Extent of IMPACT CpG island promoter methylation in mouse cell lines (KPC, 4964-POP, and 4964-HOP), and five human pancreatic liver metastases compared to three primary PDACs, and two normal pancreas and liver tissue.

GCN1 cDNA sequence in 4964-POP and 4964-HOP cells.

Figure S6. Nuclear GCN1 stabilizes HNRNPK leading to immune escape

Figure S3. IMPACT limits ATF4 activation through competitive inhibition of GCN1

Differential gene expression analysis by RNA-Seq of 4964-POP cells in comparison to 4964-HOP cells arranged in order of highest expression and significant p value.

Figure S2. GCN1 drives metastatic growth through activation of ATF4

Figure S4. IMPACT abrogates GCN1-mediated immune cell evasion

List of all reagents used in the study

Figure S7. Expression of IMPACT is lost through genomic deletion and epigenetic silencing

List of all hits identified in the cDNA screen and categorized by function. cDNAs highlighted in red were identified in the <1Kb cDNA library fraction while no cDNAs were identified from the >3 KB library fraction. The number of tumor lesions identifying the cDNA is given.

TRANSITE prediction of RNA binding proteins whose motifs are overrepresented in the 3’-UTR transcriptomes of GCN1 KD (SH1 and SH2) KLM1 cells.

Importance:Approximately 1% to 3% of gastric cancers and 5% of lobular breast cancers are hereditary. Loss of function CDH1 gene variants are the most common gene variants associated with hereditary diffuse gastric cancer and lobular breast cancer. Previously, the lifetime risk of gastric cancer was estimated to be approximately 25% to 83% and for breast cancer it was estimated to be approximately 39% to 55% in individuals with loss of function CDH1 gene variants. Objective:To describe gastric and breast cancer risk estimates for individuals with CDH1 variants. Design, Setting, and Participants:Multicenter, retrospective cohort and modeling study of 213 families from North America with a CDH1 pathogenic or likely pathogenic (P/LP) variant in 1 or more family members conducted between January 2021 and August 2022. Main Outcomes and Measures:Hazard ratios (HRs), defined as risk in variant carriers relative to noncarriers, were estimated for each cancer type and used to calculate cumulative risks and risks per decade of life up to age 80 years. Results:A total of 7323 individuals from 213 families were studied, including 883 with a CDH1 P/LP variant (median proband age, 53 years [IQR, 42-62]; 4% Asian; 4% Hispanic; 85% non-Hispanic White; 50% female). In individuals with a CDH1 P/LP variant, the prevalence of gastric cancer was 13.9% (123/883) and the prevalence of breast cancer among female carriers was 26.3% (144/547). The estimated HR for advanced gastric cancer was 33.5 (95% CI, 9.8-112) at age 30 years and 3.5 (95% CI, 0.4-30.3) at age 70 years. The lifetime cumulative risk of advanced gastric cancer in male and female carriers was 10.3% (95% CI, 6%-23.6%) and 6.5% (95% CI, 3.8%-15.1%), respectively. Gastric cancer risk estimates based on family history indicated that a carrier with 3 affected first-degree relatives had a penetrance of approximately 38% (95% CI, 25%-64%). The HR for breast cancer among female carriers was 5.7 (95% CI, 2.5-13.2) at age 30 years and 3.9 (95% CI, 1.1-13.7) at age 70 years. The lifetime cumulative risk of breast cancer among female carriers was 36.8% (95% CI, 25.7%-62.9%). Conclusions and Relevance:Among families from North America with germline CDH1 P/LP variants, the cumulative risk of gastric cancer was 7% to 10%, which was lower than previously described, and the cumulative risk of breast cancer among female carriers was 37%, which was similar to prior estimates. These findings inform current management of individuals with germline CDH1 variants.

Abstract Introduction: Metastatic outgrowth and colonization requires that disseminated tumor cells simultaneously compensate for alterations in nutrient availability, counteract oxidative stress, and evade host innate and adaptive immune surveillance. The mechanistic underpinnings of how these vital processes are coordinated is not understood. Experimental procedures: Using intrasplenic injection (liver metastases) and tail vein injection (lung metastases), we employed a gain-of-function cDNA screen in mice to identify regulators of colonization. In doing so, we identified GCN1 signaling as indispensable to the disseminated pancreatic cancer cell. Summary of unpublished data: Specifically, we demonstrate that alterations in nutrient availability encountered in target organs (liver, lung) trigger pancreatic cancer cells to utilize GCN1 stress signaling to upregulate the expression of serine, folate, and methionine pathway biosynthetic enzymes together with amino acid transporters through the integrated stress response effector ATF4. These pathways act in concert to facilitate acquisition of metabolites critical for cellular functions including maintenance of redox homeostasis. Surprisingly, we found that GCN1 also functions in the nucleus, where it interacts with HNRNPK to destabilize the transcripts encoding natural killer (NK) cell activation ligands and key regulators of major histocompatibility complex (MHC) class I molecules. Intriguingly, we identified an endogenous protein rheostat, IMPACT, for GCN1’s dual functions and show that IMPACT expression is lost in human pancreatic metastases through DNA methylation. IMPACT overexpression in pancreatic cancer cell lines inhibited the integrated stress response effector ATF4 to retard nutrient uptake and activated the expression of NK cell ligands and MHC class I molecules to fuel anti-tumor immune responses. Conversely, IMPACT knockout enabled successful acquisition of metabolic intermediaries in response to nutrient alterations and suppressed anti-tumor immune response to accelerate metastatic outgrowth. Accordingly, bioinformatic analyses of human pancreatic cancer showed that while the expression of GCN1 increases in metastatic disease, the expression of IMPACT is lost, correlating with significantly shorter survival of GCN1-high, IMPACT-low expressing tumors. Finally, IMPACT overexpression synergized with immune checkpoint inhibitors in mice to eliminate macrometastases formation. Conclusion: In summary, we have identified IMPACT as an immunometabolic checkpoint that restrains GCN1-mediated metabolic plasticity and GCN1-mediated suppression of innate and adaptive immune surveillance. We propose that drugs that can restore IMPACT expression or IMPACT mimetic agents will have therapeutic value for patients with pancreatic cancer. Citation Format: Surajit Sinha, Abir Panda, Zeribe Nwosu, Rodrigo Neves Das, Xu Ke, Elke van Beek, Alexander J. Rossi, Reed I. Ayabe, James McDonald, Michael M. Wach, Samantha Ruff, Priyanka P. Desai, David Sun, Martha E. Teke, Emily A. Verbus, Areeba Saif, Shreya Gupta, Tahsin Khan, Leila Sarvestani, Carrie E. Ryan, Jacob Lambdin, Kirsten Remmert, Emily Smith, Kenneth Luberice, Stephie Lux, Imani A. Alexander, Tracey Pu, Allen Luna, Sarfraz R. Akmal, Shahyan Rehman, Ashley Rainey, Hanna Hong, Yuri Lin, Samantha Sevilla, Gasmi Billel, Sivasish Sindiri, Todd Prickett, King Chan, Eileen Li, Xiaolin Wu, Nicholas D. Klemen, Giorgio Trinchieri, Costas A. Lyssiotis, Jeremy Davis, Pankaj K. Singh, Steven A. Rosenberg, Michael B. Yaffe, Filippo Giancotti, Ethan M. Shevach, Jonathan M. Hernandez. IMPACT restrains immuno-metabolic GCN1 signaling to govern pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C106.

Background:Treatment of advanced liver tumors remains challenging. Although immune checkpoint inhibition has revolutionized treatment for many cancers, responses in colorectal liver metastases and biliary tract cancers remain suboptimal. Investigation into additional immunomodulatory therapies for these cancers is needed. Interleukin-12 (IL-12) is a pro-inflammatory cytokine with robust anti-tumor activity, but systemic adverse effects largely terminated therapeutic development of recombinant human IL-12 (rhIL-12). PDS01ADC is a novel human monoclonal antibody (NHS76) conjugated to two IL-12 heterodimers with established safety in phase I trials. The NHS76 antibody specifically targets histone/DNA complexes which are accessible only in regions of cell death and this antibody has been shown to accumulate locally in tumors. Methods:Patients with unresectable metastatic colorectal cancer (mCRC) or unresectable intrahepatic cholangiocarcinoma (ICC) will receive synchronization of subcutaneous PDS01ADC with floxuridine delivered via a hepatic artery infusion pump (HAIP). The primary outcome measured in this study will be overall response rate as measured by Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Secondary outcomes measured in this study will include hepatic and non-hepatic progression-free survival (PFS), overall survival, and safety of PDS01ADC combination therapy with HAIP. Discussion:Poor clinical response of these liver tumors to immunotherapy is likely due to various factors, including poor immune infiltrate into the tumor and immunosuppression by the tumor microenvironment. By exploiting the tumor cell death induced by HAIP locoregional therapy in combination with systemic chemotherapy, PDS01ADC is poised to modulate the tumor immune microenvironment to improve outcomes for patients undergoing HAIP therapy. Trial Registration:ClinicalTrials.gov (ID NCT05286814 version 2023-10-18); https://clinicaltrials.gov/study/NCT05286814?term=NCT05286814&rank=1.

Background: Primary large cell neuroendocrine carcinoma (LCNEC) of the extrahepatic bile ducts is extremely rare, with seven cases reported in the English literature. The infrequency in which LCNEC occurs poses significant diagnostic and therapeutic challenges. Methods: N/A Results: A 70-year-old man with a history of colon adenocarcinoma status post partial colectomy and adjuvant FOLFOX in 2017 presented in 2021 with fever and multiple episodes of emesis one month after laparoscopic cholecystectomy for choledocholithiasis. CT and MRI imaging revealed intra/extrahepatic biliary ductal dilatation with a 2.4 x 1.5 cm mass in the common bile duct and a 2.6 cm mass in segment IVa of the liver. Pathology from biliary stricture brushings and liver core needle biopsies revealed a poorly differentiated adenocarcinoma with the following IHC profile: CDX2+, TTF-1+, weakly CK7+, CK20-, CK5/6-, p40-, Hep Par1-, and GATA3-. The patient was referred to our institution for management of metastatic extrahepatic cholangiocarcinoma and subsequently underwent four cycles of neoadjuvant cisplatin and gemcitabine. On restaging scan, four new lesions were identified in the liver. A single-patient, FDA-approved protocol (IRB # 000693-C) was granted for the use of hepatic artery infusion pump (HAIP) therapy to manage liver metastases from extrahepatic cholangiocarcinoma. The patient was then taken to the operating room and underwent resection of four liver metastases, ablation of one liver metastasis, resection of extrahepatic bile duct with Roux-en-Y hepaticojejunostomy (HJ) reconstruction, and HAIP placement. Final pathology revealed a high-grade tumor with IHC stains strongly positive for chromogranin, synaptophysin, and variably positive for CDX2 consistent with LCNEC of the proximal bile duct. Two of seventeen lymph nodes were positive. The FDA approved HAIP floxuridine as originally planned given the limited alternative systemic treatment options for LCNEC. The patient received 5 cycles of floxuridine with a maximal elevation in alkaline phosphatase of 127 IU/L (baseline 79 IU/L) while serum bilirubin remained within normal limits. The patient developed progression of disease in the liver as well as new metastases to the spleen and sternum and succumbed to his disease seven months after the operation. Conclusion: Although HAIP floxuridine failed to demonstrate efficacy for our patient with LCNEC, this case highlights several important points. To our knowledge, concomitant extrahepatic bile duct resection/Roux-en-Y HJ reconstruction with HAIP placement has rarely been undertaken. Cholecystectomy with HJ reconstruction contaminates the biliary tree and alters/accelerates bile flow into the intestines, with uncertain impact on the adverse events associated with HAIP therapy. Importantly, these changes neither negatively impacted the patient's ability to tolerate HAIP floxuridine nor resulted in damage to the liver and/or bile ducts during therapy, suggesting this surgical approach may be viable for floxuridine-sensitive cancers such as cholangiocarcinoma.