Marc Valentine

Long-term Safety and Efficacy Following Systemic Administration of a Self-complementary AAV Vector Encoding Human FIX Pseudotyped With Serotype 5 and 8 Capsid Proteins

Adeno-associated virus vectors (AAV) show promise for liver-targeted gene therapy. In this study, we examined the long-term consequences of a single intravenous administration of a self-complementary AAV vector (scAAV2/ 8-LP1-hFIXco) encoding a codon optimized human factor IX (hFIX) gene in 24 nonhuman primates (NHPs). A dose-response relationship between vector titer and transgene expression was observed. Peak hFIX expression following the highest dose of vector (2 × 10(12) pcr-vector genomes (vg)/kg) was 21 ± 3 µg/ml (~420% of normal). Fluorescent in-situ hybridization demonstrated scAAV provirus in almost 100% of hepatocytes at that dose. No perturbations of clinical or laboratory parameters were noted and vector genomes were cleared from bodily fluids by 10 days. Macaques transduced with 2 × 10(11) pcr-vg/kg were followed for the longest period (~5 years), during which time expression of hFIX remained >10% of normal level, despite a gradual decline in transgene copy number and the proportion of transduced hepatocytes. All macaques developed serotype-specific antibodies but no capsid-specific cytotoxic T lymphocytes were detected. The liver was preferentially transduced with 300-fold more proviral copies than extrahepatic tissues. Long-term biochemical, ultrasound imaging, and histologic follow-up of this large cohort of NHP revealed no toxicity. These data support further evaluation of this vector in hemophilia B patients.

CHROMOSOMAL LOCALIZATION AND CATALYTIC PROPERTIES OF THE RECOMBINANT ALPHA SUBUNIT OF HUMAN LYMPHOCYTE METHIONINE ADENOSYLTRANSFERASE

Human lymphocyte methionine adenosyltransferase (HuLy MAT) consists of heterologous subunits α and β. The cDNA sequence of the α subunit of HuLy MAT from Jurkat leukemic T cells was identical to that of the human kidney α subunit and highly homologous to the sequence of the extrahepatic MAT from other sources. The 3′-untranslated sequence was found to be highly conserved, suggesting that it may be important in regulating the expression of MAT. The extrahepatic α subunit of MAT was found to be expressed also in human liver, and no differences were found in the sequence of the α subunit from normal and malignant T cells. The sequence of two unspliced introns found in the cDNA clones from the Jurkat library enabled us to isolate genomic clones harboring the human extrahepatic α subunit gene and to localize it to the centromere on chromosome arm 2p, an area that corresponds to band 2p11.2. Expression of the α subunit cDNA in Escherichia coli yielded two peptides with the immunoreactivity and mobilities of authentic α/α‘ subunits from HuLy. The K of the recombinant α subunit was 80 μM, which is 20-fold higher than found for the (αα′) β holoenzyme purified from leukemic lymphocytes and 4-10-fold higher than found for the normal lymphocyte enzyme. The data suggest that the α/α‘ subunits mediate the enzyme catalytic activity and that the β subunit may be a regulatory subunit of extrahepatic MAT.

Targetable kinase gene fusions in high risk B-ALL: a study from the Children's Oncology Group

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by genomic alterations that activate cytokine receptor and kinase signaling. We examined the frequency and spectrum of targetable genetic lesions in a retrospective cohort of 1389 consecutively diagnosed patients with childhood B-lineage ALL with high-risk clinical features and/or elevated minimal residual disease at the end of remission induction therapy. The Ph-like gene expression profile was identified in 341 of 1389 patients, 57 of whom were excluded from additional analyses because of the presence of BCR-ABL1 (n = 46) or ETV6-RUNX1 (n = 11). Among the remaining 284 patients (20.4%), overexpression and rearrangement of CRLF2 (IGH-CRLF2 or P2RY8-CRLF2) were identified in 124 (43.7%), with concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) identified in 63 patients (50.8% of those with CRLF2 rearrangement). Among the remaining patients, using reverse transcriptase polymerase chain reaction or transcriptome sequencing, we identified targetable ABL-class fusions (ABL1, ABL2, CSF1R, and PDGFRB) in 14.1%, EPOR rearrangements or JAK2 fusions in 8.8%, alterations activating other JAK-STAT signaling genes (IL7R, SH2B3, JAK1) in 6.3% or other kinases (FLT3, NTRK3, LYN) in 4.6%, and mutations involving the Ras pathway (KRAS, NRAS, NF1, PTPN11) in 6% of those with Ph-like ALL. We identified 8 new rearrangement partners for 4 kinase genes previously reported to be rearranged in Ph-like ALL. The current findings provide support for the precision-medicine testing and treatment approach for Ph-like ALL implemented in Childrens Oncology Group ALL trials.

Transgenesis in Xenopus using the Sleeping Beauty transposon system.

Transposon‐based integration systems have been widely used for genetic manipulation of invertebrate and plant model systems. In the past decade, these powerful tools have begun to be used in vertebrates for transgenesis, insertional mutagenesis, and gene therapy applications. Sleeping Beauty (SB) is a member of Tc1/mariner class of transposases and is derived from an inactive form of the gene isolated from Atlantic salmon. SB has been used extensively in human cell lines and in whole animal vertebrate model systems such as the mouse, rat, and zebrafish. In this study, we describe the use of SB in the diploid frog Xenopus tropicalis to generate stable transgenic lines. SB transposon transgenes integrate into the X. tropicalis genome by a noncanonical process and are passed through the germline. We compare the activity of SB in this model organism with that of Tol2, a hAT (hobo, Ac1, TAM)‐like transposon system. Developmental Dynamics 238:1727–1743, 2009.

The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis

SUMMARY In the developing retina, multipotent neural progenitors undergo unidirectional differentiation in a precise spatiotemporal order. Here we profile the epigenetic and transcriptional changes that occur during retinogenesis in mice and humans. Although some progenitor genes and cell cycle genes were epigenetically silenced during retinogenesis, the most dramatic change was derepression of cell type–specific differentiation programs. We identified developmental stage–specific super-enhancers and showed that most epigenetic changes are conserved in humans and mice. To determine how the epigenome changes during tumorigenesis and reprogramming, we performed integrated epigenetic analysis of murine and human retinoblastomas and induced pluripotent stem cells (iPSCs) derived from murine rod photoreceptors. The retinoblastoma epigenome mapped to the developmental stage when retinal progenitors switch from neurogenic to a terminal patterns of cell division. The epigenome of retinoblastomas was more similar to that of normal retina than was that of retina-derived iPSCs, and we identified retina-specific epigenetic memory.

Retinal Cell Type DNA Methylation and Histone Modifications Predict Reprogramming Efficiency and Retinogenesis in 3D Organoid Cultures

SUMMARY Diverse cell types can be reprogrammed into pluripotent stem cells by ectopic expression of Oct4 (Pou5f1), Klf4, Sox3, and Myc. Many of these induced pluripotent stem cells (iPSCs) retain memory, in terms of DNA methylation and histone modifications (epigenetic memory), of their cellular origins, and this may bias subsequent differentiation. Neurons are difficult to reprogram, and there has not been a systematic side-by-side characterization of reprogramming efficiency or epigenetic memory across different neuronal subtypes. Here, we compare reprogramming efficiency of five different retinal cell types at two different stages of development. Retinal differentiation from each iPSC line was measured using a quantitative standardized scoring system called STEM-RET and compared to the epigenetic memory. Neurons with the lowest reprogramming efficiency produced iPSC lines with the best retinal differentiation and were more likely to retain epigenetic memory of their cellular origins. In addition, we identified biomarkers of iPSCs that are predictive of retinal differentiation.

CRLF2-Positive B-Cell Acute Lymphoblastic Leukemia in Adult Patients

Objectives Cytokine receptor-like factor 2 ( CRLF2 ) rearrangement is found in approximately 50% of pediatric Ph-like B-cell acute lymphoblastic leukemia (B-ALL), and around 50% of CRLF2 + cases harbor JAK mutations. We analyzed CRLF2 expression and studied its correlation with CRLF2 rearrangement in adult patients with B-ALL. Methods Multiparameter flow cytometry (MFC) was performed consecutively in 126 patients. Results CRLF2 overexpression was detected in 30 (27%) patients, 28 (41%) of 69 patients with B-ALL not otherwise specified, 14 (21%) of 67 untreated patients, and 16 (27%) of 59 patients with relapsed B-ALL, with the highest among Hispanic patients (25/55, 45%). Of CRLF2+ cases, 21 (100%) of 21 cases showed CRLF2 rearrangement by fluorescence in situ hybridization, preferentially involving IGH@CRLF2 (15/15). The entire coding region of JAK2 was sequenced in 14 patients with CRLF2+ B-ALL, and nine (64%) were positive for JAK2 mutations. Conclusions MFC allows a rapid, inexpensive, and reliable detection of B-ALL with CRLF2 rearrangement that would further facilitate testing for JAK2 mutations for targetable therapy.

ATM-deficiency increases genomic instability and metastatic potential in a mouse model of pancreatic cancer

Germline mutations in ATM (encoding the DNA-damage signaling kinase, ataxia-telangiectasia-mutated) increase Familial Pancreatic Cancer (FPC) susceptibility, and ATM somatic mutations have been identified in resected human pancreatic tumors. Here we investigated how Atm contributes to pancreatic cancer by deleting this gene in a murine model of the disease expressing oncogenic Kras (KrasG12D). We show that partial or total ATM deficiency cooperates with KrasG12D to promote highly metastatic pancreatic cancer. We also reveal that ATM is activated in pancreatic precancerous lesions in the context of DNA damage and cell proliferation, and demonstrate that ATM deficiency leads to persistent DNA damage in both precancerous lesions and primary tumors. Using low passage cultures from primary tumors and liver metastases we show that ATM loss accelerates Kras-induced carcinogenesis without conferring a specific phenotype to pancreatic tumors or changing the status of the tumor suppressors p53, p16Ink4a and p19Arf. However, ATM deficiency markedly increases the proportion of chromosomal alterations in pancreatic primary tumors and liver metastases. More importantly, ATM deficiency also renders murine pancreatic tumors highly sensitive to radiation. These and other findings in our study conclusively establish that ATM activity poses a major barrier to oncogenic transformation in the pancreas via maintaining genomic stability.

Abstract 4729: Frequency of actionable gene fusions in patients with Philadelphia chromosome-like (Ph-like) B-acute lymphoblastic leukemia (ALL): A retrospective study from the Children's Oncology Group (COG)

Introduction: While cure rates exceed 80%, many children with B-ALL still relapse. Many of these patients (pts) display a Ph-like gene expression profile (GEP), but lack canonical BCR-ABL1 fusion. We have identified alternate kinase fusions in Ph-like ALL that induce cell proliferation sensitive to tyrosine kinase inhibitors (TKI) (Roberts, NEJM 2014). We report retrospective analyses of 1390 B-ALL pts, 885 NCI high risk and 505 standard risk B-ALL pts with elevated minimal residual disease. Methods: Cases were screened using an 8-gene Taqman low-density array (LDA) PCR assay to identify the Ph-like GEP (Harvey, ASH 2013). Ph-like cases with elevated CRLF2 expression were tested for CRLF2 rearrangement (CRLF2-R; P2RY8-CRLF2 by Taqman PCR on the LDA card and IGH-CRLF2 by FISH). JAK mutations in CRLF2-R cases were tested by Sanger sequencing. Ph-like cases without CRLF2-R were tested for previously identified kinase fusions involving ABL1, ABL2, CSF1R, JAK2, NTRK3, and PDGFRB by RT-PCR. Ph-like cases without detected fusions underwent RNA-sequencing, either using standard Illumina library preparation or a customized kinome capture kit (Agilent). Results: 339 (24%) pts were Ph-like. BCR-ABL1 (N = 45) and ETV6-RUNX1 (N = 11) were excluded from further analyses, as the former already receives TKI therapy and ETV6-RUNX1 ALL lacks targetable kinase fusions (unpublished). Of the remaining 283 Ph-like cases, 153 were CRLF2high (defined by CRLF2 expression levels on the LDA card). 61 (40%) had P2RY8-CRLF2 fusion, and of the remaining 91 CRLF2high cases, 56 of 69 tested had CRLF2-R (55 to IGH, 1 to an unknown partner). Thus, 117/130 (90%) CRLF2high Ph-like cases had a documented CRLF2 genomic lesion and 52 (44%) of these had a JAK mutation. Of the 130 Ph-like CRLF2low cases, 61 (47%) had a previously reported targetable TK fusion identified by RT-PCR, kinome capture or RNA sequencing. These included: 38 ABL class fusions (17 ABL1, 5 ABL2, 3 CSF1R, 13 PDGFRB) sensitive to imatinib/dasatinib; 14 JAK2 and 8 EPOR fusions sensitive to ruxolitinib; and 1 NTRK3 fusion sensitive to crizotinib. Nine cases had known fusions with new alternate breakpoints, and an additional 9 cases had fusions of novel N-terminal partners with known actionable C-terminal kinase genes. RNA sequencing identified 8 cases with IGH-EPOR fusions not previously captured by the kinome assay, indicating the cryptic and complex nature of this rearrangement. Conclusion: Almost half of Ph-like pediatric B-ALL pts lacking CRLF2-R harbor altered TKs with compelling pre-clinical data that they are likely amenable to targeted therapy using FDA-approved TKIs. The COG will start real-time screening with this algorithm in 2015 and allocate pts with ABL class fusions to treatment with chemotherapy plus dasatinib. Citation Format: Shalini C. Reshmi, Richard C. Harvey, Amy Smith, I-Ming Chen, Marc Valentine, Yu Liu, Yongjin Li, Jinghui Zhang, Kathryn G. Roberts, Ying Shao, John Easton, Debbie Payne-Turner, Meenakshi Devidas, Nyla Heerema, Andrew J. Carroll, Elizabeth A. Raetz, Michael J. Borowitz, Brent L. Wood, Anne L. Angiolillo, Michael M. Burke, Wanda L. Salzer, Patrick A. Zweidler-McKay, Karen R. Rabin, William L. Carroll, Mignon L. Loh, Stephen P. Hunger, Charles G. Mullighan, Cheryl L. Willman, Julie M. Gastier-Foster. Frequency of actionable gene fusions in patients with Philadelphia chromosome-like (Ph-like) B-acute lymphoblastic leukemia (ALL): A retrospective study from the Children9s Oncology Group (COG). [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 4729. doi:10.1158/1538-7445.AM2015-4729

Genomic and outcome analyses of Ph-like ALL in NCI standard-risk patients: a report from the Children’s Oncology Group

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL; BCR-ABL1-like ALL) in children with National Cancer Institute (NCI) intermediate- or high-risk (HR) ALL is associated with poor outcome. Ph-like ALL is characterized by genetic alterations that activate cytokine receptor and kinase signaling and may be amenable to treatment with tyrosine kinase inhibitors. The prevalence, outcome, and potential for targeted therapy of Ph-like ALL in standard-risk (SR) ALL is less clear. We retrospectively analyzed a cohort of 1023 SR childhood B-ALL consecutively enrolled in the Childrens Oncology Group AALL0331 clinical trial. The Ph-like ALL gene expression profile was identified in 206 patients, and 67 patients with either BCR-ABL1 (n = 6) or ETV6-RUNX1 (n = 61) were excluded from downstream analysis, leaving 139 of 1023 (13.6%) as Ph-like. Targeted reverse transcription polymerase chain reaction assays and RNA-sequencing identified kinase-activating alterations in 38.8% of SR Ph-like cases, including CRLF2 rearrangements (29.5% of Ph-like), ABL-class fusions (1.4%), JAK2 fusions (1.4%), an NTRK3 fusion (0.7%), and other sequence mutations (IL7R, KRAS, NRAS; 5.6%). Patients with Ph-like ALL had inferior 7-year event-free survival compared with non-Ph-like ALL (82.4 ± 3.6% vs 90.7 ± 1.0%, P = .0022), with no difference in overall survival (93.2 ± 2.4% vs 95.8 ± 0.7%, P = .14). These findings illustrate the significant differences in the spectrum of kinase alterations and clinical outcome of Ph-like ALL based on presenting clinical features and establish that genomic alterations potentially targetable with approved kinase inhibitors are less frequent in SR than in HR ALL.