TPEN Selectively Eliminates Lymphoblastic B Cells from Pediatric Acute Lymphoblastic Lleukemia Patients

B-acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by abnormal proliferation and accumulation of immature B-lymphoblast arrested at various differentiation stages. Despite some advances in treatment, there is still an important percentage of pediatric patients with precursor-B ALL who relapsed. Therefore, alternative therapies are needed to improve cure rates for pediatric patients. TPEN is a pro-oxidant agent capable of selectively inducing apoptosis in leukemia cells. Consequently, TPEN has been suggested as a potential agent for oxidative therapy. However, it is not yet known whether TPEN can selectively destroy leukemia cells in a more disease-like milieu e.g., bloodstream and bone marrow (BM) in vivo. In this investigation, we report for the first time that TPEN significantly induces apoptosis in CD34+/CD19+ cells from whole bone marrow de novo B-ALL (n=5) and refractory B-ALL (n=6) patients by oxidative stress (OS, n=8). We found that TPEN significantly increased not only positive cell counts for the oxidation of the stress sensor protein DJ-1 as a sign of the formation of H2O2, but also significantly increased positive cell counts for the proapoptotic protein TP53, PUMA, and CASPASE-3 as indicative of apoptosis in B-ALL cells irrespective of diagnostic status (de novo or refractory) and sex. Understanding the TPEN-induced cell death in leukemia cells provides insight into more effective therapeutic prooxidant-inducing anticancer agents.

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TPEN Selectively Eliminates Lymphoblastic B cells from Bone Marrow Pediatric Acute Lymphoblastic Leukemia Patients

10.20944/preprints202103.0611.v2 ◽

2021 ◽

Author(s):

Miguel Mendivil-Perez ◽

Carlos Velez-Pardo ◽

Lina Maria Quiroz-Duque ◽

Alexandra Restrepo-Rincon ◽

Natalia Andrea Valencia-Zuluaga ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Anticancer Agents ◽

Pediatric Patients ◽

De Novo ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Leukemic Cell ◽

Leukemia Cells

B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by the abnormal proliferation and accumulation of immature B-lymphoblasts arrested at various stages of differentiation. Despite advances in treatment, a significant percentage of pediatric patients with precursor B-ALL still relapse. Therefore, alternative therapies are needed to improve the cure rates for pediatric patients. TPEN (N, N, N’, N’-tetrakis(2-pyridylmethyl)-ethylenediamine).is a pro-oxidant agent capable of selectively inducing apoptosis in leukemia cells. Consequently, it has been suggested that TPEN could be a potential agent for oxidative therapy. However, it is not yet known whether TPEN can selectively destroy leukemia cells in a more disease-like model, for example, the bloodstream and bone marrow (BM), in vitro. This investigation is an extension of a previous study that dealt with the effect of TPEN on ex vivo isolated/purified refractory B-ALL cells. Here, we evaluated the effect of TPEN on whole BM from nonleukemic patients (control) or pediatric patients diagnosed with de novo B-ALL or refractory B-ALL cells by analyzing the hematopoietic cell lineage marker CD34/CD19. Although TPEN was innocuous to nonleukemic BM (n=3), we found that TPEN significantly induced apoptosis in de novo (n = 5) and refractory B-ALL (n = 6) leukemic cell populations. Moreover, TPEN significantly increased the counts of cells positive for the oxidation of the stress sensor protein DJ-1, a sign of the formation of H2O2, and significantly increased the counts of cells positive for the pro-apoptotic proteins TP53, PUMA, and CASPASE-3 (CASP-3), indicative of apoptosis, in B-ALL cells. We demonstrate that TPEN selectively eliminates B-ALL cells independent of age, diagnosis status (de novo or refractory), sex, karyotype, or immunophenotype. Understanding TPEN-induced cell death in leukemia cells provides insight into more effective therapeutic oxidation-inducing anticancer agents.

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Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽

10.1182/blood-2020-140060 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 21-21

Author(s):

Gisele Olinto Libanio Rodrigues ◽

Julie Hixon ◽

Hila Winer ◽

Erica Matich ◽

Caroline Andrews ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cell Line ◽

Lymphoblastic Leukemia ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

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Xenografts of Pediatric Acute Lymphoblastic Leukemia Retain the Gene Expression Pattern From the Diagnostic Material They Originated From Over Serial Passages.

Blood ◽

10.1182/blood.v114.22.1629.1629 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1629-1629

Author(s):

Manon Queudeville ◽

Elena Vendramini ◽

Marco Giordan ◽

Sarah M. Eckhoff ◽

Giuseppe Basso ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Leukemia Cells ◽

Diagnostic Samples ◽

Xenotransplantation Model

Abstract Abstract 1629 Poster Board I-655 Primary childhood acute lymphoblastic leukemia (ALL) samples are very difficult to culture in vitro and the currently available cell lines only poorly reflect the heterogeneous nature of the primary disease. Many groups therefore use mouse xenotransplantation models not only for in vivo testing but also as a means to amplify the number of leukemia cells to be used for various analysis. It remains unclear as to what extent the xenografted samples recapitulate their respective primary leukemia. It has been suggested for example that transplantation may result in the selection of a specific clone present only to a small amount in the primary diagnostic sample. We used a NOD/SCID xenotransplantation model and injected leukemia cells isolated from fresh primary diagnostic material of 4 pediatric ALL patients [2 pre-B-ALL, 1 pro-B-ALL (MLL/AF4}, 1 cortical T-ALL] intravenously into the lateral tail vein of unconditioned mice. As soon as the mice presented clinical signs of leukemia, leukemia cells were isolated from bone marrow and spleen. Isolated leukemia cells were retransplanted into secondary and tertiary recipients. RNA was isolated from diagnostic material and serial xenograft passages and gene expression profiles were obtained using a human whole genome array (Affymetrix U133 2.0). Simultaneously, immunophenotypic analysis via multicolor surface and cytoplasmatic staining by flow cytometry was performed for the diagnostic samples and respective serial xenograft passages. In an unsupervised clustering analysis the diagnostic sample of each patient clustered together with the 3 derived xenograft samples, although the 3 xenograft samples clustered stronger to each other than to their respective diagnostic sample. Comparison of the 4 diagnostic samples vs. all xenograft samples resulted in a gene list of 270 genes upregulated at diagnosis and 8 genes upregulated in the xenograft passages (Wilcoxon, p< .05). The high number of genes upregulated at diagnosis is most likely due to contamination of primary patient samples with normal peripheral blood and/or bone marrow cells as 15% of genes are attributed to myeloid cells, 7% to erythroid cells, 7% to lymphoid cells, 32% to bone marrow in general as well as to innate immunity, chemokines, immunoglobulins. The remaining genes can not be attributed to a specific hematopoetic cell lineage and are not known to be related to leukemia or cancer in general. Accordingly, there are no statistically significant differences between the primary, secondary and tertiary xenograft passages. The immunophenotype analysis are also in accordance with these findings, as the diagnostic blast population retains its immunophenotypic appearance during serial transplantation, whereas the contaminating CD45-positive non- leukemic cells disappear after the first xenograft passage. The few genes upregulated in xenograft samples compared to diagnosis are mainly involved in cell cycle regulation, protein translation and apoptosis resistance. Some of the identified genes have already been described in connection with cancer subtypes, their upregulation therefore being indicative of a high proliferative state in general and could argue towards a more aggressive potential of the engrafted leukemia cells but alternatively could also simply be due to the fact that the xenograft samples are pure leukemic blasts and are not contaminated with up to 15% of non-cycling healthy bone marrow cells as in the diagnostic samples. We conclude that the gene expression profiles generated from xenografted leukemias are very similar to those of their respective primary leukemia and moreover remain stable over serial retransplantation passages as we observed no statistically significant differences between the primary, secondary and tertiary xenografts. The differentially expressed genes between diagnosis and primary xenotransplant are most likely to be due to contaminating healthy cells in the diagnostic samples. Hence, the NOD/SCID-xenotransplantation model recapitulates the primary human leukemia in the mouse and is therefore an appropriate tool for in vivo and ex vivo studies of pediatric acute leukemia. Disclosures No relevant conflicts of interest to declare.

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Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Blood ◽

10.1182/blood-2018-99-120120 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1322-1322

Author(s):

Manabu Kusakabe ◽

Ann Chong Sun ◽

Kateryna Tyshchenko ◽

Rachel Wong ◽

Aastha Nanda ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Research Funding ◽

De Novo ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Rna Seq ◽

Cell Acute Lymphoblastic Leukemia

Abstract Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

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Identification of Gene Expression Profiles in Acute Lymphoblastic Leukemia Cells That Discriminate Intracellular Thioguanine Nucleotide Accumulation in ALL Cells after In Vivo Treatment with Mercaptopurine.

Blood ◽

10.1182/blood.v104.11.453.453 ◽

2004 ◽

Vol 104 (11) ◽

pp. 453-453

Author(s):

Gianluigi Zaza ◽

Meyling Cheok ◽

Wenjian Yang ◽

Pei Deqing ◽

Cheng Cheng ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Post Treatment ◽

Leukemia Cells ◽

True Positive

Abstract Thioguanine nucleotides (TGN) are considered the principal active metabolites exerting the antileukemic effects of mercaptopurine (MP). Numerous clinical studies have reported substantial inter-patient variability in intracellular TGN concentrations during continuation therapy of acute lymphoblastic leukemia (ALL). To identify genes whose expression is related to the intracellular accumulation of TGN in leukemia cells after in vivo treatment with MP alone (MP) or in combination with MTX (MP+MTX), we used oligonucleotide microarrays (Affymetrixâ HG-U95Av2) to analyze the expression of approximately 9,670 genes in bone marrow leukemic blasts obtained at diagnosis from 82 children with ALL. TGN levels were determined in bone marrow aspirates of these patients 20 hours after mercaptopurine infusion (1 g/m2 I.V). Because, as previously reported, patients treated with MP alone achieved higher levels of intracellular TGN compared to those treated with the combination, we used Spearman’s rank correlation to identify genes associated with TGN levels separately for the 33 patients treated with MP alone and the 49 with the combination (MP: median TGN: 2.46 pmol/5x106 cells, range: 0.01–19.98; and MTX+MP: median TGN: 0.55 pmol/5x106 cells, range: 0.005–3.31). Hierarchical clustering using these selected probe sets clearly separated the 33 patients treated with MP alone into two major groups according to TGN concentration (< 2.46 and > 2.46 pmol/5x106 cells; n=60 genes) and two major branches were also found for patients treated with the combination (< 0.55 and > 0.55 pmol/5x106 cells; n=75 genes). Interestingly, there was no overlap between the two sets of genes, indicating that different genes influence the accumulation of TGN when this drug is given alone or in combination with MTX. The association between gene expression profiles and TGN levels determined by leave-one-out cross-validation using support vector machine (SVM) based on Spearman correlation, was rho=0.60 (p<0.001) for MP alone and rho=0.65 (p<0.001) for MTX+MP, with false discovery rate (FDR) computed using Storey’s q-value (MP: 50% true positive, MTX+MP: 70% true positive respectively). Genes highly associated with the post-treatment TGN level in ALL patients treated with MP alone encode transporters, enzymes involved in the MP metabolic pathway and cell proliferation. Genes associated with post-treatment levels of TGN after combined therapy have been implicated in protein and ATP biosynthesis. Together, these in vivo data provide new insights into the basis of inter-patient differences in TGN accumulation in ALL cells, revealing significant differences between treatment with MP alone or in combination with MTX.

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In Vivo Imaging In the Individualized Mouse Model of Acute Lymphoblastic Leukemia Enables Highly Sensitive and Continuous Follow up of Patient-Derived Xenografts

Blood ◽

10.1182/blood.v116.21.3259.3259 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3259-3259

Author(s):

Nadia Terziyska ◽

Katarina Farkasova ◽

Ernst Wagner ◽

Manfred Ogris ◽

Irmela Jeremias

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

In Vivo Imaging ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Lentiviral Transduction ◽

Patient Derived Xenograft ◽

Over Time

Abstract Abstract 3259 In the individualized xenograft mouse model, acute leukemia cells from patients are transplanted into severely immuno-deficient mice to serve as a preclinical animal model. The use of this mouse model for pre-clinical therapy trials is hampered by the low sensitivity of existing readouts disabling the reliable follow up of single animals. Both secretion of leukemic cells into the blood flow as well as clinical signs, if at all, occur at late states of the disease; readouts in further organs by, e.g., immunohistochemistry or flow cytometry analysis, require organ extraction and can thus be performed only once per mouse. To overcome this obstacle, we established in vivo imaging in the xenograft mouse model of acute lymphoblastic leukemia (ALL). We engrafted pediatric acute leukemia cells from patients at diagnostic bone marrow aspiration of either diagnosis or relapse. In agreement with published data, mice developed leukemia within weeks to months. Engraftment was followed by easy passaging of cells into further generations of mice. We established lentiviral transduction of xenograft cells which enabled expression of transgenes in these cells. Using lentiviral transduction, we stained patient-derived xenograft ALL-cells using luciferase as a reporter. Transgenic xenograft leukemia cells were visualized once per week by bioluminescence in vivo imaging using a charge-coupled device camera. Our first data show that in vivo imaging enabled the reliable and continuous follow up of single animals over time. The sensitivity of in vivo imaging in measuring leukemic engraftment was significantly higher compared to current readouts like examination of blood cells. In vivo imaging data support that the leukemic pattern of metastases of patient-derived xenograft ALL-cells in mice highly resembles the distribution of the disease in men. Upon intravenous injection, cells first home to the liver, where they stay alive only for a few weeks. Long-term engraftment is seen in the bone marrow of many different bones and constantly increases over time. Engraftment in spleen indicates rather late stages of disease. This kinetic of engraftment remained constant between several transgenic xenograft samples and between different mice engrafted with the same sample. Taken together, we have established molecular modulation of xenograft cells which enables expression of luciferase and in vivo bioluminescence imaging as a new sensitive and continuous in vivo readout in mice. In vivo imaging will allow realizing precise preclinical trials in the individualized mouse model in the future. Disclosures: No relevant conflicts of interest to declare.

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Functional Modulation of VLA6 in BCR-ABL1+ Pre-B Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v120.21.2565.2565 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2565-2565

Author(s):

Eun Ji Gang ◽

Yao-Te Hsieh ◽

Huimin Geng ◽

Jennifer Pham ◽

Markus Muschen ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Conditional Knockout ◽

Leukemia Cells

Abstract Abstract 2565 Chemotherapy drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem, resulting in reduced treatment efficacy and relapse. The bone marrow environment (BME) has been shown to promote resistance of leukemia cells towards chemotherapy, which has been attributed to several proteins, including integrins. Our analysis of 207 children with high-risk (BCR/ABL1−) pre-B ALL revealed that high expression of the laminin-binding integrin VLA6 (alpha6beta1) portends poor clinical outcomes in patients with minimal residual disease (MRD+) on day 29 of induction. In addition, our comparative analysis of pre-B leukemia and normal B-cells revealed that VLA6 is preferentially upregulated on BCR/ABL1+ pre-B ALL blasts. Alterations in adhesion properties have been described for BCR/ABL1+ (p210) chronic myeloid leukemia. The role of integrins and integrin VLA6 in particular for cell adhesion-mediated drug resistance (CAM-DR) in BCR/ABL1+ (p210) ALL has not been addressed. With respect to its role for normal immature hematopoietic cells, contradictory observations have been reported. Therefore, we hypothesized that VLA6-mediated adhesion of ALL cells to the bone marrow stromal niche contributes to drug resistance. We evaluated the role of VLA6 in BCR-ABL1+ leukemia using two of our established models of leukemia, a conditional knockout model of VLA6 in murine BCR-ABL1+ leukemia and a xenograft model of human BCR-ABL1+ leukemia. VLA6fl/fl cells were oncogenically transformed using BCR-ABL1 (p210) and cultured under lymphoid-skewing conditions. Induction of pre- B (B220+ CD19+) ALL was confirmed by flow cytometry. Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which VLA6 ablation could be induced (CreERT2) or not (EmptyERT2) by addition of Tamoxifen. Conditional ablation of VLA6 in vitro decreased adhesion significantly compared to undeleted controls (19.7%±8.1% vs. 87.7%±6.0%; p=0.00041) and increased apoptosis of murine BCR-ABL1+ leukemia cells as determined by analysis of Annexin V−/7-AAD− viable cells by flow cytometry (VLA6 deleted vs. undeleted: 35.3%±1.1% vs. 75.1%±1.2%; p=0.0001). Moreover, VLA6 deletion sensitized murine ALL to a tyrosine kinase inhibitor (TKI), Nilotinib (p=0.022, 45.6%±2.4% vs. 73.3%±13.0%). To test the effect of VLA6 deletion on leukemic progression in vivo, VLA6 BCR/ABL1+ pre-B (B220+ CD19+) CreERT2+ or control transduced ALL cells were transferred into NOD/SCID mice. 3 days thereafter, VLA6 deletion was induced by Tamoxifen administration to all animals in 2 cycles for 5 days. In vivo deletion of VLA6 in delayed leukemia progression compared to VLA6 competent controls from a median survival time (MST) of 30 days post-leukemia injection to a MST of 43 days post-leukemia injection (p=0.008 Log-rank test). In vivo deletion of VLA6 in combination with Nilotinib treatment delayed leukemia progression compared to VLA6 competent, as Nilotinib-treated control animals have uniformly died of leukemia with a MST of 39.5 days, however the Nilotinib treated VLA6 deleted group is completely alive and is still being monitored (p=0.0025). When VLA6 was ablated before transfer and recipients were observed for leukemia progression, the recipients of VLA6–deficient murine leukemia cells also showed attenuated leukemia progression compared to recipients of VLA6 competent cells. Moreover, we show that VLA6 blockade de-adheres primary ALL cells from their cognate counter receptor laminin in vitro, and sensitizes primary ALL cells to TKI Taken together, modulating the function of VLA6 in ALL offers a new approach to overcome drug resistance in ALL. Given that VLA6 is probably largely redundant for normal immature hematopoiesis, this approach may be preferable over targeting of other integrins in BCR/ABL1+ leukemias on which VLA6 is expressed. Disclosures: No relevant conflicts of interest to declare.

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Dual Targeting of PI3Kdelta and BRD4 Sensitizes Acute Lymphoblastic Leukemia to Chemotherapy

Blood ◽

10.1182/blood-2018-99-116310 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1420-1420 ◽

Cited By ~ 1

Author(s):

Hye Na Kim ◽

Cydney Nichols ◽

Enzi Ji Jiang ◽

Nour Abdel-Azim ◽

Ariana Coba ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Regulation Of Transcription ◽

Dual Inhibitor ◽

Bet Inhibitor ◽

Phosphorylated Akt

Abstract Introduction: Acute lymphoblastic leukemia is the most prevalent form of cancer affecting children with 2,500-3,500 new cases per year. More effective targeted therapies have yielded a current five-year survival rate of at least 85% for children; however relapsed disease, as well as harsh side effects of treatment, remain prevalent hurdles for many patients. Cells harbored by the bone marrow comprise minimal residual disease that may contribute to later re-expansion of the tumor population following treatment, also known as cell adhesion mediated drug resistance (CAM-DR). Bone marrow stromal cell contact has been shown to upregulate phosphorylated AKT, promoting survival of ALL cells. We investigate a new dual targeted therapy, Morpholinothienopyrane (SF2535), which inhibits both PI3Kdelta and BRD4, each key molecules in signal transduction pathways between microenvironment and leukemia cells. PI3Kdelta is a central nodal molecule in outside-in pathways including cell-cell interactions mediated by integrins. BRD4, a member of the bromodomain and extraterminal domain (BET) family of proteins which bind acetylated lysines at promoter and enhancer regions, is another key player involved in regulation of transcription of myc family transcription factors. In addition to transcriptional regulation by BRD4, myc is also regulated by PI3kdelta which inhibits GSK3beta-mediated degradation of myc. We hypothesize that this dual inhibitor, SF2535, will disrupt signaling between leukemia cells and the bone marrow microenvironment, thus addressing CAM-DR. Methods/Results: In vitro and in vivo BRD4 and PI3Kdelta target downregulation was measured by western blot including assessment of myc, AKT, and phosphorylated AKT. Cells were starved in vitro in serum-free conditions for 18 hours followed by treatment with SF2535 and stimulation with serum. Levels of phosphorylated AKT were noticeably decreased in SF2535 treated cells compared with DMSO control or JQ1 (BET inhibitor) treatment alone. Truncation of microenvironmental outside-in signaling via inhibition of PI3Kdelta and BRD4 was measured via transwell migration assay. Primary ALL cells were cultured on top of a porous membrane with SDF-1alpha in the chamber below. Significantly less primary ALL cells migrated toward SDF-1alpha when treated with SF2535 compared to DMSO, CAL101 (PI3Kdelta inhibitor), or JQ1 (BET inhibitor). The combination of SF2535 with vincristine, dexamethasone, and L-asparaginase (VDL), the backbone of induction therapy for many ALL patients, was evaluated in vitro. Primary tumor cells were co-cultured on irradiated OP-9 murine stromal cells and treated with SF2535, VDL, or SF2535 and VDL combined. Apoptosis in response to treatment was measured via AnnexinV and 7-AAD flow cytometry. The combination of SF2535 and VDL showed significantly more cell death compared to either SF2535 or VDL alone at three concentrations of each treatment. Additionally, CalcuSyn software was used to assess putative additive versus synergistic effects of this combination. CalcuSyn analysis of VDL and SF2535 combination compared to either SF2535 or VDL treatment alone shows synergy (defined as a combination index of 0.3-0.7) between these two therapies used on sample LAX7R after 1 day of treatment. Discussion: Thus far we have shown that this novel inhibitor decreases expression of downstream targets of PI3Kdelta (phosphorylated AKT) and BRD4 (C-myc), inhibits migration of ALL toward bone marrow stromal factors, and increases apoptosis of primary patient samples when combined with VDL compared to VDL alone. Most notably, we have determined a synergistic relationship between SF2535 and VDL in vitro, suggesting a role for this novel therapy in sensitizing cells to chemotherapy. The use of a single agent against both PI3Kdelta and BRD4 ultimately aimed at downregulation of myc levels provides the possibility of more effective, less toxic therapeutic option that addresses CAM-DR faced by many patients. While ongoing experiments in vitro and in vivo will provide a more robust picture of the efficacy of this drug, our preliminary data suggest promise for this novel application of BRD4 and PI3Kdelta inhibition in the context of acute lymphoblastic leukemia. Disclosures Durden: SignalRx Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Development of a Unique In Vitro and In Vivo Model System of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph-ALL) with Emphasis on Cell to Cell Interaction.

Blood ◽

10.1182/blood.v108.11.1845.1845 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1845-1845 ◽

Cited By ~ 1

Author(s):

Arinobu Tojo ◽

Kiyoko Izawa ◽

Rieko Sekine ◽

Tokiko Nagamura-Inoue ◽

Seiichiro Kobayashi

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Suspension Culture ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Leukemia Cells ◽

L2 Cells

Abstract Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) is one of the most intractable hematological malignancies, readily acquires resistance to chemotherapeutic drugs including imatinib mesylate (IM), and shows a high relapse rate even after allogeneic stem cell transplantation. Nevertheless, primary blast cells are generally susceptible to apoptotic cell death in sort-term suspension culture after isolation from patients with Ph-ALL. We established two Ph-ALL cell lines and characterized their growth properties supported by adhesive interaction with a murine bone marrow stromal cell line, HESS-5. IMS-PhL1 (L1) cells mainly expressed p210-type BCR-ABL mRNA with wild type sequences in the ABL kinase domain and were weakly positive for p190-type mRNA. IMS-PhL2 (L2) cells exclusively expressed p190-type transcripts with Y253H mutation and showed much lower sensitivity to imatinib than L1 cells. The growth of L1 cells was slowly autonomous in suspension culture, but became more vigorous and their apoptosis was prevented by co-culture with HESS-5 cells. In contrast, the sustained growth and survival of L2 cells was absolutely dependent on direct contact with HESS-5 cells and did not respond to soluble cytokines including SCF, IL3and IL7. Both cell lines adhered to and migrated beneath the HESS-5 cell layer, resulting in the formation of cobblestone areas. This migration was significantly inhibited by the pretreatment of those with a neutralizing antibody against α4-integrin. While non-adherent L1 cells were eradicated by 1 mM IM, a portion of adherent L1 cells could survive even at 10 mM IM. Similarly, adherent L2 cells considerably resisted prolonged exposure to 10 mM IM. Intravenous injection of both cell lines caused leukemia in NOD-SCID mice after distinct latent periods. Leukemia cells appeared in peripheral blood, bone marrow as well as spleen. Interestingly, expression of α5-integrin was significantly down-regulated in both leukemia cells collected from those tissues, but was restored after co-culture with HESS-5. The study of L1 and L2 cells in vitro and in vivo will not only contribute to further insights into microenvironmental regulation of clonal maintenance and progression of Ph-ALL but also provide a unique model for experimental therapeutics against Ph-ALL. Figure Figure

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Idh2R140Q Cooperates with NHD13 to Induce Immature T Cell Leukemia By Targeting Early T Cell Progenitors or DN2 Thymocytes

Blood ◽

10.1182/blood-2019-128675 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1346-1346

Author(s):

Vijay Negi ◽

Liat Goldberg ◽

Yang Jo Chung ◽

Masahiro Onozawa ◽

Peter D. Aplan

Keyword(s):

Bone Marrow ◽

T Cell ◽

Transgenic Mice ◽

Expression Profile ◽

Gene Set Enrichment Analysis ◽

Leukemia Cells ◽

Enzymatic Function ◽

Cell Counts ◽

Significant Difference

Introduction Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are frequently observed in patients with acute myeloid leukemia, with IDH2 mutations reported in 9- 19% of AML cases. IDH2 mutation leads to loss of normal enzymatic function and accumulation of 2-hydroxyglutarate (2-HG) through a newly gained enzyme activity. The most common IDH mutation in AML patients involves IDH2 R140. Transgenic mice that expressed an Idh2R140Q mutant in all hematopoietic tissues did not develop leukemia, nor was there a significant difference between overall survival of Idh2R140Q mice compared to wildtype mice. These findings were consistent with those from other investigators, which also suggested that expression of mutant IDH2 was not sufficient for development of leukemia, and that collaborating mutations are necessary. A recent report of whole exome sequence (WES) from mouse models of hematopoietic malignancies identified recurrent Idh1R132H mutations in NUP98-HOXD13 (NHD13) driven AML. Given that Idh1 R132 is paralogous to IDH2 R140, we considered the possibility that an Idh2R140Q mutation would collaborate with an NHD13 transgene and promote leukemic transformation. Therefore, we generated Idh2R140Q/NHD13 transgenic mice by crossing Idh2R140Q with NHD13 mice. Idh2R140Q/NHD13 transgenic mice developed a leukemia at a median of 10 months of age that was similar to human ETP in terms of immunophenotype and additional acquired cooperative mutations in genes such as Pten, N/Kras, Ptpn11, and Sh2b3. Results Gene set enrichment analysis (GSEA) based on RNA-Seq data from Idh2R140Q/NHD13 ETP leukemias and non-ETP T cell leukemias showed that Idh2R140Q/NHD13 ETP gene expression profile correlated well with human ETP leukemic expression profile. An in vitro thymocyte differentiation assay using co-culture of immature double negative (DN) 1 and DN2 thymocytes from Idh2R140Q/NHD13 mice on an OP9-DL1 stromal layer demonstrated a complete block in differentiation to double positive (DP) CD4+CD8+ thymocytes. The Idh2R140Q/NHD13 DN1/2 co-cultured cells arrested at the DN2 stage of differentiation, similar to the in-vivo phenotype of Idh2R140Q/NHD13 leukemia. In addition, Idh2R140Q/NHD13 DN1/2 cells had greater proliferative potential compared to wildtype control. We further observed that Idh2R140Q/NHD13 DN cells would proliferate indefinitely on OP9-DL1 stromal cells, and that treatment of Idh2R140Q/NHD13 thymocytes with AG-221, a potent and selective mutant IDH2 inhibitor, led to a marked decrease in cell proliferation. We developed an in vivo bone marrow transplantation (BMT) model for Idh2R140Q/NHD13 ETP leukemia to assess response to AG-221 in vivo. Primary Idh2R140Q/NHD13 ETP leukemia cells were transplanted into sub-lethally (600 cGy) irradiated recipient mice. This resulted in recipient mice that were anemic and thrombocytopenic with elevated white blood cell counts, suggesting engraftment of acute leukemia. The transplanted, secondary leukemias were consistent with the primary disease immunophenotype by flow cytometry, and tissue histology showed infiltration of blasts into the bone marrow, spleen and perivascular regions of the liver, consistent with disseminated leukemia. Blast cells were positive for both CD3 and myeloperoxidase, further highlighting an ETP phenotype. An assay for clonal T cell receptor beta rearrangement confirmed clonality of recipient leukemia cells identical to the primary leukemia cells. In vivo treatment of Idh2R140Q/NHD13 ETP recipient mice with AG221 showed a significant decrease in leukemic cell expansion compared to control mice treated by gavage with vehicle only; survival data is pending. Conclusion In summary, the IDH2 inhibitor data suggests that targeting the mutant IDH2 in Idh2R140Q/NHD13 leukemic cells can result in a significant decrease in leukemic burden in vivo. Furthermore, the Idh2R140Q/NHD13 primary ETP leukemia BMT mice serves as an excellent model for the study of ETP leukemia development and therapy. In this context, it is important to note that 14% of human ETP show mutations in IDH1/2, and although NUP98 translocations are rare but recurrent events in human ETP ALL, these NUP98 translocations lead to enforced expression of HOXA genes, which is a common event in ETP ALL. Disclosures Aplan: NIH: Patents & Royalties: royalties for the invention of NUP98-HOXD13 .

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