Citations

Abstract Triple negative breast cancer (TNBC) represents an aggressive and therapeutically challenging breast cancer subtype with higher incidence and mortality rates in non-Hispanic Black (NHB) women. This disparity persists even after adjusting for socioeconomic factors, tumor biology, and quality of care. The study aims to unravel specific molecular mechanisms underlying this disparity, with a focus on Hedgehog signaling pathways in premenopausal NHB women. Understanding these mechanisms is vital for developing targeted therapies and personalized medicine approaches to address these health disparities. Our research utilized a unique cohort of NHB and non-Hispanic White (NHW) women who underwent breast reduction surgery at the Mayo Clinic and donated breast tissue samples for our study. We isolated RNA from whole tissue sections from a cohort of 12 NHB and 40 NHW women who were parous and premenopausal at time of surgery. We performed gene expression profiling using NanoString BC360 and IO360 assays. This allowed us to identify a distinct gene expression signature characteristic of basal-like/TNBC and indicative of heightened activity in the Hedgehog signaling pathway. We also employed advanced cell culture techniques to generate tissue organoids and subculture human mammary epithelial cells (HMECs) that preserve the intrinsic characteristics of the original tissue samples. We found that significantly differential expression of IL20RA and SOX10, principal factors in the gene expression signature, were preserved in both the derived organoids and the HMECs. These results suggest a potential intrinsic difference in the activation of the Hedgehog pathway in the breast epithelial cells of NHB women. This activation may contribute to increased proliferation and transformation of basal stem cells within the breast, possibly fueling the development and progression of TNBC. The implication of activated Hedgehog signaling in NHB women opens new avenues for understanding TNBC disparities. IL20RA and SOX10 represent potential biomarkers for TNBC risk that may serve as a stratification tool for NHB women. The results of this study have the potential to improve assessment and management of TNBC risk among NHB women, thereby contributing significantly to reducing health disparities in breast cancer outcomes. Citation Format: Jennifer Vanessa Cabezas, Savanna Touré, Matthew Wilson, Melody L. Stallings Mann, Laura M. Pacheco-Spann, Joshua W. Ogony, Mark E. Sherman, Derek C. Radisky. Identifying molecular mechanisms in triple negative breast cancer disparities: Unveiling the role of Hedgehog signaling in non-Hispanic Black women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2139.

Abstract Background: Cholangiocarcinoma (CCA) is a lethal and heterogenous malignancy of the biliary tree characterized by perineural invasion. About 85% of CCA show activation of YAP signaling, which promotes proliferation, anti-apoptosis, and therapeutic resistance. YAP signaling is mediated by Src family kinases (SFK) by phosphorylation of YAP (pYAPy357). NXP900 is a first-in-class, highly selective, SFK inhibitor with a novel mechanism of action which locks SFK in its closed and inhibited conformation, in contrast to other Src inhibitors such as dasatinib. A phase 1a study of NXP900 in patients with advanced solid tumors was recently initiated. Here, we examined treatment responses of CCA to NXP900 in vitro and in vivo. Methods: Cell viability was determined in seven CCA cell lines by CellTiter-Glo. Cell death and apoptosis were assayed by PI/Hoechst and Caspase-Glo 3/7 assay. CalcuSyn software was used to determine synergistic drug effects. Immunoblot analysis, RT-PCR, and IF staining of YAP localization were used to evaluate YAP knockdown. Clones of human CCA cell lines resistant to NXP900 were generated through escalating exposure to NXP900 over 6 months. Five patient-derived xenograft (PDX) tumors were expanded into flanks of NOD/SCID mice. Tumor bearing mice were randomized 1:1 with 5 mice per arm and treated with vehicle or NXP900 (40 mg/kg) once daily for up to 4 weeks. To determine drivers of sensitivity and resistance, predicted sensitivity scores based on tumor growth were linked to multi-omics (RNA seq and [phospho-]proteomics) of our PDX models. Results: All cell lines were sensitive to NXP900 with IC50 values between 7nM-15µM; IC50’s of resistant clones were 1000 times higher. NXP900 induced more cell death compared to vehicle, which appeared to be through apoptosis. NXP900 inhibited pSrc, decreased pYAPy357, and upregulated inactive pYAPs127. Correspondingly, decreased YAP target gene (Cyr61, NUAK, and CTGF) levels and a nuclear-to-cytoplasmic translocation were observed. NXP900/GemCis combination therapy increased cell death demonstrated a synergistic effect (Combination Indices <1) at all concentrations. In our PDX models, treatment was associated with a significant decrease in tumor growth in 3 models (mean fold change 3.1 vs. 14.2; 1.0 vs. 7.6; 1.4 vs. 2.6). The major resistant signature was the TRK signaling network, involved in nerve growth factor binding activity, while drivers of sensitivity included the SRC network. Conclusion: NXP900 demonstrated therapeutic activity in vitro and in human PDX models. We are currently performing multi-omic approaches in NXP900 sensitive and resistant cell lines to unravel determinants of activity and resistance. Additional in vivo studies will be performed to determine effects of NXP900/GemCis and NXP900/anti-PDL-1 combination therapy. Citation Format: Hendrien Kuipers, Jennifer L. Tomlinson, Danielle M. Carlson, Amro M. Abdelrahman, Erik Jessen, Jack W. Sample, Nathan W. Werneburg, Hannah E. Stumpf, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 614.

Abstract Background: Nutritional and inflammatory factors collected in routine cancer labs, neutrophil percentage to albumin ratio (NPAR), neutrophil lymphocyte ratio (NLR), monocyte lymphocyte ratio (MLR), and platelet lymphocyte ratio (PLR), have been shown feasible in identification of patients at risk of increased treatment complications, decreased cognition, anxiety and depression, and increased mortality. These have proven sensitive to multiple cancer types and stages, though little is known about causal pathways. The Vitamin/Mineral and Inflammatory Correlations To Understand Chronic Stress in the U.S. (The VICTUS Study) sought to elucidate a relationship between diet and food insecurity within these panels. Methods: Using NHANES 2017/2018 surveys, adults > 18 years without renal or hepatic disorders, pregnancy, alcohol, or supplement use were included. NPAR, NLR, MLR, and PLR were categorized as quartiles and compared to demographic, food security, plasma micronutrient statuses [vitamins A, B9, C, D, E, calcium (Ca), ferritin (Fe), phosphorus (P), potassium (K), sodium (Na)], and smoking, activity level, and sun exposure, using chi-square and ANOVA. ANOVA models compared food security and poverty to NPAR, NLR, MLR, and PLR means. Results: From 3,317 participants, 50.2% were male, 54.3% non-Hispanic White, 33.7%, food insecure, 51.1% food stamp recipients, 27.7% smokers, with a mean age of 42.5 (SD 0.44), poverty to income ratio 2.79 (SD 0.08), CRP 3.90 (SD 0.18), NPAR 14.1 (SD 0.14), NLR 2.07 (SD 0.04), MLR 0.28 (SD 0.003), and PLR 118.7 (SD 1.61). CRP was found to be statistically significantly associated with all biomarker ratios (p < 0.018). Significant micronutrient biomarker-inflammatory biomarker associations were identified in Fe [NPAR, MLR, PLR (p < 0.001)], P [NPAR, NLR, MLR, PLR (p < 0.033)], C [NPAR, NLR (p < 0.008)], E [MLR, PLR (p < 0.016)], K [NPAR, NLR, MLR, PLR (p < 0.015)], Ca [NPAR, PLR (p < 0.04)], A [NPAR (p < 0.023)] and D [NLR (p < 0.035)]. Significant micronutrient intake-inflammatory biomarker associations were detected in vitamin A [NPAR, PLR (p < 0.013)], lycopene [NLR, PLR (p < 0.035)], and B12 [NLR, PLR (p < 0.031)]. Age [NPAR, NLR, MLR, PLR (p < 0.001)], gender [NPAR, MLR, PLR (p < 0.001)], ethnicity [NPAR, NLR, MLR (p < 0.006)], and sedentariness [MLR, PLR (p < 0.039)] were also associated with inflammatory markers. Poverty was significantly associated with NPAR, NLR, MLR, and CRP (p < 0.018). Food insecurity was significantly associated with NPAR (p < 0.001) and CRP (p < 0.001). Discussion: Food insecurity and poverty were significantly associated with NPAR, a ratio implicated in overall poorer cancer health outcomes. Multiple inadequate micronutrient intakes and plasma biomarkers were associated with inflammatory panels, though neither nutritional biomarker nor food insecurity statuses and their contributions to inflammatory processes are routinely surveilled within cancer disease and treatment. Citation Format: Jennifer Crook, Eunkyung Lee, Rui Xie, Opeyemi Bolajoko, Victoria Loerzel. Nutritional mediators of inflammation: A cause for increased surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2171.

Abstract Introduction: Cholangiocarcinoma (CCA) is an aggressive and heterogenous biliary malignancy. Previous therapeutic shortcomings have encouraged the investigation of new strategies, such as targeted therapeutics. YAP/TAZ, transcriptional coactivators of the hippo pathway, have been implicated in the tumorigenesis of CCA along with the Src familiar kinase, LCK. Milk-derived nanovesicles (MNVs) have emerged as a promising biologic delivery apparatus for hepatobiliary malignancy due to their predilection for hepatic uptake. We sought to investigate the utility of aptamer guided MNVs loaded with short interfering RNA (siRNA) targeting YAP, TAZ, and LCK. Methods: MNVs were prepared and loaded with species-specific siRNA prior to decoration with an epithelial cellular adhesion molecule (EpCAM) targeting aptamer. In vitro studies were performed using murine and human CCA cell lines, SB1 and HuCCT-1, respectively. In vivo studies were carried out using C57BL/6 mice following Institutional Animal Care and Use Committee protocols. Results: We previously validated EpCAM as an effective aptamer demonstrated by increased levels of expression in CCA cell lines using immunoblot, RT-PCR and immunofluorescence. Additionally, in vivo experiments demonstrated excellent biodistribution and affinity for tumor-selective uptake. To evaluate knockdown efficacy, CCA cell lines were incubated with EpCAM aptamer guided MNVs individually targeting YAP, TAZ, and LCK which resulted in downregulation of these targets on immunoblot and RT-PCR. MNVs were then loaded with equal concentrations of siRNA targeting YAP, TAZ, and LCK in combination which resulted in similar degrees of gene downregulation compared with single transcriptome targeting MNVs. Due to the role of YAP/TAZ activation in CCA systemic therapy resistance, we investigated the effect of MNVs loaded with either single target or combinatory target siRNA alongside and in combination with gemcitabine and cisplatin (GemCis). Incubation with both combinatory siRNA and GemCis significantly intensified cell death on Pi/Hoechst staining compared with single target knockdown alone. Similar findings were found using separate assays of cell viability, including ATP luminescent quantitation and caspase 3/7 activity. These data suggest MNV transcriptome targeting may induce a state of cell stress which allows for amplification of chemotherapy treatment effect. Conclusion: Aptamer guided, nanovesicle mediated transcriptome targeting is a promising therapeutic strategy in preclinical CCA models that may potentiate chemotherapy response. Our future direction involves further investigating the role of CCA target downregulation and its effect on the tumor immune microenvironment utilizing aptamer guided nanovesicle delivery and inducible knockdown cell lines. Citation Format: Jack W. Sample, Mincheng Yu, Hendrien Kuipers, Danielle M. Carlson, Nathan W. Werneberg, Jennifer L. Tomlinson, Gregory J. Gores, Rory L. Smoot. Milk-derived extracellular nanovesicles: A novel delivery strategy for transcriptome targeting therapies in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5753.

Abstract Lung cancer is the leading cause of cancer deaths in the US. Non-small cell lung cancer (NSCLC) accounts for ~80% of lung cancer cases and is further classified into adenocarcinoma (LUAD), squamous cell carcinoma (LUSC), and large cell carcinoma. New approaches targeting pathways in NSCLC have emerged, resulting in encouraging new treatments that benefit LUAD patients, but very few advances have been made in the treatment options for LUSC which accounts for ~30% of lung cancer cases. NSCLCs, including LUSCs, contain a sub-population of cells that exhibit properties of cancer stem cells (CSCs) and are responsible for tumor initiation, maintenance, therapeutic resistance, and relapse. Therefore, CSCs must be effectively targeted to elicit long lasting therapeutic results in LUSC patients. Our studies have shown that: 1) PKCι is an oncogene and prognostic marker in LUSC; 2) copy number gains (CNGs) of the PKCι gene (PRKCI) drives overexpression of PKCι in 90% of LUSCs; 3) PKCι phosphorylates SOX2, and controls SOX2-mediated transcriptional activation of Hedgehog Acyl Transferase, resulting in Hedgehog (Hh) pathway activation that drives a CSC phenotype in LUSC cells; and 4) the FDA approved compound Auranofin (ANF) is a potent inhibitor of oncogenic PKCι. In this study, we assessed the effect of ANF in combination with the Hh pathway inhibitor, LDE225, on LUSC CSCs and tumor growth. Cell viability, sphere formation and soft agar assays were used to assess the effects of ANF and LDE225 alone or in combination on the growth of LUSC CSCs with PRKCI CNGs or LUAD and LUSC CSCs without PRKCI CNG to determine if PRKCI CNG can be used to predict response to ANF and LDE225 and test the selectivity of response for LUSC. The effects of ANF and LDE225 on the levels of PKCι-Hh signaling components was assessed by qPCR and western blotting. Finally, the efficacy of ANF and LDE225 in combination was evaluated in human primary LUSC patient-derived xenograft (PDX) models that harbor PRKCI CNGs and the ability of PKCι-Hh signaling intermediates to serve as biomarkers of PKCι-Hh pathway inhibition was validated in vivo. We observed a dose-dependent decrease in cell proliferation, clonal expansion and transformed growth in CSCs treated with ANF and LDE225 alone. ANF and LDE225 combined treatment of LUSC CSCs with PRKCI CNGs (but not LUAD or LUSC CSCs without PRKCI CNG) synergistically inhibited CSC phenotypes based on results of combination index analysis. Furthermore, treatment of LUSC PDX models with ANF and LDE225 in combination had a synergistic effect on tumor growth inhibition. Lastly, ANF and LDE225 treatment led to inhibition of PKCι-Hh signaling intermediates demonstrating the on-target effects of treatment. Our data indicate that a PKCι-Hh vertical blockade strategy of combined ANF and LDE225 effectively targets LUSC CSCs and blocks tumor growth in vivo and provides compelling rationale to develop this novel combination strategy further in clinical trials. Citation Format: Dania Alqasrawi, Ryan Argo, Christine Ratliff Rang, Prita Pandya, Skyeler Klinge, Jennifer Lindemann, Nyla Searl, Alan Fields, Verline Justilien. A novel combination therapy for lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5434.

Abstract The Voyage Study is a large-scale, prospective cohort study of long-term health outcomes among participants with a valid multi-target stool DNA (mt-sDNA) test order. Recently, the Voyage Study achieved its enrollment target of 150,000 participants. The intent of this descriptive analysis is to provide baseline geographic and socio-demographic characteristics of Voyage Study participants to set the stage for future comparisons to the broader mt-sDNA user population. Participants were randomly selected from all persons in the U.S. or Puerto Rico who had a valid order for mt-sDNA screening during the enrollment period (10/18/2019 - 10/17/2023). Enrolled participants completed a baseline questionnaire that collected socio-demographic and health information. Participant characteristics and baseline survey answers were compared between regions using chi-square tests where appropriate. Regional geographic data was aggregated for the broader mt-sDNA user population during the enrollment period, though no formal comparisons were made between the broader population and the Voyage population at this time. Analyses were limited to 139,369 participants with available baseline questionnaire data. Voyage participants were more likely to reside in the Midwest, Northeast, or Southeast (29.9%, 22.5%, 22.6% respectively) and less likely to reside in the Southwest and West (11.6%, 13.4%). During the enrollment period, differential participation was also observed in the mt-sDNA user population, with the Midwest, Northeast, and Southeast having the greatest proportion (25.4%, 22.2%, 26.7%) and the Southwest and West having the lowest proportion of mt-sDNA users (13.8%, 11.9%). In the Voyage cohort, Midwest participants reported the lowest education attainment as characterized by years of school completed (14.1% high school or below, 59.2% college or greater), while participants in the West reported the highest education attainment (7.9% high school or below, 67.3% college or greater). The West also had the highest percentage of participants reporting an income of $100,000 or greater (51.6%), with the Southeast being the lowest (42.2%), as compared to the other regions. We report here early results of geographic and socio-demographic characteristics from a new research resource that will enable future analyses of health outcomes and behaviors in a population of mt-sDNA users. We observed that the greatest percentage of Voyage participants resided in the Midwest, Northeast, or Southeast, and that there was differential socio-demographic participant characteristics based on region. Future analyses will compare long-term healthcare outcomes and differences in colorectal cancer incidence and mortality between the broader mt-sDNA user population and the Voyage cohort. Citation Format: Allison M. Berry, Jessica A. Grimm, Christine R. Kirt, Emily J. Kirsch, Rachel E. Dixon, Winnie Z. Fan, Kandace A. Bohn, Jennifer St. Sauver, Kathleen J. Yost, Lila J. Finney Rutten, Janet E. Olson. U.S. Geographic and socio-demographic characteristics of Voyage Study participants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3453.

Abstract CD19-directed chimeric antigen receptor T (CART19) cell therapy has shown remarkable outcomes in B cell malignancies and was FDA approved in multiple indications, but durable remissions are limited to ~40%. Inhibitory myeloid cells in the tumor microenvironment have been found to suppress T cell expansion and contribute to CART19 failure. Here, we studied interactions between monocytes, CART19, and tumor cells to understand the impact of monocytes on CART19 effector functions. First, CD28-costimulated CART19 (CART19-28ζ) generated in the lab from healthy donors were cocultured with JeKo-1, a CD19+ mantle cell lymphoma cell line, and freshly isolated monocytes or ex vivo differentiated M2-like macrophages. M2-like macrophages were generated by incubating fresh monocytes with rhGM-CSF followed by coculturing with JeKo-1. CART19 antigen-specific proliferation was significantly inhibited by M2-like macrophages but not fresh monocytes (JeKo-1+CART19 vs JeKo-1+M2+CART19, p=0.00503). Transwell assays indicated that suppression from M2-like macrophages was not contact-dependent. IL-1 receptor antagonist (IL-1ra) was significantly elevated in M2-like macrophage coculture supernatant (JeKo-1+Mono+CART19 vs JeKo-1+M2+CART19, p=0.0292) and intracellularly in M2-like macrophages after coculture (90%+). We therefore hypothesized that M2-like macrophages inhibit CART19 by secreting IL-1ra, which blocks IL-1 signaling in CART19. We interrogated the role of IL-1ra in CART19. JeKo-1 was cocultured with CART19, supplemented with rhIL-1β only, IL-1β + IL-1ra, or IL-1β +IL-1ra + IL-1ra neutralizing antibody (neuAb). CART19 antigen-specific proliferation was improved by IL-1β (PBS vs IL-1β, p<0.0001). IL-1ra inhibited IL-1β-dependent CART19 proliferation (IL-1β vs IL-1β+IL1ra p=0.0006), which was restored by IL-1ra neutralization (IL-1β+IL-1ra vs IL-1β+IL-1ra+IL-1ra neuAb, p=0.0215). Additionally, expression of IL-1RI, the receptor of IL-1β and IL-1ra, was measured on T cells after coculturing CART19, JeKo-1, and M2-like macrophages. M2-like macrophages downregulated IL-1RI expression on CART cells, which is a potential mechanism of blocking CART response to IL-1β, resulting in further suppression of CART proliferation (JeKo-1+M2+CART19 vs JeKo-1+CART19, p=0.001). Next, we assessed the impact of IL-1ra on CART cell functions in the presence of M2-like macrophage in a mantle cell lymphoma xenograft model. NOD-SCID-γ−/- (NSG) mice were subcutaneously injected with human macrophages and luciferase+ JeKo-1 cells. Upon tumor engraftment by bioluminescent imaging, mice were randomized to treatment with CART19 + IL-1ra neuAb or control IgG for 3 weeks. CART19 + IL-1ra neuAb led to improved antitumor activity (IL-1ra neuAb vs control IgG, p=0.0033). Overall, our study revealed a role of IL-1ra in macrophage-induced CART inhibition mediated by blocking IL-1β signaling through IL-1RI. Citation Format: Kun Yun, Reona Sakemura, Truc Huynh, Claudia Manriquez Roman, Olivia Sirpilla, Carli Stewart, James Girsch, Ekene Ogbodo, Ismail Can, Jennifer Feigin, Long Mai, Hong Xia, Brooke Kimball, Makena Rodriguez, Lionel Kankeu Fonkoua, Mehrdad Hefazi, Michael Ruff, Elizabeth Siegler, Saad Kenderian. Immunosuppressive monocytes suppress CART19 functions through modulation of the IL-1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6813.

Abstract Introduction Cholangiocarcinoma (CCA) is a highly lethal, heterogenous biliary malignancy. Despite the development of targeted therapies, treatment success has been hindered by primary or acquired resistance. New therapeutic strategies are necessary to improve outcomes in these patients. NXP800 is a GCN2 small-molecule activator that phosphorylates eIF2a inducing selective ATF4 transcription which stimulates stress-induced genes leading to apoptosis. NXP800 has been previously validated in ovarian cancers with ARID1A mutations, well-known players in cholangiocarcinoma. We sought to determine the treatment efficacy of novel NXP800 in CCA utilizing patient derived xenograft (PDX) preclinical models. Methods In our preliminary studies, four human and two murine CCA cell lines were tested for cell viability using CellTiter-Glo to determine the maximal inhibitory concentration (IC50). Immunoblot analysis of HuCCT-1 cells incubated with 1 mM of NXP800 for 6 hours vs. vehicle was performed. We also evaluated response to GCN2 activation in multiple RNA sequenced PDX models. Xenografts were expanded into the flank of NOD/SCID mice and treatment begun when tumors averaged 120mm3. Tumor bearing mice were treated with NXP800 (35 mg/kg) or vehicle via oral gavage five days on, two days off, for 28 days. Tumor volume and animal weight were collected twice weekly. Predicted sensitive signatures were determined using multi-omics. Results IC50 dose response curves showed nanomolar activity in all six cell lines, ranging from 5-170 nM. Immunoblotting demonstrated an upregulation of phosphorylated eIF2a and in turn upregulation of ATF4 with treatment of NXP800 compared to vehicle. Five PDX tumors from patients with intrahepatic or distal CCA was established and validated by histologic analysis. Molecular characterization of the tumor identified tumor mutation and a total tumor mutational burden. RNA sequencing was completed and identified 21-gene signatures related to YAP activity, which were cross-referenced against our existing PDX model tumor bank. Treatment with NXP800 was associated with a statistically significant decrease in tumor size in three of five PDX models tested, which included an ARID1A, FGFR1, and ATM mutants. Preliminary multi-omics on the PDX models revealed that sensitivity mechanisms were centered around EGFR signaling. Conclusion The novel GCN2 kinase activator, NXP800, has nanomolar efficacy in both human and murine CCA cell lines in vitro. Additionally, NXP800 demonstrated therapeutic activity in multiple cholangiocarcinoma PDX models in vivo. We are opening a Phase 1b clinical trial investigating the effects of NXP800 use in patients with advanced CCA. Future studies determining the cellular effect of GCN2 activation utilizing NXP800 are being conducted. Citation Format: Danielle Marie Carlson, Hendrien Kuipers, Amro Abdelrahman, Erik Jessen, Joshua Roldan Kalil, Jack Sample, Jennifer Tomlinson, Mark Truty, Rory Smoot. A novel GCN2 kinase activator demonstrates therapeutic efficacy in preclinical PDX models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2098.

Abstract Background: Though widely recognized in cancer pathogenesis, inflammatory pathways of allostatic load remain vaguely understood. A partial explanation may be found within the realm of food insecurity and micronutrient inadequacies, defined as nutrient intake less than the estimated average requirement. Many micronutrient deficiencies contribute to pro-inflammatory and immunocompromised milieus. Inadequacies, however, which may present without overt symptoms, are not currently well studied. The purpose of this study was to examine relationships between known inflammation-associated nutritional biomarkers of micronutrients and biomarker variables of allostatic load. Methods: Using NHANES 2017/2018 surveys, adults > 18 years without renal or hepatic disorders, pregnancy, alcohol, or supplement use were included. Plasma levels of vitamins A, B9, C, D, E, calcium (Ca), ferritin (Fe), phosphorus (P), potassium (K) and sodium (Na) were grouped into deficiency, inadequate and adequate groups based on clinically recognized standards. Micronutrient prevalence was assessed and variables of allostatic load [blood pressure, glucose, albumin, creatinine, red cell distribution width (RDW), high density lipoprotein (HDL), low density lipoprotein (LDL), cholesterol and triglycerides] were analyzed for mean differences among micronutrient groups, food security and poverty. Results: From 3,317 unique participants, inadequacy prevalence percentages were quantified as: vitamins A (0.4%), B9 (12.2%), C (58.3%), D (39.6%), E (21.0%), P (11.4%), K (3.4%), Na (24.2%), Ca (59.5%), and Fe (37.4%) [p < 0.001]. Statistically significant differences were identified in age [C, D, E, Ca, Fe, P, Na (p < 0.004)], race/ethnicity [B9, C, D (p < 0.006)], gender [C, E, Ca, Fe, P, K, Na (p < 0.018)], poverty [A, B9, C, D, E, Fe, K (p < 0.025)], and food security [B9, C, D, E, P (p < 0.032)]. Associations to allostatic load were discovered in: albumin [A, C, D, Ca, Fe, P, Na (p < 0.025)], glucose [C, E, Fe, P, Na (p < 0.009)], HDL [C, D, E, Fe, P, K (p<0.045)], LDL [E, Ca, Fe, Na (p<0.028)], creatinine [C, D, Ca, Fe (p<0.001)], triglycerides [B9, D, E, Fe (p<0.033)], cholesterol [C, D, E, Ca, Fe, Na (p<0.006)], B/P [C, E, Fe, P, Na (p < 0.035)], and RDW [C, D, Ca, Fe (p < 0.001)]. Poverty was significantly associated with all variables of allostatic load (F = 2.85, p = 0.002). Food security was significantly correlated with albumin (p < 0.001), glucose (p = 0.005) and HDL (p < 0.001). Conclusion: Allostatic load, poverty, and food security were significantly associated with subclinical micronutrient statuses, which may not present with signs of deficiency and are not currently well assessed. Similarly, poverty was significantly associated with all variables of allostatic load, though food security was only identified to be associated with albumin, glucose, and HDL, suggesting that food security may not be currently adequately defined for surveillance. Citation Format: Jennifer M. Crook, Eunkyung Lee, Opeyemi Bolajoko, Rui Xie. Poverty, food insecurity, and nutritional inflammatory mediators of allostatic load [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2198.

Abstract Introduction: Despite the remarkable activity of CD19-directed chimeric antigen receptor T cell therapy (CART19), durable remissions remain low, especially in chronic lymphocytic leukemia (CLL). Ibrutinib is an oral irreversible inhibitor of Bruton’s tyrosine kinase and is a first line therapy for CLL. Preclinical and early clinical data indicate that ibrutinib plus CART19 may be synergistic through inhibition of IL2-inducible T cell kinase. To systematically study the effects of prior ibrutinib treatment on subsequent CART functions, we utilized blood samples from the pivotal clinical trial E1912 (NCT02048813). E1912 was a phase 3 randomized trial comparing treatment with ibrutinib/rituximab (IR) to fludarabine/cyclophosphamide/rituximab (FCR) in previously untreated patients with CLL, which led to the FDA approval of ibrutinib as a first-line therapy in CLL. Methods: We evaluated the ex vivo expansion and effector functions of T cells and CART19 generated from patients (n=12) with CLL progression following treatment with IR or FCR. We generated CART19 from longitudinal samples obtained at baseline (pretreatment), 6 months post-treatment, 12 months post-treatment, and disease progression. Isolated T cells were lentivirally transduced to express CD28- or 41BB-costimulated CAR19. To assess antigen specific killing and proliferation, untransduced T cells or CART19 were co-cultured at a 1:1 ratio with the CD19+ cell line, JeKo-1, as a surrogate for CLL cells. Results: Ex vivo T cell expansion increased from an average of 1.5-fold at baseline to 6.7-fold at 6 months, 11.6-fold at 12 months, and 16.9-fold at progression, following ibrutinib-based therapy (p = 0.02, 0.04, and 0.04, respectively). Improved T cell expansion was independent of disease burden (p = 0.89, 0.73, and 0.54 for expansion vs B cell count at 6 months, 12 months, and progression). T cell expansion significantly worsened following treatment with FCR-based therapy compared to ibrutinib-based therapy (0.6- vs 11.6-fold at 12 months and 0.9- vs 16.9-fold at progression; p = 0.031 and 0.039, respectively). Starting at 6 months post-IR, CART19 demonstrated improved antitumor activity and antigen-specific proliferation against JeKo-1. There was no significant difference in the expansion of 41BB- vs CD28-costimulated CART19 following ibrutinib treatment. However, 41BB-CART19 showed enhanced antigen-specific proliferation upon stimulation with JeKo-1 (p = 0.008, 0.02, and 0.02 at 6 months, 12 months, and progression). Conclusion: Our results indicate significant improvement in T cell expansion and CART19 cell functions in patients with CLL following frontline ibrutinib-based therapy, and worsening T cell functions after FCR treatment. Improvement in T cell functions is independent of malignant B cell eradication and is more significant in 41BB-costimulated CART19. Citation Format: Truc Ngoc Huynh, Victoria Wang, Tait Shanafelt, Elizabeth L. Siegler, Carli M. Stewart, Kun Yun, Long K. Mai, Olivia Sirpilla, James H. Girsch, Claudia Manriquez-Roman, Jennifer M. Feigin, Makena Rodriguez, Brooke L. Kimball, Hong Xia, Reona L. Sakemura, Ismail Can, Omar L. Gutierrez-Ruiz, Ekene J. Ogbodo, Erin E. Tapper, Lionel A. Fonkoua, Mehrdad T. Hefazi, Michael W. Ruff, Sameer Parikh, Neil E. Kay, Saad J. Kenderian. Frontline ibrutinib-based therapy enhances CART cell functions in patients with CLL: Analysis of the phase 3 randomized clinical trial ECOG-ACRIN E1912 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3849.