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

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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Efficacy of Focal Adhesion Kinase Inhibition in Combination with Dasatinib in BCR-ABL1 Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.3766.3766 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3766-3766 ◽

Cited By ~ 1

Author(s):

Michelle L. Churchman ◽

Luke Jones ◽

Kathryn Evans ◽

Jennifer Richmond ◽

Irina M Shapiro ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Leukemic Cells ◽

Employment Equity ◽

Self Renewal ◽

Equity Ownership

Abstract Introduction: BCR-ABL1+ B-progenitor acute lymphoblastic leukemia (Ph+ B-ALL) is a highly aggressive disease that is often refractory to currently available therapies. Our previous genomic profiling studies have identified loss-of-function or dominant negative mutations in IKZF1, encoding the lymphoid transcription factor Ikaros, in over 80% of Ph+ ALL. In addition, deletion of CDKN2A, which encodes the INK4A and ARF tumor suppressors, is observed in approximately half of all cases (Mullighan et al., 2008). Alterations of IKZF1 are associated with poor outcome despite the use of tyrosine kinase inhibitors (TKIs). Ikzf1 alterations, including Ikaros isoform 6 (IK6), result in the acquisition of stem cell-like features, enhanced self-renewal, expression of adhesion molecules, and transcriptional upregulation of focal adhesion kinase (FAK), resulting in increased adhesion in vitro and in vivo, and decreased sensitivity to TKIs (Churchman, Cancer Cell, in press). VS-4718 is a potent, selective, and orally bioavailable FAK inhibitor currently under evaluation in a phase 1 clinical trial in subjects with various solid tumors, however in vivo efficacy in hematological malignancies had not been evaluated. Targeting FAK with VS-4718 is an attractive approach to abrogate the adhesive phenotype of IKZF1-altered leukemic cells potentially enhancing the effects of dasatinib in the treatment of high-risk BCR-ABL1 B-ALL. Methods: We examined the efficacy and mechanisms of FAK inhibition using VS-4718 as a single agent and in combination with dasatinib in vitro and in vivo in a range of xenograft and genetically engineered mouse models of BCR-ABL1 ALL. Each model had concomitant deletion of Arf which is observed in approximately 50% of human cases. Results: A pre-clinical in vivo trial of dasatinib and VS-4718 combination therapy in a murine C57Bl/6 Arf-/- BCR-ABL1 pre-B cell model resulted in a marked increase in survival in both IK6-expressing and non-IK6 cohorts of mice, and one complete long-term remission in the IK6-expressing group. Further, we showed increased efficacy of VS-4718 and dasatinib, compared to either agent alone, against two highly aggressive human Ph+ IK6-expressing B-ALL xenografts in vivo, with decreased infiltration of leukemic cells in bone marrow and spleens demonstrating a synergistic effect of the VS-4718/dasatinib combination. In vitro cell viability was reduced with induction of apoptosis at increasing concentrations of VS-4718 as a single agent, and further potentiated the effects of dasatinib in cytotoxicity assays using human xenografted and murine leukemic cells. VS-4718 profoundly diminished the ability of BCR-ABL1-expressing cells to form cell-matrix adhesions in vitro, as evident by the reduced adherence to fibronectin monolayers and bone marrow stromal cells. VS-4718 almost completely abolished the colony-forming potential of BCR-ABL1-expressing murine pre-B cells with and without Ikzf1 alterations at drug concentrations that do not affect cell viability suggestive of a reduction in self-renewal. Calvarial imaging of mice transplanted with Ikzf1-altered BCR-ABL1 leukemic cells and treated with VS-4718 alone in vivo revealed a discernible reduction in adhesion in the intact bone marrow niche of Prrx1-Cre; LSL-tdTomato recipient mice. VS-4718 treated leukemic cells localized to Prrx1-expressing perivascular endothelial cells and exhibited round morphology in contrast to the typical spindle-like appearance of Ikzf1-altered pre-B cells adhering to the bone marrow stroma, suggesting that VS-4718 treatment abolished the aberrant leukemic cell-stromal adhesion induced by Ikaros alterations in vivo. Conclusions: Direct inhibition of FAK with VS-4718 attenuates the adhesive, stem-like properties of IKZF1-altered BCR-ABL1 leukemic cells that contribute to the poor prognosis of patients treated with currently available therapies. Targeted FAK inhibition is thus a promising avenue for improving the response of BCR-ABL1 ALL to dasatinib, particularly in refractory cases harboring IKZF1 alterations. These data support the clinical development of VS-4718 in combination with dasatinib in Ph+ B-ALL. Disclosures Shapiro: Verastem: Employment, Equity Ownership. Pachter:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding; Incyte: Consultancy, Honoraria. Off Label Use: The FAK inhibitor VS-4718 for the treatment of BCR-ABL1 acute lymphoblastic leukemia in preclinical models.

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In vitro and in vivo single-agent efficacy of checkpoint kinase inhibition in acute lymphoblastic leukemia

Journal of Hematology & Oncology ◽

10.1186/s13045-015-0206-5 ◽

2015 ◽

Vol 8 (1) ◽

Cited By ~ 16

Author(s):

Ilaria Iacobucci ◽

Andrea Ghelli Luserna Di Rorà ◽

Maria Vittoria Verga Falzacappa ◽

Claudio Agostinelli ◽

Enrico Derenzini ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Kinase Inhibition ◽

Checkpoint Kinase

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mTOR inhibitors are synergistic with methotrexate: an effective combination to treat acute lymphoblastic leukemia

Blood ◽

10.1182/blood-2008-02-137141 ◽

2008 ◽

Vol 112 (5) ◽

pp. 2020-2023 ◽

Cited By ~ 87

Author(s):

David T. Teachey ◽

Cecilia Sheen ◽

Junior Hall ◽

Theresa Ryan ◽

Valerie I. Brown ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cyclin D1 ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Mtor Inhibitors ◽

Mechanistic Explanation ◽

Effective Combination ◽

Turn Over

Abstract We have previously demonstrated that mTOR inhibitors (MTIs) are active in preclinical models of acute lymphoblastic leukemia (ALL). MTIs may increase degradation of cyclin D1, a protein involved in dihydrofolate reductase (DHFR) synthesis. Because resistance to methotrexate may correlate with high DHFR expression, we hypothesized MTIs may increase sensitivity of ALL to methotrexate through decreasing DHFR by increasing turn-over of cyclin D1. We tested this hypothesis using multiple ALL cell lines and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with human ALL. We found MTIs and methotrexate were synergistic in combination in vitro and in vivo. Mice treated with both drugs went into a complete and durable remission whereas single agent treatment caused an initial partial response that ultimately progressed. ALL cells treated with MTIs had markedly decreased expression of DHFR and cyclin D1, providing a novel mechanistic explanation for a combined effect. We found methotrexate and MTIs are an effective and potentially synergistic combination in ALL.

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Clinical Trial of Paricalcitol in Myelodysplastic Syndrome.

Blood ◽

10.1182/blood.v104.11.4720.4720 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4720-4720

Author(s):

H. Phillip Koeffler ◽

James O’Kelly ◽

Noune Aslanian ◽

Keyword(s):

Clinical Trial ◽

Myelodysplastic Syndrome ◽

Clinical Response ◽

Vitamin D3 ◽

Single Agent ◽

White Blood Cells ◽

The Elderly ◽

Leukemic Cells

Abstract Myelodysplastic syndrome is often a pernicious disorder associated with pancytopenia in the elderly. Therapeutic approaches need to balance their toxicities versus the side-effects of the disease. 1,25(OH)2-vitamin-D3 inhibits proliferation and induces differentiation of leukemic cells in vitro. Small clinical trials have shown slight efficacy in MDS. Hypercalcemia prevents the administration of doses of this seco-steroid, which have been shown to be effective in vitro. This has provided a stimulus to identify vitamin D analogs that have anti-leukemic activity with minimal hypercalcemic effects. Paricalcitol (19-nor-1,25(OH)2D2, Zemplar) has been approved by the FDA for the treatment of secondary hyperparathyroidism; the drug is unique because it has little hypercalcemic potential; but in vitro, it has strong antileukemic effects. We conducted a clinical trial of oral paricalcitol to twelve MDS patients whose disease varied between an IPSS of low to high. Therapy began at 8 μg per day and increased at two week intervals until serum calcium was slightly above normal level; at which point, dose was decreased by 4-8 μg qd. The amount of paricalcitol taken varied between 8 μg qod to 54 μg qd (average 16 μg qd). We confirmed that the drug was having biologic activity in vivo by examining a target of the activated vitamin D3 receptor, 1,25-(OH)2-vitamin-D3-24(OH)ase mRNA. Each patient had prominent induction of this transcript in his or her white blood cells. Furthermore, in selected patients serum paricalcitol was measured and confirmed to be prominently present. The drug was well tolerated in all patients. Two of the 12 patients showed a clinical response. One patient’s platelet counts rose from 50,000 to 120,000/ul blood over 5 weeks; however, the patient succumbed to a fatal fungal infection. The second patient responded by a decrease in RBC transfusions associated with a rise in his hemoglobin, which lasted for about 5 months. Eventually, his hemoglobin began to fall, and erythropoietin therapy was substituted for paricalcitol. In summary, high doses of paricalcitol were well tolerated in all patients. Two patients had a partial clinical response. In general, paricalcitol given as a single agent to individuals with MDS is not therapeutically very efficacious; further trials should examine it in combination with other approaches.

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In Vitro Cellular Drug Resistance and Prognosis in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v90.7.2723 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2723-2729 ◽

Cited By ~ 179

Author(s):

G.J.L. Kaspers ◽

A.J.P. Veerman ◽

R. Pieters ◽

C.H. Van Zantwijk ◽

L.A. Smets ◽

...

Keyword(s):

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

Clinical Response ◽

Mtt Assay ◽

Combination Chemotherapy ◽

Risk Group ◽

Lymphoblastic Leukemia ◽

Newly Diagnosed ◽

Response To Chemotherapy

Abstract As an important determinant of the response to chemotherapy, measurements of cellular drug resistance may provide prognostically significant information, which could be useful for optimal risk-group stratification. The objective of this report is to determine the relation between in vitro resistance to 12 drugs, measured with the colorimetric methyl-thiazol-tetrazolium (MTT) assay, and long-term clinical response to chemotherapy in 152 children with newly diagnosed acute lymphoblastic leukemia. At risk-group stratified analyses, in vitro resistance to prednisolone, L-asparaginase, and vincristine were each significantly (P < .01) related to the probability of disease-free survival (pDFS) after combination chemotherapy. The combination of data for prednisolone, L-asparaginase, and vincristine provided a drug-resistance profile with prognostic independent significance superior to that of any single drug or any other factor. The 3-years pDFS was 100% for the group with the most sensitive profile, 20% of all patients, 84% (SE 6%) for the group with an intermediately sensitive profile, 40% of all patients, and 43% (SE 8%) for the remaining group with the most resistant profile (P < .001). In conclusion, the extent of in vitro cellular resistance to prednisolone, L-asparaginase, and vincristine, measured using the MTT assay, was significantly related to the clinical response to combination chemotherapy. Treatment failure in newly diagnosed childhood ALL can be predicted based on cellular drug resistance data.

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In Vitro Cellular Drug Resistance and Prognosis in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v90.7.2723.2723_2723_2729 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2723-2729 ◽

Cited By ~ 5

Author(s):

G.J.L. Kaspers ◽

A.J.P. Veerman ◽

R. Pieters ◽

C.H. Van Zantwijk ◽

L.A. Smets ◽

...

Keyword(s):

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

Clinical Response ◽

Mtt Assay ◽

Combination Chemotherapy ◽

Risk Group ◽

Lymphoblastic Leukemia ◽

Newly Diagnosed ◽

Response To Chemotherapy

As an important determinant of the response to chemotherapy, measurements of cellular drug resistance may provide prognostically significant information, which could be useful for optimal risk-group stratification. The objective of this report is to determine the relation between in vitro resistance to 12 drugs, measured with the colorimetric methyl-thiazol-tetrazolium (MTT) assay, and long-term clinical response to chemotherapy in 152 children with newly diagnosed acute lymphoblastic leukemia. At risk-group stratified analyses, in vitro resistance to prednisolone, L-asparaginase, and vincristine were each significantly (P < .01) related to the probability of disease-free survival (pDFS) after combination chemotherapy. The combination of data for prednisolone, L-asparaginase, and vincristine provided a drug-resistance profile with prognostic independent significance superior to that of any single drug or any other factor. The 3-years pDFS was 100% for the group with the most sensitive profile, 20% of all patients, 84% (SE 6%) for the group with an intermediately sensitive profile, 40% of all patients, and 43% (SE 8%) for the remaining group with the most resistant profile (P < .001). In conclusion, the extent of in vitro cellular resistance to prednisolone, L-asparaginase, and vincristine, measured using the MTT assay, was significantly related to the clinical response to combination chemotherapy. Treatment failure in newly diagnosed childhood ALL can be predicted based on cellular drug resistance data.

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Specific Inhibition of BCR-ABL Gene Expression in Acute Lymphoblastic Leukemia by Catalytic DNAzymes.

Blood ◽

10.1182/blood.v106.11.148.148 ◽

2005 ◽

Vol 106 (11) ◽

pp. 148-148 ◽

Cited By ~ 1

Author(s):

Tillmann Taube ◽

Shabnam Shalapour ◽

Georg J. Seifert ◽

Madlen Pfau ◽

Guenter Henze ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Specific Treatment ◽

Protein Translation ◽

Leukemic Cells ◽

Cellular Delivery ◽

Childhood All

Abstract The BCR-ABL fusion protein p190 resulting from the translocation t(9;22) exhibits dysregulated tyrosine kinase activity and was shown to cause acute lymphoblastic leukemia (ALL). Detection of the BCR-ABL fusion gene in childhood ALL is associated with an adverse prognosis and defines a group of high risk patients. Because the BCR-ABL gene fusion is specific for leukemic cells it represents an ideal target for leukemia specific treatment approaches. Catalytic DNAzymes are able to cleave mRNA in a sequence specific manner, causing inhibition of protein translation from the DNAzyme targeted mRNA both in vitro and in vivo. In order to cut off the BCR-ABL driven malignant proliferation, we designed DNAzymes to impede the expression of p190 BCR-ABL by cleaving the BCR-ABL mRNA adjacent to the fusion site. One construct was found that cleaved the target mRNA efficiently and specifically leaving BCR and ABL, relevant for normal cell survival and proliferation, unaffected. Activity and specificity of the BCR-ABL DNAzyme was investigated in cleavage assays with in vitro transcribed BCR-ABL, BCR and ABL mRNA. DNAzymes were delivered to cultured BCR-ABL+ ALL cells by lipid transfection. The efficiency of cellular delivery reached 90% as studied by flow cytometry, fluorescence microscopy and confocal microscopy after transfection of FITC labeled DNAzymes. To control for unspecific effects of DNAzyme delivery as well as for antisense effects, a catalytically inactive DNAzyme still exhibiting BCR-ABL antisense activity was designed. Fourty-eight hours after a single treatment of BCR-ABL+ ALL-cells with DNAyzmes the BCR-ABL mRNA concentration, as measured by quantitative real-time RT-PCR, was significantly reduced by 56% and 66% compared to controls treated with the inactivated DNAzyme and to untreated cells, respectively. Western blot analysis showed a decrease in p190 protein levels after DNAzyme treatment in comparison to the control treated with inactive DNAzyme as well as to the untreated cells. Most noteworthy, four days after a single DNAzyme treatment the net growth of BCR-ABL+ ALL cells treated with the active DNAzyme was inhibited by 68% compared to the untreated control. From these data we conclude, firstly, DNAzymes targeting mRNA coding for the minor BCR-ABL variant are able to significantly reduce the amount of fusion mRNA in the cells, leading to a reduction in protein expression, followed by the inhibition of BCR-ABL driven proliferation of ALL cells. Secondly, this exemplified setting gives a hint that DNAzymes might be of therapeutic use in hematopoietic malignancies associated with specific mutations, expressing oncogenic fusion genes or overexpressing oncogenic genes.

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Effective Targeting of Leukemic Cells in Children with B-Precursor Acute Lymphoblastic Leukemia Treated with Anti-CD22 (Epratuzumab). A Children’s Oncology Group (COG) Study.

Blood ◽

10.1182/blood.v108.11.2585.2585 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2585-2585 ◽

Cited By ~ 2

Author(s):

Michael J. Borowitz ◽

William L. Carroll ◽

Peter C. Adamson ◽

Mitchell S. Cairo ◽

David M. Goldenberg ◽

...

Keyword(s):

Induction Chemotherapy ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Poor Response ◽

Antigen Expression ◽

Response To Therapy ◽

Leukemic Cells ◽

Color Flow

Abstract Identifying new approaches to treating relapsed ALL is a top priority because these patients fare poorly with current retrieval strategies. COG protocol ADVL04P2 is a phase I/II feasibility pilot study for children with relapsed B-precursor ALL that uses epratuzumab, a humanized anti-CD22 monoclonal antibody, in combination with conventional re-induction chemotherapy. Children whose blasts are CD22 positive first receive 4 doses of 360 mg/m2 epratuzumab as a single agent on days minus(−)14, −10, −6 and −2. Subsequently, they receive epratuzumab weekly in combination with standard re-induction chemotherapy starting on day 0. We assessed the effectiveness of epratuzumab targeting by by assaying CD22 expression on residual leukemic blasts. Peripheral blood was obtained from 15 patients 24 hours after administration of the first dose (day −13), and again at day −6 and day 0 and stained with the combination of CD10/CD22/CD45/CD19 in four color flow cytometry to permit assessment of CD22 on analytically isolated leukemic blasts. We determined quantitative expression of CD22 with a calibration kit (Quantibrite, BDBiosciences, San Jose, CA) using two PE-conjugated anti CD22 antibodies directed against different epitopes of the CD22 molecule: clone RFB4 (Caltag, Burlingame CA), directed against the same epitope as epratuzumab, and clone SHCL-1 (BDBiosciences), directed against a non-cross-reacting epitope of CD22. RFB4 binding was decreased by more than 99% within 24 hours after administration of the first dose in all patients, indicating rapid targeting of epratuzumab to leukemic cells. In all but 4 patients, levels remained low at all subsequent time points; in 2 patients expression was restored to about 15% of baseline levels by day 0, and 2 additional patients had very small (1–2%) subsets of blasts with significant expression, one at day −6 and one at day 0. In contrast to RFB4, SHCL-1 identified residual CD22 antigen expression, but SHCL-1 binding was decreased by an average of 70–75% in 14 of 15 patients at all three post-epratuzumab timepoints. The one patient with no change in SHCL-1 binding was a patient with MLL-rearranged ALL with very low levels of CD22 expression at diagnosis, and poor response to therapy. Because binding of epratuzumab to CD22 has been shown to result in antigen internalization in vitro, we interpret our findings as demonstrating in vivo internalization of CD22 with epratuzumab binding, though we cannot exclude either shedding of antigen or conformational change to block SHCL-1 sites. We conclude that epratuzumab rapidly targets CD22 on ALL blasts in vivo, though some blasts in some patients may escape over time. Targeting is associated with a physical change in CD22 antigen expression in most patients, most likely through internalization.

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Preclinical Activity of LBH589 Alone or in Combination with Chemotherapy in a Xenogeneic Mouse Model of Human Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.1520.1520 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1520-1520

Author(s):

Xabier Agirre ◽

Amaia Vilas-Zornoza ◽

Gloria Abizanda ◽

Cristina Moreno ◽

Victor Segura ◽

...

Keyword(s):

Cell Proliferation ◽

Acute Lymphoblastic Leukemia ◽

Mouse Model ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Regulatory Function ◽

Human Leukemia

Abstract Abstract 1520 Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here we analyzed the therapeutic effect of LBH589 or panobinostat, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced a significant dose-dependent increase in cell apoptosis and a markedly inhibition of cell proliferation, which were associated with increased H3 and H4 histone acetylation. While apoptosis of ALL cells was detected between 12 and 24 hours after treatment with LBH589, changes in acetylated H3 and H4 were detected as early as 2 hours. Phosphorylation of H2AX, as an early marker of DNA damaged, was detected 12 to 24 hours after in vitro treatment with LBH589. These results suggest that H3 and H4 acetylation precede DNA damaged and induction of apoptosis indicating that inhibition of HDAC is likely to be responsible at least in part for LBH589 induced apoptosis and inhibition of cell proliferation. The in vivo activity of LBH589 was initially examined in a subcutaneous ALL mouse model. The ALL cell lines TOM-1 and MOLT-4 were transplanted (1×106 cell per animal) subcutaneously into the left flanks of 6-week-old female BALB/cA-Rag2−/−γc−/−. These cell lines develop into a rapidly growing tumor. Treatment with 5mg/kg of LBH589 was initiated 24 hours after injection of the leukemic cells, included 3 cycles of 5 consecutive days of LBH589 with two days rest between cycles and animals were monitored for 24 days. A significant inhibition of tumor growth was demonstrated in animals treated with LBH589 compared with control animals (P <0.01). Inhibition of leukemia cell growth was associated with an increase in the levels of acetylated H3 and H4 and an increase in phosphorylated H2AX in the leukemic cells obtained after sacrifice of mice. These results suggest that LBH589 has a powerful antileukemic effect not only in vitro but also in vivo. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2−/−γc−/− mice was established, allowing continuous passages of transplanted cells to several mouse generations. A total of 10 million cells from a patient with T-ALL (ALL-T1) and a patient with B-ALL (ALL-B1) were administered intravenously into the tail vein of 6-week-old immunodeficient female BALB/cA-Rag2−/−γc−/− mice. Kinetics of engraftment of leukemic cells was monitored in PB and BM by phenotyping while organ infiltration was analyzed by immunohistochemistry. There were no significant differences in the genome, methylome or transcriptome between the original sample and the samples obtained after multiple generations on mice. To determine the efficacy of LBH589 alone or in combination with drugs currently used for treatment of ALL, BALB/cA-RAG2−/−γc−/− mice engrafted with ALL-T1 and ALL-B1 cells were treated with LBH589, Vincristine and Dexamethasone or a combination of LBH589 with Vincristine and Dexamethasone. Treatment was initiated when disease could be detected in PB by FACS (24 hours after injection of cells for ALL-T1 and between day 17 and 21 after injection for ALL-B1). LBH589 was administered i.p. on days 1–5, 8–12 and 15–19, Vincristine i.v. on days 1, 8 and 21 and Dexamethasone daily until day 21 i.p. and survival was analyzed. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving Vincristine and Dexametasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with Vincristine and Dexametasone. Our results demonstrate the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL. Disclosures: No relevant conflicts of interest to declare.

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PDE4B Modulates Glucocorticoid Sensitivity in Childhood Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.530.530 ◽

2012 ◽

Vol 120 (21) ◽

pp. 530-530

Author(s):

Jun J. Yang ◽

Shuyu E ◽

Shuhong Shen ◽

Deepa Bhojwani ◽

Huining Kang ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Molecular Mechanisms ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Lymphoid Cells ◽

Childhood Acute Lymphoblastic Leukemia ◽

Remission Induction ◽

Newly Diagnosed ◽

Glucocorticoid Sensitivity

Abstract Abstract 530 Although cure rates of childhood acute lymphoblastic leukemia (ALL) have improved dramatically, a substantial portion of children still relapse and the prognosis of relapsed ALL is extremely poor. Therefore, a better understanding of molecular determinants of drug resistance in ALL is imperative for the development of more efficacious and individualized therapy, particularly in the context of relapsed disease. In a recent genome-wide association study of 2,534 children with ALL, we identified that genetic variation in PDE4B– phosphodiesterase 4B–strongly influenced the risk of ALL relapse across various ALL treatment regimens (Nat Genet 2011: 43:237). While PDE4B is the predominant phosphodiesterase in lymphoid tissue and a major regulator of cyclic AMP, its role in ALL pathobiology is largely unknown. To this end, we sought to characterize the molecular mechanisms by which PDE4B modulates antileukemic drug sensitivity in ALL. We first characterized PDE4B expression in ALL blasts at diagnosis and its relationship with drug response in vivo in 3 independent cohorts of children with ALL. In 191 children with newly diagnosed ALL enrolled on the COG P9906 protocol, PDE4B expression in ALL blasts was positively correlated with minimal residual disease status at the end of remission induction (P=0.0096). Higher PDE4B expression was also associated with slower early response to induction therapy in COG 1961 (N=82, P=0.019). In 275 children with newly-diagnosed ALL enrolled on the Shanghai Children's Medical Center ALL05 study, we determined that PDE4B2 was the predominant isoform of PDE4B in ALL blasts (P<0.0001); there was also a trend that children with poor in vivo response to the upfront single-agent prednisone treatment had higher PDE4B2 expression in the diagnostic blasts (P=0.042). In parallel, shRNA-mediated knock-down of PDE4B in a glucocorticoid-sensitive (i.e., Nalm6) and a glucocorticoid-resistant (i.e., UOCB1) ALL cell line significantly potentiated cytotoxic effects of prednisolone, whereas Nalm6 and CEM ALL cells over-expressing PDE4B2 were significantly more resistant to prednisolone compared to cells transduced with empty vectors. Sensitization to glucocorticoid was further amplified by forskolin, a stimulator of cAMP synthesis, and was concomitant with activation of PKA as determined by CREB phosphorylation, suggesting that the effects of PDE4B on glucocorticoid sensitivity involve signaling of the cAMP-PKA cascade. Importantly, PDE4B knockdown by shRNA and pharmacologic inhibition by rolipram in Nalm6 and UOCB1 cells consistently led to upregulation of BIM, a key apoptosis regulator and a critical mediator of glucocorticoid sensitivity in lymphoid cells. In both cells lines, BIM upregulation following PDE4B inhibition was enhanced by forskolin but suppressed by PKA inhibitor H89, indicating that BIM might act as a downstream effector of cAMP-PKA signaling in ALL. Finally, we evaluated effects of small molecule regulators of the PDE-cAMP pathway (namely, forskolin and rolipram) on glucocorticoid response in primary ALL cells (N=23) in vitro. Measuring IC50 by MTT assay, increased cytotoxicity was observed in 16 (70%), 8 (35%), and 17 (74%) cases, when forskolin, rolipram, or both were added to prednisolone, respectively. In conclusion, tumor expression of PDE4B at diagnosis was associated with poorer early treatment response in ALL, particularly resistance to glucocorticoids in vivo. Inhibition of PDE4B sensitized both cultured and primary ALL cells to glucocorticoids via activating the cAMP-PKA pathway and subsequent upregulation of BIM. Disclosures: No relevant conflicts of interest to declare.

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