Acute Lymphoblastic Leukemia Xenografts Research Articles

Thioimidazoline based compounds reverse glucocorticoid resistance in human acute lymphoblastic leukemia xenografts.

Glucocorticoids form a critical component of chemotherapy regimens for pediatric acute lymphoblastic leukemia (ALL) and the initial response to glucocorticoid therapy is a major prognostic factor, where resistance is predictive of poor outcome. A high-throughput screen identified four thioimidazoline-containing compounds that reversed dexamethasone resistance in an ALL xenograft derived from a chemoresistant pediatric ALL. The lead compound (1) was synergistic when used in combination with the glucocorticoids, dexamethasone or prednisolone. Synergy was observed in a range of dexamethasone-resistant xenografts representative of B-cell precursor ALL (BCP-ALL) and T-cell ALL. We describe here the synthesis of twenty compounds and biological evaluation of thirty two molecules that explore the structure-activity relationships (SAR) of this novel class of glucocorticoid sensitizing compounds. SAR analysis has identified that the most effective dexamethasone sensitizers contain a thioimidazoline acetamide substructure with a large hydrophobic moiety on the acetamide.

Harnessing Negative B Cell Selection to Overcome Drug-Resistance in Acute Lymphoblastic Leukemia

Background: B cells are selected for an intermediate level of (pre-) B cell receptor (BCR) signaling strength: attenuation below minimum (e.g. non-functional BCR) or hyperactivation above maximum (e.g. autoreactive BCR) thresholds of signaling strength causes negative selection and cell death. About 25% of B cell acute lymphoblastic leukemia (ALL) is driven by oncogenic tyrosine kinases (e.g. BCR-ABL1 in Ph+ ALL), which mimics constitutively active pre-BCR signaling and defines the ALL subgroup with the worst clinical outcome. Currently more potent tyrosine kinase inhibitors (TKI) are developed for Ph+ ALL to suppress oncogenic signaling below a minimum threshold for survival. However Ph+ ALL cells invariably develop resistance against TKI. Here, we tested the hypothesis that targeted hyperactivation of oncogenic signaling above a maximum threshold will trigger B cell-inherent mechanisms of negative selection and selectively kill Ph+ALL cells.Results: The Ph+ ALL cells don not express a functional pre-BCR and BCR-ABL1 oncogene mimics a constitutively active pre-BCR by phosphorylating SYK, LYN, BTK and PLCg2. An incremental increase of pre-BCR downstream signaling (ITAM or SYK overexpression) was indeed sufficient to induce cell death in Ph+ ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ALL cells in this experimental setting.Ph+ ALL cells differ from normal pre-B cells by expression of high levels of ITIM containing inhibitory receptors including PECAM1 (CD31), CD300A and LAIR1. Importantly, high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials. In the COG trial (P9906; n=207) for children high-risk ALL, mRNA levels of PECAM1, CD300A and LAIR1at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p<0.0005), and negatively correlated with overall survival (OS) rate (p<0.02) or relapse free survival (RFS) rate (p<0.05). In the ECOG trial (E2993; n=215) for adults ALL, PECAM1 mRNA level negatively correlated with OS rate (p=0.0285).Genetic studies revealed that Pecam1, Cd300a and Lair1 receptors are critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases Ptpn6 (SHP1) and Inpp5d (SHIP1). Deletion of Pecam1, Cd300a or Lair1 in Ph+ ALL cells caused increased ROS levels, G0/G1cell cycle arrest, decreased colony formation capacity and cellular senescence. Phosphorylation of pre-BCR downstream molecules (SYK, LYN, BTK and PLCg2) was increased after Lair1 deletion and this hyper-signaling could not be tolerated by Ph+ ALL cells. Lair1 deletion resulted in rapid leukemia regression and prolonged survival of recipient mice in a transplant experiment. Leukemia cell death caused by Lair1-deletion could be rescued by overexpression of the active inhibitory phosphatase Ptpn6 (CD8-SHP1) or Inpp5d (CD8-SHIP1). Genetic deletion of Ptpn6 and Inpp5d caused increased pre-BCR signaling and cell death in BCR-ABL1 ALL cells but not myeloid cells (normal and BCR-ABL1-transformed), which -unlike B cells- are not subject to negative selection of auto-reactive clones. Decreasing pre-BCR signaling by SYK inhibition rescued cell death after Ptpn6- or Inpp5d- deletion.Blocking inhibitory receptors by using chimeric PECAM1, CD300A and LAIR1 receptor decoys inhibited proliferation and caused cell death in Ph+ ALL xenograft cells. More potently, a novel small molecule inhibitor of INPP5D (SHIP1) selectively killed Ph+ ALL xenograft cells through inducing hyper pre-BCR signaling, regardless of TKI resistance. We demonstrate that inhibitory phosphatase signaling represents a potential novel class of therapeutic targets for Ph+ALL.Conclusions: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified ITIM-receptors and phosphatases as members of a potential novel class of therapeutic targets. The concept of pharmacological perturbance of oncogenic signaling equilibrium in leukemia cells by inhibition (e.g. TKI-treatment) or exaggeration of signaling strength (e.g. blockade of ITIM-receptors) may lead to the discovery of multiple additional therapeutic targets and broaden our repertoire of currently available therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Antileukemic Efficacy of Continuous Vs Discontinuous Dexamethasone in Murine Xenografts of Acute Lymphoblastic Leukemia

As cure rates for acute lymphoblastic leukemia (ALL) have increased, toxic complications of chemotherapy have also increased. The incidence of osteonecrosis is high – 54% of children had asymptomatic osteonecrosis, and 18% experienced symptomatic osteonecrosis in one prospective study; osteonecrosis was positively associated with plasma dexamethasone exposure (Kawedia et al. Blood 2011 PMID: 21148812). Osteonecrosis is also more common after continuous than discontinuous dosing of dexamethasone in children (Mattano et al. JCO 2012 PMID: 22901620, Seibel et al. Blood 2008 PMID: 18039957). Many ALL clinical trials now include a discontinuous glucocorticoid schedule, in which patients receive dexamethasone daily for one week, none the second week, then daily for the third week, in an attempt to reduce the rate of osteonecrosis.Using a murine model of osteonecrosis, our group previously found that mice treated with a discontinuous regimen of dexamethasone developed less osteonecrosis than those treated with a continuous regimen (Yang 2008 PMID: 18683891), consistent with clinical data. However, it is not known whether the discontinuous schedule may also compromise the cure rate. Our goal was to use murine models of ALL to determine if there was a difference in antileukemic efficacy of the continuous versus discontinuous dexamethasone regimens.Each of six patient samples of various ALL subtypes was engrafted into immunocompromised NSG mice, by intravenous injection of 1-5 million cells. In vitro sensitivity to dexamethasone was assessed at the time of diagnosis, and all samples were sensitive to nanomolar concentrations. The samples were passaged serially: they were transduced with a lentiviral vector containing YFP and luciferase prior to the second passage, sorted for YFP+ cells prior to the third passage, and tested for in vivo efficacy of continuous (cont) and discontinuous (disc) dexamethasone on the fourth and/or fifth passage. Dexamethasone was given in the drinking water, either half the week at 8 mg/L (Disc dex) or continuously at 4 mg/L (Cont dex) starting 7-28 days from ALL injection. All mice received antibiotic prophylaxis in the drinking water. Plasma concentrations of dexamethasone in mice on treatment were similar to plasma levels (20 – 200 nM) achieved in patients with ALL (Yang et al JCO PMID: 18421047).For all ALL samples engrafted, there was improved leukemia-free survival (LFS) with dexamethasone treatment compared to no dexamethasone treatment (median LFS +4 to +129 days relative to no dexamethasone, log-rank p=0.02 to p=0.001). For five of the six samples, there were no differences in LFS between the continuous and discontinuous regimens (median LFS differed by 1 – 15 days, log-rank p=0.14 to 0.9). For one of the six samples, the continuous dexamethasone was more effective (median LFS of 111 vs 77 days, log-rank p=0.002) than the discontinuous schedule. In a second experiment with the same sample, when all the mice were sacrificed on the same day from start of therapy, ALL burden as assessed by ventral luminescence (Kruskal-Wallis p=0.004) and by spleen size (Kruskal-Wallis p=0.006) was lower in the continuous dexamethasone group vs the discontinuous dexamethasone group.Thus far, these data indicate that for most pediatric ALL, discontinuous dexamethasone is likely to be as effective as continuous dexamethasone; however, it is possible that for some ALL cases, there may be differences in ALL efficacy between the continuous and discontinuous dexamethasone regimens. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bafilomycin A1 Targets Both Autophagy and Apoptosis Pathways in Pediatric t(1;19) Pre-B Acute Lymphoblastic Leukemia Cells

The t (1; 19) subtype leukemia accounts for a quarter of pre-B acute lymphoblastic leukemia (ALL) and up to 5% of all ALL patients. Despite plausible remission rate, current treatment regimen on the pediatric pre-B ALL is associated with side effects and CNS relapse, which poses the need for more effective and safer drugs. Bafilomycin A1 (Baf-A1) is known as an inhibitor of late phase of autophagy by inhibiting fusion between autophagosomes and lysosomes as well as by inhibiting lysosomal degradation.Here we show that Baf-A1 of low concentration (1 nM) effectively and specifically inhibits and kills the pre-B ALL cells. E2A/Pbx1 fusion gene positivepre-B ALL 697 cells were used for In vitro experiments. The results of flow cytometry analysis and western blotting showed that Baf-A1 induced cell cycle arrest and proliferation inhibition of ALL cells by upregualting cell cycle negative regulators and downregulating cell cycle positive regulators. In contrast, AML and CML cell lines were insensitive to Baf-A1 treatment. Western blotting and confocal observation on protein LC3 also showed that Baf-A1 at 1 nM blocked basal autophagic flux. Baf-A1 treatment activated mTOR signaling and induced the formation of Becn1–Bcl-2 complex to inhibit the induction of autophagy. Furthermore, apoptosis was induced in ALL cells treated with Baf-A1 for 72 h. However, procaspase-3 and poly-(ADP-ribose) polymerase (PARP) were not cleaved in these cells. We observed that AIF relocalized to the nucleus after 72h Baf-A1 treatment by confocal and immunoblotting. Knockdown of AIF significantly attenuated apoptosis induced by Baf-A1. These data suggest that Baf-A1 targets mitochondria membrane to trigger apoptosis via AIF pathway. In the in vivo experiment, Baf-A1 treatment extended survival and improved pathology of 697 xenograft mice, and significantly reduced the E2A/PBX1 positive leukemia cells in the bone marrow of mice. In vivomouse toxicity assay confirms Baf-A1 as a safe compound. The bone marrow cells of pre-B ALL leukemia patients were sorted against CD133+CD19+ markers, and treatment with Baf-A1 induced a clear inhibition on the CD133+CD19+ primary cells with a significant increased cell death in the sorted B-ALL patient samples. Conversely, Baf-A1 had no inhibitory effect on the bone marrow cells isolated from acute myeloid leukemia patients and healthy people.In summary, Baf-A1 treatment at low concentration effectively and specifically inhibited autophagy by activating mTOR and inducing beclin1-Bcl-2 interaction and induced AIF-dependent apoptosis in t (1; 19) pre-B ALL 697 cells. In the pre-B ALL xenograft mouse model, Baf-A1 specifically targets the leukemia cells while sparing normal cells. More importantly, Baf-A1 potently inhibits and kills the primary cells from pediatric pre-B ALL patients both at initial diagnosis and relapse without compromising normal human hematopoietic cells, all proposing Baf-A1 as a promising drug candidate for this pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Leukemia Stem Cells in Acute Lymphoblastic Leukemia: Unveiling Hierarchical Structure at Single Cell Resolution

Leukemia stem cells (LSCs) are the root of leukemia, and are responsible for drug resistance and disease relapse. However, LSCs have not yet been identified for acute lymphoblastic leukemia (ALL). Among many challenges, lack of phenotypic markers is one of the major problems in identifying ALL LSCs. In this study, we demonstrated a novel method to isolate LSCs from both T- and B- cell ALLs and further characterized their transcriptome profile at the single cell level.We have recently identified a novel method to isolate ALL LSCs based on cellular metabolic activities. We demonstrated that these isolated LSCs had in vivo leukemia-initiating capability (LIC). We have developed a series of primary ALL xenograft mouse models using patient samples and NOD/SCID/IL2Rg-/- (NSG) mice. Leukemia cells harvested from several generations of these mice were used in this study. We isolated LSCs and non-LSCs from 4 different B-cell type ALL samples and transplanted them separately into healthy NSG mice. Cell numbers used varied between 5, 10, and 50,000 per mouse, and the number of the animals varied between three and eight per group. All the animals transplanted with LSCs developed leukemia between 5-14 weeks, whereas those transplanted with non-LSCs did not develop the disease within the same timeframe or by the end of the study, which was more than 4 months after leukemia development in the LSC group.In order to characterize and identify potential therapeutic targets in the LSCs, we investigated the transcriptome profile of these cells. First, we performed genomewide microarray gene expression profiling of RNA isolated from the LSCs and non-LSCs using 4 ALL cell lines (Reh, JM1, Jurkat, and Molt4). There were 173 genes which showed at least 2-fold difference in gene expression between the LSCs and non-LSCs. Using a panel of primer sets for the 100 genes exhibiting the highest difference in expression, we performed qRT-PCR for these genes in the isolated LSCs and non-LSCs from 11 primary ALL samples (10 B-cell and 1 T-cell type) transplanted and harvested from our NSG xenograft mouse models at different generations. There was a distinct difference in the transcriptome profile between the LSCs and non-LSCs in these primary ALL samples. Overall gene expression of 93 LSC signature genes was much lower in the LSCs than in the non-LSCs.Recent advances in microfluidic technologies allowed us to investigate cells at single cell resolution. Growing evidence suggests that cancer stem cells consist of heterogeneous cell populations (subclones). Therefore, we further investigated whether these isolated LSCs have subclones using the Fluidigm C1 and Biomark system. Preliminary results using a primary ALL sample harvested from our xenograft mouse model, indicate that there are at least two distinct subclones in the LSCs based on principal component analysis of the single cell data.In summary, we 1) developed a novel method to isolate ALL LSCs which have in vivo LIC, 2) demonstrated that isolated LSCs have a distinct transcriptome profile, and 3) discovered that the LSCs seem to consist of subclones. Currently we are in the process of performing detailed comprehensive transcriptome analyses and additional single cell transcriptome assays. DisclosuresNo relevant conflicts of interest to declare.

UNC2025, a Small Molecule MerTK and Flt3 Tyrosine Kinase Inhibitor, Decreases Disease Burden, Prolongs Survival, and Promotes Sensitivity to Chemotherapy in Xenograft Models of Acute Leukemia

Acute myeloid leukemia (AML) and relapsed or refractory acute lymphoblastic leukemia (ALL) are extremely difficult to treat and new therapies are needed to improve survival and decrease toxicities associated with conventional therapy. The MerTK receptor tyrosine kinase is aberrantly expressed in 30% of pediatric B-ALL patient samples, 50% of pediatric T-ALL samples, and 80-90% of pediatric and adult AML samples and inhibition of MerTK using shRNA prolonged median survival time in ALL and AML xenograft models. These data validate MerTK as a potential therapeutic target in acute leukemias. In AML, internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (Flt3) receptor are observed in ~20-30% of adult and ~15% of pediatric AML patient samples and are associated with poor prognosis. Flt3 inhibitors have demonstrated therapeutic efficacy in clinical studies but resistance to current Flt3-targeted TKIs and unacceptable adverse side effects have been reported, indicating a need for new therapeutic agents directed against Flt3. Here we report a novel dual MerTK/Flt3-selective small molecule TKI as a potential therapy for acute leukemias with MerTK expression and/or activating Flt3 mutation. UNC2025 mediated potent inhibition of MerTK and Flt3 in enzymatic (Ki = 0.16 nM and 0.59 nM, respectively) and cell-based (IC50 = 2.7 nM and 14 nM, respectively) assays and had limited off-target activity against 300 other kinases. Treatment with 100-200 nM UNC2025 inhibited MerTK-dependent pro-survival signaling pathways, induced apoptosis in 40-90% of cells, and reduced colony-forming potential by 80-100% in leukemia cell line cultures. Similarly, a MerTK-expressing leukemic patient sample exhibited reduced MerTK-dependent pro-survival signaling, induction of apoptosis, and decreased colony formation in semi-solid medium in response to UNC2025. In contrast, a MerTK-negative leukemic patient sample did not respond to treatment, suggesting that UNC2025-mediated anti-leukemia effects are a consequence of MerTK inhibition. UNC2025 is orally bioavailable and inhibits MerTK phosphorylation/activation in leukemic blasts in the bone marrow. Treatment with UNC2025 resulted in a dose-dependent reduction in tumor burden and increased median survival from 27 days after inoculation with tumor cells to 70 days in an orthotopic B-ALL xenograft model of minimal residual disease (p < 0.0001) and from 27.5 to 45 days in a similar model of existent disease (p < 0.0001). In a MerTK-expressing patient-derived xenograft (PDX) model of AML, short-term treatment with UNC2025 resulted in a significant decrease in peripheral disease burden (9.3% blasts versus 35.6% for vehicle-treated animals, p = 0.0225) and both the fraction of splenic blasts (28.5% versus 75.7%, p = 0.0005) and spleen mass (170 mg versus 834 mg, p < 0.0001) were dramatically decreased. UNC2025 was even more effective in a FLT3-ITD mutant, MerTK-expressing AML PDX model. In this case, treatment with UNC2025 resulted in stable disease regression (0.24% peripheral blasts after 4 weeks of treatment versus 11.4% pre-treatment, p = 0.0094) and prolonged median survival from 16 days post-treatment to > 107 days (p = 0.0114). In addition, in the B-ALL xenograft model, treatment with UNC2025 and methotrexate, a chemotherapy currently in clinical use for treatment of pediatric ALL, resulted in reduced tumor burden and increased tumor-free survival relative to mice treated with either agent alone, suggesting that addition of UNC2025 to standard therapies may be a particularly effective treatment strategy and/or may allow for chemotherapy dose-reduction and decreased toxicity. The very high potency, relative selectivity, oral bioavailability, and target inhibition and therapeutic efficacy mediated by UNC2025 in murine models, both alone and in combination with chemotherapy, support continued development of a dual MerTK/Flt3 inhibitor for treatment of patients with acute leukemia. DisclosuresDeRyckere:Meryx, Inc.: Equity Ownership. Wang:University of North Carolina Chapel Hill: UNC2025 Patents & Royalties; Meryx, Inc.: Equity Ownership. Frye:Meryx, Inc.: Equity Ownership; University of North Carolina Chapel Hill: UNC2025 Patents & Royalties. Earp:Meryx, Inc.: Equity Ownership; University of North Carolina Chapel Hill: UNC2025 Patents & Royalties. Graham:Merys, Inc.: Equity Ownership.

High-Throughput Screening of Human Leukemia Xenografts to Identify Dexamethasone Sensitizers

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Glucocorticoids (e.g., dexamethasone) form a critical component of chemotherapy regimens for pediatric ALL, and the initial response to glucocorticoid therapy is a major prognostic factor, where resistance is predictive of poor outcome. We have previously established a clinically relevant ALL xenograft model, consisting of primary pediatric ALL biopsies engrafted into immune-deficient mice, in which in vitro and in vivo dexamethasone sensitivity significantly correlated with patient outcome. In this study, we used high-throughput screening (HTS) to identify novel compounds that reverse dexamethasone resistance in a xenograft (ALL-19) derived from a chemoresistant pediatric ALL patient that is representative of the most common pediatric ALL subtype (B-cell precursor [BCP-ALL]). The compound 2-(4-chlorophenoxy)-2-methyl-N-(2-(piperidin-1-yl)phenyl)propanamide showed little cytotoxic activity alone (IC50 = 31 µM), but when combined with dexamethasone, it caused a marked decrease in cell viability. Fixed-ratio combination assays were performed against a broad panel of dexamethasone-resistant and -sensitive xenografts representative of BCP-ALL, T-cell ALL, and Mixed Lineage Leukemia–rearranged ALL, and synergy was observed in six of seven xenografts. We describe here the development of a novel 384-well cell-based high-throughput screening assay for identifying potential dexamethasone sensitizers using a clinically relevant ALL xenograft model.

Variegated clonality and rapid emergence of new molecular lesions in xenografts of acute lymphoblastic leukemia are associated with drug resistance

The use of genome-wide copy-number analysis and massive parallel sequencing has revolutionized the understanding of the clonal architecture of pediatric acute lymphoblastic leukemia (ALL) by demonstrating that this disease is composed of highly variable clonal ancestries following the rules of Darwinian selection. The current study aimed to analyze the molecular composition of childhood ALL biopsies and patient-derived xenografts with particular emphasis on mechanisms associated with acquired chemoresistance. Genomic DNA from seven primary pediatric ALL patient samples, 29 serially passaged xenografts, and six in vivo selected chemoresistant xenografts were analyzed with 250K single-nucleotide polymorphism arrays. Copy-number analysis of non-drug-selected xenografts confirmed a highly variable molecular pattern of variegated subclones. Whereas primary patient samples from initial diagnosis displayed a mean of 5.7 copy-number alterations per sample, serially passaged xenografts contained a mean of 8.2 and chemoresistant xenografts a mean of 10.5 copy-number alterations per sample, respectively. Resistance to cytarabine was explained by a new homozygous deletion of the DCK gene, whereas methotrexate resistance was associated with monoallelic deletion of FPGS and mutation of the remaining allele. This study demonstrates that selecting for chemoresistance in xenografted human ALL cells can reveal novel mechanisms associated with drug resistance.

Abstract B26: Cell and molecular determinants of in vivo efficacy of the BH3 mimetic ABT-263 against pediatric acute lymphoblastic leukemia xenografts

Abstract Introduction: Manipulation of the mitochondrial apoptosis pathway is an appealing strategy for cancer treatment using small molecule BH3 mimetics such as ABT-737, ABT-263 and ABT-199. Defining a reliable cell or molecular signature that indicates a particular cancer's susceptibility to these drugs is a high priority to optimize their use for effective cancer chemotherapy. The goal of this study was to identify cell or molecular biomarkers predictive of the in vivo responses of pediatric acute lymphoblastic leukemia (ALL) xenografts established in immune-deficient (NOD/SCID) mice to single-agent ABT-263 treatment. Methods: The in vivo efficacy of ABT-263 (100 mg/kg x 21 days) was assessed against 31 patient-derived ALL xenografts derived from infant ALL with rearrangement of the Mixed Lineage Leukemia (MLL) oncogene (infant MLL-ALL, n=9), B-cell precursor (BCP)-ALL (n=7) and T-cell ALL (T-ALL, n=15) patients. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45+ cells in the peripheral blood. Anti-leukemic efficacy was assessed using an objective response measure modeled after the clinical setting, as well as the median event-free survival (EFS) of treated or control groups post-treatment. Three methods were applied to identify correlates of in vivo ABT-263 responses: (1) analysis of differentially expressed genes by gene expression profiling (GEP, Illumina HT12 BeadChips); (2) functional assessment of the mitochondrial priming status (BH3 profiling); (3) in vitro responses to single-agent ABT263 using a co-culture system with immortalized (hTERT) human mesenchymal stromal cells. Results: ABT-263 showed impressive single-agent efficacy across all three ALL subtypes, with 29 of 31 xenografts exhibiting significant progressive delays (range: 3.1 – 78 days), and 19 achieving Objective Responses (3 Maintained Complete Responses, 11 Complete Responses, and 5 Partial Responses). GEP showed that MCL1 exhibited the strongest correlation with in vivo ABT-263 sensitivity amongst BCL2 family members across the xenograft panels. Moreover, MCL1 protein function, assessed via mitochondrial depolarization mediated by Noxa peptide, also significantly correlated with in vivo ABT-263 resistance. Using an in vitro co-culture assay and cell survival assessed by flow cytometry after ABT-263 exposure (0.001-10 µM, 72 h), a training set of 17 xenografts was used to build a prediction model for in vivo xenograft responses. Testing this model against 11 other xenografts for which the in vivo results were blinded demonstrated that the fraction of cell survival at 10 nM ABT-263 predicted the in vivo responses with high sensitivity (67%) and specificity (100%). Conclusions: ABT-263 exhibits broad in vivo efficacy against pediatric ALL xenografts with no apparent subtype specificity. Cell and molecular analysis revealed MCL1 to be an important determinant of in vivo ABT-263 sensitivity, while an in vitro co-culture cytotoxicity assay was able to predict in vivo sensitivity. These in-principle approaches could be used to predict the individual responses of pediatric ALL patients to second-generation BH3 mimetics such as ABT-199. Supported by NCI NO1CM42216. Citation Format: Santi Suryani, Hernan Carol, Viktoras Frimantas, Beat Bornhauser, Chintanu Sarmah, Triona Chonghaile, Kathryn Evans, Elise Tonna, Luke Jones, Anthony Letai, Jean-Pierre Bourquin, Peter J. Houghton, Malcolm A. Smith, Richard B. Lock. Cell and molecular determinants of in vivo efficacy of the BH3 mimetic ABT-263 against pediatric acute lymphoblastic leukemia xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B26.

Abstract 843: Methylthioadenosine phosphorylase (MTAP)-deficient T-cell ALL xenografts are sensitive to pralatrexate and 6-thioguanine alone and in combination

Abstract In T-cell acute lymphoblastic leukemia (ALL), the genes CDKN2A (p16 and p14) and MTAP are homozygously co-deleted, with a frequency of ∼35 %. MTAP cleaves the natural substrate, 5′-deoxy-5′-methylthioadenosine, to adenine and 5-methylthioribose-1-phosphate, which are converted to adenine nucleotides and methionine. Cells lacking MTAP are unable to salvage adenine through this pathway and rely on the de novo pathway for purine synthesis. This variation between normal MTAP+ cells and tumor MTAP-/- cells led to several studies showing that MTAP -/- cells are more sensitive to inhibitors of de novo purine synthesis. Pralatrexate (PDX) is a second generation antifolate that targets dihydrofolate reductase and via conversion to polyglutamylates, inhibits de novo purine synthesis. 6-Thioguanine (6TG), an antimetabolite analog of guanine, also target de novo purine synthesis. MTAP-deficient ALL cells should be more sensitive to these agents, remarkably so in combination. We tested single agent PDX and 6TG as well as a combination treatment in MTAP+ (Molt4) and MTAP-/- (CEM) ALL xenografts. CEM xenografts were more sensitive to PDX treatment than MOLT4 xenografts. CEM xenografts were also much more sensitive to 6TG, while Molt4 xenografts were resistant to 6TG. Early results of the combination of PDX and 6TG in vivo show promise as an effective therapy of MTAP-/- T-cell ALL. Citation Format: Philip M. Tedeschi, Yamini K. Kathari, Iqra Farooqi, Joseph R. Bertino. Methylthioadenosine phosphorylase (MTAP)-deficient T-cell ALL xenografts are sensitive to pralatrexate and 6-thioguanine alone and in combination. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 843. doi:10.1158/1538-7445.AM2014-843

Abstract 2930: Global chromatin profiling reveals NSD2 mutation in pediatric ALL

Abstract Epigenetic dysregulation is an emerging hallmark of cancers. We developed a high-information-content mass spectrometry approach to profile global histone modifications in human cancers. When applied to 115 lines of the Cancer Cell Line Encyclopedia1, this approach identified distinct molecular chromatin signatures. One signature was characterized by increased H3K36 dimethylation, exhibited by several lines harboring NSD2 translocations. A novel NSD2 p.E1099K variant was identified in non-translocated acute lymphoblastic leukemia (ALL) lines sharing this signature. Ectopic expression of the variant induced a chromatin signature characteristic of NSD2 hyperactivation and promoted transformation. NSD2 knockdown selectively inhibited the proliferation of NSD2-mutant lines and impaired the in vivo growth of an NSD2-mutant ALL xenograft. Sequencing analysis of &amp;gt;1000 pediatric cancer genomes identified the NSD2 p.E1099K mutation in 14% of t(12;21)[ETV6-RUNX1]-containing ALLs. These findings identify NSD2 as a potential therapeutic target for pediatric ALL and provide a general framework for the functional annotation of cancer epigenomes. 1. Barretina,J. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603-607 (2012). Citation Format: Ho Man Chan, Jacob D. Jaffe, Yan Wang, Jinghui Zhang, Robert Huether, Gregory V. Kryukov, Hyo-eun C. Bhang, Jordan E. Taylor, Min Hu, Nathan P. Englund, Feng Yan, Zhaofu Wang, E Robert McDonald III, Lei Wei, Jing Ma, John Easton, Zhengtian Yu, Rosalie deBeaumount, Veronica Gibaja, Kavitha Venkatesan, Robert Schlegel, William R. Sellers, Nicholas Keen, Jun Liu, Giordano Caponigro, Jordi Barretina, Vesselina G. Cooke, Charles Mullighan, Steven A. Carr, James R. Downing, Levi A. Garraway, Frank Stegmeier. Global chromatin profiling reveals NSD2 mutation in pediatric ALL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2930. doi:10.1158/1538-7445.AM2014-2930

Efficacy of CPX-351, (cytarabine:daunorubicin) liposome injection, against acute lymphoblastic leukemia (ALL) xenograft models of the Pediatric Preclinical Testing Program.

CPX-351, a liposomal formulation of cytarabine and daunorubicin co-encapsulated at an optimized synergistic 5:1 molar ratio, has demonstrated improved clinical outcomes over conventional cytarabine/daunorubicin treatment in a randomized phase 2 trial in patients with AML as well as superior efficacy against preclinical leukemia models when compared to the free drugs in combination. Given the promising phase 2 data, limited toxicities observed, and the known clinical activities of cytarabine/daunorubicin, we assessed the efficacy of CPX-351 against a panel of childhood ALL xenograft models. Plasma pharmacokinetics of cytarabine and daunorubicin following CPX-351 treatment were determined by HPLC in order to correlate efficacy with drug exposure. CPX-351, at a dose of 5 units/kg (corresponding to 5 mg/kg cytarabine and 2.2 mg/kg daunorubicin), was highly efficacious against all xenografts tested, inducing complete responses in four B-lineage xenografts and partial response in one T-lineage xenograft. These therapeutic responses were achieved with CPX-351 doses that provided drug exposures (based on Cmax and AUC) comparable to those observed in patients with AML. These results suggest that CPX-351 may be a promising chemotherapeutic to be utilized in the treatment of ALL and support its testing in pediatric patients with leukemia.

Cell and molecular determinants of in vivo efficacy of the BH3 mimetic ABT-263 against pediatric acute lymphoblastic leukemia xenografts.

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. 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. 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%). 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.

Highlights of This Issue

Manipulation of the apoptosis pathway is an appealing strategy for cancer treatment using BH3 mimetics such as ABT-263, although predictive biomarkers are required to identify patients who may benefit from their use. To investigate this problem, Suryani and colleagues studied the in vivo efficacy of ABT-263 against 31 pediatric acute lymphoblastic leukemia xenografts. High MCL1 expression and function correlated with in vivo ABT-263 resistance, and in vitro assays predicted in vivo ABT-263 responses. These approaches could be included in the design of prospective clinical trials to determine if they can identify patients who may respond to treatment with this class of therapeutic agents.Gastric carcinoma (GC) is the second leading cause of cancer deaths in the world, with many deaths occurring in East Asia. To identify DNA methylation biomarkers for prediction of GC metastasis, scientists, and oncologists from China, the United States, Japan, and Korea have profiled differential methylation patterns in GCs and perform an in-depth characterization of methylation changes in 73 candidate genes. The authors have established a methylation biomarker-set composed of three genes GFRA1, SRF, and ZNF382 that could be used to synergistically predict GC metastasis and patients' overall survival from multiple patient cohorts in China, Japan, and Korea.TAZ, the core component of Hippo signaling pathway, attracts increasing attention as a critical player in tumorigenesis. Using an integrated translational approach, the authors explored the role of TAZ in non-small cell lung cancer (NSCLC). Noguchi and colleagues showed that higher TAZ expression was associated with shorter patient survival, and TAZ promoted lung cancer cell proliferation in vitro and tumor development in vivo. Moreover, TAZ regulated the expression of ErbB ligands and the EGFR signaling pathway was activated in NSCLC tissues with high TAZ expression. This study offers potential therapeutic strategies, either directly against TAZ or indirectly via EGFR pathway.Activation of Wnt-signaling is a common event in colorectal carcinoma (CRC), but its role in CRC metastasis is still under extensive study. In the present study, Zhang and colleagues revealed subcellular redistribution of NAT10 (N-acetyltransferase 10) in CRCs, which correlated well with nuclear translocation of β-catenin and reflected invasive potential of CRC. They demonstrated that inhibition of GSK-3β induced NAT10 redistribution through increasing its stability and nuclear export, which consequently enhanced cytoskeletal remodeling and promoted migration, invasion, and metastasis of cancer cells. These findings suggest NAT10 could be a useful prognostic marker and potential therapeutic target in CRC.

Caspase-dependent Mcl-1 cleavage and effect of Mcl-1 phosphorylation in ABT-737-induced apoptosis in human acute lymphoblastic leukemia cell lines.

ABT-737 is a BH3-mimetic that has a wide spectrum of single-agent activity against acute lymphoblastic leukemia (ALL) cell lines and xenografts. Previously, we reported that in response to ABT-737, ABT-737-resistant ALL cell lines showed an apparent increase in Mcl-1 (an anti-apoptotic Bcl-2 family protein that is not effectively inhibited by ABT-737) while ABT-737-sensitive ALL cell lines showed decreased Mcl-1 levels. Here we explored the mechanism of Mcl-1 cleavage by ABT-737 and the effect of adjacent phosphorylation sites on Mcl-1 cleavage and apoptosis induced by ABT-737 in a human B-lineage ALL cell line. Caspase cleavage sites in Mcl-1 and the effect of mutation in Mcl-1 phosphorylation sites were determined by transducing Mcl-1 variants tagged with the V5 epitope into human ALL cells. Cytotoxicity was by fluorescence-based DIMSCAN, and changes in protein by immunoblotting. ABT-737 induced a caspase-dependent cleavage of Mcl-1. Of the two Mcl-1 caspase cleavage sites (D127 and D157), D157 was the site of ABT-737-induced cleavage in ALL cells. Cells with exogenously expressed Mcl-1 Δ157 fragment showed greater caspase-3 and caspase-9 activation when they were treated with ABT-737 compared with cells expressing wild-type or D157A mutant Mcl-1. Cells with mutated phosphorylation sites on Mcl-1 (S159A and T163A) were less susceptible to Mcl-1 cleavage and apoptosis induced by ABT-737. Our data showed that Mcl-1 is post-translationally regulated in response to ABT-737 treatment, primarily via a caspase-dependent cleavage that generates a pro-apoptotic Mcl-1 fragment.

Attenuated measles virus controls pediatric acute B-lineage lymphoblastic leukemia in NOD/SCID mice

Novel therapies are needed for pediatric acute lymphoblastic leukemia resistant to conventional therapy. While emerging data suggest leukemias as possible targets of oncolytic attenuated measles virus, it is unknown whether measles virus can eradicate disseminated leukemia, in particular pediatric acute lymphoblastic leukemia. We evaluated the efficacy of attenuated measles virus against a large panel of pediatric xenografted and native primary acute lymphoblastic leukemias ex vivo, and against four different acute lymphoblastic leukemia xenografts of B-lineage in non-obese diabetic/severe combined immunodeficient mice. Ex vivo, attenuated measles virus readily spread among and effectively killed leukemia cells while sparing normal human blood cells and their progenitors. In immunodeficient mice with disseminated acute lymphoblastic leukemia a few intravenous injections of attenuated measles virus sufficed to eradicate leukemic blasts in the hematopoietic system and to control central nervous system disease resulting in long-term survival in three of the four xenografted B-lineage leukemias. Differential sensitivity of leukemia cells did not require increased expression of the measles entry receptors CD150 or CD46 nor absence of the anti-viral retinoic acid-inducible gene I/melanoma differentiation associated gene-5 /interferon pathway. Attenuated oncolytic measles virus is dramatically effective against pediatric B-lineage acute lymphoblastic leukemia in the pre-clinical setting warranting further investigations towards clinical translation.

CD19-antigen specific nanoscale liposomal formulation of a SYK P-site inhibitor causes apoptotic destruction of human B-precursor leukemia cells.

We report the anti-leukemic potency of a unique biotargeted nanoscale liposomal nanoparticle (LNP) formulation of the spleen tyrosine kinase (SYK) P-site inhibitor C61. C61-loaded LNP were decorated with a murine CD19-specific monoclonal antibody directed against radiation-resistant CD19-receptor positive aggressive B-precursor acute lymphoblastic leukemia (ALL) cells. The biotargeted C61-LNP were more potent than untargeted C61-LNP and consistently caused apoptosis in B-precursor ALL cells. The CD19-directed C61-LNP also destroyed B-precursor ALL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. This unique nanostructural therapeutic modality targeting the SYK-dependent anti-apoptotic blast cell survival machinery shows promise for overcoming the clinical radiochemotherapy resistance of B-precursor ALL cells.

Overcoming Apoptosis Resistance In High Risk Acute Lymphoblastic Leukemia By Smac Mimetics In a Preclinical ALL Xenograft Model In Vivo

Abstract Intensified treatment of pediatric acute lymphoblastic leukemia (ALL) has lead to increased survival rates of about 80%, however therapy fails in the remaining patients leading to relapse of the disease associated with inferior prognosis. Because treatment failure is, at least in part, due to defects in apoptosis programs, novel therapeutic strategies that counter apoptosis resistance are needed. “Inhibitor of Apoptosis” (IAP) proteins block the apoptosis machinery at a central point and are highly expressed in acute leukemias, thereby providing a target structure for therapeutic intervention. Molecules antagonizing these apoptosis inhibitors, so called SMAC-mimetics, therefore provide a promising strategy to overcome apoptosis deficiency and effectively treat high risk ALL. In this study, we investigated the effects of the small molecule SMAC-mimetic BV6 (kindly provided by Genentech) in B cell precursor- (BCP-) ALL. BV6 showed a clear induction of cell death at nanomolar concentrations in ALL cell lines. ALL cells sensitive for SMAC-mimetic induced cell death showed rapid cIAP degradation, NFkB activation and secretion of TNF-alpha (TNF-a). Interestingly, mitochondrial perturbation and caspase activation could be inhibited by the soluble TNF-a receptor Etanercept indicating the induction of a TNF-a dependent feed forward loop by the SMAC-mimetic BV6. In addition to cell lines, we investigated the effects of BV6 on a series of 42 primary ALL samples isolated from ALL bearing mice of established patient derived NOD/SCID/huALL xenograft leukemias. Intriguingly, upon treatment with the small molecule SMAC mimetic BV6, induction of cell death was observed in a majority of 70% of all individual patient-derived leukemias and BV6 induced cell death was inhibited by Etanercept demonstrating TNF-a dependency also in primary ALL. We previously described that rapid engraftment of ALL cells transplanted onto NOD/SCID mice (short Time To Leukemia, TTLshort) is associated with deficient apoptosis signaling in the ALL cells and indicative for early patient relapse. Importantly, primary xenograft ALL samples with a TTLshort/early relapse phenotype showed increased cell death upon treatment with SMAC-mimetic BV6 and activation of the constitutive deficient apoptosis signaling pathway, demonstrating that SMAC-mimetics induce intact apoptosis signaling in former apoptosis resistant primary ALL cells. Based on theses findings, we further evaluated the in vivo effectivity of the SMAC-mimetic BV6 on high risk ALL using our NOD/SCID/huALL xenograft model in a preclinical setting. ALL bearing recipients were treated with either BV6 or solvent for a given time of two weeks and further investigated for the presence of leukemia. Most interestingly, a significant delay of post-treatment leukemia reoccurrence was observed upon BV6 in vivo treatment along with a profound reduction of tumor load in the recipients compared to solvent treated animals. In a clinical setting, high-risk disease is unlikely to be treated by one compound alone. Therefore, we combined BV6 with multidrug chemotherapy resembling ALL induction treatment and observed a significant delay of ALL reoccurrence and prolonged survival of animals treated with the combination of the SMAC-mimetic and chemotherapy in contrast to chemotherapy alone. Most importantly, concomitant in vivo therapy with Etanercept revoked the cell death sensitizing effect of BV6, both in single treatment and in combination with chemotherapy. This indicates that BV6 induced apoptosis sensitization involves signaling via TNF-a and thereby provides a potential biomarker for the identification of patients who would benefit from SMAC-mimetic treatment. Taken together, we show that the small molecule SMAC-mimetic BV6 induces cell death via a TNF-a loop ex vivo and in vivo in primary patient-derived ALL. Moreover, BV6 is able to overcome apoptosis deficiency of high risk ALL leading to prolonged in vivo survival in a preclinical therapy model of patient-derived ALL xenograft ALL. Thus, induction of cell death by new generation small molecule SMAC-mimetics provides a promising novel strategy for targeted therapy of high-risk acute lymphoblastic leukemia and involvement of TNF-a signaling in BV6-sensitive patients points to its potential use as biomarker indicating effective cell death sensitization. Disclosures: No relevant conflicts of interest to declare.

The Poison Oligonucleotide F10 Is Efficiently Taken Up By, and Highly Effective Against, Acute Lymphoblastic Leukemia While Sparing Normal Hematopoietic Cells

The development of imatinib to target the BCR-ABL kinase in chronic phase (CP) CML has changed the natural history of the disease. This success generated much enthusiasm for the approach in the more genetically complex acute lymphoblastic leukemia (ALL). Unfortunately, the durable responses seen in CP CML are not reproduced in BCR-ABL positive ALL. Indeed, all previous attempts to target a single oncogenic pathway in acute leukemias have resulted in transient responses with frequent relapse. An alternative approach is to use agents that target “final common pathways”, i.e. processes that must be accomplished to produce additional leukemia cells regardless of driving mutations. In this paradigm, agents are not judged by differential expression of a target, but by the degree of differential uptake. One pathway to exploit is the known increased uptake of oligonucleotides by ALL cells.F10 is a poison deoxy-oligonucleotide that is a 10mer of the thymidylate synthase (TS) inhibitory 5-fluorouracil (5-FU) metabolite, 5-fluoro-2'-deoxyuridine-5'-O-monophosphate. We sought to determine the uptake and activity of F10 against preclinical ALL models.Using fluorescently labeled F10, we determined that uptake by the human ALL cell lines DG75 and SUP-B15 was rapid and had a profound temperature dependence consistent with an active process. Both cell lines demonstrated increased uptake compared to normal, lineage- depleted marrow cells from C57Bl/6 mice. Using a syngeneic, BCR-ABL-expressing murine ALL model, we confirmed rapid uptake of F10 in vivo. Furthermore, preliminary experiments suggested normal human HSCs have decreased uptake compared to primary patient samples. Consistent with this decreased uptake, F10 treatment did not alter the ability of human HSCs to engraft in immunodeficient mice.F10 treatment resulted in robust induction of apoptosis that could not be equaled by 100 fold more 5-FU. IC50 values for F10 against B6 ALL, Jurkat, DG75, Molt-4, CCRF-CEM, and SUP-B15 were in the picomolar to nanomolar range, with an average value of 2.15 nM (range 0.12 to 5.4 nM). For comparison we also tested 5-FU, doxorubicin, and cytarabine. In all cases, F10 was the most potent agent; over 1000 times more potent than 5-FU.In vivo, F10 treatment was associated with a significant increase in survival in a BCR-ABL driven, syngeneic ALL mouse model (p= 0.0001 by log rank test). Survival was dose-dependent, with median survival extended by 3, 9 or 15 days for 4, 6, or 9 doses, respectively. F10 also protected mice from leukemia-induced weight loss during and for several days after treatment. To confirm the in vivo activity of F10, we treated a separately derived syngeneic model expressing the T315I variant of BCR-ABL. As in the previous model, F10 significantly prolonged survival (p=0.0013). To further extend these results and confirm F10's activity against human ALL cells, we treated DG75, Jurkat, and SUP-B15 xenograft models. As in the murine models, F10 caused regression of disease with several animals cured and resulted in a significant survival benefit (p=0.0112).As the uptake and mechanism of action of F10 is unique we sought to determine the in vivo efficacy of F10 on ALL previously treated with cytarabine. When mice injected with previously treated ALL cells were treated with cytarabine a median survival benefit of only one day (11 vs 12 days) was observed. In contrast F10 treatment resulted in a median survival of 31 days, similar to the survival seen in previously untreated ALL.F10 exposure results in trapped topoisomerase I (Topo1) cleavage complexes in vitro. To determine if this occurs in vivo, we treated leukemic C57Bl/6 or nude mice harboring ALL xenografts with F10. After 24 hours, leukemia cells were harvested and assayed for trapped Topo1 complexes. F10 treatment resulted in detectable trapped Topo1 complexes in both the xenograft and syngeneic models.In summary, F10 exhibited remarkable activity against human and murine ALL cells in vitro and in vivo by inducing apoptosis and trapping Topo1 complexes. These data demonstrate agents that target “final common pathways” but with differential uptake can be safe and effective, even against genetically complex and aggressive leukemias. Disclosures:Pardee:Salzburg Therapeutics: Membership on an entity's Board of Directors or advisory committees. Gmeiner:Salzburg Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Effective Targeting Of The P53/MDM2 Axis In Preclinical Models Of Infant MLL-Rearranged Acute Lymphoblastic Leukemia

IntroductionWhile the overall cure rate for the most common pediatric cancer, acute lymphoblastic leukemia (ALL) now approaches 90%, infants (<12 months) diagnosed with ALL harboring translocations in the mixed-lineage leukemia oncogene (infant MLL-ALL) experience shorter remission duration and a significantly reduced likelihood of survival (∼50%). Therefore, new treatments that can be incorporated into conventional chemotherapy regimens to extend patient remission and improve survival are urgently required. Mutations in the p53 tumor suppressor are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be of therapeutic benefit. Nutlin cis-imidazole molecules selectively inhibit p53-MDM2 binding, resulting in activation of the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenograft models. MethodsIn vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. P53 protein levels and subcellular distribution were assessed by immunoblotting. Patient-derived xenografts were established from infant MLL-ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice, and their gene expression profiles generated using Illumina Human Ref-12 Expression BeadChips. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45+ cells in the PB. Mice with established disease received vehicle, RG7112 (100 mg/kg daily x 5 p.o.), a combination of vincristine (0.15 mg/kg once i.p.) dexamethasone (5 mg/kg daily x 5 i.p.) and L-asparaginase (1,000 IU/kg daily x 5 i.p.) (VXL), or RG7112 plus VXL. Anti-leukemic efficacy was assessed using an objective response measure modeled after the clinical setting, as well as the median event-free survival (EFS) of treated or control groups from treatment initiation. Therapeutic enhancement was considered to occur when the RG7112/VXL combination significantly extended mouse EFS compared with that of both of the RG7112 and VXL treated groups. ResultsUnsupervised hierarchical clustering of gene expression profiles revealed that the MLL-ALL (n=9), BCP-ALL (n=7) and T-ALL (n=13) xenografts clustered according to leukemia subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA gene family, were significantly upregulated in MLL-ALL xenografts. The specificity of RG7112 was validated by cytotoxicity assays against leukemia cell lines of known p53 status; p53 wild-type cell lines (RS4;11, IC50 1.4 µM; NALM-6, IC50 3.0 µM) were markedly more sensitive than those with mutant p53 (CEM, IC50 >10 µM; JURKAT, IC50 >10 µM). The in vitro sensitivity of BCP-ALL (n=3) and infant MLL-ALL (n=4) xenografts was consistent with wild-type p53 status, with IC50s of 0.11 - 2.2 µM. Exposure of ALL xenograft cells to nutlin-3 (10 µM, 6h) caused marked p53 up-regulation and nuclear translocation. Since we had previously shown that RG7112 administered as a single agent for 14 days induced significant regressions [Complete Responses (CRs) or greater] in 7/7 infant MLL-ALL xenografts in vivo, we assessed its efficacy in a 5-day combination treatment with an induction-type regimen (VXL) against two infant MLL-ALL xenografts (MLL-5 and MLL-14). The RG7112/VXL combination caused a Partial Response in MLL-5 compared with Progressive Disease for both RG7112 and VXL. The efficacy of RG7112/VXL was even more pronounced against MLL-14, causing a Maintained CR compared with CRs for both RG7112 and VXL, which met the criteria for Therapeutic Enhancement (the median EFS of RG7112/VXL-treated mice, 65.0 days, was significantly greater, P< 0.0001, than that of RG7112, 22.2 days, and VXL, 28.5 days). ConclusionsRG7112 induces significant regressions in a high proportion of infant MLL-ALL xenografts and enhances the efficacy of an induction-type regimen. The utility of targeting the p53-MDM2 axis in combination with established drugs for the clinical management of infant MLL-ALL warrants further investigation.This study was supported by NCI NO1CM42216. The authors thank Roche Pharmaceuticals, Inc., for providing RG7112. Disclosures:No relevant conflicts of interest to declare.