-γ and TNF-α in the peripheral blood for up to 14 days after injection (p<0.0001 and 0.01, respectively). Despite the increase in IFN-γ and TNF-α cytokines, there was no increase in IL-6 levels.Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. DisclosuresBrentjens:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Presence of Endogenous TCR Antigen in Vivo Attenuates Efficacy of Anti-CD19 Targeted CAR T Cell Therapy

Adoptive therapy using T cells genetically engineered to express chimeric antigen receptors (CAR) has proven extremely effective against acute lymphoblastic leukemia (ALL) in clinical trials with the use of anti-CD19 CAR T cells. Most CAR T cell protocols use autologous T cells, which are then activated, transduced with the anti-CD19 CAR, and expanded ex-vivo before infusion back into the patient. This approach minimizes the risk of graft-versus-host disease (GVHD) even in allogeneic transplant recipients, due to tolerization of the donor T cell repertoire in the recipient. However, many patients have heavy disease burden and lymphopenia due to previous treatments, which makes the isolation of healthy T cells difficult. Thus, centers are exploring the potential of allogeneic T cell donors and the possibility of universal T cell donors for CAR-based therapy including the use of virus-specific T cells. In these cases, in addition to the chimeric receptor specificity, the transduced T cell population will also have reactivity against target antigens through the endogenous TCR. However, little is known about the impact of signaling of the endogenous TCR on CAR T cell activity, particularly in vivo. To test this, we used a syngeneic transplantable ALL murine model, E2aPBx, in which CD19 CAR T cells can effectively eradicate ALL. CD4 (Marilyn) and CD8 (Matahari) T cells from syngeneic HY-TCR transgenic donors specific for the minor histocompatibility male antigen, HY, were used as CAR T cell donors to control for endogenous TCR reactivity. Splenic T cells isolated from Matahari, Marilyn, or B6 mice were activated ex-vivo using anti-CD3/anti-CD28 beads, with the addition of IL2 and IL7. T cells were transduced with a retroviral vector expressing a murine CAR composed of anti-CD19 scfv/CD28/CD3ζ on days two and three. CAR T cells are evaluated in vitro by CD107a degranulation assay and INF gamma ELISA. In response to HY peptide alone or HY+CD19- line M39M, transduced CD8 HY (Matahari) cells produced IFN gamma and expressed CD107a whereas transduced CD4 HY (Marilyn) cells only produced IFN gamma. Interestingly, in response to CD19+HY- ALL, both Matahari and Marilyn expressed CD107a and produced IFN gamma indicating that CD4 T cells can acquire CD8-like lytic activity when stimulated through a CAR receptor. When CD19 CAR transduced Marilyns and Mataharis were stimulated in the presence of HY and CD19, CD8 Mataharis had an attenuated effect against CD19, suggesting that the presence of antigen activated TCR adversely affects the potency of the CAR receptor. Efficacy of the HY and polyclonal CAR T cells were next tested in-vivo in male and female B6 mice. Mice were given 1E6 E2aPBx ALL leukemia cells on day 1, and received 500 rads sub-lethal total body irradiation on day 4 as a lymphodepleting regimen. On day 5, mice were given a low (1E5) or high (5E6) dose of CAR T cells. There was a statistically significant (p=0.0177) improvement in the survival of female versus male mice after treatment with the CD4+ HY specific anti-CD19 CAR T cells, and female mice that received HY anti-CD19 CAR T cells survived longer than untreated control females (p=0.01). Remarkably, the survival of male mice that received HY anti-CD19 CAR T cells was statistically worse than untreated control males (p=0.008). This suggests that the presence of TCR antigen negatively impacts the function of CAR T cells. Furthermore, in a separate experiment using an equally mixed population of Marilyn (CD4+) and Matahari (CD8+) HY specific T cells, males has a statistically significantly (p=0.0116) worse survival compared to females after receiving 5E5 HY specific T cells. In conclusion, simultaneous stimulation through both CAR and TCR results in attenuated cytokine production and degranulation by CD8 T cells. In vivo, in the presence of the endogenous TCR antigen, both CD4 and CD8 CAR T cells are less potent at eradicating leukemia. These have implications for the development of universal donors for CAR T cell therapy. DisclosuresNo relevant conflicts of interest to declare.

Efficacy of CPX-351, (cytarabine:daunorubicin) liposome injection, against acute lymphoblastic leukemia (ALL) xenograft models of the Pediatric Preclinical Testing Program.

CPX-351, a liposomal formulation of cytarabine and daunorubicin co-encapsulated at an optimized synergistic 5:1 molar ratio, has demonstrated improved clinical outcomes over conventional cytarabine/daunorubicin treatment in a randomized phase 2 trial in patients with AML as well as superior efficacy against preclinical leukemia models when compared to the free drugs in combination. Given the promising phase 2 data, limited toxicities observed, and the known clinical activities of cytarabine/daunorubicin, we assessed the efficacy of CPX-351 against a panel of childhood ALL xenograft models. Plasma pharmacokinetics of cytarabine and daunorubicin following CPX-351 treatment were determined by HPLC in order to correlate efficacy with drug exposure. CPX-351, at a dose of 5 units/kg (corresponding to 5 mg/kg cytarabine and 2.2 mg/kg daunorubicin), was highly efficacious against all xenografts tested, inducing complete responses in four B-lineage xenografts and partial response in one T-lineage xenograft. These therapeutic responses were achieved with CPX-351 doses that provided drug exposures (based on Cmax and AUC) comparable to those observed in patients with AML. These results suggest that CPX-351 may be a promising chemotherapeutic to be utilized in the treatment of ALL and support its testing in pediatric patients with leukemia.

Caspase-dependent Mcl-1 cleavage and effect of Mcl-1 phosphorylation in ABT-737-induced apoptosis in human acute lymphoblastic leukemia cell lines.

ABT-737 is a BH3-mimetic that has a wide spectrum of single-agent activity against acute lymphoblastic leukemia (ALL) cell lines and xenografts. Previously, we reported that in response to ABT-737, ABT-737-resistant ALL cell lines showed an apparent increase in Mcl-1 (an anti-apoptotic Bcl-2 family protein that is not effectively inhibited by ABT-737) while ABT-737-sensitive ALL cell lines showed decreased Mcl-1 levels. Here we explored the mechanism of Mcl-1 cleavage by ABT-737 and the effect of adjacent phosphorylation sites on Mcl-1 cleavage and apoptosis induced by ABT-737 in a human B-lineage ALL cell line. Caspase cleavage sites in Mcl-1 and the effect of mutation in Mcl-1 phosphorylation sites were determined by transducing Mcl-1 variants tagged with the V5 epitope into human ALL cells. Cytotoxicity was by fluorescence-based DIMSCAN, and changes in protein by immunoblotting. ABT-737 induced a caspase-dependent cleavage of Mcl-1. Of the two Mcl-1 caspase cleavage sites (D127 and D157), D157 was the site of ABT-737-induced cleavage in ALL cells. Cells with exogenously expressed Mcl-1 Δ157 fragment showed greater caspase-3 and caspase-9 activation when they were treated with ABT-737 compared with cells expressing wild-type or D157A mutant Mcl-1. Cells with mutated phosphorylation sites on Mcl-1 (S159A and T163A) were less susceptible to Mcl-1 cleavage and apoptosis induced by ABT-737. Our data showed that Mcl-1 is post-translationally regulated in response to ABT-737 treatment, primarily via a caspase-dependent cleavage that generates a pro-apoptotic Mcl-1 fragment.

The addition of sirolimus to tacrolimus/methotrexate GVHD prophylaxis in children with ALL: a phase 3 Children's Oncology Group/Pediatric Blood and Marrow Transplant Consortium trial

AbstractSirolimus has activity against acute lymphoblastic leukemia (ALL) in xenograft models and efficacy in preventing acute graft-versus-host disease (aGVHD). We tested whether addition of sirolimus to GVHD prophylaxis of children with ALL would decrease aGVHD and relapse. Patients were randomized to tacrolimus/methotrexate (standard) or tacrolimus/methotrexate/sirolimus (experimental). The study met futility rules for survival after enrolling 146 of 259 patients. Rate of Grade 2-4 aGVHD was 31% vs 18% (standard vs experimental, P = .04), however, grade 3-4 aGVHD was not different (13% vs 10%, P = .28). Rates of veno-occlusive disease (VOD) and thrombotic microangiopathy (TMA) were lower in the nonsirolimus arm (9% vs 21% VOD, P = .05; 1% vs 10% TMA, P = .06). At 2 years, event free survival (EFS) and overall survival (OS) were 56% vs 46%, and 65% vs 55% (standard vs experimental), respectively (P = .28 and .23). Multivariate analysis showed increased relapse risk in children with ≥0.1% minimal residual disease (MRD) pretransplant, and decreased risk in patients with grades 1-3 aGVHD (P = .04). Grades 1-3 aGVHD were associated with improved EFS (P = .02), whereas grade 4 aGVHD and extramedullary disease at diagnosis led to inferior OS. Although addition of sirolimus decreased aGVHD, survival was not improved. This study is registered with ClinicalTrials.gov as #NCT00382109.