Autoantibodies against double-stranded DNA (anti-dsDNA) are a hallmark of systemic lupus erythematosus (SLE). It is well documented that anti-dsDNA reactive B lymphocytes are normally controlled by immune self-tolerance mechanisms operating at several levels. The evolution of high levels of IgG anti-dsDNA in SLE is dependent on somatic hypermutation and clonal selection, presumably in germinal centers from non-autoreactive B cells. Twin studies as well as genetic studies in mice indicate a very strong genetic contribution for the development of anti-dsDNA as well as SLE. Only few single gene defects with a monogenic Mendelian inheritance have been described so far that are directly responsible for the development of anti-dsDNA and SLE. Recently, among other mutations, rare null-alleles for the deoxyribonuclease 1 like 3 (DNASE1L3) and the Fc gamma receptor IIB (FCGR2B) have been described in SLE patients and genetic mouse models. Here, we demonstrate that double Dnase1l3- and FcgR2b-deficient mice in the C57BL/6 background exhibit a very early and massive IgG anti-dsDNA production. Already at 10 weeks of age, autoantibody production in double-deficient mice exceeds autoantibody levels of diseased 9-month-old NZB/W mice, a long established multigenic SLE mouse model. In single gene-deficient mice, autoantibody levels were moderately elevated at early age of the mice. Premature autoantibody production was accompanied by a spontaneous hyperactivation of germinal centers, early expansions of T follicular helper cells, and elevated plasmablasts in the spleen. Anti-dsDNA hybridomas generated from double-deficient mice show significantly elevated numbers of arginines in the CDR3 regions of the heavy-chain as well as clonal expansions and diversification of B cell clones with moderate numbers of somatic mutations. Our findings show a strong epistatic interaction of two SLE-alleles which prevent early and high-level anti-dsDNA autoantibody production. Both genes apparently synergize to keep in check excessive germinal center reactions evolving into IgG anti-dsDNA antibody producing B cells.

Pancreatic Ductal Adenocarcinoma (PDAC) is estimated to become the second leading cause of cancer-related deaths by 2030 with mortality rates of up to 93%. Standard of care chemotherapeutic treatment only prolongs the survival of patients for a short timeframe. Therefore, it is important to understand events driving treatment failure in PDAC as well as identify potential more effective treatment opportunities. PDAC is characterized by a high-density stroma, high interstitial pressure and very low oxygen tension. The aim of this study was to establish a PDAC platform that supported the understanding of treatment response of PDAC organoids in mono-, and co-culture with pancreatic stellate cells (PSCs) under hypoxic and normoxic conditions. Cultures were exposed to Gemcitabine in combination with molecules targeting relevant molecular programs that could explain treatment specific responses under different oxygen pressure conditions. Two groups of treatment responses were identified, showing either a better effect in monoculture or co-culture. Moreover, treatment response also differed between normoxia and hypoxia. Modulation of response to Gemcitabine was also observed in presence of a Hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) inhibitor and HIF inhibitors. Altogether this highlights the importance of adjusting experimental conditions to include relevant oxygen levels in drug response studies in PDAC.

Comprehensive molecular landscaping studies reveal a potentially brighter future for pancreatic ductal adenocarcinoma (PDAC) patients. Blood-borne biomarkers obtained from minimally invasive “liquid biopsies” are now being trialled for early disease detection and to track responses to therapy. Integrated genomic and transcriptomic studies using resectable tumour material have defined intrinsic patient subtypes and actionable genomic segments that promise a shift towards genome-guided patient management. Multimodal mapping of PDAC using spatially resolved single cell transcriptomics and imaging techniques has identified new potentially therapeutically actionable cellular targets and is providing new insights into PDAC tumour heterogeneity. Despite these rapid advances, defining biomarkers for patient selection remain limited. This review examines the current PDAC cancer biomarker ecosystem (identified in tumour and blood) and explores how advances in single cell sequencing and spatially resolved imaging modalities are being used to uncover new targets for therapeutic intervention and are transforming our understanding of this difficult to treat disease.

Background Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Although combined treatment with gemcitabine and albumin-bound paclitaxel has improved the prognosis of PDAC, both intrinsic and acquired chemoresistance remain as severe hurtles towards improved prognosis. Thus, new therapeutic targets and innovative strategies are urgently needed. Methods In this study, we used the KPC mouse model-derived PDAC cell line TB32047 to perform kinome-wide CRISPR-Cas9 loss-of-function screening. Next-generation sequencing and MAGeCK-VISPR analysis were performed to identify candidate genes. We then conducted cell viability, clonogenic, and apoptosis assays and evaluated the synergistic therapeutic effects of cyclin-dependent kinase 7 (CDK7) depletion or inhibition with gemcitabine (GEM) and paclitaxel (PTX) in a murine orthotopic pancreatic cancer model. For mechanistic studies, we performed genome enrichment analysis (GSEA) and Western blotting to identify and verify the pathways that render PDAC sensitive to GEM/PTX therapy. Results We identified several cell cycle checkpoint kinases and DNA damage-related kinases as targets for overcoming chemoresistance. Among them, CDK7 ranked highly in both screenings. We demonstrated that both gene knockout and pharmacological inhibition of CDK7 by THZ1 result in cell cycle arrest, apoptosis induction, and DNA damage at least predominantly through the STAT3-MCL1-CHK1 axis. Furthermore, THZ1 synergized with GEM and PTX in vitro and in vivo, resulting in enhanced antitumor effects. Conclusions Our findings support the application of CRISPR-Cas9 screening in identifying novel therapeutic targets and suggest new strategies for overcoming chemoresistance in pancreatic cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Although combined treatment with gemcitabine and albumin-bound paclitaxel has improved the prognosis of PDAC, both intrinsic and acquired chemoresistance cloud the outcomes. Hence, there is an urgent need to explore new therapeutic targets and formulate innovative strategies for the more effective treatment of PDAC.Methods: We transduced pancreatic cancer cells with a protein kinase library, and transfected cells were selected by puromycin treatment, followed by continuous exposure to vehicle control or gemcitabine/paclitaxel chemotherapy for 21 days. Next-generation sequencing as well as MAGeCK-VISPR analysis were performed to the identify candidate genes. Finally, we used the CRISPR-Cas9 to knockout the target gene, confirming that it is a viable target for overcoming chemoresistance in vivo and in vitro.Results: We identified several cell cycle checkpoint kinases and DNA damage-related kinases as targets for overcoming chemoresistance. Among them, cyclin-dependent kinase 7 (CDK7) ranked highly in both screenings. We demonstrated that both gene knockout and pharmacological inhibition of CDK7 by THZ1 result in cell cycle arrest, apoptosis induction. Furthermore, THZ1 synergized with gemcitabine and paclitaxel in vitro and in vivo, resulting in enhanced antitumor effects.Conclusions: Our findings support the application of CRISPR-Cas9 screening in identifying novel therapeutic targets and provide new strategies for overcoming chemoresistance in pancreatic cancer.

Pancreatic Ductal Adenocarcinoma (PDAC), the most common pancreatic cancer type, will become the second leading cause of cancer-related deaths by 2030 with mortality rates of up to 93%. Current standard-of-care for patients with PDAC includes chemotherapeutic regimens and pancreatic cancer surgery. However, only 20% of the patients are eligible for surgery due to late diagnosis. Although chemotherapeutic regimens are the leading treatment to PDAC patients, these are still very limited and the development of resistance to treatment is often observed. PDAC tumors are characterized by high-density stroma and hypovascularization, therefore these present high interstitial pressure and hypoxia. These features potentially interfere with the efficiency of chemotherapeutic drugs and highlight the urgent need for novel PDAC screening platforms. Here, we describe the establishment of PDAC organoid cultures in the MIMETAS OrganoPlate®. The aim of this project was to determine the treatment response of PDAC organoids in mono-, and co-culture with pancreatic stellate cells (PSCs) under hypoxic and normoxic conditions. To recapitulate in-vivo like conditions, the 2-lane OrganoPlate® from MIMETAS was used to study organoid growth and sensitivity to treatment. Several standard of care (chemo-)therapeutics were tested on PDAC organoids embedded within an extracellular matrix in the Organoplate® 2-lane. These were exposed to chemotherapeutic treatments for 72h. PDAC organoids showed an overall better survival when grown in co-culture with PSCs. Interestingly, organoids grown in co-culture showed a higher survival rate under hypoxic conditions. In contrast, when grown in monoculture cell viability was higher or similar under normoxic conditions than in hypoxia for the different chemotherapeutics. The OrganoPlate® 2-lane provides an excellent platform for (co-) cultivation and high-throughput phenotypic drug screening of PDAC organoids, thereby potentially enabling the development of novel in-vivo like model systems for efficient patient stratification and drug discovery. Citation Format: Marlene Geyer, Daniel Schreyer, Lisa-Marie Gaul, Désiréé Goubert, Susanne Pfeffer, Christian Pilarsky, Karla S. Queiroz. A novel microfluidic platform for PDAC organoid culture and drug screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4809.

Cutaneous squamous cell carcinoma (cSCC) can be a relatively low-risk cancer type as it is typically curable upon surgical resection; however, in advanced cases, treatment options are lacking and patient outcomes are poor. The molecular mechanisms that drive metastasis and the associated targets for therapeutic intervention are yet to be determined. To investigate drivers of metastatic cSCC, we undertook whole-genome sequencing of matched primary and metastatic cSCC samples from two patients. Analysis of single nucleotide variants and copy number changes revealed the high tumour mutational burden of this cancer type due to ultraviolet-induced DNA damage, highlighting the challenge faced in filtering out passenger mutations and identifying oncogenic events. Variations unique to the metastases were obtained, and their pathogenicity was assessed. Damaging mutations were found in transforming growth factor beta receptor 2 (TGFBR2) and genes in the PTEN/PI3K/Akt signalling pathway, specifically PTEN, PIK3R1 and PIK3CA, which have previously occurred in cancer samples included in COSMIC, cBioPortal, Cancer Hotspots and Genomic Data Commons. To validate functionally the role of TGFBR2 and PTEN/PI3K/Akt pathway mutations in skin cancers, we generated conditional, inducible Ptenfl/fl;K14Cre+, Pten+/fl;Tgfbr2fl/fl;K14Cre+ and Ptenfl/fl;Tgfbr2fl/fl; K14Cre+ mice. Loss of both Tgfbr2 and Pten in mice was associated with increased proliferation (P = 2.1 × 106), invasion, lymph node metastases and poorer tumour-free (P < 9.0 × 107) and overall survival (P < 2.0 × 106). These data showcase the role of transforming growth factor-β and PTEN/PI3K/Akt signalling pathways on the development of metastatic cSCC.