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

BACKGROUND: Chimeric 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. METHODS: Initially, 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). RESULTS: Following 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. CONCLUSIONS: The 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.

miR-539-5p targets BMP2 to regulate Treg activation in B-cell acute lymphoblastic leukemia through TGF-ß/Smads/MAPK.

MicroRNAs (mRNAs) were believed to play an important role in cancers, and this study aimed to explore the mechanism of miRNA regulating Treg in B-cell acute lymphoblastic leukemia (B-ALL). Firstly, the differentially expressed miRNAs and target genes significantly associated with Tregs were screened out by high-throughput sequencing, and their enrichment pathways were analyzed. The binding relationship between miRNA and target genes was further verified, and the effects of miRNA on the proliferation and apoptosis of B-ALL Nalm-6 cells and Treg activation were analyzed. Results showed that differentially expressed miR-539-5p was significantly under-expressed, and its target gene BMP2 was significantly over-expressed in B-ALL, and significantly enriched in the TGF-ß1 pathway. In addition, both miR-539-5p and BMP2 were significantly correlated with Treg activity in B-ALL. In vitro experiments further confirmed that miR-539-5p could directly target BMP2. The low expression of miR-539-5p in B-ALL significantly promoted BMP2 expression to promote the proliferation and inhibit apoptosis of Nalm-6 cells. Furthermore, the high expression of BMP2 in B-ALL could cooperate with TGF-ß1 to promote the activation of human CD4+CD25-T cells to Treg, and significantly activate the TGF-ß/Smads/MAPK pathway. In vivo experiments also confirmed that overexpression of miR-539-5p significantly inhibited BMP2 to suppress Treg activation and Smad1 and Smad2 phosphorylation, and finally inhibit the B-ALL process. In conclusion, miR-539-5p was significantly under-expressed in B-ALL and could target BMP2 to promote its expression, and the overexpressed BMP2 further promoted Treg activation in B-ALL by regulating TGF-ß/Smads/MAPK pathway.

Dihydroartemisinin Enhances the Therapeutic Efficacy of BH3 Mimetic Inhibitor in Acute Lymphoblastic Leukemia Cells via Inhibition of Mcl-1.

INTRODUCTION: Up-regulation of the anti-apoptotic proteins such as Mcl-1 is associated with the primary and secondary resistance of tumor cells to ABT-737 Bcl-2 inhibitor. The combined treatment of Bcl-2 inhibitors with Mcl-1 inhibitors has been proposed as an attractive therapeutic strategy to overcome this drug resistance. Here, we investigated the effect of dihydroartemisinin on Mcl-1 expression and sensitization of T-ALL cells to ABT-737. METHODS: The cell growth and survival were tested by the cell proliferation and MTT assays, respectively. The mRNA levels of Bcl-2, Mcl-1, Bax and P21 were examined by qRT-PCR. Apoptosis were detected by Hoechst 33342 staining and caspase-3 activity assay. RESULTS: Our data showed that combination treatment with dihydroartemisinin and ABT-737 caused a significant decrease in the IC50 value and synergistically reduced the cell survival compared with dihydroartemisinin or ABT-737 alone. ABT-737 enhanced the Mcl-1 mRNA expression. Dihydroartemisinin also down-regulated the expression of Bcl-2 and Mcl-1 and enhanced the P21 and Bax expression. Moreover, dihydroartemisinin enhanced the apoptosis induced by ABT-737 in MOLT-4 and MOLT-17 cell lines. CONCLUSION: In conclusion, dihydroartemisinin demonstrates anti-tumor activities in human ALL cells via inhibition of cell survival and growth. Dihydroartemisinin augments the apoptotic effect of ABT-737 by inhibiting the expression of Mcl-1.

Synergistic combination of isogarcinol isolated from edible fruits of Garcinia multiflora and dexamethasone to overcome leukemia glucocorticoid resistance.

Isogarcinol (ISO), a cytotoxic polycyclic polyprenylated acylphloroglucinol isolated from the edible fruits of Garcinia multiflora. However, synergistic combination of ISO and dexamethasone (DEX) to overcome leukemia glucocorticoid resistance has never been investigated. Therefore, in this study, the effects of ISO in combination with DEX was conducted on leukemia in vivo and glucocorticoid resistance in vitro. As a result, the combination of the two compounds could efficiently inhibit leukemia progression in mice and reverse DEX resistance in acute lymphoblastic leukemia (ALL) Jurkat cells. Significantly, our findings indicated that c-Myc may be a potential target of ISO, as it is involved in cell cycle arrest and apoptosis by the combination of ISO and DEX in Jurkat cells. Furthermore, western blot analysis revealed that ISO and DEX inhibits the PI3K/Akt/mTOR signaling pathway and promotes the nuclear translocation of glucocorticoid receptor (GR), which activates target genes NR3C1 and TSC22D3, leading to apoptosis in Jurkat cells. Hence, our results suggest that ISO, as a safe and effective food-derived agent, can enhance the anti-leukemia effects of DEX.

A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

Towards development of biobetter: L-asparaginase a case study.

BACKGROUND: L-asparaginase (ASNase) has played a key role in the management of acute lymphoblastic leukaemia (ALL). As an amidohydrolase, it catalyzes the hydrolysis of L-asparagine, a crucial step in the treatment of ALL. Various ASNase variants have evolved from diverse sources since it was first used in paediatric patients in the 1960s. This review describes the available ASNase and approaches being used to develop ASNase as a biobetter candidate. SCOPE OF REVIEW: The review discusses the Glycosylation and PEGylation techniques, which are frequently used to develop biobetter versions of the majority of the therapeutic proteins. Further, it explores current ASNase biobetters in therapeutic use and discusses the protein engineering and chemical modification approaches that were employed to reduce immunogenicity, extend protein half-life, and enhance protease stability of ASNase. Emerging strategies like immobilization and encapsulation are also highlighted as potential pathways for improving ASNase properties. MAJOR CONCLUSIONS: The purpose of the development of ASNase biobetter is to achieve a novel therapeutic candidate that could improve catalytic efficiency, in vivo stability with minimum glutaminase (GLNase) activity and toxicity. Modification of ASNase by immobilization and encapsulation or by fusion technologies like Albumin fusion, Fc fusion, ELP fusion, XTEN fusion, etc. can be exploited to develop a novel biobetter candidate suitable for therapeutic approaches. GENERAL SIGNIFICANCE: This review emphasizes the importance of biobetter development for therapeutic proteins like ASNase. Improved ASNase molecules have the potential to significantly advance the treatment of ALL and have broader implications in the pharmaceutical industry.

Polyoxazoline-Conjugated l-Asparaginase: An Antibody-Production-Free Therapeutic Agent for Acute Lymphoblastic Leukemia.

l-asparaginase (ASNase), an enzyme that catalyzes the hydrolysis of l-asparagine into l-aspartic acid, is frequently used as a medication for acute lymphoblastic leukemia (ALL). However, when derived from bacterial sources, this enzyme can elicit side effects, including allergic or hypersensitivity reactions, owing to immune responses. Here, we describe the synthesis of polyoxazoline-conjugated ASNase (POx-ASNase) and investigate its enzyme activity, anticancer efficacy, immunogenicity, and retention in the bloodstream. The water-soluble POx was coupled with surface lysine residues of ASNase using a bifunctional cross-linker. The average number of polymers bound to each enzyme was determined as 10. Although the enzymatic activity of POx-ASNase decreased to 56% of that of native ASNase, its temperature and pH dependencies remained unaltered. Remarkably, the lyophilized powder form of POx-ASNase retained its catalytic ability for 24 months. POx-ASNase demonstrated nearly identical anticancer efficacy compared to naked ASNase against leukemia and lymphoma cells (MOLT-4, CLBL-1, and K562) while displaying no cytotoxicity toward normal cells. Animal experiments conducted using rats revealed that the POx decoration suppressed the generation of anti-ASNase IgM and IgG antibodies with no detection of anti-POx antibodies. The half-life within the bloodstream extended to 34 h, representing a 17-fold increase compared to unmodified ASNase. These findings suggest that POx-ASNase serves as an anticancer therapeutic agent, characterized by the absence of antibody production and notably extended circulation persistence.

Blinatumomab differentially modulates peripheral blood and bone marrow immune cell repertoire: A Campus ALL study.

Blinatumomab is the first bi-specific T-cell engager approved for relapsed or refractory B-cell precursor acute lymphoblastic leukaemia (B-ALL). Despite remarkable clinical results, the effects of blinatumomab on the host immune cell repertoire are not fully elucidated. In the present study, we characterized the peripheral blood (PB) and, for the first time, the bone marrow (BM) immune cell repertoire upon blinatumomab treatment. Twenty-nine patients with B-ALL received blinatumomab according to clinical practice. Deep multiparametric flow cytometry was used to characterize lymphoid subsets during the first treatment cycle. Blinatumomab induced a transient redistribution of PB effector T-cell subsets and Treg cells with a persistent increase in cytotoxic NK cells, which was associated with a transient upregulation of immune checkpoint receptors on PB CD4 and CD8 T-cell subpopulations and of CD39 expression on suppressive Treg cells. Of note, BM immune T-cell subsets showed a broader post-treatment subversion, including the modulation of markers associated with a T-cell-exhausted phenotype. In conclusion, our study indicates that blinatumomab differentially modulates the PB and BM immune cell repertoire, which may have relevant clinical implications in the therapeutic setting.

Chimeric Antigen Receptor T Cells: Immunotherapy for the Treatment of Leukemia, Lymphoma, and Myeloma.

In immunotherapy with T cells genetically modified to express chimeric antigen receptors (CAR), autologous lymphocytes are extracted from the patient, genetically modified to obtain CAR-T cells, and reintroduced into the patient to attack cancer cells. The success of this therapy has been achieved in the area of CD19-positive leukemias and lymphomas, being approved for the treatment of non-Hodgkin's lymphomas, acute lymphoblastic leukemia, and multiple myeloma. CARs are proteins that combine antibody specificity with T-cell cytotoxicity. The most common toxicities associated with therapy were not predicted by preclinical testing and include cytokine release syndrome, neurotoxicity, and cytopenias. These toxicities are usually reversible. One of the main challenges facing the field is the high economic cost that therapy entails, so the search for ways to reduce this cost must be a priority. In addition, other challenges to overcome include the situation that not all patients are supplied with the product and the existence of long waiting times for the start of therapy. The aim of this review is to present the development of the structure of CAR-T cells, the therapies approved to date, the toxicity associated with them, and the advantages and limitations that they present as immunotherapy.

Substrate oxidation during exercise in childhood acute lymphoblastic leukemia survivors.

Children with acute lymphoblastic leukemia (ALL) are at high risk of developing long-term cardiometabolic complications during their survivorship. Maximal fat oxidation (MFO) is a marker during exercise of cardiometabolic health, and is associated with metabolic risk factors. Our aim was to characterize the carbohydrate and fat oxidation during exercise in childhood ALL survivors. Indirect calorimetry was measured in 250 childhood ALL survivors to quantify substrate oxidation rates during a cardiopulmonary exercise test. A best-fit third-order polynomial curve was computed for fat oxidation rate (mg/min) against exercise intensity (%V̇O2peak) and was used to determine the MFO and the peak fat oxidation (Fatmax). The crossover point was also identified. Differences between prognostic risk groups were assessed (ie, standard risk [SR], high risk with and without cardio-protective agent dexrazoxane [HR + DEX and HR]). MFO, Fatmax and crossover point were not different between the groups (p = .078; p = .765; p = .726). Fatmax and crossover point were achieved at low exercise intensities. A higher MFO was achieved by men in the SR group (287.8 ± 111.2 mg/min) compared to those in HR + DEX (239.8 ± 97.0 mg/min) and HR groups (229.3 ± 98.9 mg/min) (p = .04). Childhood ALL survivors have low fat oxidation during exercise and oxidize carbohydrates at low exercise intensities, independently of the cumulative doses of doxorubicin they received. These findings alert clinicians on the long-term impact of cancer treatments on childhood ALL survivors' substrate oxidation.

[Study on the Mechanism of Multi-Drug Resistance of Agaricus Blazei Extract FA-2-b-ß Mediated Wnt Signaling Pathway to Reverse Acute T Lymphoblastic Leukemia].

OBJECTIVE: To investigate the mechanism of drug reversing resistance of Agaricus blazei extract FA-2-b-ß on T cell acute lymphoblastic leukemia (T-ALL) cell lines. METHODS: Cell proliferation was detected by CCK-8 assay; the apoptosis, cell cycle mitochondrial membrane potential, and intracellular rhodamine accumulation were detected by flow cytometry, and apoptosis-related gene and protein expression were detected by qPCR and Western blot; the membrane surface protein MDR1 was observed by immunofluorescence microscopy. RESULTS: Different concentrations of FA-2-b-ß significantly inhibited proliferation and induced apoptosis of CCRF-CEM and CEM/C1 (P<0.05), and CCRF-CEM cell cycle were arrested at S phase, and CEM/C1 cells were arrested at G0/G1 phase. Western blot and qPCR results show that FA-2-b-ß inhibited ABCB1、ABCG2、CTNNB、MYC and BCL-2 expression, but upregulated Bax expression. In addition, FA-2-b-ß reversed the resistance characteristics of CEM/C1 drug-resistance cells, which decreased mitochondrial membrane potential, and significantly increased the intracellular rhodamine accumulation, and weakening of the expression of the membrane surface protein MDR1. With the Wnt/ß-catenin inhibitor (ICG001), the process was further intensified. CONCLUSION: Agaricus Blazei Extract FA-2-b-ß inhibits cell proliferation, promotes apoptosis, regulates the cell cycle, reduces mitochondrial energy supply, and down-regulate MDR1 expression to reverse the resistance of CEM/C1, which all suggest it is through regulating the Wnt signaling pathway in T-ALL.

[Expression and Clinical Significance of Exosome Derived MiR-181b-5p in Children with Acute Lymphoblastic Leukemia].

OBJECTIVE: To explore the expression level of exosome derived miR-181b-5p in different disease stages of children with acute lymphoblastic leukemia and its relationship with clinical characteristics. METHODS: Bone marrow plasma samples of 86 children with ALL were collected. Exosomes were extracted by exosome extraction kit, and RNA in exosomes was extracted by TRIzol method. The levels of miR-181b-5p in the blood plasma exosomes of the patients in the newly diagnosed group, relapse group, remission group and control group were detected by qRT- PCR. The difference of miR-181b-5p expression level in each group was compared and analyzed, and the relationship between miR-181b-5p expression level and clinical characteristics was analyzed. RESULTS: The expression level of exosomal miR-181b-5p in the newly diagnosed group and the relapsed group was significantly lower than that in the remission group and the control group (P< 0.05). The expression level of exosomal miR-181b-5p in T-ALL children was higher than that in B-ALL children (P<0.05). The expression level of plasma exosomal miR-181b-5p in male children was higher than that in female children (P<0.01). CONCLUSION: Exosome derived miR-181b-5p changes dynamically in the course of ALL children, and can be used as a marker miRNA to monitor disease status. Exosomes can transmit information in the tumor microenvironment and serve as a potential carrier for biomolecular targeted therapy.

Rapid molecular response to dasatinib in Ph-like acute lymphoblastic leukemia patients with ABL1 rearrangements: case series and literature review.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype with a poor prognosis under conventional chemotherapy. Ph-like ALL has a similar gene expression profile to Philadelphia chromosome-positive (Ph+) ALL, but is highly heterogeneous in terms of genomic alterations. Approximately 10-20% of patients with Ph-like ALL harbor ABL class (e.g. ABL1, ABL2, PDGFRB, and CSF1R) rearrangements. Additional genes that form fusion genes with ABL class genes are still being researched. These aberrations result from rearrangements including chromosome translocations or deletions and may be targets of tyrosine kinase inhibitors (TKIs). However, due to the heterogeneity and rarity of each fusion gene in clinical practice, there is limited data on the efficacy of tyrosine kinase inhibitors. Here, we report three cases of Ph-like B-ALL with ABL1 rearrangements treated with the dasatinib backbone for the CNTRL::ABL1, LSM14A::ABL1, and FOXP1::ABL1 fusion genes. All three patients achieved rapid and profound remission with no significant adverse events. Our findings suggest that dasatinib is a potent TKI for the treatment of ABL1-rearranged Ph-like ALL and can be used as a first-line treatment option for such patients.

Polygenic Pharmacogenomic Markers as Predictors of Toxicity Phenotypes in the Treatment of Acute Lymphoblastic Leukemia: A Single-Center Study.

PURPOSE: Acute lymphoblastic leukemia (ALL) is the most prevalent cause of childhood cancer and requires a long course of therapy consisting of three primary phases with interval intensification blocks. Although these phases are necessary to achieve remission, the primary chemotherapeutic agents have potentially serious toxicities, which may lead to delays or discontinuations of therapy. The purpose of this study was to perform a comprehensive pharmacogenomic evaluation of common antileukemic agents and develop a polygenic toxicity risk score predictive of the most common toxicities observed during ALL treatment. METHODS: This cross-sectional study included 75 patients with pediatric ALL treated between 2012 and 2020 at the University of Florida. Toxicity data were collected within 100 days of initiation of therapy using CTCAE v4.0 for toxicity grading. For pharmacogenomic evaluation, single-nucleotide polymorphisms (SNPs) and genes were selected from previous reports or PharmGKB database. 116 unique SNPs were evaluated for incidence of various toxicities. A multivariable multi-SNP modeling for up to 3-SNP combination was performed to develop a polygenic toxicity risk score of prognostic value. RESULTS: We identified several SNPs predictive of toxicity phenotypes in univariate analysis. Further multivariable SNP-SNP combination analysis suggest that susceptibility to chemotherapy-induced toxicities is likely multigenic in nature. For 3-SNPscore models, patients with high scores experienced increased risk of GI (P = 2.07E-05, 3 SNPs: TYMS-rs151264360/FPGS-rs1544105/GSTM1-GSTM5-rs3754446), neurologic (P = .0005, 3 SNPs: DCTD-rs6829021/SLC28A3-rs17343066/CTPS1-rs12067645), endocrine (P = 4.77E-08, 3 SNPs: AKR1C3-rs1937840/TYMS-rs2853539/CTH-rs648743), and heme toxicities (P = .053, 3 SNPs: CYP3A5-rs776746/ABCB1-rs4148737/CTPS1-rs12067645). CONCLUSION: Our results imply that instead of a single-SNP approach, SNP-SNP combinations in multiple genes in drug pathways increases the robustness of prediction of toxicity. These results further provide promising SNP models that can help establish clinically relevant biomarkers allowing for greater individualization of cancer therapy to maximize efficacy and minimize toxicity for each patient.

Opposing effects of KDM6A and JDP2 on glucocorticoid sensitivity in T-ALL.

Glucocorticoids (GCs) are the cornerstone of acute lymphoblastic leukemia (ALL) therapy. Although mutations in NR3C1, which encodes the GC receptor (GR), and other genes involved in GC signaling occur at relapse, additional mechanisms of adaptive GC resistance are uncertain. We transplanted and treated 10 primary mouse T-lineage acute lymphoblastic leukemias (T-ALLs) initiated by retroviral insertional mutagenesis with GC dexamethasone (DEX). Multiple distinct relapsed clones from 1 such leukemia (T-ALL 8633) exhibited discrete retroviral integrations that upregulated Jdp2 expression. This leukemia harbored a Kdm6a mutation. In the human T-ALL cell line CCRF-CEM, enforced JDP2 overexpression conferred GC resistance, whereas KDM6A inactivation unexpectedly enhanced GC sensitivity. In the context of KDM6A knockout, JDP2 overexpression induced profound GC resistance, counteracting the sensitization conferred by KDM6A loss. These resistant "double mutant" cells with combined KDM6A loss and JDP2 overexpression exhibited decreased NR3C1 mRNA and GR protein upregulation upon DEX exposure. Analysis of paired samples from 2 patients with KDM6A-mutant T-ALL in a relapsed pediatric ALL cohort revealed a somatic NR3C1 mutation at relapse in 1 patient and a markedly elevated JDP2 expression in the other. Together, these data implicate JDP2 overexpression as a mechanism of adaptive GC resistance in T-ALL, which functionally interacts with KDM6A inactivation.

Genomic and proteomic characterization of Philadelphia-like B-lineage acute lymphoblastic leukemia: A report of Indian patients.

BACKGROUND: The gold standard for the identification of Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) patients is gene expression profiling. Because of its diverse nature, its identification is extremely difficult and expensive. On the genomic and proteomic landscape of Ph-like ALL patients, there is a paucity of published literature from developing countries. METHODS: The authors used digital barcoded nCounter NanoString gene expression profiling for its detection, followed by molecular and proteomic characterization using fluorescence in situ hybridization and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The authors found 32.05% Ph-like ALL patients and their median age at presentation was considerably higher than Ph-negative ALL cases (p = .0306). Furthermore, we identified 20% CRLF2 overexpressed cases having 8.33% CRLF2-IGH translocation with concomitant R683S mutation and 8.33% CRLF2-P2RY8 translocation. In 80% of CRLF2 downregulated cases, we identified 10% as having JAK2 rearrangement. Minimal residual disease-positivity was more common in Ph-like ALL cases (55.55% vs. 25% in Ph-negative ALL cases). Immunoglobulin J chain (Jchain), small nuclear ribonucleoprotein SmD1 (SNRPD1), immunoglobulin κ constant (IGKC), NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 2 (NDUFA2), histone H2AX (H2AFX), charged multivesicular body protein 4b (CHMP4B), and carbonyl reductase (NADPH) (CBR1) proteins were identified to be substantially expressed in Ph-like ALL patients, using LC-MS/MS. Gene enrichment analysis indicated that involvement of spliceosomal mediated messenger RNA splicing pathway and four microRNAs was statistically significant in Ph-like ALL patients. CONCLUSIONS: For the first time, we have described incidence, molecular, and proteomic characterization of Ph-like ALL, in developing nations. PLAIN LANGUAGE SUMMARY: In developing countries, detecting Philadelphia (Ph)-like B-lineage acute lymphoblastic leukemia is complicated and challenging due to its diverse genetic landscape. There is no well-defined and cost-effective methodology for its detection. The incidence of this high-risk subtype is very high in adult cases, and there is an urgent need for its accurate detection. We have developed an online PHi-RACE classifier for its rapid detection, followed by delineating the genomic and proteomic landscape of Ph-like acute lymphoblastic leukemias for the first time in Indian patients.

[Effect of MXRA7 on the Biological Functions of Acute B Lymphoblastic Leukemia Cell Line REH].

OBJECTIVE: To discover the relationship between matrix remodeling associated 7 (MXRA7) and acute B lymphoblastic leukemia (B-ALL), and explore the effect of MXRA7 on the biological functions of B-ALL cell line REH. METHODS: The expression of MXRA7 in blood diseases was searched and analyzed through BloodSpot database. Real-time qPCR was used to detect the expression level of MXRA7 in B-ALL cell line 697 and REH cells. Lentivirus-mediated shRNA interference technology was utilized to knock down the expression of MXRA7 in REH cells. The effects of MXRA7 on the biological functions of REH cells were studied by in vitro experiments. Cell proliferation was detected by CCK-8 assay, cell cycle was detected by PI staining, cell apoptosis was detected by Annexin V and 7-AAD staining, and the expression of apoptosis pathway related proteins was detected by Western blot. RESULTS: Database analysis showed that MXRA7 was highly expressed in B-ALL patients, and real-time qPCR results showed that MXRA7 was also highly expressed in cell lines 697 and REH cells. Knockdown of MXRA7 in REH cells inhibited the cell proliferation and increased the percentage of G0/G1 phase cells. After treatment with cytarabine, the apoptotic ratio was increased in MXRA7-impaired REH cells, and the activation of caspase-3 and caspase-9 were also increased. CONCLUSION: Knockdown of MXRA7 can reduce the malignancy of REH cells by inhibiting the cell proliferation and increasing the sensitivity of REH cells to cytarabine. These results indicate MXRA7 may be as a novel target for the treatment of B-ALL, and the potential usefulness of MXRA7 in B-ALL deserves further investigation.

ERG Expression is Helpful in Differentiating T-Lymphoblastic Lymphoma from Thymoma.

ETS-related gene (ERG) is the member of ETS-family of transcription factors and is commonly expressed in Ewing sarcoma. Recently, we found that ERG can also be expressed in lymphoblastic lymphoma. The aim of this article is to explore the expression patterns of ERG in T-lymphoblastic lymphoma, and to evaluate its diagnostic value for differentiating T-lymphoblastic lymphoma and nonneoplastic T-precursor cells in thymoma via immunohistochemistry. In this study, we explored the expression pattern of ERG in T-lymphoblastic lymphoma and thymoma specimens via immunohistochemistry. Sixteen T-lymphoblastic lymphoma and 18 thymoma specimens were evaluated for the expression of ERG. Our findings showed that ERG was expressed in 10 of the 16 (63%) T-lymphoblastic lymphoma specimens, and in only 1 of the 18 (6%) thymoma specimens. The positive and negative predictive value of ERG in T-lymphoblastic lymphoma was 91% and 74%, respectively. ERG is a helpful marker for the diagnosis of T-lymphoblastic lymphoma and is a promising new method to differentiate T-lymphoblastic lymphoma and the nonneoplastic T-precursor cells in thymoma.

BCL6 inhibition ameliorates resistance to ruxolitinib in CRLF2-rearranged acute lymphoblastic leukemia.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an intractable disease and most cases harbor genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits a CRLF2 gene rearrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient as a treatment, so combinatorial therapies targeting multiple signals are needed. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, we explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and found that elevated BCL6 expression was one such mechanism. Upregulated BCL6 suppressed the expression of TP53 along with its downstream cell cycle inhibitor p21 (CDKN2A) and pro-apoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells some degree of resistance to therapy. BCL6 inhibition (with FX1) alone was able to upregulate TP53 and restore the TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis- sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged the survival of xenografted mice. These findings provide one mechanism for the insufficiency of JAK inhibition for the treatment of CRLF2-rearranged ALL and indicate BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition.