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NSG mice were injected in the tail with either ETP-ALL (TALL-x-11) or typical T-ALL (TALL-x-2) 1X106 cells and following engraftment to 65% the mice were randomized to receive vehicle, ABT-199 or ABT-263 treatment for two-weeks. The spleen weights (A) and absolute human CD45+ counts (B) along with % human CD45+ cell in the blood (C) were measured for the ETP-ALL primagraft following treatment. The leg bones were harvested and representative images are shown for the different treatments (D). Similar experiments were performed for the typical T-ALL primagraft spleen weights (E), absolute human CD45+ count in the spleen (F) with % human CD45+ cells in the blood were measured (G). Similarly the legs bones were harvested and representative images are shown (H).

Flow cytometry gating strategy to identify the early progenitor cells using CD44 and CD25 antibodies on the double negative CD4-/CD8- gate. BCL-2 expression and BCL-XL expression was measured on each of the gated regions listed.

The percentage of cells expressing CD8 is shown for both ETP-ALL and for Typical T-ALL primary human samples. The data is modified from the published online dataset (2). The majority of typical T-ALL samples are positive for CD8 expression by flow cytometry analysis.

Primary T-ALL samples were treated for six hours in short term culture with both ABT-199 and ABT-263. Apoptosis was assesed by Annexin V and Propidium Iodide staining. The calculated IC50 in μM is listed beside each of the BH3 mimetics.

T-cell ALL cell lines were treated for 48hr with five doses of ABT-199 and ABT-263. Apoptosis was assessed by Annexin V and Propidium Iodide (PI) staining. The mean % viability of three independent experiments is graphed +/- standard error. The calculated IC50 in μM for each of the BH3 mimetics is listed.

Supplementary Table 1 and 2 - PDF file 68K, Supplementary Table 1. Identity of tagSNPs, details of PCR and ASO hybridization Supplementary Table 2. Identity of polymorphisms genotyped only in controls

Purpose: Asparaginase (ASNase) is a standard and critical component in the therapy of childhood acute lymphoblastic leukemia (ALL), but it is also associated with several toxicities.Experimental design: We recently reported the results of an association study between ASNase pathway genes and event-free survival (EFS) in childhood patients with ALL. The same polymorphisms were interrogated here in relation to allergies, pancreatitis, and thrombotic events following treatment with E. coli ASNase.Results: Among patients of the discovery group, allergies, and pancreatitis were more frequent in individuals who are homozygous for the triple-repeat allele (3R) of the asparagine synthetase (ASNS) gene, resulting in remarkably higher risk of these toxicities associated with 3R3R genotype [OR for allergies, 14.6; 95% confidence interval (CI), 3.6–58.7; P < 0.0005 and OR for pancreatitis, 8.6; 95% CI, 2.0–37.3; P = 0.01]. In contrast, the ASNS haplotype *1 harboring double-repeat (2R) allele had protective effect against these adverse reactions (P ≤ 0.01). The same haplotype was previously reported to confer reduction in EFS. The risk effect of 3R3R genotype was not replicated in the validation cohort, whereas the protective effect of haplotype *1 against allergies was maintained (P ≤ 0.002). Analysis with additional polymorphisms in ASNS locus in lymphoblastoid cell lines showed that haplotype *1 is diversified in several subtypes of which one was associated with reduced in vitro sensitivity to ASNase (rs10486009, P = 0.01) possibly explaining an association seen in clinical setting.Conclusions: This finding might have implication for treatment individualization in ALL and other cancers using asparagine depletion strategies. Clin Cancer Res; 21(2); 329–34. ©2014 AACR.See related commentary by Avramis, p. 230

PDF file - 80K, Polymorphisms in ATF5, ASNS and ASS1 genes analyzed in the relation to ASNase related complications.

Purpose: Corticosteroids induce apoptosis in the malignant lymphoid cells and are critical component of combination therapy for acute lymphoblastic leukemia (ALL). Several genome-wide microarray studies showed major implication of proapoptotic Bim in mediating corticosteroid-related resistance in leukemia cells.Experimental Design: We investigated Bim gene polymorphisms and their association with childhood ALL outcome, and the mechanism underlying the observed finding.Results: Lower overall survival (OS) was associated with BimC29201T located in Bcl-2 homology 3 (BH3) domain (P = 0.01). An association remained significant in multivariate model (P = 0.007), was more apparent in high-risk patients (P = 0.004) and patients treated with dexamethasone (P = 0.009), and was subsequently confirmed in the replication patient cohort (P = 0.03). RNA analysis revealed that C29201T affects generation of γ isoforms (γ1) that lack proapoptotic BH3 domain. The phenotypic effect was minor suggesting the influence of additional factors that may act in conjunction with Bim genotype. Combined analysis with Mcl gene polymorphism (G-486T) revealed profound reduction in OS in individuals with both risk genotypes (P < 0.0005 in discovery and P = 0.002 in replication cohort) and particularly in high-risk patients (P ≤ 0.008).Conclusions: Increased expression of prosurvival Mcl1 and presence of Bim isoforms lacking proapoptotic function might explain marked reduction of OS in a disease and dose-dependent manner in ALL patients carrying Bim- and Mcl1-risk genotypes. Clin Cancer Res; 19(18); 5240–9. ©2013 AACR.

PDF file, 62K, Newly acquired unpublished clinical data for cases previously described by our group.

Supplementary Figures 1-2 from Differentiation of NUT Midline Carcinoma by Epigenomic Reprogramming

Supplementary Tables 1-3 from Gene Expression Profiling Identifies BAX-δ as a Novel Tumor Antigen in Acute Lymphoblastic Leukemia

NUT midline carcinoma (NMC) is a lethal pediatric tumor defined by the presence of BRD-NUT fusion proteins that arrest differentiation. Here we explore the mechanisms underlying the ability of BRD4-NUT to prevent squamous differentiation. In both gain-of and loss-of-expression assays, we find that expression of BRD4-NUT is associated with globally decreased histone acetylation and transcriptional repression. Bulk chromatin acetylation can be restored by treatment of NMC cells with histone deacetylase inhibitors (HDACi), engaging a program of squamous differentiation and arrested growth in vitro that closely mimics the effects of siRNA-mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of HDACi differentiation therapy was established in three different NMC xenograft models, where it produced significant growth inhibition and a survival benefit. Based on these results and translational studies performed with patient-derived primary tumor cells, a child with NMC was treated with the FDA-approved HDAC inhibitor, vorinostat. An objective response was obtained after five weeks of therapy, as determined by positron emission tomography. These findings provide preclinical support for trials of HDACi in patients with NMC. Cancer Res; 71(7); 2686–96. ©2011 AACR.

Purpose: Asparaginase (ASNase) is a standard and critical component in the therapy of childhood acute lymphoblastic leukemia (ALL), but it is also associated with several toxicities. Experimental design: We recently reported the results of an association study between ASNase pathway genes and event-free survival (EFS) in childhood patients with ALL. The same polymorphisms were interrogated here in relation to allergies, pancreatitis, and thrombotic events following treatment with E. coli ASNase. Results: Among patients of the discovery group, allergies, and pancreatitis were more frequent in individuals who are homozygous for the triple-repeat allele (3R) of the asparagine synthetase (ASNS) gene, resulting in remarkably higher risk of these toxicities associated with 3R3R genotype [OR for allergies, 14.6; 95% confidence interval (CI), 3.6–58.7; P < 0.0005 and OR for pancreatitis, 8.6; 95% CI, 2.0–37.3; P = 0.01]. In contrast, the ASNS haplotype *1 harboring double-repeat (2R) allele had protective effect against these adverse reactions (P ≤ 0.01). The same haplotype was previously reported to confer reduction in EFS. The risk effect of 3R3R genotype was not replicated in the validation cohort, whereas the protective effect of haplotype *1 against allergies was maintained (P ≤ 0.002). Analysis with additional polymorphisms in ASNS locus in lymphoblastoid cell lines showed that haplotype *1 is diversified in several subtypes of which one was associated with reduced in vitro sensitivity to ASNase (rs10486009, P = 0.01) possibly explaining an association seen in clinical setting. Conclusions: This finding might have implication for treatment individualization in ALL and other cancers using asparagine depletion strategies. Clin Cancer Res; 21(2); 329–34. ©2014 AACR. See related commentary by Avramis, p. 230

Acute lymphoblastic leukemia (ALL) is a hematopoietic malignancy derived from immature B-lymphoid and T-lymphoid cells (T-ALL). In T-ALL, there is an early T-cell progenitor (ETP) subgroup that has a very high risk for relapse. In this study, we used mitochondrial BH3 profiling to determine antiapoptotic protein dependencies in T-ALL. We found that T-ALL cell lines and primary patient samples are dependent upon BCL-XL, except when the cancer bears an ETP phenotype, in which case it is BCL-2 dependent. These distinctions directly relate to differential sensitivity to the BH3 mimetics ABT-263 and ABT-199, both in vitro and in vivo. We thus describe for the first time a change of antiapoptotic protein dependence that is related to the differentiation stage of the leukemic clone. Our findings demonstrate that BCL-2 is a clinically relevant target for therapeutic intervention with ABT-199 in ETP-ALL. Significance: ETP T-ALL is a treatment-resistant subtype of T-ALL for which novel targeted therapies are urgently needed. We have discovered, through BH3 profiling, that ETP-ALL is BCL-2 dependent and is very sensitive to in vitro and in vivo treatment with ABT-199, a drug well tolerated in clinical trials. Cancer Discov; 4(9); 1074–87. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973

Purpose: Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. We recently reported an association of DHFR promoter polymorphisms with ALL outcome. Lower event-free survival correlated with haplotype *1, defined by A317 and C1610 alleles. Haplotype *1 was also associated higher DHFR expression. Experimental Design: Here, we analyzed adjacent 400-bp region participating in DHFR regulation as both a major promoter and a noncoding minor transcript. Results: Six polymorphisms were identified, of which five were single nucleotide polymorphisms and one was length polymorphism composed of variable number of 9-bp elements and 9-bp insertion/deletion. Haplotype analysis including all promoter polymorphisms revealed diversification of haplotype *1 into five subtypes (*1a-*1e). DNA variations of major promoter/noncoding transcript region and haplotype *1 subtypes were subsequently analyzed for the association with ALL outcome. Lower event-free survival was associated with an A allele of G308A polymorphism (P = 0.02) and with *1b haplotype (P = 0.01). This association was particularly striking in high-risk patients (P = 0.001) and was subsequently confirmed in independent patient cohort (P = 0.02). Haplotype *1b was the only haplotype *1 subtype associated with higher mRNA levels. Conclusions: The study provides a new insight into DHFR regulatory variations predisposing to an event in ALL patients. (Clin Cancer Res 2009;15(22):69318)

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of the outcome of cytotoxic chemotherapy for cancer. To fully exploit this finding, it will be important to understand the molecular genetic contexts responsible for the relative mitochondrial priming of chemotherapy-sensitive versus resistant cell populations. Here we report that mitochondrial apoptosis resistance in T-cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2) and consequent downstream upregulation of the TRAP1 gene, which encodes a mitochondrial chaperone protein of the HSP90 family. In clinical samples from 47 T-ALL patients, we found that loss-of-function mutations in any of three core components of PRC2 (EZH2, EED or SUZ12) were associated with resistance to mitochondrial apoptosis, as assessed by BH3 profiling (P = 0.015). In human T-ALL cells, PRC2 depletion induced resistance to mitochondrial apoptosis induction, as assessed by caspase 3/7 activation or annexin V/PI staining, in response to multiple antileukemic drugs with distinct mechanisms of action, including dexamethasone, doxorubicin, vincristine, and asparaginase (P < 0.01). In mouse immature T-cell progenitors, haploinsufficiency for the PRC2 components Ezh2 or Eed was sufficient to induce resistance to mitochondrial apoptosis, as assessed by BH3 profiling analysis (P ≤ 0.01). PRC2 is a histone-modifying complex whose activity is strongly associated with transcriptional repression. We found that PRC2 represses transcription of TRAP1, a nuclearly encoded, mitochondrially localized chaperone of the HSP90 family. Importantly, TRAP1 overexpression was necessary to induce resistance to chemotherapy-induced apoptosis downstream of PRC2 inactivation (P < 0.001), while pharmacologic inhibition of TRAP1 synergized with antileukemic drugs in PRC2-deficient leukemic cells. These findings demonstrate the importance of relative mitochondrial apoptotic priming as a prognostic factor in T-ALL, and implicate mitochondrial chaperone function as a molecular determinant of response to cancer chemotherapy, suggesting a rationale for targeted therapeutic intervention. This abstract is also being presented as Poster 07. Citation Format: Ingrid Aries, Triona Ni Chonghaile, Salmaan Karim, Sebastian Balbach, Melissa Burns, Gayle Pouliot, Stevenson Kristen, Donna Neuberg, Meenakshi Devidas, Loh Mignon, Stephen Hunger, Stuart Winter, David Teachey, Karen Rabin, Kimberly Dunsmore, Brent Wood, Lewis Silverman, Stephen Sallan, Peter Van Vlierberghe, Stuart H. Orkin, Anthony G. Letai, Alejandro Gutierrez. Polycomb repressive complex 2 inactivation induces primary chemotherapy resistance in T-ALL by upregulating the TRAP1 mitochondrial chaperone [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR14.

Resistance to therapy is one of the major challenges in cancer treatment today, equally applicable to conventional chemotherapy as well as targeted therapy. Malignant tumors have widespread epigenetic alterations including aberrant expression of chromatin modifiers in a wide variety of tumors and chromosomal translocations involving chromatin modifiers that can drive development of some cancers. In addition, cancer genome sequencing studies have identified frequent somatic alterations in many chromatin-regulating enzymes. Moreover, epigenetic changes have been implicated in the development of drug resistance. T cell acute lymphoblastic leukemia (T-ALL) has a high rate of treatment-refractory disease and relapse that significantly lowers survival rates compared to other forms of ALL. The identification of activating somatic NOTCH1 mutations in over 50% of patients with T-ALL led to the development of γ-secretase inhibitors (GSI) that prevent cleavage and activation of NOTCH1. Although effective in vitro, the rapid development of resistance that develops with Notch inhibition in vivo has so far prevented the translation of these inhibitors into the clinical setting. We have developed a model of therapeutic resistance to inhibition of Notch signaling in T-ALL. In this model, ‘persister’ cells readily expand in the presence of GSI and the absence of Notch signaling. Rare persister cells are pre-existing in naïve T-ALL populations. Intriguingly, in vitro resistance to NOTCH1 inhibitor therapy is reversible, suggesting that it is epigenetically mediated. When compared to GSI-sensitive cells, persisters are characterized by distinct signaling and gene expression programs, and demonstrate global chromatin compaction. Using a short-hairpin knock-down screen of ∼ 300 known chromatin regulators we identified the chromatin reader BRD4 as essential for persister T-ALL cells. BRD4 expression levels are upregulated in persister T-ALL cells. Genome-wide binding studies of BRD4 show that it is found at active regulatory elements in the genome that are associated with genes known to be important for cell proliferation, survival and signaling pathways in T-ALL, e. g. MYC and BCL2. Treatment of persisters with the BRD4 inhibitor JQ1 down-regulates expression of these target genes. Functionally, JQ1 treatment leads to growth arrest and apoptosis in persister T-ALL cells, at doses well tolerated by GSI-sensitive leukemia cells. Furthermore, combination therapy of GSI and JQ1 is significantly more effective over vehicle or single agent therapy for primary human T-ALLs in vitro and in vivo. These studies demonstrate epigenetic heterogeneity as a basis of drug resistance in leukemia. We suggest that combination therapies that include targeting of chromatin regulators may hold great therapeutic promise for prevention and treatment of resistant disease. Citation Format: Birgit Knoechel, Justine Roderick, Kaylyn Williamson, Jiang Zhu, Jens Lohr, Matthew Cotton, Shawn Gillespie, Daniel Fernandez, Manching Ku, Hongfang Wang, Federica Piccioni, Serena Silver, Mohit Jain, Daniel Pearson, Michael Kluk, Christopher Ott, Dale Greiner, Michael Brehm, Leonard Shultz, Alejandro Gutierrez, Kimberly Stegmaier, Marian Harris, Lewis Silverman, Stephen Sallan, Andrew Kung, David Root, James Bradner, Jon Aster, Michelle Kelliher, Bradley Bernstein. Epigenetic resistance to Notch inhibition in T cell acute lymphoblastic leukemia. [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 4782. doi:10.1158/1538-7445.AM2014-4782