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T cells are able to recognize and eliminate a wide variety of immunologic threats while maintaining self-tolerance. Pathogen recognition and clearance is ensured by a process called V(D)J recombination, during which a T cell obtains a unique set of V, D and J gene segments for all the chains (α and β or γ and δ) that make up its T cell receptor (TCR). Each recombined TCR detects a specific disease-associated antigen peptide, which triggers the appropriate adaptive immune response. Understanding the relationship between TCR sequences (clonotypes) and T cell activation during disease pathogenesis and progression can assist in the development of next-generation therapeutics with more favorable and sustainable outcomes. Recent advances in single-cell sequencing allowed for simultaneous profiling of TCRs and full transcriptomes leading to the characterization of key T cell populations with pathogen recognition and disease clearance capabilities. Despite their success, these methods rely on microfluidics devices or plate-based protocols with limited sensitivity and throughput (1,000s-10,000s of cells) making the study of disease-relevant T cells time consuming and costly. To overcome these limitations, we have extended our split pool combinatorial barcoding technology to simultaneously characterize the TCRs alongside the full transcriptomes of up to 1 million T cells. As a proof of concept experiment, we applied our method to primary T cells from 8 healthy human donors to characterize their TCR diversity. From the 920,000 T cells assayed we were able to recover TCR sequences from at least one chain in 88% of T cells (807,000 cells) and detected hundreds of thousands of unique alpha and beta chains across all donors. Furthermore we found that most of these chains represent rare clonotypes found in 1 or 2 cells. Lastly, we performed transcriptome-based clustering analyses to identify all major T cell subsets and discovered donor and subset-specific hyper-expanded clonotypes that could help us reconstruct each donor’s recent history of infection. In summary, we report on the extension of our highly flexible and scalable combinatorial barcoding technology to allow researchers to profile up to 1 million of T cells in a single experiment and investigate their functional responses during infection, cancer, autoimmunity, or therapeutic interventions. Citation Format: Efthymia Papalexi, Grace Kim, Bryan Hariadi, Sarah Schroeder, Peter Matulich, Vuong Tran, Daniel Diaz, Charles Roco, Alexander Rosenberg. Using combinatorial barcoding to simultaneously profile the transcriptome and immune repertoire of 1 million T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 248.

In the thirteen years since its inception, single-cell RNA-sequencing (scRNA-seq) has rapidly spread across multiple fields of research, leading to many new discoveries. As technologies have matured, the number of cells that can be processed in a single experiment has seen exponential growth with workflows now assaying up to one million cells in an individual experiment. While high throughput sequencing methods have facilitated the discovery and characterization of various cell types, sequencing costs can be prohibitively high for routine use. Many applications of scRNA-seq are focused on cell type identification, gene regulatory networks, or biomarker discovery. These applications often do not require surveying the entire transcriptome, but rather require the interrogation of specific sets of well-characterized genes. In these cases, sequencing the entire transcriptome may be adding unnecessary project costs. To increase throughput and minimize sequencing costs, the development of a targeted gene enrichment method is required. Here we extend our whole transcriptome split-pool combinatorial barcoding technology, to enable enrichment of a subset of genes in the final single cell sequencing libraries. To illustrate the power of our technology, we enriched a whole transcriptome library of human bone marrow mononuclear cells (BMMCs) from four acute myeloid leukemia (AML) donors and four control donors. From a library of 27,000 cells, we used our immune gene panel to enrich 1,000 genes representing canonical immune cell markers and pathways. Our method increased the percent of reads on target from as low as 1.6% in the whole transcriptome libraries to 75% in the targeted libraries. Furthermore, despite a nearly ten-fold reduction in sequencing reads between unenriched and enriched libraries, the resulting clustering yielded very high concordance of cell type identities and preserved AML-specific signatures such as FLT3, MKI67, and CD19. Overall, we demonstrate our modular enrichment strategy preserves biological structure of the data and allows for deep characterization of gene signatures in health and disease. We envision our approach will enable researchers to simultaneously reduce sequencing costs while drastically scaling up the number of cells and samples across experiments. Citation Format: Grace Kim, Efthymia Papalexi, Peter Matulich, Ryan Koehler, Sarah Schroeder, Vuong Tran, Charles Roco, Alexander Rosenberg. Targeted transcriptome sequencing enables exponential scaling of combinatorial barcoding in AML samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 226.

Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617Fand mutant IDH1R132Hor Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617FIdh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mutand IDH2mutmutations. Taken together, these data suggest that combined JAK and IDH inhibition May offer a therapeutic advantage in this high-risk MPN subtype., Damon Runyon Cancer Research Foundation (DRG-2241-15), Howard Hughes Medical Institute (Faculty Scholars Award), Stand Up To Cancer, National Cancer Institute (U.S.) (P50CA165962), National Cancer Institute (U.S.) (P30CA14051), Koch Institute for Integrative Cancer Research ( Dana-Farber Harvard Cancer Center Bridge Project), Leukemia & Lymphoma Society of America. Specialized Center of Research (SCOR) Program, National Institutes of Health (U.S.) (grant U54OD020355-01), National Institutes of Health (U.S.) (grant NCI R01CA172636), National Institutes of Health (U.S.) (grant R35CA197594), National Cancer Institute (U.S.) (Cancer Center Support Grant (P30 CA008747).

Summary Genetic and functional studies underscore the central role of JAK/STAT signaling in myeloproliferative neoplasms (MPNs). However, the mechanisms that mediate transformation in MPNs are not fully delineated, and clinically utilized JAK inhibitors have limited ability to reduce disease burden or reverse myelofibrosis. Here we show that MPN progenitor cells are characterized by marked alterations in gene regulation through differential enhancer utilization, and identify nuclear factor κB (NF-κB) signaling as a key pathway activated in malignant and non-malignant cells in MPN. Inhibition of BET bromodomain proteins attenuated NF-κB signaling and reduced cytokine production in vivo . Most importantly, combined JAK/BET inhibition resulted in a marked reduction in the serum levels of inflammatory cytokines, reduced disease burden, and reversed bone marrow fibrosis in vivo .

The mechanisms governing how human hematopoietic stem cells (HSC) balance their capacity for maintaining long-term (LT) regeneration with meeting daily and/or stress demands are unknown. We previously linked INKA1-mediated PAK4 inhibition along with reduction of H4K16 acetylation (H4K16ac) to quiescence in leukemia stem cells. Here, we uncover mutually exclusive immunostaining for INKA1 and H4K16ac in cord blood HSC; supported by BioID and PLA data showing INKA1 interacts with the H4K16 deacetylase SIRT1. The proteomic data also pointed us to CD112, that enabled isolation of LT-HSC into transcriptionally distinct subsets that respond differently to regenerative stress: a CD112low subset (H4K16aclow, CDK6low, ROSlow, INKA1high) exhibited a transient restraint before contributing to hematopoietic reconstitution and a primed CD112high subset (H4K16achigh, CDK6high, ROShigh, INKA1low) that responded rapidly. Protein and scRNAseq pseudotime analysis of in vitro and in vivo (G-CSF-) activated HSC, respectively, confirmed upregulation of H4K16ac, PAK4, CDK6 and CD112 coinciding with cell-cycle priming. INKA1-overexpression (OE) or PAK4 knock-down (KD) induced transient restraint in reconstitution (@4w) with 20w engraftment reaching control levels; HSC frequency increased 4-8 fold as measures in secondary transplants. Thus, INKA1-OE or PAK4-KD enables HSC to resist early activation, thereby protracting their regenerative potential (a phenomenon we term latency). By elevating hematopoietic demand with 5-FU, the INKA1-OE imposed restraint was released engendering even further increased HSC frequency; in controls regenerative capacity was abolished. Collectively, our data uncover a new molecular axis for HSC self-renewal regulation and point to latency as an orchestrated physiological response that integrates quiescence control with HSC fate choices to maintain LT-HSC pool size.

Viny et al. show that depletion of the cohesin subunit Smc3 affects HSC function through disruption of chromatin structure and that cohesin haploinsufficiency cooperates with Flt3-ITD to induce transformation of stem/progenitor cells and leukemia development., Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation; however, cohesin mutant leukemias do not show genomic instability. We hypothesized that reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Biallelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation and revealed an absolute requirement for cohesin in hematopoietic stem cell (HSC) function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose dependency for cohesin in regulating chromatin structure and HSC function.

SummaryAlthough clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasm (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated whether CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2 and MPL mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients.

The identification of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) has led to the clinical development of JAK kinase inhibitors, including ruxolitinib. Ruxolitinib reduces splenomegaly and systemic symptoms in myelofibrosis and improves overall survival; however, the mechanism by which JAK inhibitors achieve efficacy has not been delineated. Patients with MPN present with increased levels of circulating proinflammatory cytokines, which are mitigated by JAK inhibitor therapy. We sought to elucidate mechanisms by which JAK inhibitors attenuate cytokine-mediated pathophysiology. Single-cell profiling demonstrated that hematopoietic cells from myelofibrosis models and patient samples aberrantly secrete inflammatory cytokines. Pan-hematopoietic Stat3 deletion reduced disease severity and attenuated cytokine secretion, with similar efficacy as observed with ruxolitinib therapy. In contrast, Stat3 deletion restricted to MPN cells did not reduce disease severity or cytokine production. Consistent with these observations, we found that malignant and nonmalignant cells aberrantly secrete cytokines and JAK inhibition reduces cytokine production from both populations. Significance: Our results demonstrate that JAK–STAT3-mediated cytokine production from malignant and nonmalignant cells contributes to MPN pathogenesis and that JAK inhibition in both populations is required for therapeutic efficacy. These findings provide novel insight into the mechanisms by which JAK kinase inhibition achieves therapeutic efficacy in MPNs. Cancer Discov; 5(3); 316–31. ©2015 AACR. See related commentary by Belver and Ferrando, p. 234 This article is highlighted in the In This Issue feature, p. 213

Reply to "Uveal melanoma cells are resistant to EZH2 inhibition regardless of BAP1 status"

The development of the dual Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib for the treatment of myeloproliferative neoplasms (MPNs) has led to studies of ruxolitinib in other clinical contexts, including JAK-mutated acute lymphoblastic leukemia (ALL). However, the limited ability of JAK inhibition to induce molecular or clinicopathological responses in MPNs suggests a need for development of better therapies for JAK kinase-dependent malignancies. Here, we demonstrate that heat shock protein 90 (HSP90) inhibition using a purine-scaffold HSP90 inhibitor in early clinical development is an effective therapeutic approach in JAK-dependent ALL and can overcome persistence to JAK-inhibitor therapy in ALL cells.

The tumor suppressors BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss in mice results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 levels seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone. Loss of BAP1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in the transcriptional regulation of EZH2, expression of SETD8-the H4K20me1 methyltransferase-reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Furthermore, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.

Recurrent somatic loss-of-function mutations in ASXL1 (Addition of sex combs-like 1) are common genetic events in a spectrum of myeloid malignancies and these alterations demarcate patients with poor outcome. ASXL1 forms a chromatin regulatory complex with the ubiquitin hydrolase BAP1 (BRCA1 associated protein-1), a protein that has been found to be transcriptionally repressed in MDS patients. These data are consistent with BAP1 having tumor suppressive activity in MDS; however, the mechanism by which disruption of the ASXL1-BAP1 axis leads to transformation is not well understood. We conditionally deleted Bap1 in the murine hematopoietic system utilizing Mx1-Cre (hereafter referred to as Bap1 KO). One hundred percent of mice with confirmed Bap1 deletion developed a fully penetrant MDS-like disease characterized by leukocytosis, anemia, and splenomegaly. Bap1 KO mice have an expansion of the granulocyte macrophage progenitor compartment (GMP; Lin- c-Kit+ Sca1- CD34+ Fcϒ+). Given the role of BAP1 in epigenetic regulation, we investigated the effect of Bap1 loss on chromatin state and transcriptional output. We first assessed epigenetic changes in Bap1 KO mice by performing histone mass spectometry in control and Bap1 KO hematopoietic stem and progenitor cells (HSPCs, c-Kit+ enriched). Bap1 loss increased H3K27me2/3 at the expense of H3K27me0/1. We confirmed that H3K27me3 was increased in Bap1 KO bone marrow cells by completing H3K27me3 ChIP-Sequencing in HSPCs. Enumeration of H3K27me3 peaks in Bap1 KO versus control cells indicated an increase in H3K27me3 domains (Figure A). We next overlaid RNA-Sequencing from GMP sorted Bap1 KO bone marrow cells with genes marked by H3K27me3, as indicated by ChIP-Sequencing. We found that Bap1 loss resulted in a global decrease in gene expression (68% downregulated, 657/968 genes, p-adj Given the alterations in H3K27me3 in Bap1 KO mice, we investigated whether Bap1- deficient transformation could be rescued by abrogation of PRC2-mediated gene repression. We developed a genetic model with compound deletion of Bap1 and Ezh2, the catalytic component of the PRC2 complex. Co-deletion of Bap1 and Ezh2 resulted in a phenotypic rescue of Bap1 KO associated splenomegaly (spleen weights, Bap1 KO avg. 541.6 mg, Bap1/Ezh2 KO avg. 157.0 mg, p EZH2 small molecule inhibitors have proven effective in EZH2-dependent models of B cell lymphoma. To determine if Ezh2 inhibition was efficacious in the setting of Bap1 loss, we treated a cohort of Bap1 KO mice with either vehicle (NaCMC) or 500 mg/kg EPZ011989, an EZH2 inhibitor with in vivo activity. Treatment of Bap1 KO mice for 16 days resulted in significant reduction of splenomegaly (spleen weights, vehicle avg. 522.0, EPZ011989 treated avg. 216.2, p Figure 1. Figure 1. Disclosures Knutson: Epizyme, Inc: Employment. Campbell:Epizyme, Inc: Employment. Keilhack:Epizyme: Employment, Equity Ownership. Melnick:Janssen: Other: Research; ROCHE: Other: Research; Genentech: Speakers Bureau; Celgene: Consultancy; Eli Lilly: Consultancy; Epizyme: Consultancy. Armstrong:Epizyme, Inc: Consultancy. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.