Identification of Patient-Specific Anti-Apoptotic Molecules As Therapeutic Targets in Poor Prognosis Acute Lymphoblastic Leukemia (ALL)

Abstract Relapse and refractory ALL shows dismal prognosis despite recent progress in intensive chemotherapy and development of molecular targeting agents. In this study, we aimed to identify vulnerabilities in genetically-diverse ALL and to find additional therapeutic targets in Philadelphia chromosome-positive (Ph+) ALL and chronic myeloid myeloid leukemia (CML) to overcome BCR-ABL tyrosine kinase inhibitor (TKI) resistance. To this end, we performed in vitro drug screening using 23 human ALL samples (9 Ph+ ALL, 5 MLL-ALL, 1 ALL with ETV-RUNX1, 5 B-ALL, not otherwise specified and 4 T-ALL), 10 CML samples and eight mixed phenotype acute leukemia (MPAL) samples (four B-Myeloid and four T-Myeloid). Based on our recent findings in AML (Hashimoto, Saito et al., Nature Cancer 2021), we treated leukemia-engrafting cells with small molecules targeting anti-apoptotic molecules (BCL-2, MCL-1 or BIRC) or molecules associated with cell division (AURKB) in addition to tyrosine kinase inhibitors. Among these compounds, BIRC inhibitor and venetoclax exhibited great efficacy. Responsiveness to each compound was: 28 of 41 for BIRC inhibitor (68.3%), 24 of 40 for venetoclax (60%), 11 of 37 for MCL-1 inhibitor (29.7%) and 4 of 37 for AURKB inhibitor (10.8%). We found differential sensitivity between T-ALL/MPAL T-myeloid and CML. Seven out of eight T-ALL and MPAL T-myeloid samples were highly sensitive to venetoclax (87.5%), while nine out of 10 CML samples were responsive to BIRC inhibition (90%). On the other hand, among Ph+ ALL/MPAL and Ph- B-ALL/MPAL B-myeloid samples, sensitivities to BIRC inhibitor and venetoclax were variable. To identify determinants of sensitivity to compounds, we examined the relation between mutational profile and in vitro leukemia elimination through targeted DNA sequencing for 79 lymphoid and myeloid malignancy-associated somatic mutations. Consistent with our previous study, CBL-mutated leukemia showed higher sensitivity to BIRC inhibitor (four of five cases) compared with venetoclax (two of five cases). Among genes related to RAS signaling pathway, KRAS mutations were most frequent (n=6). While five of six KRAS-mutated cases were BIRC inhibitor sensitive (83.3%), three of six cases were sensitive to venetoclax (50%). For cases with mutations in BCR-ABL1 kinase domain, the sensitivity to BIRC inhibitor and venetoclax was variable. Finally, we went on to setup in vivo experiments to elucidate if targeting the patient-specific vulnerabilities resulted in potent therapeutic efficacy against patient leukemic cells. We created patient-derived xenograft (PDX) models of 5 Ph+ ALL/MPAL, 2 Ph- B-ALL/MPAL, 2 T-ALL/MPAL and 2 CML cases. For Ph+ ALL/MPAL and CML, BIRC inhibitor and/or venetoclax combined with dexamethasone (DEX) and TKI achieved effective in vivo elimination of leukemic cells as predicted by in vitro experiments. Combination therapy showed almost complete elimination of Ph+ leukemic cells even in the presence of T315I mutation. For Ph- B-ALL/MPAL and T-ALL/MPAL, preliminary in vivo experiments showed additional inhibition of BIRC and BCL-2 resulted in more profound reduction of leukemic cells in BM compared with DEX alone (Combination: 2.1% [0.25-8] of hCD45+ leukemic cells, n=13 vs. DEX alone: 67.2% [59-76.5], n=11, median [IQR], p<0.001). We found targeting anti-apoptotic molecules in combination with DEX and/or TKI eradicated human genetically-diverse ALL, MPAL and CML cells both in vitro and in vivo. Inhibition of BIRC and BCL-2 overcame glucocorticoid resistance of Ph- ALL. Altogether, our results may offer precision medicine approach and contribute to improvement of clinical outcome in treatment-resistant ALL, CML and MPAL. Disclosures Uchida: Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma Inc.: Honoraria. Harigae: Ono pharma: Honoraria, Other: Subsidies or Donations; Astellas Pharma: Other: Subsidies or Donations; Kyowakirin: Other: Subsidies or Donations; Janssen Pharma: Honoraria; Chugai Pharma: Honoraria; Novartis Pharma: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria.

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Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia

Blood ◽

10.1182/blood.2019001417 ◽

2020 ◽

Vol 136 (2) ◽

pp. 210-223 ◽

Cited By ~ 2

Author(s):

Eun Ji Gang ◽

Hye Na Kim ◽

Yao-Te Hsieh ◽

Yongsheng Ruan ◽

Heather A. Ogana ◽

...

Keyword(s):

Drug Resistance ◽

Tyrosine Kinase ◽

B Cell ◽

Kinase Inhibitor ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Underlying Mechanism ◽

Conditional Knockout

Abstract Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

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Serial Transplantation of a Human Acute T Lymphoblastic Leukemia into Nude Mice

Tumori Journal ◽

10.1177/030089168607200603 ◽

1986 ◽

Vol 72 (6) ◽

pp. 553-558 ◽

Cited By ~ 4

Author(s):

Maria Giovanna Martinotti ◽

Roberto Arione ◽

Roberto Foà ◽

Luigi Pegoraro ◽

Cristina Jemma ◽

...

Keyword(s):

Nude Mice ◽

Lymphoblastic Leukemia ◽

Experimental System ◽

Leukemic Cells ◽

Immune Reactivity ◽

Chromosomal Markers ◽

Total Body ◽

Serial Transplantation

A human acute T lymphoblastic leukemia line (PF-382) was serially transplanted into nude mice. No takes were observed in untreated nude mice, whereas solid tumors were observed in splenectomized and total body, sublethally irradiated mice. The minimal tumor-inducing dose and the latency time remained unchanged after the third and fifth serial transplants. Moreover, leukemic cells recovered from the 8th in vivo passages displayed the same differentiation antigens and chromosomal markers as the in vitro PF-382 cell line used for the first transplant. This stable and well-characterized experimental system could be a new model for T-lymphocyte differentiation and immune-reactivity against human leukemias.

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Pharmacokinetic and Pharmacodynamic Effects of PEG-Asparaginase in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia: Results from a Single Agent Window Study.

Blood ◽

10.1182/blood.v110.11.860.860 ◽

2007 ◽

Vol 110 (11) ◽

pp. 860-860

Author(s):

Inge M. Appel ◽

Karin M. Kazemier ◽

Anjo J.P. Veerman ◽

Elisabeth van Wering ◽

Monique L. Den Boer ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Clinical Response ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Hazard Ratio ◽

Leukemic Cells ◽

Newly Diagnosed ◽

Pharmacodynamic Effects

Abstract L-Asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia. The effectiveness is generally thought to result from a rapid depletion of asparagine in serum and cells. Several studies have shown that in vitro resistance to this drug is an independent prognostic factor in ALL. We investigated the clinical response of one in vivo dose of 1000 IU/m2 PEG-Asparaginase and its pharmacokinetic and pharmacodynamic effects in children with newly diagnosed ALL before the start of combination chemotherapy. 57 children (36M / 21F) were enrolled in the study: 2 pro B-ALL, 38 common/ pre B-ALL and 17 T-ALL. Genotyping of precursor B-ALL revealed 11 hyperdiploid, 8 TELAML1 positive, 2 BCRABL positive, no MLL rearrangement, 8 normal, 11 others. The clinical response to PEG-Asparaginase on day 0 (5 days after the PEG-Asparaginase infusion) was defined as good when the number of leukemic cells of peripheral blood was < 1 × 109/L, as intermediate when leukemic cells were 1-10 × 109/L, and as poor when leukemic cells were > 10 × 109/L. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common / pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (p=0.01). Both BCRABL positive ALL cases showed a poor response (p=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-Asparaginase (p=0.02) and both in vitro as well as in vivo response were prognostic factors for long-term event-free survival (Hazard ratio 6.4; p=0.004, and Hazard ratio 3.7; p=0.01, respectively). The L-Asparaginase activity in the serum was >100 IU/L for at least 15 days. The asparagine levels remained below the detection limit of 0.2 mM for at least 26 days with a concomitant rise in serum aspartate and glutamate. These findings confirm that PEG-Asparaginase will yield its pharmacodynamic effects for 2-4 weeks. After administration of one in vivo dose of 1000 IU/m2 PEG-Asparaginase no changes in apoptotic parameters or changes in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. Otherwise it is possible that in vivo mesenchymal cells from the bone marrow supply leukemic blasts with asparagine in response to treatment with L-Asparaginase. Conclusion: The clinical response to one dose of 1000 IU/m2 PEG-Asparaginase intravenously is related to phenotype and genotype and predicts outcome. These results suggest that children with ALL with a poor clinical response to PEG-Asparaginase might benefit from a more intensive antileukemic therapy.

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The Bone Marrow Niche of Patients with Acute Lymphoblastic Leukemia Produces No Increased Asparagine Levels In Vivo That May Lead to Clinical Asparaginase Resistance

Blood ◽

10.1182/blood.v118.21.1505.1505 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1505-1505

Author(s):

Wing H. Tong ◽

Rob Pieters ◽

Wim C.J. Hop ◽

Claudia Lanvers-Kaminsky ◽

Joachim Boos ◽

...

Keyword(s):

Bone Marrow ◽

Aspartic Acid ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Mesenchymal Cells ◽

Leukemic Cells ◽

Significant Difference ◽

Trough Levels

Abstract Abstract 1505 Asparaginase is an essential component of combination chemotherapy of acute lymphoblastic leukemia (ALL). Asparaginase breaks down asparagine into aspartic acid and ammonia. Because asparagine is necessary for protein synthesis, its depletion leads to cell death. Recently, it has been suggested that mesenchymal cells in the bone marrow may produce asparagine and form ‘protective niches’ for leukemic cells. In vitro, this led to high levels of asparagine and asparaginase resistance of the ALL cells (Iwamoto et al. (J Clin Invest. 2007)). However, it is unknown if this holds true for the clinical in vivo situation. The aim of our study is to analyse whether mesenchymal cells or other cells in the bone marrow indeed produce significant amounts of asparagine in vivo that may lead to clinical asparaginase resistance. Ten de novo ALL patients were enrolled in this study. All children received induction chemotherapy according to protocol 1-A and 1-B of the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol. Asparaginase levels and amino acid levels (asparagine, aspartic acid, glutamine and glutamic acid) were measured in bone marrow (BM) and peripheral blood at diagnosis (day 1), days 15, 33 and 79. On days that asparaginase was administered (days 15 and 33) it was ensured that study material was obtained before the E-coli L-asparaginase infusions. Changes over time of asparaginase trough levels in BM and peripheral blood were evaluated using Mixed models ANOVA. The amino acids levels in 0.5 ml BM, 3 ml BM and peripheral blood at days 15 and 33 were also compared using Mixed models ANOVA. All these analyses were done after log transformation of measured values to get approximate normal distributions. A two-sided p-value < 0.05 was considered statistically significant. The asparaginase levels were all below detection limit (< 5 IU/L) in BM and peripheral blood at days 1 and 79. In both compartments, the median asparaginase trough levels were not significantly different at days 15 and 33. At diagnosis, no significant difference in asparagine level between 3 ml BM and peripheral blood was found (median: 44.5 μM (range 20.6–59.6 μM) and 43.9 μM (range 18.4 –58.5 μM), respectively). However, the median level of aspartic acid at diagnosis in 3 ml BM (19.2 μM; range 6.2–52.6 μM) was significantly higher as compared to median level of peripheral blood (5.7 μM; range 2.4–10.1 μM) (p=0.002). The aspartic acid levels were also higher in BM compared to peripheral blood at days 15 and 33 (both p=0.001) and at day 79 (p=0.002). Aspartic acid levels were significantly higher in 0.5 ml versus 3 ml BM (p=0.001) and this difference was also found when comparing 0.5 ml BM versus peripheral blood (p<0.001) suggesting dilution with peripheral blood when taking higher volumes of ‘bone marrow’. Asparagine levels were all below the lower limit of quantification (LLQ < 0.2 μM) in both BM and blood during asparaginase treatment at days 15 and 33. At day 79, no significant difference in asparagine levels between BM (37.7 μM; range 33.4–50.3 μM) and peripheral blood (38.9 μM; range 25.7 –51.3 μM) was seen. During the time course of asparaginase infusions, the glutamine and glutamic acid levels did not change significantly. In conclusion, we demonstrate higher aspartic acid levels in bone marrow compared to peripheral blood. The higher aspartic acid levels are detected at diagnosis, during asparaginase therapy at days 15 and 33, and also at day 79 at complete remission, showing that these do not originate from leukemic cells nor from asparagine breakdown by asparaginase but from cells in the microenvironment of the bone marrow. However, there is no increased asparagine synthesis in vivo in the bone marrow of ALL patients. Therefore, increased asparagine synthesis by mesenchymal cells may be of relevance for resistance to asparaginase of leukemic cells in vitro but not in vivo. Disclosures: No relevant conflicts of interest to declare.

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Preclinical Testing of a Novel Axl-Kinase Inhibitor in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v120.21.1799.1799 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1799-1799

Author(s):

Maria Göbel ◽

Michael Möllmann ◽

Andre Görgens ◽

Ulrich Dührsen ◽

Andreas Hüttmann ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Cell Polarization ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Blue Staining ◽

Nsg Mice

Abstract Abstract 1799 The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their potential to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models. In vitro studies: Freshly isolated PBMC (>90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Since a polarized phenotype is required for migration, cell polarization was analyzed by time-lapse video-microscopy. We detected cytotoxic effects in a patient dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (LD50= 1.4 μM vs. 5.2 μM, p<0.004, n=5). Cell polarization of the remaining viable cells was significantly reduced in a dose dependent fashion in comparison to vehicle only controls (LDC2636 IC50 = 7.2 μM, p<0.00001; BMS777607: IC50=6.2μM; p=0.0004). Of note, both Axl inhibitors exhibited significantly weaker effects on both, the viability and cell polarization of normal PBMC over the whole concentration range tested (p<0.05, n=5). In vivo studies: To verify our hypothesis that reduced cell polarization results in decreased homing of leukemic cells in vivo we employed a recently developed adoptive transfer model of CLL. In this model NOD/SCID/gcnull(NSG) mice were pre-treated with a single intraperitoneal bolus of LDC2636 or BMS777607 (20 mg/kg) and subsequently transplanted with primary CLL cells. Both Axl inhibitors significantly reduced the homing capacity of CLL cells to the bone marrow of NSG mice by 43% and 59%, respectively, compared to vehicle treated controls (LDC2636: p=0.046, BMS777607 p=0.0077; n=3). These data demonstrate that Axl inhibitors exert potent in vitro and in vivo activity against human CLL cells, which is caused at least in part by the suppression of CLL homing to their supportive stromal niches. Disclosures: No relevant conflicts of interest to declare.

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Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

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Unique Effects of p53−/− Leukemic Cells On Mesenchymal Stromal Cell Gene Expression Profile in Vitro

Blood ◽

10.1182/blood.v120.21.3468.3468 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3468-3468

Author(s):

Xiaoyang Ling ◽

Ye Chen ◽

Peter P. Ruvolo ◽

Vivian Ruvolo ◽

Zhiqiang Wang ◽

...

Keyword(s):

Gene Expression ◽

Cell Biology ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Bone Marrow Niche ◽

In Vivo Experiments ◽

Cell Gene Expression

Abstract Abstract 3468 Mesenchymal stromal cells (MSC) participate in the generation of the microenvironmental bone marrow niche which protects normal and leukemic stem cells from injuries, including chemotherapy. MSC produce numerous factors that aid in this function; however, little is known about how leukemic cells affect MSC. In this study, paired murine AML cells, MLL/ENL/FIT3-ITD/p53−/− and MLL/ENL/FIT3-ITD/p53wt, originally derived from C57BL/6 mice (Zuber et al. Genes & Dev. 2009), were co-cultured with MSC from the same strain. After 48 hrs, MSC were isolated by FACS sorting using CD45−/PDGFr+ as markers. Total RNA was profiled on Illumina WG6 mouse whole-genome bead arrays by standard procedures. The significance analysis of microarrays (SAM) method identified 429 differentially-expressed genes (DEG) whose expression in MSC differed significantly (false discovery rate, 10%) in co-cultures with p53−/− (C78) vs. p53wt (C147) leukemic cells. Differences in these DEG were highly consistent in replicates (Figure 1). The results demonstrate that: 1) p53 status (p53−/− vs. p53wt) of AML cells affects GEP patterns in co-cultured MSC. Comparison of the GEP in MSC co-cultured with p53−/− (78) or p53wt (147) (Fig 1) identified the following 5 genes that showed the most significant differences (up- or down-regulated): up-regulated: WNT16, WNT5, IGFBp5, GCNT1, ATP1B1; down-regulated: NOS2, DCN, CCL7, CCL2, CAR9, CCL4. These were selected for qPCR validation, and the results confirmed the array data. In addition, immunohistochemical staining showed that WNT16 was up-regulated in MSC co-cultured with p53wt leukemic cells. In addition, CXCL5 was found up-regulated in MSC co-cultured with p53−/− leukemic cells. These results were consistent with the GEP data. 2) Leukemic cells alter MSC Signaling proteins in vitro: Western blotting showed that Stat3, Akt, PTEN, CXCL5 and HIF-1α were up- regulated in MSC co-cultured with p53−/− leukemic cells as compared to p53wt leukemic cells (48 hrs). Additional analyses showed that the downstream targets of HIF-1α, VEGFa and VEGFc, but not VEGFb, were up-regulated. Taken together, these results suggest that 1) leukemic cells with different p53 genetic background co-cultured with normal MSC have profoundly differential effects on GEP of normal MSC; 2) MSC co-cultured with p53−/− leukemic cells resulted in increased levels of onco-proteins such as Akt and HIF-1α when compared to MSC co-cultured with p53wt leukemic cells. Results suggest, for the first time, that the genetics of leukemic cells determines gene expression in co-cultured MSC. In vivo experiments are in progress to provide in vivo evidence for the existence of a novel model of leukemia-stroma interactions where the genetics of the tumor cell impacts stromal cell biology. Disclosures: No relevant conflicts of interest to declare.

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CD7+, CD4-, CD8- acute leukemia: a syndrome of malignant pluripotent lymphohematopoietic cells

Blood ◽

10.1182/blood.v73.2.381.bloodjournal732381 ◽

1989 ◽

Vol 73 (2) ◽

pp. 381-390

Author(s):

J Kurtzberg ◽

TA Waldmann ◽

MP Davey ◽

SH Bigner ◽

JO Moore ◽

...

Keyword(s):

Lymphoblastic Leukemia ◽

Leukemic Cell ◽

Leukemic Cells ◽

Cell Phenotype ◽

Therapeutic Approaches ◽

Myeloid Leukemias ◽

Cell Gene ◽

In Vitro Data

Following our initial observation of in vivo conversion of CD7+, CD4-, CD8- acute lymphoblastic leukemia (ALL) cells from lymphoid to myeloid lineages (Proc Natl Acad Sci (USA) 81:253, 1984) we have studied eight additional cases of ALL with this leukemic cell phenotype. The CD7+, CD4-, CD8- phenotype was associated with a distinct clinical entity with those affected predominantly male (either less than 35 years or greater than 65 years of age), with frequent mediastinal and/or thymic masses, skin and CNS disease, high peripheral WBC counts, and bone marrow blasts that were morphologically L1 or not ascribable to a specific lineage. These patients did not respond to conventional chemotherapeutic regimens for either acute lymphoid or myeloid leukemias. No common karyotype or T-cell gene rearrangement pattern could be defined. Importantly, seven of eight patient's leukemic cells studied were capable of multilineage (myeloid, erythroid, monocytoid, megakaryocytoid, and lymphoid) differentiation in vitro. Data is presented suggesting that CD7+, CD4-, CD8- leukemias, in many instances, are leukemias of immature hematopoietic cells. The development of novel therapeutic approaches to this form of leukemia will be necessary to alter its poor prognosis.

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Disruption of HOX-PBX Interaction; a Potential Therapeutic Target in Leukemia.

Blood ◽

10.1182/blood.v104.11.2057.2057 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2057-2057

Author(s):

Jastinder Sohal ◽

Ruji Begum ◽

Neal Fischbach ◽

Davinder Theti ◽

Ruth Pettengell ◽

...

Keyword(s):

Target Genes ◽

Hox Genes ◽

Family Members ◽

Normal Function ◽

Leukemic Cells ◽

Blue Staining ◽

Rt Pcr ◽

In Vivo Experiments

Abstract Most cases of acute myeloid leukemia (AML) are closely associated with gene rearrangements. Appraisal of these translocations and analysis of mouse models of leukemia has revealed that several members of the homeodomain containing family of transcription factors are implicated in the pathogenesis of leukemia. Overexpression of HOXA9 in murine models leads to the development of AML. This study focuses on the role of a subset of the HOX genes and their potential as a target for therapeutic intervention. We have designed a synthetic peptide, HXP4, that disrupts the interaction between HOX and PBX leading to growth inhibition of leukemic cells. An in vitro HOX-induced AML model of leukemia was utilised to determine the efficacy of HXP4 as a therapeutic agent. Using this immortalised cell line overexpressing HOXA9 (imHOXA9), we tested the efficacy of HXP4 in vitro. Cells were treated with HXP4 for four days and analysed. All results are expressed relative to untreated control cells. Following a 60μM dose of HXP4, no viable cells were detected as determined by trypan blue staining, suggesting that HXP4 was cytotoxic. Following treatment with a lower dose of 6μM HXP4, and re-suspension in drug-free medium for a further 6 days, cell regrowth was observed, suggesting a cytostatic effect. RT-PCR was performed to identify potential downstream targets of HXP4. Qualitative analysis showed other HOX family members to be unaffected by treatment with either HXP4 dose. A more detailed study was performed using quantitative RT-PCR with imHOXA9. Cells were treated with either 60μM HXP4, 3μM etoposide, or a combination of the two agents (H+Et) and harvested after 1, 2, and 4 hours. In general, no significant change in gene expression was observed in other HOX family members. However, HOXA1 was upregulated 3-fold when treated with HXP4, and HOXA2 was downregulated 2-fold in HXP4 and H+Et treated cells. The reasons for this are as yet unclear. HXP4 also downregulated N-RAS 3.5-fold at two hours. However, complete loss of N-RAS expression following H+Et treatment suggests that HXP4 may be more effective in combination with etoposide. CDC25 expression was slightly downregulated in HXP4-treated cells. The normal function of CDC25 is to activate CDC2 kinase in the nucleus, however in the absence of CDC25, CDC2 remains inactive leading to a delay in mitosis, supporting the proposed cytostatic mode of HXP4 action. For reasons as yet unclear, CD34 expression was upregulated 4-fold and 6-fold in HXP4 and H+Et treated cells respectively. These preliminary results suggest that HXP4 is a cytostatic agent at relatively low concentrations, with a reversible antiproliferative effect. Downstream genes regulated by disrupting the HOX-PBX interaction with HXP4 have been identified by RT-PCR, but microarray analysis will provide a more comprehensive screen for target genes. In vivo experiments are currently in progress. In conclusion blocking the interaction between HOX and PBX may represent a therapeutic strategy in leukemia treatment.

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Anti-Leukemic Activity of a Novel Pegylated Recombinant Erwinia Chrysanthemi-Derived L-Asparaginase On Lymphoid Cell Lines and Leukemia-Bearing Mouse Models.

Blood ◽

10.1182/blood.v120.21.2571.2571 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2571-2571 ◽

Cited By ~ 1

Author(s):

Wei-Wen Chien ◽

Céline Lebeux ◽

Nicolas Rachinel ◽

Soraya Allas ◽

Pierre Sahakian ◽

...

Keyword(s):

Cell Lines ◽

Lymphoblastic Leukemia ◽

Lymphoma Cell ◽

Erwinia Chrysanthemi ◽

Leukemic Cells ◽

Hypersensitivity Reactions ◽

Sensitive Cell ◽

Development Fund

Abstract Abstract 2571 Background: Bacterial-derived L-asparaginase (ASNase) is an indispensable component of the therapy of acute lymphoblastic leukemia (ALL). Despite the high rate of successful treatment, hypersensitivity reactions occur in 20–40 % of patients receiving this non-human protein, which limits the use of ASNase. Native Erwinia chrysanthemi-derived ASNase (n-crisantaspase) has been used for treating patients exhibiting allergic symptoms to native and PEGylated Escherichia coli-derived ASNases (EC-ASNase). However, hypersensitivity reactions are still observed in at least 17 % of patients receiving n-crisantaspase. A PEGylated recombinant Erwinia chrysanthemi-derived ASNase (PEG-r-crisantaspase) with improved pharmacokinetic and pharmacodynamics properties and reduced immunogenicity has been developed recently (Allas et al., abstracts #2003, #2034, ASH 2009). We present here the in vitro and in vivo evaluation of PEG-r-crisantaspase on leukemia and lymphoma cell lines and on a leukemia-bearing mouse model, respectively. Material and methods: Different cell lines (ALL, B, T and NK/T lymphoma) and bone marrow aspiration samples obtained from patients with B-ALL or T-ALL were exposed in vitro to increasing doses (0.00005 to 5 U/ml) of PEG-r-crisantaspase, n-crisantaspase, or native EC-ASNase for 3 days. The cytotoxicity of each molecule was evaluated using 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. For the in vivo study, 5 millions of ASNase-sensitive cells, RS(4,11) were xenografted by intravenous injection (iv) into immunodeficient NOD/SCID mice. When 5% of leukemic blasts were observed in the blood of leukemia-bearing mice, 2, 5, 10 or 20 U/kg of PEG-r-crisantaspase or vehicle was injected intravenously in those mice. Two additional injections were performed with an interval of 7 days. Mice having significant weight loss (>20%) and/or extensive expansion of leukemic cells in blood (> 25%) and/or impaired general condition were sacrificed. Results: In vitro, PEG-r-crisantaspase and n-crisantaspase exhibited similar half maximal effective concentration (EC50) values for inhibiting the proliferation of leukemia and lymphoma cells. PEG-r-crisantaspase had a greater cytotoxicity effect on two high-sensitive cell lines than n-crisantaspase did, as shown by 4.5 and 8.1-fold-weaker EC50 values of PEG-r-crisantaspase relative to n-crisantaspase. PEG-r-crisantaspase, r-crisantaspase and n-crisantaspase were more efficient than EC-ASNase on less sensitive cell lines, which might be related to the 10-fold-greater glutaminase activity of crisantaspases than EC-ASNase. In vivo, after the first administration of PEG-r-crisantaspase in animals with ≥ 5% of leukemic cells, the leukemic cells were reduced to almost 0 % in mice within 4 days with all doses of PEG-r-crisantaspase tested, whereas the leukemic cells kept multiplying in the mice receiving vehicle. The 3 repeated injections of PEG-r-crisantaspase with an interval of 7 days delayed leukemia development for 17 days. All control mice receiving the vehicle had to be sacrificed at day 44 after leukemia inoculation, whereas of 28 mice receiving PEG-r-crisantaspase, 5 reached this endpoint at day 51 and the others at day 56. PEG-r-crisantaspase significantly improved the survival of leukemia-bearing mice for 7 to 12 days, regardless of the dose tested. Conclusions: These data provide evidence that PEG-r-crisantaspase has similar in vitro cytotoxic effect to n-crisantaspase on leukemia and lymphoma cell lines and significantly reduces the expansion of leukemic cells in leukemia-bearing mice, prolonging the survival of the animals. These results, together with preclinical PK/PD and immunogenicity data, support the clinical development of PEG-r-crisantaspase. A phase I dose escalation study in adult patients with relapsed or refractory hematological malignancies has been recently initiated. Research support from Alizé Pharma, the European Regional Development Fund (ERDF) and Grand Lyon. Disclosures: Allas: Alizé pharma: Employment. Sahakian:Alizé pharma: Employment. Julien:Alizé pharma: Employment. Abribat:Alizé pharma: Employment.

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