Cancer therapy is often limited by toxicity from pneumonitis. This often-lethal side effect is known to be impacted by innate immunity, and in particular the pathways regulated by the TRAIL death receptor DR5. We investigated whether DR5 agonists could rescue mice from the lethal effects of radiation. We found that two different agonists, parenteral PEGylated trimeric-TRAIL (TLY012) and oral TRAIL-Inducing Compound #10 (TIC10/ONC201), could achieve this goal. Both compounds could completely protect mice from lethality by reducing pneumonitis, alveolar-wall thickness, and oxygen desaturation. At the molecular level, this protection appeared to be due to the inhibition of CCl22, a macrophage-derived chemokine previously associated with radiation pneumonitis and pulmonary fibrosis. The discovery that short-term treatment with TRAIL pathway agonists effectively rescues animals from high doses of radiation exposure has important translational implications. One Sentence Summary Prevention of lethality, pneumonitis, lung fibrosis and skin dermatitis post-ψ-irradiation by short- term treatment with innate immune TRAIL pathway agonists

Radiotherapy can be limited by pneumonitis, which is impacted by innate immunity, including pathways regulated by TRAIL death receptor DR5. We investigated whether DR5 agonists could rescue mice from toxic effects of radiation and found that 2 different agonists, parenteral PEGylated trimeric TRAIL (TLY012) and oral TRAIL-inducing compound (TIC10/ONC201), could reduce pneumonitis, alveolar wall thickness, and oxygen desaturation. Lung protection extended to late effects of radiation including less fibrosis at 22 weeks in TLY012-rescued survivors versus unrescued surviving irradiated mice. Wild-type orthotopic breast tumor-bearing mice receiving 20 Gy thoracic radiation were protected from pneumonitis with disappearance of tumors. At the molecular level, radioprotection appeared to be due to inhibition of CCL22, a macrophage-derived chemokine previously associated with radiation pneumonitis and pulmonary fibrosis. Treatment with anti-CCL22 reduced lung injury in vivo but less so than TLY012. Pneumonitis severity was worse in female versus male mice, and this was associated with increased expression of X-linked TLR7. Irradiated mice had reduced esophagitis characterized by reduced epithelial disruption and muscularis externa thickness following treatment with the ONC201 analog ONC212. The discovery that short-term treatment with TRAIL pathway agonists effectively rescues animals from pneumonitis, dermatitis, and esophagitis following high doses of thoracic radiation exposure has important translational implications.

High grade serous ovarian cancer (HGSOC) is the most lethal of all gynecologic malignancies in which the majority of patients eventually develop chemoresistant recurrent disease. Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme canonically known for its involvement in neurodegeneration, but recently has been shown to play a key role in tumorigenesis. Furthermore, UCHL1 has garnered attention across a multitude of cancer subtypes as it has the ability to be targeted through small molecule inhibition. Therefore, the goal of this present study was to elucidate mechanistic consequences of small molecule UCHL1 inhibition in HGSOC. Comparative label-free proteomic analysis of HGSOC cell line, OVCAR8 revealed prominent changes in cell metabolism proteins upon treatment with UCHL1 small molecule inhibitor, LDN-57444. Further validation via Western blot analysis revealed that changes in cell metabolism proteins differed in matched chemosensitive versus chemoresistant HGSOC cells. Finally, cell viability analysis demonstrated that a combinatorial carboplatin and LDN-57444 blockade produced a promotion or conversely, inhibition of cell death, in chemoresistant, and chemosensitve HGSOC cells, respectively. This phenomenon was further corroborated by respective differences in activation levels of common tumor cell growth pathways STAT3, MAPK/ERK, and AKT in chemoresistant versus chemosensitive HGSOC cells. Overall, this investigation established that pharmacologic targeting of UCHL1 produces differential effects according to HGSOC chemosensitivity status.

Background:Representing 15-20% of all breast cancer cases, triple negative breast cancer (TNBC) is diagnosed more frequently in reproductive-age women and exhibits higher rates of disease metastasis and recurrence when compared with other subtypes. Few targeted treatments exist for TNBC, and many patients experience infertility and endocrine disruption as a result of frontline chemotherapy treatment. While they are a promising option for less toxic therapeutic approaches, little is known about the effects of immune checkpoint inhibitors on reproductive and endocrine function. Results:Our findings in a syngeneic TNBC mouse model revealed that therapeutically relevant immunotherapies targeting PD-1, LAG-3, and TIM-3 had no effect on the quality and abundance of ovarian follicles, estrus cyclicity, or hormonal homeostasis. Similarly, in a tumor-free mouse model, we found that ovarian architecture, follicle abundance, estrus cyclicity, and ovulatory efficiency remain unchanged by PD-1 blockade. Conclusions:Taken together, our results suggest that immunotherapy may be a promising component of fertility-sparing therapeutic regimens for patients that wish to retain ovarian and endocrine function after cancer treatment.

Abstract Objective: The goal of this study was to determine the effect that amphiregulin (AREG) has on the ovarian tumor immune microenvironment (TIME). Methods: OVCAR8WT cells were stimulated with recombinant AREG (rAREG) and submitted for NanoString nCounter® PanCancer IO360 analysis to identify genomic changes in over 770 genes related to tumor immunology. A commercially available phospho-kinase proteome profiler and western blot analysis was employed to determine signaling pathways changes affected by rAREG in OVCAR8WT, and the additional high grade serous ovarian cancer (HGSOC) cell line PEA1. OVCAR8WT and PEA1 cells were co-cultured with peripheral blood mononuclear cells (PBMCs) with and without the addition of rAREG and viability of the HGSOC cells was assessed. Furthermore, chemoresistant HGSOC cells PEA2 were co-treated with carboplatin and an AREG neutralizing antibody (AREG nab), and cell viability was determined. Finally, an in vivo study was performed utilizing an ID8p53-/- C57/BL6 model in which mice were treated with rAREG or saline daily for up to 6 days following tumor formation. Tumors, ascites, and serum from mice were collected at time of euthanasia, and serum and ascites fluid submitted for multiplex cytokine/chemokine analysis to Eve Technologies. Immunohistochemistry was employed to determine PD-L1 and CD8+ T cell levels in mouse tumors. Results: Nanostring analysis revealed that OVCAR8WT cells treated with rAREG led to numerous significant (p<0.006) tumor-intrinsic immune increases, notably in DUSP5 (4.1- fold), DUSP1 (1.6-fold), IL6 (1.6-fold), CXCL8 (11.2-fold), CXCL2 (3.2-fold), and CXCL1 genes (5.4- fold), Phospho-kinase proteome profiler results revealed an increase in STAT3 expression following rAREG exposure in OVCAR8WT (2.9- fold) and PEA1(1.6- fold) cells. These results were validated via western blot. Furthermore, western blot analysis also revealed that phospho-AKT, phospho-ERK, and programmed death ligand 1 (PD-L1) were increased following rAREG treatment in HGSOC cells. OVCAR8WT and PEA1 cells co-treated with PBMCs stimulated with rAREG demonstrated a significantly(p<0.05) higher cell viability than cells treated with PBMCs alone. Moreover, PEA2 cells co-treated with both carboplatin and an AREG nab synergistically and significantly (p<0.05) decreased cell viability. Furthermore, mice treated with rAREG demonstrated significantly (p<0.05) lower levels of IL-2, IL-5, and IL-11 in their ascites and higher serum levels of IL-20. Finally, intratumoral levels of PD-L1 were significantly (p<0.05) upregulated in rAREG treated mouse tumors as well as significantly (p<0.05) lower levels of CD8+ T cells. Conclusions: These findings demonstrate that increased AREG leads to tumor-intrinsic activation of key immune pathways and associated genes and promotes tumor immune evasion in an immunocompetent HGSOC mouse model. Further studies include determining the effects of targeting AREG alone and in combination with chemotherapy in vivo. Citation Format: Jasmine Ebott, Julia McAdams, Payton De La Cruz, Chloe Kim, Nicole E. James. Elucidating the role of amphiregulin in the ovarian tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B088.

e15078 Background: High grade serous ovarian cancer (HGSOC) is the most lethal of all gynecologic malignancies, due to the fact that the majority of patients are diagnosed at an advanced stage and eventually develop chemoresistant disease 12-18 months following completion of frontline therapy. Therefore, novel targeted therapies are urgently needed to combat chemoresistance and improve patient clinical outcomes. Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that plays a vital role in protein homeostasis and is highly overexpressed in HGSOC. It was previously uncovered that targeting UCHL1 with small molecule inhibition reduced cell viability in a chemoresistant ovarian cancer cell line. Methods: Comparative-label free proteomic analysis was used to uncover significant changes in HGSOC cell line OVCAR8WT following treatment with UCHL1 small molecule inhibitor, LDN-5744 or corresponding DMSO control. Significant pathway changes were determined by KEGG analysis. Matched chemosensitive and resistant HGSOC cells PEA1 and PEA2 were treated in combination with LDN-5744 and carboplatin, with cell viability was determined by an MTS assay. Furthermore, western blot analysis was employed to compare common cell growth pathway changes as a result of UCHL1 inhibition in HGSOC cells. Results: Proteomic analysis revealed a significant (p<0.05) upregulation in methyl phosphate capping enzyme (MEPCE) (4.9-fold), asparagine synthetase (ASNS) (2.5-fold), Phosphoserine aminotransferase 1 (PSAT1) (1.7-fold), and downregulation in centrosomal protein 55 (CEP55) (-7.2-fold), These identified proteomic changes were confirmed by western blot. KEGG pathways analysis using all significantly (p<0.05) differentially expressed proteins showcased significant (p<0.05) enrichment in amino acid biosynthesis, and metabolism, and chemical carcinogenesis-reactive oxygen species. Chemoresistant PEA2 cells treated in combination with LDN-5744 and carboplatin demonstrated a 59% reduction in cell viability, significantly (p<0.001) lower than the 13% and 22%, that was observed from UCHL1 and carboplatin treatment alone, respectively. Conversely, PEA1, the chemosensitive counterpart to PEA2 demonstrated a significant (p<0.001) 37% increase in cell viability upon combinatorial treatment compared with carboplatin alone, indicating that UCHL1 inhibition rescues the cells from chemotherapy induced cell death. Finally, LDN-5744 treatment in PEA2 led to a reduction in levels of phospho-(p-)AKT, p-STAT3, and p-ERK, while conversely in PEA1 cells levels of these proteins were increase or unchanged. Conclusions: Our findings demonstrate that targeting UCHL1 results in prominent metabolic changes in HGSOC cells and that the efficacy of UCHL1 inhibition is heavily dependent upon platinum status. Overall, this study highlights UCHL1’s novel therapeutic role in the recurrent HGSOC.

High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy in which patients have still yet to respond meaningfully to clinically available immunotherapies. Hence, novel immune targets are urgently needed. Our past work has identified that mast cells are significantly upregulated at the mRNA level in HGSOC patient tumors following neoadjuvant chemotherapy (NACT) exposure. Therefore, in this current investigation we sought to characterize intratumoral mast cell phenotypic changes as a result of NACT exposure and determine how these adaptations are associated with patient clinical outcomes. Hematologic immunohistochemistry was employed to determine mast cell levels in 36 matched pre- and post-NACT HGSOC patient tumors. Fluorescent Immunohistochemistry was utilized to identify Tryptase+(carboxypeptidase A3 (CPA3) + mast cells as well as histamine levels in 29 and 20, respectively, matched pre- and post-NACT HGSOC patient tumors. Finally, human immortalized mast cells, LUVA were stimulated with carboplatin and paclitaxel and genomic changes were analyzed by quantitative PCR. Hematologic labeled intratumoral mast cells were significantly upregulated in the intraepithelial and stromal regions of the tumor, post-NACT. Lower levels of pre-NACT mast cells were significantly associated with an improved progression-free survival (PFS). Histamine, a marker of mast cell degranulation was similarly upregulated in post-NACT exposed tumors. Through the characterization of mast cell specific proteases Tryptase and CPA3, it was found that Tryptase+/ CPA3 + mast cells were significantly upregulated both in the intraepithelial and stromal compartments of the tumor, while Tryptase + cells were significantly upregulated in the stromal regions of the tumor. Lower post-NACT treated levels with Tryptase+/ CPA3 + cells were significantly associated with improved overall survival (OS) and PFS while higher Tryptase + mast cells were associated with improved OS. Finally, following chemotherapy exposure mast cell activating factors AREG and CCL2 were significantly upregulated while TGFB1, an inhibitor of mast cell activation was downregulated in LUVA cells. Enhanced mast cell numbers, as well as activation and degranulation are a consequence of NACT exposure. Post-NACT mast cells displayed differing associations with survival outcomes that was dependent upon granule classification. Ultimately, mast cells represent a clinically relevant putative HGSOC immune target.

Abstract Objective: The goal of this study is to examine intratumoral expression and phenotypic changes in mast cells following neoadjuvant chemotherapy (NACT) exposure in high grade serous ovarian cancer (HGSOC). Methods: 37 HGSOC matched pre-and post-NACT patient tumors were stained with a commercially available mast cell hemotoxic-based kit and both intratumoral and stromal mast cells were quantified. Immunohistochemistry was employed to stain for histamine in 19 matched HGSOC patient tumors and mean intensity and integrated optical density was quantified using Image J. Kaplan Meier curve analysis was employed to determine the relationship between mast cells levels and progression-free survival (PFS). Immortalized human mast cells, LUVA were treated with carboplatin and paclitaxel and specific genomic changes were measured by quantitative PCR (qPCR). Results. Mast cells were more prominently expressed in intraepithelial regions compared to stromal cells, however both populations were significantly (p<0.05) increased following NACT. Total mast cells stratified by upper and lower quartile demonstrated that higher levels were significantly (p<0.05) associated with improved PFS. Furthermore, histamine, a marker of mast cell degranulation was significantly (p<0.05) increased in HGSOC tumors post-NACT and significantly correlated (r=0.553, p=0.013) with the change in total mast cells. Following chemotherapy treatment LUVA cells demonstrated an over 7-fold increase in amphiregulin (AREG) expression (p<0.01), a key marker of mast cell activation. Interestingly, angiogenic and pro-tumorigenic factors IL-10, IL-8, and VEGFA exhibited no significant differences in expression following chemotherapy exposure. Conclusions: Overall, our findings suggest a correlation in NACT exposure and intratumoral mast cell recruitment, degranulation, and activation. Further studies include investigating differences in specific tumorigenic genes and cell signaling pathways in mast cells pre- and post- NACT, as well as determining if mast cells can impact HGSOC chemotherapy response. Citation Format: Julia McAdams, Jasmine Ebott, Payton De La Cruz, Nicole E. James. Intratumoral expression analysis of mast cells in high grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B089.

High grade serous ovarian cancer (HGSOC) is a lethal gynecologic malignancy in which chemoresistant recurrence rates remain high. Furthermore, HGSOC patients have demonstrated overall low response rates to clinically available immunotherapies. Amphiregulin (AREG), a low affinity epidermal growth factor receptor ligand is known to be significantly upregulated in HGSOC patient tumors following neoadjuvant chemotherapy exposure. While much is known about AREG’s role in oncogenesis and classical immunity, it is function in tumor immunology has been comparatively understudied. Therefore, the objective of this present study was to elucidate how increased AREG exposure impacts the ovarian tumor immune microenvironment (OTIME). Using NanoString IO 360 and protein analysis, it was revealed that treatment with recombinant AREG led to prominent upregulation of genes associated with ovarian pathogenesis and immune evasion (CXCL8, CXCL1, CXCL2) along with increased STAT3 activation in HGSOC cells. In vitro co-culture assays consisting of HGSOC cells and peripheral blood mononuclear cells (PBMCs) stimulated with recombinant AREG (rAREG) led to significantly enhanced tumor cell viability. Moreover, PBMCs stimulated with rAREG exhibited significantly lower levels of IFNy and IL-2. In vivo rAREG treatment promoted significant reductions in circulating levels of IL-2 and IL-5. Intratumoral analysis of rAREG treated mice revealed a significant reduction in CD8+ T cells coupled with an upregulation of PD-L1. Finally, combinatorial treatment with an AREG neutralizing antibody and carboplatin led to a synergistic reduction of cell viability in HGSOC cell lines OVCAR8 and PEA2. Overall, this study demonstrates AREG’s ability to modulate cytotoxic responses within the OTIME and highlights its role as a novel HGSOC immune target.

Abstract Background Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. While PD-1 based immunotherapies overall have led to improved treatment outcomes for this disease, a diverse response to frontline chemotherapy and immunotherapy still exist in TNBC, highlighting the need for more robust prognostic markers. Methods Tumor-intrinsic immunotranscriptomics, serum cytokine profiling, and tumor burden studies were conducted in two syngeneic mouse models to assess differential effects in both the early-stage and metastatic setting. Bioinformatic analyses of both early and metastatic TNBC patient data were performed to assess if identified NF-κB-associated factors are associated with improved patient clinical outcomes. Results NF-κB signaling driven by lymphotoxin beta expression is associated with tumor regression in TNBC mouse models. Furthermore, lymphotoxin beta expression in patient TNBC cohorts is prognostic of improved survival outcomes. Conclusions This study highlights the potential role for NF-κB-associated factors, specifically lymphotoxin beta to be used as prognostic markers in TNBC, which could ultimately provide insight for improved targeted treatment approaches in the clinic.

OBJECTIVE:To investigate the long-term effects of in utero taxane exposure on exposed daughters' ovarian reserve and reproductive potential. DESIGN:Pregnant dams were treated with a single, human-relevant animal-equivalent dose of saline, docetaxel, or paclitaxel at embryonic day 16.5. In utero-exposed daughters were aged to multiple postnatal time points for ovarian and endocrine analysis or were bred to assess fertility and fecundity. Granddaughters of treated dams were assessed also for ovarian follicle composition and atresia. SETTING:Laboratory study. ANIMALS:C57BL/6 mice. INTERVENTION(S):In utero exposure to saline, docetaxel, or paclitaxel. MAIN OUTCOME MEASURE(S):Ovarian follicle composition, rates of follicle atresia, and rates of multioocyte follicles were analyzed in all exposure groups. Serum hormone levels and oocyte retrieval outcomes following ovarian hyperstimulation were also assessed. Finally, animals from all exposure groups were bred with the number of litters, pups per litter, live births, interlitter time interval, and age at the last litter analyzed. RESULT(S):We found that docetaxel and paclitaxel exposure in utero results in ovarian toxicity later in life, significantly affecting folliculogenesis as well as increasing the rate of follicular abnormalities, including follicle atresia and multioocyte follicles. Furthermore, viability staining indicates that the ovaries of daughters exposed to taxanes in utero demonstrate a significantly higher number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive follicles. Hormone measurements also revealed that serum follicle-stimulating hormone concentration was significantly altered in taxane-exposed daughters, with the ratio of luteinizing hormone to follicle-stimulating hormone significantly elevated, specifically after paclitaxel exposure, coincident with the inability of these animals to properly respond to ovarian stimulation. Breeding studies over the course of a year also suggest that these taxane-exposed mice are fertile, although the duration of their fertility is shortened and they produce significantly fewer litters. Finally, ovarian effects are apparent in granddaughters of mice treated with docetaxel, suggesting persistent and multigenerational effects of taxane exposure. CONCLUSION(S):Our studies demonstrate that in utero exposure to taxane-based therapy during late gestation has a significant effect on the long-term reproductive health of exposed daughters (as well as their daughters) and will be instrumental in helping clinicians better understand which chemotherapies for maternal malignancy are least detrimental to a developing fetus.

MDM2 and MDM4/MDMX have emerged as potential mediators of tumor progression, organ-specific metastasis, and hyper-progression after immune checkpoint blockade therapy. While there are currently a number of MDM2 inhibitors in clinical trials, we found no pure MDM4/MDMX inhibitors under clinical investigation. Because hormone-resistant cancers pose a significant challenge for clinical intervention, the identification of translatable novel therapeutic targets remains an important goal. Analysis of triple-negative breast cancer (TNBC) and castration-resistant prostate cancer (CRPC) reveals overexpression of p53 negative regulator MDM4/MDMX as a frequent alteration in these cancers. There is an urgent need to study and target MDM4/MDMX in cancer therapy and this may ultimately include dual MDM2 and MDM4/MDMX inhibitors. However, it is clear that MDM4/MDMX is a primary oncogenic driver that requires specific therapeutic targeting. As metastasis is often observed clinically in patients diagnosed with breast and prostate cancer, we sought to expand our understanding of the metastasis reduction potential of a previously described preclinical MDM4/MDMX inhibitor, a 4-nitrobenzofuroxan derivative, XI-006 (NSC207895). In a scratch assay using breast and prostate cancer cell lines, increasing doses of XI-006 decreased tumor cell migration in a time and dose-dependent manner, demonstrating the benefit of MDMX inhibition in preventing the induction of tumor cell migration. The exact mechanism of this result remains unclear, and further investigation is needed to elucidate the impact of XI-006 on TNBC and CRPC cell proliferation and migration. Our future directions include identifying the synergistic potential of combining MDM4/MDMX inhibitor XI-006 with other cancer therapies, assessing the impact of XI-006 on immune responses in co-culture studies, and testing therapeutic efficacy in vivo. Citation Format: Arielle De La Cruz, Andrew George, Payton De La Cruz, Maximilian P. Pinho-Schwermann, Kimberly S. Meza, Taylor Arnoff, Ilyas Sahin, Stephanie L. Graff, Benedito A. Carneiro, Wafik S. El-Deiry. Preclinical anti-tumor effects of MDM4/MDMX inhibitor XI-006 in breast cancer and prostate cancer cell lines mediated through reduced tumor cell migration. [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 3952.

Background: Few targeted treatments exist for triple negative breast cancer (TNBC), an aggressive form of breast cancer that is diagnosed more frequently in reproductive-age women. As a result, most patients receive frontline chemotherapies and often experience infertility and endocrine disruption. Immunotherapies are a promising option for less toxic therapeutic approaches, but little is known about their effects on reproductive and endocrine function. Methods: E0771 cells were injected into the bilateral mammary pads of 8-week-old female C57Bl/6J mice. After tumor development (14 days post-implantation), E0771 mice received 200 ug doses of immunotherapy (anti-PD-1, anti-LAG-3, or anti-TIM-3 antibodies) or IgG isotype control every four days, with treatments ceasing after the third dose. 8 days after the final dose, estrus cycling was monitored via vaginal cytology. 11-15 days after the final dose, mice were euthanized during proestrus stage, and serum and ovaries were collected. Serum levels of anti-Mullerian hormone (AMH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were quantified via ELISA. Ovaries were fixed in formalin and embedded in paraffin, then sectioned serially. Follicle staging and abundance was quantified via hematoxylin and eosin staining. Follicle health and apoptosis was evaluated via TUNEL staining. Results: There were no significant differences between any of the treatment and control groups in the percentage of time spent in each stage of the estrus cycle, indicating that treatment with the selected immunotherapies does not cause perturbations in reproductive cyclicity. Serum hormone analysis revealed no significant differences in AMH, LH, or FSH levels between treatment and control groups, indicating that these immunotherapies do not cause endocrine toxicity. There were no significant differences in follicle abundance between any of the treatment and control groups, suggesting that the immunotherapies are not toxic to the ovarian reserve or developing follicles. TUNEL staining revealed no significant differences in granulosa cell or oocyte apoptosis between treatment groups. Conclusion: Treatment with anti-PD-1, anti-LAG-3, or anti-TIM-3 monoclonal antibodies does not cause reproductive or endocrine toxicity in a tumor-bearing mouse model. These results demonstrate that immunotherapy may be a promising component of fertility-sparing therapeutic regimens for patients who wish to maintain ovarian and endocrine function after cancer treatment. Citation Format: Payton De La Cruz, Jennifer Ribeiro, Kathryn Grive. Monoclonal antibodies targeting PD-1, LAG-3, and TIM-3 do not cause reproductive or endocrine toxicity in a syngeneic model of triple negative breast cancer [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 3210.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy, primarily due to the fact that the majority of patients experience a chemoresistant recurrence following frontline platinum-based therapy. In recent years, immunotherapies have garnered widespread success within the field of oncology; however, response rates have remained low for HGSOC patients. Therefore, an improved understanding of how chemotherapy affects the ovarian tumor immune microenvironment (TIME) may uncover novel treatment modalities to improve patient clinical outcomes. Our prior data established that following treatment with carboplatin and paclitaxel, the transmembrane glycoprotein amphiregulin (AREG) was one of the most significantly induced genes in human HGSOC tumors post-neoadjuvant chemotherapy. Therefore, this present study sought to elucidate the role that increased AREG plays in ovarian TIME following chemotherapy.

OBJECTIVE:High grade serous ovarian cancer (HGSOC) exhibits low response rates to clinically available immunotherapies. Nevertheless, emerging research has demonstrated that certain immune factors are predictive for HGSOC patient clinical outcomes, with our own groups previous work demonstrating that intratumoral levels of the immune checkpoint receptor LAG-3 is associated with improved patient survival. In this current study we sought to uncover non-invasive circulating immune prognostic and predictive signatures in HGSOC.METHODS:A multiplex approach was employed that examined circulating levels of immune checkpoint receptors LAG-3 and PD-1 along with 48 common cytokine and chemokines in a cohort of 75 HGSOC treatment naïve patient serum samples.RESULTS:Elevated serum LAG-3 was significantly associated with improved progression-free survival (PFS) and overall survival (OS) in HGSOC, while circulating PD-1 levels were largely unrelated with patient clinical outcomes. Cytokine and chemokine analysis revealed lower IL-15 expression correlated with improved PFS and OS, while increased IL-1α, IL-1Ra, IL-6, IL8 and VEGF were significantly associated with preoperative CA-125 levels. ROC analysis demonstrated that serum LAG-3 levels exhibited consistent reasonable predictability as a single agent.CONCLUSIONS:Serum-derived LAG-3 was identified out of a diverse array of chemokine and cytokines as the immune-based factor most significantly associated with improved HGSOC survival. These findings suggest that LAG-3 could be implemented as a non-invasive patient predictive marker for improved HGSOC clinical outcomes.

The high rate of ovarian cancer recurrence and chemoresistance necessitates further research into how chemotherapy affects the tumor immune microenvironment (TIME). While studies have shown that immune infiltrate increases following neoadjuvant (NACT) chemotherapy, there lacks a comprehensive understanding of chemotherapy-induced effects on immunotranscriptomics and cancer-related pathways and their relationship with immune infiltrate and patient responses. In this study, we performed NanoString nCounter® PanCancer IO360 analysis of 31 high grade serous ovarian cancer (HGSOC) patients with matched pre-treatment biopsy and post-NACT tumor. We observed increases in pro-tumorigenic and immunoregulatory pathways and immune infiltrate following NACT, with striking increases in a cohort of genes centered on the transcription factors ATF3 and EGR1. Using quantitative PCR, we analyzed several of the top upregulated genes in HGSOC cell lines, noting that two of them, ATF3 and AREG, were consistently upregulated with chemotherapy exposure and significantly increased in platinum resistant cells compared to their sensitive counterparts. Furthermore, we observed that pre-NACT immune infiltrate and pathway scores were not strikingly related to platinum free interval (PFI), but post-NACT immune infiltrate, pathway scores, and gene expression were. Finally, we found that higher levels of a cohort of proliferative and DNA damage-related genes was related to shorter PFI. This study underscores the complex alterations in the ovarian TIME following chemotherapy exposure and begins to untangle how immunologic factors are involved in mediating chemotherapy response, which will allow for the future development of novel immunologic therapies to combat chemoresistance.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health.

ABSTRACT Ubiquitin C-Terminal Hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well-established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health. Summary sentence Ubiquitin C-Terminal Hydrolase L1 (UCHL1) is required for proper ovarian folliculogenesis, estrous cyclicity, and fertility in the female mouse.

BackgroundHigh grade serous ovarian cancer (HGSOC) is a lethal gynecologic malignancy with a five-year survival rate of only 39 percent.1 Despite the fact that ovarian tumors are considered immunologic, HGSOC patients respond poorly to PD-1 based immunotherapy. Hence, the need to identify novel prognostic and therapeutic immunologic factors is crucial. Our previous investigation uncovered intratumoral levels of immune co-inhibitory receptor LAG-3 as a marker of improved HGSOC patient survival. For this current study we sought to evaluate the prognostic utility of serum-based LAG-3, as well as determine how these circulating levels change in response to HGSOC standard of care therapy.MethodsThis study was approved by the Women and Infants Institutional Review Board; approval number 1057626. HGSOC serum samples were obtained from the Department of Special Testing and the Program in Women’s Oncology Gynecologic Tissue Bank at Women and Infants Hospital. A total of 43 HGSOC treatment naïve serum samples were tested for serum LAG-3 and in 20 of these patients, samples from on- and post- frontline platinum-based chemotherapy were also analyzed. A commercially available ELISA kit was employed to detect serum LAG-3.ResultsThere was no statistically significant change in pre-, on-, and post- serum LAG-3 levels following frontline chemotherapy, however median levels of LAG-3 decreased once patients initiated therapy and remained stable post-treatment. Spearman Rank Correlation analysis revealed a significant relationship between progression-free survival (PFS) and pre-treatment serum LAG-3 levels (r=0.36, p=0.017). Furthermore, it was found that patients with a PFS of 6 months or less exhibited a significantly (p=0.0031) lower mean rank of pre-treatment serum LAG-3 levels, compared to patients with a PFS of 18 months or longer. Finally, Kaplan Meier curve analysis revealed a statistically significant association between longer patient PFS and higher pre-treatment LAG-3 concentrations, when stratified by both median(HR=0.4916, log-rank p=0.022) and quartile LAG-3 serum levels (HR=0.2679, log-rank p=0.0031).ConclusionsThis study demonstrates for the first time that circulating immune checkpoint receptors have prognostic capabilities in ovarian cancer. Our findings suggest that LAG-3 is a marker for improved patient PFS. Future directions include an expansion of this original cohort in order to validate and further assess the clinical prognostic utility of serum LAG-3 in HGSOC.ReferenceSurvival Rates for Ovarian Cancer, American Cancer Society, [https://www.cancer.org/cancer/ovarian-cancer/detection-diagnosis-staging/survival-rates.html]