Kathryn Evans

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.

Venetoclax responses of pediatric ALL xenografts reveal sensitivity of MLL-rearranged leukemia

The clinical success of the BCL-2-selective BH3-mimetic venetoclax in patients with poor prognosis chronic lymphocytic leukemia (CLL) highlights the potential of targeting the BCL-2-regulated apoptotic pathway in previously untreatable lymphoid malignancies. By selectively inhibiting BCL-2, venetoclax circumvents the dose-limiting, BCL-XL-mediated thrombocytopenia of its less selective predecessor navitoclax, while enhancing efficacy in CLL. We have previously reported the potent sensitivity of many high-risk childhood acute lymphoblastic leukemia (ALL) xenografts to navitoclax. Given the superior tolerability of venetoclax, here we have investigated its efficacy in childhood ALL. We demonstrate that in contrast to the clear dependence of CLL on BCL-2 alone, effective antileukemic activity in the majority of ALL xenografts requires concurrent inhibition of both BCL-2 and BCL-XL We identify BCL-XL expression as a key predictor of poor response to venetoclax and demonstrate that concurrent inhibition of both BCL-2 and BCL-XL results in synergistic killing in the majority of ALL xenografts. A notable exception is mixed lineage leukemia-rearranged infant ALL, where venetoclax largely recapitulates the activity of navitoclax, identifying this subgroup of patients as potential candidates for clinical trials of venetoclax in childhood ALL. Conversely, our findings provide a clear basis for progressing navitoclax into trials ahead of venetoclax in other subgroups.

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.

AKR1C3 is a biomarker of sensitivity to PR-104 in preclinical models of T-cell acute lymphoblastic leukemia.

PR-104, a phosphate ester of the nitrogen mustard prodrug PR-104A, has shown evidence of efficacy in adult leukemia clinical trials. Originally designed to target hypoxic cells, PR-104A is independently activated by aldo-keto-reductase 1C3 (AKR1C3). The aim of this study was to test whether AKR1C3 is a predictive biomarker of in vivo PR-104 sensitivity. In a panel of 7 patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts, PR-104 showed significantly greater efficacy against T-lineage ALL (T-ALL) than B-cell-precursor ALL (BCP-ALL) xenografts. Single-agent PR-104 was more efficacious against T-ALL xenografts compared with a combination regimen of vincristine, dexamethasone, and l-asparaginase. Expression of AKR1C3 was significantly higher in T-ALL xenografts compared with BCP-ALL, and correlated with PR-104/PR-104A sensitivity in vivo and in vitro. Overexpression of AKR1C3 in a resistant BCP-ALL xenograft resulted in dramatic sensitization to PR-104 in vivo. Testing leukemic blasts from 11 patients confirmed that T-ALL cells were more sensitive than BCP-ALL to PR-104A in vitro, and that sensitivity correlated with AKR1C3 expression. Collectively, these results indicate that PR-104 shows promise as a novel therapy for relapsed/refractory T-ALL, and that AKR1C3 expression could be used as a biomarker to select patients most likely to benefit from such treatment in prospective clinical trials.

Evaluation of the In Vitro and In Vivo Efficacy of the JAK Inhibitor AZD1480 against JAK-Mutated Acute Lymphoblastic Leukemia

Genome-wide studies have identified a high-risk subgroup of pediatric acute lymphoblastic leukemia (ALL) harboring mutations in the Janus kinases (JAK). The purpose of this study was to assess the preclinical efficacy of the JAK1/2 inhibitor AZD1480, both as a single agent and in combination with the MEK inhibitor selumetinib, against JAK-mutated patient-derived xenografts. Patient-derived xenografts were established in immunodeficient mice from bone marrow or peripheral blood biopsy specimens, and their gene expression profiles compared with the original patient biopsies by microarray analysis. JAK/STAT and MAPK signaling pathways, and the inhibitory effects of targeted drugs, were interrogated by immunoblotting of phosphoproteins. The antileukemic effects of AZD1480 and selumetinib, alone and in combination, were tested against JAK-mutated ALL xenografts both in vitro and in vivo. Xenografts accurately represented the primary disease as determined by gene expression profiling. Cellular phosphoprotein analysis demonstrated that JAK-mutated xenografts exhibited heightened activation status of JAK/STAT and MAPK signaling pathways compared with typical B-cell precursor ALL xenografts, which were inhibited by AZD1480 exposure. However, AZD1480 exhibited modest single-agent in vivo efficacy against JAK-mutated xenografts. Combining AZD1480 with selumetinib resulted in profound synergistic in vitro cell killing, although these results were not translated in vivo despite evidence of target inhibition. Despite validation of target inhibition and the demonstration of profound in vitro synergy between AZD1480 and selumetinib, it is likely that prolonged target inhibition is required to achieve in vivo therapeutic enhancement between JAK and MEK inhibitors in the treatment of JAK-mutated ALL. Mol Cancer Ther; 14(2); 364–74. ©2014 AACR.

A review of new agents evaluated against pediatric acute lymphoblastic leukemia by the Pediatric Preclinical Testing Program.

Acute lymphoblastic leukemia (ALL) in children exemplifies how multi-agent chemotherapy has improved the outcome for patients. Refinements in treatment protocols and improvements in supportive care for this most common pediatric malignancy have led to a cure rate that now approaches 90%. However, certain pediatric ALL subgroups remain relatively intractable to treatment and many patients who relapse face a similarly dismal outcome. Moreover, survivors of pediatric ALL suffer the long-term sequelae of their intensive treatment throughout their lives. Therefore, the development of drugs to treat relapsed/refractory pediatric ALL, as well as those that more specifically target leukemia cells, remains a high priority. As pediatric malignancies represent a minority of the overall cancer burden, it is not surprising that they are generally underrepresented in drug development efforts. The identification of novel therapies relies largely on the reappropriation of drugs developed for adult malignancies. However, despite the large number of experimental agents available, clinical evaluation of novel drugs for pediatric ALL is hindered by limited patient numbers and the availability of effective established drugs. The Pediatric Preclinical Testing Program (PPTP) was established in 2005 to provide a mechanism by which novel therapeutics could be evaluated against xenograft and cell line models of the most common childhood malignancies, including ALL, to prioritize those with the greatest activity for clinical evaluation. In this article, we review the results of >50 novel agents and combinations tested against the PPTP ALL xenografts, highlighting comparisons between PPTP results and clinical data where possible.

Effective Targeting of the P53/MDM2 Axis in Preclinical Models of Infant MLL-Rearranged Acute Lymphoblastic Leukemia

Purpose: Although the overall cure rate for pediatric acute lymphoblastic leukemia (ALL) approaches 90%, infants with ALL harboring translocations in the mixed-lineage leukemia (MLL) oncogene (infant MLL-ALL) experience shorter remission duration and lower survival rates (∼50%). Mutations in the p53 tumor-suppressor gene are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be beneficial. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenografts. Experimental Design: Eight MLL-ALL patient-derived xenografts were established in immune-deficient mice, and their molecular features compared with B-lineage ALL and T-ALL xenografts. The sensitivity of MLL-ALL xenografts to RG7112 was assessed in vitro and in vivo, and the ability of RG7112 to induce p53, cell-cycle arrest, and apoptosis in vivo was evaluated. Results: Gene-expression analysis revealed that MLL-ALL, B-lineage ALL, and T-ALL xenografts clustered according to subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA family, were significantly upregulated in MLL-ALL xenografts, confirming their ability to recapitulate the clinical disease. Exposure of MLL-ALL xenografts to RG7112 in vivo caused p53 upregulation, cell-cycle arrest, and apoptosis. RG7112 as a single agent induced significant regressions in infant MLL-ALL xenografts. Therapeutic enhancement was observed when RG7112 was assessed using combination treatment with an induction-type regimen (vincristine/dexamethasone/L-asparaginase) against an MLL-ALL xenograft. Conclusions: The utility of targeting the p53–MDM2 axis in combination with established drugs for the management of infant MLL-ALL warrants further investigation. Clin Cancer Res; 21(6); 1395–405. ©2015 AACR.

Synergism of FAK and tyrosine kinase inhibition in Ph+ B-ALL

BCR-ABL1+ B progenitor acute lymphoblastic leukemia (Ph+ B-ALL) is an aggressive disease that frequently responds poorly to currently available therapies. Alterations in IKZF1, which encodes the lymphoid transcription factor Ikaros, are present in over 80% of Ph+ ALL and are associated with a stem cell-like phenotype, aberrant adhesion molecule expression and signaling, leukemic cell adhesion to the bone marrow stem cell niche, and poor outcome. Here, we show that FAK1 is upregulated in Ph+ B-ALL with further overexpression in IKZF1-altered cells and that the FAK inhibitor VS-4718 potently inhibits aberrant FAK signaling and leukemic cell adhesion, potentiating responsiveness to tyrosine kinase inhibitors, inducing cure in vivo. Thus, targeting FAK with VS-4718 is an attractive approach to overcome the deleterious effects of FAK overexpression in Ph+ B-ALL, particularly in abrogating the adhesive phenotype induced by Ikaros alterations, and warrants evaluation in clinical trials for Ph+ B-ALL, regardless of IKZF1 status.

Overexpression of ERG in cord blood progenitors promotes expansion and recapitulates molecular signatures of high ERG leukemias

High expression of the ETS family transcription factor ERG is associated with poor clinical outcome in acute myeloid leukemia (AML) and acute T-cell lymphoblastic leukemia (T-ALL). In murine models, high ERG expression induces both T-ALL and AML. However, no study to date has defined the effect of high ERG expression on primary human hematopoietic cells. In the present study, human CD34+ cells were transduced with retroviral vectors to elevate ERG gene expression to levels detected in high ERG AML. RNA sequencing was performed on purified populations of transduced cells to define the effects of high ERG on gene expression in human CD34+ cells. Integration of the genome-wide expression data with other data sets revealed that high ERG drives an expression signature that shares features of normal hematopoietic stem cells, high ERG AMLs, early T-cell precursor-ALLs and leukemic stem cell signatures associated with poor clinical outcome. Functional assays linked this gene expression profile to enhanced progenitor cell expansion. These results support a model whereby a stem cell gene expression network driven by high ERG in human cells enhances the expansion of the progenitor pool, providing opportunity for the acquisition and propagation of mutations and the development of leukemia.

Acute Sensitivity of Ph-like Acute Lymphoblastic Leukemia to the SMAC-Mimetic Birinapant.

Ph-like acute lymphoblastic leukemia (ALL) is a genetically defined high-risk ALL subtype with a generally poor prognosis. In this study, we evaluated the efficacy of birinapant, a small-molecule mimetic of the apoptotic regulator SMAC, against a diverse set of ALL subtypes. Birinapant exhibited potent and selective cytotoxicity against B-cell precursor ALL (BCP-ALL) cells that were cultured ex vivo or in vivo as patient-derived tumor xenografts (PDX). Cytotoxicity was consistently most acute in Ph-like BCP-ALL. Unbiased gene expression analysis of BCP-ALL PDX specimens identified a 68-gene signature associated with birinapant sensitivity, including an enrichment for genes involved in inflammatory response, hematopoiesis, and cell death pathways. All Ph-like PDXs analyzed clustered within this 68-gene classifier. Mechanistically, birinapant sensitivity was associated with expression of TNF receptor TNFR1 and was abrogated by interfering with the TNFα/TNFR1 interaction. In combination therapy, birinapant enhanced the in vivo efficacy of an induction-type regimen of vincristine, dexamethasone, and L-asparaginase against Ph-like ALL xenografts, offering a preclinical rationale to further evaluate this SMAC mimetic for BCP-ALL treatment. Cancer Res; 76(15); 4579-91. ©2016 AACR.