Rb Function Research Articles

Retinoblastoma Expression and Targeting by CDK4/6 Inhibitors in Small Cell Lung Cancer.

The canonical model of "small cell lung cancer" (SCLC) depicts tumors arising from dual inactivation of TP53 and RB1. However, many genomic studies have persistently identified tumors with no RB1 mutations. Here, we examined RB1 protein expression and function in SCLC. RB1 expression was examined by IHC analysis of 62 human SCLC tumors. These studies showed that ∼14% of SCLC tumors expressed abundant RB1 protein, which is associated with neuroendocrine gene expression and is enriched in YAP1 expression, but no other lineage proteins that stratify SCLC. SCLC cells and xenograft tumors with RB1 protein expression were sensitive to growth inhibition by the CDK4/6 inhibitor palbociclib, and this inhibition was shown to be dependent on RB1 expression by CRISPR knockout. Furthermore, a patient with biopsy-validated wild-type RB1 SCLC who received the CDK4/6 inhibitor abemaciclib demonstrated a dramatic decrease in mutant TP53 ctDNA allelic fraction from 62.1% to 0.4% and decreased tumor mass on CT scans. Importantly, IHC of the diagnostic biopsy specimen showed RB1 positivity. Finally, we identified a transcriptomics-based RB1 loss-of-function signature that discriminates between SCLC cells with or without RB1 protein expression and validated it in the patient who was responsive to abemaciclib, suggesting its potential use to predict CDK4/6 inhibitor response in patients with SCLC. Our study demonstrates that RB1 protein is an actionable target in a subgroup of SCLC, a cancer that exhibits no currently targetable mutations.

The Cyclin-G Associated Kinase (GAK) Is a Critical Dependency for Cell-Cycle Progression in Diffuse Large B-Cell Lymphoma Associated with Deregulation of the Retinoblastoma Tumor Suppressor

Background: Recurrent genomic alterations in specific cancers reveal tumor drivers that inform the development of rational therapeutic approaches. In diffuse large B-cell lymphoma (DLBCL), such oncoprotein-focused drug development has achieved only limited success. Phenotypic screening, less explored in DLBCL drug discovery, is an emerging alternative that can be leveraged especially through the application of machine-learning analyses to raw screening data. Under this paradigm, we reveal strong therapeutic potential in DLBCL of inhibiting the cyclin-G associated kinase (GAK). GAK participates in clathrin-mediated endocytosis and regulates mitotic-spindle alignment during cell division. It has been studied as an antiviral target that interrupts internalization at cell membranes but is minimally explored in cancer therapeutics and is a novel target in DLBCL. Methods and Results: We performed a first-of-its-kind screen of kinase inhibitors in DLBCL followed by machine learning-based analysis using our in-house target identification platform, idTRAX. The screen revealed GAK as a strong target for both activated B-cell (ABC) and germinal center B-cell (GCB) derived cell lines with complete sparing of peripheral-blood mononuclear cells (PBMCs), included as non-malignant controls. We analyzed gene-expression data available for 414 previously untreated DLBCL patients (Lenz cohort) and found tumors with high GAK expression had dramatically worse overall survival (OS) compared to those with low expression (hazard ratio=1.84 (95% conf. int. 1.33-2.53), p=0.0002). Mechanistically, we explored GAK's role in endocytosis but found no change in cell-surface B-cell receptor (BCR) expression after treatment with the GAK tool compound SGC-GAK-1. In addition, there was no evident perturbation of signaling pathways downstream of BCR by immunoblotting. We found instead that GAK inhibition in DLBCL results in profound disruption of progression through mitosis, potently triggering the spindle assembly checkpoint (SAC) and causing accumulation in G2 before onset of apoptosis. We explored whether GAK dependency is induced due to cell cycle deregulation. Analysis of gene-expression data in the Lenz cohort showed a striking correlation between high GAK expression and low expression of RB1, encoding the tumor suppressive master cell-cycle regulator retinoblastoma-associated protein (RB, p<0.0001). The ABC-DLBCL cell lines RIVA and U2932 completely lack RB expression and show high sensitivity and accumulation in G2 in response to SGC-GAK-1 treatment. Immunofluorescent confocal microscopy revealed profound disruption of mitotic spindles and shattering of chromosomes in these cells and, to a lesser extent, in RB-competent lines. SGC-GAK-1 is not optimized for use as a chemical probe in vivo, but target validation through shRNA knockdown in DLBCL patient-derived xenograft (PDX) models is underway and expected at time of presentation. Conclusions: GAK is a novel kinase therapeutic target in DLBCL revealed by our target identification platform, idTRAX. GAK's role in mitotic-spindle alignment represents a critical kinase dependency for DLBCL tumors, in particular those with cell-cycle deregulation due to loss of RB function. Our ongoing efforts to synthesize novel GAK inhibitors and reveal synergistic targets may reveal new therapeutic strategies for translation to clinical trials for DLBCL patients with unmet clinical needs.

CSIG-33. BCOR LOSS PROMOTES RETINOBLASTOMA GROWTH AND DISSEMINATION BY MODULATING INSULIN-LIKE GROWTH FACTOR 2

Abstract While loss of RB function is the most common genetic alteration in the pediatric embryonal eye tumor retinoblastoma, mutations leading to BCL-6 corepressor (BCOR) loss of function are the second most common alteration. BCOR mutations are found in more aggressive tumors, and are associated with metastasis and poor prognosis. We investigated the effects of BCOR loss or overexpression in retinoblastoma cells in order to better understand its functional role, and to determine if it represents a new therapeutic target in these tumors. Six established retinoblastoma cell lines were examined, and BCOR protein was most highly expressed in WERI-RB1, but absent in Y79 and two other lines. BCOR knock down in WERI-RB1 cells using shRNA caused markedly increased migration and proliferation. BCOR was also knocked out in these cells using CRSPR, resulting in more than three-fold increased size of intraocular murine xenografts and extensive extraocular extension of tumors. In terms of mechanism, BCOR loss of function induces IGF2 transcription, resulting in an activation of IGF1R and the ERK signaling pathway. In contrast, overexpressing BCOR in Y79 retinoblastoma cells with no baseline protein had opposite effects on IGF2. Targeting IGF2/IGF1R signaling pharmacologically using Lincitinib inhibited proliferation and migration in BCOR-negative retinoblastoma cells. Increased IGF2 transcription was regulated at least in part by hyper methylation of CCCTC-binding factor (CTCF)-binding regions in the IGF2/H19 imprinting control region (ICR), suggesting that BCOR knock down promotes increased methylation at this site. In summary, our studies revealed that BCOR loss promotes the growth and invasion of retinoblastoma cells in vitro and in vivo, at least in part via modulating IGF2 transcription, suggesting new potential therapeutic targets for these aggressive childhood tumors of the eye.

Rb1 negatively regulates bone formation and remodeling through inhibiting transcriptional regulation of YAP in Glut1 and OPG expression and glucose metabolism in male mice

ObjectiveBone is a highly dynamic organ that undergoes constant bone formation and remodeling, and glucose as a major nutrient is necessary for bone formation and remodeling. Retinoblastoma (Rb1) is a critical regulator of mesenchymal stem cells (MSCs) fate, but how Rb1 regulates bone formation and remodeling is poorly understood. MethodsWe generated MSCs- and osteoprogenitors-specific Rb1 knockout mouse models and utilized these models to explore the function and mechanism of Rb1 in regulating bone formation and remodeling in vivo and in vitro primary cell culture. ResultsRb1 deficiency in MSCs significantly increased bone mass and impaired osteoclastogenesis. Consistently, depletion of Rb1 in osteoprogenitors significantly promoted bone formation. Mechanistically, loss of Rb1 in MSCs elevated YAP nuclear translocation and transcriptional activity of YAP/TEAD1 complex, thereby increasing the transcriptional expression of Glut1 and OPG. Moreover Prx1-Cre; Rb1f/f mice displayed hypoglycemia with increased systemic glucose tolerance instead of increased insulin level. In vitro data revealed that Rb1-mutant MSCs enhanced glucose uptake and lactate and ATP production. Increased osteogenesis caused by increased glucose metabolism and decreased osteoclastogenesis caused by increased expression of OPG eventually resulted in increased bone formation and remodeling. ConclusionsCollectively, these findings demonstrated that Rb1 in MSCs inhibits YAP-medicated Glut1 and OPG expression to control glucose metabolism, osteogenesis and osteoclastogenesis during bone formation and remodeling, which provide new insights that controlling Rb1 signaling may be a potential strategy for osteopetrosis.

Conjugate natural convection flow of a nanofluid with oxytactic bacteria under the effect of a periodic magnetic field

Conjugate natural convection flow in a cavity is numerically investigated under the effect of a periodic magnetic field. The cavity has a vertical thermally conducting solid block and a part where the nanofluid flows. The most left wall of the cavity is the hot one and the most right one is the cold one. The problem is considered as two-dimensional and time independent, and a vertical periodic magnetic field is taken into account. Nanoparticle is chosen as SiO2 and the host fluid is water. Inside of the nanofluid, the existence of oxytactic microorganisms is also assumed. Brownian motion and thermophoresis effects are also considered in governing equations. The influence of pertinent parameters biconvection Rayleigh number Rb=1–100, Lewis number Le=1–20, Peclet number Pe=0.1–5, Brownian motion parameter Nb=0.1–1, thermophoresis parameter Nt=0.1–1, Hartmann number Ha=0–100, period of periodic magnetic field λ=0.1–1, amplitude of periodic magnetic field A=0.1–1 and conductivity ratio kr=0.1–10 are examined on fluid flow, heat and mass transfer. Numerical results in view of isolines as well as average Nusselt and Sherwood numbers are presented. Conductivity ratio kr=10 has more impact on convective heat and mass transfer. Convective mass transfer depends on the vertical wall considered as solid–fluid interface or the right cold vertical wall. It is an increasing function of Le, Pe, Ha, Nt and Nb and decreasing function of Rb in interface.

Enhancements of Second Harmonic Generation and Birefringence in Traditional RbTiOPO4‐Type Crystals by Aliovalent Substitution of Ti Site

AbstractStrong second harmonic response and large birefringence of nonlinear optical (NLO) materials have important scientific and technical significance for tunable lasers. The second harmonic generation (SHG) response in NLO crystals has been generally enhanced by improving the polarization of bridged oxygen atoms or local structural distortions, while the synergy of the two contributions to enhance the SHG response is rarely explored. To this end, new crystals with traditional RbTiOPO4 (RTP)‐structure, Rb1–xTi1–xSbxOPO4 (x = 0.01, 0.03, and 0.06), containing novel functional SbO6 octahedra and Rb vacancies are designed through a one‐site aliovalent substitution and grown in this work. Rb0.94Ti0.94Sb0.06OPO4 exhibits an enhanced SHG response, nearly 1.48 times that of the RTP crystal, accompanied by the largest birefringence in phosphates (exp. 0.202 at 546 nm, cal. 0.156 at 1064 nm). Theoretical calculations and crystal structure analysis confirm that the higher polarizability of bridged oxygen and the local structural distortions in Rb1–xTi1–xSbxOPO4 strengthen the NLO activity of neighboring units, thus significantly prompt the SHG effect and optical anisotropy. This study provides a new paradigm to deeply improve the NLO performance of crystal materials and benefits for expanding their practical applications.

Lack of Retinoblastoma Protein Shifts Tumor Metabolism from Glycolysis to OXPHOS and Allows the Use of Alternate Fuels.

Mutations in the RB1 locus leading to a loss of functional Rb protein cause intraocular tumors, which uniquely affect children worldwide. These tumors demonstrate rapid proliferation, which has recently been shown to be associated with an altered metabolic signature. We found that retinoblastoma tumors and in-vitro models lack Hexokinase 1 (HK1) and exhibit elevated fatty acid oxidation. We show that ectopic expression of RB1 induces HK1 protein in Rb null cells, and both RB1 and HK1 can mediate a metabolic switch from OXPHOS to glycolysis with increased pyruvate levels, reduced ATP production and reduced mitochondrial mass. Further, cells lacking Rb or HK1 can flexibly utilize glutamine and fatty acids to enhance oxidative phosphorylation-dependent ATP generation, as revealed by metabolic and biochemical assays. Thus, loss of Rb and HK1 in retinoblastoma reprograms tumor metabolic circuits to enhance the glucose-independent TCA (tricarboxylic acid) cycle and the intermediate NAD+/NADH ratios, with a subsequent increase in fatty-acid derived L-carnitine to enhance mitochondrial OXPHOS for ATP production instead of glycolysis dependence. We also demonstrate that modulation of the Rb-regulated transcription factor E2F2 does not result in any of these metabolic perturbations. In conclusion, we demonstrate RB1 or HK1 as critical regulators of the cellular bioenergetic profile and identify the altered tumor metabolism as a potential therapeutic target for cancers lacking functional Rb protein.

Korean Red Ginseng and Rb1 facilitate remyelination after cuprizone diet-induced demyelination

BackgroundDemyelination has been observed in neurological disorders, motivating researchers to search for components for enhancing remyelination. Previously we found that Rb1, a major ginsenoside in Korean Red Ginseng (KRG), enhances myelin formation. However, it has not been studied whether Rb1 or KRG function in remyelination after demyelination in vivo. MethodsMice were fed 0.2% cuprizone-containing chow for 5 weeks and returned to normal chow with daily oral injection of vehicle, KRG, or Rb1 for 3 weeks. Brain sections were stained with luxol fast blue (LFB) staining or immunohistochemistry. Primary oligodendrocyte or astrocyte cultures were subject to normal or stress condition with KRG or Rb1 treatment to measure gene expressions of myelin, endoplasmic reticulum (ER) stress, antioxidants and leukemia inhibitory factor (LIF). ResultsCompared to the vehicle, KRG or Rb1 increased myelin levels at week 6.5 but not 8, when measured by the LFB+ or GST-pi+ area within the corpus callosum. The levels of oligodendrocyte precursor cells, astrocytes, and microglia were high at week 5, and reduced afterwards but not changed by KRG or Rb1. In primary oligodendrocyte cultures, KRG or Rb1 increased expression of myelin genes, ER stress markers, and antioxidants. Interestingly, under cuprizone treatment, elevated ER stress markers were counteracted by KRG or Rb1. Under rotenone treatment, reduced myelin gene expressions were recovered by Rb1. In primary astrocyte cultures, KRG or Rb1 decreased LIF expression. ConclusionKRG and Rb1 may improve myelin regeneration during the remyelination phase in vivo, potentially by directly promoting myelin gene expression.

Combined TRIP13 and Aurora Kinase Inhibition Induces Apoptosis in Human Papillomavirus-Driven Cancers.

Human papillomavirus (HPV) causes >5% of cancers, but no therapies uniquely target HPV-driven cancers. We tested the cytotoxic effect of 864 drugs in 16 HPV-positive and 17 HPV-negative human squamous cancer cell lines. We confirmed apoptosis in vitro and in vivo using patient-derived xenografts. Mitotic pathway components were manipulated with drugs, knockdown, and overexpression. Aurora kinase inhibitors were more effective in vitro and in vivo in HPV-positive than in HPV-negative models. We hypothesized that the mechanism of sensitivity involves retinoblastoma (Rb) expression because the viral oncoprotein E7 leads to Rb protein degradation, and basal Rb protein expression correlates with Aurora inhibition-induced apoptosis. Manipulating Rb directly, or by inducing E7 expression, altered cells' sensitivity to Aurora kinase inhibitors. Rb affects expression of the mitotic checkpoint genes MAD2L1 and BUB1B, which we found to be highly expressed in HPV-positive patient tumors. Knockdown of MAD2L1 or BUB1B reduced Aurora kinase inhibition-induced apoptosis, whereas depletion of the MAD2L1 regulator TRIP13 enhanced it. TRIP13 is a potentially druggable AAA-ATPase. Combining Aurora kinase inhibition with TRIP13 depletion led to extensive apoptosis in HPV-positive cancer cells but not in HPV-negative cancer cells. Our data support a model in which HPV-positive cancer cells maintain a balance of MAD2L1 and TRIP13 to allow mitotic exit and survival in the absence of Rb. Because it does not affect cells with intact Rb function, this novel combination may have a wide therapeutic window, enabling the effective treatment of Rb-deficient cancers.

Ginsenoside Rb1 from Panax ginseng attenuates monoiodoacetate-induced osteoarthritis by inhibiting miR-21-5p/FGF18-mediated inflammation.

Ginsenoside Rb1 (Rb1) is a major active compound in Panax ginseng and has shown considerable anti-inflammation effects. Osteoarthritis (OA) is one of the major degenerative disorders affecting the knee. MiR-21-5p is a potential therapeutic target for OA treatment. This study explored the anti-OA effects of Rb1 by focusing on its interaction with the miR-21-5p/FGF18 axis. OA was induced in rats using monoiodoacetate (MIA) and managed with Rb1. Then, changes in the histological structure and miR-21-5p-mediated signaling pathway were measured in joint tissues. The role of miR-21-5p/FGF18 in the anti-OA effects of Rb1 was confirmed by inducing its levels in rats and chondrocytes. Rb1 improved the histological structure and suppressed the production of cytokines in joint tissues. At the molecular level, Rb1 down-regulated miR-12-5p levels and up-regulated FGF18 levels. In chondrocytes, Rb1 increased cell viability, suppressed inflammation, down-regulated miR-21-5p levels, and up-regulated FGF18 levels. The restored level of miR-21-5p compromised the anti-OA effects of Rb1. In a nutshell, our study reported that the anti-OA effects of Rb1 relied on the inhibited expression of miR-21-5p. PRACTICAL APPLICATIONS: Ginsenoside Rb1 (Rb1) is a major active compound in Panax ginseng and has shown considerable anti-osteoarthritis (OA) effects. The current study not only relates the anti-OA function of ginsenoside Rb1 with microRNA but also provides valuable information for exploring novel targets for the development the anti-OA strategies.

MYCN induces cell-specific tumorigenic growth in RB1-proficient human retinal organoid and chicken retina models of retinoblastoma

Retinoblastoma is a rare, intraocular paediatric cancer that originates in the neural retina and is most frequently caused by bi-allelic loss of RB1 gene function. Other oncogenic mutations, such as amplification and increased expression of the MYCN gene, have been found even with proficient RB1 function. In this study, we investigated whether MYCN over-expression can drive carcinogenesis independently of RB1 loss-of-function mutations. The aim was to elucidate the events that result in carcinogenesis and identify the cancer cell-of-origin. We used the chicken retina, a well-established model for studying retinal neurogenesis, and established human embryonic stem cell-derived retinal organoids as model systems. We over-expressed MYCN by electroporation of piggyBac genome-integrating expression vectors. We found that over-expression of MYCN induced tumorigenic growth with high frequency in RB1-proficient chicken retinas and human organoids. In both systems, the tumorigenic cells expressed markers for undifferentiated cone photoreceptor/horizontal cell progenitors. The over-expression resulted in metastatic retinoblastoma within 7–9 weeks in chicken. Cells expressing MYCN could be grown in vitro and, when orthotopically injected, formed tumours that infiltrated the sclera and optic nerve and expressed markers for cone progenitors. Investigation of the tumour cell phenotype determined that the potential for neoplastic growth was embryonic stage-dependent and featured a cell-specific resistance to apoptosis in the cone/horizontal cell lineage, but not in ganglion or amacrine cells. We conclude that MYCN over-expression is sufficient to drive tumorigenesis and that a cell-specific resistance to apoptosis in the cone/horizontal cell lineage mediates the cancer phenotype.

Abstract 1268: Early proliferative response via [18]FLT-PET/CT and serum TK1 activity are predictive biomarkers for synergistic effect of sequential cell cycle targeted therapy in sarcoma

Abstract Introduction: Sarcomas are rare, aggressive tumors that make up a higher percentage of overall cancer mortality in children and young adults. Primary management of sarcoma patients includes surgical resection with adjuvant or neoadjuvant radiation/chemotherapy. Even with aggressive multimodal treatment regimens, patients with high-grade sarcomas experience suboptimal prognosis and advanced disease is almost universally fatal. Innovative biomarker driven targeted therapies are in urgent need to improve survival in these patients. Discoveries in molecular and genetic basis of sarcoma have revealed new targets that deserve consideration. Inactivation of retinoblastoma (Rb) pathway, which causes unabated cell proliferation, through overexpression of cyclin D1 and/or CDK4/6 has been detected in the majority of sarcomas even when wild-type Rb protein is expressed. Hence, inhibition of cell cycle progression through reactivation of the tumor suppressor function of Rb by inhibiting CDK4/6 activity poses a potential avenue for new therapeutic options. Methods: A panel of 4 sarcoma cell lines and 4 patient derived xenograft (PDX) models were used: 2 with an intact Rb pathway (Rb+ve, p16-ve) and 2 with an altered Rb pathway (Rb low/-ve, p16+ve). CDK4/6 inhibitor, abemaciclib, was used to reactivate the Rb pathway followed by treatment with gemcitabine or WEE1-kinase-inhibitor, to target the enriched S and G2/M phase cells post-abemaciclib removal. 18F-FLT PET imaging and serum thymidine kinase 1 (TK1) were used to monitor changes in cell proliferation and treatment response in both in vitro and in vivo. Changes in cell cycle biomarkers in response to treatment were also interrogated. Results: Rb+ve sarcoma cell lines were sensitive to the anti-proliferative activity of abemaciclib leading to their reversible G1 arrest, while Rb null cells did not arrest in any phase of cell cycle in response to CDK4/6 inhibition. Following abemciclib removal, Rb+ve models (but not Rb-ve) showed significant increase in 18F-FLT biotracer uptake concomitant with elevated serum TK1 and enrichment of cells in S phase. The enrichment of cells in S phase maximized therapeutic efficacy of S-phase or G2/M targeting agents leading to synergistic cell killing in Rb+ve cell lines and PDX models. Conclusion: Our study presents an innovative treatment strategy inhibiting the Rb pathway followed by S or G2/M pathway sequentially resulting in synergistic cell killing in those cells/tumors with wild-type Rb. We also incorporate real-time PET imaging and serum TK1 as non-invasive indicators of response to the treatment strategy. The study with PDX models also identified biomarkers of cell proliferation to be used in conjunction with PET imaging to provide the pre-clinical rationale to translate our findings into clinical trials for this aggressive disease. Citation Format: Tuyen Duong Nguyen, Marc A. Pina, Yan Wang, Cansu Karakas, Tuyen N. Bui, Osama Mawlawi, Alexander J. Lazar, Davis R. Ingram, Peter J. Hougton, Kelly K. Hunt, Khandan Keyomarsi. Early proliferative response via [18]FLT-PET/CT and serum TK1 activity are predictive biomarkers for synergistic effect of sequential cell cycle targeted therapy in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1268.

HPV E7 oncogene expression impairs Rb function and confers CDK4/6 inhibitor resistance in cervical cancer.

e17504 Background: Malignant tumor proliferation is one of the important factors for poor prognosis. CDK4/6 inhibitors can delay tumor progression by inhibiting cell cycle and inducing apoptosis, so they are potentially effective broad-spectrum anti-tumor targeted drugs, but drug resistance limits their clinical applications. Our previous research found that the CDK4/6 inhibitor LEE011 has anti-tumor effects on cervical cancer C33A cells, but no anti-tumor activity against HeLa. The mechanism is not very clear, we speculate that it is related to HPV infection. In this research, our aim is to clarify the correlation between HPV and tumor drug sensitivity and explore the possible mechanisms. Methods: Two HPV positive human cervical cancer cell lines, SiHa and Caski were chosen to verify the sensitivity to LEE011 by cell morphology, cell cycle and apoptosis. The antitumor activity of CDK4/6 inhibitor LEE011 by E7 knockin C33A and E7 knock-down HeLa cell lines was assessed by in vitro clonogenic assay, flow cytometry, and target inhibition verified by immunoblotting. Effects of LEE011 and E7 knock-down combination in vivo were studied by xenograft tumor regrowth delay, and tissue section immunohistochemistry. Results: SiHa and Caski were insensitive to LEE011 antitumor activity. Enhancement of drugsensitivity was lost in cell lines with HPV positive. After down-regulation of E7, LEE011 treatment increased the inhibition cell proliferation and pro-apoptosis of HeLa. Mechanistically, the loss of E7-induced Rb inactivation and LEE011 inhibited Rb phosphorylation, leading to cell-cycle arrest. In vivo, LEE011 inhibited tumor regrowth, with sustained inhibition of cyclin D-CDK4/6-Rb-E2F1 activity in E7 knock-down HeLa. However, up-regulation of E7 can reduce the ability of LEE011 to inhibit cell proliferation and pro-apoptosis of C33A. In summary, our study signifies inhibiting the CDK4/6 pathway by LEE011 in combination with down-regulation of E7 as a promising therapeutic strategy to treat cervical cancer. Conclusions: These findings suggested that HPV can affect Rb function by expressing oncoprotein E7 leading to resistance to targeted drug therapy, which means that HPV may be a biomarkers of drug therapy efficacy.

Understanding Retinoblastoma Post-Translational Regulation for the Design of Targeted Cancer Therapies.

Simple SummaryRb1 is a regulator of cell cycle progression and genomic stability. This review focuses on post-translational modifications, their effect on Rb1 interactors, and their role in intracellular signaling in the context of cancer development. Finally, we highlight potential approaches to harness these post-translational modifications to design novel effective anticancer therapies.The retinoblastoma protein (Rb1) is a prototypical tumor suppressor protein whose role was described more than 40 years ago. Together with p107 (also known as RBL1) and p130 (also known as RBL2), the Rb1 belongs to a family of structurally and functionally similar proteins that inhibits cell cycle progression. Given the central role of Rb1 in regulating proliferation, its expression or function is altered in most types of cancer. One of the mechanisms underlying Rb-mediated cell cycle inhibition is the binding and repression of E2F transcription factors, and these processes are dependent on Rb1 phosphorylation status. However, recent work shows that Rb1 is a convergent point of many pathways and thus the regulation of its function through post-translational modifications is more complex than initially expected. Moreover, depending on the context, downstream signaling can be both E2F-dependent and -independent. This review seeks to summarize the most recent research on Rb1 function and regulation and discuss potential avenues for the design of novel cancer therapies.

Abstract P005: Establishment and characterization of a panel of advanced prostate cancer patient-derived xenograft (PDX) models for cancer therapeutic evaluation

Abstract Background: Prostate cancer is one of the most frequently diagnosed solid malignancies and is accountable for the second highest cancer-related deaths in males worldwide. Despite the success of targeting the androgen receptor signaling pathway with androgen deprivation therapy (ADT) and androgen receptor blockage (ARB), relapse and resistance to treatment leads to castration resistant prostate cancer (CRPC) or metastatic CRPC (mCRPC), resulting in poor clinical prognosis and therapeutic outcome. Prostate cancer is a highly heterogeneous disease, with different molecular pathways playing a role in the advanced disease, such as androgen receptor mutations, DNA repair gene deletions, P53 aberrations, RB1 function loss, as well as gene rearrangements, like TMPRSS2-ERG2. Preclinical models representing CRPC heterogeneity and clinically relevant responses to standard of care treatments are needed to better understand the biology and mechanisms of this malignancy. METHODS: CRPC PDX models derived from metastases of prostate cancer patients, which failed anti-androgen therapies, were established via subcutaneous engraftment in immunodeficient mice. Model classification and characterization were performed by histopathology and immunohistochemistry (IHC) with prostate-specific biomarkers. Genomic profiling of these models was performed by next generation sequencing (NGS). Chemotherapies such as docetaxel and target therapies, including anti-androgen, were evaluated in these prostate models. Tumor volume over time was used to determine growth rate and response to treatment in uncastrated and castrated mice. RESULTS: A series of CRPC PDX models derived from metastatic lesions in patients, including metastases from lymph nodes, bladder, ascites and rib/soft bone, were established and characterized. Histopathology by H&amp;E staining presented distinct features of adenocarcinoma and the immunohistochemistry data indicated various expression levels of prostate-specific markers, such as prostate-specific antigen (PSA) and androgen receptor (AR). Most of the models showed loss of PTEN expression. Sequencing data also detected different gene aberrations such as TMPRSS2-ERG fusion, TP53 deletion and mutations, RB1 mutations, and AR alterations. In vivo modeling of these models presented tumor progression under castration. Docetaxel and enzalutamide, commonly used in mCRPC patients, presented varied responses. CONCLUSIONS: Prostate cancer presents high heterogenicity in clinical and molecular features, and treatment response. A panel of mCRPC PDX models have been established and characterized to provide clinically relevant preclinical models for prostate cancer therapeutic evaluation. Citation Format: Jessie Jingjing Wang, Leilei Chen, Xueying Yang, Likun Zhang, Wubin Qian, Henry Qixiang Li. Establishment and characterization of a panel of advanced prostate cancer patient-derived xenograft (PDX) models for cancer therapeutic evaluation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P005.

Abemaciclib is synergistic with doxorubicin in osteosarcoma pre-clinical models via inhibition of CDK4/6-Cyclin D-Rb pathway.

Osteosarcoma is a prevalent type of bone tumor in children and adolescents, with limited treatment and poor prognosis. Abemaciclib, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), is approved for the treatment of advanced breast cancer as single agent therapy and is currently under investigation in clinical trials for the treatment of several solid tumors. The efficacy of abemaciclib was determined using osteosarcoma cellular assays and xenograft mouse model. The combination studies were performed based on the Chou-Talalay method. Immunoblotting analysis was performed to determine the underlying mechanisms of abemaciclib in osteosarcoma cell lines. Abemaciclib potently inhibits growth, anchorage-independent colony formation and survival of a panel of osteosarcoma cell lines, with IC50 range from 90 nM to >20 μM. In addition, the combination of abemaciclib and doxorubicin is synergistic and antagonistic in abemaciclib-sensitive (IC50<1 μM) and abemaciclib-resistant (IC50 >1 μM), respectively. Abemaciclib inhibits tumor formation and growth in a dose-dependent manner without causing significant drug toxicity in mice. The combination of abemaciclib and doxorubicin results in much greater efficacy than doxorubicin alone in inhibiting tumor growth throughout the whole treatment duration. Abemaciclib acts on osteosarcoma via suppressing CDK4/6-Cyclin D-Rb pathway. Our pre-clinical evidence provides a rationale of initializing clinical trial of investigating the efficacy of abemaciclib in combination with doxorubicin in osteosarcoma patients. Our work also highlights the therapeutic value of CDK4/6 inhibition in osteosarcoma with proper function of Rb.

In This Issue

In This Issue

Molecular alterations in retinoblastoma beyond RB1

Retinoblastoma is the most common malignant ocular tumor in children. Although RB1 alterations are most frequently involved in the etiology of retinoblastoma, candidate driver events and somatic alterations leading to cell transformation, tumor onset and progression remain poorly understood. In this study, we identified novel genomic alterations in tumors with a panel of 160 genes. Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) were initially performed for identifying patients without apparent RB1 alterations in blood DNA. Subsequently, NGS analyses of 24 paired (blood/tumor) samples of these patients were carried out for identifying somatic mutations and copy number variation in RB1 and other 159 genes. One novel pathogenic RB1 mutation and seven novel VUS were identified as well as 90 novel pathogenic mutations in 61 other genes. Twenty-three genes appeared exclusively mutated in tumors without altered RB1 alleles and three frequently affected biological pathways while five other tumors did not show pathogenic RB1 alterations or SNV/indels in 159 other genes. Curiously, deletion of GATA2, AKT1, ARID1A, DNMT3A, MAP2K2, MEN1, MTOR, PTCH1 and SUFU (in homo- or heterozygosity) were exclusively found in these tumors when compared to those with any pathogenic alterations, probably indicating genes that might be essential for the development of retinoblastoma regardless of a functional RB1. Identification of genes associated with retinoblastoma will contribute to understanding presently unknown aspects of this malignancy, which might be essential for its initiation and progression, as well as providing valuable molecular markers.

Abstract 1022: Characterization of lurbinectedin as a single agent and in combinations with DNA damage response inhibitor for the treatment and bio-marker discovery of SCLC

Abstract Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung malignancy which is primarily driven by loss of function of tumor suppressor gene TP53 and RB1 and accounts for around 13-15% of all lung cancers. For metastatic SCLC the standard-of-care, first-line therapy is combination of platinum-based therapy with etoposide or with irinotecan. Though response rate in first-line chemotherapy in SCLC is very high, relapse is almost universal. For decades topotecan, a topoisomerase-I inhibitor was the only FDA approved second-line treatment in SCLC. However, on June 15th, 2020 the FDA approved lurbinectedin for treatment of patients with metastatic SCLC with disease progression from platinum-based chemotherapy and designated it as an orphan drug. Lurbinectedin (PM01183) is a synthetic analog of the natural marine-based tetrahydroisoquinoline, trabectedin which comes from the sea-squirt species Ecteinascidia turbinate. Lurbinectedin blocks transcription by inhibiting the activity of RNA-polymerase-II and inducing its specific degradation by the ubiquitin/proteasome machinery, also inducing DNA damage. Emerging data from our group, and others supports that SCLC is transcriptionally addicted via one of three main transcription factors ASCL1 (A), NeuroD1 (N) &amp; POU2F3 (P), which may contribute to the promising results observed in SCLC trials to date with lurbinectedin. However, there are currently no established biomarkers to predict SCLC sensitivity or resistance to lurbinectedin. Furthermore, little is known regarding molecular changes in SCLC cells or other tumors upon exposure to lurbinectedin. In this preclinical study we investigated the therapeutic efficacy of lurbinectedin in large number of profiled SCLC cell lines representing the four SCLC subtypes (A, N, P and I) to identify candidate markers of drug sensitivity and resistance. In 12 human-derived and 3 mouse model-derived SCLC cell lines, the majority of cell lines were highly sensitive to lurbinectedin at a very low doses (median IC50 0.52 nM, range 0.08-1.84nM). We also observed increased markers of DNA damage following treatment in sensitive cell lines (e.g., γH2AX, ChK1, RPA32) by western blot. Notably, no subtype specific difference in lurbinectedin sensitivity was observed among the four subtypes (A, N, P and I) of SCLC cells. However, cell lines with higher SLFN11 expression were more sensitive to lurbinectedin. Western blot experiments showed significant increase in phosphorylation of γH2AX, ChK1, RPA32 in lurbinectedin treated SCLC cells compared to DMSO treatment. Together our preliminary data confirm lurbinectedin as a potent treatment option for SCLC, and support a rationale for potential combinations with other DNA damaging agents. Citation Format: Kiran Kundu, Robert J. Cardnell, Li Shen, C. Allison Stewart, Kasey Cargill, Carl M. Gay, Jing Wang, Lauren A. Byers. Characterization of lurbinectedin as a single agent and in combinations with DNA damage response inhibitor for the treatment and bio-marker discovery of SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1022.

Retinoblastoma: from discovery to clinical management.

The retinoblastoma gene (RB1) was the first tumour suppressor cloned; the role of its protein product (RB) as the principal driver of the G1 checkpoint in cell cycle control has been extensively studied. However, many other RB functions are continuously reported. Its role in senescence, DNA repair and apoptosis, among others, is indications of the significance of RB in a vast network of cellular interactions, explaining why RB loss or its malfunction is one of the leading causes of a large number of paediatric and adult cancers. RB was first reported in retinoblastoma, a common intraocular malignancy in the paediatric population worldwide. Currently, its diagnosis is clinical, and in nondeveloped countries, where the incidence is higher, it is performed in advanced stages of the disease, compromising the integrity of the eye and the patient's life. Even though new treatments are being continuously developed, enucleation is still a major choice due to the late disease stage diagnosis and treatments costs. Research into biomarkers is our best option to improve the chances of good results in the treatment and hopes of patients' good quality of life. Here, we recapitulated the history of the disease and the first treatments to put the advances in its clinical management into perspective. We also review the different functions of the protein and the progress in the search for biomarkers. It is clear that there is still a long way to go, but we should offer these children and their families a better way to deal with the disease with the community's effort.