Selective Targeting Research Articles

Abstract 5400: CD160 regulates angiogenesis in activated human endothelial cells

Abstract Angiogenesis, the process of forming new blood vessels from pre-existing ones, plays a pivotal role in various physiological and pathological conditions. CD160, a cell surface receptor known for its modulatory function in the immune response, has recently garnered attention as a surface marker for activated endothelial cells. In this study, we investigate the anti-angiogenic effects of a novel anti-CD160 antibody. Using the tube formation assay, we evaluated the novel anti-CD160 antibody's ability to inhibit neovascularization potential in activated Human umbilical vein endothelial cells (HUVEC) in vitro. Our results revealed a significant reduction in total tube length, the number of branches, and closed networks, indicating the antibody's efficacy in impeding neovascularization. In addition, we explored the molecular mechanisms underlying CD160's anti-angiogenic properties, showing its ability to selectively decrease fibroblast growth factor-2-induced angiogenesis. Furthermore, we assessed the safety profile of our new anti-CD160 antibody by examining its impact on endothelial cell apoptosis. Encouragingly, our findings demonstrated no induction of apoptosis, suggesting the antibody's selective targeting of angiogenic processes without compromising endothelial cell viability, thus supporting CD160's vascular normalization properties. In conclusion, our research provides compelling evidence for the anti-angiogenic effects of the CD160 antibody, as evidenced by its inhibition of tube formation without inducing apoptosis in activated endothelial cells. These findings hold promising therapeutic implications for targeting angiogenesis in various pathological conditions, warranting further exploration for potential clinical applications. Keywords: CD160, Angiogenesis, Endothelial cells. HUVECs. Citation Format: Abdel-ilah AZIZ. CD160 regulates angiogenesis in activated human endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5400.

Abstract 3198: An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting of triple-negative breast cancer

Abstract Background: Every breast cancer diagnosis is unique in its own way. Triple-negative breast cancer (TNBC), has a highly aggressive phenotype characterized by a rapid growth rate profile, increased risk of metastasis and recurrence. TNBC is devoid of epidermal growth factor receptor 2, estrogen, and progesterone receptors. All three are commonly present in other forms of breast cancer disease. The aim of this study was to develop a cell membrane lipid-extracted nanoliposomes (CLENs) for selective targeting compared to conventional nanoliposomal varieties. An additional objective was to investigate the role of microenvironment on targeting using cell model of TNBC, and representations of non-target tissue environments for comparison. Method: Lipid extracts (LE) obtained from 4T1, a murine mammary carcinoma cell line from a BALB/cfC3H mouse was used to formulate the CLENs (cell membrane lipid-extracted nanoliposomes) with different ratios of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a phospholipid used in most conventional drug delivery liposomes. Different lipid compositions and ratios were employed to evaluate cellular uptake in target, non-target and off-target cells. The target cell line, 4T1, off-target and non-target cell populations included CRL-2089, normal breast fibroblasts, SKBR3 a (human breast cancer), and A549 a (lung cancer) cell lines. Other studies involved determination of molecular charge and zeta potential of various nanoliposomal preparations and corresponding fluorescence studies in vitro. Results: The physicochemical properties of CLENs such as particle size and surface charge characteristics were vital when assessing benefit of the lipid-based preparations. The size of the CLENs typically ranged between 130 - 165 nm and zeta potential values were negatively-charged. The inclusion of 70 mol% of LE in preparation of 4T1-CLENs demonstrated increased cellular uptake and binding by the 4T1 target cells compared to the control DOPC (100%). The highest degree of selectivity was observed in early cell binding events, and with studies involving more extended incubation time points under different experimental conditions. Off-target cells demonstrated reduced uptake by comparison. Overall, 4T1-CLENs was most efficient when applied against intended target 4T1 cells, when compared to relevant control cell populations. In conclusion, studies performed to date suggest the notion that lipid extracts derived from TNBC improves targeting. Citation Format: Christiana Ayertey, Parmida Amid, Mohammed H. Almozain, Robert B. Campbell. An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3198.

Abstract 4536: MTAP loss alters the epigenetic landscape and demonstrates superior therapeutic sensitivity to concomitant PRMT5 and PARP inhibition in cholangiocarcinoma

Abstract 5’-methylthioadenosine phosphorylase (MTAP), is deleted in 15% of all cancers (estimated 100,000 patients per year in the United States), including 12-15% of cholangiocarcinoma (CCA) cases. MTAP deficiency accumulates 5’-methylthioadenosine (MTA) that subsequently binds protein arginine methyl transferase 5 (PRMT5) and inhibits its activity. This renders selective targeting of MTAP null tumors with agents (MRTX1719, AMG193, TNG462, TNG908) targeting MTA bound PRMT5 (PRMT5:MTA), while sparing surrounding normal MTAP wild-type (WT) tissue. Early signs of clinical activity was seen with MRTX1719, including objective responses in patients with MTAP loss tumors, including biliary tract cancer from the phase I/II study (NCT05245500). Despite these advances, prolonged targeted therapy poses the risk of development of acquired resistance demanding the need for better therapeutic strategies. To address this pressing need, we co-treated CCA cell lines with MRTX1719 and PARP inhibitor olaparib. We hypothesize that since PRMT5 is a critical regulator of splicing and DNA damage pathways, inhibition of PRMT5 disrupts these downstream processes creating an additional vulnerability to DNA damage inducing agents such as PARP inhibitors. Our preliminary data combining MRTX1719 with olaparib in three MTAP loss CCA cell lines (RBE, YSCCC, TFK1) show synergy (combination index; CI<1), and significant reduction in cell viability as well as colony formation ability when compared to monotherapy in vitro. To further gain mechanistic insights into the biology of MTAP loss, we stably expressed MTAP in MTAP null CCA cell line, RBE and demonstrated that MTAP loss drives tumorigenesis in vivo highlighting its tumor suppressor function in CCA. Mass spectrometry-based profiling of histone modifications from the isogenic RBE cell lines showed heightened methylation and reduced acetylation marks in the absence of MTAP. We also noted substantial differences in the distribution of splicing events, with increased intron retention, 3’ alternative splicing and decreased exon skipping as a result of MTAP loss. Similar differences in proportion of splicing events were also observed in MTAP loss CCA patient derived xenografts as compared to WT models. Collectively, these data implicate MTAP to be a crucial player in modulation of the chromatin and splicing events that may drive therapeutic response to PRMT5 inhibition. Citation Format: Pooja A. Shah, Ajay Kumar Saw, William Padron, Ming Zhao, Argun Akcakanat, Kurt Evans, Funda Meric-Bernstam, Milind Javle, Jordi Rodon, Kunal Rai. MTAP loss alters the epigenetic landscape and demonstrates superior therapeutic sensitivity to concomitant PRMT5 and PARP inhibition in cholangiocarcinoma [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 4536.

Abstract 1955: Novel long-acting covalent PI3Kα inhibitors boost potential action in cancer

Abstract Phosphoinositide 3-kinase (PI3K) is a key regulator of cell proliferation, survival, and metabolism. Hotspot mutations in the PIK3CA gene constitutively activate the catalytic subunit of the PI3Kα isoform and are found in 10-30% of solid tumors. The development of PI3K pathway inhibitors has been hampered by poor drug tolerance and release of negative feedback loops, leading to rapid reactivation of signaling in response to reversible inhibitors. To remedy these challenges, we developed highly selective covalent PI3Kα inhibitors that irreversibly bind to a non-conserved cysteine (C862) located 11 Å outside the ATP-binding site. Here we present the properties of an optimized drug-like covalent lead, dubbed compound 9, and describe the generation of a biotinylated probe developed to be used in conjunction with compound 9 to monitor PI3Kα target occupancy in relation to downstream PI3K signaling outputs. Biochemical investigations of compound 9 using TR-FRET assays showed high affinity binding (Ki), high reaction rates (kinact) for covalent bond formation with PI3Kα, and negligible off-target reactivity. Cellular NanoBRET assays revealed that compound 9 diffuses three times more rapidly into cells as compared to BYL719 (alpelisib), and leads to rapid and potent on-target engagement. Covalent bond formation to residue C862 of PI3Kα was further confirmed by X-ray crystallography. In PIK3CA mutant cancer cell lines, covalent binding of PI3Kα occurred at low nanomolar concentrations and led to persistent inhibition of Akt/PKB phosphorylation for more than 72 hours after drug removal. Notably, investigation of PI3Kα re-synthesis after labeling with compound 9 showed a negligible reappearance in cancer cell lines, which indicates that persistent suppression of PI3Kα signaling by compound 9 results in a much longer half-life for PI3Kα than previously reported. The irreversible inactivation of PI3Kα by compound 9 provides a marked gain in growth inhibition potency (10-600-fold) in PIK3CA mutant cancer cell lines in comparison to clinical reversible PI3Kα inhibitors and allows for intermittent dosing without compromising efficacy. These data collectively demonstrate that compound 9 enables precise and selective targeting of PI3Kα with high potency and long-lasting efficacy in cancer models. The new class of irreversible PI3Kα inhibitors have a unique pharmacology among PI3K inhibitors characterized by strong decoupling of drug exposure from efficacy which may provide opportunities to lower treatment burden in patients. Citation Format: Theodora A. Constantin, Lukas Bissegger, Erhan Keleş, Luka Raguž, Clara Orbegozo, Thorsten Schaefer, Isobel Barlow-Busch, John E. Burke, Chiara Borsari, Matthias P. Wymann. Novel long-acting covalent PI3Kα inhibitors boost potential action in 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 1955.

Abstract 198: Selective targeting of endothelial mTORC1 reduces transendothelial fatty acid delivery to improve anti-tumor immunity and suppress metastatic outgrowth

Abstract Tumor metastasis requires sufficient nutrients to support outgrowth of disseminated tumor cells. Cancer cells utilize fatty acids as a key source of energy and biomass, but these nutrients impair the cytotoxic activities of T cells, serving to support metastatic progression. Blood is a rich source of fatty acids, but the vascular endothelium serves as a barrier to limit access to the surrounding tumor tissue. However, it is unclear how long-chain fatty acid delivery to early metastatic tumors is regulated. Here, we report that endothelial mTORC1 promotes transport of fatty acids into developing metastatic tumors, leading to reduced anti-tumor immune responses and improved outgrowth. Using a murine model of metastatic outgrowth in the lung, tumor burden was significantly reduced upon targeted deletion of Raptor, an essential component of the mTORC1 complex, in endothelial cells (RptorECKO). We employed a multifaceted approach using metabolomics, transcriptomics, and molecular assays to identify a Raptor/mTORC1-mediated mechanism of transendothelial fatty acid transport via RAB and CLSTN1 cargo trafficking. In vivo uptake of a fluorescent palmitic acid analog in tumor cells and T cells was reduced in RptorECKO lung metastatic tumors, which correlated with improved markers of cytotoxicity. Combination of low-dose RAD001/everolimus, which selectively inhibits mTORC1 in endothelial cells, with anti-PD1 reduces metastatic disease and impairs long-chain fatty acid uptake in T cells, corresponding to improved anti-tumor immunity. These findings describe a novel mechanism of transendothelial fatty acid transportation during metastatic outgrowth, supporting future development of therapeutic strategies to target endothelial mTORC1 activity. Citation Format: Deanna N. Edwards, Shan Wang, Wenqiang Song, Laura C. Kim, Yoonha Hwang, Jin Chen. Selective targeting of endothelial mTORC1 reduces transendothelial fatty acid delivery to improve anti-tumor immunity and suppress metastatic outgrowth [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 198.

Abstract 192: Orchestrating CDC42 turnover to regulate membrane protrusion and tumor metastasis

Abstract F-actin cytoskeleton remodeling is essential for cell migration, organ development, and immune responses. CDC42, a factor orchestrating F-actin remodeling for membrane dynamics, switches between its inactive GDP- and active GTP-bound forms. However, the biological and clinical significance of the mechanisms regulating CDC42 protein turnover remains unclear. Here we show that KLHL23-mediated CDC42·GTP polyubiquitylation for degradation and RhoGDI-mediated CDC42·GDP sequestration away from the plasma membrane co-inactivate CDC42 in a spatiotemporal context, influencing membrane dynamics and homeostasis during migration. Via a functional genomic screen, we identified KLHL23 as a suppressor of tumor invasion. Decreased KLHL23 level was associated with tumor metastasis and poor clinical outcomes in patients with liver and pancreas cancers. KLHL23 functions as the E3 ligase responsible for CDC42 polyubiquitylation and degradation. KLHL23 shares with RhoGDI the switch II region of CDC42 for binding, enhancing selective targeting of CDC42·GTP and CDC42·GDP, respectively. KLHL23 depletion in cells leads to F-actin and membrane over-protrusion, as well as epithelial-mesenchymal transition and tumor metastasis. Fluorescence resonance energy transfer assays reveal that the KLHL23—CDC42·GTP interaction plays a primary role in quenching CDC42 activity during membrane protrusion-retraction, while the RhoGDI—CDC42·GDP interaction occurs lately. Our results demonstrate the spatiotemporal interplay between RhoGDI and KLHL23 in regulating CDC42 turnover for membrane dynamics. As KLHL23/RhoGDI—CDC42 axis dysregulation causes tumor metastasis, our findings open avenues for exploring novel therapeutics. Citation Format: Sen-Yung Hsieh. Orchestrating CDC42 turnover to regulate membrane protrusion and tumor metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 192.

Abstract 4428: Integrated multi-omics analysis reveals systemic and localized metabolic disruptions in colorectal cancer

Abstract Despite technological advances in molecular medicine over the last 30 years, no single approach has proven to be sufficient to meet the diagnostic needs in cancer care. Here we use a multiomic approach leveraging proteomic, metabolomic, and transcriptomic technologies to study the metabolic shifts that occur during colorectal cancer (CRC) progression, Our integrated analysis identified systemic changes to beta oxidation pathways and localized changes to tyrosine metabolism within the tumor, creating a mechanistic, actionable description of the core CRC metabolic program. We conducted untargeted proteomics and metabolomics profiling on serum samples from CRC patients (n=10) and healthy controls (n=10). Our serum proteomics data is generated through sample enrichment on the Seer Proteograph system followed by LC/MS analysis with the Thermo Orbitrap Astral. Our metabolomics approach employs LC/MS assays for unbiased profiling of the serum metabolome, exposome, and lipidome. We supplemented this discovery work with both targeted serum and tumor-specific approaches including targeted proteomics to quantify select targets, inflammatory profiling with Alamar proteomics, public transcriptomics data from TCGA, and in vitro metabolic flux data. These large and diverse datasets were then integrated through joint pathway analysis and network integration. The global serum proteomics and metabolomics profiles generated show distinct separation between healthy and diseased individuals. Joint pathway analysis of these discovery datasets highlighted enrichment in beta oxidation pathways and tyrosine metabolism, among others. Interestingly, the Alamar inflammatory panel revealed that many of the analytes in the tyrosine metabolism pathway were well correlated with inflammatory status, while the beta oxidation signature had a lower correlation. To determine the relationship of these systemic findings from serum to tumor metabolism itself, we integrated tumor-specific transcriptomics data and found alterations to tyrosine metabolism with differences in tyrosine aminotransferase expression. The beta oxidation related genes, on the other hand, were not concordant with the serum findings. However, our in vitro metabolic flux studies have shown beta oxidation in CRC cells is upregulated to provide additional fuel for oxidative phosphorylation. This result suggests that beta oxidation may not be transcriptionally regulated in the tumor but rather a consequence of organismal metabolic rewiring. The results of this work, which is currently expanding into a larger and more diverse patient population, underscores the power of multiomic profiling for enhancing our understanding of metabolic dysregulation in CRC. Ultimately, a comprehensive model of the molecular alterations in CRC will yield a better understanding of tumor phenotypes and inform better diagnostics and therapies. Citation Format: Gary Patti, Ethan Stancliffe, Adam Richardson, Ashima Mehta, Monil Gandhi, Kevin Cho. Integrated multi-omics analysis reveals systemic and localized metabolic disruptions in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4428.

Abstract 2914: AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation

Abstract Background: While checkpoint inhibitors have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal objective responses observed. Phenomic has developed a proprietary machine learning/artificial intelligence platform to identify novel stromal targets with superior expression profiles that enable selective targeting of immune activating agents that will relieve these immunosuppressive barriers in difficult to treat indications. Methods: Using our single cell RNA Atlas, we assessed cancer-associated fibroblasts (CAFs) in several solid tumor indications for identification of novel targets, including proteoglycans. Our Atlas was also used to identify immune activating payloads whose cognate receptors were present in indications of interest. Antibodies were generated, and lead clones who demonstrated potent ligand binding and cell staining were used to generate fusion proteins to immune activating ligands. Efficacy and PD were assessed in multiple syngeneic tumor models. Results: Bioinformatic analysis identified a unique subset of pathogenic CAFs, which are TGF beta responsive, secrete several ECM proteins, and their presence tracks with poor outcome and resistance to immunotherapy in several solid tumor types. CTHRC1 was identified as a novel matrix protein highly expressed in this CAF subtype as well as tumor epithelium and is highly selective in a range of tumor types such as ovarian cancer, triple negative breast cancer, and pancreatic ductal adenocarcinoma. While secreted, CTHRC1 is complexed on the cell surface and affords the opportunity to drug this target in a variety of ways. We identified an absence of 4-1BBL expression across indications of interest, and fusion proteins were generated to deliver 4-1BBL via CTHRC1 targeting. In checkpoint-resistant tumor models, we observed significant increases in CD8 T cells and robust anti-tumor activity with anti-CTHRC1-targeted 4-1BBL. Biodistribution studies were conducted using our targeting mAb and demonstrate uptake only in sites of primary and metastatic tumors, even at doses 20-fold higher than those that achieve maximal therapeutic activity, suggesting the potential to minimize the toxicity that has been observed with other 4-1BB agonists. Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both pathogenic CAFs and tumor cells that is highly selective for tumors, enabling the therapeutic targeting of immune activating payloads with the potential for safely delivering payloads while limiting toxicity. Given the specificity and selectivity afforded by CTHRC1 expression, ADC and CD3 engager approaches are also being pursued. These data represent novel approaches aimed at breaking down stromal barriers in tumors previously unresponsive to immunotherapies. Citation Format: Christopher Harvey, Elizabeth Koch, Amanda Hanson, Lindsey Rice, Amy Berkley, Kerry White, Reza Saberianfar, Nikolai Suslov, Sam Cooper, Michael Briskin. AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation [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 2914.

Abstract 3303: Paralogue selective p300 degraders induce synthetic lethality in pre-clinical models of CBP-deficient and p300-dependent malignancies

Abstract EP300 (or p300) acts as histone acetyltransferase (HAT) and transcriptional adapter or co-activator regulating transcription via chromatin remodeling. Both histone and non-histone proteins are acetylated by p300. In addition to HAT function, p300 has crotonyl-transferase activities, and that p300-catalyzed histone crotonylation directly stimulates transcription to a greater degree. p300 functions by scaffolding or as co-activator and enhancer of different transcription factors like HIF1a, BRCA-1, p53, NFκB, c-Myc, estrogen receptor (ER) and androgen receptor (AR) and other proteins such as PD-L1 and FOXP3. Selective targeting of p300 is expected to lead to therapeutic efficacy in CBP-mutant and p300-dependent malignancies with high degree of tolerability because of sparing of the other paralog CBP in normal cells. CBP mutant cancers comprise of several solid and hematological malignancies including 10% to 15% of non-small cell and small cell lung cancers harboring loss-of-function (LOF) aberrations. p300-dependent malignancies include prostate cancer, in which p300 plays a major role for androgen-dependent and -independent transactivation of the AR, MYCN-amplified neuroblastoma and ER+ breast cancers. Since the conventional CBP/p300 inhibitors do not discriminate between CBP and p300 proteins due to high sequence homology, we adopted a degrader approach, which has the potential to lead to paralog selectivity due to differentiated ternary complex formation. Herein we report best-in-class novel, potent and paralog selective p300 degraders with excellent selectivity over CBP and and other bromodomain containing proteins such as BRD4 by implementing molecular modeling, iterative medicinal chemistry and SAR based approaches. Synthetic lethality has been demonstrated while selectively degrading p300 in the cell lines having LOF mutation of another paralogue, CBP. Identified selective p300 degraders displayed excellent selectivity towards p300 degradation across tested cell lines and are orally bioavailable. Selective antiproliferative activity was observed in a panel of cell lines with CBP mutations or p300 dependency including AR+/ER+ cell lines along with the downregulation of target genes. In summary, we have identified highly selective degraders of p300 with desirable profile. Efforts are underway to advance them further towards nominating a development candidate. Citation Format: Saravanan Thiyagarajan, Chandrasekhar Abbineni, Krishna Chaitanya T, Achala Apte, Iram Khan Iqbal, Naveen Kumar R, Aravind A B, Avinash Kumar, Dabbeeru Madhu Babu, Shwetha Shwetha, Ankita Manna, Monalisha Mandal, Rituparna Panigrahi, Sivapriya Marappan, Suraj T Gore, Mohamad Fairus, Lim Yang Jin, Subhendu Mukherjee, Girish Daginakatte, Shekar Chelur, Kavitha Nellore, Murali Ramachandra, Susanta Samajdar. Paralogue selective p300 degraders induce synthetic lethality in pre-clinical models of CBP-deficient and p300-dependent malignancies [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 3303.

Abstract 897: Synthetic lethality screening with Recursive Feature Machines

Abstract Synthetic lethality refers to the concept that simultaneous perturbation of gene pairs leads to cell death but individual perturbation does not. Synthetically lethal gene pairs (SL pairs) provide a potential avenue for selective targeting of cancer cells based on underlying genetic vulnerabilities. The rise of large scale gene perturbation screens such as the Cancer Dependency Map (DepMap) offers the opportunity to identify SL pairs automatically using machine learning. Yet, a key difficulty in using machine learning models for this task is the lack of labeled data in the form of known SL pairs. Thus, prior approaches framed SL pair identification as a feature learning problem where the goal is to identify the genomic features most influential for predicting low cellular viability under a gene knockout. These prior approaches have primarily utilized random forests since these machine learning models are one of the only nonlinear models for which feature importances are provided explicitly. On the other hand, if we could identify features learned by state-of-the-art models on screening tasks such as kernel machines, we would be better powered in finding SL pairs. In this work, we present a computationally efficient and effective pipeline for SL pair screening built on a recently developed class of feature learning kernel machines known as Recursive Feature Machines (RFMs). We show that our pipeline more accurately recovers experimentally verified SL pairs than prior work including the best model from the DepMap portal and previous random forest based approaches (see the table below). Moreover, our pipeline identifies several new candidate SL pairs for further analysis, opening new avenues for targeting genetic vulnerabilities in cancer. Rank of verified SL pairs across unsupervised methods (lower is better with a minimum value of 1). Experimentally Verified SL Pairs Pearson Correlation PARIS DepMap RFMs (Ours) SMARCA2/SMARCA4 1 1 1 1 ARID1A/ARID1B 1 1 1 1 STAG1/STAG2 1 1 1 1 CREBBP/EP300 1 1 1 1 VPS4A/VPS4B 7 > 10 5 1 DDX17/DDX5 4 1 1 1 ENO1/ENO2 > 10 1 1 1 SMARCC1/SMARCC2 > 10 1 1 1 UBB/UBC > 10 1 > 10 1 MAGOH/MAGOHB 1 1 1 1 FAM50A/FAM50B 1 1 1 1 Citation Format: Adityanarayanan Radhakrishnan, Cathy Cai, Barbara A. Weir, Christopher Moy, Caroline Uhler. Synthetic lethality screening with Recursive Feature Machines [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 897.

Abstract 4160: V7 targeted liposomes detect pancreatic adenocarcinoma via multispectral optoacoustic tomography

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDA) remains one of the most aggressive malignancies with a 5-year survival rate of 9-10%. Insufficient early detection coupled with limitations in operating surgery renders PDA as one of the most challenging tumors to treat. Accurate localization of tumor margins can tremendously improve surgical treatment outcomes in patients. Recently, nanotechnology has emerged as a promising platform for theranostic applications in the field of drug discovery, although the clinical success is limited by their tumor-specific uptake. Hypoxia due to aberrant vascularization is a hallmark of PDA and can be exploited for active targeting. pH-low insertion peptide variant 7 (V7) is used as hypoxia and acidosis-dependent biomarker for selective targeting of pancreatic cancer tissue. This work is focused on using IR 780 contrast agent loaded, V7 peptide conjugated liposomes for the active targeting of PDA towards enhanced surgical outcomes. Methods: Liposomes encapsulating IR780 dye were prepared using thin film hydration technique. V7 peptide was conjugated to liposomes using an SMCC linker and liposomes were characterized for their size, polydispersity and surface potential. In-vitro uptake of IR780-V7-liposome by S2VP10 at pH 7.4, 6.8, and 6.6 was evaluated through NIR fluorescence imaging using an Odyssey Infrared System. Athymic mice were orthotopically implanted with S2VP10 cells. After tumors were 3 mm, V7-liposomes and untargeted liposomes (0.01 OD) were intravenously injected and biodistribution and tumor specific uptake was accessed using multispectral optoacoustic imaging (MSOT). Results: Dynamic light scattering (DLS) revealed the size of liposomes to be 109 nm with a polydispersity index of 0.198 and zeta potential of +35.8 mV. Surface conjugation of V7 peptide was confirmed using the zeta potential measurement. NIR fluorescence imaging in S2VP10 cells established enhanced tumor specific uptake of V7 conjugated liposomes than passively targeted liposomes (p<0.05). Biodistribution of V7-liposomes demonstrated tumor specific accumulation (PDAC tumor 23.7 a.u, liver 3.6 a.u., and kidney 1.1 a.u.) while untargeted liposomes had the greatest uptake in liver (PDAC tumor 2.1 a.u., liver 6.3 a.u., and kidney 0.7 a.u.) p<0.05. Conclusion: pH-specific actively targeted liposomes can be used to specifically target PDA with minimal off-target binding effects. V7-IR780-liposome formulation can be used to efficiently deliver contrast agent for imaging tumor margins and to expand the potential of MSOT towards clinical applications. Citation Format: Happy Agarwal, Phillip Chuong, William E. Grizzle, Barish H. Edil, Ajay Jain, Lacey R. McNally. V7 targeted liposomes detect pancreatic adenocarcinoma via multispectral optoacoustic tomography [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 4160.

Abstract 3309: Discovery of potent and selective bivalent CDK2 degraders that demonstrate activity in CCNE1amp driven tumors

Abstract Introduction: The Cyclin-Dependent Kinases (CDKs) with their cyclin binding partners, are associated with cell cycle progression and transcriptional regulation. Targeting CDKs is a key oncology therapeutic strategy with CDK4/6 inhibitors demonstrating significant clinical benefit in HR+/HER2− metastatic breast cancer, the most prevalent subtype. However, 30% of patients develop acquired resistance in the clinic with cyclin E1 (CCNE1) amplification/overexpression and CDK2 activation implicated as a major resistance mechanism to CDK4/6i breast cancer therapy. Additionally, CCNE1/CDK2 activation has also been associated with poor prognosis in ovarian and endometrial cancers. Selective targeting of CDK2 using small molecule inhibitor-based approaches have recently advanced into the clinic. Targeted protein degradation of CDK2 provides an alternative strategy that has the potential to eliminate the activity of the CCNE1/CDK2 complex, as well as CDK2 complexed with other cyclins, including cyclin A, and provide improved clinical benefit. Results: Discovery efforts at Plexium have identified CDK2 bivalent degraders that consist of a CDK2-binding moiety, a linker and a high affinity cereblon-binding ligand. Cereblon binding potency was found to correlate with potent and deep degradation of CDK2. Degradation was blocked in the presence of a proteasome inhibitor as well as in a cereblon knock-out cell line, confirming that CDK2 degradation is mediated by the ubiquitin proteasome system and through engaging cereblon. Proteome-wide analysis demonstrated that CDK2 was selectively depleted without significantly modulating other CDKs or known cereblon neo-substrates. Selective degradation was achieved despite lack of selectivity for binding/inhibition of other CDKs and was confirmed to be dependent on the formation of a CRBN-CDK2 ternary complex. Dose dependent CDK2 degradation resulted in dose dependent inhibition of Rb phosphorylation, cell cycle arrest, senescence-associated phenotypes and antiproliferative activity in CCNE1 amplified cancer cell lines. The in vitro data was used to evaluate the PK/PD drug exposure-response relationship in vivo and deep CDK2 degradation was demonstrated both in vitro and in vivo. Conclusions: These data provide validation for CDK2 degradation as a therapeutic approach. Potent and selective CDK2 bivalent degraders were exploited as tools for studying the sensitivity of CCNE1 amplified tumor models to CDK2 degradation and dependence on E3 ligase. This proof-of concept study supports Plexium’s current approach of discovering novel CDK2 molecular glue degraders for the treatment of CDK4/6 inhibitor-naïve and -resistant HR+/HER2− breast cancer, and CCNE1 amplified ovarian and endometrial cancers. Citation Format: Adina Gerson-Gurwitz, Pengyu Yang, Sarah Fish, Gabrielle Blanco, Hongfeng Gao, Kyohei Hayashi, Mike Hocker, Aleks Jamborcic, Andre Richters, Mary E. Spalding, Julia Toth, Duc Tran, Linette Yang, Shu You, Andrew Burritt, Alex Campos, Gregory Parker, Kevin Freeman-Cook, Peggy A. Thompson, Simon Bailey. Discovery of potent and selective bivalent CDK2 degraders that demonstrate activity in CCNE1amp driven tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3309.

Abstract 4077: T cell targeting with PD-1-selective immune cell engagers based on GlycoConnect™ (GC™-ICEs) show excellent efficacy and tolerability

Abstract Engagement of T cell cells to harness a patient’s immune system is a promising approach in immuno-oncology. An immunocytokine, i.e. a tumor-targeting antibody (fragment) genetically fused to an immunostimulatory cytokine, is specifically designed for this purpose. Fusion antibody-immunocytokines, based on molecular architectures generated by a variant of DNA recombinant technologies, have recently entered the clinic. Earlier we have demonstrated how GlycoConnect™, a site-specific conjugation technology anchoring on the native antibody glycan1, can be applied for attachment of various cytokines, including RLI or IL-15, without prior antibody engineering. The immunostimulatory activity of these GlycoConnect™ immune cell engagers (GC™-ICEs) can be modulated by tailoring linker design and payload stoichiometry. In this presentation, we demonstrate the GC™-ICE with either IL-15 or RLI can be applied to selective targeting of T cells, based on PD-1-selective binding and activation, using either protease-sensitive or non-cleavable linkers. We will show how such T cell targeting can be achieved leading to significant tumor volume reductions in syngeneic mouse models. Moreover, we have found that the GC-ICE™ can be applied without inducing systemic immune activation, thereby displaying a promising therapeutic index. In vitro and in vivo studies will be presented to showcase the biological activity of these GC™-ICEs in comparison to conventional immunocytokines obtained via genetic engineering.1Wijdeven et al., Enzymatic glycan remodeling-metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering. mAbs 2022, 14, DOI: 10.1080/19420862.2022.2078466. Citation Format: Remon van Geel, Mick Verhagen, Elias Post, Willem Vugs, Sorraya Popal, Sander van Berkel, Floris van Delft. T cell targeting with PD-1-selective immune cell engagers based on GlycoConnect™ (GC™-ICEs) show excellent efficacy and tolerability [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 4077.

Abstract 6575: Selective targeting of integrins αVβ8 and αVβ1 within the dynamic ecosystem of pancreatic cancer to improve the overall anti-tumor response

Abstract Pancreatic ductal adenocarcinoma (PDA) has a 5-year survival of less than 10% and remains the 3rd leading cause of cancer-related death in Western societies. New treatment options are urgently needed. TGF-β promotes stromal cell reprogramming, immunosuppression, and fibrinogenesis in cancers, including PDA. Integrins αVβ8 and αVβ1 are important activators of TGF-β signalling. Selective integrin blockade has recently emerged as a promising therapeutic approach to address TGF-β-mediated immunotherapy resistance and improve anti-tumour response across cancer models. The purpose of this study was to assess the in vivo efficacy of small molecule inhibitor PLN-104, a selective inhibitor of αVβ1, and PLN-101095, a dual inhibitor of αVβ8 and αVβ1, in well-annotated models of advanced PDA. We determined the preclinical efficacy and detailed anti-stromal effect of PLN-101095 inhibitor in genetically defined and patient-derived PDA models, including clinically relevant combinations with standard of care (SoC) chemotherapy and anti-programmed death receptor-1 antibody (anti-mPD-1). Mechanistic assessment of alterations in tumour cell-stromal cell crosstalk was performed using comprehensive transcriptomics and immunofluorescence approaches. Dual targeting of αVβ8 and αVβ1 with PLN-101095 in the syngeneic LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre (KPC) model effectively reduced tumour growth by 45% in comparison to the vehicle, and significantly delayed disease progression in vivo. Single-cell analysis of PLN-101095-treated KPC pancreatic tumours revealed a positive reprogramming of malignant cells from a mesenchymal to a more epithelial-like state. This effect was further associated with an upregulation of MHC type I/II markers and corresponding downregulation of pro-metastatic factors across diverse cancer sub-populations. Additionally, combining PLN-101095 with anti-mPD-1 antibody further improved survival in this aggressive model of metastatic PDA. In a second syngeneic model, Pan02, PLN-101095 in combination with anti-mPD-1 antibody significantly reduced tumour growth, while increasing CD8+ lymphocyte infiltration. The combination of PLN-101095 with Anti-PD1 increased MHC and IFN gene expression. Finally, utilizing patient-derived models of metastatic PDA revealed that both PLN-104 and PLN-101095 significantly blocked tumour growth, improved the response to SoC chemotherapy Gemcitabine/Abraxane, and reduced metastatic spread to distant sites. In conclusion, these data provide scientific rationale for the design of future PLN-101095 and SoC chemotherapy as well as immunotherapy combinations in pancreatic cancer, with Phase I first-in-human oncology studies with PLN-101095 plus ICB already underway. Citation Format: Dannielle Upton, Diego Chacon Fajardo, Sofia Omari, Sean Porazinski, Benjamin McLean, Diana Schuhmacher, Aji Istadi, Australian Pancreatic Cancer Genome Initiative, Vishal Kothari, Darren Finkelstein, Tim Machajewski, Fernando Rock, Scott Turner, Marina Pajic. Selective targeting of integrins αVβ8 and αVβ1 within the dynamic ecosystem of pancreatic cancer to improve the overall anti-tumor response [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 6575.

Limiting downstream dispersal of invasive carp egg surrogates using a laboratory-scale oblique bubble screen

We conceptualize and test a non-intrusive barrier, comprised of an oblique bubble screen (OBS) oriented at an angle to the mean flow, to prevent the downstream dispersal of invasive carp egg surrogates. Three surrogates of different densities and diameters were tested. Secondary flows created by the OBS were tuned to redirect surrogate eggs to facilitate their capture. Surface particle image velocimetry and acoustic Doppler velocimetry were used to characterize secondary flows. We assessed the influence of airflow rate, OBS angle, mean flow velocity, and surrogate density on particle redirection. In general, redirection efficiency improves by increasing the OBS angle with respect to the cross-section. At a mean flow velocity of 0.75 metres per second (m/s), the OBS system redirected up to 60% (%) of positively buoyant particles (specific gravity SG = 0.9, and diameter d = 7.09 millimetres [mm]) and 40% of semi-buoyant particles (SG = 1.001, d = 3.1 mm). Negatively buoyant particles (SG = 1.04, and d = 5.90 mm) were redirected by the physical structure of the diffuser rather than by OBS-induced flow. The study shows that an OBS system can be used to effectively redirect carp-egg surrogates over a wide range of particle sizes and densities, allowing for selective targeting of undesired particles in streams.

Augmenting Cancer Therapy with a Supramolecular Immunogenic Cell Death Inducer: A Lysosome-Targeted NIR-Light-Activated Ruthenium(II) Metallacycle.

Though immunogenic cell death (ICD) has garnered significant attention in the realm of anticancer therapies, effectively stimulating strong immune responses with minimal side effects in deep-seated tumors remains challenging. Herein, we introduce a novel self-assembled near-infrared-light-activated ruthenium(II) metallacycle, Ru1105 (λem = 1105 nm), as a first example of a Ru(II) supramolecular ICD inducer. Ru1105 synergistically potentiates immunomodulatory responses and reduces adverse effects in deep-seated tumors through multiple regulated approaches, including NIR-light excitation, increased reactive oxygen species (ROS) generation, selective targeting of tumor cells, precision organelle localization, and improved tumor penetration/retention capabilities. Specifically, Ru1105 demonstrates excellent depth-activated ROS production (∼1 cm), strong resistance to diffusion, and anti-ROS quenching. Moreover, Ru1105 exhibits promising results in cellular uptake and ROS generation in cancer cells and multicellular tumor spheroids. Importantly, Ru1105 induces more efficient ICD in an ultralow dose (10 μM) compared to the conventional anticancer agent, oxaliplatin (300 μM). In vivo experiments further confirm Ru1105's potency as an ICD inducer, eliciting CD8+ T cell responses and depleting Foxp3+ T cells with minimal adverse effects. Our research lays the foundation for the design of secure and exceptionally potent metal-based ICD agents in immunotherapy.

Polymer-Based Drug Delivery Systems for Cancer Therapeutics.

Chemotherapy together with surgery and/or radiotherapy are the most common therapeutic methods for treating cancer. However, the off-target effects of chemotherapy are known to produce side effects and dose-limiting toxicities. Novel delivery platforms based on natural and synthetic polymers with enhanced pharmacokinetic and therapeutic potential for the treatment of cancer have grown tremendously over the past 10 years. Polymers can facilitate selective targeting, enhance and prolong circulation, improve delivery, and provide the controlled release of cargos through various mechanisms, including physical adsorption, chemical conjugation, and/or internal loading. Notably, polymers that are biodegradable, biocompatible, and physicochemically stable are considered to be ideal delivery carriers. This biomimetic and bio-inspired system offers a bright future for effective drug delivery with the potential to overcome the obstacles encountered. This review focuses on the barriers that impact the success of chemotherapy drug delivery as well as the recent developments based on natural and synthetic polymers as platforms for improving drug delivery for treating cancer.

Supercharged coiled-coil protein with N-terminal decahistidine tag boosts siRNA complexation and delivery efficiency of a lipoproteoplex.

Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non-palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non-viral materials, while offering safer and non-cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled-coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C-terminal decahistidine tags on α-helical secondary structure. Cross-correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency.

Assessment of nanocarrier mediated afatinib pharmacokinetic: A comparative in-vivo study in rat model for enhanced therapeutic efficacy in lung cancer management

Nanocarrier-based drug delivery systems are at the forefront of innovative research in the field of medical science, providing potential pathways for treatments across several routes of administration. Notably, a great deal of research has gone into maximizing the potential of polymeric nanoparticles and liposomes, with a focus on addressing the intricacies of lung cancer through the pulmonary route. The present research aims to improve the pharmacokinetic profile of afatinib using a liposome and polymeric nanoparticle based dry powder approach rather than the afatinib plain drug solution to increase the survival rate and improvise the treatment of patients suffering from lung cancer. Formulated afatinib encapsulated PEGylated liposomal and afatinib loaded PLGA nanoparticles dry powder inhaler were characterized for the in-vivo pulmonary pharmacokinetics study using rat model upon intratracheal administration. The results of in-vivo pulmonary pharmacokinetics studies of both formulations indicated that it improved the pharmacokinetics profiles of the encapsulated agent (afatinib) by increasing the Cmax, AUC Total, Mean residence time (MRT) and decreasing the clearance (CL), and volume of distribution (VZ) than the afatinib plain drug solution. Results suggested that the selective targeting ability of the nanocarrier system can be a favourable approach for the management of lung cancer by the inhalation route.

Domain-Targeted Membrane Partitioning of Specific Proteins with DNA Nanodevices.

The cell membrane exhibits a remarkable complexity of lipids and proteins that dynamically segregate into distinct domains to coordinate various cellular functions. The ability to manipulate the partitioning of specific membrane proteins without involving genetic modification is essential for decoding various cellular processes but highly challenging. In this work, by conjugating cholesterols or tocopherols at the three bottom vertices of the DNA tetrahedron, we develop two sets of nanodevices for the selective targeting of lipid-order (Lo) and lipid-disorder (Ld) domains on the live cell membrane. By incorporation of protein-recognition ligands, such as aptamers or antibodies, through toehold-mediated strand displacement, these DNA nanodevices enable dynamic translocation of target proteins between these two domains. We first used PTK7 as a protein model and demonstrated, for the first time, that the accumulation of PTK7 to the Lo domains could promote tumor cell migration, while sequestering it in the Ld domains would inhibit the movement of the cells. Next, based on their modular nature, these DNA nanodevices were extended to regulate the process of T cell activation through manipulating the translocation of CD45 between the Lo and the Ld domains. Thus, our work is expected to provide deep insight into the study of membrane structure and molecular interactions within diverse cell signaling processes.