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Recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with a significant unmet need for enhancing immunotherapy response given current modest efficacy. Here, we perform an in vivo CRISPR screen in an HNSCC mouse model to identify immune evasion genes. We identify several regulators of immune checkpoint blockade (ICB) response, including the ubiquitin C-terminal hydrolase 5 (UCHL5). Loss of Uchl5 in tumors increases CD8 + T cell infiltration and improved ICB responses. Uchl5 deficiency attenuates extracellular matrix (ECM) production and epithelial-mesenchymal-transition (EMT) transcriptional programs, which contribute to stromal desmoplasia, a histologic finding we describe as associated with reduced anti-PD1 response in human HNSCCs. COL17A1, a collagen highly and specifically expressed in HNSCC, mediates in part Uchl5 -mediated immune evasion. Our findings suggest an unappreciated role for UCHL5 in promoting EMT in HNSCC and highlight ECM modulation as a strategy to improve immunotherapy responses. UCHL5 is a deubiquitinating enzyme that cleaves Lys-48-linked polyubiquitin chains. Here, the authors discover through in-vivo CRISPR-Cas9 screens that Uchl5 is involved in immune evasion and modulation of extracellular matrix deposition in head and neck squamous cell carcinoma.

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.

Approximately 14.4 million Americans are experiencing alcohol use disorder (AUD) and about two-thirds of people who experience drug addiction will relapse, highlighting the need to develop novel and effective treatments. Glucagon-like peptide-1 (GLP-1) is a peptide hormone implicated in the mesocorticolimbic reward system and has become a peptide of interest with respect to its putative inhibitory effects on drug reward. In order to further develop treatments for those diagnosed with AUD, the interplay between GLP-1 receptor signaling and ethanol consumption must be elucidated. In the present study, we investigated the ability of the GLP-1 analogue, exendin-4 (Ex-4), to alter alcohol intake and operant responding for sucrose pellets in order to further understand the role of this compound in mediating reward. We selected multiple sites throughout the prosencephalic and mesencephalic regions of the brain, where we directly administered various doses of Ex-4 to male Sprague Dawley rats. In alcohol investigations, we utilized a two-bottle choice intermittent access protocol. In separate groups of rats, we adopted an operant paradigm in order to examine the effect of Ex-4 on motivated responding for palatable food. Results indicated that GLP-1 receptor signaling effectively suppressed voluntary alcohol intake when injected into the ventral tegmental area (VTA), the accumbens core (NAcC) and shell (NAcS), the dorsomedial hippocampus (DMHipp), and the lateral hypothalamus (LH), which are all structures linked to brain reward mechanisms. The arcuate nucleus (ARcN) and the paraventricular nucleus (PVN) of the hypothalamus were unresponsive, as was the basolateral amygdala (BLA). However, Ex-4 treatment into the ArcN and PVN suppressed operant responding for sucrose pellets. In fact, the VTA, NAcC, NAcS, LH, and the DMHipp all showed comparable suppression of sucrose responding. Overall, our findings suggest that these central structures are implicated in brain reward circuitry, including alcohol and appetitive motivation, which may be mediated by GLP-1 receptor mechanisms. GLP-1, therefore, may play a critical role in modifying addictive behaviors via activation of multiple GLP-1 systems throughout the brain.

BackgroundThe tyrosine phosphatases PTPN2 and PTPN1 negatively regulate several signaling pathways in immune and tumor cells. We previously demonstrated that oral administration of our recently discovered active site PTPN2/N1 small molecule inhibitor ABBV-CLS-484 (AC-484) promotes anti-tumor immunity in several syngeneic mouse tumor models. AC-484 improves T cell activation and function upon TCR stimulation and enhances dendritic cell and macrophage activity in vitro consistent with prior findings in PTPN2 or PTPN1 genetically deficient T cells and myeloid cells. However, a role for PTPN2 or PTPN1 in NK cells has not been previously described. NK cells are essential for eliminating tumors that typically evade the adaptive T cell response and are critically important to control metastasis formation. Given the role of inhibitory signaling pathways, we hypothesized that PTPN2 and PTPN1 may also negatively regulate NK activity and therefore AC-484 should enhance NK cell function and NK-mediated anti-tumor immunity.MethodsTo understand the impact of AC-484 on NK cells, we employed cytotoxicity assays in vitro and utilized immunophenotyping and single cell RNA sequencing of tumor-infiltrating immune cells isolated from mouse syngeneic tumor models. We also assessed the contribution of NK cells to AC-484-mediated efficacy in subcutaneous primary tumor and spontaneous lung metastasis formation models.ResultsAC-484 treatment enhanced NK cell function and NK-mediated tumor cell killing in vitro. Consistent with these findings, immunophenotyping and single-cell RNAseq analyses demonstrated that in vivo AC-484 therapy increased NK cell abundance and activation in mouse tumor models with varying responsiveness to immune checkpoint blockade. Further, in tumor models that do not rely on T cells for tumor control such as those with MHCI or Jak1 deficiency, AC-484 therapy improved NK-mediated efficacy. In addition to controlling primary tumors, AC-484 also potently prevented lung metastasis formation in the B16F10 intravenous and the 4T1 orthotopic breast cancer models in an NK cell-dependent manner.ConclusionsHere, we describe for the first time a role for PTPN2 and PTPN1 in NK cells. Our findings suggest that AC-484 can both control primary tumors and prevent tumor metastasis in an NK cell-dependent manner. We further show that AC-484 treatment overcomes various common immune evasion mechanisms developed by tumors, including those acquired via mutations in Beta-2-microglobulin, HLA, and JAK1/2. These findings, along with our previous reports, underscore how AC-484 significantly promotes anti-tumor efficacy through a multifaceted mechanism by sensitizing tumor cells to inflammation and enhancing the activity of a variety of immune subsets.Ethics ApprovalHumanHuman blood samples were acquired through the internal AbbVie Inc’s blood donation program in accordance with AbbVie’s Occupational Safety and Health Administration protocols or healthy donors from Stanford University.AnimalsAll in vivo experiments conducted at AbbVie were in compliance with the NIH Guide for Care and Use of Laboratory Animals guidelines in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). All in vivo studies conducted at the Broad Institute or Calico Life Sciences were approved by the respective IACUC committees.DisclosuresC.H.P., I.S., Y.L., M.N.P., and J.D.P. are employees of Calico Life Sciences LLC. K.A.M., K.L.K., J.D.A, K.H., J.T.K., A.W.S., K.M.H, J.M.F, P.R.K, and C.K.B. are employees of AbbVie Inc. H.E., A.J.M., P.T., O.A, K.C., K.O., K.B.Y., and R.T.M. are employees of the Broad Institute. The laboratory of R.T.M. at the Broad Institute receives research funding from Calico Life Sciences LLC. R.T.M. has served as a consultant for Bristol Myers Squibb and receives research funding from Calico Life Sciences LLC.

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.

Treatment with immune checkpoint inhibitors induces remarkable clinical responses in several cancer types. However, most cancer patients fail to respond to immunotherapy, and patients who initially respond often exhibit acquired resistance. Understanding the universe of immune evasion strategies will enable design of more effective immunotherapies. Here, we identify genes that drive immune evasion using genome-scale in vivo CRISPR gain-of-function screens in tumors treated with anti-PD-1 antibodies and found that the transcription factor CREB5 drives immune checkpoint blockade resistance. Using transcriptional profiling and functional studies, we show that CREB5 promotes a mesenchymal-like phenotype in melanoma characterized by upregulation of extracellular matrix genes including collagen and collagen-stabilizing factors. Using engineered tumor models and knockout mice, we found that immunotherapy resistance is functionally mediated by tumor-intrinsic collagen deposition. Collagen is the major ligand for the inhibitory receptor LAIR1, broadly expressed on T cells, B cells, NK cells, and myeloid cells. Deletion of LAIR1 in mice or overexpression of the decoy receptor LAIR2 in tumors abrogated the resistance induced by CREB5 overexpression, demonstrating that collagen-LAIR1 inhibitory signaling drives resistance to immune checkpoint inhibitors. These observations define a transcriptional program that remodels the tumor microenvironment to promote immunotherapy resistance via extracellular matrix deposition and indicates that targeting this pathway may enhance immunotherapy efficacy. One-Sentence Summary: In vivo gain-of-function screening in immunotherapy-treated mice reveals a transcription factor, Creb5, that drives the mesenchymal state in melanoma and facilitates immune escape by promoting tumor-intrinsic collagen matrix deposition.