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Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance 1 , 2 . The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity 3 – 6 . However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8 + T cell function by enhancing JAK–STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes. An orally bioavailable small-molecule active-site inhibitor of the phosphatases PTPN2 and PTPN1, ABBV-CLS-484, demonstrates immunotherapeutic efficacy in mouse models of cancer resistant to PD-1 blockade.

During steady state, CD4 T helper (Th) cells reprogram to cytotoxic T lymphocytes (CTLs) in the small intestinal epithelium. In the present study, CD4 CTLs were evaluated for their potential to promote tolerance during steady state as well as their ability to fend off enteric pathogens during infection. The CD4 T cell adoptive transfer model of colitis was used to determine if CTLs may represent a strategy of avoiding CD4 Th-mediated inflammation. Using both an in vitro differentiation model as well as transgenic models, where CD4 T cell fates are forced into predetermined phenotypes, the conversion of Th to CTL was found to mitigate disease pathology and promote tolerogenic conditions. After defining the role of CD4 T cells during steady state, we then assessed the ability of CD4 CTLs to respond to enteric pathogens. Pre-existing CD4 CTLs were found to have the capacity to kill Salmonella enterica-infected cells, thereby preserving the integrity of the barrier and preventing bacterial dissemination. Furthermore, we identified IL-15/IL-15Ra complex trans-presentation as a mechanism of inciting CD4 CTLs to become active killers. Functionally, CD4 CTLs represent a strategy of the immune system to fortify the epithelium with quiescent but primed CTLs that can provide rapid immunity during enteric infection. The generation of CD4 CTLs during steady state and their functional relevance during infection is a novel strategy of mucosal immunity that can be defined as ‘protective tolerance’.

BackgroundImmune checkpoint blockade is effective for a subset of patients across many cancers, but most patients are refractory to current immunotherapies and new approaches are needed to overcome resistance.1 2 The protein tyrosine phosphatase PTPN2 and the closely related PTPN1 are central regulators of inflammation, and their genetic deletion in either tumor cells or host immune cells promotes anti-tumor immunity.3–6 However, phosphatases are challenging drug targets and in particular, the active site has been considered undruggable. Here, we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2/N1 active site inhibitor.MethodsIn this study, we characterize AC484 and evaluate its effects in vitro and in vivo. We conduct in vitro experiments to investigate the interferon response and the activation and function of various immune cell subsets in response to AC484. We employ murine cancer models resistant to PD-1 blockade and assess the anti-tumor efficacy of AC484 monotherapy in these models. Additionally, through single-cell transcriptional profiling of tumor-infiltrating immune cells, we examine the transcriptional and functional effects of AC484 treatment, with a focus on CD8+ T cells.ResultsAC484 treatment demonstrates the ability to amplify the response to interferon and enhance the activation and function of multiple immune cell subsets in vitro. In murine cancer models resistant to PD-1 blockade, monotherapy AC484 treatment generates robust anti-tumor immunity. Transcriptomic and functional analyses of tumor-infiltrating immune cells reveal that AC484 treatment elicits broad effects on myeloid and lymphoid compartments, particularly influencing CD8+ T cells. Surprisingly, we find that AC484 treatment induces a unique transcriptional state in CD8+ T cells mediated by enhanced JAK-STAT signaling, whereby T cells display a highly cytotoxic effector profile, increased memory signatures, and reduced exhaustion and dysfunction.ConclusionsOur results demonstrate that oral administration of small molecule inhibitors of PTPN2/N1 can induce potent anti-tumor immunity. PTPN2/N1 inhibitors offer a promising new strategy for cancer immunotherapy and are currently being evaluated clinically in patients with advanced solid tumors (NCT04777994). More broadly, our study shows that small molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics targeting this important class of enzymes.ReferencesHugo W, et al. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell. 2017;168:542.Fares CM, Van Allen EM, Drake CG, Allison JP, Hu-Lieskovan S. Mechanisms of resistance to immune checkpoint blockade: why does checkpoint inhibitor immunotherapy not work for all patients? Am Soc Clin Oncol Educ Book. 2019;39:147–164.Manguso RT, et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature. 2017;547:413–418.Wiede F, et al. PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours. EMBO J. 2020;39:e103637.LaFleur MW, et al. PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity. Nat. Immunol. 2019;20:1335–1347.Flosbach M, et al. PTPN2 deficiency enhances programmed T cell expansion and survival capacity of activated T cells. Cell Rep. 2020;32:107957.Ethics ApprovalThe protocol, under which human blood samples were acquired, was approved by and is reviewed on an annual basis by WCG IRB (Puyallup, Washington). WCG IRB is in full compliance with the Good Clinical Practices as defined under the U.S. Food and Drug Administration (FDA) Regulations, U.S. Department of Health and Human Services (HHS) regulations and the International Conference on Harmonisation (ICH) Guidelines. All human research participants signed informed consent forms. All animal studies at AbbVie, were reviewed and approved by AbbVie’s Institutional Animal Care and Use Committee and in compliance with the NIH Guide for Care and Use of Laboratory Animals guidelines. Animal studies were conducted in an AAALAC accredited program where veterinary care and oversight was provided to ensure appropriate animal care. All in vivo studies conducted at the Broad Institute were approved by the Broad Institute IACUC committee and mice were housed in a specific-pathogen free facility. All in vivo studies at Calico were conducted according to protocols approved by the Calico Institutional Animal Care and Use Committee.

The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effect s ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.

Cancer therapy-related cardiovascular toxicity (CTR-CVT) is now recognised as one of the leading causes of long-term morbidity and mortality in cancer patients. To date, potential overlapping cardiotoxicity mechanism(s) across different chemotherapeutic classes have not been elucidated. Doxorubicin, an anthracycline, and Carfilzomib, a proteasome inhibitor, are both known to cause heart failure in some patients. Given this common cardiotoxic effect of these chemotherapies, we aimed to investigate differential and common mechanism(s) associated with Doxorubicin and Carfilzomib-induced cardiac dysfunction. Primary human cardiomyocyte-like cells (HCM-ls) were treated with 1 µM of either Doxorubicin or Carfilzomib for 72 h. Both Doxorubicin and Carfilzomib induced a significant reduction in HCM cell viability and cell damage. DNA methylation analysis performed using MethylationEPIC array showed distinct and common changes induced by Doxorubicin and Carfilzomib (10,270 or approximately 12.9% of the DMPs for either treatment overlapped). RNA-seq analyses identified 5,643 differentially expressed genes (DEGs) that were commonly dysregulated for both treatments. Pathway analysis revealed that the PI3K-Akt signalling pathway was the most significantly enriched pathway with common DEGs, shared between Doxorubicin and Carfilzomib. We identified that there are shared cardiotoxicity mechanisms for Doxorubicin and Carfilzomib pathways that can be potential therapeutic targets for treatments across 2 classes of anti-cancer agents.

Graphene oxide (GO) and reduced graphene oxide (rGO) are promising materials for sustainable energy storage due to their excellent conductivity, large surface area, and chemical stability. However, conventional methods of reducing GO often involve toxic chemicals, raising environmental concerns. This study introduces a green solvothermal method to synthesize rGO using L-ascorbic acid and ammonia solution as reducing agents, producing a material referred to as G-rGO for supercapacitor applications. Theoretical calculations suggest that the reduction process significantly enhances the electronic properties and structural integrity of the material. X-ray diffraction (XRD), scanning electron microscopy (SEM), Raman spectroscopy, and energy-dispersive X-ray (EDX) analysis confirmed the reduction of GO to G-rGO. Electrochemical tests, including cyclic voltammetry (CV), galvanostatic charge/discharge (GCD), and electrochemical impedance spectroscopy (EIS), demonstrated that G-rGO performed exceptionally well. It exhibited a high specific capacitance of 401 F/g at 1 A/g and impressive cycle stability of 93% after 10,000 cycles. Theoretical results indicate that these characteristics are attributed to enhanced charge transport and a favorable surface area-to-volume ratio. Additionally, G-rGO achieved an energy density of 200.5 Wh/kg at a power density of 2.5 W/kg, underscoring its potential as an eco-friendly, high-performance electrode material for sustainable energy storage.

Squamous cell carcinomas (SqCC) of the aerodigestive tract have similar etiological risk factors. Although genetic risk variants for individual cancers have been identified, an agnostic, genome-wide search for shared genetic susceptibility has not been performed. To identify novel and pleotropic SqCC risk variants, we performed a meta-analysis of GWAS data on lung SqCC (LuSqCC), oro/pharyngeal SqCC (OSqCC), laryngeal SqCC (LaSqCC) and esophageal SqCC (ESqCC) cancers, totaling 13,887 cases and 61,961 controls of European ancestry. We identified one novel genome-wide significant ( P meta <5x10 -8 ) aerodigestive SqCC susceptibility loci in the 2q33.1 region (rs56321285, TMEM273 ). Additionally, three previously unknown loci reached suggestive significance ( P meta <5x10 -7 ): 1q32.1 (rs12133735, near MDM4 ), 5q31.2 (rs13181561, TMEM173 ) and 19p13.11 (rs61494113, ABHD8) . Multiple previously identified loci for aerodigestive SqCC also showed evidence of pleiotropy in at least another SqCC site, these include: 4q23 ( ADH1B ), 6p21.33 ( STK19 ), 6p21.32 ( HLA-DQB1 ), 9p21.33 ( CDKN2B-AS1 ) and 13q13.1( BRCA2 ). Gene-based association and gene set enrichment identified a set of 48 SqCC-related genes rel to DNA damage and epigenetic regulation pathways. Our study highlights the importance of cross-cancer analyses to identify pleiotropic risk loci of histology-related cancers arising at distinct anatomical sites.