Barbara Szymanska

The Bcl-2 Homology Domain 3 Mimetic ABT-737 Targets the Apoptotic Machinery in Acute Lymphoblastic Leukemia Resulting in Synergistic in Vitro and in Vivo Interactions with Established Drugs

Antiapoptotic Bcl-2 proteins are overexpressed in a number of cancers, including leukemias, and are frequently associated with resistance to conventional chemotherapeutic drugs. ABT-737, a Bcl-2 homology domain 3 mimetic (for structure, see Nature 435:677–681, 2005) inhibits the prosurvival function of Bcl-2, Bcl-XL, and Bcl-w. We show that ABT-737 was effective as a single agent against a panel of pediatric acute lymphoblastic leukemia (ALL) xenografts, previously established, from patient biopsies, in immunodeficient mice. Although in vitro resistance of leukemia cell lines correlated with expression of the prosurvival protein Mcl-1, there was no relationship between Mcl-1 expression and in vivo xenograft response to ABT-737. However, expression of the pro-apoptotic protein Bim, and the extent of its association with Bcl-2, significantly correlated with in vivo ABT-737 sensitivity. ABT-737 potentiated the antileukemic effects of l-asparaginase, topotecan, vincristine, and etoposide against drug-resistant xenografts in vitro and in vivo. Finally, we show that the combination of l-asparaginase (by specifically down-regulating Mcl-1 protein levels), topotecan (by activating p53 via DNA damage), and ABT-737 (by inhibiting antiapoptotic Bcl-2 family members) caused profound synergistic antileukemic efficacy both in vitro and in vivo. Rational targeting of specific components of the apoptotic pathway may be a useful approach to improve the treatment of refractory or relapsed pediatric ALL. Overall, this study supports the inclusion of the clinical derivative of ABT-737, ABT-263 (for structure, see Cancer Res 68:3421–3428, 2008), into clinical trials against relapsed/refractory pediatric ALL.

Epigenetic silencing of BIM in glucocorticoid poor-responsive pediatric acute lymphoblastic leukemia, and its reversal by histone deacetylase inhibition.

Glucocorticoids play a critical role in the therapy of lymphoid malignancies, including pediatric acute lymphoblastic leukemia (ALL), although the mechanisms underlying cellular resistance remain unclear. We report glucocorticoid resistance attributable to epigenetic silencing of the BIM gene in pediatric ALL biopsies and xenografts established in immune-deficient mice from direct patient explants as well as a therapeutic approach to reverse resistance in vivo. Glucocorticoid resistance in ALL xenografts was consistently associated with failure to up-regulate BIM expression after dexamethasone exposure despite confirmation of a functional glucocorticoid receptor. Although a comprehensive assessment of BIM CpG island methylation revealed no consistent changes, glucocorticoid resistance in xenografts and patient biopsies significantly correlated with decreased histone H3 acetylation. Moreover, the histone deacetylase inhibitor vorinostat relieved BIM repression and exerted synergistic antileukemic efficacy with dexamethasone in vitro and in vivo. These findings provide a novel therapeutic strategy to reverse glucocorticoid resistance and improve outcome for high-risk pediatric ALL.

Pronounced hypoxia in models of murine and human leukemia: high efficacy of hypoxia-activated prodrug PR-104.

Recent studies indicate that interactions between leukemia cells and the bone marrow (BM) microenvironment promote leukemia cell survival and confer resistance to anti-leukemic drugs. There is evidence that BM microenvironment contains hypoxic areas that confer survival advantage to hematopoietic cells. In the present study we investigated whether hypoxia in leukemic BM contributes to the protective role of the BM microenvironment. We observed a marked expansion of hypoxic BM areas in immunodeficient mice engrafted with acute lymphoblastic leukemia (ALL) cells. Consistent with this finding, we found that hypoxia promotes chemoresistance in various ALL derived cell lines. These findings suggest to employ hypoxia-activated prodrugs to eliminate leukemia cells within hypoxic niches. Using several xenograft models, we demonstrated that administration of the hypoxia-activated dinitrobenzamide mustard, PR-104 prolonged survival and decreased leukemia burden of immune-deficient mice injected with primary acute lymphoblastic leukemia cells. Together, these findings strongly suggest that targeting hypoxia in leukemic BM is feasible and may significantly improve leukemia therapy.

Gene therapy of diabetes: glucose-stimulated insulin secretion in a human hepatoma cell line (HEP G2ins/g).

In order to design a feasible somatic cell gene delivery system for the treatment of type I diabetes, a suitable cell type needs to be determined. We have previously shown that the stable transfection of the full-length insulin cDNA into the human liver cell line, (HEP G2ins) resulted in synthesis, storage and acute regulated release of insulin to analogues of cAMP, but not to the physiological stimulus glucose. In attempting to explain the lack of glucose responsiveness of the HEP G2ins cells we have stably transfected these cells with the human islet glucose transporter GLUT 2 (HEP G2ins/g cells). The HEP G2ins/g cell clones exhibit glucose-stimulated insulin secretion and glucose potentiation of the secretory response to nonglucose secreta- gogues. While glucose responsiveness commenced at a lower concentration than normal islets, a secretion curve approaching normal physiological conditions was generated. Immunoelectron microscopy revealed the presence of insulin-containing granules, similar in size and appearance to those of the normal beta cell. These results demonstrate that while it is most likely that the HEP G2ins/g cell line predominantly secretes insulin via the constitutive pathway, significant acute regulated release was seen in response to glucose, and thus represents significant progress in the creation of a genetically engineered ‘artificial beta cell’ from a human hepatocyte cell line.

The Anti-CD19 Antibody–Drug Conjugate SAR3419 Prevents Hematolymphoid Relapse Postinduction Therapy in Preclinical Models of Pediatric Acute Lymphoblastic Leukemia

Purpose: Relapsed or refractory pediatric acute lymphoblastic leukemia (ALL) remains a major cause of death from cancer in children. In this study, we evaluated the efficacy of SAR3419, an antibody–drug conjugate of the maytansinoid DM4 and a humanized anti-CD19 antibody, against B-cell precursor (BCP)-ALL and infant mixed lineage leukemia (MLL) xenografts. Experimental Design: ALL xenografts were established as systemic disease in immunodeficient (NOD/SCID) mice from direct patient explants. SAR3419 was administered as a single agent and in combination with an induction-type regimen of vincristine/dexamethasone/l-asparaginase (VXL). Leukemia progression and response to treatment were assessed in real-time, and responses were evaluated using strict criteria modeled after the clinical setting. Results: SAR3419 significantly delayed the progression of 4 of 4 CD19+ BCP-ALL and 3 of 3 MLL-ALL xenografts, induced objective responses in all but one xenograft but was ineffective against T-lineage ALL xenografts. Relative surface CD19 expression across the xenograft panel significantly correlated with leukemia progression delay and objective response measure scores. SAR3419 also exerted significant efficacy against chemoresistant BCP-ALL xenografts over a large (10-fold) dose range and significantly enhanced VXL-induced leukemia progression delay in two highly chemoresistant xenografts by up to 82 days. When administered as protracted therapy following remission induction with VXL, SAR3419 prevented disease recurrence into hematolymphoid and other major organs with the notable exception of central nervous system involvement. Conclusion: These results suggest that incorporation of SAR3419 into remission induction protocols may improve the outcome for high-risk pediatric and adult CD19+ ALL. Clin Cancer Res; 19(7); 1795–805. ©2013 AACR.

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic β cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose–response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for β cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic β cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic β cells and become a therapy for the treatment of type I diabetes.

Cell and Molecular Determinants of In Vivo Efficacy of the BH3 Mimetic ABT-263 against Pediatric Acute Lymphoblastic Leukemia Xenografts

Purpose: Predictive biomarkers are required to identify patients who may benefit from the use of BH3 mimetics such as ABT-263. This study investigated the efficacy of ABT-263 against a panel of patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts and utilized cell and molecular approaches to identify biomarkers that predict in vivo ABT-263 sensitivity. Experimental Design: The in vivo efficacy of ABT-263 was tested against a panel of 31 patient-derived ALL xenografts composed of MLL-, BCP-, and T-ALL subtypes. Basal gene expression profiles of ALL xenografts were analyzed and confirmed by quantitative RT-PCR, protein expression and BH3 profiling. An in vitro coculture assay with immortalized human mesenchymal cells was utilized to build a predictive model of in vivo ABT-263 sensitivity. Results: ABT-263 demonstrated impressive activity against pediatric ALL xenografts, with 19 of 31 achieving objective responses. Among BCL2 family members, in vivo ABT-263 sensitivity correlated best with low MCL1 mRNA expression levels. BH3 profiling revealed that resistance to ABT-263 correlated with mitochondrial priming by NOXA peptide, suggesting a functional role for MCL1 protein. Using an in vitro coculture assay, a predictive model of in vivo ABT-263 sensitivity was built. Testing this model against 11 xenografts predicted in vivo ABT-263 responses with high sensitivity (50%) and specificity (100%). Conclusion: These results highlight the in vivo efficacy of ABT-263 against a broad range of pediatric ALL subtypes and shows that a combination of in vitro functional assays can be used to predict its in vivo efficacy. Clin Cancer Res; 20(17); 4520–31. ©2014 AACR.

Pharmacokinetic Modeling of an Induction Regimen for In Vivo Combined Testing of Novel Drugs against Pediatric Acute Lymphoblastic Leukemia Xenografts

Current regimens for induction therapy of pediatric acute lymphoblastic leukemia (ALL), or for re-induction post relapse, use a combination of vincristine (VCR), a glucocorticoid, and l-asparaginase (ASP) with or without an anthracycline. With cure rates now approximately 80%, robust pre-clinical models are necessary to prioritize active new drugs for clinical trials in relapsed/refractory patients, and the ability of these models to predict synergy/antagonism with established therapy is an essential attribute. In this study, we report optimization of an induction-type regimen by combining VCR, dexamethasone (DEX) and ASP (VXL) against ALL xenograft models established from patient biopsies in immune-deficient mice. We demonstrate that the VXL combination was synergistic in vitro against leukemia cell lines as well as in vivo against ALL xenografts. In vivo, VXL treatment caused delays in progression of individual xenografts ranging from 22 to >146 days. The median progression delay of xenografts derived from long-term surviving patients was 2-fold greater than that of xenografts derived from patients who died of their disease. Pharmacokinetic analysis revealed that systemic DEX exposure in mice increased 2-fold when administered in combination with VCR and ASP, consistent with clinical findings, which may contribute to the observed synergy between the 3 drugs. Finally, as proof-of-principle we tested the in vivo efficacy of combining VXL with either the Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, or arsenic trioxide to provide evidence of a robust in vivo platform to prioritize new drugs for clinical trials in children with relapsed/refractory ALL.