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.

Abstract Introduction: Androgen Deprivation Therapy (ADT) is a highly effective treatment for prostate cancer. However, resistance to ADT develops in most patients, leading to a lethal state of castration-resistant prostate cancer (CRPC). Senescent prostate cancer (SPC) cells can arise after ADT and contribute to therapy resistance. SPCs can restore replicative function and alter the tumor microenvironment through pro-tumorigenic senescence-associated secretory phenotype (SASP). We aimed to characterize changes in SASP in prostate cancer cell lines and serum samples from patients receiving ADT. Methods: SASP factors (Luminex 100/200 System, xMAP Instruments) were analyzed in patients undergoing ADT. To correlate SASP with the sensitivity to ADT, we sought to evaluate the regulation of SASP in vitro using PCa cell lines. The PC3 (AR resistant) and LNCaP (AR sensitive) cell lines were used in the experiments. We performed an analysis of archival tumor tissue (paired) from 18 patients with or without alterations in DNA repair genes (BRCA1, BRCA2, and CDK12). We performed immunohistochemical (IHC) staining of known SASP markers, such as P16, uPAR, gamma-H2AX, and Lamin B1. Results: In a cohort of patients (n=8) with pre and post-ADT matched status, we demonstrate increased levels of SASP factors such as IL-6 (pre-ADT: 1.54±0.24 pg/ml, post-ADT: 5.23±1.3 pg/ml p=0.001), IL-7 (pre-ADT: 3.11±0.6 pg/ml, post-ADT: 13.5±2.7 pg/ml p=0.01), IL-1 alpha (pre-ADT: 1.06±0.3 pg/ml, post-ADT: 15.3±3.5 pg/ml p=0.001), IL-15 (pre-ADT: 15.6±3.1 pg/ml, post-ADT: 37.1±4.8 pg/ml p=0.002), MIF (pre-ADT: 1036±67.6pg/ml, post-ADT: 2362±55.5 pg/ml p=0.003), and IL-15 (pre-ADT: 5±2.1 pg/ml, post-ADT: 37.5±5.1 pg/ml p=0.002). In vitro, the AR-responsive LNCaP cell line, exposed to AR blockade, showed similar findings in upregulating IL-6, IL-7, and IL-15 but not the PC3 cell line. We also demonstrate measurable differences between the expression of u-PAR, gamma-2AX, p16, and loss of Lamin B1 expression in tumor specimens from patients with advanced castration-resistant prostate cancer (n=18). The expression of p16 was significantly upregulated post-treatment irrespective of DDR status (p=0.0417). Other SASP factors showed a trend in upregulation but were insignificant (u-PAR p=0.056, gamma-H2AX p=0.62, Lamin B1 p=0.72) Conclusion: These results provide evidence of systemic increases in SASP-related cytokines following treatment with ADT. The in vitro results suggest that secretion of SASP is promoted by an AR-mediated mechanism. Preliminary results from tumor specimens suggest increased senescence marker p16 expression induced by therapy. Ongoing experiments are further characterizing the senescence markers in prostate tumor specimens and correlated to treatment effect. Citation Format: Maximilian Schwermann, Matthew Hadfield, Shaolei Lu, Praveen Srinivasan, Vida Tajiknia, William MacDonald, Andrew George, Shengliang Zhang, Leiqing Zhang, Kimberly Meza, Andre De Souza, Dragan Golijanin, Elias Hyams, Galina Lagos, Sheldon Holder, Anthony Mega, John Sedivy, Howard Safran, Wafik El-Deiry, Benedito Carneiro. Androgen deprivation therapy (ADT) and senescence-associated secretory phenotype (SASP) in vitro: Correlation with SASP in tumor specimens as well as in the serum of patients after ADT [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 2954.

Abstract Glioblastoma multiforme (GBM) is the most common form of primary malignant brain tumor in adults. It is also the most aggressive and lethal. Standard of care therapy comprises maximal safe surgical resection followed by adjuvant alkylating agent temozolomide (TMZ) and radiotherapy (RT). There have only been five drugs and one device ever approved by the FDA for the treatment of GBM. The five-year survival rate for GBM patients has shown no notable improvement in the last three decades. We previously reported that the first-in-class imipridone small-molecule Dordaviprone (ONC201) decreases protein chaperone ClpX to unleash mitochondrial protease ClpP activity, integrated stress response and cell death. Preclinical combination of ONC201 (100 mg/kg weekly) with radiotherapy and Temozolomide in a GBM mouse orthotopic model results in reduced tumor burden and prolonged survival. The second generation imipridone ONC206 also activates integrated stress response and decreases ClpX in GBM cells but ONC206 is approximately 20 times more potent than its parent imipridone ONC201. ONC206 0.08 uM in combination with 12.5 uM TMZ and 2 Gy RT reduced the cell viability of GBM cells significantly compared to all single treatments and double treatments. During a short-term treatment of a GBM mouse orthotopic model, ONC206 (50 mg/kg weekly, half of ONC201 doses) synergizes with TMZ and RT to induce more tumor cell apoptosis and inhibits more tumor cell proliferation compared to other groups with single or double treatments. There are two ongoing clinical trials with ONC206: single weekly or multiple-day weekly dose regimens of single-agent, oral ONC206 in patients with recurrent, primary central nervous system (CNS) neoplasms (NCT04541082) and ONC206 alone or in combination with radiation therapy in treating patients with diffuse midline gliomas that is newly diagnosed or has come back (recurrent) or other recurrent primary malignant CNS tumors (NCT04732065). Future studies evaluating the role of immune function, mitochondrial metabolism, dopamine receptors, the ISR and TRAIL pathway in the synergistic effect would support further development of the triple combination regimen of ONC206, TMZ and radiation therapy for GBM clinical trial. Citation Format: Lanlan Zhou, Leiqing Zhang, Jun Zhang, Shengliang Zhang, Wafik S. El-Deiry. Preclinical combination of ONC206 with radiotherapy and temozolomide in a GBM mouse orthotopic model [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 3331.

e16331 Background: Elraglusib (9-ING-41), a potent and selective GSK-3 inhibitor, is a targeted anticancer agent with clinical activity in phase 1 trial and undergoing clinical development for the treatment of metastatic pancreatic ductal adenocarcinoma (mPDAC). Elraglusib (9-ING-41) displayed immune-modulatory activity in preclinical models and modulation of serum cytokine profile in clinical specimens. Here, we investigated the correlation between plasma levels of cytokines/chemokines/soluble cell receptors/growth factors (CCSG) and clinical outcomes of patients with mPDAC treated with elraglusib and gemcitabine/nab-paclitaxel (GnP) in an exploratory study. Methods: CCSGs were evaluated in the pre-dose plasma samples of patients with newly diagnosed mPDAC enrolled in the 1801 phase 2 study (NCT03678883) treated with GnP (n = 13) or elraglusib/GnP (n = 32). Using a Luminex immunoassay, 40 CCSGs were screened as predictive biomarkers of median overall survival (mOS) in the elraglusib/GnP arm using 5-fold cross-validation and maximally selected rank statistic cutpoint determination. A subset of CCSGs (n = 10) was remeasured using an MSD immunoassay with greater sensitivity. Maximally selected rank statistic cutpoint determinations were conducted using MSD results for elraglusib/GnP samples with mOS and median progression free survival (mPFS). P values < 0.05 were considered statistically significant. Results: Luminex screening revealed several CCSGs significantly stratified elraglusib/GnP patients by mOS. IL-6 and IL-8 were identified as top candidates with stable and significant results on cross-validation. Using the MSD assay, both were confirmed to stratify elraglusib/GnP patients by OS and extended to analyze PFS. Low level of IL-8 conferred a better OS prognosis (HR: 0.0919, 95%, CI: 0.0205-0.411, P < 0.001) than low IL-6 (HR: 0.205, 95%, CI: 0.063-0.665, P = 0.005) in the elraglusib/GnP treated patients. Correlations with mPFS were similar in the elraglusib/GnP patients for IL-6 (HR: 0.248, 95% CI: 0.072-0.848, P = 0.02) and IL-8 (HR: 0.297, 95%, CI: 0.111-0.743, P = 0.01). Conclusions: Preliminary results of our exploratory study identified low plasma levels of IL-6 and IL-8 prior to elraglusib/GnP treatment as potential predictive biomarkers associated with improved outcomes in previously untreated patients with mPDAC. The 1801 Part 3 Arm B clinical trial recruitment has been completed. We plan to analyze additional pre-dose plasma samples obtained from approximately 200 patients by April 2024. These will be analyzed using interim OS data available by April 2024 and included in the final presentation. Clinical trial information: NCT03678883 .

Androgen receptor (AR) signaling is a target in prostate cancer therapy and can be treated with non-steroidal anti-androgens (NSAA) including enzalutamide, and apalutamide for patients with advanced disease. Metastatic castration-resistant prostate cancer (mCPRC) develop resistance becomes refractory to therapy limiting patient overall survival. Darolutamide is a novel next-generation androgen receptor-signaling inhibitor that is FDA approved for non-metastatic castration resistant prostate cancer (nmCRPC). Imipridone ONC201/TIC10 is first-in-class small molecule imipridone that activates the integrated stress response (ISR), upregulates TNF-related apoptosis-inducing ligand (TRAIL) and has activity against CRPC alone or in combination with enzalutamide in preclinical models. We hypothesized that combination of imipridones with androgen receptor signaling blockers such as darolutamide may synergize in anti-tumor efficacy against mCRPC cells. mCRPC cell lines 22RV1, LNCaP, DU145 and PC3 were treated with imipridones ONC201, ONC206, apalutamide, darolutamide, or enzalutamide as single agents or in combinations. Combinations of ONC201 or ONC206 and androgen receptor signaling blockers demonstrated synergistic effects in mCRPC cells. Combinations of ONC201 and darolutamide or enzalutamide reduced PSA levels in LNCaP cells and induced of ATF4 in both LNCaP and 22RV1 cell lines. Darolutamide synergized with ONC201 regardless of AR status or castration sensitivity in vitro. Flow cytometric analysis showed increased intra-tumoral NK cells in mice treated with ONC201 and combination of ONC201 and darolutamide. Trends of increased TRAIL activation within NK cells were also observed in treatment groups. ONC201 and darolutamide demonstrated anti-tumor effects in vivo in the 22RV1 CRPC model. Our results prompt further translational and clinical studies with imipridones ONC201 or ONC206 in combination with enzalutamide or darolutamide for treatment of castrate resistant advanced or metastatic prostate cancer.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. ONC212 is a second-generation imipridone with antitumor effects in human PDAC cell lines. ONC212 has been shown to bind to mitochondrial protease ClpP, suppress ClpX, and impair oxidative phosphorylation by decreasing ATP production. While ONC212 has been evaluated in immunocompromised mice xenografted with human pancreatic tumors, ONC212 has not been evaluated, either alone or in combination with MEK and/or immune checkpoint inhibition (ICI), in immunocompetent mice bearing notoriously aggressive KrasLSL.G12D/+; Tp53LSL.R172H/+; Pdx1Cretg/+ (KPC) murine PDAC tumors. We hypothesized that, like human PDAC cells, KPC cells would demonstrate sensitivity to ONC212 both in vitro and in vivo. Our group has previously shown that ONC212 synergizes with trametinib to induce tumor cell death in human PDAC cells. We therefore hypothesized that this combination, together with ICI, would enhance KPC tumor cell death in vivo. Methods: We determined in vitro sensitivity of the KPC cells to ONC212 alone and in combination with trametinib using CellTiter-Glo® luminescent cell viability assays. Results were analyzed after 72 hours of incubation using Compusyn and Combenefit. In vivo experiments involved C57BL/6 mice that were injected subcutaneously with 3 × 105 KPCy cells in 100 mL of PBS/matrigel. To determine the ideal ONC212 dose, we tested five different doses/dosing frequencies (50 mg/kg; 25 mg/kg; 12.5 mg/kg given by oral gavage weekly or twice weekly) in tumor-bearing (50-75 mm3) mice. Mouse weight and tumor size was measured every four days. Treatment was stopped once tumor volumes reached 3,000 mm3 or if ulceration occurred. Once the ideal dose of ONC212 was determined, a study treating KPC tumor-bearing C57BL/6 mice with ONC212, trametinib, and ICI (anti-PD-1 mAb) alone and all possible doublet/triplet combinations was performed using the same methods. Results: We found that the combination of ONC212 and trametinib exhibited synergy in the KPC cell line in vitro. Our in vivo experiments revealed that ONC212 controlled KPC tumor growth in a dose-dependent manner, however, toxicity was also noted at higher, more frequent doses. While both 25 mg/kg and 50 mg/kg twice weekly were equally effective, 25 mg/kg was better tolerated and determined to be the ideal dose. In the combination therapy study, all treatments resulted in tumor reduction, as compared to the vehicle control; however, the triple therapy group had the lowest average tumor size at day twenty. Toxicity was noted in mice receiving at least two treatments, reflected by reduced weights and mobility. Further analysis of the tumor immune microenvironment using multiplex cytokine and immunofluorescence are ongoing. Citation Format: Jasper Chan, Alexis J. Lannigan, Grace Sun, Varun V. Prabhu, Robert Edwards, Leiqing Zhang, Lanlan Zhou, Wafik S. El-Deiry, Alexander Grenander Raufi. The imipridone ONC212 cooperates with MEK and immune checkpoint inhibition to elicit in vivo regression of KPC mouse pancreatic tumors [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 2078.

Abstract Metastatic colorectal cancer (mCRC) is a deadly disease with a five-year survival rate of 14%. Most patients receive 5-FU (F) and folinic acid (FOL) combined with oxaliplatin (OX), irinotecan (IRI), or both (OXIRI). Immune checkpoint inhibitors (ICIs) are effective against microsatellite instable (MSI) tumors, but 95% of mCRC tumors are microsatellite stable (MSS) and ICI-resistant. Though the primary targets of 5-FU, irinotecan, and oxaliplatin are well-understood, their effects on the tumor microenvironment (TME) remain incompletely characterized. Chemotherapy-mediated immune stimulation in MSS CRC has been observed in preclinical models and clinical trials. We hypothesize that combination chemotherapy treatment modulates the immune microenvironment of CRC -/+ ICI, with differences across subtype and treatment regimen. Chemotherapy-dependent changes in cancer cell gene expression, PD-L1 levels, cytokine secretion, and T cell activation were measured using in vitro models of MSS and MSI CRC. A preliminary evaluation of spleen and tumor T cells and dendritic cells (DCs) in murine models of MSS and MSI CRC after immunotherapy -/+ chemotherapy was conducted. CD8+ T cells, DCs, PD-L1, CD69, and GM-CSF expression were measured in clinical specimens of chemotherapy-treated MSS mCRC patients. In MSS CRC, FOX increased GM-CSF and activated CD8+ T cells in vitro and activated splenic CD8+ and CD4+ T cells in vivo. FOX and FIRI suppressed an anti-PD-1-mediated enhancement of type 1 cDCs in the tumor in vivo. FOLFOX was associated with increased tumor CD8+ T cells and decreased GM-CSF in MSS mCRC biopsies. FIRI treatment increased PD-L1 in vitro. FOLFIRI was not associated with increased tumor CD8+ T cells in MSS mCRC biopsies. In MSI CRC, FOX increased tumor CD8+ T cells in vivo. Here, we elucidate novel effects of clinically relevant chemotherapy combinations on the TME in MSS and MSI CRC. These data point to a FOX-specific mechanism by which CD8+ T cell infiltration into MSS mCRC tumors is enhanced, but their activation is halted potentially by GM-CSF suppression and/or type 1 cDC depletion in the TME. These findings contribute to our understanding of the mechanisms of chemotherapy-dependent immune modulation and bring the field closer to harnessing these effects for therapeutic gain. Citation Format: Lindsey Carlsen, Maximilian Pinho-Schwermann, Leiqing Zhang, Andrew Elliott, Kelsey E. Huntington, William J. MacDonald, Brooke Verschleiser, Laura Jinxuan Wu, Wafik S. El-Deiry. Modulation of the MSS and MSI colorectal cancer immune microenvironment with FOLFOX and FOLFIRI -/+ anti-PD-1 immunotherapy [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 1189.

Abstract Cytokines mediate strong immunomodulatory effects in cancer. Elraglusib (9-ING-41), an inhibitor of GSK-3, is a potent antitumor and immune-modulatory agent in phase 2 clinical trials for the treatment of various cancers. Here, we examined the immune environment of elraglusib-treated cancer patients by measuring peripheral blood protein levels to explore whether the expression level of cytokines/chemokines/soluble cell receptors/growth factors (CCSG) is correlated with clinical outcome. Using a customized Luminex platform, the expression of forty CCSG hypothesized to be linked to GSK-3 activity was assessed in pre-dose plasma samples obtained from 45 cancer patients with relapsed/refractory metastatic disease (NCT03678883; the 1801 trial). All patients in the 1801 trial were treated with elraglusib (dose range: 1-15 mg/kg) either as a single agent (Part 1; n=21) or in combination with chemotherapy (Part 2; n=24). Pre-dose peripheral blood protein levels were examined as binary predictors of clinical outcome using optimal cutoff points determined by maximally selected rank statistics. In the aggregate Part 1 and 2 populations (n=45), we identified 13 CCSG that significantly stratified the cohort by overall survival (OS). High pre-dose levels of CD95 and TNFRSF10C were associated with better OS. CD95 was a better prognostic of OS than TNFRSF10C (HR: 2.27, 95% CI: 1.10-4.67, p-value: 0.023). Low pre-dose levels of 11 CCSG were also significantly associated with favorable OS, with IL-8 being the most predictive (HR: 0.137, 95% CI: 0.0545-0.346, p-value: <0.0001). Part 1 and 2 predose samples were also analyzed separately, and many of the biomarkers identified in the aggregate analysis retained significance in the separated analysis. In Part 1, CXCL5, IFN-alpha, and IL-18 emerged as unique markers, and in Part 2, CCL22 was the single unique marker.The results of our exploratory study identified several putative biomarkers of elraglusib clinical benefit and demonstrated potential immunomodulatory mechanisms of elraglusib that will be used to inform the further clinical development of elraglusib for the treatment of metastatic cancer. Citation Format: Taylor Weiskittel, Kelsey Huntington, Leiqing Zhang, Austin Koukol, Benedito A. Carneiro, Hu Li, Andrey Ugolkov, Wafik El-Deiry, Andrew Mazar. Identification of potential immune biomarkers for GSK-3 inhibitor elraglusib (9-ING-41) in patients with relapsed/refractory metastatic cancer [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 6426.

Abstract PARP inhibitors (PARPi) targeting poly (ADP-ribose) polymerase are the first clinically approved drugs designed to apply synthetic lethality in BRCA1 mutant/deficient cancer. However, the treatment can cause reversion of BRCA mutation which results in drug resistance. To explore new strategies to improve antitumor efficacy of PARPi, we applied PARPi in combination with a small molecule NSC59984 for cancer therapy. NSC59984 is a small molecular targeting mutant p53 degradation and activating p73 via ROS-ERK2-MDM2 pathway. The treatments with radiation or hydrogen peroxide showed that NSC59984 enhanced DNA comet tails and gamma-H2AX, correlated to reduction of rad51 foci in cancer cells. These results suggest that NSC59984 impairs DNA damage repair via abrogation of homologous recombination (HR). We further applied the combinational treatment of NSC59984 and PARPi in BRCA1 wild-type ovarian cancer cells and found that NSC59984 synergized with PARPi to reduce cell viability, inhibit colony formation, and increase cell death. The combination treatment enhanced DNA damage and correlated with reduction of BRCA1 at the protein level. These results, taken together, suggest that the induction of BRCAness causes a synthetical lethality with PARPi in BRCA1 wild-type cancer cells. Cell cycle profiling showed that the cells were arrested at the G2/M phase in response to combinational treatment. At the G2 phase arrest, the high doses of the combinational drugs led to cell death via mitotic catastrophe, and the intermediate doses induced cellular senescence defined by senescence phenotypic hallmarks including senescence-associated beta-gal staining and a secretome consistent with senescence-associated secretory phenotype (SASP). Therapy-induced senescence (TIS) in cancer cells is considered as one of the mechanisms of tumor recurrence and drug resistance. To reduce the risk of TIS in treatment, we applied senolytic treatment to target senescent cells in the combinational treatment. The senolytic drug ABT263 treatment eliminated the senescent cancer cells from the combinational treatment. ABT263, NSC59984 + PARPi combinational treatment further reduced cell viability. Our study provides a rationale for small molecular compounds targeting HR deficiency in combination with PARPi to treat BRCA1wild-type ovarian cancer cells, and additional senolytic treatment may be required to limit resistance by removal of the TIS. Citation Format: Shengliang Zhang, Lanlan Zhou, Leiqing Zhang, Maximilian Pinho-Schwermann, Benedito Carneiro, John Sedivy, Wafik S. El-Deiry. Small molecule NSC59984 synergizes with PARP inhibitors in BRCA1 wild type ovarian cancer [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 5941.

Glioblastoma remains the most lethal common primary brain tumor in adults with limited therapeutic options. TIC10/ONC201, a first-in-class imipridone we discovered, achieved meaningful therapeutic effects in phase I/II trials in patients with diffuse gliomas harboring H3K27M mutations, and currently the drug is in randomized phase III testing (ACTION trial; [NCT05580562][1]). ONC201 targets mitochondrial protease ClpP to disrupt oxidative phosphorylation and trigger the integrated stress response (ISR), TRAIL/DR5, and tumor cell death. We hypothesized that ONC201 and its analogue ONC206 synergize with temozolomide (TMZ) and ionizing radiation (IR), standard-of-care glioblastoma therapies. ONC201 enhances TMZ or IR-induced apoptosis, and cytotoxicity. ClpP-silencing suppresses ONC201-induced cytotoxicity but not TMZ or RT. Both ONC201 and ONC206 reduce expression of TMZ-resistance mediator MGMT. Suppression of MGMT protein was observed in H3K27M-mutated DIPG cell lines following treatment with ONC201 or ONC206 with or without TMZ. Cytokine profiling indicates distinct ONC201 alterations relative to TMZ suggesting distinct anti-tumor immune mechanisms. Triple IR+TMZ+ONC201 (ITR) therapy prolongs median survival to 123 days with a tail on survival curve (3-of-7 mice alive beyond 200-days) in an orthotopic U251 GBM model versus ONC201 (44-days; p=0.000197), IR (63-days; p=0.0012), TMZ (78-days; p=0.0354), ONC201+IR (55-days; p=0.0004), ONC201+TMZ (80-days; p=0.0041) and IR+TMZ (103-days; p>0.05). By 231-days, the only surviving mice were in IRT group. Our results support investigation of ONC201/ONC206 in combination with TMZ and IR (ITR) in GBM or H3K27M mutated diffuse glioma therapy. ### Competing Interest Statement W.S.E-D. is a co-founder of Oncoceutics, Inc., a subsidiary of Chimerix. Dr. El-Deiry has disclosed his relationship with Oncoceutics/Chimerix and potential conflict of interest to his academic institution/employer and is fully compliant with NIH and institutional policy that is managing this potential conflict of interest. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT05580562&atom=%2Fbiorxiv%2Fearly%2F2024%2F09%2F27%2F2024.09.25.610187.atom

Abstract Radiation pneumonitis is one of the most common but challenging toxicities seen in patients that are receiving ionizing radiotherapy for thoracic malignancies. While radiation effectively eliminates cancer cells, the damage it causes to surrounding healthy lung tissue leads to an immune response that can impact treatment regimen and lead to long term fibrotic tissue formation. The mechanism of radiation pneumonitis and the inflammatory response in the lungs is poorly understood. However, it is known that the TRAIL pathway plays an important role in inflammatory and fibrotic response. Through our previous work, it was discovered that modulating the TRAIL pathway via pegylated recombinant long-acting TRAIL (TLY012) in both WT and TRAIL-/- C57Bl/6 could suppress radiation pneumonitis in mice that were exposed to 20 Gy of thoracic radiation with shielding of other organs as early two weeks post exposure. In addition to the TRAIL pathway, transforming growth factor-beta (TGF-β) is a cytokine that is known to play a key role in fibrosis. Broad spectrum integrin inhibitor GLPG0187 is known to inhibit the activation of TGF-β via binding to specific integrin receptors. In order to determine if the inhibition of TGF-β could suppress radiation pneumonitis without stimulation of the TRAIL pathway, DR5 null mice were utilized for the experiment. Male DR5-/- C57Bl/6 mice received a single 20 Gy thoracic radiation dose with shielding of other organs and were treated with 100 mg/kg of GLPG-0187 or control twice a week via IP injection with the first dose being administered 1 hour before radiation (n = 4/treatment/group). 13 days post-irradiation, lungs, sternal bone marrow, and peripheral serum were collected. Upon histological analysis, it was observed that there were thinner alveolar borders and lessened inflammation compared to the control mice. Mice were weighed every three days, and it was discovered that mice treated with GLPG-0187 maintained steady weight compared to the control group. Additional analysis including immunohistochemistry, cytokine profiling, and quantification needs to be conducted. As the TRAIL pathway could not be modulated in the DR5 null mice, these findings suggest that GLPG-0187 was able to suppress radiation pneumonitis via inhibition of TGF-β. While some rescue from radiation pneumonitis was observed via GLPG0187, it was to a lesser extent compared to treatment of WT and TRAIL-/- C57Bl/6 mice with TLY012. Future directions include investigating the synergistic effects between TLY012 and GLPG0187 in suppressing radiation pneumonitis and decreasing fibrotic response to radiation. Citation Format: Jillian Strandberg, Anna Louie, William MacDonald, Praveen Srinivasan, Leiqing Zhang, Lanlan Zhou, Seulki Lee, Wafik El-Deiry. Post-exposure suppression of radiation pneumonitis suggests non-redundant independent effects of TGF-beta and TRAIL/DR5 [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 710.

Abstract Pancreatic cancer is a devastating disease with an overall five-year survival rate of ~12% according to the American Cancer Society. Novel chemotherapeutics and targeted therapies in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin (PM01183; LY-01017) received accelerated FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. Lurbinectedin is a chemotherapeutic synthetic tetrahydroisoquinoline alkaloid that inhibits active transcription by binding to guanine rich sequences in the minor groove of DNA. Lurbinectedin has been shown to reduce oncogenic transcription by stalling and degrading RNA Polymerase II and inducing breaks in DNA, causing subsequent apoptosis. We now demonstrate lurbinectedin’s highly efficient killing of pancreatic tumor cells as a single agent in PANC-1, BxPC-3, and HPAF-II cell lines, with 72-hour IC-50s ranging from 0.266 – 0.964 nM. We further demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in synergistic killing of pancreatic tumor cells in vitro. Synergism was calculated using SynergyFinder, with HSA scores exceeding 15 in every cell line (synergy scores greater than 10 are considered synergistic according to the website manufacturer). Western Blot analysis of combination therapy indicates an upregulation of γH2AX and ATR, indicating DNA damage likely plays a role in the synergistic effects of the drugs. Bcl-2 downregulation in combination therapy indicates that the Bcl-2 family proteins likely play a role in the apoptosis of the cancer cells. Cytokine profiling analysis indicates an upregulation of the Fas apoptosis-inducing ligand under lurbinectedin and irinotecan monotherapy and combination therapy. VEGF and prolactin were downregulated in combination therapy. We further demonstrate that the triple combination between lurbinectedin, irinotecan, and 5-Fluorouracil (5-FU) enhances % inhibition as each drug concentration increases, and the presence of lurbinectedin enhances the synergism between irinotecan and 5-FU. Our results are developing insights regarding molecular mechanisms underlying therapeutic efficacy of a novel combination drug treatment for pancreatic cancer. Citation Format: Tej Tummala, Arielle De La Cruz, Ash Uruchurtu, Nicholas R. Liguori, Abbas E. Abbas, Leiqing Zhang, Chistopher G. Azzoli, Lanlan Zhou, Wafik S. El-Deiry. Preclinical synergistic combination therapy of lurbinectedin with irinotecan and 5-fluorouracil in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C088.

KRAS mutations occur in ∼40-50% of mCRC and are associated with aggressive disease that is refractory to anti-EGFR therapies. Pancreatic cancer harbors ∼90% KRAS driver gene mutation frequency. Small molecules targeting KRAS G12C gained FDA approval for KRAS G12C-mutated NSCLC. ONC212, a fluorinated imipridone with nM anti-cancer activity has preclinical efficacy against pancreatic cancer and other malignancies. MRTX1133, identified as a noncovalent selective KRAS G12D inhibitor that suppresses G12D signaling by binding to the switch II pocket thereby inhibiting protein-protein interactions. We investigated cell viability, drug synergies, pERK suppression and cytokine, chemokine or growth factor alterations following treatment with 5-Fluorouracil (5-FU) or ONC212 plus MRTX1133 in 6 human CRC and 4 human pancreatic cancer cell lines. IC50 sensitivities ranged from 7 to 12 μM for 5-FU, 0.2-0.8 μM for ONC212, and >100 nM to >5,000 nM for MRTX1133 (G12D N=4: LS513 >100, HPAF-II >1,000, SNUC2B >5,000, PANC-1 >5,000). For non-G12D, the range of IC50 for MRTX1133 was >1,000 to >5,000 nM for CRC lines with G12V, G13D, or WT KRAS (N=7). Synergies between MRTX1133 plus 5-FU or ONC212 were observed regardless of KRAS G12D mutation with combination indices of <0.5 indicating strong synergy. Observed synergies were greater with MRTX1133 plus ONC212 compared to MRTX1133 plus 5-FU. pERK was suppressed with mutant but not wild-type KRAS at nM MRTX1133 doses. Immunostimulatory profiles included reduction in IL8/CXCL8, MICA, Angiopoietin 2, VEGF and TNF-alpha and increase in IL-18/IL-1F4 with MRTX treatments and combinations. Our studies reveal preclinical activity of MRTX1133 alone or synergies when combined with 5-FU or ONC212 against mCRC and pancreatic cancer cells regardless of KRAS G12D mutation. The results suggest that KRAS G12V and KRAS G13D should be further considered in clinical trials including combination therapies involving MRTX1133 and 5-FU or ONC212.

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer death among men in the United States. There is an estimated 10% to 50% of cases progress to metastatic castration resistant prostate cancer (mCRPC) state within 3 years of diagnosis. mCPRC remains lethal despite therapeutic advances. There is approximately 20% of mCRPC patients present somatic DNA damage repair (DDR) gene mutations. Ceralasertib, formerly known as AZD6738, is a potent and selective orally bioavailable inhibitor of the ataxia tenlangiectasia and Rad3-related (ATR) kinase, which is involved in DNA repair in response to DNA damage and replication stress. Preclinical studies have demonstrated ATRi sensitizing alterations in DNA damage response (DDR) genes. Ceralasertib’s antitumor activity as a monotherapy in treating prostate cancer is moderate. Thus, we combined ceralasertib with imipridones (ONC201 and ONC206), which target mitochondrial caseinolytic protease P (ClpP) and the integrated stress response, resulting in enhanced antitumor efficacy in vitro. Prior data have demonstrated sensitivity of 4 prostate cancer lines to ceralasertib and imipridones monotherapies, synergistic activities with the combination treatments, and immune enhancement effects with the combination treatments when co-cultured with NK92MI cell line. We proceeded to a short term in-vivo study validating the combination treatment efficacy. Preliminary results include cytokine profiling using the Luminex 200 technology to understand treatment induced changes in the tumor microenvironment (TME) from both in vitro and in vivo studies. Our results guide novel clinical trials for effective clinical responses in mCPRC patients. Citation Format: Yutong Linda Xia, Leiqing Zhang, Maryam Ghandali, Maximilian P. Schwermann, Wafik El-Deiry. The anti-tumor efficacy and immune effects of combining of ceralasertib, an oral ATR kinase inhibitor, with imipridones, in prostate cancer treatment in vitro and in vivo [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 5559.

Abstract Introduction: Approximately 30% of advanced bladder cancer (BCa) tumors exhibit MTAP gene deletion, which has been associated with luminal subtype BCa, poor response to chemotherapy, and shorter progression to metastatic disease (De Souza 2023). MTAP deletion has been proposed as a potential biomarker for response to pemetrexed and an emerging class of PRMT5 and MAT2A inhibitors in development for MTAP-deleted tumors. Here, we describe the first patient-derived 3D model of BCa with MTAP deletion and its translational potential as a drug sensitivity platform. Methods: A patient (pt) with metastatic BCa harboring MTAP deletion underwent a pelvic lymph node biopsy. A fresh tumor sample was processed under collagenase/dispase solution for 30 minutes and filtered through a 70-um cell strainer. Cells were cultures in low attachment plates with MammoCult™ Basal Medium (Stem Cell Technologies). After 5 passages and 3D spheroid formation, immunostaining for cytokeratin (CK) 5 and 6, GATA3, CK20, and Ki67 staining were performed to confirm the urothelial origin. The MTAP-del was confirmed by a western blot of the organoid specimen and compared to other BLCa cell lines (UMUC, RT4, J82, and 5637). The viability assays were performed using the CellTiter-Glo® 3D Cell kit. Next-generation sequencing (NGS) of primary BCa tumor revealed deletion of MTAP, CDKN2A, CDKN2B, and mutations in p53, TERT, and KDM6A. Results: The patient organoid sample was positive for the luminal CK5/6 markers and negative for GATA3. Ki67 and CK20 were primarily expressed in the nucleus of the same sample. The organoid retained morphologic and immunohistochemical features of the patient’s primary bladder tumor, showing the same squamous differentiation and similar Ki-67 proliferative index. The organoid displayed no evidence of the MTAP protein by western blot (similar findings with the MTAP-del cell lines UMUC and RT4). The organoid showed sensitivity to pemetrexed [pemetrexed IC50=0.12 uM (other MTAP-del BLCa IC50: 0.1-0.23 uM)] and resistance to gemcitabine and cisplatin [Gem IC50=55 nM (other BLCa IC50: 1-29 nM), Cis IC50=90.5 uM (other BLCa IC50: 2-17 uM). Conclusions: We describe the first patient-derived MTAP-deleted BCa organoid recapitulating the features of the primary tumor. The organoid displayed sensitivity to pemetrexed as described with MTAP-deleted BCa. The organoid represents a unique platform for testing novel therapeutic strategies for MTAP-deleted BCa. Citation Format: Maximilian Schwermann, Benedito Carneiro, Shaolei Lu, Andre De Souza, Matthew Hadfield, Anthony Mega, Galina Lagos, Sari Khaleel, Dragan Golijanin, Sheldon Holder, Praveen Srinivasan, William MacDonald, Andrew George, Lanlan Zhou, Leiqing Zhang, Wafik El-Deiry. Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development [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 4248.

Pancreatic cancer is a devastating disease with a poor prognosis. Novel chemotherapeutics in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin (PM01183; LY-01017) received FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. Lurbinectedin stalls and degrades RNA Polymerase II and introduces breaks in DNA, causing subsequent apoptosis. We now demonstrate lurbinectedin’s highly efficient killing of human-derived pancreatic tumor cell lines PANC-1, BxPC-3, and HPAF-II as a single agent. We further demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in the synergistic killing of pancreatic tumor cells. Western blot analysis of combination therapy indicates an upregulation of γH2AX, a DNA damage marker, and the Chk1/ATR pathway, which is involved in replicative stress and DNA damage response. We further demonstrate that the triple combination between lurbinectedin, irinotecan, and 5-fluorouracil (5-FU) results in a highly efficient killing of tumor cells. Our results are developing insights regarding molecular mechanisms underlying the therapeutic efficacy of a novel combination drug treatment for pancreatic cancer.

Pancreatic cancer is a devastating disease with a five-year survival rate below 10% according to the American Cancer Society. Novel chemotherapeutics and targeted therapies in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin is a chemotherapeutic synthetic tetrahydroisoquinoline alkaloid that inhibits active transcription by binding to guanine rich sequences in the minor groove of DNA. Lurbinectedin has been shown to reduce oncogenic transcription by stalling and degrading RNA Polymerase II while also inducing single- and double-stranded breaks to DNA, causing subsequent apoptosis. Lurbinectedin received accelerated FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. We recently described a synergistic interaction between lurbinectedin and ONC201/TIC10, a compound that induces the TRAIL pathway, in killing SCLC cell lines associated with activation of p-Chk1 and the integrated stress response. We now demonstrate lurbinectedin’s efficient killing of pancreatic tumor cells as a single agent in PANC-1, BxPC-3, and HPAF-II cell lines, with IC-50s corresponding to sub-nanomolar concentrations. We also demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in synergistic killing of pancreatic tumor cells in vitro. We further demonstrate a combination of lurbinectedin and ONC212, an imipridone, the same class of compounds as ONC201, also results in synergistic killing of pancreatic tumor cells. Cell viability was measured using the CellTiterGlo assay at varying drug concentrations. We hypothesize a combination therapy of lurbinectedin and irinotecan or ONC212 can enhance immune cell killing of pancreatic tumor cells. This is being explored by co-culturing CD8+ T lymphoblast cells with pancreatic tumor cells treated with lurbinectedin, irinotecan, ONC212, and combination, along with molecular mechanisms of cell death and effects on cytokines in the tumor microenvironment. Our results are developing insights regarding molecular mechanisms underlying therapeutic efficacy of a novel combination drug treatment for pancreatic cancer. Citation Format: Tej Tummala, Arielle De La Cruz, Ash Uruchurtu, Nicholas R. Liguori, Abbas E. Abbas, Leiqing Zhang, Christopher G. Azzoli, Lanlan Zhou, Wafik S. El-Deiry. Synergistic combinations of lurbinectedin with irinotecan and ONC212 in pancreatic 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 2674.

Abstract Glioblastoma (GBM) is the most common and lethal primary malignancy of the central nervous system. It is estimated that more than 13,000 new cases of GBM will be diagnosed this year in the United States. Despite multidisciplinary treatments such as surgery, chemotherapy, and radiotherapy, the five-year survival rate for GBM patients is only 6.8 percent and has shown no notable improvement in the last three decades. There have only been five drugs and one device ever approved by the FDA for the treatment of GBM since it was first identified in the scientific literature in the 1920’s. Our in vitro studies suggested that first-in-class small-molecule imipridone TIC10/ONC201 can inactivate ERK/AKT, induce the integrated stress response (ISR), upregulate pro-apoptotic TRAIL receptor DR5, deplete cancer stem cells, and induce mitochondrial dysfunction, growth arrest or cell death in GBM cells. Knockdown of ClpP protects GBM cells from ONC201 but not TMZ. ONC201 crosses the blood-brain barrier and has induced durable tumor regressions in adult and pediatric H3K27M-mutant diffuse midline glioma patients. We hypothesized that ONC201 may synergize with radiotherapy and temozolomide in GBM. GBM mouse orthotopic models were established through intracranial injection of luciferase expressing U251 GBM cells with a KOPF model 940 small animal stereotaxic frame and a Stoelting Quintessential Stereotaxic Injector. Tumor formation and growth was confirmed with bioluminescence imaging. Randomized treatment group mice received weekly treatment of ONC201 (100 mg/kg p.o.) and/or radiotherapy (2 Gy local irradiation) and/or TMZ (20 mg/kg i.p.) for four weeks for long term survival and tumor monitoring or one week for short term biomarker studies. We observed that the triple combination of ONC201, radiotherapy and TMZ significantly prolongs survival and reduces tumor burden compared to single treatment and dual combinations. Short term biomarker studies demonstrated that triple combination treatment decreases tumor cell proliferation, induces more apoptosis and inhibits ClpX to unleash mitochondrial ClpP. Our data support further development of the triple combination regimen of ONC201, TMZ and radiation therapy for GBM first-line treatment. Citation Format: Lanlan Zhou, Laura Jinxuan Wu, Jun Zhang, Andrew George, Marina Hahn, Leiqing Zhang, Attila A. Seyhan, Wafik S. El-Deiry. Preclinical combination of ONC201 with radiotherapy and Temozolomide in a GBM mouse orthotopic model results in reduced tumor burden and prolonged survival. [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 5494.

Pancreatic cancer is a devastating disease with an overall five-year survival rate of ~12% according to the American Cancer Society. Novel chemotherapeutics and targeted therapies in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin (PM01183; LY-01017) received accelerated FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. Lurbinectedin is a chemotherapeutic synthetic tetrahydroisoquinoline alkaloid that inhibits active transcription by binding to guanine rich sequences in the minor groove of DNA. Lurbinectedin has been shown to reduce oncogenic transcription by stalling and degrading RNA Polymerase II and inducing breaks in DNA, causing subsequent apoptosis. We now demonstrate lurbinectedin’s highly efficient killing of pancreatic tumor cells as a single agent in PANC-1, BxPC-3, and HPAF-II cell lines, with 72-hour IC-50s ranging from 0.266 – 0.964 nM. We further demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in synergistic killing of pancreatic tumor cells in vitro. Synergism was calculated using SynergyFinder, with HSA scores exceeding 15 in every cell line (synergy scores greater than 10 are considered synergistic according to the website manufacturer). Western Blot analysis of combination therapy indicates an upregulation of γH2AX and ATR, indicating DNA damage likely plays a role in the synergistic effects of the drugs. Bcl-2 downregulation in combination therapy indicates that the Bcl-2 family proteins likely play a role in the apoptosis of the cancer cells. Cytokine profiling analysis indicates an upregulation of the Fas apoptosis-inducing ligand under lurbinectedin and irinotecan monotherapy and combination therapy. VEGF and prolactin were downregulated in combination therapy. We further demonstrate that the triple combination between lurbinectedin, irinotecan, and 5-Fluorouracil (5-FU) enhances % inhibition as each drug concentration increases, and the presence of lurbinectedin enhances the synergism between irinotecan and 5-FU. Our results are developing insights regarding molecular mechanisms underlying therapeutic efficacy of a novel combination drug treatment for pancreatic cancer. Citation Format: Tej Tummala, Arielle De La Cruz, Ash Uruchurtu, Nicholas R Liguori, Abbas E Abbas, Leiqing Zhang, Christopher Azzoli, Lanlan Zhou, Wafik S El-Deiry. Preclinical synergistic combination therapy of lurbinectedin with irinotecan and 5-fluorouracil in pancreatic cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B026.