Pancreatic Ductal Adenocarcinoma Cancer Research Articles

GJB3 promotes pancreatic cancer liver metastasis by enhancing the polarization and survival of neutrophil.

Connexins are membrane expressed proteins, which could assemble into hexamers to transfer metabolites and secondary messengers. However, its roles in pancreatic cancer metastasis remains unknown. In this study, by comparing the gene expression patterns in primary pancreatic cancer patients primary and liver metastasis specimens, we found that Gap Junction Protein Beta 3 (GJB3) significantly increased in Pancreatic ductal adenocarcinoma (PDAC) liver metastasis. Animal experiments verified that GJB3 depletion suppressed the hepatic metastasis of PDAC cancer cells. Further, GJB3 over expression increased the neutrophil infiltration. Mechanistic study revealed that GJB3 form channels between PDAC tumor cells and accumulated neutrophil, which transfer cyclic adenosine monophosphate (cAMP) from cancer to neutrophil cells, which supports the survival and polarization. Taken together, our data suggesting that GJB3 could act as a potential therapeutic target of PDAC liver metastasis.

Niraparib-induced STAT3 inhibition increases its antitumor effects.

Recently, poly(ADP-ribosyl)ation polymerase inhibitors (PARPis), which induce synthetic lethality of tumor cells with DNA damage repair defects, have emerged as a promising therapy for ovarian, breast, and pancreatic cancer. Although the PARPi Olaparib is limited to treating cancer patients with DNA repair deficiencies, the PARPi Niraparib is FDA approved to treat ovarian cancer patients regardless of their status in DNA repair pathways. Despite differences in the affinity to PARP enzymes, the rationale behind the clinical use of Niraparib in patients without DNA repair deficiencies is still lacking. Moreover, only Olaparib has been approved for pancreatic ductal adenocarcinoma (PDAC) patients with BRCA mutations, accounting for only 5-7% of total PDACs. It remains unclear whether Niraparib could be beneficial to PDACs without BRCA mutations. We found that Niraparib inhibits ovarian and PDAC tumor cell growth, regardless of BRCA mutational status, more effectively than Olaparib. Unlike Olaparib, which is known to activate STAT3, Niraparib inhibits STAT3 activity in ovarian and PDAC cancer cell lines and patient tumors. Moreover, Niraparib regulates the expression of several STAT3 downstream genes involved in apoptosis. Overexpression of a constitutively activated STAT3 mutant rescues Niraparib-induced cancer cell apoptosis. Our results suggest that Niraparib inhibits pSTAT3 by interfering with SRC tyrosine kinase. Collectively, our studies provide a mechanism underlying Niraparib's ability to induce tumor cell apoptosis without BRCA mutations, suggesting the potential use of Niraparib for treating PDAC patients regardless of BRCA status.

Cystathionine β-synthase overexpression drives metastatic dissemination in pancreatic ductal adenocarcinoma via inducing epithelial-to-mesenchymal transformation of cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of all cancer types with a constant rise in global incidence. Therefore, better understanding of PDAC biology, in order to design more efficient diagnostic and treatment modalities, is a priority.Here we found that the expression levels of cystathionine β-synthase (CBS), a transsulfuration enzyme, is markedly elevated in metastatic PDAC cells compared to cell lines isolated from non-metastatic primary tumors. On human immunohistochemical samples from PDAC patients we also found higher CBS staining in cancerous ductal cells compared to in non-tumor tissue, which was further elevated in the lymph node metastasis of the same patients. In mice, orthotopically injected CBS-silenced T3M4 cells induced fewer liver metastases compared to control cells indicating important roles for CBS in PDAC cancer cell invasion and malignant transformation. Wound healing and colony formation assays in cell culture confirmed that CBS-deficient metastatic T3M4 and non-metastatic BxPC3 primary tumor cells migrate slower and have impaired anchorage-independent growth capacities compared to control T3M4 cells. CBS silencing in T3M4 cells lowered WNT5a and SNAI1 gene expression down to levels that were observed in BxPC3 cells as well as resulted in an increase in E-cadherin and a decrease in Vimentin signals in mouse tumors when injected orthotopically. These observations suggested a primary role for the epithelial to mesenchymal transformation of cancer cells in CBS-mediated tumor aggressiveness. Under normal conditions, STAT3, an upstream regulator of Wnt signaling pathways, was less phosphorylated and more oxidized in shCBS T3M4 and BxPC3 compared to control T3M4 cells, which is consistent with decreased transcriptional activity at lower CBS levels due to less protection against oxidation. Sulfur metabolome analyses suggested that this CBS-mediated protection against oxidative modifications is likely to be related to persulfide/sulfide producing activities of the enzyme rather than its canonical function to produce cystathionine for cysteine synthesis. Taken together, CBS overexpression through regulation of the EMT plays a significant role in PDAC cancer cell invasion and metastasis.

SMAD2/3 mediate oncogenic effects of TGF-β in the absence of SMAD4

TGF-β signaling is involved in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis, representing one of the four major pathways genetically altered in 100% of PDAC cases. TGF-β exerts complex and pleiotropic effects in cancers, notably via the activation of SMAD pathways, predominantly SMAD2/3/4. Though SMAD2 and 3 are rarely mutated in cancers, SMAD4 is lost in about 50% of PDAC, and the role of SMAD2/3 in a SMAD4-null context remains understudied. We herein provide evidence of a SMAD2/3 oncogenic effect in response to TGF-β1 in SMAD4-null human PDAC cancer cells. We report that inactivation of SMAD2/3 in SMAD4-negative PDAC cells compromises TGF-β-driven collective migration mediated by FAK and Rho/Rac signaling. Moreover, RNA-sequencing analyses highlight a TGF-β gene signature related to aggressiveness mediated by SMAD2/3 in the absence of SMAD4. Using a PDAC patient cohort, we reveal that SMAD4-negative tumors with high levels of phospho-SMAD2 are more aggressive and have a poorer prognosis. Thus, loss of SMAD4 tumor suppressive activity in PDAC leads to an oncogenic gain-of-function of SMAD2/3, and to the onset of associated deleterious effects.

54 (PB044) - Non-clinical identification and characterization of KRAS G12D inhibitors

KRAS G12D is the most prevalent KRAS oncogenic mutant and occurs frequently in pancreatic ductal adenocarcinoma (PDAC), colorectal adenocarcinoma (CRC), and non-small cell lung cancer (NSCLC; 37%, 12% and 4%, respectively). KRAS G12D cycles between inactive (GDP) and active (GTP) states to regulate the RAS/MAPK pathway. Although the rate of intrinsic and GAP (GTPase activating protein) mediated hydrolysis is significantly slower in KRAS G12D than in KRAS wild-type (KRAS WT), we were able to build upon success of targeting the GDP state of KRAS G12C and designed novel KRAS G12D lead compounds with promising in vitro and in vivo activity. Our KRAS G12D inhibitors preferentially bind to KRAS G12D GDP as indicated by differential scanning fluorimetry (DSF), block KRAS G12D SOS1-mediated nucleotide exchange, and inhibit KRAS G12D binding to the RAS binding domain of C-RAF (RAF-RBD) with single digit nanomolar potency. Our leads effectively inhibit ERK1/2 phosphorylation (pERK) in AsPC-1 PDAC cells and inhibit cell proliferation in 3-dimensional Cell-Titer Glo assays in a panel of KRAS G12D mutant PDAC, CRC, and NSCLC cell lines. This in vivo activity translates in vitro where robust pharmacodynamic modulation in the PDAC AsPC-1 CDX model was induced. Tumor growth inhibition studies in this model revealed robust dose-dependent tumor growth inhibition and tumor regression in the absence of overt toxicity. In summary, we have identified potent and selective KRAS G12D inhibitors with robust dose-dependent tumor growth inhibition and regression activity in a PDAC CDX model. Lead optimization is ongoing with the aim of identifying a development candidate. No conflict of interest.

241 (PB021) - Development of a novel antibody-drug conjugate targeting both CEACAM5 and CEACAM6

Background: Carcinoembryonic antigen cell adhesion molecule (CEACAM) family members 5 and 6 are tumor-associated antigens frequently upregulated in epithelial cancers, including pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC), where they contribute to invasion, metastasis, survival, resistance to chemotherapy, and immune escape. CT109 is a novel antibody with dual specificity for both CEACAM5 and 6, mediated by binding to a shared glycoepitope. CT109 exhibits a high affinity (in the single nM range) to the target.

6P Sensitization of pancreatic and colorectal cancer to radiotherapy, chemotherapy and inhibition of PD-1 expression by newly developed proprotein convertase inhibitors

The pancreatic ductal adenocarcinoma (PDAC) and advanced colorectal cancer (CRC) have a bad prognosis with delayed diagnosis that significantly impedes the use of therapeutic resection. In these situation, palliative chemotherapy has modest activity, and Immune checkpoint inhibitors or radiotherapy failed to improve survival. The proprotein convertases (PCs) are enzymes involved in the biological activation/maturation of various protein precursors crucial for the malignant phenotype of PDAC and CRC tumors, and the resistance to various anticancer treatments.

480P CATRIPCA – A phase I of pembrolizumab (P) combined with Xevinapant (Debio 1143, (X)) in patients (pts) with non MSI-high advanced/metastatic pancreatic ductal adenocarcinoma (PDAC) or colorectal cancer (CRC)

X is an orally available inhibitor of apoptosis proteins (IAP) inhibitor. Preclinical data suggest that IAP inhibition may synergize with immune checkpoint blockers (ICB) by modulating the NF-KB pathway in immune cells.

489P Analysis of 3-dimensional volumetric distribution and dispersion of the radioenhancer NBTXR3 in various solid malignancies

NBTXR3, a first-in-class radioenhancer composed of functionalized hafnium oxide nanoparticles, is administered by one-time intratumoral (IT) injection and activated by radiotherapy (RT). It is designed to locally amplify the tumor-killing effect of RT without additional toxicity to surrounding healthy tissue. EU marketing approval was obtained in preoperative treatment of locally advanced soft tissue sarcomas (STS). Investigation in multiple tumor types is ongoing. Here we report feasibility and intra-tumor dispersion of IT NBTXR3 injection in patients with various solid malignancies: STS, head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), liver and lung metastases, pancreatic ductal adenocarcinoma (PDAC), esophageal cancer, and intra-prostate injection in patients with prostate adenocarcinoma. Patients with STS received NBTXR3+RT or RT alone followed by tumor resection in the phase II/III randomized Act.in.Sarc [NCT02379845] trial. NBTXR3 IT injection feasibility and safety is evaluated in phase I trials: HNSCC [NCT01946867], HCC or liver metastases [NCT02721056], advanced cancers in combination with anti-PD-1 [NCT03589339], PDAC [NCT04484909], esophageal cancer [NCT04615013], and prostate adenocarcinoma [NCT02805894]. IT injection in patients with STS, HNSCC, HCC, liver and lung metastases, PDAC, esophageal cancer, and intra-prostate injection is feasible. CT scan did not detect leakage in surrounding healthy tissues. IT dispersion ranged from 4.9-28.2% of tumor volume, depending on tumor size, with NBTXR3 treatment doses ranging from 5-22% in HNSCC, HCC, lung metastasis, STS, and prostate. Tumor responses were observed indicating the potential for NBTXR3 to improve patient outcomes at the doses tested and with dispersion within the tumor of up to 28.3%. Results from PDAC and esophageal cancer will be presented. IT injection of NBTXR3 is feasible in multiple tumor types and has shown favorable safety in several phase I studies. NBTXR3 demonstrated efficacy in a phase II/III study in patients with STS, and promising signs of efficacy have been observed in other tumor types.

Clinico-genomic Characterization of ATM and HRD in Pancreas Cancer: Application for Practice.

Characterizing germline and somatic ATM variants (gATMm, sATMm) zygosity and their contribution to homologous recombination deficiency (HRD) is important for therapeutic strategy in pancreatic ductal adenocarcinoma (PDAC). Clinico-genomic data for patients with PDAC and other cancers with ATM variants were abstracted. Genomic instability scores (GIS) were derived from ATM-mutant cancers and overall survival (OS) was evaluated. Forty-six patients had PDAC and pathogenic ATM variants including 24 (52%) stage III/IV: gATMm (N = 24), and sATMm (N = 22). Twenty-seven (59%) had biallelic, 15 (33%) monoallelic, and 4 indeterminate (8%) variants. Median OS for advanced-stage cohort at diagnosis (N = 24) was 19.7 months [95% confidence interval (CI): 12.3-not reached (NR)], 27.1 months (95% CI: 22.7-NR) for gATMm (n = 11), and 12.3 months for sATMm (n = 13; 95% CI: 11.9-NR; P = 0.025). GIS was computed for 33 patients with PDAC and compared with other ATM-mutant cancers enriched for HRD. The median was lower (median, 11; range, 2-29) relative to breast (18, 3-55) or ovarian (25, 3-56) ATM-mutant cancers (P < 0.001 and P = 0.003, respectively). Interestingly, biallelic pathogenic ATM variants were mutually exclusive with TP53. Other canonical driver gene (KRAS, CDKN2A, SMAD4) variants were less frequent in ATM-mutant PDAC. ATM variants in PDAC represent a distinct biologic group and appear to have favorable OS. Nonetheless, pathogenic ATM variants do not confer an HRD signature in PDAC and ATM should be considered as a non-core HR gene in this disease.

ID1 marks the tumorigenesis of pancreatic ductal adenocarcinoma in mouse and human

Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly disease that has an increasing death rate but no effective treatment to now. Although biological and immunological hallmarks of PDAC have been frequently reported recently, early detection and the particularly aggressive biological features are the major challenges remaining unclear. In the current study, we retrieved multiple scRNA-seq datasets and illustrated the genetic programs of PDAC development in genetically modified mouse models. Notably, the transcription levels of Id1 were elevated specifically along with the PDAC development. Pseudotime trajectory analysis revealed that Id1 was closely correlated with the malignancy of PDAC. The gene expression patterns of human PDAC cells were determined by the comparative analysis of the scRNA-seq data on human PDAC and normal pancreas tissues. ID1 levels in human PDAC cancer cells were dramatically increased compared to normal epithelial cells. ID1 deficiency in vitro significantly blunt the invasive tumor-formation related phenotypes. IPA analysis on the differentially expressed genes suggested that EIF2 signaling was the core pathway regulating the development of PDAC. Blocking EFI2 signaling remarkably decreased the expression of ID1 and attenuated the tumor-formation related phenotypes. These observations confirmed that ID1 was regulated by EIF2 signaling and was the critical determinator of PDAC development and progression. This study suggests that ID1 is a potential malignant biomarker of PDAC in both mouse models and human and detecting and targeting ID1 may be a promising strategy to treat or even rescue PDAC.

"Management of hospitalised adults with coronavirus disease 2019 (COVID-19): a European Respiratory Society living guideline." James D. Chalmers, Megan L. Crichton, Pieter C. Goeminne, et al. Eur Respir J 2021; 57: 2100048.

<h3>Background</h3> Cancers affect a large number of people worldwide and sometimes have mortality rates of over 90% (pancreatic ductal adenocarcinoma (PDAC), high grade glioblastomas). Currently, immunotherapeutic strategies fail in number of cancer cases due to a lack of understanding of the molecular mechanisms operated by these cancers to evade immune surveillance. Recent evidence demonstrated that the protein galectin-9 is highly expressed in majority of cancer cells. Importantly, this protein is a crucial part of the immune evasion machinery operated by human malignant tumours. Understanding the biochemistry of this immune evasion machinery is required for designing highly efficient and affordable personalised targeted immunotherapy of human cancers. <h3>Materials and Methods</h3> We used, glioblastoma, PDAC, breast and renal cancer cell lines as cancer cell models. Jurkat T lymphocytes, TALL-104 cytotoxic T cells and primary human and mouse CD3-positive T lymphocytes were employed for immune evasion studies. C57 BL16 mice were used for <i>in vivo</i> studies. Western blot analysis, on-cell and in-cell Western, ELISA, qRT-PCR, chromatin immunoprecipitation (ChIP) assays as well as a variety of biochemical tests were used to conduct the studies. <h3>Results</h3> We found that T cells trigger galectin-9 expression and secretion (in most cases requires Tim-3 protein (T cell immunoglobulin and mucine domain containing protein 3)) in a variety of human cancer cells including but not limited to glioblastoma, PDAC, breast and renal cancers. Galectin-9 triggers exhaustion and often death of cytotoxic T cells. Other immune checkpoint proteins including VISTA (V-domain Ig-containing suppressor of T cell activation) and PD-L1 (ligand of programmed cell death protein 1) promote apoptotic death of cytotoxic T cells induced by galectin-9. Activity of T helpers, required to induce cytotoxic T lymphocytes is also suppressed by galectin-9 in cooperation with VISTA and PD-L1. We discovered that expression of all these immune checkpoint proteins is differentially controlled by the autocrine transforming growth factor beta type 1 (TGF-β)-Smad3 pathway as well as several types of interferons. <h3>Conclusions</h3> The immune evasion machinery triggered by TGF-β-Smad3 and interferon pathways and operated by galectin-9, Tim-3, VISTA and PD-L1 should be considered as a potential target for personalised immunotherapy of cancer. Importantly, blood and tumour sample tests could help mapping the exact immune evasion networks employed by each tumour and the pathways responsible to produce respective immune checkpoint proteins. As such, personalised targeted immunotherapy could be adapted. Further investigations required to map the immune evasion machinery for each type of cancer and design the best targets for personalised immunotherapy of human malignancies. <h3>Disclosure Information</h3> <b>V.V. Sumbayev:</b> None. <b>I. Yasinska:</b> None. <b>S. Schlichtner:</b> None. <b>G.S. Lall:</b> None. <b>E. Fasler-Kan:</b> None. <b>B.F. Gibbs:</b> None.

Potential of Gold Nanoparticle in Current Radiotherapy Using a Co-Culture Model of Cancer Cells and Cancer Associated Fibroblast Cells.

Many cancer therapeutics are tested in vitro using only tumour cells. However, the tumour promoting effect of cancer associated fibroblasts (CAFs) within the tumour microenvironment (TME) is thought to reduce cancer therapeutics' efficacy. We have chosen pancreatic ductal adenocarcinoma (PDAC) as our tumor model. Our goal is to create a co-culture of CAFs and tumour cells to model the interaction between cancer and stromal cells in the TME and allow for better testing of therapeutic combinations. To test the proposed co-culture model, a gold nanoparticle (GNP) mediated-radiation response was used. Cells were grown in co-culture with different ratios of CAFs to cancer cells. MIA PaCa-2 was used as our PDAC cancer cell line. Co-cultured cells were treated with 2 Gy of radiation following GNP incubation. DNA damage and cell proliferation were examined to assess the combined effect of radiation and GNPs. Cancer cells in co-culture exhibited up to a 23% decrease in DNA double strand breaks (DSB) and up to a 35% increase in proliferation compared to monocultures. GNP/Radiotherapy (RT) induced up to a 25% increase in DNA DSBs and up to a 15% decrease in proliferation compared to RT alone in both monocultured and co-cultured cells. The observed resistance in the co-culture system may be attributed to the role of CAFs in supporting cancer cells. Moreover, we were able to reduce the activity of CAFs using GNPs during radiation treatment. Indeed, CAFs internalize a significantly higher number of GNPs, which may have led to the reduction in their activity. One reason experimental therapeutics fail in clinical trials relates to limitations in the pre-clinical models that lack a true representation of the TME. We have demonstrated a co-culture platform to test GNP/RT in a clinically relevant environment.

Independent Validation and Assay Standardization of Improved Metabolic Biomarker Signature to Differentiate Pancreatic Ductal Adenocarcinoma From Chronic Pancreatitis

Pancreatic ductal adenocarcinoma cancer (PDAC) is a highly lethal malignancy requiring efficient detection when the primary tumor is still resectable. We previously developed the MxPancreasScore comprising 9 analytes and serum carbohydrate antigen 19-9 (CA19-9), achieving an accuracy of 90.6%. The necessity for 5 different analytical platforms and multiple analytical runs, however, hindered clinical applicability. We therefore aimed to develop a simpler single-analytical run, single-platform diagnostic signature. We evaluated 941 patients (PDAC, 356; chronic pancreatitis [CP], 304; nonpancreatic disease, 281) in 3 multicenter independent tests, and identification (ID) and validation cohort 1 (VD1) and 2 (VD2) were evaluated. Targeted quantitative plasma metabolite analysis was performed on a liquid chromatography-tandem mass spectrometry platform. A machine learning-aided algorithm identified an improved (i-Metabolic) and minimalistic metabolic (m-Metabolic) signatures, and compared them for performance. The i-Metabolic Signature, (12 analytes plus CA19-9) distinguished PDAC from CP with area under the curve (95% confidence interval) of 97.2% (97.1%-97.3%), 93.5% (93.4%-93.7%), and 92.2% (92.1%-92.3%) in the ID, VD1, and VD2 cohorts, respectively. In the VD2 cohort, the m-Metabolic signature (4 analytes plus CA19-9) discriminated PDAC from CP with a sensitivity of 77.3% and specificity of 89.6%, with an overall accuracy of 82.4%. For the subset of 45 patients with PDAC with resectable stages IA-IIB tumors, the sensitivity, specificity, and accuracy were 73.2%, 89.6%, and 82.7%, respectively; for those with detectable CA19-9 >2 U/mL, 81.6%, 88.7%, and 84.5%, respectively; and for those with CA19-9 <37 U/mL, 39.7%, 94.1%, and 76.3%, respectively. The single-platform, single-run, m-Metabolic signature of just 4 metabolites used in combination with serum CA19-9 levels is an innovative accurate diagnostic tool for PDAC at the time of clinical presentation, warranting further large-scale evaluation.

Abstract 3195: Tumor inhibitory CAFs in CNS metastases

Abstract The 2-year survival rate for patients with central nervous system (CNS) metastases (CMs) remains below 2%. With the aim to define new therapeutic options, researchers have investigated how interactions between CM cells and the surrounding tumor microenvironment (TME) support metastatic progression. As an important component of the TME, cancer associated fibroblasts (CAFs) have largely been reported as tumor supportive. But new evidence for the high heterogeneity of the CAF compartment, including in vivo studies from our lab, showed that certain CAF subpopulations could exhibit tumor inhibitory capabilities. Interestingly, recent reports revealed that CAFs showing high expression of Immunoglobulin Superfamily containing Leucin Rich Repeat (ISLR) could show tumor inhibitory capabilities in both pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). Yet, despite the invaluable clinical potential of tumor inhibitory CAFs, the CAF compartment in the TME of CMs is still largely under described. Using CM patient tissue and CM patient-derived cell lines established in our lab, we performed single cell RNA sequencing (scRNA-seq) to define CAF subpopulations present in the TME of CMs. We identified distinct clusters characterized by differential expression of ISLR and tumor supportive CAF marker Actin Alpha 2, Smooth Muscle (ACTA2/α-SMA). CAF subpopulations were thus defined as follows: ISLRhigh/α-SMAlow, ISLRlow/α-SMAhigh, ISLRlow/α-SMAlow. Altogether, we hypothesized that ISLRhigh/α-SMAlow CAFs could act to restrain tumor progression in the TME of CMs, as opposed to potentially tumor supportive ISLRlow/α-SMAhighCAFs. ISLRhigh and ISLRlow CAFs were obtained through fluorescence-activated cell sorting (FACS) performed on one of our patient-derived CAF cell lines represented in all defined scRNA-seq clusters. ISLRhigh CAFs were validated to express lower levels of α-SMA (ISLRhigh/α-SMAlow CAFs) compared to ISLRlow cells (ISLRlow/α-SMAhigh CAFs). This mutually exclusive expression of ISLR and α-SMA in different CAF subpopulations is in accordance with recent data in PDAC and CRC and could indicate common underlying mechanisms driving the phenotype of each subpopulation. Further in vitro and in vivo experiments are currently underway in our lab to determine the tumor supportive capabilities of both ISLRhigh/α-SMAlow and ISLRlow/α-SMAhigh CAFs. Ultimately, we expect the current study to unravel the diverse and dynamic nature of CAFs in the TME of CMs, which is still largely unknown. Citation Format: Thomas Simon, David Buckley, Bodour Salhia. Tumor inhibitory CAFs in CNS metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3195.

Abstract 328: LCB84, a TROP2-targeted ADC, for treatment of solid tumors that express TROP-2 using the hu2G10 tumor-selective anti-TROP2 monoclonal antibody, a proprietary site-directed conjugation technology and plasma-stable tumor-selective linker chemistry

Abstract LCB84 is a human Trop-2-targeting antibody drug conjugate (ADC) composed of monomethyl auristatin E (MMAE) as payload and the Hu2G10 (by Mediterranea Theranostic) humanized IgG1 antibody that selectively targets the ADAM10-activated Trop-2 protein selectively expressed in transformed cancer cells (1). LCB84 was prepared using ConjuAll࣪, a proprietary site-directed conjugation technology of LegoChem Biosciences, which incorporates a conjugation ‘handle’ joined by enzymatic prenylation to a specifically engineered recognition sequence (CaaX) on antibody light chains. This conjugation handle facilitates simple versatile chemical conjugation to the linker-payload. A proprietary plasma-stable cleavable linker that is recognized and cleaved by a cancer-associated lysosomal enzyme, β-glucuronidase, was used to enable efficient and traceless payload release in a cancer-specific manner. LCB84 has been evaluated for anti-tumor activity and showed superior anticancer efficacy in triple-negative breast cancer (TNBC), pancreatic ductal adenocarcinoma (PDAC), gastric cancer and non-small cell lung cancer (NSCLC) cell line-derived xenograft (CDX) models compared to the ADC competitors Trodelvy and DS-1062. The LCB84 treatments were well tolerated, with no changes in body weight compared to control animals, for all dosing groups. LCB84 has robust cross-reactivity against primate Trop-2, which allows rigorous toxicity studies in monkeys. Remarkably, preliminary toxicity studies using cynomolgus monkeys showed that LCB84 is well tolerated, with calculated therapeutic index (TI, MTD/MED) of ~30 for single dosing and ~40 for repeat dosing. In conclusion, LCB84 is highly effective against Trop-2-positive CDX models in mice at doses that are well tolerated in mice and in primate models. Use of this proprietary plasma-stable cancer-selective linker technology and the Hu2G10 anti-Trop-2 monoclonal antibody that targets cancer-activated Trop-2 has led to a greatly improved next generation ADC for the treatment of various Trop-2-positive solid cancers including TNBC, PDAC, NSCLC and gastric cancer. Citation Format: Hyejung Kim, Emanuela Guerra, Eunji Baek, Yeojin Jeong, Hyogeun You, Byeongjun Yu, Taeik Jang, Alberti Saverio, Chul-Woong Chung, Changsik Park. LCB84, a TROP2-targeted ADC, for treatment of solid tumors that express TROP-2 using the hu2G10 tumor-selective anti-TROP2 monoclonal antibody, a proprietary site-directed conjugation technology and plasma-stable tumor-selective linker chemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 328.

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-03.

Abstract 2119: KRAS and fungi cooperate to drive IL33 secretion and type 2 immunity in the pancreatic cancer tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, the five-year survival rate of PDAC patient is around ten percent. It is fourth largest cause of cancer related death and is projected to become the second deadliest cancer by 2030. PDAC patients generally present with advanced stage, metastatic disease that is mostly resistant to chemotherapy, radiation, and immunotherapy. A significant finding is that mutant KrasG12D (Kras*) is the primary oncogenic driver of PDAC development, and therefore efforts have been devoted to target KRAS. Here, we show in pancreatic ductal adenocarcinoma (PDAC), oncogenic KrasG12D (Kras*) increases the expression of IL33, a damage-associated molecular pattern (DAMP) molecule, member of IL1 cytokine family protein and localized in the PDAC cell nucleus, which upon secretion, chemoattracts the type 2 immunocytes. Type 2 immune cells—TH2, eosinophils and innate lymphoid cells (ILC)2, stimulate tumor growth by secreting pro-tumorigenic cytokines such as IL4, IL5 and IL13. However, the mechanisms by which type 2 immune cells traffic to the tumor microenvironment (TME) are unknown. Cancer cell-specific deletion of IL33 leads to a significant reduction in TH2 and ILC2 recruitment and lead to tumor regression. Further, we discovered that the release of IL33 requires interactions between cancer cells and the intratumoral mycobiome. PDA tumors harbors a ~3000-fold higher fungal load compared to normal pancreas in both mouse and humans. However, the mechanism of fungal mediated cytokine release is still unknown and represents a target for limiting IL33 release, and lessening PDAC tumor burden. Genetic deletion of IL33 alone or fungal depletion leads to a decrease in TH2 and ILC2 infiltration along with robust tumor regression. Moreover, repopulation of the PDAC TME with fungal species such as Alternaria and Malassezia lead to a re-infiltration of type 2 immunocytes that reinvigorates tumor growth. Notably, transplantation of tumor-derived ILC2s into mice accelerates PDAC growth. Mechanistically fungus activates dectin-1 pathway in PDAC cancer cells, mediating the release of IL33. Consistent with the murine data, IL33 expression is upregulated in approximately 20% of human PDAC, which is mainly restricted to cancer cells. Collectively, our work shows that the intratumoral mycobiome stimulates PDAC to secrete IL33, which plays a leading role in facilitating the recruitment of type 2 immunocytes that promote tumor progression. Citation Format: Aftab Alam, Eric Levanduski, Maulasri Bhatta, Sharon Senchanthisai, Sheila Jani Sait, Jianmin Wang, Scott I. Abrams I. Abrams, Prasenjit Dey. KRAS and fungi cooperate to drive IL33 secretion and type 2 immunity in the pancreatic cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2119.

Sorafenib Chemosensitization by Caryophyllane Sesquiterpenes in Liver, Biliary, and Pancreatic Cancer Cells: The Role of STAT3/ABC Transporter Axis.

A combination of anticancer drugs and chemosensitizing agents has been approached as a promising strategy to potentiate chemotherapy and reduce toxicity in aggressive and chemoresistant cancers, like hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and pancreatic ductal adenocarcinoma (PDAC). In the present study, the ability of caryophyllane sesquiterpenes to potentiate sorafenib efficacy was studied in HCC, CCA, and PDAC cell models, focusing on the modulation of STAT3 signaling and ABC transporters; tolerability studies in normal cells were also performed. Results showed that the combination of sorafenib and caryophyllane sesquiterpenes synergized the anticancer drug, especially in pancreatic Bx-PC3 adenocarcinoma cells; a similar trend, although with lower efficacy, was found for the standard ABC transporter inhibitors. Synergistic effects were associated with a modulation of MDR1 (or Pgp) and MRP transporters, both at gene and protein level; moreover, activation of STAT3 cascade and cell migration appeared significantly affected, suggesting that the STAT3/ABC-transporters axis finely regulated efficacy and chemoresistance to sorafenib, thus appearing as a suitable target to overcome drawbacks of sorafenib-based chemotherapy in hepato-biliary-pancreatic cancers. Present findings strengthen the interest in caryophyllane sesquiterpenes as chemosensitizing and chemopreventive agents and contribute to clarifying drug resistance mechanisms in HCC, CCA, and PDAC cancers and to developing possible novel therapeutic strategies.

P-274 The prognostic significance of early expression of epithelial-to-mesenchymal transition markers in resectable pancreatic cancer patients

Pancreatic ductal adenocarcinoma cancer (PDAC) is the seventh leading cause of cancer death worldwide, mainly due to late-stage presentation, therapy resistance and early metastasis. E-cadherin (E-cad), one of the cell-cell adhesion molecules, is regarded as a major suppressor of metastasis and is typically downregulated in most pancreatic tumors. Epithelial to mesenchymal transition (EMT) is characterized by a downregulation in epithelial markers such as E-cad and an upregulation of mesenchymal markers such as Vimentin (Vim).