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Many xenobiotic metabolizing enzymes, such as P450s, SULTs, UGTs, and sulfatases, are expressed in the brain, including astrocytes, the most abundant cell type in the brain. To obtain a purified culture of primary astrocytes, a method was developed and confirmed through flow cytometry. Primary glial cells are isolated separately from male and female postnatal day 1/2 (PND1/2) rat pups to isolate developing astrocytes. Sex is first determined by the anogenital distance and then confirmed through PCR followed by gel electrophoresis. Once isolated, the medium is changed every 3-4 days with additional incubation at room temperature for 10 min on a plate shaker before aspirating media to release microglia. When flasks reach confluency, cells are passaged and reseeded with 2 x 105 cells per flask. Once the cells reached passage 3, or appear to be confluent with only astrocytes, flow cytometry is performed utilizing Anti-Iba1 and GFAP primary antibodies.

Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully in vitro system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL.Significance:Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully in vitro nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development.This article is highlighted in the In This Issue feature, p. 1861

Supplementary Data from Surface Proteomics Reveals CD72 as a Target for In Vitro–Evolved Nanobody-Based CAR-T Cells in KMT2A/MLL1-Rearranged B-ALL

Supplementary Figure from Surface Proteomics Reveals CD72 as a Target for In Vitro–Evolved Nanobody-Based CAR-T Cells in KMT2A/MLL1-Rearranged B-ALL

Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully in vitro system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL. Significance: Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully in vitro nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development. This article is highlighted in the In This Issue feature, p. 1861

We present a case of a 65-year-old woman with a persistently positive nasopharyngeal swab for severe acute respiratory syndrome coronavirus 2 PCR who developed new complications of coronavirus disease 2019 (COVID-19) 63 days from illness onset. She presented with intermittent fevers, fluctuating disorientation, gait instability, diffuse corticospinal tract signs, and acute venous thromboembolism. No alternate diagnosis was identified. This case highlights the potential for prolonged SARS-CoV-2 PCR positivity and persistent multisystem complications (particularly neurological), even after several months of initial COVID-19 diagnosis.

The 22q11.2 deletion syndrome (22q11.2DS), associated with congenital and neuropsychiatric anomalies, is the most common copy number variant (CNV)-associated syndrome. Patient-derived, induced pluripotent stem cell (iPS) models have provided important insight into the mechanisms of phenotypic features of this condition. However, patient-derived iPS models may harbor underlying genetic heterogeneity that can confound analysis of pathogenic CNV effects. Furthermore, the ∼1.5 Mb “A-B” deletion at this locus is inherited at higher frequency than the more common ∼3 Mb “A-D” deletion, but remains under-studied due to lack of relevant models. To address these issues, here we leveraged a CRISPR-based strategy in Cas9-expressing iPS cells to engineer novel isogenic models of the 22q11.2 “A-B” deletion. After in vitro differentiation to excitatory neurons, integrated transcriptomic and cell surface proteomics identified deletion-associated alterations in surface adhesion and cell signaling. Furthermore, implantation of iPS-derived neuronal progenitor cells into the cortex of neonatal mice found accelerated neuronal maturation within a relevant microenvironment. Taken together, our results suggest pathogenic mechanisms of the 22q11.2 “A-B” deletion in driving neuronal and neurodevelopmental phenotypes, both in vitro and in vivo. We further propose that the isogenic models generated here will provide a unique resource to study this less-common variant of the 22q11.2 microdeletion syndrome.

Background: CD38 is a surface ectoenzyme expressed at high levels on myeloma plasma cells and is the target for the monoclonal antibodies (mAbs) daratumumab and isatuximab. These antibodies have multiple mechanisms of action, primarily involving recruiting and modulating components of the immune system, but they may also carry direct anti-tumor effects. CD38 density on tumor cells is an important determinant of mAb efficacy while CD38 is lost after mAb treatment. Several small molecules have been found to increase tumor surface CD38, with the goal of boosting mAb efficacy in a co-treatment strategy. However, we do not yet have a broad global sense of the transcriptional or post-transcriptional networks that most strongly impact CD38 expression. There may be alternate strategies to even more potently increase CD38 expression that have not yet been identified. Furthermore, prior clinical studies showed that CD38 downregulation after daratumumab treatment was accompanied by increases in the complement inhibitors CD55 and CD59. Are there other features of myeloma surface remodeling driven by CD38 downregulation? Here we sought to extend our currently limited insight into CD38 surface expression by using a multi-omics approach. Methods: Genome-wide CRISPR interference screening was performed in RPMI-8226 cells stably expressing the dCas9-KRAB fusion protein. Cells were grown for 14 days after library transduction, flow-sorted on the top and bottom 25% of CD38 surface expression, and sgRNA's deep sequenced. Antibody-dependent cellular cytoxicity assays were performed with NK92-CD16 cells. Cell surface proteomics was performed using N-glycoprotein cell surface capture in triplicate. Phosphoproteomics was performed used immobilized metal affinity chromatography in triplicate. Murine studies were performed in NSG mice under approved IACUC-approved institutional protocols. Results: A genome-wide CRISPR-interference screen in RPMI-8226 cells demonstrated that transcriptional and epigenetic factors played the most prominent role in surface CD38 regulation (Fig. 1A). One of the genes that when knocked down led to greatest surface CD38 increase was RARA. This finding supports the promise of all-trans retinoic acid (ATRA), which leads to RARA degradation, as a potent agent to induce CD38 upregulation. Validation of additional screen hits TLE3 and HEXIM1 also illustrated that these negative regulatory transcription factors suppress CD38 expression at baseline (not shown). We found the transcription factor SPI1 to be a prominent positive regulator of CD38. SPI1 knockdown led to daratumumab resistance both in vitro and in vivo, similar to the resistance observed after CD38 knockdown (not shown). Analysis of myeloma patient ATAC-seq data, assessing transcription factor motifs present at the CD38 locus, combined with a predictive machine learning model, further identified XBP1 as one of the most potent transcriptional regulators of CD38 (Fig. 1B). We next used "antigen escape profiling" - knockdown of CD38 followed by unbiased cell surface proteomics - to mimic surface alterations in the context of CD38 loss. We found minimal changes in other cell surface proteins beyond CD38 (Fig. 1C), indicating the CD38 loss alone is not sufficient to remodel the myeloma surfaceome. This finding also supports the hypothesis that complement or other immune system interactions are necessary to lead to other myeloma surface protein alterations. In a parallel analysis of pharmacologic regulation, we also used cell surface proteomics integrated with RNA-seq to demonstrate that ATRA leads to few other surface protein changes beyond CD38 (not shown). In contrast, other molecules, such as azacytidine and panobinostat, led to broader changes across many more surface proteins, showing a lack of specificity when driving CD38 upregulation. Finally, unbiased phosphoproteomics revealed partial inhibition of the MAP kinase pathway after daratumumab binding (Fig. 1D). This result may comprise a direct anti-proliferative effect of anti-CD38 therapeutic antibody engagement in myeloma. Conclusions: Our work provides a resource to design strategies to enhance efficacy of CD38-targeting immunotherapies in myeloma. Our approach also outlines a broad multi-omic strategy to evaluate surface and transcriptional regulation of other key immunotherapeutic targets in hematologic malignancies. Figure 1 Figure 1. Disclosures Choudhry: Genentech: Current Employment, Current equity holder in publicly-traded company. Ramkumar: Senti Biosciences: Current Employment, Current holder of individual stocks in a privately-held company.

Background Identifying factors that can predict severe disease in patients needing hospitalization for COVID-19 is crucial for early recognition of patients at greatest risk. Objective (1) Identify factors predicting intensive care unit (ICU) transfer and (2) develop a simple calculator for clinicians managing patients hospitalized with COVID-19. Methods A total of 2,685 patients with laboratory-confirmed COVID-19 admitted to a large metropolitan health system in Georgia, USA between March and July 2020 were included in the study. Seventy-five percent of patients were included in the training dataset (admitted March 1 to July 10). Through multivariable logistic regression, we developed a prediction model (probability score) for ICU transfer. Then, we validated the model by estimating its performance accuracy (area under the curve [AUC]) using data from the remaining 25% of patients (admitted July 11 to July 31). Results We included 2,014 and 671 patients in the training and validation datasets, respectively. Diabetes mellitus, coronary artery disease, chronic kidney disease, serum C-reactive protein, and serum lactate dehydrogenase were identified as significant risk factors for ICU transfer, and a prediction model was developed. The AUC was 0.752 for the training dataset and 0.769 for the validation dataset. We developed a free, web-based calculator to facilitate use of the prediction model (https://icucovid19.shinyapps.io/ICUCOVID19/). Conclusion Our validated, simple, and accessible prediction model and web-based calculator for ICU transfer may be useful in assisting healthcare providers in identifying hospitalized patients with COVID-19 who are at high risk for clinical deterioration. Triage of such patients for early aggressive treatment can impact clinical outcomes for this potentially deadly disease.

Alternate strategies are needed for B-cell malignancy patients relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-ALL, which we further found to be expressed in other B-cell malignancies. Using a recently-described, fully-in vitro system we selected synthetic CD72-specific nanobodies, incorporated them into CARs, and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of CD72’s role in inhibiting B-cell receptor signaling, we found that pharmacologic SHIP1 inhibition increased CD72 surface density. We establish CD72-nanobody CAR T’s as a promising therapy for MLLr B-ALL.

Eukaryotic DNA replication begins from multiple origins. The origin recognition complex (ORC) binds origin DNA and scaffolds assembly of a prereplicative complex (pre-RC), which is subsequently activated to initiate DNA replication. In multicellular eukaryotes, origins do not share a strict DNA consensus sequence, and their activity changes in concert with chromatin status during development, but mechanisms are ill-defined. Previous genome-wide analyses in Drosophila and other organisms have revealed a correlation between ORC binding sites and the histone variant H3.3. This correlation suggests that H3.3 may designate origin sites, but this idea has remained untested. To address this question, we examined the enrichment and function of H3.3 at the origins responsible for developmental gene amplification in the somatic follicle cells of the Drosophila ovary. We found that H3.3 is abundant at these amplicon origins. H3.3 levels remained high when replication initiation was blocked, indicating that H3.3 is abundant at the origins before activation of the pre-RC. H3.3 was also enriched at the origins during early oogenesis, raising the possibility that H3.3 bookmarks sites for later amplification. However, flies null mutant for both of the H3.3 genes in Drosophila did not have overt defects in developmental gene amplification or genomic replication, suggesting that H3.3 is not essential for the assembly or activation of the pre-RC at origins. Instead, our results imply that the correlation between H3.3 and ORC sites reflects other chromatin attributes that are important for origin function.

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.

Transplanting islets serves best option for restoring lost beta cell mass and function. Small bio-chemical agents do have the potential to generate new islets mass, however lack of understanding about mechanistic action of these small molecules eventually restricts their use in cell-based therapies for diabetes. We recently reported “Swertisin” as a novel islet differentiation inducer, generating new beta cells mass more effectively. Henceforth, in the present study we attempted to investigate the molecular signals that Swertisin generate for promoting differentiation of pancreatic progenitors into islet cells. To begin with, both human pancreatic progenitors (PANC-1 cells) and primary cultured mouse intra-islet progenitor cells (mIPC) were used and tested for Swertisin induced islet neogenesis mechanism, by monitoring immunoblot profile of key transcription factors in time dependent manner. We observed Swertisin follow Activin-A mediated MEPK-TKK pathway involving role of p38 MAPK via activating Neurogenin-3 (Ngn-3) and Smad Proteins cascade. This MAP Kinase intervention in differentiation of cells was confirmed using strong pharmacological inhibitor of p38 MAPK (SB203580), which effectively abrogated this process. We further confirmed this mechanism in-vivo in partial pancreatectomised (PPx) mice model, where we could show Swertisin exerted potential increase in insulin transcript levels with persistent down-regulation of progenitor markers like Nestin, Ngn-3 and Pancreatic Duodenal Homeobox Gene-1 (PDX-1) expression, within three days post PPx. With detailed molecular investigations here in, we first time report the molecular mode of action of Swertisin for islet neogenesis mediated through MAP Kinase (MEPK-TKK) pathway involving Ngn-3 and Smad transcriptional regulation. These findings held importance for developing Swertisin as potent pharmacological drug candidate for effective and endogenous differentiation of islets in cell based therapy for diabetes.