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Introduction: Graft-versus-host-disease (GVHD) arises from the inflammatory cascade triggered by alloreactive T cell following allogeneic hematopoietic cell transplantation (allo-HCT). The tissues most frequently involved by GVHD include the small and large intestines, skin, and liver. Prior studies of T cell receptor (TCR) sequencing in diagnostic biopsies and blood have identified dominant T cell clones in GVHD-affected tissues that were not abundant in circulation or shared across multiple patients, but this has not been studied in sites poorly accessible to biopsy nor in lymphoid tissues in humans. We hypothesized that the GVHD-affected tissues have distinct TCR repertoires reflecting the differential expression of allo-antigens at different anatomic sites. Methods: We performed rapid autopsies on patients whose post allo-HCT course was complicated by GVHD to profile the TCR repertoire in tissues inaccessible to biopsy. Tissues were obtained from seven patients (HLA-identical allografts with a variety of graft sources and GVHD-prophylaxis regimens), all with active GVHD and/or on immunosuppression for GVHD control. Spleen, liver, skin, and multiple sites along the gastrointestinal (GI) tract were sampled, when possible, from the esophagus to the rectum. When available, blood and bone marrow mononuclear cells were also viably preserved. T cell receptors were sequenced from 38 different snap-frozen tissues from five patients via genomic DNA based next-generation sequencing of the TCR-beta CDR3 (ImmunoSeq, Adaptive Biotechnologies). Four out of five subjects sequenced had skin and GI GVHD, one only GI. In parallel, TCRs were sequenced from GVHD-affected tissues from a major and minor mouse model of GVHD: BALB/c 7-14 days after HCT with C57BL/6 T cells and C57BL/6 mice > 30 days after HCT with LP/J T cells. Results: Sequences representing 264,678 productive TCRs were recovered from the human autopsy samples with over 100 unique clones for nearly all tissues (mean 1539; standard deviation 1626). We found virtually no TCR clones defined by nucleotide sequence shared across patients, even in the context of shared HLA haplotypes (4/5 patients shared HLA-A*02-01). This is consistent with prior studies of diagnostic biopsies describing a paucity of clones shared across patients. We observed greater repertoire overlap between sites within the GI tract compared to other tissues in a given patient, as measured by the Jensen Shannon Diversity (JSD) index, especially compared to the skin, another GVHD-affected tissue. Despite differences in global repertoires across tissues, some clones were shared across all samples for a given patient, with at least one clone present among the top twenty clones by frequency for each tissue with sufficient sampling. A strikingly similar pattern of repertoire sharing across tissues was observed in the TCR repertoires of a major and minor mouse model of GVHD. While tissues within the GI tract and mesenteric lymph nodes shared the most clones in the mice, there were also dominant clones shared across tissues outside the GI tract. Individual mice with GVHD had highly divergent repertoires from each other in spite of the markedly controlled setting including inbred mice with the same donor T cell pool for each transplant, consistent with the notion that T cells that mediate GVHD may be present at very low frequency in the pool of naïve T cells in the allograft. Conclusion: This work characterizes T cell repertoire in GVHD in human tissues that have not been previously available, including lymphoid tissues and small intestine samples. Combined with the mouse data, this study reveals the relationships between TCR repertoires across different tissues, highlighting the striking diversity of clones driving the GVHD process across tissues and individuals. Although some even dominant clones are shared across all tissues within an individual, each sampled site had a tissue-specific clonal composition. While GVHD-directed therapy focuses primarily on the inhibition of the T cells themselves, additional attention must be devoted to understanding the pattern of target tissue-specific antigen expression in order to identify the key drivers of GVHD. Disclosures Giardina: Seres Therapeutics: Other: salary support. Slingerland:Seres Therapeutics: Other: salary support. Jenq:Kaleido Biosciences: Membership on an entity's Board of Directors or advisory committees; Karius Dx: Speakers Bureau; Merck: Consultancy; MicrobiomeDx: Consultancy; Prolacta: Consultancy; Seres Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: January 1, 2040. Giralt:Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; PFIZER: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees, Research Funding; Actinuum: Other: Advisory board, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Other: Advisory Board, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees; SPECTRUM Pharma: Membership on an entity's Board of Directors or advisory committees; Jensenn: Membership on an entity's Board of Directors or advisory committees, Research Funding; JAZZ Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; MILTENYI: Research Funding; TAKEDA: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; OMEROS: Research Funding. Perales:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Medigene: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding; Kite/Gilead: Honoraria, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Cidara Therapeutics: Other; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Servier: Membership on an entity's Board of Directors or advisory committees, Other; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Iacobuzio-Donahue:BMS: Research Funding. van den Brink:Seres Therapeutics: Consultancy, Patents & Royalties, Research Funding; DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Forty-Seven, Inc: Consultancy; Juno Therapeutics: Patents & Royalties; WindMIL Therapeutics: Honoraria; Rheos: Honoraria; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Magenta: Honoraria; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Peled:Seres Therapeutics: Patents & Royalties, Research Funding; Davolterra: Consultancy.

TO THE EDITOR: Recent advances in gene editing technologies using CRISPR/Cas9 allow precise genome editing at a site of interest and have accelerated human disease modeling and the development of corrective gene therapies for various genetic disorders.[1][1],[2][2] We adapted CRISPR/Cas9 editing of

Targeting the BCL11A erythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A +58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2 promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A +58 editing alone, 29.1%±3.9%; BCL11A +55 editing alone, 34.8±5.1%; HBG1/2 promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A +58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed. Together these results suggest that combined BCL11A +58 and +55 erythroid enhancer editing produces highly efficient on-target allelic disruption, erythroid-specific BCL11A downregulation, heightened HbF induction capacity compared to alternative approaches, preserved long-term multilineage engraftment potential by both human xenotransplant and rhesus autotransplant assays, and absence of evident genotoxicity, under clinically relevant SpCas9 RNP electroporation conditions. Disclosures No relevant conflicts of interest to declare.

Introduction: Individuals with sickle cell anemia (SCA) are at increased risk for deficits in multiple domains of neurocognitive functioning, including executive functions. In addition to assessing the effects of silent cerebral infarcts (SCI) and stroke on cognition, prior research has focused on hemoglobin and transcranial Doppler velocity as hemodynamic correlates. Recent studies have begun to use more precise measures of blood delivery to the brain (e.g., cerebral blood flow; CBF) to determine more sensitive indicators of cognitive risk prior to neurological injury. Nevertheless, empirical and meta-analytic findings suggest that these deficits increase with age, which can have broad impact on psychosocial functioning, including self-management and navigation through the transition from pediatric to adult medical care. This study aimed to assess brain volume as a mediator of the association between CBF and executive functioning in a sample of individuals with SCA. The secondary aim was to assess age as a moderator of hemodynamic and structural correlates of executive function. Methods: Children, adolescents, and young adults with SCA were enrolled prospectively. Each participant received a 3-Tesla non-contrast magnetic resonance imaging and magnetic resonance angiography of the brain, and a neurological examination by the study neurologist. Gray matter CBF was calculated from pseudo-continuous arterial spin labeling using the solution to the flow-modified Bloch equation after correcting for individual hematocrit. Three measures of brain volume were also computed from 3D-T1 images using Freesurfer version 7.1.1: total brain volume, gray matter volume, and white matter volume was calculated as the difference between the two. At a separate study visit, participants completed an age-appropriate Wechsler Working Memory Index (WMI). Pearson correlations assessed bivariate associations among variables, SPSS PROCESS macro was used to test gray matter volume as a mediator in the relation between CBF and working memory, and multiple linear regression analyses tested age as a moderator of the impact of CBF and brain volume on working memory. Results: Twenty-nine children and adolescents (ages 6 to 17 years) and 25 adults (ages 18 to 31 years) were enrolled. Five participants were excluded from analyses due to history of overt stroke that resulted in significant brain volume loss. Of 49 included participants, 20 had SCIs. Working memory was inversely correlated with age (r = -.30, p = .037) and CBF (r = -.36, p = .013), such that WMI decreased cross-sectionally with older age and higher CBF. Working memory was positively correlated with gray matter volume (r = .42, p = .002); however, it was not related to white matter volume (r = -.05, p = .715) or total brain volume (r = -.07, p = .642). Finally, patient age was positively correlated with CBF (r = .36, p = .014), but the association of age with gray matter volume did not reach statistical significance (r = -.27, p = .065). Analyses in Figure 1 show that although CBF and gray matter were directly related to working memory (path c and path b, respectively), gray matter volume did not mediate the association between CBF and working memory (path a*b). However, regression analyses (Table 1) showed that age moderated the association between gray matter volume and working memory, such that there was only a significant relation in children and adolescents. This association did not exist for young adults (Figure 2). Conclusions: Neurocognitive assessments has been cited as an important standard of care for children and adolescents with SCA. Given the increase in deficits with age, and the increase in mortality after transferring from pediatric to adult care, monitoring executive function abilities and potential impact on self-management should continue into adulthood. Findings from the current study provide preliminary evidence that cerebral hemodynamic compensation with elevated CBF may be insufficient to prevent gray matter volume loss in children and adolescents and decline in working memory ability. Some limitations of the current study include small sample size and whole brain gray and white matter volumes as opposed to specific regions relevant to executive functions (e.g., prefrontal cortex); however, findings from global measures provide promising evidence for future research on hemodynamic and structural predictors of executive function in SCA. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cell (HSC) gene therapy is now curative for multiple genetic diseases; however, it is limited by morbidity and mortality from cytotoxic chemotherapy-based conditioning. To overcome these limitations, we developed an antibody drug conjugate (ADC) targeting CD117 (c-Kit) to specifically deplete both HSCs and progenitor cells. In our preliminary study, 0.2 mg/kg CD117-ADC conditioning resulted in >99% bone marrow depletion, detectable engraftment of gene-modified cells (vector copy number per cell (VCN) ~0.01), and minimal toxicities in a rhesus HSC gene therapy model (ASH 2019). In this study, we investigated escalating doses of CD117-ADC to determine the optimum conditioning dose to enable engraftment of gene-modified CD34+ HSCs in rhesus macaques. We evaluated autologous CD34+ cell transplantation with lentiviral gene marking following conditioning using a single injection of CD117-ADC at the 0.3 mg/kg dose for ZL13 and ZJ62, and the 0.4 mg/kg dose for H635 and H96G. The extent of gene marking was compared with myeloablative busulfan conditioning (5.5 mg/kg x 4 days) for 12U018 and 12U020. Mobilized rhesus CD34+ cells (ADC 3.8±1.9x10e7 vs. Busulfan 2.9±0.2x10e7, n.s.) were transduced with a lentiviral vector encoding BCL11A-targeting microRNA-adapted short hairpin RNA (shmiR-BCL11A) co-encoding a truncated human erythropoietin receptor (thEpoR) for stable fetal hemoglobin (HbF) induction (Sci Transl Med. 2021). These cells (in vitro VCN 10.1±3.8 vs. 10.2±7.3, n.s.) were transplanted into autologous animals 6 or 10 days after ADC conditioning (0.3 or 0.4 mg/kg, respectively) or 1 day after busulfan conditioning. Blood counts, gene-marking levels, and HbF induction were evaluated for 0.3-1.2 years post-transplant in ADC conditioning and for 1.5 years in busulfan conditioning. After a reduction of blood counts post-transplantation with ADC or busulfan conditioning, all lineages recovered. Granulocyte (>500/μl, day 6-9 vs. day 8-9), reticulocyte (>50,000/μl, day 10-14 vs. day 11), and platelet (>30,000/μl, day 2-8 vs. no reduction) recoveries were similar for ADC and busulfan conditioning, respectively. Only ADC conditioning resulted in a reduction of platelet counts as well as a novel transient rebound in all major lineages. Two months post-transplant, efficient gene marking (VCN in granulocytes 0.28±0.16 vs. 0.44±0.17, n.s.) was observed in 3 of 4 animals in ADC-conditioning (ZJ62 with 0.3 mg/kg ADC, and H635 and H96G with 0.4 mg/kg ADC). This marking level was similar to busulfan conditioning (Left panel in Figure). Robust and durable HbF induction was also detected by both HbF-positive percentages (F-cell 8.5±1.8% vs. 13.7±5.8%, n.s.) and HPLC-quantitated HbF amounts (8.0±2.9% vs. 11.1±5.2%, n.s.) in these 3 animals, similar to busulfan conditioning (Right panel in Figure). In ZL13 (1 of 2 animals in 0.3 mg/kg ADC), lower gene marking (VCN in granulocytes 0.02) was obtained, along with low HbF induction (F-cell 1.0% and HbF amounts 0.9%), suggesting that 0.3 mg/kg ADC is marginal and 0.4 mg/kg ADC is sufficient for robust engraftment of gene-modified cells. Importantly, CD117-ADC conditioning resulted in minimal toxicities unlike busulfan conditioning. In summary, we demonstrated that a single dose of CD117-ADC allows for efficient engraftment of gene-modified CD34+ HSCs in a rhesus gene therapy model, achieving a similar level as myeloablative busulfan conditioning. Robust HbF induction was also confirmed at the protein levels in this rhesus gene therapy model with ADC conditioning. This targeted approach for safer conditioning could improve the risk benefit profile in HSC gene therapy. Figure 1 Figure 1. Disclosures Latimer: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bhattarai: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Yoder: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Palchaudhuri: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Li: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bertelsen: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Olson: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

By virtue of their direct cytotoxicity to transformed and virus infected cells, Natural Killer (NK) cells play crucial roles in immunity. NK cells modulate and coordinate innate and adaptive responses through the release of chemokines and cytokines. Although NK cells are endowed only with germ-line encoded receptors, evidence has been accumulating, that subsets of NK cells can bestow adoptively transferable, long-lasting and antigen-specific immune responses to certain haptens and viruses. Growing evidence suggests that adaptive immune responses lie on a spectrum. Rechallenge of cells, canonically belonging to the innate immune system, can result in enhanced responsiveness - a process termed 'trained immunity' and thought to be maintained by epigenetic and metabolic reprogramming. In previous work, our lab studied the role of NK cell responses to rhesus cytomegalovirus (rhCMV) in a genetic barcoding model. We found that new clones arose in the CD16 + NK compartment after primary rhCMV infection. There was rapid clearance without the emergence of new clones in subsequent rechallenge with rhCMV. In this study we used 3'-end single cell RNA-seq (3'-scRNA-seq) with CITE-seq to profile NK and T cells from an initially CMV-naïve rhesus macaque (RM) at four time points before and after primary and secondary infections with rhCMV. We immunophenotypically sorted NK and T cells from peripheral Blood (PB) samples at 'baseline', 30 days after initial rhCMV infection ('primary infection'), ca. 500 days after initial rhCMV infection ('steady state') and 10 days after rmCMV reinfection ('secondary infection'). Alongside the PB samples at 'steady state' and after 'secondary infection', we also sorted NK and T cells from lymph nodes (LN). We applied CD16 and CD56 CITE-seq antibodies to NK cells from all samples; NK cells from the 'steady state' and 'secondary infection' samples were also labeled with CX3CL1 CITE-seq antibodies. We multiplexed NK and T cells from each time point in 4:1 ratios before preparing 3'-scRNA-seq libraries. We used scanpy and scvi-tools as well as custom python code to demultiplex NK from T cells, harmonize 3'-scRNA-seq with CITE-seq data and integrate the 6 different samples. We used scvelo and cellrank to compute RNA velocities and infer trajectories, respectively. We obtained a total of 35,523 high-quality cells. We identified 20 clusters of NK and T cells, on the basis of community detection via the Leiden algorithm. All clusters contained cells from both tissue sources. The 4 clusters characterized by expression of CD56 exhibit higher expression of KLRC1 (protein: NKG2A), IL7R and the transcription factors LEF1 and MYC. The 8 clusters of CD16 + cells are distinguished by high expression of the transcription factors ZEB2 and TBX21/T-BET, cytotoxicity markers, GZMB and PRF1, and activating receptors, KLRC2 (protein: NKG2C), KLRC3 (protein: NKG2E) and NCR3 (protein: NKp30). An adaptive population of NK cells is identified on the basis of high KLRC2 and low FCER1G expression. We analyzed changes in the proportions of cells in each cluster of the time course of CMV infection using a binomial generalized linear model. Clusters associated with proliferation and acute inflammation were increased in proportion after primary rhCMV infection; the proportion of the adaptive population did not significantly change during the acute phase of primary infection but increased markedly by the later 'steady state' samples. RNA velocity and inferred developmental trajectories suggest transitions between the adaptive, proliferating CD16 + and mature effector subsets; the predominant path into the adaptive population occurring from the proliferating CD16 + subset after primary infection. There is a notable paucity of inferred transitions between the CD56 + and CD16 +subpopulations under all the experimental conditions we observed. We have characterized the single cell transcriptional states and dynamics of RM NK cells in response to rhCMV infection. We focus on a subset transcriptionally resembling a previously identified subset with adaptive function and find it arises from a proliferating population of effector cells after primary infection. This may be analogous to the dedifferentiation of effector CD8 T cells into memory T cells proposed by Youngblood et al. Confirmatory experiments to analyze the reconstitution of the CD16 + compartment after treatment with a depleting antibody are on-going. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

A series of large-scale genomic studies has reported that clonally expanded hematopoietic cells bearing somatic mutations are increasingly prevalent with age, even in the absence of cytopenias, myelodysplasia, or leukemia. Individuals with acquired somatic mutations at a variant allele frequency (VAF) of at least 2% in genes recurrently mutated in hematologic malignancies not meeting criteria for any known hematologic disorders have been labeled as manifesting "age-related clonal hematopoiesis (ARCH)". Dominant negative or loss-of-function (LOF) mutations in genes encoding for epigenetic modifier enzymes such as DNMT3A, TET2, and ASXL1 are most common in ARCH, and individuals with ARCH are at a greater risk for cardiovascular diseases as well as hematologic malignancies. However, the relationships between these mutations, clonal expansion, and clinical outcomes are not fully elucidated due to difficulties in studying individuals with ARCH longitudinally over time in the absence of an overt clinical abnormality, and extrapolating from murine models that may not closely recapitulate human hematopoietic physiology. Since non-human primates (NHP) have a high similarity in HSPC and marrow properties to humans, and we have identified typical spontaneous ARCH mutations in aged macaques not yet identified in aged mice, we sought to generate a rhesus macaque model of human ARCH utilizing CRISPR/Cas9 technology to investigate clonal behavior and intervention strategies. We delivered a gRNA pool targeting the three most frequently mutated genes in human ARCH with Cas9 in the form of ribonucleoprotein (RNP) into HSPCs obtained from three young adult macaques, targeting a low efficiency, and the edited HSPCs were reinfused into autologous animals following total body irradiation. All macaques engrafted promptly after transplantation and maintained normal blood counts. Up to three years of long-term follow-up revealed reproducible and significant expansion of multiple HSPC clones with heterozygous TET2 LOF mutations, compared to the limited expansion of clones carrying DNMT3A and ASXL1 mutations, reaching a VAF almost 25% with doubling time of 7.5 months in circulating granulocytes of the first macaque (ZL26, Fig 1A). Although there were differences in population doubling rates between individuals, the three macaques shared the general pattern of a gradual but dramatic expansion of TET2-mutated clones, with most of the expanding indels resulting in frameshifts predicted to result in LOF. These data suggest a single mutation in TET2 is sufficient for clonal expansion, and that other intrinsic and/or extrinsic factors can regulate the pace of TET2 clonal expansion. Bone marrow of these macaques exhibited hypercellularity and myeloid-predominant skewing without dysplastic changes compared with macaques of similar age previously transplanted with HSPCs edited at non-ARCH loci. Furthermore, RNA-seq indicated that TET2-disrupted myeloid colony-forming units (CFUs) and mature cells exhibited a distinct hyperinflammatory gene expression profile. Indeed, CD14+CD163+ macrophages purified from all three ARCH macaques exhibited hyperinflammatory function, with upregulated NLRP3 inflammasome activity and increased IL-6 signaling. We hypothesized that interrupting the vicious cycle of clonal expansion driven by and driving inflammation could halt the expansion of TET2-mutated clones. To address this, we treated the animal with the fastest TET2-mutant clonal expansion (ZH63) with tocilizumab, an antibody blocking IL-6 signaling, starting 13 months after transplantation and continuing for 4 months. The TET2 mutated allele frequency in granulocytes declined by 30% by the end of the treatment and began to increase again after withdrawal (Fig 1B), suggesting that interruption of the IL-6 axis removes the selective advantage of mutant HSPCs and this repressive effect is specific to the TET2-mutant genotype. In summary, our CRISPR/Cas9-engineered rhesus macaque ARCH model recapitulates human ARCH and uncovers the impact of TET2 LOF on hematopoiesis and inflammation, as well as demonstrates the suppressive effect of IL-6 axis blockade in TET2-mutant clonal expansions. This robust NHP model will be further utilized for examining the pathophysiology of ARCH and testing of potential therapeutic interventions. Disclosures Dunbar: Novartis: Research Funding.

PURPOSE : The World Trade Center (WTC) attack of September 11, 2001 created an unprecedented environmental exposure to known and suspected carcinogens. A higher incidence of multiple myeloma (MM) and precursor disease has been reported among first responders to the WTC disaster compared to the unexposed population (Landgren, 2018). To expand on prior screening studies, and to characterize the genomic impact of the exposure to known and potential carcinogens in the WTC debris, we were motivated to perform whole genome sequencing (WGS) of WTC first responders and recovery workers who were diagnosed with a plasma cell disorder after the attack. PATIENTS AND METHODS: We performed WGS of 9 CD138-positive bone marrow mononuclear samples from patients who were diagnosed with plasma cell disorders after exposure to the WTC disaster: 4 monoclonal gammopathy of undetermined significance (MGUS), 2 smoldering multiple myeloma (SMM), 2 MMs, and 1 patient with plasma cell leukemia (PCL). Eight patients (88%) were first responders and one was a recovery worker. Peripheral blood mononuclear cells were used as normal match. Median coverage for tumor and normal samples was 50.9X (range 47-76) and 37X (range 35-41), respectively. The landscape of genomic drivers and complex structural events was compared to 752 MM patients enrolled in the CoMMpass trial with available whole exome and low-coverage long-insert WGS data (IA15; NCT01454297). To characterize the mutational signature landscape we combined the WTC cohort with 110 whole genomes from 56 patients with multiple myeloma and myeloma precursor disease (Rustad et al. 2020; Landau et al. 2020) and we ran our three-step workflow: de novo extraction (i.e. sigprofiler), assignment, and fitting (i.e. mmsig). To exclude contribution of any environmental agents in the WTC debris with known mutational signatures (Kucab et al., 2019), we ran our fitting algorithm mmsig in each post-WTC case, including and forcing the extraction of these mutational signatures. RESULTS: No significant differences were observed in comparing the post-WTC driver and mutational signatures landscape with 110 previously published WGS from 56 patients with MM and the CoMMpass WGS cohort (n=752). Likewise, we did not observe any new or distinct mutational signatures among WTC-exposed patients. Following forced extraction of 5 mutational signatures associated with environmental agents detected in the WTC debris (e.g. PAHs), we did not find significant contributions from any of these described environmental mutational signatures. To reconstruct the temporal activity of each mutational process we divided all single nucleotide variants into subclonal and clonal. Clonal mutations were further subdivided into duplicated (acquired before a chromosomal gain) and unduplicated (Rustad et al. 2020). WTC-exposed patients had differing patterns in mutational signature timelines of AID and APOBEC activity. Overall, the mutational signature activity over time in post-WTC plasma cell dyscrasia reflects what has been previously observed in multiple myeloma without WTC-exposure (Rustad et al., 2020). Finally, leveraging constant activity of the clock-like single base substitution mutational signatures 1 and 5 over time and our molecular time workflow (Rustad et al., 2020), we estimated the age at which each evaluable patient acquired a tumor-initiating chromosomal gain and found that they were windowed to both pre- and post-WTC exposure across neoplasms (Figure 1). In some cases, clonal multi-chromosomal gain events were acquired decades before both the diagnosis and the WTC exposure. Specifically, of 6 patients with large clonal chromosomal gains, 1 MM case, 1 SMM, and 1 MGUS showed evidence of a pre-existing clone prior to WTC exposure, two MGUS showed evidence of multi-gain events following the exposure, and one MM case had a 1q gain in the same time window as the attack. CONCLUSIONS: Post-WTC plasma cell neoplasms had similar genomic landscapes to non-exposed cases. Although limitations in sample size preclude any definitive conclusions, our findings suggest that the observed increased incidence of plasma cell neoplasms in this population is due to complex and heterogeneous effects of the WTC exposure that may have initiated or contributed to progression of malignancy. The existence of pre-malignant clonal entities at time of WTC exposure may therefore be relevant for future WTC-related study. Figure 1 Disclosures Hultcrantz: Intellisphere LLC: Consultancy; Amgen: Research Funding; Daiichi Sankyo: Research Funding; GSK: Research Funding. Shah:Physicians Education Resource: Honoraria; Celgene/BMS: Research Funding. Iacobuzio-Donahue:BMS: Research Funding. Papaemmanuil:Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Kyowa Hakko Kirin: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Prime Oncology: Consultancy, Honoraria; MSKCC: Patents & Royalties. Verma:BMS: Consultancy, Research Funding; acceleron: Consultancy, Honoraria; stelexis: Current equity holder in private company; Janssen: Research Funding; Medpacto: Research Funding. Dogan:National Cancer Institute: Research Funding; EUSA Pharma: Consultancy; Takeda: Consultancy; Seattle Genetics: Consultancy; Corvus Pharmaceuticals: Consultancy; Physicians Education Resource: Consultancy; Roche: Consultancy, Research Funding; AbbVie: Consultancy. Landgren:Celgene: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Cellectis: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Merck: Other; Karyopharma: Research Funding; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Pfizer: Consultancy, Honoraria; Merck: Other; Karyopharma: Research Funding; Binding Site: Consultancy, Honoraria; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Seattle Genetics: Research Funding; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Adaptive: Consultancy, Honoraria; Cellectis: Consultancy, Honoraria; Glenmark: Consultancy, Honoraria, Research Funding; Binding Site: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Glenmark: Consultancy, Honoraria, Research Funding.

Momelotinib (MMB), a JAK1, JAK2 and ACVR1 inhibitor, has demonstrated clinically comparable splenic and symptomatic benefits to ruxolitinib (RUX), the standard-of-care JAK1/JAK2 inhibitor for myelofibrosis (MF), a condition marked by splenomegaly, constitutional symptoms and progressive anemia and thrombocytopenia. MMB also uniquely restores iron homeostasis and red blood cell production, reduces or eliminates the need for transfusions and improves or maintains platelet (PLT) counts. Consistent with MMB's differentiated biological profile, low myelosuppressive potential and favorable hematological tolerability, prolonged, near-maximal MMB dose intensity can be maintained regardless of underlying PLT values. In contrast, RUX's hematological toxicity profile necessitates attenuated starting doses for thrombocytopenic (TCP) patients with PLTs A retrospective analysis of spleen, symptom and TI response rates at week 24 was conducted in the TCP and ITT groups from SIMPLIFY-1 (S1), a double-blind Phase 3 study in intermediate/high risk MF patients randomized 1:1 to MMB or RUX over a 24-week treatment period, and SIMPLIFY-2 (S2), a Phase 3 study comparing MMB to best available therapy (BAT) in previously RUX-exposed MF patients. A baseline PLTs ≥50 × 109/L was required in S1, while there was no lower PLT limit for S2. Most subjects randomized to BAT (88%) received RUX during the 24-week randomized period. In S1, 9.5% and 24% of 432 subjects randomized had a PLT count of In S2, 44% of 156 subjects randomized had a PLT count of These retrospective analyses of data from the two Phase 3 SIMPLIFY studies demonstrate that MMB efficacy is maintained in TCP patients and is comparable to that observed in the broader JAKi-naïve and previously JAKi-treated ITT populations in S1 and S2. These data contrast with RUX data from S1 where the SRR was markedly decreased and the TSS moderately decreased in TCP patients, consistent with reduced RUX dose intensity and higher rates of early discontinuation in this subset. Consequently, for patients in S1 with low PLTs, MMB and RUX demonstrated similar symptomatic benefit, while MMB had a more favorable profile for splenic volume reduction and TI. Response rates for the three parameters in the MMB arm of S2 were comparable between the TCP and ITT groups. Response rates in the control arm were low and not substantially different between the TCP and ITT groups, with most patients receiving very low dose intensity of RUX. Together, the findings of comparable spleen, symptom and TI response in the TCP and ITT groups treated with MMB suggest that the compound could become the optimal JAK inhibitor therapy for intermediate/high risk MF patients with underlying disease-related or prior RUX-induced thrombocytopenia. Disclosures Kiladjian: Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Amgen: Honoraria, Research Funding; Geron: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Mayer:Principia Biopharma: Research Funding; AbbVie: Research Funding. Illés:Novartis, Janssen, Pfizer, Roche;: Other: Travel, Accommodations, Expenses; Takeda, Seattle Genetics: Research Funding; Celgene, Janssen, Novartis,Roche, Takeda: Consultancy; Janssen, Celgene, Takeda, Novartis Pharma SAS, Pfizer Pharmaceuticals Israel, Roche;: Consultancy, Honoraria. Vannucchi:AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Nagy:MorphoSys AG: Patents & Royalties. Yoon:Novartis: Consultancy, Honoraria; Janssen: Consultancy; F. Hoffmann-La Roche: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland., Research Funding; Amgen: Consultancy, Honoraria; Kyowahako Kirin: Research Funding; YuhanPharma: Research Funding. Von Bubnoff:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Clinical biomarker research, steering committee, Patents & Royalties: Research support, Research Funding; Astra Zeneca: Honoraria, Other: Lectures, Patents & Royalties: Astra Zeneca. Verstovsek:Promedior: Research Funding; PharmaEssentia: Research Funding; AstraZeneca: Research Funding; Celgene: Consultancy, Research Funding; Protagonist Therapeutics: Research Funding; CTI Biopharma Corp: Research Funding; Sierra Oncology: Consultancy, Research Funding; Gilead: Research Funding; Genentech: Research Funding; Incyte Corporation: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding; ItalPharma: Research Funding; Blueprint Medicines Corp: Research Funding; NS Pharma: Research Funding. Klencke:Sierra Oncology Inc.: Current Employment, Current equity holder in publicly-traded company. Donahue:Sierra Oncology Inc.: Current Employment, Current equity holder in publicly-traded company. Mesa:Incyte: Research Funding; Bristol Myers Squibb: Research Funding; AbbVie: Research Funding; Sierra Oncology: Consultancy; Novartis: Consultancy; LaJolla Pharmaceutical Company: Consultancy; Samus Therapeutics: Research Funding; Promedior: Research Funding; Genentech: Research Funding; CTI BioPharma: Research Funding.