#381 HIV Integrates in Regulating Cell Cycle, DNA Damage, and Viral Transport

1a 2a 1 2 1 1 1 1 1b 2,3b Presenter: Sulggi A. Lee , Kevin Haworth , Cassandra Thanh , Lauren E. Schefter, Zachary K. Norgaard , Christopher Pilcher , Frederick Hecht , Jeffrey N. Martin , Steven G. Deeks , Timothy Henrich , and Hans-Peter Kiem Sulggi Lee, MD PhD 995 Potrero Avenue 1 University of California San Francisco, CA, USA; 2Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 3University of Washington, Seattle, WA, USA. a Co-first authors and b co-senior authors. Bldg 80, Box 0874 San Francisco, CA 94110 (415) 735-5127 [email protected] Background Results Delayed ART Initiation is #Unique IS is Inversely Correlated Potential Effects of Timing of ART and Insertion in Cell Cycle Genes on Latency Descriptive Characteristics associated with ↓ # Unique IS with Viral Transcription • HIV integration is a key step in the viral replication cycle. N=22, Spearman R = -0.44, P=0.039 Spearman R = -0.51, P=0.0075 N = 26 A. 10000 100000 • Prior in vivo studies disagree on whether HIV preferentially B. integrates in oncogenes leading to clonal proliferation (Maldarelli et. Male (%)a 25 (96%)

al., Science 2014; Symons, et. al., IAS Conference, Paris 2017, Age (years) 46 (43 – 54) 10000 1000 HIV Antigen #MOAA0104). Nadir CD4+ T cell count (cells/mm3) 368 (252 – 500) CD4+ cells) T 6 Pre-ART maximum HIV RNA (log10copies/mL) 4.6 (4.0 – 5.3) Methods 100 1000 Timing of ART initiation (years from HIV infection) 3.0 (1.3 – 4.2) (copies/10 • HIV integration site (IS) analysis from 26 HIV+ antiretroviral therapy Duration of ART suppression (years) 5.1 (3.6 – 7.6) (N) Sites Integration Unique Cell-associated unspliced HIV-1 RNA HIV-1 unspliced Cell-associated (ART)-suppressed participants from the UCSF SCOPE cohort was 10 100 Proximal CD4+ T cell count (cells/mm3) 647 (550 – 886) 0 2 4 6 8 10 100 1000 10000 performed and compared to HIV IS analysis from a novel humanized mouse model challenged with a CCR5-tropic virus. Abbreviations: antiretroviral therapy (ART). Median and interquartile ranges shown. a Frequency Timing of ART (years) Unique Integration Sites (N) and percent shown. Delayed ART may promote selection of HIV insertions may promote latency, • Cryopreserved DNA from PBMCs were enriched for CD4+ T cells, clones over time that favor persistence. reflected as reduced viral transcription. HIV Preferentially Inserts into Delayed ART Initiation Clones Harboring HIV in Non-Random Pattern of HIV Insertion in Human Regions of Accessible Chromatin Favors Accumulation of Genes Promoting Latency and quantified cell-associated HIV total DNA and unspliced RNA by Regression estimates for the above: There was a 1.0-fold decrease in unique integration sites per fold-increase of years delay in ART and/or Active Transcription Cells with Defective Virus are Selected for Over Time and Humanized Mouse Genomes initiation, P=0.050 (A), and a 0.5-fold decrease in copies of HIV RNA for each fold-increase in unique integration sites, P=0.0051 (B). qPCR. These estimates did not change considerably after adjustment for total HIV DNA, age, nadir CD4+ T cell count, or pre-ART HIV RNA.

Humanized Mouse HIV Dataset 225 Ini@al HIV Infec@on ART Ini@a@on Long-Term ART Suppression Black Data Set 200 • HIV IS were identified using nested PCR (Illumina MiSeq) with 175 150 Mouse Bin Start Bin End Total IS # 125 100 Top 10 Frequently Observed Genes in hs19 49550001 49575000 220 primers for HIV genome and linker sequences, as previously 75 hs16 3075001 3100000 213 50 25 hs19 8450001 8475000 210 10 26 HIV+ ART-Suppressed Participants Conclusions described (Beard et. al., Methods Mol Biol 2014). hs19 1075001 1100000 208 20 hs16 2250001 2275000 196 30 a b c d e hs17 81875001 81900000 195 40 Summary N Unique IS Oncogene Cell Cycle Regulation hs16 88575001 88600000 192 50 • We observed consistent integration site patterns hs20 63700001 63725000 191 60 hs11 66100001 66125000 185 Regulates spindle disassembly during mitosis and STEP 1: Randomly shear hs8 144300001 144325000 181 NPLOC4 type I IFN to promote degradation of RIG-I 19 116 N Y between HIV+ participants and in vivo humanized mice. DNA samples from Human infected cell popula=ons LTR-genome junc.on MROH1 Involved in actin-regulated late vesicular sorting 18 202 N N • HIV insertion may not be random but may STEP 2: Ligate linker segment FANCA DNA repair post-replication, cell cycle checkpoint 16 115 Y Y Human HIV Samples preferentially occur in specific regions of genome Red Data Set PPP6R2 Essential mitotic kinase, responds to TNF-alpha 16 91 N N Chromosome Bin Start Bin End Total IS # hs16 200001 225000 58 (e.g., open chromatin sites, suggested by pathway- hs1 875001 900000 55 MIR1268A Unknown function microRNA 15 74 N N STEP 3: Nested PCR amplifica=on hs9 136800001 136825000 53 based analysis results). hs8 144225001 144250000 52 DNMT1 Essential in DNA methylation 15 58 N N HIV LTR primers Linker primers hs8 144475001 144500000 52 hs8 144200001 144225000 49 hs16 750001 775000 47 Contains caspase activation recruitment domain, • The majority of insertions occurred in cell cycle genes, hs8 144150001 144175000 47 QRICH1 may be involved in inflammation, apoptosis 14 54 N N 300-800base pair hs19 49600001 49625000 46 bands extracted which may serve to promote viral latency hs8 144275001 144300000 46 PTK2 Critical for cell growth and signal transduction 14 68 N Y HIV integration site analysis of 26 HIV+ patient samples (red Circos plot) and humanized mouse Role in mitotic progression, binds to HIV-1 long • A higher number of unique integration sites samples (black Circos plot) demonstrate similar genomic loci and frequencies of integration. HSF1 terminal repeat to reactivate transcription 14 56 N Y Concentric rings denote axis increments of 15 integration sites. STEP 4: Band extrac=on correlated with lower levels of residual viral and purifica=on Controls cell growth, apoptosis, development, and transcription AXIN1 enhances TGF-b signaling 13 27 Y Y General Qmeline Humanized Mouse Model aSummary adapted from Gene Cards (www..org). bNumber of participants. cTotal number of unique integration sites within the specific gene. Blood Collections HIV Challenge Necropsy dOncogene defined as genes in which a single allele variant contribute to oncogenesis; the COSMIC: Cancer Gene database • Integrations were enriched in genes associated monitor engraftment Tissue Analysis STEP 5: Illuminna MiSeq Analysis (http://cancer.sanger.ac.uk/cosmic). eGenes involved in cell cycle regulation as defined by the database (http://www.geneontology.org). CD34+ with cell cycle, response to DNA damage, and viral cells • Sequences were aligned using UCSC Genome BLAT (human hg38 injected transport. 1-3 day neonate Top 5 Biologic Pathways Enriched for HIV Insertion Genes HIV Challenge assembly). 108 7 10 GO ID GO Biological Process Fold-Enrichment P-Value Example of flow analysis gaQng This work was 106 • Spearman correlations, multivariate negative binomial (for cell- Peripheral Blood mCD45+ vs. hCD45+ CD20 B-cell vs CD3 T-cell CD8 vs CD4 T-cell GFP Modified Cells 105 supported by: GO:0031572 G2 DNA damage checkpoint 2.68 9.6 x 10-3 associated HIV RNA or DNA), and log-transformed multivariate 104 U19 AI096109 103 GO:0051081 Nuclear envelope disassembly 2.38 1.2 x 10-2 K23GM112526 regression (for unique number of HIV integration sites) were copies/mL viralHIV RNA 102 R21 AI110154 16 18 20 22 24 26 28 GO:0030397 Membrane disassembly 2.38 1.2 x 10-2 SSC GFP CD8 CD20 performed to link clinical, HIV reservoir, and HIV IS data. FSC mouseCD45 humanCD45 CD3 CD4 CD45 Age of Mice (weeks) P30 MH59037 GO:0075733 Intracellular transport of virus 2.29 9.7 x 10-3 Eight neonatal mice were engrafted with human CD34+ hematopoietic stem and progenitor cells, giving rise to AI069994 • Gene set enrichment analyses were performed using COSMIC human CD3+CD4+ T cells in the peripheral blood. These cells were capable of initiating HIV+ infection, resulting GO:0032508 DNA duplex unwinding 2.26 2.2 x 10-4 UL1 RR024131 Cancer Gene and Gene Ontology Consortium databases. in detectable plasma HIV RNA in the peripheral circulation and multiple lymphoid compartments. Infected cells Analyses performed using Panther version 11 (http://pantherdb.org/geneListAnalysis.do; Mi et. al., Nuc Acid Res 2017). amfAR 106710-40 were then analyzed at necropsy for integration site studies (Haworth et. al., Mol Ther Methods Clin Dev 2017).