Patient-derived Cells Research Articles

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.

Sustained Release Formulation of Hydroxypropyl-β-cyclodextrin Eye Drops Using Xanthan Gum.

Bietti's crystalline dystrophy (BCD) is an autosomal recessive chorioretinal degeneration caused by mutations in the CYP4V2 gene. It is characterized by cholesterol accumulation and crystal-like deposits in the retinas. Hydroxypropyl-β-cyclodextrin (HP-β-CyD) exerts therapeutic effects against BCD by reducing lysosomal dysfunction and inhibiting cytotoxicity in induced pluripotent stem cell (iPSC)-RPE cells established from patient-derived iPS cells. However, the ocular retention of HP-β-CyD is low and needs to be improved. Therefore, this study used a viscous agent to develop a sustained-release ophthalmic formulation containing HP-β-CyD. Our results suggest that HP-β-CyD-containing xanthan gum has a considerably higher sustained release capacity than other viscous agents, such as methylcellulose and sodium alginate. In addition, the HP-β-CyD-containing xanthan gum exhibited pseudoplastic behavior. It was less cytotoxic to human retinal pigment epithelial cells compared with HP-β-CyD alone. Furthermore, the slow release of HP-β-CyD from xanthan gum caused a sustained decrease in free intracellular cholesterol. These results suggest that xanthan gum is a useful substrate for the sustained release formulation of HP-β-CyD, and that HP-β-CyD-containing xanthan gum has potential as an eye drop for BCD treatment.

The Recombinant Oncolytic Virus VV-GMCSF-Lact and Chemotherapy Drugs against Human Glioma

Virotherapy is one of the perspective technologies in the treatment of malignant neoplasms. Previously, we have developed oncolytic vaccinia virus VV-GMCSF-Lact and its high cytotoxic activity and antitumor efficacy against glioma was shown. In this work, using immortalized and patient-derived cells with different sensitivity to VV-GMCSF-Lact, we evaluated the cytotoxic effect of chemotherapy agents. Additionally, we studied the combination of VV-GMCSF-Lact with temozolomide which is the most preferred drug for glioma treatment. Experimental results indicate that first adding temozolomide and then the virus to the cells is inherently more efficient than dosing it in the reverse order. Testing these regimens in the U87 MG xenograft glioblastoma model confirmed this effect, as assessed by tumor growth inhibition index and histological analysis. Moreover, VV-GMCSF-Lact as monotherapy is more effective against U87 MG glioblastoma xenografts comparing temozolomide.

C9ORF72 patient-derived endothelial cells drive blood-brain barrier disruption and contribute to neurotoxicity

The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream, playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS); however, the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression, compromised barrier integrity, and increased P-glycoprotein transporter activity. In addition, mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover, our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally, C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.Graphical

Feasibility of functional precision medicine for guiding treatment of relapsed or refractory pediatric cancers.

Children with rare, relapsed or refractory cancers often face limited treatment options, and few predictive biomarkers are available that can enable personalized treatment recommendations. The implementation of functional precision medicine (FPM), which combines genomic profiling with drug sensitivity testing (DST) of patient-derived tumor cells, has potential to identify treatment options when standard-of-care is exhausted. The goal of this prospective observational study was to generate FPM data for pediatric patients with relapsed or refractory cancer. The primary objective was to determine the feasibility of returning FPM-based treatment recommendations in real time to the FPM tumor board (FPMTB) within a clinically actionable timeframe (<4 weeks). The secondary objective was to assess clinical outcomes from patients enrolled in the study. Twenty-five patients with relapsed or refractory solid and hematological cancers were enrolled; 21 patients underwent DST and 20 also completed genomic profiling. Median turnaround times for DST and genomics were within 10 days and 27 days, respectively. Treatment recommendations were made for 19 patients (76%), of whom 14 received therapeutic interventions. Six patients received subsequent FPM-guided treatments. Among these patients, five (83%) experienced a greater than 1.3-fold improvement in progression-free survival associated with their FPM-guided therapy relative to their previous therapy, and demonstrated a significant increase in progression-free survival and objective response rate compared to those of eight non-guided patients. The findings from our proof-of-principle study illustrate the potential for FPM to positively impact clinical care for pediatric and adolescent patients with relapsed or refractory cancers and warrant further validation in large prospective studies. ClinicalTrials.gov registration: NCT03860376 .

Antitumoral Activity of the Universal Methyl Donor S-Adenosylmethionine in Glioblastoma Cells

Glioblastoma (GBM), the most frequent and lethal brain cancer in adults, is characterized by short survival times and high mortality rates. Due to the resistance of GBM cells to conventional therapeutic treatments, scientific interest is focusing on the search for alternative and efficient adjuvant treatments. S-Adenosylmethionine (AdoMet), the well-studied physiological methyl donor, has emerged as a promising anticancer compound and a modulator of multiple cancer-related signaling pathways. We report here for the first time that AdoMet selectively inhibited the viability and proliferation of U87MG, U343MG, and U251MG GBM cells. In these cell lines, AdoMet induced S and G2/M cell cycle arrest and apoptosis and downregulated the expression and activation of proteins involved in homologous recombination DNA repair, including RAD51, BRCA1, and Chk1. Furthermore, AdoMet was able to maintain DNA in a damaged state, as indicated by the increased γH2AX/H2AX ratio. AdoMet promoted mitotic catastrophe through inhibiting Aurora B kinase expression, phosphorylation, and localization causing GBM cells to undergo mitotic catastrophe-induced death. Finally, AdoMet inhibited DNA repair and induced cell cycle arrest, apoptosis, and mitotic catastrophe in patient-derived GBM cells. In light of these results, AdoMet could be considered a potential adjuvant in GBM therapy.

NMR Metabolomics of Primary Ovarian Cancer Cells in Comparison to Established Cisplatin-Resistant and -Sensitive Cell Lines

Cancer cell lines are frequently used in metabolomics, such as in vitro tumor models. In particular, A2780 cells are commonly used as a model for ovarian cancer to evaluate the effects of drug treatment. Here, we compare the NMR metabolomics profiles of A2780 and cisplatin-resistant A2780 cells with those of cells derived from 10 patients with high-grade serous ovarian carcinoma (collected during primary cytoreduction before any chemotherapeutic treatment). Our analysis reveals a substantial similarity among all primary cells but significant differences between them and both A2780 and cisplatin-resistant A2780 cells. Notably, the patient-derived cells are closer to the resistant A2780 cells when considering the exo-metabolome, whereas they are essentially equidistant from A2780 and A2780-resistant cells in terms of the endo-metabolome. This behavior results from dissimilarities in the levels of several metabolites attributable to the differential modulation of underlying biochemical pathways. The patient-derived cells are those with the most pronounced glycolytic phenotype, whereas A2780-resistant cells mainly diverge from the others due to alterations in a few specific metabolites already known as markers of resistance.

Dynamic molecular network analysis of iPSC-Purkinje cells differentiation delineates roles of ISG15 in SCA1 at the earliest stage

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1), a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However, the regulatory network of SCA1 pathology, especially central regulators of the earliest developmental stages and inflammatory events, remains incompletely understood. Here, we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development, and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients.

Suppression of Glioblastoma Stem Cell Potency and Tumor Growth via LRRK2 Inhibition.

Leucine-rich repeat kinase 2 (LRRK2), a large GTP-regulated serine/threonine kinase, is well-known for its mutations causing late-onset Parkinson's disease. However, the role of LRRK2 in glioblastoma (GBM) carcinogenesis has not yet been fully elucidated. Here, we discovered that LRRK2 was overexpressed in 40% of GBM patients, according to tissue microarray analysis, and high LRRK2 expression correlated with poor prognosis in GBM patients. LRRK2 and stemness factors were highly expressed in various patient-derived GBM stem cells, which are responsible for GBM initiation. Canonical serum-induced differentiation decreased the expression of both LRRK2 and stemness factors. Given that LRRK2 is a key regulator of glioma stem cell (GSC) stemness, we developed DNK72, a novel LRRK2 kinase inhibitor that penetrates the blood-brain barrier. DNK72 binds to the phosphorylation sites of active LRRK2 and dramatically reduced cell proliferation and stemness factors expression in in vitro studies. Orthotopic patient-derived xenograft mouse models demonstrated that LRRK2 inhibition with DNK72 effectively reduced tumor growth and increased survival time. We propose that LRRK2 plays a significant role in regulating the stemness of GSCs and that suppression of LRRK2 kinase activity leads to reduced GBM malignancy and proliferation. In the near future, targeting LRRK2 in patients with high LRRK2-expressing GBM could offer a superior therapeutic strategy and potentially replace current clinical treatment methods.

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.

Abstract CT151: Trial in progress:A phase 1 trial of TSC-100 and TSC-101, engineered T cell therapies that target minor histocompatibility antigens to eliminate residual disease after hematopoietic cell transplantation

Abstract Engineered T cell therapies such as CAR-T cell therapies have transformed the treatment of B-cell but not non-B cell hematologic malignancies. Allogeneic hematopoietic cell transplantation (HCT) remains the best curative option for hematologic malignancies but ~40% of patients relapse post-HCT with up to 90% mortality due to residual disease post-HCT. A key challenge in non-B cell malignancies is identifying the right antigens for T cell targeting. Cancer-associated antigens are heterogenous, enabling rapid escape of malignant cells with low antigens, while targeting lineage-specific antigens without distinct expression in malignant versus normal myeloid cells can cause prolonged neutropenia. A potential solution is targeting minor histocompatibility antigens (MiHAs) that are homogenously expressed on all hematopoietic cells and are genetically mismatched between donors and patients undergoing HCT. These mismatches enable T cells to selectively eliminate residual patient hematopoietic cells, normal or malignant, leaving donor cells untouched. TScan has developed allogeneic donor derived T-cell products TSC-100 and TSC-101, targeting MiHAs HA-1 and HA-2 respectively, both presented on HLA-A*02:01. By choosing HCT patients who are HLA-A*02:01 positive (&amp;gt;98% of whom are either HA-1 or HA-2 positive) and donors who are either HLA-A*02:01 or MiHA negative, TSC-100 or TSC-101 can potentially eliminate all residual patient-derived hematopoietic cells after HCT, to prevent disease relapse.Study NCT05473910 is a multi-center, multi-arm, non-randomized controlled Phase 1 umbrella study evaluating the feasibility, safety and preliminary efficacy of TSC-100 and TSC-101. Inclusion criteria (Figure 1) include adults with AML, MDS or ALL eligible for reduced intensity conditioning-based haploidentical donor transplantation from HLA or MiHA mismatched donors. HLA-A*02:01-positive patients undergo HA-1/HA-2 testing and are assigned to either TSC-100 or TSC-101 treatment arms in addition to HCT. HLA-A*02:01-negative patients in the control arm receive HCT alone. Upon count recovery after HCT, patients in treatment arms receive either TSC-100 or TSC-101, administered as single or two doses. Primary endpoints include adverse event profiles and dose limiting toxicities. Secondary endpoints include relapse rates, disease-free survival and overall survival. Exploratory endpoints include surrogates of efficacy such as minimal residual disease (MRD) rates and donor chimerism rates and kinetics. MRD is measured before and after HCT using flow cytometry, NGS and ddPCR. Donor chimerism is measured by standard STR-based and novel high-sensitivity NGS-based assays to quantify residual patient-derived hematopoietic cells. Together, these assays measure elimination of residual patient hematopoietic cells, malignant or normal, and could provide early evidence of biological activity. Citation Format: Monzr Al Malki, Alla Keyzner, Hyung Suh, Uday Popat, Chen Yi-Bin, Saar Gill, Melhem Solh, Joseph Uberti, Iris Isufi, Erica Buonomo, Yun Wang, Jim Murray, Gavin MacBeath, Debora Barton, Shrikanta Chattopadhyay, Ran Reshef. Trial in progress:A phase 1 trial of TSC-100 and TSC-101, engineered T cell therapies that target minor histocompatibility antigens to eliminate residual disease after hematopoietic cell transplantation [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 CT151.

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 LB452: Effects of AKT1, 2, and 3 knock-out by CRISPR-Cas9 in CD133+human melanoma stem cells treated with MEK and AKT inhibitors

Abstract Melanoma-initiating stem cells are involved in tumorigenesis, invasion, and drug resistance in malignant melanoma, and are characterized by increased expression of the stem cell marker CD133. We previously showed that CD133 knockout (KO) by CRISPR-Cas9 enhanced MEK inhibitor trametinib (T) -induced apoptosis in patient-derived BAKP melanoma cells by reducing anti-apoptotic p-AKT and p-BAD and increasing pro-apoptotic BAX. Dox-induced CD133 expression, in contrast, diminished apoptosis in T-treated cells, and exhibited elevated p-AKT, p-BAD, and decreased activation of BAX and caspases-3 and -9. This is the first time that CD133 is shown to activate a survival pathway wherein (1) CD133 increases AKT phosphorylation and activation, which induces (2) BAD phosphorylation and inactivation, and (3) reduces caspases-3 and -9 activity leading to apoptosis suppression, increased cell survival and drug resistance in melanoma. We investigated the effects of MEK inhibition with T in combination with the pan-AKT inhibitor capivasertib (AZD5363; C) in BAKP melanoma cells, with or without Dox-induced CD133 expression. Kinetic XTT cell viability and Annexin-Sytox Blue apoptosis assays revealed significantly reduced survival of BAKP cells exposed to a combination of T+C, compared to T alone. To determine the relative contribution of each of the three AKT genes to increased drug resistance of CD133-inducible melanoma stem cells, AKT 1, 2, and/or 3 were knocked out using sgRNAs specific to each of the three AKTs in a CRISPR-Cas9 lentiviral vector, followed by puromycin selection. Gene knockout was confirmed by immunoblot analysis using antibodies specific to each of the three AKTs, as well as by DNA sequence analysis. BAKP control or AKT KO cells were then exposed to T or T+C, and subjected to apoptosis assays, as well as immunoblot analysis with antibodies to apoptosis markers. Single KO of AKT 2 or 3, but not AKT 1, sensitized BAKP cells exposed to T alone, partially obviating the requirement for C to inhibit AKT. Triple KO of AKT 1, 2, and 3 rendered cells even more sensitive to T alone. Targeting nodes of the AKT and MAPK survival pathways with trametinib and AZD5363 highlights the potential for combination therapies for melanoma stem cells increasing the arsenal of more effective treatments for patients with high-risk melanoma. Targeting AKT 1, 2, and/or 3 might be effective in eliminating recalcitrant CD133-positive stem-like cells that may be responsible for tumor recurrence. Citation Format: Cynthia M. Simbulan-Rosenthal, Nusrat Islam, Ryyan Alobaidi, Alejandro Moreno, Veerupaxagouda Patil, Amal Alzharani, Peter Sykora, Dean S. Rosenthal. Effects of AKT1, 2, and 3 knock-out by CRISPR-Cas9 in CD133+human melanoma stem cells treated with MEK and AKT inhibitors [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 LB452.

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

Abstract Pancreatic cancer (PDAC) is a lethal disease in part because tumor cells exist in distinct transcriptional states (e.g. basal/mesenchymal v.s. classical/epithelial) with unique phenotypic properties that contribute to tumor growth and treatment resistance. Two major mechanisms have been suggested for treatment evasion: (1) the intrinsic resistance of an existing state to a therapy regimen and (2) plasticity of therapy-sensitive states to adopt more resistant states. The relative contribution of these mechanisms to treatment resistance is still poorly understood. Historically, measurement of plasticity in both human patients and mouse models has involved one of three principles: (1) observing a redistribution of cell states in tissue across timepoints or conditions; (2) identifying cells that have genomic, epigenetic or proteomic features of more than one state (mixed states); and (3) performing single-cell cloning of cells and observing the cell states adopted by clonal progeny. While these approaches are observationally consistent with the notion of plasticity, they either fail to definitively prove the existence of plasticity, are restricted in measurements of plasticity outside of native tissues or are unable to quantify the role of plasticity in treatment resistance. Amongst the most well described forms of plasticity in human development and cancer is epithelial-mesenchymal plasticity (EMP), which includes epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET). To better understand and quantify the role of EMP in driving treatment resistance of human PDAC, we have developed single-cell multiomic, functional genomic and computational methods applied to patient-derived models and clinical biopsies. We first profiled twelve patient-derived PDAC cell lines by single-cell RNA-seq (scSeq) and learned convergent epithelial and mesenchymal gene programs that were consistent with programs observed in patient samples. We next performed lineage tracing experiments in three PDAC cell lines using an expressed lentiviral barcoding system (ClonMapper). By performing scSeq on these barcoded lines at weekly timepoints over four weeks, we proved the presence of EMP by showing a single cell can produce progeny in both epithelial and mesenchymal states. We next developed a generative probabilistic model of our lineage tracing data. This demonstrated that clones (cells sharing a barcode) had different transition matrices (different EMT and MET rates), thus suggesting each clone has a distinct level of plasticity. Having established this, we focused on identifying genes that might explain the differing plasticity properties of clones. Using elastic net regression we identified 50 transcription factors (TFs) whose expression significantly explained the propensity for EMP over time across clonal populations. Among these were were several known EMP TFs (Zeb1 and Gata6), understudied TFs (Elf3, Sox2, Sox4, Klf3, Klf5 and Atf4) and novel TFs (Meis2, Meis3, FoxA1 and the interferon regulatory factors Irf6, Irf7 and Irf9). Using single-cell multiomics (paired scSeq and single-cell ATAC-seq) on our barcoded population, we found that 9 of the 50 predicted TFs, including Elf3, had differential accessibility between clones with different plasticity properties, suggesting a role for epigenetic regulation of these TFs in facilitating EMP. Importantly, we leveraged our multiomic data to infer gene regulatory networks influenced by these TFs and found an enrichment of binding motifs for these TFs in enhancer regions of genes in epithelial and mesenchymal programs. To study the role of these predicted TFs in modulating EMP, we developed a CRISPRi system that enabled gene perturbations alongside lineage tracing. We performed a CRISPRi perturb-seq experiment (CRISPR perturbation with scSeq readouts), perturbing the 50 predicted TFs above and 10 control genes, and collected 1.5 million single-cell transcriptomic profiles, in addition to two other CRISPR KO perturb-seq experiments. We performed a negative binomial regression to estimate effect sizes of guide RNAs on all genes. 60% of our guides had significant perturbation effects on their target gene. We subsequently found Klf3 as an important regulator of PDAC proliferation independent of cell state. Importantly, we found that knockdown (KD) of several factors, such has Grhl2 and FoxA1, bias towards mesenchymal cell states, whereas KD of others such as Batf2, Snai1, Rel, Zeb1, Nr2f1 and Sox4 led to a bias towards epithelial cell states. Interestingly, KD of several TFs influenced transition properties of cells by decreasing rates of EMT and MET across barcodes without biasing the overall clonal distribution towards a single cell state. This suggests a role for these TFs in enabling plasticity and facilitating state transitions. To study the effect of plasticity in treatment resistance, we treated four barcoded cell lines with the first-line chemotherapy combination FOLFIRINOX (5-fluorouracil, oxaliplatin and SN-38, the active metabolite of irinotecan) or targeted therapy and performed scSeq yielding over 600,00 single-cell transcriptomic profiles. We found an enrichment after treatment with FOLFIRINOX of barcodes that were biased for cells in mesenchymal states, consistent with selection against epithelial cells, but also of those barcodes with the highest inferred state transition rates. With targeted therapies, we found selective depletion of mesenchymal states. We next treated our CRISPR perturbed cell lines and found overall a significantly more restricted barcode diversity in cells containing guide RNAs targeting plasticity factors compared to non-targeting controls, suggestive of the role of plasticity in facilitation resistance. To validate the role of these proposed plasticity factors in human patients, we collected paired biopsy samples from 23 patients in a phase 2 clinical trial of metastatic PDAC patients being treated with radiation therapy and dual checkpoint blockade (NCT03104439). We performed scSeq on these samples, and used a supervised Bayesian matrix factorization approach (Spectra) to learn epithelial and mesenchymal gene programs within tumor cells. We subsequently classified cells as epithelial, mesenchymal and intermediate cell types using a gaussian mixture model on gene expression features. We found the intermediate states were enriched in expression of our proposed plasticity factors, and importantly high expression of these factors in baseline samples correlated with a redistribution of states in follow-up biopsies. Our efforts define a robust experimental and quantitative framework for studying tumor cell plasticity in patient-derived model systems with validation in human patient samples using single-cell and spatial transcriptomics. Collectively, we nominate several regulators that alter the propensity of EMP in PDAC, thus posing a paradigm whereby perturbations may be used to homogenize tumor populations towards treatment-sensitive phenotypes for combination therapy. Citation Format: Arnav Mehta, Lynn Bi, Deepika Yeramosu, Michael Bogaev, Martin Jankowiak, Abigail Collins, Aziz Al'Khafaji, Milan Parikh, Mehrtash Babadi, Kyle Evans, Alex Bloemendal, Russell Kunnes, Marc Schwartz, Glen Munson, Elisa Donnard, Thouis R. Jones, Ben Z. Stanger, Jay Shendure, Jonathan Weissman, David T. Ting, Andrew Aguirre, Nir Hacohen, Dana Pe'er, Eric S. Lander. Dissecting and quantifying pancreatic cancer plasticity using single-cell multiomics, lineage tracing and functional genomics reveals novel mediators of therapy resistance [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 NG08.

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 LB026: Exploring MCT4 inhibitor Ful-23 and cytarabine synergy in acute myeloid leukemia therapeutics

Abstract In the ongoing pursuit of addressing the formidable challenge presented by Acute Myeloid Leukemia (AML) treatment, this comprehensive study delves into the intricate antileukemic effects of Ful-23, our newly developed Monocarboxylate Transporter 4 (MCT4) inhibitor. Positioned as a promising candidate, Ful-23's unique mechanisms and potential synergies with established chemotherapeutic agents, such as Cytarabine, are elucidated in this research. Intracellular alkalinization stands as a crucial factor contributing to the evasion of chemotherapy by cancer cells. It serves as the primary determinant for the heightened resistance exhibited by cancer cells against chemotherapy interventions. The investigation reveals the multifaceted impact of Ful-23 on AML cells, showcasing its capacity to induce intracellular lactate accumulation and acidification. These metabolic alterations correlate with a substantial inhibition of proliferation, as evidenced by an IC50 of approximately 0.55 μM, and a marked induction of apoptosis, particularly notable in the MV411 AML cell line. These findings underscore Ful-23's potential as a potent antileukemic agent with distinct cellular effects. A collaborative approach involves the combination of low concentrations of Ful-23 (0.312 μM) with Cytarabine (0.312 μM), revealing a significant enhancement in cytarabine-induced proliferation inhibition and apoptosis when Ful-23 is introduced concurrently. Importantly, the translational impact of these findings extends to a zebrafish model, utilizing both MV411 cells and transplanted primary AML patient-derived bone marrow cells. In the zebrafish AML model, the combination treatment of Ful-23 and Cytarabine demonstrates a significant enhancement in their respective antitumor effects compared to monotherapy, indicating a pronounced synergistic effect. This additional validation strengthens the clinical relevance of the observed synergistic antileukemic interactions between Ful-23 and Cytarabine. Essentially, this investigation advances our understanding of AML treatment dynamics, emphasizing the crucial role of MCT4 inhibition and elucidating the mechanism by which it leads to intracellular acidification in synergistic therapeutic approaches for this intricate and formidable disease. Citation Format: Yong Wu, Ke Wu, Jay Vadgama. Exploring MCT4 inhibitor Ful-23 and cytarabine synergy in acute myeloid leukemia therapeutics [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 LB026.

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.

Autophagy dysregulation via the USP20-ULK1 axis in the HERC2-related neurodevelopmental disorder

Sequence variants in the HERC2 gene are associated with a significant reduction in HERC2 protein levels and cause a neurodevelopmental disorder known as the HERC2-related disorder, which shares clinical features with Angelman syndrome, including global developmental delay, intellectual disability, autism, and movement disorders. Remarkably, the HERC2 gene is commonly deleted in individuals with Angelman syndrome, suggesting a potential contribution of HERC2 to the pathophysiology of this disease. Given the known critical role of autophagy in brain development and its implication in neurodevelopmental diseases, we undertook different experimental approaches to monitor autophagy in fibroblasts derived from individuals affected by the HERC2-related disorder. Our findings reveal alterations in the levels of the autophagy-related protein LC3. Furthermore, experiments with lysosomal inhibitors provide confirmation of an upregulation of the autophagy pathway in these patient-derived cells. Mechanistically, we corroborate an interaction between HERC2 and the deubiquitylating enzyme USP20; and demonstrate that HERC2 deficiency leads to increased USP20 protein levels. Notably, USP20 upregulation correlates with enhanced stability of the autophagy initiating kinase ULK1, highlighting the role of HERC2 as an autophagy regulator factor through the USP20-ULK1 axis. Moreover, we show that p38 acts as a modulator of this pathway, since p38 activation disrupts HERC2-USP20 interaction, leading to increased USP20 and LC3-II protein levels. Together, these findings uncover a previously unknown role for HERC2 in autophagy regulation and provide insights into the pathomolecular mechanisms underlying the HERC2-related disorder and Angelman syndrome.