Patient-derived Primary Cells Research Articles

A synthetic lethal dependency on casein kinase 2 in response to replication-perturbing therapeutics in RB1-deficient cancer cells.

Resistance to therapy commonly develops in patients with high-grade serous ovarian carcinoma (HGSC) and triple-negative breast cancer (TNBC), urging the search for improved therapeutic combinations and their predictive biomarkers. Starting from a CRISPR knockout screen, we identified that loss of RB1 in TNBC or HGSC cells generates a synthetic lethal dependency on casein kinase 2 (CK2) for surviving the treatment with replication-perturbing therapeutics such as carboplatin, gemcitabine, or PARP inhibitors. CK2 inhibition in RB1-deficient cells resulted in the degradation of another RB family cell cycle regulator, p130, which led to S phase accumulation, micronuclei formation, and accelerated PARP inhibition-induced aneuploidy and mitotic cell death. CK2 inhibition was also effective in primary patient-derived cells. It selectively prevented the regrowth of RB1-deficient patient HGSC organoids after treatment with carboplatin or niraparib. As about 25% of HGSCs and 40% of TNBCs have lost RB1 expression, CK2 inhibition is a promising approach to overcome resistance to standard therapeutics in large strata of patients.

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis.

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Methods for assessing and removing non-specific photoimmunotherapy damage in patient-derived tumor cell culture models.

Tumor-targeted, activatable photoimmunotherapy (taPIT) has been shown to selectively destroy tumor in a metastatic mouse model. However, the photoimmunoconjugate (PIC) used for taPIT includes a small fraction of non-covalently associated (free) benzoporphyrin derivative (BPD), which leads to non-specific killing invitro. Here, we report a new treatment protocol for patient-derived primary tumor cell cultures ultrasensitive to BPD photodynamic therapy (BPD-PDT). Based on free BPD efflux dynamics, the updated invitro taPIT protocol precludes non-specific BPD-PDT by silencing the effect of free BPD. Following incubation with PIC, incubating cells with PIC-free medium allows time for expulsion of free BPD whereas BPD covalently bound to PIC fragments is retained. Administration of the light dose after the intracellular free BPD drops below the threshold for inducing cell death helps to mitigate non-specific damage. In this study, we tested two primary ovarian tumor cell lines that are intrinsically chemoresistant, yet ultrasensitive to BPD-PDT such that small amounts of free BPD (afew percent of the total BPD dose) lead to potent induction of cell death upon irradiation. The modifications in the protocol suggested here improve invitro taPIT experiments that lack invivo mechanisms of free BPD clearance (i.e., lymph and blood flow).

A chemically adjustable BMP6-IL6 axis in mesenchymal stem cells drives acute myeloid leukemia cell differentiation

Chemotherapy alone or in combination with allogeneic stem cell transplantation has been the standard of care for acute myeloid leukemia (AML) for decades. Leukemia relapse with limited treatment options remains the main cause of treatment failure. Therefore, an effective and safe approach to improve treatment outcomes is urgently needed for most AML patients. Mesenchymal stem cells (MSCs) have been reported to efficiently induce apoptosis and shape the fate of acute myeloid leukemia cells. Here, we identified LG190155 as a potent compound that enhances the antileukemia efficiency of MSCs. Pretreatment of MSCs with LG190155 significantly provoked differentiation in both AML patient-derived primary leukemia cells and AML cell lines and reduced the tumor burden in the AML mouse model. Using the quantitative proteomic technique, we discovered a pivotal mechanism that mediates AML cell differentiation, in which autocrine bone morphogenetic protein 6 (BMP6) in MSCs boosted IL-6 secretion and further acted on leukemic cells to trigger differentiation. Furthermore, the activity of the BMP6-IL6 axis was dramatically enhanced by activating vitamin D receptor (VDR) in MSCs. Our data illustrated an effective preactivated approach to reinforcing the antileukemia effect of MSCs, which could serve as an effective therapeutic strategy for AML.

Abstract PO4-18-07: A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in triple negative breast cancer and transcriptionally addicted relapsed or refractory solid tumors

Abstract Background: MYC deregulation is a hallmark of triple negative breast cancer (TNBC) and is associated with aggressive tumors and poor clinical outcomes. Although MYC remains undrugged, targeting of its cofactors has emerged as an attractive strategy to inhibit MYC oncogenic activity. Cyclin-dependent kinase 9 (CDK9) is a critical regulator of oncogenic MYC expression and an important MYC cofactor. KB-0742 is an oral CDK9 inhibitor that demonstrates promising preclinical activity against TNBC. In a real-world cohort, TNBCs have higher MYC expression and higher rates of MYC genomic amplification than other breast cancer subtypes. In primary patient-derived cell lines, KB-0742 treatment results in stronger cytotoxic effects in TNBC as compared to other subtypes. In patient-derived organoids and patient-derived xenografts, CDK9 inhibition by KB-0742 drives antiproliferative and anti-tumor growth effects, including in models that are resistant to standard of care. KB-0742 strongly downregulates MYC protein levels at doses that only partially inhibit CDK9 activity, suggesting a therapeutic window for tumor-specific activity (Saffran, D.C. et al, 2021). KB-0742 is currently being evaluated in a phase 1/2 dose escalation and cohort expansion study in patients with TNBC and other transcriptionally addicted tumors (NCT04718675). Trial design: This phase 1/2 study includes two parts: dose escalation (part 1) and cohort expansion (part 2). Part 2 includes cohort A (solid tumors with high prevalence of MYC overexpression including TNBC, non-small cell lung cancer and ovarian) and cohort B (other transcriptionally addicted tumor types including sarcomas, adenoid cystic carcinoma, nut midline carcinoma and small cell lung cancer). KB-0742 is dosed orally once daily for 3 consecutive days, followed by 4 days, off on a weekly basis in 28-day cycles until unacceptable toxicity or disease progression. Eligibility criteria: Part 1 dose escalation is open to patients with relapsed or refractory solid tumors. Part 2 is defined by tumor indications in cohorts A and B. Eligibly criteria include age > 18 years (≥ 12 years old and with a body weight ≥ 40 kg part 2 for cohort B), acceptable organ function, and ECOG PS < 2. Specific aims: Primary objectives include evaluation of pharmacokinetics (PK), pharmacodynamics (PD), safety, tolerability, preliminary anti-tumor activity, and identifying a maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). PK measurements include Cmax, tmax, AUC0-last, accumulation ratio (Racc) and t1/2. Safety data will be evaluated per the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0. The Modified Continuous Reassessment Method (mCRM) (Goodman et al., Stat Med 1995) will guide dose escalation and MTD. RP2D nomination is informed by PD in peripheral blood mononuclear cells using assays to evaluate phosphorylation of the CDK9 substrate serine 2 on the RNA Polymerase II C-terminal domain (pSER2) and CDK9-responsive gene expression. Radiographic tumor response to KB-0742 in patients is assessed every other cycle starting from cycle two after treatment using RECIST 1.1 criteria. Target accrual: Targeted total enrollment is 170 patients. For additional information, contact clinicaltrials@kronosbio.com Citation Format: Monica Mita, Alain Mita, Miguel Villalona-Calero, Noah Federman, Drew Rasco, David Spigel, Jia Luo, Gregory Cote, Richard Cutler, Pavan Kumar, Crystal MacKenzie, Charles Lin, Jorge DiMartino, Elizabeth Olek, Brian Van Tine. A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in triple negative breast cancer and transcriptionally addicted relapsed or refractory solid tumors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-18-07.

Click-chemistry mediated synthesis of OTBN-1,2,3-Triazole derivatives exhibiting STK33 inhibition with diverse anti-cancer activities

There is a continuous and pressing need to establish new brain-penetrant bioactive compounds with anti-cancer properties. To this end, a new series of 4′-((4-substituted-4,5-dihydro-1H-1,2,3-triazol-1-yl)methyl)-[1,1′-biphenyl]-2-carbonitrile (OTBN-1,2,3-triazole) derivatives were synthesized by click chemistry. The series of bioactive compounds were designed and synthesized from diverse alkynes and N3-OTBN, using copper (II) acetate monohydrate in aqueous dimethylformamide at room temperature. Besides being highly cost-effective and significantly reducing synthesis, the reaction yielded 91–98 % of the target products without the need of any additional steps or chromatographic techniques. Two analogues exhibit promising anti-cancer biological activities. Analogue 4l shows highly specific cytostatic activity against lung cancer cells, while analogue 4k exhibits pan-cancer anti-growth activity. A kinase screen suggests compound 4k has single-digit micromolar activity against kinase STK33. High STK33 RNA expression correlates strongly with poorer patient outcomes in both adult and pediatric glioma. Compound 4k potently inhibits cell proliferation, invasion, and 3D neurosphere formation in primary patient-derived glioma cell lines. The observed anti-cancer activity is enhanced in combination with specific clinically relevant small molecule inhibitors. Herein we establish a novel biochemical kinase inhibitory function for click-chemistry-derived OTBN-1,2,3-triazole analogues and further report their anti-cancer activity in vitro for the first time.

Therapeutic potential of targeting Nrf2 by panobinostat in pituitary neuroendocrine tumors

We aimed to identify the druggable cell-intrinsic vulnerabilities and target-based drug therapies for PitNETs using the high-throughput drug screening (HTS) and genomic sequencing methods. We examined 9 patient-derived PitNET primary cells in HTS. Based on the screening results, the potential target genes were analyzed with genomic sequencing from a total of 180 PitNETs. We identified and verified one of the most potentially effective drugs, which targeted the Histone deacetylases (HDACs) both in in vitro and in vivo PitNET models. Further RNA sequencing revealed underlying molecular mechanisms following treatment with the representative HDACs inhibitor, Panobinostat. The HTS generated a total of 20,736 single-agent dose responses which were enriched among multiple inhibitors for various oncogenic targets, including HDACs, PI3K, mTOR, and proteasome. Among these drugs, HDAC inhibitors (HDACIs) were, on average, the most potent drug class. Further studies using in vitro, in vivo, and isolated PitNET primary cell models validated HDACIs, especially Panobinostat, as a promising therapeutic agent. Transcriptional surveys revealed substantial alterations to the Nrf2 signaling following Panobinostat treatment. Moreover, Nrf2 is highly expressed in PitNETs. The combination of Panobinostat and Nrf2 inhibitor ML385 had a synergistic effect on PitNET suppression. The current study revealed a class of effective anti-PitNET drugs, HDACIs, based on the HTS and genomic sequencing. One of the representative compounds, Panobinostat, may be a potential drug for PitNET treatment via Nrf2-mediated redox modulation. Combination of Panobinostat and ML385 further enhance the effectiveness for PitNET treatment.

Abstract LB272: Sequential inhibition of PARP and BET as a rational therapeutic strategy for glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM), characterized as one of the most aggressive brain cancers, presents significant treatment challenges due to its rapid growth, heterogeneity, and robust defensive mechanisms. Despite advancements in neuro-oncology, GBM's prognosis remains grim, with the median survival of approximately 15 months, underscoring the urgent need for innovative therapeutic strategies. Facing these challenges, PARP inhibitors (PARPi) hold substantial promise in treating GBM, evidenced by over 20 ongoing clinical trials. However, their application has been limited by side effects such as nausea, fatigue, and particularly anemia, as well as a narrow therapeutic spectrum focusing mainly on patients with homologous recombination deficiency (HRDness). Through unbiased transcriptomic and proteomic sequencing, and employing Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA), and Transcriptome-Proteome Correlation Analysis, we discovered that the BET inhibitor (BETi) Birabresib profoundly alters the processes of DNA replication and cell cycle progression in GBM cells, extending beyond the previously known impact of BET inhibition on homologous recombination repair. In vitro experiments using GBM cell lines and patient-derived primary GBM cells demonstrated that concurrent administration of PARPi and BETi can synergistically inhibit GBM. Intriguingly, we observed that DNA damage persists after the discontinuation of PARPi monotherapy. Leveraging these residual effects of PARPi, subsequent BETi administration elevated replication stress levels, leading to the inactivation of GBM cell cycle checkpoints, with efficacy comparable to concurrent treatment. The treatment-induced prolonged stalling of replication forks resulted in their collapse and the formation of double-ended double-strand breaks (DSBs). In GBM cells with elevated baseline replication stress, the sequential regimen exhibits comparable efficacy to concurrent treatment, while protecting normal glial cells with lower baseline replication stress from DNA toxicity and subsequent death. In vivo orthotopic transplantation tumor experiments in zebrafish and nude mice demonstrated consistent effects, indicating that sequential administration of PARPi followed by BETi maintained anti-tumor efficacy, similar to concurrent treatment, while reducing toxicity, as evident by the reduced impact on hemoglobin levels. Our findings provide a broader and deeper understanding of the synergistic interaction between BETi and PARPi than previously recognized. This study offers compelling preclinical evidence supporting the development of innovative drug administration strategies focusing on PARPi for GBM therapy. Citation Format: Xin Peng, Xin Huang, Shaolu Zhang, Naixin Zhang, Shengfan Huang, Yingying Wang, Zhenxing Zhong, Shan Zhu, Haiwang Gao, Zixiang Yu, Xiaotong Yan, Zhennan Tao, Yuxiang Dai, Zhe Zhang, Xi Chen, Feng Wang, Francois X. Claret, Ning Ji, Yuxu Zhong, Dexin Kong. Sequential inhibition of PARP and BET as a rational therapeutic strategy for glioblastoma multiforme [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 LB272.

Abstract LB290: Whole exome analysis of oral squamous cell carcinoma in local patients at tertiary care hospitals in Peshawar, Pakistan

Abstract The Precision Medicine Lab (PML) has been mandated by the Federal Government in 2018 to pilot precision medicine in Pakistan using genomics in oral cancer. With the Indian subcontinent as its capital, oral squamous cell carcinoma (OSCC), also dubbed as ‘poor man’s cancer', constitutes 90% of all head and neck cancers and is the highest incidence cancer found in Pakistani males. Backed by a bio-repository of clinical samples as well as a catalogue of primary cell clines, PML takes a multi-omic approach to discovering new, actionable biomarkers for diagnostic, prognostic and therapeutic applications. With all necessary ethical approvals obtained from IRBs at each site along with written informed consent from each patient, treatment-naive biopsy-confirmed patients of OSCC were recruited in two main tertiary care hospitals in Peshawar, Pakistan. Our cohort was predominantly older with 37.84% between 41-60 years, late-stage with 78.46% presenting in stage IV and self-reportedly non-alcohol consuming (96.9%). About 44.6% reported the use of smokeless tobacco while 30.8% reported the use of both smoked and smokeless tobacco. Majority of the tumours were located in the buccal mucosa (29.2%), alveolar region (27.7%) or the vestibular region (24.6%). We obtained blood and tumour samples for matched-pair whole exome sequencing, transcriptomics and metabolomics as well as generation of primary cell lines. We also obtained saliva samples for oral microbiome analysis. Combined with the patient’s clinical history and digital histopathology images, this helps us create a multi-omic stack that we intend to make available for integrated machine learning experiments. With our whole exome sequencing now complete, we undertook a preliminary analysis for this late breaking abstract. Raw reads were aligned to the GRCh38.p14 reference genome and were called for germline and somatic SNPs and INDELs using GATK (v4.5). Mutational analysis was conducted using the maftools (v2.18.0) package in R (v4.3) by comparing against the COSMIC SBS Signature database (v3.2). We report MUC12 (100%), ZNF717 (100%), FOLR3 (86%), EPPK1 (79%) and SPATA31A6 (79%) as the top mutated genes in the subset of exomes analysed thus far for our cohort. Genes reported previously including TP53, NOTCH1 and PIK3CA appear to rank lower on the list. In terms of cancer-related pathways, RTK-RAS (92.8%), Hippo (92.8%) and Notch (78.5%) were affected in most of the samples. To our knowledge, we describe for the first time the mutational landscape in an OSCC cohort from the Pakistani population. We also go beyond whole exomes as our multi-omic approach offers the potential on machine learning experiments to identify putative mutational signatures and biomarkers for OSCC that can be of diagnostic, prognostic and therapeutic value to the local (Pakistani and Afghan) patient population. With the availability of primary cell lines for every patient, we also intend to undertake experimental validation of high priority targets using primary patient-derived cell lines in the lab. Citation Format: Hina Zamir, Khudeja Salim, Madina Sherdil, Aqsa Hussain, Yusra Ilyas, Arsalan Riaz, Momal Agha, Zainab Jahan, Khadim Shah, Yasir Rehman Khattak, Fahim Uddin, Zahid Qayyum Shah, Zubair Durrani, Muhammad Mushtaq Khattak, Johar Ali, Faisal F. Khan. Whole exome analysis of oral squamous cell carcinoma in local patients at tertiary care hospitals in Peshawar, Pakistan [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 LB290.

Targeting the EGFR pathway: An alternative strategy for the treatment of tuberous sclerosis complex?

Tuberous sclerosis complex (TSC) is caused by variants in TSC1/TSC2, leading to constitutive activation of the mammalian target of rapamycin (mTOR) complex 1. Therapy with everolimus has been approved for TSC, but variations in success are frequent. Recently, caudal late interneuron progenitor (CLIP) cells were identified as a common origin of the TSC brain pathologies such as subependymal giant cell astrocytomas (SEGA) and cortical tubers (CT). Further, targeting the epidermal growth factor receptor (EGFR) with afatinib, which is expressed in CLIP cells, reduces cell growth in cerebral TSC organoids. However, investigation of clinical patient-derived data is lacking. Observation of EGFR expression in SEGA, CT and focal cortical dysplasia (FCD) 2B human brain specimen and investigation of whether its inhibition could be a potential therapeutic intervention for these patients. Brain specimens of 23 SEGAs, 6 CTs, 20 FCD2Bs and 17 controls were analysed via immunohistochemistry to characterise EGFR expression, cell proliferation (via Mib1) and mTOR signalling. In a cell-based assay using primary patient-derived cells (CT n= 1, FCD2B n= 1 and SEGA n= 4), the effects of afatinib and everolimus on cell proliferation and cell viability were observed. EGFR overexpression was observed in histological sections of SEGA, CT and FCD2B patients. Both everolimus and afatinib decreased the proliferation and viability in primary SEGA, tuber and FCD2B cells. Our study demonstrates that EGFR suppression might be an effective alternative treatment option for SEGAs and tubers, as well as other mTOR-associated malformations of cortical development, including FCD2B.

Synergistic effects of the combination of trametinib and alpelisib in anaplastic thyroid cancer with BRAF and PI3KCA co-mutations

BackgroundAnaplastic thyroid cancer (ATC), a rare and aggressive malignancy with a poor prognosis, has shown promise with the approved dabrafenib/trametinib combination for BRAFV600E mutation. Co-occurring PI3KCA mutations, identified as negative prognostic factors in lung cancer with BRAFV600E mutation, emphasize the need to target both pathways. Exploring trametinib and alpelisib combination becomes crucial for ATC. MethodsA patient-derived xenograft (PDX) and primary cell line were obtained from an ATC patient with BRAF and PI3KCA co-mutation. Individual testing of targeted therapies against BRAF, MEK, and PI3KCA was followed by a combination treatment. Synergistic effects were evaluated using the combination index. Immunoblotting assessed the efficacy, with validation performed using a PDX model. ResultsIn this study, the ATC0802 cell line and PDX were established from a refractory ATC patient. NGS revealed BRAF and PI3KCA co-mutations pre- and post-dabrafenib/trametinib treatment. Trametinib/alpelisib combination showed synergy, suppressing both pERK and pAKT levels, unlike monotherapies or BRAF knockdown. The combination induced apoptosis and, in the PDX model, demonstrated superior tumor growth inhibition compared to monotherapies. ConclusionsThe combination of trametinib and alpelisib showed promise as a strategy for treating ATC with co-mutations in BRAF and PI3KCA, both in vitro and in vivo. This combination offers insights into overcoming resistance to BRAF-targeted treatments in ATC with mutations in BRAF and PI3KCA.

Abstract 6956: Role of soluble CD109 in the progression of head and neck squamous cell carcinoma

Abstract Introduction: About five percent of global cancer deaths are attributed to Head and Neck Squamous Cell Carcinoma (HNSCC), and its annual incidence is predicted to skyrocket by thirty percent by 2030 (according to GLOBOCAN estimates). HNSCC originates from squamous cells in the oral cavity, lips, tongue, larynx, pharynx, and salivary glands. Despite the comparatively easy tumor diagnosis, the number of patients diagnosed at an advanced stage with metastatic spread is substantial. Multiple cell-signaling pathways have been proven to be dysregulated in HNSCC, including the epidermal growth factor receptor (EGFR). Our group has identified CD109, a GPI-anchored membrane-associated protein, as a TGF-β co-receptor that inhibits TGF-β signaling. CD109 expression is upregulated in SCC, and its elevated expression in premalignant lesions is associated with an increased risk of metastatic progression. Recent studies by our team have revealed that CD109 promotes tumor growth in vivo, likely by enhancing EGFR signaling. Interestingly, we have also discovered that membrane CD109 (mCD109) is endogenously released from the cell surface; however, the mechanism of action of the released/soluble form of CD109 (sCD109) remains unknown. In the current study, we examined whether sCD109 opposes the mCD109/EGFR-mediated pro-tumorigenic signaling and HNSCC progression. Research Questions: 1.Does sCD109 regulate EGF-induced EGFR signaling and downstream stem cell marker expression?2.Does sCD109 regulate EGFR expression levels on the HNSCC cell surface?3.Does sCD109 affect the nuclear localization of EGFR?4.Does sCD109 promote EGFR internalization and degradation? Methodology: HNSCC cells and patient-derived primary cells were treated with/without sCD109 recombinant protein (1nM, 5nM, and 10nM) without and with the EGF induction (10ng/ml for 15 mins) and were analyzed by western blotting, immunofluorescence, and immunocytochemistry. The role of sCD109 in regulating membrane EGFR levels was assessed by flow cytometry and surface protein biotinylation assay. Results: Our findings demonstrated that sCD109 inhibited EGF-induced phosphorylation of EGFR (Y1068), STAT3, AKT, and ERK. The sCD109 suppressed the stem cell marker expression (Nanog, OCT4, SOX2). Additionally, sCD109 hindered the nuclear localization of activated EGFR. The sCD109 decreased the membrane EGFR expression levels and activated the phosphorylation of EGFR (Y1045), suggesting a role for sCD109 in promoting EGFR degradation. Conclusion: Our team has previously reported that mCD109 is pro-tumorigenic, and our current findings suggest that sCD109 opposes the pro-tumorigenic effects of mCD109. This study aimed to determine how sCD109 regulates cellular signaling pathways and will provide key insights into the molecular mechanisms underlying the progression of HNSCC, a highly prevalent malignancy. Citation Format: Varsha Reddy Durgempudi, Tenzin Kungyal, Anie Philip. Role of soluble CD109 in the progression of head and neck squamous cell 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 6956.

Abstract 941: Patient-derived model systems of endometrial cancers for disease modeling and drug sensitivity testing

Abstract Background: Endometrial cancer (EC) is the most common gynecological cancer in the US, with increasing incidence and mortality rates. The survival rate lags behind four decades ago (81% vs 87%), underscoring the critical need for more effective treatment strategies. The heterogeneous nature of EC contributes to varied outcomes with current treatments. This project aims to establish reliable EC models for characterizing each individual tumor, distinguish optimal drug treatment, and determine specific drug effect mechanisms for personalized therapy. Methods: Freshly collected tumors were used for patient-derived xenograft models (PDXs) and patient-derived primary cancer cells (PDCs). Immunohistochemistry (IHC) staining was used to confirm the tumors grown in mice faithfully replicate the characteristics of patient tumor tissues. FDA-approved anticancer drugs were used to screen the optimal drugs to inhibit tumor cell proliferation and further utilized to inhibit tumor growth using PDXs. Results: We have collected 26 EC tumors and characterize them by DNA mutation and RNA-seq analysis. We implanted them in NSG mice, and 16 tumor samples have successfully grown in mice with a success rate of 61%. Most PDXs can be faithfully passaged to three generations. From the 16 PDXs, we created six novel PDCs. Using 2D and 3D spheroid cell culture, we screened 133 FDA-approved oncology drugs in the EC-PDC models. The drug screening results clearly highlighted several standout drugs, including CUDC-907 (a dual PI3K/HDAC inhibitor), two histone deacetylase (HDAC) inhibitors including romidepsin and panobinostat, four topoisomerase II (TOP II) inhibitors including mitoxantrone, daunorubicin, doxorubicin, and epirubicin, two proteasome inhibitors including carfilzomib and bortezomib, 2 DNA-directed RNA synthesis inhibitor dactinomycin and plicamycin, omacetaxine mepesuccinate (protein synthesis inhibitor), and valrubicin (DNA synthesis inhibitor). These drugs have demonstrated a significant capacity to inhibit tumor cell proliferation. Our pilot studies using single drugs, CUDC-907, romidepsin and mitoxantrone, achieved moderate drug effects. Combine mitoxantrone with romidepsin or CUDC-907 show surprisingly synergistic effects in multiple PDC models. The combination strategy will be tested in PDXs in the near future. Conclusion: Our unique PDXs and PDCs are excellent models for representing various characteristics of EC and testing novel therapeutics. This current study presents a promising direction for developing personalized therapy options for EC patients and provides a platform for further investigation of drug mechanisms and tumor development. Future studies will also involve etiology, such as chronic psychological stress, DNAm age and intratumoral microbiome. This information will help with endometrial cancer prevention, diagnosis, and prognosis. Citation Format: Tianyue Li, Xiaohao Huang, Riley Rosenmeyer, Samuel Robinson, Kazi Salsabil, Yiqin Xiong, Xiangbing Meng, Shujie Yang. Patient-derived model systems of endometrial cancers for disease modeling and drug sensitivity testing [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 941.

Abstract 4621: Dual targeting as an effective therapeutic strategy for malignant brain tumors

Abstract Glioblastoma, a type of brain cancer known for its aggressive nature and resistance to traditional treatments, remains a significant challenge for the medical community. Despite advances in surgical, radiation, and chemotherapy strategies, patients with glioblastoma typically have a poor prognosis, with a median survival time of only 15 months. While temporary symptom relief may be possible for some patients, complete tumor remission is often not achieved. Given the complexity of this disease, effective treatment options for glioblastoma are highly sought after. In this study, we have evaluated the effectiveness of a combinatorial approach using stem cell-derived exosomes enriched with miR-124 cargo paired with viral oncolysis for treating glioblastoma. Human Umbilical Cord Stem cells (hUCs) are genetically modified with specific miR plasmids, and then cultivated in a specialized medium that has been depleted of exosomes. The exosomes (exo) that contain the target miR are subsequently extracted, purified, quantified, and characterized. These exosomes are then utilized for all subsequent investigations. Using a combination of cell viability assays and microscopy, the overall effect of the exo-miR-124 and oncolytic virus on both established and patient-derived primary GBM cells is analyzed. Additionally, a time course western blot analysis is conducted to comprehend the molecular mechanisms underlying the effects of the miR modification and viral oncolysis on cell proliferation and death pathways. Orthotopic animal models of GBM will be utilized to assess the effects of miR modulation and viral oncolysis in vivo. Our research has shown that stem cell-derived exosomes are an effective way to deliver microRNA to specific cells, including tumor cells. This is because they can evade the immune system and target cells directly. Our experiments have shown that combining miR modulation paired with viral oncolysis as a promising approach for treating glioblastoma. Specifically, we have found that miR modulation and viral oncolysis can halt tumor cell proliferation in vitro by targeting multiple pathways involved in GBM growth and progression. Citation Format: Elle Y. Magnan, Karthik Gourishetti, Sydney Thomas, Deepak Bhere. Dual targeting as an effective therapeutic strategy for malignant brain 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 4621.

Abstract 259: Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) patients who experience relapse have a dismal prognosis, with 5-year overall survival rates of 10% for adults and 36% for children. Relapse is attributed to cytotoxic chemotherapy’s inability to target Leukemia Initiating Cells (LICs) that have the potential to re-populate the cancer. There is a need for novel therapeutic strategies to target LIC self-renewal in T-ALL with improved safety profiles and better chances of transition to clinical development. This project utilized a transgenic zebrafish model that accurately recapitulates the most aggressive form of the human T-ALL. This model offered several advantages, including a high frequency of LICs and suitability for drug testing. Over 770 FDA-approved drugs were screened, in vivo using >2,500 syngeneic CG1 zebrafish. Further secondary screening in human T-ALL cells narrowed down the potential hits into 4 compounds that specifically interfere with self-renewal. Finally, the hit drug Amiloride was confirmed by a limiting dilation assay (LDA), resulting in a more than 4-fold decrease in the LIC frequency (p=0.0026). The top hit, Amiloride, which reduced the frequency of LICs in zebrafish and human T-ALL cells, is an inhibitor of the Sodium Hydrogen Exachanger-1 (NHE1). NHE1 has been previously linked to the proliferation, migration, and drug resistance of different cancers. In addition, Amiloride treatment was found to impair mitochondrial function in cancer cells, offering a possible mechanism for the inhibition of LICs. Research has not yet investigated how NHE1, and the altered mitochondrial dynamics related to its inhibition affect the development and persistence of LICs in T-ALL. In our human T-ALL cells, we found that Amiloride significantly reduced mitochondrial basal respiration (p=0.0074), ATP production (p=0.0008) and increased the expression of genes associated with mitochondrial stress such as TFAM, PINK1, PARKIN and MT-CO1. Experiments using shRNA mediated knock down of the NHE1 in human T-ALL cells are underway and will be followed by investigating the impact of NHE1 inhibition on primary patient-derived T-ALL cells in a mouse model. Overall, this project showcases of the power of the zebrafish in cancer research in an efficient and accurate large-scale screen for drugs that impact LIC self-renewal. We have identified Amiloride as a top hit that reduces the frequency of LIC in vivo and in vitro. Finally, we are continuing to investigate the role of the NHE1 in self-renewal in human T-ALL and the associated mitochondrial alterations. Citation Format: Majd Al-Hamaly, Yelena Chernyavskaya, Emma Arvin, Logan Turner, Meghan Haney, Jessica Blackburn. Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating 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 259.

Abstract 5906: A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer cells

Abstract Despite advances in immune and targeted therapies, chemotherapy remains the primary treatment for ovarian cancer (OvCa) patients. Unfortunately, most OvCa patients develop chemotherapy-resistant disease that is inevitably fatal. Quiescence is an important yet poorly understood mechanism underlying chemotherapy resistance. Therefore, we aimed to characterize quiescent ovarian cancer (qOvCa) cells to identify novel therapeutic targets to improve OvCa patient outcomes. Primary patient-derived qOvCa cells and proliferating cells were isolated using a single-cell microfluidics culture device and collected for single-cell RNA-seq. scRNA-seq of qOvCa cells identified (i) downregulation of MBD3 and CHD4, which are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, and (ii) upregulation of proteasome-associated genes. Knockdown of either MBD3 or CHD4 by shRNA, or NuRD complex pharmacologic inhibition with the HDAC inhibitors such as Vorinostat resulted in significant cell growth arrest without cell death. Standard and Fucci reporter-based cell cycle analysis further confirmed that inhibition of the NuRD complex with Vorinostat induced 94.47% cells in the G0 phase (p<0.0001), suggesting a dense quiescent state. Through cell cycle phase maps produced by iterative immunofluorescence of 30 core cell cycle regulators and manifold learning, we found Vorinostat-treated OvCa cells demonstrated a completely different cell cycle structure from control cells, with most of the cells in an extreme G0 phase. Bulk RNA-seq of Vorinostat-treated cells in three OvCa cell lines validated the downregulation of pathways relating to cell cycle, cell division, and DNA replication (LFC<-1, p<0.05), which is consistent with stem cells. Upregulation of several pathways involved in proteostasis were also noted. We therefore performed a targeted drug screen for combinatorial therapy approaches to eradicate qOvCa. We found that Vorinostat combined with the proteasome inhibitor Carfilzomib demonstrated the most profound synergistic cell death (Loewe index synergy score 23-73). Increased proteasome activity was also found in both primary and Vorinostat-induced qOvCa, suggesting the essential role of the proteasome in qOvCa. Tumor xenograft studies confirmed that Vorinostat alone can delay tumor growth (p<0.05) and combinatorial therapy of Vorinostat and Carfilzomib is more efficacious (p=0.0049). Taken together, NuRD complex inhibition induces quiescence. Combination therapy with Vorinostat to induce quiescence combined with proteasome inhibitors resulted in the profound death of therapy-resistant quiescent cells. Our work suggests this is a novel therapeutic approach to eradicate chemoresistant qOvCa cells and increase cure rates. Citation Format: Qi Jiang, Michelle Ertel, Santiago Panesso- Gómez, Sara Sannino, April Sagan, Alexander J. Cole, Stacy C. McGonigal, Betsy Ann Varghese, Wayne Stallaert, Jeffrey L. Brodsky, Hatice U. Osmanbeyoglu, Ronald J. Buckanovich. A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer 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 5906.

Abstract 2500: Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine

Abstract Introduction: Next generation sequencing (NGS) has revolutionized precision oncology by enabling identification of cancer patients harboring actionable mutations who may benefit from corresponding targeted therapies. However, sequencing-based biomarkers are not detected for most patients and imperfectly predict response to therapy even when present. To address this unmet need, we have developed a CRISPR-based diagnostics platform for ‘personalized functional genomics’, allowing identification of patient-specific therapeutic vulnerabilities irrespective of mutation status. We have previously applied this approach to primary hematologic malignancies, and here report application to solid tumor-derived samples. Methods: Patient-derived solid tumor samples from several tumor types including breast, ovarian, colon and lung were obtained from 3 sources: fresh tissue (obtained from patients via core biopsy, surgical resection, or malignant fluid sampling), primary Patient-Derived Cell Lines (PDCLs, generated from surgical material and adapted for ex vivo culture), and commercially obtained viably frozen dissociated tumor cells (DTCs). Samples were dissociated to single cell suspensions and transduced with a lentivirus encoding Cas9 and an sgRNA library covering a custom targeted oncology gene panel. The panel consists of genes encoding targets of FDA-approved oncology drugs, as well as reference genes. After a short culture period, transduced cells were harvested and relative sgRNA distribution was measured by amplicon NGS. Changes in sgRNA abundance were collapsed into gene-level log2-fold changes, and significantly depleted genes scored as functional dependencies. For select PDCLs, cells were treated with cognate small molecule inhibitors over the course of 4 days. Cell viability was read out using CTG assay and a calculated IC50 lower than 500nM inferred as inhibitor sensitive. Results: In a double-blind study using primary PDCLs, functional dependencies identified by CRISPR perfectly discriminated alpelisib sensitive (2/2) from resistant samples (6/6). Using fresh primary samples from varying cancer types and specimen sources, we achieve a technical success rate >70%. Among gene dependencies identified, targets of FDA-approved oncology drugs and those under late-stage clinical investigation were both observed including BRAF, CDK4/6, VEGFR1/2 and PARP1/2. Conclusion: We demonstrate technical proof of concept of the FX platform for CRISPR-based personalized functional genomics. This approach is feasible when applied to primary patient samples from a variety of tumor types and tissue sources, and results are validated by comparison with orthogonal methods. CRISPR-based functional profiling represents a powerful adjunct to traditional genomic profiling, for the first time allowing drug target dependency mapping in a patient-specific and mutation-agnostic manner. Citation Format: Karl Hodel, Andreas Varkaris, Nicholas Chevalier, Maci Meyers, Allison Kehlmann, Stephane Corbel, Anastasia Stavridi, Peter Kilfeather, Leonardo Hagmann, Kathleen Tran, Yali Li, Christian Schmedt, Ioannis Sanidas, Srinath Sampath, Srihari Sampath, Dejan Juric. Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine [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 2500.

Abstract 7593: Photodynamic therapy improves therapeutic management of glioblastoma

Abstract Glioblastoma (GBM) remains a formidable challenge in the realm of oncology, primarily due to the limitations of the standard of care therapies. Photodynamic therapy (PDT), which exploits the light-activated potential of 5-aminolevulinic acid (5-ALA), emerges as a promising avenue to combat GBM. In this study, primary patient-derived cell lines, Ox5 core, and G7, served as critical subjects, exposed to light excitation at wavelengths of 410 nm, 528 nm, and 810 nm, both with and without 5-ALA (protoporphyrin IX). The central objective was to rigorously assess the efficacy of PDT, either with or without 5-ALA, in reducing cell viability and curtailing clonogenic expansion. This evaluation was performed within the context of the existing standard of care for GBM, encompassing Temozolomide (TMZ) and Radiotherapy. Our results unveiled striking insights into the potential of PDT as an innovative therapeutic strategy. Notably, Ox5 cells exhibited a remarkable reduction in viability, exceeding 70%, following exposure to PDT with 410 nm light and 5-ALA at a concentration of 50μM. A similar, albeit slightly less profound, effect was observed in G7 cells, with a viability decrease of more than 53%. These outcomes, meticulously quantified through the MTT viability assay (n=4), were reinforced by statistical significance, as indicated by p-values falling below 0.05 and, lower 0.001. Furthermore, clonogenic assays employing G7 cells provided an additional layer of evidence, demonstrating a substantial inhibition of clonogenic potential following PDT treatment with 410 nm light and 5-ALA. Immunohistochemistry techniques, particularly focusing on the cleaved caspase-3 marker, in both Ox5 core and G7 cells, underscoring the mechanistic basis of the observed cell death. In stark contrast, the conventional standard of care, involving TMZ monotherapy, was tested, and the results across four biological replicates were less significant. The relative viability of Ox5 and G7 cells exhibited no statistical significance, with p-values exceeding 0.05, implying a limited impact of this regimen on cell viability. Moreover, clonogenic assays conducted under the standard of care protocol (TMZ+/-Radiotherapy) unveiled the persistence of colonies, even at higher radiation doses of 4 and 6 Gy.In summary, PDT, specifically when implemented with 410 nm light and 5-ALA, represents a breakthrough strategy that leads to substantial cell death in both Ox5 and G7 cells. This compelling alternative therapy shines a hopeful light on the horizon of GBM treatment, providing a potentially transformative approach that can augment the current standard of care. Citation Format: Nazar Vasyliv, Karin Williams. Photodynamic therapy improves therapeutic management of glioblastoma [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 7593.

N6-isopentenyladenosine inhibits aerobic glycolysis in glioblastoma cells by targeting PKM2 expression and activity.

Glioblastoma (GBM) is a primary tumor in the central nervous system with poor prognosis. It exhibits elevated glucose uptake and lactate production. This metabolic state of aerobic glycolysis is known as the Warburg effect. N6-isopentenyladenosine (iPA), a natural cytokine modified with an isopentenyl moiety derived from the mevalonate pathway, has well-established anti-tumor activity. It inhibits cell proliferation in glioma cells, inducing cell death by apoptosis and/or necroptosis. In the present study, we found that iPA inhibits aerobic glycolysis in unmodified U87MG cells and in the same cell line engineered to over-express wild-type epidermal growth factor receptor (EGFR) or EGFR variant III (vIII), as well as in a primary GBM4 patient-derived cell line. The detection of glycolysis showed that iPA treatment suppressed ATP and lactate production. We also evaluated the response of iPA treatment in normal human astrocyte primary cells, healthy counterpart cells of the brain. Aerobic glycolysis in treated normal human astrocyte cells did not show significant changes compared to GBM cells. To determine the mechanism of iPA action on aerobic glycolysis, we investigated the expression of certain enzymes involved in this metabolic pathway. We observed that iPA reduced the expression of pyruvate kinase M2 (PKM2), which plays a key role in the regulation of aerobic glycolysis, promoting tumor cell proliferation. The reduction of PKM2 expression is a result of the inhibition of the inhibitor of nuclear factor kappa-B kinase subunit, beta/nuclear factor-kappa B pathway upon iPA treatment. In conclusion, these experimental results show that iPA may inhibit aerobic glycolysis of GBM in stabilized cell lines and primary GBM cells by targeting the expression and activity of PKM2.

A first-in-class TIMM44 blocker inhibits bladder cancer cell growth

Mitochondria play a multifaceted role in supporting bladder cancer progression. Translocase of inner mitochondrial membrane 44 (TIMM44) is essential for maintaining function and integrity of mitochondria. We here tested the potential effect of MB-10 (MitoBloCK-10), a first-in-class TIMM44 blocker, against bladder cancer cells. TIMM44 mRNA and protein expression is significantly elevated in both human bladder cancer tissues and cells. In both patient-derived primary bladder cancer cells and immortalized (T24) cell line, MB-10 exerted potent anti-cancer activity and inhibited cell viability, proliferation and motility. The TIMM44 blocker induced apoptosis and cell cycle arrest in bladder cancer cells, but failed to provoke cytotoxicity in primary bladder epithelial cells. MB-10 disrupted mitochondrial functions in bladder cancer cells, causing mitochondrial depolarization, oxidative stress and ATP reduction. Whereas exogenously-added ATP and the antioxidant N-Acetyl Cysteine mitigated MB-10-induced cytotoxicity of bladder cancer cells. Genetic depletion of TIMM44 through CRISPR-Cas9 method also induced robust anti-bladder cancer cell activity and MB-10 had no effect in TIMM44-depleted cancer cells. Contrarily, ectopic overexpression of TIMM44 using a lentiviral construct augmented proliferation and motility of primary bladder cancer cells. TIMM44 is important for Akt-mammalian target of rapamycin (mTOR) activation. In primary bladder cancer cells, Akt-S6K1 phosphorylation was decreased by MB-10 treatment or TIMM44 depletion, but enhanced after ectopic TIMM44 overexpression. In vivo, intraperitoneal injection of MB-10 impeded bladder cancer xenograft growth in nude mice. Oxidative stress, ATP reduction, Akt-S6K1 inhibition and apoptosis were detected in MB-10-treated xenograft tissues. Moreover, genetic depletion of TIMM44 also arrested bladder cancer xenograft growth in nude mice, leading to oxidative stress, ATP reduction and Akt-S6K1 inhibition in xenograft tissues. Together, targeting overexpressed TIMM44 by MB-10 significantly inhibits bladder cancer cell growth in vitro and in vivo.