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

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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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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Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽

10.1182/blood.v120.21.5028.5028 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5028-5028 ◽

Cited By ~ 1

Author(s):

Deepak Sampath ◽

Elizabeth Punnoose ◽

Erwin R. Boghaert ◽

Lisa Belmont ◽

Jun Chen ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Cell Lines ◽

Single Agent ◽

Employment Equity ◽

Bone Marrow Biopsies ◽

Xenograft Models ◽

Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

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AT1412, a Patient-Derived Antibody in Development for the Treatment of CD9 Positive Precursor B-Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-127894 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4461-4461

Author(s):

Greta De Jong ◽

Sophie E Levie ◽

Remko Schotte ◽

Wouter Pos ◽

Daniel Go ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Melanoma Cells ◽

Employment Equity ◽

Equity Ownership

Despite rapid advances in immunotherapeutic options for precursor B-acute lymphoblastic leukemia (ALL), outcomes remain poor especially for adult ALL and relapsed pediatric ALL. With conventional chemotherapy, remission percentages in adult ALL range from 75 to 90%, but relapse rates are high and long-term leukemia-free survival ranges between 35-70% depending on age and risk group. The introduction of CD19 targeting immunotherapy has significantly improved patient outcomes in (relapsed) B-ALL. However, tumor escape via downregulation of CD19 occurs in a significant number of patients. Therefore an ongoing urgency remains for the identification of additional or alternative immunotherapeutic targets for the treatment of ALL. AT1412 is an antibody that was identified from the peripheral blood memory B cell pool of a patient cured of metastatic melanoma after adoptive T-cell therapy, using a B cell immortalization technology (AIMSelect) with ectopic Bcl-6 and Bcl-xL expression as described previously [Kwakkenbos et al. Nat. Med. 2010]. The antibody was selected based on differential binding to melanoma cells as compared to healthy melanocytes and was shown to be successful in killing melanoma cells in vitro and in vivo [manuscript submitted]. In addition to melanoma, AT1412 binds other tumor types including B-ALL, gastric, colon- and pancreatic cancer. The target of AT1412 is the tetraspanin CD9, which is expressed by more than half of all B-ALL. Expression of CD9 has been correlated with adverse prognosis [Liang et al. Cancer Biomark. 2018]. We assessed binding of this human CD9 antibody to a panel of ALL cell lines using flow cytometry. Binding of AT1412 to the B-ALL cell lines SUP-B15, MHH-CALL-2 and CCRF-SB varied as expected based on the CD9 levels that we detected using a commercial CD9 antibody. AT1412 induced antibody dependent cellular cytotoxicity (ADCC) on these cells, in line with the level of AT1412 binding. No binding was seen to the T-ALL cell line Jurkat. Importantly, these findings were confirmed in primary ALL samples, obtained prospectively at diagnosis from a cohort of patients with T- or B-ALL (n=30). AT1412 showed binding to 61% of B-ALL samples but not to T-ALL samples. The potential of AT1412 to induce ADCC was tested on patient samples from the same panel. Remarkably, AT1412 induced ADCC of all B-ALL samples it bound to (8 out of 14) and of none of the T-ALL samples. Cytotoxicity significantly correlated with the level of AT1412 binding. These findings were supported by the observation that AT1412 induced B-ALL cell death when a freshly drawn whole bone marrow sample from a patient with newly diagnosed B-ALL was cocultured with AT1412. AT1412-induced cell death of B-ALL blasts occurred without affecting the monocytic, granulocytic and lymphocytic populations. This cell death was not observed when this patient's ALL blasts were incubated with AML-targeting antibodies. Remarkably, AT1412 induced cell death in the absence of added effector cells or other (chemo)therapeutic agents, while the bone marrow sample contained over 80% blasts and as little as 3% lymphocytes. We are currently investigating the in vivo efficacy of the antibody in a humanized immune system mouse model with human B-ALL. Taken together, the majority of precursor B-ALL blasts express CD9 and expression of CD9 is associated with a dismal outcome. Our data demonstrate that CD9 can be successfully targeted by the human CD9 antibody AT1412, suggesting that AT1412 has the potential to be developed as a therapeutic antibody for B-ALL. AT1412 is currently being advanced through preclinical development. Disclosures De Jong: AIMM Therapeutics: Employment. Levie:AIMM Therapeutics: Employment. Schotte:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. Pos:AIMM Therapeutics: Patents & Royalties: Patent WO2017119811A1. Go:AIMM Therapeutics: Employment, Patents & Royalties: Patent WO2017119811A1. Yasuda:AIMM Therapeutics: Employment, Equity Ownership. Cercel:AIMM Therapeutics: Employment. van Hal-van Veen:AIMM Therapeutics: Employment. Frankin:AIMM Therapeutics: Employment. Villaudy:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. van Helden:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. van Eenennaam:AIMM Therapeutics: Employment. Spits:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. Hazenberg:AIMM Therapeutics: Other: Employment/equity of partner/spouse.

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The First Fully Experimental In Vivo Model of Human Leukemogenesis: Human Hematopoietic Stem Cells Infected with MLL-ENL Induce Pro-B Acute Lymphoblastic Leukemia in NOD/SCID Mice.

Blood ◽

10.1182/blood.v106.11.238.238 ◽

2005 ◽

Vol 106 (11) ◽

pp. 238-238

Author(s):

Frederic Barabe ◽

James A. Kennedy ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Scid Mice ◽

Leukemic Cells ◽

In Vivo Model ◽

Self Renewal ◽

Genetic Event ◽

Hematopoietic Stem

Abstract Identification of genes and translocations involved in human leukemia, as well as classification and clustering by gene arrays, have greatly evolved in the past years. However, the mechanisms of human leukemogenesis remain to be elucidated and the failure to develop an in vivo model where primary human hematopoietic cells are transformed into leukemic cells represents a significant limitation. Using a retrovirus encoding the oncogene MLL-ENL resulting from the t(11;19)(q23;p13.3) translocation found in acute myeloid leukemias (AML) as well as in acute lymphoblastic leukemias (ALL) of B or T cell origin, we infected lineage-negative cord blood cells and injected those cells into sub-lethally irradiated NOD/SCID mice. 15 to 20 weeks after injection, all the mice developed an aggressive pro-B acute lymphoblastic leukemia characterized by immature B cells (CD10+, CD19+, CD20−, IgD−, IgM−) involving more than 90% of bone marrow. Spleen and thymus were increased in size and infiltrated with >90% leukemic cells. Furthermore, analysis of the lungs and liver showed significant infiltration of these organs. Transplantation of leukemic cells from primary mice to secondary recipients was able to recapitulate the disease with the same phenotype and the same organ involvement in a shorter period of time. If MLL-ENL transduced cells are grown in suspension culture with IL-3 and SCF, there is massive proliferation of cells blocked in differentiation along the monocytic lineage. In contrast to untransduced cells, colony-forming progenitors were maintained long term in these cultures and could be serially replated, suggestive of an enhanced capacity for self-renewal. After 50 to 70 days in culture, these cells were injected in NOD/SCID mice and mice were analyzed after 12 to 15 weeks. Monoblastic cells were engrafted in the bone marrow and spleen with the same phenotype of the cultured cells (CD33+, CD11b+, CD15+, HLA DR+). These cells were able to engraft secondary and tertiary recipients formally demostrating increased self-renewal capacity of the transformed stem cell. In a limited number of primary mice, transplanted with high cell doses, AML developed at 15 weeks post-transplant. To our knowledge, these results provide the first in vivo model where human hematopietic stem/progenitor cells are transformed into leukemia. Remarkably, depending on the cellular environment, MLL-ENL can induce ALL or AML in primary cells as a sole genetic event, although we cannot rule out the spontaneous acquistion of additional co-operating genetic or epigenetic abnormalities. This model provides a significant step forward to understand the mechanisms involved in human leukemogenesis.

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Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽

10.1182/blood.v52.4.712.712 ◽

1978 ◽

Vol 52 (4) ◽

pp. 712-718 ◽

Cited By ~ 28

Author(s):

SD Smith ◽

EM Uyeki ◽

JT Lowman

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Bone Marrow Cells ◽

Lymphoblastic Leukemia ◽

Lymphoid Cells ◽

Assay System ◽

Leukemic Cells ◽

Specific Cell ◽

Specific Cell Surface

Abstract An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

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Bone marrow from children in relapse with pre-B acute lymphoblastic leukemia proliferates and disseminates rapidly in scid mice

Blood ◽

10.1182/blood.v78.11.2973.bloodjournal78112973 ◽

1991 ◽

Vol 78 (11) ◽

pp. 2973-2981 ◽

Cited By ~ 2

Author(s):

S Kamel-Reid ◽

M Letarte ◽

M Doedens ◽

A Greaves ◽

B Murdoch ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Poor Prognosis ◽

Lymphoblastic Leukemia ◽

Growth Characteristics ◽

Scid Mice ◽

Leukemic Cells ◽

Murine Bone Marrow ◽

In Vivo Growth

Bone marrow samples from patients with pre-B acute lymphoblastic leukemia (pre-B ALL), either at diagnosis or at relapse, were transplanted into scid mice to determine whether these freshly obtained leukemic cells could proliferate in vivo and whether there were any differences in their in vivo growth characteristics. Cells from three patients who relapsed within 13 months of diagnosis proliferated rapidly in the murine bone marrow, spleen, and thymus, invaded peripheral organs, and resulted in morbidity and mortality of the animals within 4 to 16 weeks. Cells from two patients who relapsed 3.5 years after diagnosis grew much slower than the early relapse samples, taking up to 30 weeks to infiltrate the bone marrow of recipient mice. In contrast, leukemic cells were absent or were detected at low numbers in scid mice transplanted with cells obtained at diagnosis from three patients who have not yet relapsed. These results show an increased ability of leukemic cells from patients with aggressive lymphoblastic leukemia of poor prognosis to proliferate in scid mice.

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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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Serotherapy of acute lymphoblastic leukemia with monoclonal antibody

Blood ◽

10.1182/blood.v58.1.141.141 ◽

1981 ◽

Vol 58 (1) ◽

pp. 141-152 ◽

Cited By ~ 201

Author(s):

J Ritz ◽

JM Pesando ◽

SE Sallan ◽

LA Clavell ◽

J Notis-McConarty ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Chemotherapeutic Agents ◽

Leukemic Cells ◽

Antigenic Modulation ◽

Multiple Intravenous Infusions ◽

Marrow Cellularity

Abstract We tested the efficacy of passive serotherapy in the treatment of acute lymphoblastic leukemia in four patients who had relapsed while receiving standard chemotherapeutic agents. Each patient received multiple intravenous infusions of J-5 monoclonal antibody specific for common acute lymphoblastic leukemia antigen (CALLA). In the three patients with circulating leukemic cells, there was a rapid decrease in circulating blasts that began immediately after antibody infusion, but not all leukemic cells were cleared, and remaining cells appeared to be resistant to further serotherapy. Although J-5 antibody was also demonstrable on bone marrow lymphoblasts immediately after antibody infusion in one patient, there was no change in bone marrow cellularity or differential during serotherapy. Analysis of the cell surface phenotype of leukemic cells during serotherapy and in vitro studies with patient cells suggests that resistance to serotherapy was mediated in part by antigenic modulation of CALLA in response to J-5 antibody.

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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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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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