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T-cell-based immunotherapies are promising treatments for cancer patients. Although durable responses can be achieved in some patients, many patients fail to respond to these therapies, underscoring the need for improvement with combination therapies. From a screen of 850 bioactive compounds, we identify HSP90 inhibitors as candidates for combination with immunotherapy. We show that inhibition of HSP90 with ganetespib enhances T-cell-mediated killing of patient-derived human melanoma cells by their autologous T cells in vitro and potentiates responses to anti-CTLA4 and anti-PD1 therapy in vivo. Mechanistic studies reveal that HSP90 inhibition results in upregulation of interferon response genes, which are essential for the enhanced killing of ganetespib treated melanoma cells by T cells. Taken together, these findings provide evidence that HSP90 inhibition can potentiate T-cell-mediated anti-tumor immune responses, and rationale to explore the combination of immunotherapy and HSP90 inhibitors. Many patients fail to respond to T cell based immunotherapies. Here, the authors, through a high-throughput screening, identify HSP90 inhibitors as a class of preferred drugs for treatment combination with immunotherapy.

The pathway of V(D)J recombination was discovered almost three decades ago. Yet it continues to baffle scientists because of its inherent complexity and the multiple layers of regulation that are required to efficiently generate a diverse repertoire of T and B cells. The non-homologous end-joining (NHEJ) DNA repair pathway is an integral part of the V(D)J reaction, and its numerous players perform critical functions in generating this vast diversity, while ensuring genomic stability. In this review, we summarize the efforts of a number of laboratories including ours in providing the mechanisms of V(D)J regulation with a focus on the NHEJ pathway. This involves discovering new players, unraveling unknown roles for known components, and understanding how deregulation of these pathways contributes to generation of primary immunodeficiencies. A long-standing interest of our laboratory has been to elucidate various mechanisms that control RAG activity. Our recent work has focused on understanding the multiple protein–protein interactions and protein–DNA interactions during V(D)J recombination, which allow efficient and regulated generation of the antigen receptors. Exploring how deregulation of this process contributes to immunodeficiencies also continues to be an important area of research for our group.

BackgroundCurrent immunotherapies fail to provide benefit to tumors poorly infiltrated with T and NK cells. However, activation of myeloid cells and B cells can lead to recruitment of functional T and NK cells turning the ‘cold’ tumor microenvironment (TME) into ‘hot’. CLEC2D is broadly expressed on germinal center B cells, activated primary plasmacytoid DCs (pDC) and tumor associated macrophages (TAMs). Upon internalization, CLEC2D acts as a vehicle to deliver histone/CpG complexes to endosomal TLR9, stimulating an inflammatory response. Furthermore, CLEC2D is the ligand for CD161 which is an immune checkpoint expressed on both T and NK cells, making it a target for additional immunotherapeutic intervention.MethodsWe have developed a fully human anti-CLEC2D monoclonal antibody that binds and triggers receptor internalization on CLEC2D+ cells. Next, we conjugated this antibody to a CpG oligonucleotide to produce an anti-CLEC2D-TLR9-ISAC (Immune Stimulating Antibody Complex) molecule for systemic delivery of TLR9 agonist to CLEC2D+ cells. We have utilized multiple assays to get functional proof of concept (POC) of triggering TLR9 activation in myeloid cells, B cells and pDCs by this ISAC moleculeResultsFirst, we have used THP1-TLR9 reporter cell lines to monitor successful activation of TLR9 pathway. We have shown that anti-CLEC2D-TLR9-ISAC treatment will induce both IRF and NFkB reporter activation over CpG alone. Next, treatment of in vitro generated human pDCs with this ISAC molecule dramatically increased production of IFN-a, a critical cytokine for induction of anti-tumor T cells. To test functionality on primary B cells, we have developed an in vitro assay with CLEC2D expressing primed B cells. Treatment of these B cells with the ISAC molecule results in sustained B cell proliferation and upregulation of co-stimulatory molecules such as CD80, CD86 and CD40 enabling stronger induction of T cell immune responses. Many tumors are highly infiltrated by immunosuppressive macrophages. We have shown that there is CLEC2D expression on TAMs within tumors, as well as on in vitro generated TAM like macrophages. Treatment of TAMs with the ISAC reversed TAM mediated suppression of T cell proliferation and activation, indicating that TLR9 agonism can reprogram TAMs towards inflammatory state. Finally, we show that treatment of normal human PBMCs with anti-CLEC2D-ISAC molecules did not trigger release of inflammatory cytokines providing preliminary safety data.ConclusionsOverall, anti-CLEC2D antibody is a novel molecule that can effectively deliver CpG to endosomal TLR9. This results in activation of both myeloid and B cells enabling induction of sustained T cell immunity.

Treatment options are limited in well differentiated (WD) and dedifferentiated (DD) retroperitoneal liposarcoma. We sought to study the intratumoral adaptive immune response and explore the potential feasibility of immunotherapy in this disease. Tumor-infiltrating lymphocytes (TILs) were isolated from fresh surgical specimens and analyzed by flow cytometry for surface marker expression. Previously reported immune cell aggregates known as tertiary lymphoid structures (TLS) were further characterized by immunohistochemistry. In all fresh tumors, TILs were found. The majority of TILs were CD4 T cells; however cytotoxic CD8 T cells were also seen (average: 20% of CD3 T cells). Among CD8 T cells, 65% expressed the immune checkpoint molecule PD-1. Intratumoral TLS may be sites of antigen presentation as DC-LAMP positive, mature dendritic cells were found juxtaposed next to CD4 T cells. Clinicopathologic correlation, however, demonstrated that presence of TLS was associated with worse recurrence-free survival in WD disease and worse overall survival in DD disease. Our data suggest that an adaptive immune response is present in WD/DD retroperitoneal liposarcoma but may be hindered by TLS, among other possible microenvironmental factors; further investigation is needed. Immunotherapy, including immune checkpoint blockade, should be evaluated as a treatment option in this disease.

Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)–JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58 . Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion. Pooled CRISPR perturbation screens with multimodal RNA and protein single-cell profiling readout (Perturb-CITE-seq) applied to patient-derived melanoma and tumor-infiltrating lymphocyte co-cultures identifies new tumor immune evasion mechanisms.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.

Resistance to immune checkpoint inhibitors (ICR) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-seq, enabling pooled CRISPR-Cas9 perturbations with single-cell transcriptome and protein read-outs. In patient-derived melanoma cells and autologous tumor infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICR. We recover known mechanisms of resistance, including defects in the IFNγ-JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and novel ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 expression was not significantly regulated by IFNγ and CD58 loss conferred immune evasion without compromising MHC expression, suggesting that it acts orthogonal to known mechanisms of ICR. This work provides framework for deciphering complex mechanisms by large-scale perturbation screens with multi-modal single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion.

Treatment options are limited in well differentiated (WD) and dedifferentiated (DD) retroperitoneal liposarcoma. We sought to study the intratumoral adaptive immune response and explore the potential feasibility of immunotherapy in this disease. Tumorinfiltrating lymphocytes (TILs) were isolated from fresh surgical specimens and analyzed by flow cytometry for surface marker expression. Previously reported immune cell aggregates known as tertiary lymphoid structures (TLS) were further characterized by immunohistochemistry. In all fresh tumors, TILs were found. The majority of TILs were CD4 T cells; however cytotoxic CD8 T cells were also seen (average: 20% of CD3 T cells). Among CD8 T cells, 65% expressed the immune checkpoint molecule PD-1. Intratumoral TLS may be sites of antigen presentation as DC-LAMP positive, mature dendritic cells were found juxtaposed next to CD4 T cells. Clinicopathologic correlation, however, demonstrated that presence of TLS was associated with worse recurrence-free survival in WD disease and worse overall survival in DD disease. Our data suggest that an adaptive immune response is present in WD/DD retroperitoneal liposarcoma but may be hindered by TLS, among other possible microenvironmental factors; further investigation is needed. Immunotherapy, including

V(D)J recombination is a site specific recombination reaction that occurs in developing B and T cells and is essential for their survival and function. ARTEMIS was discovered as a factor critical for this process through presence of its mutations in human radiosensitive severe combined immune deficiency (RS-SCID) syndrome. It is known to open hairpin coding end intermediates during V(D)J recombination, and is also a Non-Homologous End-Joining (NHEJ) factor required for DNA repair. During our search for novel interactions of Artemis with other NHEJ factors, we found that Ligase IV, another critical player of these processes interacts with Artemis in human pre B cells. This interaction was mediated by Artemis’ C-terminal region and the DNA binding domain of Ligase IV. Amino acids within the C-terminal region of Artemis that are critical for this interaction were identified and shown to be required for efficient V(D)J recombination and resistance to ionizing radiations. The hypomorphic human mutations within the C-terminal region of Artemis that cause combined SCID plus development of B cell lymphomas also fail to interact with Ligase IV. Thus, for the first time we show a direct role of the C-terminal region of Artemis in V(D)J recombination and NHEJ and the mechanism by which these hypomorphic mutations of Artemis may cause immunodeficiency and cancer. Sequence analysis of Artemis has predicted a number of residues within its N-terminal region to be critical for its endonuclease function. We have found another highly conserved residue within Artemis, threonine 65, by identification of its missense mutation in a patient suffering from radiosensitive Atypical SCID/Omenn Syndrome. This mutation (T65I) results in decreased Artemis protein levels in vivo, defective endonuclease function in vitro and significant reduction in levels of V(D)J recombination on a plasmid substrate based assay. Coding joints generated in presence of this mutation and a SCID mutation of Artemis, D165N showed decreased junctional processing, providing evidence for Artemis’ role as the nuclease that trims the coding ends before joining. Thus, highly reduced V(D)J recombination and defective generation of antigen receptors that may induce deletion of immature lymphocytes and/or receptor editing, may be the mechanisms explaining the etiology of this Artemis mediated immunodeficiency.

Adoptive T cell therapy (ACT) is a promising treatment for melanoma patients with a clinical response rate of about 50%. However, half of patients treated do not respond to this therapy, underlining the need for improvement . One of the limitations of ACT is the poor effector function of transferred T cells influenced by the immunosuppressive tumor microenvironment. In order to identify pathways which may contribute to this observation, we used a murine ACT model in which mice bearing established B16 tumors were treated with Pmel T cells which recognize the melanoma antigen gp100 in the context of H-2Db. Pmel T cells were recovered on day 6 and 13, after transfer, from the tumor and spleen of treated mice and their gene expression patterns were compared. We found that 720 genes were differentially expressed by T cells recovered from the tumor site compared to those recovered from the spleen. Amongst the differentially expressed genes were several transcription factors, including Runx2, Rora, E2F1 and Tcf7. After an initial in vivo screen, Runx2 overexpressing Pmels conferred a worse antitumor effect when compared to the control Pmels (median tumor size 30.7 vs 20.7 mm2 respectively on day 7 after T cell transfer, p<0.05). We also found fewer numbers of circulating Pmels in mice that received Runx2 overexpressing Pmels when compared to mice that received control Pmels. In addition, there was decreased accumulation of Runx2 overexpressing Pmels at the tumor site when compared to the control Pmel (Median luciferase output of 2.0X107 vs 9.0X107 photons/s/cm2/sr, respectively on day 6 after T cell transfer, p value =0.042). To further interrogate the role of Runx2 in T cells, we assessed the production of IFN-γ after stimulation in vitro with either plate bound anti-CD3 or gp100 expressing tumor cells. We found that IFN-γ production was comparable between Runx2 overexpressing Pmels and control Pmels after anti-CD3 stimulation. However, IFN-γ production was impaired in Runx2 overexpressing Pmels upon stimulation with tumor cells. Furthermore, in vitro characterization also revealed that Runx2 overexpressing Pmels have decreased proliferation and display an apoptotic phenotype. Taken together, our studies suggest that Runx2 regulates apoptosis, proliferation and IFN-γ production in tumor reactive T cells. Further studies to mechanistically understand these findings are ongoing.