Trevor Tivey

Rapid Evolution of HIV-1 to Functional CD8+ T Cell Responses in Humanized BLT Mice

Humanized BLT mice accurately develop human HIV-specific CD8+ T cell responses capable of rapidly selecting for CTL escape mutations. Mirror, Mirror One limitation of using animal models of disease is that there’s no magic mirror to tell you which one best reflects human disease. Instead, most animal disease models mimic some aspects of the human condition, but may not recapitulate the disease in its entirety. This limitation is especially true for HIV infection because the virus does not naturally infect mice—the model of choice for biomedical research. Attempts to “humanize” immunodeficient mice through grafting of human immune cells may reconfigure the mouse from a distorting funhouse mirror into a well-lit vanity one. Now, Dudek et al. use humanized BLT (brain, liver, thymus) mice to study human immune responses to HIV. The authors found that HIV-1–specific immune responses in BLT mice mimicked those in humans in terms of specificity, kinetics, and dominant target. Importantly, HIV adapted to the immune responses in these mice just as it does in humans, evolving rapidly to escape from the selective pressure. Indeed, an HLA allele that is protective in humans induced similar protective immune responses in these mice. Although no animal model may perfectly reflect human disease, for HIV infection, humanized BLT mice may be one of the fairest of them all. The development of mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT (bone marrow, liver, thymus) mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exist regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. We infected humanized BLT mice with HIV-1 as a model pathogen and characterized HIV-1–specific immune responses and viral evolution during the acute phase of infection. HIV-1–specific CD8+ T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics, and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication and restricted the same CD8+ T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen–specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the use of a small-animal model for human pathogens.

Graft versus Host Disease in the Bone Marrow, Liver and Thymus Humanized Mouse Model

Mice bearing a “humanized” immune system are valuable tools to experimentally manipulate human cells in vivo and facilitate disease models not normally possible in laboratory animals. Here we describe a form of GVHD that develops in NOD/SCID mice reconstituted with human fetal bone marrow, liver and thymus (NS BLT mice). The skin, lungs, gastrointestinal tract and parotid glands are affected with progressive inflammation and sclerosis. Although all mice showed involvement of at least one organ site, the incidence of overt clinical disease was approximately 35% by 22 weeks after reconstitution. The use of hosts lacking the IL2 common gamma chain (NOD/SCID/γc−/−) delayed the onset of disease, but ultimately did not affect incidence. Genetic analysis revealed that particular donor HLA class I alleles influenced the risk for the development of GVHD. At a cellular level, GVHD is associated with the infiltration of human CD4+ T cells into the skin and a shift towards Th1 cytokine production. GVHD also induced a mixed M1/M2 polarization phenotype in a dermal murine CD11b+, MHC class II+ macrophage population. The presence of xenogenic GVHD in BLT mice both presents a major obstacle in the use of humanized mice and an opportunity to conduct preclinical studies on GVHD in a humanized model.

Mutations in G protein β subunits promote transformation and kinase inhibitor resistance

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4–5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Abstract PR07: Novel oncogenic mutations in the beta subunit of heteromeric G-proteins identified by functional cDNA library screening.

Although next-generation sequencing can delineate the genetic alterations within a primary tumor specimen, it can be difficult to distinguish the small number of driver mutations from the large number of passenger mutations. To overcome this issue, we developed a system for identifying oncogenic alterations directly from tumor cells. In this system, retroviral cDNA libraries built from cancer cell lines and directly from primary cancer samples are transduced into BaF3 cells, an IL3-dependent B cell line. Transformation by oncogenes promotes IL3-independent survival, allowing for the isolation of individual transformed clones and sequencing of the integrated cDNA. In the past, we identified CRLF2 as a novel oncogene in acute lymphoblastic leukemia (Yoda et al. PNAS 2010). We have improved the method and demonstrated 100% sensitivity for isolating well-characterized oncogenes, including EGFR, HER2, RAS and ALK (Shindoh et al. PLoS One 2012). Recently, we isolated a mutated GNB1 K89E allele from a cDNA library generated from a primary blastic plasmacytoid dendritic cell neoplasm (BPDCN). BPDCN is a rare and aggressive leukemia with a dismal prognosis. GNB1 encodes the beta subunit of the heterotrimeric G-protein, a binding complex that transduces signals from G-protein coupled receptors to multiple downstream pathways. Gain-of-function mutations have been reported in alpha subunits of the G-protein, however, the contributions of beta subunits to cancer remains undefined. To investigate downstream signaling from GNB1 K89E, we performed gene expression profiling and mass spectrometry (MS)-based phosphoproteomics and found significant activation of RAS/MAPK and PI3K/AKT pathways in GNB1 K89E-expressing cells compared to isogenic cells expressing wild-type GNB1. To target GNB1 K89E signaling, we screened kinase inhibitors using a multiplex panel of small molecules and found selective sensitivity of GNB1 K89E cells to MEK and pan-PI3-kinase inhibitors. Next, we transduced GNB1 alleles into bone marrow cells from Cdkn2a-deficient mice and transplanted into wild-type recipient mice. Within 4 months after transplantation, all mice (n=10) that received bone marrow transduced with GNB1 K89E developed a lethal dendritic cell malignancy, confirming the transforming effects of GNB1 K89E in vivo. A search of published cancer mutations identified four cases with GNB1 I80T/N in chronic lymphocytic leukemia or B-cell lymphomas, five cases with GNB1 K57E/T in myeloid malignancies, one case of GNB1 K89E in acute lymphoblastic leukemia, and two cases with GNB2 M101T/V in ovarian cancer. All of these alleles promoted GM-CSF-independent growth in human TF1 cells. Interestingly, the mutated codons are all located on the GNB1 molecular surface that is critical for interactions between GNB1 and both alpha subunits and downstream effectors. Immunoprecipitation followed by MS demonstrated that GNB1 K89E and I80T mutants failed to bind inhibitory G alpha subunits GNAI2 and GNAI3 as well as GNA11 that are bound by wild-type GNB1. Thus, gain-of-function mutations in G-protein beta subunits occur across a broad range of malignancies, can drive in vivo transformation, and activate targetable downstream kinases by modifying essential interactions with partner proteins. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR07. Citation Format: Akinori Yoda, Guillaume Adelmant, Nobuaki Shindoh, Bjoern Chapuy, Yuka Yoda, Oliver Weigert, Nadja Kopp, Shuo-Chieh Wu, Sunhee S. Kim, Huiyun Liu, Trevor Tivey, Jeffrey W. Tyner, Jason Gotlib, Michael W. Deininger, Shannon Turley, Jarrod A. Marto, Andrew A. Lane, David M. Weinstock. Novel oncogenic mutations in the beta subunit of heteromeric G-proteins identified by functional cDNA library screening. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR07.

Parp3 promotes long-range end joining in murine cells

Significance Chromosomal rearrangements are early and essential events in the formation of many tumors. Two distinct end-joining pathways, classic and alternative nonhomologous end joining, can mediate rearrangement formation. Previous studies have shown that genetic factors mediating rearrangements differ significantly between mouse and human cells. Here we show that poly(ADP)ribose polymerase 3 (Parp3) uniquely promotes chromosomal rearrangements in both species. Using next-generation sequencing of rearrangement junctions, we investigated the mechanistic contribution of Parp3 and a closely related enzyme, Parp1, that is also known to promote rearrangements in murine cells. We find differences in the phenotypes of rearrangements in cells lacking Parp3, Parp1, or both, suggesting that these enzymes promote rearrangements through distinct mechanisms and providing insight into this essential mechanism of tumorigenesis. Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class–switch recombination in primary B cells, and inversions in tail fibroblasts that generate Eml4–Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4–Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

Corrigendum: PARP3 is a promoter of chromosomal rearrangements and limits G4 DNA

This corrects the article DOI: 10.1038/ncomms15110.

Abstract 433: Triplication of HMGN1 promotes B cell acute lymphoblastic leukemia (B-ALL) through suppression of H3K27me3

Our goal is to identify oncogenic loci in regions of recurrent DNA copy number alterations in cancer. Constitutional trisomy 21 (Down syndrome) carries a 20-fold increased risk of B-ALL, and chr.21 gains are the most common acquired aneuploidy in B-ALL. Interstitial amplification in the chr.21q22 region (iAMP21) is also a recurrent finding in B-ALL and carries a poor prognosis. However, the gene(s) on chr.21 responsible for this association remain unclear. We studied the Ts1Rhr mouse, which carries germline triplication of 31 genes homologous to human chr.21q22. Chr.21q22 triplication was sufficient to promote B cell autonomous self-renewal and maturation defects, and cooperated with BCR-ABL or CRLF2 with JAK2 R683G to accelerate leukemogenesis. Chr.21q22 triplication also resulted in histone H3K27 hypomethylation at gene promoters, and the expression signature of triplicated B cells was enriched for genes targeted by polycomb repressor complex 2 (PRC2), which trimethylates H3K27. Thus, chr.21q22 triplication may deregulate B cell development by causing H3K27 hypomethylation at genes critical for progenitor cell growth. In support of this hypothesis, pharmacologic inhibition of PRC2 function was sufficient to confer self-renewal in wild-type B cells, while inhibition of H3K27 demethylases blocked self-renewal induced by chr.21q22 triplication. In three independent B-ALL cohorts, PRC2/H3K27 gene signatures distinguished leukemias with +21 from those without, validating the same biology in human disease. One of the 31 triplicated genes, HMGN1, encodes a nucleosome binding protein known to modulate chromatin structure and facilitate transcriptional activation. When we overexpressed HMGN1 in BaF3 proB cells, H3K27me3 decreased proportionally to the level of overexpression. We next knocked down each of the 31 triplicated genes with lentivirally-expressed shRNAs (5 per gene) and assessed the effects on growth of Ts1Rhr and wild-type primary B cells. Strikingly, Hmgn1 was the top scoring gene and all 5 hairpins targeting Hmgn1 were depleted in the assay. Finally, we studied transgenic mice (HMGN1_OE) that overexpress human HMGN1 (∼2-fold total overexpression). HMGN1_OE mice had a defect in B cell maturation, increased proB colony forming capacity, and a transcriptional signature overlapping with that of triplication of all 31 Ts1Rhr genes. In a bone marrow transplant model driven by BCR-ABL, recipients of HMGN1_OE bone marrow developed B-ALL with decreased latency (median 33 days vs not reached) and increased penetrance (17/18 vs 4/17 mice died by 80 days; leukemia-free survival difference P Citation Format: Andrew A. Lane, Bjoern Chapuy, Charles Y. Lin, Trevor Tivey, Hubo Li, Elizabeth Townsend, Diederik van Bodegom, Tovah A. Day, Shuo-Chieh Wu, Huiyun Liu, Akinori Yoda, Gabriela Alexe, Anna Schinzel, Timothy J. Sullivan, Sebastien Malinge, Jordan Taylor, Kimberly Stegmaier, Jacob Jaffe, Michael Bustin, Geertruy te Kronnie, Shai Izraeli, Marian Harris, Kristen Stevenson, Donna Neuberg, Lewis B. Silverman, Steven E. Sallan, James E. Bradner, William C. Hahn, John D. Crispino, David Pellman, David M. Weinstock. Triplication of HMGN1 promotes B cell acute lymphoblastic leukemia (B-ALL) through suppression of H3K27me3. [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 433. doi:10.1158/1538-7445.AM2014-433