pre-B Cell Acute Lymphoblastic Leukemia Research Articles

Murine pre–B-cell ALL induces T-cell dysfunction not fully reversed by introduction of a chimeric antigen receptor

AbstractAdoptive transfer of patient-derived T cells modified to express chimeric antigen receptors (CARTs) has demonstrated dramatic success in relapsed/refractory pre–B-cell acute lymphoblastic leukemia (ALL), but response and durability of remission requires exponential CART expansion and persistence. Tumors are known to affect T-cell function, but this has not been well studied in ALL and in the context of chimeric antigen receptor (CAR) expression. Using TCF3/PBX1 and MLL-AF4–driven murine ALL models, we assessed the impact of progressive ALL on T-cell function in vivo. Vaccines protect against TCF3/PBX1.3 but were ineffective when administered after leukemia injection, suggesting immunosuppression induced early during ALL progression. T cells from leukemia-bearing mice exhibited increased expression of inhibitory receptors, including PD1, Tim3, and LAG3, and were dysfunctional following adoptive transfer in a model of T-cell receptor (TCR)–dependent leukemia clearance. Although expression of inhibitory receptors has been linked to TCR signaling, pre–B-cell ALL induced inhibitory receptor expression, at least in part, in a TCR-independent manner. Finally, introduction of a CAR into T cells generated from leukemia-bearing mice failed to fully reverse poor in vivo function.

Characterizing Deformability of Drug Resistant Patient-Derived Acute Lymphoblastic Leukemia (ALL) Cells Using Acoustic Tweezers

The role of cell mechanics in cancer cells is a novel research area that has resulted in the identification of new mechanisms of therapy resistance. Single beam acoustic (SBA) tweezers are a promising technology for the quantification of the mechanical phenotype of cells. Our previous study showed that SBA tweezers can be used to quantify the deformability of adherent breast cancer cell lines. The physical properties of patient-derived (primary) pre-B acute lymphoblastic leukemia (ALL) cells involved in chemotherapeutic resistance have not been widely investigated. Here, we demonstrate the feasibility of analyzing primary pre-B ALL cells from four cases using SBA tweezers. ALL cells showed increased deformability with increasing acoustic pressure of the SBA tweezers. Moreover, ALL cells that are resistant to chemotherapeutic drugs were more deformable than were untreated ALL cells. We demonstrated that SBA tweezers can quantify the deformability of nonadherent leukemia cells and discriminate this mechanical phenotype in chemotherapy-resistant leukemia cells in a contact- and label-free manner.

A Comparison of Two Chemotherapeutic Regimes in Children with B-Cell Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood, and B-cell is the most common type of ALL. In childhood ALL, the most important prognostic factor is treatment and without effective treatment, ALL is a fatal illness. So far, different treatment protocols are employed for ALL chemotherapy. Each of these treatment protocols has different side effects and prognosis. Methods: In the current study, the children oncology group (COG) protocol was compared with the modified protocols, based on the Berlin-Frankfurt-Munster (BFM) protocol, called the Mofid Children’s Hospital protocol (MCHP). The current study was conducted on 108 patients; 51 patients with COG protocol and 57 with MCHP; 61.1% of patients had pre B-cell ALL type and 38.9% had early pre B-cell ALL type. Results: Induction failures in the COG and MCHP groups were 5.9% and 10.5%, respectively (P = 0.390). In the two groups, the most common recurrence sites were bone marrow (BM) and central nervous system (CNS). Moreover, the incidence of recurrence was significantly higher in the MCHP group than the COG (P = 0.262). In terms of complications, bleeding was significantly higher in the COG group than the MCHPs (P = 0.016). There was no significant difference in mortality rate between the two groups (P = 0.489). Conclusions: Comparison between treatment results of these two protocols can lead to finding a better treatment protocol to treat ALL.

The Formin mDia1 Regulates Acute Lymphoblastic Leukemia Engraftment, Migration, and Progression in vivo.

Leukemias typically arise in the bone marrow and then spread to the blood and into other tissues. To disseminate into tissues, leukemia cells migrate into the blood stream and then exit the circulation by migrating across vascular endothelial barriers. Formin proteins regulate cytoskeletal remodeling and cell migration of normal and malignant cells. The Formin mDia1 is highly expressed in transformed lymphocytes and regulates lymphocyte migration. However, the role of mDia1 in regulating leukemia progression in vivo is unknown. Here, we investigated how mDia1 mediates the ability of leukemia cells to migrate and disseminate in vivo. For these studies, we used a mouse model of Bcr-Abl pre-B cell acute lymphoblastic leukemia. Our data showed that mDia1-deficient leukemia cells have reduced chemotaxis and ability to complete transendothelial migration in vitro. In vivo, mDia1 deficiency reduced the ability of leukemia cells to engraft in recipient mice. Furthermore, leukemia dissemination to various tissues and leukemia progression were inhibited by mDia1 depletion. Finally, mDia1 depletion in leukemia cells resulted in prolonged survival of recipient mice in a leukemia transfer model. Overall, our data show that the Formin mDia1 mediates leukemia cell migration, and drives leukemia engraftment and progression in vivo, suggesting that targeting mDia1 could provide a new method for treatment of leukemia.

Abstract A50: Trivalent CAR T cells mitigate CD19-negative relapse and improve tumor control in primary pre-B cell acute lymphoblastic leukemia (B-ALL)

Abstract B-acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children, and limited treatment options exist for patients with relapsed or refractory disease. Cellular immunotherapy, specifically chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in treating B-ALL. However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens (TAAs). Here, we hypothesize that targeting three distinct leukemia antigens CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse. We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19 (FMC63 ScFv), CD20 (Rituximab ScFv), and CD22 (m971 ScFv) fused to the intracellular signaling domains of the co-stimulatory molecule 4-1BB and the T-cell receptor zeta chain (2nd generation). Using viral 2A intervening sequences for near equal expression, the first T cell product expresses the three CARs individually on the surface of a single T cell (TriCAR). The second T cell product expresses a traditional single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 through a tandem arrangement (SideCAR). Donor T cells were successfully engineered to express the CARs using a retroviral system and the surface expression of these CAR molecules was confirmed by flow cytometry. Using a target expression validated panel of patient derived B-ALL cells (US7 CD19/CD20/CD22 +++/++/++, LAX-56 +++/+/+, TXL-2 +++/++/+++), we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low effector to target ratios (E:T) in a 51Cr release cytotoxicity assay. TriCAR and SideCAR T cells secreted similar levels of IFN-gamma; when compared to CD19 CAR T cells demonstrating a safety profile very similar to the CD19 CAR T cells, but with enhanced killing. Further, we tested the efficacy of TriCAR and SideCAR T cells against primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of the three primary ALL samples. Using these models of CD19 escape we demonstrated that trivalent CAR T cells effectively mitigated CD19 negative relapse, producing IFN-gamma; and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. In conclusion, trivalent CAR T cells are effective at targeting primary ALL cells of varying antigen profiles and mitigating CD19 negative relapse. This strategy has potential for use as a front-line therapy for primary ALL as well as a salvage therapy for patients with CD19-negative disease relapse. Citation Format: Kristen Fousek, Junji Watanabe, Xingyue An, Ann George, Heba Samaha, Shoba Navai, Tiara T. Byrd, Jonathan Kirzner, Hye Na Kim, Albert Jang, Sujith Joseph, Matthew Baker, Meenakshi Hegde, Navin Varadarajan, Nora Heisterkamp, Hisham Abdel-Azim, Nabil Ahmed. Trivalent CAR T cells mitigate CD19-negative relapse and improve tumor control in primary pre-B cell acute lymphoblastic leukemia (B-ALL) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A50.

The telomerase inhibitor MST-312 synergistically enhances the apoptotic effect of doxorubicin in pre-B acute lymphoblastic leukemia cells

BackgroundThere have been consistent efforts in concomitant administration of chemotherapeutic agents such as doxorubicin beside other more tumor-specific drugs in order to increase sensitivity of tumor cells to the cytotoxicity of doxorubicin and reduce necessary chemotherapeutic dosage. Telomerase-targeted therapy for cancer has received great attention because telomerase is expressed in almost all cancer cells but is inactive in most normal somatic cells. The present study was aimed to investigate the effect of telomerase inhibitor MST-312, a chemically modified derivative of epigallocatechin gallate (EGCG), on doxorubicin-induced apoptosis in pre-B acute lymphoblastic leukemia cells. Materials and methodsThe pre-B ALL cell lines (NALM-6 and REH) were cultured and treated with MST-312 and doxorubicin, separately and in combination. Cell viability was measured by trypan blue staining and MTT assay. Annexin-V/7-AAD staining by flow cytometry was used for evaluation of apoptosis. Gene expression of hTERT, c-Myc, Bax and Bcl-2, was detected by the quantitative Real-Time PCR. ResultsOur results showed that MST-312 exerted dose-dependent short-term cytotoxic and apoptotic effects on pre-B ALL cells. Combination of MST-312 and doxorubicin synergistically enhanced the cytotoxicity and apoptosis of both NALM-6 and REH cells. Consistently, apoptosis induction by the combined treatment was associated with significant reduction of Bcl-2, c-Myc and hTERT, and a marked increase of Bax mRNA expression levels, compared with the single treatments. ConclusionsOur data suggest that the synergistic anticancer effect induced by combination of MST-312 and doxorubicin represents a novel treatment strategy for pre-B ALL.

Novel vitexin-inspired scaffold against leukemia

Acute lymphoblastic leukemia (ALL) is the most common type of leukemia in children. Up to a quarter of ALL patients relapse and face poor prognosis. To identify new compound leads, we conducted a phenotypic screen using terrestrial natural product (NP) fractions against immortalized ALL cellular models.We identified vitexin, a flavonoid, as a promising hit with biological activity (EC50 = 30 μM) in pre-B cell ALL models with no toxicity against normal human tissue (BJ cells) at the tested concentrations. To develop more potent compounds against ALL and elucidate its potential mode of action, a vitexin-inspired compound library was synthesized. Thus, we developed an improved and scalable protocol for the direct synthesis of 4-quinolone core heterocycles containing an N-sulfonamide using a one-pot condensation reaction protocol. The newly generated compounds represent a novel molecular scaffold against ALL as exemplified by compounds 13 and 15, which demonstrated EC50 values in the low micromolar range (0.3–10 μM) with little to no toxicity in normal cellular models. Computational studies support the hypothesis that these compounds are potential CDK inhibitors. The compounds induced apoptosis, caused cell arrest at G0/G1 and G2/M, and induced ROS in cancer cells.

Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. DisclosuresNo relevant conflicts of interest to declare.

TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance.

Chimeric antigen receptor (CAR)-expressing T cells induce durable remissions in patients with relapsed/refractory B cell malignancies. CARs are synthetic constructs that, when introduced into mature T cells, confer a second, non-major histocompatibility complex-restricted specificity in addition to the endogenous T cell receptor (TCR). The implications of TCR activation on CAR T cell efficacy has not been well defined. Using an immunocompetent, syngeneic murine model of CD19-targeted CAR T cell therapy for pre-B cell acute lymphoblastic leukemia in which the CAR is introduced into T cells with known TCR specificity, we demonstrate loss of CD8 CAR T cell efficacy associated with T cell exhaustion and apoptosis when TCR antigen is present. CD4 CAR T cells demonstrate equivalent cytotoxicity to CD8 CAR T cells and, in contrast, retain in vivo efficacy despite TCR stimulation. Gene expression profiles confirm increased exhaustion and apoptosis of CD8 CAR T cells upon dual receptor stimulation compared to CD4 CAR T cells and indicate inherent differences between CD4 and CD8 CAR T cells in the use of T cell-associated signaling pathways. These results provide insights into important aspects of CAR T cell immune biology and indicate opportunities to rationally design CAR constructs to optimize clinical efficacy.

CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy

Chimeric antigen receptor (CAR) T-cells targeting CD19 mediate potent effects in relapsed/refractory pre-B cell acute lymphoblastic leukemia (B-ALL) but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed on most B-ALL and usually retained following CD19 loss. We report results from a phase I trial testing a novel CD22-CAR in twenty-one children and adults, including 17 previously treated with CD19-directed immunotherapy. Dose dependent anti-leukemic activity was observed with complete remission in 73% (11/15) of patients receiving ≥ 1 × 106 CD22-CART cells/kg, including 5/5 patients with CD19dim/neg B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted escape from killing by CD22-CART cells. These results are the first to eastablish the clinical activity of a CD22-CAR in pre-B cell ALL, including in leukemia resistant to anti-CD19 immunotherapy, demonstrating comparable potency to CD19-CART at biologically active doses in B-ALL. They also highlight the critical role played by antigen density in regulating CAR function. (Funded by NCI Intramural Research Program)

Blinatumomab Induced Response of Multiply Refractory Multiple Myeloma in the Context of Secondary Pre-B Cell Acute Lymphoblastic Leukemia

Blinatumomab Induced Response of Multiply Refractory Multiple Myeloma in the Context of Secondary Pre-B Cell Acute Lymphoblastic Leukemia

Treatment of immune thrombocytopenic purpura associated with cytomegalovirus infection in a child with pre-B cell acute lymphoblastic leukaemia after central nervous system relapse

A 13-year-old male patient with a history of pre-B cell acute lymphoblastic leukaemia (ALL) with isolated central nervous system relapse on maintenance chemotherapy presented with severe thrombocytopenia refractory to platelet...

Abstract 93: Transcriptional control of glucocorticoid responses in leukemia

Abstract Glucocorticoids (GCs) are central to all major therapy regimens for pre-B cell-derived acute lymphoblastic leukemia (ALL), but have no activity in myeloid leukemia. Such divergent responses represent an empirically established clinical standard; however, neither the mechanism by which GCs induce cell death nor the biological basis for the distinct responses in B-cell and myeloid leukemias is clear. Studying patient-derived samples revealed that NR3C1 (glucocorticoid receptor) levels were 6- to 20-fold higher in pre-B ALL compared to chronic myeloid leukemia (CML). High levels of Nr3c1 were reduced upon B- to myeloid-lineage conversion, suggesting that regulation of NR3C1 expression and GC responsiveness depend on a B-cell transcriptional program. B-cell transcription factors (e.g. PAX5, IKZF1) are critical for B-cell development, yet they are genetically lesioned in more than 80% of pre-B ALL cases. Despite such high frequency, the significance of these inactivating lesions remains elusive. Combining ChIP-seq and RNA-seq analyses, we identified a novel B-cell transcriptional program for activation of NR3C1 and its transcriptional target TXNIP (a negative regulator of glucose uptake). Reconstitution of PAX5 or IKZF1 expression in haploinsufficient patient-derived pre-B ALL cells increased NR3C1 and TXNIP levels. Conversely, expression of dominant negative mutant of PAX5 or IKZF1 abolished NR3C1 expression. Loss of Nr3c1 or Txnip in murine BCR-ABL1-driven pre-B ALL cells resulted in survival advantage in competitive growth assays. Importantly, loss of Nr3c1 or Txnip significantly elevated glucose uptake, lactate production and cellular ATP levels. These findings suggest that GCs induce cell death by exacerbating glucose and energy depletion. Notably, reconstitution of PAX5 or IKZF1 rendered haploinsufficient patient-derived pre-B ALL cells more sensitive to dexamethasone (dex) treatment. In contrast, dominant-negative PAX5 or IKZF1 largely de-sensitized pre-B ALL cells expressing wildtype PAX5 or IKZF1. These findings suggest that B-cell transcription factors set the threshold for GC responsiveness in pre-B ALL. Since relapsed ALL cells often acquire GC resistance, drug-combinations may be useful to prevent GC-resistance. As expected, loss of Nr3c1 abrogated responses to GCs. Interestingly, loss of Txnip also largely rescued GC-induced cell death in pre-B ALL cells. On this basis, we tested drug interactions between GCs and TXNIP agonists, 3-O-methylglucose (3-OMG) and D-allose. Treating patient-derived GC-refractory pre-B ALL cells with 3-OMG or D-allose strongly synergized with GC-treatment. Collectively, our findings provide a mechanistic explanation for the empiric finding that GCs are effective in the treatment of B-cell but not myeloid malignancies, and identify TXNIP as a novel therapeutic target in pre-B ALL. Note: This abstract was not presented at the meeting. Citation Format: Lai N. Chan, Zhengshan Chen, Gang Xiao, Jae Woong Lee, Kadriye Nehir Cosgun, Huimin Geng, Valeria Cazzaniga, Hilde Schjerven, Ross A. Dickins, Markus Muschen. Transcriptional control of glucocorticoid responses in 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 93. doi:10.1158/1538-7445.AM2017-93

RNA methylation in lymphoid malignancies

N 6 -methyl-adenosine (m 6 A) modification is the most abundant internal modification in mammalian RNA and plays an important role in gene expression mechanisms such as mRNA splicing, nuclear export, transcript stability and translational efficiency. The extent and the significance of m 6 A modifications in pathological conditions, especially hematological malignancies such as lymphomas are not known. In this study, the global m 6 A methylation by an immunocapture method in normal B cells (GMO 6990), and in the B cell neoplastic cells such as diffuse large B cell lymphoma (DB), Burkitt’s lymphoma (Raji) and a relapsed pre-B cell acute lymphoblastic leukemia (ALL) (NALM6) cells are discussed. Variable mRNA expression of RNA methylases such as methyltransferase like 3 (METTL3) and Wilms tumor 1 associated protein (WTAP) that methylates internal adenosine residues in mRNA was observed in normal and lymphoma cells as quantified by qRT-PCR. Further, an mRNA demethylase, Fat mass and obesity associated enzyme (FTO) mRNA expression was found to be high in lymphoma cells compared to normal B cells. A similar trend was also observed between normal and ALL patients at diagnosis. Overall, our studies suggest that altered RNA methylation and enzymes controlling this process may be of significance in the pathology of lymphoid malignancies.

ABCC4 functional SNP in the 3' splice acceptor site of exon 8 (G912T) is associated with unfavorable clinical outcome in children with acute lymphoblastic leukemia.

ATP-binding cassette subfamily C member 4 (ABCC4) encoding MRP4 protein is involved in pediatric acute lymphoblastic leukemia (ALL) drug resistance. The nonsynonymous single nucleotide polymorphism (SNP) rs2274407 (G912T; K304N) is located in the 3' splice acceptor site of exon 8 of ABCC4 pre-mRNA. The aim of this study was to investigate the prognostic value of rs2274407 in childhood ALL and its possible functional effect on MRP4. ABCC4 G912T SNP was genotyped in 145 Iranian Philadelphia-negative (Ph-) children with ALL using modified tetra-primer ARMS PCR and evaluated for possible association with 3-year disease-free survival (3DFS). In addition, functional impact of rs2274407 on the MRP4 activity and possible post-transcriptional modifications were bioinformatically and experimentally studied. ABCC4 912T allele carriers (G/T and T/T genotypes) are associated with worse 3DFS in Pre-B cell ALL [P=0.00019, OR (95% CI)=13.17 (2.55-68.11)]. In addition, computational studies showed that K304N alteration has no impact on the MRP4 activity. However, it may disrupt the normal splicing process of ABCC4 pre-mRNA. To date, this is the first study that shows the potential functional impact of rs2274407 SNP on the aberrant splicing of ABCC4 mRNA. We also demonstrated a robust association between G912T and pediatric ALL negative outcome, which may be explained by the novel computational studies performed in this study.

Anticancer effect and enhancement of therapeutic potential of Vincristine by extract from aerial parts of Juniperus excelsa on pre-B acute lymphoblastic leukemia cell lines

The natural products and conventional chemotherapeutic drugs combination can increase the efficacy of anticancer treatment through their potential synergistic effects and reduce its toxicity. The current study investigates the effect of methanolic extract from aerial parts of Juniperus excelsa on cell death activities induced by vincristine in acute lymphoblastic leukemia cells.Cytotoxic activity of J. excelsa extract and vincristine in Nalm-6 and Reh cells was determined using MTT assay and synergism was evaluated using the CompuSyn software. Apoptosis was assessed by caspase 3 activity assay and flow cytometry. The expression levels of some apoptosis-related genes, Caspase 3, BAX and BCL-2 were determined by Real-time PCR. Statistical analysis was assessed by one-way ANOVA and Tukey's tests.The combined treatment of VCR and J. excelsa extract showed a synergistic cytotoxic effect on both Nalm-6 and Reh cells at low doses of vincristine (CI<1).J. excelsa extract also significantly increased VCR-induced apoptosis (P<0.001). Expression of CASP3 and BAX genes were upregulated, while BCL-2 gene was downregulated in both cells (P<0.05).Our study suggested the combined use of lower doses of VCR and J. excelsa extract promote its effects by apoptosis induction and this combination could potentially decrease the side effects of the drug.

Childhood pre-B cell acute lymphoblastic leukemia with translocation t(1;19)(q21.1;p13.3) and two additional chromosomal aberrations involving chromosomes 1, 6, and 13: a case report

BackgroundThe translocation t(1;19)(q23;p13), which results in the TCF3-PBX1 chimeric gene, is one of the most frequent rearrangements observed in B cell acute lymphoblastic leukemia. It appears in both adult and pediatric patients with B cell acute lymphoblastic leukemia at an overall frequency of 3 to 5%. Most cases of pre-B cell acute lymphoblastic leukemia carrying the translocation t(1;19) have a typical immunophenotype with homogeneous expression of CD19, CD10, CD9, complete absence of CD34, and at least diminished CD20. Moreover, the translocation t(1;19) correlates with known clinical high risk factors, such as elevated white blood cell count, high serum lactate dehydrogenase levels, and central nervous system involvement; early reports indicated that patients with translocation t(1;19) had a poor outcome under standard treatment.Case presentationWe report the case of a 15-year-old Syrian boy with pre-B cell acute lymphoblastic leukemia with abnormal karyotype with a der(19)t(1;19)(q21.1;p13.3) and two yet unreported chromosomal aberrations: an interstitial deletion 6q12 to 6q26 and a der(13)t(1;13)(q21.1;p13). According to the literature, cases who are translocation t(1;19)-positive have a significantly higher incidence of central nervous system relapse than patients with acute lymphoblastic leukemia without the translocation. Of interest, central nervous system involvement was also seen in our patient. ConclusionsTo the best of our knowledge, this is the first case of childhood pre-B cell acute lymphoblastic leukemia with an unbalanced translocation t(1;19) with two additional chromosomal aberrations, del(6)(q12q26) and t(1;13)(q21.3;p13), which seem to be recurrent and could influence clinical outcome. Also the present case confirms the impact of the translocation t(1;19) on central nervous system relapse, which should be studied for underlying mechanisms in future.

Inhibition of telomerase using BIBR1532 enhances doxorubicin-induced apoptosis in pre-B acute lymphoblastic leukemia cells

ABSTRACTObjectives: Interest into targeting telomerase in cancer has increased by the recent disclosure that elevated telomerase activity is associated with disease recurrence and poor outcome in cancers. In addition, cellular acquisition of unlimited replicative potential, which is closely related to the maintenance of telomeres mostly via the reactivation of telomerase, has been shown to confer loss of sensitivity to a wide range of anti-neoplastic agents.Methods: To evaluate whether telomerase inhibition using non-nucleosidic inhibitor of telomerase BIBR1532 could enhance cytotoxic effect of doxorubicin in acute lymphoblastic leukemia, Nalm-6 pre-B ALL cells were subjected to combination treatment and subsequent cell viability, growth kinetics, caspase-3 activity, and transcriptional alteration of p73, p21, FOXO3a, c-Myc, hTERT, and other apoptosis-related target genes were investigated.Results: Combination of BIBR1532 with doxorubicin produced a synergistic anticancer effect probably through induction of p73. Transcription factor p73 not only suppressed the proliferative capacity of the cells through induction of p21-mediated G1 arrest, but also down-regulated the mRNA level of hTERT and c-Myc. Our results also report that BIBR1532 induced a caspase-dependent apoptosis, at least partially, through heightened ROS levels, and noteworthy enhanced the pro-oxidant property of doxorubicin. In harmony, transcriptional repression of survivin could be a probable underlying mechanism for the induction of apoptosis through shifting the ratio of death promoters to death repressors via alteration of Bax and Bcl2 expression.Conclusions: Overall, it seems that combination of BIBR1532 and doxorubicin could be a novel therapeutic strategy for acute lymphoblastic leukemia that may be clinically accessible in the near future.

CREB knockdown inhibits growth and induces apoptosis in human pre-B acute lymphoblastic leukemia cells through inhibition of prosurvival signals.

A majority of acute lymphoblastic leukemia patients overexpress CREB in the bone marrow. However, the functional significance of this up-regulation and the detailed molecular mechanism behind the regulatory effect of CREB on the growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells has not been elucidated. We demonstrated here that CREB knockdown induced apoptosis and impaired growth of BCP-ALL NALM-6 cells which was associated with caspase activation. The gene expression levels of prosurvival signals Bcl-2, Mcl-1, Bcl-xL, survivin and XIAP were down-regulated upon CREB suppression. These findings indicate a critical role for CREB in proliferation, survival, and apoptosis of BCP-ALL cells. The data also suggest that CREB could possibly serve as potential therapeutic target in BCP-ALL.

An uncommon cause of right heart failure: primary cardiac pre-B cell acute lymphoblastic leukemia (RCD code: VI-2C.1).

Hematological malignancies are protean in their presentations. Primary cardiac tumors are exceedingly rare, and primary cardiac leukemia presenting as an isolated mass is even rare. Here, we present a case of a young man who presented with one month history of fever and shortness of breath. His examination revealed signs of right sided heart failure. On workup he was found to have a mass in the heart on CT scan of the chest, his transthoracic echocardiography showed an isolated right atrioventricular groove mass. Surgical biopsy confirmed it to be an extremely rare presentation of Pre B Acute Lymphoblastic leukemia(ALL). Though the patient underwent surgical excision of the lesion yet, he developed bone marrow leukemic involvement confirmed by bone marrow biopsy and died of overwhelming sepsis secondary to pancytopenia. This case highlights that hematological malignancies can present as cardiac masses and should be considered during workup of patients presenting with chronic fever and right sided heart failure.