Precision Therapies Research Articles

Abstract LB413: A newly developed patient-derived culture panel identifies precision therapies and pan-effective agents for head and neck squamous cell carcinoma

Abstract Head and neck squamous carcinoma (HNSCC) is an aggressive cancer with few treatment options. Its well-known genetic heterogeneity presents a major challenge for precision medicine development. We report a newly developed panel of 20 HNSCC patient-derived cultures (HNSCC-PDCs) from advanced or recurrent HNSCC, with comprehensive somatic and germline characterization for precision medicine (PM) discovery. The PDCs retain genetic heterogeneity of the original patient tumors, and xenografts of which also phenocopied HNSCC patient tumors. Importantly, somatic and germline events that were never or seldom captured by long-established HNSCC cell lines are represented by the PDC panel, including HRAS-CASP8 co-mutations, EGFR-AS1 germline mutation, exonic germline PIK3CA mutation, sonic hedgehog (SHH) pathway and FGFR pathway mutations. We hypothesize that drug screening with this newly developed HNSCC-PDC panel that recapitulates HNSCC patient genetics may identify new gene-drug sensitivity relationships for precision medicine development in advanced HNSCC. Here, data from our pilot drug screen of 38 selected FDA approved/Phase I-II clinical trial agents first reveal: 1) HNSCC-PDC with the hotspot HRASp.G13R, together with CASP8p.L183S, is sensitive to tipifarnib vs. tipifarnib-resistance in a long-established HNSCC cell line, SNU899 with the same HRAS mutation. Subsequent high-content imaging cell death assay further demonstrates a dose-dependent tipifarnib-induced cell death in HRASp.G13R-mutated PDC, but not in SNU-899; 2) TP53-EP300 co-mutated HNSCC-PDCs are most sensitive to cisplatin with ~340 nM sensitivity (mean IC50) among all PDCs tested, which is 22 times more sensitive than that reported in Genomics of Drug Sensitivity in Cancer (GDSC) HNSCC cell lines; 3) EGFR-AS1 (c.2361G>A)-germline mutated PDCs, and MAPK1-mutated PDCs, are both hypersensitive to EGFR inhibitors (EGFRi: erlotinib and mobocertinib), which independently credentialed gene-drug sensitivity relationships reported in exceptional responders in HNSCC clinical trials. Further signaling investigation in PDC models demonstrates phospho-MAPK activation as a reliable surrogate biomarker for EGFRi sensitivity in HNSCC; 4) FRS3-mutated PDCs are sensitive to FDA-approved FGFR pathway inhibitors; and 5) TP53-wildtype HNSCC-PDCs are 150-fold more sensitive to novel p53-MDM2 interaction inhibitors vs. TP53-mutated PDCs, a phenomenon that cannot be captured in long-established TP53-wildtype HNSCC cell lines. Lastly, 4 pan-PDC effective agents are identified with nM sensitivities and high drug activities (i.e. drug-induced growth inhibition) in more than half of our PDC models. These include 3 FDA-approved agents, TAK-981, Selinexor (a novel XPO1 inhibitor), and trametinib, as well as the preclinical agent JQ1. In conclusion, our pilot HNSCC-PDC drug screen identifies new gene-drug sensitivity relationships for HNSCC PM development and new pan-effective agents for advanced HNSCC. Citation Format: Yuen Keng Ng, Hui Li, Wenying Piao, Cecilia Pik Yuk Lau, Peony Hiu Yan Poon, Tin Yu Samuel Law, Jason Ying Kuen Chan, Yuxiong Su, Chin Wang Lau, Zigui Chen, Laura Ren Huey Ip, Thomas Chung-Kit Yeung, Sai Fung Yeung, Jason Hoi-Lam Ngan, Zoey Wing Yee Liu, Noah R. Wiese, Vivie Vo, Zoey Patel, Jeremia Onyekachi, Laili Afzali, Jasmine Zohal Afzali, Jack Nimitz Smith, Vivian Wai Yan Lui. A newly developed patient-derived culture panel identifies precision therapies and pan-effective agents for head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB413.

Cyclophilin D knockout significantly prevents HCC development in a streptozotocin-induced mouse model of diabetes-linked NASH.

A family of Peptidyl-prolyl isomerases (PPIases), called Cyclophilins, localize to numerous intracellular and extracellular locations where they contribute to a variety of essential functions. We previously reported that non-immunosuppressive pan-cyclophilin inhibitor drugs like reconfilstat (CRV431) or NV556 decreased multiple aspects of non-alcoholic fatty liver disease (NAFLD) in mice under two different non-alcoholic steatohepatitis (NASH) mouse models. Both CRV431 and NV556 inhibit several cyclophilin isoforms, among which cyclophilin D (CypD) has not been previously investigated in this context. It is unknown whether it is necessary to simultaneously inhibit multiple cyclophilin family members to achieve therapeutic benefits or if loss-of-function of one is sufficient. Furthermore, narrowing down the isoform most responsible for a particular aspect of NAFLD/NASH, such as hepatocellular carcinoma (HCC), would allow for more precise future therapies. Features of human diabetes-linked NAFLD/NASH can be reliably replicated in mice by administering a single high dose of streptozotocin to disrupt pancreatic beta cells, in conjunction with a high sugar, high fat, high cholesterol western diet over the course of 30 weeks. Here we show that while both wild-type (WT) and Ppif-/- CypD KO mice develop multipe severe NASH disease features under this model, the formation of HCC nodules was significantly blunted only in the CypD KO mice. Furthermore, of differentially expressed transcripts in a qPCR panel of select HCC-related genes, nearly all were downregulated in the CypD KO background. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NAFLD/NASH. This study highlights the impact of CypD loss-of-function on the development of HCC, one of the most severe disease outcomes.

Precision Endpoints for Contemporary Precision Oncology Trials.

Traditional endpoints such as progression-free survival and overall survival do not fully capture the pharmacologic and pharmacodynamic effects of a therapeutic intervention. Incorporating mechanism-driven biomarkers and validated surrogate proximal endpoints can provide orthogonal readouts of anti-tumor activity and delineate the relative contribution of treatment components on an individual level, highlighting the limitation of solely relying on aggregated readouts from clinical trials to facilitate go/no-go decisions for precision therapies.

Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy.

Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell-cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation. To overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs). We found that PCDH19 is highly expressed in early (days 20-35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells. Our results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.

Prevalence and Factors Affecting the Optimal and Non-optimal Peak Inspiratory Flow Rate in Stable and Exacerbation Phases of Chronic Obstructive Pulmonary Disease and Bronchial Asthma in India.

Chronic obstructive pulmonary disease (COPD) and bronchial asthma pose significant threats and challenges to global health care, emphasizing the need for precise inhaler therapies to overcome this burden. The optimal peak inspiratory flow rate (PIFR) is a crucial determinant for the right selection and effective use of an inhaler device. It also helps to improve the treatment effectiveness of obstructive airway diseases worldwide as it allows effective drug delivery to distal airways and lung parenchyma. It is used as a selection criterion by physicians around the worldfor selecting personalized inhaler devices. To find out the optimal and non-optimal PIFR prevalence and its influencing factors in stable and exacerbation phases of COPD and bronchial asthma in Tamil Nadu, India. It is a single-center, observational, cross-sectional study conducted from February 2022 to August 2023. The patients who meet the diagnostic criteria specified by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines for COPD and the Global Initiative for Asthma (GINA) guidelines for bronchial asthma are enrolled in our study. The PIFR was measured using a hand-held digital spirometry device, along with demographic data collection. Statistical analyses, including t-tests and chi-square tests, were performed using SPSS version 21 (IBM Corp., Armonk, NY). Gender, height, and disease severity significantly impacted the PIFR. Females, normal BMI individuals, and those with moderate disease severity exhibited higher optimal PIFR rates. Stable or exacerbation phases, disease, and smoking status do not influence either optimal or non-optimal PIFR. Notably, substantial differences in lung function parameters were observed between optimal (60-90 L/min) and non-optimal PIFR (insufficient: <30 L/min, suboptimal: 30-60 L/min, excessive: >90 L/min) groups, highlighting their impact on respiratory health. This study emphasizes the importance of personalized inhaler strategies, considering gender, height, and disease severity. Proper inhaler device selection, continuous monitoring of inhaler technique, and tailored inhaler education at every OPD visit are vital for optimizing effective COPD and bronchial asthma management and improving adherence to treatment.

Sera from people with HIV and depression induce commensurate metabolic alterations in astrocytes: toward precision diagnoses and therapies.

People with HIV (PWH) have high rates of depression and neurocognitive impairment (NCI) despite viral suppression on antiretroviral therapy (ART). Mounting evidence suggests that immunometabolic disruptions may contribute to these conditions in some PWH. We hypothesized that metabolic dysfunction in astrocytes is associated with depressive symptoms and cognitive function in PWH. Human astrocytes were exposed to sera from PWH (n=40) with varying degrees of depressive symptomatology and cognitive function. MitoTrackerTM Deep Red FM (MT) was used to visualize mitochondrial activity and glial fibrillary acidic protein (GFAP) as an indicator of astrocyte reactivity using the high-throughput fluorescent microscopy and image analyses platform, CellInsight CX5 (CX5). The Seahorse platform was used to assess glycolytic and mitochondrial metabolism. More severe depression, as indexed by higher Beck's Depression Inventory (BDI-II) scores, was associated with lower MT signal measures. Better cognitive function, as assessed by neuropsychiatric testing t-scores, was associated with increased MT signal measures. GFAP intensity negatively correlated with several cognitive t-scores. Age positively correlated with (higher) MT signal measures and GFAP intensity. Worse depressive symptoms (higher BDI-II scores) were associated with decreased oxygen consumption rate and spare respiratory capacity, concomitant with increased extracellular acidification rate in astrocytes. These findings show that factors in the sera of PWH alter mitochondrial activity in cultured human astrocytes, suggesting that mechanisms that alter mitochondrial and astrocyte homeostasis can be detected peripherally. Thus, in vitro cultures may provide a model to identify neuropathogenic mechanisms of depression or neurocognitive impairment in PWH and test personalized therapeutics for neurologic and psychiatric disorders.

Virus-Induced Epilepsy vs. Epilepsy Patients Acquiring Viral Infection: Unravelling the Complex Relationship for Precision Treatment.

The intricate relationship between viruses and epilepsy involves a bidirectional interaction. Certain viruses can induce epilepsy by infecting the brain, leading to inflammation, damage, or abnormal electrical activity. Conversely, epilepsy patients may be more susceptible to viral infections due to factors, such as compromised immune systems, anticonvulsant drugs, or surgical interventions. Neuroinflammation, a common factor in both scenarios, exhibits onset, duration, intensity, and consequence variations. It can modulate epileptogenesis, increase seizure susceptibility, and impact anticonvulsant drug pharmacokinetics, immune system function, and brain physiology. Viral infections significantly impact the clinical management of epilepsy patients, necessitating a multidisciplinary approach encompassing diagnosis, prevention, and treatment of both conditions. We delved into the dual dynamics of viruses inducing epilepsy and epilepsy patients acquiring viruses, examining the unique features of each case. For virus-induced epilepsy, we specify virus types, elucidate mechanisms of epilepsy induction, emphasize neuroinflammation's impact, and analyze its effects on anticonvulsant drug pharmacokinetics. Conversely, in epilepsy patients acquiring viruses, we detail the acquired virus, its interaction with existing epilepsy, neuroinflammation effects, and changes in anticonvulsant drug pharmacokinetics. Understanding this interplay advances precision therapies for epilepsy during viral infections, providing mechanistic insights, identifying biomarkers and therapeutic targets, and supporting optimized dosing regimens. However, further studies are crucial to validate tools, discover new biomarkers and therapeutic targets, and evaluate targeted therapy safety and efficacy in diverse epilepsy and viral infection scenarios.

Mitochondrial energy metabolism in diabetic cardiomyopathy: Physiological adaption, pathogenesis, and therapeutic targets.

Diabetic cardiomyopathy is defined as abnormal structure and function of the heart in the setting of diabetes, which could eventually develop heart failure and leads to the death of the patients. Although blood glucose control and medications to heart failure show beneficial effects on this disease, there is currently no specific treatment for diabetic cardiomyopathy. Over the past few decades, the pathophysiology of diabetic cardiomyopathy has been extensively studied, and an increasing number of studies pinpoint that impaired mitochondrial energy metabolism is a key mediator as well as a therapeutic target. In this review, we summarize the latest research in the field of diabetic cardiomyopathy, focusing on mitochondrial damage and adaptation, altered energy substrates, and potential therapeutic targets. A better understanding of the mitochondrial energy metabolism in diabetic cardiomyopathy may help to gain more mechanistic insights and generate more precise mitochondria-oriented therapies to treat this disease.

An anti-GD2 aptamer-based bifunctional spherical nucleic acid nanoplatform for synergistic therapy targeting MDM2 for retinoblastoma

Retinoblastoma (RB) is a type of pediatric solid tumor in the fundus. The lack of precision therapies combined with the difficulty of delivering small interfering RNA (siRNA) into the eyes means that there is currently no nucleic acid-based therapy for RB in clinical practice. Here, we reported on anti-GD2 and glutathione-responsive spherical nucleic acids (SNAs), loaded with siRNA and the inhibitor NVP-CGM097, which jointly blocked the oncogenic factor n in RB cells (Y79 and WERI-RB-1). The SNAs were formed through the self-assembly of bifunctional cholesterol amphiphiles containing aptamers that specifically targeted GD2-positive RB cells, allowing for the formation of an SNA with a dense DNA shell. The aptamer/siRNA component functioned both as a carrier and a payload, enhancing the specific recognition and delivery of both components and constituting an active agent for MDM2 regulation. Following SNA endocytosis by RB cells, siRNA and NVP-CGM097 were released from the SNA particles by glutathione, which synergistically blocked the MDM2-p53 pathway, increasing p53 protein content and inducing cell apoptosis. This study showed a potent antitumor effect following intravitreal injection of SNAs in Y79 tumor-bearing mice through clinical manifestation and tumor pathological analysis. In hematological analysis and hepatotoxicity assays, SNAs were safer for mice than melphalan, the favored drug for treating RB in clinical practice. Our results illustrated the potential of intravitreally injected SNAs as a precision medicine for treating RB.

Abstract 2703: Targeting STING pathway as a therapeutic strategy against β-catenin driven immune escape and resistance to anti-PD1 therapy in hepatocellular carcinoma

Abstract β-catenin mutation occurs in around 40% of hepatocellular carcinoma(HCC); yet no precision therapies are available to treat this tumor subset. Immune checkpoint inhibitors (ICIs) are now widely used for the treatment of patients with advanced HCC. Unfortunately, preliminary evidence showed that β-catenin mutation drives resistance to anti-PD1 immunotherapy in HCC. In this study, we studied the molecular mechanisms underlying β-catenin mutation during HCC pathogenesis to develop novel therapeutics targeting β-Catenin-driven HCC. Firstly, three HCC cohorts were examined, including 70 untreated, 20 treated patients with nivolumab, and patients from a public TCGA database. By single-cell RNA sequencing analysis, we identified that anti-PD-1 treatment displayed effectiveness with the increase of CD27+CD8+ T-cell infiltration, correlating to improved prognosis with longer progression-free survival (PFS) and OS. Importantly, multiplex IHC staining showed that β-catenin activation is associated with significantly reduced CD8+CD27+ T cell infiltration in tumors (n=70), demonstrating that β-catenin promoted immune escape and resistance to anti-PD-1 treatment involved defective cytotoxic T-cell activity. Subsequently, we found that the interaction of β-Catenin with sirtuin 1 (SIRT1) mediated suppression of IRF3/IRF7 activity, resulting in inhibition of Type I interferon (IFN-I) production. However, by CRISPRi screening, we found that disruption of β-catenin-SIRT1 interaction via STING caused persistent high-level IFN-I response which increased the transcription of T-cell costimulatory molecule CD27 via IL10/STAT3/BATF signaling on infiltrated CD8+ T cells. This effect was also confirmed in the STING null mice, emphasizing that STING signaling is a therapeutic target to overcome β-catenin driven immune escape. Further, using hydrodynamic tail-vein injection murine HCC models, we found that the STING agonist inhibited β-catenin-SIRT1 activity, restored IFN signaling to rescue T-cell cytotoxic capacities, and enhanced the anti-PD-1 therapy efficacy. In conclusion, we demonstrated that STING reprogrammed β-Catenin mutated the HCC tumor microenvironment and a combination of STING and anti-PD-1 treatment represents an effective novel therapeutic strategy for HCC patients with β-Catenin mutation. Citation Format: Qingmei Zhang, Jiacheng Bi, Oscar Wai-Ho Yeung, Tao Ding, Wing-Lim Chan, Yueqin Zhu, Jiang Liu, Kevin Tak-Pan Ng, Kwan Man. Targeting STING pathway as a therapeutic strategy against β-catenin driven immune escape and resistance to anti-PD1 therapy in hepatocellular carcinoma [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 2703.

Abstract 2552: Real-world analysis of biomarker testing and use of targeted therapies in metastatic non-small cell lung cancer (mNSCLC) in the United States (US)

Abstract Background: Between 1998 and 2022, over 40% of new oncology drugs approved by the US FDA were precision therapies. In nonsquamous mNSCLC, detecting biomarkers where targeted therapies exist (EGFR, ALK, ROS1, BRAF V600E variant, MET exon 14 skipping variant, RET and PD-L1) has become essential for adequate clinical care. This study investigated real-world use of these biomarker tests, their characteristics, and associated use of targeted therapies in mNSCLC patients in the US. Methods: Adults ≥18 years old with stage IV mNSCLC from the nationwide Flatiron Health electronic health record-derived de identified database (01/2011 - 04/2023). The proportion of patients receiving biomarker tests, timing, characteristics, test type (single, multiple or next-generation sequencing), and proportion of patients receiving guideline recommended associated targeted therapies (i.e. actionable treatment) was assessed. Actionable treatment was defined in study years where at least one actionable therapy was FDA approved (biomarker, therapy, date: ALK, crizotinib, Aug 2011; BRAF V600E, dabrafenib+trametinib, Jul 2015; EGFR, afatinib, Jul 2013; MET exon 14 skipping, capmatinib, May 2020; PD-L1, pembrolizumab, Oct 2015; RET, pralsetinib, Sep 2020; ROS-1, crizotinib, Mar 2016). Results: In 42,037 patients with mNSCLC, the proportion of patients receiving each test were: ALK: 71.6%, BRAF: 48.8%, EGFR: 74.9%, MET: 41.4%, PD-L1: 49.3%, RET: 42.3%, and ROS1: 54.2%. Median (Q1, Q3) time from mNSCLC diagnosis to first biomarker result was 21 (12, 37) days. Tissue was the most common sample type for biomarker testing, although sampling by blood has increased in recent years. Multiple testing methods were more common in ALK and PD-L1 (48.1% and 50.3%, respectively) and next generation sequencing was more common in the other biomarkers (BRAF: 77.7%, EGFR: 57.1%, MET: 74.1%, RET: 70.7%, ROS1: 54.0%). Testing rates increased from 2011 through 2023 for each biomarker, with the highest rates in 2023: ALK (87.7%), BRAF (85.2%), EGFR (88.7%), MET (85.7%), PDL1 (78.7%), RET (84.8%), and ROS1 (86.9%). Testing rates were similar when timing was restricted to a 1-year window from mNSCLC diagnosis. Receipt of actionable treatment varied by biomarker, in order of increasing frequency: RET+ (23.0%), MET+ (34.0%), BRAF+ (42.7%), PDL1+ (56.3%), ROS1+ (59.8%), EGFR+ (70.6%), and ALK+ (89.0%). Generally, across all biomarkers, receipt of actionable therapy increased in more recent years. Conclusion: Current real-world biomarker testing rates and use of actionable treatments may reflect increased adoption of clinical guideline recommendations. Despite increased testing, use of actionable therapy remains low for certain biomarkers. Further improvements in the implementation of robust biomarker testing to use appropriate therapies are needed, which can lead to better patient outcomes. Citation Format: Michael J. Dennis, Devin Abrahami, Maria Cecilia Vieira, Darrin Benjumea, Marley Boyd, Anran Shao, Kirsten Duncan, John Kelton, Sandip P. Patel. Real-world analysis of biomarker testing and use of targeted therapies in metastatic non-small cell lung cancer (mNSCLC) in the United States (US) [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 2552.

Abstract 937: Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe

Abstract Translation of precision oncology data into feasible precision therapies for hard-to-cure childhood, adolescent and young adult malignancies remains a significant challenge. Identifying therapeutic targets at the protein and pathway level and demonstrating treatment response in personalized models hold great promise, particularly for combination therapies, but may be considered complex and time consuming. Here, we present the case of an adolescent with metastatic, progressive spindle epithelial tumor with thymus-like differentiation (SETTLE) and evaluate how proteomics combined with rapid patient-derived models can identify treatment options not apparent at the genome or transcript level. Mass spectrometric proteome analysis of macro-dissected tumor and adjacent normal from formalin fixed paraffin embedded sections was completed within two weeks of biopsy and identified key proteins involved in one-carbon metabolism, including SHMT2 and DHFR as possible targets for single or combination therapy. Elevated SHMT2 levels were validated by immunohistochemistry and compared to levels across AYA tumors. Based on the suitability for an innovative therapy trial, we prioritized single-agent sertraline, a commercially available anti-depressant medication that inhibits SHMT2, and confirmed a positive drug response in both chicken chorioallantoic membrane (CAM) and larval zebrafish xenografts generated from the patient. Retrospective expansion in a murine xenograft enabled metabolic tracing on isolated SETTLE- patient-derived xenograft cells using 13C6-glucose confirming SHMT2 activity and response to in vitro treatment. Following failure of cytotoxic chemotherapy and second-line sorafenib treatment, a monotherapy trial of sertraline was initiated by the patient but stopped after 8 weeks after evidence of progressive disease. Possible combination therapies were evaluated further in the patient-derived models. Combining sertraline with the common antibiotic trimethoprim, resulted in enhanced growth inhibition of SETTLE cells in the larval zebrafish xenografts. Significance: Overall, we demonstrate that proteomics and personalized xenograft models may provide supportive pre-clinical data in a clinically meaningful timeframe to support medical decision-making and impact clinical practice. Citation Format: Georgina D. Barnabas, Tariq A. Bhat, Verena Goebeler, Pascal Leclair, Nadine Azzam, Nicole Melong, Jason N. Berman, Jennifer A. Chan, Donna L. Senger, Seth Parker, Christopher A. Maxwell, Gregor S. Reid, Jonathan Bush, Caron Strahlendorf, Rebecca Deyell, C James Lim, Philipp F. Lange. Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe [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 937.

Abstract 5830: A first-of-its-kind model that reconstitutes targeted drug-induced cellular transdifferentiation

Abstract Introduction: Lung tumors treated with targeted therapies ultimately relapse due to intra-genic resistance mutations, bypass pathway activation, or cellular lineage changes. Newer precision therapies have higher selectivity and potency against both resistant and activating gene mutations, resulting in a significant increase in incidence of cellular transdifferentiation. The most common form of transdifferentiation is the change from lung adenocarcinomas (LUADs) to small cell lung cancer (t-SCLC) upon drug treatment which remains very poorly understood. Methods: We have combined patient derived xenografts (PDX) and ex vivo lines with multi-omic characterization, high-content image-based morphometry and fluorescence tracking, and single-cell barcode tracing to better understand the process of lung tumor transdifferentiation. Results: We have developed a first-of-its-kind t-SCLC model that reconstitutes ex vivo LUAD to SCLC transdifferentiation. Genomic and transcriptomic analyses of our NSCLC PDX collection identified tumors with mutation in TP53 and RB1, two genes that are altered in virtually all de novo SCLCs. PDX sample CBX336 was identified as having mutations in both TP53 and RB1, in addition to expressing the KRASG12C oncogene. Upon treatment with Sotorasib, CBX336 tumors initially demonstrated slow tumor growth, followed by a dramatic increase in the growth after prolonged drug exposure. Our experimental and computational data suggest a highly coordinated procession toward transdifferentiation with the parallel upregulation of the neuroendocrine transcription factor NEUROD1 across numerous epithelial cellular clones. Conclusions: Our new experimental system allows characterization at the single-cell level. Ongoing work includes the integration of new resources to study lineage reprogramming, and advancing new therapeutic strategies. Citation Format: Priyanka Gopal, Mohamed Abazeed. A first-of-its-kind model that reconstitutes targeted drug-induced cellular transdifferentiation [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 5830.

Abstract 138: UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma

Abstract PAX3-FOXO1 positive rhabdomyosarcoma (RMS) is an aggressive pediatric sarcoma with a terrible prognosis. The resultant fusion acts as a chimeric transcription factor, in part remodeling chromatin to inhibit terminal differentiation and maintain cancer cell proliferation. As this oncoprotein is not directly druggable, we have used genetic screens to identify dependencies downstream of PAX3-FOXO1 to improve our understanding of pathogenesis and develop new therapeutic targets. We performed pooled CRISPRi screening in an isogenic system of a patient-derived RMS cell line with or without knockdown of PAX3-FOXO1. Loss of the ubiquitin hydroxylase UCHL5 led to selective depletion of PAX3-FOXO1 positive Rh30 cells compared to PAX3-FOXO1 knockdown cells, suggesting that the fusion creates UCHL5 dependence. We validated this finding in competition growth assays in additional fusion positive (FP) and fusion negative (FN) RMS cell lines, and observed UCHL5 dependency only in the former. By contrast, UCHL5 was dispensable for cell growth and survival in FN RMS cell lines. These data were recapitulated in the large DepMap dataset, which also identified the top genetic co-dependencies of UCHL5 as INO80-complex (INO80c) members. We therefore investigated whether the fusion selective effects of UCHL5 were mediated by the INO80c, of which UCHL5 is an N-terminal member. We observed that knockdown of another N-terminal component of INO80c, NFRKB, phenocopies the fusion-selective effects of UCHL5 loss. Conversely, the catalytic ATPase INO80 was pan-essential, suggesting that only N-terminal subunits of INO80c regulate PAX3-FOXO1 specific functions. INO80c plays an essential role in mitigating DNA replication stress. Indeed, loss of UCHL5 led to DNA damage as measured by γH2AX and slowing of replication forks as measured by fiber assays in FP, but not FN RMS. Unexpectedly, acute degradation of PAX3-FOXO1 also slowed DNA replication fork speed, suggesting a genetic model in which UCHL5 and PAX3-FOXO1 cooperate to resolve replication stress in FP RMS. Finally, in vivo experiments using cell line xenografts confirms that loss of UCHL5 slows the growth of FP RMS, but not FN RMS. These findings suggest that loss of UCHL5 induces replication stress in a fusion-specific manner, and offers both an opportunity to study the mechanisms by which the PAX3-FOXO1 oncoprotein mitigates replication stress, and to develop UCHL5 inhibitors as precision therapies for FP RMS patients. Citation Format: Amit J. Sabnis, Pushpendra K. Sahu, Yue Pan. UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma [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 138.

Abstract 5996: Genome-wide CRISPR screening reveals a central role for ferroptotic cell death in the anti-tumor response to mTOR inhibitors in HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, resulting in more than 300,000 deaths each year worldwide. Treatment options for HNSCC patients include surgery, radiation, chemotherapy and molecularly targeted therapies, and although immunotherapies have recently revolutionized the treatment landscape, &amp;lt;20% of HNSCC patients respond to immune check point blockade (ICB) therapies. The survival rate of HNSCC patients has changed only modestly over the past decades, and therefore HNSCC is a significant global health problem with high mortality and morbidity. Genomic alterations converging in the persistent activation of the PI3K/mTOR pathway (&amp;gt;80% cases) represent one of the most frequently altered signaling circuitries in HNSCC. This overreliance on PI3K/mTOR signaling for tumor growth may expose a cancer vulnerability that can be exploited therapeutically, as revealed in recent clinical trials using mTOR inhibitors (mTORi) in HNSCC in the adjuvant and neoadjuvant setting. Here, we took advantage of a whole-genome CRISPR screening approach to identify mechanisms of sensitivity and resistance to mTORi, aimed at increasing their clinical activity. We found that the activation of autophagy, a biological process that plays a paradoxical pro-survival or antitumoral role in a cancer specific fashion, is strictly required for mTORi cell growth inhibition in HNSCC. In depth analysis of our CRISPR screening also revealed multiple hits suggestive of a novel role for iron metabolism and ferroptosis downstream from mTORi. Ferroptosis is iron-dependent regulated cell death process caused by the peroxidation of polyunsaturated fatty acids. Indeed, we found that mTORi induces ferroptosis in HNSCC cells, and that inhibition of ferroptosis reduces the effect of mTORi. Evidence will be presented supporting a novel mechanism linking mTORi-triggered autophagy and the activation of ferroptotic death programs, as well as new synergistic combinations with mTORi by repurposing approved drugs that disable cellular ferroptotic defense mechanisms. Our studies uncovered how mTORi act in HNSCC, thereby revealing new multimodal precision therapies for HNSCC and many human malignancies displaying overactive PI3K/mTOR signaling. Citation Format: Keiichi Koshizuka, Xingyu Wu, Kuniaki Sato, Pham Thuy Vo, Gosia M. Murawska, Tomohiko Ishikawa, Zhiyong Wang, Alfredo A. Molinolo, Edward A. Dennis, Prashant Mali, J. Silvio Gutkind. Genome-wide CRISPR screening reveals a central role for ferroptotic cell death in the anti-tumor response to mTOR inhibitors in HNSCC [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 5996.

Functional Consequences of CFTR Interactions in Cystic Fibrosis.

Cystic fibrosis (CF) is a fatal autosomal recessive disorder caused by the loss of function mutations within a single gene for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CFTR is a chloride channel that regulates ion and fluid transport across various epithelia. The discovery of CFTR as the CF gene and its cloning in 1989, coupled with extensive research that went into the understanding of the underlying biological mechanisms of CF, have led to the development of revolutionary therapies in CF that we see today. The highly effective modulator therapies have increased the survival rates of CF patients and shifted the epidemiological landscape and disease prognosis. However, the differential effect of modulators among CF patients and the presence of non-responders and ineligible patients underscore the need to develop specialized and customized therapies for a significant number of patients. Recent advances in the understanding of the CFTR structure, its expression, and defined cellular compositions will aid in developing more precise therapies. As the lifespan of CF patients continues to increase, it is becoming critical to clinically address the extra-pulmonary manifestations of CF disease to improve the quality of life of the patients. In-depth analysis of the molecular signature of different CF organs at the transcriptional and post-transcriptional levels is rapidly advancing and will help address the etiological causes and variability of CF among patients and develop precision medicine in CF. In this review, we will provide an overview of CF disease, leading to the discovery and characterization of CFTR and the development of CFTR modulators. The later sections of the review will delve into the key findings derived from single-molecule and single-cell-level analyses of CFTR, followed by an exploration of disease-relevant protein complexes of CFTR that may ultimately define the etiological course of CF disease.

Enhancing Drug Delivery Precision: Development and Optimization of Nanoparticle-Based Formulations for Targeted Therapy in Preclinical Models

In recent years, the utilization of nanoparticles has proliferated across a wide spectrum of clinical domains. Nanoparticles have been engineered to surmount the constraints associated with free therapeutics and negotiate biological barriers—systemic, microenvironmental, and cellular—that exhibit heterogeneity across diverse patient cohorts and diseases. Mitigating this patient heterogeneity has also been facilitated through precision therapeutics, where tailored interventions have augmented therapeutic effectiveness. Nonetheless, current nanoparticle development predominantly emphasizes the refinement of delivery platforms with a uniform approach. As lipid-based, polymeric, and inorganic nanoparticles undergo increasingly nuanced engineering, there arises the potential for tailoring them to drug delivery in a more personalized manner, ushering in the era of precision medicine. In this Review, we deliberate on sophisticated nanoparticle designs employed in both generalized and precision applications, offering insights into their potential for enhancing precision therapies. We concentrate on advancements in nanoparticle design that surmount heterogeneous barriers to delivery, positing that intelligent nanoparticle design can enhance efficacy in broad delivery applications while facilitating customized designs for precision applications, thereby ultimately enhancing overall patient outcomes.

Angiopep-2-Functionalized Lipid Cubosomes for Blood-Brain Barrier Crossing and Glioblastoma Treatment.

Glioblastoma multiforme (GBM) is an aggressive brain cancer with high malignancy and resistance to conventional treatments, resulting in a bleak prognosis. Nanoparticles offer a way to cross the blood-brain barrier (BBB) and deliver precise therapies to tumor sites with reduced side effects. In this study, we developed angiopep-2 (Ang2)-functionalized lipid cubosomes loaded with cisplatin (CDDP) and temozolomide (TMZ) for crossing the BBB and providing targeted glioblastoma therapy. Developed lipid cubosomes showed a particle size of around 300 nm and possessed an internal ordered inverse primitive cubic phase, a high conjugation efficiency of Ang2 to the particle surface, and an encapsulation efficiency of more than 70% of CDDP and TMZ. In vitro models, including BBB hCMEC/D3 cell tight monolayer, 3D BBB cell spheroid, and microfluidic BBB/GBM-on-a-chip models with cocultured BBB and glioblastoma cells, were employed to study the efficiency of the developed cubosomes to cross the BBB and showed that Ang2-functionalized cubosomes can penetrate the BBB more effectively. Furthermore, Ang2-functionalized cubosomes showed significantly higher uptake by U87 glioblastoma cells, with a 3-fold increase observed in the BBB/GBM-on-a-chip model as compared to that of the bare cubosomes. Additionally, the in vivo biodistribution showed that Ang2 modification could significantly enhance the brain accumulation of cubosomes in comparison to that of non-functionalized particles. Moreover, CDDP-loaded Ang2-functionalized cubosomes presented an enhanced toxic effect on U87 spheroids. These findings suggest that the developed Ang2-cubosomes are prospective for improved BBB crossing and enhanced delivery of therapeutics to glioblastoma and are worth pursuing further as a potential application of nanomedicine for GBM treatment.

An iPSC line (FINi003-A) from a male with late-onset developmental and epileptic encephalopathy caused by a heterozygous p.E1211K variant in the SCN2A gene encoding the voltage-gated sodium channel Nav1.2

Many developmental and epileptic encephalopathies (DEEs) result from variants in cation channel genes. Using mRNA transfection, we generated and characterised an induced pluripotent stem cell (iPSC) line from the fibroblasts of a male late-onset DEE patient carrying a heterozygous missense variant (E1211K) in Nav1.2(SCN2A) protein. The iPSC line displays features characteristic of the human iPSCs, colony morphology and expression of pluripotency-associated marker genes, ability to produce derivatives of all three embryonic germ layers, and normal karyotype without SNP array-detectable abnormalities. We anticipate that this iPSC line will aid in the modelling and development of precision therapies for this debilitating condition.

APOL1 nephropathy - a population genetics success story.

More than a decade ago, apolipoprotein L1 ( APOL1 ) risk alleles designated G1 and G2, were discovered to be causally associated with markedly increased risk for progressive kidney disease in individuals of recent African ancestry. Gratifying progress has been made during the intervening years, extending to the development and clinical testing of genomically precise small molecule therapy accompanied by emergence of RNA medicine platforms and clinical testing within just over a decade. Given the plethora of excellent prior review articles, we will focus on new findings regarding unresolved questions relating mechanism of cell injury with mode of inheritance, regulation and modulation of APOL1 activity, modifiers and triggers for APOL1 kidney risk penetrance, the pleiotropic spectrum of APOL1 related disease beyond the kidney - all within the context of relevance to therapeutic advances. Notwithstanding remaining controversies and uncertainties, promising genomically precise therapies targeted at APOL1 mRNA using antisense oligonucleotides (ASO), inhibitors of APOL1 expression, and small molecules that specifically bind and inhibit APOL1 cation flux are emerging, many already at the clinical trial stage. These therapies hold great promise for mitigating APOL1 kidney injury and possibly other systemic phenotypes as well. A challenge will be to develop guidelines for appropriate use in susceptible individuals who will derive the greatest benefit.