Efficacy Of Immune Checkpoint Blockade Research Articles

Readily available drugs and other interventions to potentially improve the efficacy of immune checkpoint blockade in cancer.

To improve the efficacy of immune checkpoint inhibitors (ICIs) for cancer treatment, various strategies, including combination therapies with repurposed drugs, are being explored. Several readily available interventions with potential to enhance programmed death 1 (PD-1) blockade have been identified. However, these interventions often remain overlooked due to the lack of financial incentives for their development, making them financial orphans. This review summarizes current knowledge regarding off-label drugs, supplements, and other readily available interventions that could improve the efficacy of PD-1 blockade. The summary of each intervention includes the proposed mechanism of action for combination with checkpoint inhibitors and data from animal and human studies. Additionally, we include summaries of common interventions to be avoided by patients on PD-1 blockade. Finally, we present approaches for conducting further studies in patients, with the aim of expediting the clinical development of these interventions. We strive to increase awareness of readily available combination therapies that may advance cancer immunotherapy and help patients today.

Multiomics characterization of pyroptosis in the tumor microenvironment and therapeutic relevance in metastatic melanoma

BackgroundPyroptosis, mediated by gasdermins with the release of multiple inflammatory cytokines, has emerged as playing an important role in targeted therapy and immunotherapy due to its effectiveness at inhibiting tumor growth. Melanoma is one of the most commonly used models for immunotherapy development, though an inadequate immune response can occur. Moreover, the development of pyroptosis-related therapy and combinations with other therapeutic strategies is limited due to insufficient understanding of the role of pyroptosis in the context of different tumor immune microenvironments (TMEs).MethodsHere, we present a computational model (pyroptosis-related gene score, PScore) to assess the pyroptosis status. We applied PScore to 1388 melanoma samples in our in-house cohort and eight other publicly available independent cohorts and then calculated its prognostic power of and potential as a predictive marker of immunotherapy efficacy. Furthermore, we performed association analysis for PScore and the characteristics of the TME by using bulk, single-cell, and spatial transcriptomics and assessed the association of PScore with mutation status, which contributes to targeted therapy.ResultsPyroptosis-related genes (PRGs) showed distinct expression patterns and prognostic predictive ability in melanoma. Most PRGs were associated with better survival in metastatic melanoma. Our PScore model based on genes associated with prognosis exhibits robust performance in survival prediction in multiple metastatic melanoma cohorts. We also found PScore to be associated with BRAF mutation and correlate positively with multiple molecular signatures, such as KRAS signaling and the IFN gamma response pathway. Based on our data, melanoma with an immune-enriched TME had a higher PScore than melanoma with an immune-depleted or fibrotic TME. Additionally, monocytes had the highest PScore and malignant cells and fibroblasts the lowest PScore based on single-cell and spatial transcriptome analyses. Finally, a higher PScore was associated with better therapeutic efficacy of immune checkpoint blockade, suggesting the potential of pyroptosis to serve as a marker of immunotherapy response.ConclusionsCollectively, our findings indicate that pyroptosis is a prognostic factor and is associated with the immune response in metastatic melanoma, as based on multiomics data. Our results provide a theoretical basis for drug combination and reveal potential immunotherapy response markers.

Role of stromal PD-L1 expression in colorectal liver metastasis

Background and AimThe outcomes of immune checkpoint blockade for colorectal cancer (CRC) treatment are unsatisfactory. Furthermore, the efficacy of immune checkpoint blockade for liver metastasis of various cancer is poor. Here, we investigated the relationship between stromal programmed death-ligand 1 (PD-L1) expression and the prognosis of patients with colorectal cancer liver metastasis (CRLM).MethodsThe present study enrolled 84 CRLM patients who underwent surgery (R0) for CRC. Immunohistochemistry was performed to analyze stromal PD-L1 expression in CRLM.ResultsStromal PD-L1 was expressed in 52.3% of CRLM samples, which was associated with fewer not optimally resectable metastases (p = 0.04). Stromal PD-L1 also tended to associate with a lower tumor grade (p = 0.08). Stromal PD-L1-positive patients had longer overall survival (p = 0.003). Multivariate analysis identified stromal PD-L1 expression (p = 0.008) and poorer differentiation (p < 0.001) as independent prognostic indicators. Furthermore, stromal PD-L1 expression was correlated to a high number of tumor-infiltrating lymphocytes (TILs). Stromal PD-L1– and low TIL groups had shorter OS than stromal PD-L1 + and high TIL groups (46.6% vs. 81.8%, p = 0.05) Stromal PD-L1-positive patients had longer disease-free survival (DFS) (p = 0.03) and time to surgical failure (p = 0.001). Interestingly, stromal PD-L1 expression was positively related to the desmoplastic subtype (p = 0.0002) and inversely related to the replacement subtype of the histological growth pattern (p = 0.008).ConclusionsStromal PD-L1 expression may be a significant prognostic marker for CRLM.

Tumor-associated neutrophils suppress CD8+ T cell immunity in urothelial bladder carcinoma through the COX-2/PGE2/IDO1 Axis.

Many urothelial bladder carcinoma (UBC) patients don't respond to immune checkpoint blockade (ICB) therapy, possibly due to tumor-associated neutrophils (TANs) suppressing lymphocyte immune response. We conducted a meta-analysis on the predictive value of neutrophil-lymphocyte ratio (NLR) in ICB response and investigated TANs' role in UBC. We used RNA-sequencing, HALO spatial analysis, single-cell RNA-sequencing, and flow cytometry to study the impacts of TANs and prostaglandin E2 (PGE2) on IDO1expression. Animal experiments evaluated celecoxib's efficacy in targeting PGE2 synthesis. Our analysis showed that higher TAN infiltration predicted worse outcomes in UBC patients receiving ICB therapy. Our research revealed that TANs promote IDO1 expression in cancer cells, resulting in immunosuppression. We also found that PGE2 synthesized by COX-2 in neutrophils played a key role in upregulating IDO1 in cancer cells. Animal experiments showed that targeting PGE2 synthesis in neutrophils with celecoxib enhanced the efficacy of ICB treatment. TAN-secreted PGE2 upregulates IDO1, dampening T cell function in UBC. Celecoxib targeting of PGE2 synthesis represents a promising approach to enhance ICB efficacy in UBC.

The Tumor Microenvironment Affects Circulating Tumor Cells Metastasis and the Efficacy of Immune Checkpoint Blockade in Non-small Cell Lung Cancer

Abstract: Lung cancer is one of the most lethal malignancies, with non-small cell lung cancer (NSCLC) being the most common histologic subtype. Metastasis leads to poor prognosis for patients with cancer. Tumor cells leave the tumor lesions, invade the surrounding stroma, and enter the bloodstream as circulating tumor cells (CTCs). The development of CTCs is the beginning of metastasis. The internal environment in which tumor cells grow and survive is called the tumor microenvironment (TME). It includes tumor cells, fibroblasts, immune cells, and the extracellular matrix. The TME is complex and dynamic. Moreover, the TME plays an important role in tumor development and metastasis and significantly impacts therapeutic outcomes. Immune checkpoint blockade (ICB) aims to inhibit the interaction of ligands with their corresponding receptors. ICB has the function of restoring the anti-tumor effect of immune cells. This review examines how TME interacts with CTCs, allowing CTCs to evade immunity and facilitating CTC metastasis. TME not only affects the progression of tumor metastasis but also interacts with tumor cells, which may affect the efficacy of immunotherapy.

Genetic insights into carbohydrate sulfotransferase 8 and its impact on the immunotherapy efficacy of cancer

Immune checkpoint blockade (ICB) is a promising therapy for solid tumors, but its effectiveness depends on biomarkers that are not precise. Here, we utilized genome-wide association study to investigate the association between genetic variants and tumor mutation burden to interpret ICB response. We identified 16 variants (p<5×10-8) probed to 17 genes on 9 chromosomes. Subsequent analysis of one of the most significant loci in 19q13.11 suggested that the rs111308825 locus at the enhancer is causal, as its A allele impairs KLF2 binding, leading to lower carbohydrate sulfotransferase 8 (CHST8) expression. Breast cancer cells expressing CHST8 suppress Tcell activation, and Chst8 loss attenuates tumor growth in a syngeneic mouse model. Further investigation revealed that programmed death-ligand 1 (PD-L1) and its homologs could be sulfated by CHST8, resulting in M2-like macrophage enrichment in the tumor microenvironment. Finally, we confirmed that low-CHST8 tumors have better ICB response, supporting the genetic effect and clinical value of rs111308825 for ICB efficacy prediction.

Retracted: Computational Biology Predicts the Efficacy of Tumor Immune Checkpoint Blockade.

[This retracts the article DOI: 10.1155/2022/6087751.].

Comparison of primary and metastatic site-related PD-L1 expression in predicting ORR in patients with advanced NSCLC who received ICB-based therapy.

Whether the value of PD-L1 expression from metastatic sites to predict the efficacy of immune checkpoint blockade (ICB)-based treatment is equivalent to that from a primary tumor is uncertain. This study aimed to compare the utility of PD-L1 TPS from a primary lung tumor and metastatic sites to predict the overall response rate (ORR) of first-line ICB-based treatment. This study included 249 patients with advanced non-small cell lung cancer (NSCLC) who received first-line ICB-based treatment. All subjects underwent PD-L1 testing prior to ICB-based treatment and were divided into two cohorts corresponding to the different biopsy sites: lung primary site-sampled cohort (PT cohort, n = 167) and metastatic site-sampled cohort (MT cohort, n = 82). There was no statistical significance in PD-L1 TPS distribution between the two cohorts (p = 0.742). PD-L1 TPS ≥50% was also related to high ORR compared with PD-L1 < 50% in the PT cohort (34.3% vs. 14.1%, p = 0.004). In contrast, ICB-based therapy could bring comparable ORR (35.1% vs. 33.3%, p = 0.871) in the MT cohort regardless of PD-L1 TPS status (≥50%, or <50%). As supported, when the cutoff value of TPS was selected as 50%, it was suggested that PT-related PD-L1 was the independent predictor of ORR (OR 2.870, 95% CI: 1.231-6.694, p = 0.015) rather than MT-related PD-L1 (OR 0.689, 95% CI: 0.236-2.013, p = 0.495). Furthermore, ROC proved that PT-related PD-L1 expression manifested a better AUC of 0.621 (p = 0.026) than that of MT-related PD-L1 (AUC = 0.565, p = 0.362). Compared with PT-related PD-L1 expression, MT-related PD-L1 expression showed limited value in predicting ORR in patients with advanced NSCLC receiving ICB-based therapy. It was concluded that even patients with low MT-related PD-L1 expression could benefit from ICB-based therapy.

Protein kinase C delta regulates mononuclear phagocytes and hinders response to immunotherapy in cancer.

Mononuclear phagocytes (MPs) play a crucial role in tissue homeostasis; however, MPs also contribute to tumor progression and resistance to immune checkpoint blockade (ICB). Targeting MPs could be an effective strategy to enhance ICB efficacy. We report that protein kinase C delta (PKCδ), a serine/threonine kinase, is abundantly expressed by MPs in human and mouse tumors. PKCδ-/- mice displayed reduced tumor progression compared to wild types, with increased response to anti-PD-1. Tumors from PKCδ-/- mice demonstrated TH1-skewed immune response including increased antigen presentation and T cell activation. Depletion of MPs in vivo altered tumor growth in control but not PKCδ-/- mice. Coinjection of PKCδ-/- M2-like macrophages with cancer cells into wild-type mice markedly delayed tumor growth and significantly increased intratumoral T cell activation compared to PKCδ+/+ controls. PKCδ deficiency reprogrammed MPs by activating type I and type II interferon signaling. Thus, PKCδ might be targeted to reprogram MPs to augment ICB efficacy.

Harnessing p53 to Improve Immunotherapy for Lung Cancer Treatment.

Immunotherapy, especially immune checkpoint blockade (ICB), has become a critical therapy for lung cancer treatment in recent years. Tumor mutational burden (TMB) is one of the decisive biomarkers for predicting ICB effect. Writing in Cancer Cell, Zhu and colleagues use autochthonous and syngeneic mouse models to show that p53 mutation and tumor heterogeneity may be responsible for resistance in patients with lung cancer. Pole-induced high TMB shows enhanced immunogenicity in KrasG12D mice, however, loss of p53 in KrasG12D PoleP286/+ mice can lead to an immune suppressive profile of lung tumors, which diminishes immune response to ICB. Moreover, high TMB causes high shared mutations, which helps promote immune protection and immune memory. Heterogeneity can drive immune escape to tumor cells causing resistance to ICB. Decreased cGAS/STING signaling may explain possible resistance to ICB. On the basis of the new model found by Zhu and colleagues for lung cancer, combined ICB with STING agonists or p53 inducers may be new therapeutic options to improve the efficacy of ICB for patients with lung cancer with high TMB.

Re-"Formate" T-cell Antitumor Responses.

Rowe and colleagues discover that one-carbon (1C) metabolism rewiring occurs upon T-cell activation to support proliferation and cytolytic activity in CD8+ T cells and that supplementation of 1C donor formate rescues the dysfunctional T cells and their responsiveness to anti-PD-1 in selective tumor-infiltrated T-cell subsets. This finding represents an attractive strategy to overcome a metabolic vulnerability in the tumor microenvironment and improve the efficacy of immune checkpoint blockade. See related article by Rowe et al., p. 2566 (8).

CHEK2 deficiency increase the response to PD-1 inhibitors by affecting the tumor immune microenvironment

Immune checkpoint blockade (ICB) therapy has improved treatment effects in multiple cancers. Gene mutations in the DNA damage repair pathway (DDR) may cause genomic instability and may relate to the efficacy of ICB. Checkpoint kinase 2 (CHEK2) and polymerase epsilon (POLE) are important genes in the DDR. In this study, we aimed to study the impact of CHEK2 deficiency mutations on the response to ICB. We found that tumors with CHEK2 mutations had a significantly higher tumor mutational burden (TMB) compared to those with CHEK2-WT in a pancancer database. We noted that CHEK2 deficiency mutations potentiated the anti-tumor effect of anti-PD-1 therapy in MC38 and B16 tumor-bearing mice with the decrease of tumor volume and tumor weight after anti-PD-1 treatment. Mechanistically, CHEK2 deficiency tumors were with the increased cytotoxic CD8+ T-cell infiltration, especially cytotoxic CD8+ T cells, and modulated the tumor-immune microenvironment with an upregulated immune inflammatory pathway and antigen presentation pathway after anti-PD-1 treatment. Furthermore, murine models with POLE mutations confirmed that CHEK2 deficiency shaped similar mutational and immune landscapes as POLE mutations after anti-PD-1 treatment. Taken together, our results demonstrated that CHEK2 deficiency mutations may increase the response to ICB (eg. anti-PD-1) by influencing the tumor immune microenvironment. This indicated that CHEK2 deficiency mutations were a potentially predictive biomarker and CHEK2 deficiency may potentiate response to immunotherapy.

TLR9 plus STING Agonist Adjuvant Combination Induces Potent Neopeptide T Cell Immunity and Improves Immune Checkpoint Blockade Efficacy in a Tumor Model.

Immune checkpoint blockade (ICB) immunotherapies have emerged as promising strategies for the treatment of cancer; however, there remains a need to improve their efficacy. Determinants of ICB efficacy are the frequency of tumor mutations, the associated neoantigens, and the T cell response against them. Therefore, it is expected that neoantigen vaccinations that boost the antitumor T cell response would improve ICB therapy efficacy. The aim of this study was to develop a highly immunogenic vaccine using pattern recognition receptor agonists in combination with synthetic long peptides to induce potent neoantigen-specific T cell responses. We determined that the combination of the TLR9 agonist K-type CpG oligodeoxynucleotides (K3 CpG) with the STING agonist c-di-AMP (K3/c-di-AMP combination) significantly increased dendritic cell activation. We found that immunizing mice with 20-mer of either an OVA peptide, low-affinity OVA peptides, or neopeptides identified from mouse melanoma or lung mesothelioma, together with K3/c-di-AMP, induced potent Ag-specific T cell responses. The combined K3/c-di-AMP adjuvant formulation induced 10 times higher T cell responses against neopeptides than the TLR3 agonist polyinosinic:polycytidylic acid, a derivative of which is the leading adjuvant in clinical trials of neoantigen peptide vaccines. Moreover, we demonstrated that our K3/c-di-AMP vaccine formulation with 20-mer OVA peptide was capable of controlling tumor growth and improving survival in B16-F10-OVA tumor-bearing C57BL/6 mice and synergized with anti-PD-1 treatment. Together, our findings demonstrate that the K3/c-di-AMP vaccine formulation induces potent T cell immunity against synthetic long peptides and is a promising candidate to improve neoantigen vaccine platform.

Abstract C167: An SN38 dendrimer nanoparticle, DEP irinotecan (SN38-SPL9111), demonstrates efficacy in mouse models of gastrointestinal cancer and augments anti-tumor effects of immune checkpoint blockade and PARP inhibition

Abstract Background: Irinotecan is a topoisomerase 1 inhibitor pro-drug used to treat gastrointestinal (GI) cancers including as first line for colorectal cancer (CRC) as part of the FOLFIRI plus monoclonal antibody (mAb) regimen, and for pancreatic cancer in the FOLFIRINOX regimen. However, irinotecan causes severe and dose-limiting side effects, including diarrhea and neutropenia. Starpharma has developed a highly optimized polylysine dendrimer enhanced (DEP) nanoparticle conjugate of the irinotecan active metabolite, SN38 (DEP SN38 or DEP irinotecan). DEP SN38 avoids the need for liver conversion of irinotecan to SN38 and achieves preferential tumor targeting. DEP SN38 is in phase 2 clinical investigation as monotherapy or combination therapy in patients with solid tumors (EudraCT no: 2019-001318-40). SN38 is known to enhance the efficacy of immune checkpoint blockade (ICB) via effects on immune cells in the tumor microenvironment. Here, we evaluated the anti-tumor effects of DEP SN38 alone or in combination with either ICB (anti- programmed cell death-1 [PD1] mAb) or poly adenosine diphosphate (ADP) ribose polymerase (PARP) inhibition (olaparib) in mouse models of GI cancer. Methods: HT29 (human CRC) and CAPAN-1 (human pancreatic cancer) cell line xenografts were established subcutaneously in BALB/c nude or NOD-scid-Gamma immunodeficient mice, respectively. Three doses of DEP SN38 (at 5-10mg/kg SN38) or irinotecan (35-80 mg/kg) were administered intravenously (IV) on days 1, 8, 15. Olaparib (50 mg/kg) was delivered by daily IV injection for 5 days/week over 3 weeks. DEP SN38 (IV on days 1, 8, 15 at 18-35 mg/kg SN38) or irinotecan (75 mg/kg) plus rat anti-PD1 mAb therapy or control mAb (intraperitoneally, 100 mg/kg then 50 mg/kg on days 5, 8, 12) was evaluated in both MC38 (C57BL/6) and CT26 (BALB/c) allograft models of CRC. Toleration of drug was bodyweight loss ≤15%. Results: DEP SN38 treatment of HT29 or CAPAN-1 xenografts led to tumor regressions, prolonged control of tumor growth and improved survival. In contrast, conventional irinotecan had minimal effect on tumor growth versus vehicle control. DEP SN38 was well tolerated by mice with minimal bodyweight loss. Administration of anti-PD1 mAb alone confirmed the known modest sensitivity of MC38, and resistance of CT26 syngeneic tumors, to ICB. DEP SN38 plus anti-PD1 mAb, enhanced anti-tumor responses in the MC38 model beyond those seen for each agent alone and also resulted in tumor eradication and prolonged survival. In the CT26 model, anti-PD1 mAb was also augmented by addition of DEP SN38 but not irinotecan alone, despite resistance of CT26 tumors to anti-PD1 mAb alone. The HT29 CRC model was resistant to Olaparib alone. In contrast, anti-tumor effects and prolonged survival were observed with olaparib plus DEP SN38. Conclusions: DEP SN38 demonstrated superior efficacy compared to irinotecan when used in models of GI cancer. The anti-tumor efficacy of both ICB and PARP inhibition was augmented by DEP SN38 in models of CRC, and these combinations should be investigated clinically. Citation Format: Benjamin J Blyth, Brian D Kelly, Michael Giannis, Anne Cargill, Aynaz Seta, Graham P Heery, Anthony Eglezos, Cameron N Johnstone, Jeremy R A Paull. An SN38 dendrimer nanoparticle, DEP irinotecan (SN38-SPL9111), demonstrates efficacy in mouse models of gastrointestinal cancer and augments anti-tumor effects of immune checkpoint blockade and PARP inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C167.

Abstract C079: Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy

Abstract Immuno-oncology has revolutionized cancer treatment by harnessing the immune system to target and eliminate tumor cells. In this study, we employed a combination of fluorescence-activated cell sorting (FACS) and Cytek full spectrum flow cytometry to investigate the impact of anti-PD1 (programmed cell death protein 1) and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) treatments on the 4T1 breast cancer model. We comprehensively analyzed 25 surface markers on immune cells to gain insights into the dynamic changes induced by immunotherapy. The 4T1 murine breast cancer model is known for its aggressive and metastatic nature, resembling triple-negative breast cancer in humans. We established a syngeneic orthotopic tumor graft of 4T1 cells in immunocompetent mice and treated them individually with anti-PD1 or anti-CTLA4 antibodies and compared the results to the respective isotype controls. Using FACS, we characterized immune cell populations within the tumor microenvironment and spleenocytes at multiple time points (before treatment, day 5, and day 17) during the treatment course. The Cytek full spectrum flow cytometry platform allowed simultaneous measurement of 25 surface markers, enabling a detailed analysis of immune cell phenotypes and functional states in a single experiment. Our findings revealed significant alterations in the immune landscape following anti-PD1 or anti-CTLA4 treatments. We observed an increase in tumor-infiltrating lymphocytes (TILs) and specifically CD4+ as well as CD8+ T cell infiltration within the tumor, accompanied by upregulation of T cell activation markers, such as CD69, CD38, CD44, PD1 and CD25. The immune cell composition within 4T1 tumors included CD4+ and CD8+ TILs, as well as increased populations of monocytes and macrophages. Additionally, a decrease in regulatory T cells (TRegs) was observed, indicating a potential restoration of immune balance. Moreover, F4/80+ CD206+ tumor-associated macrophages (TAMs) were identified in the 4T1 tumors vs. the spleen. This was associated with increased granzyme B production by CD8+ T cells and a shift towards an effector memory phenotype, suggesting improved cytotoxic function. To gain insights into spatial infiltration patterns of immune cells, we performed Immunohistochemistry (IHC) stainings of various immune cell markers in the 4T1 tumors. In line with the FACS results, we observed increased immune cell infiltration in tumors treated with anti-PD1 and anti-CTLA4 antibodies. Our comprehensive immunophenotypic analysis, utilizing Cytek full spectrum flow cytometry and IHC sheds light on the dynamic interplay between immune cells and the 4T1 breast cancer model during anti-PD1 and anti-CTLA4 immunotherapy. These findings provide valuable insights into the mechanisms underlying the therapeutic efficacy of immune checkpoint blockade in this aggressive cancer model. Citation Format: Christos Nikolaou, Simon Heller, Stefan Kaulfuss, Carlo Stresemann, Alexandra Eichten, Oliver von Ahsen, Martin Lange. Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C079.

Exploiting the Gut Microbiota to Boost Immunotherapy and Switch the Trajectory of Multiple Myeloma

Clonal proliferation of neoplastic plasma cells in the bone marrow (BM) and organ dysfunction characterizes multiple myeloma (MM), a treatable but incurable disease. Although asymptomatic smoldering MM (SMM) often precedes full blown MM, most patients affected by SMM are only offered active observation, which increases their frustration and anxiety. In transgenic Vk*MYC mice developing de novo MM, we found a direct link between gut microbiota composition and T helper 17 (Th17) lymphocytes, which migrated from the gut to the BM during asymptomatic MM (Early-MM), and favored the expansion of neoplastic plasma cells, thus fueling progression to symptomatic disease (Late-MM) [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Similarly, in SMM patients, higher levels of BM Interleukin-17 (IL-17) predicted accelerated disease [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Interestingly, Prevotella melaninogenica ( P.m.), a human commensal, limited MM aggressiveness in mice challenged with Vk*MYC-derived MM cells (t-Vk*MYC) by restraining Th17 cell expansion [Calcinotto et al. Nat Commun (2018) 9(1):4832]. Because the gut microbiota also contributes to the therapeutic efficacy of immune checkpoint blockade (ICB) and human neoplastic plasma cells express PD-L1, we hypothesized that modulation of the gut microbiota by P.m. could limit Th17 cell expansion in mice affected by Early-MM, thus fully exploiting the therapeutic potential of anti-PD-L1 against MM. Clinical trials combining anti-PD-1/PD-L1 antibodies, dexamethasone and immune modulatory drugs in MM were halted by US FDA because of unfavorable benefit-risk profile and life-threatening immune related adverse events (irAEs). Because IL-17 is involved in irAEs [Johnson et al. Cancer Immunol Res (2019) 7 (6): 860-865], we also hypothesized treatment with P.m. could restrain ICB-induced expansion of Th17 cells while unleashing CD8-mediated antitumor immunity. Early-MM Vk*MYC mice and t-Vk*MYC, the latter mimicking aggressive MM, were treated with P.m. and/or anti-PD-L1 and disease progression was monitored by paraprotein quantification in blood. Anemia provided clinical evidence of symptomatic disease. At sacrifice, gut, spleen and BM were analyzed by flow cytometry. In vitro assays were conducted to clarify the mechanism by which the gut microbiota influenced the induction of Th17 cells. To mimic cutaneous irAE, the skin of treated mice was exposed to imiquimod, which induces Th17-mediated inflammation [van der Fits et al. J Immunol (2009 )182(9): 5836-5845]. Body weight loss, skin inflammation, and inflammatory infiltrate into secondary lymphoid organs were taken as measure of imiquimod-induced toxicity. In Vk*MYC mice, all neoplastic plasma cells expressed PD-L1. Treatment of Early-MM Vk*MYC mice with anti-PD-L1 antibodies delayed progression to Late-MM, thus extending previous findings on the use of ICB in this model to anti-PD-L1 antibodies and Early-MM [Guillerey et al. J Clin Invest (2015 )125(5): 2077-2089]. Treatment with P.m. exerted a similar effect on disease progression, suggesting that a treatment as simple as taking a probiotic has an impact on MM evolution. When P.m. was combined with anti-PD-L1 antibodies, evolution to Late-MM was further delayed or even blocked. Single and combined treatments also reduced MM aggressiveness in t-Vk*MYC mice. Mechanistically, P.m. limited the expansion of gut-born Th17 cells and their accumulation in the BM of treated mice without dampening the antitumor cytotoxic response elicited by the ICB. Thus, only the combined treatment provided the most favorable CD8/Th17 and CD8/T regulatory cell ratios. Ex vivo, P.m.-conditioned BM DCs showed a limited propensity to produce Th17-polarizing cytokines. In vitro, stimulation of both human and mouse DCs with P.m. reduced polarization of naïve T cells toward a Th17 phenotype. Skin application of imiquimod in C57BL/6 mice induced psoriasis-like lesions and expansion of Th17 cells both in draining lymph nodes and spleen. The contextual administration of anti-PD-L1 antibodies worsened imiquimod-induced inflammation. An ongoing experiment will clarify if mice receiving P.m. and ICB experience reduced skin lesions. Taken together, our data support the development of microbiota-based strategies in combination with ICB to treat full-blown MM and to prevent progression of asymptomatic SMM to full-blown disease.

Contrasting Features of T-Cell Expansion in Classical Hodgkin Lymphoma: Tumor Microenvironment Vs. Peripheral Blood and before Vs. during Anti-PD1 Treatment

Introduction The Tumor Microenvironment (TME) in classical Hodgkin Lymphoma (HL) contains abundant CD4+ and CD8+ T-cells and only few Hodgkin-Reed-Sternberg cells (HRSC). Despite their low abundance, HRSC comprise the neoplastic cell population that intensively interacts with cells of the TME. Understanding these interactions is crucial to the further development of immune checkpoint blockade (ICB) based treatment options. Clinical trials have shown high efficacy of ICB with anti-PD1 antibodies in relapsed HL and more recently promising results of anti-PD1 antibodies in combination with conventional chemotherapy were reported in the first-line setting. (NIVAHL trial, NCT03004833) In solid cancers, anti-PD1 ICB was shown to revert tumor-induced exhaustion of CD4+ and CD8+ T-cells, thereby enabling T-cell activation and a tumor-directed immune response. Since HL differs in many aspects from non-lymphoid tumors due to e.g. loss of HLA-expression, the exact mechanisms of action of ICB in HL is not fully understood and T-cell expansion after anti-PD1 first-line treatment not yet studied. Methods To characterize T-cell activation at different timepoints and to investigate a possible T-cell mediated immune response in HL, we analyzed T-cell Receptor (TCR) repertoires of the NIVAHL study cohort before (tissue: n=90; blood: n=9) and during anti-PD1 treatment (tissue: n=4; blood: n=9). The final cohort comprised additional TCR repertoires of in-house tissue biopsies of treatment-naïve HL (n=18), relapsed HL after chemotherapy (HL-R; n=18)) and publicly available TCR repertoires of the blood of treatment-naïve HL (n=11), HL-R (n=20) and HL-R patients during anti-PD1 treatment (n=20). (Cader et al. Nat Med. 2020 Sep;26(9):1468-1479) TCR repertoires of in-house reactive lymph nodes (n=8, healthy control) and breast cancer (BC; n=6, positive control) patients served as controls for T-cell activation in the tissue, publicly available TCR repertoires of CMV- (n=22, healthy control) and CMV+ otherwise healthy (n=9, positive control) people as controls in the blood. (De Neuter et al. Genes Immun. 2019 Mar;20(3):255-260) TCR repertoires were sequenced by Adaptive Biotechnologies. For each sample, TCR sequences with the same amino acid sequence were aggregated by the sum of their counts. We computed three measures to describe T-cell expansion: debiased Simpson's Clonality (dSC), Percentage of Singletons (PoS), and clonal expansion. Singletons have recently been defined as TCR sequences detected only once in a given sample. Clonal expansion is computed as the percentage of TCR sequences that increase their frequency in a patient from one timepoint to a following by ≥ 2. Results In tissue biopsies, treatment-naïve HL showed a significantly lower dSC compared to treatment-naïve BC and significantly higher dSC compared to RLN from healthy controls. No significant differences could be observed comparing HL with different treatments (during anti-PD1 vs. after chemotherapy) and treatment-naïve HL vs. HL during anti-PD1 treatment. HL and HL-R biopsies showed a significantly lower clonal expansion of Non-Singletons than BC tumors. In peripheral blood, healthy controls, treatment-naïve HL and HL patients during anti-PD1 treatment showed significantly lower dSC in their CD4+ T-cells compared to CMV+ controls and also compared to HL-R patients. We identified significantly higher dSC in CD8+ T-cells compared to CD4+ T-cells in the blood of HL patients before and during any treatment and in relapsed HL. During anti-PD1 treatment, CD8+ T-cells showed a significantly higher clonal expansion of Non-Singletons than CD4+ T-cells in HL-R and a similar but not significant trend in HL. All observed differences in dSC were significant for the PoS too, but as expected by their respective definitions with an opposing pattern (i.e. dSC high = PoS low). Discussion We did not observe features of intratumoral T-cell expansion in primary HL samples, early during anti-PD1 treatment or at relapse after chemotherapy, suggesting that within the HL tissue, T-cell expansion is hampered. However, patterns of T-cell expansion differed between TME and the peripheral blood, where we observed features of CD8+ T-cell clonal expansion, already prior to any treatment. In summary, our findings suggest a possible anti-tumor immune response of CD8+ T-cells in the peripheral blood of HL patients, that is not found in the TME.

PAK in Pancreatic Cancer-Associated Vasculature: Implications for Therapeutic Response.

Angiogenesis has been associated with numbers of solid tumours. Anti-angiogenesis drugs starve tumours of nutrients and oxygen but also make it difficult for a chemo reagent to distribute into a tumour, leading to aggressive tumour growth. Anti-angiogenesis drugs do not appear to improve the overall survival rate of pancreatic cancer. Vessel normalisation is merging as one of the new approaches for halting tumour progression by facilitating the tumour infiltration of immune cells and the delivery of chemo reagents. Targeting p21-activated kinases (PAKs) in cancer has been shown to inhibit cancer cell growth and improve the efficacy of chemotherapy. Inhibition of PAK enhances anti-tumour immunity and stimulates the efficacy of immune checkpoint blockades. Inhibition of PAK also improves Car-T immunotherapy by reprogramming the vascular microenvironment. This review summarizes current research on PAK's role in tumour vasculature and therapeutical response, with a focus on pancreatic cancer.

PRMT1 Inhibition Activates the Interferon Pathway to Potentiate Antitumor Immunity and Enhance Checkpoint Blockade Efficacy in Melanoma.

Targeting PRMT1 stimulates interferon signaling by increasing expression of endogenous retroviral elements and double-stranded RNA through repression of DNMT1, which induces antitumor immunity and synergizes with immunotherapy to suppress tumor progression.

Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer

CD8+ cytotoxic T cells (CTLs) orchestrate antitumour immunity and exhibit inherent heterogeneity1,2, with precursor exhausted T (Tpex) cells but not terminally exhausted T (Tex) cells capable of responding to existing immunotherapies3–7. The gene regulatory network that underlies CTL differentiation and whether Tex cell responses can be functionally reinvigorated are incompletely understood. Here we systematically mapped causal gene regulatory networks using single-cell CRISPR screens in vivo and discovered checkpoints for CTL differentiation. First, the exit from quiescence of Tpex cells initiated successive differentiation into intermediate Tex cells. This process is differentially regulated by IKAROS and ETS1, the deficiencies of which dampened and increased mTORC1-associated metabolic activities, respectively. IKAROS-deficient cells accumulated as a metabolically quiescent Tpex cell population with limited differentiation potential following immune checkpoint blockade (ICB). Conversely, targeting ETS1 improved antitumour immunity and ICB efficacy by boosting differentiation of Tpex to intermediate Tex cells and metabolic rewiring. Mechanistically, TCF-1 and BATF are the targets for IKAROS and ETS1, respectively. Second, the RBPJ–IRF1 axis promoted differentiation of intermediate Tex to terminal Tex cells. Accordingly, targeting RBPJ enhanced functional and epigenetic reprogramming of Tex cells towards the proliferative state and improved therapeutic effects and ICB efficacy. Collectively, our study reveals that promoting the exit from quiescence of Tpex cells and enriching the proliferative Tex cell state act as key modalities for antitumour effects and provides a systemic framework to integrate cell fate regulomes and reprogrammable functional determinants for cancer immunity.