Resistant Cell Lines Research Articles

Activated interferon response from DNA damage in multiple myeloma cells contributes to the chemotherapeutic effects of anthracyclines

IntroductionMultiple myeloma (MM) is a malignant plasma cell disease caused by abnormal proliferation of clonal plasma cells in bone marrow. Upfront identification of tumor subgroups with specific biological markers has the potential to improve biologically-driven therapy. Previously, we established a molecular classification by stratifying multiple myeloma into two subtypes with a different prognosis based on a gene module co-expressed with MCL-1 (MCL1-M).MethodsGene Ontology (GO) analysis with differentially expressed genes was performed to identify signal pathway. Drug sensitivity was analyzed using the OncoPredict algorithm. Drug sensitivity of different myeloma cell lines was detected by CCK8 and flow cytometry. RNA-seq was performed on drug-sensitive cell lines before and after adriamycin treatment. RT-qPCR was used to further verify the sequencing results. The expression of γ-H2AX and dsDNA in sensitive and resistant cell lines was detected by immunofluorescence method.ResultsIn our study, we demonstrated that MCL1-M low MM were more sensitive to anthracyclines. We treated different myeloma cell lines with doxorubicin in vitro and discovered the association of drug sensitivity with IFN signaling. Herein, we demonstrate that the doxorubicin-sensitive myeloma cell line showed significant DNA damage and up-regulated expression of genes related to the IFN response, which was not observed in drug-insensitive cell lines.DiscussionOur results suggest that the active IFN signaling pathway may serve as a marker for predicting chemotherapy sensitivity in patients with myeloma. With our MCL1-M molecular classification system, we can screen patients with a potentially good response to the interferon signaling pathway and provide individualized treatment for MM. We propose IFN-a as adjuvant therapy for patients with myeloma sensitive to anthracyclines to further improve the therapeutic effect and prolong the survival of patients.

Cyclic Sesquiterpene-Flavanone [4+2] Hybrids, Syzygioblanes A-C, Found in an Indonesian Traditional Medicine, "Jampu Salo" (Syzygium oblanceolatum).

A plant used in an Indonesian traditional herbal medicine as a diabetes treatment and known locally as "Jampu Salo" was collected on Sulawesi Island, Indonesia. It was identified as Syzygium oblanceolatum (C. B. Rob.) Merr. (Myrtaceae) and found for the first time in Sulawesi; it was previously reported only in the eastern Philippines and Borneo. A phytochemical study of S. oblanceolatum led to the isolation of three unprecedented meroterpenoids, syzygioblanes A-C (1-3, respectively). These compounds might be biosynthesized through [4+2] cycloaddition of various germacrane-based cyclic sesquiterpenoids with the flavone desmethoxymatteucinol to form a spiro skeleton. The unique and complex structures were elucidated by microcrystal electron diffraction analysis in addition to general analytical techniques such as high-resolution mass spectrometry, various nuclear magnetic resonance methods, and infrared spectroscopy. Synchrotron X-ray diffraction and calculations of electronic circular dichroism spectra helped to determine the absolute configurations. The newly isolated compounds exhibited collateral sensitivity to more strongly inhibit the growth of a multidrug resistant tumor cell line compared to a chemosensitive tumor cell line.

Transposable elements alter gene expression and may impact response to cisplatin therapy in ovarian cancer.

Cisplatin is widely employed for cancer treatment; therefore, understanding resistance to this drug is critical for therapeutic practice. While studies have delved into differential gene expression in the context of cisplatin resistance, findings remain somewhat scant. We performed a comprehensive investigation of Transposable Elements (TEs) expression and their impact in host genes in two cisplatin-treated ovarian cancer cell lines. RNA-seq, ATAC-seq, and in-depth bioinformatics analysis were used to compare cisplatin-sensitive and -resistant ovarian cancer cell lines. Our results reveal that cisplatin therapy alters the expression of protein-coding genes, but also key TEs, including LINE1, Alu, and endogenous retroviruses, in both cisplatin-sensitive and -resistant cell lines. By co-expressing with downstream genes or by creating chimeric transcripts with host genes at their insertion sites, these TEs seem to control the expression of protein-coding genes, including tumor-related genes. Our model uncovers TEs influencing the expression of cancer genes and cancer pathways. Collectively, our findings indicate that TEs alterations associated with cisplatin treatment occur in critical cancer genes and cellular pathways synergically. This research highlights the importance of considering the entire spectrum of transcribed elements in the genome, especially TE expression, for a complete understanding of complex models like cancer response to treatment.

CircHIF1A induces cetuximab resistance in colorectal cancer by promoting HIF1α-mediated glycometabolism alteration

Epidermal growth factor receptor (EGFR)-targeted therapy is an important treatment for RAS wild-type metastatic colorectal cancer (mCRC), but the resistance mechanism remains unclear. Here, the differential expression of circRNAs between Cetuximab sensitive and resistant cell lines was analyzed using whole-transcriptome sequencing. We identified that the expression of circHIF1A was significantly higher in LIM1215-R than in LIM1215. When treated with Cetuximab, downregulation of circHIF1A level weakened the proliferation and clonal formation ability of LIM1215-R, caused more cells to enter G0-G1 phase, and significantly reduced the basal respiration, ATP production, and maximal respiration, as well as the glycolytic capacity and glycolytic reserve. The response rate and prognosis of circHIF1A-positive patients were inferior to those of negative patients. Mechanistically, circHIF1A can upregulate the level of hypoxia-inducible factor 1 A (HIF1A) by competitively binding to miR-361-5p, inducing the overexpression of enzymes such as glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA). In a xenograft model, inhibition of circHIF1A expression increased the sensitivity to Cetuximab treatment. In conclusion, circHIF1A can promote HIF1α-mediated glycometabolism alteration to induce Cetuximab resistance in CRC. It has the potential to become a screening indicator for the Cetuximab beneficial population in mCRC and a new therapeutic target for enhancing treatment efficacy.

Modulation of Epithelial–Mesenchymal Transition Is a Possible Underlying Mechanism for Inducing Chemoresistance in MIA PaCa-2 Cells against Gemcitabine and Paclitaxel

To elucidate the currently unknown molecular mechanisms responsible for the similarity and difference during the acquirement of resistance against gemcitabine (GEM) and paclitaxel (PTX) in patients with pancreatic carcinoma, we examined two-dimensional (2D) and three-dimensional (3D) cultures of parent MIA PaCa-2 cells (MIA PaCa-2-PA) and their GEM resistance cell line (MIA PaCa-2-GR) and PTX resistance (MIA PaCa-2-PR). Using these cells, we examined 3D spheroid configurations and cellular metabolism, including mitochondrial and glycolytic functions, with a Seahorse bio-analyzer and RNA sequencing analysis. Compared to the MIA PaCa-2-PA, (1) the formation of the 3D spheroids of MIA PaCa-2-GR or -PR was much slower, and (2) their mitochondrial and glycolytic functions were greatly modulated in MIA PaCa-2-GR or -PR, and such metabolic changes were also different between their 2D and 3D culture conditions. RNA sequencing and bioinformatic analyses of the differentially expressed genes (DEGs) using an ingenuity pathway analysis (IPA) suggested that various modulatory factors related to epithelial –mesenchymal transition (EMT) including STAT3, GLI1, ZNF367, NKX3-2, ZIC2, IFIT2, HEY1 and FBLX, may be the possible upstream regulators and/or causal network master regulators responsible for the acquirement of drug resistance in MIA PaCa-2-GR and -PR. In addition, among the prominently altered DEGs (Log2 fold changes more than 6 or less than −6), FABP5, IQSEC3, and GASK1B were identified as unique genes associated with their antisense RNA or pseudogenes, and among these, FABP5 and GASK1B are known to function as modulators of cancerous EMT. Therefore, the observations reported herein suggest that modulations of cancerous EMT may be key molecular mechanisms that are responsible for inducing chemoresistance against GEM or PTX in MIA PaCa-2 cells.

Abstract PO3-14-05: Integration of metabolomics and transcriptomics to Reveal Potential Biomarkers Associated with Treatment Response of Neoadjuvant Therapy in HER2+ Breast Cancer

Abstract Background: Neoadjuvant therapy (NAT) has become the standard treatment for early human epidermal growth factor receptor 2 positive (HER2+) breast cancer. For patients with HER2+ breast cancer, achieving a pathologic complete response (pCR) is highly indicative of their prognosis. However, not all HER2+ breast cancer patients could benefit from the NAT, about 40%-50% patients could not achieve pCR at surgery, even if they used the present standard neoadjuvant regime of TCbHP (taxane, carboplatin, trastuzumab and pertuzumab). This study was conducted to explore potential indicators associated with neoadjuvant efficacy of TCbHP in HER2+ breast cancer. Methods: Consecutive HER2+ breast cancer patients, who received and completed NAT with TCbHP regime and subsequent surgery at Chongqing University Cancer Hospital were prospectively enrolled. LC-MS and GC-MS platform-based untargeted metabolomics were performed to determine the metabolic profiles of plasma samples from these patients at different time points during treatment period. Random forest (RF) was used to establish predictive models based on pre-therapeutic metabolic traits. The potential monitors for the treatment response were obtained by time series analysis. Transcriptome analysis was performed in available samples to identify differentially expressed genes (DEGs) before treatment. qRT-pCR was used to detect DEGs in tastuzumab-sensitive and -resistant cell lines. Metabolic and transcriptomic data were integrated to explore substantially altered pathways that might be responsible for drug resistance. Results: From July 20, 2020 to May 28, 2021, a total of 40 HER2+ breast cancer patients with 120 plasma samples were eligible and recruited for this study. Of whom 21 (52.5%) patients achieved pCR and 19 (47.5%) achieved non-pCR, there are no significant difference in baseline clinicopathological features between pCR and non-pCR patients. There were significant differences in plasma metabolic profiles between non-pCR and pCR groups before and during treatment. A total of 100 differential metabolites were identified between pCR and non-pCR patients at pre-therapeutic period, which were markedly enriched in 40 metabolic pathways. Four key metabolites [sophorose, N-(2-acetamido)iminodiacetic acid, taurine and 6-hydroxy-2-aminohexanoic acid] were selected by RF analysis. The AUC value to discriminate pCR and non-PCR group to NAT of the single potential metabolite or combined panel of these metabolites were more than 0.910. 18 metabolites exhibit a potential for monitoring efficacy. Among 163 DEGs identified by RNA-seq, some genes might associate with trastuzumab resistance detected by qRT-PCR in cell lines. 39 altered pathways were found abnormally expressed at both the transcriptional and metabolic levels, including ABC transporters, protein digestion and absorption, mineral absorption, et al. Conclusion: By metabolomics analysis, we have both offered a road map of the molecular alterations underlying NAT of TCbHP regime in HER2+ breast cancer and a preliminary analysis of a unique metabolic signature that might be used to distinguish non-pCR from pCR patients. Metabolomics integrated with transcriptomics analysis could assist for gaining new insights into biochemical pathophysiology and might facilitate the development of new treatment targets for insensitive patients. Citation Format: Ningning Zhang, Yuxin Huang, Guanwen Wang, Yimei Xiang, Zhouhong Jing, Junjie Zeng, Feng Yu, Xianjun Pan, Wenqi Zhou, Xiaohua Zeng. Integration of metabolomics and transcriptomics to Reveal Potential Biomarkers Associated with Treatment Response of Neoadjuvant Therapy in HER2+ Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-05.

Abstract PO4-23-11: Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing

Abstract Background Understanding the mechanisms of resistance to CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is pivotal in exploring new therapeutic strategies for hormone receptor-positive (HR+), HER2-negative metastatic breast cancer. To decipher these resistance mechanisms, we analyzed the alterations in comprehensive RNA-seq (coding and non-coding RNAs) of HR+ HER2-negative BC cell lines via thermostable group II intron reverse transcriptase sequencing (TGIRT-seq). Methods We established Tamoxifen-resistant (TMR), Abemaciclib-resistant (ACR), Palbociclib-resistant (PCR), Tamoxifen/Abemaciclib double-resistant (TMR-ACR), and Tamoxifen/Palbociclib double-resistant (TMR-PCR) BC cell lines from MCF7 and T47D HR+/HER2- BC cell lines through stepwise dose-escalation continuous drug exposure. We performed a TGIRT-seq transcriptomic analysis using a protocol that allows the sequencing of both long and short non-coding and protein-coding RNAs in a single library. All libraries were sequenced using paired-end 150 bp on the Novaseq platform, resulting in an average of 50 million reads per library. For analysis, raw reads underwent adapter trimming, small RNA mapping, whole genome mapping, and the generation of annotated genes' read count. We used raw counts to detect differentially expressed genes (DEGs) with DESeq2 in R using the cut-off (log2 [fold change] > 1, FDR < 1e-3). We then used the selected DEGs for Gene Set Enrichment Analysis (GSEA) and Kaplan-Meier survival analysis from the TCGA database. Results The TGIRT-seq analysis identified 1171 to 3472 DEGs in different drug resistance and cell line combinations. Most DEGs in resistant cells were consistently downregulated compared to parent cells. Seventy-five percent of DEGs were protein-coding genes, with the rest being non-coding RNAs, such as small nucleolar RNAs, microRNAs, long non-coding RNAs, and transposable element RNAs, which conventional RNA-seq poorly detects. In principal component analysis (PCA) plots, replicates per drug resistance clustered clearly in both cell line backgrounds. PCA also indicated distinctive pathways for acquiring drug resistance in MCF7 and T47D cells. The estrogen receptor (ER) expression decreased, while HER2 increased in resistant cell lines. DEGs identified were enriched in ESR1-related pathways in all MCF7 and T47D cell lines resistant models. Several ER-regulating genes like IL-1R1 and RET were upregulated, while ADCY1 was consistently downregulated across different resistance types. A significant overlap with single-resistance DEGs was observed among the DEGs in double-resistance cell lines, but 20-50% of up/down-regulated DEGs in double-resistance cell lines were uniquely altered. Several previously identified DEGs (e.g., SALL4, TOP2A) and 21 novel candidate genes correlated with poor survival outcomes. Conclusion The analysis identified unique DEGs in double resistance cell lines, suggesting that double resistance mechanisms may not merely be a cumulative effect of single resistance mechanisms, necessitating further investigation and validation. CDK4/6i and/or ET-resistant BC cell lines displayed significant transcriptome reprogramming during the development of drug resistance. In previously published research, ESR1-related pathway alterations were proposed in tamoxifen resistance cell lines. Here we find that CDK4/6i-resistant cells also modify these ESR1-related pathways. Additionally, we identified several targetable genes, such as IL-1R1 and RET, involved in ESR1-related pathways that could pave the way for developing new treatment strategies. Citation Format: Toshiaki Iwase, Hengyi Xu, Nakyung Oh, Jangsoon Lee, Alan M Lambowitz, Naoto Ueno. Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-11.

Abstract PO5-18-07: A Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits TNBC Lung Metastases

Abstract Introduction: A promising novel target for breast cancer is secreted frizzled-related protein 2 (SFRP2). SFRP2 protects tumors against apoptosis, promotes T-cell exhaustion, and induces angiogenesis. SFRP2 is expressed in breast cancer but has not been specifically evaluated in triple negative breast cancer. We previously developed a humanized monoclonal antibody to SFRP2, (hSFRP2 mAb) that inhibits primary breast cancer growth in mice without toxicity. The purpose of this study is to establish the presence of SFRP2 in human TNBC, evaluate the efficacy of hSFRP2 mAb in metastatic TNBC in vivo, and in a cell line resistant to chemotherapy. Methods: Immunohistochemistry (IHC) with SFRP2 antibody on a human TNBC tissue microarray (TMA) containing 88 FFPE cores. After IHC with primary antibody to SFRP2, the TMA was scanned and staining intensity was quantified using spatial analysis. Results for SFRP2 staining were analyzed as previously described for analyses of estrogen and progesterone receptor positivity; categories are: 0-absence of staining; low positive (0-10%), and positive >10%. Development of a doxorubicin (dox)-resistant cell line and induction of apoptosis with hSFRP2 mAb. MDA-MB-231 cells were treated with 4nM dox and increased 2nM at a time until the cells were resistant to 10µM dox after 12 months. WT and dox-resistant cells were treated for 4 hours with either hSFRP2 mAb (10uM,) or IgG control (10uM) (n=6). Apoptosis was detected with Promokine Apoptosis/Necrosis/Healthy cells kit. Cells were imaged using the EVOS FLc and counted using Imagej software. Differences between treated and control were compared with two-tailed T-test. hSFRP2 mAb treatment of mice with metastatic TNBC. PY8119 and E0771 TNBC murine cells (5 × 105/100 μL) were injected iv into 6–8-week-old C57BL/6 female mice. The next day the mice were treated with IgG1 control 8 mg/kg iv or hSFRP2 mAb 8 mg/kg iv every 3 days. After four weeks, the lungs were resected, and surface metastases were counted. Differences between treated and control were compared with Poisson distributions. FFPE sections of lungs were stained with ApopTag kit. Three metastases per lung were counted at 20 X, and the difference between IgG1 and hSFRP2 mAb groups compared with two-tailed T-test. Results: SFRP2 protein is present in human TNBC. Out of 88 cores, 83 cores (94%) were positive, 4 cores (4.5%) were minimally positive, and 1 core (1.1%) was negative for SFRP2. Development of a doxorubicin (dox)-resistant cell line and induction of apoptosis with hSFRP2 mAb treatment in vitro. The number of apoptotic cells in IgG1 control-treated MDA-MB-231 cells was 11.5 ± 3.7, and 76.6 ± 5.0 for hSFRP2 mAb-treated tumors (p< 0.001, n=9). The mean number of apoptotic MDA-MB-231 dox-resistant cells in IgG1 control was 16.7 ± 6.0, and 68.4 ± 6.4 for hSFRP2 mAb-treated tumors, (p< 0.001, n=9). hSFRP2 mAb inhibits the growth of metastatic TNBC in vivo and increases apoptosis. For E0771, the number of surface metastases was 8.9 ±2.8 in the IgG1 control group and 4.9 ± 1.1 in the hSFRP2 mAb treated group (p< 0.05, n=15). The number of apoptotic cells/HPF was 9.8 ± 1.8 for the IgG1 control group and 21.0 ± 4.6 for the hSFRP2 mAb group (n=15, p< .01). For Py8119 tumors there were 6.4 ± 1.0 metastases in the IgG1 control group and 3.6 ± 0.9 in the hSFRP2 mAb group (p< 0.05, n=11). The number of apoptotic cells/HPF was 6 ± 1.8 for the IgG1 control group and 14 ±1.3 for the hSFRP2 mAb group (n=10, p< 0.001). Conclusions: SFRP2 protein is abundantly expressed in human TNBC. A humanized monoclonal antibody to SFRP2 reduces TNBC lung metastases growth in two in vivo models and increases tumor apoptosis. TNBC resistant to doxorubicin remains sensitive to apoptosis induced by the hSFRP2 mAb. Citation Format: Lillian Hsu, Patrick Nasarre, Julie Siegel, Eleanor Hilliard, Rupak Mukherjee, Nancy Klauber-Demore. A Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits TNBC Lung Metastases [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-18-07.

Abstract PO5-27-09: Pharmacological eIF4E inhibition suppresses anti-tumor activity in ER+ breast cancer

Abstract Elevated levels of eukaryotic initiation factor 4E (eIF4E) are found in a broad range of cancers, including breast, and have been associated with aggressive, drug resistant tumors. Furthermore, elevated eIF4E activity is sufficient to cause transformation in various cancers, while inhibition suppresses tumor growth. eIF4E, the main regulator and rate limiting factor of protein synthesis, is the downstream integrator of several important oncogenic signaling pathways (PI3K/AKT/mTOR, Ras/Raf/MEK, and Myc). Activation of eIF4E results in increased protein levels of key proliferation and metabolism proteins such as Cyclin D1 (CCND1) and Ornithine Decarboxylase 1 (ODC1), as well as an overall increase in cellular synthesis machinery, causing cellular growth and proliferation. Here, we describe the development and characterization of novel, potent, and selective eIF4E inhibitors. Compounds from this chemical series have nanomolar activity in multiple biochemical and biophysical m7G cap-competition assays as well as potent inhibition of translation in cellular and biochemical assays. In cells, these compounds rapidly and reversibly decrease protein levels of several oncogenes, including CCND1, leading to a G1 cell cycle arrest. We demonstrate that eIF4E inhibitors cause growth inhibition in a variety of breast cancer cell lines, including ER+ breast cancer. These inhibitors also inhibit growth of breast cancer lines with acquired resistance to Palbociclib with the same potency as the parental cell lines. Additionally, eIF4E inhibition shows an increased sensitivity in resistant cell lines when combined with standard of care (SOC), including Palbociclib. Select analogs from the series demonstrate favorable ADMET/PK properties with good oral bioavailability and low safety risk. Lastly, these compounds have demonstrated near complete tumor growth inhibition in non-breast cancer in vivo models. Ongoing experiments will address in vivo efficacy in ER+ breast cancer in both monotherapy and in combination with SOC. Citation Format: Matthew Friedersdorf, Devon Blake, Sarah Thompson, Krista Marran, Jessica Sorrentino. Pharmacological eIF4E inhibition suppresses anti-tumor activity in ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-09.

Abstract PO3-28-12: Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance

Abstract Standard of care for breast cancer (BC) treatment including Selective-Estrogen Receptor (ER)-modulators and/or degraders (SERM; tamoxifen, SERD;Fulvestrant) or cyclin-dependent kinases CDK4 and CDK6 (palbociclib) can be effective therapeutic strategies. However, drug toxicity and/or drug resistance severely limit these approaches and patients succumb to the disease. Therefore, there is an urge to develop new therapeutic strategies against ER+-BC. Ion channels are general pharmacological targets however, little is known about the role of this class of proteins in cancer biology. We discovered that expression of the KCa3.1 potassium ion channel is downregulated in ER+-BC. Also, patients expressing high level of KCa3.1 present a better overall survival (OS) and relapse free survival when compared with patients with low expression. Thus, these data indicate that KCa3.1 is a prognostic factor in ER+-BC and that high activity of this channel plays a fundamental role in an oncosuppressor signature. Our central hypothesis is that pharmacological targeting KCa3.1 with activator molecules arrests growth of ER+-BC. We discovered that ER+-BC patient-derived organoids (PDO) and cell lines treated with KCa3.1 agonists including the FDA approved Chlorzoxazone, (CZX) arrests significantly inhibit tumor growth. Activation of KCa3.1 arrested the cell cycle in the G0/G1 phase by activating a senescent-like phenotype. Furthermore, our studies on the biochemical signaling that underlines this event demonstrated that increased expression level of KCa3.1 protein associated with lower expression level of ERa in a cell cycle phase-dependent manner. Furthermore, use of KCa3.1activator produced a proteasomal-dependent ERa degradation. To better characterize the therapeutic benefit of targeting the KCa3.1, we assessed the effects of CZX on developed several tamoxifen and/or palbociclib resistant cell lines and PDO models. We found that the combination of CZX strongly potentiated the lethal effects of both tamoxifen and palbociclib in the relative resistant models. We conclude that pharmacological targeting KCa3.1 potassium channel in ER+-BC can be considered a novel, safe and efficacious therapeutic strategy. Citation Format: Davide Delisi, Metin Cetin, Ozge Saatci, Ozgur Sahin, Saverio Gentile. Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-28-12.

Abstract PO2-14-10: A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model

Abstract Background Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses but up to 30% of breast cancer-related deaths. Similarly, Inflammatory breast cancer (IBC) accounts for 2-4% of breast cancer diagnosis but causes 8%–10% of all breast cancer-related deaths. The myeloid cell leukemia-1 (MCL-1) is a member of the BCL-2 family of proteins, which is highly amplified in numerous human cancers and associated with cell immortalization, transformation, and chemoresistance. In fact, MCL-1 is amplified in 55% of TNBC after preoperative chemotherapy. Amongst the MCL-1 inhibitors that have been developed, AZD5991 showed high selectivity and affinity for MCL-1. In myeloma and acute myeloid leukemia cells AZD5991 induces apoptosis and showed anti-tumor effects in in vivo models. In the present study, we evaluated the biological role of the MCL-1 inhibitor AZD5991 in IBC and TNBC in vitro and in vivo models. Materials and methods The expression of the MCL-1 gene was analyzed in the World Consortium IBC patient dataset. Next, we screened a panel of TNBC and IBC cell lines for MCL-1 expression by western blot analysis. The effect of AZD5991 on TNBC and IBC cells was analyzed via cell titer-blue proliferation, clonogenic assays and soft agar assay. To further validate these findings, we used siRNA to knockdown MCL-1 and performed cell proliferation, clonogenic and migration assays. To identify signaling pathways involved in MCL-1 Inhibitor’s effects we performed a proteome profiler human apoptosis array in TNBC cells in the presence of AZD5991. The anti-tumor effect of the MCL-1 inhibitor was evaluated using a SUM-149 (TN-IBC) orthotopic xenograft mouse model. The mice were treated with AZD5991 at doses of 15 and 30mg/kg once weekly via tail vein injection for a duration of 4 weeks. Results The mean expression level of MCL-1 was higher in patients with TNBC than in those with non-TNBC. Further, compared to the patients with ER positive, HER-2 negative tumors those with TNBC showed an increased in the mean expression of MCL-1 gene (p value= 0.05). We screened a panel of TNBC and IBC cell lines in which most of the cell lines showed high levels of MCL-1. The cell proliferation assay showed IC50 ranging from 0.27 – 19.6 μM. The SUM149, SUM190, FCIBC02, MDA-IBC3 and MDA-MB-468 cells showed high sensitivity and showed significant decrease in the proliferation rate while BCX010, KPL4, MDA-MB-231, 4T1.2 and E0771 cells as well as the normal breast cells MCF10A exhibited resistance with no significant decrease in proliferation. The clonogenic and soft agar assays with these sensitive and resistant cell lines showed similar results. Further, these findings were validated via genetic knockdown of MCL-1 which resulted in a significant decrease in cell proliferation, colony formation and migration capability of the TNBC cells. To identify signaling pathways involved in MCL-1 Inhibitor’s phenotypic changes, we performed a proteome profiler human apoptosis array which showed a decreased in BCLx expression, and an increase in expression of BAX and phospho-Rad17 proteins in the AZD5991 treated TN-IBC cells. Finally, in a SUM-149 (TN-IBC) orthotopic xenograft mouse model, mice injected with 30mg/kg showed a significant tumor regression compared to the vehicle-treated mice (vehicle vs 30 mg/kg, p value=0.0006). Conclusions and Future directions Our data demonstrates that AZD5991 inhibits tumorigenesis in an inflammatory breast cancer xenograft model. We are currently validating several significant targets to understand the biological mechanisms related to our candidate drug. Our long-term goal is to develop MCL-1 targeted therapy in combination with standard-of-care treatment for inflammatory and triple-negative breast cancers. Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Michael White, Bisrat Debeb, Emilly Villodre, Xiaoding Hu, Debu Tripathy, Wendy Woodward, Rachel Layman, Geoffrey Bartholomeusz, Chandra Bartholomeusz. A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-10.

Abstract PO4-23-10: Restoring the efficacy of standard of care in treatment-refractory ER+ breast cancer by re-activation of cAMP-ROS-DNA damage axis

Abstract Estrogen receptor (ER)-positive (ER+) breast cancer has the highest incidence rate and accounts for around 75% of all cases. Endocrine therapy has been the mainstay therapy for the treatment of both early and late-stage ER+ breast cancer for decades. Although most ER+ breast cancer patients initially respond well to endocrine therapy, resistance is common. The addition of CDK4/6 inhibitors to endocrine therapy led to significant improvements in clinical outcome, and they are now considered one of the standard of care (SOC) therapies. However, a significant proportion of patients still suffer from disease relapse upon prolonged use of endocrine therapy in combination with CDK4/6 inhibitors, representing a major clinical challenge that reduces the long-term benefit and patient survival. Therefore, elucidating the mechanisms of sensitivity and resistance to SOC therapy and identifying novel actionable targets are urgently needed. Here, we show that endocrine therapies and CDK4/6 inhibitors cause toxic PARP1 trapping and generation of a functional BRCAness phenotype by downregulating key DNA repair proteins that ultimately result in increased histone parylation and reduced H3K9 acetylation, leading to transcriptional blockage and cell death. Mechanistically, we found that SOC therapy downregulates phosphodiesterase 4D (PDE4D), resulting in increased cAMP levels, PKA-dependent phosphorylation of mitochondrial COXIV-I, generation of mitochondrial reactive oxygen species (ROS), and DNA damage. Importantly, we identified PDE4D as a novel ER target gene that in turn stimulates ER activity in a feedforward loop in endocrine-responsive models and regulates BRCA1 expression. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun. Inhibition of PDE4D using BPN14770, the first-in-class PDE4D allosteric inhibitor that has successfully completed Phase II trials in Fragile X syndrome, or its upstream EGFR in combination with SOC therapies in drug-resistant settings, including multiple acquired SOC resistant cell line models, primary cultures and organoids of endocrine resistant ER+ PDXs reinstates PARP1 trapping and BRCAness, leading to drug sensitization in vitro and in vivo. Notably, we demonstrated that high PDE4D mRNA and protein expression is associated with dramatically worse disease-free survival and overall survival in endocrine therapy-treated ER+ breast cancer. Considering the availability of potent and non-toxic PDE4D inhibitors, clinically approved EGFR and PARP1 inhibitors, our findings have great translational potential. Citation Format: Ozge Saatci, Metin Cetin, Meral Uner, Unal Metin Tokat, Ioulia Chatzistamou, Pelin Gulizar Ersan, Elodie Montaudon, Aytekin Akyol, Sercan Aksoy, Aysegul Uner, Elisabetta Marangoni, Sajish Mathew, Ozgur Sahin. Restoring the efficacy of standard of care in treatment-refractory ER+ breast cancer by re-activation of cAMP-ROS-DNA damage axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-10.

Bioinformatics and In Vitro Study Reveals ERα as The Potential Target Gene of Honokiol to Enhance Trastuzumab Sensitivity in HER2+ Trastuzumab-Resistant Breast Cancer Cells

Trastuzumab resistance presents a significant challenge in the treatment of HER2+ breast cancer, necessitating the investigation of combination therapies to overcome this resistance. Honokiol, a compound with broad anticancer activity, has shown promise in this regard. This study aims to discover the effect of honokiol in increasing trastuzumab sensitivity in HER2+ trastuzumab-resistant breast cancer cell line HCC1954 and the underline mechanisms behind. A bioinformatics study performed to explore the most potential target hub-gene for honokiol in HER2+ breast cancer. Honokiol, trastuzumab and combined treatment cytotoxicity activity was then evaluated in both parental HCC1954 and trastuzumab resistance (TR-HCC1954) cells using MTT assay. The expression levels of these hub-genes were then analysed using qRT-PCR and those that couldn’t be analysed were subjected to molecular docking to determine their potential. Honokiol showed a potent cytotoxicity activity with an IC50 of 41.05μM and 69.61μM in parental HCC1954 and TR-HCC1954 cell line respectively. Furthermore, the combination of honokiol and trastuzumab resulted in significant differences in cytotoxicity in TR-HCC1954 cells at specific concentrations. Molecular docking and the qRT-PCR showed that the potential ERα from the bioinformatics especiallystudy were affected by the treatment. Our results show that honokiol has the potential to increase the sensitivity of trastuzumab in HER2+ trastuzumab resistant breast cancer cell line HCC1954 by affecting regulating estrogen receptor signaling. Further research with expanded data sets is necessary to validate these findings.

Mcl-1 mediates intrinsic resistance to RAF inhibitors in mutant BRAF papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is the most frequent form of thyroid cancer. PTC commonly presents with mutations of the serine/threonine kinase BRAF (BRAFV600E), which drive ERK1/2 pathway activation to support growth and suppress apoptosis. PTC patients often undergo surgical resection; however, since the average age of PTC patients is under 50, adverse effects associated with prolonged maintenance therapy following total thyroidectomy are a concern. The development of mutant-selective BRAF inhibitors (BRAFi), like vemurafenib, has been efficacious in patients with metastatic melanoma, but the response rate is low for mutant BRAF PTC patients. Here, we assay the therapeutic response of BRAFi in a panel of human PTC cell lines and freshly biopsied patient samples. We observed heterogeneous responses to BRAFi, and multi-omic comparisons between susceptible and resistant mutant BRAF PTC revealed overrepresented stress response pathways and the absence of compensatory RTK activation – features that may underpin innate resistance. Importantly, resistant cell lines and patient samples had increased hallmarks of failed apoptosis; a cellular state defined by sublethal caspase activation and DNA damage. Further analysis suggests that the failed apoptotic phenotypes may have features of “minority mitochondrial outer membrane permeabilization (MOMP)” – a stress-related response characterized by fragmented and porous mitochondria known to contribute to cancer aggressiveness. We found that cells presenting with minority MOMP-like phenotypes are dependent on the apoptotic regulator, Mcl-1, as treatment with the Mcl-1 inhibitor, AZD5991, potently induced cell death in resistant cells. Furthermore, PI3K/AKT inhibitors sensitized resistant cells to BRAFi; an effect that was at least in part associated with reduced Mcl-1 levels. Together, these data implicate minority MOMP as a mechanism associated with intrinsic drug resistance and underscore the benefits of targeting Mcl-1 in mutant BRAF PTC.

Abstract P09: AN EX-VIVO COMBINATION DRUG SENSITIVITY PLATFORM FOR PREDICTING SENSITIVITY TO FLT3 INHIBITOR-BASED COMBINATION IN ACUTE MYELOID LEUKEMIA

Abstract FMS-like tyrosine kinase-3 (FLT3) mutations are often linked with poor prognosis in Acute myeloid leukemia (AML). Despite increasing availability of targeted therapy, such as FLT3 inhibitors (FLT3i), remission is often short-term and followed by relapse and drug resistance. With varying response rates to standard treatments, the need to understand the molecular mechanisms driving drug resistance and accurately predict responses to treatment is crucial. Here, we present the ability of Quadratic phenotypic optimization platform (QPOP), in accurately identifying potential responders to possible combination therapies in AML. AML cell lines and AML patient samples were treated with a set panel of drugs including FLT3i. QPOP drug combination scores and ranking were derived from cell viability response following treatment. 40 patients were recruited for the study, of which 20 patients were evaluated for their clinical concordance. RNA sequencing and molecular analysis was further conducted to determine the accuracy of QPOP predicted response and to determine mechanism of resistance to FLT3i. QPOP predicted response was concordant to patient treatment outcome with 80% sensitivity and specificity. QPOP analysis on AML cell lines accurately identified responders to FLT3i including the differential response between a FLT3- ITD isogenic parental and FLT3i resistant cell line. Serial QPOP analysis in a FLT3 mutant patient sample indicated a decrease in FLT3i sensitivity concordant with an increasing FLT3 allelic burden and the development of resistance. The development of resistance to FLT3i based combinations was correlated to an increase in phosphorylated AKT expression. Activation of AKT is associated with the upregulation of Forkhead box M1 (FoxM1) in the resistant cell line. Co-treatment with an AKT inhibitor restored sensitivity to FLT3i treatment in the resistant cells. We demonstrate that QPOP analysis accurately predicts sensitivity to drug-combinations and can be used as a tool to identify sensitivity to FLT3i-based combination therapy in AML. Citation Format: Noor Rashidha Bte Meera Sahib, Jen Wei Ying, Melissa Ooi, Yen Lin Chee, Jayalakshmi, Jameelah Bte Sheik Mohamed, Masturah Rashid, Edward KH Chow. AN EX-VIVO COMBINATION DRUG SENSITIVITY PLATFORM FOR PREDICTING SENSITIVITY TO FLT3 INHIBITOR-BASED COMBINATION IN ACUTE MYELOID LEUKEMIA [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P09.

Abstract P07: Extracellular Vesicles Secreted from Daratumumab Resistant Cells Promote Resistance and Proliferation of Daratumumab Sensitive cells, possibly Through the Transfer of miRNA Cargo

Abstract Daratumumab (dara) is an anti-CD38 monoclonal antibody used for Multiple Myeloma (MM) treatment, but responses are heterogeneous, and resistance is inevitable. We established three dara resistant (dara R) cell lines in Natural Killer T-cell Lymphoma (NKTL), T-cell acute lymphoblastic leukaemia (T-ALL) and MM via long-term exposure to increasing concentrations of dara. We have previously demonstrated that the exosome biogenesis pathway plays a significant role in the survival of dara R cell lines. Coculture of dara sensitive (dara S) cell lines with dara R cell lines separated by a cell-impermeable transwell insert promoted cell proliferation and reduced response to dara treatment in dara S cell lines thereby suggesting a potential role for extracellular vesicles (EVs) in mediating these responses. To assess the specific role of EVs, we isolated dara R EVs and added it to the culture of dara S cells. We observed that uptake of dara R EVs directly promote proliferation and reduced cytotoxic response to dara treatment in dara S cells. In this study, we seek to investigate the role of EVs secreted by dara R cells, in particular, that of EV-derived miRNAs. Exosomal miRNAs are known to play a role in regulating cellular pathways driving cancer cell resistance and survival. To evaluate the potential role of EV-derived miRNA cargo, we performed miRNA sequencing of EVs isolated from the plasma fraction of bone marrow samples obtained from MM patients. We found that the EVs from patients with a progression free survival (PFS) of less than 2 years after dara based therapy had an enrichment of mir-155 and mir-181a as compared to those with a PFS longer than 2 years. These data align with our findings in the cell lines and suggest that these miRNAs may be utilised as potential biomarkers for daratumumab resistance. Citation Format: Muhamad Irfan Bin Azaman, Wee Joo Chng, Sanjay de Mel, Nurulhuda Mustafa. Extracellular Vesicles Secreted from Daratumumab Resistant Cells Promote Resistance and Proliferation of Daratumumab Sensitive cells, possibly Through the Transfer of miRNA Cargo [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P07.

Novel STAT3 oligonucleotide compounds suppress tumor growth and overcome the acquired resistance to sorafenib in hepatocellular carcinoma.

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the occurrence and progression of tumors, leading to resistance and poor prognosis. Activation of STAT3 signaling is frequently detected in hepatocellular carcinoma (HCC), but potent and less toxic STAT3 inhibitors have not been discovered. Here, based on antisense technology, we designed a series of stabilized modified antisense oligonucleotides targeting STAT3 mRNA (STAT3 ASOs). Treatment with STAT3 ASOs decreased the STAT3 mRNA and protein levels in HCC cells. STAT3 ASOs significantly inhibited the proliferation, survival, migration, and invasion of cancer cells by specifically perturbing STAT3 signaling. Treatment with STAT3 ASOs decreased the tumor burden in an HCC xenograft model. Moreover, aberrant STAT3 signaling activation is one of multiple signaling pathways involved in sorafenib resistance in HCC. STAT3 ASOs effectively sensitized resistant HCC cell lines to sorafenib in vitro and improved the inhibitory potency of sorafenib in a resistant HCC xenograft model. The developed STAT3 ASOs enrich the tools capable of targeting STAT3 and modulating STAT3 activity, serve as a promising strategy for treating HCC and other STAT3-addicted tumors, and alleviate the acquired resistance to sorafenib in HCC patients. A series of novel STAT3 antisense oligonucleotide were designed and showed potent anti-cancer efficacy in hepatocellular carcinoma in vitro and in vivo by targeting STAT3 signaling. Moreover, the selected STAT3 ASOs enhance sorafenib sensitivity in resistant cell model and xenograft model.

CDK4/6 inhibition sensitizes MEK inhibition by inhibiting cell cycle and proliferation in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is not sensitive to most chemotherapy drugs, leading to poor chemotherapy efficacy. Recently, Trametinib and Palbociclib have promising prospects in the treatment of pancreatic cancer. This article aims to explore the effects of Trametinib on pancreatic cancer and address the underlying mechanism of resistance as well as its reversal strategies. The GDSC (Genomics of Drug Sensitivity in Cancer) and CTD2 (Cancer Target Discovery and Development) were utilized to screen the potential drug candidate in PDAC cell lines. The dose-increase method combined with the high-dose shock method was applied to induce the Trametinib-resistant PANC-1 and MIA PaCa-2 cell lines. The CCK8 proliferation assay, colony formation assay, flow cytometry, and western blot were conducted to verify the inhibitory effect of Trametinib and Palbociclib. RNA-seq was performed in resistant PDAC cell lines to find the differential expression genes related to drug resistance and predict pathways leading to the reversal of Trametinib resistance. The GDSC and CTD2 database screening revealed that Trametinib demonstrates a significant inhibitory effect on PDAC. We found that Trametinib has a lower IC50 than Gemcitabine in PDAC cell lines. Both Trametinib and Gemcitabine can decrease the proliferation capacity of pancreatic cells, induce cell cycle arrest, and increase apoptosis. Simultaneously, the phosphorylation of the AKT and ERK pathways were inhibited by the treatment of Trametinib. In addition, the RNA-seq of Trametinib-induced resistance PDAC cell lines reveals that the cyclin-dependent kinase (CDK)-RB-E2F regulatory axis and G2/M DNA damage checkpoint might lead the drug resistance. Besides, the combination of Trametinib with Palbociclib could inhibit the proliferation and cell cycle of both resistant cells lines and also restore the sensitivity of drug-resistant cells to Trametinib. Last but not least, the interferon-α and interferon-γ expression were upregulated in resistance cell lines, which might lead to the reversal of drug resistance. The study shows Trametinib has a critical inhibitory effect on PDAC. Besides, the combination of Trametinib with Palbociclib can inhibit the proliferation of PDAC-resistant cells.

Phenylspirodrimane with Moderate Reversal Effect of Multidrug Resistance Isolated from the Deep-Sea Fungus Stachybotrys sp. 3A00409.

Two new phenylspirodrimanes, stachybotrins K and L (1 and 2), together with eight known analogues (3-10), were isolated from deep-sea-derived Stachybotrys sp. MCCC 3A00409. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Absolute configurations of new compounds were determined through a comparison of their circular dichroism (CD) spectra with other reported compounds. The possible reversal effects of all compounds were assayed in the resistant cancer cell lines. Stachybotrysin B (8) can reverse multidrug resistance (MDR) in ABCB1-overexpression cells (KBv200, Hela/VCR) at the non-cytotoxic concentration. Doxorubicin accumulation assay and molecular-docking analysis reveal that the mechanism of its reversal MDR effect may be related to the increase in the intracellular concentration of substrate anticancer drugs.

Abstract ND06: First disclosure of AZD8421, a highly selective CDK2 inhibitor to address resistance to CDK4/6 inhibitors in breast and CCNE1-high cancers

Abstract Cyclin-dependent kinases (CDKs) are an attractive class of cancer targets due to their role in cell-cycle regulation. CDK2 is a Ser/Thr kinase activated via interaction with its cyclin partners CCNE and CCNA, driving G1/S progression of the cell cycle. CDK2 inhibition has the potential to address multiple resistance mechanisms to CDK4/6 inhibitors in breast cancer. In addition, CCNE1 is frequently amplified and over-expressed in cancers including ovarian, with pre-clinical data linking CDK2 inhibition sensitivity to high CCNE1. First-generation inhibitors targeting CDK2 suffered from poor tolerability in the clinic likely due to off-target activities across both the CDK family and across the protein kinome. As well as achieving CDK family selectivity in cells versus key off-targets (CDK1, CDK4/6, CDK9), it’s noteworthy that AZD8421 had no significant kinase inhibition outside the CDK family. X-ray co-crystal structures of AZD8421 with CDK2 provided the basis for the observed selectivity among the CDK-family. For example, hydrogen bonding interaction of AZD8421 with Lys89 (a CDK2-specific residue) near the solvent region of the ATP-binding pocket was the key requisite for achieving >300-fold selectivity over CDK9. In nanoBRET assays measuring cellular target engagement, AZD8421 had an IC50 against CDK2 of 9nM with selectivity over CDK1, CDK4 and CDK6. Further kinetic characterization of AZD8421 binding revealed a long residence time at CDK2 (2 hours) but not CDK1 (7 minutes). These kinetic studies showed that AZD8421 has a low Kd,app for CDK2 and thus even higher selectivity vs CDK1 than was measured by single timepoint assays. In a CCNE1 amplified cell line, AZD8421 potently inhibited cell proliferation (69nM, OVCAR3), correlated with inhibition of pRB, arrest in G1/S phase of the cell cycle and induction of senescence. In vitro combination assays in CDK4/6 inhibitor resistant breast cancer cell lines showed combination benefit with AZD8421 plus approved CDK4/6 inhibitors. In vivo, AZD8421 potently suppressed phosphorylation of Rb, and demonstrated robust monotherapy and CDK4/6i combination activity in breast and ovarian in vivo models. In vivo PD marker suppression and efficacy was demonstrated in CDK4/6 inhibitor resistant breast PDXs in combination with palbociclib (CDK4/6i). AZD8421 showed robust monotherapy activity in a CCNE1 amplified ovarian model OVCAR3 with regressions seen with monotherapy and in combination with palbociclib. These data, along with suitable physical and pharmacokinetic properties, supported progression of AZD8421 into clinical studies [NCT06188520]. Citation Format: Christopher R. Denz, Michael Grondine, Jun Fan, Jessie Hao-Ru Hsu, Anne Jackson, James Robinson, Grace Guo, Wen Li, Yanjun Wang, Maryann San Martin, Laura Prickett, Jeffrey Johannes, Avipsa Ghosh, Kun Song, Dhivya Sudhan, Christina Vasalou, Ryan Richards, Kevin Beaumont, Matthew Peters, Lisa Drew, Stephen Fawell, Frederick W. Goldberg. First disclosure of AZD8421, a highly selective CDK2 inhibitor to address resistance to CDK4/6 inhibitors in breast and CCNE1-high cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND06.