Loss Of Rb Research Articles

Osimertinib, platinum, etoposide as initial treatment for patients with EGFR mutant lung cancers with TP53 and RB1 alterations.

8565 Background: EGFR-mutant lung adenocarcinomas (EGFR+LC) transform to small cell lung cancer (SCLC) in up to 14% of cases, which creates an aggressive disease phenotype that is treated like de novo small lung cancer with platinum/etoposide chemotherapy. Transformed EGFR+LC molecularly mimic de novo small cell lung cancers and harbor TP53and RB1 loss. We hypothesized that if small cell directed chemotherapy is utilized prior to histologic transformation in EGFR/TP53/RB1 mutant LC, we may eradicate any pre-existing small cell clones, prevent small cell histologic transformation, and improve patient outcomes. Methods: We administered osimertinib, platinum, and etoposide in patients with newly diagnosed EGFR+LC with TP53and RB1alterations. Patients received 3 months of osimertinib induction, then 4 cycles of osimertinib, platinum and etoposide followed by osimertinib until progression. Objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety were assessed. Patients were required to have an on-treatment biopsy prior to initiation of chemotherapy upon which single cell RNA sequencing (scRNAseq) was performed. All patients had pre-treatment and post-progression biopsies that underwent next-generation sequencing. Patients were evaluated for acquired resistance (classified as on-target, off-target, transformation, and unknown). Among patients that transformed, markers of SCLC (ASCL1, NEUROD1, POU2F3, YAP1, synaptophysin, chromogranin, and Ki-67) were analyzed. Results: Eleven patients were enrolled [median age 58 years old range (49-81), 45% female, 73% EGFRexon 19 deletions and 27% EGFR L858R]. Three patients received cisplatin/etoposide, six patients carboplatin/etoposide, and two patients progressed two months on induction osimertinib before they could start chemotherapy. The ORR was 82%, median PFS 15.6 months and median OS was 37.9 months. All patients retained EGFR, TP53, and RB1mutations on post-treatment biopsy. Acquired on-target resistance mechanisms were identified in 9% of patients ( EGFR C797S), off-target in 9% of patients ( CDKN2A), and small cell transformation in 46% of patients. Among the transformed patients, all had high Ki-67 (70-95%) and 80% were positive for synaptophysin and chromogranin. 40% of cases were ASCL1-dominant, 40% were NEUROD1-dominant, and 20% were unknown. Additionally, we utilized copy number inference and CytoTrace to identify subclones within pre-transformed EGFR+LC that potentially harbor features of early plasticity. Further scRNAseq and whole exome sequencing data are forthcoming. Conclusions: In patients with EGFR+LCs with concurrent TP53and RB1alterations, osimertinib plus platinum and etoposide demonstrated activity and no new safety signals were seen. Early addition of small cell directed chemotherapy did not prevent histologic transformation. Clinical trial information: NCT03567642 .

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

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

Comprehensive genomic and transcriptomic characterization of high-grade gastro-entero-pancreatic neoplasms

BackgroundHigh-grade gastro-entero-pancreatic neoplasms (HG GEP-NENs) can be stratified according to their morphology and Ki-67 values into three prognostic classes: neuroendocrine tumors grade 3 (NETs G3), neuroendocrine carcinomas with Ki-67 < 55% (NECs <55) and NECs with Ki-67 ≥ 55% (NECs ≥55).MethodsWe analyzed a cohort of 49 HG GEP-NENs by targeted Next-Generation Sequencing (TrueSight Oncology 500), RNA-seq, and immunohistochemistry for p53, Rb1, SSTR-2A, and PD-L1.ResultsFrequent genomic alterations affected TP53 (26%), APC (20%), KRAS and MEN1 (both 11%) genes. NET G3 were enriched in MEN1 (p = 0.02) mutations, while both NECs groups were enriched in TP53 (p = 0.001), APC (p = 0.002) and KRAS (p = 0.02) mutations and tumors with TMB ≥ 10 muts/Mb (p = 0.01). No differentially expressed (DE) gene was found between NECs <55% and NECs ≥55%, while 1129 DE genes were identified between NET G3 and NECs. A slight enrichment of CD4+ and CD8+ T cells in NECs and of cancer-associated fibroblasts and macrophages (M2-like) in NET G3. Multivariate analysis identified histologic type and Rb1 loss as independent prognostic factors for overall survival.ConclusionsThis study showed that GEP-NET G3 and GEP-NECs exhibit clear genomic and transcriptomic differences, differently from GEP-NECs <55% and GEP-NECs ≥55%, and provided molecular findings with prognostic and potentially predictive value.

Reproducible preclinical models of androgen receptor driven human prostate cancer bone metastasis.

Preclinical models recapitulating the metastatic phenotypes are essential for developing the next-generation therapies for metastatic prostate cancer (mPC). We aimed to establish a cohort of clinically relevant mPC models, particularly androgen receptor positive (AR+) bone metastasis models, from LuCaP patient-derived xenografts (PDX) that reflect the heterogeneity and complexity of mPC. PDX tumors were dissociated into single cells, modified to express luciferase, and were inoculated into NSG mice via intracardiac injection. The progression of metastases was monitored by bioluminescent imaging. Histological phenotypes of metastases were characterized by immunohistochemistry and immunofluorescence staining. Castration responses were further investigated in two AR-positive models. Our PDX-derived metastasis (PDM) model collection comprises three AR+ adenocarcinomas (ARPC) and one AR- neuroendocrine carcinoma (NEPC). All ARPC models developed bone metastases with either an osteoblastic, osteolytic, or mixed phenotype, while the NEPC model mainly developed brain metastasis. Different mechanisms of castration resistance were observed in two AR+ PDM models with distinct genotypes, such as combined loss of TP53 and RB1 in one model and expression of AR splice variant 7 (AR-V7) expression in another model. Intriguingly, the castration-resistant tumors displayed inter- and intra-tumor as well as organ-specific heterogeneity in lineage specification. Genetically diverse PDM models provide a clinically relevant system for biomarker identification and personalized medicine in metastatic castration-resistant prostate cancer.

Heterozygous RB1 mutation enhanced ATP production in human iPSC-derived retinal organoids.

Recent in vitro studies using RB1+/- fibroblasts and MSCs have shown molecular and functional disruptions without the need for biallelic loss of RB1. However, this was not reflected in the recent in vitro studies employing RB1+/- retinal organoids. To gain further insights into the molecular disruptions in the RB1+/- retinal organoids, we performed a high throughput RNA sequencing analysis. iPSCs were generated from RB1+/+ and RB1+/- OAMSCs derived from retinoblastoma patients. RB1+/+ and RB1+/- iPSCs were subjected to a step-wise retinal differentiation protocol. Retinal differentiation was evaluated by Real-time PCR and flow cytometry analysis of the retinal markers. To gain further insights into the molecular differences in RB1+/- retinal organoids, a high throughput RNA sequencing followed by differential gene expression analysis and gene set enrichment analysis (GSEA) was performed. The analysis revealed a shift from the regular metabolic process of glycolysis to oxidative phosphorylation in the RB1+/- retinal organoids. To investigate further, we performed assays to determine the levels of pyruvate, lactate and ATP in the retinal organoids. The results revealed significant increase in ATP and pyruvate levels in RB1+/- retinal organoids of day 120 compared to that of the RB1+/+. The results thus revealed enhanced ATP production in the RB1+/- retinal organoids. The study provides novel insights into the metabolic phenotype of heterozygous RB1 mutant suggesting dysregulation of energy metabolism and glycolytic pathways to be first step even before the changes in cellular proliferation or other phenotypic consequences ensue.

Abstract PO2-16-02: RBsig gene-expression signature in patients with endocrine resistant metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA trial

Abstract Background: We have previously identified RBsig, a gene-expression signature of resistance to CDK4/6 inhibitors composed of E2F1/E2F2 dependent genes, which is associated with genetic loss of RB1. Although RBsig has been previously tested in vitro and in clinical trials with CDK4/6 inhibitors, further clinical validation is needed. Additionally, data on the correlation of RBsig with other prognostic/predictive biomarkers for CDK4/6 inhibitors such as serum Thymidine Kinase activity (sTKa), is lacking. Here, we aimed to investigate RBsig in patients (pts) treated with palbociclib (P) + fulvestrant (F) within the PYTHIA trial (IBCSG 53- 14/BIG 14-04; NCT02536742), a downstream trial of the AURORA molecular program (BIG 14-01; NCT02102165). Methods: PYTHIA is a biomarker discovery phase II trial which enrolled 122 pts with endocrine-resistant ER+ and HER2 - metastatic breast cancer (MBC) who received P+F at standard schedule and dose. We analyzed a subset of 38 pts enrolled in PYTHIA and successfully included in AURORA who had available RNAseq from tumor tissue samples (24 primary, 24 metastatic, 10 both). Ki67 by IHC was also available. The PYTHIA biomarker assessment included sTKa at baseline and during treatment (D15 and D28) measured using DiviTumÒ TKa (Biovica International). Evaluation of the prognostic role of RBsig was a pre-specified primary objective of PYTHIA. RBsig is composed of 87 genes and was calculated as previously described. Wilcoxon rank sum test was used to compare continuous RBsig values between groups defined by sTKa. Median progression free survival (mPFS) was estimated using Kaplan-Meier method for groups defined by dichotomizing RBsig at zero. Results: Median RBsig was higher in metastatic than in primary samples; in paired samples, RBsig increased in metastatic for 7/10 pts vs primary. When evaluated as continuous variables, RBsig correlated with tissue Ki67 both in primary (Spearman R 0.8) and in metastatic (R 0.68) samples. Additionally, RBsig was higher in Basal and Luminal B intrinsic subtypes in metastatic samples. RBsig was significantly higher in pts with high baseline sTKa (median cut-off) (Wilcoxon test, p=0.04 and p=0.03 in primary and metastatic samples respectively), with a similar direction, albeit not statistically significant, observed in the group of pts with no sTKa clearance at D15 (below limit of detection cut-off). When evaluated in the combined cohort, where RBSig values were taken from metastatic samples when available and from primary samples otherwise, higher RBsig values were associated with poorer PFS as compared to lower RBsig values [mPFS 5.5 months (95% CI 3.7, 14) vs 13 months (95% CI 8.6, 28); Log-rank p-value=0.004]. Conclusions: Higher RBsig was associated with higher tumor proliferation (measured by both tissue ki67 and sTKa), luminal B status, and shorter PFS on treatment with P+F. This analysis reinforces prior data on RBsig prognostic significance in pts treated with CDK4/6 inhibitors. Further investigation on the added value of RBsig as a novel biomarker for patients’ stratification, including in the early setting, is warranted. Citation Format: Matteo Benelli, Svitlana Tyekucheva, Thayane Crestani, Michail Ignatiadis, Patrick Neven, Marco Colleoni, Stéphanie Henry, Konstantinos Papadimitriou, Antonio Bernardo, Elena Seles, François Duhoux, Iain Macpherson, Alastair Thomson, David Mark Davies, Ilenia Migliaccio, Gabriele Zoppoli, Roswitha Kammler, Heidi De Swert, Barbara Ruepp, Philippe Aftimos, Jose Seoane, Dario Romagnoli, David Venet, Theodora Goulioti, David Cameron, Sherene Loi, Martine Piccart, Meredith Regan, Luca Malorni. RBsig gene-expression signature in patients with endocrine resistant metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA trial [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 PO2-16-02.

Abstract PO2-27-05: Composite biomarkers for the prediction of progression-free survival with CDK4/6 inhibitors in metastatic HR+/HER2- breast cancer

Abstract CDK4/6 inhibitors are widely used in combination with endocrine therapy in the treatment of HR+/HER2- metastatic breast cancer. Generally the CDK4/6 inhibitors lead to a doubling of progression-free survival (PFS) over single agent endocrine therapy. In spite of a known mechanism of action, biomarkers to define the duration of response to CDK4/6 inhibitors with either aromatase inhibitors (AI) or fulvestrant (FUL) remain lacking. A cohort of over 300 patients receiving standard of care CDK4/6 inhibitor combination therapy for metastatic disease were consented for participation (NCT04526587). The duration of PFS in both AI and FUL treated sub-groups were comparable to that observed in randomized clinical trials. In this cohort progesterone receptor-status (HR 0.70, p=0.0123), visceral disease (HR 1.55, p=0.0013), prior endocrine therapy (HR 2.34, p&amp;lt; 0.001), and type of endocrine therapy (HR 2.16, p&amp;lt; 0.001) were associated with duration of PFS. To develop biomarkers, gene expression analyses was performed on 313 tumor samples representing over 200 individual patients. Focusing on the pre-treatment metastatic biopsy, established signatures associated with prognosis in HR+/HER2- breast cancer were employed to determine association with response to combination therapy in the metastatic setting. Proliferation-associated signatures were associated with shorter PFS. For example, an established RB loss signature was associated with PFS in both AI (HR 1.9, p=0.009) and FUL (HR 2.8, p=0.007) treated patients. Clinically employed signatures (e.g. OncotypeDx and Mammaprint) harbored variable associations with outcomes. Random Survival Forest feature selection was applied to individual and combined prognostic signatures. This approach resulted in a potent classifier for progression-free survival (HR 10, p&amp;lt; 1E-20). To enable applicability to different manifestations of disease, significant clinical pathological variables were incorporated into the model so that there was equivalent performance in AI and FUL treated patients for prediction of PFS. This predictive algorithm was also strongly associated with overall survival (HR 4.6, p=2.7E-8). Ongoing studies are evaluating the algorithms prospectively in NCT04526587. Citation Format: Agnieszka Witkiewicz, Jianxin Wang, Erik Knudsen, Ellis Levine, Tracey O'Connor. Composite biomarkers for the prediction of progression-free survival with CDK4/6 inhibitors in metastatic HR+/HER2- 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 PO2-27-05.

Pathogenic Mechanisms Activated by NSD2 Translocations in Multiple Myeloma

The NSD2 gene is overexpressed in multiple cancers and is associated with cancer cell proliferation, invasion, and metastasis. NSD2 gene is universally present in multiple myeloma (MM) patients with t(4;14) (p16;q32) translocation, a high-risk subtype. NSD2 is characterized by different breakpoints. NSD2Δ3 breakpoint is associated with shorter survival than other breakpoints. NSD2Δ3 contains non-coding RNA ACA11 within intron 18. Additionally, 80% of t(4;14) MM patients lack the retinoblastoma tumor suppressor gene (RB1). We hypothesize that ACA11 cooperates with RB1 loss in MM pathogenesis. Also, we hypothesize that NSD2Δ3 increases protein expression driving myeloma growth. To overexpress NSD2WT, snoACA11, and NSD2Δ3 in cells, we cloned NSD2WT, and NSD2Δ3 plasmid constructs in a murine stem cell virus (MSCV)-IRES- GFP vector. We isolated primary B cells from RB1 wild-type and knock-out mice by CD43 microbeads and transduced those cells using a virus containing ACA11 and NSD2WT. ACA11 overexpression significantly increased B cell proliferation in Rb1dKO mice. This hyperproliferative phenotype is induced by reactive oxygen species (ROS) levels. However, we did not find a further increase in B cell proliferation with co-overexpression of NSD2WT and ACA11 in Rb1dKO mice. We demonstrated increased protein stability by finding higher NSD2Δ3 protein expression compared to NSD2WT protein expression in HEK293T cells. These results support our hypothesis and demonstrate oncogenic role of ACA11 in t(4;14) MM. Future work will be done to determine the cell biological effects of NSD2Δ3 and establish its role in myeloma pathogenesis. Completion of this study will advance understanding of this epigenetic NSD2 gene and potentially develop targeted drugs for NSD2, providing more individualized treatment options for these patients. University of Iowa Department of Internal Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Therapeutic Strategies for RB1-Deficient Cancers: Intersecting Gene Regulation and Targeted Therapy.

The retinoblastoma (RB) transcriptional corepressor 1 (RB1) is a critical tumor suppressor gene, governing diverse cellular processes implicated in cancer biology. Dysregulation or deletion in RB1 contributes to the development and progression of various cancers, making it a prime target for therapeutic intervention. RB1's canonical function in cell cycle control and DNA repair mechanisms underscores its significance in restraining aberrant cell growth and maintaining genomic stability. Understanding the complex interplay between RB1 and cellular pathways is beneficial to fully elucidate its tumor-suppressive role across different cancer types and for therapeutic development. As a result, investigating vulnerabilities arising from RB1 deletion-associated mechanisms offers promising avenues for targeted therapy. Recently, several findings highlighted multiple methods as a promising strategy for combating tumor growth driven by RB1 loss, offering potential clinical benefits in various cancer types. This review summarizes the multifaceted role of RB1 in cancer biology and its implications for targeted therapy.

Molecular characterization and survival analysis of a cohort of glioblastoma, IDH-wildtype

Glioblastoma, IDH-wild type, the most common malignant primary central nervous system tumor, represents a formidable challenge in clinical management due to its poor prognosis and limited therapeutic responses. With an evolving understanding of its underlying biology, there is an urgent need to identify prognostic molecular groups that can be subject to targeted therapy. This study established a cohort of 124 sequential glioblastomas from a tertiary hospital and aimed to find correlations between molecular features and survival outcomes. Comprehensive molecular characterization of the cohort revealed prevalent alterations as previously described, such as TERT promoter mutations and involvement of the PI3K-Akt-mTOR, CK4/6-CDKN2A/B-RB1, and p14ARF-MDM2-MDM4-p53 pathways. MGMT promoter methylation is a significant predictor of improved overall survival, aligned with previous data. Conversely, age showed a marginal association with higher mortality. Multivariate analysis to account for the effect of MGMT promoter methylation and age showed that, in contrast to other published series, this cohort demonstrated improved survival for tumors harboring PTEN mutations, and that there was no observed difference for most other molecular alterations, including EGFR amplification, RB1 loss, or the coexistence of EGFR amplification and deletion/exon skipping (EGFRvIII). Despite limitations in sample size, this study contributes data to the molecular landscape of glioblastomas, prompting further investigations to examine these findings more closely in larger cohorts.

An E7-retinoblastoma protein pathway mechanism may account for the higher carcinogenic ability of HPV16 over HPV58 in cervical cancer.

Among human papillomavirus (HPV) type, HPV16 displays the strongest carcinogenic capacity for cervical cancer, but the mechanism underlying this phenomenon remains unclear. We investigated the effect and the underlying mechanism of HPV16 on higher carcinogenic capacity than HPV58. We collected 4,030 cervical exfoliated cell samples for genotyping HPV using HybriBio's proprietary flow-through hybridization technique, liquid-based cytology (LBC), colposcopy, and biopsies if indicated. Four plasmids containing E6 and E7 of HPV16 and 58 were constructed and transfected into 293T and U2OS cells. We detected the cell phenotype using Cell Counting Kit 8 (CCK8) assay, Transwell assay, flow cytometry, and apoptosis assay; the expression of retinoblastoma protein (Rb) and phosphorylated Rb (pRb) was determined via Western blot; and the cell activity was determined via a zebrafish model treated with or without roscovitine. The positive rates of HPV16 and 58 were, respectively, 18.9% and 19.7% in the ≤ low-grade squamous intraepithelial lesion (LSIL) group, 49.5% and 19.6% (P<0.001) in the high-grade squamous intraepithelial lesion (HSIL) group, 65.3% and 9.0% (P<0.001) in the cancer group. In vitro, both 293T and U2OS cells with overexpressed HPV16 E6 and E7 displayed significantly higher cell proliferation, faster cell invasion, decreased cell apoptosis, and accelerated cell cycle from G1 phase to S phase compared to those with overexpressed HPV58 E6 and E7 (all P values <0.05). Rb loss of function was observed in cells with HPV16 E7 overexpression, while a greater level of phosphorylated Rb was observed in cells with HPV58 E7 overexpression. Roscovitine restored Rb expression and decreased the cell activity in zebrafish. HPV16 possesses a stronger carcinogenic ability than does HPV 58, and the mechanism underlying this effect may be the impairment of the E7-Rb pathway.

Abstract 5719: Targeting CDK4/6 and BET proteins for the treatment of RB+ and RB- osteosarcoma

Abstract Osteosarcoma (OS) is the most common malignant bone cancer characterized by heterogeneity and genomic instability. Treatment and survival of patients have not improved in several decades, making it urgent to develop safe and effective therapies. Dysregulation of cell cycle regulators CDKN2A, cyclins, and CDK4/6 represent high-risk signatures in aggressive OS and can be targeted via CDK4/6 inhibitors (CDK4/6i). However, monotherapy often fails due to resistance from speculated RB loss as well as hyperactivation of compensatory signaling pathways in RB proficient cancers. In addition, &amp;gt;70% of OS patients have RB loss which is viewed as a biomarker of therapeutic response that negates the use of CDK4/6i. However, more research is needed in RB-deficient preclinical models to clearly define and validate the utility of RB as a biomarker for patient stratification. Based on our screening data, we hypothesize that combining CDK4/6i with bromodomain and extraterminal domain inhibitors (BETi) will potentiate anti-tumorigenic effects regardless of RB status in OS. BETi creates imbalances between transcription and replication which worsens oncogenic stress. In OS cell lines, combination index and Bliss analysis indicated additive-to-synergistic growth inhibition using CDK4/6i (abemaciclib or palbociclib) and BETi (AZD5153). Dual therapy also resulted in increased apoptosis at clinically relevant concentrations in an OS xenoline (TT2) compared to monotherapy. To determine potential for emergence of therapeutic resistance, an in-vitro longitudinal growth delay assay was established. While single agent therapies resulted in transient decreases in cell numbers over time, combination CDK4/6i+BETi resulted in cell death (p&amp;lt;0.05). To determine the effect of RB loss on therapeutic response to CDK4/6i, CRISPR mediated RB-knockout (KO) clones were developed in 143B human and K7M2 murine OS cell lines. RB KO cells exhibited sensitivity to CDK4/6i irrespective of RB status. However, levels of CDK4/6i required for 50% growth inhibition in RB KO were 1-3-fold higher using palbociclib, with negligible differences observed with abemaciclib in RB KO vs WT clones. To determine if therapeutic response is achievable in-vivo, a single-agent dosing study was conducted using palbociclib (40mg/kg, SID) and AZD5153 (1mg/kg, BID) in patient-derived xenograft (PDX) model derived from a metastatic lesion in the pelvis (TT2 PDX) harboring a CDK4/6 hyperactivation signature and monoallelic RB loss. Tumor growth reduction and survival was significantly higher in monotherapy groups compared to vehicle. However, one week after dosing was stopped, survival rates based on tumor volume endpoint were 40% and 100% for palbociclib and AZD5153 respectively. In-vivo efficacy of dual CDK46i+BETi is underway. This data provides rationale for further study of novel therapeutic options which may expand the clinical utility of CDK4/6i regardless of RB status. Citation Format: Rada Malko, Pankita H. Pandya, Harlan E. Shannon, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Farinaz Barghi, Nikan Riyahi, Ryli Justice, Melissa A. Trowbridge, Kathryn L. Coy, Anthony L. Sinn, Khadijeh Bijangi-Vishehsaraei, Micheal J. Ferguson, M. Reza Saadatzadeh, Karen E. Pollok. Targeting CDK4/6 and BET proteins for the treatment of RB+ and RB- osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5719.

Abstract 940: Integrated analysis of cfDNA fragmentomics, DNA methylation and cfRNA transcription in metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background Liquid biopsies based on cell-free DNA (cfDNA) analysis provide non-invasive clinical diagnostic insights. Recently, cfDNA fragmentomics has emerged as a tool for inferring epigenomic and transcriptional information from tumor-derived cfDNA. We conducted a comprehensive profiling of cfDNA fragmentomics, whole transcriptome sequencing (WTS), and genome-wide DNA methylation on two metastatic castration-resistant prostate cancer (mCRPC) samples to systematically investigate molecular aberrations in mCRPC. Methods Two mCRPC patient plasma samples were extensively profiled using high-depth 150x whole-genome sequencing (WGS), 30x whole-genome methylation, and cfRNA WTS. CfDNA fragmentomics analysis was performed to assess androgen receptor (AR) transcriptional activity. We calculated fragment coverage around AR binding sites (ARBS) and quantified ARBS nucleosome profiling abnormality scores (ARBS scores) by comparing centric fragment coverage with normal plasma background. Additionally, we calculated fragment size entropy around AR promoter and enhancer regions to reflect AR gene expression activity. Fragmentomics-inferred gene activity was validated through promoter-targeted panels and WTS profiling of matched mCRPC samples. Results Both mCRPC plasma samples exhibited significant ARBS nucleosome depletion. Sample A showed a higher ARBS score compared to Sample B. Interestingly, AR enhancer activity inferred from high-depth WGS and targeted panels was high in both samples, while AR promoter activity was high only in Sample A. Genome-wide DNA methylation revealed hypo-methylation in AR enhancer and ARBS regions in both samples, with Sample A showing more pronounced hypo-methylation. Copy number variation analysis indicated AR amplification in Sample A and RB1 loss in Sample B. Collectively, these findings suggest that Sample A may represent androgen receptor-dependent prostate cancer (ARPC), while Sample B may have developed resistance to androgen therapy, possibly indicating neuroendocrine prostate cancer (NEPC). Conclusions This study, to our knowledge, is the first to combine high-depth WGS, promoter-targeted panels, WTS, and genome-wide DNA methylation to systematically study epigenomic and transcriptional dysregulation in mCRPC. The integration of mutation, copy number variation, fragmentomics, and DNA methylation profiling holds potential clinical utility for inferring prostate cancer subtypes, facilitating patient stratification, and guiding treatment selection. Citation Format: Chao Dai, Lisha Zhu, Yong Huang, Giancarlo Bonora, Kemin Zhou, Shidong Jia, Pan Du. Integrated analysis of cfDNA fragmentomics, DNA methylation and cfRNA transcription in metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 940.

Abstract 948: A novel combinatorial therapy for lethal neuroendocrine prostate cancer

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer that can arise de novo, but more commonly develops after hormone therapies for advanced prostate adenocarcinoma. Current treatment options for NEPC are only palliative, and most patients die within several months. Additionally, single-agent clinical trials targeting NEPC so far have only produced disappointing results, highlighting the clear need to develop effective combinatorial therapies for NEPC. RB1 loss, a pivotal event in the development of NEPC, can sensitize cancer cells to ferroptosis in multiple cancer cell types, including prostate cancer. Here, we aim to identify an optimal combinatorial therapy for NEPC based on targeting ferroptosis. Methods: We determined the cell-killing efficacy of ferroptosis inducers and several current and emerging treatment regimens for NEPC as single agents, then performed drug synergism analyses of the above two potent treatments. Lastly, we investigated the molecular mechanisms of drug synergism by examining the major types of cell death induced by combinatorial treatment through molecular and biochemical assays. Results: Our results demonstrated that both ferroptosis inducers and BCL2 inhibitors as single agents led to robust cell death in NEPC cell lines tested. In contrast, cisplatin, the standard of care for NEPC, and Aurora kinase A inhibitor showed modest to no cell-killing effect. Furthermore, suboptimal doses of ferroptosis inducers and BCL-2 inhibitors synergistically induced cell death in NEPC cell lines. Unexpectedly, we found that low-dose ferroptosis inducer led to increased production of mitochondria ROS, which in turn exacerbated BCL-2 suppression-induced apoptosis. Conclusions: Our findings reveal a novel combinatorial therapy for NEPC. Based on our in vitro data, we will further test the in vivo therapeutic efficacy of ferroptosis inducers combined with BCL-2 inhibitors against NEPC growth. Citation Format: Mu-En Wang, Wei-Ling Tu, Yi Lu, Jinjin Wu, Alyssa Bawcom, Andrew J. Armstrong, Qianben Wang, Yuzhuo Wang, Jiaoti Huang, Ming Chen. A novel combinatorial therapy for lethal neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 948.

Abstract 5897: Understanding and targeting fatty acid CoA ligase ACSL4 in human prostate cancer

Abstract Prostate cancer (PCa) is the most common non-cutaneous malignancy among men. Although localized prostate cancer is curable, recurrence of metastatic castration-resistant prostate cancer (mCRPC) is almost inevitable and is the major cause of death in human PCa. Recent findings from our lab and others demonstrate that RB1 loss drives lineage plasticity, metastasis, and lethality of prostate tumors initiated by PTEN loss. A downstream target upregulated by RB1 loss is acyl CoA synthetase long chain family member 4 (ACSL4), a fatty acid ligase with an essential role in utilization of long chain fatty acids. ACSL4 has been reported to be upregulated in cancers of various histological origins, including prostate cancer, and its high expression is correlated with aggressiveness of cancer. We hypothesize that ACSL4 is a critical downstream effector of RB1 loss-driven prostate tumorigenesis and pharmacological inhibition of ACSL4 could represent a novel therapeutic approach to the treatment of lethal RB1 deficient or ACSL4 overexpressing prostate cancer. Here we aim to develop novel ACSL4 inhibitors for preclinical studies. Through a structure based virtual screening that targeted ATP binding domains of ACSL4, we successfully identified 15 potential ACSL4 inhibitors. To validate the result, we performed an in vitro screening using various parental and isogenic human PCa cell lines, and found that 4 out of 15 compounds effectively inhibited PCa cell proliferation in a dose-response manner and their inhibitory effects positively correlated with cellular levels of ACSL4. Next, we will perform isotope-labeling enzyme activity assays to determine the specificity of the 4 candidate drugs, and PDPK analyses followed by characterizing their in vivo efficacies in preclinical studies, with a final goal to develop a well-validated ACSL4 inhibitor. Successful completion of this work will provide great insights into contributions of ACSL4 to prostate tumorigenesis as well as preclinical data of ACSL4 inhibitors to treat lethal prostate cancer. Citation Format: Jinjin Wu, Mu-En Wang, Ming Chen, Pyeong Hwa Jeong, Jiyong Hong, Lixin Wan. Understanding and targeting fatty acid CoA ligase ACSL4 in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5897.

Abstract 6947: Exploring the single-cell dynamics of FOXM1 under cell cycle perturbations

Abstract The cell cycle plays a critical role in maintaining normal cellular functions and preventing abnormal cell growth. The transcription factor FOXM1 has emerged as a crucial regulator of cell cycle progression and has been implicated in various physiological and pathological processes. Notably, FOXM1 overexpression has been observed in numerous cancer types, including liver, prostate, breast, lung, and colon cancer. Despite its significance, our understanding of how FOXM1 dynamic behaves at the single-cell level under different cell cycle perturbations and its link to cell behavior heterogeneity remains limited. Here, we investigated the dynamic behavior of FOXM1 in individual cells exposed to various cell cycle perturbations, focusing on understanding heterogeneity in cell behavior and improving disease treatment. At the single-cell level, our investigation reveals six distinct dynamic features of FOXM1 in response to various therapeutic interventions, each of which corresponds to a unique cell phenotype. Our result further demonstrates that for cells exhibiting a high FOXM1 pattern, targeting aurora kinase and PLK1 may hold promise as a therapeutic approach for cancer suggesting that FOXM1 is a strong biomarker, and its pattern can be predictive of drug response, especially in cases with G1 checkpoint defects like Rb1 loss or p53 deficiency. Citation Format: Tooba Jawwad, Maliwan Kamkaew, Kriengkrai Phongkitkarun, Porncheera Chusorn, Somponnat Sampattavanich. Exploring the single-cell dynamics of FOXM1 under cell cycle perturbations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6947.

Abstract 5708: Silence exportin 7 sensitizes esophageal squamous cell cancer to CDK4/6 inhibition through block the cell cycle coresectory

Abstract Introduction: There is a lack of effective target therapies for esophageal squamous cell cancer. Our previous studies have demonstrated that CDK4/6 is a potency target for ESCC and combination with ERBB inhibitors is sufficient to achieve durable response. The aim of the current study is to prospectively map the landscape of sensitivity and resistance to CDK4/6 inhibition and develop novel combination therapies in ESCC. Experimental Procedures: We performed a genome-wide functional genetic screening and mini-pool designed CRISPR screen approaches to identify the modulators whose depletion sensitize CDK4/6 inhibition or confer resistance to the CDK4/6 inhibitor Palbociclib and elucidate new therapeutic strategies. Results: Our screen identified RB as the most enriched sgRNA, while guides targeting CCNE1 and CDK2 as the most depletion sgRNAs after CDK4/6 inhibition. These results validates our experimental approach as RB loss and CCNE1/CDK2 reactivation is the main mechanisms of CDK4/6 resistance. After gathering potential candidates from both screens, we found that genetically ablation of Exportin 7, a nuclear export factor, strongly sensitize CDK4/6 inhibition both in vitro and in vivo. RNAseq revealed a further repression of transcriptional activity of E2F and G2M by addition of XPO7depletion under CDK4/6 inhibitor treatment. To increase our insight in the molecular mechanism that could explain the synergy between XPO7 and CDK4/6, we further performed proteomics of nuclear and cytoplasm in ESCC lines with or without CDK4/6 inhibition. We found that silence XPO7 result in nuclear accumulation of mitosis-regulated genes such as CENPM, KIF4A and WEE1. Combined CDK4/6 inhibition with XPO7 depletion results in further downregulate of CENPM and KIF4A expression in nucleus, abrogating compensatory activation of cell cycle regulation and promote the cytostatic effect in ESCC. We further validated that CENPM and KIF4A depletion also sensitize CDK4/6 inhibition, and consequentially use WEE1 inhibitor after CDK4/6 inhibitor also effectively block the growth of ESCC lines. Conclusion: Overall, our comprehensive functional genetic screening approach revealed modulation of sensitive and resistance to the inhibition of CDK4/6 in ESCC. XPO7 was identified as a driver to change cell cycle landscape and merits as a therapeutic target combined with CDK4/6 inhibition in ESCC, which is beneficial to provide a novel treatment strategy. Citation Format: Zhong Wu, Lin Xu, Chuang Jiang, Adam Bass, Jin Zhou. Silence exportin 7 sensitizes esophageal squamous cell cancer to CDK4/6 inhibition through block the cell cycle coresectory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5708.

Abstract 6925: Integrative single-cell tracking of genome evolution and tumor cell plasticity in small cell lung cancer (SCLC)

Abstract SCLC is the deadliest type of lung cancer, which is mostly diagnosed at late stages and accounts for 15% of pulmonary tumors. Even though chemotherapy is initially effective, patients with SCLC relapse quickly and develop resistant tumors. Genome studies revealed a universal loss of TP53 and RB1; recent transcriptional studies categorized SCLC based on the expression of 4 lineage transcription factors (TFs). We aimed to dissect how genomic cues impact transcriptional phenotypes, tumor cell plasticity and the dynamics of tumor and immune cell interactions, to thus decipher molecular mechanisms of phenotypic divergence and therapy resistance in SCLC. We performed genome sequencing and single cell transcriptome profiling of 55 tumors from patients with SCLC, including primary tumors, local and distant metastases and of paired relapsing tumors acquired after chemotherapy. Single cell sequencing was performed with the 10x Genomics platform and yielded &amp;gt; 200.000 cells. Transcriptome profiles at single cell levels revealed co-expression of at least two of the lineage TFs ASCL1, NEUROD1 or POU2F3 in each tumor. Copy number (cn) estimation for transcriptome data pointed to genomic events impacting transcriptional heterogeneity within the tumor. Furthermore, our data pointed to a marked intra- and interpatient, as well as to inter-metastatic heterogeneity. To specifically investigate how underlying genetic alterations affect molecular phenotypes, integrative studies across all patients were performed. This revealed shared transcriptional programs in tumors with amplifications of MYC family members; trajectory inference for cases with low and high-level copy gains of MYCN pointed to distinct transcriptional states. We performed multi-regional and longitudinal studies of matched patient cases to reconstruct the genomic patterns of clonal evolution, and projected genomic subsets to transcriptional profiles at single cell level, thus analyzing the effect of genome diversity on the transcriptional landscape. Additionally, tumor intrinsic profiles of heterogeneity were mapped to single cell data of patient tumors to elucidate the effects on tumor-immune cell interactions. All together our data points to a concerted regulation of a multitude of lineage factors and transcriptional programs in each SCLC tumor and provides a first comprehensive framework for the study of underlying genetic alterations that shape the transcriptional landscape and phenotypic plasticity in SCLC. Citation Format: Marcel Schmiel, Laura Kaiser, Maria Cartolano, Martin Peifer, Roman K. Thomas, Julie George. Integrative single-cell tracking of genome evolution and tumor cell plasticity in small cell lung cancer (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6925.

Abstract 6941: Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA)

Abstract Retinoblastoma is a pediatric retinal cancer that initiates in response to biallelic RB1 loss or MYCN amplification in a cone precursor cell of origin. After initial emergence, RB1-/- retinoblastomas progress to more aggressive forms by accumulation of single nucleotide variants (SNVs) and highly recurrent somatic copy number alterations including 1q+, 2p+, 6p+ and 16q- (RB SCNAs), resulting in heterogeneous mixtures of subclonal cell populations. SCNAs are enriched in highly evolved and aggressive tumor subtypes and show prognostic value in retinoblastoma liquid biopsies. However, the transcriptomic effects of retinoblastoma SCNAs are poorly understood. Single cell methods may enable comparison of tumor subclones within emerging retinoblastoma tumors and thereby provide insight into tumor progression. We retrieved single cell RNA sequencing data for 25 retinoblastoma tumors and inferred SCNAs and subclone phylogenies from scRNAseq information using a recently published tool, Numbat. We identified tumors with clonal variation in RB SCNAs and excluded samples with poor transcriptome quality or excessive additional SCNAs. We proceeded with analysis of 11 tumors with subclonal heterogeneity for an SCNA of interest, including 10 with 1q+, eight with 2p+, four with 6p+, and six with 16q-. After removal of nontumor cells, we defined cell clusters, cluster marker genes, and cell cycle phase gene signatures, and we compared cell state distributions for subclones with an acquired SCNA relative to their immediate antecedents. These analyses revealed recurrently discriminated transcriptomic clusters corresponding to six discrete cell cycle states (post-mitotic G1, G1, G1/S, S, S/G2, G2/M) and variable cell stress states, including a cluster with hypoxia and heat shock related marker gene expression in all samples. In each tumor, SCNA-defined tumor subclones had a distinct cell state distribution. Tumor subclones with acquired 1q+ were enriched in G2 states and depleted in G1 states. Subclones with 2p+ were enriched in G2 states. Subclones with 6p+ were enriched in S phase states. Subclones with 16q- were diminished in post-mitotic cell states. Tumor clones with 1q+ and 16q- were most strongly enriched for G2 phase cells. Such 1q+/16q- tumor cells were significantly differentially distributed in three tumor replicates. Tumor clones with other RB SCNAs were enriched over diploid clones in proliferating G2 cells without SCNA specific effects. To identify candidate drivers of SCNA-specific transcriptomic effects we measured RB SCNA clonal variation in gene expression within and across transcriptomic states. We identified recurrently differentially expressed in-segment genes in 1q+, 2p+, 6p+, and 16q-, as well as out-of-segment genes. Finally, we propose molecular mechanisms underlying SCNA recurrence that may enable tumor evolution in retinoblastoma. Citation Format: Kevin Stachelek, David E. Cobrinik. Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6941.

Abstract 554: Determining the role of PROX1 in prostate cancer neuroendocrine trans-differentiation

Abstract Nonbiased mutational screening to identify essential genes and pathways in cancer is a powerful approach for understanding underlying biology as well as the identification of potential therapeutic targets. Epigenetic (EPI) and transcriptional factors (TF) play an important role in the change of phenotype and lineage plasticity from adenocarcinoma to castration resistant prostate cancer (CRPC), double negative (DN) or treatment-induced neuroendocrine prostate cancer (NEPC). When RB1 loss occurs in combination with TP53 alterations, stemness and lineage plasticity increase with one consequence being differentiation to a neuroendocrine (NE) lineage. Here, we are identifying and testing the functional role of epigenetic and transcription factors required for the viability of CRPC, DN and NEPC relative to a matrix of genotypic and phenotypic characteristics. We applied CRISPR-CAS9 dropout screens in prostate organoids and cell lines to identify gene/s associated with CRPC, DN and NEPC viability. We successfully CRISPR-engineered LNCaP clones of single (LNCaP-TP53-SKO, LNCaP-RB1-SKO) and double (LNCaP-TP53-RB1-DKO) gene knockouts, which were subsequently characterized for immuno-phenotype and transcriptome. Our dropout screens, targeting all TFs and EPIs, identified 117 TF and 40 EPI genes responsible for the viability of DN, NEPC and LNCaP models. We found known genes such as FOXA1 and ASCL1, as well as novel genes including PROX1 and KLF3, which were validated through guide RNA competition assays. Our results showed PROX1 is essential for cell viability in a DN organoid model, and its expression is associated with increased aggressiveness, indicating PROX1 has role in stemness/lineage plasticity in treatment resistant adenocarcinoma. RNA-seq analysis following overexpression of PROX1 in the LNCaP-TP53-RB1 null adenocarcinoma model, with and without enzalutamide, identified pathways enriched for cell proliferation, differentiation, neuronal development, and prostate gland development. In summary, our study has suggested an important role for PROX1 in the growth and differentiation of poorly differentiated prostate cancer, which may lead to the identification of potential therapeutic targets. Citation Format: Sonam Raj, Brian Capaldo, Joel Bowman, Margaret White, Xavier Moore, Berkley Gryder, Aian N. Alilin, David Takeda, Kathleen Kelly. Determining the role of PROX1 in prostate cancer neuroendocrine trans-differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 554.