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 ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1845-1845 ◽  
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

Leptomycin B Overcomes Imatinib Resistance Mediated by Stromal Cells and Mutant BCR-ABL in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2089-2089
Author(s):  
Arinobu Tojo ◽  
Kiyoko Izawa ◽  
Rieko Sekine ◽  
Tokiko Nagamura ◽  
Minoru Yoshida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Layer ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Leptomycin B ◽  
Imatinib Resistance ◽  
L2 Cells

Abstract Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) is one of the most intractable hematological malignancies, and readily acquires resistance to chemotherapeutic drugs including imatinib mesylate. We hypothesized that the adhesive interaction of Ph-ALL cells with bone marrow stromal cells might cause their escape from drug-induced apoptosis and subsequent minimal residual disease, resulting in the generation of a chemoresistant clone such as a clone harboring mutant BCR-ABL. To gain insight into this possibility and a novel strategy against imatinib resistance, we used two Ph-ALL cell lines designated as IMS-PhL1 (L1) and IMS-PhL2 (L2). L1 cells had wild type BCR-ABL, whereas L2 cells had Y253H mutant and revealed 10-fold or more resistance to imatinib, compared with L1 cells. The growth of L1 cells was autonomous and their spontaneous apoptosis was suppressed by co-culture with a murine bone marrow stromal cell line, HESS-5. In contrast, the sustained growth and survival of L2 cells was absolutely dependent on direct contact with HESS-5. Both cell lines adhered to and migrated beneath the HESS-5 cell layer, resulting in the formation of cobblestone areas (CA). While floating L1 cells were eradicated by 1 mM imatinib, a portion of adherent L1 cells could survive even at 10 μM imatinib. Similarly, L2 cells forming CA beneath the HESS-5 cell layer considerably resisted prolonged exposure to 10 μM imatinib. Leptomycin B (LMB), a potent inhibitor of CRM1/exportin-1, can trap BCR-ABL in the nucleus and can aggressively eliminate BCR-ABL+ cells in combination with imatinib (Wang et al., 2001). We tested LMB for its ability to eliminate CA or adherent Ph-ALL cells in combination with imatinb. The result for L2 cells was shown in Figure. Dramatically, combined use of 10 μM imatinib and 1 nM LMB for 7 days exerted a synergistic effect on reduction in the number of CA. L1 cells were also susceptible to the combination of imatinib and LMB. Our results suggest that nuclear entrapment of BCR-ABL may be a promising strategy for overcoming imatinib resistance mediated by stromal cells as well as a certain BCR-ABL mutant. Figure Figure

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

Blood ◽  
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.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1420-1420 ◽  
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.

In vitro cytotoxicity of docetaxel in childhood acute leukemias.

1998 ◽  
Vol 16 (3) ◽  
pp. 907-913 ◽  
Author(s):  
R Consolini ◽  
C H Pui ◽  
F G Behm ◽  
S C Raimondi ◽  
D Campana
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Leukemic Cells ◽  
Allogeneic Bone ◽  
Acute Leukemias ◽  
Growing Cell

PURPOSE In seeking to identify novel effective antileukemic agents, we assessed the in vitro activity of the taxoid docetaxel (Taxotere; Rhone-Poulenc Rorer, Antony, France) in primary leukemic cells supported in culture by bone marrow-derived stromal layers. MATERIALS AND METHODS Bone marrow samples from children with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were cultured on allogeneic bone marrow-derived stromal layers and exposed to various concentrations of docetaxel. After 7 days of culture, the number of viable leukemic cells were counted by flow cytometry and compared with that in parallel cultures without drugs. RESULTS In 20 samples tested (15 B-lineage ALL, one T-lineage ALL, and four AML), the median cytotoxicity was 78% after a 7-day culture in the presence of 100 ng/mL docetaxel (range, 54% to 95%). The effects were dose-dependent and extended to all five ALL samples with the t(9;22)(q34;q11) (Philadelphia chromosome) or 11q23 abnormalities, karyotypes associated with an unfavorable outcome. Studies with continuously growing cell lines demonstrated that docetaxel exerted its cytotoxic effect by inducing apoptosis, and was consistently more effective than paclitaxel (Taxol; Bristol-Myers Squibb, Wallingford, CT) (mean 50% cell kill [LC50], 6.93 v 12.86 ng/mL in six leukemic cell lines). The antileukemic activities of docetaxel and vincristine were synergistic. While the mean (+/- SD) cytotoxicity of vincristine (0.1 ng/mL) was 11.2% +/- 7.3% and that of docetaxel (10 ng/mL) was 19.3% +/- 17.5% in CEM-C7 cells after 24 hours, combining the two agents increased the cytotoxicity to 62.5% +/- 20.7% (P = .003). CONCLUSION Docetaxel, at concentrations achievable in vivo, is cytotoxic to ALL and AML cells. These results provide a rationale for clinical trials of docetaxel in patients with acute leukemia.

Activity of vincristine, L-ASP, and dexamethasone against acute lymphoblastic leukemia is enhanced by the BH3-mimetic ABT-737 in vitro and in vivo

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 2057-2066 ◽  
Author(s):  
Min H. Kang ◽  
Yun Hee Kang ◽  
Barbara Szymanska ◽  
Urszula Wilczynska-Kalak ◽  
Michael A. Sheard ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Clinical Investigation ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Cytochrome C Release ◽  
Synergistic Cytotoxicity ◽  
Bh3 Mimetic

Abstract Defects in apoptosis signaling contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), and overexpression of antiapoptotic Bcl-2 (Bcl-2 and Bcl-XL) family proteins has been observed in ALL. ABT-737 is a small-molecule BH3-mimetic that inhibits the antiapoptotic Bcl-2 family proteins. We evaluated the cytotoxicity of ABT-737 in combination with vincristine, dexamethasone, and L-asparaginase (VXL) in 7 ALL cell lines. Multilog synergistic cytotoxicity was observed in all 7 cell lines with ABT-737 plus L-asparaginase or vincristine, and in 5 of 7 cell lines with ABT-737 plus dexamethasone or VXL. In leukemia cells, but not in normal lymphocytes, ABT-737 plus L-asparaginase induced greater mitochondrial depolarization (JC-1 staining); mitochondrial cytochrome c release; activation of Bax, Bid, and caspases (immunoblotting); and eventually apoptosis (annexin V staining) than did either drug alone. In mouse xenografts derived from patients with ALL at diagnosis (ALL-7) or at relapse (ALL-19), event-free survival (EFS) was significantly enhanced with ABT-737 plus VXL relative to VXL or ABT-737 alone (P ≤ .02). Thus, ABT-737 synergistically enhanced VXL cytotoxicity in ALL cell lines via a mitochondrial death pathway and enhanced EFS in VXL-treated mice bearing ALL xenografts. Combining VXL with a BH3-mimetic warrants clinical investigation in ALL at relapse and potentially in chemotherapy-resistant ALL subgroups.

scholarly journals Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3569-3569
Author(s):  
Ye Shen ◽  
Chun Shik Park ◽  
Koramit Suppipat ◽  
Takeshi Yamada ◽  
Toni-Ann Mistretta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Brdu Incorporation ◽  
Jnk Pathway

Abstract Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies. KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL. To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status. Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. Disclosures No relevant conflicts of interest to declare.

Live Cell Imaging Captures Immune Cell-Mediated Killing of Precursor-B Acute Lymphoblastic Leukemia Cells Targeted with Anti-CD19 (Medi-551) Antibodies: Support for Macrophage and NK Cell In Vivo Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1522-1522
Author(s):  
Ksenia Matlawska-Wasowska ◽  
Dennis Cook ◽  
Samuel R. Stevens ◽  
Elizabeth K. Ward ◽  
Ronald Herbst ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Nk Cells ◽  
Lymphoblastic Leukemia ◽  
Live Cell ◽  
Fluorescent Imaging ◽  
Leukemia Cells ◽  
Strong Activity ◽  
Effector Cells

Abstract Abstract 1522 Precursor-B acute lymphoblastic leukemia (pre-B ALL) is the most common malignancy in children and can be cured in a majority of patients. However, cure remains elusive in approximately 20% of patients for reasons that are not well understood. Importantly, survivors commonly develop morbidities that result from dose-intensified treatment with cytotoxic drugs. Here, we investigate the tumoricidal effects of a novel humanized anti-CD19 monoclonal antibody (Medi-551). The a-fucosylated form of this antibody has increased affinity to human FcgammaRIII (CD16) receptor, present on the surface of NK cells and macrophages, mediating antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly and thus remain accessible for effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human NK cells and macrophages (effector cells) against four pre-B ALL cell lines (697, Nalm 6, MHH-Call 3, RS 4;11), as well as freshly isolated patient blasts. We report results of live cell fluorescent imaging studies, characterizing the formation of immunological synapses between Medi 551-bound target leukemia cells and effector cells, as well as the kinetics of both NK-mediated killing and macrophage phagocytosis. The number of the CD19 receptors present on the cell surface is shown to be a factor in effector-mediated killing of Medi-551 targeted leukemia cells. Further, genetic polymorphisms in FcgammaRIII (158 F/V, V/V or F/F) affected in vitro ADCC and ADCP activities with FcgammaRIII 158 V homo- or heterozygotes showing the strongest activity. We also evaluated the efficacy of Medi-551 in a human pre-B ALL murine xenograft model. SCID mice were engrafted with 697 pre-B ALL cells and received either vehicle alone or Medi-551 (3 mg/kg; twice weekly for a total of 5 doses); treatment was started at day 5 after engraftment. Medi-551 treatment markedly lowered disease burden in blood, liver and bone marrow. The lack of cure is consistent with impaired roles for NK cells in this model, since murine NK cells lack FcgammaRIV. Experiments are in progress to improve the model through adoptive transfer of human NK cells. Taken together, the in vitro and in vivo data show that Medi-551 has strong activity against pre-B ALL and support a move forward to early phase trials in this disease. Disclosures: No relevant conflicts of interest to declare.

Role of the Histone Deacetylase Inhibitor Givinostat (ITF2357) in Treatment of CRLF2 Rearranged Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2534-2534 ◽  
Author(s):  
Angela Maria Savino ◽  
Jolanda Sarno ◽  
Luca Trentin ◽  
Margherita Vieri ◽  
Grazia Fazio ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Combined Treatment ◽  
Cytokine Receptor ◽  
Human Leukemia ◽  
Xenograft Models ◽  
Stat Pathway

Abstract B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) represents 35% of all cancers in pediatric age group. The cure rate for this disease approaches 90% with current treatment regimens, however only a third of patients with relapse are cured. Therefore, there is an urgent need to focus on subgroups of patients with hallmarks of bad prognosis that could benefit from novel therapeutic approaches. Alterations of Cytokine Receptor-like Factor 2 (CRLF2), a negative prognostic factor in pediatric BCP-ALL, have been identified in up to 10% of patients. However these patients represent half of the high risk Ph-like ALL and of Down Syndrome-associated BCP-ALL. Rearrangements of CRLF2 result in the overexpression of this component of the heterodimeric cytokine receptor for thymic stromal lymphopoietin (TSLP) and is associated with activating mutations of the JAK-STAT pathway. Together these cause hyperactivation of JAK/STAT and PI3K/mTOR signaling. Inhibition of CRLF2/JAK2 signaling has the potential to become a therapeutic targeted intervention for this subgroup of poor prognostic patients. Previous studies have shown that the HDAC inhibitor Givinostat/ITF2357 has potent anti-tumor activity against hematological malignancies, particularly JAK2V617F mutated myeloproliferative neoplasms (MPN) such as polycythemia vera, for which it has already a clinic application and established safety profile. We therefore studied the in vitro and in vivo efficacy of Givinostat in cases with CRLF2 rearrangements. Here we demonstrated that Givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged MHH-CALL4 and MUTZ5 cell lines positive for exon 16 JAK2 mutations. Of note, the observed IC50 values for MHH-CALL4 were lower than those for the SET2 cell line positive control bearing JAK2V617F mutation, both for proliferation (IC50: 0.08±0.05µM vs. 0.14±0.03µM) and apoptosis (IC50: 0.17±0.03µM vs. 0.22±0.04µM). We next investigated the effect of Givinostat on blasts from CRLF2 rearranged BCP-ALL patient samples. For this purpose we developed xenograft models of human CRLF2 rearranged ALL to expand cells from patients and to recapitulate human leukemia in recipient mice. ALL blasts isolated from xenografts were co-cultured on OP9 stroma to perform ex vivo assays. Consistent with our findings in cell lines, Givinostat (0.2µM) reduced the % of live cells (Annexin V/Sytox negative) in all xenografts treated with the drug. In particular, after 72 hours, Givinostat was able to kill up to >90% of blast cells in all xenografts in contrast with the vehicle-treated samples which showed 25-60% of blasts still alive after treatment. The induction of cell death in Givinostat treated primografts was confirmed on primary samples from diagnosis using CyTOF which allowed us to observe that CD10+/CRLF2+ blasts were preferentially killed by the drug whereas CD45 high expressing cells (normal residue) remained unaffected by the treatment. Moreover, at low doses (0.2 µM), Givinostat downregulated genes of the JAK/STAT pathway (STAT5A, JAK2, IL7Rα, CRLF2, BCL2L1 and cMYC) and inhibited the basal and ligand induced signaling, reducing the phoshporylation of STAT5 in all tested primografts (mean fold decrease of pSTAT5: 2.4+0.6). Most importantly, to understand if the transcriptional downregulation of CRLF2 resulted in a functional effect, the downmodulation of CRLF2 protein was observed by flow cytometry (mean fold decrease 3.55+1.38). In vivo, Givinostat significantly reduced engraftment of human blasts in xenograft models of CRLF2 positive BCP-ALL (ranging from 1.9 to 34 fold decrease in bone marrow). Furthermore, Givinostat augmented the effect of chemotherapy in inhibiting proliferation and inducing apoptosis in CRLF2 rearranged cell lines and in primografts, in vitro. After 72 hours, the combined treatment reached 4.6-8.8 fold lower % of remaining viable blasts than chemotherapy alone (6.3-35.3% viable cells in chemotherapy-treated samples vs 1.4-4.3% of combination), 2.5-8.5 fold lower than Givinostat alone (4.3-36.4% vs 1.4-4.3%) and 2.4-13 fold lower than Methyl-prednisolone (5.2-39.1 vs 1-16.3%). In conclusion, Givinostat may represent a novel and effective tool, in combination with current chemotherapy, to treat this difficult to handle subset of ALL and these data strongly argue for the translation of Givinostat in combination with conventional therapy into human trials. Disclosures Davis: Fluidigm, Inc: Honoraria. Nolan:Fluidigm, Inc: Equity Ownership.

scholarly journals 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 ◽  
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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