Cabozantinib and nivolumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial.

Supplementation with CBM588, a bifidogenic live bacterial product, has been associated with improved clinical outcomes in persons with metastatic renal cell carcinoma (mRCC) receiving nivolumab and ipilimumab. However, its effect on those receiving tyrosine kinase inhibitor-based combinations is unknown. In this open-label, randomized, investigator-initiated, phase 1 study, 30 participants with locally advanced or mRCC with histological confirmation of clear cell, papillary or sarcomatoid component were randomized in a 2:1 fashion to receive cabozantinib (an inhibitor of vascular endothelial growth factor receptor, MET and AXL) and nivolumab (anti-programmed cell death protein 1) with or without CBM588 as first-line treatment. Metagenomic sequencing was performed on stool samples to characterize their gut microbiome at baseline and 13 weeks into treatment. The primary endpoint was a change in the relative abundance of Bifidobacterium spp.; secondary endpoints included objective response rate (ORR), progression-free survival (PFS) and toxicity profile. The primary endpoint of the study was not met and the addition of CBM588 to cabozantinib and nivolumab did not result in a difference in the relative abundance of Bifidobacterium spp. or alpha diversity (as measured by the Shannon index). However, ORR was significantly higher in participants treated with CBM588 compared to those in the control arm (14 of 19, 74% versus 2 of 10, 20%; P = 0.01). PFS at 6 months was 84% (16 of 19) and 60% (6 of 10) in the experimental and control arms, respectively. No significant difference in toxicity profile was seen between the study arms. Our results provide a preliminary signal of improved clinical activity with CBM588 in treatment-naive participants with mRCC receiving cabozantinib and nivolumab. Further investigation is needed to confirm these findings and better characterize the underlying mechanism driving this effect.ClinicalTrials.gov identifier: NCT05122546.

Oligo-PROTAC strategy for cell-selective and targeted degradation of activated STAT3.

Decoy oligodeoxynucleotides (ODNs) allow targeting undruggable transcription factors, such as STAT3, but their limited potency and lack of delivery methods hampered translation. To overcome these challenges, we conjugated a STAT3-specific decoy to thalidomide, a ligand to cereblon in E3 ubiquitin ligase complex, to generate a proteolysis-targeting chimera (STAT3DPROTAC). STAT3DPROTAC downregulated STAT3 in target cells, but not STAT1 or STAT5. Computational modeling of the STAT3DPROTAC ternary complex predicted two surface lysines, K601 and K626, in STAT3 as potential ubiquitination sites. Accordingly, K601/K626 point mutations in STAT3, as well as proteasome inhibition or cereblon deletion, alleviated STAT3DPROTAC effect. Next, we conjugated STAT3DPROTAC to a CpG oligonucleotide targeting Toll-like receptor 9 (TLR9) to generate myeloid/B cell-selective C-STAT3DPROTAC. Naked C-STAT3DPROTAC was spontaneously internalized by TLR9+ myeloid cells, B cells, and human and mouse lymphoma cells but not by T cells. C-STAT3DPROTAC effectively decreased STAT3 protein levels and also STAT3-regulated target genes critical for lymphoma cell proliferation and/or survival (BCL2L1, CCND2, and MYC). Finally, local C-STAT3DPROTAC administration to human Ly3 lymphoma-bearing mice triggered tumor regression, while control C-STAT3D and C-SCR treatments had limited effects. Our results underscore the feasibility of using a PROTAC strategy for cell-selective, decoy oligonucleotide-based STAT3 targeting of and potentially other tumorigenic transcription factors for cancer therapy.

Oligo-PROTAC strategy for cell-selective and targeted degradation of activated STAT3.

Decoy-oligodeoxynucleotides (D-ODNs) can target undruggable transcription factors, such as STAT3. However, challenges in D-ODN delivery and potency hampered their translation. To overcome these limitations, we conjugated STAT3-specific D-ODN to thalidomide (Tha), a known ligand to cereblon (CRBN, a component of E3 ubiquitin ligase) to generate a proteolysis-targeting chimera (STAT3D PROTAC ). STAT3D PROTAC downregulated STAT3, but not STAT1 or STAT5, in target cells. Computational modeling of the STAT3D PROTAC ternary complex predicted two surface lysines on STAT3, K601 and K626 as potential ubiquitination sites for the PROTAC bound E3 ligase. Accordingly, K601/K626 point mutations in STAT3, as well as proteasome inhibitors, and CRBN deletion alleviated STAT3D PROTAC effect. Next, we conjugated STAT3D PROTAC to a CpG ligand targeting Toll-like receptor 9 (TLR9) to generate myeloid/B-cell-selective C-STAT3D PROTAC conjugate. Naked C-STAT3D PROTAC was spontaneously internalized by TLR9 + myeloid cells, B cells as well as human Ly18 and mouse A20 lymphoma cells, but not by T cells. C-STAT3D PROTAC decreased STAT3 levels to 50% at 250 nM and over 85% at 2 µM dosing in myeloid cells. We also observed significantly improved downregulation of STAT3 target genes involved in lymphoma cell proliferation and/or survival ( BCL2L1, CCND2, MYC ). Finally, we assessed the antitumor efficacy of C-STAT3D PROTAC compared to C-STAT3D or scrambled control (C-SCR) against human lymphoma xenotransplants. Local C-STAT3D PROTAC administration triggered lymphoma regression while control treatments had limited effects. Our results underscore feasibility of using PROTAC strategy for cell-selective, decoy oligonucleotide-based targeting of STAT3 and potentially other tumorigenic transcription factors for cancer therapy.

Targeting STAT3 in tumor-associated antigen-presenting cells as a strategy for kidney and bladder cancer immunotherapy.

Introduction: Immune checkpoint blockade (ICB) improved clinical outcomes in renal and bladder cancer patients, but the response rates remain limited especially in metastatic disease. While STAT3 transcription factor is well-known master regulator of tumor immune evasion, little is known about the role of STAT3 in the resistance of renal or bladder cancers to immunotherapy. Methods: To better understand immune alterations associated with ICB resistance, we assessed blood biomarkers in renal cancer patients classified as responders or non-responders to first line nivolumab/ipilimumab immunotherapy. Results: We observed that non-responders showed elevated levels of proinflammatory mediators, such as IL-1RA, IL-6, IL-8 and to lesser extent IL-10, which are associated with STAT3 activation and tumor immunosuppression. In addition, we found STAT3 activation primarily in circulating myeloid immune cells such as tolerogenic MDSCs. To assess whether STAT3 inhibition within these cell subsets can promote antitumor immune responses and/or enhance sensitivity to ICB in vivo, we used an original antisense oligonucleotide (ASO) strategy for myeloid-cell selective STAT3 knockdown (CpG-STAT3ASO). Our results in syngeneic models of renal and bladder cancers in mice demonstrated potent antitumor activity of CpG-STAT3ASO alone in contrast to PD1 blockade alone in both models. The CpG-STAT3ASO/anti-PD1 combination improved therapeutic efficacy especially against bladder tumors. Therapeutic efficacy correlated with activation of dendritic cells (DCs) and M1 macrophages in the tumor microenvironment, reduced percentages of regulatory T cells (Tregs) and the expansion of CD8 T cells in both tumor models. Discussion/Conclusion: Our study underscores the potential of using myeloid-cell targeted CpG-STAT3 inhibitors for genitourinary cancer therapy to disrupt tolerogenic signaling, restore immune cell activity and sensitivity to immune checkpoint inhibitors and/or T cell-based immunotherapies.

Nivolumab plus ipilimumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial.

Previous studies have suggested that the gut microbiome influences the response to checkpoint inhibitors (CPIs) in patients with cancer. CBM588 is a bifidogenic live bacterial product that we postulated could augment CPI response through modulation of the gut microbiome. In this open-label, single-center study (NCT03829111), 30 treatment-naive patients with metastatic renal cell carcinoma with clear cell and/or sarcomatoid histology and intermediate- or poor-risk disease were randomized 2:1 to receive nivolumab and ipilimumab with or without daily oral CBM588, respectively. Stool metagenomic sequencing was performed at multiple timepoints. The primary endpoint to compare the relative abundance of Bifidobacterium spp. at baseline and at 12 weeks was not met, and no significant differences in Bifidobacterium spp. or Shannon index associated with the addition of CBM588 to nivolumab-ipilimumab were detected. Secondary endpoints included response rate, progression-free survival (PFS) and toxicity. PFS was significantly longer in patients receiving nivolumab-ipilimumab with CBM588 than without (12.7 months versus 2.5 months, hazard ratio 0.15, 95% confidence interval 0.05-0.47, P = 0.001). Although not statistically significant, the response rate was also higher in patients receiving CBM588 (58% versus 20%, P = 0.06). No significant difference in toxicity was observed between the study arms. The data suggest that CBM588 appears to enhance the clinical outcome in patients with metastatic renal cell carcinoma treated with nivolumab-ipilimumab. Larger studies are warranted to confirm this clinical observation and elucidate the mechanism of action and the effects on microbiome and immune compartments.

Circulating cytokines associated with clinical response to systemic therapy in metastatic renal cell carcinoma.

BACKGROUND: Circulating cytokines and angiogenic factors have been associated with clinical outcomes in patients with metastatic renal cell carcinoma (RCC) receiving systemic therapy. However, none have yet examined cytokine concentrations in parallel cohorts receiving either immunotherapy or targeted therapy. METHODS: In this prospective correlative study, we enrolled 56 patients who were planned for treatment with either a vascular endothelial growth factor-tyrosine kinase inhibitor (VEGF-TKI) or immune checkpoint inhibitor (ICI). Eligibility requirements permitted any RCC histologic subtype, International Metastatic Renal Cell Carcinoma risk classification, and line of therapy. Immunologic profile was assessed at baseline and after 1 month on treatment using a Human Cytokine 30-plex protein assay (Invitrogen). Clinical benefit was defined as complete response, partial response, or stable disease ≥6 months per RECIST (Response Evaluation Criteria in Solid Tumors) V.1.1 criteria. RESULTS: Clinical benefit was similar between VEGF-TKI and ICI arms (65% vs 54%). Patients with clinical benefit from VEGF-TKIs had lower pretreatment levels of interleukin-6 (IL-6) (p=0.02), IL-1RA (p=0.03), and granulocyte colony-stimulating factor (CSF) (p=0.02). At 1 month, patients with clinical benefit from ICIs had higher levels of interferon-γ (IFN-γ) (p=0.04) and IL-12 (p=0.03). Among patients on VEGF-TKIs, those with clinical benefit had lower 1 month IL-13 (p=0.02) and granulocyte macrophage CSF (p=0.01) as well as higher 1 month VEGF (p=0.04) compared with patients with no clinical benefit. CONCLUSION: For patients receiving VEGF-TKI or ICI therapy, distinct plasma cytokines were associated with clinical benefit. Our findings support additional investigation into plasma cytokines as biomarkers in metastatic RCC.

Targeted In Vivo Delivery of NF-κB Decoy Inhibitor Augments Sensitivity of B Cell Lymphoma to Therapy.

Despite recent advances, non-Hodgkin's B cell lymphoma patients often relapse or remain refractory to therapy. Therapeutic resistance is often associated with survival signaling via nuclear factor κB (NF-κB) transcription factor, an attractive but undruggable molecular target. In this study, we describe a bipartite inhibitor comprising a NF-κB-specific decoy DNA tethered to a CpG oligodeoxynucleotide (ODN) targeting Toll-like receptor-9-expressing B cell lymphoma cells. The Bc-NFκBdODN showed efficient uptake by human diffuse large B cell (U2932, OCI-Ly3), Burkitt (RaJi), and mantle cell (Jeko1) lymphomas, respectively. We confirmed that Bc-NFκBdODN inhibited NF-κB nuclear translocation and DNA binding, resulting in CCND2 and MYC downregulation. Bc-NFκBdODN enhanced radiosensitivity of lymphoma cells in vitro. In xenotransplanted human lymphoma, local injections of Bc-NFκBdODN reduced NF-κB activity in whole tumors. When combined with a local 3-Gy dose of radiation, Bc-NFκBdODN effectively arrested OCI-Ly3 lymphoma progression. In immunocompetent mice, intratumoral injections of Bc-NFκBdODN suppressed growth of directly treated and distant A20 lymphomas, as a result of systemic CD8 T cell-dependent immune responses. Finally, systemic administration of Bc-NFκBdODN to mice bearing disseminated A20 lymphoma induced complete regression and extended survival of most of the treated mice. Our results underscore clinical relevance of this strategy as monotherapy and in support of radiation therapy to benefit patients with resistant or relapsed B cell lymphoma.

Myeloid cell-targeted STAT3 inhibition sensitizes head and neck cancers to radiotherapy and T cell-mediated immunity.

The tumor microenvironment affects the outcome of radiotherapy against head and neck squamous cell carcinoma (HNSCC). We recently found that tolerogenic myeloid cells accumulate in the circulation of HNSCC patients undergoing radiotherapy. Here, we analyzed tumor-containing lymph node biopsies collected from these patients. After 2 weeks of radiotherapy, we found an increase in tumor-associated macrophages (TAMs) with activated STAT3, while CD8+ T cells were reduced as detected using multiplex IHC. Gene expression profiling indicated upregulation of M2 macrophage-related genes (CD163, CD206), immunosuppressive mediators (ARG1, LIF, TGFB1), and Th2 cytokines (IL4, IL5) in irradiated tumors. We next validated STAT3 as a potential target in human HNSCC-associated TAMs, using UM-SCC1 xenotransplants in humanized mice. Local injections of myeloid cell-targeted STAT3 antisense oligonucleotide (CpG-STAT3ASO) activated human DCs/macrophages and promoted CD8+ T cell recruitment, thereby arresting UM-SCC1 tumor growth. Furthermore, CpG-STAT3ASO synergized with tumor irradiation against syngeneic HPV+ mEERL and HPV- MOC2 HNSCC tumors in mice, triggering tumor regression and/or extending animal survival. The antitumor immune responses were CD8+ and CD4+ T cell dependent and associated with the activation of antigen-presenting cells (DCs/M1 macrophages) and increased CD8+ to regulatory T cell ratio. Our observations suggest that targeted inhibition of STAT3 in tumor-associated myeloid cells augments the efficacy of radiotherapy against HNSCC.

An emerging role for immune regulatory subsets in chronic lymphocytic leukaemia.

The last few years has seen the burgeoning of a new category of therapeutics for cancer targeting immune regulatory pathways. Antibodies that block the PD-1/PD-L1 interaction are perhaps the most prominent of these new anti-cancer therapies, but several other inhibitory receptor ligand interactions have also shown promise as targets in clinical trials, including CTLA-4/CD80 and Lag-3/MHC class II. Related to this is a rapidly improving knowledge of 'regulatory' lymphocyte lineages, including NKT cells, MAIT cells, B regulatory cells and others. These cells have potent cytokine responses that can influence the functioning of other immune cells and many researchers believe that they could be effective targets for therapies designed to enhance immune responses to cancer. This review will outline our current understanding of FOXP3+ 'Tregs', NKT cells, MAIT cells and B regulatory cells immune regulatory cell populations in cancer, with a particular focus on chronic lymphocytic leukaemia (CLL). We will discuss evidence linking CLL with immune regulatory dysfunction and the potential for new therapies targeting regulatory cells.

Pigment epithelium-derived factor upregulates collagen I and downregulates matrix metalloproteinase 2 in osteosarcoma cells, and colocalises to collagen I and heat shock protein 47 in fetal and adult bone.

OBJECTIVE: Pigment epithelium-derived factor (PEDF) has proven anti-osteosarcoma activity. However, the mechanism(s) underpinning its ability to reduce primary bone tumour (osteosarcoma) metastasis is unknown. METHODS: Adult and fetal murine bone were immunostained for PEDF, collagen I (major protein in bone) and its processing proteins, heat shock protein 47 (HSP47, a chaperone protein for collagen I), membrane type I matrix metalloproteinase (MT1-MMP, a collagenase), and matrix metalloproteinase 2 (MMP-2, which is activated by MT1-MMP). Immunoblotting and immunocytochemistry were used to observe levels of the above biomarkers when human osteosarcoma cells were treated with PEDF. KEY FINDINGS: Immunohistochemical staining in adult and fetal bone mirrors collagen I. PEDF localised to ridges of trabecular bone in tibial cortex and to megakaryocytes within bone marrow. Second, we observed that PEDF upregulates collagen I, HSP47 and MT1-MMP, while downregulating MMP-2 in osteosarcoma cells in vitro. CONCLUSION: PEDF is a promising antagonist to osteosarcoma cell metastasis via downregulation of MMP-2, and can induce tumour cells to further adopt differentiative properties, thereby possibly reducing their aggressive growth in vitro and in vivo.

Pigment epithelium-derived factor as a natural matrix metalloproteinase inhibitor: a comparison with classical matrix metalloproteinase inhibitors used for cancer treatment.

OBJECTIVES: In the 1990s, the discovery of the important role of matrix metalloproteinases (MMPs) in cancer angiogenesis, growth and metastasis galvanised research efforts to search for ways to inhibit these MMPs. To date, this has resulted in the investigation of approximately 50 MMPIs which have undergone various phases of clinical trials. However, despite a large body of research being devoted to discovery and development of MMPIs, results have largely not been supportive of this approach to anticancer treatment. KEY FINDINGS: The reasons for the general failure of these drugs in clinical trials include various unwanted side-effects, the use of healthy volunteers to provide drug dosages which did not correctly reflect dosages for cancer patients, and the exclusion of patients with early stage cancer in clinical trials despite MMPs being determined to be critical for the angiogenic switch, a process associated with early tumour growth. In contrast, a naturally-occurring endogenous protein and a non-functional serine protease inhibitor (serpin), pigment epithelium-derived factor (PEDF), has been proposed for cancer therapy partly due to its ability to regulate specific MMPs central to cancer progression. SUMMARY: PEDF has been found to specifically downregulate membrane-type I matrix metalloproteinase (MT1-MMP) and furthermore, potentially matrix metalloproteinase-2 (MMP-2), two of the most commonly implicated MMPs in neoplasia.

Regulation of MT1-MMP and MMP-2 by the serpin PEDF: a promising new target for metastatic cancer.

The balance of endogenous angiogenic factors in the body is responsible for the homeostatic control of angiogenesis during normal physiological circumstances, with the disruption of this fragile balance leading to pathologic angiogenic events such as those involved in cancer progression. This review focuses regulation of the pro-angiogenic factors membrane type-1 matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) by the anti-angiogenic factor pigment epithelium-derived factor (PEDF) in the contexts of angiogenesis, cancer cell proliferation and metastasis. Understanding the role of PEDF in the regulation of MT1-MMP and MMP-2 as it pertains to cancer control is important in order to understand whether and how such associations provide a novel target for cancer therapy.