Acute Lymphoid Leukemia Cells Research Articles

The Impact of ZNF384 Rearranged on Antigen Editing during Treatment-Specific Selective Pressures in Adult B Cell Acute Lymphoid Leukemia

Objectives: Recent evidence shows that zinc finger protein 384 rearrangement (ZNF384r) was associated with the aberrant myeloid marker expression at diagnosis, which has been identified as a distinct subset of B cell acute lymphoid leukemia(B-ALL) related to an intermediate or even good prognosis during traditional therapy. However, there exists an indeterminate understanding of whether ZNF384r has a potential impact on antigen editing during treatment-specific selective pressures and shares the same prognostic significance in immunotherapy, such as chimeric antigen receptor T-cell therapy (CAR-T therapy). Methods: In this study, we first evaluated the immunophenotype changes of 31 de novo adult B-ALL patients who harbor ZNF384r and received chemotherapy from Nov 2020 to May 2023. Then, 49 r/r B-ALL patients who received the CD19 or CD19/CD22-based CAR-T infusion in our institute were included in the analysis to further investigate the prognosis effects of ZNF384r on CAR-T therapy. At the same time, the next-generation sequencing and RNA sequencing data of the patients with ZNF384r were analyzed to explore the potential mechanisms behind it. Results: First, we investigated the potential effects of ZNF384r in antigen editing after chemotherapy. Our results showed that 100% (31/31) of the patients express lymphoid lineage marker CD19 and 96.4% (27/28) of patients with CD22 expression at diagnosis. In addition, the majority of them had myeloid lineage marker expression, such as 96.2% (26/27) of patients with CD33 expression, and 96.5% with CD123 expression. After chemotherapy, 11 patients remained MRD positive after induction therapy, and 5 patients relapsed. In them, there were 18.8% (3/16) of patients lost CD19 expression, 12.5% of patients lost or reduced CD22 expression, while 100% of patients kept CD33 expression. For the adult r/r B-ALL patients who received the CD19 or CD19/CD22-based CAR-T infusion, there were 3 patients identified with EP300-ZNF384. Notably, all these three patients harboring ZNF384r relapsed within one year after the CAR-T therapy. Two of them experienced CD19-negative relapse, and one patient even had a myeloid transformation. These results indicated ZNF384r might associate with antigen editing during treatment-related selective pressures. The next-generation sequencing was performed in 25 B-ALL patients harboring ZNF384r before chemotherapy or CAR-T therapy, the results showed that in addition to CREBBP, NRAS, ETV6, NOTCH1/3, FLT3-ITD mutation was also the most common co-mutation with ZNF384r. Furthermore, the transcriptome analysis showed the Jak-STAT signaling pathway and the hematopoietic cell lineage pathway were highly enriched in the group with EP300::ZNF384 fusion compared with TCF3::PBX1 fusion. The expression of CD33, CD34, CD123, and IL5 were also significantly up-regulated in the EP300::ZNF384 group. All these results indicated ZNF384r might be involved in the regulation of the hematopoietic cell lineage differentiation pathway and myeloid lineage skewing. Conclusions: Our findings demonstrated that ZNF384 rearrangement might induce antigen editing during treatment-related selective pressures in adult B cell acute lymphoid leukemia, especially in CAR-T therapy. More efforts are needed to reveal mechanisms behind it to help reduce antigen loss and relapse rate after CAR-T therapy.

P19 a Parthenin Analog Induces Cell Lineage Dependent Apoptotic and Immunomodulatory Signaling in Acute Lymphoid Leukemia Cells.

Leukemia is a type of cancer that affects the blood and bone marrow. Acute lymphoid leukaemia, also known as ALL, is regarded as one of the deadliest forms of cancer. Due to the rapid increase in various cancer cases and the development of resistance in cancer cells, it is necessary to identify novel lead molecules with more potent anticancer properties. There is a growing interest in using herbal products/analogs as multi-component agents (as anticancer agents and immunomodulators) for cancer treatment. In the present investigation, an attempt has been made to explore the anticancer and immunomodulatory activity of P19, an analog of parthenin in ALL. P19 was reported to exhibit anticancer efficacy by triggering apoptotic signaling events in human leukaemia HL-60 cells by significant NO production. In contrast to this finding, ROS and NO were not required for P19-mediated apoptosis in Raji cells. The mechanism of action of P19 was observed to be cancer cell lineage dependent. P19 demonstrated very effective anticancer properties against ALL (IC50 3µM). Molecular investigations revealed that P19 induced mitochondrion mediated apoptosis by Bax localization to mitochondria and enhanced cytosolic calcium in the cytoplasm. Further activation of the caspase 3, caspase 8 and PARP cleavage suggested the involvement of the caspase-mediated apoptosis. Anti-proliferative activity revealed the telomerase inhibition and cell cycle arrest in G0/G1 phase after P19 treatment. Immunomodulatory effects of the P19 revealed the enhanced INFɣ and NO production in Jurkat and THP cells. Owing to its antiproliferative and immunomodulatory potential against leukemia cells P19 can further be explored as effective therapeutics against leukemia.

S6.1d First case report of pediatric blood stream infection by Candida magnoliae in a known case of B cell ALL post -induction chemotherapy in Central India

S6.1 Antifungal Prophylaxis in Children with Cancer and HSCT, September 22, 2022, 4:45 PM - 6:15 PM ObjectivesDocumentation and dissemination of findings of a rare fungal isolate in an immunosuppressed child.MethodsA case study with rare fungal isolate in correlation to age, clinical condition, sample, and comorbidity was done. A 6-year-old male child was admitted for routine management of B cell acute lymphoid leukemia. The patient completed induction chemotherapy in July 2021. The patient was planned for consolidation in the last week of July and to rule out any infection blood and urine samples were sent. Paired blood samples were received in pediatric automated BacT/Alert blood culture bottles. After 8 days both the blood culture bottles flashed positive. On gram stain, budding yeast cells oval to globose were seen. No pseudohyphae were seen. Nigrosin staining result was negative. It was processed further on HiCrome™ agar showing cream-colored colonies at 370 C, cornmeal agar with 1% tween 80 for Dalmau technique showed only oval to globose yeast cells with blastoconidia, enlarged cells appearing as chlamydoconidia without pseudohyphae or true hyphae were seen. Glucose and sucrose were fermented and trehalose was weakly fermented. Urease was negative. Isolate was identified as Candida glabrata/Candida auris. Antifungal susceptibility showed elevated MIC for fluconazole but susceptible to amphotericin B, voriconazole, and caspofungin. As part of routine collaboration with reference center PGIMER, bloodstream Candida isolates were sent for confirmation, and quality control.ResultsThe isolate phenotypically suspected as C. glabrata causing fungemia was confirmed by the reference center as C. magnoliae. Currently, patient is on routine follow-up and doing well. On reviewing of available literature on C. magnoliae; bloodstream infections in two low birth weight neonates from Brazil, one immunocompetent child with tenosynovitis from the USA, and a terminal oncology patient from Italy were noted.In a Chinese study of 2007, phylogenetic analysis showed a close relationship of C. magnoliae to Candida krusei.ConclusionImmunosuppression with longstanding or repeated hospital admissions is a risk for nosocomial fungal infections, especially, bloodstream infections. Already confusing phenotypic identification among C. glabrata, C. auris, C. haemoulonii, and now the current isolate C. magnoliae further complicates and challenges diagnostic workflow impacting timely management of cases. Further studies and more documentation of such findings in literature are necessary for newer insights.

PaDef (Persea americana var. drymifolia), a Plant Antimicrobial Peptide, Triggers Apoptosis, and Induces Global Epigenetic Modifications on Histone 3 in an Acute Lymphoid Leukemia Cell Line.

In recent years, it has been recognized that epigenetic alterations play an important role in the development and maintenance of cancer, including leukemias. Furthermore, it is known that these alterations are involved in the emergence of resistance to conventional chemotherapeutics. Consequently, molecules with an anticancer activity whose activity is ruled by epigenetic modifications are attractive to search for new therapies against cancer. The plant antimicrobial peptides have been widely evaluated as molecules with anticancer activity; however, the analysis of the epigenetic regulation induced by these molecules associated with this activity is scarce and still is an unexplored field. In this work, we show that the PaDef defensin, a plant antimicrobial peptide from Mexican avocado fruit (Persea americana var. drymifolia) is cytotoxic for Jurkat cell line from acute lymphoid leukemia cells, through an apoptotic process. PaDef inhibited cell viability in a concentration-dependent manner, with an IC50 = 47.3 μM. Treatment of Jurkat cells with PaDef (IC50) induced cell death by apoptosis dependent on caspases 8 and 9; besides, it was related to an increase in the production of reactive oxygen species and the loss of mitochondrial membrane potential. Interestingly, the inhibition of caspase activation by inhibitors of caspases 8 and 9 does not revert the reduction in viability, suggesting that other mechanisms, in addition to caspase activity, could be participating in the PaDef cytotoxic effect. Also, the modifications in the histone 3 tails induced by PaDef in Jurkat cells were evaluated, specifically acetylation and methylation. PaDef increased global histone 3 acetylation and lysine 9 specific marks (2-fold and up to 4-fold, respectively). These effects correlated with the reduction of the Histone Deacetylase activity (HDAC, ∼50%). Based on methylation marks, PaDef treatment increased lysine 9 di- and tri-methylation tags (2-fold in both cases). The epigenetic modulation induced by PaDef on Jurkat cells could be related to the chromatin compaction-decompaction promoting gene expression or repression; however, further studies are necessary to correlate these marks with the transcription of specific genes. Therefore, the study of new molecules that may have anticancer activity through epigenetic modulation is interesting.

Switch receptor T3/28 improves long-term persistence and antitumor efficacy of CAR-T cells

BackgroundChimeric antigen receptor (CAR) T cells have been successfully used in tumor immunotherapy due to their strong antitumor responses, especially in hematological malignancies such as B cell acute lymphoid leukemia....

Catheter Related Bloodstream Infection by Brevibacterium casei in a Patient with B Cell Acute Lymphoid leukemia - A Case Report

Patients with long term indwelling catheters with underlying immunosuppression or comorbid conditions are predisposed to develop catheter related blood stream infections with unusual organisms. Brevibacterium spp. are catalase-positive, non spore-forming, non-motile, aerobic, Gram-positive bacteria. Brevibacterium spp werenot considered a human pathogen, until recently few infections were noted. We report a case of catheter related blood stream infection by Brevibacterium casei in 17 year old young adult with B cell acute lymphoid leukemia. Patient was treated successfully with intravenous Vancomycin and Piperacillin-tazobactam along with peripherally inserted central catheter removal. Keywords: Brevibacterium casei, Catheter related blood stream infections, Sepsis, Immunocompromised

The lncRNA TEX41 is upregulated in pediatric B-Cells Acute Lymphoblastic Leukemia and it is necessary for leukemic cell growth

BackgroundLong non-coding RNAs (lncRNAs) represent a diverse class of RNAs involved in the regulation of various physiological and pathological cellular processes, including transcription, intracellular trafficking, and chromosome remodeling. LncRNAs deregulation was linked to the development and progression of various cancer types, such as acute leukemias. In this context, lncRNAs were also evaluated as a novel class of biomarkers for cancer diagnosis and prognosis. Here, we analyzed TEX41 in childhood B cell acute lymphoid leukemia (B-ALL).MethodsTotal RNA was extracted from pediatric B-ALL patients (at diagnosis and after induction of therapy) and from healthy subjects. Total RNA was also extracted from different leukemia cell line models. The expression level of TEX41 was evaluated by q-RT-PCR. Also, the dataset deposited by St. Jude Children’s Research Hospital was consulted. Furthermore, the silencing of TEX41 in RS4;11 cell line was obtained by 2′-Deoxy, 2′Fluroarabino Nucleic Acids (2′F-ANAs) Oligonucleotides, and the effect on cell proliferation was evaluated. Cell cycle progression and its regulators were analyzed by flow cytometry and immunoblotting.ResultsWe exploited the St Jude Cloud database and found that TEX41 is a lncRNA primarily expressed in the case of B-ALL (n = 79) while its expression levels are low/absent for T-cell ALL (n = 25) and acute myeloid leukemia (n = 38). The association of TEX41 with B-ALL was confirmed by real-time PCR assays. TEX41 disclosed increased expression levels in bone marrow from patients with B-ALL at diagnosis, while its expression levels became low or absent when retested in Bone Marrow cells of the same patient after 1 month of induction therapy. Also, silencing experiments performed on RS4;11 cells showed that TEX41 downregulation impaired in vitro leukemic cell growth determining their arrest in the G2-M phase and the deregulation of cell cycle proteins.ConclusionsOur findings highlight that TEX41 is an upregulated lncRNA in the case of B-ALL and this feature makes it a novel potential biomarker for the diagnosis of this leukemia subtype in pediatric patients. Finally, TEX41 expression seems to be critical for leukemic proliferation, indeed, silencing experiments targeting TEX41 mRNA in the RS4;11 cell line hampered in vitro cell growth and cell cycle progression, by inducing G2-M arrest as confirmed propidium iodide staining and by the upregulation of p53 and p21 proteins.

Anti-CD22 CAR-T Cell Therapy as a Salvage Treatment in B Cell Malignancies Refractory or Relapsed After Anti-CD19 CAR-T therapy.

BackgroundTo observe efficacy of the anti-CD22 chimeric antigen receptor modified (anti-CD22-CAR) T cell salvage therapy in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and B cell acute lymphoid leukemia (B-ALL) patients whose disease did not reach CR or progressed again after anti-CD19-CAR T cell therapy.MethodsIn our study, seven R/R DLBCL patients reached stable disease (SD) or progression of disease (PD) after their anti-CD19-CAR T cell therapy. Only three in all the six R/R B-ALL patients obtained complete response (CR)/CR with incomplete count recovery (Cri) in their anti-CD19-CAR T cell therapy, but they relapsed again in the following three, six and one months. Then, all these thirteen R/R DLBCL and B-ALL patients received anti-CD22 CAR-T cell salvage therapy because their disease did not reach CR or progressed again.ResultsFour R/R DLBCL patients obtained CR, while two R/R DLBCL patients achieved PR and one patient achieved SD. But only two R/R B-ALL patients obtained Cri in their anti-CD22 CAR-T cell salvage therapy. The overall survival (OS) of R/R DLBCL patients after the anti-CD22 CAR-T cell therapy was 6.142±3.395 months until August 31, 2020. There was no different of the median expansion peaks of the two kinds of CAR T cells (P=0.920). The time of anti-CD22-CAR T cell proportion peak days was later than that of the time of anti-CD19-CAR T cell peak days post infusion (P=0.022). Their cytokine release syndrome (CRS) was graded 2–4 in their anti-CD19-CAR T cell therapy, while the notable CRS was graded 1–2 in their anti-CD22-CAR T cell therapy. But there was no difference in the CRS and the immune effect or cell associated neurotoxic syndrome (ICANS) grades in the two kinds of therapies. And there was no difference in the hematological toxicity grades in the two kinds of therapies.ConclusionThe anti-CD22-CAR T cell salvage therapy is highly effective in R/R DLBCL patients than in R/R B-ALL patients who failed in anti-CD19-CAR T cell therapy before. We need to expand the number of R/R DLBCL or B-ALL patients and continue to observe.Trial RegistrationChiCTR-ONN-16009862 and ChiCTR1800019298.

Clonal Competition of Cancer Cells During Acute Lymphoblastic Leukemia Treatment

The importance of cancer cell heterogeneity is increasingly recognized. However, the interaction of different cancer cells remains poorly understood, particularly in response to therapeutic treatment. Past studies used naturally occurring genetic mutations to examine the heterogeneity and evolutionary trajectories of cancer cells. As these mutations occur at different times, clones marked by these mutations cannot be directly compared to assess cellular interactions. In this study, we use engineered genetic barcodes to track patient derived acute lymphoid leukemia (ALL) cells in a novel mouse model that closely mimics the ‘intensive’ and ‘maintenance’ chemo-therapy regiments used in clinical treatment.Intensive and maintenance chemotherapy are commonly combined together in ALL treatment. After the combination treatment, ALL patients who relapse early (<36 months from diagnosis) are particularly resistant to additional chemotherapy and have a very poor prognosis. In contrast, a fraction of ALL patients who relapse late (>36 months from diagnosis) have a much better prognosis, and remain chemo-sensitive. It is unclear why ALL patients who relapse late are chemo-sensitive while patients who relapse early are chemo-resistant. We hypothesize that low intensity maintenance therapy allows “beneficial competitor cells” to survive and outcompete chemo resistant cancer clones. These beneficial competitor cells expand slowly during the maintenance treatment. They are chemo sensitive and are easily eradicated by intensive therapy.To test our hypothesis, we took primary human B-ALL cells and genetically barcoded them before transplantation into immune deficient NSG mice. We established a chemotherapy treatment protocol in mice that closely resembles the ‘intensive’ and ‘maintenance’ phase of ALL treatment currently used in clinic. In our experiment, mice were first treated with a four-week ‘intensive’ phase of chemotherapy consisting of Vincristine, Dexamethasone and PEG L-Asparaginase. This intensive phase was followed by a long-term ‘maintenance’ chemotherapy phase consisting of Methotrexate.We found that ‘intensive’ therapy did not significantly change the clonal composition in mice. However, ‘new’ leukemia clones arose during ‘maintenance’ treatment. These clones might have been suppressed during the undisturbed progression of the disease, and they may play a crucial role in the relapse of the disease during the long-term ‘maintenance’ treatment.To investigate the growth phenotype of chemotherapy surviving ALL clones, we used a competitive transplantation assay. We took surviving ALL clones from ‘intensive’ only and ‘intensive + maintenance’ treated xenografts and co-transplanted these cells with untreated ALL cells. As predicted by our hypothesis, we found that ALL clones that survived ‘intensive’ only treatment exhibited a more aggressive growth phenotype compared to untreated ALL clones. Conversely, ALL clones that survived ‘intensive + maintenance’ treatment grew more slowly than untreated clones. More importantly, the growth of the co-transplanted untreated ALL clones was significantly suppressed by the presence of ALL clones that had survived intensive treatment. In contrast, the growth of the co-transplanted untreated ALL clones was significantly accelerated in the presence of ALL clones that survive the ‘intensive + maintenance’ treatment, suggesting that new clones that expanded during the long-term maintenance treatment were outcompeted by other clones in the absence of maintenance treatment.Together, our findings suggest that the clonal competition of cancer cells plays an important role in determining treatment outcome. In particular, the benefit of maintenance therapy to ALL patients may arise from cellular competition. Further characterization of the beneficial competitor cells may help to improve ALL treatment and may also help to improve the treatment of other types of cancer. DisclosuresMerchant:Pfizer: Consultancy, Research Funding.

In Search of an Ideal CAR-T Cell Antigen Target.

Chimeric antigen receptor T (CAR-T) cells are proving their value in hematological cancers such as B cell acute lymphoid leukemia (B-ALL). This success is in great part due to the chosen target antigen, the lineage marker CD19, that is expressed by leukemia blasts and B lymphocytes, which are not crucial. For solid tumors, the challenge is greater because antigen expression is highly heterogeneous within the tumor and even an efficient CAR-T strategy would not kill all tumor cells. Also, many antigens are shared between solid tumors and healthy cells, causing off-target cell lysis and dangerous collateral damage. New antigen sources are emerging as targets, such as viruses, endogenous viruses, and immune checkpoint molecules. New technologies are in search of the ideal target, with antigen combinations the leading candidates.

Urtica dioica agglutinin (a plant lectin) has a caspase-dependent apoptosis induction effect on the acute lymphoblastic leukemia cell line

Urtica dioica agglutinin (UDA) is a very small plant lectin with anti-prostatic activity. In this study, we investigated the effect of UDA on proliferation and apoptosis induction in human acute lymphoid leukemia (ALL) cell lines. The effect of UDA on Jurkat and Raji cell proliferation was examined by MTS assay. Distribution of cell cycle phases was determined by PI staining and apoptosis was examined with annexin V/PI and western blot. Results showed UDA treatment reduced cell proliferation in cells by inducing apoptosis. PI staining was associated with a higher percentage of the cell population in sub G1. Caspase-8 and caspase-9 dependent apoptosis occurred in Jurkat cells. Generally, UDA treatment resulted in cell death in ALL cell lines and induced apoptosis in the T-ALL cell line, Jurkat, through extrinsic and intrinsic pathways. These results may be considered as a guide to working on UDA as an anti-leukemic drug in the future.

Anti-leukemic activity of semisynthetic derivatives of lupeol

In our previous work, lupeol was isolated from aerial parts of V. scorpioides and modified by semisynthetic approach. The purpose of this study was to investigate the cytotoxicity of lupeol and its derivatives previously prepared on the human K562 acute myeloid leukemia cell and human Jurkat acute lymphoid leukemia cell in vitro. Compounds 3β-hydroxylup-20(29)-en-30-al (2), lup-20(30)-en-3β,29-diol (3), 3β-acetoxylup-20(29)-en-30-al (5) and 3β-acetoxy-30-hydroxylup-20(29)-ene (6) presented cytotoxicity with IC50 ranging from 11.72 to 56.15 µM at 24 h of incubation for both cell lines. Most of the active compounds (3, 5 and 6) were selective to leukemia cells, in compare with healthy cells. The hemolysis assay showed high blood compatibility of the cytotoxic lupeol derivatives which makes possible an intravenous administration of these compounds aiming to the potential to development of anti-leukemic drugs.

N-MYC INDUCES VARIOUS TYPES OF MOUSE ACUTE LEUKEMIA

N-Myc plays a role in the regulation of proliferation, differentiation, and survival of hematopoietic stem cells (HSCs) and leukemogenesis. We studied the cell of origin in the mouse acute leukemia model by N-Myc-overexpression. Our single cell RNA-seq data showed that N-Myc is restrictively expressed in HSCs and hematopoietic progenitor cells (HPCs) whereas c-Myc, but not L-Myc, is widely expressed in a variety of hematopoietic cells. HSC1, HSC2, HPC1, HPC2, and HPC3 with different self-renewal and differentiation potentials were isolated from the bone marrow of adult B6 mice. These cells were retrovirally overexpressed with N-Myc and transplanted into lethally irradiated mice with competitor cells. Acute leukemia developed from all these populations. Notably, not only B cell acute lymphoid leukemia (B-ALL), but also T cell ALL (T-ALL), acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and mixed phenotype acute leukemia (MPAL) developed. Metascape analysis of RNA-seq data showed that RNA profiles each from B-ALL, T-ALL, AML and AUL samples were enriched in special cellular signaling pathways. Collectively, the multiple cells of origin suggested that the self-renewal and differentiation potentials of cells were not related to leukemogenesis induced by N-Myc-overexpression. In addition, whole genome sequencing data suggested that retroviral integration sites play a role in the development of different types of leukemia.

Abstract B035: Enhancing antitumoral activity of cell-based immunotherapies by modulating the JAK-STAT axis

Abstract Mounting a T helper 1 (Th1) type of response is required for the successful priming of antigen specific CD8 T-cells which ultimately lead to effective antitumoral responses. Several cellular components of the immune system participate of the Th1 decision-making process. They include innate cells sensing the transformed cells, mainly dendritic cells (DCs), and the further involvement of antigen specific CD4 T-cells promoting direct activation of CD8 T-cells through cross-presentation (1). The correct stimulation of DCs by type I interferons and molecular pattern sensors as STING is required to drive their differentiation to conventional DC 1 (cDC1) which through the secretion of large amounts of IL-12 and the expression of CD40 skew the CD4 T-cells differentiation to Th1 (2). However, this system is sensitive to modifications of the tumor cytokine microenvironmen,t leading to failure. Adoptive cell transfer (ACT) cancer immunotherapies are promising treatments for advanced malignancies. These therapies attempt to supply key cellular actors missing and induce proper antitumoral immunity. They include mainly the in vitro modification of autologous cells, from DCs loaded with tumor antigens to the expression of chimeric antigen receptor (CAR) on T and NK cells. Constant development of these technologies has given successful results as clinical trials have already showed up to 90 % of complete remission in relapsed/refractory B cell acute lymphoid leukemia (B-ALL) (3), treatment now approved for commercial use. However, there is still lack of consistency in the responses obtained from different individuals and in different trials. Previously our laboratory has already shown that modulation of the JAK-STAT inhibitory protein tyrosine phosphatases (PTPs), PTPN1 and PTP-N2, enhance proinflammatory type I interferon signalling while decreases the effects of immunosuppressive cytokines acting through STAT3 signaling (4, 5). Specifically, we have demonstrated that partial inhibition of these phosphatases in immune cells enhances the secretion of IL-12p70 by DCs[6] and increases the cytotoxic activity of antigen specific CD8 T-cells [Pérez-Quintero LA, Tremblay ML, unpublished results]. Moreover, the partial inhibition of these phosphatases in CD8 T-cells enhance the acquisition of a central memory (Tcm) phenotype, which has been found to be associated with better remission in CAR-T-cells therapies. Nevertheless, alternative methods to enrich this phenotype, as cytokine cocktails, have proven expensive and ineffective. Having this in mind, we propose here the use of small-molecule inhibitors specific for PTPN1 and PTPN2 as a simple and cost-effective method to enhance antitumoral responses. By treating ex vivo the cellular products in animal models for DCs and CD8 ACT therapies we show, as a proof of concept, a marked improvement on the reduction of tumor burden and remission, with the late goal of translating these findings into a clinical setup.

Memantine potentiates cytarabine-induced cell death of acute leukemia correlating with inhibition of Kv1.3 potassium channels, AKT and ERK1/2 signaling

BackgroundTreatment of acute leukemia is challenging and long-lasting remissions are difficult to induce. Innovative therapy approaches aim to complement standard chemotherapy to improve drug efficacy and decrease toxicity. Promising new therapeutic targets in cancer therapy include voltage-gated Kv1.3 potassium channels, but their role in acute leukemia is unclear. We reported that Kv1.3 channels of lymphocytes are blocked by memantine, which is known as an antagonist of neuronal N-methyl-D-aspartate type glutamate receptors and clinically applied in therapy of advanced Alzheimer disease. Here we evaluated whether pharmacological targeting of Kv1.3 channels by memantine promotes cell death of acute leukemia cells induced by chemotherapeutic cytarabine.MethodsWe analyzed acute lymphoid (Jurkat, CEM) and myeloid (HL-60, Molm-13, OCI-AML-3) leukemia cell lines and patients’ acute leukemic blasts after treatment with either drug alone or the combination of cytarabine and memantine. Patch-clamp analysis was performed to evaluate inhibition of Kv1.3 channels and membrane depolarization by memantine. Cell death was determined with propidium iodide, Annexin V and SYTOX staining and cytochrome C release assay. Molecular effects of memantine co-treatment on activation of Caspases, AKT, ERK1/2, and JNK signaling were analysed by Western blot. Kv1.3 channel expression in Jurkat cells was downregulated by shRNA.ResultsOur study demonstrates that memantine inhibits Kv1.3 channels of acute leukemia cells and in combination with cytarabine potentiates cell death of acute lymphoid and myeloid leukemia cell lines as well as primary leukemic blasts from acute leukemia patients. At molecular level, memantine co-application fosters concurrent inhibition of AKT, S6 and ERK1/2 and reinforces nuclear down-regulation of MYC, a common target of AKT and ERK1/2 signaling. In addition, it augments mitochondrial dysfunction resulting in enhanced cytochrome C release and activation of Caspase-9 and Caspase-3 leading to amplified apoptosis.ConclusionsOur study underlines inhibition of Kv1.3 channels as a therapeutic strategy in acute leukemia and proposes co-treatment with memantine, a licensed and safe drug, as a potential approach to promote cytarabine-based cell death of various subtypes of acute leukemia.

KML001 and doxercalciferol induce synergistic antileukemic effect in acute lymphoid leukemia cells.

KML001 (NaAsO2, sodium metaarsenite, KOMINOX), a kind of arsenic compound, that has shown promising efficacy in non-Hodgkin's lymphoma(NHL) both invitro and invivo. In our study, the antileukemic effect of KML001 on acute lymphoid leukemia(ALL) and its mechanism of action were investigated. The results showed that KML001 inhibited cell proliferation in two types of ALL cell lines, CCRF-CEM and Molt-4. Exposure of ALL cells to KML001 induced apoptosis in a time-dependent manner. KML001 caused cell cycle arrest at G2/M phase instead of G0/G1 phase shown in other leukemia cells. In addition, we also tested the possibility of synergy of KML001 with doxercalciferol, a vitaminD2 derivative. Also, we found that a combination of KML001 with doxercalciferol showed a synergistic effect on ALL cell lines and this could be due to its different mechanism of action. Overall, our findings demonstrated KML001 could be a promising antileukemic agent especially when it is combined with doxercalciferol in ALL treatment.

CD47 Blockade Enhances Therapeutic Activity of TCR Mimic Antibodies to Ultra-Low Density Cancer Epitopes through Cytokine Feed Forward Mechanisms

INTRODUCTIONT Cell Receptor mimic (TCRm) antibodies to low-density peptide epitopes from undruggable intracellular proteins presented in the context of major histocompatibility (MHC) molecules are therapeutically effective in mouse models of human cancers. CD47 blockade by use of a high affinity SIRPα variant protein (CV1) has been shown to improve the effects of monoclonal antibodies to high-density antigens in tumor models by enhancement of antibody dependent cellular phagocytosis (ADCP). We asked if combination therapy with a TCRm antibody to Preferentially Expressed Antigen in Melanoma (PRAME) could enhance activity of both drugs in vitro and in vivo. Additionally, we explored the role of macrophage-secreted cytokines in the enhanced in vivo activity.METHODSWe performed in vitro ADCP assays with human acute myeloid and acute lymphoid leukemia cell lines containing antigens of interest using the two agents alone and in combination. We performed therapy experiments in NSG mice using the same leukemia cell lines transformed with a luciferase vector and measured tumor burden through bioluminescent imaging. Survival was measured. We examined cell-surface expression of epitopes of interest and HLA on cell lines in vitro after incubation with IFNγ and TNFα using flow cytometry and performed in vitro ADCP assays with the leukemia cell lines after pretreatment with IFNγ.RESULTSCV1 and TCRm antibody showed additive effects in vitro with a statistically significant increase in phagocytosis in both antigen positive cell lines with combination therapy versus single agent therapy. CV1 and TCRm antibody showed greater than additive therapeutic effects in vivo with a 3-log reduction in leukemia burden relative to control untreated mice and a 5-10 fold reduction relative to single agent groups. After therapy was stopped, mice treated with the combination had statistically significant increases in survival (p<0.0001). IFNγ and TNFα led to up-regulation of cell surface HLA-A*02:01. Additionally, the cytokines led to up-regulation of the PRAME derived epitope of interest. Pretreatment of human leukemia cell lines with IFNγ led to statistically significant increases in ADCP in vitro.CONCLUSIONSThe elimination of anti-phagocytic signal produced by CD47 blockade with the high affinity SIRPα variant CV1, combined with the pro-phagocytic signal of Fc receptor engaging TCRm was effective even with an ultra-low density epitope (700-3000 sites per cell) in vitro and in vivo. A greater than additive effect was seen in two tumor models. These results support the potency of this drug combination. The large enhancement in activity in vivo vs. in vitro may be explained by macrophage-released cytokines leading to increased presentation of epitopes of interest and increased tumor kill. DisclosuresLiu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties. Garcia:Alexo Therapeutics: Equity Ownership.

Targeting Replicative Stress to Treat Hematological Disorders

▪Background: Combinations of chromosomal translocations, copy-number variations, somatic mutations, and clonal heterogeneity that characterize hematological cancers make every patient unique from a genetic point of view. This variety creates a true challenge for tailored therapy. We have previously described that myeloma (MM) cells present signs of ongoing DNA damage, and activate an ATM/ABL1-dependent DNA damage response (DDR) without overt apoptosis. Here we further characterize the mechanisms of DNA damage and replicative stress in MM, and we extend this knowledge to other hematological malignancies to evaluate a novel and possibly shared approach to synthetic lethality (1).Results: We studied a panel of MM cell lines together with acute myeloid, lymphoid leukemia and lymphoma cell lines. Several cell lines have demonstrable ongoing DNA damage, activate ATR and CHK1 and also present with signs of replicative stress, such as 53BP1, RPA and RAD51 foci. We next evaluated a gene expression signature specific for increased chromosomal instability and DNA damage in a cohort of MM patients, comparing them with normal plasma cells. Specifically, we identified a subset of patients, representing around 20 percent of individuals with MM that show this signature and also present with an unfavorable prognosis due to a more aggressive disease. Interestingly, in a multivariate analysis, this signature was independent from other poor prognostic criteria, including proliferation and the presence of MMSET/FGFR3 or MAF translocations (2), hence representing a potential novel prognostic signature (3). Gene-set enrichment studies are ongoing in other hematological disorders. However, preliminary data show that subsets of patients with other hematological malignancies also present with intense over expression of genes belonging to the instability signature when compared to normal B cells, in a very similar fashion to that seen with MM. An intact ATR/CHK1 pathway is crucial for the survival of tumor cells in vivo, especially in the presence of activated oncogenes. For instance, Em-myc transgenic mice develop B-cell lymphomas with intense replicative stress that can be blocked by crossing the Em-myc transgenic mice with a hypomorphic Atr mouse strain (Atr-Seckel; Atr S/S). We therefore decided to exploit the concept of replicative stress overload, impeding the capacity of the cells for repairing the excess of damaged DNA. We then silenced ATR using shRNAs, the upstream protein involved in the control of stalled replication origins, in two MM cell lines (H929 and OPM-2) and in the Jurkat cell line. Critically, inhibition of ATR caused a reduction in cellular growth and induction of apoptosis. A similar phenotype was observed using VE-821, a specific ATR inhibitor. Finally, a broad panel of MM cells and leukemia cell lines was used to confirm these growth inhibitory effects.Conclusion: Replicative stress is present in multiple groups of patients with aggressive types of MM or leukemia. Strategies which couple pre-existing high rates of DNA damage with reduced DNA repair can specifically cause apoptosis of malignant cells and encouragingly spare normal ones, thus providing a strong rationale for potential clinical benefit to those cohorts of patients with otherwise very unfavorable outcomes.(1) Cottini F, Hideshima T, Xu C, Sattler M, Dori M, Agnelli L, et al. Rescue of Hippo coactivator YAP1 triggers DNA damage-induced apoptosis in hematological cancers. Nature medicine. 2014;20(6):599-606.(2) Zhan F, Huang Y, Colla S, Stewart JP, Hanamura I, Gupta S, et al. The molecular classification of multiple myeloma. Blood. 2006;108(6):2020-8.(3) Cottini F, Teru Hideshima, Rikio Suzuki, Yu-Tzu Tai, et al. Synthetic lethal approaches exploiting DNA damage in aggressive myeloma. Cancer Discovery ahead of print. DisclosuresRichardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene Corporation: Consultancy; Oncocorp: Equity Ownership; acetylon pharmaceuticals: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy.

Hydrodynamic self-focusing in a parallel microfluidic device through cross-filtration.

The flow focusing is a fundamental prior step in order to sort, analyze, and detect particles or cells. The standard hydrodynamic approach requires two fluids to be injected into the microfluidic device: one containing the sample and the other one, called the sheath fluid, allows squeezing the sample fluid into a narrow stream. The major drawback of this approach is the high complexity of the layout for microfluidic devices when parallel streams are required. In this work, we present a novel parallelized microfluidic device that enables hydrodynamic focusing in each microchannel using a single feed flow. At each of the parallel channels, a cross-filter region is present that allows removing fluid from the sample fluid. This fluid is used to create local sheath fluids that hydrodynamically pinch the sample fluid. The great advantage of the proposed device is that, since only one inlet is needed, multiple parallel micro-channels can be easily introduced into the design. In the paper, the design method is described and the numerical simulations performed to define the optimal design are summarized. Moreover, the operational functionality of devices tested by using both polystyrene beads and Acute Lymphoid Leukemia cells are shown.

Blinatumomab: a bispecific T cell engager (BiTE) antibody against CD19/CD3 for refractory acute lymphoid leukemia.

Targeted therapy has been the forefront of cancer treatment. Cancer immunotherapy is the most recent focus. In addition, novel immunotherapeutics targeting B cell receptor signaling (e.g., ibrutinib), T cell receptor ( e.g., CART19), and NK cells (e.g., AFM13) are being developed. This review summarized the new development in blinatumomab (MT103/MEDI-538), a first-in-class bispecific T engager (BiTE) antibody against CD19/CD3 in patients with relapsed/refractory precursor B cell acute lymphoid leukemia.