Subtypes Of Pediatric Acute Lymphoblastic Leukemia Research Articles

Abstract P30: Therapeutic activation of pre-B-cell receptor signaling in high-risk pediatric ALL: exploiting TCF3 and MYC-linked vulnerabilities

Abstract P30: Therapeutic activation of pre-B-cell receptor signaling in high-risk pediatric ALL: exploiting TCF3 and MYC-linked vulnerabilities

In vivo activity of the dual SYK/FLT3 inhibitor TAK-659 against pediatric acute lymphoblastic leukemia xenografts.

While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains dismal. Spleen tyrosine kinase (SYK) is a prominent cytosolic nonreceptor tyrosine kinase in pediatric B-lineage ALL (B-ALL). Activating mutations or overexpression of Fms-related receptor tyrosine kinase 3 (FLT3) are associated with poor outcome in hematological malignancies. TAK-659 (mivavotinib) is a dual SYK/FLT3 reversible inhibitor, which has been clinically evaluated in several other hematological malignancies. Here, we investigate the in vivo efficacy of TAK-659 against pediatric ALL patient-derived xenografts (PDXs). SYK and FLT3mRNA expression was quantified by RNA-seq. PDX engraftment and drug responses in NSG mice were evaluated by enumerating the proportion of human CD45+ cells (%huCD45+ ) in the peripheral blood. TAK-659 was administered per oral at 60mg/kg daily for 21 days. Events were defined as %huCD45+ ≥ 25%. In addition, mice were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival and stringent objective response measures. FLT3 and SYK mRNA expression was significantly higher in B-lineage compared with T-lineage PDXs. TAK-659 was well tolerated and significantly prolonged the time to event in six out of eight PDXs tested. However, only one PDX achieved an objective response. The minimum mean %huCD45+ was significantly reduced in five out of eight PDXs in TAK-659-treated mice compared with vehicle controls. TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric ALL PDXs representative of diverse subtypes.

Abstract 3039: Pediatric preclinical testing consortium evaluation of the dual SYK/FLT3 inhibitor TAK-659 in xenograft models of pediatric acute lymphoblastic leukemia

Abstract Introduction: While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains poor. Spleen Tyrosine Kinase (SYK), a cytosolic nonreceptor tyrosine kinase, is primarily expressed in the hematopoietic lineage and is essential for B-cell receptor signaling. It is associated with malignant transformation, cancer cell proliferation, and is a prominent tyrosine-phosphorylated protein in B-lineage pediatric ALL (Dolai et al, Cancer Res 76:2766-77, 2016). Fms Related Receptor Tyrosine Kinase 3 (FLT3) is a Class III receptor tyrosine kinase that regulates hematopoiesis. While uncommon in ALL, activating FLT3 mutations and internal tandem duplications are associated with poor outcome. TAK-659 is a dual SYK/FLT3 reversible inhibitor currently undergoing clinical trials against B-cell lymphoma, acute myeloid leukemia and solid tumors. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium to test TAK-659 in vivo against its patient-derived xenograft (PDX) models of pediatric ALL. Methods: SYK and FLT3 mRNA expression in ALL PDXs was quantified by RNAseq (https://pedcbioportal.org) and 8 B-lineage pediatric ALL PDXs (3 B-cell precursor, BCP; 4 mixed-lineage leukemia-rearranged, MLLr; one Philadelphia Chromosome-like, Ph-like) were selected for testing. Engraftment was evaluated by enumerating the proportion of human CD45+ cells (%huCD45+) in the peripheral (PB) blood at weekly intervals. TAK-659 was tested at 60 mg/kg by oral gavage daily for 21 days. Events were defined as %huCD45+ ≥ 25%. At Day 28 post-treatment initiation or event (whichever occurred first), 4 mice/group were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival (EFS) of treated (T) and control (C) groups by T-C, T/C and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). Results: The in vivo efficacy of TAK-659 was evaluated against B-lineage PDXs as FLT3 and SYK mRNA expression was higher in the PDXs from this lineage compared with PDXs from T-ALL. TAK-659 was well tolerated and significantly prolonged the time to event in 6/8 PDXs (T-C 0-25.5 days, T/C 1.0-2.2). Only one MLLr-ALL obtained an objective response (partial response) with all other models exhibiting progressive disease. The minimum mean %huCD45+ was significantly reduced in 5/8 PDXs in TAK-659 treated mice compared to control. Despite significant reductions in %huCD45+ in the PB for 5/8 PDXs, TAK-659 did not significantly reduce the spleen or BM infiltration of any PDXs. Conclusion: TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric B-ALL PDXs representative of diverse disease subtypes. (Supported by NCI Grants CA199000 and CA199922) Citation Format: Richard B. Lock, Kathryn Evans, Narimanne El-Zein, Eric J. Earley, Stephen W. Erickson, Beverly A. Teicher, Malcolm A. Smith. Pediatric preclinical testing consortium evaluation of the dual SYK/FLT3 inhibitor TAK-659 in xenograft models of pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3039.

Abstract 4181: The XPO1 inhibitior, eltanexor, exhibits potent in vivo activity against a broad range of pediatric acute lymphoblastic leukemia subtypes

Abstract Introduction: Exportin 1 (XPO1/CRM1) plays a central role in the export of proteins from the nucleus including those with tumor suppressor and growth regulatory activity. Eltanexor (KPT-8602) is a second-generation Selective Inhibitor of Nuclear Export (SINE) that specifically blocks XPO1 cargo interactions and is under clinical evaluation for adults with cancer (NCT02649790). In previous Pediatric Preclinical Testing Consortium (PPTC) studies, eltanexor showed potent in vivo activity against acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs). In an effort to understand whether eltanexor exhibited subtype-specific activity in pediatric ALL we carried out a single-mouse trial (SMT) against an intended 90 pediatric ALL PDXs. Methods: Pediatric ALL PDXs, an orthotopic model of the disease, were inoculated into NSG mice in an SMT format (a single mouse inoculated with a single PDX was treated with a single drug). Engraftment and drug responses were assessed by weekly enumeration of the % human leukemic blasts in the peripheral blood (%huCD45+/HLA-ABC+). Treatment commenced when the median %huCD45+/HLA-ABC+ exceeded 1%, and mice received eltanexor at 12.5 mg/kg per oral daily x 5 for 4 weeks. The baseline level of the %huCD45+/HLA-ABC+ in each mouse served as its own control. PDX responses to treatment were assessed by time to event, the maximum decrease in %huCD45+/ABC+ at any point after treatment initiation, and by stringent Objective Response Measures (ORMs) modeled after the clinical setting (Houghton et al, Pediatr Blood Cancer, 2007, 49:928-40). Results: Eltanexor was well tolerated with a mean maximum weight loss of 5.5% across all mice. SMT testing was successful in all 90 pediatric ALL PDXs representative of B-ALL (typical B-ALL, Philadelphia chromosome-positive ALL [Ph+-ALL] and Ph-like ALL), T-ALL (typical T-ALL and early T-cell precursor ALL [ETP-ALL]), and MLL-rearranged ALL (MLLr-ALL). Event-free survival (EFS) of mice ranged from 2.6-137 days, and 61 (68%) of mice experienced EFS that extended beyond the treatment window. Regressions of ≥50% from baseline were observed in 56 (62%) of mice, and these were most pronounced in T-ALL (14/18, 78%) and Ph-like ALL (11/19, 58%). ORMs classified as Complete Response (CR) or Maintained CR (MCR) were elicited in 43 (48%) of PDXs, and again these were more common in T-ALL (12/18, 67%). The ALL subtypes that exhibited the poorest responses to eltanexor were Ph+-ALL and ETP-ALL, although with relatively small sample size (n=3 and 6, respectively). ORMs in the SMT correlated highly with historical conventional drug testing (R=0.94, P<0.001, n=12). Conclusions: Eltanexor exhibits potent single-agent in vivo activity against PDXs derived from a broad range of ALL subtypes including T-ALL and Ph-like ALL and warrants further investigation in pediatric ALL. (Supported by NCI Grants CA199222 & CA199000) Citation Format: Richard B. Lock, Kathryn Evans, Connor D. Jones, Stephen W. Erickson, Beverly A. Teicher, TJ Unger, Yosef Landesman, Malcolm A. Smith. The XPO1 inhibitior, eltanexor, exhibits potent in vivo activity against a broad range of pediatric acute lymphoblastic leukemia subtypes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4181.

Pediatric Preclinical Testing Consortium Evaluation of the Novel Anti-Microtubule Drug E7130 in Xenograft Models of Early T-Cell Precursor Acute Lymphoblastic Leukemia

Introduction: While children diagnosed with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes as well as adult ALL remains poor. Several standard-of-care drugs used in multi-agent treatment protocols for ALL (including vincristine, daunorubicin and methotrexate) are substrates for the ATP-dependent drug efflux pump P-glycoprotein (P-gp), encoded by the ABCB1 gene, although there are limited reports of ABCB1 gene expression being associated with poor outcome in ALL (Carrillo et al, Hematol. 22:286-91, 2017). A previously identified high-risk subtype of T-ALL (early T-cell precursor ALL, ETP-ALL) characterized by poor early response to conventional induction treatment, expresses significantly higher levels of the ABCB1 gene compared with typical T-ALL (1.97-fold; false discovery rate=0.0026; P=0.00029; Zhang et al, Nature 481:157-63, 2012). E7130 is a novel anti-microtubule drug that is a less potent substrate for P-gp compared with other anti-microtubule drugs such as vincristine, and it has shown significant preclinical activity against patient-derived xenograft (PDX) models of adult malignancies. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium (PPTC) to test the in vivo activity of E7130 against its PDX models of pediatric ETP-ALL. Methods: ABCB1 mRNA expression in ALL PDXs was quantified by RNAseq (https://pedcbioportal.org) and qRT-PCR. P-gp protein expression was assessed by immunoblotting, while its activity was measured by the Rhodamine-123 efflux assay in the absence or presence of the P-gp inhibitor tariquidar. E7130 and vincristine were evaluated in vivo against 6 ETP-ALL PDXs. Each PDX was inoculated into 6-8 immune-deficient (NSG) mice per treatment group (2-5 x 106 cells/mouse). Engraftment and drug responses were evaluated by enumerating the proportion of human CD45+ cells in the peripheral blood (%huCD45+) at weekly intervals. E7130 was tested at 2 dose levels (0.09 and 0.135 mg/kg IV), while vincristine was evaluated at 1 mg/kg IP. Both drugs were administered weekly x 3. Events were defined as the %huCD45+ ≥25%. Drug efficacy was assessed by event-free survival of treated (T) and control (C) groups by T-C, T/C and stringent objective response criteria (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). Results: RNAseq analysis of the 6 ETP-ALL PDXs in the PPTC panel of 90 pediatric ALL PDXs revealed 3 with high ABCB1 expression (FPKM 7.1-13.6; ETP-2, -3 and -6) and 3 with low expression (FPKM 0-0.15; ETP-1, -4 and -5), which was confirmed by qRT-PCR and immunoblotting. Moreover, high levels of tariquidar-sensitive Rhodamine-123 efflux activity were confirmed in the 2 high ABCB1 expressing PDXs tested (ETP-2 and -3). E7130 was generally well tolerated in NSG mice, with maximum average weight loss of 2.7-17.6% in the groups treated with the highest dose compared with 3.2-12.7% in the vincristine treated groups. E7130 (0.09 mg/kg) significantly (P<0.05) delayed the progression of all 6 PDXs (T-C 10.5-41.3 days, T/C 1.8-5.9) and elicited objective responses in 2/6 PDXs (1 Complete Response, CR; 1 Maintained CR, MCR). The higher dose of E7130 (0.135 mg/kg) significantly delayed the progression of all 5 evaluable PDXs (T-C 18.1-49.5 days, T/C 2.3-8.8) and elicited 4 objective responses (1 CR, 3 MCRs). In contrast, vincristine significantly delayed the progress of 5/6 PDXs (T-C 3.5-37.7 days, T/C 1.3-6.6) and elicited 2 objective responses (1 CR, 1 MCR). When the PDXs were stratified around ABCB1 expression there was a trend for reduced vincristine activity against high (T-C 3.5-18.0 days, T/C 1.3-2.1) versus low (T-C 21.4-37.7 days, T/C 2.7-6.6) expressing PDXs. In contrast, the activity of E7130 was maintained regardless of ABCB1 expression at both the 0.09 mg/kg dose (low ABCB1, T-C 17.6-41.3 days, T/C 2.4-5.9; high ABCB1, T-C 10.5-32.0 days, T/C 1.8-3.0) and the 0.135 mg/kg dose (low ABCB1, T-C 30.7-46.2 days, T/C 3.5-8.8; high ABCB1, T-C 18.1-49.5 days, T/C 2.9-4.0). Conclusions: E7130 exhibits significant in vivo activity against pediatric ETP-ALL PDXs, regardless of their levels of ABCB1 expression. Our results support further evaluation of E7130 in pediatric ALL to determine whether it represents an alternative treatment option in ALL with high ABCB1 expression. (Supported by NCI Grants CA199000 and CA199922) Disclosures No relevant conflicts of interest to declare.

A Single Mouse Trial Platform for Evaluation of Novel Agents in Acute Lymphoblastic Leukemia By the Pediatric Preclinical Testing Consortium

Introduction: While children diagnosed with the most common pediatric malignancy, acute lymphoblastic leukemia (ALL), now experience close to a 90% likelihood of cure, the outcome for several high-risk subtypes remains poor. Furthermore, since standard-of-care drugs are extremely effective in this disease, and there are relatively few patients eligible for early phase clinical trials, only the most promising new agents are advanced for clinical evaluation following rigorous preclinical testing. However, conventional preclinical testing of novel agents is not sufficiently resourced to be able to encompass the vast heterogeneity of pediatric ALL, and new approaches to preclinical testing are required in this disease. The purpose of this study was to evaluate the utility of a single mouse trial (SMT) platform for preclinical assessment of novel agents on an almost clinical trial scale, to encompass the broad heterogeneity of pediatric ALL in a single experiment, and to identify molecular biomarkers associated with in vivo drug responses when carried out using molecularly-annotated patient-derived xenografts (PDXs).Methods: Eighty pediatric ALL PDXs broadly representative of all pediatric ALL subtypes were characterized in terms of engraftment kinetics in immune-deficient NSG mice, and molecularly annotated by RNA-seq, exome-seq and DNA copy number analysis. Between 2-5 million cells from each PDX were inoculated via the tail vein into 2 NSG mice/PDX. Starting at 2 weeks post inoculation engraftment was monitored by flow cytometric enumeration of the proportion of human CD45+cells in the murine peripheral blood (%huCD45+). When the %huCD45+ for each PDX reached >1% one mouse/PDX was treated with the established drug and topoisomerase I inhibitor topotecan (Tpt, 0.6 mg/kg IP daily x 5 x 2 weeks, repeated at 21 days) and the other mouse was treated with the experimental drug and second mitochondria-derived activator of caspases (SMAC)-mimetic birinapant (Bpt, 15 mg/kg IP every 3 days x 5). Treatment response was monitored using stringent objective response criteria modeled after the clinical setting, by mouse event-free survival (EFS where an event was defined as 25% huCD45+), and by waterfall plots comparing the maximum decrease in %huCD45+at any point post treatment initiation. The authenticity of each PDX was verified using a 60-allele SNP array both at the time of inoculation and at relapse post drug treatment for all mice.Results: Retrospective analysis of 1,000 samples of “single mouse” data previously showed that the single mouse results predicted the overall group response from conventional testing 75.3% of the time, which increased to 94.3% if a deviation of ± one response category was allowed. SMT results were achieved for 71 (88.8%) and 73 (91.3%) of the intended 80 mice for Tpt and Bpt, respectively. Waterfall plots revealed that 60/71 (84.5%) and 30/73 (41.1%) of PDXs achieved regressions in response to Tpt and Bpt treatment, respectively. When compared with historical objective response measures from conventional drug testing carried out by the Pediatric Preclinical Testing Consortium (PPTC) the SMT results showed high concordance for both Tpt (R=0.904; P=0.014; n=7 PDXs) and Bpt (R=0.804; P<0.0001; n=20 PDXs). Moreover, the SMT confirmed the preferential in vivo efficacy of Bpt against B-ALL compared with T-ALL, early T-cell precursor ALL (ETP-ALL) and ALL with mixed linage leukemia gene rearrangements (MLLr-ALL) that was previously revealed by conventional testing carried out by the PPTC. Analysis of divergent responses observed within the MLLr-ALL subpanel (n=9 PDXs) to Tpt revealed a potential 40 gene signature model, with up-regulated genes in Responders being associated with transcription regulation, cellular proliferation and differentiation.Conclusions: This study has shown that SMTs provide an accurate and cost-effective platform for preclinical drug testing in pediatric ALL on an almost clinical trial scale. Moreover, SMTs can almost encompass the heterogeneity of pediatric ALL in a single experiment, and are likely to be useful for large-scale correlations with in vitro drug sensitivity data. Finally, SMTs have the power to identify molecular biomarkers of in vivo response to established and novel drugs in pediatric ALL when combined with exome-seq, RNA-seq and DNA copy number analysis of molecularly-annotated PDXs. Supported by U01CA199000 from the NCI. DisclosuresNo relevant conflicts of interest to declare.

NGS-based methylation profiling differentiates TCF3-HLF and TCF3-PBX1 positive B-cell acute lymphoblastic leukemia.

To determine whether methylation differences between mostly fatal TCF3-HLF and curable TCF3-PBX1 pediatric acute lymphoblastic leukemia subtypes can be associated with differential gene expression and remission. Five (extremely rare) TCF3-HLF versus five (very similar) TCF3-PBX1 patients were sampled before and after remission and analyzed using reduced representation bisulfite sequencing and RNA-sequencing. We identified 7000 differentially methylated CpG sites between subtypes, of which 78% had lower methylation levels in TCF3-HLF. Gene expression was negatively correlated with CpG sites in 23 genes. KBTBD11 clearly differed in methylation and expression between subtypes and before and after remission in TCF3-HLF samples. KBTBD11 hypomethylation may be a promising potential target for further experimental validation especially for the TCF3-HLF subtype.

Abstract 1942: The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, OT-82, exhibits in vitro and in vivo efficacy against patient-derived xenograft models of high-risk acute lymphoblastic leukemia

Abstract Cancer cells are highly dependent on nicotinamide phosphoribosyltransferase (NAMPT) for the biosynthesis of nicotinamide adenine dinucleotide (NAD). Besides its role in energy metabolism, NAMPT influences the activity of NAD-dependent enzymes, including poly (ADP-ribose) polymerase-1 (PARP-1) and sirtuins, and thereby regulates cellular survival and stress response. Disruption of NAD synthesis through NAMPT inhibition represents a potential therapeutic strategy for treating cancer. The aim of this study was to evaluate the efficacy of the novel NAMPT inhibitor OT-82, initially isolated for its selective toxicity against a panel of adult leukemia cell lines, in a diverse panel of leukemia cell lines in vitro and pediatric acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs) in vivo, and to identify potential biomarkers predictive of OT-82 response. OT-82 demonstrated low nanomolar IC50 values (0.9 – 3.4 nM) in 3 ALL and 3 acute myeloid leukemia cell lines as determined by resazurin reduction assays. In vivo efficacy of OT-82 (40 mg/kg x 3 days x 3 weeks, p.o.) was evaluated as a single agent against pediatric ALL PDXs, including B-cell precursor ALL (n = 3), Philadelphia chromosome (Ph) positive ALL (n = 2), Ph-like ALL (n = 2), T-cell ALL (n = 3), and early T-cell precursor ALL (n = 3) in immune deficient (NSG) mice. Response to treatment was assessed by time to event or stringent objective response criteria modeled after the clinical setting. OT-82 was well tolerated, significantly increased event-free survival (EFS) relative to control mice in 11/13 ALL PDXs, and elicited objective responses in 11/13 (85%) PDXs [3 Partial Responses, 4 Complete Responses (CRs) and 4 Maintained CRs (MCRs)]. Analysis of basal protein expression revealed elevated levels of poly (ADP-ribosyl)ated (PARylated) PARP-1 in 4/5 responders versus 0/2 non-responders. In vitro studies examining various chemotherapeutic agents used for childhood leukemia showed synergy between cytarabine (AraC) and OT-82 in an ALL cell line. In an OT-82 sensitive Mixed Lineage Leukemia (MLL) PDX, treatment with AraC (25 mg/kg x 5 days x 2 weeks, i.p.) and OT-82 (40 mg/kg x 3 days x 2 weeks, p.o.) significantly increased EFS compared to OT-82 (P&amp;lt;0.0001) or AraC (P&amp;lt;0.0001) alone. Moreover, the OT-82/AraC combination elicited an MCR compared to OT-82 (CR) and AraC (Progressive Disease) alone. The results herein demonstrate significant activity of OT-82 against leukemia cell lines and a range of pediatric ALL subtypes in vivo, and the potential for PARylated PARP-1 expression as a biomarker for predicting OT-82 response. Moreover, the enhanced activity of OT-82 in combination with AraC over single agent therapy further suggests NAMPT inhibition as an attractive strategy for treating high-risk pediatric ALL. Supported by U01CA199000 from the NCI. Citation Format: Kathryn Evans, Tara Pritchard, Michelle J. Henderson, Klaartje Somers, Mawar Karsa, Leanna Cheung, Raymond Yung, Stephen W. Erickson, Lioubov Korotchkina, Olga Chernova, Andrei Gudkov, Malcolm A. Smith, Richard B. Lock. The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, OT-82, exhibits in vitro and in vivo efficacy against patient-derived xenograft models of high-risk acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1942. doi:10.1158/1538-7445.AM2017-1942

Outcome and Prognostic Factors for ETV6/RUNX1 Positive Pediatric Acute Lymphoblastic Leukemia Treated at a Single Institution in Korea.

PurposeETV6/RUNX1 (+) acute lymphoblastic leukemia (ALL), which is the most common genetic subtype of pediatric ALL, has a favorable prognosis. In this study, we analyzed the outcome of ETV6/RUNX1 (+) ALL patients treated at our institution with the aim of identifying significant prognostic variables.Materials and MethodsSixty-three patients were diagnosed with ETV6/RUNX1 (+) ALL from 2005 to 2011. Prognostic variables studied included minimal residual disease (MRD) as detected by ETV6/RUNX1 (+) fusion, and the presence of additional cytogenetic abnormalities.ResultsThe 5-year event-free survival was 84.1±4.6%, with 10 patients relapsing at a median of 28.3 months from diagnosis for a 5-year cumulative incidence of relapse of 15.9±4.6%. Multivariate analysis revealed that the presence MRD, as detected by real-time quantitative-polymerase chain reaction or fluorescence in situ hybridization for ETV6/RUNX1 fusion at end of remission induction, and the presence of additional structural abnormalities of 12p (translocations or inversions) negatively affected outcome. Despite treatment such as allogeneic hematopoietic cell transplantation, eight of the 10 relapsed patients died from disease progression for overall survival of 82.5±6.9%.ConclusionETV6/RUNX1 (+) ALL may be heterogeneous in terms of prognosis, and variables such as MRD at end ofremission induction or additional structural abnormalities of 12p could define a subset of patients who are likely to have poor outcome.

Abstract 1620: In vivo and in vitro efficacy of birinapant in preclinical models of Ph-like pediatric acute lymphoblastic leukemia

Abstract Despite the success of current therapies for pediatric acute lymphoblastic leukemia (ALL) more effective treatments are required for the management of high-risk subtypes. Ph-like ALL is a high-risk subtype defined by a gene expression signature similar to that of BCR-ABL1-positive ALL despite the absence of the BCR-ABL1 translocation. Approximately 50% of Ph-like pediatric ALLs harbor mutations in Janus kinases (JAKs). Birinapant is a small molecule SMAC (second mitochondria-derived activator of caspase) mimetic that potently and specifically antagonizes inhibitors of apoptosis proteins (IAPs), resulting in IAP degradation, inactivation of NF-κB survival signaling and tumor necrosis factor (TNF)-dependent apoptosis. The combination of birinapant plus azacitidine is being evaluated in a Phase 2 clinical trial for the treatment of high-risk myeloid dysplastic syndrome. The aim of this study was to evaluate the efficacy of birinapant against patient-derived xenografts (PDXs) of pediatric ALL subtypes. Birinapant (30 mg/kg IP Q3 days × 5) significantly delayed the progression of 17/19 PDXs derived from Ph-like ALL (n = 7), B-cell precursor ALL (BCP-ALL, n = 8), and infant MLL¬-rearranged ALL (MLL-ALL, n = 4) by between 2 and 80 days compared with vehicle-treated controls. Using stringent objective response criteria modeled after the clinical setting, birinapant induced objective responses in 12/19 PDXs, including 7/7 Ph-like ALL (5 complete responses, CRs; 2 maintained CRs, MCRs), which was significantly better than BCP-ALL (4/8; 2 partial responses, PRs; 1 CR; 1 MCR) or infant MLL-ALL (1/4; 1 CR) PDXs (P&amp;lt;0.05). Birinapant induced a CR in a Ph-like PDX at a dose 1/8th (3.8 mg/kg) of its maximum tolerated dose (30 mg/kg). Analysis at 14 days following treatment initiation revealed &amp;gt;98% clearance of human leukemia cells from the bone marrow, spleen and peripheral blood of mice treated with birinapant doses that achieve drug levels attainable in humans. In vitro apoptosis assays confirmed the greater sensitivity of the Ph-like ALL PDX panel. Moreover, the cIAP1 protein was rapidly degraded in PDXs upon birinapant treatment both in vitro and in vivo, regardless of their relative sensitivity. Microarray analysis of gene expression revealed a significant correlation between baseline TNFα expression and in vivo birinapant sensitivity across 19 PDXs (P = 0.002; R2 = 0.46). While exogenously-added TNFα did not potentiate apoptosis induced by birinapant, a TNFα blocking antibody partially reversed apoptosis in 3/4 Ph-like PDXs. These results show that birinapant exerts profound single-agent in vivo efficacy against Ph-like pediatric ALL PDXs, indicate a role for endogenous TNFα in the birinapant mechanism of action against this high-risk pediatric ALL subtype, and support further evaluation of birinapant in the treatment of Ph-like ALL. Supported by NCI NO1CM42216. Citation Format: Jennifer Richmond, Kathryn Evans, Alissa Robbins, Raushan T. Kurmasheva, Peter J. Houghton, Malcolm A. Smith, Richard B. Lock. In vivo and in vitro efficacy of birinapant in preclinical models of Ph-like pediatric acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1620. doi:10.1158/1538-7445.AM2015-1620

Abstract 3276: Evaluation of the Bcl-2 inhibitor ABT-199 in xenograft models of acute lymphoblastic leukemia by the pediatric preclinical testing program

Abstract Targeting the apoptosis pathway with BH3 mimetics such as ABT-263 (navitoclax) and ABT-199 is an appealing strategy for cancer therapy. While the Bcl-2/Bcl-w/Bcl-xL inhibitor navitoclax has shown promising activity in adults with lymphoid malignancies and in preclinical models of pediatric acute lymphoblastic leukemia (ALL), its application in pediatric patients has been limited by Bcl-xL-mediated thrombocytopenia. The specific Bcl-2 inhibitor ABT-199 has shown remarkable efficacy against adult chronic lymphocytic leukemia in recent clinical trials, although its activity against pediatric ALL is yet to be tested. The aim of this study was to evaluate the in vivo efficacy of ABT-199 against a large panel of pediatric ALL patient-derived xenografts (PDXs), and to identify biomarkers predictive of ABT-199 response. Immune-deficient (NOD/SCID or NSG) mice inoculated with PDXs derived from patients with infant ALL harboring rearrangement of the Mixed Lineage Leukemia (MLL) oncogene (infant MLL-ALL, n = 4), B-cell precursor ALL (BCP-ALL, n = 6), BCP-ALL categorized as Ph-like (n = 4), T-cell ALL (T-ALL, n = 4) or early T-cell precursor ALL (ETP-ALL, n = 2) were treated with ABT-199 (100 mg/kg x 21 days, p.o.) or vehicle control. Responses were assessed by time to event measurements or stringent objective response criteria modeled after the clinical setting. ABT-199 significantly delayed the progression of 12/20 PDXs by between 0.4 and 28 days, and elicited objective responses in 6/20 (30%) of PDXs (4 complete responses and 2 partial responses). No objective responses were observed in the T-ALL or ETP-ALL PDXs. By comparison, navitoclax administered on the same dose and schedule as ABT-199 elicited objective responses in 19/31 (61%) of PDXs derived from the same pediatric ALL subtypes. Analysis of basal gene and protein expression revealed that high Bcl-xL and low Bcl-2 expression were significantly associated with in vivo ABT-199 resistance. Moreover, the Bcl-2/Bcl-xL gene (P = 0.03) and protein (P = 0.002) expression ratios were significantly elevated in PDXs that responded to ABT-199 in vivo compared with non-responders. Notably, Mcl-1 expression at the gene or protein level showed no significant correlation with in vivo ABT-199 sensitivity. In conclusion, the inferior objective response rate observed for ABT-199 (30%) compared with navitoclax (61%) indicates that pediatric ALL PDXs are less dependent on Bcl-2 for cell survival compared with adult chronic lymphocytic leukemia. Moreover, Bcl-xL appears to be a more significant ABT-199 resistance factor than Mcl-1 in pediatric ALL. While ABT-199 has the potential to exert significant efficacy in the treatment of aggressive and chemoresistant pediatric ALL, the prospective identification of patients who might benefit from such treatment is of paramount importance. Supported by NCI NO1CM42216. Citation Format: Santi Suryani, Kathryn Evans, Jennifer Richmond, Alissa Robbins, Lauryn Bracken, Raushan Kurmasheva, Peter J. Houghton, Malcolm A. Smith, Richard B. Lock. Evaluation of the Bcl-2 inhibitor ABT-199 in xenograft models of acute lymphoblastic leukemia by the pediatric preclinical testing program. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3276. doi:10.1158/1538-7445.AM2015-3276

POL5551, a Novel and Potent CXCR4 Antagonist, Enhances Sensitivity to Chemotherapy in an in Vivo Model of High-Risk (HR) Pediatric Acute Lymphoblastic Leukemia (ALL)

Background: Interaction between surface receptor CXCR4 (s-CXCR4) and chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow (BM) microenvironment. We previously demonstrated: 1) chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) and ALL enhances stromal protection from chemotherapy-induced apoptosis; 2) the FDA-approved CXCR4 inhibitor plerixafor reverses stromal protection and chemotherapy resistance both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged (MLL-R) ALL; 3) the novel Protein Epitope Mimetic POL5551, a selective and potent antagonist of CXCR4, blocks the SDF-1-binding site of CXCR4, inhibits SDF-1-induced chemotaxis, and reverses stromal-mediated protection from chemotherapy in vitro in pre-B and T ALL cell lines. Here, we further characterize the effects of POL5551 (POL) on surface adhesion molecule expression in ALL, and its in vivo effects in a xenograft model of HR pediatric ALL.In Vitro Methods/Results: We have previously shown that POL inhibits 12G5 antibody binding to s-CXCR4 in ALL cell lines, suggesting an overlapping of the two binding sites. We sought to verify these results using primary samples of pediatric ALL. We treated 3 pre B and 3 T cell ALL primary samples with a dose range of POL and measured s-CXCR4 by FACS at multiple time points. POL inhibition of 12G5 binding was potent (average IC50 at 2 hours pre B 8.3 nM, T 1.4 nM), rapid (<1 hour) and sustained (>24 hours). Further, POL was significantly more potent at inhibiting 12G5 binding than plerixafor (average IC50 at 2 hours pre B 18.4 nM, T 8.4 nM). To further characterize POL’s effects in ALL, we treated 2 pre B and 2 T cell ALL cell lines with POL or vehicle control and then treated with SDF-1α or vehicle control. Treatment with POL inhibited SDF-1α-induced phosphorylation of ERK1/2 in a dose-dependent manner. In parallel, we measured POL-induced compensatory upregulation of the alternative surface adhesion molecules CXCR7 and VLA-4 (CD49d), and found that POL led to increased CXCR7 expression at early time points that began to decrease after 24 hours. We did not find a consistent effect of POL on CD49d surface expression.Xenograft Methods/Results: Infant MLL-R ALL primary samples (n=4) were transplanted into sublethally irradiated NSG mice. After 2 weeks, mice were treated on days 1-3 of 2 consecutive weeks with 1) vehicle control (C), 2) POL (5 mg/kg SC), 3) AraC (200 mg/kg IP), or 4) POL followed by AraC 4 hours later (POL+AraC). One week after treatment, cells were harvested from BM, spleen, and peripheral blood (PB). Leukemic blasts were defined as human CD19+ and CD45+. Overall leukemic burden (average % blasts in BM+spleen+PB) did not differ between mice treated with either C (56.2%) or POL (49.5%, p=0.12). However, treatment with AraC (36.7%, p=3E-07) or POL+AraC (26.3%, p=4E-15) significantly decreased total leukemic burden compared to C. Notably, POL+AraC significantly decreased total leukemic burden compared to AraC alone (p=0.001), demonstrating that POL increased overall sensitivity to AraC. When analyzed by organ-specific leukemic burden, POL+AraC resulted in decreased leukemic burden compared to AraC alone in BM (42.8 vs. 49.8%, p=0.27), spleen (16.9 vs. 30.8%, p=0.002), and PB (19.3 vs. 29.6%, p=0.008). Interestingly, AraC and POL+AraC led to significantly increased CXCR7 expression (blasts from BM p<0.01, spleen p<0.04, and PB p<0.03 vs. C) as well as a trend toward increased CD49d expression compared to C.Conclusions: POL5551 is a potent antagonist of s-CXCR4 in pediatric ALL cell lines and primary samples. Surface expression of adhesion molecules in ALL blasts is dynamic and affected by anti-leukemic therapy. Importantly, POL5551 enhances sensitivity to AraC in a xenograft model of infant MLL-R ALL, a HR subtype of pediatric ALL. Our findings support the continued development of BM microenvironment-targeted agents as a therapeutic strategy for pediatric ALL. DisclosuresRomagnoli:Polyphor Ltd.: Employment. Douglas:Polyphor Ltd.: Employment. Tuffin:Polyphor Ltd.: Employment. Zimmermann:Polyphor Ltd.: Employment.

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.

Discovery of Subtype-Specific Mirnas and New MiR-Genes In Pediatric Acute Lymphoblastic Leukemia

AbstractAbstract 3232MicroRNAs (miRNAs) are non-coding RNAs that regulate the activity of protein-coding genes and some miRNAs have been shown to be dysregulated in cancer. The function of miRNAs differs per cell type and prominent miRNAs in e.g. B-cell lymphoma may not be important in acute lymphoblastic leukemia (ALL). To identify which miRNAs may be relevant in pediatric ALL the expression level of 397 different miRNAs was measured in leukemic cells (>90% purity) of 81 pediatric ALL and 17 normal hematopoietic control samples by stem-loop reverse-transcriptase (RT) quantitative real-time PCR. Except for BCRABL1-positive and B-other ALL, all major subtypes including T-ALL, MLL-rearranged, TELAML1-positive, E2APBX1-positive and hyperdiploid (>50 chromosomes) ALL presented with unique miRNA signatures that differ from each other and from those in healthy hematopoietic cells. The expression levels highly varied amongst subtypes and, for example, differed by 4000-fold for miR-708 in T-ALL (P<0.0001) and 1700-fold for miR-383 in TELAML1-positive ALL (P<0.0001) compared to other precursor B-ALL cases. Some subtypes shared similarities in their miRNA expression signatures suggesting a common underlying biology; e.g. miR-196b was highly expressed in 9/12 MLL-rearranged and 14/22 T-ALL patients. In particular, all T-ALL cases carrying CALM-AF10, MLL-AF6, SET-NUP214 or an inversion of chromosome 7 expressed high levels of miR-196b similar to MLL-rearranged ALL. These genetic subtypes have all been functionally linked to upregulation of HOXA cluster genes. In correspondence, miR-196b expression levels were also highly correlated with HOXA (Rs>0.7, p<0.003), which was not seen for HOXB and HOXC cluster genes. High levels of miR-196b coincide with reduced methylation of the DNA at CpG islands directly upstream of miR-196b in MLL-rearranged cases compared to non-MLL precursor B-ALL and normal bone marrow cases, suggesting an epigenetic origin for miR-196b overexpression in these patients.High-throughput sequencing (Solexa technology) of small RNA libraries representing 7 subtypes of ALL and 3 normal hematopoietic tissue types followed by application of stringent computational miRNA precursor prediction algorithms resulted into the identification of 28 novel and 431 candidate novel miR-genes (besides 554 known miR-genes) for which the expression levels differed between genetic subtypes of pediatric ALL and normal hematopoietic cells. Stem-loop RT-quantitative real-time PCR confirmed aberrant expression levels of newly discovered miR-genes in a different set of patients. These data may point to leukemia-associated miRNAs for which the (aberrant) expression level and activity may be cell type (e.g. MLL-subtype) specific. These subtype-specific (known and novel) miRNAs may have stayed unrecognized when analyzed in a set of genetically unspecified ALL cases.In conclusion, genetic subtypes of ALL display unique miRNA expression signatures. Functional studies are in progress for these discriminative miRNAs since this may provide new insights into the biology of ALL subtypes. Disclosures:No relevant conflicts of interest to declare.

Pharmacogenomics of pediatric acute lymphoblastic leukemia

Importance of the field: Pediatric acute lymphoblastic leukemia (ALL) represents one of the best examples of progress in disease treatment and improved outcome based in part upon the incorporation of the principles of pharmacogenomics. Throughout the past several decades, clinical scientists have continued to refine risk stratification in clinical trials with the understanding that individual patients have different subtypes of pediatric ALL that will respond to therapy in different, but predictable ways.Areas covered in this review: Discussed in this review are the most significant findings from pharmacogenomic studies of pediatric ALL from 1989 to the present. Pharmacogenomic studies related to the drugs commonly used to treat pediatric ALL are covered in detail, including an emphasis on both genome-wide and candidate gene/pathway approaches.What the reader will gain: Readers of this paper will acquire a detailed understanding of how pharmacogenomic studies can be integrated into clinical trials, in addition to some of the discrepancies still present in the field.Take home message: The outcome for children with pediatric ALL has improved greatly, and this is in part due to the successful integration of data from pharmacogenomic studies into clinical trials.

Genetic Abnormalities Underlying B-Cell Development Are Characteristic for a New Subgroup of Childhood Precursor B-ALL with a Very Poor Prognosis

Gene expression signatures are a promising tool to predict lineage and genetic subtypes of acute lymphoblastic leukemia (ALL) and other types of cancer. Using a 110 probe set signature, we correctly identified patients belonging to the 4 major subtypes of pediatric ALL (T-ALL, TELAML1-positive, hyperdiploid and E2A-rearranged ALL). In addition, we identified a novel subtype of precursor B-ALL. The signature of this group was most similar to true BCRABL-positive cases, whereas these cases did not have the BCRABL-translocation. We therefore describe this group as BCRABL-like. The existence of BCRABL-like patients was confirmed in an independent validation cohort of 107 patients. Most importantly, they represent 15–20% of precursor B-ALL cases with a highly unfavorable prognosis (5-year disease-free survival 60±11%) compared to other precursor B-ALL cases (84±4%; p=0.009), resembling that of true BCRABL-positive cases. To investigate the genetic abnormalities in BCRABL-like patients, we applied array-comparative genomic hybridization analysis. In 44 BCRABL-like patients, we found seven recurrent deletions. Most abnormalities were found in chromosome 9p, while other chromosomes affected included 20q, 22q11, 7p12, 11q, 13q14 and 19p13. Interestingly, many of these deleted regions harbor genes that play an important role in B-cell development. In total, 82% of BCRABL-like patients had abnormalities in one or more genes that are involved in B-cell development including PAX 5 (16 patients), VpreB1 (17 patients), Ikaros (17 patients), EBF1 (6 patients) and TCF3/E2A (3 patients). Bi-allelic loss was frequently found in VpreB1 and PAX5, including inactivating point mutations in exon 2 (V26G) in PAX5. The frequency of B-cell development gene deletions was also high in BCRABL-positive cases (80%), but was significant lower in a control group of precursor B-ALL, including TELAML1-positive, hyperdiploid, MLL-rearranged en B-other patients (36%, p=0.0002). In summary, these results indicate that the BCRABL-like group is a novel high-risk subtype of precursor B-ALL with a high percentage of deletions in B-cell development genes. Currently experiments are being done to determine the biological significance of the recurrent changes in the BCRABL-like group.