Multiplexed spatial profiling of Hodgkin Reed-Sternberg cell neighborhoods in classic Hodgkin lymphoma.

PURPOSE: Classic Hodgkin lymphoma (cHL) is a B cell lymphoma that occurs primarily in young adults and, less frequently, in elderly individuals. A hallmark of cHL is the exceptional scarcity (1-5%) of the malignant Hodgkin Reed-Sternberg (HRS) cells within a network of non-malignant immune cells. Molecular determinants governing the relationship between HRS cells and their proximal microenvironment remain largely unknown. EXPERIMENTAL DESIGN: We performed spatially resolved multiplexed protein imaging and transcriptomic sequencing to characterize HRS cell states, cellular neighborhoods, and gene expression signatures of 23.6 million cells from 36 newly diagnosed Epstein-Barr virus (EBV) positive and EBV-negative cHL tumors. RESULTS: We show that MHC-I expression on HRS cells is associated with immune inflamed neighborhoods containing CD8+ T cells, MHC-II+ macrophages, and immune checkpoint expression (i.e., PD-1 and VISTA). We identified spatial clustering of HRS cells, consistent with the syncytial variant of cHL, and its association with T cell excluded neighborhoods in a subset of EBV-negative tumors. Finally, a subset of both EBV-positive and EBV-negative tumors contained regulatory T cells high neighborhoods harboring HRS cells with augmented proliferative capacity. CONCLUSIONS: Our study links HRS cell properties with distinct immunophenotypes and potential immune escape mechanisms in cHL.

Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation.

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants is exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupt broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss leads to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrate impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype.

ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease, the pathophysiology and genetic basis of which are incompletely understood. Using a forward genetic screen in multiplex families with systemic lupus erythematosus (SLE) we identified an association between SLE and compound heterozygous deleterious variants in the non-receptor tyrosine kinases (NRTKs) ACK1 and BRK. Experimental blockade of ACK1 or BRK increased circulating autoantibodies in vivo in mice and exacerbated glomerular IgG deposits in an SLE mouse model. Mechanistically, non-receptor tyrosine kinases (NRTKs) regulate activation, migration, and proliferation of immune cells. We found that the patients' ACK1 and BRK variants impair efferocytosis, the MERTK-mediated anti-inflammatory response to apoptotic cells, in human induced Pluripotent Stem Cell (hiPSC)-derived macrophages, which may contribute to SLE pathogenesis. Overall, our data suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis in macrophages.

Antigen escape as a shared mechanism of resistance to BCMA-directed therapies in multiple myeloma.

B-cell maturation antigen (BCMA)-targeting therapeutics have dramatically improved outcomes in relapsed/refractory multiple myeloma (RRMM). However, whether the mechanisms of resistance between these therapies are shared and how the identification of such mechanisms before therapy initiation could refine clinical decision-making remains undefined. We analyzed outcomes for 72 RRMM patients treated with teclistamab, a CD3 x BCMA bispecific antibody (BsAb), 42% (30/72) of whom had prior BCMA-directed therapy exposure. Malignant plasma cell BCMA expression was present in all BCMA therapy-naïve patients. Prior therapy-mediated loss of plasma cell BCMA expression before teclistamab treatment, measured by immunohistochemistry, was observed in 3 patients, none of whom responded to teclistamab, and one of whom also did not respond to ciltacabtagene autoleucel. Whole exome sequencing of tumor DNA from one patient revealed biallelic loss of TNFRSF17 following treatment with belantamab mafodotin. Low-to-undetectable peripheral blood soluble BCMA levels correlated with the absence of BCMA expression by bone marrow plasma cells. Thus, although rare, loss of BCMA expression following TNFRSF17 gene deletions can occur following any BCMA-directed therapy and prevents response to subsequent anti-BCMA-directed treatments, underscoring the importance of verifying the presence of a target antigen.

Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer.

Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance.

Childhood cancer mutagenesis caused by transposase-derived PGBD5.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

A microglia clonal inflammatory disorder in Alzheimer's Disease.

Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however few studies have investigated its role in neurodegenerative processes such as Alzheimer's Disease (AD). Here we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in human, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients.

PDGFRß Signaling Cooperates with ß-Catenin to Modulate c-Abl and Biologic Behavior of Desmoid-Type Fibromatosis.

PURPOSE: This study sought to identify ß-catenin targets that regulate desmoid oncogenesis and determine whether external signaling pathways, particularly those inhibited by sorafenib (e.g., PDGFRß), affect these targets to alter natural history or treatment response in patients. EXPERIMENTAL DESIGN: In vitro experiments utilized primary desmoid cell lines to examine regulation of ß-catenin targets. Relevance of results was assessed in vivo using Alliance trial A091105 correlative biopsies. RESULTS: CTNNB1 knockdown inhibited hypoxia-regulated gene expression in vitro and reduced levels of HIF1α protein. ChIP-seq identified ABL1 as a ß-catenin transcriptional target that modulated HIF1α and desmoid cell proliferation. Abrogation of either CTNNB1 or HIF1A inhibited desmoid cell-induced VEGFR2 phosphorylation and tube formation in endothelial cell co-cultures. Sorafenib inhibited this activity directly but also reduced HIF1α protein expression and c-Abl activity while inhibiting PDGFRß signaling in desmoid cells. Conversely, c-Abl activity and desmoid cell proliferation were positively regulated by PDGF-BB. Reduction in PDGFRß and c-Abl phosphorylation was commonly observed in biopsy samples from patients after treatment with sorafenib; markers of PDGFRß/c-Abl pathway activation in baseline samples were associated with tumor progression in patients on the placebo arm and response to sorafenib in patients receiving treatment. CONCLUSIONS: The ß-catenin transcriptional target ABL1 is necessary for proliferation and maintenance of HIF1α in desmoid cells. Regulation of c-Abl activity by PDGF signaling and targeted therapies modulates desmoid cell proliferation, thereby suggesting a reason for variable biologic behavior between tumors, a mechanism for sorafenib activity in desmoids, and markers predictive of outcome in patients.

Immune-Related Colitis Is Associated with Fecal Microbial Dysbiosis and Can Be Mitigated by Fecal Microbiota Transplantation.

Colitis induced by treatment with immune-checkpoint inhibitors (ICI), termed irColitis, is a substantial cause of morbidity complicating cancer treatment. We hypothesized that abnormal fecal microbiome features would be present at the time of irColitis onset and that restoring the microbiome with fecal transplant from a healthy donor would mitigate disease severity. Herein, we present fecal microbiota profiles from 18 patients with irColitis from a single center, 5 of whom were treated with healthy-donor fecal microbial transplantation (FMT). Although fecal samples collected at onset of irColitis had comparable α-diversity to that of comparator groups with gastrointestinal symptoms, irColitis was characterized by fecal microbial dysbiosis. Abundances of Proteobacteria were associated with irColitis in multivariable analyses. Five patients with irColitis refractory to steroids and biologic anti-inflammatory agents received healthy-donor FMT, with initial clinical improvement in irColitis symptoms observed in four of five patients. Two subsequently exhibited recurrence of irColitis symptoms following courses of antibiotics. Both received a second "salvage" FMT that was, again, followed by clinical improvement of irColitis. In summary, we observed distinct microbial community changes that were present at the time of irColitis onset. FMT was followed by clinical improvements in several cases of steroid- and biologic-agent-refractory irColitis. Strategies to restore or prevent microbiome dysbiosis in the context of immunotherapy toxicities should be further explored in prospective clinical trials.

Tissue-specific features of the T cell repertoire after allogeneic hematopoietic cell transplantation in human and mouse.

T cells are the central drivers of many inflammatory diseases, but the repertoire of tissue-resident T cells at sites of pathology in human organs remains poorly understood. We examined the site-specificity of T cell receptor (TCR) repertoires across tissues (5 to 18 tissues per patient) in prospectively collected autopsies of patients with and without graft-versus-host disease (GVHD), a potentially lethal tissue-targeting complication of allogeneic hematopoietic cell transplantation, and in mouse models of GVHD. Anatomic similarity between tissues was a key determinant of TCR repertoire composition within patients, independent of disease or transplant status. The T cells recovered from peripheral blood and spleens in patients and mice captured a limited portion of the TCR repertoire detected in tissues. Whereas few T cell clones were shared across patients, motif-based clustering revealed shared repertoire signatures across patients in a tissue-specific fashion. T cells at disease sites had a tissue-resident phenotype and were of donor origin based on single-cell chimerism analysis. These data demonstrate the complex composition of T cell populations that persist in human tissues at the end stage of an inflammatory disorder after lymphocyte-directed therapy. These findings also underscore the importance of studying T cell in tissues rather than blood for tissue-based pathologies and suggest the tissue-specific nature of both the endogenous and posttransplant T cell landscape.

A transposase-derived gene required for human brain development.

DNA transposable elements and transposase-derived genes are present in most living organisms, including vertebrates, but their function is largely unknown. PiggyBac Transposable Element Derived 5 (PGBD5) is an evolutionarily conserved vertebrate DNA transposase-derived gene with retained nuclease activity in cells. Vertebrate brain development is known to be associated with prominent neuronal cell death and DNA breaks, but their causes and functions are not well understood. Here, we show that PGBD5 contributes to normal brain development in mice and humans, where its deficiency causes disorder of intellectual disability, movement and seizures. In mice, Pgbd5 is required for the developmental induction of post-mitotic DNA breaks and recurrent somatic genome rearrangements in neurons. Together, these studies nominate PGBD5 as the long-hypothesized neuronal DNA nuclease required for brain function in mammals.

Moving Spatially Resolved Multiplexed Protein Profiling toward Clinical Oncology.

The shift in cancer therapy from broadly cytotoxic agents toward "personalized" treatments that target specific alterations in each patient's tumor requires diagnostic pathology approaches that are quantitative and biospecimen-friendly. Novel multiplexed antibody-based imaging technologies can measure single-cell expression of over 60 proteins in intact tumor sections and hold promise for clinical oncology.

Overcoming Barriers to Tumor Genomic Profiling through Direct-to-Patient Outreach.

PURPOSE: To overcome barriers to genomic testing for patients with rare cancers, we initiated a program to offer free clinical tumor genomic testing worldwide to patients with select rare cancer subtypes. EXPERIMENTAL DESIGN: Patients were recruited through social media outreach and engagement with disease-specific advocacy groups, with a focus on patients with histiocytosis, germ cell tumors (GCT), and pediatric cancers. Tumors were analyzed using the MSK-IMPACT next-generation sequencing assay with the return of results to patients and their local physicians. Whole-exome recapture was performed for female patients with GCTs to define the genomic landscape of this rare cancer subtype. RESULTS: A total of 333 patients were enrolled, and tumor tissue was received for 288 (86.4%), with 250 (86.8%) having tumor DNA of sufficient quality for MSK-IMPACT testing. Eighteen patients with histiocytosis have received genomically guided therapy to date, of whom 17 (94%) have had clinical benefit with a mean treatment duration of 21.7 months (range, 6-40+). Whole-exome sequencing of ovarian GCTs identified a subset with haploid genotypes, a phenotype rarely observed in other cancer types. Actionable genomic alterations were rare in ovarian GCT (28%); however, 2 patients with ovarian GCTs with squamous transformation had high tumor mutational burden, one of whom had a complete response to pembrolizumab. CONCLUSIONS: Direct-to-patient outreach can facilitate the assembly of cohorts of rare cancers of sufficient size to define their genomic landscape. By profiling tumors in a clinical laboratory, results could be reported to patients and their local physicians to guide treatment. See related commentary by Desai and Subbiah, p. 2339.

The endogenous repertoire harbors self-reactive CD4+ T cell clones that adopt a follicular helper T cell-like phenotype at steady state.

The T cell repertoire of healthy mice and humans harbors self-reactive CD4+ conventional T (Tconv) cells capable of inducing autoimmunity. Using T cell receptor profiling paired with in vivo clonal analysis of T cell differentiation, we identified Tconv cell clones that are recurrently enriched in non-lymphoid organs following ablation of Foxp3+ regulatory T (Treg) cells. A subset of these clones was highly proliferative in the lymphoid organs at steady state and exhibited overt reactivity to self-ligands displayed by dendritic cells, yet were not purged by clonal deletion. These clones spontaneously adopted numerous hallmarks of follicular helper T (TFH) cells, including expression of Bcl6 and PD-1, exhibited an elevated propensity to localize within B cell follicles at steady state, and produced interferon-γ in non-lymphoid organs following sustained Treg cell depletion. Our work identifies a naturally occurring population of self-reactive TFH-like cells and delineates a previously unappreciated fate for self-specific Tconv cells.

Innate immune signaling drives late cardiac toxicity following DNA-damaging cancer therapies.

Late cardiac toxicity is a potentially lethal complication of cancer therapy, yet the pathogenic mechanism remains largely unknown, and few treatment options exist. Here we report DNA-damaging agents such as radiation and anthracycline chemotherapies inducing delayed cardiac inflammation following therapy due to activation of cGAS- and STING-dependent type I interferon signaling. Genetic ablation of cGAS-STING signaling in mice inhibits DNA damage-induced cardiac inflammation, rescues late cardiac functional decline, and prevents death from cardiac events. Treatment with a STING antagonist suppresses cardiac interferon signaling following DNA-damaging therapies and effectively mitigates cardiac toxicity. These results identify a therapeutically targetable, pathogenic mechanism for one of the most vexing treatment-related toxicities in cancer survivors.

Molecular Characterization of Acquired Resistance to KRASG12C-EGFR Inhibition in Colorectal Cancer.

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance. SIGNIFICANCE: Clinical resistance to KRASG12C-EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches. This article is highlighted in the In This Issue feature, p. 1.

Increased tumor glycolysis is associated with decreased immune infiltration across human solid tumors.

Response to immunotherapy across multiple cancer types is approximately 25%, with some tumor types showing increased response rates compared to others (i.e. response rates in melanoma and non-small cell lung cancer (NSCLC) are typically 30-60%). Patients whose tumors are resistant to immunotherapy often lack high levels of pre-existing inflammation in the tumor microenvironment. Increased tumor glycolysis, acting through glucose deprivation and lactic acid accumulation, has been shown to have pleiotropic immune suppressive effects using in-vitro and in-vivo models of disease. To determine whether the immune suppressive effect of tumor glycolysis is observed across human solid tumors, we analyzed glycolytic and immune gene expression patterns in multiple solid malignancies. We found that increased expression of a glycolytic signature was associated with decreased immune infiltration and a more aggressive disease across multiple tumor types. Radiologic and pathologic analysis of untreated estrogen receptor (ER)-negative breast cancers corroborated these observations, and demonstrated that protein expression of glycolytic enzymes correlates positively with glucose uptake and negatively with infiltration of CD3+ and CD8+ lymphocytes. This study reveals an inverse relationship between tumor glycolysis and immune infiltration in a large cohort of multiple solid tumor types.

FOXA1 repression drives lineage plasticity and immune heterogeneity in bladder cancers with squamous differentiation.

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors.

Ordered and deterministic cancer genome evolution after p53 loss.

Although p53 inactivation promotes genomic instability1 and presents a route to malignancy for more than half of all human cancers2,3, the patterns through which heterogenous TP53 (encoding human p53) mutant genomes emerge and influence tumorigenesis remain poorly understood. Here, in a mouse model of pancreatic ductal adenocarcinoma that reports sporadic p53 loss of heterozygosity before cancer onset, we find that malignant properties enabled by p53 inactivation are acquired through a predictable pattern of genome evolution. Single-cell sequencing and in situ genotyping of cells from the point of p53 inactivation through progression to frank cancer reveal that this deterministic behaviour involves four sequential phases-Trp53 (encoding mouse p53) loss of heterozygosity, accumulation of deletions, genome doubling, and the emergence of gains and amplifications-each associated with specific histological stages across the premalignant and malignant spectrum. Despite rampant heterogeneity, the deletion events that follow p53 inactivation target functionally relevant pathways that can shape genomic evolution and remain fixed as homogenous events in diverse malignant populations. Thus, loss of p53-the 'guardian of the genome'-is not merely a gateway to genetic chaos but, rather, can enable deterministic patterns of genome evolution that may point to new strategies for the treatment of TP53-mutant tumours.