Patient-derived Cell Lines Research Articles

Gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) promotes oral cancer stemness by acting as a molecular sponge of miR331-3p

Background/purposeAccumulating evidence has suggested that treatment failure of cancer therapy can be attributed to cancer stem cells (CSCs). Among numerous regulators of cancer stemness, non-coding RNAs (ncRNAs) have gained significant attention recently. In this study, we examined the role of gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) in oral CSCs (OCSCs). Materials and methodsRNA Sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the expression of GAPLINC. Flow cytometry and sphere-forming assay were exploited to isolate OCSCs. Measurement of aldehyde dehydrogenase 1 (ALDH1) activity, CD44 expressing cells, and various phenotypic assays, such as self-renewal, migration, invasion, and colony-forming abilities, were conducted in CSCs of two types of oral cancer cells (SAS and GNM) following the knockdown of GAPLINC. A luciferase reporter was also carried out to validate the direct interaction between GAPLINC and microRNA (miR)-331-3p. ResultsOur results showed that GAPLINC was overexpressed in OCSCs from patient-derived and oral cancer cell lines. We demonstrated that silencing of GAPLINC in OCSCs downregulated various CSC hallmarks, such as ALDH1 activity, percentage of CD44-expressing cells, self-renewal capacity, and colony-forming ability. Moreover, our results revealed that the effect of GAPLINC on cancer stemness was mediated by direct repression of miR-331-3p. ConclusionThese data have potential clinical implications in that we unraveled the aberrant upregulation of GAPLINC and demonstrated that suppression of GAPLINC may reduce cancer stemness via sequestering miR-331-3p.

Zebrafish Avatars: Toward Functional Precision Medicine in Low-Grade Serous Ovarian Cancer.

Ovarian cancer (OC) is an umbrella term for cancerous malignancies affecting the ovaries, yet treatment options for all subtypes are predominantly derived from high-grade serous ovarian cancer, the largest subgroup. The concept of "functional precision medicine" involves gaining personalized insights on therapy choice, based on direct exposure of patient tissues to drugs. This especially holds promise for rare subtypes like low-grade serous ovarian cancer (LGSOC). This study aims to establish an in vivo model for LGSOC using zebrafish embryos, comparing treatment responses previously observed in mouse PDX models, cell lines and 3D tumor models. To address this goal, a well-characterized patient-derived LGSOC cell line with the KRAS mutation c.35 G>T (p.(Gly12Val)) was used. Fluorescently labeled tumor cells were injected into the perivitelline space of 2 days' post-fertilization zebrafish embryos. At 1 day post-injection, xenografts were assessed for tumor size, followed by random allocation into treatment groups with trametinib, luminespib and trametinib + luminespib. Subsequently, xenografts were euthanized and analyzed for apoptosis and proliferation by confocal microscopy. Tumor cells formed compact tumor masses (n = 84) in vivo, with clear Ki67 staining, indicating proliferation. Zebrafish xenografts exhibited sensitivity to trametinib and luminespib, individually or combined, within a two-week period, establishing them as a rapid and complementary tool to existing in vitro and in vivo models for evaluating targeted therapies in LGSOC.

Generation of a healthy heavy smoker patient-derived induced pluripotent stem cell line UHOMi007-A from peripheral blood mononuclear cells

Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized healthy heavy smoker male donor free from emphysema or tobacco related disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers.The reported UHOMi007-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing.

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 PO3-28-12: Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance

Abstract Standard of care for breast cancer (BC) treatment including Selective-Estrogen Receptor (ER)-modulators and/or degraders (SERM; tamoxifen, SERD;Fulvestrant) or cyclin-dependent kinases CDK4 and CDK6 (palbociclib) can be effective therapeutic strategies. However, drug toxicity and/or drug resistance severely limit these approaches and patients succumb to the disease. Therefore, there is an urge to develop new therapeutic strategies against ER+-BC. Ion channels are general pharmacological targets however, little is known about the role of this class of proteins in cancer biology. We discovered that expression of the KCa3.1 potassium ion channel is downregulated in ER+-BC. Also, patients expressing high level of KCa3.1 present a better overall survival (OS) and relapse free survival when compared with patients with low expression. Thus, these data indicate that KCa3.1 is a prognostic factor in ER+-BC and that high activity of this channel plays a fundamental role in an oncosuppressor signature. Our central hypothesis is that pharmacological targeting KCa3.1 with activator molecules arrests growth of ER+-BC. We discovered that ER+-BC patient-derived organoids (PDO) and cell lines treated with KCa3.1 agonists including the FDA approved Chlorzoxazone, (CZX) arrests significantly inhibit tumor growth. Activation of KCa3.1 arrested the cell cycle in the G0/G1 phase by activating a senescent-like phenotype. Furthermore, our studies on the biochemical signaling that underlines this event demonstrated that increased expression level of KCa3.1 protein associated with lower expression level of ERa in a cell cycle phase-dependent manner. Furthermore, use of KCa3.1activator produced a proteasomal-dependent ERa degradation. To better characterize the therapeutic benefit of targeting the KCa3.1, we assessed the effects of CZX on developed several tamoxifen and/or palbociclib resistant cell lines and PDO models. We found that the combination of CZX strongly potentiated the lethal effects of both tamoxifen and palbociclib in the relative resistant models. We conclude that pharmacological targeting KCa3.1 potassium channel in ER+-BC can be considered a novel, safe and efficacious therapeutic strategy. Citation Format: Davide Delisi, Metin Cetin, Ozge Saatci, Ozgur Sahin, Saverio Gentile. Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance [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 PO3-28-12.

CD133 Stimulates Cell Proliferation via the Upregulation of Amphiregulin in Melanoma.

CD133, a cancer stem cell (CSC) marker in tumors, including melanoma, is associated with tumor recurrence, chemoresistance, and metastasis. Patient-derived melanoma cell lines were transduced with a Tet-on vector expressing CD133, generating doxycycline (Dox)-inducible cell lines. Cells were exposed to Dox for 24 h to induce CD133 expression, followed by RNA-seq and bioinformatic analyses, revealing genes and pathways that are significantly up- or downregulated by CD133. The most significantly upregulated gene after CD133 was amphiregulin (AREG), validated by qRT-PCR and immunoblot analyses. Induced CD133 expression significantly increased cell growth, percentage of cells in S-phase, BrdU incorporation into nascent DNA, and PCNA levels, indicating that CD133 stimulates cell proliferation. CD133 induction also activated EGFR and the MAPK pathway. Potential mechanisms highlighting the role(s) of CD133 and AREG in melanoma CSC were further delineated using AREG/EGFR inhibitors or siRNA knockdown of AREG mRNA. Treatment with the EGFR inhibitor gefitinib blocked CD133-induced cell growth increase and MAPK pathway activation. Importantly, siRNA knockdown of AREG reversed the stimulatory effects of CD133 on cell growth, indicating that AREG mediates the effects of CD133 on cell proliferation, thus serving as an attractive target for novel combinatorial therapeutics in melanoma and cancers with overexpression of both CD133 and AREG.

Abstract PO5-27-02: A Tumor Emboli Platform for Morphometric Analysis, Drug screening and for Intravital Optical Imaging in a Murine Model to Simulate Clinicopathological Features of Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is distinguished by the presence of tumor emboli, clusters of tumor cells, found within breast parenchyma and dermal lymphatic vessels. These emboli serve as a crucial clinicopathological hallmark, believed to underlie the characteristic diffuse growth pattern and aggressive progression observed in IBC patients, with nearly 30% of patients presenting with metastatic disease at diagnosis. However, the rarity of this cancer and the absence of a solid tumor mass pose challenges in obtaining biospecimens, particularly those with evidence of tumor emboli. To address this pressing need for preclinical models that replicate this distinctive tumor growth pattern, we describe a tumor emboli culture platform that facilitates live imaging, enables compound screening by assessing various phenotypes of the emboli, and permits evaluation of emboli in an in vivo murine model. Methods: The tumor emboli culture system was evaluated for physiological measurements corresponding to dermal lymphatic vessels and subsequently employed to generate tumor emboli from diverse patient-derived cell lines and PDX. Confocal microscopy was employed to characterize cellular information across multiple slices of the tumor emboli. This provided a comprehensive, multi-parametric analysis of single-cell and spheroid phenotypes, including measurements of number, size, shape, and viability of the organoids. Furthermore, we developed imaging algorithms to evaluate phenotypic changes in tumor emboli in response to anti-cancer drug treatment. We also optimized a technique that allows for in vivo tumor emboli implantation including in a transgenic mice with red fluorescent lymphatic vasculature that were surgically implanted with a dorsal skin window chamber. This allowed for intravital optical imaging of the local tumor microenvironment and adjacent skin. Results: Our culture platform enables live imaging of the tumor emboli generated over a span of 7-10 days, maintaining key parameters including kinematic and dynamic viscosity, density along with sheer wall stress within the reported physiological range for lymphatics. Through higher resolution confocal image acquisition and multi-parametric analysis, we conducted particle counting and classification, providing statistically significant insights into the integrity of tumor emboli over time. Additionally, the developed algorithms allowed for quantitative analysis of images from tumor emboli cultures subjected to anti-cancer drugs, revealing disparities in drug efficacy on tumor emboli integrity and dispersion including inter-cluster space within the emboli. Intravital optical imaging, coupled with quantitative analysis of dispersion characteristics of the tumor emboli implanted in the murine model over 5-6 days, demonstrated a diffuse spread reminiscent of observations in IBC patients. Furthermore, immunohistochemistry staining showed marked Ki67, CD45, F4-80 revealing a proliferative tumor microenvironment with high levels of macrophage infiltration. Conclusions: As of now specific targeted therapies for IBC patients remain elusive. Our culture platform for tumor emboli, when combined with advanced imaging techniques both in vitro and in vivo and traditional approaches like immunohistochemistry and genomic evaluation, forms a powerful toolkit for assessing the phenotype and dispersion characteristics of this unique pathological feature in IBC. This preclinical model offers crucial insights, potentially paving the way for precision medicine strategies in the treatment of this understudied cancer. Funding in part from American Cancer Society Mission Boost MBG-20-141-01-MBG grant (GRD), Department of Defense W81XWH-20-1-0153 (GRD), Duke Consortium for Inflammatory Breast Cancer Education (AB, CW, RE). Citation Format: Dorababu Sannareddy, Theresa Charity, Alexandra Bennion, Caroline Way, Edwin Yu, Ralph Erdmann, Gregory M Palmer, Gayathri R Devi. A Tumor Emboli Platform for Morphometric Analysis, Drug screening and for Intravital Optical Imaging in a Murine Model to Simulate Clinicopathological Features of Inflammatory Breast Cancer [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 PO5-27-02.

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.

Antiretroviral Drug Repositioning for Glioblastoma.

Outcomes for glioblastoma (GBM) remain poor despite standard-of-care treatments including surgical resection, radiation, and chemotherapy. Intratumoral heterogeneity contributes to treatment resistance and poor prognosis, thus demanding novel therapeutic approaches. Drug repositioning studies on antiretroviral therapy (ART) have shown promising potent antineoplastic effects in multiple cancers; however, its efficacy in GBM remains unclear. To better understand the pleiotropic anticancer effects of ART on GBM, we conducted a comprehensive drug repurposing analysis of ART in GBM to highlight its utility in translational neuro-oncology. To uncover the anticancer role of ART in GBM, we conducted a comprehensive bioinformatic and in vitro screen of antiretrovirals against glioblastoma. Using the DepMap repository and reversal of gene expression score, we conducted an unbiased screen of 16 antiretrovirals in 40 glioma cell lines to identify promising candidates for GBM drug repositioning. We utilized patient-derived neurospheres and glioma cell lines to assess neurosphere viability, proliferation, and stemness. Our in silico screen revealed that several ART drugs including reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs) demonstrated marked anti-glioma activity with the capability of reversing the GBM disease signature. RTIs effectively decreased cell viability, GBM stem cell markers, and proliferation. Our study provides mechanistic and functional insight into the utility of ART repurposing for malignant gliomas, which supports the current literature. Given their safety profile, preclinical efficacy, and neuropenetrance, ARTs may be a promising adjuvant treatment for GBM.

Pannexin 1 dysregulation in Duchenne muscular dystrophy and its exacerbation of dystrophic features in mdx mice

BackgroundDuchenne muscular dystrophy (DMD) is associated with impaired muscle regeneration, progressive muscle weakness, damage, and wasting. While the cause of DMD is an X-linked loss of function mutation in the gene encoding dystrophin, the exact mechanisms that perpetuate the disease progression are unknown. Our laboratory has demonstrated that pannexin 1 (Panx1 in rodents; PANX1 in humans) is critical for the development, strength, and regeneration of male skeletal muscle. In normal skeletal muscle, Panx1 is part of a multiprotein complex with dystrophin. We and others have previously shown that Panx1 levels and channel activity are dysregulated in various mouse models of DMD.MethodsWe utilized myoblast cell lines derived from DMD patients to assess PANX1 expression and function. To investigate how Panx1 dysregulation contributes to DMD, we generated a dystrophic (mdx) mouse model that lacks Panx1 (Panx1−/−/mdx). In depth characterization of this model included histological analysis, as well as locomotor, and physiological tests such as muscle force and grip strength assessments.ResultsHere, we demonstrate that PANX1 levels and channel function are reduced in patient-derived DMD myoblast cell lines. Panx1−/−/mdx mice have a significantly reduced lifespan, and decreased body weight due to lean mass loss. Their tibialis anterior were more affected than their soleus muscles and displayed reduced mass, myofiber loss, increased centrally nucleated myofibers, and a lower number of muscle stem cells compared to that of Panx1+/+/mdx mice. These detrimental effects were associated with muscle and locomotor functional impairments. In vitro, PANX1 overexpression in patient-derived DMD myoblasts improved their differentiation and fusion.ConclusionsCollectively, our findings suggest that PANX1/Panx1 dysregulation in DMD exacerbates several aspects of the disease. Moreover, our results suggest a potential therapeutic benefit to increasing PANX1 levels in dystrophic muscles.

Aurora kinase A inhibition plus Tumor Treating Fields suppress glioma cell proliferation in a cilium-independent manner

Tumor Treating Fields (TTFields) extend the survival of glioblastoma (GBM) patients by interfering with a broad range of tumor cellular processes. Among these, TTFields disrupt primary cilia stability on GBM cells. Here we asked if concomitant treatment of TTFields with other agents that interfere with GBM ciliogenesis further suppress GBM cell proliferation in vitro. Aurora kinase A (AURKA) promotes both cilia disassembly and GBM growth. Inhibitors of AURKA, such as Alisertib, inhibit cilia disassembly and increase ciliary frequency in various cell types. However, we found that Alisertib treatment significantly reduced GBM cilia frequency in gliomaspheres across multiple patient derived cell lines, and in patient biopsies treated ex vivo. This effect appeared glioma cell-specific as it did not reduce normal neuronal or glial cilia frequencies. Alisertib-mediated depletion of glioma cilia appears specific to AURKA and not AURKB inhibition, and attributable in part to autophagy pathway activation. Treatment of two different GBM patient-derived cell lines with TTFields and Alisertib resulted in a significant reduction in cell proliferation compared to either treatment alone. However, this effect was not cilia-dependent as the combined treatment reduced proliferation in cilia-depleted cell lines lacking, ARL13B, or U87MG cells which are naturally devoid of ARL13B+ cilia. Thus, Alisertib-mediated effects on glioma cilia may be a useful biomarker of drug efficacy within tumor tissue. Considering Alisertib can cross the blood brain barrier and inhibit intracranial growth, our data warrant future studies to explore whether concomitant Alisertib and TTFields exposure prolongs survival of brain tumor-bearing animals in vivo.

Molecular clues unveiling spinocerebellar ataxia type-12 pathogenesis

Spinocerebellar Ataxia type-12 (SCA12) is a neurodegenerative disease caused by tandem CAG repeat expansion in the 5'-UTR/non-coding region of PPP2R2B. Molecular pathology of SCA12 has not been studied in the context of CAG repeats, and no appropriate models exist. We found in human SCA12-iPSC-derived neuronal lineage that expanded CAG in PPP2R2B transcript forms nuclear RNA foci and were found to sequester variety of proteins. Further, the ectopic expression of transcript containing varying length of CAG repeats exhibits non-canonical repeat-associated non-AUG (RAN) translation in multiple frames in HEK293T cells, which was further validated in patient-derived neural stem cells using specific antibodies. mRNA sequencing of the SCA12 and control neurons have shown a network of crucial transcription factors affecting neural fate, in addition to alteration of various signaling pathways involved in neurodevelopment. Altogether, this study identifies the molecular signatures of SCA12 disorder using patient-derived neuronal cell lines.

Abstract P15: Aberrant Non-canonical NF-κB Signalling Reprograms the Epigenome Landscape to Drive Oncogenic Transcriptomes in Multiple Myeloma

Abstract Activation of the NF-κB pathway is recognized to be necessary for the survival and progression of haematological cancers including Multiple Myeloma (MM). In particular, recurrent genetic lesions resulting in the chronic induction of non-canonical NF-κB (ncNF-κB) signalling have been exclusively linked to MM. Such mutations among patients are associated with significant proliferative and metastatic advantages and have been causally linked to treatment resistance. How the constitutive activity of ncNF-κB transcription factors can support such adaptations in this common haematological malignancy has been a fundamental question in cancer. In this study, we captured the epigenomic landscape of patient-derived MM cell lines harbouring recurrent NF-κB activating mutations through perturbation of nfkb2/p52 and integrated these cis-regulatory changes with MM patient epigenome data. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a novel p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression. Citation Format: Daniel A. Ang, Jean-Michel Carter, Kamalakshi Deka, Joel H.L. Tan, Jianbiao Zhou, Qingfeng Chen, Wee Joo Chng, Nathan Harmston, Yinghui Li. Aberrant Non-canonical NF-κB Signalling Reprograms the Epigenome Landscape to Drive Oncogenic Transcriptomes in Multiple Myeloma [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P15.

Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

Abstract LB215: Polymerase θ (POLθ) inhibitors (novobiocin, ART-558, RP6685) were tested with 22 approved and investigational agents in multi-cell type spheroids of genetically selected patient-derived human tumor cell lines

Abstract The potential of novobiocin to augment cancer chemotherapy was explored in the late 1980s and early 1990s in tumor cells and tumor-bearing mice and in Phase 1 clinical trials with cyclophosphamide or cisplatin. The molecular target of novobiocin was identified to be DNA POLθ which has a role in repair of DNA double strand breaks (DSB) during mitosis. Genetic alterations which may increase or decrease POLθ inhibitor effects have been elucidated. Thirty patient-derived tumor cell lines with known BRCA, ATM, ATR, POLQ, XRCC1, PALB2, PARP1, and LIG3 alterations were screened in a complex spheroid assay (tumor cells, endothelial cells, mesenchymal stem cells) with a POLθ inhibitor (novobiocin, ART-558, or RP6685), alone or in simultaneous combination with a FDA approved or investigational anticancer small molecule, using a 7-day exposure and a CellTiter-Glo 3D luminescence endpoint. As single agents, the POLθ inhibitors had little or no cytotoxicity. Potentiation of the topoisomerase II inhibitors doxorubicin (DOX) and etoposide by the POLθ inhibitors ART-558 or RP6685 was observed in spheroids grown from the serous endometrial adenocarcinoma 922993-354-T-J3, which has ATM and BRCA2 variants. The 379773-124-R-J2 endometrioid endometrial adenocarcinoma which harbors mutant POLθ demonstrated antagonism with ART-558 in combination with PARP inhibitors and less than additive to additive cytotoxicity in combination with DOX. Activity of the Chk1/2 inhibitor prexasertib was potentiated by either ART-558 or RP6685 in the 922993-354-T-J3 complex spheroids. The combination of POLθ inhibitor ART-558 and the Chk1/2 inhibitor prexasertib produced up to 1 log increase in cytotoxicity in the 922993-354-T-J3 complex spheroids. KRAS G12D inhibitor MRTX-133 cytotoxicity against 186277-243-T-J2 colon adenocarcinoma with the KRAS G12D variant was potentiated by either ART-558 or RP6685. Potential combinations to advance into in vivo studies will be presented. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Beverly A. Teicher, Thomas S. Dexheimer, Thomas Silvers, Nathan P. Coussens, J Paul Eder, James H. Doroshow. Polymerase θ (POLθ) inhibitors (novobiocin, ART-558, RP6685) were tested with 22 approved and investigational agents in multi-cell type spheroids of genetically selected patient-derived human tumor cell lines [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 LB215.

Upregulation of the Renin-Angiotensin System Is Associated with Patient Survival and the Tumour Microenvironment in Glioblastoma.

Glioblastoma is a highly aggressive disease with poor survival outcomes. An emerging body of literature links the role of the renin-angiotensin system (RAS), well-known for its function in the cardiovascular system, to the progression of cancers. We studied the expression of RAS-related genes (ATP6AP2, AGTR1, AGTR2, ACE, AGT, and REN) in The Cancer Genome Atlas (TCGA) glioblastoma cohort, their relationship to patient survival, and association with tumour microenvironment pathways. The expression of RAS genes was then examined in 12 patient-derived glioblastoma cell lines treated with chemoradiation. In cases of glioblastoma within the TCGA, ATP6AP2, AGTR1, ACE, and AGT had consistent expressions across samples, while AGTR2 and REN were lowly expressed. High expression of AGTR1 was independently associated with lower progression-free survival (PFS) (p = 0.01) and had a non-significant trend for overall survival (OS) after multivariate analysis (p = 0.095). The combined expression of RAS receptors (ATP6AP2, AGTR1, and AGTR2) was positively associated with gene pathways involved in hypoxia, microvasculature, stem cell plasticity, and the molecular characterisation of glioblastoma subtypes. In patient-derived glioblastoma cell lines, ATP6AP2 and AGTR1 were upregulated after chemoradiotherapy and correlated with an increase in HIF1A expression. This data suggests the RAS is correlated with changes in the tumour microenvironment and associated with glioblastoma survival outcomes.

Abstract NG08: Dissecting and quantifying pancreatic cancer plasticity using single-cell multiomics, lineage tracing and functional genomics reveals novel mediators of therapy resistance

Abstract NG08: Dissecting and quantifying pancreatic cancer plasticity using single-cell multiomics, lineage tracing and functional genomics reveals novel mediators of therapy resistance

Abstract LB069: Repurposing anti-mesothelin CAR-NK immunotherapy against colorectal cancer

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide, with highly variable prognosis and response to treatment. A large subset of patients does not respond to standard treatments or develops resistance. As an alternative, adoptive immunotherapy based on chimeric antigen receptor (CAR)-transduced immune cells has been proposed, however with significant adverse events. We therefore evaluated alternative CAR targets already tested in other tumour types, and employed the natural killer cell line NK-92 for CAR transduction because of its more favourable toxicity profile. As an alternative antigen, we considered mesothelin (MSLN), the most represented in CAR-based clinical studies for solid tumours. MSLN RNA expression was analysed in large series of CRC patient-derived xenografts (PDXs, n=500), cell lines (n=150) and tumours (n=640), to evaluate its distribution and to identify MSLN-overexpressing models. NK-92 cells were transduced with anti-MSLN CAR, and subsequently sorted and cloned. Activity of CAR-NK-92 cells against target-expressing ovarian and CRC cells was assessed in vitro and in vivo. Statistical significance of efficacy was evaluated by t-test. The large-scale expression analysis highlighted that about 10% of CRCs overexpress MSLN at levels comparable to those of ovarian cancer, a typical target of MSLN-CAR-based therapy. Intriguingly, MSLN overexpression is more frequent in the CRIS-B poor prognosis subtype and KRAS/BRAF-mutant CRC. Lentiviral transduction of NK-92 cells with the MSLN-CAR, followed by sorting and cloning, led to the identification of one clone, NK-92cl45, stably expressing the CAR and retaining the NK phenotype. As expected, the clone demonstrated significant in vitro and in vivo activity against ovarian cancer cells. When repurposed against models of CRC expressing high MSLN levels, it displayed comparable efficacy, both in vitro and in vivo. Specificity of NK-92cl45 was confirmed by the absence of activity on control models with low or absent MSLN. Our results provide preclinical evidence that a subset of colorectal cancers expressing high mesothelin levels can be effectively targeted by MSLN-CAR-based immunotherapy. The potential therapeutic impact of these findings is enhanced by the fact that frequently MSLN-overexpressing CRCs display worse prognosis and resistance to standard care. Citation Format: Erica Torchiaro, Marco Cortese, Alice D'Andrea, Consalvo Petti, Enzo Medico. Repurposing anti-mesothelin CAR-NK immunotherapy against colorectal cancer [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 LB069.

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.

Matched Three-Dimensional Organoids and Two-Dimensional Cell Lines of Metastatic Melanoma to the Brain Mirrors Response to Targeted Molecular Therapy

INTRODUCTION: Metastatic melanoma to the brain (MMB) represents a significant treatment challenge. Limitations in preclinical models, namely the lack of heterogeneity in cell lines and low throughput of xenograft models, have thwarted novel therapeutic development. Surgically eXplanted Organoids (SXOs) are ex vivo, three-dimensional models that recapitulate genotypic and phenotypic features of parent tumors with minimal extracranial processing. We aim to develop the first known matched patient-derived SXO and cell line of BRAF-mutant MMB to investigate responses to targeted therapy. METHODS: MMB SXOs were created by a novel protocol incorporating techniques for creating glioma and cutaneous melanoma organoids. A BRAFV600K-mutant MMB sample was collected and processed within 30 minutes of resection. SXOs were created using a 1.0 mm biopsy punch. Organoids were cultured in low-adherence plates in an optimized culture medium under continuous orbital rotation without an artificial extracellular scaffold. Organoid media was changed every 48 hours. Concurrently, matched patient-derived cell lines were created and passaged weekly. Drug screens were conducted with the CellTiter-Glo Luminescence Viability Assay (cell line) and ReadyProbe Cell Viability Imaging Kit (SXOs). RESULTS: After an initial shedding period, organoid growth was observed within 3-4 weeks. MMB SXOs retained histological phenotypes of the parent tissue, including pleomorphic epithelioid cells with abundant cytoplasm, large nuclei, focal melanin accumulation, strong Sox10 positivity, and HMB45 positivity. After sufficient growth, organoids could be manually parcellated to increase the number of replicates. Matched SXOs and cell lines demonstrated sensitivity to BRAF-V600 and MEK inhibitors. CONCLUSIONS: Here, we outline the creation of the first known scaffold-free organoid model of MMB. Further study using SXOs will improve the translational relevance of preclinical studies and enable the study of the melanoma tumor microenvironment.