ATR inhibition facilitates targeting of leukemia dependence on convergent nucleotide biosynthetic pathways.

Leukemia cells rely on two nucleotide biosynthetic pathways, de novo and salvage, to produce dNTPs for DNA replication. Here, using metabolomic, proteomic, and phosphoproteomic approaches, we show that inhibition of the replication stress sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) reduces the output of both de novo and salvage pathways by regulating the activity of their respective rate-limiting enzymes, ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), via distinct molecular mechanisms. Quantification of nucleotide biosynthesis in ATR-inhibited acute lymphoblastic leukemia (ALL) cells reveals substantial remaining de novo and salvage activities, and could not eliminate the disease in vivo. However, targeting these remaining activities with RNR and dCK inhibitors triggers lethal replication stress in vitro and long-term disease-free survival in mice with B-ALL, without detectable toxicity. Thus the functional interplay between alternative nucleotide biosynthetic routes and ATR provides therapeutic opportunities in leukemia and potentially other cancers.Leukemic cells depend on the nucleotide synthesis pathway to proliferate. Here the authors use metabolomics and proteomics to show that inhibition of ATR reduced the activity of these pathways thus providing a valuable therapeutic target in leukemia

T cell antigen discovery via signaling and antigen-presenting bifunctional receptors

CD8^+ T cells recognize and eliminate tumors in an antigen-specific manner. Despite progress in characterizing the antitumor T cell repertoire and function, the identification of target antigens remains a challenge. Here we describe the use of chimeric receptors called signaling and antigen-presenting bifunctional receptors (SABRs) in a cell-based platform for T cell receptor (TCR) antigen discovery. SABRs present an extracellular complex comprising a peptide and major histocompatibility complex (MHC), and induce intracellular signaling via a TCR-like signal after binding with a cognate TCR. We devised a strategy for antigen discovery using SABR libraries to screen thousands of antigenic epitopes. We validated this platform by identifying the targets recognized by public TCRs of known specificities. Moreover, we extended this approach for personalized neoantigen discovery

T cell antigen discovery via trogocytosis

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide–major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide–MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy

PD-1 Blockade Expands Intratumoral Memory T Cells

Tumor responses to PD-1 blockade therapy are mediated by T cells, which we characterized in 102 tumor biopsies obtained from 53 patients treated with pembrolizumab, an antibody to PD-1. Biopsies were dissociated and single cell infiltrates were analyzed by multicolor flow cytometry using two computational approaches to resolve the leukocyte phenotypes at the single cell level. There was a statistically significant increase in the frequency of T cells in patients who responded to therapy. The frequency of intratumoral B cells and monocytic myeloid-derived suppressor cells (moMDSCs) significantly increased in patients’ biopsies taken on treatment. The percentage of cells with a T regulatory phenotype, monocytes, and NK cells did not change while on PD-1 blockade therapy. CD8(+) T memory cells were the most prominent phenotype that expanded intratumorally on therapy. However, the frequency of CD4(+) T effector memory cells significantly decreased on treatment, whereas CD4(+) T effector cells significantly increased in nonresponding tumors on therapy. In peripheral blood, an unusual population of blood cells expressing CD56 were detected in two patients with regressing melanoma. In conclusion, PD-1 blockade increases the frequency of T cells, B cells, and MDSCs in tumors, with the CD8(+) T effector memory subset being the major T-cell phenotype expanded in patients with a response to therapy

T cell antigen discovery via trogocytosis

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide–major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide–MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy

Sensitive Detection and Analysis of Neoantigen-Specific T Cell Populations from Tumors and Blood

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines

Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma

BACKGROUND: Approximately 75% of objective responses to anti–programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti–PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor–associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.

Identification of Features of Response and Resistance to Anti-Programmed-Death-1 Immunotherapy in Melanoma

Checkpoint blockade immunotherapy takes advantage of an endogenous anti-cancer immune response and promotes tumor killing by altering the balance of signals that control T-cell activity. Dramatic and durable responses have led to FDA approvals for anti-programmed- death-1 (anti-PD1) therapy across a broad range of cancer types, however only a fraction of patients benefit and some with an initial response will later progress. The specific mechanisms of tumor-immune evasion or resistance in this setting are not well established, but understanding them will be critical for accurate prognosis, proper therapy selection, and identification of next- generation drug targets.To investigate mechanistic correlates of anti-PD1 response and resistance in melanoma, we used high-throughput sequencing to analyze genomic, transcriptomic, and T-cell data in whole-tumor biopsies and early passage cell lines from 1) longitudinally sampled lesions from patients with an initial response followed by late progression (acquired resistance) and 2) pre-treatment lesions in cohorts of patients with or without response (primary resistance).Whole exome sequencing from four cases of late acquired resistance revealed evidence of in-situ clonal selection for new homozygous loss-of-function mutations in the interferon-receptor associated kinases JAK1 and JAK2 and in beta-2-microglobulin (B2M, a co-factor required for class I antigen presentation). These JAK mutations abolished sensitivity to interferon-gamma (IFNγ), including IFNγ-induced PD-L1 expression and growth arrest. In complementary work, we evaluated the frequency of interferon insensitivity (or lack of PD-L1 inducibility) and interferon pathway mutations in PD1-therapy na�ve cohorts. Among the handful of JAK homozygous loss-of-function mutations we identified from patients with response data, all were found in non-responders.Two other retrospective cohort studies revealed additional response-correlated insights. Transcriptomic data from pre-treatment biopsies showed enrichment for a coordinated set of genes associated with epithelial-to-mesenchymal transition (EMT), wound-healing, angiogenesis, and hypoxia in PD1 non-responders. By contrast, we found the rare desmoplastic melanoma subtype (DM) was unusually sensitive to anti-PD1 therapy, with a 70% response rate double that of unselected cutaneous melanoma. While often a histological diagnosis, we observed our DM cohort was characterized by a high mutational load and NF1 driver subtype.Finally, we worked with colleagues at Caltech on enabling studies with a new technology for sensitive identification of neo-antigen specific T-cell responses that is optimized for downstream T-cell capture and receptor cloning. Here, the goal was to aid in assessment of the diversity and dynamics of defined tumor antigen-specific T-cells and to allow for their ex-vivo production