Aggressive Type Research Articles

Abstract 1650: Characterization of nucleolar biological functions via Auxin inducible degron-mediated acute depletion in triple negative breast cancer cells

Abstract Nucleoli are large nuclear sub-compartments where vital cellular processes, such as rRNA production and ribosome assembly, take place. Aggressive tumor types, such as Triple Negative Breast Cancer (TNBC), require increased ribosome biogenesis to support their enhanced proliferation. Therefore, it has been suggested that the nucleolus could be a valid anti-neoplastic target. However, we still have a limited understanding of the cancer-specific functions of the nucleolar components, preventing the effective development of therapeutic agents. The characterization of nucleolar proteins is technically challenging. Several nucleolar proteins are essential, and their complete abrogation cannot be achieved through conventional RNAi or CRISPR-KO approaches. Additionally, many nucleolar proteins display peculiar biophysical properties, and their functions are connected to their abundance. Therefore, artificial overexpression systems might not fully recapitulate their endogenous biological functions. To overcome these challenges, we implemented an Auxin Inducible Degron (AID) system to acutely abrogate endogenous nucleolin, one of the most abundant nucleolar proteins, and asses its impact on the proliferation of TNBC cell lines. We used CRISPR/Cas9 editing in TNBC cell lines to modify the endogenous NCL gene, fusing it with an AID tag. Then, we stably transduced the cells with a modified Oryza Sativa TIR1 (OsTIR1-F74G) E3 ubiquitin ligase. In response to the synthetic phytohormone phenyl-auxin, OsTIR1 leads to the proteasomal degradation of AID-containing endogenous nucleolin in less than 6h, sparing any other human protein. RNA sequencing analysis showed that nucleolin degradation significantly alters the expression of genes involved in cell cycle progression. Accordingly, nucleolin degradation reduces cancer cell proliferation in vitro, associated with increased propidium iodide staining, suggestive of a G2/M cell cycle arrest. However, immunofluorescence analyses revealed that nucleolin abrogation leads to the accumulation of bi-nucleated cells with actin patches. These findings are indicative of potential cytokinesis defects due to altered spindle tension during mitosis. Notably, nucleolin abrogation increased the efficacy of chemical inhibitors of the Anaphase Promoting Complex on suppression of cell proliferation. To the best of our knowledge, this study is the first reporting the acute abrogation of an endogenous, highly abundant nucleolar protein, using the AID system. This approach allowed the characterization of nucleolin biological functions with unprecedented temporal resolution, shedding light on new biological functions in the regulation of cell division. Finally, our results suggest that nucleolin inhibition could enhance the therapeutic activities of drugs affecting cell cycle progression in TNBC. Citation Format: Nastaran Samadi Rad, Joseph Mills, Anna Tessari, Damu Sunil Kumar, Vollter Anastas, Saranya Lamba, Ashley B Reers, Ilaria Cosentini, Zirui Zhu, Thomas J Magliery, Emanuele Cocucci, Lara Rizzotto, Dario Palmieri. Characterization of nucleolar biological functions via Auxin inducible degron-mediated acute depletion in triple negative breast cancer cells [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 1650.

Abstract 375: Lipogenic phenotype is an adaptation to nutrient stress in the tumor microenvironment

Abstract The nutrient and oxygen poor microenvironment combined with acidosis arising from fermentative metabolism imparts strong selection pressure on cancer cells that promotes evolution of more aggressive cancer types. Poor nutrient availability in the tumor microenvironment forces cancer cells to adapt to alternate routes of metabolism, nutrient scavenging and autophagy leading to a shift in their metabolic phenotype. These adaptations promote evolution of invasive and aggressive phenotypes. To test this hypothesis and to characterize the evolution of cancer cells under nutrient stress, we generated nutrient stress adapted cells from MCF7-GFP cells by subjecting them to multiple cycles of nutrient starvation and repletion. Distinct clones that outgrew were selected as nutrient stress adapted cells (NSA). Here we show that MCF7-NSA cells that are adapted to grow in low nutrient conditions exhibited robust accumulation of lipid droplets compared to parental MCF7-GFP cells as shown by confocal microscopy after neutral lipid staining as well as immunofluorescence staining for PLIN2 (Perilipin 2), a lipid droplet coat protein. In addition, MCF7-NSA cells showed elevated expression of the nuclear receptor PPARγ and the adipokine FABP4 indicating an adipogenic/lipogenic signature. Strikingly, these nutrient stress adapted cells were capable of nutrient scavenging by macropinocytosis compared to control cells even under nutrient replete conditions. Macropinocytosis was assessed by DQ-BSA processing and TMR-Dextran uptake. NHE inhibitor EIPA (ethyl isopropyl amiloride), that blocks macropinocytosis, but not receptor mediated endocytosis confirmed dextran uptake through macropinocytosis. Similarly, macropinocytosis inhibitor KH7 blocked dextran uptake in these adapted cells. Furthermore, MCF7-NSA cells showed high expression of TFEB and TFE3, master regulators of autophagy and lysosomogenesis suggesting that the necrotic cell debris scavenged through macropinocytosis and processed through autolysosome machinery might be contributing to de novo lipogenesis. Although control and adapted cells exhibited similar growth kinetics in vitro, when implanted in mammary fat pads (MFP) of NSG mice, NSA cells formed significantly smaller and slow growing primary tumors. Interestingly, 80% of these mice showed metastasis to other organs (lung, liver, kidney) whereas control mice did not develop metastasis as parental MCF7 is not an aggressive cell line in xenograft models. MCF7-NSA cells were more migratory in vitro in wound healing assays and were capable of developing lung metastases in a mouse model where primary tumor from mammary fat pad was resected to allow for metastases to develop. Taken together, our data suggest that lipogenic phenotype confers survival advantage in the harsh tumor microenvironment and selects for more invasive and aggressive phenotypes. Citation Format: Crystal Griffith, Dominique Abrahams, Antonio Ortiz, Alexandra Tassielli, Joseph Johnson, Robert Gatenby, Smitha Pillai. Lipogenic phenotype is an adaptation to nutrient stress in the tumor microenvironment [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 375.

Abstract 4714: Mechanisms by which the Rac & Cdc42 inhibitor MBQ-167 overcomes the adverse effects of paclitaxel in TNBC

Abstract Triple-negative breast cancer (TNBC) is the most aggressive, recurrent, and metastatic type of BC. Since there are limited targeted therapies available, the standard treatment is taxane-based chemotherapy, which still results in recurrence and metastasis. Therefore, we developed MBQ-167, a clinical-stage Rac/Cdc42 inhibitor that reduces tumor progression and metastasis in TNBC mouse models. In vitro studies show that PXL increased Rac activation, which MBQ-167 reduced in combination with PXL. In mouse models of TNBC, we found that PXL increases metastasis and that MBQ-167 prevents it. Therefore, this study aimed to identify potential mechanisms to explain the antimetastatic effects of MBQ-167 in combination with PXL. Previous studies suggest that PXL may act as an agonist to the Toll-Like 4 Recceptor (TLR4) and activate NfkB signaling to promote metastasis. We hypothesize that MBQ-167 chemosensitizes TNBC cells to PXL and reduces metastasis by blocking Rac/Cdc42 and TLR4/NF-kB signaling. Treatment of SCID mice with orthotopic MDA-MB-468 TNBC tumors with MBQ-167, PXL, or the combination increased lung metastases in mice treated with PXL, which MBQ-167 suppressed. Flow cytometry of spleens harvested from the mice with or without tumors demonstrated increased M2 immunosuppressive macrophages (F480+, CD206+) compared to M1 macrophages (F480+, CD206-), where macrophage differentiation has been implicated with TLR4 signaling. To determine the role of TLR4 signaling, we treated MDA-MB-231 cells stably expressing scrambled or TLR4 shRNA with MBQ-167, PXL, or the combination. Treated cells were evaluated for cell viability, apoptosis, migration, mammosphere assay, and NF-kB p65 activation. Results show that MBQ-167 reduced cell viability stem-cell-like features and increased apoptosis comparable to vehicle or PXL. TLR4 knockdown (kd) did not change cell viability, apoptosis, or migration in vehicle-treated cells. However, TLR4 kd further reduced the decreased cell viability and increased apoptosis in response to MBQ-167, PXL, or the combination, indicating that PXL effects on cell growth are at least partially dependent on TLR4. PXL treatment did not affect cell migration in the control cells but reduced migration in TLR4 kd cells. TLR4 kd exacerbated the inhibitory effects of MBQ-167 or the combination. MBQ-167, but not PXL, reduced LPS-induced (i.e., TLR4-regulated) NfkB translocation to the nucleus, with a similar effect as the combination. Therefore, MBQ-167 may prevent PXL-induced metastasis by partially inhibiting the NF-kB pathway in TNBC tumors and reducing inflammation and immunosuppression from M1 and M2 macrophages in vivo. Citation Format: Nilmary Grafals-Ruiz, Patricia Sánchez-Orive, Anamaris Torres-Sánchez, Ailed Cruz-Collazo, Derealise García-Almedina, Stephanie Dorta-Estremera, Suranganie Dharmawardhane. Mechanisms by which the Rac & Cdc42 inhibitor MBQ-167 overcomes the adverse effects of paclitaxel in TNBC [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 4714.

Abstract 7550: KB-0742, an oral highly selective CDK9 inhibitor, demonstrates preclinical activity in transcription factor fusion driven adenoid cystic carcinoma patient-derived models

Abstract Adenoid Cystic Carcinoma (ACC) is an aggressive rare type of cancer of the secretory glands with no approved targeted therapy and high unmet clinical need. Deregulated transcription driven by MYB-NFIB or MYBL1-NFIB transcription factor (TF) fusions are a hallmark of ACC pathogenesis. More aggressive disease and resistance to therapy has been associated with co-mutation in NOTCH. Although direct targeting of oncogenic TF fusions has remained challenging, targeting of their activity via transcriptional cofactors has emerged as an attractive and clinically actionable strategy. Cyclin-dependent kinase 9 (CDK9) is a key potentiator of TF activity via its ability to act both as an upstream regulator of TF expression and a downstream cofactor. KB-0742 is a potent, selective, and orally bioavailable inhibitor of CDK9 with a long plasma half-life. KB-0742 is being studied in an ongoing Phase 1/2 study (NCT04718675) in advanced solid tumors including ACC. KB-0742 has demonstrated on-mechanism, single agent anti-tumor activity and a manageable safety profile in heavily pre-treated patients (0-11 prior lines of therapy) with transcriptionally addicted solid tumors. Here we present the preclinical rationale for targeting of oncogenic fusion TF activity in ACC. XenoSTART Patient-Derived Xenograft (XPDX) models were established from viable human tumor tissue for in vivo and ex vivo testing. Cell viability was assessed by CellTiter GLO reagent and phase imaging. Changes in MYB-fusion transcripts were assessed by RNA-Seq. Tumor Fragments (~70 mg) from ACCx5M1, ACCx6, ACCx9, ACCx11 models were implanted into athymic nude mice. Animals were treated with either vehicle (normal saline) or 60 mg/kg KB-0742 on a Q3D/week until tumors reached 2500 mm3 or up to 60 days post start of treatment. In a retrospective analysis using real world data, ACC has a low incidence of genomic instability and high rates of MYB fusions detectable by RNA-seq, making immunotherapy challenging but creating an opportunity for targeting MYB transcription. In primary MYB-fusion positive and NOTCH co-mutated patient-derived spheroid models, KB-0742 treatment induced strong antiproliferative activity and cytotoxicity. Importantly, KB-0742 demonstrated stronger cytotoxic effects when compared to historical control agents. In XPDXs, CDK9 inhibition with KB-0742 resulted in antiproliferative activity and stronger tumor growth inhibition in MYB-fusion positive and NOTCH co-mutated tumor models compared to MYB-fusion positive only models.These data demonstrate KB-0742 is effective in preclinical models of ACC suggesting KB-0742 may be a promising therapeutic option for ACC patients. KB-0742 is currently being evaluated in a phase 1/2 dose-escalation and cohort expansion trial in patients with ACC and other transcriptionally addicted tumors (NCT04718675). Citation Format: Michael R. McKeown, Luis A. Carvajal, Tressa R. Hood, Nicole S. Burr, Jeffrey Kaufman, Adam Boynton, Kameron Mori, Tessa DesRochers, Michael Wick, Charles Y. Lin, Jorge F. DiMartino. KB-0742, an oral highly selective CDK9 inhibitor, demonstrates preclinical activity in transcription factor fusion driven adenoid cystic carcinoma patient-derived models [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 7550.

Loss of PTPRS elicits potent metastatic capability and resistance to temozolomide in glioblastoma.

Glioblastoma (GBM) is the most aggressive brain tumor type with worse clinical outcome due to the hallmarks of strong invasiveness, high rate of recurrence, and therapeutic resistance to temozolomide (TMZ), the first-line drug for GBM, representing a major challenge for successful GBM therapeutics. Understanding the underlying mechanisms that drive GBM progression will shed novel insight into therapeutic strategies. Receptor-type tyrosine-protein phosphatase S (PTPRS) is a frequently mutated gene in human cancers, including GBM. Its role in GBM has not yet been clarified. Here, inactivating PTPRS mutation or deficiency was frequently found in GBM, and deficiency in PTPRS significantly induced defects in the G2M checkpoint and limited GBM cells proliferation, leading to potent resistance to TMZ treatment in vitro and in vivo. Surprisingly, loss of PTPRS triggered an unexpected mesenchymal phenotype that markedly enhances the migratory capabilities of GBM cells through upregulating numerous matrix metalloproteinasesvia MAPK-MEK-ERK signaling. Therefore, this work provides a therapeutic window for precisely excluding PTPRS-mutated patients who do not respond to TMZ.

Abstract 1692: MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal type of pancreatic neoplasm. Unfortunately, most patients are diagnosed with unresectable or metastatic disease, making current treatments insufficient. As a result, the 5-year survival rate for PDAC is below 10%.Pancreatic neoplasms are primarily initiated by the KRAS mutation, accounting for 95% of cases. However, the progression of PDAC to a highly aggressive and therapy-resistant cancer is accelerated by chronic inflammation. The role of mutant KRAS signaling and NF-κB inflammatory signaling in promoting PDAC progression has been extensively studied. Yet, the transcriptional mechanism of their cooperation remains unclear. Additionally, the interaction between PDAC tumor cells and signals in the tumor microenvironment (TME) is not yet fully understood. Our study demonstrates that the simultaneous activation of KRAS/MAPK and NF-κB by epidermal growth factor (EGF) and TNFα, respectively, increases cell migration as shown through live-cell imaging analysis. An integrated analysis of epigenetic and transcriptomic data (ChIP-seq and RNA-seq) was performed after dual stimulation of both pathways. This revealed a high occupancy of the active mark, H3K27ac at transcription start sites of a subset of genes involved in cell migration and subtype switch. Through gain and loss-of-function studies, FOSL1 and RELA were identified as the key transcription factors. The epigenome mapping of FOSL1 and RELA after the stimulation of both pathways revealed their occupancy at RELA-dominant and FOSL1/RELA co-occupied regions. Inhibitor treatments and loss-of-function studies revealed that RELA binding controls RNA polymerase recruitment for transcription initiation and enhances cell migration. Further analysis of RNA-seq data on PDAC cell lines and cells from patient-derived xenografts highlighted that active inflammatory signaling contributes to a subtype switch from the classical to the more migratory basal subtype. Our study, combined with single-cell RNA-seq analysis of published data, shows that macrophages in the tumor microenvironment supply TNFα that activates NF-κB signaling, leading to changes in cell migration. Our study has provided a comprehensive understanding of the mechanism by which mutant KRAS and inflammatory signaling contribute to the progression of PDAC. These findings could offer a new perspective in the development of mechanism-based therapeutic approaches that can improve the efficacy of chemotherapeutic agents and make tumors more amenable to surgical resection. Citation Format: Joana E. Aggrey-Fynn, Meghana Manjunath, Steven A. Johnsen. MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis [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 1692.

Abstract 1863: α-ABN501, a novel antibody targeting claudin-3, demonstrates the potential to be a new therapeutic option for small cell lung cancer

Abstract Small Cell Lung Cancer (SCLC) is the most aggressive and lethal type of lung cancer, characterized by a poor prognosis, with a 5-year survival rate of only 7% for overall SCLC patients. Limited treatment options such as chemotherapy and immune-checkpoint inhibitor therapy are available for SCLC patients, very often followed by rapid recurrence and metastasis. Thus, new effective treatment options for SCLC remain high unmet medical needs. Overexpression of claudin-3 (CLDN3), one of the tight junction proteins, is observed in various types of cancers. The abnormal conformation of tumor cell growth leads to the external exposure of CLDN3, in contrast, the normal cells that maintain high polarity do not expose CLDN3 externally. Therefore, CLDN3 could be a potential therapeutic target and biomarker. Based on the analysis of the expression pattern of CLDN3 using a single-cell RNA sequencing dataset derived from SCLC patients, we found that CLDN3 could be a superior and unique target of SCLC compared to others (e.g., DLL3 and B7H3). Hence, we developed a fully human monoclonal antibody, afucosylated ABN501 (α-ABN501), which has high specificity and strong affinity to CLDN3-expressing cells. The α-ABN501 antibody showed CLDN3-dependent killing effects against the SCLC cells, expressing different levels of CLDN3, as assessed via the antibody-dependent cellular cytotoxicity (ADCC) assay wherein using NK cells expressing CD16a (NK-92MI-CD16a), peripheral blood mononuclear cells (PBMC), and primary NK cells. Further, the efficacy study in the CLDN3-expressing SCLC cell derived xenograft mouse model revealed that α-ABN501 was highly efficacious in inhibition of tumor growth. Collectively, we elucidated that the ADCC activity of α-ABN501 was dependent on the expression level of CLDN3 and especially specific to the SCLC cells, which underlines significant suppression of tumor growth. Present study data sets suggest that the α-ABN501 can be a potential new therapeutic option for SCLC. In addition, the α-ABN501 antibody is currently under way of development into new drug platforms for further applications. Citation Format: Jae Choi, Sunghyun Hong, Ha Yeon Park, Jaemun Kim, Sangin Lee, Myeung-Ryun Seo, Sang-Beom Bang, Hyo-Jin Shin, Kwang-il Jeong, Hobin Yang, Sungyoul Hong, Jun Young Choi, Na Young Kim, Young Kee Shin, Saehyung Lee. α-ABN501, a novel antibody targeting claudin-3, demonstrates the potential to be a new therapeutic option for small cell lung 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 1863.

Abstract 1218: CDK9 inhibitors modulate the transcriptional landscape of colorectal cancer to suppress MAPK signaling and synergizes with BRAF inhibitors to treat BRAF-mutant colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US, and it remains a significant public health burden. Metastatic CRC (mCRC) is usually treated with combination chemotherapy and targeted therapy regimens, but eventually becomes chemotherapy refractory. Thus, novel and more effective treatments for mCRC are urgently needed. Cyclin-dependent kinase 9 (CDK9) is a key activator of RNA Pol II transcription and promotes the expression of many cancer driver genes, making CDK9 a promising target for cancer therapy. Methods: Human CRC cell lines, patient-derived organoids (PDOs), cell line xenografts, and patient-derived xenografts were used to investigate the efficacy and mechanisms of action of the CDK9 inhibitors AZD4573, enitociclib, and NVP-2, as well as the BRAF inhibitors encorafenib and dabrafenib. In vitro viability was measured by chemical cell proliferation assays and in vivo tumor sizes were measured by calipers. In vitro mechanisms of action were assessed by RNA sequencing, real time polymerase chain reaction, and Western blotting. In vivo mechanisms of action were assessed by immunohistochemistry. Results: CDK9 inhibitors potently inhibited CRC cells and PDOs. In contrast to hematologic malignancies, we did not observe consistent suppression of the oncogenes c-MYC and MCL-1 by CDK9 inhibitor treatment. Instead, CDK9 inhibitors suppressed several other key cancer pathways in CRC, such as MAPK, mTOR, and PI3K signaling pathways. Importantly, multiple targets within the MAPK pathway were strongly suppressed, including EGFR, KRAS, and BRAF. We hypothesized that combination treatment with CKD9 inhibitors and MAPK pathway inhibitors can synergistically treat CRC. As proof-of-concept, we investigated the combination of CDK9 and BRAF inhibitors in models of BRAF-mutant CRC, a particularly aggressive type of CRC. We found this combination to synergistically suppress CRC growth in vitro and in vivo. Compared to single agents, combination treatment led to significantly stronger induction of apoptosis and suppression of MAPK pathway signaling. Our results suggest that concurrent treatment with CDK9 inhibitors plus established MAPK pathway inhibitors, such as BRAF inhibitors, can significantly improve upon single agent treatment. Conclusions: We have found CDK9 inhibitors to potently suppress CRC growth and survival through a unique mechanism of action, by vertical suppression of the MAPK signaling pathway. We demonstrate that CDK9 inhibitors can synergize with BRAF inhibitors in the treatment of BRAF-mutant CRC models. Thus, CDK9 inhibitors are a promising class of drugs warranting further investigation, including early-phase clinical trials, in mCRC. Citation Format: Chaoyuan Kuang, Ning Wei, Mahshid Mohammadi, Muzaffer A. Bhat, Terence Li, Priyanka Patil, Othon Wiltz, Renee Huang, Kohtaro Ooka, Melanie Quintal, Edward Chu. CDK9 inhibitors modulate the transcriptional landscape of colorectal cancer to suppress MAPK signaling and synergizes with BRAF inhibitors to treat BRAF-mutant colorectal 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 1218.

Abstract 2092: Genomic and proteomic analysis of glioblastoma recurrences during TTFields exposure

Abstract Glioblastoma (GB) is the most aggressive type of brain tumor, and current treatments are generally ineffective in preventing recurrence. Tumor Treating Fields (TTFields) are an innovative treatment that has been shown to improve patients' life expectancy in the last two decades. TTFields therapy has an antitumoral effect that involves different mechanisms and is an optional add-on to standard maintenance temozolomide. Although TTFields therapy initially slows tumor progression, it does not prevent tumor relapse in most patients. In the present investigation, we run a genomic and proteomic analysis of surgical material from 10 patients treated with TTFields. GB tissues were obtained from the primary tumor mass and from a second surgery after tumor relapse. All the patients, in addition to brain tumor standard therapies, were treated with TTFields for eighteen hours a day, the time span of treatment application was dependent on GB recurrence. Control samples were obtained from primary and secondary surgeries of 5 patients treated with standard therapy alone. The GB tissue extracts were analyzed with an RNAseq routine, and the results from the secondary surgery were compared with the data obtained from tumor specimens of the primary surgery. A common feature of transcripts among all the patients was the alterations of several pathways promoting the increase of intracellular oxidation and cell cycle regulation. In parallel with the analysis of patient specimens, we developed an in vitro GB cell relapse model. This was instrumental in investigating the beneficial contribution of TTFields action during tumor relapse, but also in uncovering the limitation of this therapeutic procedure. GB primary cultures obtained from patients' surgery were exposed for up to 12 days with 200 kHz TTFields stimulation using the inovitro™ system. After 12 days of continuous TTFields application, GB cells showed an average depolarization of the resting membrane potential, increasing oxidation, and acidification of the cytoplasm. Genomic analysis of treated cells compared with wild-type primary culture, denoted an increase of tumor stem cell markers, activation of several metabolic pathways, and upregulation of different ion channel protein transcripts. Our final goal is to identify specific molecular features enhanced by TTFields treatment to be used as a parallel therapy to fight GB recurrence. Citation Format: Francesca Cianci, Guido Rey, Dietmar Krex, Davide Ceresa, Paolo Malatesta, Michele Mazzanti. Genomic and proteomic analysis of glioblastoma recurrences during TTFields exposure [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 2092.

Abstract 5697: MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production

Abstract Despite the progress in endocrine therapy, targeted therapy and immunotherapy, the treatment of triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer, is challenging. This is closely associated with the lack of effective molecular targets in TNBC cells for therapeutic intervention. Here we report that the long noncoding RNA (lncRNA) MILIP drives TNBC cell survival, proliferation and tumorigenicity through complexing with transfer RNAs (tRNAs) to promote protein production, and thus represents a potential therapeutic target in TNBC. MILIP was expressed at high levels in TNBC cells that commonly harbor loss-of-function mutations of the tumor suppressor p53, yet MILIP silencing diminished TNBC cell viability and retarded TNBC xenograft growth, indicating that MILIP functions distinctively in TNBC beyond its well-established role in repressing p53 in other types of cancers. Mechanistical investigations revealed that MILIP interacted with eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) and formed an RNA-RNA duplex with tRNALeu and tRNASer, type II tRNAs, through their variable loops, thus facilitating the binding of eEF1α1 to these tRNAs. Disrupting the interaction between MILIP and eEF1α1 or tRNAs diminished protein production and cell viability, with therapeutic potential revealed where targeting MILIP using Gapmers inhibited TNBC growth and cooperated with the clinically available protein synthesis inhibitor omacetaxine mepesuccinate in vivo. Collectively, these results identified MILIP as an RNA translation elongation factor to promote protein production in TNBC cells and demonstrated that experimental therapy with MILIP Gapmers inhibits TNBC growth, indicating MILIP targeting is a potential avenue for developing improved TNBC treatment through blocking protein synthesis. Citation Format: Yuchen Feng, Simin Zheng, Xiaohong Zhao, Yuanyuan Zhang, Ran Xu, Liang Xu, Thomas Preiss, Lei Jin, Jinnan Gao, Xu Dong Zhang. MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production [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 5697.

Abstract 5935: Non-allosteric inhibition of enolase 1 sensitizes triple-negative breast cancer for checkpoint inhibitors

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer that commonly returns and spreads. Currently, very few options are available for TNBC treatment. Cancer cells exploit glycolytic machinery for the Warburg effect and aerobic glycolysis. Enolase 1 (ENO1) is the glycolytic enzyme expressed in most tissues, and many cancer cells have higher expression. Apart from the glycolytic role in the cytosol, ENO1 also plays different roles in cancer cells, including as a surface receptor. Method: non-allosteric chemical inhibition was developed and tested in vitro for its efficacy and target validation. Subsequently, in vivo examinations were conducted on emerging small molecules in syngeneic and patient-derived murine models of breast cancer to unveil their anti-tumor efficacy. Results: Our non-allosteric small molecule inhibitor, SU212, binds to ENO1 and restricts its movement within cells. SU212 treatment leads to the accumulation of ENO1 in the nucleus where it restrains the activity of c-Myc. The treatment causes the production of truncated ENO1 called myc promoter-binding protein 1 (MBP-1), which limits the activity of c-Myc. Stand-alone treatment of SU212 restrains the tumor progression and metastasis in orthotopic syngeneic and PDX mouse models. Combining SU212 and Anti-PD1 significantly inhibits tumor growth and metastasis in mouse models. C-Myc is linked to protein transcription that aids immune resistance and tumor evasion of immune surveillance. Suppression of c-Myc activity via SU212 treatment sensitizes the TNBC tumors for anti-PD1 therapy. Conclusions: This study provides compelling preclinical data for further development of SU0212 as an immune therapy sensitizer for the treatment of TNBC. Citation Format: Dhanir Tailor, Fernando Jose Garcia-Marques, Abel Bermudez, Wenqi Li, Arpit Dheeraj, Sharon J. Pitteri, Sanjay V. Malhotra. Non-allosteric inhibition of enolase 1 sensitizes triple-negative breast cancer for checkpoint inhibitors [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 5935.

Abstract 6951: The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is a rare and aggressive type of breast cancer in which the lymphatic vessels become blocked by cancer cells in the breast tissue. The five-year survival rate for this type of cancer is around 40%, one of the worst among breast cancer. Most IBCs are estrogen receptor (ER) and progesterone receptor (PR) negative and HER2 positive, with some presenting an amplification of HER2 while others are ER, PR, and HER2 negative, formally known as the triple-negative (TNBC) subtype. However, it has been reported that treatment with 17β-Estradiol (E2) of ER-negative IBC and non-IBC cells can exert rapid non-genomic signaling mediated by ERα-36, GPR30, and/or estrogen receptor beta, activating MAPK pathways involved in oncogenic phenotypes. Previous data generated in our lab showed that when cells were treated with estradiol it led to increased phosphorylation levels of p38. However, the specific role p38 MAPK signaling takes on in IBC is unknown. Thus, this study centers on examining the role of estradiol and p38 regulation in the triple-negative IBC cell line model, SUM149PT. We hypothesize that upon estradiol treatment p38 signaling pathway is activated resulting in the over-expression of pro-inflammatory cytokines which regulate pro-tumorigenic phenotypes including the growth and survival of IBC cells. Inflammatory cytokines can promote pro-oncogenic features by promoting cell proliferation, angiogenesis, and immune suppression. We treated SUM149 IBC cells with 10nM of estradiol and measured the levels of human cytokines using a Human Cytokine Antibody Array. In addition, treatment using SB202190, a potent p38 inhibitor, caused a significant decrease in cell viability of several breast cancer cell lines in a dose-dependent manner. Ongoing studies include assessing the effects of p38 inhibition proliferation, invasion, and migration. In summary, a better characterization of the effect of E2 and p38 activation in ER-negative cell lines may provide great insight into novel targeted therapies for IBC. Citation Format: Keyla V. Ramirez, Adrian D. Bonilla-González, Jeisac Guzman-Rivera, Esther A. Peterson-Peguero. The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast 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 6951.

Abstract 6933: Decoding the archetypes and eco-hubs of triple-negative breast cancer in responses to chemotherapy

Abstract Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer commonly treated with neoadjuvant chemotherapy (NAC). While about 50% of patients achieve a pathological complete response (pCR), the remainder often develop resistance and metastasis, leading poor survivals. Currently, the factors linking pre-treatment cancer cells and the tumor microenvironment (TME) to NAC response are unclear. To investigate this question, we conducted single-cell RNA sequencing on fresh pre-treatment core biopsy samples collected from patients in the ARTEMIS clinical trial. We identified 13 gene expression meta-programs of cancer cells and about 50 cell states of immune and stromal cell types. Based on the cancer cells alone, we identified four TNBC archetypes at patient-level: luminal secretory-associated (LSA), basal-associated (BA), immunoreactive (IR), and luminal androgen receptor (LAR). Notably, the archetype BA and IR were associated with non-pCR and pCR, respectively. Additionally, we found the TNBC ecosystem was composed of eight eco-hubs, reflecting the co-occurrence patterns of cancer and TME cell states. These eco-hubs revealed different cell community across archetypes and NAC outcomes. For example, an eco-hub with a co-occurrence of interferon response-related cancer cells and immune cells was prevalent in the archetype IR and pCR patients. To facilitate a clinical application, we further developed a 13-gene-based machine learning model to predict NAC response. In summary, these results provide new insights into the cellular determinants of TNBC biology and chemotherapeutic response. Citation Format: Yun Yan, Yiyun Lin, Tapsi Kumar, Shanshan Bai, Jianzhuo Li, Tuan Tran, Min Hu, Elizabeth Ravenberg, Maia Rauch, Alyson Clayborn, Alastair Thompson, Bora Lim, Lei Huo, Stacy Moulder, Clinton Yam, Nicholas Navin. Decoding the archetypes and eco-hubs of triple-negative breast cancer in responses to chemotherapy [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 6933.

Abstract 4567: Dual targeting of HSP70 and AURKA improves treatment in neuroendocrine prostate cancer

Abstract Background: Neuroendocrine prostate cancer (NEPC) is the most aggressive type of prostate cancer with no effective treatments. Therefore, there is an urgent need to develop new treatment strategies. N-Myc plays a key role in driving NEPC progression, and Aurora kinase A (AURKA) prevents N-Myc degradation by interact with N-Myc and disallowing the intervention of ubiquitin ligases. The AURKA inhibitor Alisertib inhibits NEPC tumor growth by disrupting N-Myc signaling, but it is failed in a phase II clinical trial. In this study, we tested the synergy of novel HSP70 allosteric inhibitor JG231 and Alisertib in NEPC cells, organoids, and patient derived xenograft (PDX) models, which will provide new therapeutic strategies for the NEPC treatment. Methods: RT-PCR, western blotting, or immunohistochemistry (IHC) were used to determine the expression of N-Myc, neuroendocrine maker (NSE, SYP, CHGA, etc) in different prostate cancer cell lines and PDX tumors. After the knockdown of HSP70 and AURKA by siRNA transfection, cell proliferation and N-Myc expression were assessed by using cell viability assay, RT-PCR and Western blotting respectively. The co-immunoprecipitation assay was performed to detect the N-Myc ubiquitination. RNA-sequencing, gene set enrichment analysis was employed to determine the changes of JG231 on gene programs. The effects of JG231 and Alisertib on the proliferation were examined in NEPC cell lines and PDX organoids models. Results: We determined that PC3, CWR22Rv1, H660 cell lines, LuCaP49, and LuCaP93 PDX tumors had significantly increased the expression of N-Myc and neuroendocrine makers. Knockdown of HSP70 or using the HSP70 inhibitor, JG231, significantly inhibit the growth of H660 and CWR22Rv1 cells and leads to a synergistic effect with Alisertib (p<0.001). JG231 combined with Alisertib also inhibits growth and induces the death of LuCaP93 and H660 tumor derived organoids in a dose-dependent manner (p<0.001). Mechanistically, JG231 inhibits the protein expression of N-Myc through the ubiquitin-proteasome system and promote STUB1 to enter the nucleus to bind to N-Myc. Conclusions: JG231, a novel HSP70 inhibitor, can improves the treatment efficacy of AURKA inhibitor Alisertib against NEPC through the regulation of N-Myc protein homeostasis. Citation Format: Pengfei Xu, Joy C. Yang, Bo Chen, Shu Ning, Leyi Wang, Christopher Nip, Christopher P. Evans, Marc A. Dall'Era, Allen C. Gao, Jason Gestwicki, Chengfei Liu. Dual targeting of HSP70 and AURKA improves treatment in neuroendocrine prostate 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 4567.

RFC3 drives the proliferation, migration, invasion and angiogenesis of colorectal cancer cells by binding KIF14.

Colorectal cancer (CRC) is a deadly and aggressive type of cancer that has a high fatality rate. The expression levels of replication factor C subunit 3 (RFC3) and kinesin family member 14 (KIF14) have been reported to be increased in CRC. The current study aimed to explore the effects of RFC3 on the malignant behaviors of CRC cells and its possible underlying mechanism involving KIF14. RFC3 and KIF14 expression levels in CRC tissues were analyzed using TNMplot database and Gene Expression Profiling Interactive Analysis database bioinformatics tools. RFC3 and KIF14 levels in CRC cells were examined using reverse transcription-quantitative PCR and western blotting. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were performed to assess cell proliferation. Cell apoptosis was determined using flow cytometric analysis. Wound healing and Transwell assays were adopted for the evaluation of cell migration and invasion. Tube formation assay in human umbilical vein endothelial cells was used to measure angiogenesis. Western blotting analysis was performed to determine the expression of apoptosis-, migration- and angiogenesis-associated proteins. Additionally, bioinformatics tools predicted the co-expression and interaction of RFC3 and KIF14, which was verified by a co-immunoprecipitation assay. RFC3 displayed elevated expression in CRC tissues and cells, and depletion of RFC3 halted the proliferation, migration, invasion and angiogenesis, while increasing the apoptosis of CRC cells; this was accompanied by changes in the expression levels of related proteins. In addition, RFC3 bound to KIF14 and interference with RFC3 reduced KIF14 expression. Moreover, KIF14 upregulation reversed the effects of RFC3 depletion on the aggressive cellular behaviors in CRC. In conclusion, RFC3 might interact with KIF14 to function as a contributor to the malignant development of CRC.

Enhanced laser-induced fluorescence and Raman spectroscopy with gold nanoparticles for the diagnosis of oral squamous cell carcinoma

BackgroundThe incidence, mortality, and recurrence rates of oral cancer are high worldwide. It is a common and aggressive type of tumor. Owing to the challenges associated with early illness diagnosis, squamous cell carcinoma, a kind that is prevalent of oral cancer, has an unacceptably high fatality rate. The management of the condition and the prevention of cancer, on the other hand, depend greatly on early detection. Therefore, alternative methods for the treatment and early diagnosis are essential for oral cancer. The detection of tongue squamous cell carcinoma is aided by coupled surface plasmon resonance, which can occur in gold nanoparticles (AuNPs). Compared to the currently utilized imaging contrast chemicals, AuNPs are more biocompatible and capable of targeting specific surface molecules. In the current study, AuNPs were synthesized in one step via citrate reduction and applied to tongue samples of a Caucasian man's Homo sapiens (Squamous cell carcinoma from ATCC cell-lines) in order to improve early detection using and laser-induced fluorescence and Raman spectroscopy.ResultsUV–visible spectroscopy, Zeta potential, TEM, and FTIR spectroscopic technique were used to characterize the synthesized nanoparticles. The synthesized AuNPs measured 13 ± 3 nm with uniform size distribution and high stability. Results demonstrate the significance of AuNPs in improving the identification of tongue squamous cell carcinoma.ConclusionObtained results revealed that the use of AuNPs modifies the emitted spectra in the two employed spectroscopic techniques and provides more significant receiver operating characteristic curve parameters, hence a higher detection rate of cancer.

Abstract 3764: Spatially resolved immune landscape revealed sex-biased T-cell infiltration in glioblastoma

Abstract Glioblastoma (GBM) multiforme is the most aggressive type of brain cancer. These lethal brain tumors are characterized by the inter- and intra-tumor molecular heterogeneity and demonstrated cell plasticity. A male:female distribution of 1.6:1; survival outcome from treatment (debulking, radiation, and temozolomide) benefits females nearly twice that for males. Through spatial transcriptomics, we interrogated the immune landscape of GBM, seeking to tease out whether distinct biological and therapeutic niches in GBM were evident, and to understand how the tumor microenvironments contributed to the heterogeneity of GBM. We designed a set of RNA hybridization probes specifically for portraying the cell identities and cell functional states within GBM tumor. The probe set was used to profile six human GBM samples from male and female cohorts on the Vizgen MERSCOPE platform. An integrated analysis of 505,614 cells from the six samples yielded a rich and varied spatial cell atlas of glioblastoma. Our preliminary findings depict distinct spatial niches of different tumor cell subpopulations that are associated with local environmental factors such as hypoxia. We identified a small cluster of cells that are in neuronal-like state, which could potentially drive invasion. We found spatial proximity of CD4+ cells and cells that express HIF1A, suggesting the dynamic interactions between tumor cells, regulatory T cells and tumor microenvironment. We also observed significant difference in the degree of CD8+ T-cell infiltration in female samples versus male samples. The average CD4+/CD8+ T-cell ratio in males is twice as high as that of females, suggesting intrinsic sex-based differences in immune response to tumor. Expansion of these studies in additional specimens will further illuminate microenvironmental influences on glioma progression and may instruct specific interventions leading to more effective and predictable immune-oncology of this disease. Citation Format: Yue Hao, George Reid, Angela Baker, Karen L. Fink, George J. Snipes, Bruce E. Mickey, Andrew E. Sloan, Jill S. Barnholtz-Sloan, Michael Berens. Spatially resolved immune landscape revealed sex-biased T-cell infiltration in glioblastoma [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 3764.

Abstract 5681: Identification of dysregulated miRNAs involved in TNBC progression

Abstract Triple negative breast cancer (TNBC) is the most aggressive type of all breast cancer subtypes. Although various studies have been performed to identify prognostic and therapeutic targets for TNBC, the TNBC specific biomarkers have not yet been elucidated. Herein, we identified dysregulated miRNAs in TNBC via analysis of miRNA microarray in breast cancer tissues and found the oncogenic RNA (miR-374a-5p) and tumor suppressive miRNAs (miR-320c, miR-371b-5p). These miRNAs were closely related to overall survival of TNBC patients and status and involved in TNBC progression or drug responsiveness. Collectively, these results suggest that dysregulated miRNAs might serve as potential prognostic marker or therapeutic target for TNBC. Citation Format: Minyoung Lee, Yesol Kim, Dasom Son, Sera Lim, Jong Hoon Park. Identification of dysregulated miRNAs involved in TNBC progression [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 5681.

Abstract 1458: Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents a very aggressive type of pancreatic cancer (9% survival rate at 5 years). PDX1 is an important transcription factor for embryonic development of the pancreas, endocrine lineage differentiation and the maintenance of mature beta cells. Notably, in PDAC patients, the expression of PDX1 is downregulated in tumor cells compared to adjacent non-tumoral tissue. Interestingly, increasing tumor PDX1 expression has been shown to enhance the overall survival rate in PDAC patients. Therefore, our aim was to analyze the role of PDX1 on tumor aggressiveness of PDAC cells. To induce PDX1 expression, PANC1 cells were treated with BRD7552 (a PDX1 inducer) for 9 days. Overexpression of PDX1 was confirmed by Western Blot analysis and no cytotoxic effects were observed by MTT or Trypan Blue exclusion assays on treated cells compared to control. Wound healing and transwell migration assays showed a significant reduction in the migration rate compared to the control group. A higher proportion of cells in G1 phase was observed in treated cells compared to control by propidium iodide staining followed by flow cytometry assay. Furthermore, the cell confluence assay showed a significant reduction in proliferation rate in treated cells compared to control, but no differences were observed on the proportion of KI67+ and PH3+ cells by immunostaining. To assess the in ovo effects of BRD7552, treated PANC1 cells were implanted onto the chorioallantoic membrane of chick embryos and tumor growth was measured at different stages observing a significant reduction in tumor growth in PANC1 cells compared to control between days 3 and 8 post implantation. No significant differences in morphology or color were observed between treated and control implanted cells. Hematoxylin-eosin staining showed reduced invasion of treated cells into the chorioallantoic membrane compared to control. In conclusion, the overexpression of PDX1 affects cell cycle, reduces proliferation rate and inhibits migratory potential in vitro. Moreover, in an in ovo model this overexpression diminished tumor growth and invasion in PANC1 cells. In conclusion, the overexpression of PDX1 induced by BRD7552 drives pancreatic ductal adenocarcinoma to a less aggressive tumoral phenotype. Citation Format: Jimena Mosna, Federico Garde, Marcelo Stinson, Candela Pastore, Pablo Sanchis, Abel Carcagno. Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness [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 1458.

Abstract 2521: New predictive markers for dasatinib responsiveness in triple-negative breast cancer: A biomarker discovery study

Abstract Triple negative breast cancer is the most heterogeneous and aggressive type of breast tumor type and is characterized by the lack of expression of clinical biomarkers (ER, PR, and HER2) and targeted therapies. In this regard, different clinical trials have failed, unable to stratify these patients to find an effective response to specific therapy. This study aimed to identify biomarkers exclusively expressed in the basal mammary compartment that can help us to subclassify the triple-negative breast cancer subtype. Based on computational analysis from single-cell RNA sequencing data, we have identified a list of basal identity-associated genes (BC-markers). Subsequent histological validation confirmed the expression pattern of these markers in a cohort of 50 samples, which has allowed us to stratify triple-negative breast cancer patients into BC-TNBC and luminal-like subtypes. Importantly, the expression of these markers correlated with poorer prognosis in TNBC patients. Functional analysis revealed that TAGLN played a significant role in cell proliferation and migration, potentially impacting tumor aggressiveness. Moreover, the expression of these BC-markers is associated with a mesenchymal phenotype which corresponds to the previously described Lehmannn’s mesenchymal-like signature. In this study, TAGLN appeared to influence dasatinib sensitivity, suggesting its potential as a predictive biomarker for dasatinib response in TNBC patients. Citation Format: Daniel Ortega-Álvarez, David Tebar, Marta Casado, Elena Castillo, Cristina Guardia, David Olivares-Osuna, Eva Musulén, Elisabetta Mereu, Ginés Luengo, Eva M. Galán-Moya, Veronica Rodilla. New predictive markers for dasatinib responsiveness in triple-negative breast cancer: A biomarker discovery study [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 2521.