Decrease In Tumor Volume Research Articles

Trastuzumab deruxtecan (DS-8201a), a HER2-Targeting Antibody-Drug Conjugate with Topoisomerase I Inhibitor Payload, Shows Antitumor Activity in Uterine and Ovarian Carcinosarcoma with HER2/neu Expression (046)

<b>Objectives:</b> Carcinosarcomas (CSs) are highly aggressive gynecologic malignancies containing both carcinomatous and sarcomatous elements with heterogeneous HER2/neu expression. We evaluated the preclinical <i>in vitro</i> and <i>in vivo</i> activity of trastuzumab deruxtecan (DS-8201a), a novel HER2/neu-targeting antibody-drug conjugate (ADC) against primary uterine and ovarian CSs overexpressing HER2/neu<i>.</i> <b>Methods:</b> Twelve primary uterine and ovarian CS cell lines were evaluated for HER2/neu surface expression by immunohistochemistry (IHC) and by flow cytometry, and gene amplification by ¹³cytotoxicity, viability, and bystander killing effect assays. <i>In vivo</i> activity was studied in mouse CS xenograft models. <b>Results:</b> DS-8201a, as compared to MAAA-9199 control ADC, was found to be highly effective against uterine and ovarian CS cell lines demonstrating 3+ HER2/neu expression. Specifically, the mean half-maximal inhibitory concentrations (IC50's) were 0.02931 μg/mL and 9.75 μg/mL (p<0.0001) against 3+ HER2/neu uterine cell line, and 0.0285 μg/mL and 20.9 μg/mL (p<0.0001) against 3+ ovarian HER2/neu cell lines for DS-8201a versus MAAA-9199, respectively. There was no significant difference between DS-8201a and MAAA-9199 against CS cell lines with low/negligible HER2/neu expression. Importantly, DS-8201a induced an efficient bystander killing effect of HER2/neu negative tumor cells when admixed with 3+ HER2/neu cells. <i>In vivo</i> studies confirmed that DS-8201a was significantly more effective than MAAA-9199 in decreasing tumor volume and prolonging life in 3+ HER2/neu-expressing CS xenografts (attached Figure). There was no significant toxicity demonstrated between treatment and control groups. <b>Conclusions:</b> Trastuzumab deruxtecan (DS-8201a) is a novel ADC with remarkable activity against CS with strong (3+) HER2/neu expression. Clinical studies with DS-8201a in patients harboring chemotherapy-resistant CS with high HER2/neu expression are warranted. DS-8201a showing significant tumor growth inhibition in 3+ HER2/neu expression mice. Day 8 mean tumor volumes in treated mice with DS-8201a (0.271 cm3) versus ADC isotope control (0.859 cm3, p=0.0011) and vehicle control (0.804 cm3, p=0.0047). Day 11 mean tumor volumes in treated mice with DS-8201a (0.179 cm3) versus ADC isotope control (1.166 cm3, p <0.0001) and vehicle control (1.151 cm3, p <0.0001).Fig. 1

Small molecules to regulate the GH/IGF1 axis by inhibiting the growth hormone receptor synthesis.

Growth hormone (GH) and insulin‐like growth factor‐1 (IGF1) play an important role in mammalian development, cell proliferation and lifespan. Especially in cases of tumor growth there is an urgent need to control the GH/IGF1 axis. In this study we screened a 38,480-compound library, and in two consecutive rounds of analogues selection, we identified active lead compounds based on the following criteria: inhibition the GH receptor (GHR) activity and its downstream effectors Jak2 and STAT5, and inhibition of growth of breast and colon cancer cells. The most active small molecule (BM001) inhibited both the GH/IGF1 axis and cell proliferation with an IC50 of 10‐30 nM of human cancer cells. BM001 depleted GHR in human lymphoblasts. In preclinical xenografted experiments, BM001 showed a strong decrease in tumor volume in mice transplanted with MDA‐MB‐231 breast cancer cells. Mechanistically, the drug acts on the synthesis of the GHR. Our findings open the possibility to inhibit the GH/IGF1 axis with a small molecule.

IKKε Inhibitor Amlexanox Promotes Olaparib Sensitivity through the C/EBP-β-Mediated Transcription of Rad51 in Castrate-Resistant Prostate Cancer.

Simple SummaryMost men with advanced hormone-sensitive prostate cancer (HSPC) treated with androgen deprivation therapy will develop castrate resistant prostate cancer (CRPC), a lethal form of prostate cancer (PC). Our group has previously shown that IKKε expression is stronger in CRPC tumors and correlates with aggressive PC. Moreover, we have shown that IKKε depletion or inhibition (BX795, Amlexanox) decrease CRPC cell proliferation and tumor volume in an in vivo mouse model. We also demonstrate that IKKε inhibitors specifically target CRPC to induce a senescent phenotype as well as DNA damage and genomic instability. In this study, we demonstrated that IKKε depletion or inhibition block C/EBP-β recruitment on Rad51 promoter to decrease promoter activity. We have also shown that Amlexanox treatment sensitizes CRPC cells to Olaparib in vitro and in mouse models. Taken together, targeting IKKε with Amlexanox combined with Olaparib may lead to additional effective therapeutic strategies in the management of patients with CRPC.The progression of prostate cancer (PC) is often characterized by the development of castrate-resistant PC (CRPC). Patients with CRPC are treated with a variety of agents including new generation hormonal therapies or chemotherapy. However, as the cancer develops more resistance mechanisms, these drugs eventually become less effective and finding new therapeutic approaches is critical to improving patient outcomes. Previously, we have shown that IKKε depletion and IKKε inhibitors, BX795 and Amlexanox, decrease CRPC cell proliferation in vitro and in vivo and that IKKε inhibitors induce a senescence phenotype accompanied by increased DNA damage and genomic instability in CRPC cells. Here, we describe a new role for IKKε in DNA damage repair involving Rad51 and examine the therapeutic potential of Amlexanox combined with the PARP inhibitor Olaparib in CRPC cell lines. Combining Amlexanox with Olaparib decreased CRPC cell proliferation and enhanced DNA damage through the inhibition of Olaparib-induced Rad51 recruitment and expression in CRPC cells or IKKε-depleted PC-3 cells. We demonstrated that Rad51 promoter activity, measured by luciferase assay, was decreased with Amlexanox treatment or IKKε depletion and that Amlexanox treatment decreased the occupancy of transcription factor C/EBP-β on the Rad51 promoter. Our mouse model also showed that Amlexanox combined with Olaparib inhibited tumor growth of CRPC xenografts. Our study highlights a new role for IKKε in DNA damage repair through the regulation of Rad51 transcription and provides a rationale for the combination of Amlexanox and Olaparib in the treatment of patients with CRPC.

Radiofrequency Hyperthermia Enhances Locally Delivered Oncolytic Immuno-Virotherapy for Pancreatic Adenocarcinoma.

To investigate the effect of radiofrequency hyperthermia (RFH)-enhanced oncolytic immuno-virotherapy on in vitro pancreatic adenocarcinoma cell line and in vivo rat pancreatic cancer model. Rat pancreatic adenocarcinoma cell line and 24 Lewis rats with orthotopic pancreatic adenocarcinomas underwent treatment with either (1) oncolytic virotherapy (talimogene laherparepvec [T-VEC]) plus RFH at 42 °C for 30min; (2) oncolytic virotherapy-only; (3) RFH-only; or (4) saline (control). MTS assays and flow cytometry were used to analyze tumor cell viability and apoptosis levels 24h after treatment. In the in vivo studies, bioluminescence optical/x-ray imaging and ultrasound imaging was used to assess tumor viability and size 7 and 14days after treatment. Histopathologic analysis was performed after hematoxylin and eosin staining, TUNEL, Ki-67, and immunohistochemical staining with CD8 and ANK61. Combination therapy (T-VEC + RFH) induced decreased cell viability and increased cell apoptosis compared to T-VEC alone, RFH alone, or control. Optical/x-ray imaging and ultrasound imaging demonstrated decreased tumor bioluminescent signal and tumor volume relative to baseline after combination therapy compared to T-VEC alone, RFH alone, or control. Histopathology demonstrated decreased tumor volume and cell proliferation, increased CD8+ T cell and NK cell infiltration in tumors treated with the combination therapy compared to other three groups. RFH enhances locally delivered oncolytic immuno-virotherapy for pancreatic adenocarcinoma, with decreased cell viability and increased apoptosis observed after combination therapy in vitro, and decreased cell viability and tumor volume and increased immune cell infiltrate observed after combination therapy in vivo.

Unfolding the Potency of Adenosine in Targeting Triple Negative Breast Cancer via Paclitaxel-Incorporated pH-Responsive Stealth Liposomes.

Triple-negative breast cancer (TNBC) belongs to the category of the most destructive forms of breast cancer. Being a highly potent chemotherapeutic agent, paclitaxel (PTX) is extensively utilized in the management of various cancers. Commercially available PTX formulations contain non-targeted drug carriers that result in low antitumor activity because of non-specific tissue distribution. Thus, to resolve this issue, we designed PTX-loaded pH-sensitive liposomes (pH Lipos) in the present investigation and used adenosine (ADN) as a targeting ligand. Further, d-α-tocopheryl polyethylene glycol succinate (TPGS) was incorporated into the liposomes to impart a stealth effect to the system. For the development of these pH Lipos, different conjugates were synthesized (ADN-CHEMS and TPGS-ADN) and further utilized for the preparation of ADN-PEG-pH Lipo and ADN-pH Lipo by a thin-film hydration method. DOPE:HSPC:CHEMS:cholesterol at a molar ratio of 3:3:2:2 was selected for the preparation of pH-Lipo possessing 7.5% w/w drug loading. They showed a particle size below 140 nm, a PDI below 0.205, and a % EE greater than 60%. All of the pH Lipos displayed a biphasic pattern of PTX release at pH 7.4 and 5.5. However, the percent drug release at pH 5.5 was substantially greater because of the pH-sensitive nature of the liposomes. The MDA MB 231 and 4T1 cell lines depicted improvement in the qualitative as well as quantitative cellular uptake of PTX ADN-PEG-pH Lipo with a substantial decrease in the IC50 value. Moreover, a higher apoptotic index was observed with pH Lipo compared to free PTX. PTX ADN-PEG-pH Lipo revealed a 3.98- and 3.41-fold rise in the AUC and t1/2 values of PTX compared to Intaxel, respectively. Overall, characteristic decreases in tumor volume and serum toxicity marker levels were observed, which confirmed the development of an efficient and safe formulation.

Modern approaches to the treatment of relapsed follicular lymphoma

Follicular lymphoma is a tumor of mature B-lymphocytes, characterized by a predominantly indolent course. Despite advances in first-line therapy, disease relapses still occur, and with an increase in the follow-up period, the risk of transformation into diffuse large B-cell lymphoma also increases. The choice of a further anticancer therapy is based on the results of a comprehensive examination and repeated biopsy. In addition, previous remission duration, previous treatment, clinical manifestation of relapse, the patient's somatic condition and comorbidity are taken into account. A clinical observation of a 57-year-old patient with relapsed follicular lymphoma, who received various chemotherapy regimens in combination with rituximab in the 1st and subsequent lines, is presented. After repeated biopsy and exclusion of transformation into diffuse large B-cell lymphoma, the patient received lenalidomide therapy in combination with obinutuzumab. A partial antitumor response after 4 cycles with decrease in tumor volume by more than 80 % by the end of induction was achieved. The absence of severe adverse events, together with a pronounced antitumor effect, significantly improved the patient's quality of life.

Role of phloretin as a sensitizer to TRAIL-induced apoptosis in colon cancer.

Phloretin is one of the apple polyphenols with anticancer activities. Since tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves important roles in inducing apoptosis, the present study examined the effect of phloretin on TRAIL-induced apoptosis in colon cancer cells. Treatment with both phloretin and TRAIL markedly suppressed the survival of cancer cells from several colon cancer cell lines compared with that of cells treated with either TRAIL or phloretin. Additionally, decreased numbers of colonies were observed following addition of phloretin and TRAIL. Furthermore, TRAIL- and phloretin-treated HT-29-Luc cells exhibited decreased luciferase activity. Increased apoptosis was observed in phloretin- and TRAIL-treated HT-29-Luc colon cancer cells, accompanying elevated levels of cleaved poly(ADP-ribose) polymerase, and caspase-3, −8 and −9. The expression levels of MCL1 apoptosis regulator BCL2 family member (Mcl-1) were decreased following addition of phloretin in colon cancer cells. In addition, overexpression of Mcl-1 in phloretin- and TRAIL-treated HT-29-Luc cells resulted in increased cell survival. Treatment of HT-29-Luc cells with a combination of cycloheximide (CHX) and phloretin led to a more prominent decrease in Mcl-1 expression compared with that in cells treated with CHX alone, while Mcl-1 expression was recovered by treatment with MG132. Binding of ubiquitin with Mcl-1 was verified using immunoprecipitation. Intraperitoneal injection of both TRAIL and phloretin into tumor xenografts was associated with a decreased tumor volume compared with that following injection with either TRAIL or phloretin. Overall, the present results suggest a synergistic effect of phloretin on TRAIL-induced apoptosis in colon cancer cells.

Response to immune checkpoint blockade improved in pre-clinical model of breast cancer after bariatric surgery.

Bariatric surgery is a sustainable weight loss approach, including vertical sleeve gastrectomy (VSG). Obesity exacerbates tumor growth, while diet-induced weight loss impairs progression. It remains unknown how bariatric surgery-induced weight loss impacts cancer progression or alters response to therapy. Using a pre-clinical model of obesity followed by VSG or diet-induced weight loss, breast cancer progression and immune checkpoint blockade therapy were investigated. Weight loss by VSG or weight-matched dietary intervention before tumor engraftment protected against obesity-exacerbated tumor progression. However, VSG was not as effective as diet in reducing tumor burden despite achieving similar weight and adiposity loss. Leptin did not associate with changes in tumor burden; however, circulating IL-6 was elevated in VSG mice. Uniquely, VSG tumors displayed elevated inflammation and immune checkpoint ligand PD-L1+ myeloid and non-immune cells. VSG tumors also had reduced T lymphocytes and markers of cytolysis, suggesting an ineffective anti-tumor microenvironment which prompted investigation of immune checkpoint blockade. While obese mice were resistant to immune checkpoint blockade, anti-PD-L1 potently impaired tumor progression after VSG through improved anti-tumor immunity. Thus, in formerly obese mice, surgical weight loss followed by immunotherapy reduced breast cancer burden. Finally, we compared transcriptomic changes in adipose tissue after bariatric surgery from patients and mouse models. A conserved bariatric surgery-associated weight loss signature (BSAS) was identified which significantly associated with decreased tumor volume. Findings demonstrate conserved impacts of obesity and bariatric surgery-induced weight loss pathways associated with breast cancer progression.

Red Light Phototherapy Using Light-Emitting Diodes Inhibits Melanoma Proliferation and Alters Tumor Microenvironments.

BackgroundTotal annual cancer rates have decreased due to improved treatment and prevention. However, the incidence of melanoma is rising, and not all patients respond to immune and targeted approaches. Therefore, we sought to determine the efficacy of red light (RL) phototherapy in preclinical models of melanoma.MethodsMelanoma cells (A375, B16F10, MNT-1) were irradiated with RL. Melanoma proliferation, apoptosis, oxidative stress, and p53 phosphorylation were measured in vitro. In C57BL/6 mice, phototherapy safety, B16F10 tumor growth, and immunocyte infiltration were assessed following RL.ResultsIn vitro, 640 J/cm2 RL decreased cellular proliferation without increasing apoptosis, while 1280 J/cm2 increased apoptosis. RL increased intracellular reactive oxygen species generation and p53 phosphorylation. In animal models, 2560 J/cm2 RL significantly prevented melanoma growth and increased the expression of CD103+ dendritic cells. 1280 and 1920 J/cm2 RL decreased tumor volume, but not significantly. RL did not cause skin inflammation or erythema in normal skin.ConclusionRL represents a potentially safe and effective melanoma therapeutic. RL prevented tumor growth and increased the expression of immune markers, such as CD103, that are associated with favorable melanoma outcomes. Further research is needed to determine the optimal clinical treatment regimen for melanoma using RL.

A Therapeutic Whole-Tumor-Cell Vaccine Covalently Conjugated with a TLR7 Agonist.

A single-protein or -peptide vaccine is not sufficient to arouse immune responses in cancer therapy. A whole-tumor-cell vaccine with complete cancer cell antigens and all conformations elicits robust immune responses and is a promising method for the treatment of advanced malignant tumors. In this study, we used 5-azacitidine to stimulate B16-F10 melanoma cells to express toll-like receptor (TLR) 3 on the cell surface and then chemically linked SZU-106, a small-molecule TLR7 agonist, to the cell surface with a pegylated linker to produce a novel whole-tumor-cell vaccine, abbreviated as Aza-BFcell-106. The vaccine stimulated mouse splenic lymphocytes and bone marrow-derived dendritic cells to secrete cytokines, promoted the maturation of dendritic cells and enhanced the capability of dendritic cells to present antigens. In a mouse model of melanoma, the vaccine effectively inhibited tumor growth, decreased tumor volume and prolonged survival. Further combination of the vaccine with a chemokine inhibitor, reparixin, significantly increased the infiltration of CD4+ and CD8+ T cells into the tumor environment, while the antitumor effect was significantly enhanced. The whole-tumor-cell vaccine Aza-BFcell-106 induced immune-activating responses in both in vitro and in vivo experiments, indicating that this vaccine has great potential to treat advanced malignant tumors.

Abstract 3541: A method to visualize and quantify effects of immunotherapies in a physiologically relevant 3D TME model

Abstract INTRODUCTION: In vitro assays based on 3D co-cultures of cancer and immune cells present an opportunity to test diverse immunotherapies and understand their mechanisms of action in boosting anti-tumor immune responses in cancer patients. Incorporation of diverse cellular players, including cancer associated fibroblasts (CAFs) and suppressive myeloid cell populations, allows for faithful recapitulation of complex cellular interactions that occur in the tumor micro-environment (TME). The application of automation results in a high throughput platform which combines physiological relevance with assay robustness and reproducibility. Functional read-outs, such as migration of immune cells towards tumoroids, infiltration into the tumoroids and their killing are obtained by applying high content imaging, allowing for better understanding of the immune-modulatory profile of IO drugs. Here, we have applied image analysis to quantify the effects of different antibodies and small molecules targeting both the tumor and the T cells. MATERIAL and METHODS: Tumoroids generated from breast and colon cancer organoids and cell lines were cultured in protein hydrogel. Partially HLA-matching T cells isolated from healthy PBMCs donors were added with or without activation to the 3D culture after incorporation of different suppressive populations, including CAFs and M2 macrophages. The capacity of effector T cells to infiltrate and subsequently kill tumor structures was visualized using high-content microscopy and quantified with morphometric image analysis software after reconstitution of 3D image stacks. RESULTS and DISCUSSION: Automated 3D image and data analysis enabled discrimination of cellular interactions in a complex TME model. Depending on the activation status of T cells and the presence of suppressive cells in the environment, different levels of infiltration and tumor killing by effector T cells were measured. The suppression induced on the T cells by the environment was partially reversed by immune-modulators (e.g. CSF1R inhibitor) added to the co-culture, which was confirmed by increased IFNγ levels in culture supernatants. Two quantitative read outs that are strictly dependent on the 3D environment; T cell infiltration into tumor and decrease in tumor volume were combined to demonstrate immune suppression release induced by different immuno-modulators. CONCLUSION: Our image-based platform described here allows for analysis of immunotherapy effects on cells that engage in a physiologically relevant spatial setting and in the presence of suppressive TME elements. Visualization and quantification of these complex cellular interactions within the TME empowers immunotherapy drug developers with a highly powerful tool to select the most promising candidates and better understand their mechanism of action, which ultimately will have an impact in the clinic. Citation Format: Lidia Daszkiewicz, Gera Goverse, Nataliia Beztsinna, Saskia de Man, Tomas Veenendaal, Michelle Klop, Daniel Okkes, Ashgard Weterings, Kuan Yan, Leo Price. A method to visualize and quantify effects of immunotherapies in a physiologically relevant 3D TME model [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 3541.

Abstract 2043: The role of CCL20 in response to radiation in head and neck squamous cell carcinomas

Abstract Background: Radioresistance is responsible for most cases of tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC). Combined radiotherapy (RT) and immunotherapy (IT) treatment has improved survival for a minority of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To enhance the response of radioresistant tumors to radiation, an understanding of the factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs and their depletion improves tumor response to RT. In this study, we investigated how radiation modulates Treg infiltration through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity and that blocking CCL20 could lead to improved response to RT. Methods: Human (Cal27, SCC9) and mouse (MOC1, MOC2) cell lines with different immune phenotypes were irradiated at doses of 2Gy or 10Gy. Conditioned media (CM), RNA and protein were collected for the assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cell were were implanted into the right buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for the analysis of serum levels of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Results: Cal27 and MOC2 tumors had a gene signature associated with immunosuppression and Treg infiltration whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed microenvironment. In vitro, high-dose radiation significantly induced CCL20 in Cal27 and MOC2 cells relative to control. Multiplex analysis of chemokines from conditioned media samples showed that induction of CCL20 was associated with increased secretion of GM-CSF by MOC2 and Cal27 cells. In vivo, inhibition of CCL20 alone resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. The combination of RT and anti-CCL20 showed a significant decrease in tumor volume compared to RT alone. Conclusion: Our data suggests that high-dose radiation promotes the induction of CCL20 secretion by tumor cells which is responsible for attraction of Tregs. Inhibition of CCL20 secretion using anti-CCL20 antibodies can slow down tumor growth and enhance the response to radiation. Citation Format: Cleopatra Rutihinda, Ayman J. Oweida, Patrick Delage, Léanie Moreau, Safia Chelighem, Nour Elhouda Saidi. The role of CCL20 in response to radiation in head and neck squamous cell carcinomas [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 2043.

Abstract 254: Dietary fish oil and aspirin to augment anti-PD-1 immunotherapy in metastatic melanoma

Abstract Introduction: First line anti-PD-1 immune checkpoint inhibition (αPD-1 ICI) induces objective responses in less than 50% of metastatic melanoma patients. Aspirin (ASA) use is associated with improved responsiveness to ICI, with inhibition of myeloid-induced immunosuppression in the tumor microenvironment (TME) a purported mechanism. Cyclooxygenase modulators ASA (pharmacologic inhibitor) and dietary fish oil (FO, substrate) shift myeloid and T cell populations to anti-inflammatory, pro-resolution phenotypes in immunity and cancer. Methods: C57-BL6/J mice were injected with YUMM 1.7 melanoma in the flank. Mice were fed control chow and water (CTL) or omega-3 rich (FO) chow (10% w/w, 30%kcal/kcal), given ASA in drinking water (600 ug/mL), or FO/ASA combined (COMBO) starting 12 days post tumor implantation, the same day as starting intraperitoneal αPD1 or IgG2a vehicle (q3-4 days). An alternative cohort received pre-αPD-1 treatment ASA and FO starting day 7. Tumors were assessed for growth, harvested, and characterized via flow cytometry on day 32. Results: When diets and water were started concurrent with αPD-1/IgG2a, FO decreased tumor volume vs. CTL for both vehicle (28%) and αPD-1 (27%) treatments; ASA and COMBO (p = 0.0501) did not. Compared to CTL, FO and COMBO increased monocytes (CD45+, CD11b+, Ly6C+, Ly6G-) and PD-L1+ monocytes in the TME with no effect on tumor associated macrophages (TAMS); ASA decreased the percent of Arginase 1+, CD206+ TAMs. ASA increased PD1+ CD8 T cells, while COMBO increased CD4 and CD8 T cells including LAG3+ and PD1+ CD8 T cells. When diets and water were started on day 7, FO + αPD-1 decreased tumor volume (21%) vs. αPD-1 controls; ASA and COMBO did not. Compared to CTL, FO and COMBO similarly altered immune populations as above, with FO additionally increasing total CD3+ T-cells, PD-L1+ CD4 T cells, and PD-1+ CD8 T cells; COMBO increased dendritic cells (CD11c+, MHCII+, F4/80-). TAMS were not assessed. Results significant (p&amp;lt; 0.05) by ANOVA or t-test unless specified. Food eaten, water imbibed, and weight did not vary between groups. Conclusions: Dietary fish oil impaired in vivo melanoma tumor growth with and without αPD-1 ICI despite increases in classically deleterious monocyte populations, while aspirin decreased M2-like macrophages in the TME without alterations in tumor growth. Modulation of the cyclooxygenase axis may be a cost-effective method to skew TME immune populations to favor αPD-1 immunotherapy responses in metastatic melanoma. Citation Format: Alexander C. Chacon, Shuyang S. Qin, Alexa D. Melucci, Katherine M. Jackson, Paul R. Burchard, Yatee A. Dave, Rachel Jewell, Brian A. Belt, William Tabayoyong, David C. Linehan, Peter A. Prieto. Dietary fish oil and aspirin to augment anti-PD-1 immunotherapy in metastatic melanoma [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 254.

Abstract 653: Molecular pathology of metastatic prostatic adenocarcinoma treated with bipolar androgen therapy (BAT) reveals a correlation between MYC mRNA and protein

Abstract Molecular analysis of tissue samples from metastatic cancer lesions can improve our understanding of drug response and resistance. Since prostate cancer cells initially require androgen signaling through the androgen receptor (AR) for proliferation, the treatment of metastatic prostate cancer involves surgical or chemical castration. While most patients respond initially, nearly all develop acquired resistance to combined androgen blockade using both first and second line agents. This resistance is often related to overexpression of AR, which is frequently driven by AR gene amplification. In some patients, this increased AR appears to render tumor cells sensitive to high dose androgens in a manner that paradoxically inhibits their growth. Bipolar Androgen Therapy (BAT) was introduced several years ago at our institution in which patients with castration resistant prostate cancer (CRPC) are treated intermittently with high dose testosterone. Prior clinical trials have shown that BAT can produce biochemical and objective responses, and may re-sensitize prostate cancer to subsequent second generation AR inhibitors. To define the mechanism(s) by which BAT results in tumor regression, we performed a clinical trial (NCT03554317) for patients with CRPC that included biopsies of soft tissue metastases before BAT and after 3 cycles of BAT (C4D1). Up to four tissue cores were taken per biopsy with two cores for formalin fixation and paraffin embedding (FFPE) and the others snap frozen. Histologically viable tumor cells were present for both the pretreatment and C4D1 time points for 24 of the 42 enrolled patients. The FFPE samples were stained for H&amp;E and by IHC for AR, MYC and Ki67. MYC and Ki67 IHC stained slides were subjected to quantitative image analysis using HALO software (Indica Labs). Adequate frozen tumor tissue was available for laser capture microdissection and adequate amounts of RNA were obtained from 15 patients with paired pretreatment and C4D1 biopsies. RNAseq libraries were prepared using the NuGEN Ovation RNA-Seq System V2 and barcoded libraries were sequenced to an average depth of &amp;gt; 100 million reads per sample on an Illumina NovaSeq. Gene expression values were obtained with RSEM using Star aligner with GRCh38 (human). When comparing the C4D1 samples to the pretreatment samples, there were marked decreases in tumor MYC protein levels in approximately half of the treated patients, which was associated with decreased Ki67, decreased tumor volume, and prolonged progression-free survival on BAT. Using an image analysis computed H-SCORE for MYC, there was a strong correlation between MYC protein levels and MYC mRNA using RNAseq (r=0.81, p = 2.3 x 10-7). This indicates that MYC protein levels correlate with MYC mRNA and that reductions of MYC protein by BAT are likely related to reductions of MYC mRNA in clinical metastatic tissue samples. Citation Format: Carolina Gomes Alexandre, Tracy Jones, Jessica L. Hicks, John T. Isaacs, Anuj Gupta, Alyza Skaist, Laura Sena, Jennifer Meyers, Emmanuel Antonarakis, Mark Markowski, Samuel Denmeade, Srinivasan Yegnasubramanian, Angelo Michael De Marzo. Molecular pathology of metastatic prostatic adenocarcinoma treated with bipolar androgen therapy (BAT) reveals a correlation between MYC mRNA and protein [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 653.

Abstract LB217: Ascorbic acid rescues antitumor immune response by reversing hypoxia-induced CD8+T cell dysfunction

Abstract Growing evidence suggests that hypoxia is one of the most important phenomena of solid tumors that supports tumor progression as well as therapeutic resistance. When T cells infiltrate the tumor microenvironment (TME), they encounter hypoxia and, in this condition, cells upregulate an evolutionary conserved Hypoxia-inducible factor 1 (HIF-1) transcription factor to adapt to the hypoxic stress. Hypoxia has been reported to alter specific aspects of CD8+ T cell biology through various mechanisms but in large part by the HIF1-α, leading to immune suppression. Therefore, identification and development of drugs that target hypoxia is critical to augment immune cells' function and enhance the anti-tumor response of immunotherapy. Over the past decades, several cellular and pre-clinical studies have shown that ascorbic acid (AA) possesses anti-tumor properties and acts as an adjuvant to various cancer therapies. AA, as a proven redox potential modulator, has proved to also be effective in targeting oxygen sensing regulator (reducing Hif1-α levels) and epigenetic reprogramming (increasing ten-eleven translocation (TET) methylcytosine dioxygenase enzymatic activity) in cancer cells. Most recently, immunomodulating potential of AA has been recognized as it enhanced the efficacy of checkpoint blockade by increasing immune cell infiltration within the TME. However, the exact mechanisms by which AA exerts immunotherapeutic effects in cancer have not been fully understood. Here, we hypothesized that AA exerts its immunotherapeutic effects by targeting tumor-induced hypoxic stress in CD8 T cells. Indeed, we found that AA induces a robust anti-tumor response by decreasing tumor volume and increasing mice survival. Immune profiling in the TME revealed that AA increases the infiltration of CD8+ T cells with enhanced cytotoxic cytokine production and proliferation. To further evaluate the underlying mechanism, we challenged CD8+ T cells with a cognate antigen ex vivo under the hypoxic/normoxic conditions. We found that hypoxia negatively affected the effector functions and proliferation of CD8 T cells. Interestingly, AA could reverse these effects, bringing the function and proliferation of cells back to the normoxic level by increasing the expression of Granzyme B, Interferon gamma and Ki-67. This reversal of hypoxia-induced adverse effects on CD8 T cells was achieved by AA-mediated decrease in the Hif1-α levels (a hypoxia mediator) and increase in TET-2 protein expression and function (characterized by increased 5-hydroxymethylcytosine (5-hmc) levels). Moreover, AA treatment could significantly enhance the metabolic fitness of CD8+ T cells (characterized by improved glycolytic ability and mitochondrial function) that were exposed to severe hypoxia. In conclusion, our data indicate that AA treatment shows enhanced anti-tumor effects via restoring cytotoxic T cell functions under hypoxic TME. These results also provide new insights into the immunomodulatory effects of AA via targeting hypoxia in CD8 T cells. Given that hypoxia in the TME is a barrier for generation of effective immune response, our results suggest that AA treatment is an effective strategy against cancer and may further enhance the anti-tumor effects of immunotherapeutic agents. Citation Format: Nazli Jafarzadeh, Pankaj Gaur, Dareen Sarhan, Nour Shobaki, Wael Traboulsi, Zainab Ramlaoui, Vivek Verma, Seema Gupta, Samir N. Khleif. Ascorbic acid rescues antitumor immune response by reversing hypoxia-induced CD8+T cell dysfunction [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 LB217.

Abstract 4005: Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma

Abstract Background and Aims: Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative platin-based regimens, commonly cisplatin. Here, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in HB cell lines, organoids and murine models. Methods: Paired tumor and adjacent tissues from 31 resected HBs and a validation set of 50 HBs were analyzed at the transcriptomic, genomic and epigenomic level using RNAseq, SNP and methylation arrays, respectively. The main targetable driver in HB was identified by gene co-expression network analysis (GCN) and its overexpression was confirmed by qRT-PCR. The anti-tumor effect of driver inhibition with molecular therapies alone or in combination with cisplatin was assessed in cell lines, patient-derived HB organoids and in a HB xenograft murine model. Results: IGF2 overexpression (FC&amp;gt; 4 vs adjacent tissue) was identified as the top targetable HB driver (71%, 22/31). IGF2high tumors displayed progenitor cell features and were significantly enriched in molecular classes with aggressive phenotypes and CTNNB1 mutations, while IGF2low tumors were enriched in inflammatory and TGF-β signaling. IGF2high tumors correlated with shorter recurrence-free survival after resection (median 34 months vs not reached for IGF2low; p = 0.02). IGF2 overexpression correlated in most cases (86%) with fetal promoter hypomethylation (50%), 11p15.5 loss of heterozygosity (LOH, 57%) or overexpression of miR483 (55%). Xentuzumab (anti-IGF1/2 mAb) alone or combined with cisplatin reduced proliferation and clonogenic capacity in IGF2high cell lines. The combination of xentuzumab and cisplatin exhibited synergistic effects in terms of cell viability in organoids derived from IGF2high human HBs. In mice, this combination induced a significant decrease in the viable tumor volume and extended survival compared to cisplatin alone (n = 13-14 per arm, p = 0.04). Conclusion: IGF2 is an actionable driver in HB and its overexpression was associated with fetal promoter hypomethylation, LOH or miR483 overexpression. The combination of a mAb against IGF1/2 (xentuzumab) with cisplatin led to remarkable anti-tumoral effects in pre-clinical models, providing the rationale for exploring this regimen in IGF2high HB patients. Citation Format: Jordi Abril-Fornaguera, Laura Torrens, Juan Carrillo-Reixach, Alexander Rialdi, Ugne Balaseviciute, Carla Montironi, Philipp Haber, Álvaro Del Río-Álvarez, Montserrat Domingo-Sàbat, Laura Royo, Nicholas Akers, Catherine E. Willoughby, Judit Peix, Miguel Torres-Martin, Marc Puigvehi, Roser Pinyol, Stefano Cairo, Margaret Childs, Rudolf Maibach, Rita Alaggio, Piotr Czauderna, Bruce Morland, Bojan Losic, Vincenzo Mazzaferro, Ernesto Guccione, Daniela Sia, Carolina Armengol, Josep M. Llovet. Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma [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 4005.

Abstract 1815: AVA6000, a novel Precision medicine, targeted to the tumor microenvironment via Fibroblast Activation Protein (FAP) mediated cleavage

Abstract AVA6000 is a therapeutic product based on proprietary pre|CISION™ technology which incorporates a substrate that is sensitive to cleavage by FAP. The pre|CISION™ substrate can be utilized in a drug conjugate linker or to generate chemotherapy prodrugs that are only activated in the tumor microenvironment. AVA6000 consists of a doxorubicin molecule covalently bonded to a dipeptide (pyridine-4-carbonyl)-D-Ala-L-Pro), which is designed to be susceptible to hydrolysis by Fibroblast Activation Protein α (FAP) but is resistant to hydrolysis by both closely related and wider mammalian peptidases. FAP, a post-prolyl endopeptidase, is overexpressed on the surface of activated fibroblastic cells which are abundant in the supporting stroma of over 90% of malignant epithelial cancers, as well as in bone and soft tissue sarcoma. While FAP is also present both in normal tissues and as a soluble enzyme in plasma, levels are significantly lower than those present in malignant epithelial cancers. Consequently, AVA6000 has the potential to deliver doxorubicin directly to the tumor microenvironment, while exposing the patient to a lesser degree of doxorubicin-associated toxicities. The primary mechanism of action of doxorubicin is thought to involve stabilisation of a topoisomerase-II-DNA cleavable complex through non-specific DNA-intercalation. The non-specific DNA-intercalation causes a number of downstream effects, which may ultimately result in apoptotic cell death. Although doxorubicin has been one of the most effective and widely used chemotherapeutic agents for the treatment of various solid malignancies for over 40 years, its clinical utility is limited by dose-limiting toxicities, including myelosuppression and cardiotoxicity. The unique FAP specificity of the N-(pyridine-4-carbonyl)-D-Ala-L-Pro leaving group conjugated to doxorubicin in AVA6000 is supported by the absence of cleavage of the fluorogenic analogue, 3114-AMC, in FAP gene-knockout mice (Fap-/-). In vitro cytotoxicity assessments involving human tumor cell lines showed that AVA6000 was between 80-fold to 4,000-fold less cytotoxic compared to doxorubicin. In several in vivo efficacy studies in tumours with high FAP levels, AVA6000 significantly decreased tumor volume and increased survival in a dose-dependent manner. In a PDX model of osteosarcoma, AVA6000 significantly decreased tumor volume while doxorubicin had no significant effect. The efficacy and tolerability profile of AVA6000 strongly support its clinical development, and a Phase I trial in patients with locally advanced or metastatic selected solid tumours in underway. Citation Format: Fiona McLaughlin, Sarah E. Poplawski, David G. Sanford, Andrew Saunders, Jack H. Lai, Matthew Vincent, William W. Bachovchin, Neil Bell. AVA6000, a novel Precision medicine, targeted to the tumor microenvironment via Fibroblast Activation Protein (FAP) mediated cleavage [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 1815.

Abstract 2155: PMR-116, a novel inhibitor of ribosome biogenesis with antitumor activity in preclinical models of prostate cancer

Abstract Advanced prostate cancer is characterised by mutations and amplifications of genes involved in regulating protein synthesis. PTEN-loss stimulates activity of the mTOR pathway, while amplification of MYC leads to increased ribosome biogenesis and elevated mRNA translation rate. Our previous work has demonstrated the efficacy of co-targeting ribosome biogenesis, via inhibition of RNA Pol I activity, and 4E-BP1 phosphorylation to suppress prostate cancer growth in vivo in GEMM of PCa and in patient-derived xenografts (Rebello et al., Clinical Cancer Research, 2016; Lawrence et al., European Urology, 2018). In a collaboration with Pimera Inc., we investigated the efficacy of their new lead RNA Pol I inhibitor PMR-116 in models of prostate cancer. PMR-116 is well tolerated in vivo in mice and can be given at 300mg/kg weekly. Using the Hi-MYC mouse model of PCa we show that dosing 6 month old mice once weekly for 4 weeks can decrease the incidence of invasive lesions by up to 85% compared to vehicle control while reverting glands to patterns of low grade intraepithelial neoplasia. PMR-116 rapidly inhibits proliferation in the Hi-MYC model with a 50% decrease in Ki67 observed 12 hours after oral administration. Conversely, PMR-116 showed minimal anti-tumour efficacy in the PTEN-null model of PCa suggesting that elevated MYC signalling may be required for optimal response. To further validate our promising GEMM results in more clinically relevant human-derived models, we used patient-derived xenografts lines we established from multidrug-resistant, metastatic PCa (Lawrence et al, European Urology, 2018). PMR-116 treatment decreased tumour volume in all PDX tested including complete response in a line in which tumour volume decreased by ~90% compared to baseline. We believe this new RNA Pol I inhibitor shows promising results in a wide range of preclinical models and may exert higher efficacy in tumours expressing high levels of MYC. PMR-116 is currently in Phase I dose escalation trial in patient with solid tumours (ACTRN12620001146987). Citation Format: Alisee Huglo, Richard Rebello, Mitchell Lawrence, Gail Risbridger, Denis Drygin, Mustapha Haddach, Katherine Hannan, Ross Hannan, Luc Furic. PMR-116, a novel inhibitor of ribosome biogenesis with antitumor activity in preclinical models of prostate cancer [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 2155.

Abstract 2566: Pre-clinical efficacy of the Wnt pathway inhibitor RXC004 in combination with anti-cancer therapies

Abstract Background: RXC004, a potent and selective inhibitor of the Wnt pathway regulator Porcupine, is being investigated in phase 2 studies in patients with advanced cancers (NCT04907851 and NCT04907539). Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Frizzled (Fzd) on the cell surface increasing Wnt-ligand dependent signaling. These alterations, which have been shown to be sensitive to Porcupine inhibitors, are implicated in colorectal, gastric and pancreatic cancer. Pre-clinical in vivo data on the effects of RXC004 in combination with clinically relevant anti-cancer therapies have not previously been described in genetically-defined models of cancer. Methods: RXC004 was evaluated as a monotherapy, and in combination with clinically relevant chemotherapy regimens, in an RSPO-fusion colorectal xenograft model (SNU-1411). RXC004, dosed at 5mg/kg QD (5 days on/2 days off), was evaluated in combination with either the clinically relevant colorectal triplet chemotherapy regimen (5-fluorouracil 5-FU (25mg/kg), irinotecan (50mg/kg) and oxaliplatin (5mg/kg)) or doublet chemotherapy regimen (5-FU (25mg/kg), irinotecan (50mg/kg)). Each chemotherapy regimen was dosed once a week for 3 to 4 weeks either alone or in combination with RXC004, which was dosed from day 8 onwards at 5mg/kg QD (5 days on/2 days off). Efficacy was measured by tumor volume and survival endpoint. In addition, RXC004 was studied in vitro, as monotherapy and in combination with a PARP inhibitor in genetically selected cell lines with both RSPO-fusion and RNF43 mutation. Combination and monotherapy effects on proliferation and effects on downstream signaling were investigated. Results: RXC004 in combination with the triplet chemotherapy regimen led to significantly increased survival compared to either treatment alone (p&amp;lt;0.005) and significant tumor volume reduction measured at Day 22 (p&amp;lt;0.001). RXC004 in combination with the doublet chemotherapy also increased survival compared to either treatment alone (p&amp;lt;0.05), and the combination led to significantly decreased tumor volume measured at Day 25 (p&amp;lt;0.05). RXC004 treatment in genetically defined cell lines with RSPO fusions or RNF43 mutations led to a significant reduction in BRCA1 and BRCA2 gene expression. In in vitro proliferation studies a synergistic effect of RXC004 and a PARP inhibitor was demonstrated in genetically defined cell lines. Conclusion: These data demonstrate that RXC004 in combination with clinically relevant standard of care chemotherapy regimens can lead to potential benefit over chemotherapy alone. The RXC004 induced downregulation of DNA repair pathway genes observed in vitro could contribute to the beneficial effect seen in combination with a PARP inhibitor in vitro as well as the combination effect observed in vivo with standard of care chemotherapy agents. Citation Format: James Kelly, Simon Woodcock, Inder Bhamra, Cliff Jones, Richard Armer, Jane Robertson, Caroline Phillips. Pre-clinical efficacy of the Wnt pathway inhibitor RXC004 in combination with anti-cancer therapies [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 2566.

Abstract 3316: Tumor Treating Fields in combination with radiation cause significant delay in tumor growth in in-vivo mice modelsignificant delay in tumor growth in in-vivo mice model

Abstract New physical cancer treatment modality called Tumor Treating Fields (TTFields) deliver low-intensity, intermediate frequency, alternating electric fields non-invasively to the tumor. TTFields is believed to inhibit mitosis as its primary mechanism of action. We previously showed that TTFields exposure decreased the expression of FANC/BRCA1 pathway genes, which play an essential role in DNA double strand break (DSB) repair. As a result, DNA DSB repair under IR exposure is downregulated, and most importantly, the TTFields alone increased gH2AX foci and chromatid aberrations as a function of TTFields time exposure. The length of newly replicated DNA decreased and R-loop formation increased after TTFields treatment, suggesting replication stress is induced by TTFields. Our research revealed that TTFields are involved in DNA damage and replication stress pathways besides mitosis, thus, we predicted that, by applying TTFields first, a conditional vulnerability environment would be created, rendering cells more susceptible to agents like radiation and chemotherapy. In accordance with our hypothesis, radiation susceptibility increased when cells were exposed to TTFields prior to IR treatment compared to IR treatment followed by TTFields. The combination of TTFields together with Radiation has been tested in-vivo with the newly designed Inovivo system. To measure tumor growth delay caused by TTFields treatment, three distinct syngenic tumor mouse models were utilized, namely LLC (Murine Lewis Lung Carcinoma), KPC63 (Pancreatic Cancer) and MC38 (Colon Cancer). Although TTField exposure decreased tumor volume in the three mouse models tested, there was no statistically significant difference in tumor growth between the heat and TTField groups. In IR combination experiments, the MC38 mouse model was chosen since TTFields induced a greater TGD effect in this model. In the first Inovivo experiment, we combined TTFields and radiation therapy, which delayed tumor growth significantly. Following that, we tested the combined efficacy of two rounds of TTFields and radiation treatment. The tumor growth delay was more pronounced after two rounds of TTFields treatment compared to one, which confirms the hypothesis that TTFields treatment induces conditional vulnerability. The results are consistent with observations made by the researchers in the clinic that high compliance patients (who have received more exposure to TTFields) have better prognosis compared to low compliance patients. Our inovivo experiment results not only confirm our earlier in vitro results, but also support the use of TTFields in IR combination therapies. Citation Format: Narasimha Kumar Karanam, Zengfu Shang, Michael D. Story, Debabrata Saha. Tumor Treating Fields in combination with radiation cause significant delay in tumor growth in in-vivo mice modelsignificant delay in tumor growth in in-vivo mice model [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 3316.