Reverse Phase Protein Array Research Articles

Abstract 4756: NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC)

Abstract Members of the NSD protein family (NSD1, NSD2, and NSD3) are histone methyltransferases (HMTs) that catalyze lysine 36 dimethylation (K36me2) at histone H3. H3K36 modifications play an important role in regulating the function and structure of chromatin, affecting transcription, replication, and repair. Abnormal H3K36 methylation is often detected during tumor development and progression. Inactivating NSD1 mutations are frequent in head and neck squamous cell carcinoma (HNSCC). They commonly occur in HPV-negative oropharyngeal (OP) and laryngeal (LC) carcinomas, and define a prognostic subtype in LC, associated with significantly improved overall and progression-free survival. Notably, the SCC4 cell line, carrying a damaging mutation in the NSD1 gene demonstrated reduced dimethylation level of H3K36 compared to NSD1 wild-type HNSCC counterparts. To explore the biological impact of the NSD1/NSD2 loss of function in HNSCC, we established cell lines with doxycycline-inducible shRNA knockdown of NSD1 and NSD2 in the set of HNSCC cell lines originating from different sites (JHU011, JHU022, Cal27, and FaDu cell lines). The depletion of NSD1 and NSD2 led to reduction of K36me2, significant decrease in cell growth as measured by cell titer blue (CTB) and clonogenic assays. NSD1/NSD2 depletion in these HNSCC cells also caused a significant increase in apoptosis. Gene Set Enrichment Analysis (GSEA) of RNA-seq for NSD1 wt versus knockdown cells indicates that NSD1 knockdown reduced expression of E2F target genes. Among the E2F transcription factor family, E2F2 gene expression was significantly decreased in all NSD1 knockdown cell lines. NSD1 knockdown also activated gene pathways related to autophagy and response to starvation. NSD1 knockdown reduced the levels of autophagy initiation gene ULK1 at both mRNA and protein levels. We also probed for protein signaling in HNSCC cells following NSD1 depletion using a reverse protein phase array (RPPA) approach, and validated Phosphatidylinositol-5-Phosphate 4-Kinase Type 2 Beta (PIP4K2B), but not other members of this family (PIP4K2A and PIP4K2C), as NSD1-regulated. PIP4K2B was regulated at the mRNA level by NSD1 as well. The CHIP-qPCR assay demonstrated the loss of H3K36me2 at the promoter of the PIP4K2B gene in NSD1 knockdown cells, suggesting direct regulation by NSD1. Moreover, PIP4K2B siRNA depletion has also led to a significant decrease in HNSCC proliferation, which suggests that the NSD1 may regulate proliferative activity through PIP4K2B. Taken together, while this data supports the suggestion that NSD histone methyltransferases have multiple downstream targets, the underlying mechanism remain to be investigated in more detail. Further, NSD proteins are attractive targets for drug development for improving treatment strategies for HNSCC. Citation Format: Iuliia Topchu, Igor Bychkov, Petr Makhov, Evgeny Izumchenko, John Karanicolas, Jindan Yu, Jochen Lorch, Yanis Boumber. NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4756.

Abstract 3854: TNIK inhibition as a novel therapeutic in cMyc high/TTF1 low SCLC

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy, with rapidly acquired chemotherapy resistance. Some studies have reported that Wnt signaling pathway activation promoted cell proliferation and was correlated with chemo-resistance in SCLC. None of the therapies targeting Wnt pathway components of the transmembrane and the cytoplasm have been successful in a clinical application due to toxicity and insufficient efficacy. However, targeting Wnt signaling inside the nucleus has been drawing increasing attention as cancer therapeutics. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers for cell proliferation, thus TNIK is expected as a novel druggable target. On the other hand, the question remains whether TNIK is a critical target in SCLC. We hypothesize that a TNIK inhibitor has potent anti-tumor effects in SCLC and its promising biomarkers exist. Methods: We used 29 SCLC cell lines including all four subtypes defined by differential expression of transcription factors ASCL1, NEUROD1, POU2F3, and inflamed gene signature (SCLC-A, N, P, and I, respectively) to evaluate the effect of a TNIK inhibitor, NCB-0846 in vitro. We correlated NCB-0846 IC50 values with proteomic profiling (Reverse Phase Protein Array, RPPA) data. Protein expression was examined by western blotting. Results: NCB-0846 markedly reduced cell proliferation in SCLC-N and P cell lines. There was a strong positive correlation between cMyc and the efficacy of NCB-0846 (r=-0.484, P<0.01), while a negative correlation between TTF1 and the efficacy (r=0.569, P<0.01). Additionally, NCB0846 decreased the expression of cMyc in cMyc-high/TTF-1 low SCLC cells. Conclusions: These findings indicate that TNIK inhibitors may be a new personalized molecular-targeted therapy in cMyc-high/TTF-1-low SCLC. Citation Format: Azusa Tanimoto, Robert J. Cardnell, Benjamin B. Morris, Kavya Ramkumar, Shen Li, Qi Wang, Allison C. Stewart, Carl Michael Gay, Jing Wang, Lauren Averett Byers. TNIK inhibition as a novel therapeutic in cMyc high/TTF1 low SCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3854.

Abstract 5310: An expanded quantitative protein expression atlas of human cancers

Abstract The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) are the most widely used and deeply characterized resources for cancer research. Despite rich molecular and phenotypic data available for these cohorts, large-scale proteomic data across cancer lineages remain limited. Here we expanded our previous effort to generate high-quality protein expression data of 447 clinically relevant proteins for ~8,000 TCGA patient samples and ~900 CCLE cell line samples using reverse phase protein arrays. We show that the protein expression profiles provide deeper mechanistic insights into cancer dependency and serve as a sensitive functional readout for the fitness effect of somatic mutations, e.g., BRAF mutations. We develop a protein-centered strategy to identify synthetic lethality pairs with high confidence. We identify pro- and anti-metastasis protein markers and demonstrate their prognostic relevance in patient cohorts. Collectively, this dataset provides a valuable resource for elucidating cancer mechanisms, identifying protein biomarkers, and developing therapeutic strategies. Citation Format: Han Liang. An expanded quantitative protein expression atlas of human cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5310.

Abstract 3864: Targeting IL-6/STAT3 signaling in EGFR-mutant drug tolerant persister cells

Abstract While the majority of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients initially respond to treatment with an EGFR tyrosine kinase inhibitor (TKI), most will eventually develop acquired resistance to these agents. The evolution of tumor cells with acquired resistance to targeted agents initially involves the persistence of a drug-tolerant subpopulation of tumor cells which display a transient and flexible resistant state. These persister cells can remain quiescent for prolonged periods of time, but eventually give rise to fully drug resistant tumor cells that resume growth and metastatic spread. The signaling pathways that allow persister cells to remain viable or to re-activate expansive growth are poorly understood. We and others have reported that EGFR TKI resistance that occurs independent of secondary EGFR mutations or MET amplification is associated with an epithelial to mesenchymal transition (EMT). Our analysis of tumor cells that had undergone EMT-mediated resistance to the EGFR TKI osimertinib revealed that interleukin-6 (IL-6) was highly upregulated in resistant cells as compared to parental cells. We find that in the setting of EGFR mutant NSCLC, IL-6 suppresses the anti-tumor cell activity of T cells and NK cells. To assess the role of IL-6 in the early events of EGFR TKI resistance, we generated drug-tolerant persister cells by treating EGFR mutant HCC4006 and HCC827 NSCLC cells with the EGFR TKI osimertinib for 10 days and then assessed IL-6 expression. We observed that IL-6 was upregulated at both the RNA and protein levels in EGFR-TKI persister cells as compared to parental cells. While the cell surface expression of IL6R was not significantly increased in EGFR-TKI persister cells as compared to parental cells, we observed increased secretion of soluble IL6R in persister cells by ELISA assay. We next assessed changes in protein expression and activation in drug tolerant persister cells by reverse phase protein array (RPPA) and observed that p-STAT3, a key component of the IL-6 signaling pathway, was significantly elevated in persister cells as compared parental cells or cells treated with osimertinib for 24 hours. Using genetically engineered mouse models (GEMM) of EGFRL858R mutant lung cancer, we also observed a similar upregulation of both IL-6 and p-STAT3 expression at the RNA and protein levels in EGFR-mutant tumors treated with osimertinib for 2 or 4 weeks. Additionally, circulating levels of IL-6 also are elevated in EGFRL858R GEMMs treated with osimertinib for 2 or 4 weeks. Collectively, our data indicates that upregulation of IL-6 is an early event in the evolution of drug resistance. Given the role of IL-6 in promoting EGFR TKI resistance, further studies investigating whether targeting of IL-6 signaling may impair the survival or outgrowth of persister populations are warranted. Citation Format: Sonia A. Patel, Monique Nilsson, Yan Yang, Li Shen, Jing Wang, Alissa Poteete, Xiaoxing Yu, Xiaoyang Ren, Xiuning Le, John Heymach. Targeting IL-6/STAT3 signaling in EGFR-mutant drug tolerant persister cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3864.

Abstract 6192: MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC

Abstract Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide, accounting for >2.2 million of new cancer diagnoses and 1.8 million cancer-related deaths in 2020. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, including adenocarcinomas and squamous cell carcinomas. Many lung adenocarcinomas have activating driver mutations in KRAS (~32% tumors), often co-occurring with mutations inactivating the TP53 tumor suppressor. Expression of the RNA-binding protein Musashi-2 (MSI2) MSI2 regulates target mRNA translation, and its expression is progressively elevated along with tumor stage in lung cancers. We previously showed that MSI2 actively promotes lung cancer growth, invasion, and metastasis, in part through promoting expression and activity of the TGFβR1/SMAD3 and EGFR signaling cascades. We now find that genetic MSI2 deficiency in a murine lung tumor model, Krasmut/Trp53KO/Msi2KO (KPM2) decreases both the total tumor number and tumor burden in comparison to control Krasmut/Trp53KO (KP) mice. Using a panel of KPM2 and KP cell lines we established from tumors from these mice, we found KPM2 cell lines demonstrate a significant decrease in proliferation compared to KP cell lines. Interestingly, reverse phase protein array (RPPA) comparison analysis of KPM2 and KP cell lines showed that MSI2 strongly and positively regulates expression of Ataxia-Telangiectasia Mutated (ATM) protein, with lower levels observed in KPM2 cells. The ATM serine/threonine protein kinase is a key transducer of DDR signaling in cases of double-strand breaks; ATM activation causes G1/S cell cycle arrest, contributes to efficiency of DNA repair, and triggers apoptosis in cases of repair failure. Notably, KPM2 cells showed significantly higher levels of γH2AX, suggesting inefficient DDR. Finally, untreated or cisplatin-treated KPM2 cells demonstrated G2/M arrest and had increased level of apoptosis as demonstrated by increase in caspase 3 cleavage, consistent with ATM loss and compensatory activation of Ataxia-telangiectasia and Rad3-related protein (ATR). Taken together, our data suggests that one of the consequences of MSI2 upregulation in NSCLC includes maintenance of an efficient DDR. Targeting MSI2 and its downstream signaling would be of therapeutic interest. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Rajendra P. Pangeni, Christopher Lengner, Evgeny Izumchenko, Jyoti Patel, John Karanicolas, Petr Makhov, Yanis Boumber. MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6192.

Abstract 6793: In vivo effects and molecular mechanisms of Minnelide mediated enhancement of measles virus oncolysis in human colorectal cancer

Abstract Background: The development of novel biotherapies for metastatic colorectal cancer (CRC), a fatal disease when advanced, is an urgent medical need. Oncolytic viruses (OVs) are novel cancer biotherapies, and the oncolytic measles virus (MV) has demonstrated safety and antitumor activity in early clinical studies. Triptolide (TRP), a diterpenoid epoxide extracted from the thunder god vine, and Minnelide (MN), its water-soluble compound, are novel agents associated with potent antitumor, anti-proliferative, pro-apoptotic, antiangiogenic, and ER stress inducing effects. The objectives of this study are to characterize the in vitro and in vivo effects and molecular mechanisms of measles virotherapy in combination with TRP and MN in CRC. Methods: The in vitro effects of TRP, MV-GFP (Edmonston strain of Measles virus expressing eGFP), MV-CD46-muPA (dual targeted MV against the murine uPA receptor and human CD46), or virus-triptolide combinations on tumor cell cytotoxicity were assessed on HT-29, HCT116 and SW620 CRC cells. Molecular and mechanistic characterization of TRP’s effects alone and combination with MV in HT29 cells was analyzed by the Reverse Phase Protein Array (RPPA) and validated by western blot analysis. In vivo effects (tumor progression and survival) of MN alone and in combination with MV vectors using different treatment schemes were assessed in HT-29 tumor bearing NSG mice, and tumor correlative studies were performed. Results: MV and TRP had single agent antitumor activity against human CRC cell lines. MV-TRP combinations were associated with increased in vitro cytotoxicity compared to single agent treatments. Functional proteomics analysis showed that TRP-MV combinations were associated with inhibition of survival and proliferation pathways, such as AKT, ERK, inhibition of HSP70 as well as induction of autophagy, at a higher level than each agent alone. In vivo, both a 7-days MN treatment and a 21-days MN treatment significantly improved long term tumor control and significantly improved survival, compared to each agent alone. Tumor studies showed that MN increased MV tumor deposition, as well as enhancement in apoptosis and inhibition of proliferation. Molecular characterization of oncogenic pathways modulated by MV-Minnelide combinations (Nanostring) are underway and will be presented at the meeting. Conclusions: Our results demonstrated that, in human CRC xenografts, Minnelide safely and significantly enhances measles virus oncolysis in vivo, leading to improved antitumor effects and prolonged survival. These effects were associated with enhanced antiproliferative and pro-apoptotic effects with the MV-Minnelide combination, suggesting in vivo synergy. Studies characterizing the molecular mechanisms of in vivo synergy and assessing this promising combination in immunocompetent CRC models are underway. Citation Format: Valery A. Chavez, Floritza Bustamante, Carolina Merchan-Mendes, Ashok Saluja, Jaime Merchan. In vivo effects and molecular mechanisms of Minnelide mediated enhancement of measles virus oncolysis in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6793.

Abstract 6504: Germline genetically predicted body mass index is associated with endometrial cancer somatic transcriptomic, immune, and mutational signatures in The Cancer Genome Atlas

Abstract High body mass index (BMI) is a causal risk factor for endometrial cancer but the molecular mechanisms underlying this association remain elusive. Here we sought to characterize the tumor genomic landscape of endometrial cancers that have developed on a germline genetic background of predisposition to elevated BMI. We built a polygenic score (PGS) for adult BMI in women using effect size estimates and allele information on 242 independent (r2<0.05) variants associated with BMI at genome-wide significance (P<5x10-9) in 379,501 women of European ancestry. We performed sample and germline (blood) genotype quality control and imputation into the 1000 Genomes reference panel on data from 354 endometrial cancer cases of genetically inferred European ancestry from The Cancer Genome Atlas (TCGA). We assigned each woman in this TCGA cohort her genetically predicted life-course BMI based on the BMI PGS and found this to be modestly correlated with BMI at the point of diagnosis (r2=0.23; P=2.1x10-5). Multivariable linear (default) and quasi-Poisson (for zero-inflated counts) regression models were used to test for associations between the BMI germline PGS and endometrial cancer tumor genomic, transcriptomic, proteomic, and immune traits in TCGA. All analyses were adjusted for age, stage, microsatellite status and 10 genetic principal components. Mutational signature models were also adjusted for signature accuracy. We ranked 18,458 genes based on the association between their tumor expression and the BMI PGS and performed gene set enrichment analysis to identify associations between the BMI PGS and upregulation of genes in the IL6-JAK-STAT3 signaling (false discovery rate (FDR)=8.50x10-7), inflammatory response (FDR=7.03x10-6), interferon gamma response (FDR=5.49x10-5) and glycolysis (FDR=3.28x10-4) pathways. High BMI PGS had an inverse association with endometrial tumor EGFR (FDR=0.07) protein levels of the 131 tumor proteins profiled by reverse phase protein array. Endometrial tumors that had developed on a germline background predictive of high BMI were also associated with increased infiltration of activated mast cells (FDR=9.55x10-3) in our evaluation of 22 tumor immune cell infiltrates quantified by the CIBERSORT algorithm, as well as the mitotic and aging clock-like single base substitution (SBS) signatures 1 (FDR=0.01) and 5 (FDR=0.04). The two SBS signature associations and the activated mast cell association with the BMI PGS were substantially more pronounced in the subgroup of endometrial cancers with microsatellite instability. Thus, we combined germline and somatic data using a novel approach to identify endometrial cancer tumor molecular features associated with genetically predicted higher BMI, providing precision multi-omic portraits of endometrial cancers that develop on a background of adiposity. Citation Format: George Richenberg, Victoria Gray, Carina Owen, Tom Gaunt, Caroline Relton, Emma Vincent, Siddhartha Kar. Germline genetically predicted body mass index is associated with endometrial cancer somatic transcriptomic, immune, and mutational signatures in The Cancer Genome Atlas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6504.

Abstract 5248: The use of reverse phase protein array (RPPA) to identify new tumor vulnerability and actionable drug targets against human cancer cell lines

Abstract Introduction: The EGFR/K-RAS pathway is a major tumor driver whose hyperactivation is associated with malignant tumor growth, multidrug resistance, early tumor relapse, chemo-resistance, and metastasis. Despite more than 40 years of research, oncogenic K-RAS activation remains an undruggable target in clinical oncology. Seven-In-Absentia (SINA) homologues (SIAH) are conserved E3 ubiquitin ligases that play a critical gatekeeper role downstream of the EGFR/K-RAS pathway. Our prior studies have shown that tumor growth was abolished in human tumor cell lines following SIAH inhibition; however, the underpinning molecular mechanisms that give rise to this anti-EGFR/K-RAS and anticancer phenotype remain unclear. Specific Objectives: to delineate the molecular mechanism(s) of why anti-SIAH2PD targeted therapy is so effective in impeding growth, we conducted reverse-phase protein array (RPPA)-based kinomic analysis to discover how major cancer signaling pathways and K-RAS-dependent signaling networks are remodeled in response to SIAH2 inhibition. Ongoing studies aim to identify and validate dysregulated phospho-proteins identified by RPPA-based kinomic analysis. Methods: 300 proteins/phosphoproteins were quantitatively measured by the RPPA platform to identify new tumor vulnerabilities, actionable targets, and compensatory signaling network alterations in response to anti-SIAH targeted therapies in five cancer cell lines in triplicate. Doxycycline (DOX)-inducible Tet-ON MiaPaCa, MDA-MB-231, MDA-MB-468, HeLa, and A459 cell lines were generated from a single cell, and DOX-induced SIAH2PD expression was confirmed. Reverse Phase Protein Array (RPPA) was conducted to quantify fold-changes of proteins whose expression was significantly (p<0.05) altered in response to SIAH inhibition. Ongoing Western Blot and Immunofluorescence (IF) studies are being used to validate our key RPPA findings. Results: Supported by statistical analyses, we identified 6 unique proteins that were significantly up-or down-regulated in response to SIAH inhibition. These proteins play a role in controlling and regulating cell growth, cell death, NFκB signaling, stress response, DNA damage, immune function, and cell attachment pathways. Thus, our data provide additional evidence supporting our tumor eradication phenotype observed in these human cancer cell lines lacking functional SIAH. Western Blot and IF validation studies have yielded further supporting evidence to confirm our RPPA results. Conclusion: Validation studies are being conducted to gain insight into pathway alterations and dynamic rewiring in order to uncover SIAH as a major tumor vulnerability in multiple malignant cancers. Our data supports the explanation as to why anti-SIAH targeted therapy works so effectively to shut down malignant tumor growth, as reported in the literature. Citation Format: Andrew Howell, Julia Wulfkhule, Rosa Gallagher, Jonathan Baker, Ashleigh Hannah, Emanuel F. Petricoin, Amy H. Tang. The use of reverse phase protein array (RPPA) to identify new tumor vulnerability and actionable drug targets against human cancer cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5248.

Abstract 4675: Chemoresistant patient-derived xenografts to identify treatment options in breast cancer patients not responding to standard chemotherapy

Abstract Resistance to chemotherapy is a major clinical challenge in breast cancer (BC), and patients developing resistance need treatment alternatives. In this project we utilize patient-derived xenografts (PDXs) from triple negative BC (TNBC), as these models better reflect the human tumor complexity and heterogeneity, compared to cell line-based- and transgene animal models. An in-house established orthotopically growing PDX, MAS98.12, has gained resistance to chemotherapy due to prolonged exposure to paclitaxel (MAS98.12-PR), a microtubule-targeting chemotherapeutic agent. As the resistance was obtained in animals, we also have the paclitaxel sensitive equivalent (MAS98.12-PS). By utilizing the pair of sensitive and resistant tumors we can study the corresponding mechanism evolving in patients who initially respond to chemotherapy before resistance and disease progression. This model will aid in identifying key mechanisms of survival in the resistant tumors, and eventually in identifying targets for use as novel therapeutic opportunities in TNBC patients resistant to standard therapy. Finally, we have samples collected early and late after resistance development, allowing us to characterize mechanisms involved in stepwise progression of paclitaxel resistance. We are currently performing molecular profiling to reveal the mechanisms involved in development of resistance. Data from bulk RNAseq, immune profiling of myeloid cells, exome sequencing, secretome profiling and reverse-phase protein array (RPPA) allow for characterizing the isogenic pair of tumors on multiple levels. Preliminary observations include aberrant expression of ABCB1/MDR1, normally involved in translocating drugs across membranes, and previously described as a mechanism involved in pertaining resistance in tumor cells. As multiple attempts to target MDR1 itself has failed, we hypothesize that our PS and PR pair will identify vulnerable/targetable signaling axis in the resistant tumors. Additionally, both PDX models have been assessed for their sensitivity to various chemo-/targeted-drugs and their combinations, revealing alternative means of treating the resistant tumors. Altogether, the molecular profiles and the sensitivity data will give novel knowledge and treatment options for patients failing to respond to standard chemotherapy. Citation Format: Eivind Valen Egeland, Kotryna Seip, Geir Frode Øy, Eleni Skourti, Solveig J Pettersen, Siri Juell, Mads Haugland Haugen, Olav Engebraaten, Lina Prasmickaite, Gunhild M Maelandsmo. Chemoresistant patient-derived xenografts to identify treatment options in breast cancer patients not responding to standard chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4675.

Abstract 528: Mechanisms and rational combinations with gp-2250, a novel oxathiazine derivative, in ovarian cancer

Abstract Background: GP-2250, a novel analog of taurultam (TRLT), has emerged as a potent anti-neoplastic drug; however, the mechanisms underlying its effects are not well understood. Here, we investigated the mechanism of action and the biological effects of GP-2250 using in vitro and in vivo models. Methods: We carried out a series of in vitro experiments including MTT assay, Annexin V/PI assay, colony formation assay, reverse-phase protein array (RPPA), and HRLC/IC analysis to determine the biological activity of GP-2250 and investigate the mechanism of action. In vivo experiments were carried out to determine the therapeutic efficacy of GP-2250 alone and in combination with standard-of-care drugs (e.g., paclitaxel, cisplatin topotecan, and poly ADP-ribose polymerases (PARP) inhibitors. Results: We investigated the cytotoxic effect of GP-2250 in 10 ovarian cancer cell lines and found that HRD ovarian cancer cells (e.g., Kuramochi, OVCAR4, and OVCAR8) were more vulnerable to GP-2250 than HRP ovarian cancer cells (e.g., A2780 and OVCAR5). In addition, GP-2250 combination with a PARP inhibitor showed the most synergistic effects. There was no difference among the PARP inhibitors (e.g., olaparib, niraparib, and rucaparib) with regard to the combinatorial effect with GP-2250. RPPA analyses revealed that GP-2250 inhibited hypoxia-inducible factor-1α, AKT, and mTOR activation and expression level. Ultra-high resolution mass spectrometry (HRMS) analysis also revealed that hexokinase2 activity and expression was significantly reduced by GP-2250 treatment. Furthermore, GP-2250 also reduced glycolysis and ATP synthesis in cancer cells. In vivo pharmacodynamic experiment using the OVCAR8 mouse model demonstrated that a dose of 500 mg/kg GP-2250 was the most effective in downregulating AKT and mTOR activation and expression In the in vivo therapy experiment using an orthotopic mouse model, a combination of GP-2250 and PARP inhibitors (olaparib, niraparib, or rucaparib) or bevacizumab showed a significant reduction of tumor weights (0.16 ± 0.05 g, 0.13 ± 0.06 g, 0.29 ± 0.05 g, and 0.07 ± 0.03 g, respectively) and nodules (1.56 ± 0.44, 1.89 ± 0.59, 3.11 ± 0.59, and 0.78 ±0.2, respectively) compared to those treated with a vehicle (tumor weight, 0.95 ± 0.1 g and nodules, 8.4 ± 0.65) or the monotherapy groups, GP-2250 (tumor weight, 2.9 ± 0.48 g, and nodules, 2.9 ± 0.48), olaparib (tumor weight, 0.53 ± 0.09 g, and nodules, 3.3 ± 0.64), niraparib (tumor weight, 0.38 ± 0.05 g, and nodules, 3.4 ± 0.44), rucaprib, (tumor weight, 0.52 ± 0.1 g, and nodules, 4.85 ± 0.79), and bevacizumab (tumor weight, 0.43 ± 0.08 g, and nodules, 3.8 ± 0.71), respectively. Conclusions: Taken together, our data indicate that GP-2250 exerts profound effects on tumor metabolism and combination with PARP inhibitors or bevacizumab showed promising anti-tumor efficacy. These findings could have implications for the clinical development of GP-2250. Citation Format: Mark S. Kim, Deanna Glassman, Adrian Lankenau Ahumada, Emine Bayraktar, Nicholas B. Jennings, Robiya Joseph, Sanghoon Lee, Robert L. Coleman, Anil K. Sood. Mechanisms and rational combinations with gp-2250, a novel oxathiazine derivative, in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 528.

Abstract 2170: Pre-treatment protein landscape in HER2-negative breast cancer treated with carboplatin in a neoadjuvant chemotherapy setting

Abstract Background and Purpose: Neoadjuvant treatment regimens containing the DNA-targeting chemotherapy carboplatin has been shown to increase the pathological complete response (pCR) rate in triple negative (TN) breast cancer. In addition, the response to such therapy among patients with aggressive hormone receptor positive (HR+) cancer should be investigated. Neither the molecular mechanisms leading to good response, nor the criteria for selection of optimal treatment groups are known. Therefore, predictive biomarkers that identify responders to carboplatin in breast cancer are needed. Materials and Methods: In the I-BCT phase II clinical trial (ClinicalTrials.gov identifier: NCT02546232), HER2-negative breast cancer patients (N = 187) were randomized (1:1) to receive neoadjuvant chemotherapy with or without carboplatin. Pre-treatment tumor biopsies (N = 178, N = 89 in each arm) were analyzed by reverse-phase protein array (RPPA) for expression levels of 494 breast cancer relevant (phospho-) proteins. Change in tumor size during treatment was used as continuous response measure. In addition, response was dichotomized with 30 % tumor shrinkage as cutoff. For the patients that received carboplatin, differential expression analysis between responders and non-responders was performed to identify proteins that may predict response to carboplatin. Gene set enrichment analysis on differentially expressed proteins was performed to identify biologically upregulated pathways. Results: We found differentially expressed proteins related to response to carboplatin in treatment naive tumors. Biologically upregulated pathways have been identified in patients with high tumor shrinkage after treatment with carboplatin. The results demonstrate the importance of the pre-treatment protein landscape in prediction of benefit to carboplatin. Studies regarding biological mechanisms related to response in both TN and HR+ breast cancer, and differences among these subtypes, are ongoing. Conclusion: Our study provides new insight into the mechanisms of response and resistance to carboplatin treatment in the neoadjuvant setting for HER2-negative breast cancer patients. The results support further studies on the protein landscape of tumors as a complementary method to gene expression analysis to unravel the molecular mechanisms of sensitivity and resistance to treatment in breast cancer. Citation Format: Maria Aanesland Dahle, Eivind Valen Egeland, Lina Prasmickaite, Ole Christian Lingjærde, Hege Russnes, Øystein Garred, Marianne L. Smebye, Helle Skjerven, Ellen Schlichting, Bjørn Naume, Gunhild Mælandsmo, Olav Engebraaten, Mads H. Haugen. Pre-treatment protein landscape in HER2-negative breast cancer treated with carboplatin in a neoadjuvant chemotherapy setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2170.

Abstract 4487: Effective anti-tumor effect in a rare metastatic Wilms tumor xenograft by inhibition of RAS/PI3K hyperactivation

Abstract Wilms Tumor (WT) is an embryonal renal tumor that accounts for more than 90% of kidney tumors in children. Significant advances in multidisciplinary therapeutic approaches have increased survival rates up to 90%. However, approximately 15% of patients with favorable histology and 50% of patients with anaplastic WT experience recurrence of disease. Thus, there is still a critical need to develop therapies that are effective and safe for these patients with progressive disease. To address this, we have developed a pipeline of patient derived xenograft (PDX) models from single academic institution patients with metastatic and aggressive cancers. Among these we have developed 3 xenograft models derived from patients with WT. To gain a detailed understanding of the complex array of mutations and identify therapeutic targets, molecular signatures were obtained at the DNA and protein levels via whole genome sequencing and reverse phase protein array analyses respectively. While PDX derived from encapsulated WT specimens exhibited basal levels of PI3K and MAPK pathway activation, which are well-known for promoting tumor survival, growth, and metastasis, no mutations that drive hyperactivation of these pathways were identified in these PDX. In contrast, a rare WT xenograft (PDX120) was established from a female patient with recurrent disease and metastatic cancer in the liver. -OMICS data indicated hyperactivation of PI3K (PIK3CA p.H1047R) and MAPK (KRAS p.G12_G10ins) which was validated by qPCR and immunoblotting in original tumor sample and serially passaged PDX120 xenografts. -OMICS data also confirmed the fidelity of the molecular signature between the original tumor sample and the serially passaged PDX120. The metastatic WT model was screened with two FDA approved drugs as single agents: Trametinib (MAPK pathway inhibitor; BID M-F 1mg/kg PO Day 1-35) or Alpelisib (PI3K pathway inhibitor; QD M-F 50mg/kg PO: Mice were dosed Day 1-29, followed by a gap period, and then dosing re-initiated, Day 86-103). Trametinib therapy did not effectively attenuate PDX120 tumor growth. In contrast, the PI3K inhibitor Alpelisib was well tolerated and significantly decreased PDX120 growth and prolonged survival compared to the vehicle control (Two-way ANOVA, Holm-Sidak, p<0.05). At the end of the study, histological analyses indicated a complete lack of Ki67+ and tumorigenic cells in the Alpelisb-treated tumors compared to vehicle controls, indicating a dominant growth dependency on PI3K pathway activation. While efficacy data generated in PDX derived from patients is not used to guide clinical decisions at this time, it can validate therapeutic approaches and provide rationale for future clinical trials. In conclusion, these studies provide critical feasibility data that has high translational value and great potential to improve survival and quality of life for children suffering with progressive WT. Citation Format: Mohammad Reza Saadatzadeh, Khadijeh Bijangi Vishehsaraei, Erika A. Dobrota, Barbara J. Bailey, Lauren E. Hein, Kathryn L. Coy, Melissa A. Trowbridge, Anthony L. Sinn, Troy A. Markel, Michael J. Ferguson, Pankita H. Pandya, Karen E. Pollok. Effective anti-tumor effect in a rare metastatic Wilms tumor xenograft by inhibition of RAS/PI3K hyperactivation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4487.

Abstract 3492: The small GTPase ARF6 augments BRAFV600E-MAPK signaling in melanomagenesis

Abstract ARF6 is a member of the Ras superfamily of small GTPases and has critical functions in endo-membrane trafficking pathways. We previously reported that ARF6 is aberrantly activated in metastatic melanoma and that activation of ARF6 stimulates WNT5A/beta-catenin and PI3K/AKT signaling to promote invasion and accelerate metastasis. Herein, we discovered an ARF6 activation signature in primary melanomas that is associated with inferior overall survival, supporting a novel role for ARF6 in early-stage progression. Conversely, melanocyte-specific deletion of Arf6 in Dct;TVA::BrafV600E;Cdkn2aNull murine melanoma significantly reduced tumorigenesis and progression, and improved survival. Importantly, bulk tumor transcriptome analysis showed reduced expression of ERK-induced genes in these BRAFV600E tumors. Likewise, immunofluorescent staining of tumor sections showed a significant reduction of cytoplasmic and nuclear p-ERK levels. Together these data demonstrate that loss of ARF6 reduces BRAFV600E-MAPK signaling output in vivo. This is a provocative finding because it implies that endomembrane trafficking has a major role in promoting BRAFV600E-driven tumorigenesis. More specifically, ARF6 may be critical for augmenting BRAFV600E signaling. Consistent with this hypothesis, expression of activated ARF6(ARF6Q67L) in our murine BRAFV600E melanoma cells was associated with upregulation of BRAFV600E, p-MEK, and p-ERK levels, detected by Reverse Phase Protein Array analysis. In addition, ARF6Q67L tumor cells showed an increase in anti-apoptotic proteins with a reciprocal decrease in pro-apoptotic proteins. Knockdown of Arf6 in BRAFV600E human melanoma cells significantly reduced proliferation and sensitized cells to vemurafenib-induced apoptosis. Overall, our data reveal that ARF6 augments MAPK signaling in BRAFV600E-driven tumorigenesis. Further work is needed to understand if ARF6 mediates endomembrane-dependent signaling of the BRAFV600E-MAPK pathway. Citation Format: Junhua Wang, Aaron Rogers, Coulson Rich, Lise Sorensen, Sheri Holmen, Roger Wolff, Allie Grossmann. The small GTPase ARF6 augments BRAFV600E-MAPK signaling in melanomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3492.

Combining MEK and SRC inhibitors for treatment of colorectal cancer demonstrate increased efficacy in vitro but not in vivo.

Metastatic colorectal cancer (mCRC) is the second leading cause of cancer deaths in the United States. More than 50% of patients with mCRC harbor mutations of the oncogenic driver RAS (KRAS or NRAS). Because directly targeting most mutations of RAS is technically challenging, researchers have concentrated on targeting MEK, a downstream mediator of RAS. However, targeting MEK as single-agent therapy is ineffective in patients with mCRC. We hypothesize that combining a MEK inhibitor with other agents can enhance the efficacy of MEK targeting in mCRC. Unbiased high-throughput screening (HTS) was performed to identify drugs that enhance the efficacy of MEK inhibitors. HTS was performed with KRAS-mutated CRC cells using the MEK inhibitor trametinib as a "backbone" and two "clinically ready" compound libraries approved by the U.S. Food and Drug Administration or in clinical trials. HTS demonstrated that the combination of the SRC inhibitor dasatinib and trametinib was synergistic in CRC cells in vitro (MTT and colony formation assays). Analysis of markers for cell proliferation and apoptosis using fluorescence-activated cell sorting, reverse-phase protein array, or Western blotting demonstrated decreased cell proliferation and increased cell death when targeting both SRC and MEK as compared to single agents in multiple CRC cell lines. However, combining dasatinib and trametinib in vivo at doses in mice equivalent to doses used in humans failed to significantly enhance the antitumor activity of trametinib when compared to that of trametinib alone. These results underscore the importance of performing careful preclinical in vivo validation studies using clinically relevant doses as a prerequisite for translating in vitro findings to the clinic.

DNA Damage Response-Related Proteins Are Prognostic for Outcome in Both Adult and Pediatric Acute Myelogenous Leukemia Patients: Samples from Adults and from Children Enrolled in a Children's Oncology Group Study.

The survival of malignant leukemic cells is dependent on DNA damage repair (DDR) signaling. Reverse Phase Protein Array (RPPA) data sets were assembled using diagnostic samples from 810 adult and 500 pediatric acute myelogenous leukemia (AML) patients and were probed with 412 and 296 strictly validated antibodies, respectively, including those detecting the expression of proteins directly involved in DDR. Unbiased hierarchical clustering identified strong recurrent DDR protein expression patterns in both adult and pediatric AML. Globally, DDR expression was associated with gene mutational statuses and was prognostic for outcomes including overall survival (OS), relapse rate, and remission duration (RD). In adult patients, seven DDR proteins were individually prognostic for either RD or OS. When DDR proteins were analyzed together with DDR-related proteins operating in diverse cellular signaling pathways, these expanded groupings were also highly prognostic for OS. Analysis of patients treated with either conventional chemotherapy or venetoclax combined with a hypomethylating agent revealed protein clusters that differentially predicted favorable from unfavorable prognoses within each therapy cohort. Collectively, this investigation provides insight into variable DDR pathway activation in AML and may help direct future individualized DDR-targeted therapies in AML patients.

Reverse Phase Protein Array Profiling Identifies Recurrent Protein Expression Patterns of DNA Damage-Related Proteins across Acute and Chronic Leukemia: Samples from Adults and the Children's Oncology Group.

DNA damage response (DNADR) recognition and repair (DDR) pathways affect carcinogenesis and therapy responsiveness in cancers, including leukemia. We measured protein expression levels of 16 DNADR and DDR proteins using the Reverse Phase Protein Array methodology in acute myeloid (AML) (n = 1310), T-cell acute lymphoblastic leukemia (T-ALL) (n = 361) and chronic lymphocytic leukemia (CLL) (n = 795) cases. Clustering analysis identified five protein expression clusters; three were unique compared to normal CD34+ cells. Individual protein expression differed by disease for 14/16 proteins, with five highest in CLL and nine in T-ALL, and by age in T-ALL and AML (six and eleven proteins, respectively), but not CLL (n = 0). Most (96%) of the CLL cases clustered in one cluster; the other 4% were characterized by higher frequencies of deletion 13q and 17p, and fared poorly (p < 0.001). T-ALL predominated in C1 and AML in C5, but both occurred in all four acute-dominated clusters. Protein clusters showed similar implications for survival and remission duration in pediatric and adult T-ALL and AML populations, with C5 doing best in all. In summary, DNADR and DDR protein expression was abnormal in leukemia and formed recurrent clusters that were shared across the leukemias with shared prognostic implications across diseases, and individual proteins showed age- and disease-related differences.

Mitotic CDK1 and 4E-BP1 II: A single phosphomimetic mutation in 4E-BP1 induces glucose intolerance in mice.

Cyclin-dependent kinase 1 (CDK1)/cyclin B1 phosphorylates many of the same substrates as mTORC1 (a key regulator of glucose metabolism), including the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Only mitotic CDK1 phosphorylates 4E-BP1 at residue S82 in mice (S83 in humans), in addition to the common 4E-BP1 phospho-acceptor sites phosphorylated by both CDK1 and mTORC1. We examined glucose metabolism in mice having a single aspartate phosphomimetic amino acid knock in substitution at the 4E-BP1 serine 82 (4E-BP1S82D) mimicking constitutive CDK1 phosphorylation. Knock-in homozygous 4E-BP1S82D and 4E-BP1S82A C57Bl/6N mice were assessed for glucose tolerance testing (GTT) and metabolic cage analysis on regular and on high-fat chow diets. Gastrocnemius tissues from 4E-BP1S82D and WT mice were subject to Reverse Phase Protein Array analysis. Since the bone marrow is one of the few tissues typically having cycling cells that transit mitosis, reciprocal bone-marrow transplants were performed between male 4E-BP1S82D and WT mice, followed by metabolic assessment, to determine the role of actively cycling cells on glucose homeostasis. Homozygous knock-in 4E-BP1S82D mice showed glucose intolerance that was markedly accentuated with a diabetogenic high-fat diet (p = 0.004). In contrast, homozygous mice with the unphosphorylatable alanine substitution (4E-BP1S82A) had normal glucose tolerance. Protein profiling of lean muscle tissues, largely arrested in G0, did not show protein expression or signaling changes that could account for these results. Reciprocal bone-marrow transplantation between 4E-BP1S82D and wild-type littermates revealed a trend for wild-type mice with 4E-BP1S82D marrow engraftment on high-fat diets to become hyperglycemic after glucose challenge. 4E-BP1S82D is a single amino acid substitution that induces glucose intolerance in mice. These findings indicate that glucose metabolism may be regulated by CDK1 4E-BP1 phosphorylation independent from mTOR and point towards an unexpected role for cycling cells that transit mitosis in diabetic glucose control.

Abstract P4-08-13: The Use of Reverse Phase Protein Arrays (RPPA), Principal Component Analysis (PCA), and CScape Pathway Mapping to Identify New Tumor Vulnerability and Actionable Targets in Human Malignant Triple-Negative Breast cancer cell lines

Abstract Breast cancer is the major leading cause of cancer-related fatalities nationally and globally. There will be 287,850 new breast cancer cases and 43,250 breast cancer related deaths in the United States in 2022. The relapsed, treatment-resistant, undruggable, and incurable breast cancers are often associated with EGFR/HER2/K-RAS/SIAH pathway activation. The EGFR/HER2/K-RAS/SIAH pathway is a major tumor-driver whose hyperactivation is associated malignant tumor growth, multidrug-resistant phenotypes, early tumor relapse, and systematic metastasis. Seven-In-Absentia (SINA) homologues (SIAH) are extraordinarily evolutionarily-conserved E3 ubiquitin ligases that play a critical gatekeeper role downstream of the EGFR/HER2/K-RAS pathway. SIAH is a major tumor vulnerability that is ideally positioned to become an attractive target for innovative targeted therapy development against metastatic breast cancer (MBC). Prior studies have shown that tumor growth was abolished in malignant tumor cell lines such as MDA-MB-231, MDA-MB-468, MiaPaCa, A459, and HeLa following SIAH inhibition in xenograft models; however, the underpinning molecular mechanisms that give rise to this striking anti-EGFR/K-RAS and anticancer phenotype remain unclear. Specific objectives: to delineate the molecular mechanism(s) of why anti-SIAH2PD targeted therapy is so effective in impeding and eradicating the stage IV and aggressive tumors, we conducted reverse phase protein array (RPPA)-based kinomic analysis to delineate how major cancer signaling pathways and EGFR/HER2/K-RAS/SIAH-dependent signaling networks are rewired and remodeled in response to anti-SIAH2 targeted therapy. Brief statement of methods: About 300 proteins/phosphoproteins were quantititatively measured by the RPPA platform to identify new tumor vulnerabilities and actionable targets, compensatory signaling network activation/inhibition in response to anti-SIAH targeted therapies in five highly malignant cancer cell lines. Doxycycline (DOX)-inducible Tet-ON MDA-MB-231, MDA-MB-468, MiaPaCa, HeLa and A459 cell lines were amplified from single cell and DOX-induced SIAH2PD expression was confirmed. Each of the cell lines was then subjected to one of four experimental conditions: Tet-ON control cells without DOX induction (group A), Tet-ON control cells with DOX induction (group B), Tet-ON-SIAH2PD cancer cells without DOX-induction (no SIAH2PD inhibitor) (group C), Tet-ON-SIAH2PD cancer cells with DOX-induction (SIAH2PD inhibitor) (group D). Reverse Phase Protein Array (RPPA) in conjunction with Principal Component Analysis (PCA) was conducted to quantify fold-changes of proteins/phosphoproteins in response to SIAH inhibition. The ratios of D/C/B/A, D/C, D/B, C/A, and B/A were calculated using GAPDH normalized data. Summary of results: Supported by statistical analyses, we identified 6 unique phospho-proteins that were either up- or down-regulated in response to SIAHLoss-of-function. Many have known roles in controlling and regulating cell growth, cell death, NFκB signaling, stress response, DNA damage, and cell attachment pathways, supporting a tumor eradication phenotype in the absence of SIAH function in these cancer cell lines. Conclusion: Cancer landscape (CScape) functional protein pathway mapping has categorized the synergistic feedforward, feedback, and compensatory signaling pathway activation/inactivation in response to SIAH blockade in these EGFR/HER2/K-RAS-driven malignant human cell lines. Further validation analysis will be conducted to gain better insight into the global cancer pathway alterations to reveal the molecular mechanism(s) of why SIAH inhibition works so effectively to shut down malignant tumor growth in the preclinical models. Citation Format: Andrew P. Howell, Julia Wulfkuhle, Rosa I. Gallagher, Emanuel F. Petricoin, Amy H. Tang. The Use of Reverse Phase Protein Arrays (RPPA), Principal Component Analysis (PCA), and CScape Pathway Mapping to Identify New Tumor Vulnerability and Actionable Targets in Human Malignant Triple-Negative Breast cancer cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-13.

Abstract HER2-17: HER2-17 Novel Quantitative HER2 Assay for Determining Dynamic HER2 Expression in the HER2 IHC 0 “Ultra-Low” Setting: Implications for Precision Therapy in HER2- Breast Cancer

Abstract Background: The HER2 antibody drug conjugate (ADC) fam-trastuzumab deruxtecan-nxki (T-DXd) significantly improves outcomes over standard chemotherapy in pts with HER2-LOW (IHC 1+ and IHC 2+ FISH-) metastatic breast cancer (MBC). Data from the DAISY (NCT04132960) trial in pts with HER2 IHC 0 or “ultra low” MBC revealed median progression-free survival (PFS) of 4.2 mos vs 6.7 mos in HER2-LOW and 11.1 mos in HER2 3+ pts (Diéras V, et al. Cancer Res. 2022;82(4_Suppl):PD8-02). There is an urgent need to develop methods to accurately discern HER2 LOW expression versus HER2 IHC 0/ultra-low expression so appropriate pts may receive T-DXd. We have developed a quantitative HER2 assay using reverse phase protein array (RPPA) technology with laser capture microdissection (LCM) enrichment of tumor epithelium that measures HER2 expression over a 65-fold dynamic range in HER2 IHC 0 to 3+ breast cancers. In I-SPY 1 and 2 trials we found that RPPA-assessed quantitative HER2 and activated/phosphorylated HER2 expression predicted for pathologic complete response in pts with HER2 IHC 0, HER2-LOW and HER2 3+ disease with various HER2 and other targeted agents. We then developed and validated the first CLIA/CAP-accredited quantitative HER2 protein expression and activation assay, and now explore quantitative HER2 expression in HER2 IHC 0/ultra low disease. Given the recent approval of sacituzumab govitecan-hziy in TNBC and the results of the TROPiCS-02 (NCT03901339), we also evaluated quantitative RPPA-based TROP-2 expression levels in HER2 IHC 0 breast cancer. Methods: LCM-enriched tumor epithelium was obtained from freshly cut FFPE core needle and resected breast cancer samples from 175 pts with pathology-determined HER2 IHC 0 status (N=68 ER+ and N=107 ER-). Quantitative HER2 output is generated as a relative intensity unit (RU) that is interpolated to an internal calibrator curve and a population referent comprised of known HER2+ (IHC 3+ and 2+/FISH+), HER2-LOW and HER2 IHC 0/ultra-low tumors to generate population-based cut-points as a referent. RPPA-based quantitative HER2 expression are reported as one of four levels of expression across the HER2 dynamic range observed: “non/extremely low”, “modest“, “moderate”, and “high”. Quantitative TROP2 protein expression levels were also measured for each case by the RPPA assay on the same lysate using the same adjectival determinants of relative expression as HER2. Results: For the HER2 IHC 0 ER+ cohort, we observed HER2 protein expression over a 25-fold dynamic range and that 57% (N=39) had HER2 non/extremely low, 37% (N=25) modest, and 4 (6%) moderate/high HER2 expression by RPPA. For the HER2 IHC 0 ER- cohort, we observed HER2 protein expression over a 15-fold dynamic range and that 71% (N=76) had non/extremely low, and 31% (N=29%) modest HER2 expression. We observed TROP-2 protein expression over a 35-fold dynamic range across all tumors measured, and it was found to be at least modestly expressed in 95% (37/39) ER+ and 95% (72/76) ER- of the IHC 0/ultra-low tumors that were also found to be HER2 non/extremely low by RPPA. Conclusions: LCM-RPPA quantitative assessment of HER2 expression showed that nearly 40% of ER+ IHC 0 and 30% of ER- IHC 0 “ultra-low” breast cancers actually had modest to moderate HER2 expression. Interestingly, this frequency approximates the response rate of 30% seen with T-DXd in HER2 IHC 0 pts in the DAISY trial. Quantitatively measured TROP2 is at least modestly expressed in the vast majority of RPPA-assessed HER2 IHC 0 cancers regardless of ER status, making a TROP2-directed ADC an attractive therapeutic option for these pts. If validated in larger studies, LCM-RPPA-based HER2 expression could provide a better understanding of the potential for therapeutic efficacy with T-DXd in patients with HER2 IHC “ultra-low” disease, and may better define true ultra-low HER2 expression in HER2 IHC 0 tumor biopsies at baseline and refine the lower limit of the HER2-LOW designation. Citation Format: Emanuel F. Petricoin, Brian A. Corgiat, Joyce O’Shaughnessy, Patricia LoRusso, Kris Weinberg, Justin Davis, Chelsea Gawryletz. HER2-17 Novel Quantitative HER2 Assay for Determining Dynamic HER2 Expression in the HER2 IHC 0 “Ultra-Low” Setting: Implications for Precision Therapy in HER2- Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-17.

Abstract P6-11-15: Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which limited targeted therapies are available. Therefore, conventional chemotherapy remains the backbone of standard neoadjuvant treatment (NACT) for TNBC patients. Unfortunately, ~45% of patients will have substantial residual tumor burden post neoadjuvant chemotherapy, leading to poor prognoses (PMID: 28135148). Recently, it has been demonstrated that mitochondrial oxidative phosphorylation (oxphos) is upregulated and is a therapeutic vulnerability in chemoresistant TNBC (PMID: 30996079; Baek et al., BioRxiv doi.org/10.1101/2022.02.25.481996). However, mechanisms driving increased oxphos in chemoresistant TNBC are not understood. Upregulated fatty acid (FA) metabolism is a common adaptation in tumors, providing an energy source through fatty acid β-oxidation (FAO), and promoting lipid accumulation after fatty acid synthesis (FAS) when energy needs are met. Chemotherapy can induce oxidative stress through the generation of reactive oxygen species. Cancer cells adapt to these damaging molecules by increasing de novo lipogenesis, resulting in the accumulation of lipid droplets (LDs) in the cytosol (PMID: 32782526, 20876798). We hypothesize that TNBC cells metabolically adapt to the stress of NACT by upregulating lipid metabolic pathways, providing highly energetic molecules that can be utilized to drive oxphos in chemoresistant TNBC. Methods: Using orthotopic patient-derived xenograft (PDX) models of TNBC (PIM001-P, PMID: 30996079, HCI-010, PMID: 22019887; WHIM14, PMID:24055055), we are measuring protein levels of fatty acid synthase (FASN) in vehicle tumors vs residual tumors surviving treatment with the standard front-line neoadjuvant chemotherapy regimens (Adriamycin plus cyclophosphamide (AC), docetaxel, carboplatin, or docetaxel+carboplatin) using immunohistochemistry (IHC). Vectra 3 microscopy (Akoya) is being used to quantify tumor cell-specific staining. We complemented our IHC analysis with reverse-phase protein array (RPPA). To assess LD accumulation in residual PDX tumors, we conducted transmission electron microscopy (TEM). To complement these PDX studies, we modeled the residual tumor metabolic state in cultured human TNBC cells. Following treatment with the IC50 of standard chemotherapeutic agents (AC, carboplatin, paclitaxel, docetaxel), we assessed oxphos by measuring oxygen consumption rate (OCR) using a Seahorse Bioanalyzer (Agilent). Further, we tested LD accumulation using LipidTOX staining. In ongoing studies, we are measuring incorporation of 13C palmitate into the tricarboxylic acid cycle (TCA) prior to and following chemotherapy treatments to assess if lipids fuel mitochondrial metabolism in residual TNBC cells. Results/Discussion: IHC in the PIM001-P PDX model after in vivo AC treatment revealed increased levels of FASN in post-AC residual tumors compared to the treatment-naive tumors. Further, key proteins involved in fatty acid synthesis, FASN and Acetyl-CoA carboxylase, were significantly increased in residual PIM001-P cells that survived AC compared to vehicle by RPPA. TEM analysis of the HCI-010 PDX revealed significantly more LDs in carboplatin-treated tumors compared to vehicle. This finding was supported by increased LDs observed in TNBC cell lines treated with NACT compared to vehicle in our LipidTOX analyses. Taken together, these data indicate that NACT induces increased expression of key lipid metabolism proteins and accumulation of cytosolic LDs. Our future experiments will reveal if chemoresistant TNBC cells preferentially utilize and incorporate lipids into the tricarboxylic acid cycle, in turn driving oxphos. These data have the potential to provide rationale for the incorporation of FAO/LD inhibitors in sequential combinations with conventional chemotherapies to more effectively kill TNBC cells that are chemo-refractory. Citation Format: Katherine E. Pendleton, Mokryun L. Baek, Junegoo Lee, Lin Tan, Hannah L. Johnson, Lacey E. Dobrolecki, James P. Barrish, Michael T. Lewis, Philip L. Lorenzi, Fabio Stossi, Gloria V. Echeverria. Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-15.