Development and In Vivo Evaluation of Small-Molecule Ligands for Positron Emission Tomography of Immune Checkpoint Modulation Targeting Programmed Cell Death 1 Ligand 1.

A substantial portion of patients do not benefit from programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) checkpoint inhibition therapies, necessitating a deeper understanding of predictive biomarkers. Immunohistochemistry (IHC) has played a pivotal role in assessing PD-L1 expression, but small-molecule positron emission tomography (PET) tracers could offer a promising avenue to address IHC-associated limitations, i.e., invasiveness and PD-L1 expression heterogeneity. PET tracers would allow for improved quantification of PD-L1 through noninvasive whole-body imaging, thereby enhancing patient stratification. Here, a large series of PD-L1 targeting small molecules were synthesized, leveraging advantageous substructures to achieve exceptionally low nanomolar affinities. Compound 5c emerged as a promising candidate (IC50 = 10.2 nM) and underwent successful carbon-11 radiolabeling. However, a lack of in vivo tracer uptake in xenografts and notable accumulation in excretory organs was observed, underscoring the challenges encountered in small-molecule PD-L1 PET tracer development. The findings, including structure-activity relationships and in vivo biodistribution data, stand to illuminate the path forward for refining small-molecule PD-L1 PET tracers.

Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis.

ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

A ventricular fibrillation cardiac arrest model with extracorporeal cardiopulmonary resuscitation in rats: 8 minutes arrest time leads to increased myocardial damage but does not increase neuronal damage compared to 6 minutes.

Introduction: Extracorporeal cardiopulmonary resuscitation (ECPR) is an emerging strategy in highly selected patients with refractory cardiac arrest (CA). Animal models can help to identify new therapeutic strategies to improve neurological outcome and cardiac function after global ischemia in CA. Aim of the study was to establish a reproducible ECPR rat model of ventricular fibrillation CA (VFCA) that leads to consistent neuronal damage with acceptable long-term survival rates, which can be used for future research. Materials and methods: Male Sprague Dawley rats were resuscitated with ECPR from 6 min (n = 15) and 8 min (n = 16) VFCA. Animals surviving for 14 days after return of spontaneous resuscitation (ROSC) were compared with sham operated animals (n = 10); neurological outcome was assessed daily until day 14. In the hippocampal cornu ammonis 1 region viable neurons were counted. Microglia and astrocyte reaction was assessed by Iba1 and GFAP immunohistochemistry, and collagen fibers in the myocardium were detected in Azan staining. QuPath was applied for quantification. Results: Of the 15 rats included in the 6 min CA group, all achieved ROSC (100%) and 10 (67%) survived to 14 days; in the 8 min CA group, 15 (94%) achieved ROSC and 5 (31%) reached the endpoint. All sham animals (n = 10) survived 2 weeks. The quantity of viable neurons was significantly decreased, while the area displaying Iba1 and GFAP positive pixels was significantly increased in the hippocampus across both groups that experienced CA. Interestingly, there was no difference between the two CA groups regarding these changes. The myocardium in the 8 min CA group exhibited significantly more collagen fibers compared to the sham animals, without differences between 6- and 8-min CA groups. However, this significant increase was not observed in the 6 min CA group. Conclusion: Our findings indicate a uniform occurrence of neuronal damage in the hippocampus across both CA groups. However, there was a decrease in survival following an 8-min CA. Consequently, a 6-min duration of CA resulted in predictable neurological damage without significant cardiac damage and ensured adequate survival rates up to 14 days. This appears to offer a reliable model for investigating neuroprotective therapies.

Decoding molecular programs in melanoma brain metastases.

Melanoma brain metastases (MBM) variably respond to therapeutic interventions; thus determining patient's prognosis. However, the mechanisms that govern therapy response are poorly understood. Here, we use a multi-OMICS approach and targeted sequencing (TargetSeq) to unravel the programs that potentially control the development of progressive intracranial disease. Molecularly, the expression of E-cadherin (Ecad) or NGFR, the BRAF mutation state and level of immune cell infiltration subdivides tumors into proliferative/pigmented and invasive/stem-like/therapy-resistant irrespective of the intracranial location. The analysis of MAPK inhibitor-naive and refractory MBM reveals switching from Ecad-associated into NGFR-associated programs during progression. NGFR-associated programs control cell migration and proliferation via downstream transcription factors such as SOX4. Moreover, global methylome profiling uncovers 46 differentially methylated regions that discriminate BRAFmut and wildtype MBM. In summary, we propose that the expression of Ecad and NGFR sub- classifies MBM and suggest that the Ecad-to-NGFR phenotype switch is a rate-limiting process which potentially indicates drug-response and intracranial progression states in melanoma patients.

Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma.

Leukemic cutaneous T-cell lymphomas (L-CTCL) are lymphoproliferative disorders of skin-homing mature T-cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L-CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L-CTCL, which correlated with the increased clonal T-cell count in the blood. Dual inhibition of STAT3/5 using small-molecule degraders and multi-kinase blockers abolished L-CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L-CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti-leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L-CTCL.

KMT2C methyltransferase domain regulated INK4A expression suppresses prostate cancer metastasis.

BACKGROUND: Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated. METHODS: To investigate the functional effects of these mutations, we deleted the C-terminal catalytic core motif of Kmt2c specifically in mouse prostate epithelium. We analysed the effect of Kmt2c SET domain deletion in a Pten-deficient PCa mouse model in vivo and of truncation mutations of KMT2C in a large number of prostate cancer patients. RESULTS: We show here for the first time that impaired KMT2C methyltransferase activity drives proliferation and PIN formation and, when combined with loss of the tumour suppressor PTEN, triggers loss of senescence, metastatic dissemination and dramatically reduces life expectancy. In Kmt2c-mutated tumours we show enrichment of proliferative MYC gene signatures and loss of expression of the cell cycle repressor p16INK4A. In addition, we observe a striking reduction in disease-free survival of patients with KMT2C-mutated prostate cancer. CONCLUSIONS: We identified truncating events of KMT2C as drivers of proliferation and PIN formation. Loss of PTEN and KMT2C in prostate cancer results in loss of senescence, metastatic dissemination and reduced life expectancy. Our data demonstrate the prognostic significance of KMT2C mutation status in prostate cancer patients. Inhibition of the MYC signalling axis may be a viable treatment option for patients with KMT2C truncations and therefore poor prognosis.

Active immunization with a Her-2/neu-targeting Multi-peptide B cell vaccine prevents lung metastases formation from Her-2/neu breast cancer in a mouse model.

In pre-clinical and clinical settings, active immunization with a Her-2/neu vaccine (HerVaxx), comprising B-cell peptide from Trastuzumab binding site, has been shown to reduce primary tumor growth via induction of polyclonal anti-tumor immune responses and immunological memory. Here, we tested the combination of HerVaxx and the recently identified B-cell epitope/mimotope of Pertuzumab, i.e. a multi-peptide B-cell vaccine, for preventing Her-2/neu lung metastases formation in a mouse model. Active immunization with the multi-peptide vaccine was associated with decreased lung weights, and histological evaluation of the lungs showed that the significant reduction of lung metastases was associated with increased CD4+ and CD8+ T cell infiltration. Notably, along with the overall reduction of lungs weights and Her-2 positive metastases, a formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our results might pave the way to a multi-peptide B-cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Moreover, combination therapy targeting PD-L1 may result in total remission of metastases. Such a therapy may be used clinically to alternately target Her-2/neu and PD-L1 in metastatic breast cancer.

STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer.

Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co-expression network of transcriptomics data in addition to laser-microdissected proteomics from human and murine prostate FFPE samples. We show up-regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up-regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis.

The targetable kinase PIM1 drives ALK inhibitor resistance in high-risk neuroblastoma independent of MYCN status.

Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status.

Interferon gamma-induced human guanylate binding protein 1 inhibits mammary tumor growth in mice.

Interferon gamma (IFN-gamma) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-gamma are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-gamma-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-gamma-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-gamma-induced antitumoral defense system.

Reconstituting retroviral (ReCon) vectors facilitating delivery of cytotoxic genes in cancer gene therapy approaches.

BACKGROUND: We have previously described the generation of reconstituting retroviral (ReCon) vectors designed for cancer gene therapy using cytotoxic gene products. The unique vector structure with a promoter physically separated from the transgene allows generation of stable virus producer cells irrespective of the toxic gene. The mechanism of synthesis of DNA from retroviral RNA dictates that infection leads to the reconstitution of functional expression cassettes in the target cell. METHODS: To improve vector titres, a cytomegalovirus enhancer was inserted upstream of the 5'-long-terminal repeat (LTR); the Woodchuck hepatitis virus post-transcriptional regulatory element and an elongated attachment site upstream of the 3'-LTR were included. In addition, a bacterial origin of replication was deleted and a functional internal polyadenylation signal mutated. Transcriptional targeting was attempted by introducing mammary tissue-specific promoters such as the U3 region of mouse mammary tumour virus or the promoter of the whey acidic protein encoding gene. All modifications were analysed in detail with respect to virus production and infectivity. Finally, the vector was armed with the lambda-holin encoding gene and transduced cells were analysed for cytotoxic effects. RESULTS: Distinct modifications of the vector resulted in a titre improvement of more than 560-fold. Compatibility of the optimized vector with targeted cellular promoters was demonstrated. When equipped with the cytotoxic gene, stable producer cells could be successfully established and high titre virus infection resulted in rigorous target cell killing. CONCLUSIONS: The ReCon vector in its optimized form is an attractive tool for cancer gene therapy approaches.

Quantification and characterization of autotransduction in retroviral vector producer cells.

Gene therapy has evolved into a tempting strategy for the management of cancer and other life-threatening diseases. Various approaches employ retroviral vectors to deliver the therapeutic gene. The profound knowledge about retrovirus biology allows the generation of increasingly advanced vector systems as well as an accurate assessment and management of potential safety risks. This study focuses on the genetic stability of retrovirus producer cells as a basic safety requirement and its compromise by autotransduction. It has been shown previously that protection of retroviral packaging systems by superinfection interference is not guaranteed. The current study provides insight into the extent of autotransduction and the time point at which it occurs, and examines strategies to antagonize it. Therefore, a reconstituting vector system was used that obviates transgene expression in virus producer cells by physically separating transgene and promoter. Just on infection two functional expression cassettes are reconstituted, causing highly efficient transgene expression in transduced cells. Equipped with an enhanced green fluorescent protein-encoding gene, this vector allowed accurate quantification of autotransduced cells, which were then isolated by fluorescence-activated cell sorting and further characterized. Sequencing of recloned integrated vector copies demonstrated that high transgene expression levels were strictly associated with the presence of reverse-transcribed vector copies. Envelope protein expression levels, however, were found to be equal in autotransduced and noninfected virus producer cells. Finally, the occurrence of autotransduction could be assigned to an early time point after transfection and was successfully blocked by azidothymidine treatment, yielding a stable and homogeneous population of noninfected retrovirus producer cells.

The cytotoxic activity of the bacteriophage lambda-holin protein reduces tumour growth rates in mammary cancer cell xenograft models.

BACKGROUND: The potential use of gene therapy for cancer treatment is being intensively studied. One approach utilises the expression of genes encoding cytotoxic proteins. Such proteins can affect cellular viability, for example by inhibiting the translation machinery or disturbing membrane integrity. The bacteriophage Lambda (lambda)-holin protein is known to form a lesion in the cytoplasmic membrane of E. coli, triggering bacterial cell lysis and thereby enabling the release of new bacteriophage particles. The aim of this study was to evaluate whether the lambda-holin protein has a cytotoxic impact on eukaryotic cells and whether it holds potential as a new therapeutic protein for cancer gene therapy. METHODS: To explore this possibility, stably transfected human cell lines were established that harbour a tetracycline (Tet)-inducible system for controlled expression of the lambda-holin gene. The effect of the lambda-holin protein on eukaryotic cells was studied in vitro by applying several viability assays. We also investigated the effect of lambda-holin gene expression in vivo using a human breast cancer cell tumour xenograft as well as a syngeneic mammary adenocarcinoma mouse model. RESULTS: The lambda-holin-encoding gene was inducibly expressed in eukaryotic cells in vitro. Expression led to a substantial reduction of cell viability of more than 98%. In mouse models, lambda-holin-expressing tumour cell xenografts revealed significantly reduced growth rates in comparison to xenografts not expressing the lambda-holin gene. CONCLUSIONS: The lambda-holin protein is cytotoxic for eukaryotic cells in vitro and inhibits tumour growth in vivo suggesting potential therapeutic use in cancer gene therapy.

HIV-1 Rev can specifically interact with MMTV RNA and upregulate gene expression.

We present evidence that the HIV-1 Rev protein can heterologously regulate expression of the simple beta retrovirus mouse mammary tumour virus (MMTV). Up to 10-fold upregulation was seen in a functional assay system when specific MMTV sequences were substituted for the HIV-1 Rev responsive element (RRE). RNA gel shift analysis showed that purified recombinant Rev could specifically bind to MMTV unique region 3 prime (U3) RNA and that these sequences could compete for wild-type Rev-RRE binding approximately 20-fold more efficiently than a non-specific competitor RNA. Using a combination of in silico and deletion mutation analyses, it was not possible to define any single specific secondary structure responsive to Rev, suggesting that a structure or combination of structures that only form in the context of the complete U3 transcript is/are required to interact with Rev. Taken together, these results suggest that HIV-1 Rev can directly bind to MMTV RNA as well as mediate upregulation of MMTV gene expression.