Targeted Cancer Therapy Research Articles

Synthesis, in silico studies and in vitro cytotoxicity evaluation of novel posaconazole derivative as a ALK TK inhibitor

Oncology research progresses, yet cancer remains the largest medical need. The use of a medication with an accessible lead molecule shows great potential for long-term cancer therapy. It may help target cancer therapy and drug resistance. This work uses FDA-approved, clinically proven compounds to build novel cancer therapeutic ligands to prevent treatment resistance. Based on the reported findings, an in-silico docking experiment was performed using FDA-licensed fungus medication posaconazole. The ALK TK pdb:4cmu ligand and posaconazole docked similarly in the research. Posaconazole's in silico docking findings were then tested against A549 lung cancer cells. Posaconazole, the lead, underwent an ADME investigation. Given in silico and experimental confirmation, acetyl, benzyl, and benzoyl derivatives of the lead molecule posaconazole was synthesized. IR, mass, 1H NMR, 13C NMR, and elemental analysis were performed on the synthesized compounds. Next, the three variants were evaluated against A549 lung cancer cells. They were then examined by utilizing computational approaches such as molecular docking, DFT analysis, MD simulation, and MMGBSA analysis. The ADME studies were conducted on posaconazole and its derivatives based on the in-vitro cell line investigation. Based on the results of an ADME study, two new ALK TK inhibitors that don't work as Pgp substrates are good lead candidates. Thus, the present investigation fruitfully reported two lead drug candidates against the issue of resistance in cancer therapy.

Identifying an immunogenic cell death-related gene signature contributes to predicting prognosis, immunotherapy efficacy, and tumor microenvironment of lung adenocarcinoma.

Immunogenic cell death (ICD) is a regulated form of cell death that triggers an adaptive immune response. The objective of this study was to investigate the correlation between ICD-related genes (ICDGs) and the prognosis and the immune microenvironment of patients with lung adenocarcinoma (LUAD). ICD-associated molecular subtypes were identified through consensus clustering. Subsequently, a prognostic risk model comprising 5 ICDGs was constructed using Lasso-Cox regression in the TCGA training cohort and further tested in the GEO cohort. Enriched pathways among the subtypes were analyzed using GO, KEGG, and GSVA. Furthermore, the immune microenvironment was assessed using ESTIMATE, CIBERSORT, and ssGSEA analyses. Consensus clustering divided LUAD patients into three ICDG subtypes with significant differences in prognosis and the immune microenvironment. A prognostic risk model was constructed based on 5 ICDGs and it was used to classify the patients into two risk groups; the high-risk group had poorer prognosis and an immunosuppressive microenvironment characterized by low immune score, low immune status, high abundance of immunosuppressive cells, and high expression of tumor purity. Cox regression, ROC curve analysis, and a nomogram indicated that the risk model was an independent prognostic factor. The five hub genes were verified by TCGA database, cell sublocalization immunofluorescence analysis, IHC images and qRT-PCR, which were consistent with bioinformatics analysis. The molecular subtypes and a risk model based on ICDGs proposed in our study are both promising prognostic classifications in LUAD, which may provide novel insights for developing accurate targeted cancer therapies.

Folic Acid-Conjugated Brush Polymers Show Enhanced Blood-Brain Barrier Crossing in Static and Dynamic In Vitro Models Toward Brain Cancer Targeting Therapy.

Over the past decades, evidence has consistently shown that treatment of central nervous system (CNS)-related disorders, including Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis, and brain cancer, is limited due to the presence of the blood-brain barrier (BBB). To assist with the development of new therapeutics, it is crucial to engineer a drug delivery system that can cross the BBB efficiently and reach target cells within the brain. In this study, we present a potentially efficient strategy for targeted brain delivery through utilization of folic acid (FA)-conjugated brush polymers, that specifically target the reduced folate carrier (RFC, SLC19A1) expressed on brain endothelial cells. Here, azide (N3)-decorated brush polymers were prepared in a straightforward manner coupling a heterotelechelic α-NH2, ω-N3-poly(2-ethyl-2-oxazoline) (NH2-PEtOx-N3) to N-acylated poly(amino ester) (NPAE)-based brushes. Strain-promoted azide-alkyne cycloaddition (SPAAC) 'click chemistry' with DBCO-folic acid (FA) yielded FA-brush polymers. Interestingly, while azide functionalization of the brush polymers dramatically reduced their association to brain microvascular endothelial cells (hCMEC/D3), the introduction of FA to azide led to a substantial accumulation of the brush polymers in hCMEC/D3 cells. The ability of the polymeric brush polymers to traverse the BBB was quantitatively assessed using different in vitro BBB models including static Transwell and microfluidic platforms. FA-brush polymers showed efficient transport across hCMEC/D3 cells in a manner dependent on FA composition, whereas nonfunctionalized brush polymers exhibited limited trafficking under the same conditions. Further, cellular uptake inhibition studies suggested that the interaction and transport pathway of FA-brush polymers across BBB relies on the RFC-mediated pathways. The potential application of the developed FA-brush polymers in brain cancer delivery was also investigated in a microfluidic model of BBB-glioblastoma. Brush polymers with more FA units successfully presented an enhanced accumulation into U-87 MG glioma cells following its BBB crossing, compared to controls. These results demonstrate that FA-modified brush polymers hold a great potential for more efficient delivery of future brain therapeutics.

Development of an efficient liposomal DOX delivery formulation for HCC therapy by targeting CK2α.

Hepatocellular carcinoma (HCC) is a digestive tract cancer with high mortality and poor prognosis, especially in China. Current chemotherapeutic drugs lead to poor prognosis, low efficacy, and high side effects due to weak targeting specificity and rapidly formed multidrug resistance (MDR). Based on the previous studies on the doxorubicin (DOX) formulation for cancer targeting therapy, we developed a novel DOX delivery formulation for the targeting chemotherapy of HCC and DOX resistant HCC. HCSP4 was previously screened and casein kinase 2α (CK2α) was predicted as its specific target on HCC cells in our lab. In the study, miR125a-5p was firstly predicted as an MDR inhibiting miRNA, and then CK2α was validated as the target of HCSP4 and miR125a-5p using CK2α-/-HepG2 cells. Based on the above, an HCC targeting and MDR inhibiting DOX delivery liposomal formulation, HCSP4/Lipo-DOX/miR125a-5p was synthesized and tested for its HCC therapeutic efficacy in vitro. The results showed that the liposomal DOX delivery formulation targeted to HCC cells specifically and sensitively, and presented the satisfied therapeutic efficacy for HCC, particularly for DOX resistant HCC. The potential therapeutic mechanism of the DOX delivery formulation was explored, and the formulation inhibited the expression of MDR-relevant genes including ATP-binding cassette subfamily B member 1 (ABCB1, also known as P-glycoprotein), ATP-binding cassette subfamily C member 5 (ABCC5), enhancer of zeste homolog 2 (EZH2), and ATPase Na+/K+ transporting subunit beta 1 (ATP1B1). Our study presents a novel targeting chemotherapeutic drug formulation for the therapy of HCC, especially for drug resistant HCC, although it is primarily and needs further study in vivo, but provided a new strategy for the development of novel anticancer drugs.

Design, synthesis, and evaluation of purine and pyrimidine-based KRAS G12D inhibitors: Towards potential anticancer therapy

Oncogenic RAS mutations, commonly observed in human tumors, affect approximately 30% of cancer cases and pose a significant challenge for effective cancer treatment. Current strategies to inhibit the KRAS G12D mutation have shown limited success, emphasizing the urgent need for new therapeutic approaches. In this study, we designed and synthesized several purine and pyrimidine analogs as inhibitors for the KRAS G12D mutation. Our synthesized compounds demonstrated potent anticancer activity against cell lines with the KRAS G12D mutation, effectively impeding their growth. They also exhibited low toxicity in normal cells, indicating their selective action against cancer cells harboring the KRAS G12D mutation. Notably, the lead compound, PU1-1 induced the programmed cell death of KRAS G12D-mutated cells and reduced the levels of active KRAS and its downstream signaling proteins. Moreover, PU1-1 significantly shrunk the tumor size in a pancreatic xenograft model induced by the KRAS G12D mutation, further validating its potential as a therapeutic agent. These findings highlight the potential of purine-based KRAS G12D inhibitors as candidates for targeted cancer therapy. However, further exploration and optimization of these compounds are essential to meet the unmet clinical needs of patients with KRAS-mutant cancers.

The Interplay between Glioblastoma Cells and Tumor Microenvironment: New Perspectives for Early Diagnosis and Targeted Cancer Therapy.

Glioblastoma multiforme (GBM) stands out as the most tremendous brain tumor, constituting 60% of primary brain cancers, accompanied by dismal survival rates. Despite advancements in research, therapeutic options remain limited to chemotherapy and surgery. GBM molecular heterogeneity, the intricate interaction with the tumor microenvironment (TME), and non-selective treatments contribute to the neoplastic relapse. Diagnostic challenges arise from GBM advanced-stage detection, necessitating the exploration of novel biomarkers for early diagnosis. Using data from the literature and a bioinformatic tool, the current manuscript delineates the molecular interplay between human GBM, astrocytes, and myeloid cells, underscoring selected protein pathways belonging to astroglia and myeloid lineage, which can be considered for targeted therapies. Moreover, the pivotal role of extracellular vesicles (EVs) in orchestrating a favorable microenvironment for cancer progression is highlighted, suggesting their utility in identifying biomarkers for GBM early diagnosis.

Immune Response and Metastasis-Links between the Metastasis Driver MACC1 and Cancer Immune Escape Strategies.

Metastasis remains the most critical factor limiting patient survival and the most challenging part of cancer-targeted therapy. Identifying the causal drivers of metastasis and characterizing their properties in various key aspects of cancer biology is essential for the development of novel metastasis-targeting approaches. Metastasis-associated in colon cancer 1 (MACC1) is a prognostic and predictive biomarker that is now recognized in more than 20 cancer entities. Although MACC1 can already be linked with many hallmarks of cancer, one key process-the facilitation of immune evasion-remains poorly understood. In this review, we explore the direct and indirect links between MACC1 and the mechanisms of immune escape. Therein, we highlight the signaling pathways and secreted factors influenced by MACC1 as well as their effects on the infiltration and anti-tumor function of immune cells.

Magnetically actuated sonodynamic nanorobot collectives for potentiated ovarian cancer therapy.

Ovarian cancer presents a substantial challenge due to its high mortality and recurrence rates among gynecological tumors. Existing clinical chemotherapy treatments are notably limited by drug resistance and systemic toxic side effects caused by off target drugs. Sonodynamic therapy (SDT) has emerged as a promising approach in cancer treatment, motivating researchers to explore synergistic combinations with other therapies for enhanced efficacy. In this study, we developed magnetic sonodynamic nanorobot (Fe3O4@SiO2-Ce6, FSC) by applying a SiO2 coating onto Fe3O4 nanoparticle, followed by coupling with the sonosensitizer Ce6. The magnetic FSC nanorobot collectives could gather at fixed point and actively move to target site regulated by magnetic field. In vitro experiments revealed that the magnetic FSC nanorobot collectives enabled directional navigation to the tumor cell area under guidance. Furthermore, under low-intensity ultrasonic stimulation, FSC nanorobot collectives mediated sonodynamic therapy exhibited remarkable anti-tumor performance. These findings suggest that magnetically actuated sonodynamic nanorobot collectives hold promising potential for application in target cancer therapy.

Development of a hypoxia-activated red-emission fluorescent probe for in vivo tumor microenvironment imaging and anti-tumor therapy.

Hypoxia, a significant feature of the tumor microenvironment, is closely associated with tumor growth, metastasis, and drug resistance. In the field of tumor microenvironment analysis, accurately imaging and quantifying hypoxia - a critical factor associated with tumor progression, metastasis, and resistance to therapy - remains a significant challenge. Herein, a hypoxia-activated red-emission fluorescent probe, ODP, for in vivo imaging of hypoxia in the tumor microenvironment is presented. Among various imaging methods, optical imaging is particularly convenient due to its rapid response and high sensitivity. The ODP probe specifically targets nitroreductase (AzoR), an enzyme highly expressed in hypoxic cells, playing a vital role by catalyzing the cleavage of azo bonds. The optical properties of ODP exhibited excellent performance in terms of fluorescence enhancement, fluorescence lifetime (0.81 ns), and detection limit (0.86 µM) in response to SDT. Cell imaging experiments showed that ODP could effectively detect and image intracellular hypoxia and the imaging capability of ODP was studied under various conditions including cell migration, antioxidant treatment, and different incubation times. Through comprehensive in vitro and in vivo experiments, including cellular imaging and mouse tumor models, this work demonstrates the efficacy of ODP in accurately detecting and imaging hypoxia. Moreover, ODP's potential in inducing apoptosis in cancer cells offers a promising avenue for integrating diagnostic and therapeutic strategies in cancer treatment. This innovative approach not only contributes to the understanding and assessment of tumor hypoxia but also opens new possibilities for targeted cancer therapy.

Abstract 5967: Blockade of Wnt5A/ROR1 signaling as cancer stem cell targeting therapy for metastatic prostate cancer

Abstract BACKGROUND– Wnt5A and its non-canonical Wnt receptor, ROR1, have emerged as a promising new signaling pathway target for lethal, metastatic prostate cancer. Wnt5A has emerged as a significant marker of poor prognosis in circulating tumor cells (CTCs) of metastatic castration resistant prostate cancer (mCRPC) patients. ROR1 is expressed in lethal types of mCRPC, especially neuroendocrine prostate cancer (NEPC). A therapeutic ROR1 antibody, Zilovertamab, has been clinically tested in chronic lymphocytic leukemia (CLL) and metastatic breast cancer and shown to be safe. We sought to investigate Zilovertamab-based therapies for metastatic prostate cancer in pre-clinical model systems. HYPOTHESIS – Wnt5A/ROR1 signaling may mediate a cancer stem cell program which makes cells resistant to therapies which target the cell cycle and proliferation. Blocking ROR1 may reveal vulnerabilities which sensitize cancer cells to chemotherapies like docetaxel. METHODS– ROR1 expression was determined using RNASeq, qRT-PCR, FACS, IHC and Western blotting. ROR1 signaling was blocked using the anti-ROR1 therapeutic antibody, Zilovertamab, or CRISPRCas9 ROR1 knock out. Cell growth was measured using an Incucyte real time imaging system. Stem cell activity was assessed in 3D organoids. Cell cycle analysis was performed in live cells in 2D cultures and 3D organoids using the Fucci2BL bicistronic Fluorescent, Ubiquitination-based Cell Cycle Indicator reporter system. PDX PCSD13 tumor growth was measured via calipers and IVIS. RNASeq was performed on tumors. RESULTS– We showed that ROR1 was expressed at high levels in mCRPC cell lines: PC3, DU145, and in the bone metastatic prostate cancer PDX: PCSD13. CRISPR/Cas9 Knock out of ROR1 in PC3 and DU145 cells showed increased sensitivity to docetaxel inhibition of proliferation in vitro in 2D real time Incucyte proliferation assays and in 3D organoids. Organoid formation was significantly reduced in ROR1 KO cells. Cells expressing the FUCCI live cell cycle tracker showed docetaxel led to G2 arrest and ROR1 signaling inhibition increased efficacy of docetaxel induced cell cycle arrest. Treatment of PCSD13 PDX in vivo with Zilovertamab plus docetaxel synergistically increased tumor growth inhibition in vivo and modulated cancer stem cell and cell cycle expression profiles. CONCLUSIONS– Cancer stem cells represent the fundamental precursors from which all diverse tumor subpopulations evolve. Thus, therapeutic targeting of these tumor initiating stem/progenitor cells may prevent the evolutionary diversification of a tumor and overcome a critical clinical barrier to cancer treatment. We showed synergistic response in our PDX and PCs cell line models to Zilovertamab plus docetaxel. We are now conducting a phase 1b clinical trial with zilovertamab plus docetaxel in patients with metastatic CRPC (CirmD, NCT05156905, PI R Mckay). Citation Format: Christina A.M. Jamieson, Jamillah Murtadha, Christopher S. Oh, Michelle T. Muldong, Niloofar Etemadfard, Evodie Koutouan, Young E. Yoon, Sanghee Lee, Oh Kwon, Jong J. Oh, Min S. Kim, Karl Willert, Nicholas A. Cacalano, Catriona H.M. Jamieson, Terry Gaasterland, Rana R. Mckay, Christopher J. Kane, Anna A. Kulidjian, Charles E. Prussak. Blockade of Wnt5A/ROR1 signaling as cancer stem cell targeting therapy for metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5967.

Abstract 3412: Benralizumab for treatment of eosinophil-related cutaneous adverse events associated with checkpoint inhibitors and anticancer targeted therapies: Phase 2 study

Abstract Background: Eosinophil-related cutaneous adverse events (ercAEs) are common toxicities of PI3K inhibitors, antibody drug conjugates (ADCs), checkpoint inhibitors (CPIs), and targeted cancer therapies. While systemic corticosteroids are effective, their use is limited by additional toxicities and high recurrence. Benralizumab is an anti-IL-5Rα monoclonal antibody that induces eosinophil depletion via antibody-dependent cellular cytotoxicity, and is FDA-approved for severe eosinophilic asthma but has not been studied for ercAEs. Methods: We conducted a single-center, single-arm, Phase 2 trial (NCT04552288) of benralizumab in cancer patients with Grade 2/3 ercAEs from PI3K inhibitors, ADCs, CPIs or targeted therapies. Patients received benralizumab 30 mg subcutaneously every 4 weeks for 3 doses, then every 8 weeks for 3 doses. The primary endpoint was clinical response, defined as reduction in ercAE Grade to 0/1 at Week 4. Secondary endpoints included changes in quality of life, rash body surface area (BSA), cytokines and eosinophil biomarkers, as well as adverse events (AEs). Results: Forty-seven patients were enrolled, predominantly with breast (55.3%), bladder (10.6%), melanoma (10.6%), or kidney (6.4%) cancer. Baseline ercAEs were Grade 2 (48.9%) and Grade 3 (51.1%), most often from PI3K inhibitors (46.8%), CPIs (21.3%), tyrosine kinase inhibitors (8.5%) or ADCs (8.5%). Overall, 32 of 42 evaluable patients (76.2%) had clinical response by Week 4, with decreased ercAE grade (mean 2.4 to 0.9, p<0.0001), improved quality of life, reduced rash BSA, decreased peripheral and skin eosinophils (all p<0.0001), and cytokine changes. Clinical response by Week 4 was seen in all patients with ercAEs from alpelisib (PI3K inhibitor; n=18) or enfortumab vedotin (ADC; n=4); p-value for both: p<0.05. Mild-to-moderate mucositis, diarrhea, and xerosis were the most common AEs, all unrelated to benralizumab. Conclusions: Benralizumab demonstrated favorable safety and efficacy for Grade 2/3 ercAEs from cancer therapies, especially alpelisib and enfortumab vedotin. Larger placebo-controlled trials are warranted to confirm these preliminary findings. Citation Format: Mario E. Lacouture, Tara Maier, Alexander Pan, Anna Chen, George Dranitsaris, Neil J. Shah, Sarat Chandarlapaty, Chau Dang, Devika Gajria, Allison Gordon, Neil Iyengar, Pedram Razavi, Mark Robson, Ezra Rosen, Serena Wong, Ucalene Harris, Kwami Ketosugbo, Cindy Bravo, Manu Jain, Alina Markova. Benralizumab for treatment of eosinophil-related cutaneous adverse events associated with checkpoint inhibitors and anticancer targeted therapies: Phase 2 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3412.

Abstract 5891: An image-based phenotypic screen identified CBP/p300 as new cancer drug resistance target and enabled the development of the clinical candidate TT125-802

Abstract Resistance to targeted therapies is a major challenge in oncology. Genetic mechanisms of resistance such as gatekeeper mutations in the primary drug target can be readily identified. However, drug targets responsible for non-genetic resistance mechanisms such as transcriptional reprogramming remain elusive. Here we describe an innovative phenotypic screening approach that led to the development of the clinical candidate TT125-802 and identification of its target CBP/p300 which we discovered as a novel regulator of transcriptional resistance to a range of targeted therapies. Targeted cancer therapies have been reported to induce profound transcriptional reprogramming leading to drug resistance. For instance, inhibition of oncogenic MAPK signaling using a BRAF/MEK inhibitor combination in BRAF-V600E mutated melanoma, triggers expression of the pluripotency transcription factor SOX2 along with broader expression of associated stemness and EMT genes. We used this system as a model to screen for small molecules that prevent transcriptional escape mechanisms to targeted cancer therapies. For this, we developed an automated high-throughput immunofluorescence staining and analysis method of SOX2 as a surrogate marker for resistance-conferring transcriptional reprogramming. A hit-like high diversity library of 16’000 small molecules was screened using an image-based multi-parameter readout with an integrated counter-screen for general cell toxicity. A battery of downstream assessments including patentability, synthetic tractability, physico-chemical properties, and dose-dependent modulation of transcriptional resistance signatures was used to select the chemical scaffold TT125 for further hit to lead optimization. Hit to lead optimization was initially target agnostic. To de-orphan the scaffold, a chemical probe of TT125 was developed in house and used in a chemoproteomics assay. The transcriptional co-activators CBP/p300 were identified as the target of TT125. Biochemical assays revealed that the TT125 scaffold binds specifically to the bromodomain of CBP/p300 but not the histone acetyl transferase domain. Moreover, TT125 was confirmed to bind highly selectively to the bromodomain of CBP/p300 but not other bromodomain-containing proteins such as BRD2 or BRD4. Structure-based drug design along with in vivo PD models using transcriptional short-term readouts were used for lead optimization that resulted in the development of the potent and highly selective clinical candidate TT125-802. Validating our phenotypic approach to drug discovery and development, TT125-802 is currently being evaluated in a phase I oncology trial. The modular setup of our phenotypic screening system has the potential to power the discovery of additional novel modulators of transcriptional drug resistance. Citation Format: Dorothea Gruber, Charles-Henry Fabritius, Thomas Bohnacker, Martin Schwill, Sara Laudato, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Stefanie Flückiger-Mangual. An image-based phenotypic screen identified CBP/p300 as new cancer drug resistance target and enabled the development of the clinical candidate TT125-802 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5891.

Abstract 5708: Silence exportin 7 sensitizes esophageal squamous cell cancer to CDK4/6 inhibition through block the cell cycle coresectory

Abstract Introduction: There is a lack of effective target therapies for esophageal squamous cell cancer. Our previous studies have demonstrated that CDK4/6 is a potency target for ESCC and combination with ERBB inhibitors is sufficient to achieve durable response. The aim of the current study is to prospectively map the landscape of sensitivity and resistance to CDK4/6 inhibition and develop novel combination therapies in ESCC. Experimental Procedures: We performed a genome-wide functional genetic screening and mini-pool designed CRISPR screen approaches to identify the modulators whose depletion sensitize CDK4/6 inhibition or confer resistance to the CDK4/6 inhibitor Palbociclib and elucidate new therapeutic strategies. Results: Our screen identified RB as the most enriched sgRNA, while guides targeting CCNE1 and CDK2 as the most depletion sgRNAs after CDK4/6 inhibition. These results validates our experimental approach as RB loss and CCNE1/CDK2 reactivation is the main mechanisms of CDK4/6 resistance. After gathering potential candidates from both screens, we found that genetically ablation of Exportin 7, a nuclear export factor, strongly sensitize CDK4/6 inhibition both in vitro and in vivo. RNAseq revealed a further repression of transcriptional activity of E2F and G2M by addition of XPO7depletion under CDK4/6 inhibitor treatment. To increase our insight in the molecular mechanism that could explain the synergy between XPO7 and CDK4/6, we further performed proteomics of nuclear and cytoplasm in ESCC lines with or without CDK4/6 inhibition. We found that silence XPO7 result in nuclear accumulation of mitosis-regulated genes such as CENPM, KIF4A and WEE1. Combined CDK4/6 inhibition with XPO7 depletion results in further downregulate of CENPM and KIF4A expression in nucleus, abrogating compensatory activation of cell cycle regulation and promote the cytostatic effect in ESCC. We further validated that CENPM and KIF4A depletion also sensitize CDK4/6 inhibition, and consequentially use WEE1 inhibitor after CDK4/6 inhibitor also effectively block the growth of ESCC lines. Conclusion: Overall, our comprehensive functional genetic screening approach revealed modulation of sensitive and resistance to the inhibition of CDK4/6 in ESCC. XPO7 was identified as a driver to change cell cycle landscape and merits as a therapeutic target combined with CDK4/6 inhibition in ESCC, which is beneficial to provide a novel treatment strategy. Citation Format: Zhong Wu, Lin Xu, Chuang Jiang, Adam Bass, Jin Zhou. Silence exportin 7 sensitizes esophageal squamous cell cancer to CDK4/6 inhibition through block the cell cycle coresectory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5708.

Abstract 251: MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis

Abstract To establish and maintain functional and properly formed tissues, cells must integrate cues from their environment into fate decisions. External cues—from other cells and the extracellular matrix for instance—must be met with appropriate responses to ensure cell fate decisions that control lineage plasticity and favor proper development, maintenance, or regeneration of tissues while suppressing neoplastic transformation. MCAM (CD146; MUC18; S-endo-1; and Gicerin) is an integral membrane glycoprotein whose misexpression is associated with a variety of epithelial cancers, including breast cancer. However, the mechanisms by which MCAM conveys extracellular cues into malignant cellular outputs is not well understood. Our observation that MCAM is normally expressed in transplantable fetal and adult mammary stem cells (MaSCs) with multilineage potential, suggested it could control mammary cell state plasticity in both neoplastic and normal mammary tissue. Using a plastic, mouse mammary tumor cell line derived from the MMTV-PyMT mouse (Py230) that has been reported to exhibit mammary stem cell and luminal progenitor features, we tested the role of Mcam in controlling the tumorigenicity and lineage/cell-state relationships of mammary tumor cells. We found that Mcam knockdown in Py230 cells increases STAT3 expression, activation, and downstream target transcription, leading to a shift in the predominant cell state away from a luminal progenitor state toward more differentiated alveolar and basal phenotypes. Mechanistically, these changes involve altered activity of casein kinase II (CK2) against multiple substrates including STAT3 and PTEN. This effect may be mediated by direct coupling and sequestration of CK2 to various substrates via the cytoplasmic tail of MCAM. We have also found that inhibition of Ck2 or Stat3 reversed the transcriptional state change elicited by Mcam knockdown. In grafted tumors in vivo, this state switch correlates with reduced overall tumorigenicity and attenuated expression of Sox10, a gatekeeper of neural crest-like invasive features. In the normal gland, we hypothesize that this activity regulates multilineage stem cell potential upon transplantation and multilineage regenerative potential across lactation/involution cycles as we found reciprocal expression patterns for MCAM and STAT3 in alveolar mammary epithelial cells. These data point to an integral role for MCAM in lineage plasticity of normal mammary epithelial cells that is co-opted during transformation to potentiate tumor cell plasticity, progression, and evasion of targeted cancer therapies. Citation Format: Brooke L. Gates, Ozlen Balcioglu, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 251.

Abstract 4700: Induced phenotype targeted ADC by using caspase enzymatic cleavable peptide linker to overcome tumor heterogeneity

Abstract Antibody-drug conjugates (ADCs) have ushered in a new era of precision cancer therapy, and the caspase-3-cleavable linker has demonstrated its potential as a game-changing addition to the ADC toolkit. The DEVD peptide, specifically cleaved by caspase and lysosomal enzymes, in combination with the KG peptide, provides the necessary flexibility to achieve targeted cancer cell apoptosis. This research explores the key attributes and actions of the linker, aiming to highlight its potential for revolutionizing ADC therapy. The caspase-3-cleavable linker operates through caspase-3 activation during apoptosis. After targeting cancer cells with specific antigens, the ADC is internalized into the cells, and the linker is cleaved by caspase-3, releasing the drug and initiating apoptosis. The specificity of caspase-3 extends the drug’s effect to neighboring cells, even those lacking the target antigen. This cascade effect ensures the continuous activation of ADCs until all cancer cells are eradicated. Firstly, it overcomes tumor heterogeneity by activating the drug outside of the cell. Traditional bystander killing effects rely on internalization followed by drug release and penetration of neighbor cancer cells. However, this linker's unique mechanism ensures that the drug is activated by caspase-3 outside the cell without the need for internalization, effectively targeting all cancer cells within the tumor tissue. This is achieved through the induced phenotype targeting effect (IPTE), which involves caspase-3 cleavage of the linker, inducing a phenotype in the tumor tissue that targets all cancer cells, regardless of their specific markers. Secondly, it maintains therapeutic efficacy even as the target antigen decreases. Current targeted therapies face a challenge as target antigens may diminish during treatment, leading to reduced drug effectiveness. However, the unique mechanism of the linker ensures that the drug continues to be activated as long as there is caspase-3 secretion, resulting in continuous activation of ADCs in the tumor, with a feedback amplification loop that ensures therapeutic efficacy until all cancer cells are eliminated. Lastly, the linker's superior physicochemical properties allow for a maximum drug-to-antibody ratio (DAR). Its hydrophilic nature enables independent drug conjugation, increasing therapeutic potential and mitigating aggregation issues. This innovation addresses the current limitations in ADC conjugation technologies. In summary, the caspase-3-cleavable linker represents a significant leap forward in the field of ADCs. Its unique attributes offer promising prospects for more effective and targeted cancer treatments. This linker is poised to revolutionize the landscape of targeted cancer therapy, bringing us one step closer to the dream of eradicating cancer cells with precision and efficiency. Citation Format: Ha Kyeong Lee, Byoungmo Kim, Hyo Won Chang, Sang Yoon Kim, Youngro Byun. Induced phenotype targeted ADC by using caspase enzymatic cleavable peptide linker to overcome tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4700.

Abstract 5963: VGLL1-derived peptides demonstrated anticancer effect by inhibiting VGLL1-TEAD4 interaction

Abstract YAP/TAZ, cofactors of TEA domain-containing transcription factor TEADs (TEAD1-4), play a key role in the HIPPO pathway, which is involved in various function including embryonic development, organ size, cell proliferation, tissue regeneration, and the development and progression of several cancers. YAP/TAZ is therefore considered as master regulators of various cancers. However, we are interested in VGLL1, a cofactor of TEAD4, because it is mainly involved in the proliferation and metastasis of various cancers, including gastric, breast, pancreatic, and ovarian cancers. In gastric cancer, VGLL1 expression is regulated by the PI3K/AKT/β-catenin pathway. VGLL1 is then phosphorylated at S84 by TGF-β/ribosomal S6 kinase 2 (RSK2) and interacts with TEAD4 to express target genes such as MMP9, cyclin D1, IGFBP3, IGFBP5 and ITGαV. which are involved in cancer malignancy. We designed S84-containing VGLL1-derived peptides (VGLL1S84 peptides, SCVP), which consist of 7 to 15 amino acids linked to CPP, a cell-penetration peptide. We evaluated SCVP to inhibit the VGLL1-TEAD4 interaction and their therapeutic potential as anticancer agents. SCVP suppressed the growth of gastric and breast cancer cells expressing high levels of VGLL1, EGFR, and/or HER2. In the RNA-seq analysis of NUGC3 to elucidate the molecular mechanisms of SCVP induced cell death, SCVP-treated cells showed gene expression changes in metabolic pathway, RAS signaling, PI3K-AKT signaling, Wnt signaling, TNF signaling, and mTOR signaling. However, RNA-seq analysis of breast cancer cells treated with SCVP showed changes in the expression of genes involved in cell cycle, DNA replication, DNA repair, and apoptosis. Then, we performed CRISPR/Cas9 screening using SCVP and sgRNA library to explore companion biomarkers and genetic interactors of VGLL1 for targeted cancer therapy. As a result, we identified several candidate genes, including ZNF292 and PLK1, as synthetic lethal genes of VGLL1. As expected, HCC70 cells with ZNF292 mutation showed increased sensitivity to SCVP. We also found that SCVP in combination and the PLK inhibitor volasertib resulted in a synergistic effect on the growth inhibition of breast cancer cells. In conclusion, we demonstrated VGLL1 as a novel target for anticancer drug development, and elucidated the molecular mechanism and therapeutic potential of the SCVP in gastric and breast cancers. Citation Format: Sangmi Ock, Changsub Lee, Wongi Jung, Ina Whang, Kyung-Soo Kim, Jongmin Han, Sujin Yim, Bo-Kyung Kim, Hong-Sub Lee, Kwan-Young Chang, Misun Won. VGLL1-derived peptides demonstrated anticancer effect by inhibiting VGLL1-TEAD4 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5963.

Abstract 441: Bioluminescent dehydrogenase assays: A novel approach for metabolic profiling and inhibitor screening in cancer biology

Abstract Dehydrogenases play a pivotal role in cellular metabolism, acting as gatekeepers in key metabolic pathways. Their activity reflects the metabolic state of the cell, which is particularly relevant in the context of cancer and the immune response, where metabolic reprogramming is a hallmark of tumor progression and differentiation stages. Here, we present the development and application of novel bioluminescent dehydrogenase assays for direct measurement of metabolic activity in cell lysates. Our assays are highly sensitive, requiring as few as 100-500 cells, and have been successfully miniaturized to a 384-well plate format conducive to high-throughput screening. This technological advance allows for the precise monitoring of dehydrogenase activity across the pentose phosphate pathway, glycolysis, and the mitochondrial tricarboxylic acid (TCA) cycle. We demonstrate how shifts in dehydrogenase activity can serve as indicators of the metabolic status of cells, and their modulation during differentiation. Moreover, our bioluminescent assays provide a rapid screening platform for pathway-specific metabolic inhibitors, with potential applications in identifying new therapeutic targets within cancer metabolism. Our data reveal a dynamic landscape of metabolic activity during T cell maturation, underscoring the potential of dehydrogenase activity profiling in cancer immunobiology and the development of targeted cancer therapies. Citation Format: Kim Haupt, Natasha Karassina, Anthony Lauer, Kayla Sylvester, Jolanta Vidugiriene. Bioluminescent dehydrogenase assays: A novel approach for metabolic profiling and inhibitor screening in cancer biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 441.

Abstract 7254: Evaluation of scFv and diabody pseudotyped Sindbis engineered retrovectors for targeted cancer therapy

Abstract Engineering viral vectors to target specific cells and organs after intravenous delivery remains a challenge. Transcriptional targeting with cell-specific promoters is a commonly used approach but does not change vector biodistribution. We have developed a novel gene delivery system targeted to tumor cells by pseudotyping the vector with Sindbis Virus (SB) envelope bearing an antigen-binding domain. Prototype vectors were generated by inserting a universal IgG binding domain from Protein A into the SB envelope (SB-ZZ). Antibodies (Abs) against various cell-surface targets (HLA, HER2, CD47, EGFR, Nectin-4), were conjugated to SB-ZZ vectors delivering a luciferase (Luc) reporter gene. Targeted transduction was confirmed on cells expressing the corresponding antigens by significantly higher Luc activity compared to SB-ZZ vectors without Abs. SB envelopes were further engineered by adding or removing furin cleavage sites and linkers, transduction-enhancing point mutations, and direct incorporation of single-chain variable fragment (scFv) in different VH and VL orientations, or diabody (DB) sequences, against the same targets. The specificity of optimized HER2-targeting vectors delivering the Luc reporter was evaluated in SKBR3 (HER2 over-expressing) and MDA-MB-468 (HER2-null) cells. Robust Luc activity was observed in SKBR3 cells, but only negligible activity in MDA-MB-468 cells. In MDA-MB-468 cells overexpressing HER2, significantly higher transduction was observed compared to the wild-type cells. When comparing HER2-targeting vectors with scFv vs. DB sequences based on the same antibody, transduction capability was higher with the DB motif in HER2-positive cell lines. Targeting vectors delivering HSV-eTK were also constructed, and ganciclovir (GCV)-mediated cell killing was evaluated in vitro. SKBR3 cells transduced with HER2-targeting vectors showed significantly more cell killing after GCV treatment than after transduction with an amphotropic reference vector. Our results indicate a method for directing gene therapy vectors to specific populations of tumor cells, and provide a framework for highly modular targeted gene therapy and immunotherapeutics. Experiments are underway to examine in vivo delivery of targeted vs amphotropic vectors. Citation Format: Makoto Sato, Alexander Cunha, Sara Collins, Noriyuki Kasahara, Cecilia Roh, Robert G. Johnson. Evaluation of scFv and diabody pseudotyped Sindbis engineered retrovectors for targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7254.

Abstract 413: Aneuploid cells depend on the RAF/MEK/ERK pathway and on RNA degradation for overcoming DNA damage and transcriptional burden

Abstract Aneuploidy, a state of karyotype imbalance, is a hallmark of cancer. Understanding the broad implications of aneuploidy at molecular, cellular, and physiological levels is crucial for effectively targeting aneuploid cancer cells. Aneuploidy often results in specific stress responses within cells, which must adapt to for survival and growth. Exploring these adaptive mechanisms to aneuploidy open avenues for targeted cancer therapies. Here, we induced aneuploidy in non-transformed RPE1-hTERT cells, generating multiple stable clones each exhibiting varying degrees of chromosomal imbalances. Employing a comprehensive genomic profiling strategy, we analyzed six isogenic clones using whole-exome and RNA sequencing. We uncovered a complex landscape, where each degree of aneuploidy brought its own set of changes and challenges. Additionally, we explored their cellular dependency landscapes, performing genome-wide CRISPR/Cas9 screenings along with extensive drug screens. As previously reported, we observed and confirmed increased DNA damage and p53 pathway activation in the aneuploid cells. We also found that the aneuploid clones triggered the DNA damage response (DDR), leading to an increased resistance to further DNA damage. Interestingly, we observed that aneuploid cells showed increased activity in the RAF/MEK/ERK pathway. Moreover, these cells demonstrated a heightened sensitivity to a range of clinically significant drugs that target this pathway, in particular in response to both genetic and chemical inhibition of CRAF. The activity of CRAF was found to be functionally connected to the resistance against DNA damage induction, and the inhibition of CRAF making aneuploid cells more susceptible to chemotherapies that induce DNA damage. Next, we observed increased of RNA synthesis and degradation in the aneuploid cells, along with heightened activity in both the nonsense-mediated decay (NMD) and the microRNA-mediated mRNA silencing processes. As a result, aneuploid cells displayed increased sensitivity to the perturbation of several key components of RNA degradation pathways. Furthermore, the dependencies of aneuploid cells on RAF/MEK/ERK activity and on intact RNA degradation were confirmed through examining a broad range of human cancer cell lines, emphasizing their significance in human cancer. Altogether, our results provide a comprehensive resource of karyotypically stable cells representing a range of aneuploidy states, and they reveal previously unidentified weaknesses in aneuploid cells that may be crucial for therapeutic developments. Citation Format: Marica Rosaria Ippolito, Johanna Zerbib Zerbib, Yonatan Eliezer, Eli Reuveni, Sonia Viganò, Giuseppina De Feudis, Anouk Savir Kadmon, Alice Ratti, Sara Martin, Kathrin Laue, Yael Cohen-Sharir, Simone Scorzoni, Francisca Vazquez, Uri Ben-David, Stefano Santaguida. Aneuploid cells depend on the RAF/MEK/ERK pathway and on RNA degradation for overcoming DNA damage and transcriptional burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 413.

Abstract 2623: Enabling 5T4 for targeted cancer therapy: TUB-030, a novel ADC built with ethynylphosphonamidate conjugation chemistry, shows long-lasting anti-tumor activity via Topoisomerase-I inhibition with an optimized therapeutic index

Abstract TUB-030 is a novel Antibody-Drug Conjugate (ADC) targeting the oncofetal 5T4 antigen, a glycoprotein, that is functionally involved in regulation of the Wnt signalling pathway and promoting cellular motility and adhesion. Immunohistochemical analysis identified 5T4 overexpressed in many types of cancers, including bladder, lung, breast, stomach, esophagus, head and neck, colon, and ovarian. Analysis in fresh frozen healthy human tissue with the therapeutic mAb candidate of TUB-030 revealed limited cross reactivity. Ethynylphosphonamidate conjugation chemistry was used to build TUB-030 with a homogenous DAR (drug-antibody-ratio) of 8 and the Topoisomerase I inhibitor Exatecan as payload, which is connected to a humanized, Fc-silenced IgG1 antibody targeting 5T4 using a cleavable linker system. Overall, TUB-030 contrasts previous ADC designs targeting 5T4, implementing several layers of differentiation in terms of antibody, payload and conjugation technology. In vitro, TUB-030 is characterized by sub-nanomolar affinity to 5T4, efficient target-mediated internalization and strong cytotoxicity towards cancer cells from different tumor indications with a broad range of 5T4 expression levels. Differently to previous 5T4-targeting ADCs, TUB-030 has efficient bystander activity against co-cultured target-negative cells, which facilitates targeting of tumors with heterogenous 5T4 expression. Pharmacokinetic analysis showed that TUB-030 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation and antibody-like clearance profile contrasting maleimide-conjugated ADCs and enabling most efficient and continued delivery of Exatecan to the tumor site. Single-dose treatment with TUB-030 results in long-lasting tumor regression with high complete remission rates across a variety of both cell-line derived xenograft (CDX) and patient-derived xenograft (PDX) cancer models of multiple tumor indications in vivo, including models with low target expression. Preliminary repeat-dose toxicological assessment of TUB-030 in a pharmacologically relevant non-human primate species demonstrated that TUB-030 is well tolerated. The preclinical results underline that TUB-030 with novel ADC technology has the potential to enable 5T4 as a therapeutic target and provide treatment options in numerous solid cancer indications with high unmet medical need supporting further development towards the clinic. Citation Format: Saskia Schmitt, Isabelle Mai, Paul Machui, Sarah Herterich, Danila Hauswald, Philipp Ochtrop, Philipp Cyprys, Izabela Kozlowska, Annabel Kitowski, Marcus Gerlach, Olivier Marcq, Pamela A. Trail, Dominik Schumacher, Marc-André Kasper, Günter Fingerle-Rowson, Björn Hock, Jonas Helma-Smets, Annette M. Vogl. Enabling 5T4 for targeted cancer therapy: TUB-030, a novel ADC built with ethynylphosphonamidate conjugation chemistry, shows long-lasting anti-tumor activity via Topoisomerase-I inhibition with an optimized therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2623.