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[This corrects the article DOI: 10.1371/journal.pbio.1000428.].

Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1(-/-) DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.

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.

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.

BackgroundAdoptive cellular therapy (ACT) in the form of CAR-T cell therapy has demonstrated potent clinical efficacy in hematological malignancies, however, its success in treating solid tumors has been very limited. The tyrosine phosphatases PTPN2 and PTPN1 negatively regulate several signaling pathways in immune and tumor cells. We previously demonstrated that oral administration of the PTPN2/N1 small molecule inhibitor ABBV-CLS-484 (AC484) promotes anti-tumor immunity in several syngeneic mouse tumor models and is now being explored in a Phase I clinical trial (NCT04777994). ABBV-CLS-484 acts by both enhancing anti-tumor immune responses as well as in a cell intrinsic fashion by enhancing the susceptibility of tumor cells to immune-mediated killing. Considering this dual mechanism of action, we hypothesized that AC484 therapy would enhance CAR-T cell function and sensitize solid tumors to be more sensitive to ACT.MethodsTo test this hypothesis, we tested the ability of AC484 to enhance inhibition of tumor growth in syngeneic murine CAR-T and TCR ACT models to solid tumors. We also utilized RNA sequencing and flow cytometry to phenotype tumor-infiltrating immune and tumor cells post-treatment.ResultsAC484 treatment enhanced ACT efficacy to solid tumors. Consistent with these findings, immunophenotyping analyses demonstrated that in vivo AC484 therapy with CAR-T cells increased donor and endogenous T cell homeostatic proliferation, abundance, cytotoxicity, reprogrammed metabolism, persistence and reduced exhaustion. AC484 also enhanced IFNγ signaling in solid tumors to promote more ICAM, cytokine and chemokine expression to support T cell expansion and recruitment to the tumor.ConclusionsHere, we describe a novel role for the PTPN2/PTPN1 inhibitor AC484 during adoptive cellular therapy. Our findings suggest that AC484 can positively impact both donor and endogenous T cells to eradicate solid tumors. We further show that AC484 treatment overcomes various common immune evasion mechanisms presented by solid tumors for successful ACT. These findings, along with our previous reports, underscore how AC484 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. These data suggest that AC484 therapy can be deployed in the clinic to markedly enhance adoptive T cell therapy for solid tumors.AcknowledgmentsC.H.P., I.S., Y.L., M.N.P., and J.D.P. are employees of Calico Life Sciences LLC. Q.Z., J.H., J.J.E, K.A.M., J.M.F, P.R.K, and C.K.B. are employees of AbbVie Inc.Ethics ApprovalAnimals: All 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 Calico Life Sciences was approved by the respective IACUC committee.

Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1-/- DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here we present the discovery and characterization of a novel pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

The phosphoinositide 3-kinase, p110α, is an essential mediator of insulin signaling and glucose homeostasis. We systematically interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate and completely inhibit its activity. This inhibitory state corresponds to a conformational change of a membrane binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases, MST1/2, using forskolin or epinephrine is associated with phosphorylation and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules. Our study reveals a novel inhibitory pathway of PI3K signaling and a previously unappreciated link between epinephrine and insulin signaling.

Insulin stimulates conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), which mediates downstream cellular responses. PI(4,5)P2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and byphosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Here we show that deletion of the three genes that encode PIP4Ks (PIP4K2A, PIP4K2B and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P2 and a subsequent increase in insulin-stimulated production of PI(3,4,5)P3. Surprisingly, reintroduction of either wildtype or kinase-dead forms of the PIP4Ks restored cellular PI(4,5)P2 levels and insulin stimulation of the PI3K pathway. These effects are explained by an increase in PIP5K activity upon deletion of PIP4Ks, which we demonstrate can suppress PIP5K activity in vitro through a direct binding interaction. Collectively, our work reveals an important non-catalytic function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3 by PI3K enzymes and suggests that pharmacological depletion of PIP4K enzymes using emerging degrader technologies could represent a novel strategy for stimulating insulin signaling.