Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis 1 . These lesions are precursors for blood cancers 2 – 6 , but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2 , ASXL1 , SF3B1 and SRSF2 , but this effect was not seen in clones with driver mutations in DNMT3A . TCL1A was not expressed in normal or DNMT3A -mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

Epigenetic dysregulation is frequently observed in the disease pathology of pediatric cancers, including neuroblastoma, the most common extracranial solid tumor in pediatric patients. Neuroblastoma tumors co-opt developmentally linked adrenergic or mesenchymal super-enhancer landscapes that rewire their transcriptional programs. Here, we describe that the lineage commitment to a mesenchymal epigenetic state is an important mechanism of resistance to anti-GD2 therapy through loss of GD2 antigen, a ganglioside glycolipid expressed on the cell surface. Low GD2 expression was significantly correlated with the mesenchymal state in a large panel of neuroblastoma cell lines and a forced adrenergic-to-mesenchymal transition conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2 expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Genome-wide CRISPR/Cas9 screening to identify regulators of GD2 in neuroblastoma revealed that the ablation of the polycomb repressive complex 2 (PRC2) significantly upregulates GD2 expression in GD2-low cells. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells into an adrenergic-like state, re-expressed ST8SIA1, and restored surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential PRC2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma. Citation Format: Nathaniel W. Mabe, Min Huang, Daniel A. Schaefer, Guillermo N. Dalton, Giulia Digiovanni, Gabriela Alexe, Anna C. Geraghty, Delan Khalid, Marius M. Mader, Michal Sheffer, Miles H. Linde, Nghi Ly, Maria Caterina Rotiroti, Benjamin A. H. Smith, Marius Wernig, Carolyn R. Bertozzi, Michelle Monje, Constantine Mitsiades, Ravindra Majeti, Ansuman T. Satpathy, Kimberly Stegmaier, Robbie G. Majzner. Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR003.

T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly un-derstood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to system-atically discover regulators of T cell exhaustion, which identified an enrichment of epigenetic factors. In vivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved T cell persistence in tumors. In vivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating T cells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chro-matin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of T cell exhaustion and demonstrate that modulation of epigenetic state can improve T cell responses in cancer immunotherapy.

Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1 , restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

Abstract Epigenetic dysregulation is frequently observed in the disease pathology of pediatric cancers, including neuroblastoma, the most common extracranial solid tumor in pediatric patients. Neuroblastoma tumors co-opt developmentally linked adrenergic or mesenchymal super-enhancer landscapes that rewire their transcriptional programs. Here, we describe that the lineage commitment to a mesenchymal epigenetic state is an important mechanism of resistance to anti-GD2 therapy through loss of GD2 antigen, a ganglioside glycolipid expressed on the cell surface. Low GD2 expression was significantly correlated with the mesenchymal state in a large panel of neuroblastoma cell lines and a forced adrenergic-to-mesenchymal transition conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2 expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Genome-wide CRISPR/Cas9 screening to identify regulators of GD2 in neuroblastoma revealed that the ablation of the polycomb repressive complex 2 (PRC2) significantly upregulates GD2 expression in GD2-low cells. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells into an adrenergic-like state, re-expressed ST8SIA1, and restored surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential PRC2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma. Citation Format: Nathaniel W. Mabe, Min Huang, Daniel A. Schaefer, Guillermo N. Dalton, Giulia Digiovanni, Gabriela Alexe, Anna C. Geraghty, Delan Khalid, Marius M. Mader, Michal Sheffer, Miles H. Linde, Nghi Ly, Maria Caterina Rotiroti, Benjamin A. H. Smith, Marius Wernig, Carolyn R. Bertozzi, Michelle Monje, Constantine Mitsiades, Ravindra Majeti, Ansuman T. Satpathy, Kimberly Stegmaier, Robbie G. Majzner. Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR003.

The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2(Delta-165)mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2(Delta-165) mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2(Delta-165) mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at +164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.